WO2018078643A1 - Monoclonal antibodies neutralizing the tetanus toxin - Google Patents

Abstract

Anti-tetanus monoclonal antibodies and methods for the production thereof.

Description

MONOCLONAL ANTIBODIES NEUTRALIZING THE TETANUS TOXIN

FIELD OF THE INVENTION The present invention relates to monoclonal antibodies and compositions comprising the same that are capable of neutralizing the tetanus toxin (also known as tetanospasmin), and to the production and use thereof.

BACKGROUND

Tetanus is a medical condition, characterised by prolonged and severe contractions of skeletal muscle fibres, which is in many cases fatal if left untreated. It is caused by a toxin (tetanospasmin) which is produced by the anaerobic bacterium Clostridium tetani. C. tetani is widely distributed in the environment, especially in soil, and can infect a host through an open or contaminated wound. Once an infection is established, the bacterium releases tetanospasmin which is distributed by the blood and lymphatic systems of the host.

Tetanospasmin, also referred to as tetanus toxin, is a neurotoxin that acts on the central nervous system, and is one of the most potent toxins currently known. It binds to gangliosides on peripheral nerves, and is subsequently internalised. It is then moved from the peripheral to the central nervous system by retrograde axonal transport. The toxin is internalised into presynaptic cells and cleaves synaptobrevin. This in turn prevents the release of the inhibitory neurotransmitter GABA, thereby preventing inhibitory control of motor neurons and so causing the muscular spasms characteristic of tetanus.

The toxin is synthesised as a single 150kDa polypeptide chain which is converted into its active form by cleavage by a bacterial protease. The protease cleaves the polypeptide into two chains, a 50kDa light chain and a lOOkDa heavy chain, which remain held together by a disulfide linkage. The light chain, also referred to as the A- fragment of the toxin, is responsible for the cleavage of synaptobrevin. The amino- terminal portion of the heavy chain, also referred to as the B -fragment of toxin, is involved in internalisation of the toxin. The carboxyl-terminal portion of the heavy chain, also referred to as the C-fragment of the toxin, mediates binding of the toxin to the neuronal gangliosides.

Tetanus is typically prevented by providing a patient with active immunity against the tetanus toxin by administering a tetanus toxoid vaccine. However, in some circumstances the patient may have insufficient time or ability to develop an effective immune response of his or her own (i.e. effective levels of active immunity) against the tetanus toxin. For example, the patient may be immunocompromised, may be at imminent risk of C. tetani infection, or (in particular) may already be suffering from tetanus. In such circumstances, the established treatment is to administer anti-tetanus antibodies to the patient so as to provide the patient with passive immunity against tetanus. In cases where the patient already has tetanus, antibiotics are usually administered alongside the anti-tetanus antibodies, so as to treat the C. tetani infection at the same time as neutralising the tetanus toxin with the anti-tetanus antibodies.

Traditionally, the anti-tetanus antibodies that have been used have been polyclonal antibodies obtained from the blood plasma of volunteers who have been hyperimmunized against tentanus. However, the use of polyclonal antibodies has a number of recognized drawbacks, not least of which are the continuing need for a number of volunteer donors sufficient to meet the demand for antibody, and the risk of contamination of the antibody preparation with any toxins, bacteria, viruses or other pathogens that may be present in the donor's blood.

Whereas polyclonal antibodies constitute antibodies secreted by a mixture of different plasma cells, and therefore constitute a heterogeneous mixture of antibodies of unknown composition secreted against a specific antigen and typically recognizing a variety of epitopes, monoclonal antibodies are produced from cells that are all clones of a single parent cell (i.e. from cells that are all of the same cell line), and thus are produced as a homogeneous population of antibodies, as is well known in the art. The cell lines from which monoclonal antibodies are produced can be developed and cultured in-vitro, and this means monoclonal antibodies have the potential to be produced as and when required, both in large amounts and at high levels of purity. Accordingly, monoclonal anti-tetanus antibodies have a number of potential advantages over the polyclonal antitetanus antibody preparations that have traditionally been used. A number of techniques for producing monoclonal antibodies in general, and monoclonal anti-tetanus antibodies in particular, have been described. For example: US-B-6,475,787 discloses a method for preparing monoclonal antibodies, in which a suitable eukaryotic host cell is transformed with a DNA sequence encoding an antibody heavy chain and a DNA sequence encoding an antibody light chain, the two sequences being linked to different amplifiable marker genes so as to allow differential amplification of the heavy and light chain DNAs in order to optimize the relative gene copy numbers of the heavy and light chain DNAs. In a preferred embodiment the host cell is a Chinese Hamster Ovary (CHO) cell which is DHFR deficient (i.e. incapable of producing dihydrofolate reductase), one of the amplifiable marker genes is an adenosine deaminase (ADA) gene, and the other is a DHFR gene. Amplification of the DNA encoding one antibody chain and linked in the ADA gene can then be achieved by treating the recombinant cells with increasing concentrations of 2'-deoxycoformycin, whilst amplification of the DNA encoding the other antibody chain and linked in the DHFR gene is achieved by treating the cell with increasing concentrations of methotrexate (MTX). EP-B-0562132 describes the preparation of monoclonal anti-tetanus toxin antibodies and pharmaceutical compositions comprising the same. Mononuclear cells expressing anti-tetanus toxin antibody were obtained from volunteers immunized with tetanus toxoid. The mononuclear cells were EBV-transformed and subcultured, and positive cultures (expressing anti-tetanus toxin antibodies) were fused with a human heteromyeloma cell line. Hybridomas secreting anti-tetanus toxin antibodies were then cloned to obtain a number of cell lines expressing monoclonal anti-tetanus toxin antibodies. Monoclonal antibodies from 100 different cell lines were tested, of which the monoclonal antibodies from 7 cell lines were found to have tetanus toxin neutralising capacity in mice. The monoclonal antibodies from 5 of these cell lines were specific for the A-fragment of tetanus toxin, and the monoclonal antibodies from the other two had binding affinity for both the A- and C-fragments of tetanus toxin.

Nevertheless, there remains a need for further anti-tetanus monoclonal antibodies, compositions comprising the same, and methods for the production thereof. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts the amino acid sequence of the heavy chain variable region of monoclonal antibody 191.2D6, in which the complementarity determining regions have been highlighted in bold and underlined;

Figure 2 depicts the amino acid sequence of the light chain variable region of monoclonal antibody 191.2D6, in which the complementarity determining regions have been highlighted in bold and underlined; Figure 3 depicts the amino acid sequence of the heavy chain variable region of monoclonal antibody 215.6G7, in which the complementarity determining regions have been highlighted in bold and underlined;

Figure 4 depicts the amino acid sequence of the light chain variable region of monoclonal antibody 215.6G7, in which the complementarity determining regions have been highlighted in bold and underlined;

Figure 5 is a map of plasmid vector pSBYL3; Figure 6 is a map of plasmid vector pSB YL11 ;

Figure 7 is a detailed scheme of a heavy chain insert subcloned into the expression vector PSBYL3 using the Xbal and BamHI restriction sites; Figure 8 is a detailed scheme of a light chain insert subcloned into the expression vector PSBYL11 using the Xbal and BamHI restriction sites;

Figure 9 is a detailed scheme of a heavy chain insert containing an internal BamHI site subcloned into the expression vector PSBYL3 using the Xbal restriction site and a blunt end (created by digesting the insert by EcoRV restriction enzyme and by cutting the vector by BamHI restriction enzyme followed by polishing the end with the Klenow fragment of DNA polymerase); and Figure 10 is a detailed scheme of a light chain insert containing an internal BamHI site subcloned into the expression vector PSBYL11 using the Xbal restriction site and a blunt end (created by digesting the insert by EcoRV restriction enzyme and by cutting the vector by BamHI restriction enzyme followed by polishing the end with the Klenow fragment of DNA polymerase).

DETAILED DESCRIPTION

According to a first aspect of the present invention, there is provided an anti-tetanus monoclonal antibody comprising: (a) a heavy chain variable region having first, second and third complementarity determining regions (CDRs) that are substantially identical or identical to the respective first, second and third CDRs of the heavy chain variable region of monoclonal antibody (mAb) 191.2D6; and (b) a light chain variable region having first, second and third CDRs that are substantially identical or identical to the respective first, second and third CDRs of the light chain variable region of mAb 191.2D6.

Monoclonal antibody 191.2D6 is described in further detail in the Examples that follow. It binds to the C-fragment of the tetanus toxin, and accordingly the monoclonal antibody according to the first aspect of the invention likewise binds to the C-fragment of the tetanus toxin. The amino acid sequences of the heavy chain and light chain variable regions of mAb 191.2D6, and the location and sequence of the first, second and third CDRs (designated CDR1, CDR2 and CDR3) of each variable region, are set out in the accompanying sequence listing and depicted in Figures 1 and 2. In the sequence listing, the amino sequence of the heavy chain variable region of mAb 191.2D6 is set out in SEQ ID NO: 2, and the amino sequence of the light chain variable region of mAb 191.2D6 is set out in SEQ ID NO: 4. Thus, alternatively stated, the anti-tetanus monoclonal antibody according to the first aspect of the present invention comprises: (a) a heavy chain variable region having first, second and third complementarity determining regions (CDRs) that are substantially identical or identical to the respective first, second and third CDRs of SEQ ID NO: 2; and (b) a light chain variable region having first, second and third CDRs that are substantially identical or identical to the respective first, second and third CDRs of the light chain variable region of SEQ ID NO: 4. According to a second aspect of the present invention, there is provided an anti-tetanus monoclonal antibody comprising: (a) a heavy chain variable region having first, second and third complementarity determining regions (CDRs) that are substantially identical or identical to the respective first, second and third CDRs of the heavy chain variable region of monoclonal antibody (mAb) 215.6G7; and (b) a light chain variable region having first, second and third CDRs that are substantially identical or identical to the respective first, second and third CDRs of the light chain variable region of mAb 215.6G7.

Monoclonal antibody 215.6G7 is also described in further detail in the Examples that follow. It does not recognise the C-fragment of the tetanus toxin, and instead binds to another fragment of the tetanus toxin. Accordingly, the monoclonal antibody according to the second aspect of the invention likewise binds to a fragment of tetanus toxin other than the C-fragment. The amino acid sequences of the heavy chain and light chain variable regions of mAb 215.6G7, and the location and sequence of the first, second and third CDRs (designated CDR1, CDR2 and CDR3) of each variable region, are set out in the accompanying sequence listing and depicted in Figures 3 and 4. In the sequence listing, the amino sequence of the heavy chain variable region of mAb 215.6G7 is set out in SEQ ID NO: 6, and the amino sequence of the light chain variable region of mAb 191.2D6 is set out in SEQ ID NO: 8. Thus, alternatively stated, the anti-tetanus monoclonal antibody according to the second aspect of the present invention comprises: (a) a heavy chain variable region having first, second and third complementarity determining regions (CDRs) that are substantially identical or identical to the respective first, second and third CDRs of SEQ ID NO: 6; and (b) a light chain variable region having first, second and third CDRs that are substantially identical or identical to the respective first, second and third CDRs of the light chain variable region of SEQ ID NO: 8.

As used herein, the term "anti-tetanus antibody" refers to both whole antibodies and to fragments thereof that bind to and are capable of neutralising tetanus toxin (tetanospasmin). The binding specificity/affinity and neutralising potency of an antibody can be measured in various ways, suitable assays for which will be known to and can be routinely implemented by one of ordinary skill in the art. For example, antibodies recognising and specifically binding to tetanus toxin, or a specific fragment thereof, can be determined using one or more standard techniques as known to one of ordinary skill in the art, such as but not limited to EIA / ELISA techniques. A protocol for determining the neutralisation potency of anti-tetanus antibodies is, for example, described in European Pharmacopoeia 4.0. Exemplary ELISA and neutralisation protocols are described in further detail in the Examples that follow.

As is well known in the art, whole antibodies are typically formed of one or two heavy and one or two light chains. The heavy and light chains each comprise a variable region and a constant region. The variable regions (also referred to as the variable domains) dictate the antibody's antigen binding specificity. Each variable domain is composed of complementarity determining regions (CDRs, of which there are typically three, designated CDR1, CDR2 and CDR3) interspersed with more conserved regions known as framework regions. On folding of the antibody to adopt the correct quaternary structure, the CDRs of a heavy and light chain together form the antigen binding site. The constant region of the heavy chain is composed of three or more constant domains and is dependent on the class (eg. IgA, IgD, IgE, IgG, or IgM) and isotype (eg. IgAl, IgA2, IgGl, IgG2, IgG3, IgG4) of the antibody. It is identical in all antibodies of the same class and isotype, but differs in antibodies of different isotypes. The light chain constant region is composed of a single constant domain which is of one of two isotypes, kappa or lambda, and is likewise identical in all antibodies of the same isotype. The constant regions of the antibodies typically mediate binding of the antibody to host tissues or factors.

Antibody fragments according to the present invention include at least the CDRs and sufficient of the framework regions to bind tetanus toxin. Exemplary types of fragment include, but are not limited to, a Fab' fragment (consisting of the variable domain and a constant domain of both the light and heavy chains), a F(ab')2 fragment (two Fab' fragments linked by a disulfide bridge at the hinge region), a Fv fragment (consisting of the variable domains only of the light and heavy chains), and other types of fragment as known to one skilled in the art. As the term is used herein, two CDRs are "substantially identical" if they have amino acid sequences that preferably are at least 80% identical and/or differ in no more than one amino acid. More preferably the sequences are at least 90% identical and/or differ in no more than one amino acid. Where the CDRs of two antibodies are at least substantially identical, it is reasonable to predict that the resulting antigen binding site of the two antibodies will have similar antigen binding properties. For example, each of the corresponding CDRs of mAbs 191.2D6 and 191.8H2 described below were found to be either identical or to differ in no more than one amino acid, and both were found to have good neutralizing potency against tetanus toxin (as is described in the Examples that follow).

It is preferred that the CDRs of the monoclonal antibody according to the first aspect of the invention are identical to the respective CDRs of mAb 191.2D6. Likewise, it is preferred that the CDRs of the monoclonal antibody according to the second aspect of the invention are identical to the respective CDRs of mAb 215.6G7.

However, where one or more of the CDRs of the monoclonal antibody of the first aspect are substantially identical, rather than identical, to those of mAb 191.2D6, or where one or more of the CDRs of the monoclonal antibody of the second aspect are substantially identical, rather than identical, to those of mAb 215.6G7, it is preferable that the difference or differences in amino acid sequence that exist constitute semi-conservative or, more preferably still, conservative substitutions. Conservative and semi-conservative substitutions can be identified using the Clustal series of programs (Multiple sequence alignment with the Clustal series of programs. (2003) Chenna, Ramu, Sugawara, Hideaki, Koike,Tadashi, Lopez, Rodrigo, Gibson, Toby J, Higgins, Desmond G, Thompson, Julie D. Nucleic Acids Res 31 (13):3497-500. PubMedID: 12824352).

In a preferred embodiment of the first aspect of the invention, the heavy chain variable region and the light chain variable region of the monoclonal antibody according to the first aspect of the invention are at least 75% identical, more preferably at least 80% identical, more preferably at least 85% identical, more preferably at least 90% identical, more preferably at least 95% identical, more preferably at least 98% identical and most preferably are identical to the respective heavy chain and light chain variable regions of mAb 191.2D6.

Likewise, in a preferred embodiment of the second aspect of the invention, the heavy chain variable region and the light chain variable region of the monoclonal antibody according to the second aspect of the invention are at least 75% identical, more preferably at least 80% identical, more preferably at least 85% identical, more preferably at least 90% identical, more preferably at least 95% identical, more preferably at least 98% identical and most preferably are identical to the respective heavy chain and light chain variable regions of mAb 215.6G7.

Techniques for identifying antibody variable regions and CDRs, comparing and aligning amino acid sequences, and determining the % identity between two amino acid sequences are well known in the art. For example, the CDRs, variable regions, and constant regions of an antibody can be determined using software such as IMGT/V- QUEST tool (Brochet, X. et al., Nucl. Acids Res. 36, W503-508 (2008). PMID: 18503082) using default settings, and/or via comparison with databases of known immunoglobulin sequences such as IMGT/GENE-DB (Giudicelli V., Chaume, D. and Lefranc M.-P., 'IMGT/GENE-DB: a comprehensive database for human and mouse immunoglobulin and T cell receptor genes' Nucleic Acids Res., 33, D256-D261 (2005) PMID: 15608191) or V-BASE. Amino acid or nucleic acid sequence sequences, whether for whole antibodies or specific parts thereof, can be aligned and their % identity determined using Clustal programs such as ClustalW or ClustalW2 (Multiple sequence alignment with the Clustal series of programs. (2003) Chenna, Ramu, Sugawara, Hideaki, Koike,Tadashi, Lopez, Rodrigo, Gibson, Toby J, Higgins, Desmond G, Thompson, Julie D. Nucleic Acids Res 31 (13):3497-500 PubMedID: 12824352) using default parameters, or using proprietary software such as Vector NTI (Invitrogen, Carlsbad, CA). In preferred embodiments of the first and second aspects of the invention, the monoclonal antibodies of the present invention further comprise a light chain constant domain and at least one heavy chain constant domain. In these embodiments the monoclonal antibody may for example be a Fab' or F(ab')2 fragment, as discussed above, or a whole antibody. The light chain constant domain is preferably of the kappa isotype. The heavy chain constant domain is preferably an IgG class constant domain. If the monoclonal antibody is a whole antibody, preferably all the heavy chain constant domains are IgG domains (i.e. the antibody comprises an IgG heavy chain constant region). In a particularly preferred embodiment, the constant domain or region is an IgG 1 constant domain or region. Preferably all constant domains (both light and heavy) are human constant domains.

The monoclonal antibodies according to the first and second aspects of the invention are, of course, produced by monoclonal techniques. Thus, the monoclonal antibody according to the first aspect of the invention is produced as a homogeneous population of antibodies produced by cells that are all clones of the same parent cell, and likewise the monoclonal antibody according to the second aspect of the invention is produced as a homogeneous population of antibodies produced by cells that are all clones of the same parent cell. In preferred embodiments, the monoclonal antibodies according to the first or second aspects of the invention are produced by a method according to the sixth aspect of the present invention, as described in further detail below.

In a particularly preferred embodiment, the anti-tetanus monoclonal antibody according to the first aspect of the invention is the monoclonal antibody 191.2D6, and the anti-tetanus monoclonal antibody according to the second aspect of the invention is the monoclonal antibody 215.6G7.

According to a third aspect of the present invention, there is provided an isolated polynucleotide encoding the light and/or heavy chain of an antibody according to the first or second aspect.

As used herein, the term an "isolated polynucleotide" refers to a polynucleotide that has been isolated from a cellular environment (i.e. it is not present in a cell or organism), and it can be in purified form (i.e. substantially free of other polynucleotides, proteins, and cellular components) or form part of composition containing other polynucleotides and/or compounds. The term "encoding a light chain" refers not only to sequences encoding whole light chains, but also to sequences encoding fragments thereof (such as the variable domain only) where the antibody to be expressed is an antibody fragment as described above. Similarly, the term "encoding a heavy chain" refers not only to sequences encoding whole heavy chains, but also to sequences encoding fragments thereof (such as the variable domain only or the variable domain plus one or more but not all of constant domains) where the antibody to be expressed is an antibody fragment as described above.

Exemplary nucleic acid sequences include the relevant coding sequences of the nucleic acid sequences, set out in the accompanying sequence listing, that encode mAbs 191.2D6 and 215.6G7. The nucleic acid sequences may be modified for optimised expression (i.e. transcription and/or translation) in the desired host cell, for example via techniques known to one of skill in the art. For example, optimization of a nucleic acid sequence may comprise one or more of: optimizing the GC distribution and AT/GC stretches (to enhance the stability of mRNA); removing inhibitory motifs (such as premature polyA signals); removing cryptic splice sites (to prevent alternative, incorrect splicing of mRNA); optimizing mRNA secondary structure (to avoid tight hairpins possibly stalling translation); optimizing open reading frames (to avoid secondary or alternative reading frames); and optimizing codon usage (to avoid rare codons that can slow down translation). According to a fourth aspect of the present invention, there is provided an expression system comprising one or more expression vectors and including coding sequences encoding the light and heavy chains of an antibody according to the first or second aspect. The expression vector(s) may be of any type used in the art, such as for example plasmids or viral vectors. The expression vectors of the present invention are preferably plasmids. In addition to the antibody chain coding sequences, the vector(s) will include the necessary regulatory sequences for proper transcription and translation of the coding sequences in the intended host cell, such as for example a suitable promoter and polyadenylation (polyA) sequence. The vector(s) may further comprise a Kozak sequence for increased efficiency of expression, and/or a sequence encoding for a signal peptide for post translational transport of the antibody chains (for example for secretion of the antibodies). A further preferred feature is the presence of one or more antibiotic resistance genes and/or other forms of selection marker, allowing for selection of cells that have been stably transfected with the vector, and/or that display stronger expression of the antibody coding sequences, as discussed below in more detail.

The promoters and poly(A) sequences used to drive expression of the light and heavy chain coding sequences may be of any type used in the art. A variety of different promoters and poly(A) sequences are known, the selection of appropriate promoters and poly(A) sequences for use in the chosen host cell being well within the abilities of one of ordinary skill in the art. For example, suitable promoters for use in a mammalian host cell include the SV40 early and late, elongation factor 1 (EF-1), and cytomegalovirus (CMV) promoters. Suitable poly(A) sequences include those from SV40 poly(A), bovine growth hormone (BGH), thymidin kinase (TK), and human growth hormone (hGH). In a preferred embodiment, the light and heavy chain coding sequences are driven by the human elongation factor 1 alpha (hEF- 1 alpha) promoter and BGH poly(A) sequence.

In one embodiment, the expression system comprises an expression vector that includes both the coding sequence for the light chain and the coding sequence for the heavy chain. In an alternative embodiment, the light and heavy chain coding sequences are carried by separate vectors, the expression system comprising: a first expression vector including the coding sequence encoding the light chain; and a second expression vector including the coding sequence encoding the heavy chain. In this embodiment, one or both of said first and second expression vectors may include a dihydrofolate reductase (DHFR) selection marker. This marker comprises a coding sequence for DHFR, which is coupled to suitable promoter and polyadenylation sequences, preferably the SV40 early (SV40E) promoter and poly(A) sequences. DHFR allows de novo synthesis of the DNA precursor thymidine. Therefore, by transfecting a host cell-line which is DHFR deficient (i.e. which is itself incapable of producing DHFR), one can then select for cells which have stably integrated the vector into their genome by growing the cells in a medium deficient in deoxyribonucleosides and ribonucleosides. Moreover, once the successfully transfected cells have been isolated, the expression of the desired coding sequence(s) (i.e. the light and/or heavy chain) can be amplified by using the DHFR inhibitor methotrexate (MTX), which causes some cells to react by amplifying large regions of DNA surrounding the DHFR gene.

In a preferred embodiment, one of said first and second expression vectors includes an antibiotic resistance gene (a nucleic acid sequence that imparts resistance to the antibiotic in question) but does not include the DHFR coding sequence, and the other of said expression vectors includes the DHFR coding sequence but does not include a gene providing resistance to the same antibiotic as said antibiotic resistance gene. The antibiotic resistance gene may be of any type used in the art. For example, suitable antibiotic resistance genes for imparting resistance to a mammalian host cell include: aminoglycoside (e.g. neomycin, hygromycin B) resistance genes, such as neomycin phosphotransferase {npt) and hygromycin B phosphotransferase (hpt, hph); aminonucleoside (eg. puromycin) resistance genes such as puromycin N- acetyltransferase (pac); glycopeptide (e.g. bleomycin, phleomycin) resistance genes such as the ble gene; and peptidyl nucleoside (eg. blasticidin) resistance genes such as the bis, bsr or bsd genes. As with the DHFR selection marker, the antibiotic resistance gene may as needed be coupled to any suitable promoter and polyadenylation sequences. Preferred are the SV40 early (SV40E) promoter and poly(A) sequences. In a particularly preferred embodiment, the antibiotic resistance gene comprises a neomycin phosphotransferase (Npt) coding sequence. The cells stably transfected with the vector including the Npt coding sequence can then be selected for by growing the cells in a medium containing neomycin, or a neomycin analog such as G418, the toxic effects of which are neutralized by Npt.

Thus, the above described embodiment, in which one vector has the DHFR selection marker and the other has the antibiotic selection gene, allows for selection of only those cells which have stably integrated both vectors into their genome by growing the cells in a medium deficient in deoxyribonucleosides and ribonucleosides and containing the relevant antibiotic (such as neomycin or a suitable analogue where the antibiotic resistance gene is the npt gene). Cells that were not transfected or were transfected with only one plasmid will not survive the selection process. Moreover, because the co-transfected plasmids often integrate into one spot of the genome, subsequent growth of the successfully transfected cells in increasing concentrations of MTX can still be used to effectively amplify expression of the antibody chains encoded by both vectors (i.e. to amplify expression of both the heavy and light chain sequences).

It should be noted that while, in this embodiment, the vector carrying the DHFR selection marker does not include a gene providing resistance to the same antibiotic as the antibiotic resistance gene carried by the other vector, it and indeed both vectors may further comprise a different antibiotic resistance gene providing resistance against a further antibiotic. Again, the additional antibiotic gene may be of any type used in the art. For example, where one but not both vectors carries an Npt coding sequence (providing resistance against neomycin and analogues thereof) both vectors may usefully additionally comprise an ampicillin resistance (AmpR) gene, for the purpose of providing ampicillin resistance when incorporated into a bacterial host cell. Antibiotic resistance genes that are commonly used to impart resistance in bacterial hosts include: □ lactamase genes (providing resistance to□ lactam antibiotics such as ampicillin and other penicillins), such as TEM-1 β-lactamase; genes providing resistance to aminoglycosides such as streptomycin, kanamycin, tobramycin, and amikacin; and tetracycline (e.g. tetracycline, doxycycline, minocycline, oxtetracycline) resistance genes, such as the tetA genes. According to a fifth aspect, the present invention provides a cell transformed with an expression system according to the fourth aspect.

The host cells for use in the present invention may be of any suitable type.

However, in a preferred embodiment the host cell (cell to be transfected) is a eukaryotic cell, more preferably a vertebrate cell, most preferably a mammalian cell. A variety of suitable mammalian host cells are available. Preferred mammalian host cells include: all variants of CHO cells, such as CHO Kl and DHFR-deficient CHO (DG44, DXB 11);

HEK293; BHK; COS-1 and COS-7; NSO; and PER.C6. The preferred host cells are

Chinese Hamster Ovary (CHO) cells, in particular DHFR-deficient CHO cells (DHFR- CHO cells). The host cells may be transfected with the expression vectors using standard techniques and transfection conditions, such as are known in the art.

Exemplary transfection conditions are provided in the Examples that follow. According to a sixth aspect, the present invention provides a method of manufacturing monoclonal antibodies, comprising cultivating cells according to the fifth aspect, and recovering the antibodies from the culture medium. Exemplary growth media and conditions are provided in the Examples that follow, but any suitable growth conditions and commercial or custom growth media can be used, as are routinely employed in the art. Likewise, any standard technique for purifying secreted antibodies from growth media can be employed, exemplary techniques being again outlined below.

According to a seventh aspect, the present invention provides a pharmaceutical composition comprising an anti-tetanus monoclonal antibody according to the first aspect of the invention and/or an anti-tetanus monoclonal antibody according to the second aspect of the invention. Typically, the pharmaceutical compositions according to this aspect of the invention will also comprise a pharmaceutically acceptable carrier. The monoclonal antibodies can be formulated as desired dependent on the intended route of administration. For example, the monoclonal antibodies may be formulated for injection (for example intra-muscularly) as a suspension of the antibodies in a suitable liquid carrier, analogous to conventional polyclonal anti-tetanus formulations. Exemplary concentrations of anti-tetanus antibody range from 250 to 1000 IU/ml (as measured by bioassay in mice as described below in further detail). Exemplary liquid carriers include buffer solutions such as: phosphate-buffered saline; and glycine saline buffer. An exemplary phosphate-buffered saline solution would, for example, be 20 mM phosphate buffer containing 150 mM NaCl, adjusted to pH 6.8. An exemplary glycine saline buffer would, for example, be 0.3 M glycine containing 0.15 M NaCl, adjusted to pH 6.5.

The composition may comprise monoclonal antibodies of a single type only, i.e. only a monoclonal antibody according to the first aspect of the invention or only a monoclonal antibody according to the second aspect of the invention, but more preferably the composition comprises a combination of more than one type of monoclonal antibody. In particular, it is preferred that the composition comprises an anti-tetanus monoclonal antibody that binds to the C-fragment of tetanus toxin and an anti-tetanus monoclonal antibody that binds to a fragment of tetanus toxin other than the C-fragment of tetanus toxin. Thus, where the composition comprises an anti-tetanus monoclonal antibody according to the first aspect of the invention (which antibodies, as noted above, bind to the C-fragment of tetanus toxin), the composition preferably further comprises an anti- tetanus monoclonal antibody that binds to a fragment of tetanus toxin other than the C-fragment of tetanus toxin. In a preferred embodiment, the two types of antibody are present in a 3: 1 to 1:3 ratio, and most preferably are present in a 1: 1 ratio (as measured on a w/w basis). As is demonstrated in the Examples that follow, the anti-tetanus monoclonal antibody according to the first aspect of the invention has a synergistic effect in neutralising tetanus toxin when used in combination with an anti-tetanus monoclonal antibody that binds to a fragment of tetanus toxin other than the C-fragment of tetanus toxin.

Likewise, where the composition comprises an anti-tetanus monoclonal antibody according to the second aspect of the invention (which antibodies, as noted above, bind to a fragment of tetanus toxin other than the C-fragment of tetanus toxin), the composition preferably further comprises an anti-tetanus monoclonal antibody that binds to the C-fragment of tetanus toxin. In a preferred embodiment, the two types of antibody are present in a 3: 1 to 1:3 ratio, and most preferably are present in a 1: 1 ratio (as measured on a w/w basis). As is demonstrated in the Examples that follow, the antitetanus monoclonal antibody according to the second aspect of the invention has a synergistic effect in neutralising tetanus toxin when used in combination with an anti-tetanus monoclonal antibody that binds to the C-fragment of tetanus toxin. In a particularly preferred embodiment, the composition comprises a mixture of both an anti-tetanus monoclonal antibody according to the first aspect of the invention and an anti-tetanus monoclonal antibody according to the second aspect of the invention. In a preferred embodiment, the two types of antibody (i.e. the antibody according to the first aspect and the antibody according to the second aspect) are present in a 3: 1 to 1:3 ratio, and most preferably are present in a 1: 1 ratio (as measured on a w/w basis). As is demonstrated in the Examples that follow, the anti-tetanus monoclonal antibodies according to the first and second aspects of the invention show a particularly marked synergistic effect in neutralising tetanus toxin when they are used in combination with each other. Where the composition comprises a combination of more than one type of monoclonal antibody, it preferably contains only the two types of monoclonal antibody as defined above. However, if desired addition types of monoclonal antibody could be included in the composition, such as where the compositions comprises at most 3, 4, 5, 10, 15, 20 25 or 50 different types of monoclonal antibody.

According to an eighth aspect, the present invention provides a method of providing a patient with passive immunity against tetanus, comprising administering an effective amount of a monoclonal antibody according to the first or second aspect or a pharmaceutical composition according to the seventh aspect.

According to a ninth aspect, the present invention provides a monoclonal antibody according to the first or second aspect, or a pharmaceutical composition according to the seventh aspect, for use in a method of providing passive immunity against tetanus.

According to a tenth aspect, the present invention provides the use of a monoclonal antibody according to the first aspect and/or a monoclonal antibody according to the second aspect in the manufacture of a medicament for providing passive immunity against tetanus.

The invention is further illustrated in the following non-limiting Examples, with reference also to the accompanying drawings.

Examples Immunization of mice

As a first step in generating murine hybridomas producing antibodies against tetanus toxin, Balb/c mice divided into three groups were immunized according to the scheme presented in Table 1. The antigen was freshly prepared before each immunization and was typically injected in a total volume of 200 Dl per mouse. Tetanus toxoid and tetanus toxin were products of List Biological Laboratories (Cat. No. 191B and 190, respectively).

Assessment of the immunization efficiency was performed by determining the titer of the anti-tetanus antibodies in the sera of the immunized mice. The immune sera were diluted 5,000x, ΙΟ,ΟΟΟχ, and 20,000x and assayed by ELISA (below). The mice with the highest titers were used as a source of splenocytes for preparing the hybridomas. Some mice received another dose of tetanus toxoid in FIA followed later by a booster injection of toxin before being used for generating hybridomas.

Table 1. Scheme of immunization of mice against tetanus toxin. Abbreviations: FCA, Freund's complete adjuvant; FIA, Freund's incomplete adjuvant; RAS, Ribi Adjuvant System; s.c, subcutaneously; i.p., intraperitoneally)

Enzyme-linked Immunosorbent Assay (ELISA) for determining the concentration of mouse anti-tetanus antibodies One day before the assay, Immulon 4HBX Microtiter® Immunoassay plates

(DYNEX Technologies) were coated with 50 μΙ/well of the reconstituted tetanus toxoid (Table 2) dissolved in coating buffer (0.5 μg/ml final concentration) and incubated overnight at 4°C. Next day, the plates were washed and blocked for at least 1 hour at room temperature. The blocking solution was aspirated and the blanks, standards, and samples (undiluted supernatants or serially diluted sera) were pipetted into the wells at 50 μΙ/well and incubated for 1 hour at room temperature. After the incubation the plates were washed and the anti-mouse HRP conjugate, diluted 1000-5000x in sample diluent, was added to the wells at 50 μΙ/well. The plates were incubated for 1 hour at room temperature and then thoroughly washed. 100 μΙ of the reconstituted substrate was pipetted into each well and the plate was incubated at room temperature in the dark until color developed (approx. 15 min). The absorbance was read at 650 nm without stopping the reactions. The results were analyzed with the SOFTmax PRO software (Molecular Devices). Table 2. Reagents and buffers for ELISA assay. Abbreviations: PBS, phosphate-buffered saline; BSA, bovine serum albumin; TMB, 3 ,3 ' ,5,5 ' -tetramethylbenzidine.

Antigen: Lyophilized tetanus toxoid (List Biological Laboratories, Cat. No. 191B)

Coating buffer: 50 mM carbonate buffer pH 8.5

^"Sample diluent and blocking buffer: PBS + 1% BSA

Wash buffer: PBS + 0.2% Tween-20

Standards: Monoclonal anti-tetanus toxin fragment C (Roche, Cat. No. 1131621) serially diluted from 1 to 0.01 μg/ml

Secondary antibody: Sheep anti-mouse IgG HRP conjugate (Sigma, Cat. No. A6782) Substrate: TMB (ScyTek TM1 Standard sensitivity soluble TMB substrate)

Screening of anti-tetanus antibodies for binding to tetanus toxin fragment C

Determination whether the antibodies bind to fragment C of the tetanus toxin was performed in an ELISA assay that was essentially the same as the protocol above, with the only difference being that the Immulon 4HB plates were coated with the reconstituted tetanus toxin fragment C (List Biological Laboratories, Cat. No. 193) at ^g/ml.

Estimation of the neutralization potency of anti-tetanus antibodies in mouse sera

The protocol for determination of the potency of human tetanus immunoglobulin is provided in European Pharmacopoeia. To estimate the neutralization capacity of mouse or chimeric anti-tetanus antibodies in sera of mice and in cell culture supernatants of hybridomas and recombinant CHO cell lines, a modified version of the protocol from European Pharmacopoeia 4.0 was established. The main modification was the adjustment of the test dose of the tetanus toxin. A 100-fold lower test dose (Lp/1000) was used compared to the Pharmacopoeia protocol (Lp/10). The Lp/1000 dose was determined in the same way as in the Pharmacopoeia protocol; however, a 100-fold lower amount of the reference preparation of human tetanus immunoglobulin (NIBSC), calibrated in International Units, was used. To simplify the test and lower its cost, two mice per group were employed instead of six specified in the Pharmacopoeia protocol. For the toxin from List Biological Laboratories (Cat. No. 190), the Lp/1000 test dose was established to be 20 ng. For estimating the neutralizing capacity of antibodies in sera of immunized mice, the Lp/1000 dose was mixed with 0.5 ml of 1000-5000x diluted antisera, incubated for 60 min at room temperature, and injected s.c. into the neck region of test mice (two mice per dilution). The mice were observed for signs of paralysis over the period of four days and the observations were recorded on a daily basis. Mice that became paralyzed were promptly euthanized by C02 asphyxiation. Diluent only (0.2% gelatin in sterile PBS) served as a negative control; diluent plus tetanus toxin but with no antibody served as a positive control.

Fusion

Three days before fusion, each mouse received a booster shot of ^g tetanus toxin i.p. As expected for animals that acquired a substantial immunity against the toxin, the mice did not develop any symptoms of paralysis over the three-day period. Each animal was euthanized by C02 asphyxiation and disinfected by swabbing with 70% ethanol. The spleen was dissected out under aseptic conditions and placed in a small Petri dish in 5 ml of serum-free medium (SFM, Table 3). The splenocytes were isolated by mincing the spleen between two sterile, frosted glass slides, and the cell suspension was transferred to a 15 ml tube. Cells still remaining in the Petri dish were recovered in additional 5 ml of SFM and added to the tube. After centrifugation (5 min at 400 x g), the contaminating red blood cells were lysed by 5 ml ammonium chloride solution (Table 3) for 1 min. A small sample was set aside and used for counting. During the above procedure, the Sp2/0 myeloma cells were harvested, counted, and centrifuged (5 min at 400 x g). The number of Sp2/0 cells that would yield an approximate ratio 1:2 to the splenocytes was resuspended in 20 ml of SFM in a 50 ml tube. The splenocytes were added to the tube, along with 20 ml of 3% dextran (Table 3). The tube was gently mixed and spun down for 5 min at 400 x g.

The supernatant was carefully removed and the cell pellet was dispersed by flicking the tube. 1 ml of 35% PEG solution (Table 3) was added and gently mixed with the cells, at which point the cells agglutinated. After one minute, 20 ml of SFM was added, followed by 20 ml of growth media containing 20% FBS (Table 3). The tube was gently mixed by inversion and spun down for 5 min at 400 x g. The supernatant was aspirated, the cell pellet was gently resuspended in 150-300 ml of selective media, and plated into 10-20 96- well cell culture plates at 150 μΐ of cell suspension per well. The plates were incubated in a humidified C02 incubator set to 7.0- 8.5% C02. The media in the plates was changed after four days and then as needed, depending on the growth of hybridoma colonies.

Table 3. Media and reagents for hybridoma fusions. Abbreviations: FBS, fetal bovine serum; PBS, phosphate-buffered saline; PEG, polyethylene glycol; SFM, serum-free medium.

Serum-free medium: RPMI or DMEM/F12 + 15 mM HEPES

Growth medium: DMEM/F12 + 20% FBS + 2 mM glutamine + penicillin/streptomycin

Selective medium: Growth medium + 0.1 mM hypoxanthine + 200 μg/ml azaserine 0.17 M NH₄C1

3% dextran in PBS

PEG 1500, 50% w/v solution (Roche) diluted to 35% in SFM

Screening of hybridomas

The growth of the hybridomas was monitored under a microscope. When the colonies became visible, the supernatants from wells in which the medium started to turn yellow were collected and the wells were re-fed with a fresh medium. The supernatants were assayed by ELISA (above) in order to identify the colonies producing antibodies recognizing tetanus toxoid and exclude the colonies either not producing any antibodies or producing antibodies with different specificities. Colonies producing anti-tetanus antibodies were expanded, cryopreserved, and subcloned.

Subcloning of hybridomas

In order to select single-cell clones, hybridomas were plated in an appropriate number of flat-bottom 96-well plates at 0.5-1 cell per well. During the process, the cell growth and health was monitored under the microscope. Cells were cultured for approximately two weeks prior to selection of the best producing clones by screening with ELISA. Isotype determination

The isotypes of all mouse monoclonal antibodies were determined with the help of a commercial kit (IsoStrip Mouse Monoclonal Antibody Isotyping Kit; Roche Cat. No. 1-493-027). The assay was performed directly on hybridoma cell culture supernatants (diluted 20x in PBS) or on purified antibodies diluted to 0.1 μg/ml. The diluted antibodies were incubated with the test strips according to manufacturer's instructions.

Estimation of the neutralization potency of anti-tetanus antibodies in hybridoma supernatants For measuring the neutralizing capacity of antibodies in cell culture supernatants of hybridomas and recombinant CHO cell lines, only 5 ng (1/4 of the Lp/1000 dose) was routinely used. The toxin was mixed with 0.5 ml of undiluted supernatant; the rest of the procedure was the same as described above for the test of the antisera.

Hybridoma clones selected for development of recombinant cell lines

Mouse monoclonal antibodies and their corresponding hybridoma clones selected for development of recombinant chimeric antibodies are listed in Table 4.

Table 4. Designation of anti-tetanus antibodies

Antibody Hybridoma Isotype Binds to fragment C of clone tetanus toxin

190 15A5 190 15A5.G2 IgGl, kappa Yes

190 16F3 190 16F3.D5 IgGl, kappa Yes

190 3H6 190 3H6.G3 IgGl, kappa Yes

191 2D6 191 2D6.E8 IgGl, kappa Yes

191 8H2 191 8H2.H9.1G7 IgGl, kappa Yes

207 10A3 207 10A3.H11 IgGl, kappa No

208 14F9 208 14F9.E9 IgGl, kappa No

209 1C7 209 1C7.H10 IgGl, kappa No

215 6G7 215 6G7.D12 IgGl, kappa No RNA isolation

Total RNA from the hybridoma cells was purified using Trizol reagent (Invitrogen) according to the protocol suggested by the manufacturer with the additional step of RNA extraction with chloroform to remove traces of phenol. Spectrophotometrical RNA quantification was carried out at 260 nm assuming 1 OD to be equivalent to 40 μg/ml RNA.

First strand synthesis

The first strand of cDNA was synthesized using the Super Script III First-Strand System for RT-PCR (Invitrogen) according to the protocol suggested by the supplier. The reactions were primed by primers IgGl R_ HC (SEQ ID NO: 13) and MKC-R1 (SEQ ID NO: 14) that contain sequences complementary to the constant region of murine gammal and kappa chains, respectively. These primers were designed based on the sequence information acquired from public databases.

RNA hydrolysis

The removal of RNA molecules from reverse transcription reaction was carried out by RNaseH digestion (Super Script III First-Strand System for RT-PCR) according to manufacturer's instructions. First-strand cDNA was cleaned using QIAquick PCR Purification Kit (Qiagen).

Tailing of first-strand cDNA

To facilitate amplification of first-strand cDNA with unknown 3' sequence, poly(A) tail was appended to the 3' end of each cDNA to create a defined priming site. For this purpose, recombinant Terminal Deoxynucleotidyl Transferase (Invitrogen) was used. The reaction was carried out according to manufacturer's recommendations. Reaction product was cleaned using QIAquick PCR Purification Kit. PCR amplification of Ig heavy- (HC) and light chain (LC) variable regions

The primers (SEQ ID NOs: 15 to 17) used for PCR amplification of the heavy- and light chain variable regions from the first- strand cDNA are listed below (the gene- specific portion of the primer sequence is underlined).

Degenerate forward primer OligoDTF (compatible with the poly(A) extension of the first strand of cDNA): 5 ' -GACTGAATTC AAGCTTTTTTTTTTTTTTTTTTTTNN-3 '

Reverse primer MHCnest (specific for murine gammal constant region):

5'- AGTCGTCGACGGAGTTAGTTTGGGCAGCAGATCCAGG-3'

Reverse primer MKCnest (specific for murine kappa constant region):

5'- CAGTGAATTC GGAAGATGGATACAGTTGGTGCAGCATCAG-3' PCR was carried out using PfuUltra high-fidelity thermostable DNA-polymerase

(Stratagene). The first five cycles were primed with the forward primer only at annealing temperature 45°C. The reverse, gene-specific primer was then added to the reaction and the PCR was extended for another 30-35 cycles at annealing temperature 50-65°C. Resulting fragments were gel purified using QIAquick Gel Extraction Kit (Qiagen), subcloned into a cloning vector, and sequenced.

Subcloning of PCR products into a cloning vector The purified PCR products were phosphorylated and re-purified. pBluescript cloning vector (Stratagene) was cut with EcoRV restriction enzyme, dephosphorylated, and purified. Both DNA fragments were ligated using the Quick Ligation Kit (NEB). DH5 D bacterial cells were transformed with the resulting DNA and spread onto LB plates supplemented with 40 μg/ml ampicillin and pre-treated with 50μ1 of 20mg/ml Xgal and 25μ1 of 200mg/ml Isopropyl β-D-l-thiogalactopyranoside (IPTG). Blue color of colonies signals the presence of a vector without an insert. Therefore, only white colonies containing the vector with an insert were selected for further processing.

Isolation of plasmid DNA and sequencing

Ten white colonies were picked and expanded. The plasmid DNA was isolated from the bacterial clones with QIAprep Spin Miniprep Kit (Qiagen). A control digest was performed with Hindlll plus EcoRI. Inserts in plasmids yielding the expected digestion pattern were sequenced (Biotech Core) using standard sequencing primers annealing to the pBluescript vector.

Sequences of the variable regions

The sequence data were analyzed with the help of IMGT databases and software (imgt.cines.fr). More specifically, the exact sequences of variable regions and the CDRs were determined using IMGT/V-QUEST tool (Brochet, X. et al., Nucl. Acids Res. 36, W503-508 (2008). PMID: 18503082)), by selecting the immunoglobulin species (mouse), uploading the available nucleotide sequence spanning the variable region and a part of the constant region, in FASTA format, and analyzing the sequence using IMGT/V-QUEST default settings. It will be apparent from the IMGT analysis what segments are employed in a given antibody chain. Under Alignments, one can find germline sequences of V- segments, D- segments (if applicable), and J- segments of all heavy and light chains. The sequences of the signal peptides are determined as the coding sequences (starting with the ATG codon) preceding the V-segment in each antibody chain. For further information on IMGT/V-QUEST tool and IMGT/GENE- DB see also:

Lefranc M.-P., Giudicelli V., Kaas Q., Duprat E., Jabado-Michaloud J., Scaviner D., Ginestoux C, Clement O., Chaume D. and Lefranc G. IMGT, the international ImMunoGeneTics information system. Nucl. Acids Res., 2005, 33, D593-D597;

Giudicelli V., Chaume D. and Lefranc M.-P. IMGT/V-QUEST, an integrated software for immunoglobulin and T cell receptor V-J and V-D-J rearrangement analysis. Nucl. Acids Res. 2004, 32, W435-W440; and, Giudicelli V., Chaume D. and Lefranc M.-P. IMGT/GENE-DB: a comprehensive database for human and mouse immunoglobulin and T cell receptor genes. Nucl. Acids Res. 2005, 33, D256-D261. The amino acid sequences of the heavy chain (HC) and light chain (LC) variable regions for mAb 191.2D6 and mAb 215.6G7, and the corresponding nucleotide sequences encoding said heavy and light chain variable regions, are set out in the accompanying sequence listing. More specifically: SEQ ID NO: 1 is the nucleotide sequence encoding the heavy chain signal peptide (nucleotides 1-54) and variable region (nucleotides 55-408) of 191.2D6. Nucleotides 130-156 encode CDR1, nucleotides 208-228 encode CDR2, and nucleotides 343-375 encode CDR3. The amino acid sequence of the heavy chain signal peptide and variable region of 191.2D6 is given as SEQ ID NO: 2.

SEQ ID NO: 3 is the nucleotide sequence encoding the light chain signal peptide (nucleotides 1-60) and variable region (nucleotides 61-381) of 191.2D6. Nucleotides 139-156 encode CDR1, nucleotides 208-216 encode CDR2, and nucleotides 325-351 encode CDR3. The amino acid sequence of the light chain signal peptide and variable region of 191.2D6 is given as SEQ ID NO: 4.

SEQ ID NO: 5 is the nucleotide sequence encoding the heavy chain signal peptide (nucleotides 1-57) and variable region (nucleotides 58-414) of 215.6G7. Nucleotides 133-156 encode CDR1, nucleotides 208-231 encode CDR2, and nucleotides 346-381 encode CDR3. The amino acid sequence of the heavy chain signal peptide and variable region of 215.6G7 is given as SEQ ID NO: 6.

SEQ ID NO: 7 is the nucleotide sequence encoding the light chain signal peptide (nucleotides 1-60) and variable region (nucleotides 61-381) of 215.6G7. Nucleotides 139-156 encode CDR1, nucleotides 208-216 encode CDR2, and nucleotides 325-351 encode CDR3. The amino acid sequence of the light chain signal peptide and variable region of 215.6G7 is given as SEQ ID NO: 8. The amino acid sequences of the heavy chain (HC) and light chain (LC) variable regions of mAb 191.2D6 and mAb 215.6G7 are also depicted in Figures 1 to 4, in which the complementarity determining regions have been highlighted in bold and underlined. More specifically:

Figure 1 depicts the HC variable region of mAb 191.2D6, with the locations of

CDR1, CDR2 and CDR3 highlighted;

Figure 2 depicts the LC variable region of mAb 191.2D6, with the locations of CDR1, CDR2 and CDR3 highlighted;

Figure 3 depicts the HC variable region of mAb 215.6G7, with the locations of CDR1 , CDR2 and CDR3 highlighted; and

Figure 4 depicts the LC variable region of mAb 215.6G7, with the locations of CDR1, CDR2 and CDR3 highlighted.

Alignments of amino acid sequences

The amino acid sequences of all variable regions were aligned with the ClustalW program (Multiple sequence alignment with the Clustal series of programs. (2003) Chenna, Ramu, Sugawara, Hideaki, Koike,Tadashi, Lopez, Rodrigo, Gibson, Toby J, Higgins, Desmond G, Thompson, Julie D. Nucleic Acids Res 31 (13):3497-500 PubMedID: 12824352), using the default parameters from the website.

All five fragment C-binding antibodies were found to have highly similar variable regions; their HC variable regions are derived from the same VH gene, and their LC variable regions utilize the same Vn gene. Each of the CDRs of the heavy and light chains of mAb 191.8H2 were found to be either identical or substantially identical (differed in no more than one amino acid) to the corresponding CDR of mAb 191.2D6. The CDRs of the remaining three mAbs (190.15A5, 190.16F3 and 190.3H6) differed from the CDRs of mAb 191.2D6 to a greater extent. Of the antibodies not binding the fragment C, the HC variable region of mAb

207.10A3 was found to exhibit high homology (78% at the amino acid level) to the HC variable region of mAb 215.6G7, whereas the homology of the LC variable region of 207.10A3 to the LC variable region of 215.6G7 was somewhat lower (58%). The other two mAbs (208.14F9 and 209.1C7) had heavy and light chain variable regions that differed more markedly from those of 215.6G7. None of mAbs 207.10A3, 208.14F9 and 209.1C7 had CDRs that were each identical or substantially identical to the corresponding CDRs of 215.6G7.

Expression vectors

Two plasmid expression vectors, designated pCB3 and pCB 11, were constructed for expressing the antibody heavy and light chains in CHO dhfr- cells.

pSBYL3

This plasmid is illustrated in Figure 5. The map shows the vector after insertion of a generic antibody heavy chain between the Xbal and BamHI cloning sites. The components of this plasmid are as listed in Table 5.

Table 5. Components of expression vector pSBYL3

pSBYLll

This plasmid is illustrated in Figure 6. The map shows the vector after insertion of a generic antibody light chain between the Xbal and BamHI cloning sites. The components of this plasmid are as listed in Table 6.

Table 6. Components of expression vector pSBYLll

Construction of chimeric IgG molecules

Chimerization of the antibodies was achieved by assembling mouse variable regions and human constant regions in the pBluescript SK cloning vector (Stratagene). This assembly was mediated by a common restriction site engineered through silent mutations into the 3' end of each mouse variable region and the 5' end of the corresponding human constant region. For heavy chains, this restriction site was Nhel; for light chains, the site was BsiWI.

The mouse variable regions including the signal peptides were amplified with primers specific for each nucleotide sequence. The forward primers comprised the restriction site Xbal, the Kozak motif (CCACC), known to increase the efficiency of eukaryotic translation, and, immediately downstream of the Kozak motif, the sequence matching the beginning of the signal peptide of each variable region starting with an ATG codon. To enable the in-frame connection with the heavy chain constant region, the reverse primers specific for the 3' end of each heavy chain variable region were designed to add six nucleotides coding for the first two amino acids of the human gammal constant region downstream of the variable region. In primer sequences, these two codons were silently mutated in such a way that they created the Nhel restriction site. Similarly, the reverse primers specific for the 3' end of each light chain variable region were designed to add six nucleotides coding for the first two amino acids of the human kappa constant region. Again, those two codons were silently mutated, this time to introduce the BsiWI restriction site.

Human gammal and kappa constant regions (SEQ ID NO. 9-12) were amplified from cDNA prepared from human peripheral blood lymphocytes (Clontech). The nucleotide sequence encoding the human gammal constant region is shown in SEQ ID NO: 9; the amino acid sequence of the human gammal constant region is shown in SEQ ID NO: 10; the nucleotide sequence encoding the human kappa constant region is shown in SEQ ID NO: 11; and the amino acid sequence of the human kappa constant region is shown in SEQ ID NO: 12. The design of the primers was based on immunoglobulin constant region sequences available from public databases. The first two codons of the gammal and kappa constant regions were silently mutated in the forward primer sequences to create the Nhel and BsiWI restriction sites, respectively, for appending the constant regions in-frame behind the variable regions. Additionally, the forward primers carried an upstream Xbal site and the reverse primers contained a downstream BamHI site for convenient cloning of the amplified constant regions into pBluescript. Cloning of the human constant regions into pBluescript was the first step of the chimerization process. In the second step, the resulting plasmids were cut with Xbal and Nhel (gamma 1 -containing vector) or Xbal and BsiWI (kappa-containing vector) and the corresponding heavy chain or light chain variable regions were inserted. This convenient method produced chimeras where all mouse- and human-derived amino acid sequences were authentic, i.e. no unnatural mutations on the amino acid level occurred. All plasmids were sequenced to confirm the accuracy of the inserts.

Subcloning of chimeric immunoglobulin cDNA genes into mammalian expression vectors

For mammalian expression, the heavy and light chain genes were transferred from pBluescript into pSBYL3 (carrying the dhfr selection marker) and pSBYLl l (carrying the neo selection marker) mammalian expression vectors, respectively. Expression of genes of interest in these vectors is driven by a highly effective promoter derived from the human EF-l D gene. DNA fragments were excised from pBluescript by Xbal and BamHI restriction enzymes and were inserted between the same sites in the pSBYL vectors (Figs. 7 and 8). In cases where the inserts contained an internal BamHI site, the fragments were cut out from pBluescript by Xbal and EcoRV and inserted between the Xbal and blunted BamHI sites (Figs. 9 and 10). The vectors were transformed into DH5 D bacterial cells. Plasmid DNA from six colonies of each construct was prepared and the presence of inserts was verified by digestion of the purified plasmids either by Xbal and BamHI, or by Xbal and Notl.

DNA mini- and maxipreps

In order to confirm the presence and accuracy of the vectors in bacterial colonies, the plasmid DNA was routinely isolated from 3 ml cultures with the QIAprep Spin Miniprep Kit (Qiagen). Large-scale DNA preparations needed for transfections were prepared from 100 ml cultures using Qiagen Plasmid Maxi Kits. Cell culture Growth media

MEMa growth medium was used at all stages of recombinant CHO cell line development work. The components, formulation, and material sources are shown in Table 7. After the addition of all components, the complete medium was filtered through a 0.22 Dm filter (Stericup-GP 0.22 Dm filter unit, Millipore or equivalent).

Table 7 - Culture media

Medium Components Vendors Catalog # Final

concentration

CHO DXB 11 MEMa Gibco or 32561-037 or lx

Host Cell without Cellgro CV2561-049 lx

Growth ribonucleosides and

Medium 1 deoxyribonucleosides

HT, 250x Gibco 31985-070 lx

Gamma-irradiated Hyclone SH30079.03 7.5% dialyzed fetal bovine

serum (dFBS)

GlutaMax, lOOx Gibco 35050-061 lx

CHO DXB 11 MEMa Gibco 32571-036 lx

Host Cell with ribonucleosides

Growth and

Medium 2 deoxyribonucleosides

Gamma-irradiated Hyclone SH30070.03 7.5% fetal bovine serum

(FBS)

GlutaMax, lOOx Gibco 35050-061 lx

Transfectant MEMa Gibco or 32561-037 or lx

Selection without Cellgro CV2561-049 lx

Medium ribonucleosides and deoxyribonucleosides

Gamma-irradiated Hyclone SH30079.03 7.5% dFBS

GlutaMax, lOOx Gibco 35050-061 lx

Geneticin (a G-418 Gibco 10131-027 0.5 mg/ml formulation)

Freezing media The composition of the freezing media used for cryopreservation of cells is given in Table 8.

Table 8 - Components of freezing media Freezing medium 1 :

Freezing medium 2:

Maintenance of cells

Dihydrofolate reductase (DHFR)-deficient CHO DXB l l cells were grown in Host Cell Growth Medium 1 or 2 (Table 7) and were split every 3-4 days. Cell density and viability measurements

Viable cell density and cell viability was determined using the Trypan Blue exclusion method and a hemocytometer (Hausser Scientific). Transfection of CHO DUX Bll Cells

Exponentially growing CHO cells were trypsinized, resuspended in MEMa medium (Gibco) containing 7.5% FBS, and counted. 3xl0⁵ cells were seeded in each well of a six-well plate. The transfection was performed 36 hours later in the following manner:

For each well of a six-well plate, 1 μg of DNA (single transfection) or 1+1 μg of DNA (co-transfection) was diluted in 100 μΐ of MEMa medium without FBS. 12 μΐ of Plus reagent (Gibco) was added and the mixture was incubated at room temperature for 15 minutes. 8 μΐ of Lipofectamine reagent (Gibco) was diluted in 100 μΐ of MEMa medium without FBS, added to the DNA/Plus reagent mixture, and again incubated at room temperature for 15 minutes. During the second incubation, the medium in CHO cell cultures seeded in six-well plates was removed and replaced with MEMa medium without FBS (1 ml per well). The DNA solution was added drop wise to the cells and mixed with the medium by tilting the plate. After incubating the cells for 3-4 hours at 37°C, the medium was changed to MEMa with 7.5% FBS.

Every transfection was accompanied by a mock transfection performed exactly in the same manner, but with the DNA omitted, to assure the efficiency of the selection process.

Selection and screening of stable transfectants 48 hours post-transfection, the cells were harvested, resuspended in the DHFR selection medium [MEMa without ribonucleosides and deoxyribonucleosides, with 7.5% dialyzed FBS, with 0.5 mg/ml G418 (Gibco), with or without 27.5 nM methotrexate (Calbiochem)], and re-seeded in 10 cm dishes at several different concentrations. A sample of the original supernatant was saved for the determination of transient expression of IgG by ELIS A. Chimeric IgG expression analysis was performed by ELIS A (below).

Dishes that developed well-separated colonies were selected; the individual colonies were picked under the microscope and transferred to 96-well plates. The medium in the wells in which the cells became confluent was changed and the supernatants were tested 24 hours later by ELISA. Clones with the highest secretion were expanded, archived, and subjected to increasing concentration of methotrexate in order to achieve gene amplification and enhanced protein production.

Single cell cloning

In order to select single-cell clones, stably transfected cells were plated in an appropriate number of flat-bottom 96-well plates at 0.5-1 cell per well. During the process, the cell growth and health was monitored under the microscope. Cells were cultured for approximately two weeks prior to selection of the best producing clones by screening with ELISA.

ELISA for determining the concentration of chimeric antibodies

The titers of chimeric antibodies during all stages of cell line development were evaluated with the Human IgG ELISA Quantitation Kit (Bethyl Laboratories) according to manufacturer's instructions. Shortly, the Nunc Maxisorp ELISA plates were coated with Fc-specific goat anti-human IgG polyclonal antibody in phosphate -buffered saline (PBS). Plates were incubated overnight at 4°C. Next day, the plates were washed three times and blocked for 1 hour with powdered non-fat milk dissolved in the wash buffer. After a washing step, samples and standards were pipetted onto the plates and incubated at room temperature for 1 hour, followed by three washes. Secondary antibody conjugated to horseradish peroxidase (HRP) was then added to each well and the plates were incubated again at room temperature for 1 hour. Plates were washed three times with wash buffer, rinsed once with distilled water, and tapped dry. Tetramethylbenzidine (TMB)-containing substrate was added to each well and color was allowed to develop for 15 minutes at room temperature. The reaction was stopped by sulfuric acid and the plates were read on a plate reader (Bio-Rad, Molecular Dynamics, or Dynex Technologies) at 450nm. The data was analyzed with a software package supplied with the plate reader.

Estimation of the neutralization potency of chimeric anti-tetanus antibodies

The neutralizing capacity of chimeric antibodies was determined in a similar way to mouse monoclonal antibodies. The concentration of the chimeric antibody in cell culture supernatant was determined by ELISA. The supernatant was diluted with cell culture medium to a final concentration of antibody 1 μg/ml. 0.5 ml of this solution was mixed with 10 ng (1/2 of the Lp/1000 dose) of tetanus toxin, incubated for 60 min at room temperature, and injected s.c. so that each mouse received 500 ng of the antibody and 10 ng of the toxin. Culture medium only served as a negative control; culture medium plus tetanus toxin but with no antibody served as a positive control. The mice were observed for signs of paralysis over the period of 12 days and the observations were recorded daily (Table 9). Mice that became paralyzed were promptly euthanized by C02 asphyxiation.

In case a mixture of two antibodies was tested, 0.5 ml of each antibody was added to a tube, mixed with 20 ng (one Lp/1000 dose) of toxin, and incubated as above. Only 0.5 ml of this mixture was injected in each mouse making the dose of the antibody and toxin the same as for single antibodies.

Table 9. Estimation of neutralization activity of either individual antibodies or of mixtures of one fragment C-binding and one fragment C-non-binding antibody. Shown are average survival times (in days) of two mice per datapoint.

Antibody purification

The pH of the culture supernatants was adjusted to pH 7.2 with 1M NaOH. Each supernatant was filtered through a 0.2μ filter and loaded on a protein A column pre- equilibrated in phosphate -buffered saline (PBS). The column was washed with PBS to remove all the unbound material from the culture supernatant. The antibody bound to the protein A column was eluted with 0.1M Glycine (pH 2.5). The eluate was neutralized with 2M Tris buffer adjusted to pH 8.0. The eluate containing monoclonal antibody was dialyzed against PBS. The antibody concentration was determined spectrophotometrically at 280 nm using an optical density value of 1.4 OD for a 1 mg/ml solution based on the molar extinction coefficient for human monoclonal antibody.

Measurement of neutralization potency of purified chimeric anti-tetanus antibodies

Based on the estimated neutralization potency, eight chimeric antibodies were selected for more accurate measurements. Since it was clear from the previous experiments that the antibodies can act synergistically and that the best effect can be achieved by combining an antibody recognizing fragment C of the tetanus toxin with an antibody not binding to this fragment, such combinations were tested along the individual antibodies. The neutralization activity was compared to the reference preparation of human tetanus immunoglobulin (NIBSC) and expressed in international units per milligram of antibody (IU/mg; Table 10). The protocol described in European Pharmacopoeia 4.0 was strictly followed. As can be seen from table, the combination of mAbs 191.2D6 and 215.6G7 was found to have the highest neutralization potency for neutralizing tetanus toxin.

Table 10. Neutralization potencies of either individual antibodies or of combinations of one fragment C-binding and one fragment C-non-binding antibody. The potencies are expressed in IU/mg of antibody.

Chimeric

antibody; 190 15A5 190 3H6 191 2D6 191 8H2 None

Fragment Yes Yes Yes Yes N/A

C binding

207 10A3

220.0 122.0 250.0 166.0 2.5 No 208 14F9

34.0 4.4 110.0 30.5 1.4 No

209 1C7

55.0 55.0 137.0 125.0 0.2 No

215 6G7

220.0 122.0 275.0 183.0 0.2 No

None

27.5 15.7 50.0 50.0 N/A N/A

Clinical Formulations Based on the above results, it was decided to prepare a formulation comprising a 1: 1 mixture (as measured on a weight/weight (w/w) basis) of the two mAbs 191.2D6 and 215.6G7 (hereinafter referred to as "m-TIG") for use in further clinical trials.

The 1: 1 mixture (m-TIG) was prepared by culturing a CHO cell line expressing 191.2D6 and separately culturing a CHO cell line expressing 215.6G7, purifying the two mAbs separately as two separate active pharmaceutical ingredients (APIs), and then combining two mAbs in a 1: 1 proportion.

More specifically, the steps involved in preparing each mAb as a separate API were as follows:

culture CHO cells from CHO cell line expressing desired mAb (i.e. 191.2D6 or 215.6G7) in a bioreactor (in complete CD media);

harvest and clarification operations to separate and remove cells and cellular debris from the cell supernatant;

- capture mAbs from the cell supernatant using affinity chromatography; and further purification and concentration of the mAbs via, sequentially, anion exchange chromatography, nanofiltration, diafiltration, and 0.2 um filtration.

The purified liquid bulk of 191.2D6 produced by the above process and the purified liquid bulk of 215.6G7 produced by the above process were then blended in a 1: 1 w/w ratio to provide m-TIG (i.e. the 1: 1 mixture of 191.2D6 and 215.6G7). For example, if the purified liquid bulk of 191.2D6 contains 1 mg/ml of antibody, and the purified liquid bulk of 215.6G7 contains 1 mg/ml of antibody, then a 1: 1 mixture (m- TIG) can be prepared by mixing 1 volume (e.g. 1 ml) of the purified liquid bulk of 191.2D6 with 1 volume (e.g. 1 ml) of the purified liquid bulk of 215.6G7.

A pharmaceutical formulation suitable for administration via injection was prepared suspending m-TIG in a glycine saline buffer, having a pH of between 6 and 7. Vials containing 250 IU, 500 IU or 1000 IU of m-TIG in glycine saline buffer were prepared for clinical use. In each case, the vials contained (in addition to m-TIG) 30 mg glycine and 5.8 mg of NaCl. Clinical Trials

A prospective, open-label, comparative study was conducted to evaluate the safety and efficacy of m-TIG in treating confirmed cases of tetanus. 97 patients, male and female, with ages between 18 and 70 years of age

(inclusive), and with clinical manifestation of tetanus (grade I, II, III or IV, as per Ablett's classification of severity of Tetanus) were included in the study. 72 were treated with m-TIG, and the remaining 25 were treated with a reference preparation of polyclonal human anti-tetanus immunoglobulin, this being the polyclonal human anti- tetanus immunoglobulin "Tetglob"™ manufactured by Bharat Serums and Vaccines.

The treatment regimen was as follows. On day 1 of admission into the study, the patient was treated with a dose of 3000 IU-5000 IU of anti-tetanus immunoglobulin (either m-TIG or Tetglob). Additional does (maximum dose 2000 IU per day) were then administered daily from days 2 to 4 based on whether convulsions/spasms persist, and subject to documented progression or insufficient/non-response to first dose. The maximum total dose of anti-tetanus immunoglobulin (either m-TIG or Tetglob) allowed for the duration of the the study (days 1 to inclusive) was 8000 IU. On each occasion the anti-tetanus immunoglobulin (either m-TIG or Tetglob) was administered by intramuscular injection.

The results of the study are set out below in Tables 11 to 14. Table 11 - Efficacy Results

Visit Severity Grade n(%)

Table 12 - Serious Adverse Events (Death)

i Proportion of subjects 25% 44% j 0.0713

Table 13 - Adverse Events

ADVERSE m-TIG pTIG Total (N=97) EVENTS (Tetglob) n (%)

(N=72) (N=25)

n (%) n (%)

DISEASE 19 ( 35.8) 6 ( 11.3) 25 ( 47.2) PROGRESSION

ENCEPHALITIS 1 ( 1.9) 0 1 ( 1.9) VIRAL

ORAL 1 ( 1.9) 0 1 ( 1.9)

CANDIDIASIS

DYSPNOEA 1 ( 1.9) 4 ( 7.5) 5 ( 9.4)

COUGH 1 ( 1.9) 0 1 ( 1.9) PNEUMONIA 0 1 ( 1.9) 1 ( 1.9)

ASPIRATION

TACHYPNOEA 1 ( 1.9) 0 1 ( 1.9)

CONVULSION 0 3(5.7) 3 ( 5.7)

ENCEPHALOPAT 1 ( 1.9) 0 1 ( 1.9)

HY

HYPOTENSION 0 2 ( 3.8) 2 ( 3.8)

ISCHAEMIA 0 1 ( 1.9) 1 ( 1.9)

TACHYCARDIA 0 2 ( 3.8) 2 ( 3.8)

DIARRHOEA 2 ( 3.8) 0 2 ( 3.8)

PYREXIA 2 ( 3.8) 0 2 ( 3.8)

DECUBITUS 1 ( 1.9) 1 ( 1.9) 2 ( 3.8)

ULCER

HYPOKALAEMIA 1 ( 1.9) 0 1 ( 1.9)

TRISMUS 0 1 ( 1.9) 1 ( 1.9)

URINARY 1 ( 1.9) 0 1 ( 1.9)

RETENTION

TOTAL 32 ( 60.4) 21 ( 39.6) 53 (100.0)

Table 14 - Results Summary

Sr. No. Secondary Endpoints mTIG PTIG p Value

(Tetglob)

1 Duration of Hospital 10.3 (±5.84) 10.2 (± 6.51) 0.23 (>0.05)

Stay:

2 Duration of 46.9 (±14.45) 63.2 0.55

Occurrence of seconds (±23.34)

Spasms seconds

3 Duration of 44.9 (± 2.33) 43.6 (±3.89) 0.78

Respiratory minutes minutes

Assistance:

4 Proportion of 11% 28% 0.0415

Respiratory

Assistance [Stratified

by severity group

(Group 1,11)

Vs. Group IILIV 18% 16% 1.000

5 Proportion of Clinical 25 (35%) 11 (44%) 0.41

Progression

6 Proportion of Deaths 25% 44% 0.07 As can be seen from the above, 29 subjects died during this study after receiving more than 1 dose of immunoglobulin: 18 subjects in the m-TIG group and 11 subjects in the Tetglob group. These deaths were due to progression of underlying disease (tetanus), and were not considered related to the immunoglobulin being administered.

Thus, m-TIG was as safe as Tetglob in treating subjects suffering from Tetanus.

The incidence of other adverse events was less in the m-TIG group than in the Tetglob group, although the difference was not statistically significant.

The treatment with m-TIG also effectively reduced mortality in subjects with Tetanus (Grades I to IV) as compared to standard therapy using polyclonal human antitetanus immunoglobulin (Tetglob).

Claims

CLAIMS What is claimed is:

1. An anti-tetanus monoclonal antibody comprising:

(a) a heavy chain variable region having first, second and third CDRs that are identical or substantial identical to the respective first, second and third CDRs of the heavy chain variable region of monoclonal antibody 191.2D6; and

(b) a light chain variable region having first, second and third CDRs that are identical or substantial identical to the respective first, second and third CDRs of the light chain variable region of monoclonal antibody 191.2D6.

2. The anti-tetanus monoclonal antibody of Claim 1, wherein the heavy chain variable region of said antibody is at least 75% identical the heavy chain variable region of monoclonal antibody 191.2D6, and wherein the light chain variable region of said antibody is at least 75% identical the light chain variable region of monoclonal antibody 191.2D6.

3. The anti-tetanus monoclonal antibody of Claim 1 or 2, wherein said CDRs of said antibody are identical to the respective CDRs of monoclonal antibody 191.2D6.

4. The anti-tetanus monoclonal antibody of Claim 3, wherein the heavy chain variable region and the light chain variable region of said antibody are identical to the respective heavy chain and light chain variable regions of monoclonal antibody 191.2D6.

5. The anti-tetanus monoclonal antibody of any one of the preceding claims, wherein said antibody further comprises a light chain constant domain and a heavy chain constant domain or region.

6. An anti-tetanus monoclonal antibody comprising:

(a) a heavy chain variable region having first, second and third CDRs that are identical or substantial identical to the respective first, second and third CDRs of the heavy chain variable region of monoclonal antibody 215.6G7; and (b) a light chain variable region having first, second and third CDRs that are identical or substantial identical to the respective first, second and third CDRs of the light chain variable region of monoclonal antibody 215.6G7.

7. The anti-tetanus monoclonal antibody of Claim 6, wherein the heavy chain variable region of said antibody is at least 75% identical the heavy chain variable region of monoclonal antibody 215.6G7, and wherein the light chain variable region of said antibody is at least 75% identical the light chain variable region of monoclonal antibody 215.6G7.

8. The anti-tetanus monoclonal antibody of Claim 6 or 7, wherein said CDRs of said antibody are identical to the respective CDRs of monoclonal antibody 215.6G7.

9. The anti-tetanus monoclonal antibody of Claim 8, wherein the heavy chain variable region and the light chain variable region of said antibody are identical to the respective heavy chain and light chain variable regions of monoclonal antibody 215.6G7.

10. The anti-tetanus monoclonal antibody of any one of Claim 6 to 10, wherein said antibody further comprises a light chain constant domain and a heavy chain constant domain or region.

11. An isolated polynucleotide encoding the light and/or heavy chain of an antibody according to any one of Claims 1 to 10.

12. An expression vector including coding sequences encoding the light and heavy chains of an antibody according to any one of Claims 1 to 10.

13. An expression system including coding sequences encoding the light and heavy chains of an antibody according to any one of Claims 1 to 10, wherein the expression system comprises:

a first expression vector including the coding sequence encoding the light chain; and a second expression vector including the coding sequence encoding the heavy chain.

14. A cell transformed with an expression vector or system according to Claim 12 or 13.

15. The cell of Claim 14, wherein the cell is a mammalian cell.

16. A method of manufacturing monoclonal antibodies, comprising cultivating cells according to Claim 14 or 15, and recovering the antibody from the culture medium.

17. A pharmaceutical composition comprising an anti-tetanus monoclonal antibody according to any one of Claims 1 to 10.

18. The pharmaceutical composition of claim 17, wherein the pharmaceutical composition comprises: an anti-tetanus monoclonal antibody according to any one of Claims 1 to 5; and an anti-tetanus monoclonal antibody that binds to a fragment of tetanus toxin other than the C -fragment of tetanus toxin.

19. The pharmaceutical composition of claim 17, wherein the pharmaceutical composition comprises: an anti-tetanus monoclonal antibody according to any one of Claims 6 to 10; and an anti-tetanus monoclonal antibody that binds to the C-fragment of tetanus toxin.

20. The pharmaceutical composition of claim 17, wherein the pharmaceutical composition comprises: an anti-tetanus monoclonal antibody according to any one of Claims 1 to 5; and an anti-tetanus monoclonal antibody according to any one of Claims 6 to 10.

21. A method of providing a patient with passive immunity against tetanus, comprising administering an effective amount of a monoclonal antibody according to any one of Claims 1 to 10 or pharmaceutical composition according to any one of Claims 17 to 20.

22. A monoclonal antibody according to any one of Claims 1 to 10, or a pharmaceutical composition according to any one of Claims 17 to 20, for use in a method of providing passive immunity against tetanus.

23. Use of a monoclonal antibody according to any one of Claims 1 to 5 and/or a monoclonal antibody according to any one of Claims 6 to 10 in the manufacture of a medicament for providing passive immunity against tetanus.