WO2022263574A1 - Crm197 protein carrier - Google Patents

Abstract

This invention relates to a Cross-Reactive Material (CRM) 197 polypeptide engineered to include a cysteine residue at a position corresponding to position 496 of SEQ ID NO: 1. The additional cysteine residue introduced into the CRM 197 polypeptide provides a free thiol group that can be used to conjugate a functional or cargo moiety (e.g. an immunogen or a drug). CRM197 polypeptides, CRM197 conjugates, methods of producing CRM197 conjugates, and therapeutic compositions, such as vaccines, comprising CRM 197 conjugates are provided.

Description

CRM 197 protein carrier

Field

The present invention relates to carrier proteins and their uses in conjugates, such as vaccines. In particular, the invention relates to Cross-Reactive Material (CRM) 197 variants and their uses in conjugates.

Background

The low inherent immunogenicity of some isolated, pathogen-derived polysaccharides can lead to suboptimal immune responses to said polysaccharides when administered as a vaccine. To overcome this, it has long been recognised that coupling polysaccharides to immunogenic carrier proteins such as Cross- Reactive Material 197 (CRM197) can increase the immunogenicity of pathogen-derived polysaccharides. This approach leads to the stimulation of not only a B-cell dependent antibody response but also T-cell dependent immunological memory formation critical for vaccine efficacy.

Cross-reactive material 197 (CRM 197) is a genetically detoxified variant of the diphtheria toxin (DT), isolated from a group of six different nitrosoguanidine-treated DT mutants first described in the early 1970s by the Alwin Max Pappenheimer Jr’s Lab at Harvard University (Pappenheimer, Uchida and Harper (1972), Immunochem. 9(1); Uchida, Pappenheimer and Harper (1972), Science 175(1): 901-903; Pappenheimer AM (1977), Annu. Rev. Biochem. 46(1): 69-94).

CRM197 is a 535 amino acid (aa) protein that can be cleaved by trypsin-like proteases in two fragments: fragment A (~21 kDa, containing the catalytic domain) and fragment B (~38 kDa, containing the transmembrane and receptor domains) (Bigio et al. (1987), FEBS Letters 218(1): 271-276; Giannini et al. (1984), Nucleic Acids Res. 12(1): 4063-4069; Malito et al. (2012), PNAS 109(1): 5229-5234). The protein contains two disulphide bonds: a more exposed bond at Cys186-201 , and another at Cys461-471 that appears to be more buried inside the protein, and therefore could be less accessible to chemical modifications (Malito et al. (2012), PNAS 109(1): 5229-5234). From the sequence first described by G. Giannini in 1984, to X-ray crystallography and MD dynamics studies, throughout the years it has become clear that a single substitution in position 52 (due to a mutation of the wild-type codon “GGG” for Gly into “GAG” for Glu) is enough to render the protein non-toxic. This mutation seems to increase the flexibility of the active-site loop that covers the NAD-binding pocket of CRM197, when compared to that in DT (Malito et al. (2012), PNAS 109(1): 5229-5234).

Besides the genetic mutation that makes this protein safe without the need for often destructive chemical treatments, very early on it was observed that CRM 197 is capable of eliciting a consistent and memory- inducing immune response in children and toddlers, making it ideal as a carrier protein in paediatric vaccines (Shinefield HR. (2010), Vaccine 28(1): 4335-4339).

Interestingly, CRM197 has also been shown to have great potential as an anti-tumour agent, and could be effective in the treatment of atherosclerosis (since both cancer and vascular plaques overexpress HB-EGF, the specific receptor for DT and CRM197) (Buzzi et al. (2004), Therapy 1(1): 61-66; Buzzi et al. (2004), Cancer Immunol. Immunother. 53(1): 1041-1048; Hu et al. (2015), J. Cell. Physiol. 230(1): 1713-1728). The importance of the native disulphides for the maintenance of CRM197’s structure, and thus its immunogenicity, makes cysteine-directed site-selective conjugation difficult to achieve without tightly targeted modifications or extensive characterization of the modified protein.

Summary

The present inventors have recognised that introducing a mutation at position 496 of CRM197 to replace a serine residue with a cysteine residue does not significantly alter the three-dimensional structure of CRM197, which maintains its ability to elicit a consistent and memory-inducing immune response. The additional cysteine residue introduced by this mutation provides a free thiol group that can be used to conjugate a functional or cargo moiety (e.g. an immunogen or a drug).

Accordingly, a first aspect of the invention provides a CRM197 polypeptide comprising a cysteine residue at a position corresponding to position 496 of SEQ ID NO: 1.

A suitable CRM197 polypeptide may comprise the amino acid sequence of SEQ ID NO: 1 or a variant of SEQ ID NO: 1 that is modified to comprise an additional cysteine residue at a position corresponding to position 496 of SEQ ID NO: 1. For example, the CRM197 polypeptide may include a S496C mutation.

A preferred CRM197 polypeptide of the first aspect may comprise the amino acid sequence of SEQ ID NO: 2 or a variant thereof. For example, a suitable variant may comprise an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 2 and comprises a cysteine residue at a position that corresponds to position 496 of SEQ ID NO: 2.

CRM197 polypeptides of the first aspect may be useful for example as a carrier protein.

A second aspect of the invention provides a conjugate comprising a CRM197 polypeptide according to the first aspect covalently linked to a cargo moiety, wherein the cargo moiety is covalently linked to at least the cysteine residue at the position corresponding to position 496 of SEQ ID NO: 1.

Suitable cargo moieties include peptides, proteins, glycoproteins, glycans, carbohydrates, small organic molecules, immunogens, antigens, drugs, such as chemotherapeutic or anti-Alzheimer’s drugs, haptens, or detectable labels, such as fluorophores. Preferred cargo moieties may include an antigenic polysaccharide derived from Haemophilus influenza type b ( Hib ), pneumococcus, or meningococcus, or cancer- associated antigens. Suitable antigenic polysaccharides include Pneumococcal polysaccharide serotype (PPS) 1, PPS 3, PPS 4, PPS 5, PPS 6A, PPS 6B, PPS 7F, PPS 9V, PPS 14, PPS 18C, PPS 18C1, PPS 19A, PPS 19F, PPS 19F1, PPS 23F, Meningococcal group A oligosaccharide, Meningococcal group C oligosaccharide, Meningococcal group W-135 oligosaccharide, and Meningococcal group Y oligosaccharide. Suitable cancer-associated antigens include mucins. Suitable chemotherapeutic drugs include oxaliplatin, bevacizumab, and leuprorelin. Suitable anti-Alzheimer’s drugs include memantine and donepezil. A third aspect of the invention provides a method of producing a conjugate, for example a conjugate of the second aspect, wherein the method comprises reacting a cargo moiety with at least the thiol group of the cysteine residue at a position corresponding to position 496 of a CRM197 polypeptide according to the first aspect.

A fourth aspect of the invention provides a nucleic acid encoding an isolated CRM 197 polypeptide according to the first aspect of the invention. In a preferred embodiment, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 3.

A fifth aspect of the invention provides an expression vector comprising the nucleic acid according to the fourth aspect of the invention.

A sixth aspect of the invention provides a recombinant cell comprising one or more heterologous nucleic acids encoding one or more isolated carrier proteins according to the first aspect of the invention.

A seventh aspect of the invention provides a pharmaceutical composition comprising the CRM 197 polypeptide of the first aspect or the conjugate of the second aspect and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition may be a vaccine composition. Suitable vaccine compositions may further comprise an adjuvant.

An eight aspect of the invention provides a method of inducing an immune response comprising administering a CRM197 polypeptide of the first aspect, conjugate of the second aspect or pharmaceutical composition of the seventh aspect to an individual in need thereof.

The immune response may be an immune response against the CRM197 polypeptide or the functional moiety.

The immune response may be a B cell mediated immune response and/or a T cell mediated immune response.

A ninth aspect of the invention provides a method of treating cancer, Alzheimer’s disease, addiction or atherosclerosis comprising administering a CRM197 polypeptide of the first aspect, conjugate of the second aspect or pharmaceutical composition of the seventh aspect to an individual in need thereof.

A tenth aspect of the invention provides a CRM197 polypeptide of the first aspect, conjugate of the second aspect or pharmaceutical composition of the seventh aspect for use in a method of the eighth or ninth aspect.

A eleventh aspect of the invention provides the use of a CRM197 polypeptide of the first aspect, conjugate of the second aspect or pharmaceutical composition of the seventh aspect in the manufacture of a medicament for use in a method of the eighth or ninth aspect. Other aspects and embodiments of the invention are described in more detail below.

Brief Description of the Figures

Figure 1 shows the design and expression of i5CysCRM197. A) Representative SDS-PAGE gel in which 20 pL of the flowthrough, wash, and elution fractions were analysed. B) Representative gel showing efficiency of protease-dependent Strep-tag® II removal, analysed under reducing (with b-mercaptoethanol) or nonreducing (without b-mercaptoethanol) conditions. The tagged protein could be collected and re-subjected to protease digestion. C) Overlapped predictions of secondary structures of CRM197 (in black) and i5CysCRM197 (in orange), based on the Garnier-Osguthorpe-Robson method. For all three graphs, values above the axis line represent regions which are likely to form helix, sheet or turn structures.

Figure 2 shows i5CysCRM197 purification and secondary structure characterisation. A) Representative photo of western-blot analysis of initial purification of i5CysCRM197. The membrane was probed with an antibody against CRM197. B) Far-UV CD spectra of commercial-CRM197, i5CysCRM197 and iCRM197. C) Results obtained from Capita and K2D3 CD analysis online-tools. In the case of Capita, alpha-helix includes a, 310, and p-helix; b-sheet includes b-bridge, and bonded turn; and bends, and loops are included in the structural feature irregular. For K2D3, helical and b-strand contents were subtracted from 100 to obtain percentage of irregular. All values are presented as percentage.

Figure 3 shows CRM197 conjugation with AlexaFluor488 fluorophores for internalisation assays. A) Schematics of the conjugation of CRM 197 and variants with a fluorophore (Alexa-Fluor 488), for protein tracking in live cells. B) SDS-PAGE gel for confirmation of specificity of the 1 h click-reaction, for samples of unmodified or CAA-alkyne modified samples of CRM197 and variants. The same Coomassie-stained lane (left) is shown next to the corresponding fluorescence detection (right). Marker is annotated with the molecular weights corresponding to each band.

Figure 4 shows characterisation of i5CysCRM197 as a protein carrier. A) Confocal imaging detection of the internalized proteins in Raji cells. Scale bar is 10 pm. B) Imaging flow cytometry results for detection of fluorescently labelled proteins inside Raji cells (at 30 min after start of incubation), including photos corresponding to the GFP-positive population. C) Anti CRM197 specific IgG levels of Balb/CByJ mouse groups immunized with commercial-CRM197 (blue), iCRM197 (green), and i5CysCRM197 (orange) after first and second boost immunizations, administered two weeks apart. Data is shown as ELISA units per mL of serum. D) ELISA raw data obtained from immunization experiment with vehicle control (black), commercial- CRM197 (blue), iCRM197 (green) or i5CysCRM197 (orange). Absorbance at 450 nm plotted against serial serum dilutions. “TO” refers to timepoint at the time of the first injection (left panel); “post I” refers to timepoint after the first booster injection (middle panel); “post II” refers to timepoint after the second booster injection (right panel). Graphs show mean and standard error of the mean.

Detailed Description

CRM197 stimulates B-cell dependent antibody production and T-cell dependent immunological memory formation when conjugated to an immunogen. This activity depends on the secondary and tertiary structure of CRM197. Conjugation of an immunogen to the native cysteine residues of CRM197 necessitates the disruption of disulphide bridges, which are important in the maintenance of the secondary and tertiary structure of CRM197. As a result, the ability of previous CRM197 conjugates to stimulate B- and T-cell mediated responses is reduced.

The present inventors have found that an additional cysteine residue can be introduced into Cross-Reactive Material (CRM) 197 polypeptide (SEQ ID NO: 1) without disrupting the structure of the CRM197 polypeptide by replacing the serine residue at position 496 with the additional cysteine residue. The additional cysteine provides an accessible free thiol group that can be used to conjugate functional or cargo moieties to the CRM197 polypeptide without affecting its biological activities, such as the ability to stimulate B- and T-cell mediated responses

Cross-reactive material 197 (CRM 197) is a diphtheria toxin (DT) that is detoxified by the presence of a Gly to Glu mutation at a position corresponding to position 52 of the wild type CRM197 sequence shown in SEQ ID NO: 1 (Broker et al (2011) Biologicals 39(4) 195-204).

A CRM 197 polypeptide as described herein comprises an additional cysteine residue at a position corresponding to position 496 of SEQ ID NO: 1. This additional cysteine residue provides free thiol group for conjugation without affecting the structure or properties of CRM197 and is not present in the wild-type CRM 197. The additional cysteine is therefore useful for the attachment of cargo moieties and may thus be described as an attachment cysteine residue. The additional cysteine residue is introduced into CRM197 by modifying or mutating a CRM197 coding sequence by recombinant means to produce the CRM197 polypeptide described herein. Since it is not present in natural or wild-type CRM197, the additional cysteine residue may be described as heterologous or exogenous. The additional cysteine residue may be introduced by substitution of the serine residue at the position corresponding to position 496 of SEQ ID NO:

1 (i.e. a S496C mutation).

The CRM197 polypeptide may further comprise cysteine residues at positions corresponding to positions 186, 201 , 461 , and 471 of SEQ ID NO: 1. These cysteine residues may form intramolecular disulphide linkages. For example, the CRM197 polypeptide may comprise disulphide linkages at positions corresponding to positions Cys186-201 and Cys461-471 of SEQ ID NO: 1. In some embodiments, a CRM197 polypeptide may comprise an oxetane graft at the Cys186-201 disulphide bridge. This may be useful for example in improving the stability of the polypeptide and increase its immunogenicity.

The CRM 197 polypeptide further comprises a Glu residue at a position corresponding to position 52 of SEQ ID NO: 1.

Preferably, the additional cysteine residue at the position corresponding to position 496 of SEQ ID NO: 1 is the only free reactive thiol group in the CRM197 polypeptide. The site-selective conjugation of a cargo moiety to this free reactive thiol group allows the generation of CRM197 conjugates with predictable reaction outcomes and low batch-to-batch variability. A suitable CRM197 polypeptide may comprise the amino acid sequence of SEQ ID NO: 1 or variant thereof that is modified to contain a cysteine residue at a position in the amino acid sequence corresponding to position 496 of SEQ ID NO: 1. For example, a CRM197 polypeptide may comprise the amino acid sequence of SEQ ID NO: 2 or a variant thereof.

A variant of a reference sequence set out herein, such as a reference CRM197 sequence may comprise an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% sequence identity to the reference sequence. Particular amino acid sequence variants may differ from a reference sequence shown herein by insertion, addition, substitution or deletion of 1 amino acid, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more than 10 amino acids.

Sequence similarity and identity are commonly defined with reference to the algorithm GAP (Wisconsin Package, Accelerys, San Diego USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences that maximizes the number of matches and minimizes the number of gaps. Generally, default parameters are used, with a gap creation penalty = 12 and gap extension penalty = 4. Use of GAP may be preferred but other algorithms may be used, e.g. BLAST (which uses the method of Altschul et al. (1990) J. Mol. Biol. 215: 405-410), FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448), or the Smith-Waterman algorithm (Smith and Waterman (1981) J. Mol Biol. 147: 195- 197), or the TBLASTN program, of Altschul et al. (1990) supra, generally employing default parameters. In particular, the psi-Blast algorithm (Nucl. Acids Res. (1997) 253389-3402) may be used.

Sequence comparison may be made over the full-length of the relevant sequence described herein.

A CRM 197 polypeptide described herein may be useful as a carrier protein or protein carrier.

A carrier protein is a protein moiety that it is linked to cargo moiety in a conjugate. For example, a carrier protein may be linked to a cargo moiety (e.g. a drug) in a conjugate for example for use as a therapeutic agent. The carrier protein may improve the immunogenic, pharmacokinetic or other properties of the cargo moiety. In some embodiments, a conjugate may be used to deliver the cargo moiety to a target cell, such as a tumour cell, in vivo. In other embodiments, a carrier protein may be immunogenic and may be linked to an immunogen cargo moiety (such as an immunogenic protein, glycan or carbohydrate) in a conjugate for example for use as a vaccine. The carrier protein may increase the immunogenicity of the linked immunogen cargo moiety.

The free thiol of the additional cysteine residue of CRM197 polypeptides described herein may be useful in covalently attaching a cargo moiety to the CRM197 polypeptide to produce a conjugate. For example, a CRM197 polypeptide as described herein may be subjected to site-selective conjugation at the additional cysteine residue to produce a conjugate.

A conjugate described herein may comprise a CRM197 polypeptide as described herein covalently linked to a cargo moiety. A cargo moiety is a functional moiety that is covalently attached to the CRM 197 polypeptide of the invention to produce a conjugate. Cargo moieties may include glycans, carbohydrates, proteins, such as antibodies, peptides, small organic molecules, or glycoproteins.

Suitable cargo moieties include immunogens or antigens, small organic molecules, detectable labels, and drugs.

In some embodiments, the cargo moiety may be an immunogen or antigen. For example, the cargo moiety may be an immunogenic glycan, carbohydrate, oligosaccharide, polysaccharide, glycoprotein, hapten, protein, or peptide.

Suitable immunogenic glycans, oligosaccharides, polysaccharides or carbohydrates include pathogen antigens, such as those derived from Haemophilus influenza type b (Hib), pneumococcus, or meningococcus. For example, suitable immunogenic polysaccharides include Pneumococcal polysaccharide serotype (PPS) 1, PPS 3, PPS 4, PPS 5, PPS 6A, PPS 6B, PPS 7F, PPS 9V, PPS 14, PPS 18C, PPS 18C1 , PPS 19A, PPS 19F, PPS 19F1, and PPS 23F. Other suitable immunogenic oligosaccharides include Meningococcal group A oligosaccharide, Meningococcal group C oligosaccharide, Meningococcal group W-135 oligosaccharide, and Meningococcal group Y oligosaccharide.

For example, a conjugate may comprise a CRM197 polypeptide linked to a cargo moiety comprising two or more covalently linked serotype glycans. Multiple serotype glycans can be used in vaccine formulations to give broad protection against various pathogen serotypes. For example, a cargo moiety may comprise two or more covalently linked glycans selected from PPS 1, PPS 3, PPS 4, PPS 5, PPS 6A, PPS 6B, PPS 7F, PPS 9V, PPS 14, PPS 18C, PPS 18C1, PPS 19A, PPS 19F, PPS 19F1, PPS 23F, Meningococcal group A oligosaccharide, Meningococcal group C oligosaccharide, Meningococcal group W-135 oligosaccharide, and Meningococcal group Y oligosaccharide.

Other suitable antigens include cancer-associated antigens. Suitable cancer-associated antigens include glycoproteins such as mucins, including Mucin 1 (MUC1), or fragments thereof. Additional suitable cancer- associated antigens include proteins and peptides, including HER2, or fragments thereof.

Other suitable antigens include haptens. For example, opioid haptens such as heroin haptens. CRM197 conjugated to a heroin hapten induces antibodies capable of binding heroin in the blood circulation thereby preventing heroin from crossing the blood-brain barrier, thereby blocking the biological effects. Conjugates comprising a CRM197 polypeptide linked to a heroin hapten may be useful in treating a subject with heroin addiction.

Conjugates comprising an immunogen linked to a CRM197 polypeptide as described herein may be internalised by B cells and may be useful, for example in the generation of B- and T-cell mediated immune responses against the immunogen. In other embodiments, the cargo moiety may be a drug or other therapeutic agent. Suitable therapeutic agents include anti-cancer and anti-atherosclerotic compounds. CRM 197 binds to HB-EGFR, a receptor commonly upregulated in various cancers and atherosclerotic plaques. Conjugates comprising a CRM 197 polypeptide linked to an anti-cancer or anti-atherosclerotic compound may be useful in treating cancer or atherosclerosis, respectively. Conjugates comprising a CRM197 polypeptide linked to an anti-cancer compound may be particularly useful at treating ovarian cancers, oral cancers, colon carcinoma, triplenegative breast cancer, metastatic breast carcinoma, neuroblastoma, and brain tumours. Suitable anticancer compounds include oxaliplatin, bevacizumab, and leuprorelin.

Other suitable therapeutic agents include drugs for delivery across the blood-brain barrier, for example anti- Alzheimer’s and anti-brain tumour compounds. CRM197 has been shown to cross the blood-brain barrier and promote drug delivery to the brain. Conjugates comprising a CRM197 polypeptide linked to an anti-brain tumour compound or an anti-Alzheimer’s compound may be useful in treating brain tumours or Alzheimer’s disease, respectively. Suitable anti-Alzheimer’s compounds include memantine and donepezil.

A conjugate described herein may comprise a CRM197 polypeptide linked to a cargo moiety comprising two or more covalently linked therapeutic compounds. For example, a conjugate may comprise a cargo moiety of two or more covalently linked drug molecules for delivering high doses of a drug to a target cell.

A conjugate described herein may comprise a CRM 197 polypeptide linked to a cargo moiety comprising a therapeutic compound covalently linked to a detectable probe for disease monitoring. For example, a cargo moiety may comprise a drug covalently linked to a fluorescent molecule suitable for in vivo bioimaging.

In other embodiments, the cargo moiety may be a detectable label or probe. Any suitable detectable probe may be used as a cargo moiety, for example, fluorescent proteins, fluorescent molecules, chemiluminescent probes, chromogenic probes, and enzymatic probes. For example, in some embodiments, the detectable probe is a fluorescent molecule. Suitable fluorescent molecules include near-infrared and infrared probes useful for in vivo bioimaging and analysis. Other suitable fluorescent molecules include visible (e.g. AlexaFluor 488) and ultraviolet probes particularly useful for in vitro imaging and analysis. Such fluorescent probes are commercially available and well known in the art.

The cargo moiety of a conjugate described herein may be linked to the free thiol group of the additional cysteine residue of the CRM197 polypeptide through a covalent bond. The covalent bond may be formed by any suitable means known in the art. For example, suitable thiol-specific reactions useful for conjugation to the additional cysteine residue include disulphide exchange, alkylation, or conjugate addition to a Michael acceptor. In some embodiments, additional cargo moieties may be linked to one or more of the natural or wild type cysteine residues of the CRM197 polypeptide through a covalent bond.

In some embodiments, the cargo moiety may be linked to the additional cysteine residue of the CRM197 polypeptide through a thioether or thioester bond. For example, the cargo moiety may comprise a maleimide group, haloacetyl group, or carbonacrylic group that reacts with the thiol group of the additional cysteine residue to form the thioether bond. Examples of suitable carbonylacryclic reagents can be found in Bernardim, B et al (2016) Nat. Comms. 7, 13128. Other suitable thiol-reactive groups that form thioether or thioester bonds are known in the art. Additional cargo moieties may be linked to one or more of the natural or wild type cysteine residues of the CRM197 polypeptide through a thioether or thioester bond.

In other embodiments, the cargo moiety may be linked to the additional cysteine residue of the CRM197 polypeptide through a disulphide bond. For example, the cargo moiety may comprise a pyridyl group that can react with the thiol group of the additional cysteine residue to form the disulphide bond. In another example, the cargo moiety may comprise a disulphide bond that can react with the free thiol group of the additional cysteine residue to undergo disulphide exchange thereby forming a disulphide bond between the cargo moiety and the additional cysteine residue. Other suitable thiol-reactive groups that form disulphide bonds are known in the art. Additional cargo moieties may be linked to one or more of the natural or wild type cysteine residues of the CRM197 polypeptide through a disulphide bond.

A conjugate as described herein may be produced by a method comprising reacting a cargo moiety with the thiol group of the additional cysteine residue of a CRM197 polypeptide as described herein, such that the cargo moiety is covalently linked to the cysteine residue. In some embodiments, additional cargo moieties may be reacted with the thiol group of one or more of the natural or wild type cysteine residues of the CRM197 polypeptide. In some embodiments, the cargo moiety may be reacted with the CRM197 polypeptide under non-reducing conditions.

The cargo moiety may be reacted with the additional cysteine residue by any convenient chemistry to covalently link the cargo moiety and the CRM 197 polypeptide. For example, the cargo moiety may comprise a thiol-reactive group that reacts with the free thiol group of the additional cysteine residue to form a covalent bond, such as a thioether or disulphide bond.

A method of producing a conjugate may comprise; providing a cargo moiety comprising a thiol-reactive group; and reacting the thiol-reactive group with the thiol group of the additional cysteine residue of a CRM 197 polypeptide to covalently link the cargo moiety to the CRM197 polypeptide through a covalent bond.

Suitable thiol-reactive groups are well known in the art and include maleimide groups, haloacetyl groups, carbonylacrylic groups, disulphide groups, and pyridyl groups.

In some embodiments, the thiol-reactive group may form a thioether bond with the additional cysteine. For example, a method of producing a conjugate may comprise; providing a cargo moiety comprising a maleimide group, haloacetyl group, or carbonylacrylic group; and reacting the maleimide group, haloacetyl group, or carbonylacrylic group of the cargo moiety with the thiol group of the additional cysteine residue of a CRM197 polypeptide to covalently link the cargo moiety to the CRM197 polypeptide through a thioether bond. Suitable reaction conditions may vary depending on the chosen reactive groups. Optimisation of the reaction conditions is standard practice and well known in the art.

In other embodiments, the thiol-reactive group may form a disulphide bond with the additional cysteine. A method may comprise providing a cargo moiety comprising a pyridyl group or disulphide group; reacting the pyridyl group or disulphide group of the cargo moiety with the thiol group of the additional cysteine residue of a CRM 197 polypeptide to covalently link the cargo moiety to the CRM 197 polypeptide through a disulphide bond.

Suitable reaction conditions may vary depending on the chosen reactive groups. Optimisation of the reaction conditions for specific reactive groups is standard practice and well known in the art.

Techniques for the introduction of a suitable thiol reactive group onto a cargo moiety (i.e. functionalisation of the cargo moiety) will depend on the chemical nature of the cargo moiety. Methods for addition of suitable thiol reactive groups to cargo moieties such as glycans, peptides, proteins, drugs etc., are well known in the art.

A conjugate described herein may comprise one, two or more cargo moieties. For example, a first cargo moiety may be covalently linked to the CRM 197 polypeptide and a second cargo moiety may be covalently linked to the first cargo moiety. Suitable first and second cargo moieties and reaction chemistries are described above. For example, a method may comprise the steps of; i) reacting the thiol group of the addition cysteine residue of a CRM 197 polypeptide with a carbonylacrylic group of a first cargo moiety to form a thioether bond between the first cargo moiety and the carrier protein; and ii) reacting an alkyne group of the first cargo moiety with an azide group on a second cargo moiety to link the first cargo moiety and the second cargo moiety by a 5 membered heteroatom ring.

Steps i) and step ii) may be performed in any order or simultaneously.

A CRM197 polypeptide as described above may be encoded by an isolated nucleic acid. The nucleic acid may comprise the nucleotide sequence of SEQ ID NO:3 or may be a variant thereof.

A variant of a reference nucleotide sequence set out herein, such as a reference CRM197 coding sequence may comprise a nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% sequence identity to the reference sequence. Particular nucleotide sequence variants may differ from a reference sequence shown herein by insertion, addition, substitution or deletion of 1 amino acid, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more than 10 nucleotides. Nucleic acid molecules may comprise DNA and/or RNA and may be partially or wholly synthetic. Reference to a nucleotide sequence as set out herein encompasses a DNA molecule with the specified sequence, and encompasses a RNA molecule with the specified sequence in which U is substituted for T, unless context requires otherwise.

A nucleic acid may be codon optimised for expression in a prokaryotic system, such as E. coli.

Further provided are constructs in the form of vectors (e.g. expression vectors), or transcription or expression cassettes which comprise such nucleic acids. Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes, origins of replication and other sequences as appropriate. A vector will typically contain expression control sequences compatible with the prokaryotic host cell (e.g., an origin of replication). In addition, any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda. The promoters will typically control expression, optionally with an operator sequence, and have ribosome binding site sequences and the like, for initiating and completing transcription and translation. Vectors for use in prokaryotic cells may also require an origin of replication component.

Vectors may be plasmids e.g. phagemid, or viral e.g. 'phage, as appropriate. For further details see, for example, Sambrook & Russell (2001) Molecular Cloning: a Laboratory Manual: 3rd edition, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulation of nucleic acid, for example in the preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Ausubel et al. (1999) 4^th eds., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, John Wiley & Sons.

A method of producing a CRM 197 polypeptide as described herein may comprise introducing a nucleic acid or vector as described herein may be introduced into a host cell. For example, the vector or nucleic acid may be transformed into a host cell in which the vector is functional.

Techniques for the introduction of nucleic acid into host cells (generally referred to without limitation as “transformation”) are well established in the art and any suitable technique may be employed, in accordance with the circumstances. Suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage (see for example, Sambrook & Russell (2001) supra ; Ausubel et al. (1999) supra). In addition, the vector may comprise a viral vector, such as an adenovirus, retrovirus, lentivirus, adeno-associated virus, baculovirus, vaccinia virus or herpes simplex virus vector. A viral vector may comprise a viral particle, comprising a nucleic acid and one or more viral proteins.

The introduced nucleic acid may be on an extra-chromosomal vector within the cell or the nucleic acid may be integrated into the genome of the host cell. Integration may be promoted by inclusion of sequences within the nucleic acid or vector which promote recombination with the genome, in accordance with standard techniques.

Marker genes such as antibiotic resistance or sensitivity genes may be used in identifying clones containing nucleic acid of interest, as is well-known in the art.

A range of host cells suitable for the production of recombinant CRM 197 polypeptides are known in the art. Suitable host cells may include bacterial cells, such as Escherichia coli, Corynebacterium diphtheriae, Lactococcus lactis; bacilli, such as Bacillus subtilis, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species including Pseudomonas fluorescens. Preferably, the host cell is an E coli cell.

The introduction may be followed by expression of the nucleic acid encoding the CRM 197 polypeptide in the host cell to produce the encoded CRM197 fusion protein. In some embodiments, the host cell (which may include cells actually transformed although more likely the cells will be descendants of the transformed cells) may be cultured in vitro under conditions for expression of the nucleic acid, so that the encoded CRM197 polypeptide is produced. Suitable conditions are well known in the art. When an inducible promoter is used, expression may require the activation of the inducible promoter. For example, a bacterial strain, such as E. coli B121 (DE3) which comprises T7 polymerase linked to an inducible lac UV5 promoter may be used.

Following expression, the CRM197 polypeptide may be isolated and/or purified. Suitable techniques are well known in the art. CRM197 polypeptides are described herein may be useful in the production of conjugates for therapeutic applications.

The isolated CRM197 polypeptide may be conjugated to a cargo moiety as described herein to produce a conjugate. Conjugates as described herein may be useful in therapeutic applications.

A method may further comprise admixing a conjugate as described herein with a pharmaceutically acceptable excipient to produce a pharmaceutical composition.

The term “pharmaceutically acceptable” relates to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound veterinary or medical judgement, suitable for use in contact with the tissues of a subject (e.g. human or other mammal) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation.

Suitable excipients and carriers include, without limitation, water, saline, buffered saline, phosphate buffer, alcoholic/aqueous solutions, emulsions or suspensions. Other conventionally employed diluents, adjuvants, and excipients may be added in accordance with conventional techniques. Such carriers can include ethanol, polyols, and suitable mixtures thereof, vegetable oils, and injectable organic esters. Buffers and pH- adjusting agents may also be employed, and include, without limitation, salts prepared from an organic acid or base. Representative buffers include, without limitation, organic acid salts, such as salts of citric acid (e.g., citrates), ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, phthalic acid, Tris, trimethylamine hydrochloride, or phosphate buffers. Parenteral carriers can include sodium chloride solution, Ringer's dextrose, dextrose, trehalose, sucrose, lactated Ringer's, or fixed oils. Intravenous carriers can include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives such as, for example, antimicrobials, antioxidants, chelating agents (e.g., EGTA; EDTA), inert gases, and the like may also be provided in the pharmaceutical carriers. The pharmaceutical compositions described herein are not limited by the selection of the carrier.

The preparation of these pharmaceutically-acceptable compositions, from the above-described components, having appropriate pH, isotonicity, stability and other conventional characteristics, is within the skill of the art.

Suitable carriers, excipients, etc. may be found in standard pharmaceutical texts, for example, Remington’s Pharmaceutical Sciences and The Handbook of Pharmaceutical Excipients, 4th edit., eds. R. C. Rowe et al, APhA Publications, 2003.

The pharmaceutical composition of the invention may be a vaccine composition. The pharmaceutical composition or vaccine composition may further comprise an adjuvant.

An adjuvant is a compound that enhances or augments the immune response to an immunogenic compound. Suitable adjuvants for use in vaccine compositions include but are not limited to inorganic compounds, e.g. aluminium salt, oils, bacterial products, e.g. toxoids, and cytokines, e.g. IL-1 and IL-2.

Vaccines and pharmaceutical compositions may conveniently be presented in unit dosage form and may be prepared by any methods well-known in the art of pharmacy. Such methods include the step of bringing the one or more isolated conjugates/immunogenic polypeptides into association with a carrier or excipient as described above which may constitute one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active compound with liquid carriers or finely divided solid carriers or both.

Vaccines and pharmaceutical compositions may be made in the form of sterile aqueous solutions or dispersions, suitable for injectable use, or made in lyophilized forms using freeze-drying techniques. Lyophilized pharmaceutical compositions are typically maintained at about 4°C, and can be reconstituted in a stabilizing solution, e.g., saline or HEPES, with or without adjuvant.

Vaccines and pharmaceutical compositions may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections immediately prior to use.

Vaccines and therapeutic pharmaceutical compositions according to the present invention may be formulated for administration by a number of routes, including but not limited to, parenteral, intravenous, intra-arterial, intramuscular, intratumoural, oral and nasal. Also provided herein are methods of stimulating an immune response in a subject, wherein the method comprises administering a conjugate or pharmaceutical composition described herein to a subject in need thereof; and conjugates or pharmaceutical compositions described herein for use in such methods.

Immune responses include B cell mediated immune responses and T cell mediated immune responses.

Also provided herein are methods of treating cancer, Alzheimer’s disease, heroin addiction or atherosclerosis in a subject, wherein the method comprises administering a conjugate or pharmaceutical composition described herein a subject in need thereof; and conjugates or pharmaceutical compositions described herein for use in such methods. For example, the method may comprise administering a conjugate or pharmaceutical composition comprising an anti-cancer cargo moiety to a subject having ovarian cancer, oral cancer, glioma, colon carcinoma, triple-negative breast cancer, metastatic breast carcinoma, or neuroblastoma. In other examples, the method may comprise administering a conjugate or pharmaceutical composition comprising a heroin hapten cargo moiety to a subject having a heroin addiction.

Other aspects and embodiments of the invention provide the aspects and embodiments described above with the term “comprising” replaced by the term “consisting of and the aspects and embodiments described above with the term “comprising” replaced by the term ’’consisting essentially of”.

It is to be understood that the application discloses all combinations of any of the above aspects and embodiments described above with each other, unless the context demands otherwise. Similarly, the application discloses all combinations of the preferred and/or optional features either singly or together with any of the other aspects, unless the context demands otherwise.

Modifications of the above embodiments, further embodiments and modifications thereof will be apparent to the skilled person on reading this disclosure, and as such, these are within the scope of the present invention.

All documents and sequence database entries mentioned in this specification are incorporated herein by reference in their entirety for all purposes.

“and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, “A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

Experimental

Identifying suitable sites for Cys amino acid substitution

The Garnier-Osguthorpe-Robson method was used to predict secondary protein structure of CRM197. This method allows for determination of the relative probability for the presence of an alpha-helix, beta-sheet or random-coil, based on the primary structure. The same method was then used to analyse the secondary structure of various mutants, and these predictions were compared to those obtained for CRM 197. It is important to note that substitution-type mutations in CRM 197 should not be performed in proximity to the active-site loop that covers the NAD-binding pocket of CRM 197, because changes in this region might revert the protein’s toxicity. Therefore, substitutions assessed in silico were constrained to certain regions of fragment B of CRM 197 as this would most likely not affect the catalytic domain or the NAD-binding pocket. The final mutant sequence comprising an additional cysteine at position 496 i showed least differences in terms of both the percentage and the location of secondary structures (herein referred to as 5CysCRM197). In fact, i5CysCRM197 shows virtually no differences when compared to CRM197, when prediction of secondary structures is overlapped (Figure 1C).

Generation and purification of i5CysCRM197

A plasmid encoding 5CysCRM197 was used for the expression and purification of the fusion protein herein called i5CysCRM197.

For the expression protocol, all growths are performed in standard Erlenmeyer type flasks, using orbital shakers with adjustable temperature. Cultures are grown in Lima's broth (Miller’s formulation) low salt medium (Sigma, Cat# L3397) with (solid) or without (liquid) 10 g/L agar. Growth medium is always supplemented with ampicillin or carbenicillin (at 100 pg/mL), to maintain selective pressure (the latter for liquid media).

Growth for protein expression was performed directly from frozen vials of transformants bearing the i5CysCRM197 plasmid. Bacteria can be recovered from the glycerol stocks using a pipette, removing a portion of the upper surface of the frozen stock and inoculate a starter culture. This culture is then grown overnight, at 37 °C, under 150-250 rpm agitation. The starter culture was then used to inoculate (at 1 :50 inoculum) carbenicillin-supplemented LB. The ratio of liquid to flask capacity should be up to 25%. This culture was maintained at 37 °C under mild-shaking conditions (up to 180 rpm) until OD600 reaches mid-log phase (0.5-0.7), at which point cultures were removed from the shaker, and IPTG was added to the medium. Cultures are then cultured at 16-18 °C, for another 5 h, at 120 rpm. Collection of bacterial cells was performed at 4 °C, at 8000 xg, for 20 min. Pellets were frozen at -20 °C, until the protocol for isolation of the soluble fraction was performed.

For the isolation of soluble fractions, all flasks were kept on ice. When it was necessary to store solutions overnight, and to avoid freeze-thaw cycles, flasks were kept on ice, and the containers lidded and placed inside a cold-chamber (room temperature of 4 °C). Isolation of whole protein content of the soluble fraction was performed by resuspending the bacterial pellet directly in a standard binding buffer (100 mM Tris-HCI, 150 mM NaCI, 1 mM EDTA, pH 8) appropriate for purification using a Strep-Tactin® High-capacity Fast Flow column. To process bacterial pellet corresponding to up to 500 mL of culture, 10 mL of buffer solution was used. The cell suspension was then sonicated, at 14 mV, for 4 min (20” ON + 20” OFF). The preparation is then centrifuged at 18000 xg, for 30 min, at 4 °C. Supernatant (hereafter named “soluble fraction”) was then kept on ice for purification of i5CysCRM197. When required, DNase treatment can be performed, but DNA contaminations are not common in this protocol.

Soluble fraction was loaded into a pre-equilibrated Strep-Tactin® column, until the volume of flow-through matches the volume of column input, and was fully collected in a Falcon tube that is kept on ice. At this point, 6 column volumes of binding buffer were used to wash unbound proteins (and the fractions collected into a new collection tube). Bound i5CysCRM197 is then eluted with 6 column volumes of elution buffer (similar to binding buffer, but with 2.5 mM of desthiobiotin). SDS-PAGE was performed with up to 10 pg of protein content from each of these fractions to confirm protein presence, and stability by comparing reduced samples with non-reduced samples (See “Biophysical characterisation” below).

Cleavage of the Strep-tag® II was performed using a commercial enteropeptidase obtained from Abeam (Cat# ab2007001), according to the manufacturer’s instructions, at 20 ^°C, for 4h.

Biophysical characterisation

Next, SDS-PAGE analysis was performed on i5CysCRM197 under both non-reducing (-b-mercaptoethanol) and reducing (+ b-mercaptoethanol). SDS-PAGE analysis under non-reducing conditions allows detection of covalent and noncovalent aggregation in the samples, and impurities. Gels run in reducing conditions allow detection of the proteolytically nicked form (if any) of the protein. i5CysCRM197 appears as a ~60 kDa band in a SDS-PAGE (consistently with the theoretical mass of 60475 Da), both under reducing and non-reducing conditions (Figure 1B). Post-purification aggregation was not detected in any of the batches. The lower weight band present in Figure 1 B corresponds to the enteropeptidase used for cleavage of the Strep-tag® from purified i5CysCRM197, which was later removed in the polishing step.

The identity of i5CRM197 was confirmed by western-blot assays performed using samples collected at different stages of the purification protocol. Membranes were probed with an antibody against Strep-tag® II, as well as an antibody against full-length CRM197 (Figure 2A).

Characterization of protein secondary structure was performed through circular dichroism analysis. i5CysCRM197 was compared directly to commercial CRM 197 (COM-CRM197) and tested under the same experimental conditions.

Quantification of the prevalence of secondary structures was done by direct comparison of the CD spectra (Figure 2B), as well as by blind-analysis of the molar ellipticity using two different CD analysis online-tools: Capito14 and K2D315 (Figure 2C). Both Capito14 and K2D315 use the spectra of proteins that have been characterized by x-ray crystallography as standards.

Capita is a web-tool for estimating secondary structure content and analyzing far-UV CD data based on a selected set of far-UV CD data as available from the Protein Circular Dichroism Data Bank (PCDDB), and is especially suited for analysis of mutants of the same protein in multiple conditions. K2D3 is a tool that includes data not just from experiment far-UV CD data available in the PCDDB databases, but also uses DichroCalc to calculate the theoretical CD spectra of a non-redundant set of structures, thus adding to this database. It is described as particularly useful for analysis of proteins with a high percentage of beta-sheets in their structure (or less globular proteins, which is the case with CRM 197). Direct analysis of the far-UV spectra did not reveal significant differences between the CRM197 standard and the variant produced in this work. Quantification of the percentage of helical content, beta-strands, and irregular structures confirms this observation (Figure 2C). Quantification of the beta-strand content using Capito14 and K2D315 provide different values for each of COM-CRM and i5CysCRM197. However, percentages of the categories are similar when comparing the proteins amongst each other, using the same tool. While analysis with K2D3 raises the possibility of some loss of structure (increase of prevalence of random coils and reduction of helical content) in the i5CysCRM197 protein mutant this loss apparent loss appears to be minor.

Cys-directed modification

Native CRM 197 has been shown to be amenable to Cys-directed reactions. Therefore, thiol availability was assessed for i5CysCRM197. For i5CysCRM197 a faint change in colour was observed in both TCEP- reduced and (more faint in) non-reduced protein samples, so it seems apparent that there is an available thiol in the native protein that should be provided by the engineered Cys. i5CysCRM197 as a protein carrier

To determine if i5CysCRM197 can function as a protein carrier its ability to be internalised into B cells was assessed. To assess this, it was necessary to be able to track the protein inside live cells. Thus, i5CysCRM197 and the corresponding commercial control were modified with a carbonylacrylic reagent bearing an alkyne moiety (Bernardim, B., et al (2016) Nat. Comms. 7:13128), after which these modified proteins (CAA-modified proteins) were conjugated by click chemistry to a fluorescent molecule (Figure S3A). The addition of the fluorophore was shown to be specific for the CAA-modified proteins, as shown by detection of fluorescence on a SDS-PAGE gel (Figure 3B).

Both proteins were tagged using the same reaction conditions, and these now fluorescent proteins were incubated with Raji cells for up to 2h. Fluorescence inside the cells could be detected already at 30 min after the start of incubation, both by confocal microscopy (Figure 4A) and imaging flow-cytometry (Figure 4B). Overall, this data indicates internalization of i5CysCRM197 (and E. coli-derived iCRM197) with internalization kinetics identical to wild-type CRM197 as previously described for this CRM197 (Lai, Z. and Schreiber, JR., (2009) Vaccine 27(24): 3137-3144.

For i5CysCRM197s to be useful as protein carrier, it is essential that in vivo it has the capability to not only trigger antibody production, but also that internalization is successful and activates T-cell dependent responses, such as generation of immunological memory. To test this, groups of 4 Balb/C mice were immunized with either a protein solution, or the same volume of vehicle. Each group received 3 injections, 2 weeks apart, and sera was collected every two weeks after each immunization to determine total specific- CRM197 IgG production.

ELISA results showed that i5CysCRM197 is comparable to CRM 197 and iCRM197 in terms of specific antibody responses (Figure 4C, 4D). Moreover, a booster effect was observable, which confirms its capacity to trigger immunological memory (Figure 4C, 4D). If indeed i5CysCRM197 is structurally less stable (as CD data analysis with K2D3 might suggest), then this was not reflected in the ability of this protein to generate specific anti-CRM 197 IgG in vivo. Taken together, this data clearly shows that i5CysCRM197 can induce not only specific antibody responses, but also generation of immunological memory, both features of an effective vaccine protein carrier.

5

Reference Sequences

SEQ ID NO: 1 - CRM197 amino acid sequence

GADDWDSSKSFVMENFSSYHGTKPGYVDS IQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGV VKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRWLSLPFAEGSSSVEYINNWEQAKALS VELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSE EKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALS ILPGIGSVMGIADGA VHHNTEEIVAQS IALSSLMVAQAI PLVGELVDIGFAAYNFVES I INLFQWHNSYNRPAYSPGHKTQPFLHDGYAVSWNT VEDS I IRTGFQGESGHDIKITAENTPLPIAGVLLPTI PGKLDVNKSKTHI SVNGRKIRMRCRAIDGDVTFCRPKSPVYVG NGVHANLHVAFHRSSSEKIHSNEI SSDS IGVLGYQKTVDHTKVNSKLSLFFEIKS

SEQ ID NO: 2 - 5CysCRM197 amino acid sequence (496C underlined)

GADDWDSSKSFVMENFSSYHGTKPGYVDS IQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGV VKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRWLSLPFAEGSSSVEYINNWEQAKALS VELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSE EKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALS ILPGIGSVMGIADGA VHHNTEEIVAQS IALSSLMVAQAI PLVGELVDIGFAAYNFVES I INLFQWHNSYNRPAYSPGHKTQPFLHDGYAVSWNT VEDS I IRTGFQGESGHDIKITAENTPLPIAGVLLPTI PGKLDVNKSKTHI SVNGRKIRMRCRAIDGDVTFCRPKSPVYVG NGVHANLHVAFHRSSCEKIHSNEI SSDS IGVLGYQKTVDHTKVNSKLSLFFEIKS

SEQ ID NO: 3 - 5CysCRM197 nucleic acid sequence.

GGTGCAGATGATGTTGTTGATAGCAGCAAAAGCTTTGTGATGGAAAACTTTAGCAGCTACCATGGCACCAAACCGGGTTA

TGTTGATAGCATTCAGAAAGGTATTCAGAAACCGAAAAGCGGCACCCAGGGTAATTATGATGATGATTGGAAAGAGTTCT

ACAGCACCGATAACAAATATGATGCAGCAGGTTATAGCGTGGATAATGAAAATCCGCTGAGCGGTAAAGCCGGTGGTGTT

GTTAAAGTTACCTATCCGGGTCTGACCAAAGTTCTGGCACTGAAAGTTGATAATGCCGAAACCATCAAAAAAGAACTGGG

TCTGAGCCTGACCGAACCGCTGATGGAACAGGTTGGCACCGAAGAATTTATCAAACGTTTTGGTGATGGTGCAAGCCGTG

TTGTTCTGAGCCTGCCGTTTGCAGAAGGTAGCAGCAGCGTTGAATATATCAATAATTGGGAACAGGCAAAAGCCCTGAGC

GTTGAACTGGAAATCAATTTTGAAACCCGTGGTAAACGTGGTCAGGATGCAATGTATGAATACATGGCACAGGCATGTGC

AGGTAATCGTGTTCGTCGTAGCGTTGGTAGCAGCCTGAGCTGTATTAATCTGGATTGGGATGTGATTCGCGACAAAACCA

AAACCAAAATCGAAAGCCTGAAAGAACATGGTCCGATTAAAAACAAAATGAGCGAAAGCCCGAATAAAACCGTGAGCGAA

GAAAAAGCAAAACAGTATCTGGAAGAATTTCATCAGACCGCACTGGAACATCCGGAACTGAGCGAACTGAAAACCGTTAC

CGGCACCAATCCGGTTTTTGCCGGTGCAAATTATGCAGCATGGGCAGTTAATGTTGCACAGGTTATTGATAGCGAAACCG

CAGATAATCTGGAAAAAACCACCGCAGCACTGAGCATTCTGCCTGGTATTGGTAGCGTTATGGGTATTGCAGATGGTGCA

GTTCATCATAACACCGAAGAAATTGTTGCACAGAGCATTGCACTGAGCAGCCTGATGGTTGCACAGGCAATTCCGCTGGT

TGGTGAACTGGTTGATATTGGTTTTGCAGCCTATAACTTTGTCGAGAGCATTATCAACCTGTTTCAGGTTGTGCATAACA

GCTATAATCGTCCGGCATATAGTCCGGGTCATAAAACCCAGCCGTTTCTGCATGATGGTTATGCAGTTAGCTGGAATACC

GTTGAAGATAGCATTATTCGTACCGGTTTTCAGGGTGAAAGCGGTCATGATATCAAAATTACCGCAGAAAATACACCGCT

GCCGATTGCCGGTGTTCTGCTGCCGACCATTCCGGGTAAACTGGATGTGAATAAAAGCAAAACCCATATCAGCGTGAACG

GTCGTAAAATTCGTATGCGTTGTCGTGCAATTGATGGTGATGTTACCTTTTGTCGTCCGAAAAGTCCGGTTTATGTTGGT

AATGGTGTTCATGCAAATCTGCATGTTGCATTTCATCGTAGCTCCTGCGAAAAAATTCATAGCAATGAAATTAGCAGCGA

TAGCATTGGTGTTCTGGGTTATCAGAAAACCGTTGATCATACCAAAGTGAACAGCAAACTGAGCCTGTTTTTTGAAATCA

AAAGC

Claims

Claims:

1. A Cross-Reactive Material (CRM) 197 polypeptide comprising a cysteine residue at a position corresponding to position 496 of SEQ ID NO: 1.

2. A CRM197 polypeptide according to claim 1 comprising the amino acid sequence of SEQ ID NO: 2 or a variant thereof.

3. A conjugate comprising a CRM197 polypeptide according to claim 1 or claim 2 covalently linked to a cargo moiety, wherein the cargo moiety is covalently linked to at least said cysteine residue at a position corresponding to position 496 of SEQ ID NO: 1.

4. A conjugate according to claim 3 wherein the cargo moiety is covalently linked to at least said cysteine residue at a position corresponding to position 496 of SEQ ID NO: 1 through a thioether or disulfide bond

5. A conjugate according to claim 3 or claim 4 wherein the cargo moiety is a glucan, carbohydrate, protein, peptide, small organic molecule, immunogen, antigen, drug, hapten, or a detectable label.

6. A conjugate according to any one of claims 3 to 5, wherein the cargo moiety is an immunogen from a pathogen.

7. A conjugate according to claim 6, wherein the cargo moiety is an immunogenic glycan or carbohydrate derived from Haemophilus influenza type b {Hib), pneumococcus, or meningococcus.

8. A conjugate according to claim 7, wherein the cargo moiety is selected from Pneumococcal polysaccharide serotype (PPS) 1, PPS 3, PPS 4, PPS 5, PPS 6A, PPS 6B, PPS 7F, PPS 9V, PPS 14, PPS 18C, PPS 18C1 , PPS 19A, PPS 19F, PPS 19F1, PPS 23F, Meningococcal group A oligosaccharide, Meningococcal group C oligosaccharide, Meningococcal group W-135 oligosaccharide, or Meningococcal group Y oligosaccharide

9. A method of producing a CRM197 conjugate comprising; reacting a cargo moiety with the free thiol group of the cysteine residue of a CRM197 polypeptide according to claim 1 or claim 2, such that the cargo moiety is linked to the cysteine residue by a covalent bond.

10. A method according to claim 9, wherein the cargo moiety comprises a maleimide group, haloacetyl group, or carbonylacrylic group that reacts with the thiol group to link the cargo moiety to the cysteine residue by a thioether bond.

11. A method according to claim 9, wherein the cargo moiety comprises a pyridyl group that reacts with the thiol group to link the cargo moiety to the cysteine residue by a disulfide bond.

12. An isolated nucleic acid encoding a CRM 197 polypeptide according to claim 1 or claim 2.

13. An isolated nucleic acid according to claim 12 comprising the nucleotide sequence of SEQ ID NO: 3 or a variant thereof.

14. An expression vector comprising a nucleic acid according to claim 12 or claim 13.

15 A recombinant host cell comprising a nucleic acid according to claim 12 or claim 13 or an expression vector according to claim 14.

16. A pharmaceutical composition comprising a conjugate according to any one of claims 3 to 8 and a pharmaceutically acceptable excipient.

17. A pharmaceutical composition according to claim 16, wherein the composition is a vaccine composition.

18. A method of making a pharmaceutical composition comprising; admixing a conjugate according to any of claims 3 to 8 with a pharmaceutically acceptable excipient and optionally an adjuvant.

19. A pharmaceutical composition of claim 16 or claim 17 for use in a method of inducing an immune response in a subject.

20. A therapeutic composition of claim 16 or claim 17 for use in a method of treating cancer, Alzheimer’s disease, addiction, or atherosclerosis in a subject.