WO2020010016A1 - Conjugués immunogènes auto-adjuvés - Google Patents

Conjugués immunogènes auto-adjuvés Download PDF

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WO2020010016A1
WO2020010016A1 PCT/US2019/040184 US2019040184W WO2020010016A1 WO 2020010016 A1 WO2020010016 A1 WO 2020010016A1 US 2019040184 W US2019040184 W US 2019040184W WO 2020010016 A1 WO2020010016 A1 WO 2020010016A1
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antigen
nnaa
conjugate
kda
protein
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PCT/US2019/040184
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English (en)
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Jeffery Fairman
Jon Heinrichs
Wei Chan
Neeraj Kapoor
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Sutrovax, Inc.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • A61K39/092—Streptococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/0208—Specific bacteria not otherwise provided for
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/0216—Bacteriodetes, e.g. Bacteroides, Ornithobacter, Porphyromonas
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/095—Neisseria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6415—Toxins or lectins, e.g. clostridial toxins or Pseudomonas exotoxins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6087—Polysaccharides; Lipopolysaccharides [LPS]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • A61K2039/627—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier characterised by the linker

Definitions

  • the immune response to a“weak” saccharide antigen can be amplified by conjugation to a known“strong” carrier polypeptide antigen such as diphtheria toxoid, tetanus toxoid,
  • H. influenzae protein D or CRM 197.
  • the PedvaxHIBTM, MenjugateTM, PrevnarTM, and SynflorixTM products all include bacterial capsular saccharide antigens which are conjugated to such carrier polypeptides.
  • the use of conjugation is particularly important in pediatric vaccines or else the saccharides does not elicit a memory immune response.
  • Each of these vaccines also includes an aluminum salt adjuvant.
  • the adjuvant enhances the immunogenicity of the immunogenic saccharide-polypeptide conjugates.
  • Aluminum salts are effective adjuvants but are not ideal because of concerns about potential toxicity, particularly when multiple doses are administered ( e.g . see Vecchi et al.
  • a further object of the invention is to provide improved immunogenicity over saccharide-polypeptide conjugates.
  • immunogenic conjugates comprising a carrier polypeptide, an antigen, and an immunostimulator, wherein the carrier polypeptide includes at least one non-natural amino acid residue via which the carrier polypeptide is covalently linked to the antigen and/or the immunostimulator.
  • the antigen may be site-specifically conjugated to a reactive group of nnAAs in an unconjugated carrier protein through a chemical handle introduced into the antigen.
  • the carrier protein comprises (a) at nn sites an nnAA residue wherein each nnAA residue (i) comprises a reactive group that provides site-specific conjugation of an antigen to the carrier protein, and (ii) was introduced site-specifically during synthesis of the carrier protein, wherein nn is an integer greater than or equal to 2; and (b) at least one T-cell activating epitope that has not been inactivated by the presence of an nnAA residue.
  • nnAA residue(s), the antigen, and the immunostimulator can be arranged in various ways.
  • a first nnAA residue can be covalently linked to the
  • immunostimulator and a second nnAA residue in the same carrier polypeptide as the first nnAA residue can be covalently linked to the antigen; a nnAA residue can be covalently linked to the immunostimulator, and the antigen can be covalently linked to the immunostimulator, such that the antigen is joined to the nnAA via the immunostimulator; or a nnAA residue can be covalently linked to the antigen, and the immunostimulator can be covalently linked to the antigen, such that the immunostimulator is joined to the nnAA via the antigen. Certain arrangements involve covalently linking an antigen to both a nnAA residue and an antigen to the immunostimulator, such that the immunostimulator is joined to the nnAA via the antigen.
  • immunostimulator These arrangements can conveniently be achieved by providing an antigen having multiple conjugation sites and reacting some of these with nnAA residues and others with an immunostimulator. Where a single carrier polypeptide includes multiple nnAA residues this arrangement can advantageously lead to the formation of cross-linked conjugates.
  • an immunogenic conjugate comprising a carrier polypeptide, an antigen, and an immunostimulator, wherein: (i) the carrier polypeptide includes multiple non natural amino acid residues which are covalently linked to the antigen; and (ii) the antigen is also covalently linked to the immunostimulator.
  • the carrier protein comprises 4 to 9 nnAA residues.
  • the antigen is a bacterial or viral antigen.
  • An exemplary antigen is a bacterial saccharide selected from the group consisting of capsular polysaccharides from Streptococcus pneumoniae, capsular polysaccharides from Streptococcus pyogenes, group-A- strep cell wall polysaccharides from Streptococcus pyogenes, capsular polysaccharides of Streptococcus agalactiae, capsular polysaccharides of Haemophilus influenzae, capsular polysaccharides of Neisseria meningitidis, and capsular polysaccharides from Porphyromonas gingivalis.
  • Exemplary capsular polysaccharides suitable as antigens herein are capsular polysaccharides of a Streptococcus pneumoniae serotype selected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7F, 7A, 7B, 7C, 8, 9A, 9L, 9N, 9V, 10F, 10A, 10B, 10C,
  • the carrier protein has at least 80% sequence identity with respect to SEQ ID NO: 1 or 11. In some embodiments, the carrier protein has at least 80% sequence identity with respect to SEQ ID NO: 1 or 11. In some embodiments, the carrier protein has at least 80% sequence identity with respect to SEQ ID NO: 11.
  • the carrier protein comprises amino acid SEQ ID NO: 13.
  • a variety of immunostimulators are useful herein, including TLR agonists,
  • immunostimulatory oligonucleotides immunostimulatory oligonucleotides, CLR agonists, and CDld agonists.
  • Also provided are processes for preparing an immunogenic conjugate comprising a carrier polypeptide, an antigen, and an immunostimulator comprising the steps of: (i) providing an antigen having at least two instances of a chemical handle (or "first functional group", a carrier polypeptide having a reactive group (or “second functional group”), and an immunostimulator having a third functional group, wherein the chemical handle can react to form covalent bonds with the reactive group and with the third functional group; and (ii) mixing the antigen, carrier polypeptide and immunostimulator, in any order, under conditions such that the first functional groups reacts with the second and third functional groups to give the immunogenic conjugate.
  • the reactive group and the third functional group may be either the same or different.
  • the second and third functional groups are, in some embodiments, the same (e.g. azido).
  • the process may involve a step of introducing the first functional groups into the antigen prior to step (i).
  • the antigen and immunostimulator are mixed (conjugated) and the resulting conjugate then reacted with the carrier polypeptide to form the conjugate comprising polypeptide carrier, antigen, and immunostimulator.
  • Also provided are processes for preparing an immunogenic conjugate comprising a carrier polypeptide, an antigen, and an immunostimulator wherein the process comprises steps of: (i) providing an antigen-immunostimulator conjugate having a first functional group and a carrier polypeptide having a second functional group, wherein the first functional group can react to form covalent bonds with the second functional group; and (ii) mixing the antigen- immunostimulator conjugate with the carrier polypeptide under conditions such that the first functional groups reacts with the second functional group to give the immunogenic conjugate.
  • compositions including one or more immunogenic conjugates of the invention.
  • Pharmaceutical compositions can include, for instance, between 3 and 50 different conjugates of the invention (e.g. 14, 15, 20, 21, 24, 25, or more).
  • each of these conjugates can include a capsular saccharide from a different serotype or serogroup of the same bacterial species (e.g. multiple meningococcal serogroups or multiple pneumococcal serotypes).
  • compositions can also include one or more immunogenic conjugates which include a carrier polypeptide and an antigen but do not include an immunostimulator.
  • immunogenic conjugates which include a carrier polypeptide and an antigen but do not include an immunostimulator.
  • conjugate mixtures can be created in which only particular antigens of interest are enhanced by the presence of the immunostimulator.
  • compositions including (i) one or more
  • immunogenic conjugates comprising a carrier polypeptide and an antigen and (ii) one or more immunogenic conjugates comprising a carrier polypeptide, an antigen, and an
  • FIG. 1 compares the immunogenicity of a conjugate of the invention with an analogous conjugate not containing an immunostimulator (as described in the examples herein).
  • Recombinant protein production allows the optimization of antigenicity and nontoxicity of carrier proteins, but the existing carrier proteins are difficult to produce in recombinant cells and wholly engineered proteins are difficult to produce in high yields.
  • Gentler conjugation reactions minimize the denaturation/obstruction of carrier and antigen epitopes, but the lower efficiency of these reactions results in less loading of the antigen on the carrier protein and more complicated purification schemes.
  • relatively lower antigen to carrier results in a higher likelihood of immune“interference” by antibody responses to the carrier protein itself, or the recognized phenomenon of carrier-induced epitopic suppression.
  • polypeptides including enhanced carrier proteins, comprising non-natural amino acids
  • antigens that are suitable to conjugate to polypeptides, including enhanced carrier proteins, comprising non-natural amino acids
  • vaccine compositions comprising the foregoing; and (5) methods of making and using the foregoing.
  • suppression codon refers to a nucleotide triplet that is introduced into a polynucleotide at a predetermined location and is recognized by a specific tRNA that can recognize a stop codon (e.g., an amber, ochre or opal stop codon) and allows translation to read through the codon to produce the protein, thereby suppressing the stop codon.
  • a stop codon e.g., an amber, ochre or opal stop codon
  • A“non-natural amino acid” refers to an amino acid that is not one of the 20 common amino acids or pyrolysine or selenocysteine; other terms that are used synonymously with the term“non-natural amino acid” are "non-naturally encoded amino acid,”“unnatural amino acid,”“non-naturally occurring amino acid,” and variously hyphenated and non- hyphenated versions thereof.
  • Non-natural amino acids with bio-orthogonal reactive chemical side chains are able to be used as a chemical“handle” to conjugate various payloads to discrete sites in a protein.
  • sequence identity or“percent identity” in the context of two or more nucleic acids or polypeptide sequences, refers to two or more sequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using a sequence comparison algorithm (e.g., BLASTP for amino acid sequences).
  • sequence comparison algorithm e.g., BLASTP for amino acid sequences.
  • the percent identity is determined over the full-length sequence, such as the reference sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 11.
  • the method for calculating the sequence identity as provided herein is the BLASTP program having its defaults set at a wordlength (W) of 3, an expectation (E) of 10, and the BLOSEIM62 scoring matrix (see, e.g, Henikoff & Henikoff, 1989, Proc Natl Acad Sci USA 89: 10915). See e.g., the BLAST alignment tool available on the WWW at blast.ncbi.nlm.nih.gov/Blast.cgi or elsewhere.
  • antigen refers to any molecule or a linear molecular fragment that is able to be recognized by the highly variable antigen receptors (B-cell receptors, T-cell receptors, or both) of the adaptive immune system.
  • antigens include
  • T-cell activating epitope refers to a structural unit of molecular structure which is capable of inducing T-cell immunity.
  • carrier proteins which include T-cell activating epitopes is well known and documented for conjugates.
  • a T-cell activating epitope in the carrier protein enables the covalently-attached antigen to be processed by antigen-presenting cells and presented to CD4 +ve T cells to induce immunological memory against the antigen.
  • B-cell epitope refers generally to those features of a macromolecular structure which are capable of inducing a B cell response.
  • a B-cell epitope need not comprise a peptide, since processing by antigen-presenting cells and loading onto the peptide-binding cleft of MHC is not required for B-cell activation.
  • carrier protein refers to a non-toxic or detoxified polypeptide containing a T-cell activating epitope which is able to be attached to an antigen (e.g., a polysaccharide) to enhance the humoral response to the conjugated antigen in a subject.
  • an antigen e.g., a polysaccharide
  • the term includes any of the bacterial proteins used as epitope carriers in FDA-approved vaccines.
  • the carrier protein is Corynebacterium diphtheriae toxin, Clostridium tetani tetanospasmin, Haemophilus influenzae protein D (PD, HiD), outer membrane protein complex of serogroup B meningococcus (OMPC), CRM197, or malaria ookinete specific surface protein Pfs25.
  • the carrier protein is BB, derived from the G protein of
  • A“native carrier protein” has only naturally occurring amino acids.
  • An“enhanced carrier protein” has at least one non-natural amino acid replaced for a naturally occurring amino acid in the carrier protein.
  • the terms“carrier protein” and“carrier polypeptide” are used interchangeably herein.
  • immunologic polypeptide refers to a polypeptide comprising at least one T-cell activating epitope, wherein the T-cell epitope is derived from a protein capable of inducing immunologic memory in animals.
  • eCRM or“enhanced CRM” as used interchangeably herein refers to a modified version of the G52E codon variant of diphtheria toxin, wherein at least one of the natural amino acid residues is substituted for a non-natural amino acid and the polypeptide retains at least one T-cell activating epitope.
  • the terms“modified,”“replaced,”“enhanced,” and“substituted” are considered synonymous when used to describe residues of a polypeptide, and in all cases refer to the replacement of a non-natural amino acid for a naturally occurring amino acid within a polypeptide chain.
  • T-independent antigen refers to an antigen that induces the features of B-cell mediated immunity, or which does not induce processes associated with helper T-cell mediated immunity such as isotype switching or immunologic memory.
  • polysaccharide as used herein, is used in its ordinary sense, including, without limitation, saccharides comprising a plurality of repeating units, including, but not limited to polysaccharides having 50 or more repeat units, and oligosaccharides having 50 or less repeating units. Typically, polysaccharides have from about 50, 55, 60, 65, 70, 75, 80, 85,
  • Oligosaccharides typically have from about 6, 7, 8, 9, or 10 repeating units to about 15, 20, 25, 30, or 35 to about 40 or 45 repeating units.
  • glycan refers to any linear or branched polymer consisting of monosaccharide (e.g. glucose) residues joined to each other by glycosidic linkages.
  • monosaccharide e.g. glucose
  • examples of glycans include glycogen, starch, hyaluronic acid, and cellulose.
  • Other examples of“glycans” include bacterial capsular polysaccharides.
  • molecular weight of a polysaccharide or of a carrier protein- polysaccharide conjugate refers to molecular weight calculated by size exclusion
  • SEC chromatography
  • MALS multiangle laser light scattering
  • lower alkyl refers to a saturated straight or branched hydrocarbon having one to six carbon atoms, i.e., Ci to C6 alkyl.
  • the lower alkyl group is a primary, secondary, or tertiary hydrocarbon.
  • the term includes both substituted and unsubstituted moieties. See also ETS-2014/0066598.
  • the term“lower alkylene” refers to an alkylene radical of a lower alkyl. [0040]
  • the term“about” in relation to a numerical value x is optional and means, for example, c+10%.
  • the word“substantially” does not exclude“completely” e.g. a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the invention.
  • nnAA used herein are generally a-amino acids with a chiral center at the a-carbon, and they are preferably (L) isomers.
  • PCR amplification methods are described, for example, in Innis et al. , PCR Protocols: A Guide to Methods and Applications, Academic Press Inc. San Diego, Calif., 1990 and
  • An amplification reaction typically includes the DNA that is to be amplified, a thermostable DNA polymerase, two oligonucleotide primers, deoxynucleotide triphosphates (dNTPs), reaction buffer and magnesium. Typically, a desirable number of thermal cycles is between 1 and 25.
  • Methods for primer design and optimization of PCR conditions are found in molecular biology texts such as Ausubel et al., Short Protocols in Molecular Biology, 5th Edition, Wiley, 2002, and Innis et al. , PCR Protocols, Academic Press, 1990. Computer programs are useful in the design of primers with the required specificity and optimal amplification properties (e.g., Oligo Version 5.0 (National Biosciences)).
  • the PCR primers additionally contain
  • the PCR primers also contain an RNA polymerase promoter site, such as T7 or SP6, to allow for subsequent in vitro transcription. Methods for in vitro transcription are found in sources such as Van Gelder et al. , Proc. Natl. Acad. Sci. U.S.A. 87 : 1663-1667 , 1990; Eberwine et al., Proc. Natl. Acad. Sci. U.S.A. 89:3010-3014, 1992.
  • the molecular weight of a polysaccharide or of a carrier protein-polysaccharide conjugate is measured by size exclusion chromatography (SEC) combined with multiangle laser light scattering (MALS).
  • SEC MALS-UV-RI setup consists of an Agilent HPLC 1100 (including degasser, quaternary pump, temperature-controlled auto-sampler, temperature controlled column compartment and ETV-VIS diode array detector) in line with a DAWN- HELEOS multi-angle laser light scattering detector and Optilab T-rEX differential refractive interferometer (Wyatt Technology, Santa Barbara, CA) for the detection of eluting species.
  • the column compartment is set to 25 °C and the sample compartment is set to 4 °C.
  • a mobile phase consisting of 0.2 pm filtered lx PBS with 5% v/v acetonitrile is used at a 0.5 mL/min flow rate.
  • Samples are injected within a concentration range of 0.2-1.5 mg/mL polysaccharide and the injected volume is adjusted to yield a total injected mass of 30-40 pg.
  • Agilent Open Lab software is used to control the HPLC, and Wyatt Astra 7 software is used for data collection and analysis. The technique reveals the distribution of absolute molecular weights for conjugates in a sample, and results for a population are expressed as an average value.
  • S. pneumoniae isolated capsular polysaccharides are obtained directly from bacteria using isolation procedures known to one of ordinary skill in the art (see for example methods disclosed in U.S. Patent App. Pub. Nos. 2006/0228380, 2006/0228381, 2007/0184071, 2007/0184072, 2007/0231340, and 2008/0102498 and WO 2008/118752).
  • S. pneumoniae isolated capsular polysaccharides are obtained from a commercial source (e.g., ATCC).
  • the invention generally concerns immunogenic conjugates and methods for the preparation of immunogenic conjugates.
  • conjugates comprise a carrier polypeptide, an antigen, and an immunostimulator.
  • Linking of the antigen to the carrier polypeptide can convert a T-cell independent immunogen (such as a saccharide) into a T-cell dependent immunogen, thereby enhancing the immune response which is elicited by that immunogen (particularly in children).
  • the conjugates prepared using the present methods contain covalent linkages that are formed between an antigen and a non-natural amino acid (nnAA) residue within the carrier polypeptide, as well as containing linkages that are formed between (a) the antigen and the immunostimulator, (b) the carrier polypeptide and the immunostimulator, or (c) both (a) and (b).
  • nnAA residues can provide functional groups which facilitate reactivity with an antigen of interest.
  • Conjugates used herein include covalent linkages that are formed between a non natural amino acid (‘nnAA’) residue within the carrier polypeptide and (i) the antigen and/or (ii) an immunostimulator.
  • nnAA residue(s) in the carrier polypeptide provide functional groups which facilitate reactivity with the antigen of interest and/or immunostimulator.
  • the individual components in the conjugates i.e. carrier polypeptide, antigen, and immunostimulator
  • a single carrier polypeptide can include multiple nnAA residues, with antigen and immunostimulator being attached to separate nnAA residues; an immunostimulator can be attached to the nnAA and an antigen can be attached to a different site on the immunostimulator; or, in an exemplary embodiment, an antigen can be attached to the nnAA and an immunostimulator can be attached to a different site on the antigen.
  • antigens usefully include multiple conjugation sites, permitting it to form covalent bonds with immunostimulator molecules and with nnAA residues.
  • conjugation occurs sequentially (e.g . antigen is first conjugated to immunostimulator, and then to nnAA) via the same functional groups (e.g. via alkynes in a derivatized antigen) it is necessary to have spare functional groups after the first conjugation.
  • the first conjugation uses from 5-25% of the conjugation sites (e.g. from 10-15%).
  • Antigens can have a single linking group per molecule (e.g. the reducing terminus of a saccharide) for conjugation, or can have multiple linking groups (e.g. multiple aldehyde or cyanate ester groups). Where an antigen molecule has multiple linking groups this can permit linkage to both nnAA residues and to immunostimulatory molecules. Moreover, when used in conjunction with a carrier polypeptide that includes multiple nnAA residues this can lead to the formation of high molecular weight cross-linked or lattice conjugates, involving links between multiple carrier polypeptides and multiple antigens.
  • Cross-linked conjugates are preferred herein (particularly for pneumococcus).
  • antigens with multiple conjugation sites are also preferred.
  • a single carrier polypeptide includes multiple nnAA residues (e.g. up to 10).
  • Covalent linkages are formed between a nnAA residue within the carrier polypeptide and the antigen and/or immunostimulator.
  • conjugation does not occur not via a lysine residue in the carrier polypeptide; more preferably, conjugation does not occur via a natural amino acid residue in the carrier polypeptide.
  • polypeptides comprising at least one nnAA residue, wherein nnAA residues are defined and discussed in detail in Section III. A.2, infra.
  • suitable polypeptides are biologically active peptides, such as those inducing a T-cell immune response in mammals, particularly humans, as well as domesticated animals, such as cattle, horses, sheep, dogs and cats; in general, any polypeptide that contains a T-cell epitope can be used as a carrier protein.
  • the T-cell epitope can bind to MHC class II and interact with T-cell receptors on the surface of CD4+ T-cells, thereby enhancing antibody responses against antigens or haptens conjugated thereto (see, e.g, Costantino et al. 2011, Expert Opin Drug Discov 6: 1045-66).
  • Tontini et al. (2016) Vaccine 34:4235-42 discuss pre-clinical studies of 28 carrier polypeptides, including tests of their ability to induce antibodies against saccharide antigens.
  • Poly epitope carrier polypeptides containing multiple broadly-reactive i.e.
  • T-cell epitopes found within known carriers e.g. Tt, PD, CRM197
  • Various detoxified bacterial toxins have been successfully used as carriers e.g. Tt, Dt, the P. aeruginosa exotoxin, the C.difflcile A and B toxins, etc.
  • diphtheria toxoid chemically treated toxin from Corynebacterium diphtheriae;‘Dt’
  • tetanus toxoid chemically treated tetanospasmin toxin from Clostridium tetani;‘Tt’
  • protein D from Haemophilus influenzae (‘PD’ or‘HiD’)
  • OMPC outer membrane protein complex of serogroup B meningococcus
  • diphtheriae toxin can use any of these numerous carrier polypeptides as starting points to design carrier proteins according to the present invention, modifying them to include at least one nnAA.
  • Useful carrier polypeptides contain a T-cell epitope.
  • Various such carrier polypeptides are known in the art, and within approved vaccines it is known to use diphtheria toxoid
  • tetanus toxoid chemically treated tetanospasmin toxin from Clostridium tetani;‘Tt’)
  • protein D from Haemophilus influenzae (‘PD’ or‘HiD’)
  • OMPC outer membrane protein complex of serogroup B meningococcus
  • CRM197 An exemplary carrier polypeptide upon which to base the carriers used with the invention is CRM197.
  • CRM197 is well-known in the art (e.g. see Broker el al. 2011 Biologicals 39: 195-204) and has the following amino acid sequence (SEQ ID NO: 11), where the underlined residue (Glu-52) differs from the natural diphtheria toxin, whereby the substitution of Gly Glu leads to the loss of toxic enzymatic activity in the protein:
  • the invention does not use native CRM197. Instead of using CRM197 comprising SEQ ID NO: 11, a modified amino acid sequence is used which contains at least one nnAA. These modified CRM197 carrier polypeptides are described infra. Modified CRM197 may also include, for example, an N-terminal methionine (e.g., such as in SEQ ID NO: 1, below) or other modifications described herein.
  • Another carrier polypeptide of interest is PD from H.influenzae , which naturally has the following amino acid sequence (SEQ ID NO: 8):
  • a modified amino acid sequence which contains at least one nnAA.
  • one or more Lys residues within SEQ ID NO: 8 can be replaced with a nnAA.
  • T-cell epitope prediction and recognition for PD has been reported by Hua et al. (2016) Clin Vaccine Immunol 23: 155-61.
  • An additional carrier polypeptide of interest is Tt from C.tetani , which naturally has the amino acid sequence SEQ ID NO: 15. Rather than using native Tt, a modified amino acid sequence is used which contains at least one nnAA. For instance, one or more Lys residues within SEQ ID NO: 15 can be replaced with a nnAA. There are 109 Lys residues within SEQ ID NO: 15, so several can be replaced by nnAA and then used for conjugation.
  • the polypeptide should also be detoxified prior to use as a carrier e.g. by formaldehyde or glutaraldehyde treatment.
  • polypeptide will typically be cleaved between residues 457-458, with the two fragments being held together by a disulfide bond between residues 439 & 467.
  • MPITINNFRYSDPVNNDTI IMMEPPYCKGLDIYYKAFKITDRIWIVPERYEFGTKPEDF NPPSSLIEGASEYYDPNYLRTDSDKDRFLQTMVKLFNRIKNNVAGEALLDKI INAIPYL GNSYSLLDKFDTNSNSVSFNLLEQDPSGATTKSAMLTNLI IFGPGPVLNKNEVRGIVLR VDNKNYFPCRDGFGSIMQMAFCPEYVPTFDNVIENITSLTIGKSKYFQDPALLLMHELI HVLHGLYGMQVSSHEI IPSKQEIYMQHTYPISAEELFTFGGQDANLISIDIKNDLYEKT LNDYKAIANKLSQVTSCNDPNIDIDSYKQIYQKYQFDKDSNGQYIV
  • any polypeptide including a T-cell epitope can be used as a carrier polypeptide.
  • the T-cell epitope can bind to MHC class II and interact with T-cell receptors on the surface of CD4+ T-cells, thereby enhancing antibody responses against antigens or haptens conjugated thereto ( e.g . see Costantino et al. 2011, Expert Opin Drug Discov 6:1045-66).
  • CRM197 can be used.
  • Various detoxified bacterial toxins have been successfully used as carriers e.g. Tt, Dt, the P. aeruginosa exotoxin, the C.difficile A & B toxins, etc.
  • Many different carrier polypeptides have been used for pneumococcal saccharides e.g. CRM 197 in PrevnarTM, PD, Tt and Dt in SynflorixTM, and various peptides in Velasco el al. (1995) Infect Immun 63 :961-8.
  • the invention can use any of these numerous carrier polypeptides as starting points to design carrier proteins according to the present invention, modifying them to include at least one nnAA, to enhance the immunogenicity of antigens of interest.
  • nnAA-containing carrier polypeptides to be used with the invention can be prepared using the techniques disclosed in section III.C below. Certain carriers contain nnAAs outside of at least one T-cell epitope of the carrier. If the T-cell epitope regions for a carrier are unknown then one can identify the epitopes using standard techniques e.g. see Reece el al. (1993) IJ Immunol 151 :6175-84, Beissbarth et al. (2005) Bioinformatics 21 Suppl 1 : ⁇ 29-37, Maciel Jr et al. (2008) Virol 378: 105-17, Fridman et al.
  • any particular modification of a carrier polypeptide’s sequence does not eliminate the desired T-cell response to a conjugated antigen, such as the saccharides herein.
  • An exemplary group of carriers do not contain any modification, including insertion or substitution of a nnAA, within a T-cell epitope.
  • Another exemplary group of carriers do not contain any modification, including insertion or substitution of a nnAA, within multiple T-cell epitopes.
  • Yet another exemplary group of carriers do not contain any modification, including insertion or substitution of a nnAA, within any T-cell epitope.
  • the polypeptide is an immunogenic polypeptide.
  • the nnAA residue is substituted for a native residue of a specified polypeptide.
  • the nnAA residue is incorporated by insertion, or by C-terminal or N- terminal extension.
  • the polypeptide comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 nnAA residues.
  • the polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 nnAA residues.
  • Some carriers contain as least 2, at least 3, at least 4, at least 5, or at least 6 nnAAs.
  • Certain carriers may also have a maximum of 10, 9, 8, 7 or 6 nnAAs.
  • carrier polypeptides with fewer than 10 nnAA residues are may be utilized.
  • Exemplary ranges of nnAAs in a carrier polypeptide include 2-10, 2-9, 2-8, 2-7, 2-6, 3-10, 3-9, 3-8, 3-7, 3-6, 4-10, 4-9, 4-8, 4-7, and 4-6 nnAAs, with 2-9 nnAAs, e.g., 4-6 nnAAs.
  • the carrier polypeptide may comprise two or more nnAA residues that are chemically distinct, i.e., the two or more nnAA residues comprise at least two different non-natural amino acids, such as those identified herein.
  • the polypeptide may comprise a T-cell activating epitope of a carrier protein, and may be conjugated to an antigen, such as through an nnAA within the polypeptide structure, where the antigen may be a T-cell independent antigen such as a hapten, a bacterial capsular polysaccharide, a bacterial lipopolysaccharide, or a tumor-derived glycan (for example, the antigen may comprise a bacterial non-capsular polysaccharide such as an exopolysaccharide, e.g. the S. aureus exopolysaccharide).
  • the nnAA are not in a T-cell activating epitope of the carrier polypeptide.
  • the nnAA can be substituted for a lysine residue in the native polypeptide.
  • the substitution can occur at one or more of positions K24, K33, K37, K39, K212, K214, K227, K244, K264, K385, K522 and K526 in SEQ ID NO: 11 or 12.
  • An exemplary modified CRM may include substitution of a nnAA (e.g. pAMF) at each of K33, K212, K244, K264, K385, and K526 (and in some embodiments at no other positions) according to the number of SEQ ID NO: 11 or 12.
  • nnAA substitutions to incorporate nnAA are not limited to lysine positions, however, and it is also possible to substitute other amino acids with a nnAA e.g. Phe, Asp, Asn, Glu, Gln, Arg, Ser, and/or Thr.
  • the carrier proteins described herein have a solubility of at least 50mg/L (e.g, at least 100 mg/L, at least 150 mg/L, at least 200 mg/L, or at least 250 mg/L) when expressed in a cell-free protein synthesis system.
  • a carrier protein described herein includes more than one nnAA residue
  • some embodiments include only a single species of nnAA (e.g, the only nnAA in the carrier is pAMF). This permits the same conjugation chemistry to be used simultaneously at each nnAA.
  • each conjugate includes the same single species of nnAA.
  • a composition includes multiple different conjugates (e.g., different pneumococcal serotypes) it is may be advantageous that each conjugate includes the same carrier protein.
  • the disclosure provides a polynucleotide encoding the polypeptides described herein. In certain embodiments, the disclosure provides for an expression vector comprising the polynucleotide encoding the polypeptide described herein. In certain embodiments, the disclosure provides for a host cell comprising the expression vector.
  • conjugates used herein may include covalent linkages between an antigen and a functional group within a nnAA residue in the carrier polypeptide.
  • the side chains of nnAA residues can provide reactive functional groups which are useful for conjugating antigens to discrete sites in the carrier polypeptide.
  • the nnAA can be any non-natural amino acid that can be incorporated into a polypeptide during translation but is not one of the 20 common amino acids.
  • a nnAA can be incorporated into a polypeptide by converting a tRNA molecule such that its codon incorporates the nnAA rather than the natural cognate amino acid.
  • One technique for achieving this involves using a“suppression codon” i.e.
  • nucleotide triplet that is introduced into a coding sequence at a desired position and is recognized by a specific tRNA that can recognize a natural stop codon (e.g., an amber, ochre or opal stop codon) but allows translation to continue, with incorporation of the nnAA (thereby suppressing the natural stop codon).
  • a natural stop codon e.g., an amber, ochre or opal stop codon
  • the nnAA residue can be any of the nnAA residues described herein, or any other that has been identified as compatible with cell-based or cell-free protein synthesis (see, e.g., Schultz et al. (2010) Annu Rev Biochem. 79:413-44, particularly at pp.418-420; and Chin et al. (2014) Annu Rev Biochem. 83:5.1-5.30, which are hereby incorporated by reference).
  • the nnAA is an amino acid that is not one of the 20 common amino acids or pyrolysine or selenocysteine.
  • non-natural amino acids examples include, without limitation: a non-natural analog of a tyrosine amino acid; a non- natural analog of a glutamine amino acid; a non-natural analog of a phenylalanine amino acid; a non-natural analog of a serine amino acid; a non-natural analog of a threonine amino acid; an alkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or amino-substituted amino acid, or any combination thereof; an amino acid with a photo
  • nnAA for use as described herein are those which can be incorporated during translation (in a cellular or a cell-free system) with a side chain that provides a functional group which is not found in the side chain of any of the 20 naturally occurring amino acids (e.g., azido).
  • Various techniques for incorporating such amino acids into polypeptides are known e.g. see Young & Schultz (2010) J Biol Chem 285: 11039-44, Maza et al. (2015) Bioconjugate Chem. 26: 1884-9, and Zimmerman et al. (2014) Bioconjugate Chem. 25:351-61. Additionally, described herein are methods whereby nnAA residues can be incorporated into carrier polypeptides e.g.
  • the nnAA can include a chemical group suitable for a "click” chemistry reaction with a corresponding group on an antigen of interest or hapten.
  • the nnAA can be any of 2-amino-3-(4-azidophenyl)propanoic acid (para-azido-L- phenylalanine or pAF), 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid (para-azidomethyl-L- phenylalanine or pAMF), 2-amino-3-(5-(azidomethyl)pyridin-2-yl)propanoic acid, 2-amino-3- (4-(azidomethyl)pyridin-2-yl)propanoic acid, 2-amino-3-(6-(azidomethyl)pyri din-3 -yl)propanoic acid, or 2-amino-5-azidopentanoic acid.
  • 2-amino-3-(4-azidophenyl)propanoic acid para-azido-L- phenylalanine or pAF
  • the present disclosure provides a polypeptide comprising at least one nnAA replaced for a naturally occurring amino acid within the polypeptide according to SEQ ID NO: 1, wherein the at least one nnAA is replaced for K25, K34, K38, K40, K213, K215, K228, K245, K265, K386, K523, or K527 of SEQ ID NO: 1, wherein, in some
  • the nnAA comprises a linking moiety.
  • the nnAA is selected from 2-amino-3-(4-azidophenyl)propanoic acid (pAF), 2-amino-3-(4- (azidomethyl)phenyl)propanoic acid (pAMF), 2-amino-3-(5-(azidomethyl)pyridin-2- yl)propanoic acid, 2-amino-3-(4-(azidomethyl)pyri din-2 -yl)propanoic acid, 2-amino-3-(6- (azidomethyl)pyridin-3-yl)propanoic acid, 2-amino-5-azidopentanoic acid, and 2-amino-3-(4- (azidomethyl)phenyl)propanoic acid, or any combination thereof.
  • K265 of SEQ ID NO:l is replaced.
  • K386 of SEQ ID NO:l is replaced.
  • K265 and K386 of SEQ ID NO: l are replaced.
  • the polypeptide comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 nnAAs.
  • the nnAA is selected from 2-amino-3-(4- azidophenyl)propanoic acid (pAF), 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid (pAMF), 2-amino-3-(5-(azidomethyl)pyridin-2-yl)propanoic acid, 2-amino-3-(4-(azidomethyl)pyridin-2- yl)propanoic acid, 2-amino-3-(6-(azidomethyl)pyri din-3 -yl)propanoic acid, 2-amino-5- azidopentanoic acid, and 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid, or any combination thereof.
  • pAF 2-amino-3-(4- azidophenyl)propanoic acid
  • pAMF 2-amino-3-(4-(azidomethyl)phenyl)
  • nnAA for use herein is pAMF: pAMF provides very favorable reaction kinetics for producing conjugates (e.g. much faster than using pAF when reacting with an alkyne-containing carbohydrate antigen in a SPAAC method).
  • the nnAA can be a 2,3-disubstituted propanoic acid bearing: an amino substituent at the 2-position; and an azido-containing substituent, a l,2,4,5-tetrazinyl-containing substituent, or an ethynyl-containing substituent at the 3-position.
  • the substituent at the 3-position is an azido-containing substituent, particularly an azido-containing substituent comprising a terminal azido group bound to the carbon atom at the 3-position through a linking group.
  • the linking group may comprise an arylene moiety that is optionally substituted and optionally heteroatom-containing.
  • the linking group may comprise a 5- or 6-membered arylene moiety containing 0 to 4 heteroatoms and 0 to 4 non-hydrogen ring substituents.
  • nnAA can have the structure of formula (XII):
  • Ar comprises a 5-membered or 6-membered aromatic ring optionally containing at least one heteroatom
  • W 5 is selected from Ci-Cio alkylene, -NH-, -O- and -S-;
  • Ql is zero or 1;
  • W 6 is selected from azido, l,2,4,5-tetrazinyl optionally C-substituted with a lower alkyl group, and ethynyl
  • R 4 (XIII) in which R 3 is OH or an amino acid residue of the carrier protein, and R 4 is H or an amino acid residue of the carrier protein.
  • Ar in formulae (XII) and (XIII) does not contain any heteroatoms, in which case a preferred linker is an unsubstituted phenylene group (i.e. Ar is -C6H4-).
  • Ar contains a nitrogen heteroatom and at least one additional heteroatom selected from N, O, and S.
  • Exemplary nitrogen heterocycles are described infra and Ar may be e.g. a pyridine or a pyridazine.
  • Ql is 1, W 5 is lower alkylene, and W 6 is azido.
  • the nnAA residue comprises an azido-containing nnAA, such as an azido-containing nnAA of formula (I):
  • D is— Ar— W3— or— Wi— Yi— C(O)— Y2— W2— ; each of Wi, W2, and W3 is independently a single bond or lower alkylene; each Xi is independently -NH-, -O-, or -S-; each Yi is independently a single bond, -NH-, or -O-; each Y2 is independently a single
  • Zi, Z2, and Z3 are independently -CH-.
  • the nnAA residue comprises an azido-containing amino acid of formula (II):
  • W 4 is C1-C10 alkylene
  • the nnAA residue may comprise an azido-containing amino acid selected from the group consisting of 2-amino-3-(4-azidophenyl)propanoic acid (pAF), 2- amino-3-(4-(azidomethyl)phenyl)propanoic acid (pAMF), 2-amino-3-(5-(azidomethyl)pyridin-2- yl)propanoic acid, 2-amino-3-(4-(azidomethyl)pyridin-2-yl)propanoic acid, 2-amino-3-(6- (azidomethyl)pyridin-3-yl)propanoic acid, 2-amino-5-azidopentanoic acid, or 2-amino-3-(4- (azidomethyl)phenyl)propanoic acid, and any combination thereof.
  • pAF 2-amino-3-(4-azidophenyl)propanoic acid
  • pAMF 2- amino-3-(
  • the nnAA residue comprises 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid (pAMF).
  • pAMF provides very favorable reaction kinetics for producing conjugates (e.g ., much faster than using pAF when reacting with an alkyne-containing carbohydrate antigen in a SPAAC method).
  • the non-natural amino acid residue comprises a l,2,4,5-tetrazine containing nnAA.
  • the non-natural amino acid comprises a 1, 2,4,5- tetrazine containing nnAA of formula (III):
  • V is a single bond, lower alkylene, or -W1-W2-; one of Wi and W2 is absent or lower alkylene, and the other is -NH-, -0-, or -S-; each one of Zi, Z2, and Z3 is independently -CH- or -N-; and Xi is independently -NH-, -0-, or -S-; and R is lower alkyl,
  • the nnAA residue comprises an alkyne-containing nnAA, such as a propargyl group.
  • alkyne-containing nnAA such as a propargyl group.
  • propargyl-containing amino acids and syntheses thereof are found in Beatty et al. (2006) Angew. Chem. Int. Ed. 45: 7364-7; Beatty et al. (2005) J. Am.
  • the nnAA residue comprises a propargyl-containing nnAA selected from the group consisting of homopropargylglycine, ethynylphenylalanine, and N6-[(2- propy ny 1 oxy )carb ony 1 ] -L-ly sine .
  • nnAA are generally a-amino acids with a chiral center at the a-carbon, and they are preferably L-stereoisomers.
  • useful carrier polypeptides contain a T-cell epitope, and an exemplary carrier polypeptide upon which to base the modified carriers of the present invention is
  • an exemplary carrier polypeptide for use in conjunction with the present invention is CRM197.
  • the polypeptide comprising at least one nnAA residue is a modified version of a native carrier protein (e.g., referred to as an“enhanced” or “eCRM” for an enhanced CRM197), or a polypeptide comprising one or a plurality of T-cell activating epitopes of a native carrier protein.
  • Carrier proteins suitable for such modifications include, but are not limited to, proteins used in conjugate vaccines such as Corynebacterium diphtheriae toxin, Clostridium tetani tetanospasmin, Haemophilus influenzae protein D (PD, HiD), the outer membrane protein complex of serogroup B meningococcus (OMPC), and CRM197.
  • the carrier protein may also be ovalbumin.
  • the amino acid sequences of many native carrier proteins are publicly available, as are the nucleic acid sequences of the DNA encoding them. Native carrier proteins and other unmodified carrier proteins have limitations, including non-discriminate antigen conjugation to any surface-exposed amino acid.
  • the immunogenic polypeptide is a carrier protein modified by the inclusion of at least one nnAA residue for use as a site of conjugation.
  • the nnAA can be substituted for a native residue or added to the polypeptide by appending before, appending after, or inserting within the sequence of the polypeptide.
  • the use of non-natural amino acids, as described herein, allows the selective placement of non-natural amino acids for conjugation and as a result the T-cell activating epitopes of the enhanced carrier protein can be avoided in antigen conjugation.
  • a number e.g., 2-9) of nnAA may replace naturally occurring amino acids within a polypeptide sequence.
  • Table 1 shows the amino acid and nucleic acid sequences (SEQ ID NOs: 1 and 2) of an exemplary modified carrier protein: CRM197.
  • SEQ ID NOs: 1 and 2 amino acid and nucleic acid sequences
  • Those of skill in the art will recognize the addition of an N-terminal methionine to the amino acid sequence of conventional CRM 197 produced by fermentation of C. diphtheriae , and the resulting addition of 1 to the conventional amino acid residue position numbering.
  • the methionine is present because of the inclusion of a start codon in the cell-free protein synthesis method which was used to produce these carriers a described herein.
  • the enhanced carrier protein comprising the nnAA residues has at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity to a homologous native or non-toxic carrier protein used in a conjugate vaccine.
  • Carrier proteins having significant sequence identity to SEQ ID NO: 11 include other mutant diphtheria toxin proteins, such as the non-toxic K51E/E148K double mutant which has also been used as a carrier protein in conjugates (Pecetta et al. 2016 Vaccine 34: 1405-11).
  • the natural toxicity of wild-type diphtheria toxin is absent (via the G52E mutation (bolded below) in CRM197, or the K51E/E148K mutations of Pecetta et al.
  • Table 1 also shows the amino acid sequence of protein D (SEQ ID NO:8) from H. influenzae.
  • the enhanced carrier protein comprising nnAA residues may have at least 80% sequence identity to SEQ ID NO: 8. At least one Lys residue in SEQ ID NO: 8 can be replaced by a nnAA. There are 36 Lys residues within SEQ ID NO: 8, so several can be replaced by nnAA and then used for conjugation.
  • sequence identity is determined relative to diphtheria or tetanus toxin, it should be determined relative to the processed heavy chain sequence e.g. relative to amino acids 226- 567 of P00588-1, or to amino acids 458-1315 of P04958-1 (UniProt sequences).
  • the enhanced carrier protein comprising the nnAA residues comprises less than the full native sequence of the carrier protein, and instead comprises at least one or a plurality of T-cell activating epitopes from Corynebacterium diphtheriae toxin, Clostridium tetani tetanospasmin, Haemophilus influenzae protein D (PD, HiD), outer membrane protein complex of serogroup B meningococcus (OMPC), CRM197, Pfs25, or another suitable native or non-toxic carrier protein.
  • the toxicity of the enhanced carrier protein is limited by treatment with paraformaldehyde (or by treatment with formaldehyde or glutaraldehyde) followed by a quenching agent.
  • the enhanced carrier protein comprising the nnAA residues is a polypeptide comprising a plurality of T-cell activating epitopes of native CRM197 (SEQ ID NO: 11). Table 1:
  • CRM197 cross-reacting material 197
  • cross-reacting material 197 is a non-toxic mutant of diphtheria toxin which is used in many approved gly coconjugate vaccines (e.g. see Broker et al. (2011) Biologicals 39: 195-204).
  • exemplary carrier proteins for use with the invention comprise an amino acid sequence which has at least 90% sequence identity to SEQ ID NOs: 1 or 11.
  • the carrier protein can comprise the amino acid sequence SEQ ID NOs: lor 11 except for the presence of one or more nnAA (which may be inserted within SEQ ID NOs: 1 or 11 or may be substituted for one or more amino acid residues within SEQ ID NOs: l or 11 e.g. substituted for Lys and/or Phe).
  • At least one Lys and/or at least one Phe residue in SEQ ID NOs: 1 or 11 is substituted by a nnAA residue. It may be preferred to substitute more than one residue in SEQ ID NOs: 1 or 11 with a nnAA and, in some embodiments, only one species of residue in SEQ ID NOs: 1 or 11 is substituted by a nnAA e.g. only Lys residues are substituted. Where more than one residue in SEQ ID NOs: 1 or 11 is substituted for a nnAA it may be advantageous that the same nnAA is used at each position e.g. pAMF at each substitution position.
  • Carrier proteins with from 2-9 nnAA residues within SEQ ID NOs: 1 or 1 1 are useful, for example with from 4-9, 4-8, or 4-6 nnAA residues e.g. 4, 5 or 6 nnAA residues. This may permit more extensive attachment of antigens to the carrier than using a single nnAA, thereby increasing the antigen: carrier ratio, while avoiding excessive disruption of the native sequence and structure, which can result in insolubility.
  • Exemplary modified CRM avoid introducing nnAA within these regions of SEQ ID NOs: 1 or 11. These regions include F274, F356, F361, F369, K420, K441, K446, K448, and K457 (numbered according to SEQ ID NO: 1), so these are the Phe and Lys residues which are less preferred for nnAA substitution in CRM197 or a modified CRM.
  • Exemplary Lys residues for substitution by a nnAA in SEQ ID NO: 11 are K24, K33, K37, K39, K212, K214, K227, K244, K264, K385,
  • Useful Lys residues for substitution by a nnAA in SEQ ID NO: l are K25, K34, K38, K40, K213, K215, K228, K245, K265, K386, K523, or K527.
  • Other useful Lys residues for substitution by a nnAA are Kl l, K38, K83, K104, K105, K126, K158, K173, K222, K237, K243, K475, and
  • Useful Phe residues for substitution by a nnAA are F13, F54, F124, F128, F141, F168, F251, F390, F531, or F532 (of SEQ ID NO: 1).
  • Exemplary carriers used with the invention may include at least one nnAA in the first region and at least one nnAA in the second region e.g. at least two nnAA in each region, or at least 3 nnAA in each region. This may permit conjugated antigens to be spatially separated when attached to the carrier.
  • a carrier with 3 nnAA in the first region and 3 nnAA in the second region is useful.
  • the first region contains 27 Lys residues, and the second region contains 12 Lys residues.
  • one or more (e.g. 3) Lys residues within the N-terminal 374 amino acids and one or more (e.g. 3) Lys residues within the C-terminal 162 amino acids of SEQ ID NO: 1 can be substituted with a nnAA e.g. within pAMF.
  • Exemplary embodiments of nnAA-containing carriers based on CRM197 have the amino acid sequence of SEQ ID NO: 11 in which one or more of residues K24, K33, K37, K39, K212, K214, K227, K244, K264, K385, K522 and/or K526 is/are replaced by a nnAA.
  • Exemplary embodiments of nnAA-containing carriers based on N-terminally modified CRM197 have the amino acid sequence of SEQ ID NO: 1 in which one or more of residues K25, K34, K38, K40, K213, K215, K228, K245, K265, K386, K523, and/or K527 is/are replaced by a nnAA.
  • One such sequence is SEQ ID NO:9, in which each X represents a nnAA (preferably the same nnAA, such as pAMF):
  • This carrier protein has been found to be very well-expressed in a cell-free protein synthesis system, while retaining good solubility and providing good immunogenic responses when conjugated to pneumococcal capsular polysaccharides.
  • compositions including multiple different conjugates (e.g. different pneumococcal serotypes) in which each conjugate includes a carrier protein having amino acid sequence SEQ ID NO:9 (for example, in which each X residue is the same nnAA, preferably pAMF).
  • each conjugate includes a carrier protein having amino acid sequence SEQ ID NO:9 (for example, in which each X residue is the same nnAA, preferably pAMF).
  • SEQ ID NO: 1 has a N-terminal methionine (which will typically be formylated) that is not present in wild-type CRM197 (SEQ ID NO: 11) but is included for initiating translation without requiring the whole native leader sequence.
  • the carrier protein used herein lacks a N-terminal methionine e.g. the N-terminus methionine of SEQ ID NO: 1 or SEQ ID NO:9 may be absent.
  • a carrier protein based on CRM197 includes no natural amino acids (and more preferably no amino acids) upstream of the N- terminus of SEQ ID NO: 1 or downstream of the C-terminus of SEQ ID NO: 1.
  • the invention also provides a protein for preparing an immunogenic polysaccharide- protein conjugate, wherein the protein has an amino acid sequence which has at least 80% sequence identity to SEQ ID NO: 1 ( e.g . at least 85%, at least 90%, or at least 95%) and includes at least one nnAA, wherein the protein has a N-terminal methionine.
  • the invention also provides an immunogenic polysaccharide-protein conjugate prepared by conjugating a polysaccharide to at least one nnAA in the protein.
  • the invention also provides a protein for preparing an immunogenic polysaccharide- protein conjugate, wherein the protein comprises the amino acid sequence SEQ ID NO: l except that at least one (e.g. 2-9) lysine residues is a nnAA.
  • the nnAA is ideally an azido-containing nnAA (such as pAMF), a l,2,4,5-tetrazinyl-containing nnAA, or an alkenyl-containing nnAA.
  • the invention also provides a conjugate comprising such a protein conjugated to a
  • polysaccharide antigen via at least one of its nnAA.
  • the invention also provides an immunogenic polysaccharide-protein conjugate, wherein the protein is a modified CRM197 having a N-terminal methionine.
  • nnAA-containing modified CRMl97-based carriers are typically present in monomeric form when used for preparing conjugates, rather than being associated with other modified CRMl97-based carrier subunits to form modified CRMl97-based multimers.
  • carrier polypeptides of interest herein are modified forms of CRM197.
  • carrier polypeptides for use with the invention may comprise an amino acid sequence that has at least 80% sequence identity (e.g. >85%, >90%, >95%, >96%, >97%, or preferably >98%) to SEQ ID NOs: 1 or 11.
  • the carrier polypeptide can comprise the amino acid sequence SEQ ID NOs: 1 or 11 except for the presence of up to 10 nnAA, as discussed above.
  • SEQ ID NOs: 1 and 11 include an Arg-Arg dipeptide sequence at positions 192-193 according to SEQ ID NO: 11 or 193-914 according to SEQ ID NO: l.
  • This sequence can be subject to proteolytic cleavage in some circumstances. If desired, this site can be modified to prevent cleavage and improve yield.
  • a modified CRM197 carrier polypeptide used herein is free from an Arg-Arg dipeptide sequence. For instance, Arg-l92 and/or Arg-l93 of SEQ ID NO: 11 (or Arg-l93 and/or Arg-l94 of SEQ ID NO: 1) can be deleted or can be substituted with a different amino acid.
  • a useful carrier polypeptide may comprise an amino acid sequence which (i) has at least 80% (e.g. >85%, >90%, >95%, >96%, >97%, or preferably >98%) sequence identity to SEQ ID NO: 1 orl 1; (ii) is free from an Arg-Arg dipeptide sequence; and (iii) includes at least one (e.g. at least 2, and preferably more, as discussed above) nnAA residue.
  • SEQ ID NO: 12 which differs from SEQ ID NO: 11 by having an Arg Asn substitution at position 193:
  • SEQ ID NO: 13 Another such amino acid sequence is SEQ ID NO: 13, which differs from SEQ ID NO: 1 by having an Arg Asn substitution at position 194:
  • SEQ ID NO: 12 wherein SEQ ID NO: 12 has been modified to include from 1-10 ( e.g . from 3-9 or from 2-8, or from 2-6, or from 3-6, or from 4-6) nnAA residues. These nnAA residue modifications can be incorporated into SEQ ID NO: 12 as insertions and/or substitutions (e.g. 6 Lys nnAA substitutions (e.g., pAMF) at positions K33, K212, K244, K264, K385, K526). In certain variations, residue Asn-l93 of SEQ ID NO: 12 is not substituted by a nnAA.
  • This carrier polypeptide can be used to prepare immunogenic conjugates (e.g. of saccharide antigens) via the nnAA residue(s) therein.
  • a carrier polypeptide comprising amino acid sequence SEQ ID NO: 13, wherein SEQ ID NO: 13 has been modified to include from 1-10 (e.g. from 3-9 or from 2-8, or from 2-6, or from 3-6, or from 4-6) nnAA residues. These nnAA residue modifications can be incorporated into SEQ ID NO: 13 as insertions and/or substitutions (e.g. SEQ ID NO: 14, which includes 6 Lys nnAA substitutions e.g, pAMF). In certain variations, Asn-l94 of SEQ ID NO: 13 is not substituted by a nnAA.
  • This carrier polypeptide can be used to prepare immunogenic conjugates (e.g. of saccharide antigens) via the nnAA residue(s) therein.
  • these carrier polypeptides include amino acid sequences upstream and/or downstream of SEQ ID NO: 11 or 12.
  • they can include a methionine residue upstream of the N-terminus amino acid residue of SEQ ID NO: 11 or 12.
  • This methionine residue may be formylated.
  • a methionine residue is not present at this position in wild-type CRM197 but it can be included herein for initiating translation (e.g. in a cell-free polypeptide synthesis system) without requiring the whole native leader sequence.
  • a carrier polypeptide includes (i) no amino acids upstream of the N-terminus of SEQ ID NO: 11 or 12, except for an optional methionine, and (ii) no amino acids downstream of the C-terminus of SEQ ID NO: 11 or 12.
  • these carrier polypeptides include amino acid sequences upstream and/or downstream of SEQ ID NO: 1 or 13.
  • a carrier polypeptide includes (i) no amino acids upstream of the N-terminus of SEQ ID NO: 1 or 13, except for an optional methionine, and (ii) no amino acids downstream of the C-terminus of SEQ ID NO: 1 or 13.
  • at least one Lys residue in SEQ ID NO: 11 or 12 is substituted by a nnAA residue.
  • nnAA it is useful to substitute more than one residue in SEQ ID NO: 11 or 12 with a nnAA and, in some embodiments, only one species of residue in SEQ ID NO: 11 is substituted by a nnAA e.g. only Lys residues are substituted. Where more than one residue in SEQ ID NO: 11 is substituted for a nnAA it is may be preferred that the same nnAA is used at each position e.g. pAMF at each substitution position. As noted above, in some embodiments residues other than Lys are substituted.
  • At least one Lys residue in SEQ ID NO: l or 13 is substituted by a nnAA residue. It can be useful to substitute more than one residue in SEQ ID NO: 1 or 13 with a nnAA and, in some embodiments, only one species of residue in SEQ ID NO: l is substituted by a nnAA e.g. only Lys residues are substituted. Where more than one residue in SEQ ID NO: 1 is substituted for a nnAA it may be preferred that the same nnAA is used at each position e.g. pAMF at each substitution position. As noted above, in some embodiments residues other than Lys are substituted.
  • Carrier polypeptides comprising amino acid sequence SEQ ID NO: 11 or 12 with from 2-9 substitutions by nnAA residues (e.g. Lys— nAA substitutions, such as Lys pAMF) are provided, and for example with from 2-8, 2-6, 3-8, 3-6, 4-9, 4-8, or 4-6 nnAA substitutions e.g.
  • Carrier polypeptides comprising amino acid sequence SEQ ID NO: 1 or 13 with from 2-9 substitutions by nnAA residues (e.g. Lys— nAA substitutions, preferably Lys pAMF) are provided, and for example with from 2-8, 2-6, 3-8, 3-6, 4-9, 4-8, or 4-6 nnAA substitutions e.g. 4, 5 or 6 nnAA residues.
  • This permits more extensive attachment of antigens to the carrier than using a single nnAA, thereby increasing the antigenxarrier ratio, while avoiding excessive disruption of the native sequence and structure, which can result in insolubility.
  • the two general 3D regions evidenced by the structural studies of CRM197 mentioned earlier herein may be within SEQ ID NO:l 1 or 12, wherein, again, the first region runs from the N-terminus to Asn-373, and the second region runs from Ser-374 to the C-terminus.
  • nnAA-containing carriers based on CRM197 have the amino acid sequence of SEQ ID NO:l 1 or SEQ ID NO: 12 in which one or more of residues K24, K33, K37, K39, K212, K214, K227, K264, K385, K522 and K526 is/are replaced by a nnAA (such as pAMF).
  • a nnAA such as pAMF
  • K33, K212, K244, K264, K385, K526 are replaced by a nnAA.
  • each nnAA is same nnAA, such as pAMF).
  • nnAA-containing carriers based on CRM 197 have the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 13 in which one or more of residues K25, K343, K38, K40, K213, K215, K228, K265, K386, K523 and K527 is/are replaced by a nnAA (such as pAMF).
  • a nnAA such as pAMF
  • K343, K213, K245, K265, K386, K527 are replaced by a nnAA.
  • One such sequence is SEQ ID NO:9, in which each X represents a nnAA (preferably the same nnAA, such as pAMF).
  • SEQ ID NO: 14 containing the above-mentioned Arg-Asn substitution and in which each X represents a nnAA (preferably the same nnAA, such as pAMF):
  • SEQ ID NOs: 9 and 14 can be very well-expressed in a cell-free protein synthesis system, while retaining good solubility and providing good immunogenic responses when conjugated to pneumococcal capsular saccharides.
  • SEQ ID NO: 14 lacks the native Arg-Arg dipeptide.
  • polypeptide comprising an amino acid sequence which (i) has at least 80% (e.g. >85%, >90%, >95%, >96%, >97%, or preferably >98%) sequence identity to SEQ ID NO: 1 or 11; (ii) is free from an Arg-Arg dipeptide sequence; and (iii) includes at least one nnAA residue; and wherein the polypeptide has a N-terminus methionine and/or is in monomeric form.
  • polypeptide comprising an amino acid sequence which (i) has at least 80% (e.g.
  • sequence identity to SEQ ID NO: 1; (ii) is free from an Arg-Arg dipeptide sequence; and (iii) includes at least one nnAA residue; and wherein the polypeptide has a N-terminal methionine and/or is in monomeric form.
  • modified CRMl97-derived carrier polypeptides can be used in the same manner for conjugation as CRM197 has been used in the prior art (e.g. see Broker el al. 2011 supra , WO2015/117093, etc.), but with the improvement of permitting site-specific conjugation via the nnAA residue(s). They will generally be used in monomeric form, rather than being associated with other CRM197 or CRMl97-derived subunits to form polypeptide multimers. Similarly, they will generally include at least one disulfide bridge e.g. between Cys-l86 & Cys-20l (numbered according to SEQ ID NO: 11) and, optionally, between Cys-46l & Cys-47l.
  • an immunogenic conjugate comprising any of these various carrier polypeptides conjugated to a saccharide antigen via at least one of its nnAA.
  • the carrier polypeptides are particularly useful for conjugating to pneumococcal capsular saccharides and/or antigens via the nnAA residue(s) therein. Immunogenic conjugates prepared in this way can be combined to form multivalent compositions as discussed elsewhere herein.
  • the invention additionally provides an immunogenic conjugate comprising a carrier polypeptide, a saccharide antigen and an immunostimulator, wherein (i) the carrier polypeptide has amino acid sequence SEQ ID NO: 14, e.g.
  • each X is pAMF; and (ii) the saccharide antigen is covalently bonded to the carrier polypeptide via at least one nnAA residue within SEQ ID NO: 14, and (iii) wherein the immunostimulator is conjugated to the antigen and/or the polypeptide.
  • a multivalent pharmaceutical composition comprising two or much such immunogenic conjugates.
  • the immunostimulator is conjugated to the antigen and the antigen further conjugated to the polypeptide.
  • the invention additionally provides a pharmaceutical composition including multiple different conjugates (e.g. different pneumococcal serotypes) in which each conjugate includes a carrier polypeptide having amino acid sequence SEQ ID NO: 14.
  • each conjugate includes a carrier polypeptide having amino acid sequence SEQ ID NO: 14.
  • an immunogenic conjugate comprising a carrier polypeptide and a saccharide antigen, wherein (i) the carrier polypeptide has amino acid sequence SEQ ID NO: 14; (ii) the saccharide antigen is covalently bonded to the carrier polypeptide via at least one nnAA residue within SEQ ID NO: 14; and (iiia) the saccharide antigen is a capsular saccharide from any of pneumococcal serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11 A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F or (iiib) the saccharide antigen is a capsular saccharide from any of pneumococcal serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9N, 9V, 10A,
  • the serotype 20 saccharide is 20A, 20B or a mixture thereof.
  • the disclosure provides for an expression vector comprising the polynucleotide. In certain embodiments, the disclosure provides for a host cell comprising the expression vector. III. C. Carrier Protein Production Methods
  • the enhanced carrier protein is produced by any method described for production of polypeptides.
  • Methods suitable for production of polypeptides include, but are not limited to, solid phase chemical peptide synthesis, cell-based recombinant protein expression (in E. coli or a native host), and cell-free protein expression, and any combination thereof (e.g . expressed protein ligation using a combination of synthetic and recombinant peptide components).
  • the nnAA- bearing enhanced carrier protein is produced by a method that comprises“codon reassignment”.
  • nnAAs that are close structural analogs of the 20 canonical amino acids (e.g. homoallylglycine, fluorinated leucine, azidohomoalanine) are used.
  • the nnAA is loaded onto its corresponding tRNA using wild-type aminoacyl-tRNA synthetases, and the nnAA completely replaces one of the 20 canonical amino acids specified in a template DNA sequence.
  • this generally requires use of a bacterial expression strain that is auxotrophic for the native amino acid being replaced. This strategy is amino acid rather than residue-specific, since all AA residues of a certain type are replaced with the nnAA.
  • the nnAA- bearing enhanced carrier protein is produced by a strategy that comprises“nonsense
  • the“nonsense suppression” approach involves isolating a tRNA/aaRS pair, modifying the tRNA at the anti-codon loop to recognize an orthogonal codon (e.g. the amber codon TAG, the opal codon TGA, or another codon or base sequence not commonly used to specify amino acids in translation), and modifying the aaRS to prefer the nnAA over the aminoacyl-tRNAs native amino acid.
  • an orthogonal codon e.g. the amber codon TAG, the opal codon TGA, or another codon or base sequence not commonly used to specify amino acids in translation
  • modifying the aaRS to prefer the nnAA over the aminoacyl-tRNAs native amino acid.
  • the tRNA/aminoacyl-tRNA synthetase pair is from the same organism as the translation machinery used for polypeptide synthesis. In other embodiments, the tRNA/aminoacyl-tRNA synthetase pair is from a different
  • production of the enhanced carrier protein does not involve the use of an engineered aminoacyl-tRNA synthetase.
  • an orthogonal tRNA alone is isolated and modified at the anti-codon loop to recognize an orthogonal codon (e.g. the amber codon TAG, or another codon or base sequence not commonly used to specify amino acids in translation).
  • the orthogonal engineered tRNA is then acylated in vitro by a suitable chemical method (e.g., the method of Heckler et al.
  • nnAA-containing carrier proteins use cell-free protein synthesis.
  • cell-free protein expression techniques are known in the art and various nnAA can be incorporated in this way (e.g. see Table 1 of Quast el al. (2015) FEBS Letters 589: 1703-12) while avoiding potential cytotoxic effects of nnAA.
  • the enhanced carrier protein is produced by cell-free extract-based protein synthesis.
  • the cell-free extract comprises an extract of rabbit reticulocytes, wheat germ, or A. coli.
  • the cell-free extract is supplemented with amino acids, energy sources, energy regenerating systems, or cation cofactors, and any combination thereof.
  • the extract comprises exogenously supplemented mutant tRNA or mutant aaRS (aminoacyl tRNA synthetase), and any combination thereof.
  • the extract comprises lysates from E. coli strains genetically encoding mutant tRNA or mutant aaRS, and any combination thereof.
  • the E. coli strains used for lysates are RF-l attenuated strains.
  • Compatible cell-free protein synthesis systems have been described for the insertion of formulas I, II, and III into recombinant polypeptides (e.g., US8715958B2,
  • US8715958B2 demonstrates a regenerating cell-free E. coli based system whereby the tRNA Ty 7Tyrosine-synthetase pair from Methanococcus jannaschii (Wang et al. (2001) Science 292(5516):498-500) is used to introduce the non-natural amino acid p-azido- L-phenylalanine (pAF) into recombinant chloramphenicol acetyltransferase (CAT), GM-CSF, and TetA.
  • pAF non-natural amino acid p-azido- L-phenylalanine
  • CAT chloramphenicol acetyltransferase
  • TetA TetA
  • US20160257946A1 demonstrates: (a) how the Methanococcus jannaschii Tyrosine-synthetase above is adapted using mutagenesis so that it preferentially loads p-azidomethyl-L-phenylalanine (pAMF) onto an amber-recognizing tRNA, and (b) how a cell- free synthesis system comprising the modified synthetase/tRNA pair is used to selectively incorporate pAMF into antibodies such as trastuzumab.
  • pAMF p-azidomethyl-L-phenylalanine
  • US20160257945A1 demonstrates: (a) how th Q Methanococcus jannaschii Tyrosine-synthetase above is adapted using mutagenesis so that it preferentially loads (S)-2-amino-3-(5-((6-methyl-l,2,4,5-tetrazin-3-ylamino)methyl)pyridin-2-yl)propanoic acid (a pyridyl tetrazine amino acid derivative) onto an amber-recognizing tRNA, and (b) how a cell- free synthesis system comprising the modified synthetase/tRNA pair is used to selectively incorporate (S)-2-amino-3-(5-((6-methyl- 1,2,4, 5-tetrazin-3-ylamino)methyl)pyridin-2- yl)propanoic acid into recombinant GFP.
  • the disclosure provides for methods of producing
  • polypeptides in a cell-free extract containing two or more non-natural amino acids in a cell-free extract containing two or more non-natural amino acids.
  • the polypeptides also have biological activity comparable to the native protein.
  • the polypeptides have improved or enhanced biological activity comparable to the native protein.
  • a functional assay e.g. immunostaining, ELISA, quantitation on coomassie or silver stained gel, etc.
  • the specific activity as thus defined will be at least about 5% that of the native protein, usually at least about 10% that of the native protein, and optionally is about 20%, about 40%, about 60% or greater.
  • the methods of producing the nnAA-containing polypeptides involve altering the concentrations of nnAA-specific tRNA, nnAA-specific synthetase, nnAA itself, or translation temperature, and any combination thereof. Such conditions optionally allow for fewer translational errors, improved rate of incorporation of the nnAA, improved activity of chaperones necessary for protein folding with incorporation of the nnAA, decreased activity of cellular factors that interfere with nnAA incorporation, or any combination of the
  • nnAA-specific tRNA concentration is increased to a concentration above about 20 mM, leading to an increased fraction of soluble or active polypeptide.
  • the tRNA concentration is increased while the nnAA concentration is kept below about 2mM and the nnAA synthetase is maintained below about 5mM.
  • the translation mix incubation temperature is between 20 degrees and 30 degrees Celsius, about 20 degrees Celsius, or below 20 degrees Celsius. In some variations, these temperature modifications are independently combined with modifications to the nnAA-specific tRNA concentrations, nnAA concentrations, or nnAA synthetase concentrations described in the preceding paragraph.
  • the improved carrier proteins of the present disclosure comprise one or more nnAA substituted at any position within the polypeptide as long as the immunogenic function of one or more T-cell epitopes of the polypeptide is preserved.
  • nnAAs may be inserted internally or at a terminus as additions to the starting carrier sequence.
  • the at least one nnAA in the improved carrier protein e.g., eCRM
  • the at least one nnAA in the improved carrier protein is not present within one or more regions of the protein that comprise a T-cell epitope.
  • no nnAA in the enhanced immunogenic polypeptide is present within one or more regions of the protein that comprise a T-cell epitope.
  • the nnAA residue is substituted for one or more of the twenty naturally-encoded amino acids, including alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine,
  • nnAA residue is substituted for one or more of a specific class of natural amino acid residue, such as aliphatic, aromatic, acidic, basic, hydroxylic, sulfur-containing, or amidic (containing amide group).
  • a specific class of natural amino acid residue such as aliphatic, aromatic, acidic, basic, hydroxylic, sulfur-containing, or amidic (containing amide group).
  • only one specific amino acid e.g., lysine
  • two or more different amino acids e.g., lysine, phenylalanine, etc.
  • Polypeptides in which only a single species of amino acid is substituted for a nnAA may be advantageous e.g. in which only Lys residues are substituted.
  • the nnAA residue is substituted for at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 natural amino acid residues of a carrier protein. In some embodiments, the nnAA residue is substituted for at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 natural amino acid residues of a carrier protein.
  • the nnAA residue is substituted for at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 natural amino acid residues of SEQ ID NO: 1 or SEQ ID NO: 11.
  • nnAA is substituted for one or more amino acid residues within a carrier protein.
  • the specific amino acid residue that is selected to create single- or multiple- substituted nnAA variants described herein is optionally determined by dividing the protein into subdomains and choosing for substitution a single amino acid or sets of amino acid residues that do not sterically obstruct each other (e.g. such that there is a multi-angstrom distance between the substitution sites). Division of CRM197 into two structural regions is discussed below.
  • the nnAA is substituted for a charged amino acid residue.
  • a nnAA can be substituted for an aspartate, glutamate, lysine, arginine or histidine amino acid residue.
  • the nnAA is substituted for a negatively-charged amino acid residue e.g. for an aspartate or glutamate residue.
  • the nnAA is substituted for a positively-charged amino acid residue e.g. for a lysine, arginine or histidine residue.
  • the nnAA is substituted for one or more lysine residues within an immunogenic polypeptide.
  • an enhanced version of SEQ ID NO: 11 is generated by substituting an nnAA for lysine in the following manner: 1) one residue from the group consisting of K24, K33, K37, and K39; 2) one residue selected from the group consisting of K212 and K214; and 3) 2 to 4 residues selected from the group consisting of K227, K244, K264, K385, K522, and K526.
  • the one or more of a specific class of natural amino acid residue substituted is selected from the group consisting of K24, K33, K37, K39, K212, K214, K227, K244, K264, K385, K522 and K526, and any combination thereof of SEQ ID NO: 1.
  • the nnAA substitution in SEQ ID NO: 11 is selected from one or more of K24, K33, K37, K39, K212, K214, K227, K244, K264, K385, K522 and K526.
  • the nnAA substitution comprises six residues consisting of K24, K214, K227, K264, K385, and K522 of SEQ ID NO: 11.
  • the nnAA substitution in SEQ ID NO: 11 comprises K264. In other embodiments, the nnAA substitution in SEQ ID NO: 11 comprises K385. In certain embodiments, the nnAA substitutions in SEQ ID NO: 11 comprise K264 and K385.
  • an enhanced version of SEQ ID NO: 1 may also be generated by substituting an nnAA for lysine in the following manner: 1) one residue from the group consisting of K25, K34, K38, and K40; 2) one residue selected from the group consisting of K213 and K215; and 3) 2 to 4 residues selected from the group consisting of K228, K245, K265, K386, K523, and K527.
  • the one or more of a specific class of natural amino acid residue substituted is selected from the group consisting of K25, K34, K38, K40, K213, K215, K228, K265, K386, K523 and K527, and any combination thereof of SEQ ID NO: l.
  • the nnAA substitution in SEQ ID NO: l is selected from one or more of K25, K34, K38, K40, K213, K215, K228, K245, K265, K386, K523, and K527.
  • the nnAA substitution comprises six residues consisting of K25, K215, K228, K265, K386, and K523 of SEQ ID NO: l.
  • the nnAA substitution in SEQ ID NO: l comprises K265. In other embodiments, the nnAA substitution in SEQ ID NO:l comprises K386. In another embodiment, the nnAA substitutions in SEQ ID NO: l comprise K265 and K386. In a further embodiment, the nnAA is substituted for a phenylalanine.
  • Exemplary phenylalanines for substitution include F13, F54, F124, F128, F141, F168, F251, F390, F531, or F532 of SEQ ID NO: 1. Because of their proximity, it is generally advantageous to not substitute at both F531 and F532.
  • the binding epitopes for human CD4+ cells on diphtheria toxin that are recognized by most subjects tested encompass residues 271-290, 321-340, 331-350, 351-370, 411-430, or 431- 450 (see, Raju et al. (1995) Eur J Immunol. 25(l2):3207-l4). Therefore, in some embodiments the one or more nnAA substituted is not within residues 270-289, 320-339, 330-349, 350-360, 410-429, and/or 430-449 of SEQ ID NO: 11. In some embodiments, the one or more nnAA substituted is not within residues 330-349 of SEQ ID NO:l. In certain embodiments, the one or more nnAA substituted is not within residues 320-339 of SEQ ID NO: l. In certain embodiments,
  • the one or more nnAA substituted is not within residues 432-449 of SEQ ID NO: 11. Also, in some embodiments the one or more nnAA substituted is not within residues 271-290, 321-340, 331-350, 351-370, 411-430, and/or 431-450 of SEQ ID NO: l. In some embodiments, the one or more nnAA substituted is not within residues 331-350 of SEQ ID NO: 1. In other embodiments, the one or more nnAA substituted is not within residues 321-340 of SEQ ID NO:l. In yet other embodiments, the one or more nnAA substituted is not within residues 431-450 of SEQ ID NO: 1.
  • the binding epitopes for human CD4+ cells on tetanus toxin that are recognized by all subjects tested encompass heavy chain residues H176-195, IDKISDVSTIVPYIGPALNI [SEQ ID NO:3], and H491-510, NNFTVSFWLRVPKVSASHLE [SEQ ID NO:4] (see, Diethelm-Okita et al., J Infect Dis. 1997 Feb;l75(2):382-9l).
  • the one or more nnAA substituted is not within residues 176-195 and/or 491-510 of the heavy chain peptide component of the tetanus toxin precursor protein.
  • the one or more nnAA substituted is not within residues 176-195 of the heavy chain peptide component of the tetanus toxin precursor protein. In yet certain embodiments, the one or more nnAA substituted is not within residues 491-510 of the heavy chain peptide component of the tetanus toxin precursor protein.
  • the binding epitopes for human CD4+ cells on Neisseria meningitidis outer membrane protein (OMP or PorA) that are recognized by most subjects tested encompass immunodominant T-cell epitopes, which are mostly located outside the variable regions and are conserved among different meningococcal (and gonococcal) strains, e.g., corresponding to conserved putative /ra//.s-membrane regions of OMP (Wiertz et al. J Exp Med. 1992; 176(1): 79-88).
  • the one or more nnAA substituted is not within a conserved region of OMP.
  • the binding epitopes for human CD4+ cells on BB a carrier protein derived from the G protein of Streptococcus strain G148, that are recognized by most subjects tested encompass amino acids 25-40 (VSDYYKNLINNAKTVE [SEQ ID NO:5]), 63-78 (DGLSDFLKSQTPAEDT [SEQ ID NO:6]), and 74-89 (AEDTVKSIELAEAKVL [SEQ ID NO:7]) in the BB sequence (Goetsch et al., Clin Diagn Lab Immunol. 2003 Jan; 10(1): 125-32).
  • the one or more nnAA substituted is not within residues 25-40, 63-78, and/or 74-89 of the BB sequence.
  • the immunogenic polypeptide comprising at least one non natural amino acid residue further comprises at least one antigen.
  • the immunogenic polypeptide comprising at least one non-natural amino acid is an enhanced carrier protein and further comprises at least one antigen.
  • the immunogenic polypeptide comprising at least one non-natural amino acid is an enhanced carrier protein and further comprises at least one antigen.
  • T-cell epitopes of a carrier protein are optionally determined by any of the known methods.
  • T-cell binding epitopes in proteins are predicted using algorithms that take into account various factors, such as amphipathicity profiles of proteins, sequence motifs, quantitative matrices (QM), artificial neural networks (ANN), support vector machines (SVM), quantitative structure activity relationship (QSAR) and molecular docking simulations, etc. (see, Desai et al. Methods Mol Biol. 2014;1184:333-64).
  • the T-cell binding epitopes in diphtheria toxin/CRM have been predicted using the DeLisi & Berzofsky algorithm (see, Bixler et al. WO89/06974 and PNAS 82:7848, 1985). Predicted T-cell epitopes can be experimentally confirmed.
  • the T-cell epitopes of an immunogenic polypeptide of interest can be experimentally determined by synthesizing partially overlapping peptide fragments corresponding to the complete sequence of the immunogenic polypeptide (or predicted regions) and performing proliferation assays of CD4+ cell lines (e.g., peripheral blood mononuclear cells (PBMC)) in the presence of each fragment.
  • CD4+ cell lines e.g., peripheral blood mononuclear cells (PBMC)
  • the conjugation scheme generally involves cell-free synthesis of the selected protein carrier (e.g., CRM197 or a modified version thereof) followed by activation of the polysaccharide to enable conjugation to the polypeptide, and, finally, conjugation.
  • An immunostimulator is covalently incorporated into the conjugate, such that the conjugate comprises the protein carrier, the antigen, and the immunostimulator.
  • a mechanical sizing step may be used to reduce the average fragment size of the polysaccharide antigen as will be explained in detail infra.
  • the protein carrier a polypeptide comprising an nnAA
  • a cell-free expression mixture maintained at a temperature between about 10 degrees Celsius and about 30 degrees Celsius. In certain embodiments, the temperature is above about 20 degrees Celsius. In certain embodiments, the temperature is below about 20 degrees Celsius. In certain embodiments, the temperature is between about 14 degrees Celsius and about 18 degrees Celsius.
  • the polypeptide is encoded by a nucleic acid comprising a suppression codon.
  • the cell-free expression mixture comprises an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the nnAA. In certain embodiments, the tRNA concentration is at least 20 mM.
  • the nnAA concentration is less than about 2mM and the concentration of the aminoacyl-tRNA synthetase is less than about 5 mM.
  • the method comprises conjugating the polypeptide to an active moiety.
  • the active moiety is selected from the group consisting of a hapten, a bacterial antigen, a viral antigen, a tumor-derived glycan, a peptide toxin, a macrolide, a polyether, and any combination thereof.
  • the polypeptide is selected from the group consisting of a growth hormone, a clotting factor, a plasma protein, an interleukin, a T-cell receptor extracellular domain, a growth factor extracellular domain, a bacterial antigen, a viral antigen, and any combination thereof.
  • the expression mixture comprises a cellular extract of E. coli , wheat germ, or rabbit reticulocyte. In certain embodiments, the expression mixture comprises at least 30% cellular extract. In certain embodiments, the polypeptide comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 nnAAs.
  • the nnAA is selected from the group consisting of 2-amino-3-(4-azidophenyl) propanoic acid (pAF), 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid (pAMF), 2-amino-3-(5-(azidomethyl)pyridin- 2-yl)propanoic acid, 2-amino-3-(4-(azidomethyl)pyridin-2-yl)propanoic acid, 2-amino-3-(6- (azidomethyl)pyridin-3-yl)propanoic acid, 2-amino-5-azidopentanoic acid, 2-amino-3-(4- (azidomethyl)phenyl)propanoic acid, and any combination thereof.
  • pAF 2-amino-3-(4-azidophenyl) propanoic acid
  • pAMF 2-amino-3-(4-(azidomethyl
  • the polypeptide produced comprises both a soluble and an insoluble fraction, wherein the ratio of the soluble fraction to the insoluble fraction is at least 40% (w/w). In certain embodiments, the polypeptide produced comprises both a soluble and an insoluble fraction, wherein the ratio of the soluble fraction to the insoluble fraction is at least 60% (w/w).
  • the polypeptide produced by cell-free expression comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 nnAAs and the ratio of the soluble fraction to the insoluble fraction is at least at least 20% (w/w), at least 30% (w/w), at least 40% (w/w), at least 50% (w/w), 60% (w/w), at least 70% (w/w), at least 80% (w/w), at least 90% (w/w).
  • the antigens are any purified natural, synthetic, or recombinantly produced macromolecule or fragment thereof. Examples include, but are not limited to lipids, polysaccharides, nucleic acids, or polypeptides, and any combination thereof (e.g. glycoproteins, glycolipoproteins, glycolipids). For instance, the glycolipid optionally is glycophosphatidylinositol.
  • the antigen is a T-independent or T-activating antigen (usually a weak T- activating antigen) selected from the group consisting of a bacterial polysaccharide, a bacterial lipopolysaccharide, a tumor-derived glycan, or a hapten.
  • a T-independent or T-activating antigen usually a weak T- activating antigen selected from the group consisting of a bacterial polysaccharide, a bacterial lipopolysaccharide, a tumor-derived glycan, or a hapten.
  • An antigen comprising a polysaccharide is optionally an oligosaccharide.
  • Oligosaccharides have a low number of repeat units, e.g., 5-15 repeat units, and are typically derived synthetically or by hydrolysis of higher molecular weight polysaccharides.
  • the antigen comprising a polysaccharide generally has a molecular weight of between about lOkDa and about 10,000 kDa. In other such embodiments, the polysaccharide has a molecular weight of between 50 kDa and 10,000 kDa. In further such embodiments, the polysaccharide has a molecular weight of between 50 kDa and 10,000 kDa; between 50 kDa and
  • an antigen comprising a polysaccharide has a molecular weight of between about 50kDa and about 1,400 kDa, such as between about 500kDa and about 3,000 kDa.
  • antigens can be included within the present immunogenic conjugates described herein. These can be bacterial, fungal, viral, or parasitic antigens, and they may be saccharides, lipids, lipopolysaccharides, or polypeptides. It is also possible to use haptens e.g. that mimic drugs of abuse such as nicotine or cocaine.
  • the antigen is a saccharide, where the term“saccharide” includes polysaccharides having 50 or more repeat units, and oligosaccharides having fewer than 50 repeating units.
  • polysaccharides have from about 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 repeating units to about 2,000 (sometimes more) repeating units, and optionally from about 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900 or 1000 repeating units to about, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, or 1900 repeating units.
  • Oligosaccharides typically have from about 6, 7, 8, 9, or 10 repeating units to about 15, 20, 25, 30, or 35 to about 40 or 45 repeating units.
  • Useful saccharides for incorporation into immunogenic conjugates include those found in bacteria. These can be non-capsular saccharides (such as an exopolysaccharide e.g. the S. aureus exopolysaccharide) but are preferably bacterial capsular saccharides.
  • Bacterial capsular saccharides are high molecular weight saccharides found in the capsule of Gram-positive or Gram-negative bacteria and they can be used as vaccine antigens. Such capsular saccharides are generally prepared from whole cell lysates or culture supernatant of the corresponding bacterium via processes that involve diafiltration, protein removal, ethanol precipitation, nucleic acid removal, and freeze drying. Bacterial saccharides used with the invention can be intact as found in the bacteria, or can be fragments obtained from intact saccharides e.g. obtained by hydrolysis of saccharides purified from the bacteria.
  • Saccharide antigens of particular interest include, but are not limited to:
  • Saccharides of Streptococcus pyogenes The antigen can be a saccharide from
  • the antigen is the capsular saccharide of S. pyogenes, which is composed of hyaluronic acid, a high molecular weight polymer where the repeating unit has the structure:
  • the antigen is a non-capsular saccharide from S. pyogenes, such as the group-A-strep cell wall saccharide, which comprises a backbone of poly-L-rhamnopyranosyl units connected by alternating a-L-(l 3) and a-L-(l 2) linkages, to which N-acetyl-P-D- glucosamine residues are attached at the 3 -position of the rhamnose backbone.
  • S. pyogenes such as the group-A-strep cell wall saccharide, which comprises a backbone of poly-L-rhamnopyranosyl units connected by alternating a-L-(l 3) and a-L-(l 2) linkages, to which N-acetyl-P-D- glucosamine residues are attached at the 3 -position of the rhamnose backbone.
  • the antigen can be a capsular saccharides of Streptococcus agalactiae
  • GBS Group B Streptococcus
  • serotypes with distinct capsular saccharide repeating units (la, lb, II-IX), but only a few serotypes are commonly responsible for disease. These include serotypes la, lb, II, III, and V, and conjugates of; capsular saccharides from these serotypes can be prepared.
  • Capsular saccharides of Haemophilus influenzae The antigen can be a capsular saccharide from H. influenzae. There are at least 6 serotypes of H. influenzae with distinct capsular saccharide chemical structures (types a-f). However, only type a and type b are considered“high-virulence” strains, and the preferred type of H. influenzae capsular saccharide for use with the invention is type b (Hib).
  • the antigen can be a capsular saccharide from N.meningitidis. There are at least 13 serogroups of N. meningitidis with distinct capsular saccharide chemical structures (serogroups A, B, C, E-29, H, I, K, L, W-135, X, Y, Z, and Z'), but only six (A, B, C, W-135, X, Y) are thought to be life-threatening.
  • the saccharide antigen is usefully derived from any of serogroups A, C, W135, X, or Y.
  • the antigen can be a capsular
  • the antigen can be a Vi saccharide.
  • Vi is the capsular saccharide of Salmonella typhi (the typhi serovar of S.enterica).
  • the Vi saccharide is a linear homopolymer of a hexosaminuronic acid, al,4-/V-acetylgalactos- aminouronic acid, which is 60 - 90% acetylated at the C-3 position.
  • the antigen can be a saccharide from S. aureus.
  • the saccharide can be the exopolysaccharide of S.aureus , which is a poly-N- acetylglucosamine (PNAG), or a capsular saccharide of S.aureus , which can be e.g. serotype 5, serotype 8 or serotype 336.
  • PNAG poly-N- acetylglucosamine
  • the antigen can be a surface glycan from C.difficile , such as PS-I or PS-II.
  • Glucans The antigen can be a glucan containing b- l ,3-linkages and/or b-l, 6-linkages. These conjugated glucans can be useful for raising an anti-fungal immune response, for example against Candida albicans also tumor-derived glycans; and haptens.
  • the arrangement of antigen, immunostimulator and carrier polypeptide has an antigen molecule linked to both an immunostimulator and a nnAA.
  • the antigen should include multiple conjugation sites, permitting it to form covalent bonds both with immunostimulator molecules and with nnAA residues.
  • the inclusion of multiple conjugation sites in a saccharide antigen is straightforward because of their repeating nature.
  • the functional groups used for conjugation to the antigen can sometimes be present within the natural antigenic structure but typically they will be introduced by activating the antigen, optionally followed by derivatization, as discussed elsewhere herein.
  • aldehyde or cyanate ester groups can be introduced at multiple sites within a saccharide antigen, and these aldehyde groups can then be derivatized e.g. to introduce a reactive cyclooctyne which can then react with azido groups in in the nnAA and in the
  • an antigen comprising a polysaccharide comprises a bacterially derived or synthetic polysaccharide, such as a capsular polysaccharide.
  • a capsular polysaccharide is high molecular mass polymers of gram-positive or gram-negative bacteria that function to protect the microorganisms against immune responses, and as such represent appealing vaccine targets when the goal is production of neutralizing antibodies.
  • Such capsular polysaccharides are generally prepared from whole cell lysates or culture supernatant of the corresponding bacterium via processes that involve diafiltration, protein removal, ethanol precipitation, nucleic acid removal, and freeze drying.
  • Examples include, but are not limited to, the Merieux protocol (Institut Merieux (1980) Brevet Belge 80:26320) and the Yavordios protocol (Yavordios et al. ER0071515A1(1983)).
  • Exemplary antigens for use with the invention are capsular saccharides from
  • S.pneumoniae is an encapsulated Gram-positive bacterium that can cause pneumonia, bacteremia, and meningitis.
  • There are at least 90 distinct documented serotypes of S.pneumoniae see e.g. Kalin, M. Thorax 1998;53: 159-162) which bear capsular saccharides with serotype-specific repeating unit structures.
  • S.pneumoniae serotype 20 is actually made up of two closely related serotypes, the capsular polysaccharides of which are largely cross-protecting (Calix et al. 2012 JBiol Chem 287:27885-94).
  • serotype 20 refers to a saccharide that would have previously been classified in the field as serotype 20, and could therefore structurally be either 20A or 20B (from a strain which would have previously been classified in the field as serotype 20, but could genotypically be either 20A or 20B) as disclosed by Calix et al.
  • the strain used to produce serotype 20 polysaccharide in PneumovaxTM (Merck) is now believed to be serotype 20A.
  • 20A may be utilized.
  • 20B may be utilized.
  • Prevalence in a target population could be a basis for selecting between these serotypes. Nevertheless, because of strains classified as 20, 20A and 20B are serologically similar, they are largely cross-protective in a vaccine and the choice among strain may not be critical.
  • the antigen used with the invention can be a capsular saccharide from any of
  • 17F, 17 A, 18F, 18A, 18B, 18C, 19F, 19A, 19B, 19C, 20 e.g ., 20A, 20B), 21, 22F, 22A, 23F,
  • the antigen can be a capsular saccharide from any of S.pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N,
  • Serotypes 6C, 7C, 15 A, 15C, 16F, 20A, 20B, 23 A, 23B, 24B, 31, 34, 35B, 35F, 37 and 38 have also become of clinical concern, so the antigen can be a capsular saccharide from one of these S.pneumoniae serotypes.
  • the invention uses conjugates from different pneumococcal serotypes, it may be advantageous to include saccharides from at least 14 different S.pneumoniae serotypes (e.g. from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more) Where a composition includes 14 or more serotypes, these may include the 13 serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F.
  • a compositions may include one or more of serotypes 2, 8, 9N, 10A, 11 A, 12F, 15B, 17F, 20 (20A and/or 20B; e.g., 20B), 22F, and/or 33F.
  • a composition may include one or more S.pneumoniae serotypes 2, 6C, 8, 9N, 10A, 12F, 15A, 15B, 15C, 16F, 17F, 20 (e.g, 20A, 20B), 22F, 23 A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F and 38.
  • a useful combination of 15 or more (e.g., 16 or more) serotypes includes each of S.pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F, and may also include serotype 8.
  • a useful combination of 20 or more (e.g. 21 or more) S.pneumoniae serotypes includes each of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10 A, 11 A, 12F, 14, 15B, 18C, 19 A, 19F, 22F, 23F and 33F.
  • Serotype 20 may refer to 20A, 20B, or a combination thereof.
  • a useful combination of 32 or more serotypes includes each of S.pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9N, 9V, 10A, 11 A, 12F, 14, 15 A, 15B, 16F, 17F, 18C, 19A, 19F, 20 (including 20A and/or 20B), 22F, 23 A, 23B, 23F, 31, 33F, and 35B..
  • a useful combination of 24 or more serotypes includes each of S.pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11 A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F.
  • Serotype 20 may refer to 20A, 20B, or a combination thereof.
  • a useful combination of 32 or more serotypes includes each of S.pneumoniae serotypes 1, 2, 3, 4, 5, 6 A, 6B, 6C, 7F, 8, 9N, 9V, 10A, 11 A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20, 22F, 23 A, 23B, 23F, 31, 33F, and 35B.
  • the capsular saccharide can be O-acetylated.
  • the degree of O-acetylation is greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, or about 100%.
  • the degree of O-acetylation of the saccharide can be determined by proton NMR (see for example Lemercinier & Jones (1996) Carbohydrate Research 296:83-96; Jones et al. (2002) J. Pharmaceutical and Biomedical Analysis 30: 1233-1247). Normally the saccharide used to prepare a conjugate will retain at least 50% (e.g. 75%, or even 100%) of the O-acetylation levels seen in the starting capsular saccharide purified from a bacterium.
  • S.pneumoniae capsular saccharides can be obtained directly from bacteria using isolation procedures known to one of ordinary skill in the art (see for example methods disclosed in U.S. Patent App. Pub. Nos. 2006/0228380, 2006/0228381, 2007/0184071, 2007/0184072, 2007/0231340, and 2008/0102498 and WO 2008/118752). As an alternative, they may be obtained from a commercial source (e.g, ATCC) or synthetically produced.
  • ATCC ATCC
  • a pneumococcal capsular saccharide antigen used with the invention can usefully have a molecular weight between lOkDa and 4,000kDa e.g. between 50 kDa and 3,000 kDa, or between 100 kDa and 2,000 kDa.
  • the molecular weight can be between 100 kDa and 2,000 kDa; between 100 kDa and 1,750 kDa; between 100 kDa and 1,500 kDa; between 100 kDa and 1,250 kDa; between 100 kDa and 1,000 kDa; between 100 kDa and 750 kDa; between 100 kDa and 500 kDa; between 200 kDa and 4,000 kDa; between 200 kDa and 3,500 kDa; between 200 kDa and 3,000 kDa; between 200 kDa and 2,500 kDa; between 200 kDa and 2,000 kDa; between 200 kDa and 2,000 kDa; between 200 kDa and 1,750 kDa; between 200 kDa and 1,500 kDa; between 200 kDa and 1,250 kDa; between 200 kDa and 1,000 kDa; between 200 kDa and 750 kD
  • the capsular saccharide is optionally chemically modified relative to the capsular saccharide found in nature.
  • the saccharide is optionally de-O-acetylated (partially or fully), de-N-acetylated (partially or fully), N-propionated (partially or fully), etc.
  • De-acetylation optionally occurs before, during or after activation, derivatization, or conjugation, but typically occurs before conjugation.
  • Some embodiments of the invention involve the use of two or more different conjugates.
  • this means (when using a single type of carrier polypeptide for each conjugate) that each‘different’ conjugate has a saccharide from a different pneumococcal serotype.
  • the antigen can be a saccharide from S. pyogenes.
  • S. pyogenes is a gram-positive bacterium (also known as group A streptococcus or‘GAS’) responsible for a wide array of infections in humans, including pharyngitis, tonsillitis, scarlet fever, cellulitis, erysipelas, rheumatic fever, post-streptococcal glomerulonephritis, necrotizing fasciitis, myonecrosis and lymphangitis.
  • the polysaccharide is the capsular polysaccharide of S. pyogenes , which is composed of hyaluronic acid, a high molecular weight polymer where the repeating unit has the structure:
  • the capsular polysaccharide from S. pyogenes has a molecular weight of between lOkDa and 4,000 kDa, for example between 50 kDa and 4,000 kDa; between 50 kDa and 3,500 kDa; between 50 kDa and 3,000 kDa; between 50 kDa and 2,500 kDa;
  • the polysaccharide is a non-capsular polysaccharide from S. pyogenes.
  • Non-capsular polysaccharides include the group-A-strep cell wall polysaccharide, which comprises a backbone of poly-L-rhamnopyranosyl units connected by alternating a-L- (l 3) and a-L-(l 2) linkages, to which N-acety 1 -b-D-gl ucosam i ne residues are attached at the 3-position of the rhamnose backbone.
  • the group-A-strep cell wall polysaccharide from S. pyogenes has a molecular weight of between lOkDa and 4,000 kDa. In other such embodiments, the polysaccharide has a molecular weight of between 50 kDa and 4,000 kDa.
  • the polysaccharide has a molecular weight of between 50 kDa and 3,500 kDa; between 50 kDa and 3,000 kDa; between 50 kDa and 2,500 kDa; between 50 kDa and 2,000 kDa; between 50 kDa and 1,750 kDa; between 50 kDa and 1,500 kDa; between 50 kDa and 1,250 kDa; between 50 kDa and 1,000 kDa; between 50 kDa and 750 kDa; between 50 kDa and 500 kDa; between 100 kDa and 4,000 kDa; between 100 kDa and 3,500 kDa; 100 kDa and 3,000 kDa; 100 kDa and 2,500 kDa; 100 kDa and 2,000 kDa; between 100 kDa and 2,000 kDa;
  • the antigen can be a capsular polysaccharide derived from S.agalactiae (Group B Streptococcus or GBS), a gram-positive bacterium commonly commensal with mammals that causes septicemia, pneumonia, and meningitis in immunologically vulnerable humans and bovine mastitis in dairy cows.
  • S.agalactiae serotypes There are at least 10 S.agalactiae serotypes with distinct capsular polysaccharide repeating units (la, lb, II-IX), but only a few serotypes are commonly
  • serotypes la, lb, II, III, and V responsible for disease.
  • conjugates of capsular polysaccharides from these serotypes can be prepared.
  • the structures for the capsular polysaccharide repeating units of common S. agalactiae serotypes have been determined and are:
  • the antigen comprising a polysaccharide comprises a capsular polysaccharide derived from H. influenzae.
  • H. influenzae is a gram-negative, anaerobic pathogenic bacterium responsible for a wide range of localized and invasive infections including pneumonia, bacteremia, meningitis, epiglottitis, cellulitis and infectious arthritis.
  • serotypes of H. influenzae with distinct capsular polysaccharide chemical structures types a-f.
  • type a and type b are considered“high-virulence” strains of H.
  • H. influenzae polysaccharide for use with the invention.
  • the structure of the repeating unit of the type b capsular polysaccharide has been determined and is:
  • the antigen comprising a polysaccharide comprises a capsular polysaccharide derived from N. meningitidis.
  • N. meningitidis is a gram negative bacterium that is a major causative agent of meningitis and meningococcal septic infection.
  • serogroups A, B, C, E-29, H, I, K, L, W-135, X, Y, Z, and Z’ (29E) serogroups A, B, C, W-135, X, Y, Z, and Z’ (29E)
  • serogroups A, B, C, W-135, X, Y
  • the structures of the repeating unit of the capsular polysaccharide for the five main life threatening serogroups of interest for conjugate preparation have been determined and are:
  • Type W-135 [ 6)-a-D-Gal >-(l 4)-a-D-Neu >5Ac(90Ac)-a-(2—]
  • the antigen is a capsular polysaccharide derived from one of the six serotypes of Porphyromonas gingivalis (e.g, Kl, K2, K3, K4, K5 and/or K6). See Van Winkelhoff et al. (1993) Oral Microbiol. Immunol. 8:259-265; and Laine et al. (1996) J.
  • Periodontal Res. 31 278-84.
  • the antigen is a Vi polysaccharide.
  • Vi is the capsular polysaccharide of Salmonella typhi (previously classified as a species itself, but now referred to as the typhi serovar of S.enterica). Vi may also be found in other serovars of Salmonella (such as S.enterica serovar paratyphi C or serovar dublin ) and in other bacteria, such as Citrobacter (e.g. C.freundii and C.youngae).
  • the Vi polysaccharide is a linear homopolymer of a hexosaminuronic acid, al,4-/V-acetylgalactos-aminouronic acid, which is 60 - 90% acetylated at the C-3 position.
  • the O-acetyl substitution on Vi is a factor in its ability to elicit a protective immune response.
  • the immunogenicity of Vi is closely related to its degree of O-acetylation. Partial de-O-acetylation can slightly increase immunogenicity; complete de-O-acetylation eliminates the immunogenicity of Vi.
  • the Vi polysaccharide used in the present invention may be chemically modified relative to the capsular polysaccharide as found in nature.
  • the Vi polysaccharide may be partially de-O-acetylated, de-N-acetylated (partially or fully), N-propionated (partially or fully), etc.
  • De-acetylation may occur before, during or after conjugation, but preferably occurs before conjugation. The effect of de-acetylation etc. can be assessed by routine assays.
  • the antigen is a polysaccharide from S.aureus.
  • polysaccharide can be the exopolysaccharide of S.aureus , which is a poly-N-acetylglucosamine (PNAG), or the capsular polysaccharide of S.aureus , which can be e.g. type 5, type 8 or type 336.
  • PNAG poly-N-acetylglucosamine
  • the antigen is a surface glycan from C.difflcile , such as PS-I or PS-II.
  • the antigen is a glucan containing b-l, 3-linkages and/or b-1,6- linkages.
  • These conjugated glucans can be useful for raising an anti-fungal immune response, for example against Candida albicans.
  • Glucans are glucose-containing polysaccharides found inter alia in fungal cell walls b-glucans include one or more b-linkages between glucose subunits.
  • a glucan used in accordance with the invention includes b-linkages, and may contain only b-linkages (i.e. no a linkages).
  • the glucan may comprise one or more b-l, 3-linkages and/or one or more b-l, 6-linkages.
  • the glucan may also comprise one or more b-l, 2-linkages and/or b-l, 4-linkages, but normally its only b linkages will be b- 1,3 -linkages and /or b-l, 6-linkages.
  • the glucan may be branched or linear.
  • the glucan may be a fungal glucan.
  • A‘fungal glucan’ will generally be obtained from a fungus but, where a particular glucan structure is found in both fungi and non fungi ( e.g . in bacteria, lower plants or algae) then the non-fungal organism may be used as an alternative source.
  • the glucan may be derived from the cell wall of a Candida , such as C .
  • albicans or from Coccidioides immitis, Trichophyton verrucosum, Blastomyces dermatidis, Cryptococcus neoformans, Histoplasma capsulatum, Saccharomyces cerevisiae,
  • b-glucans can be purified from fungal cell walls in various ways.
  • the glucan is a b-1,3 glucan with some b-1,6 branching, as seen in e.g. laminarins.
  • Laminarins are found in brown algae and seaweeds. The b(1-3):b(1-6) ratios of laminarins vary between different sources e.g.
  • the glucan used with the invention may have a b(1-3):b(1-6) ratio of between 1.5: 1 and 7.5: 1 e.g. about 2: 1, 3: 1, 4: 1, 5: 1, 6: 1 or 7: 1.
  • the glucan has exclusively or mainly b-1,3 linkages, as seen in curdlan.
  • the glucan may be made solely of b-1,3-1p ⁇ b ⁇ glucose residues (e.g. linear b-D-glucopyranoses with exclusively 1,3 linkages).
  • the glucan may include monosaccharide residues that are not b-1,3-1p ⁇ b ⁇ glucose residues e.g. it may include b-I, ⁇ -Ip ⁇ b ⁇ glucose residues.
  • the ratio of b- 1,3 -linked glucose residues to these other residues should be at least 8: 1 (e.g. >9: 1, >10: 1, >11 : 1, >12: 1, >13: 1, >14: 1, >15: 1, >16: 1, >17: 1, >18: 1, >19: 1, >20:1, >25: 1, >30: 1, >35: 1, >40: 1, >45: 1, >50: 1, >75: 1, >100: 1, etc.).
  • an antigen comprising a polysaccharide comprises a
  • an antigen comprises a hapten: a non-polymeric synthetic moiety of molecular weight less than 1,000 Da.
  • haptens that mimic drugs of abuse, e.g., nicotine or cocaine (see, e.g., Berkowitz & Spector. Science. 1972(178): 1290-1292 for morphine; Kosten et al. Vaccine. 2002(20): 1196- 1204 for cocaine; and Hatsukami et al. Clin Pharmacol Ther. 2005(78):456-467).
  • Antigens often do not intrinsically contain functional groups that are suitable or ideal for conjugation. Thus an antigen might need to be functionalized prior to its conjugation to the nnAA. Further details of such functionalization are given below.
  • the immunogenic conjugates provided herein include an immunostimulator.
  • the immunostimulator may be a toll-like receptor (TLR) agonist, a C-type lectin receptor (CLR) agonist, or a CDld agonist.
  • TLR toll-like receptor
  • CLR C-type lectin receptor
  • CDld agonist CDld agonist
  • Other small molecule immunopotentiators (SMIPs) can also be used.
  • Immunostimulatory oligonucleotides are exemplary immunostimulators (see below).
  • Immunostimulators might need to be functionalised prior to their conjugation if they do not already include an appropriate functional group. For instance, an azido or an alkyne (e.g. DBCO) group may be incorporated, permitting click chemistry conjugation.
  • CuAAC chemistry for oligonucleotides is disclosed by Jawalekar et al. (2013) Molecules 13:7346-63.
  • An alkyne can conveniently be used to conjugate to an azido-containing nnAA in a carrier polypeptide, whereas an azido can conveniently be used to conjugate to an alkyne-containing antigen.
  • Many immunostimulators include free hydroxy groups which may be modified by cyanylation (e.g. with CDAP, as disclosed elsewhere herein) for subsequent reductive amination.
  • Vaccine compositions comprising Toll-like receptor (TLR) agonists are already licensed for human use.
  • a conjugate of the invention may include a TLR agonist i.e. a compound which can agonise a Toll-like receptor.
  • a TLR agonist is an agonist of a human TLR.
  • the TLR agonist can activate any of TLR1, TLR2, TLR3, TLR4, TLR5,
  • TLR6, TLR7, TLR8, TLR9 or TLR11 preferably it can activate human TLR4, human TLR7, or human TLR9.
  • Agonist activity of a compound against any particular Toll-like receptor can be determined by standard assays. Companies such as Imgenex and Invivogen supply cell lines which are stably co-transfected with human TLR genes and NFKB, plus suitable reporter genes, for measuring TLR activation pathways. They are designed for sensitivity, broad working range dynamics and can be used for high-throughput screening. Constitutive expression of one or two specific TLRs is typical in such cell lines. See also Rosenberg et al. (2010) J Immunol 184 (1 supplement) 136.20.
  • TLR receptors are known in the art e.g. see WO2014/118305.
  • Typical TLR1 agonists are triacylated lipopeptides e.g. including a Pam3Cys group.
  • Typical TLR2 agonists are various types of lipid e.g. including a Pam2Cys group, lipoteichoic acid, lipoarabinomannan, or palmitoyl-Cys(2[R],3-dilauroyloxy-propyl)-Abu-D- Glu-NEb (where: Cys is a cysteine residue, Abu is an aminobutyric acid residue and Glu is a glutamic acid residue).
  • Typical TLR3 agonists are double-stranded RNAs.
  • Typical TLR4 agonists are lipopolysaccharides or fragments e.g. monophosphoryl lipid A or its detoxified derivates. 3dMPL is a preferred TLR4 agonist (see below).
  • Other useful TLR4 agonists include any of: glucopyranosyl lipid A (GLA) or its ammonium salt; an aminoalkyl glucosaminide phosphate, such as RC-529 or CRX-524; E5564; compounds containing lipids linked to a phosphate-containing acyclic backbone; or any of compounds‘ER 803058’,‘ER 803732’,‘ER 804053’,‘ER 804058’,‘ER 804059’,‘ER 804442’,‘ER 804680’, ‘ER 803022’,‘ER 804764’ or‘ER 804057’ (also known as E6020) disclosed in WO03/011223.
  • GLA glucopyranosyl lipid A
  • a conjugate of the invention can include an analog of monophosphoryl lipid A (MPL), such as 3-O-deacylated MPL (3dMPL) or an aminoalkyl glucosaminide phosphate (e.g. RC-529 or CRX-524; Bazin et al. 2006 Tetrahedron Lett 47:2087-92), both of which act as TLR4 agonists.
  • MPL monophosphoryl lipid A
  • 3dMPL 3-O-deacylated MPL
  • aminoalkyl glucosaminide phosphate e.g. RC-529 or CRX-524; Bazin et al. 2006 Tetrahedron Lett 47:2087-92
  • Typical TLR5 agonists are flagellins.
  • Typical TLR6 agonists are diacylated lipopeptides.
  • Typical TLR7 agonists are guanosine analogs and imidazoquinolines e.g. including imiquimod (‘R-837’), resiquimod (‘R-848’), loxoribine, and benzonaphthyridines.
  • Typical TLR8 agonists are imidazoquinolines.
  • Typical TLR9 agonists are immunostimulatory oligonucleotides, such as CpG oligonucleotides (see section IV.C.2., below).
  • 3dMPL is a mixture of 3 de-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains. Any of these individual compounds can be used in a conjugate of the invention, or a mixture or more than one. It is referred to by the trade name‘MPL®’ and further details are disclosed in Baldrick et al. (. Regulatory Toxicol Pharmacol (2002) 35:398-413). Methods for synthesizing 3dMPL are disclosed in Johnson et al. (1999) JMed Chem 42:4640-9.
  • Immunostimulatory oligonucleotides are well known in the art. Generally these include nucleotide sequences containing a CpG motif (a dinucleotide sequence containing an unmethylated cytosine nucleotide linked to a guanosine nucleotide), but oligonucleotides containing palindromic or poly(dG) sequences have also been shown to be immunostimulatory, as have Cpl motifs (i.e. a cytosine-inosine dinucleotide).
  • the oligonucleotides can include nucleotide modifications such as base modifications ( e.g .
  • Immunostimulatory CpG sequences have been classified as CpG-A, CpG-B, or CpG-C depending on the cytokine response they elicit.
  • the CpG is a CpG-B.
  • a CpG oligonucleotide is constructed so that its 5' end is accessible for receptor recognition.
  • the immunostimulatory oligonucleotide will typically be shorter than 50 nucleotides in length e.g. between 15-35 or between 18-28 nucleotide in length.
  • Two sequences for inclusion within an immunostimulatory oligonucleotide include GTCGTT and TTCGTT.
  • a preferred immunostimulatory oligonucleotide has the DNA sequence 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' (SEQ ID NO: 16). This sequence is known in the art as‘CpG 7909’ or ODN 2006’ (with a full phosphorothioate backbone) and has been tested in humans (including in pneumococcal conjugate vaccines: Sogaard et al. 2009 Clin Infect Dis 51 :42-50.
  • sequences which are useful in mice include TCCATGACGTTCCTGATGCT (SEQ ID NO: 21) and
  • TCCATGACGTTCCTGACGTT SEQ ID NO: 22
  • sequence TCGTCGTTGTCGTTTTGTCGTT SEQ ID NO: 23
  • TCGACGTTCGTCGTTCGTCGTTC SEQ ID NO: 24
  • TCGCGACGTTCGCCCGACGTTCGGTA SEQ ID NO: 25
  • Immunostimulatory oligonucleotides can usefully be derivatized to include at azido group in their 3' terminal nucleotide (e.g. see Kumar et al. 2007 JACS 129:6859-64 and
  • Azido-modified oligonucleotides can be prepared by attaching a N-hydroxysuccinimide (NHS) ester to any nucleotide (including the 3' nucleotide) and this NHS group can then be used to attach an azido group.
  • NHS N-hydroxysuccinimide
  • the oligonucleotide having SEQ ID NO: 25 can be azido-derivatized to provide the sequence TCGCGACGTTCGCCCGACGTTCGGTA-N3 (SEQ ID NO: 26).
  • the azido group can be linked to antigens using the same‘click’ chemistry as a pAMF nnAA.
  • CLR agonists include, but are not limited to, trehalose-6, 6'-dimycolate (TDM), its synthetic analog D-(+)-trehalose-6,6'-dibehenate (TDB), and other 6,6'-diesters of trehalose and fatty acids.
  • TDM trehalose-6, 6'-dimycolate
  • TDB D-(+)-trehalose-6,6'-dibehenate
  • other 6,6'-diesters of trehalose and fatty acids are 6,6'-diesters of trehalose and fatty acids.
  • TDM trehalose-6, 6'-dimycolate
  • TDB D-(+)-trehalose-6,6'-dibehenate
  • CLR agonists may have formula (C):
  • R'C(O)- and R 2 C(0)- are the same or different and are acyl groups.
  • Suitable acyl groups may be saturated or unsaturated. They may be selected from the acyl residues of a mycolic acid, a corynomy colic acid, a strengeanic acid, a 2-tetradecyl-3 -hydroxy octadecanoic acid, a 2- eicosyl-3-hydroxytetracosanoic acid, a behenic acid, a palmitic acid, e/c.
  • Useful mycolic acids include alpha-, methoxy-, and keto- mycolic acids, in cis- and or trans- forms.
  • CDld agonists include, but are not limited to, a-glycosylceramides, such as a-galactosylceramides.
  • a-GalCer a-galactosylceramide
  • the invention can be applied to glycosylceramides which are CDld agonists, including a-galactosylceramide (a-GalCer), phytosphingosine-containing a- glycosylceramides, [(2S,3S,4R)-l-0-(a-D-galactopyranosyl)-2-(N-hexacosanoylamino)-l,3,4- octadecanetriol], OCH, KRN7000 CRONY-101, 3"-0-sulfo-galactosylceramide, etc.
  • a-GalCer a-galactosylceramide
  • Conjugation involves formation of covalent linkages between the nnAA residue, an antigen, and an immunostimulator. This requires reactive functional groups in the nnAA, the antigen, and the immunostimulator.
  • a nnAA for the carrier polypeptide will generally be chosen because it already has a suitable functional group (e.g . the azido group of pAMF), but antigens and immunostimulators may not intrinsically contain functional groups that are suitable or ideal for conjugation. Thus an antigen and an immunostimulator might need to be functionalised prior to their conjugation to the nnAA. With appropriate functional groups in the nnAA, antigen, and immunostimulator conjugation is achieved in general terms by permitting these groups to react with each other to form the desired covalent links.
  • nnAA include a functional group (e.g. an azido group) which is suitable for a“click” chemistry reaction with a functional group on the antigen.
  • a functionalised antigen ideally includes a group suitable for such“click” reactions.
  • the immunostimulator includes the same functional group as the nnAA.
  • a useful way of obtaining cross-linked conjugates is to utilize the same reactive group in both the immunostimulator and the nnAA (e.g. an azido group).
  • This reactive group can form bonds with reactive groups which are present at multiple sites within the antigen (e.g. an alkyne group).
  • reactive groups which are present at multiple sites within the antigen (e.g. an alkyne group).
  • conjugation of an antigen or an immunostimulator involves three steps of (a) activation (b) optional derivatisation and (c) covalent linkage. Steps (a) and (b) ensure that the moiety being conjugated has the functional groups which are necessary for step (c) to occur.
  • Activation can introduce a group which is already suitable for conjugation, or to which a group can be attached (derivatised) which is suitable for conjugation.
  • Step (a) may also include an initial step of removing a blocking group, such that certain functional groups (e.g. hydroxyls, amines, thiols) are more accessible to activation.
  • the steps (a)-(c) can occur essentially simultaneously (e.g. where a reactive moiety such as N-hydroxysuccinimide is added to the antigen), but in other embodiments two or more of steps (a)-(c) are discrete, with optional purification between steps.
  • cross-linked conjugates are advantageous, so it is also useful to introduce multiple reactive functional groups per antigen molecule.
  • multiple aldehyde or cyanate ester groups groups can be introduced when activating a saccharide molecule. These groups can then be derivatised e.g. to introduce a reactive cyclooctyne which can then react with azido groups in in the nnAA and in the immunostimulator.
  • the antigen Prior to functionalization of a saccharide antigen to provide an activated antigen capable of conjugation to a carrier polypeptide, the antigen may be subject to a process that reduces molecular weight.
  • the process may be a mechanical sizing step, in which case the polysaccharide is subjected to shearing forces (as may accomplished using a high shear homogenizer or the like), and/or the process may involve treatment with heat or mild acid.
  • the molecular weight of the antigen following this initial treatment will approximate, within 25-30%, the native molecular weight of the corresponding serotype.
  • a mechanical sizing method involves use of a PandaPLUS 2000 homogenizer (available from GEA Niro Soavi) at a pressure in the range of about 200 psi to about 25,000 psi and a heat exchanger temperature setting in the range in of about 4°C to l2°C (e.g, 8°C to l2°C), with a polysaccharide concentration of about 1 g/L to about 4 g/L and a temperature range during processing of about 8°C to l5°C.
  • a PandaPLUS 2000 homogenizer available from GEA Niro Soavi
  • the selected antigen can be activated using any suitable method, including, without limitation: periodate oxidation (e.g. to oxidize hydroxyl groups on adjacent carbon atoms to give reactive aldehyde groups, for instance as disclosed in WO2011/110531); unmasking of an intrinsic aldehyde (e.g. a reducing terminus of a polysaccharide); cyanylation using, e.g., 1- cyano-4-dimethylaminopyridinium tetrafluorob orate (CDAP) activation; and hydroxyl activation with l,l'-carbonyldiimidazole (CDI) followed by nucleophilic addition.
  • periodate oxidation e.g. to oxidize hydroxyl groups on adjacent carbon atoms to give reactive aldehyde groups, for instance as disclosed in WO2011/110531
  • unmasking of an intrinsic aldehyde e.g. a reducing terminus of a polysaccharide
  • Activation can also involve the use of p-nitrophenylcyanate, N-cyanotriethylammonium tetrafluoroborate, active esters, carbodiimides, hydrazides, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU, etc.
  • Periodate oxidation has been shown to be useful for activating, inter alia , pneumococcal serotypes 1, 2, 3, 7F, 8, 9N, and 11 A.
  • CDAP cyanylation has been shown to be useful for activating, inter alia, pneumococcal serotypes 3, 7F, and 10 A.
  • One technique for activating saccharide antigens herein involves treatment with a periodate reagent followed by reductive deamination, with the activated antigen so provided then available for conjugation to an appropriately functionalized polypeptide carrier.
  • Periodate activation is thus a two-step method, with an initial periodate oxidation reaction followed by purification of the oxidation product and then a second reaction involving reductive amination as will be explained infra.
  • Periodate oxidation as is understood in the field, involves the cleavage of adjacent hydroxyl groups, i.e., hydroxyl groups in the form of a "vicinal" diol in which two adjacent carbon atoms are each substituted with a hydroxyl group.
  • Periodate cleavage of such a diol results in the breakage of the carbon-carbon bond and formation of an aldehyde moiety at each carbon atom, e.g., a -CH2(OH)-CH2(OH)- motif is converted upon periodate oxidation to - C(CO)H at each carbon atom.
  • Periodate activation thus activates carbohydrate sugar residues bearing adjacent hydroxyl moieties; periodate can also be used to activate amino acids containing the 2-amino alcohol moiety, i.e., N-terminal threonine or serine residues.
  • antigens activated by this method are optionally chromatographically purified and/or lyophilized after activation.
  • antigens are dissolved in a solution, e.g., in water or an aqueous buffer;
  • a source of periodate is added to the antigen from a concentrated stock solution to form an oxidation mixture;
  • the reaction mixture is incubated; and
  • (d) (optional) excess periodate is removed.
  • Deionized water or a suitable buffered solution is optionally used for the oxidation reaction.
  • the solution in step (a) is deionized water.
  • the solution in step (a) comprises an effective amount of a buffer with a pKa around physiological pH. In some embodiments, the solution in step (a) comprises an effective amount of a buffer with a pKa around physiological pH. In some embodiments, the buffer does not comprise an amine group. Examples of amine-free buffers include, but are not limited to acetate, formate, and phosphate. In some embodiments, an amine buffer is employed in step (a).
  • Suitable amine buffers generally comprise a combination of a tertiary amine or an N- heterocyclic compound with a weak acid (e.g., pyridine and acetic acid; pyridine and formic acid; N-ethylmorpholine and acetic acid; trimethylamine and carbonic acid; triethanolamine and phosphoric acid; etc.), or they can be a zwitterionic amine buffer such as 4-(2 -hy droxy ethyl)- 1- piperazineethanesulfonic acid (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), or 3-[4- (2-hydroxyethyl)piperazin-l-yl]propane-l -sulfonic acid (HEPPS).
  • a weak acid e.g., pyridine and acetic acid; pyridine and formic acid; N-ethylmorpholine and acetic acid; trimethylamine and carbonic acid; tri
  • the periodate source in step (b) is optionally selected from any periodate source with appropriate stability in aqueous solution.
  • periodate sources include, but are not limited to, sodium periodate, potassium periodate, tetrabutylammonium (meta)periodate, barium periodate, sodium hydrogen periodate, sodium (para)periodate, and tetraethylammonium (meta)periodate.
  • the level of periodate addition and reaction conditions are adjusted to convert all available diols on a polysaccharide to aldehydes.
  • the level of periodate addition and reaction conditions are adjusted to introduce a low amount of oxidation/aldehyde formation into the polysaccharide chain.
  • Less than stoichiometric amounts of sodium periodate (e.g. ⁇ 1.0 equivalents) in the oxidation reaction favor low amounts of polysaccharide chain oxidation.
  • a bacterial saccharide is activated by 0.001-0.7, 0.005-0.5, 0.01-0.5, 0.1-1.2, 0.1-0.5, 0.1-0.2, 0.5- 0.8, 0.1-0.8, 0.3-1.0 or 0.4-0.9 molar equivalents of periodate (see WO2011/110531).
  • 0.4 molar equivalent of periodate is added to a pH 6.0 solution containing a pneumococcal capsular polysaccharide and incubated for 17 hrs at 25°C (see WO2011/110531).
  • the relative amount of periodate reagent added into the saccharide antigen solution is typically, although not necessarily, in the range of 0.1 equivalents to 0.5 equivalents of periodate, with “equivalents” being relative to individual saccharide units (i.e., saccharide "monomer” units in a polysaccharide).
  • Reaction conditions for periodate oxidation of a saccharide antigen can vary, but in certain embodiments are as follows: a reaction pH in the range of 5 to 7, such as 5 to 6 or 5.4 to 5.9, e.g., 5.4; a reaction temperature in the range of 4 °C to 25 °C; a reaction time in the range of 2 to 30 hours, e.g., 2 to 24 hours, 14 to 30 hours, 14 to 24 hours, 18 to 30 hours, 18 to 24 hours, 24 hours, and the like; and polysaccharide- aldehyde (PS-aldehyde) purification carried out using dialysis, size exclusion chromatography (SEC), ultrafiltration/diafiltration (UF/DF), or the like.
  • SEC size exclusion chromatography
  • UF/DF ultrafiltration/diafiltration
  • the PS-aldehyde is maintained in an aqueous buffer throughout the activation process, typically a buffer having a concentration in the range of about 25 mM to about 150 mM, such as about 50 mM to about 110 mM, including 50 mM and 110 mM (e.g., 110 mM for serotypes 6B and 23F and 50 mM for other periodate- activatable serotypes, and is not "isolated" from aqueous buffer at any point during the activation reactions.
  • a buffer having a concentration in the range of about 25 mM to about 150 mM such as about 50 mM to about 110 mM, including 50 mM and 110 mM (e.g., 110 mM for serotypes 6B and 23F and 50 mM for other periodate- activatable serotypes, and is not "isolated" from aqueous buffer at any point during the activation reactions.
  • Periodate treatment can also be used as a way to decrease the molecular weight of polysaccharides that have a interchain glycerol phosphate linkage as periodate tends to cleave such linkages. If the purpose of the periodate treatment is both sizing and activation, larger amounts of the periodate reagent can be used.
  • the PS-aldehyde is dissolved in an aqueous buffer, typically maintained at a pH in the range of 5 to 7, such as 5 to 6.7, 5 to 6.5, 5.5 to 6.9, 5.5 to 6.7, 5.5 to 5.9, or 5.7. Or pH in the range of 5.1 to 5.9, e.g., 5.2 to 5.9, 5.3 to 5.7, 5.4 to 5.6, 5.4 to 5.9.
  • the second step of periodate activation, reductive amination is carried out by adding to the PS-aldehyde solution an activating reagent in the form of a reactive moiety coupled to a primary amino group, followed by admixture with sodium cyanoborohydride, for a time period effective to transfer the reactive moiety to the cyanate-substituted saccharide, thereby providing an activated saccharide antigen.
  • an activating reagent in the form of a reactive moiety coupled to a primary amino group
  • sodium cyanoborohydride for a time period effective to transfer the reactive moiety to the cyanate-substituted saccharide, thereby providing an activated saccharide antigen.
  • 2 to 12 equivalents of the activating reagent in the form of a reactive moiety coupled to a primary amino group
  • reaction temperature for reductive amination can vary, as can reaction time, but typical reaction temperatures are in the range of about 20 °C to about 25 °C, and typical reaction times are in the range of about 6 to 48 hours, e.g., 24 hours.
  • DBCO dibenzylcyclooctyne
  • n is an integer in the range of 2 to 12.
  • m is 1 and n is an integer in the range of 2 to 12, e.g., 2 to 8, 2 to 6, 2 to 4, or 4.
  • m is zero.
  • the reactive moiety in this case is the alkyne functionality in the eight- membered ring, the reactivity of which is enhanced by the strain imposed by the adjacent phenyl rings.
  • the amount of the DBCO derivative used in the reaction is usually in the range of 1 to 3 equivalents, e.g., at least 2 equivalents, such as 2 equivalents or 3 equivalents, again relative to the individual saccharide units, with the DBCO dissolved in a suitable solvent such as DMSO.
  • the amount of sodium cyanoborohydride employed is typically in the range of 2.0 to 12.0 equivalents, again relative to the saccharide units.
  • the amount of sodium cyanoborohydride is in the range of in the range of 8.0 to 12.0 equivalents, as this excess can increase the "degree of the activation reaction," or "DBCO%,” (i.e., the mol amount of DBCO incorporated per mole of polysaccharide repeating unit).
  • DBCO% degree of the activation reaction
  • the reaction is allowed to proceed, with stirring, for 6 to 48 hours at a reaction temperature typically in the range of 20 °C to 25 °C, with the DBCO-derivatized antigen then purified using any conventional technique, e.g., dialysis, SEC, UF/DF, or the like.
  • a periodate activation method that can be used across a plurality of antigen serotypes to provide a target DBCO%, wherein the only variable that may need to be adjusted for a specific serotype is the relative amount of periodate used per polysaccharide unit, i.e., periodate molar equivalents.
  • DBCO% refers to one measure of the yield of the activation reaction and is defined the amount of DBCO derivative that reacts with the saccharide relative to the total DBCO derivative employed.
  • Other variables can be kept constant regardless of the specific serotype undergoing activation, i.e., reaction pH, activation time, reaction temperature, molar equivalents of DBCO, and overall reaction time.
  • DBCO% typically in the range of 3% to 15%, e.g., 3% to 5%, 5% to 15%, 5% to 7%, such as 3% to 10%, e.g., 3% to 5%, 5% to 10%, 5% to 7%, and the like.
  • an optional sizing step carried out mechanically, thermally or chemically as described above, providing the saccharide antigen as a solution in an aqueous buffer, e.g., a phosphate buffer, having a pH in the range of 5.1 to 5.9 (e.g., 5.2-5.9, 5.3-5.7, 5.4-5.6, 5.4-5.9);
  • an aqueous buffer e.g., a phosphate buffer, having a pH in the range of 5.1 to 5.9 (e.g., 5.2-5.9, 5.3-5.7, 5.4-5.6, 5.4-5.9);
  • an aqueous buffer e.g., an acetate buffer (e.g., about 110 mM-l20 mM), having a pH of about 5.4;
  • a capping step follows reductive amination in order to cap unreacted aldehyde moieties on the polysaccharide; in other embodiments, capping is optional, e.g., when reductive amination is carried out using a higher ratio of the cyanoborohydride reagent to saccharide units. See Section IV.E.l, infra.
  • fewer than 0.001%, 0.01%, 0.1%, 0.5%, 1 %, 2%, 5%, 10%, 30% or 50% of the vicinal diols of a bacterial saccharide become oxidized during periodate activation (see WO2011/110531) e.g. between 5-10%.
  • Low reaction temperatures also favor lower amounts of polysaccharide chain oxidation.
  • low periodate concentrations ⁇ 0. l eq
  • polysaccharide chain oxidation of particular capsular polysaccharides such as S. pneumoniae 19F.
  • the level of periodate addition and reaction conditions are adjusted to direct cleavage to selective sugars a polysaccharide chain.
  • lmM NalCri at 4 degrees Celsius is used in the literature to selectively oxidize sialic acid residues at carbons 7, 8, or 9, while lOmM NalCri at room temperature is used to oxidize a wide variety of sugar residues, including sialic acid, galactose, and mannose residues.
  • step (b) comprises adding sodium periodate to a final concentration of 2.5 mM and step (c) comprises incubating the reaction mixture at 25 degrees Celsius for 3 minutes.
  • excess periodate is optionally removed in step (d).
  • excess periodate in some embodiments, is removed by size exclusion, dialysis, or diafiltration against water or buffer solution using a medium with a suitable molecular weight cutoff or exclusion limit.
  • removal of periodate in step (d) comprises adding a quenching agent.
  • Excess periodate is optionally quenched by the addition of glycerol (10% (v/v)), the addition of a molar excess of sodium sulfite, or the addition of a molar excess of N-acetylmethionine.
  • a polysaccharide or protein antigen is deprotected to increase accessibility of hydroxyl or amine groups for periodate activation.
  • O- acetyl or N-acetyl groups on polysaccharides are removed to increase reactivity of adjacent hydroxyls to periodate.
  • de-O-acetylation or de-N-acetylation is optionally accomplished by incubation in a mild acid (e.g. low concentration HC1) or alkaline (e.g. sodium bicarbonate) solution, followed by optional heating and adjustment back to physiological pH.
  • mild acid treatment ⁇ 0.lM HC1 or ⁇ 0.2M AcOH
  • heating and neutralization is used to partially hydrolyze (“size”) polysaccharides of high molecular weight.
  • mild acid treatment e.g. ⁇ 0.1M HC1 or ⁇ 0.2M AcOH
  • heating 45-95°C
  • neutralization to pH 5.5-6.0
  • serotypes 3, 4, 18C, and 11 A are treated by such an acid/heating/neutralization process to deprotect the polysaccharide, size the
  • S. pneumoniae serotype 3 polysaccharide is treated with 0.18M acetic acid, followed by heating at 85°C for 1 hour.
  • S. pneumoniae serotype 4 polysaccharide is treated with 0.01M HC1 followed by heating at 45°C for 1 hour.
  • S. pneumoniae 18C polysaccharide is treated with 0.18M acetic acid, followed by heating at 95°C for 40 minutes.
  • S.pneumoniae serotype 11 A polysaccharide is treated by 0.18M acetic acid, followed by heating at 80°C for 1 hour.
  • N-formyl groups on purified proteins are removed/amine groups are de-formylated by treatment with a formyl-L-methionyl peptide amidohydrolase in deionized water or a physiological pH buffered solution.
  • N-formyl groups on purified proteins are removed by treatment of lyophilized protein with anhydrous hydrazine vapor at -5°C (Miyataki et al. Eur. J. Biochem. 212, 785-789 (1993)).
  • Periodate activation is a useful technique used to activate Streptococcus pneumoniae serotypes 5, 6A, 6B, 7F, 12F, 14, 20, and 23F; see Table 2, infra.
  • a different method is used to activate the saccharide antigen in preparation for conjugation.
  • the antigen is also functionalized with a reactive moiety capable of participating in a click chemistry reaction with a bio-orthogonal reactive moiety on the polypeptide carrier, but functionalization is carried out by cyanylating the antigen with a cyanylating reagent to provide cyanate (-0-CoN) group in place of hydroxyl groups, and thereafter, in a "one pot” reaction, contacting the cyanylated antigen (PS-0-CoN) with an activating reagent as was done in periodate activation.
  • the activating reagent again comprises the reactive moiety coupled to a primary amino group, and may be a DBCO derivative as discussed in the preceding section.
  • the saccharide antigen is provided at the outset in any suitable solvent (e.g., an aqueous solution, an organic solvent such as DMSO or acetonitrile, or an organic solvent mixture), preferably an aqueous buffer, typically having a pH in the range of 7 to 11, e.g., greater than 7 and up to 11, such as 8.5 to 10, 8.5. to 9.5, or 8.5 to 9.0, or 8.9, or 9.0.
  • any suitable solvent e.g., an aqueous solution, an organic solvent such as DMSO or acetonitrile, or an organic solvent mixture
  • an aqueous buffer typically having a pH in the range of 7 to 11, e.g., greater than 7 and up to 11, such as 8.5 to 10, 8.5. to 9.5, or 8.5 to 9.0, or 8.9, or 9.0.
  • a CDAP activation method is provided that can be used across a plurality of antigen serotypes to provide a target DBCO%, wherein the only variable that may need to be adjusted for a specific serotype is the relative amount of CDAP used per
  • CDAP molar equivalents polysaccharide unit
  • Other variables can be kept constant regardless of the specific serotype undergoing activation, i.e., reaction pH, activation time, reaction temperature, and molar equivalents of DBCO.
  • These standardized conditions provide a target DBCO%, typically in the range of 3% to 10%, e.g., 3% to 5%, 5% to 10%, 5% to 7%, and the like.
  • supra- or sub-stoichiometric (with respect to polysaccharide) amounts of CDAP are used for activation, e.g., about 0.1 to about 3 eq, about 0.2 to about 0.8 eq CDAP is used for activation of a polysaccharide.
  • S. pneumoniae serotype 3 capsular polysaccharide is activated using 2.0 eq CDAP.
  • S. pneumoniae serotype 3 capsular polysaccharide is activated using 2.0 eq CDAP.
  • S. pneumoniae serotype 3 capsular polysaccharide is activated using 2.0 eq CDAP.
  • pneumoniae serotype 10A capsular polysaccharide is activated using 0.8 eq CDAP.
  • the addition of a buffering agent dramatically increases the efficiency of CDAP activation, e.g., about 1 to about 4 eq of TEA (relative to the
  • TEA TEA polysaccharide
  • about 1 to about 4 eq TEA is used as a buffering agent for a CDAP activation reaction involving S. pneumoniae serotype 7F polysaccharide.
  • 2.5 eq of TEA is used as a buffering agent.
  • 2.5 eq TEA is used as a buffering agent for a CDAP activation reaction involving S. pneumoniae serotype 7F polysaccharide.
  • the buffering agent is sodium borate, sodium carbonate, or sodium hydroxide, or any combination thereof.
  • the buffering agent has a pKa of between about 8.0 to about 11.0 or the buffering agent is used to adjust the pH of the reaction solution to between about 8.0 to about 11.0. In some embodiments, the buffering agent has a pKa of between about 9.0 to about 9.5 or the buffering agent is used to adjust the pH of the reaction solution to between about 9.0 to about 9.5.
  • sodium hydroxide adjustment of pH to 9.5 is used for a CDAP activation reaction involving S. pneumoniae serotype 3 polysaccharide. In some embodiments, sodium hydroxide adjustment of pH to 9.5 is used for a CDAP activation reaction involving S. pneumoniae serotype 10A polysaccharide.
  • CDAP activation is useful technique used to activate Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 8, 9N, 9V, 10A, 11 A, 15B, 17F, 18C, 19A, 19F, 22F, and 33F (note that serotype 5 can be readily activated effectively using either periodate or CDAP chemistry).
  • Exemplary activation technique(s) for various pneumococcal serotypes are set forth in Table 2 (where serotype 20 includes 20 A and 20B):
  • the antigen is activated with carbonyldiimidazole (CDI) or carbonylditri azole (CDT).
  • CDI and CDT are capable of activating hydroxyl groups on an antigen to form a transient reactive moiety; in this case it is an unstable carbamate (
  • CDI/CDT activation is performed in anhydrous dimethylsulfoxide (DMSO).
  • DMSO dimethylsulfoxide
  • CDI/CDT activation is performed by adding a molar excess of CDI/CDT with respect to the antigen.
  • CDI/CDT activation is performed by adding a molar amount of CDI/CDT approximately equal to the molar amount of the antigen.
  • endogenous amines or other nucleophilic moieties e.g. a primary amine
  • a deprotection step e.g. as discussed above
  • nucleophilic moieties can be conveniently reacted with a variety of common electrophilic conjugation reagents like succinate derivatives (e.g. N-hydroxysuccinimide (NHS) or sulfo-NHS esters).
  • succinate derivatives e.g. N-hydroxysuccinimide (NHS) or sulfo-NHS esters.
  • N-hydroxysuccinimide (NHS) or sulfo-NHS esters e.g. N-hydroxysuccinimide (NHS) or sulfo-NHS esters.
  • N-hydroxysuccinimide (NHS) or sulfo-NHS esters e.g. N-hydroxysuccinimide (NHS) or sulfo-NHS esters.
  • N-hydroxysuccinimide (NHS) or sulfo-NHS esters e.g. N-hydroxysuccinimide (NHS) or sulfo-
  • pneumoniae serotype 1 polysaccharide is treated with between about 0.05 to about 0.25 eq of sodium periodate at room temperature for between about 12 to about 14 hours, followed by treatment with between about 5eq to about l5eq of sodium borohydride.
  • pneumoniae serotype 1 polysaccharide is treated with 0.15 eq of sodium periodate at room temperature for 18 hours, followed by treatment with lOeq of sodium borohydride.
  • the activated antigen can be conjugated to a nnAA and/or an immunostimulator directly, but usually the activated group is used to introduce a functional group that exhibits better reactivity towards the relevant functional group in the nnAA or agonist.
  • an alkynyl group can be introduced.
  • a bifunctional reagent with an amino group and an alkyne group can react with an aldehyde group which has been introduced into an antigen ( e.g . via reductive amination) thereby leaving a pendant alkyne which can react with a nnAA and/or an immunostimulator.
  • bifunctional reagents including amino and DBCO functional groups can be used.
  • an azido group in the nnAA or immunostimulator reacts with an alkynyl group in the antigen (e.g. a propargyl group).
  • An alkyne group in an antigen is ideal for reacting with an azido group e.g. using the reactions known in the art as copper-catalyzed azide- alkyne cycloaddition (CuAAC), ruthenium-catalyzed azide-alkyne cycloaddition (RuAAC), or Huisgen azide-alkyne l,3-dipolar cycloaddition.
  • the alkynyl group may have a molecular context that increases its reactivity e.g.
  • the alkylene can be within a ring.
  • the alkylene can be within a cyclooctyne ring (optionally including a heteroatom), such as a diaryl-strained cyclooctyne ring (e.g. DBCO).
  • This reaction can be a [3+2] cycloaddition referred to in the art as strain-promoted azide-alkyne cycloaddition (SPAAC). DIFO- and DBCO-based reagents are readily available for these reactions.
  • Alkyne-containing rings useful in SPAAC reactions include difluorinated cyclooctyne (DIFO) and dibenzocyclooctynes. These are available with pendant functional groups for linking to activated antigens (e.g. with a pendant amino for linking to an aldehyde or a cyanate ester), for instance using any of the following reagents:
  • n in‘PEGn’ represents the number of oxyethylene repeat units.
  • the value of n is in the range 1-20 e.g. within 2-18, 3-16, or 4-14. Thus n can be, for example, any of 4, 5, 11, 12 or 13.
  • Other click chemistry reactions which can be used for conjugation to an antigen include, but are not limited to, tetrazine-alkene ligation and Staudinger ligation between a phosphine and an azide.
  • the antigen is conjugated to the chemical handle using any chemical method compatible with the activation methods described above. Such methods include, but are not limited to, Schiff-base formation with synthetic antigen aldehydes followed by reductive amination, hydrazone formation, oxime formation, direct nucleophilic addition, and Schiff-base formation with native antigen aldehydes followed by reductive amination.
  • the absolute polysaccharide concentration in a conjugation reaction with a chemical handle is important to minimize aggregation or cross-reactivity of the polysaccharide.
  • the absolute polysaccharide/antigen concentration in a conjugation reaction with DBCO (a dibenzocyclooctyne) or a DBCO derivative is important for
  • polysaccharides activated with periodate or CDAP as described above.
  • the polysaccharide concentration in a DBCO/DBCO-derivative conjugation reaction is less than 2, less than 5, less than 7, less than 10, less than 15, less than 17.5, or less than 20 pmol/mL.
  • the polysaccharide concentration in a DBCO/DBCO-derivative conjugation reaction is about 1.5 to about 17.5 pmol/mL
  • the chemical handle is conjugated to a polypeptide or polysaccharide antigen activated as described above using the periodate methodology.
  • unreacted aldehyde moieties remaining on the activated antigen can be capped using conventional aldehyde capping chemistry (e.g., treatment with sodium borohydride, glutamic acid, or the like).
  • Aldehyde capping is optional, however, insofar as unreacted aldehydes on the polysaccharide following periodate activation can be capped to a sufficient degree during reductive amination, particularly when higher equivalents of the cyanoborohydride reagent are used, e.g., 6 to 12 equivalents, or 8 to 12 equivalents.
  • a chemical handle comprising a functional group that forms a stable or semi-stable adduct with aldehydes is combined with the periodate activated antigen, followed by optional reduction to convert semi-stable adducts to stable adducts (see, e.g., WO2014/111344; Wu et al. Vaccine 31(2013): 5623-2626; Hermanson, G.T., Bioconjugate Techniques, Second Edition, 2008).
  • the chemical handle is added at a large molar excess with respect to the aldehyde groups on the activated antigen, such that all the aldehydes are consumed in the chemical handle/antigen conjugation reaction.
  • the chemical handle is added at a lower molar ratio with respect to the aldehydes groups on the activated antigen, and excess unreacted aldehydes on the activated antigen are consumed by further reaction with an excess of an inexpensive aldehyde-reactive nucleophile (e.g. ethanolamine), or by treatment with a reducing agent strong enough to reduce aldehydes to hydroxyl groups (e.g. NaBEE).
  • an inexpensive aldehyde-reactive nucleophile e.g. ethanolamine
  • a reducing agent strong enough to reduce aldehydes to hydroxyl groups e.g. NaBEE
  • the chemical handle is conjugated to the antigen by Schiff-base formation with synthetic antigen aldehydes followed by reductive amination.
  • This embodiment results in an end-product that has secondary amine linkage between the chemical handle and the antigen: a direct N-C bond between the amine of the chemical handle and a carbon atom on antigen.
  • the chemical handle comprises an amine.
  • the conjugation method comprises: combining the amine-containing handle with periodate-activated antigen in DI water or buffered solution containing DMSO; incubating to form a Schiff base; reducing the Schiff base to a secondary amine using sodium cyanoborohydride (NaBEECN); and optionally quenching unreacted aldehydes with NaBEE.
  • the chemical handle and antigen are combined at or near 1 : 1 stoichiometry.
  • the chemical handle and antigen are combined with a molar excess of chemical handle.
  • the chemical handle and antigen are combined with a molar excess of antigen.
  • the chemical handle can be conjugated to the antigen via hydrazone formation.
  • the chemical handle is conjugated to the antigen by oxime formation.
  • the chemical handle comprises an aminooxy (-O-NH2) group.
  • the chemical handle is conjugated to a polypeptide or polysaccharide antigen activated as described above (“CDAP activation”) with CDAP.
  • CDAP activation a polypeptide or polysaccharide antigen activated as described above
  • CDAP conjugation of chemical handles hydroxyl groups on the antigen are activated as described above ("CDAP activation"), and a chemical handle comprising an amine is additionally added to the activation mixture. Because the cyanato group is labile, the chemical handle is generally added shortly (within minutes) after activation of the antigen. In some embodiments, the antigen is added 2.5 minutes after CDAP is introduced. In some embodiments, a large molar excess of the amine-containing chemical handle with respect to activated hydroxyl groups on the antigen is added.
  • the chemical handle is added at a concentration closer to 1 : 1 molar ratio with respect to the activated hydroxyl groups on the antigen, and excess unreacted cyanato groups are exhausted by addition of an excess of an inexpensive amine (e.g. ethanolamine or hexanediamine).
  • an inexpensive amine e.g. ethanolamine or hexanediamine.
  • the chemical handle is conjugated to a polypeptide or polysaccharide antigen activated as described above (“Carbonyldiimidazole
  • CDI carbonylditri azole
  • CDT carbonylditri azole
  • a large molar excess of the amine/thiol-containing chemical handle with respect to activated hydroxyl groups on the antigen is added.
  • the chemical handle is added at a concentration closer to 1 : 1 molar ratio with respect to the activated hydroxyl groups on the antigen.
  • residual CDI/CDT in the reaction is further inactivated by treatment with sodium tetraborate.
  • the chemical handle is conjugated to an endogenous amine or other nucleophilic moiety (e.g. a primary amine) either naturally present or the result of a deprotection step from a polypeptide or polysaccharide antigen as described above.
  • an electrophilic group e.g. an NHS or sulfo-NHS ester
  • a carboxylic acid group on a chemical handle is reacted with a primary amine group on the antigen in the presence of standard peptide coupling reagents and conditions to produce an amide linkage between the chemical handle and the antigen amine.
  • the chemical handle comprises a moiety that allows for a“click” chemistry reaction with a corresponding group on nnAA residue of a polypeptide.
  • One such moiety is an alkyne group, which is capable of reacting with a nnAA residue comprising an azido group.
  • this is a propargyl group, such that an alkyne group on an antigen comprises a structure of formula IV:
  • L22 is C1-C10 alkyl
  • Ui is at least one moiety of an antigen.
  • an alkyne group on an antigen comprises a structure of formula
  • L22 is -(CH2CH20)I-IO-
  • Ui is at least one moiety of an antigen.
  • the alkyne group further comprises additional features that accelerate or facilitate the reaction of the alkyne with an azido group.
  • An example of one such feature is an 8-membered ring structure (e.g., cyclo-octyne), such that an alkyne group on an antigen further comprises a DIFO or DBCO group.
  • an alkyne group on an antigen comprises a structure of formula V, formula VI, or Via:
  • Li is independently a bond, -NH-, -0-, -S-, -NH(Li2)-, -0(Li2)-, or -S(Li2)-;
  • L12 is independently L22 or L22NH-
  • L22 is independently C1-1 0 alkyl or -(CH2CH20)I-IO-;
  • Ui is independently at least one moiety of an antigen.
  • structures of formula V and Via are conveniently formed from an antigen comprising a nucleophilic group (e.g. a primary amine) and the NHS or sulfo-NHS ester of the corresponding DIFO or DBCO carboxylic acids of structures V and Via.
  • structures of formula VI are conveniently formed from an activated antigen, and a DBCO derivative such as DBCO-NH2 or DBCO-PEGn-NEb.
  • DBCO- PEGn-NEb is DBCO-PEG4-NH2.
  • n in‘PEGn’ represents the number of oxy ethylene repeat units e.g. in the structure shown above, or within formula VII, formula Vllb, formula XI, or moiety‘A’, or within the poly(alkyloxy) of L22.
  • the value of n is in the range 1-20 e.g. within 2-18, 3-16, or 4- 14. Thus n can be, for example, any of 4, 5, 11, 12 or 13.
  • the moiety of Ui is at least one polyol of a polysaccharide. In some embodiments the moiety of Ui is at least one polyol of a lipopolysaccharide. In some embodiments the moiety of Ui is at least one amino acid of an antigenic polypeptide.
  • an antigen comprising an alkyne comprises a structure of formula VII or Vila:
  • X is independently at least one polyol of a polysaccharide; and n is at least 1.
  • a group e.g. X, Y or Ui
  • this can refer to a chemical attachment to a polyol within the polysaccharide (e.g. to a monosaccharide within the polysaccharide, which monosaccharide is a polyol).
  • the attachment itself can be to any suitable functional group (e.g. to an aldehyde, which may arise from oxidation of a vicinal diol).
  • an antigen comprising an alkyne comprises a structure of formula Vllb or Vile
  • X is independently an amine of at least one aminosugar of a polysaccharide; and n is at least 1.
  • an antigen comprising an alkyne comprises a polysaccharide according to (A-X)z-Y, wherein:
  • X is independently at least one polyol
  • an antigen comprises a polysaccharide which further comprises a DBCO group, with at least 1.5%, at least, 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20% (w/w) covalently attached DBCO.
  • the antigen comprises greater than about 1.5% (w/w) DBCO. In some embodiments, the antigen comprises greater than 3% (w/w) DBCO. In some embodiments the antigen comprises at most 20% at most 19%, at most 18%, at most 17%, at most 16%, at most 15%, at most 14%, at most 13%, at most 12%, at most 11%, at most 10%, at most 9%, at most 8%, at most 7%, at most 6%, at most 5%, at most 4%, at most 3.5% , at most 3.0%, at most 2.5%, at most 2.0%, or at most about 1.7% (w/w) covalently attached DBCO.
  • the antigen comprises less than 20% (w/w) covalently attached DBCO. In other embodiments the antigen comprises less than 10% (w/w) covalently attached DBCO. In some embodiments the antigen comprises between about 1.5 and 20%, 3% and 20%, 3% and 18%, 3% and 16%, 3% and 14%, 3% and 12%, 3% and 10%, 3% and 8%, 3% and 6%, or 3% and 4% , or 1.5 and 9% (w/w) covalently attached DBCO.
  • an antigen comprises a polysaccharide which further comprises a DBCO group comprises at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20% DBCO molecules per 100 polysaccharide repeating units.
  • the antigen comprises greater than 3% DBCO molecules per polysaccharide 100 repeating units.
  • the antigen comprises at most 20% at most 19%, at most 18%, at most 17%, at most 16%, at most 15%, at most 14%, at most 13%, at most 12%, at most 11%, at most 10%, at most 9%, at most 8%, at most 7%, at most 6%, at most 5%, at most 4%, or at most 3.5% covalently attached DBCO molecules per 100 polysaccharide repeating units. In some embodiments the antigen comprises less than 20% covalently attached DBCO per polysaccharide repeating unit. In other
  • the antigen comprises less than 10% covalently attached DBCO molecules per 100 polysaccharide repeating units. In some embodiments the antigen comprises between about 3% and 20%, 3% and 18%, 3% and 16%, 3% and 14%, 3% and 12%, 3% and 10%, 3% and 8%, 3% and 6%, or 3% and 4% covalently attached DBCO molecules per 100 polysaccharide repeating units.
  • the chemical handle comprises a moiety that allows for a“click” chemistry reaction with a corresponding group on nnAA residue of a polypeptide.
  • One such moiety is an azido group, which is capable of reacting with a nnAA residue comprising an alkyne group or a phosphine on a polypeptide.
  • an azido group on an antigen comprises a structure of formula VIII:
  • L22 is a bond, alkyl, or poly(alkyloxy), and Ui is independently at least one moiety of an antigen.
  • the chemical handle comprises a moiety that allows for a“click” chemistry reaction with a corresponding group on nnAA residue of a polypeptide.
  • a moiety is an alkene group, which is capable of reacting with a nnAA residue comprising an l,2,4,5-tetrazine group. In the simplest embodiments, this is a vinyl group.
  • an alkene group on an antigen comprises a structure of formula IX: wherein:
  • Ui is independently at least one moiety of an antigen.
  • an alkene group on an antigen comprises a structure of formula
  • L22 is Ci-10 alkyl or -(CH2CH20)I-IO-;
  • Ui is independently at least one moiety of an antigen.
  • a method for producing a glycoconjugate in which the chemical handle is an azide group, provided by 4-azidomethylphenylalanine (pAMF) as the nnAA comprises: (a) providing a nucleic acid encoding a carrier protein, wherein the nucleic acid comprises a suppression codon; (b) creating a reaction mixture by combining the nucleic acid with a cell-free bacterial extract comprising 4-azidomethylphenylalanine (pAMF), a tRNA complementary to the suppression codon, and an aminoacyl-tRNA synthetase; (c) incubating the reaction mixture of (b) under conditions sufficient to selectively incorporate pAMF at a site corresponding to the suppression codon in the carrier protein; and (d) conjugating the pAMF to a polysaccharide by a [2+3] cycloaddition.
  • the [2+3] cycloaddition comprises the reaction
  • the method additionally comprises purifying the carrier protein immediately after (c).
  • the suppression codon is selectively substituted at codon 25, 34,
  • the reaction mixture in (b) further comprises biological components necessary for protein synthesis.
  • the tRNA in (b) is capable of being charged with pAMF.
  • the aminoacyl-tRNA synthetase in (b) preferentially aminoacylates the tRNA with pAMF compared to the 20 natural amino acids.
  • the alkyne group comprises a DBCO moiety conjugated to the polysaccharide.
  • the polysaccharide is a capsular polysaccharide of Streptococcus pneumoniae , Neisseria
  • the polysaccharide is a capsular polysaccharide of a Streptococcus pneumoniae serotype selected from the group consisting of 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9 V, 9N,
  • the antigen is a capsular polysaccharide derived from one of the six serotypes of Porphyromonas gingivalis (e.g., Kl, K2, K3, K4, K5 and/or K6).
  • the disclosure provides a gly coconjugate prepared by a process comprising steps (a)-(d).
  • the pAMF is conjugated to the polysaccharide to generate a conjugate of formula X, Xa, XI, or XIa.
  • the disclosure provides for a vaccine comprising the glycoconjugate prepared by steps (a)-(d).
  • a conjugate of the invention can have a molecular weight of at least about 750 kDa, at least about 1,000 kDa, or at least about 1,500 kDa, or more. In some embodiments, the conjugate has a molecular weight of between about 750 kDa and about 5,000 kDa. In some embodiments, the conjugate has a molecular weight of between about 800 kDa and about 2,800 kDa. In some embodiments, the conjugate has a molecular weight of between about 850 kDa and about 2,800 kDa. In some embodiments, the conjugate has a molecular weight of between about 900 kDa and about 2,800 kDa.
  • the conjugate has a molecular weight of between about 950 kDa and about 2,800 kDa. In some embodiments, the conjugate has a molecular weight of between about 1,000 kDa and about 2,800 kDa.
  • the molecular weight of a conjugate is calculated by size exclusion chromatography (SEC) combined with multiangle laser light scattering (MALS).
  • Conjugates of the invention include antigen (e.g, saccharide) and carrier polypeptide, and the weight ratio of these two components can be used as a parameter to define the conjugate.
  • Antigen e.g, saccharide
  • carrier polypeptide e.g, poly(ethylene glycol)
  • the ratio is typically in the range 0.3-3.0, but this can vary with the serotype and aspects of the conjugation chemistry (Annex 2: Recommendations for the production and control of pneumococcal conjugate vaccines; WHO Technical Report Series, No. 927, 2005).
  • the ratio of the commercial vaccine Prevnar-l3TM is 0.9.
  • compositions which include conjugates of multiple pneumococcal serotypes e.g. more than 13 serotypes
  • the ratio for the complete composition is ideally above 1.0 (i.e. a weight excess of pneumococcal saccharide antigen) and is preferably 1.5 or more (e.g. within the range 1.5-3.0, or preferably 1.5-2.0).
  • Some embodiments of the invention involve the use of two or more different conjugates.
  • licensed meningococcal conjugate vaccines include capsular saccharides from 4 different serogroups
  • licensed pneumococcal conjugate vaccines include capsular saccharides from 7, 10, or 13 different serotypes.
  • a pharmaceutical composition including between 3 and 50 different conjugates ( e.g . 14, 15, 20, 21, 24, 25, or more).
  • compositions of the invention involve the use of two or more different conjugates e.g. within a single pharmaceutical composition. These embodiments are also referred to as multivalent. When any two conjugates are described as‘different’, or provide different valencies in a‘multivalent’ composition, this refers to a difference between the combination of carrier polypeptide and antigen in those two conjugates. For example, when a single type of modified CRM197 (e.g.
  • SEQ ID NOs: 1, 13, 14, etc. is conjugated to a capsular saccharide from a single serotype of pneumococcus, the reaction product will contain many different types of molecule (different molecular weights, different patterns of linkages within each molecule, etc.), but are considered as a single conjugate herein.
  • Those of skill in the art are familiar with this heterogeneity at the molecular level and similarly define individual conjugates of a vaccine by the antigen-carrier combination of the particular conjugate, with other properties (such as molecular weight) being an average within the conjugate composition.
  • Two‘different’ conjugates have a different carrier polypeptide (i.e. having a different amino acid sequence) and/or a different antigen (i.e. having a different antigenic structure).
  • capsular saccharide antigens may be purified from two different serotypes of pneumococcus. These two different capsular saccharides can be separately conjugated to a carrier polypeptide (which may be the same or different) to provide two different conjugates.
  • a carrier polypeptide which may be the same or different
  • the difference between two ‘different’ conjugates will typically be that one contains capsular saccharide from a first serotype or serogroup of a bacterial species whereas the other contains capsular saccharide from a second serotype or serogroup of that bacterial species e.g.
  • capsular saccharides from different serotypes of S.pneumoniae or capsular saccharides from different serogroups of N.meningitidis .
  • Two conjugates would also be‘different’ if they included antigenically distinct capsular saccharides from multiple bacterial species e.g. a Hib saccharide conjugate and a meningococcal saccharide conjugate.
  • Exemplary multivalent compositions of the invention include n different immunogenic saccharide conjugates, wherein the saccharide antigen in each of the n immunogenic conjugates is distinct from the saccharide antigen of the other n-1 immunogenic conjugates.
  • the composition includes antigens from a single bacterial species, there can be capsular saccharides from n different serotypes or n different serogroups of that species.
  • EP-A-2932979 refers to‘an immunogenic composition comprising 13 different polysaccharide- protein conjugates’.
  • the PCV7 PrevnarTM vaccine has 7 different conjugates
  • the PCV13 PrevnarTM vaccine has 13 different conjugates
  • the MenveoTM vaccine has 4 different conjugates
  • the MenactraTM vaccine has 4 different conjugates
  • the NimenrixTM vaccine has 4 different conjugates
  • the MenitorixTM vaccine has 2 different conjugates
  • the MenhibrixTM vaccine has 3 different conjugates
  • the SynflorixTM vaccine has 10 different conjugates, etc.
  • Multivalent compositions of pneumococcal conjugates preferably include more than 13 different conjugates e.g. 14, 15, 20, 21, 24, 25, or more. Suitable choices of serotypes for these >l3-valent compositions are discussed herein.
  • a first carrier polypeptide is conjugated to n-y different antigens (e.g, the capsular saacharides from different bacterial serotypes or serogroups) and a second polypeptide carrier is conjugated to the remaining y antigens.
  • n-y different antigens e.g, the capsular saacharides from different bacterial serotypes or serogroups
  • second polypeptide carrier is conjugated to the remaining y antigens.
  • three, four or more carriers could be used with the n antigens divided among them.
  • at least the first carrier is a nnAA-containing carrier polypeptide according to the present invention.
  • at least the first and second carriers are nnAA-containing carrier polypeptide s according to the present invention
  • a multivalent composition of the invention can include one or more immunogenic conjugates which include a carrier polypeptide and an antigen but do not include an immunostimulator.
  • a multivalent composition of the invention can comprise x immunogenic conjugates comprising a carrier polypeptide, an antigen, and an immunostimulator (as disclosed herein) andy immunogenic conjugates comprising a carrier polypeptide and an antigen but no immunostimulator, provided that x_l and x+y>2.
  • Conjugates of the invention include an intrinsic immunostimulatory, which means that it may be possible to use them without an extrinsic adjuvant.
  • a composition comprising a conjugate of the invention may be adjuvant-free.
  • an adjuvant may nevertheless be useful (e.g . to further enhance immunogenicity).
  • the inclusion of an immunostimulator can be used to enhance the immune response against specific antigens within a multivalent combination, beyond a general enhancement which is provided by an adjuvant.
  • a composition may thus include an adjuvant such as an oil-in-water emulsion adjuvant or an aluminum salt adjuvant.
  • Oil-in-water emulsion adjuvants may include squalene. Emulsions with droplets having a diameter less than 220nm are preferred as they can be subjected to filter sterilization. Two such approved squalene-containing adjuvants are‘AS03’ and‘MF59’.
  • Useful aluminum salt adjuvants include, but are not limited to, aluminum hydroxide adjuvants and aluminum phosphate adjuvants. These adjuvants are described e.g. in chapters 8 & 9 of Vaccine Design... (1995) eds. Powell & Newman. ISBN: 030644867X. Plenum.
  • the adjuvants commonly known as“aluminum hydroxide” are typically aluminum oxyhydroxide salts, which are usually at least partially crystalline.
  • Aluminum oxyhydroxide which can be represented by the formula AlO(OH)
  • AlO(OH) 3 can be distinguished from other aluminum compounds, such as Al(OH) 3 , by infrared (IR) spectroscopy, in particular by the presence of an adsorption band at l070cm _1 and a strong shoulder at 3090-3 lOOcm -1 (chapter 9 of Powell & Newman).
  • the degree of crystallinity of an aluminum hydroxide adjuvant is reflected by the width of the diffraction band at half height (WHH), with poorly-crystalline particles showing greater line broadening due to smaller crystallite sizes.
  • WHH half height
  • the surface area increases as WHH increases, and adjuvants with higher WHH values have been seen to have greater capacity for antigen adsorption.
  • a fibrous morphology e.g . as seen in transmission electron micrographs
  • the pi of aluminum hydroxide adjuvants is typically about 11 i.e. the adjuvant itself has a positive surface charge at physiological pH. Adsorptive capacities of between 1.8-2.6 mg protein per mg Al +++ at pH 7.4 have been reported for aluminum hydroxide adjuvants.
  • the adjuvants commonly known as“aluminum phosphate” are typically aluminum hydroxyphosphates, often also containing a small amount of sulfate (i.e. aluminum
  • hydroxyphosphate sulfate They may be obtained by precipitation, and the reaction conditions and concentrations during precipitation influence the degree of substitution of phosphate for hydroxyl in the salt.
  • Hydroxyphosphates generally have a PCri/Al molar ratio between 0.3 and 1.2. Hydroxyphosphates can be distinguished from strict AlPCri by the presence of hydroxyl groups. For example, an IR spectrum band at 3164cm 1 (e.g. when heated to 200°C) indicates the presence of structural hydroxyls (chapter 9 of Powell & Newman).
  • the P0 4 /Al 3+ molar ratio of an aluminum phosphate adjuvant will generally be between 0.3 and 1.2, preferably between 0.8 and 1.2, and more preferably 0.95+0.1.
  • the aluminum phosphate will generally be amorphous, particularly for hydroxyphosphate salts.
  • a typical adjuvant is amorphous aluminum hydroxyphosphate with PCri/Al molar ratio between 0.84 and 0.92, included at 0.6mg Al 3+ /ml.
  • the aluminum phosphate will generally be particulate (e.g. plate-like morphology as seen in transmission electron micrographs, with primary particles in the range of 50nm). Typical diameters of the particles are in the range 0.5-20pm (e.g. about 5-l0pm) after any antigen adsorption.
  • Adsorptive capacities of between 0.7-1.5 mg protein per mg Al +++ at pH 7.4 have been reported for aluminum phosphate adjuvants.
  • Aluminum phosphates used according to the invention will generally have a PZC of between 4.0 and 7.0, more preferably between 5.0 and 6.5 e.g. about 5.7.
  • a composition can include a mixture of both an aluminum hydroxide adjuvant and an aluminum phosphate adjuvant.
  • the concentration of aluminum ions in a composition for administration to a patient is preferably less than lOmg/ml e.g. ⁇ 5 mg/ml, ⁇ 4 mg/ml, ⁇ 3 mg/ml, ⁇ 2 mg/ml, ⁇ 1 mg/ml, etc.
  • a preferred maximum concentration is ⁇ 2.5mg/mL, and more preferably ⁇ l.7mg/mL.
  • the range of Al +++ in a composition of the invention can be between 0.3-lmg/ml or between 0.3-0.5mg/ml. A maximum of 0.85mg/dose is preferred.
  • Conjugates within the composition may be adsorbed to the aluminum salt adjuvant. Where a composition includes multiple conjugates and each of these is adsorbed to an aluminum salt adjuvant, each conjugate can be adsorbed to an aluminum salt individually and then mixed, or they can each be added in sequence to an aluminum salt, thereby forming the mixed conjugate composition. A mixture of both approaches can also be used.
  • compositions of the invention will generally include one or more pharmaceutically acceptable excipient(s).
  • excipients ed. Rowe el al. 6th edition 2009.
  • compositions are preferably in aqueous form, particularly at the point of administration, but they can also be presented in dried forms (e.g. as lyophili sates, etc.) which can be converted into aqueous forms for administration.
  • compositions can include a buffer or pH adjusting agent.
  • the buffer can be selected from the group consisting of a phosphate buffer, an acetate buffer, a histidine buffer, a citrate buffer, a succinate buffer, a Tris buffer, a HEPES buffer, etc. Buffer salts will typically be included in the 5-20mM range.
  • Pharmaceutical compositions can include a physiological salt, such as a sodium salt e.g. to control tonicity. Sodium chloride (NaCl) is typical, which may be present at between 1-20 mg/ml e.g. 10+2 mg/ml or 9 mg/ml.
  • salts that may be present include potassium chloride, potassium dihydrogen phosphate, disodium phosphate dehydrate, magnesium chloride, calcium chloride, etc.
  • Other useful salts may have sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions.
  • compositions can include an organic acid, such as acetic acid or succinic acid. This may be part of a buffer system.
  • compositions can include a sugar alcohol such as mannitol or sorbitol.
  • Pharmaceutical compositions can include a sugar such as sucrose or glucose.
  • compositions can include a surfactant.
  • Suitable surfactants include, but are not limited to, polysorbate 20, polysorbate 80, and sodium dodecyl sulfate (SDS).
  • the surface active agent is present at a concentration between 0.0003% and 0.3% (w/w) e.g. between 0.01-0.03%.
  • Polysorbate 80 is a preferred surfactant.
  • compositions can include a preservative such as thiomersal or
  • compositions containing no mercury are more preferred.
  • a preservative can be particularly useful when a composition includes an aluminum salt adjuvant because their insolubility means that the composition is generally a suspension having a cloudy appearance which can mask contaminating bacterial growth. In other embodiments, however, a pharmaceutical composition is preservative-free.
  • compositions can have an osmolality of between 200 mOsm/kg and 400 mOsm/kg, e.g. between 240-360 mOsm/kg, or between 290-310 mOsm/kg.
  • compositions typically have a pH between 5.0 and 9.5 e.g. between 6.0 and 8.0.
  • compositions are preferably non-pyrogenic e.g. containing ⁇ 1 EU (endotoxin unit, a standard measure) per dose, and preferably ⁇ 0.1 EU per dose.
  • Pharmaceutical compositions are preferably gluten free.
  • compositions are suitable for administration to animal (and, in particular, human) patients, and thus include both human and veterinary uses.
  • compositions may be prepared in unit dose form. In some embodiments,
  • a unit dose may have a volume of between 0.1-1.0ml e.g. about 0.25mL or preferably about 0.5ml. Such volumes are ideal for injection in humans.
  • An immunogenic conjugate can be administered to a mammalian subject to elicit a protective immune response against the antigen in that conjugate. They will be administered in the form of a pharmaceutical composition.
  • the composition can include multiple immunogenic conjugates as described elsewhere herein, so protective immune responses can be elicited against many antigens simultaneously.
  • conjugates as disclosed herein for use in eliciting a protective antibody response.
  • conjugates as disclosed herein in the manufacture of a medicament for eliciting a protective antibody response.
  • the protective immune response means that the conjugates can be used, for example, to provide active immunization for the prevention of invasive disease caused by S.pneumoniae , for the prevention of otitis media caused by S.pneumoniae, for the prevention of pneumonia caused by S.pneumoniae , for active immunization of subjects at risk of exposure to
  • compositions can be prepared in various forms.
  • the compositions may be prepared as injectables e.g. as liquid solutions or suspensions.
  • injectables for intramuscular administration are typical.
  • An injection volume of about 0.5ml is preferred for humans.
  • a preferred unit dose volume is about 0.5ml.
  • Administration by intramuscular injection is typical e.g. in anterolateral aspect of the thigh in infants, or the deltoid muscle of the upper arm in toddlers, children and adults.
  • Conjugates will typically be administered according to a multiple dose schedule.
  • Multiple doses may be used in a primary immunization schedule and/or in a booster
  • immunization schedule administration of more than one dose (typically two doses) is particularly useful in immunologically naive patients.
  • Multiple doses will typically be administered at least 1 week apart (e.g. about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, etc.).
  • composition“comprising” encompasses“including” as well as“consisting” e.g. a composition“comprising” X may consist exclusively of X or may include something additional e.g X + Y.
  • sequence identity in the context of two amino acid sequences refers to two sequences that are the same or have a specified percentage of amino acid residues that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using a sequence comparison algorithm (e.g., BLASTP). The percent identity is determined over the full-length reference sequence disclosed herein, such as the reference sequence set forth in SEQ ID NO: 1 or 2.
  • the method for calculating the sequence identity as provided herein is the BLASTP program having its defaults set at a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see, e.g., Henikoff & Henikoff, 1989, Proc Natl Acad Sci USA 89: 10915). See e.g., the BLAST alignment tool available on the WWW at blast.ncbi.nlm.nih.gov/Blast.cgi or elsewhere.
  • lower alkyl refers to a saturated straight or branched hydrocarbon having one to six carbon atoms, i.e., Ci to C6 alkyl.
  • the lower alkyl group is a primary, secondary, or tertiary hydrocarbon.
  • the term includes both substituted and unsubstituted moieties. See also US-2014/0066598.
  • the term“lower alkylene” refers to an alkylene radical of a lower alkyl.
  • Embodiment I Some embodiments of this disclosure relate to Embodiment I, as follows:
  • Embodiment 1-1 An immunogenic conjugate comprising a carrier polypeptide, an antigen, and an immunostimulator, wherein the carrier polypeptide includes at least one non natural amino acid residue via which the carrier polypeptide is covalently linked to the antigen and/or the immunostimulator.
  • Embodiment 1-2 The conjugate of embodiment 1-1, wherein the antigen is covalently linked to both a nnAA residue in the carrier polypeptide and an immunostimulator.
  • Embodiment 1-3 The conjugate of any preceding embodiment, wherein the carrier polypeptide includes multiple nnAA residues.
  • Embodiment 1-4 The conjugate of embodiment 1-3, wherein the carrier polypeptide comprises 4 to 9 nnAA residues.
  • Embodiment 1-5 The conjugate of any preceding embodiment, wherein: (i) the carrier polypeptide includes multiple non-natural amino acid residues which are covalently linked to the antigen; and (ii) the antigen is also covalently linked to the immunostimulator.
  • Embodiment 1-6 The conjugate of any preceding embodiment, wherein at least one nnAA is substituted for a lysine in the native carrier polypeptide.
  • Embodiment 1-7 The conjugate of any preceding embodiment, wherein the carrier polypeptide has at least 80% sequence identity to SEQ ID NO: 1.
  • Embodiment 1-8 The conjugate of embodiment 1-4, wherein at least one nnAA is substituted for K24, K33, K37, K39, K212, K214, K227, K244, K264, K385, K522 and K526 in SEQ ID NO: 1
  • Embodiment 1-9 The conjugate of any preceding embodiment, wherein the carrier protein comprises amino acid sequence SEQ ID NO: 4.
  • Embodiment I- 10 The conjugate of any preceding embodiment, wherein the at least one nnAA is a 2, 3 -di substituted propanoic acid bearing: an amino substituent at the 2-position; and an azido-containing substituent, a l,2,4,5-tetrazinyl substituent, or an ethynyl-containing substituent at the 3-position.
  • the at least one nnAA is a 2, 3 -di substituted propanoic acid bearing: an amino substituent at the 2-position; and an azido-containing substituent, a l,2,4,5-tetrazinyl substituent, or an ethynyl-containing substituent at the 3-position.
  • nnAA is selected from 2-amino-3-(4-azidophenyl)propanoic acid (pAF), 2-amino-3-(4- (azidomethyl)phenyl)propanoic acid (pAMF), 2-amino-3-(5-(azidomethyl)pyridin-2- yljpropanoic acid, 2-amino-3-(4-(azidomethyl)pyri din-2 -yljpropanoic acid, 2-amino-3-(6- (azidomethyl)pyridin-3-yl)propanoic acid, 2-amino-5-azidopentanoic acid, and 2-amino-3-(4- (azidomethyl)phenyl)propanoic acid, or any combination thereof.
  • pAF 2-amino-3-(4-azidophenyl)propanoic acid
  • pAMF 2-amino-3-(4-(azidomethyl)pheny
  • Embodiment 1-12 The conjugate of any preceding embodiment, wherein the antigen is a bacterial capsular saccharide; for example, a capsular saccharide from a bacterium selected from the group consisting of Streptococcus pneumoniae , Neisseria meningitidis , Haemophilus influenzae , Streptococcus pyogenes, Streptococcus agalactiae, and Porphyromonas gingivalis.
  • a bacterial capsular saccharide for example, a capsular saccharide from a bacterium selected from the group consisting of Streptococcus pneumoniae , Neisseria meningitidis , Haemophilus influenzae , Streptococcus pyogenes, Streptococcus agalactiae, and Porphyromonas gingivalis.
  • Embodiment 1-13 The conjugate of embodiment 1-12, wherein the antigen is a capsular saccharide of a S.pneumoniae serotype selected from the group consisting of 1, 2, 3, 4,
  • Embodiment 1-14 The conjugate of any preceding embodiment, wherein the ratio of saccharide to carrier protein (w/w) is greater than 1.
  • Embodiment 1-15 The conjugate of any preceding embodiment, wherein the immunostimulator is a TLR agonist such as an immunostimulatory oligonucleotide.
  • Embodiment 1-16 The conjugate of any preceding embodiment, wherein the polypeptide includes 3 or more nnAA residues and the conjugate has a molecular weight of at least 500kDa.
  • Embodiment 1-17 The conjugate of embodiment 1-16, wherein the polypeptide is a CRM197 containing 3-9 nnAA residues.
  • Embodiment 1-18 The conjugate of any preceding embodiment, wherein the conjugate is crosslinked through protein-antigen-protein linkages.
  • Embodiment 1-19 The conjugate of any preceding embodiment, with molecular weight between 900kDa and 5MDa.
  • Embodiment 1-20 A process for preparing an immunogenic conjugate comprising a carrier polypeptide, an antigen, and an immunostimulator, wherein the process comprises steps of: (i) providing an antigen having at least two instances of a first functional group, a carrier polypeptide having a second functional group, and an immunostimulator having a third functional group, wherein the first functional group can react to form covalent bonds with the second functional group and with the third functional group; and (ii) mixing the antigen, carrier polypeptide and immunostimulator, in any order, under conditions such that the first functional groups reacts with the second and third functional groups to give the immunogenic conjugate.
  • Embodiment 1-21 The process of embodiment 1-20, wherein the second and third functional groups are the same.
  • Embodiment 1-22 The process of embodiment 1-21, wherein the second and third functional groups are azido.
  • Embodiment 1-2 A pharmaceutical composition including one or more immunogenic conjugates according to any one of embodiments 1-1 to 1-19.
  • Embodiment 1-24 The composition of embodiment 1-23, wherein each immunogenic conjugate according to any of embodiments I- 1 to 1-18 includes a capsular saccharide from a different pneumococcal serotype.
  • Embodiment 1-25 The composition of embodiment 1-23 or 1-24, further including one or more immunogenic conjugates which include a carrier polypeptide and an antigen but do not include an immunostimulator.
  • Embodiment 1-26 The composition of any one of embodiments 1-23 to 1-25, comprising multiple conjugates according to embodiments 1-1 to 1-18, wherein each of the multiple conjugates comprises a different antigen.
  • Embodiment 1-27 The composition of embodiment 1-26, comprising: conjugates of capsular saccharides from 2 or more different pneumococcal serotypes selected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11 A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F; conjugates of capsular saccharides from 14 or more different pneumococcal serotypes selected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11 A, 12F, 13, 14, 15B, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 31, and 33F; conjugates of capsular saccharides from 15 or more different pneumococcal serotypes selected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B
  • Embodiment 1-28 A method of eliciting an immunoprotective antibody response to an antigen in a subject, comprising administering to the subject a conjugate according to any of embodiments 1-1 to 1-19 or a composition accoriding to any of embodiments 1-23 to 1-27, in an excipient suitable for parenteral administration.
  • Some embodiments of this disclosure relate to Embodiment II, as follows:
  • Embodiment II- 1 An immunogenic conjugate comprising a carrier polypeptide, an antigen, and an immunostimulator, wherein the carrier polypeptide includes at least one non natural amino acid residue via which the carrier polypeptide is covalently linked to the antigen and/or the immunostimulator.
  • Embodiment II-2 The immunogenic conjugate of embodiment II- 1, wherein the antigen has been site-specifically conjugated to a reactive group of nnAAs in the unconjugated carrier polypeptide through a chemical handle introduced into the antigen.
  • Embodiment II-3 The conjugate of embodiment II-1 or II-2, wherein the carrier polypeptide comprises (a) at nn sites a non-natural amino acid (nnAA) residue wherein each said nnAA residue (i) comprises a reactive group that provides site-specific conjugation of an antigen to the carrier polypeptide, and (ii) was introduced site-specifically during synthesis of the carrier polypeptide, wherein nn is an integer greater than or equal to 2; and (b) at least one T-cell activating epitope that has not been inactivated by the presence of an nnAA residue.
  • nnAA non-natural amino acid
  • Embodiment II-4 The conjugate of any one of embodiments II- 1 to II-3, wherein the antigen is covalently linked to both a nnAA residue in the carrier polypeptide and the immunostimulator.
  • Embodiment II-5 The conjugate of any preceding embodiment, wherein the carrier polypeptide comprises 4 to 9 nnAA residues.
  • Embodiment II-6 The conjugate of any preceding embodiment, wherein the antigen is a bacterial or viral antigen.
  • Embodiment II-7 The conjugate of embodiment II-5, wherein the antigen is a bacterial polysaccharide selected from the group consisting of capsular polysaccharides from Streptococcus pneumoniae , capsular polysaccharides from Streptococcus pyogenes , group-A- strep cell wall polysaccharides from Streptococcus pyogenes , apsular polysaccharides of Streptococcus agalactiae , capsular polysaccharides of Haemophilus influenzae , capsular polysaccharides of Neisseria meningitidis , and capsular polysaccharides from Porphyromonas gingivalis.
  • the antigen is a bacterial polysaccharide selected from the group consisting of capsular polysaccharides from Streptococcus pneumoniae , capsular polysaccharides from Streptococcus pyogenes , group-A
  • Embodiment II-8 The conjugate of embodiment II-6 wherein the antigen is a capsular polysaccharide of a Streptococcus pneumoniae serotype selected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7F, 7A, 7B, 7C, 8, 9A, 9L, 9N, 9V, 10F, 10A, 10B, 10C,
  • Embodiment II-9 The conjugate of any preceding embodiment, wherein the carrier polypeptide has at least 80% sequence identity to SEQ ID NO: 11.
  • Embodiment II- 10 The conjugate of any preceding embodiment, wherein at least one nnAA is substituted for a lysine in the native carrier protein.
  • Embodiment II- 11 The conjugate of any preceding embodiment, wherein the carrier polypeptide comprises amino acid sequence SEQ ID NO: 14.
  • Embodiment 11-12 The conjugate of any preceding embodiment, wherein the at least one nnAA is a 2, 3 -di substituted propanoic acid bearing: an amino substituent at the 2-position; and an azido-containing substituent, a l,2,4,5-tetrazinyl substituent, or an ethynyl-containing substituent at the 3-position.
  • the at least one nnAA is a 2, 3 -di substituted propanoic acid bearing: an amino substituent at the 2-position; and an azido-containing substituent, a l,2,4,5-tetrazinyl substituent, or an ethynyl-containing substituent at the 3-position.
  • Embodiment 11-13 The conjugate of any preceding embodiment, wherein the nnAA is selected from 2-amino-3-(4-azidophenyl)propanoic acid (pAF), 2-amino-3-(4- (azidomethyl)phenyl)propanoic acid (pAMF), 2-amino-3-(5-(azidomethyl)pyridin-2- yljpropanoic acid, 2-amino-3-(4-(azidomethyl)pyri din-2 -yljpropanoic acid, 2-amino-3-(6- (azidomethyl)pyridin-3-yl)propanoic acid, 2-amino-5-azidopentanoic acid, and 2-amino-3-(4- (azidomethyl)phenyl)propanoic acid, or any combination thereof.
  • pAF 2-amino-3-(4-azidophenyl)propanoic acid
  • pAMF
  • Embodiment 11-14 The conjugate of any preceding embodiment, wherein the ratio of antigen to carrier protein (w/w) is greater than 1.
  • Embodiment 11-15 The conjugate of any preceding embodiment, wherein the immunostimulator is a TLR agonist.
  • Embodiment 11-16 The conjugate of embodiment 11-14, wherein the TLR agonist comprises an immunostimulatory oligonucleotide.
  • Embodiment 11-17 The conjugate of any preceding embodiment, wherein the polypeptide includes 3 or more nnAA residues and the conjugate has a molecular weight of at least 500kDa.
  • Embodiment 11-18 The conjugate of any preceding embodiment, wherein the conjugate is crosslinked through protein-antigen-protein linkages.
  • Embodiment 11-19 The conjugate of any preceding embodiment, having a molecular weight between 900kDa and 5MDa.
  • Embodiment 11-20 The conjugate of embodiment II-4 wherein:
  • the carrier protein (i) comprises at least 80% sequence identity to SEQ ID NO: 1, (ii) does not contain an Arg-Arg dipeptide sequence, and at least one of the nnAAs is a 2,3- disubstituted propanoic acid bearing an azido-containing substituent;
  • nnAAs are 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid (pAMF) residues;
  • Embodiment 11-21 An immunogenic composition comprising at least two protein- antigen conjugates and at least one excipient suitable for parenteral administration wherein the at least two protein-antigen conjugates comprise multiple antigens and further wherein:
  • the antigen of each of the at least two protein-antigen conjugates is distinct from the antigens of the other protein-antigen conjugates;
  • At least one of the at least two proteinpolypeptide-antigen conjugates comprises is an immunogenic conjugate according to any one of embodiments II- 1 to 11-20.
  • polysaccharide(s) selected from the group consisting of capsular polysaccharides from
  • Streptococcus pneumoniae capsular polysaccharides from Streptococcus pyogenes , group-A- strep cell wall polysaccharides from Streptococcus pyogenes , apsular polysaccharides of Streptococcus agalactiae , capsular polysaccharides of Haemophilus influenzae , capsular polysaccharides of Neisseria meningitidis , and capsular polysaccharides from Porphyromonas gingivalis.
  • Embodiment 11-23 The immunogenic composition of embodiment 11-22, wherein the antigens are capsular polysaccharides from Streptococcus pneumoniae and comprise at least 4, 11, 14, 15, 16, 20, 21, or 24 protein-antigen conjugates.
  • Embodiment 11-24 The immunogenic composition of embodiment 11-23, wherein the at least 4, 11, 14, 15, 16, 20, 21, or 24 protein-antigen conjugates are i immunogenic conjugates.
  • Embodiment 11-25 The immunogenic composition of embodiment 11-24, wherein the antigens are capsular polysaccharides of a Streptococcus pneumoniae serotype selected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7F, 7A, 7B, 7C, 8, 9A, 9L, 9N, 9 V, 10F, 10A, 10B, 10C, 11F, 11 A, 11B, 11C, 11D, 12F, 12A, 12B, 13, 14, 15F, 15A, 15B, 15C, 16, 16F, 16 A, 17F, 17 A, 18F, 18 A, 18B, 18C, 19F, 19A, 19B, 19C, 20A, 20B, 21, 22F, 22A, 23F, 23 A, 23B, 24F, 24A, 24B, 25F, 25A, 27, 28F, 28A, 29, 31, 32F, 32A, 33F, 33 A, 33B, 33C, 33D, 34, 35
  • Embodiment 11-26 A method of eliciting an immunoprotective antibody response to an antigen in a subject, comprising administering to the subject an immunogenic composition according to any of embodiments 11-21 to 11-25.
  • Embodiment 11-27 A method of making an immunogenic conjugate according to any one of embodiments II-4 to 11-20, wherein the process comprises steps of:
  • Embodiment 11-28 The method of embodiment 11-27, wherein step (b) results in the immunostimulator being covalently linked to an antigen that is covalently linked to the carrier polypeptide.
  • Embodiment 11-29 The method of embodiment 11-28, wherein the antigens are (ii) viral antigen(s) or (ii) a bacterial polysaccharide(s) selected from the group consisting of capsular polysaccharides from Streptococcus pneumoniae , capsular polysaccharides from Streptococcus pyogenes , group-A-strep cell wall polysaccharides from Streptococcus pyogenes , apsular polysaccharides of Streptococcus agalactiae , capsular polysaccharides of Haemophilus influenzae , capsular polysaccharides of Neisseria meningitidis , and capsular polysaccharides from Porphyromonas gingivalis.
  • the antigens are (ii) viral antigen(s) or (ii) a bacterial polysaccharide(s) selected from the group consisting of capsular polysacc
  • Embodiment 11-30 The method of embodiment 11-29, wherein nn is selected from the group consisting of > 3, >4, 3-9, 4-8, and 4-6.
  • Embodiment II-31 The method of embodiment 11-30, wherein the carrier protein comprises a plurality plurality of T-cell activating epitopes are from a native protein carrier selected from the group consisting of Corynebacterium diphtheriae toxin, Clostridium tetani tetanospasmin, Haemophilus influenzae protein D, and CRM197.
  • a native protein carrier selected from the group consisting of Corynebacterium diphtheriae toxin, Clostridium tetani tetanospasmin, Haemophilus influenzae protein D, and CRM197.
  • Embodiment 11-32 The method of embodiment 11-31, wherein the reactive group comprises a bio-orthogonal reactive moiety.
  • WO2018/ 126229 describe in full detail the synthesis of single-site eCRM moieties K11TAG, K25TAG, K34TAG, K38TAG, K40TAG, K52TAG, K60TAG, K77TAG, K83TAG, K91TAG, K96TAG, and K103TAG. These were expressed in a cell-free protein synthesis (CFPS) extract, and pAMF was incorporated in place of natural Lys.
  • CFPS cell-free protein synthesis
  • WO2018/126229 further describe general protocols for saccharide activation with sodium meta-periodate, for periodate-oxidized polysaccharide derivatization with DBCO, for saccharide activation with CDAP, and for conjugation of saccharide-DBCO with eCRM.
  • Pneumococcal serotype 3 (PS3) saccharide was used as a test example, together with SEQ ID NO: 14 as the carrier polypeptide.
  • a starting conjugate was formed using the carrier and PS3 saccharide with no added CpG DNA, and then the azido-DNA was added in steps. Progress of the conjugation was followed by absorbance at 309nm (DBCO-NEb absorbs at this wavelength, but this signal is lost after conjugation to azido).
  • polysaccharide binding antibody IgG was measured according to the methods described in Yu et al. (2011), "Development of an Automated and Multiplexed Serotyping Assay for
  • Binding epitope for human CD4+ cells on BB (from G protein of Strep G148 at AA 25-40):
  • Binding epitope for human CD4+ cells on BB (from G protein of Strep G148 at AA 63-78):
  • Binding epitope for human CD4+ cells on BB (from G protein of Strep G148 at AA 74-89):
  • CRM 197 with 6 preferred nnAA sites ("X") and N-terminus Met:

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Abstract

La présente invention concerne diverses améliorations concernant des conjugués immunogènes qui comprennent une protéine porteuse, un antigène saccharide et un immunostimulateur, l'antigène saccharide ayant été conjugué en un site spécifique à un groupe réactif d'acides aminés non naturels dans une protéine porteuse non conjuguée par l'intermédiaire d'une poignée chimique introduite dans l'antigène.
PCT/US2019/040184 2018-07-04 2019-07-01 Conjugués immunogènes auto-adjuvés WO2020010016A1 (fr)

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