HK1210021B

HK1210021B - Glycoconjugation processes and compositions

Info

Publication number: HK1210021B
Application number: HK15110760.0A
Authority: HK
Inventors: J.谷; J-H．金; A．K．普拉萨德; Y-Y．杨
Original assignee: 辉瑞公司
Priority date: 2012-08-16
Filing date: 2013-08-12
Publication date: 2018-09-07

Description

Glycoconjugation methods and compositions

Technical Field

The present invention relates generally to glycoconjugates comprising a saccharide covalently conjugated to a carrier protein through a (2- ((2-oxoethyl) thio) ethyl) carbamate (eTEC) spacer, immunogenic compositions comprising such glycoconjugates, and methods of making and using such glycoconjugates and immunogenic compositions.

Background

Methods to increase the immunogenicity of weakly immunogenic molecules by conjugating these molecules to "carrier" molecules have been used successfully for decades (see, e.g., Goebel et al (1939) j.exp.med.69: 53). For example, many immunogenic compositions have been described in which purified capsular polymers have been conjugated to carrier proteins to produce more effective immunogenic compositions by exploiting this "carrier effect". Schnerson et al (1984) feed. Immun.45: 582-591). Conjugation has also been shown to circumvent the weak antibody response typically observed in infants when immunized with free polysaccharide (Anderson et al (1985) J.Peditar.107: 346; Insel et al (1986) J.exp.Med.158: 294).

Conjugates have been successfully generated using a variety of crosslinking or coupling reagents, such as homobifunctional (homobifunctional) crosslinkers, heterobifunctional (heterobifunctional) crosslinkers, or zero-length (zero-length) crosslinkers. A number of methods are currently available for coupling immunogenic molecules (e.g., sugars, proteins, and peptides) to peptides or protein carriers. Most processes produce amines, amides, polyurethanes, isothioureas, or disulfide bonds, or in some cases thioethers. A disadvantage of using a cross-linking or coupling reagent that introduces reactive sites into the side chains of the reactive amino acid molecules on the carrier and/or immunogenic molecule is that the reactive sites, if not neutralized, are free to react with any undesired molecules, either in vitro (thereby possibly adversely affecting the functionality or stability of the conjugate) or in vivo (thereby constituting a risk of potential adverse events in humans or animals immunized with the formulation). Such excess reactive sites can be reacted or "capped" using a variety of known chemical reactions to inactivate such sites, but such reactions may otherwise disrupt the functionality of the conjugate. This can be particularly problematic when attempting to create conjugates by introducing reactive sites into the carrier molecule, as the large size and more complex structure of the conjugate (relative to the immunogenic molecule) can make it more susceptible to the destructive effects of chemical treatment. Thus, there remains a need for new methods of preparing appropriately capped carrier protein conjugates such that the functionality of the carrier is maintained and the conjugate retains the ability to elicit a desired immune response.

Disclosure of Invention

The present invention relates to a method of preparing a glycoconjugate comprising a saccharide covalently conjugated to a carrier protein via a heterobifunctional bivalent linker, referred to herein as a (2- ((2-oxoethyl) thio) ethyl) carbamate (eTEC) spacer. The eTEC spacer comprises 7 linear atoms (i.e., -C (O) NH (CH)₂)₂SCH₂C (O) -), and provides stable thioether and amide linkages between the saccharide and the carrier protein. The invention also provides eTEC-linked glycoconjugates, immunogenic compositions comprising them, and methods of using such glycoconjugates and immunogenic compositions.

In one aspect, the present invention provides a glycoconjugate comprising a saccharide conjugated to a carrier protein through an eTEC spacer, wherein the saccharide is covalently attached to the eTEC spacer through a carbamate linkage, and wherein the carrier protein is covalently attached to the eTEC spacer through an amide linkage.

In some embodiments, the saccharide is a polysaccharide, such as a capsular polysaccharide derived from a bacterium, particularly from a pathogenic bacterium. In other embodiments, the saccharide is an oligosaccharide or a monosaccharide.

The eTEC-linked glycoconjugates of the invention can be represented by the general formula (I):

wherein the atoms comprising the eTEC spacer are contained in a central box.

The carrier protein incorporated in the glycoconjugates of the invention is selected from carrier proteins generally suitable for such purposes as further described herein or known to those skilled in the art. In a specific embodiment, the carrier protein is CRM₁₉₇。

in another aspect, the present invention provides a method of preparing a glycoconjugate comprising a saccharide conjugated to a carrier protein through an eTEC spacer, comprising the steps of a) reacting a saccharide with a carbonic acid derivative in an organic solvent to produce an activated saccharide, b) reacting the activated saccharide with cystamine or cysteamine or a salt thereof to produce a thiolated saccharide, c) reacting the thiolated saccharide with a reducing agent to produce an activated thiolated saccharide comprising one or more free thiol residues, d) reacting the activated thiolated saccharide with an activated carrier protein comprising one or more α -haloacetamide groups to produce a thiolated saccharide-carrier protein conjugate, and e) reacting the thiolated saccharide-carrier protein conjugate with (i) a first capping reagent capable of capping the uncapped α -haloacetamide groups of the activated carrier protein, and/or (ii) a second capping reagent capable of capping the uncapped free thiol residues of the activated thiolated saccharide, thereby producing an eTEC conjugate.

In a common embodiment, the carbonic acid derivative is 1,1 '-carbonyl-bis- (1,2, 4-triazole) (CDT) or 1, 1' -Carbonyldiimidazole (CDI). Preferably, the carbonic acid derivative is CDT and the organic solvent is a polar aprotic solvent, such as dimethyl sulfoxide (DMSO). In a preferred embodiment, the thiolated saccharide is produced by reacting an activated saccharide with a bifunctional symmetric thioalkylamine reagent, cystamine, or a salt thereof. Alternatively, the thiolated saccharide may be formed by reacting an activated saccharide with cysteamine or a salt thereof. The eTEC-linked glycoconjugates produced by the methods of the invention can be represented by general formula (I).

in a common embodiment, the first capping reagent is N-acetyl-L-cysteine that reacts with an unconjugated α -haloacetamide group on a lysine residue of the carrier protein to form an S-carboxymethylcysteine (CMC) residue that is covalently attached to the activated lysine residue through a thioether linkage.

In some embodiments, capping step e) further comprises reaction with a reducing agent such as DTT, TCEP, or mercaptoethanol after reaction with the first and/or second capping reagents.

in some embodiments, step d) further comprises providing an activated carrier protein comprising one or more α -haloacetamide groups, and then reacting the activated thiolated saccharide with the activated carrier protein.

In another aspect, the invention provides an eTEC-linked glycoconjugate comprising a saccharide conjugated to a carrier protein through an eTEC spacer produced according to any method disclosed herein.

For the various aspects of the present invention, in particular embodiments of the methods and compositions described herein, the eTEC-linked glycoconjugate comprises a saccharide that is a bacterial capsular polysaccharide, particularly a capsular polysaccharide derived from a pathogenic bacterium.

In some such embodiments, the eTEC-linked glycoconjugate comprises a pneumococcal (Pn) capsular polysaccharide derived from Streptococcus pneumoniae (Streptococcus pneumoniae). In a particular embodiment, the Pn capsular polysaccharide is selected from Pn-serotype 1,3, 4,5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F capsular polysaccharides.

In other such embodiments, the eTEC-linked glycoconjugate comprises a meningococcal (Mn) capsular polysaccharide derived from Neisseria meningitidis (Neisseria meningitidis). In particular embodiments, the Mn capsular polysaccharide is selected from Mn-serotype A, C, W135 and Y capsular polysaccharides.

In a particularly preferred embodiment, the saccharide is a bacterial capsular polysaccharide, covalently conjugated to CRM, for example via an eTEC spacer₁₉₇Pn or Mn capsular polysaccharides of (a).

The compositions and methods described herein are useful in a variety of applications. For example, the glycoconjugates of the invention can be used to produce immunogenic compositions comprising eTEC-linked glycoconjugates. Such immunogenic compositions are useful for protecting a subject against bacterial infection, for example, bacterial infection caused by pathogenic bacteria such as streptococcus pneumoniae or neisseria meningitidis.

Thus, in another aspect, the invention provides an immunogenic composition comprising an eTEC-linked glycoconjugate and a pharmaceutically acceptable excipient, carrier or diluent, wherein the glycoconjugate comprises a saccharide covalently conjugated to a carrier protein through an eTEC spacer as described herein.

In a common embodiment, the immunogenic composition comprises an eTEC-linked glycoconjugate, wherein the glycoconjugate comprises a bacterial capsular polysaccharide, and a pharmaceutically acceptable excipient, carrier or diluent.

In some such embodiments, the immunogenic composition comprises an eTEC-linked glycoconjugate comprising a Pn capsular polysaccharide derived from streptococcus pneumoniae. In some particular embodiments, the Pn capsular polysaccharide is selected from Pn-serotype 1,3, 4,5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F capsular polysaccharides.

In other such embodiments, the immunogenic composition comprises an eTEC-linked glycoconjugate comprising a Mn capsular polysaccharide derived from neisseria meningitidis. In some particular embodiments, the Mn capsular polysaccharide is selected from Mn-serotype A, C, W135 and Y capsular polysaccharide.

In a preferred embodiment, the immunogenic composition comprises an eTEC-linked glycoconjugate comprising a covalent conjugation to CRM through an eTEC spacer₁₉₇A bacterial capsular polysaccharide (e.g. a Pn or Mn capsular polysaccharide).

In some embodiments, the immunogenic composition comprises an adjuvant. In some such embodiments, the adjuvant is an aluminum-based adjuvant selected from the group consisting of aluminum phosphate, aluminum sulfate, and aluminum hydroxide. In one embodiment, the immunogenic composition described herein comprises an aluminum phosphate adjuvant.

In another aspect, the invention provides a method of preventing, treating or ameliorating a bacterial infection, disease or disorder in a subject, comprising administering to the subject an immunologically effective amount of an immunogenic composition of the invention, wherein the immunogenic composition comprises an eTEC-linked glycoconjugate comprising a bacterial antigen (e.g., a bacterial capsular polysaccharide).

In one embodiment, the infection, disease or disorder is associated with streptococcus pneumoniae bacteria and the glycoconjugate comprises a Pn capsular polysaccharide. In another embodiment, the infection, disease or disorder is associated with neisserial bacteria and the glycoconjugate comprises a Mn capsular polysaccharide.

In other aspects, the invention provides methods of inducing an immune response against a pathogenic bacterium; a method for preventing, treating or ameliorating a disease or disorder caused by a pathogenic bacterium; and reducing the severity of at least one symptom of an infection, disease or disorder caused by the pathogenic bacteria, in each case by administering to the individual an immunologically effective amount of an immunogenic composition comprising an eTEC-linked glycoconjugate comprising a bacterial antigen, such as a bacterial capsular polysaccharide derived from the pathogenic bacteria, and a pharmaceutically acceptable excipient, carrier or diluent.

In another aspect, the present invention provides a method of inducing an immune response in an individual comprising administering to the individual an immunologically effective amount of an immunogenic composition comprising an eTEC-linked glycoconjugate, wherein the glycoconjugate comprises a bacterial antigen, such as a bacterial capsular polysaccharide, and a pharmaceutically acceptable excipient, carrier or diluent. In preferred embodiments, the methods relate to generating a protective immune response in an individual, as further described herein.

In another aspect, the invention provides methods of administering to a subject an immunologically effective amount of an immunogenic composition comprising an eTEC-linked glycoconjugate to generate a protective immune response in the subject, as further described herein.

In yet another aspect, the invention provides antibodies generated in response to the eTEC-linked glycoconjugates of the invention, or immunogenic compositions comprising such glycoconjugates. Such antibodies can be used in research and clinical laboratory assays, such as bacterial detection and serotyping, or can be used to confer passive immunity to an individual.

In a further aspect, the present invention provides an immunogenic composition comprising the eTEC-linked glycoconjugate of the invention for use in the prevention, treatment or amelioration of a bacterial infection, for example an infection caused by streptococcus pneumoniae or neisseria meningitidis.

In another aspect, the invention provides the use of an immunogenic composition comprising the eTEC-linked glycoconjugate of the invention in the manufacture of a medicament for the prevention, treatment or amelioration of a bacterial infection (e.g. an infection caused by streptococcus pneumoniae or neisseria meningitidis).

In certain preferred embodiments of the above therapeutic and/or prophylactic methods and uses, the immunogenic composition comprises an eTEC-linked glycoconjugate comprising a bacterial capsular polysaccharide covalently linked to a carrier protein through an eTEC spacer. In a common embodiment of the methods and uses described herein, the bacterial capsular polysaccharide is a Pn capsular polysaccharide or a Mn capsular polysaccharide. In some such embodiments, the Pn capsular polysaccharide is selected from Pn-serotype 1,3, 4,5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F capsular polysaccharides. In other such embodiments, the Mn capsular polysaccharide is selected from Mn-serotype A, C, W135 and Y capsular polysaccharides.

In certain preferred embodiments, the carrier protein is CRM₁₉₇. In a particularly preferred embodiment, the immunogenic composition comprises an eTEC-linked glycoconjugate comprising covalent conjugation to CRM through an eTEC spacer₁₉₇A bacterial capsular polysaccharide (e.g. a Pn or Mn capsular polysaccharide).

Drawings

FIG. 1 shows an eTEC-linked glycoconjugate (e.g., comprising covalent conjugation to CRM) useful for preparing an eTEC-linked glycoconjugate of the invention₁₉₇Glycoconjugates of polysaccharides of (ii) are used.

FIG. 2 shows the repeating polysaccharide structure of Streptococcus pneumoniae serotype 33F (Pn-33F) capsular polysaccharide.

FIG. 3 shows the repeating polysaccharide structure of Streptococcus pneumoniae serotype 22F (Pn-22F) capsular polysaccharide.

FIG. 4 shows the repeating polysaccharide structure of Streptococcus pneumoniae serotype 10A (Pn-10A) capsular polysaccharide.

FIG. 5 shows the repeating polysaccharide structure of Streptococcus pneumoniae serotype 11A (Pn-11A) capsular polysaccharide.

FIG. 6 shows representative structures of Pn-33F glycoconjugates incorporating eTEC linker (A) and potentially capped and uncapped free thiol sites (B).

FIG. 7 shows preparation of conjugates to CRM₁₉₇Representative process flow diagrams of the activation method (A) and conjugation method (B) for Pn-33F glycoconjugates of (A).

Figure 8 shows the thiolation level of Pn-33F capsular polysaccharide as a function of molar equivalents of CDT used for the activation step.

Detailed Description

The present invention may be understood more readily by reference to the following detailed description of preferred embodiments of the invention and the examples included herein. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although certain preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. In describing embodiments and claiming the present invention, certain terminology will be used in accordance with the definitions set out below.

As used herein, the singular forms "a," "an," and "the" include plural references unless otherwise specified. Thus, for example, reference to "the method" includes one or more methods and/or one or more steps of the type described herein, and reference to "the eTEC spacer" is a reference to one or more eTEC spacers, as will be understood by those of skill in the art upon reading this disclosure.

The term "about" as used herein means within a statistically significant range of a value, such as the concentration range, time range, molecular weight, temperature, or pH. Such ranges may be within an order of magnitude of the indicated value or range, typically within 20%, more typically within 10%, even more typically within 5%. Sometimes, such ranges can be within the typical experimental error of standard methods for measuring and/or determining a given value or range. The allowable variations encompassed by the term "about" will depend on the particular system under study and can be readily understood by one skilled in the art. Whenever a range is recited within this application, every integer within the range is also contemplated as an embodiment of the invention.

It should be noted that in this disclosure, terms such as "comprising," "including," and the like can have the meaning attributed to them in U.S. patent law; for example, they may mean "including" and the like. Such terms are meant to encompass a particular ingredient or group of ingredients without excluding any other ingredient. Terms such as "consisting essentially of … …" have the meaning ascribed to them in U.S. patent law, for example, they allow the inclusion of other ingredients or steps that do not detract from the novel or essential features of the invention, i.e. they exclude other ingredients or steps not listed which do not detract from the novel or essential features of the invention. The term "consisting of … …" has the meaning ascribed to them in U.S. patent law; that is, these terms are closed. Thus, these terms are meant to include a particular ingredient or group of ingredients and to exclude all other ingredients.

The term "saccharide" as used herein may refer to a polysaccharide, an oligosaccharide or a monosaccharide. Generally, saccharides refer to bacterial capsular polysaccharides, particularly capsular polysaccharides derived from pathogenic bacteria such as streptococcus pneumoniae or neisseria meningitidis.

The terms "conjugate" or "glycoconjugate" are used interchangeably herein and refer to a saccharide covalently conjugated to a carrier protein. Glycoconjugates of the invention, sometimes referred to herein as "eTEC-linked" glycoconjugates, comprise a saccharide covalently conjugated to a carrier protein through at least one eTEC spacer. The eTEC-linked glycoconjugates of the invention and immunogenic compositions comprising them may contain an amount of free saccharide.

The term "free saccharide" as used herein means either a saccharide that is not covalently conjugated to a carrier protein but is still present in the glycoconjugate composition or a saccharide that is covalently linked to very few carrier proteins (linked at a high saccharide to protein ratio (>5: 1)) but is still present in the glycoconjugate composition. The free saccharide may be non-covalently associated with (i.e., non-covalently bound to, adsorbed to, or entrapped in or entrapped with) the conjugated saccharide-carrier protein glycoconjugate. The terms "free polysaccharide" and "free capsular polysaccharide" may be used herein to convey the same meaning with respect to glycoconjugates, wherein the saccharide is a polysaccharide or a capsular polysaccharide, respectively.

"conjugation" as used herein refers to a process whereby a saccharide, such as a bacterial capsular polysaccharide, is covalently attached to a carrier molecule or carrier protein. In the methods of the invention, the saccharide is covalently conjugated to the carrier protein through at least one eTEC spacer. Conjugation can be performed according to the methods described below or by other methods known in the art. Conjugation to a carrier protein enhances the immunogenicity of the bacterial capsular polysaccharide.

Glycoconjugates

The present invention relates to glycoconjugates comprising a saccharide covalently conjugated to a carrier protein through one or more eTEC spacers, wherein the saccharide is covalently conjugated to the eTEC spacers through a carbamate linkage, and wherein the carrier protein is covalently conjugated to the eTEC spacers through an amide linkage.

Novel features of glycoconjugates of the invention include, in addition to the presence of one or more eTEC spacers, the molecular weight distribution (profile) of the saccharide and the resulting eTEC-linked glycoconjugate, the ratio of lysine conjugated per carrier protein to the number of lysines covalently linked to the polysaccharide through the one or more eTEC spacers, the number of covalent bonds between the carrier protein and the saccharide as a function of repeat units of the saccharide, and the relative amount of free saccharide to the total amount of saccharide.

the eTEC spacer comprises 7 linear atoms (i.e., -C (O) NH (CH)₂)₂SCH₂the synthesis of eTEC-linked saccharide conjugates includes reacting an activated hydroxyl group of a saccharide with an amino group of a thioalkylamine reagent (e.g., cystamine or cysteamine or a salt thereof) to form a carbamate bond linked to the saccharide to provide a thiolated saccharide.

In the glycoconjugates of the invention, the saccharide may be a polysaccharide, an oligosaccharide or a monosaccharide, and the carrier protein may be selected from any suitable carrier as further described herein or known to those of skill in the art. In a common embodiment, the saccharide is a bacterial capsular polysaccharide. In some such embodiments, the carrier protein is CRM₁₉₇。

In some such embodiments, the eTEC-linked glycoconjugate comprises a Pn capsular polysaccharide derived from streptococcus pneumoniae. In a particular embodiment, the Pn capsular polysaccharide is selected from Pn-serotype 1,3, 4,5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F capsular polysaccharides. In other embodiments, the capsular polysaccharide is selected from Pn-serotypes 10A, 11A, 22F, and 33F capsular polysaccharides. In one such embodiment, the capsular polysaccharide is a Pn-33F capsular polysaccharide. In another such embodiment, the capsular polysaccharide is a Pn-22F capsular polysaccharide. In another such embodiment, the capsular polysaccharide is a Pn-10A capsular polysaccharide. In yet another such embodiment, the capsular polysaccharide is a Pn-11A capsular polysaccharide.

In other embodiments, the eTEC-linked glycoconjugate comprises a Mn capsular polysaccharide derived from neisseria meningitidis. In particular embodiments, the Mn capsular polysaccharide is selected from Mn-serotype A, C, W135 and Y capsular polysaccharides. In one such embodiment, the capsular polysaccharide is a Mn-A capsular polysaccharide. In another such embodiment, the capsular polysaccharide is a Mn-C capsular polysaccharide. In another such embodiment, the capsular polysaccharide is a Mn-W135 capsular polysaccharide. In yet another such embodiment, the capsular polysaccharide is a Mn-Y capsular polysaccharide.

In particularly preferred embodiments, the eTEC-linked glycoconjugate comprises covalent conjugation to CRM through an eTEC spacer₁₉₇A bacterial capsular polysaccharide of Pn or Mn, for example a Pn-serotype 1,3, 4,5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F or 33F capsular polysaccharide, or a Mn-serotype A, C, W135 or Y capsular polysaccharide.

In some embodiments, the eTEC-linked glycoconjugates of the invention comprise a saccharide covalently conjugated to a carrier protein through an eTEC spacer, wherein the saccharide has a molecular weight of 10kDa to 2,000 kDa. In other such embodiments, the saccharide has a molecular weight of 50kDa to 2,000 kDa. In other such embodiments, the saccharide has a molecular weight of 50kDa to 1,750 kDa; 50kDa to 1,500 kDa; 50kDa to 1,250 kDa; 50kDa to 1,000 kDa; 50kDa-750 kDa; 50kDa-500 kDa; 100kDa to 2,000 kDa; 100kDa to 1,750 kDa; 100kDa to 1,500 kDa; 100kDa to 1,250 kDa; 100kDa to 1,000 kDa; 100kDa-750 kDa; 100kDa-500 kDa; 200kDa to 2,000 kDa; 200kDa to 1,750 kDa; 200kDa to 1,500 kDa; 200kDa to 1,250 kDa; 200kDa to 1,000 kDa; 200kDa-750 kDa; or a molecular weight of 200kDa to 500 kDa. In some such embodiments, the saccharide is a bacterial capsular polysaccharide, for example a Pn-serotype 1,3, 4,5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, or 33F capsular polysaccharide, or a Mn-serotype A, C, W135 or Y capsular polysaccharide, wherein the capsular polysaccharide has a molecular weight that falls within any of the molecular weight ranges described.

In some embodiments, the eTEC-linked glycoconjugates of the invention have a molecular weight of 50kDa to 20,000 kDa. In other embodiments, the eTEC-linked glycoconjugate has a molecular weight of 500kDa to 10,000 kDa. In other embodiments, the eTEC-linked glycoconjugate has a molecular weight of 200kDa to 10,000 kDa. In other embodiments, the eTEC-linked glycoconjugate has a molecular weight of 1,000kDa to 3,000 kDa.

In other embodiments, the eTEC-linked glycoconjugates of the invention have a molecular weight of 200kDa to 20,000 kDa; 200kDa to 15,000 kDa; 200kDa to 10,000 kDa; 200kDa to 7,500 kDa; 200kDa to 5,000 kDa; 200kDa to 3,000 kDa; 200kDa to 1,000 kDa; 500kDa-20,000 kDa; 500kDa to 15,000 kDa; 500kDa to 12,500 kDa; 500kDa to 10,000 kDa; 500kDa to 7,500 kDa; 500kDa to 6,000 kDa; 500kDa to 5,000 kDa; 500kDa to 4,000 kDa; 500kDa to 3,000 kDa; 500kDa to 2,000 kDa; 500kDa to 1,500 kDa; 500kDa to 1,000 kDa; 750kDa to 20,000 kDa; 750kDa to 15,000 kDa; 750kDa to 12,500 kDa; 750kDa to 10,000 kDa; 750kDa to 7,500 kDa; 750kDa to 6,000 kDa; 750kDa to 5,000 kDa; 750kDa to 4,000 kDa; 750kDa to 3,000 kDa; 750kDa to 2,000 kDa; 750kDa to 1,500 kDa; 1,000kDa to 15,000 kDa; 1,000kDa to 12,500 kDa; 1,000kDa to 10,000 kDa; 1,000kDa to 7,500 kDa; 1,000kDa to 6,000 kDa; 1,000kDa to 5,000 kDa; 1,000kDa to 4,000 kDa; 1,000kDa to 2,500 kDa; 2,000kDa to 15,000 kDa; 2,000kDa to 12,500 kDa; 2,000kDa to 10,000 kDa; 2,000kDa to 7,500 kDa; 2,000kDa to 6,000 kDa; 2,000kDa to 5,000 kDa; 2,000kDa to 4,000 kDa; or a molecular weight of 2,000kDa to 3,000 kDa.

Another way to characterize the eTEC-linked glycoconjugates of the invention is via the number of lysine residues in the carrier protein that become conjugated to the saccharide through the eTEC spacer, which can be characterized as a range of conjugated lysines.

In a common embodiment, the carrier protein is covalently conjugated to the eTEC spacer through an amide bond to one or more epsilon-amino groups of lysine residues on the carrier protein. In some such embodiments, the carrier protein comprises 2-20 lysine residues covalently conjugated to a saccharide. In other such embodiments, the carrier protein comprises 4-16 lysine residues covalently conjugated to a saccharide.

In a preferred embodiment, the carrier protein comprises a polypeptide comprisingCRM with 39 lysine residues₁₉₇. In some such embodiments, the CRM₁₉₇The lysine residues may comprise 4-16 lysine residues covalently linked to the sugar among the 39 lysine residues. Another way to express this parameter is about 10% to about 41% CRM₁₉₇Lysine is covalently linked to the sugar. In another such embodiment, the CRM₁₉₇2-20 lysine residues covalently linked to the sugar may be included among the 39 lysine residues. Another way to express this parameter is about 5% to about 50% CRM₁₉₇Lysine is covalently linked to the sugar.

The eTEC-linked glycoconjugates of the invention can also be characterized by the ratio of saccharide to carrier protein (weight/weight). In some embodiments, the ratio of sugar to carrier protein (w/w) is 0.2 to 4. In other embodiments, the ratio of sugar to carrier protein (w/w) is 1.0 to 2.5. In other embodiments, the ratio of sugar to carrier protein (w/w) is 0.4 to 1.7. In some such embodiments, the saccharide is a bacterial capsular polysaccharide, and/or the carrier protein is CRM₁₉₇。

Glycoconjugates can also be characterized by the number of covalent bonds between the carrier protein and the saccharide as a function of the repeat unit of the saccharide. In one embodiment, the glycoconjugate of the invention comprises at least one covalent bond between the carrier protein and the polysaccharide for every 4 saccharide repeat units of the polysaccharide. In another embodiment, the covalent bonding between the carrier protein and the polysaccharide occurs at least once in every 10 saccharide repeat units of the polysaccharide. In another embodiment, the covalent bonding between the carrier protein and the polysaccharide occurs at least once in every 15 saccharide repeat units of the polysaccharide. In yet another embodiment, the covalent bonding between the carrier protein and the polysaccharide occurs at least once in every 25 saccharide repeat units of the polysaccharide.

In a common embodiment, the carrier protein is CRM₁₉₇And at least one pass through CRM occurs in every 4, 10, 15, or 25 saccharide repeat units of the polysaccharide₁₉₇Covalent bonding with the eTEC spacer between the polysaccharides.

An important consideration during conjugation is the development of conditions that allow the retention of potentially sensitive non-saccharide substituent functional groups of the individual components (e.g. O-acyl, phosphate or phosphoglyceride side chains that may form part of the saccharide epitope).

In one embodiment, the glycoconjugate comprises a saccharide having a degree of O-acetylation of 10-100%. In some such embodiments, the saccharide has a degree of O-acetylation of 50-100%. In other such embodiments, the saccharide has a degree of O-acetylation of 75-100%. In other embodiments, the saccharide has a degree of O-acetylation of greater than or equal to 70% (. gtoreq.70%).

The eTEC-linked glycoconjugates and immunogenic compositions of the invention can contain free saccharide that is not covalently conjugated to a carrier protein, but is still present in the glycoconjugate composition. The free saccharide can be non-covalently associated with the glycoconjugate (i.e., non-covalently bound to, adsorbed to, or entrapped in the glycoconjugate with it).

In some embodiments, the eTEC-linked glycoconjugate comprises less than about 45% free saccharide, less than about 40% free saccharide, less than about 35% free saccharide, less than about 30% free saccharide, less than about 25% free saccharide, less than about 20% free saccharide, less than about 15% free saccharide, less than about 10% free saccharide, or less than about 5% free saccharide, relative to the total amount of saccharides. Preferably, the glycoconjugate comprises less than 15% free saccharide, more preferably less than 10% free saccharide, even more preferably less than 5% free saccharide.

In certain preferred embodiments, the present invention provides eTEC-linked glycoconjugates comprising capsular polysaccharides (preferably Pn or Mn capsular polysaccharides) covalently conjugated to a carrier protein through an eTEC spacer, said glycoconjugates having one or more of the following features alone or in combination: the polysaccharide has a molecular weight of 50kDa to 2,000 kDa; the glycoconjugates have a molecular weight of 500kDa to 10,000 kDa; the carrier protein comprises 2-20 lysine residues covalently linked to the saccharide; the ratio of sugar to carrier protein (w/w) is 0.2-4; for every 4 of said polysaccharide(ii) one, 10, 15 or 25 saccharide repeat units, said saccharide conjugate comprising at least one covalent bond between a carrier protein and a polysaccharide; the saccharide has a degree of O-acetylation of 75-100%; the conjugate comprises less than about 15% free polysaccharide relative to total polysaccharide; the carrier protein is CRM₁₉₇(ii) a The capsular polysaccharide is selected from Pn-serotype 1,3, 4,5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F or 33F capsular polysaccharide, or the capsular polysaccharide is selected from Mn-serotype A, C, W135 or Y capsular polysaccharide.

The eTEC-linked glycoconjugates can also have a distribution of molecular sizes (K) through them_d) To characterize. The molecular size of the conjugate was determined by Sepharose CL-4B stationary phase Size Exclusion Chromatography (SEC) media using a high pressure liquid chromatography system (HPLC). For K_dFirst calibrating the column to determine V₀(which represents an empty volume or total exclusion volume) and V_i(i.e., the volume of the sample at which the smallest molecule elutes, also known as interparticle volume). All SEC separations occurred at V₀And V_iIn the meantime. K for each fraction collected_dThe value is given by the following expression K_d＝(V_e-V_i)/(V_i-V₀) Is determined in which V_eThe retention volume of the compound is indicated. Elution ≦ 0.3 fraction% (main peak) K for defined conjugate_d(molecular size distribution). In some embodiments, the invention provides a molecular size distribution (K) having ≧ 35%_d) The eTEC linked glycoconjugate of (a). In other embodiments, the invention provides a molecular size distribution (K) having ≥ 15%, ≥ 20%, > 25%, > 30%, > 35%, > 40%, > 45%, > 50%, > 60%, > 70%, > 80% or ≥ 90 ≥ of the molecular size distribution_d) The eTEC linked glycoconjugate of (a).

The eTEC-linked glycoconjugates and immunogenic compositions of the invention can contain free sulfhydryl residues. In some cases, the activated thiolated saccharide formed by the methods provided herein will contain multiple free thiol residues, some of which may not undergo covalent conjugation to a carrier protein during the conjugation step. Such residual free thiol residues are capped by reaction with a thiol-reactive capping reagent, such as Iodoacetamide (IAA), to cap potentially reactive functional groups. Other thiol-reactive capping reagents are also included, for example, maleimide-containing reagents and the like.

In addition, the eTEC-linked glycoconjugates and immunogenic compositions of the invention can contain residual unconjugated carrier proteins, which can include activated carrier proteins that have undergone modification during the capping process step.

The glycoconjugates of the invention are useful in the production of immunogenic compositions to protect a subject against bacterial infection, for example caused by pathogenic bacteria such as streptococcus pneumoniae or neisseria meningitidis. Thus, in another aspect, the invention provides an immunogenic composition comprising an eTEC-linked glycoconjugate and a pharmaceutically acceptable excipient, carrier or diluent, wherein the glycoconjugate comprises a saccharide covalently conjugated to a carrier protein through an eTEC spacer as described herein.

In a particularly preferred embodiment, the immunogenic composition comprises an eTEC-linked glycoconjugate comprising covalent conjugation to CRM through an eTEC spacer₁₉₇A bacterial capsular polysaccharide (e.g. a Pn or Mn capsular polysaccharide).

In some embodiments, the immunogenic composition comprises an adjuvant. In some such embodiments, the adjuvant is an aluminum-based adjuvant selected from the group consisting of aluminum phosphate, aluminum sulfate, and aluminum hydroxide. In one embodiment, the immunogenic composition comprises an aluminum phosphate adjuvant.

The eTEC-linked glycoconjugates of the invention and immunogenic compositions comprising them may contain an amount of free saccharide. In some embodiments, the immunogenic composition comprises less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5% free polysaccharide relative to the total amount of polysaccharide. Preferably, the immunogenic composition comprises less than 15% free sugar, more preferably less than 10% free sugar, even more preferably less than 5% free sugar.

In another aspect, the glycoconjugates or immunogenic compositions of the invention can be used to generate functional antibodies as measured by killing of bacteria in an animal efficacy model or opsonophagocytosis killing assay. Glycoconjugates of the invention comprising bacterial capsular polysaccharides may be used to produce antibodies against such bacterial capsular polysaccharides. Such antibodies can then be used in research and clinical laboratory assays, such as bacterial detection and serotyping. Such antibodies may also be used to confer passive immunity to an individual. In some embodiments, the antibodies generated against bacterial polysaccharides are functional in an animal efficacy model or in an opsonophagocytosis killing assay.

The eTEC-linked glycoconjugates and immunogenic compositions described herein can also be used in a variety of therapeutic or prophylactic methods to prevent, treat, or ameliorate a bacterial infection, disease, or disorder in a subject. In particular, eTEC-linked glycoconjugates comprising a bacterial antigen (e.g., a bacterial capsular polysaccharide from a pathogenic bacterium) are useful for preventing, treating, or ameliorating a bacterial infection, disease, or disorder caused by a pathogenic bacterium in a subject.

Accordingly, in one aspect, the present invention provides a method of preventing, treating or ameliorating a bacterial infection, disease or disorder in a subject, comprising administering to the subject an immunologically effective amount of an immunogenic composition of the invention, wherein the immunogenic composition comprises an eTEC-linked glycoconjugate comprising a bacterial capsular polysaccharide.

In one embodiment, the infection, disease or disorder is associated with streptococcus pneumoniae bacteria and the glycoconjugate comprises a Pn capsular polysaccharide. In some such embodiments, the infection, disease, or disorder is selected from the group consisting of pneumonia, sinusitis, otitis media, meningitis, bacteremia, sepsis, pleural effusion, conjunctivitis, osteomyelitis, septic arthritis, endocarditis, peritonitis, pericarditis, mastoiditis, soft tissue infections, and brain abscesses.

In another embodiment, the infection, disease or disorder is associated with neisserial bacteria and the glycoconjugate comprises a Mn capsular polysaccharide. In some such embodiments, the infection, disease, or disorder is selected from meningitis, meningococcemia, bacteremia, and sepsis.

In another aspect, the present invention provides a method of inducing an immune response in an individual comprising administering to the individual an immunologically effective amount of an immunogenic composition comprising an eTEC-linked glycoconjugate and a pharmaceutically acceptable excipient, carrier or diluent, wherein the glycoconjugate comprises a bacterial capsular polysaccharide.

In a further aspect, the present invention provides a method of preventing, treating or ameliorating a disease or disorder caused by a pathogenic bacterium in a subject, comprising administering to the subject an immunologically effective amount of an immunogenic composition comprising an eTEC-linked glycoconjugate, wherein the glycoconjugate comprises a bacterial capsular polysaccharide, and a pharmaceutically acceptable excipient, carrier or diluent.

In another aspect, the present invention provides a method of reducing the severity of at least one symptom of a disease or disorder caused by an infection with a pathogenic bacterium, comprising administering to the subject an immunologically effective amount of an immunogenic composition comprising an eTEC-linked glycoconjugate, wherein the glycoconjugate comprises a bacterial capsular polysaccharide, e.g., a Pn or Mn capsular polysaccharide, and a pharmaceutically acceptable excipient, carrier or diluent.

In another aspect, the present invention provides methods of administering to a subject an immunologically effective amount of an immunogenic composition comprising an eTEC-linked glycoconjugate of the present invention to generate a protective immune response in the subject, as further described herein.

In a further aspect, the present invention provides an immunogenic composition comprising the eTEC-linked glycoconjugate of the invention as described herein for use in the prevention, treatment or amelioration of a bacterial infection, for example an infection caused by streptococcus pneumoniae or neisseria meningitidis.

In another aspect, the invention provides the use of an immunogenic composition comprising an eTEC-linked glycoconjugate of the invention as described herein in the manufacture of a medicament for the prevention, treatment or amelioration of a bacterial infection (e.g. an infection caused by streptococcus pneumoniae or neisseria meningitidis).

In the above therapeutic and/or prophylactic methods and uses, the immunogenic composition typically comprises an eTEC-linked glycoconjugate comprising a bacterial capsular polysaccharide covalently linked to a carrier protein by an eTEC spacer. In a common embodiment of the methods and uses described herein, the bacterial capsular polysaccharide is a Pn capsular polysaccharide or a Mn capsular polysaccharide. In some such embodiments, the capsular polysaccharide is selected from Pn-serotype 1,3, 4,5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F capsular polysaccharides. In other such embodiments, the capsular polysaccharide is selected from Mn-serotype A, C, W135 and Y capsular polysaccharide.

In addition, the invention provides methods of inducing an immune response against a streptococcus pneumoniae or neisseria meningitidis bacterium in an individual; a method of preventing, treating or ameliorating an infection, disease or condition caused by a streptococcus pneumoniae or neisseria meningitidis bacterium in an individual; and reducing the severity of at least one symptom of an infection, disease or condition caused by infection with streptococcus pneumoniae or neisseria meningitidis in an individual by administering to the individual an immunologically effective amount of an immunogenic composition comprising an eTEC-linked glycoconjugate comprising a bacterial capsular polysaccharide derived from streptococcus pneumoniae or neisseria meningitidis, respectively, in combination with a pharmaceutically acceptable excipient, carrier or diluent.

Candy

The sugars may include polysaccharides, oligosaccharides and monosaccharides. In a common embodiment, the saccharide is a polysaccharide, in particular a bacterial capsular polysaccharide. Capsular polysaccharides are prepared by standard techniques known to those skilled in the art.

In the present invention, capsular polysaccharides may be prepared, for example, from Pn-serotypes 1,3, 4,5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F of streptococcus pneumoniae. In one embodiment, each pneumococcal polysaccharide serotype may be grown in soy-based media. The individual polysaccharides are purified by centrifugation, precipitation, ultrafiltration and/or column chromatography. The purified polysaccharide may be activated to enable it to react with the eTEC spacer prior to incorporation into the glycoconjugates of the invention, as further described herein.

The molecular weight of the capsular polysaccharide is a consideration for use in immunogenic compositions. High molecular weight capsular polysaccharides are capable of inducing certain antibody immune responses due to the higher valency of the epitopes present on the surface of the antigen. Isolation and purification of high molecular weight capsular polysaccharides is contemplated for use in the conjugates, compositions and methods of the invention.

In some embodiments, the saccharide has a molecular weight of 10kDa to 2,000 kDa. In other such embodiments, the saccharide has a molecular weight of 50kDa to 2,000 kDa. In other such embodiments, the saccharide has a molecular weight of 50kDa to 1,750 kDa; 50kDa to 1,500 kDa; 50kDa to 1,250 kDa; 50kDa to 1,000 kDa; 50kDa-750 kDa; 50kDa-500 kDa; 100kDa to 2,000 kDa; 100kDa to 1,750 kDa; 100kDa to 1,500 kDa; 100kDa to 1,250 kDa; 100kDa to 1,000 kDa; 100kDa-750 kDa; 100kDa-500 kDa; 200kDa to 2,000 kDa; 200kDa to 1,750 kDa; 200kDa to 1,500 kDa; 200kDa to 1,250 kDa; 200kDa to 1,000 kDa; 200kDa-750 kDa; or a molecular weight of 200kDa to 500 kDa. In some such embodiments, the saccharide is a bacterial capsular polysaccharide, for example a Pn-serotype 1,3, 4,5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F or 33F capsular polysaccharide, or a Mn-serotype A, C, W135 or Y capsular polysaccharide, wherein the capsular polysaccharide has a molecular weight that falls within one of the molecular weight ranges.

In some embodiments, the saccharide of the invention is O-acetylated. In some embodiments, the glycoconjugate comprises a saccharide having a degree of O-acetylation of 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 75-100%, 80-100%, 90-100%, 50-90%, 60-90%, 70-90%, or 80-90%. In other embodiments, the degree of O-acetylation is greater than or equal to 10%, greater than or equal to 20%, greater than or equal to 30%, greater than or equal to 40%, greater than or equal to 50%, greater than or equal to 60%, greater than or equal to 70%, greater than or equal to 80%, or greater than or equal to 90% or about 100%.

In some embodiments, the capsular polysaccharides, glycoconjugates, or immunogenic compositions of the invention are used to generate functional antibodies as measured by killing of bacteria in an animal efficacy model demonstrating antibody killing of bacteria or an opsonophagocytic killing assay.

Capsular polysaccharides can be obtained directly from bacteria using isolation procedures known to those skilled in the art. See, e.g., Fournier et al (1984), supra; fournier et al (1987) Ann. Inst. Pasteur/Microbiol.138: 561-567; U.S. patent application publication No. 2007/0141077; and international patent application publication No. WO 00/5656363; each of these references is incorporated herein by reference in its entirety). In addition, synthetic protocols can be used to produce capsular polysaccharides. In addition, capsular polysaccharides can be produced recombinantly using genetic engineering procedures also known to those skilled in the art (see Sau et al (1997) Microbiology143: 2395-2405; and U.S. Pat. No. 6,027,925; each of which is incorporated herein by reference in its entirety).

The streptococcus pneumoniae or neisseria meningitidis strains used to prepare the corresponding polysaccharides for use in the glycoconjugates of the invention may be obtained from established culture collections or clinical samples.

Carrier proteins

Another component of the glycoconjugates of the invention is a carrier protein conjugated to the saccharide. The terms "protein carrier" or "carrier protein" or "carrier" are used interchangeably herein. The carrier protein is preferably a non-toxic and non-reactogenic (non-reactive) protein that is available in sufficient quantity and purity. The carrier protein should be suitable for standard conjugation procedures. In the novel glycoconjugates of the invention, the carrier protein is covalently linked to the saccharide through an eTEC spacer.

Conjugation to a carrier can enhance the immunogenicity of an antigen (e.g., a bacterial antigen, such as a bacterial capsular polysaccharide). Preferred protein carriers for the antigen are toxins, toxoids or any mutant cross-reactive material (CRM) from the toxins of tetanus, diphtheria, pertussis, Pseudomonas (Pseudomonas), escherichia coli (e.coli), Staphylococcus (Staphylococcus) and Streptococcus (Streptococcus). In one implementationIn this embodiment, a particularly preferred vector is derived from the production of CRM₁₉₇diphtheria toxoid CRM of corynebacterium diphtheria (C. diphtheriae) strain C7(β 197) for protein₁₉₇. This strain has ATCC accession number 53281. Generating CRM₁₉₇Is described in U.S. patent No. 5,614,382, which is incorporated herein by reference in its entirety.

Alternatively, fragments or epitopes of protein carriers or other immunogenic proteins may be used. For example, the hapten can be coupled to a T cell epitope of a bacterial toxin, toxoid, or CRM. See U.S. patent application No. 150,688 entitled "synthetic peptides reproducing a T-Cell epitopes as a Carrier Molecule For ConjunatateVaccines" filed on 1.2.1988; this U.S. patent application is incorporated herein by reference in its entirety. Other suitable carrier proteins include inactivated bacterial toxins, such as cholera toxoid (e.g., as described in international patent application No. WO 2004/083251), escherichia coli LT, escherichia coli ST, and exotoxin a from pseudomonas aeruginosa (pseudomonas aeruginosa). Bacterial outer membrane proteins such as outer membrane complex c (OMPC), porin, transferrin binding protein, pneumolysin, pneumococcal surface protein a (PspA), pneumococcal adhesion protein (PsaA), or Haemophilus influenzae (Haemophilus influenzae) protein D may also be used. Other proteins, such as ovalbumin, Keyhole Limpet Hemocyanin (KLH), Bovine Serum Albumin (BSA) or purified tuberculin protein derivative (PPD), may also be used as carrier proteins.

Thus, in a common embodiment, the eTEC-linked glycoconjugate comprises CRM as a carrier protein₁₉₇Wherein the capsular polysaccharide is covalently attached to the eTEC spacer through a carbamate linkage, and wherein the CRM₁₉₇Covalently attached to the eTEC spacer via an amide bond formed by an activated amino acid residue of the protein, typically via the epsilon-amine group of one or more lysine residues.

The number of lysine residues in the carrier protein that become conjugated to the saccharide can be characterized as a range of conjugated lysines. For example, in immunogenic compositionsIn some embodiments, the CRM₁₉₇The lysine residues may comprise 4-16 lysine residues covalently linked to the sugar among the 39 lysine residues. Another way to express this parameter is about 10% to about 41% CRM₁₉₇Lysine is covalently linked to the sugar. In other embodiments, the CRM₁₉₇2-20 lysine residues covalently linked to the sugar may be included among the 39 lysine residues. Another way to express this parameter is about 5% to about 50% CRM₁₉₇Lysine is covalently linked to the sugar.

The frequency of lysine with which the carbohydrate chain is linked to the carrier protein is another parameter used to characterize the glycoconjugates of the invention. For example, in some embodiments, for every 4 saccharide repeat units of the polysaccharide, there is at least one covalent bond between the carrier protein and the polysaccharide. In another embodiment, the covalent bonding between the carrier protein and the polysaccharide occurs at least once in every 10 saccharide repeat units of the polysaccharide. In another embodiment, the covalent bonding between the carrier protein and the polysaccharide occurs at least once in every 15 saccharide repeat units of the polysaccharide. In yet another embodiment, the covalent bonding between the carrier protein and the polysaccharide occurs at least once in every 25 saccharide repeat units of the polysaccharide.

In a common embodiment, the carrier protein is CRM₁₉₇And at least one pass through CRM occurs in every 4, 10, 15, or 25 saccharide repeat units of the polysaccharide₁₉₇Covalent bonding with the eTEC spacer between the polysaccharides. In some such embodiments, the polysaccharide is a bacterial capsular polysaccharide derived from streptococcus pneumoniae or neisseria meningitidis.

In other embodiments, for every 5-10 saccharide repeat units; every 2-7 saccharide repeat units; every 3-8 saccharide repeat units; every 4-9 saccharide repeat units; every 6-11 saccharide repeat units; every 7-12 saccharide repeat units; every 8-13 saccharide repeat units; every 9-14 saccharide repeat units; every 10-15 saccharide repeat units; every 2-6 saccharide repeat units, every 3-7 saccharide repeat units; every 4-8 saccharide repeat units; every 6-10 saccharide repeat units; every 7-11 saccharide repeat units; every 8-12 saccharide repeat units; every 9-13 saccharide repeat units; every 10-14 saccharide repeat units; every 10-20 saccharide repeat units; or every 4-25 saccharide repeat units, the conjugate comprising at least one covalent bond between the carrier protein and the saccharide.

In another embodiment, at least one linkage between the carrier protein and the saccharide occurs for every 2, 3,4,5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 saccharide repeat units of the polysaccharide.

Method for preparing eTEC linked glycoconjugates

The present invention provides methods of making eTEC-linked glycoconjugates comprising a saccharide covalently conjugated to a carrier protein through a (2- ((2-oxoethyl) thio) ethyl) carbamate (eTEC) spacer. The eTEC spacer contains 7 linear atoms (i.e., -C (O) NH (CH)₂)₂SCH₂C (O) -) comprising stable thioether and amide bonds and used to covalently link the saccharide to the carrier protein. One end of the eTEC spacer is covalently bound to the hydroxyl group of the sugar through a carbamate linkage. The other end of the eTEC spacer is covalently bound to an amino group-containing residue, typically an epsilon-lysine residue, of the carrier protein via an amide bond.

Preparation of CRM formulations comprising conjugation to an activated carrier protein₁₉₇A representative route for glycoconjugates of the invention of polysaccharides is shown in figure 1. The chemical structures of representative bacterial capsular polysaccharides from streptococcus pneumoniae, pneumococcal serotypes 33F, 10A, 11A and 22F polysaccharides (with potential modification sites using the eTEC spacer approach) are shown in fig. 2, 3,4 and 5, respectively.

Comprising chemical covalent conjugation to CRM using an eTEC linker₁₉₇The structure of a representative eTEC-linked glycoconjugate of the invention of streptococcus pneumoniae serotype 33F polysaccharide is shown in figure 6 (a). For illustrative purposes, potential capped and uncapped free thiol sites are shown in fig. 6 (B). Polysaccharides usually contain multiple hydroxyl groups, thus eThe site of the TEC spacer that is attached to a particular hydroxyl group within the polysaccharide repeat unit via a carbamate linkage can vary.

in one aspect, the method comprises the steps of a) reacting a saccharide with a carbonic acid derivative (e.g., 1 '-carbonyl-bis- (1,2, 4-triazole) (CDT) or 1, 1' -Carbonyldiimidazole (CDI)) in an organic solvent to produce an activated saccharide, b) reacting the activated saccharide with cystamine or cysteamine or a salt thereof to produce a thiolated saccharide, c) reacting the thiolated saccharide with a reducing agent to produce an activated thiolated saccharide comprising one or more free thiol residues, d) reacting the activated thiolated saccharide with an activated carrier protein comprising one or more α -haloacetamide groups to produce a thiolated saccharide-carrier protein conjugate, and e) reacting the thiolated saccharide-carrier protein conjugate with (i) a first capping reagent capable of capping unconjugated α -haloacetamide groups of the activated carrier protein, and/or (ii) a second capping reagent capable of capping unconjugated α -haloacetamide groups of the activated carrier protein, thereby producing a TEC conjugate.

in a particularly preferred embodiment, the process comprises the steps of a) reacting Pn-33F capsular polysaccharide with CDT or CDI in an organic solvent to produce activated Pn-33F polysaccharide, b) reacting the activated Pn-33F polysaccharide with cystamine or a salt thereof to produce thiolated Pn-33F polysaccharide, c) reacting the thiolated Pn-33F polysaccharide with a reducing agent to produce activated thiolated Pn-33F polysaccharide comprising one or more free thiol residues, d) reacting the activated thiolated Pn-33F polysaccharide with activated CRM comprising one or more α -bromoacetamides₁₉₇Reaction of the carrier protein to produce thiolated Pn-33F polysaccharide-CRM₁₉₇A conjugate; and e) thiolating the Pn-33F polysaccharide-CRM₁₉₇reacting the conjugate with (i) N-acetyl-L-cysteine as a first capping reagent capable of capping unconjugated α -bromoacetamido groups of the activated carrier protein, and (ii) iodoacetamide as a second capping reagent capable of capping unconjugated free thiol residues of the activated thiolated Pn-33F polysaccharide;thus generating eTEC-linked Pn-33F polysaccharide-CRM₁₉₇A glycoconjugate.

In a common embodiment, the carbonic acid derivative is CDT or CDI. Preferably, the carbonic acid derivative is CDT and the organic solvent is a polar aprotic solvent, such as dimethyl sulfoxide (DMSO). The activated saccharide need not be lyophilized prior to the thiolation and/or conjugation step.

In a preferred embodiment, the thiolated saccharide is produced by reacting an activated saccharide with the bifunctional symmetric thioalkylamine reagent, cystamine, or a salt thereof. A potential advantage of this reagent is that the symmetric cystamine linker can react with two molecules of activated sugar, thereby forming two molecules of thiolated sugar/molecule of cystamine upon reduction of the disulfide bond. Alternatively, the thiolated saccharide may be formed by reacting an activated saccharide with cysteamine or a salt thereof. The eTEC-linked glycoconjugates produced by the methods of the invention can be represented by general formula (I).

It will be appreciated by those skilled in the art that step c) is optional when the activated saccharide is reacted with cysteamine or a salt thereof containing a free sulfhydryl residue. In practice, the thiolated saccharide comprising cysteamine is conventionally reacted in step c) with a reducing agent to reduce any oxidized disulfide by-products that may be formed during the reaction.

in some embodiments of this aspect, step d) further comprises providing an activated carrier protein comprising one or more α -haloacetamide groups, and then reacting the activated thiolated saccharide with the activated carrier protein to produce α thiolated saccharide-carrier protein conjugate.

The thiolated saccharide-carrier protein conjugate may be reacted with one or more capping reagents capable of reacting with residual activated functional groups present in the reaction mixture. Such residual reactive groups may be present on unreacted saccharide or carrier protein components due to incomplete conjugation or the presence of an excess of one of the components in the reaction mixture. In this case, capping may aid in the purification or isolation of the glycoconjugate. In some cases, residual activated functional groups may be present in the glycoconjugate.

for example, the excess α -haloacetamido groups on activated carrier proteins can be capped by reaction with low molecular weight thiols (e.g., N-acetyl-L-cysteine), which can be used in excess to ensure complete capping.capping with N-acetyl-L-cysteine also allows confirmation of conjugation efficiency by detection of the unique amino acid S-carboxymethyl cysteine (CMC) from the cysteine residues at the capped sites, which CMC can be determined by acid hydrolysis and amino acid analysis of the conjugate product.

in a preferred embodiment, the first capping reagent is N-acetyl-L-cysteine, which reacts with unconjugated alpha-haloacetamido groups on the carrier protein.

In some embodiments, the method further comprises the step of purifying the eTEC-linked glycoconjugate by, for example, ultrafiltration/diafiltration.

In a preferred embodiment, the bifunctional symmetric thioalkylamine reagent is cystamine or a salt thereof, which is reacted with an activated saccharide to provide a thiolated saccharide containing a disulfide moiety or a salt thereof.

Such thiolated saccharide derivatives are reacted with a reducing agent to produce activated thiolated polysaccharides containing one or more free sulfhydryl residues. Such activated thiolated sugars can be isolated and purified by, for example, ultrafiltration/diafiltration. Alternatively, the activated thiolated saccharide may be isolated and purified by, for example, standard Size Exclusion Chromatography (SEC) or ion exchange chromatography (e.g., DEAE, as is known in the art).

In the case of cystamine-derived thiolated saccharides, reaction with a reducing agent cleaves the disulfide bond to provide an activated thiolated saccharide comprising one or more free sulfhydryl residues. In the case of cysteamine-derived thiolated sugars, reaction with a reducing agent is optional and can be used to reduce disulfide bonds formed by oxidation of the reagent or product.

Reducing agents useful in the methods of the invention include, for example, tris (2-carboxyethyl) phosphine (TCEP), Dithiothreitol (DTT), or mercaptoethanol. However, any suitable disulfide reducing agent may be used.

in some embodiments, the method further comprises providing an activated carrier protein comprising one or more α -haloacetamide groups (preferably one or more α -bromoacetamide groups).

the reaction of the activated thiolated saccharide with the activated carrier protein comprising one or more α -haloacetamide moieties results in nucleophilic displacement of the α -halo group of the activated carrier protein by one or more free thiols of the activated thiolated saccharide, thereby forming a thioether bond of the eTEC spacer.

the α -haloacetylated amino acid residues of the carrier protein are typically linked to the epsilon amino group of one or more lysine residues of the carrier protein in common embodiments, the carrier protein contains one or more α -bromoacetylated amino acid residues.

in one embodiment, the method comprises the steps of providing an activated carrier protein comprising one or more α -haloacetamide groups, and reacting the activated thiolated polysaccharide with the activated carrier protein to produce a thiolated polysaccharide-carrier protein conjugate, thereby producing a glycoconjugate comprising a polysaccharide conjugated to a carrier protein through an eTEC spacer.

In some preferred embodiments of the methods described herein, the bacterial capsular polysaccharide is a Pn capsular polysaccharide derived from streptococcus pneumoniae. In some such embodiments, the Pn capsular polysaccharide is selected from Pn-serotype 1,3, 4,5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F capsular polysaccharides. In certain preferred embodiments, the carrier protein is CRM₁₉₇And the Pn capsular polysaccharide is selected from Pn-serotype 1,3, 4,5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F capsular polysaccharides.

In other preferred embodiments of the methods provided herein, the bacterial capsular polysaccharide is a Mn capsular polysaccharide derived from neisseria meningitidis. In some such embodiments, the Mn capsular polysaccharide is selected from Mn-serotype A, C, W135 and Y capsular polysaccharide. In certain preferred embodiments, the carrier protein is CRM₁₉₇And the capsular polysaccharide is selected from the group consisting of Mn-serotype A, C, W135 and Y capsular polysaccharides.

In some embodiments of each of the methods provided herein, the saccharide is complexed with an imidazole or triazole, and then reacted with a carbonic acid derivative, such as CDT, in an organic solvent (e.g., DMSO) containing about 0.2% w/v water to produce an activated saccharide. The use of complex sugars in the activation step increases the solubility of the sugars in organic solvents. Typically, the saccharide is complexed with 10 grams of 1,2, 4-triazole excipient per gram of polysaccharide, followed by mixing at ambient temperature to provide a complexed saccharide.

Thus, in certain embodiments, the method further comprises the step of complexing the saccharide with a triazole or imidazole to give a complexed saccharide prior to activation step a). In some such embodiments, the complex sugar shell is frozen (shell-frezen), lyophilized and reconstituted in an organic solvent (e.g., DMSO), and about 0.2% w/v water is added, followed by activation with a carbonic acid derivative (e.g., CDT).

In one embodiment, the thiolated saccharide reaction mixture is optionally treated with N-acetyl-lysine methyl ester to cap any unreacted activated saccharide. In some such embodiments, the capped thiolated saccharide mixture is purified by ultrafiltration/diafiltration.

In a common embodiment, the thiolated sugar is reacted with a reducing agent to produce an activated thiolated sugar. In some such embodiments, the reducing agent is tris (2-carboxyethyl) phosphine (TCEP), Dithiothreitol (DTT), or mercaptoethanol. In some such embodiments, the activated thiolated saccharide is purified by ultrafiltration/diafiltration.

In one embodiment, the method of producing the eTEC-linked glycoconjugate comprises the step of adjusting the pH of the reaction mixture of activated thiolated saccharide and carrier protein to a pH of about 8 to about 9 at about 5 ℃ and maintaining for about 20 hours.

In one embodiment, the method of producing the glycoconjugates of the invention comprises the step of isolating the thiolated saccharide-carrier protein conjugate after it has been produced. In a common embodiment, the glycoconjugates are isolated by ultrafiltration/diafiltration.

In another embodiment, the method of producing an eTEC-linked glycoconjugate of the invention comprises the step of isolating the isolated saccharide-carrier protein conjugate after it is produced. In a common embodiment, the glycoconjugates are isolated by ultrafiltration/diafiltration.

In yet another embodiment, a method of producing activated sugars comprises the step of adjusting the water concentration of a reaction mixture comprising sugars and CDT in an organic solvent to between about 0.1% and 0.4%. In one embodiment, the water concentration of the reaction mixture comprising the sugar and CDT in the organic solvent is adjusted to about 0.2%.

In one embodiment, the step of activating the saccharide comprises reacting the polysaccharide with an amount of CDT in an organic solvent in about 5 molar excess relative to the amount of polysaccharide present in the reaction mixture comprising capsular polysaccharide and CDT.

In another embodiment, the method of producing a glycoconjugate of the invention comprises the step of determining the water concentration of the reaction mixture comprising the saccharide. In one such embodiment, the amount of CDT added to the reaction mixture to activate the saccharide is provided in the organic solvent in about the following amounts of CDT: the amount is equimolar to the amount of water present in the reaction mixture comprising the sugar and CDT.

In another embodiment, the amount of CDT added to the reaction mixture to activate the saccharide is provided in the organic solvent in about the following amounts of CDT: the molar ratio of the amount relative to the amount of water present in the reaction mixture comprising sugar and CDT is about 0.5: 1. In one embodiment, the amount of CDT added to the reaction mixture to activate the saccharide is provided in the organic solvent as about the following amount of CDT: the molar ratio of the amount to the amount of water present in the reaction mixture comprising sugar and CDT was 0.75: 1.

In one embodiment, the method comprises the step of isolating the thiolated polysaccharide by diafiltration. In another embodiment, the method comprises the step of isolating the activated thiolated polysaccharide by diafiltration.

In one embodiment, the carrier protein used in the method of producing an isolated Pn capsular polysaccharide-carrier protein conjugate comprises CRM₁₉₇. In addition toIn one embodiment, the carrier protein used in the method for producing an isolated Mn capsular polysaccharide-carrier protein conjugate comprises CRM₁₉₇。

In some embodiments, the ratio of sugar to activated carrier protein (w/w) is 0.2 to 4. In other embodiments, the ratio of sugar to activated carrier protein (w/w) is 1.0 to 2.5. In other embodiments, the ratio of sugar to activated carrier protein (w/w) is 0.4 to 1.7. In other embodiments, the ratio of sugar to activated carrier protein (w/w) is about 1: 1. In some such embodiments, the saccharide is a bacterial capsular polysaccharide and the activated carrier protein is through CRM₁₉₇Activation (bromoacetylation) of (c).

In another embodiment, the method of producing an activated saccharide comprises the use of an organic solvent. In a common embodiment, the organic solvent is a polar aprotic solvent. In some such embodiments, the polar aprotic solvent is selected from dimethyl sulfoxide (DMSO), Dimethylformamide (DMF), Dimethylacetamide (DMA), N-methyl-2-pyrrolidone (NMP), acetonitrile, 1, 3-dimethyl-3, 4,5, 6-tetrahydro-2 (1H) -pyrimidinone (DMPU), and Hexamethylphosphoramide (HMPA), or mixtures thereof. In a preferred embodiment, the organic solvent is DMSO.

In a common embodiment, the separation of the eTEC-linked glycoconjugate comprises an ultrafiltration/diafiltration step.

In one embodiment, the saccharide used in the method of producing the glycoconjugate of the invention has a molecular weight of about 10kDa to about 2,000 kDa. In another embodiment, the saccharide used in the method of producing a glycoconjugate of the invention has a molecular weight of about 50kDa to about 2,000 kDa.

In one embodiment, the glycoconjugate produced in the method of producing a capsular polysaccharide-carrier protein glycoconjugate has a size of about 50kDa to about 20,000 kDa. In another embodiment, the glycoconjugate produced in the method of producing a capsular polysaccharide-carrier protein glycoconjugate has a size of about 500kDa to about 10,000 kDa. In one embodiment, the glycoconjugate produced in the method of producing a capsular polysaccharide-carrier protein glycoconjugate has a size of about 1,000kDa to about 3,000 kDa.

In another aspect, the invention provides an eTEC-linked glycoconjugate comprising a saccharide conjugated to a carrier protein through an eTEC spacer produced by any of the methods disclosed herein.

In another aspect, the invention provides an immunogenic composition comprising an eTEC-linked glycoconjugate produced by any of the methods described herein.

The degree of O-acetylation of the saccharide can be determined by any method known in the art, for example, by proton NMR (Lemercinier and Jones (1996)Carbohydrate Research296; 83-96, Jones and Lemercinier (2002)J.Pharmaceutical and Biomedical Analysis30, of a nitrogen-containing gas; 1233-1247, WO 05/033148 or WO 00/56357). Another common method is by Hestrin (1949)J.Biol.Chem180; 249-261. Yet another method is based on HPLC-ion exclusion chromatography. The degree of O-acetylation is determined by evaluating the amount of free acetate present in the sample and comparing this value with the amount of acetate released after hydrolysis with a weak base. Acetate was resolved from other components of the sample using Ultraviolet (UV) detection at 210nm and quantified. Another method is based on HPLC-ion exclusion chromatography. The O-acetyl group is determined by evaluating the amount of free acetate present in the sample and comparing this value with the amount of acetate released after hydrolysis with a weak base. Acetate was resolved from the other components of the sample and quantified using Ultraviolet (UV) detection at 210 nm.

Determination of the degree of conjugation by amino acid analysis

Acid hydrolysis of "pre-IAA-capped" conjugate samples generated using bromoacetyl activation chemistry resulted in the formation of acid stable S-carboxymethylcysteamine (CMCA) from cystamine at the conjugation site and acid stable S-carboxymethylcysteine (CMC) from cysteine at the capped site. Acid hydrolysis of the "IAA-capped" (final) conjugate generated using bromoacetyl activation chemistry results in the formation of acid stable S-carboxymethylcysteamine (CMCA) from the cystamine at the conjugation site and the IAA capping site and acid stable S-carboxymethylcysteine (CMC) from the cysteine at the capped site. All unconjugated and uncapped lysines were converted back to lysine and detected as such. All other amino acids were hydrolyzed back to the free amino acids except tryptophan and cysteine, which were destroyed by the hydrolysis conditions. Asparagine and glutamine are converted to aspartic acid and glutamic acid, respectively.

The amino acids of each hydrolyzed sample and control were separated using ion exchange chromatography and subsequently reacted with Beckman ninhydrin NinRX solution at 135 ℃. The derivatized amino acids were then detected at 570nm and 440nm in the visible range (see Table 1). A standard amino acid group containing 500 pmoles of each amino acid [ Pierce amino acid standard H ] was subjected to each group analysis along with the sample and control. S-carboxymethylcysteine [ Sigma-Aldrich ] was added to the standard.

TABLE 1

Retention time of amino acids obtained using gradient program 1 on Beckman 6300 amino acid Analyzer

Lysine was selected for evaluation based on its covalent attachment to cysteine and cysteamine and expected similar hydrolysis. The resulting number of moles of amino acids was then compared to the amino acid composition of the protein and reported along with the values for CMC and CMCA. The CMCA values were used directly to assess the degree of conjugation and the CMC values were used directly to assess the degree of capping.

In one embodiment, by molecular size distribution (K)_d) To characterize the glycoconjugates. Immobilization by Sepharose CL-4BPhase Size Exclusion Chromatography (SEC) media high pressure liquid chromatography systems (HPLC) were used to determine the molecular size of the conjugates. For K_dFirst calibrating the column to determine V₀(which represents empty volume or total excluded volume) and V_i(i.e., the volume of the sample at which the smallest molecule elutes, also referred to as the interparticle volume). All SEC separations occurred at V₀And V_iIn the meantime. K for each fraction collected_dThe value is given by the following expression K_d＝(V_e-V_i)/(V_i-V₀) Is determined in which V_eThe retention volume of the compound is indicated. Elution ≦ 0.3 fraction% (main peak) K for defined conjugate_d(molecular size distribution).

Immunogenic compositions

The term "immunogenic composition" refers to any pharmaceutical composition containing an antigen (e.g., a microorganism or component thereof) that can be used to elicit an immune response in an individual.

As used herein, "immunogenic" means the ability of an antigen (or epitope of an antigen), such as a bacterial capsular polysaccharide or glycoconjugate comprising a bacterial capsular polysaccharide or immunogenic composition, to elicit a humoral or cell-mediated immune response, or both, in a host individual (e.g., a mammal).

The glycoconjugates can be used to sensitize a host by providing an antigen associated with an MHC molecule on the surface of the cell. In addition, antigen-specific T cells or antibodies can be generated to allow future protection of the immunized host. Thus, the glycoconjugates can protect the host from one or more symptoms associated with bacterial infection, or can protect the host from death due to bacterial infection associated with capsular polysaccharides. Glycoconjugates can also be used to generate polyclonal or monoclonal antibodies that can be used to confer passive immunity to an individual. Glycoconjugates can also be used to generate functional antibodies, as measured by killing of bacteria in an animal efficacy model or by an opsonophagocytic killing assay.

as used herein, unless the context indicates otherwise, the term is intended to encompass not only intact polyclonal or monoclonal antibodies, but also engineered antibodies (e.g., chimeric, humanized and/or derivatized to alter effector function, stability and other biological activity) and fragments thereof (e.g., Fab ', F (ab') 2, Fv), single chain (ScFv) and domain antibodies (including shark and camel antibodies) and fusion proteins comprising an antibody portion, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies, so long as they exhibit the desired biological activity) and antibody fragments as described herein, as well as any other modified configurations of immunoglobulin molecules comprising an antigen recognition site.

An "antibody fragment" comprises only a portion of an intact antibody, wherein the portion, when present in the intact antibody, preferably retains at least one, preferably most or all of the functions normally associated with the portion.

The term "antigen" generally refers to a biomolecule, typically a protein, peptide, polysaccharide or conjugate, in an immunogenic composition, or an immunogenic substance that stimulates the production of antibodies or a T cell response or both in an individual, including compositions that are injected or absorbed into an individual. An immune response can be generated against the entire molecule or against various portions of the molecule (e.g., epitopes or haptens). The term may be used to refer to a single molecule or a population of homologous or heterologous antigen molecules. Antigens are recognized by antibodies, T cell receptors, or other components with specific humoral and/or cellular immunity. "antigen" also includes all relevant epitopes. Epitopes of a given antigen can be identified using any number of epitope mapping techniques well known in the art. See, e.g., epipope Mapping Protocols in Methods in Molecular Biology, volume 66 (GlennE. Morris eds., 1996) Humana Press, Totowa, N.J. For example, a linear epitope can be determined, for example, by: a plurality of peptides corresponding to portions of a protein molecule are synthesized simultaneously on a solid support, and the peptides are reacted with an antibody while still attached to the support. Such techniques are known in the art and are described, for example, in U.S. Pat. nos. 4,708,871; geysen et al (1984) Proc.Natl.Acad.Sci.USA81: 3998-4002; geysen et al (1986) molecular. Immunol.23: 709-715; each of these documents is incorporated herein by reference in its entirety. Similarly, conformational epitopes can be identified by determining the spatial conformation of amino acids, for example, by x-ray crystallography and two-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols, supra. Furthermore, for the purposes of the present invention, "antigen" may also be used to refer to a protein that includes modifications (e.g., deletions, additions, and substitutions) made to the native sequence (which are generally conserved in nature, but which may not be conserved) so long as the protein maintains the ability to elicit an immune response. These modifications may be artificial, such as by site-directed mutagenesis, or by specific synthetic procedures, or by genetic engineering methods, or may be episodic, such as by mutation of the host producing the antigen. Furthermore, the antigen may be derived, obtained, or isolated from a microorganism (e.g., a bacterium), or may be the entire organism. Similarly, oligonucleotides or polynucleotides expressing antigens in applications such as nucleic acid immunization are also included in the definition. Synthetic antigens also include, for example, polyepitopes, flanking epitopes (flanking epitopes) and other recombinant or synthetically derived antigens (Bergmann et al (1993) eur.j. immunol.23: 27772781; Bergmann et al (1996) j. immunol.157: 3242-3249; Suhrbier (1997) immunol.cell biol.75: 402408; Gardner et al (1998) 12th World AIDS Conference (12th World AIDS Conference, sweden geneva, 1998 from 28 days 6 to 3 days 7).

A "protective" immune response refers to the ability of an immunogenic composition to elicit a humoral or cell-mediated immune response or both that is used to protect an individual from infection. The protection provided need not be absolute, i.e., need not completely prevent or eradicate infection, if there is a statistically significant improvement over a control population of individuals (e.g., infected animals not administered a vaccine or immunogenic composition). Protection may be limited to mitigating the severity or rapidity of onset of symptoms of infection. In general, a "protective immune response" will include inducing an increase in the level of antibodies specific for a particular antigen in at least 50% of individuals, including some level of increase in a measurable functional antibody response to each antigen. In particular instances, a "protective immune response" can include inducing a two-fold increase in antibody levels or a 4-fold increase in antibody levels specific for a particular antigen in at least 50% of individuals, including some level of increase in a measurable functional antibody response to each antigen. In certain embodiments, the opsonophagocytic antibody is associated with a protective immune response. Thus, a protective immune response can be determined by measuring the percent reduction in bacterial count in an opsonophagocytosis assay (e.g., the assay described below). Preferably, the bacterial count is reduced by at least 10%, 25%, 50%, 65%, 75%, 80%, 85%, 90%, 95% or more.

The terms "immunogenic amount" and "immunologically effective amount" are used interchangeably herein and refer to an amount of an antigen or immunogenic composition sufficient to elicit an immune response, which can be a cellular (T cell) response or a humoral (B cell or antibody) response or both, wherein such immune response can be measured by standard assays known to those skilled in the art. Typically, an immunologically effective amount will elicit a protective immune response in the individual.

The immunogenic compositions of the invention may be used to prophylactically or therapeutically protect or treat an individual susceptible to bacterial infection by, for example, streptococcus pneumoniae or neisseria meningitidis bacteria, by means of administering the immunogenic composition by systemic, cutaneous or mucosal route, or may be used to generate polyclonal or monoclonal antibody preparations that can be used to confer passive immunity to another individual. These administrations may include injection by intramuscular, intraperitoneal, intradermal or subcutaneous routes; or by mucosal administration to the oral/digestive, respiratory or genitourinary tract. Immunogenic compositions can also be used to generate functional antibodies, as measured by killing of bacteria in an animal efficacy model or by an opsonophagocytic killing assay.

The optimal amounts of components for a particular immunogenic composition can be determined by standard studies involving the observation of an appropriate immune response in an individual. After the initial vaccination, the individual may receive one or several booster immunizations at sufficient intervals.

In certain embodiments, the immunogenic composition comprises one or more adjuvants. As defined herein, an "adjuvant" is a substance used to enhance the immunogenicity of the immunogenic composition of the invention. Thus, adjuvants are typically administered to boost the immune response and are well known to those skilled in the art. Suitable adjuvants to enhance the effectiveness of the composition include, but are not limited to:

(1) aluminum salts (alum) such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, and the like;

(2) oil-in-water emulsion formulations (with or without other specific immunostimulants such as muramyl peptides (defined below) or bacterial cell wall components), for example,

(a) MF59 formulated as submicron particles using a microfluidizer (e.g., 110Y Microfluidics, Newton, Mass.) (PCT publication No. WO 90/14837) containing 5% squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing different amounts of MTP-PE (see below, although not required)),

(b) SAF microfluidized as a submicron emulsion or vortexed to produce a larger particle size emulsion containing 10% squalene, 0.4% Tween 80, 5% Pluronic blocked polymers Ll21 and thr-MDP (see below), and

(c)Ribi^TMadjuvant System (RAS) (Corixa, Hamilton, Mont.), containing 2% squalene, 0.2% Tween 80 and one or more 3-O-deacylated monophosphoryl lipid A (MPL) from U.S. Pat. No. 4,912,094 (Corixa)^TM) Trehalose Dimycolate (TDM) and Cell Wall Skeleton (CWS) (preferably MPL + CWS (Detox)^TM) A bacterial cell wall component of);

(3) saponin adjuvant, such as Quil A or STIMULON^TMQS-21 (antibiotics, framingham. masses.) (U.S. patent No. 5,057,540), or particles produced therefrom, such as ISCOMs (immune stimulating complexes);

(4) bacterial lipopolysaccharides, synthetic lipid a analogs (e.g., aminoalkyl glucosamine phosphate compounds (AGPs)) or derivatives or analogs thereof, which are available from Corixa and described in U.S. patent No. 6,113,918; one such AGP is 2- [ (R) -3-tetradecanoyloxytetradecanoylamino ] ethyl 2-deoxy-4-O-phosphono-3- [ (R) -3-tetradecanoyloxytetradecanoyl ] -2- [ (R) -3-tetradecanoyloxytetradecanoylamino ] -b-D-glucopyranoside, also known as 529 (formerly RC529), which is formulated in an aqueous form or stable emulsion, synthetic polynucleotide, e.g., an oligonucleotide containing a CpG motif (U.S. Pat. No. 6,207,646);

(5) cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, IL-18, etc.), interferons (e.g., gamma interferon), granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), Tumor Necrosis Factor (TNF), co-stimulatory molecules B7-1 and B7-2, etc.;

(6) detoxified mutants of bacterial ADP-ribosylating toxins, such as Cholera Toxin (CT), Pertussis Toxin (PT), or E.coli heat-Labile Toxin (LT), especially LT-K63, LT-R72, CT-S109, PT-K9/G129 (see, e.g., WO 93/13302 and WO 92/19265), in wild-type or mutant form (e.g., according to published International patent application No. WO 00/18434 (see also WO 02/098368 and WO 02/098369) wherein the glutamic acid at amino acid position 29 is replaced by another amino acid, preferably histidine); and

(7) other substances that act as immunostimulants to enhance the effectiveness of the composition.

Muramyl peptides include, but are not limited to, N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyldesmosomyl (normuramyl) -L-alanine-2- (1'-2' dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) -ethylamine (MTP-PE), and the like.

In certain embodiments, the adjuvant is an aluminum-based adjuvant, such as an aluminum salt. In particular embodiments, the aluminum-based adjuvant is selected from the group consisting of aluminum phosphate, aluminum sulfate, and aluminum hydroxide. In certain embodiments, the adjuvant is aluminum phosphate.

The immunogenic composition may optionally comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier includes a carrier that is approved by or listed in the U.S. pharmacopeia (u.s.pharmacopeia) or another generally recognized pharmacopeia for use in a subject, including humans and non-human mammals, by a regulatory agency of the federal or a state government or another regulatory agency. The term carrier may be used to refer to a diluent, excipient or vehicle with which the pharmaceutical composition is administered. Water, saline solutions, and aqueous dextrose and glycerol solutions can be employed as the liquid carrier for injectable solutions, among others. Examples of suitable Pharmaceutical carriers are described in e.w. martin, "Remington's Pharmaceutical Sciences". The formulation should be suitable for the mode of administration.

In addition to the plurality of capsular polysaccharide-protein conjugates, the immunogenic compositions of the invention may also comprise one or more preservatives. The FDA requires that bioproducts in multi-dose (multi-dose) vials contain preservatives with few exceptions. Vaccine products containing preservatives include vaccines containing benzethonium chloride (anthrax), 2-phenoxyethanol (DTaP, HepA, Lyme disease (Lyme), poliomyelitis (parenteral)), phenol (pneumonia (Pneumo), typhoid (parenteral), vaccinia disease) and thimerosal (DTaP, DT, Td, HepB, Hib, influenza, JE, meningitis (Mening), pneumonia, rabies). Preservatives approved for use in injectable pharmaceuticals include, for example, chlorobutanol, m-cresol, methyl paraben, propyl paraben, 2-phenoxyethanol, benzethonium chloride, benzalkonium chloride, benzoic acid, benzyl alcohol, phenol, thimerosal, and phenylmercuric nitrate.

In certain embodiments, formulations of the invention compatible with parenteral administration comprise one or more nonionic surfactants including, but not limited to, polyoxyethylene sorbitan fatty acid esters, polysorbate-80 (Tween 80), polysorbate-60 (Tween 60), polysorbate-40 (Tween 40), and polysorbate-20 (Tween 20), polyoxyethylene alkyl ethers including, but not limited to, Brij 58, Brij 35, and others, such as Triton X-100, Triton X-114, NP40, Span 85, and the Pluronic series of nonionic surfactants (e.g., Pluronic 121), with a preferred component being polysorbate-80 at a concentration of about 0.001% to about 2% (preferably up to about 0.25%) or polysorbate-40 at a concentration of about 0.001% to 1% (preferably up to about 0.5%).

Packaging and administration forms

Delivery of the immunogenic compositions of the invention directly to an individual may be by parenteral administration (intramuscular, intraperitoneal, intradermal, subcutaneous, intravenous or to the interstitial space); or by mucosal administration to the oral/digestive, respiratory or genitourinary tract; or by topical, transdermal, intranasal, ocular, otic, pulmonary or other mucosal administration.

In one embodiment, parenteral administration is by intramuscular injection, for example, into the thigh or upper arm of the individual. The injection may be through a needle (e.g., a hypodermic needle), or a needle-free injection may be used. A typical intramuscular dose is 0.5 mL. In another embodiment, intranasal administration is used to treat pneumonia or otitis media (since nasopharyngeal carriage by pneumococci can be more effectively prevented, thereby attenuating infection at its earliest stage).

The compositions of the present invention may be prepared in a variety of forms, for example, as liquid solutions or suspensions for injection. In certain embodiments, the compositions may be prepared as a powder or spray for pulmonary administration, e.g., in the form of an inhaler. In other embodiments, the composition may be formulated as a suppository or pessary, or for nasal, aural or ocular administration, e.g., as a spray, drop, gel or powder.

The amount of glycoconjugate in each dose of immunogenic composition is selected to be an amount that induces an immune protective response without significant adverse effects. Such amounts may vary depending on the bacterial serotype present in the glycoconjugate. Typically, each dose comprises 0.1. mu.g to 100. mu.g, particularly 0.1. mu.g to 10. mu.g, more particularly 1. mu.g to 5. mu.g of polysaccharide.

In a particular embodiment of the invention, the immunogenic composition is conjugated individually to CRM via eTEC linker₁₉₇A sterile liquid formulation of the Pn or Mn capsular polysaccharide of (a), wherein each 0.5mL dose is formulated to contain 1-5 μ g of the polysaccharide, which may further contain 0.125mg of elemental aluminum (0.5mg of aluminum phosphate) adjuvant; and sodium chloride and sodium succinate buffers as excipients.

The immunogenic compositions of the invention can be packaged in unit-dose or multi-dose form (e.g., 2,4, or more doses). For multi-dose forms, vials are generally, but not necessarily, preferred over pre-filled syringes. Suitable multi-dose forms include, but are not limited to: 2-10 doses per container, 0.1-2 mL per dose. In certain embodiments, the dose is a 0.5mL dose. See, for example, international patent application WO 2007/127668, which is incorporated herein by reference.

The composition may be provided in a vial or other suitable storage container, or may be provided in a pre-filled drug delivery device, such as a single or multi-component syringe that may be supplied with a needle or needle-free. Syringes typically, but not necessarily, contain a single dose of the preservative-containing immunogenic composition of the invention, but also include pre-filled multi-dose syringes. Likewise, a vial may include a single dose, but may also include multiple doses.

The effective dose volume can be routinely determined, but a typical injected dose of the composition has a volume of 0.5 mL. In certain embodiments, the dosage is formulated for administration to a human subject. In certain embodiments, the dosage is formulated for administration to a human subject who is an adult, juvenile, adolescent, toddler, or infant (i.e., no more than 1 year old) and in preferred embodiments may be administered by injection.

The liquid immunogenic compositions of the invention are also suitable for reconstitution of other immunogenic compositions provided in lyophilized form. Provided that the immunogenic composition is to be used for such immediate reconstitution, the invention provides a kit having one or more of two or more vials, two or more ready-to-fill syringes, or each, wherein the contents of the syringes are used to reconstitute the contents of the vials prior to injection, or vice versa.

In yet another embodiment, the container in a multi-dose form is selected from, but not limited to, one or more of the following: common laboratory glassware, flasks, beakers, graduated cylinders, fermenters, bioreactors, tubing, tubes, bags, jars, vials, vial caps (e.g., rubber stoppers, screw caps (screw on caps)), ampoules, syringes, dual or multi-chamber syringes, syringe stoppers, syringe plungers, rubber caps, plastic caps, glass caps, cartridges, disposable pens, and the like. The container of the present invention is not limited by the material of manufacture and includes materials such as glass, metals (e.g., steel, stainless steel, aluminum, etc.), and polymers (e.g., thermoplastics, elastomers, thermoplastic elastomers). In a specific embodiment, the form of the container is a 5mL Schott type 1 glass vial with a butyl stopper. Those skilled in the art will appreciate that the above forms are by no means exhaustive lists, but merely serve as guidance to the skilled person as to the variety of forms that can be used in the present invention. Other formats for use in the present invention can be found in published catalogues from laboratory equipment suppliers and manufacturers (e.g., United States plastics, Lima, OH), VWR).

Methods of inducing immune responses and protecting against infection

The invention also includes methods of using the eTEC linked glycoconjugates and immunogenic compositions comprising them prophylactically or therapeutically. For example, one aspect of the invention provides a method of inducing an immune response against a pathogenic bacterium (e.g. a streptococcus pneumoniae or meningococcal bacterium) comprising administering to an individual an immunologically effective amount of any of the immunogenic compositions described herein comprising a bacterial antigen, e.g. a bacterial capsular polysaccharide derived from a pathogenic bacterium. One embodiment of the invention provides a method of protecting a subject from an infection by a pathogenic bacterium, or a method of preventing, treating or ameliorating an infectious disease or condition associated with a pathogenic bacterium, or a method of reducing the severity of or delaying the onset of at least one symptom associated with an infection by a pathogenic bacterium, in each case comprising administering to the subject an immunologically effective amount of any of the immunogenic compositions described herein comprising a bacterial antigen, e.g., a bacterial capsular polysaccharide derived from a pathogenic bacterium.

One embodiment of the invention provides a method of preventing, treating or ameliorating a bacterial infection, disease or disorder in a subject, the method comprising administering to the subject an immunologically effective amount of an immunogenic composition of the invention, wherein the immunogenic composition comprises an eTEC-linked glycoconjugate comprising a bacterial antigen (e.g., a bacterial capsular polysaccharide).

In some embodiments, the method of preventing, treating, or ameliorating a bacterial infection, disease, or disorder comprises human, veterinary, animal, or agricultural treatment. Another embodiment provides a method of preventing, treating or ameliorating a bacterial infection, disease or disorder associated with a pathogenic bacterium in an individual, the method comprising producing a polyclonal or monoclonal antibody preparation from an immunogenic composition described herein and using the antibody preparation to confer passive immunity to the individual. One embodiment of the present invention provides a method of preventing a bacterial infection in a subject undergoing a surgical procedure, said method comprising the step of administering to said subject prior to said surgical procedure a prophylactically effective amount of an immunogenic composition described herein.

In a preferred embodiment of each of the above methods, the pathogenic bacterium is a streptococcus pneumoniae or meningococcal bacterium, such as a streptococcus pneumoniae or neisseria meningitidis bacterium. In some such embodiments, the bacterial antigen is a capsular polysaccharide selected from Pn-serotype 1,3, 4,5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F capsular polysaccharides. In other such embodiments, the bacterial antigen is a capsular polysaccharide selected from Mn-serotype A, C, W135 and Y capsular polysaccharides.

The immune response to an antigen or immunogenic composition is characterized by the generation of a humoral and/or cell-mediated immune response in an individual to molecules present in the antigen or immunogenic composition of interest. For the purposes of the present invention, a "humoral immune response" is an antibody-mediated immune response and involves the introduction and generation of antibodies that recognize and bind with some affinity to antigens in the immunogenic compositions of the invention, while a "cell-mediated immune response" is an immune response mediated by T cells and/or other leukocytes. A "cell-mediated immune response" is induced by providing an epitope associated with a Major Histocompatibility Complex (MHC) class I or II molecule, CD1, or other atypical MHC-like molecules. This activates antigen-specific CD4+ T helper cells or CD8+ cytotoxic T lymphocytes ("CTLs"). CTLs are specific for peptide antigens that exhibit association with proteins encoded by classical or non-classical MHC and expressed on the surface of cells. CTLs help to induce and promote intracellular destruction of intracellular microorganisms, or lysis of cells infected with such microorganisms. Another aspect of cellular immunity relates to antigen-specific reactions by helper T cells. Helper T cells are used to help stimulate the function of, and focus on the activity of, non-specific effector cells against cells that display peptides or other antigens associated with typical or atypical MHC molecules on their surfaces. "cell-mediated immune response" also refers to the production of cytokines, chemokines and other such molecules produced by activated T cells and/or other leukocytes, including those derived from CD4+ and CD8+ T cells. The ability of a particular antigen or composition to stimulate a cell-mediated immune response can be determined by a variety of assays, for example by lymphoproliferation (lymphocyte activation) assays, CTL cytotoxic cell assays, by assaying for T-lymphocytes specific for the antigen in sensitized individuals, or by measuring cytokine production by T-cells in response to antigen restimulation. Such assays are well known in the art. See, e.g., Erickson et al (1993) J.Immunol.151: 4189-4199; and Doe et al (1994) Eur.J.Immunol.24: 2369-2376.

The immunogenic compositions and methods of the invention may be used in one or more of the following: (i) preventing infection or reinfection, as in a traditional vaccine, (ii) reducing the severity of symptoms or eliminating symptoms, and/or (iii) substantially or completely eliminating the pathogen or disorder of interest. Thus, treatment can be effected either prophylactically (prior to infection) or therapeutically (after infection). In the present invention, prophylactic treatment is a preferred mode. According to particular embodiments of the present invention, there are provided compositions and methods for treating a bacterial infection caused by, for example, streptococcus pneumoniae or neisseria meningitidis in a host individual, including prophylactically and/or therapeutically immunizing the host individual against a bacterial infection caused by, for example, streptococcus pneumoniae or neisseria meningitidis. The methods of the invention are useful for conferring prophylactic and/or therapeutic immunity to an individual. The methods of the invention may also be carried out on individuals for biomedical research applications.

The term "individual" as used herein means a human or non-human animal. More specifically, an individual refers to any animal classified as a mammal, including humans, domestic and farm animals, as well as research, zoo, sports, and pet companion animals, such as domestic pets and other domestic animals, including but not limited to cattle, sheep, ferrets, pigs, horses, rabbits, goats, dogs, cats, and the like. Preferred companion animals are dogs and cats. Preferably, the subject is a human.

The amount of a particular conjugate in a composition is typically calculated based on the total amount of polysaccharide (both conjugated and unconjugated) used for the conjugate. For example, a conjugate containing 20% free polysaccharide would contain about 80 μ g of conjugated polysaccharide and about 20 μ g of unconjugated polysaccharide in a 100 μ g polysaccharide dose. The effect of the protein on the conjugate is generally not considered when calculating the dose of the conjugate. The immunogenic amount of the conjugate or immunogenic composition can vary depending on the bacterial serotype. Typically, each dose comprises 0.1. mu.g to 100. mu.g, particularly 0.1. mu.g to 10. mu.g, more particularly 1. mu.g to 10. mu.g of polysaccharide. The immunogenic amounts of the different polysaccharide components in the immunogenic composition can vary and each can comprise 1 μ g, 2 μ g, 3 μ g, 4 μ g, 5 μ g, 6 μ g, 7 μ g, 8 μ g, 9 μ g, 10 μ g, 15 μ g, 20 μ g, 30 μ g, 40 μ g, 50 μ g, 60 μ g, 70 μ g, 80 μ g, 90 μ g, or about 100 μ g of any particular polysaccharide antigen.

The term "invasive disease" refers to the isolation of bacteria from a generally sterile site where relevant signs/symptoms of clinical disease are present. Typically sterile body sites include blood, CSF, pleural fluid, pericardial fluid, peritoneal fluid, joint/synovial fluid, bone, internal body sites (lymph nodes, brain, heart, liver, spleen, vitreous fluid, kidney, pancreas, ovary), or other typically sterile sites. Clinical conditions characterized by invasive disease include bacteremia, pneumonia, cellulitis, osteomyelitis, endocarditis, septic shock, and the like.

The effectiveness of an antigen as an immunogen can be measured by a proliferation assay, by a cytolytic assay (e.g., a chromium release assay to measure the ability of T cells to lyse their specific target cells), or by measuring the level of B cell activity (which is measured by measuring the level of circulating antibodies specific for the antigen in serum). As described herein, the immune response can also be detected by measuring the serum levels of antigen-specific antibodies induced following administration of the antigen, and more particularly, by measuring the ability of such induced antibodies to enhance the opsonophagocytic capacity of particular leukocytes. The level of protection of the immune response can be measured by challenging the immunized host with the administered antigen. For example, if the antigen for which an immune response is desired is a bacterium, the level of protection induced by an immunogenic amount of the antigen is measured by measuring the percent survival or percent death following challenge of the animal with the bacterial cells. In one embodiment, the protective amount can be measured by measuring at least one symptom associated with the bacterial infection (e.g., fever associated with the infection). The amount of each antigen in a multi-antigen or multi-component vaccine or immunogenic composition will vary with respect to each other component and can be determined by methods known to those skilled in the art. Such methods include procedures for measuring immunogenicity and/or in vivo efficacy.

In another aspect, the invention provides antibodies and antibody compositions that specifically and selectively bind to a capsular polysaccharide or glycoconjugate of the invention. In some such embodiments, the invention provides antibodies and antibody compositions that specifically and selectively bind to Pn-serotype 1,3, 4,5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, or 33F capsular polysaccharides or glycoconjugates comprising the same. In other such embodiments, the invention provides antibodies and antibody compositions that specifically and selectively bind to Mn-serotype A, C, W135 or Y capsular polysaccharide or glycoconjugates comprising the same. In some embodiments, the antibody is generated upon administration of a capsular polysaccharide or glycoconjugate of the invention to a subject. In some embodiments, the invention provides purified or isolated antibodies against one or more capsular polysaccharides or glycoconjugates of the invention. In some embodiments, the antibodies of the invention are functional as measured by the killing of bacteria in an animal efficacy model or by an opsonophagocytic killing assay. The antibodies or antibody compositions of the invention are useful in methods of treating or preventing a bacterial infection, disease or disorder associated with a pathogenic bacterium (e.g., a streptococcus pneumoniae or neisseria meningitidis bacterium) in an individual, comprising generating a polyclonal or monoclonal antibody preparation, and using the antibodies or antibody compositions to confer passive immunity to the individual. The antibodies of the invention may also be used in diagnostic methods, for example, to detect the presence of or to quantify the levels of capsular polysaccharides or glycoconjugates thereof. For example, the antibodies of the invention may also be used to detect the presence or quantify the level of Pn or Mn capsular polysaccharides or glycoconjugates thereof, wherein the glycoconjugates comprise bacterial capsular polysaccharides conjugated to a carrier protein by an eTEC spacer.

Several assays and animal models known in the art can be used to evaluate the efficacy of any of the immunogenic compositions described herein. For example, Chiavolini et al,Clin.Microbiol.Rev.(2008) 21(4):666-685) describe animal models of Streptococcus pneumoniae disease. Gorringe et al, METHODS IN MOLECULAR MEDINE, Vol.66 (2001), Chapter 17, Pollard and Maiden eds (Humana Press company) describe animal models for meningococcal disease.

Opsonophagocytic Activity (OPA) assay

OPA assay procedure was based on previous assays performed by Hu et al (Clin.Diagn.Lab.Immunol.2005; 12(2):287-95), with the following modifications. Heat inactivated serum was serially diluted 2.5-fold in buffer. Target bacteria were added to the assay plate and incubated at 25 ℃ for 30min on a shaker. Young rabbit complement (3-4 weeks old, Pel-Freez, 12.5% final concentration) and differentiated HL-60 cells were then added to each well at an approximate effector to target organ ratio of 200: 1. The assay plates were incubated at 37 ℃ for 45min on a shaker. To stop the reaction, 80 μ L of 0.9% NaCl was added to all wells, mixed, and 10 μ L aliquots were transferred to the wells of Millipore, MultiScreenHTSHV filter plates containing 200 μ L of water. The liquid was filtered through the plate under vacuum and 150 μL HySoy medium was added to each well and filtered. The filter plate was then filtered at 37 ℃ with 5% CO₂Incubate overnight and then fix with destaining solution (Bio-Rad). The plates were then stained with Coomassie Blue (Coomassie Blue) and destained once. Colonies were imaged and plated on Cellular Technology Limited (CTL) ImmunoSpotAnd (4) counting up. OPA antibody titers were interpolated from the reciprocal of two serum dilutions covering the point where the bacterial colony number decreased by 50% relative to the control well without immune serum.

The foregoing disclosure outlines the invention. A more complete understanding can be obtained by reference to the following specific examples. These examples are described for the purpose of illustration only and are not intended to limit the scope of the invention.

Examples

Example 1 general procedure for the preparation of eTEC-linked glycoconjugates

Sugar activation and thiolation with cystamine dihydrochloride

The sugars were reconstituted in anhydrous dimethyl sulfoxide (DMSO). The moisture content of the solution was determined by Karl Fischer (Karl Fischer) (KF) analysis and adjusted to achieve a moisture content of 0.1% to 0.4% (typically 0.2%).

To initiate activation, solutions of 1,1 '-carbonyl-bis-1, 2, 4-triazole (CDT) or 1, 1' -Carbonyldiimidazole (CDI) were freshly prepared in DMSO at a concentration of 100 mg/mL. The sugars were activated with varying amounts of CDT/CDI (1-10 molar equivalents) and the reaction was allowed to proceed at 23. + -. 2 ℃ for 1 hour. The level of activation can be determined by HPLC. Cystamine dihydrochloride was freshly prepared in anhydrous DMSO at a concentration of 50 mg/mL. The activated saccharide was reacted with 1 molar equivalent of cystamine dihydrochloride. Alternatively, the activated saccharide is reacted with 1 molar equivalent of cysteamine hydrochloride. The thiolation reaction was allowed to proceed at 23. + -. 2 ℃ for 21. + -.2 hours to yield the thiolated sugar. The level of thiolation was determined by the amount of CDT/CDI added.

The residual CDT/CDI in the activation reaction solution was quenched by the addition of 100mM sodium tetraborate pH9.0 solution. Calculations were performed to determine the amount of tetraborate added and to adjust the final moisture content to a total moisture content of at most 1-2%.

Reduction and purification of activated thiolated sugars

The thiolated sugar reaction mixture was diluted 10-fold by addition to pre-cooled 5mM sodium succinate in 0.9% saline (pH6.0) and filtered through a 5 μm filter. Diafiltration of the thiolated sugar was performed against a 40 diafiltration volume (diavolme) of WFI. To the retentate was added a solution of tris (2-carboxyethyl) phosphine (TCEP) (1-5 molar equivalents) after dilution by 10% volume of 0.1M sodium phosphate buffer (pH 6.0). This reduction was allowed to proceed at 5. + -. 3 ℃ for 20. + -. 2 hours. The purification of the activated thiolated sugar is preferably performed by ultrafiltration/diafiltration against pre-cooled 10mM sodium dihydrogen phosphate (pH 4.3). Alternatively, the thiolated saccharide is purified by standard Size Exclusion Chromatography (SEC) procedures or ion exchange chromatography methods. Aliquots of the activated thiolated sugar retentate were aspirated (pull) to determine sugar concentration and thiol content (Ellman) measurements.

Alternative reduction and purification of activated thiolated saccharides

As an alternative to the purification operation described above, the activated thiolated sugar is also purified as follows.

To the thiolated sugar reaction mixture was added a solution of tris (2-carboxyethyl) phosphine (TCEP) (5-10 molar equivalents) and allowed to proceed at 23 ± 2 ℃ for 3 ± 1 hour. The reaction mixture was then diluted 5-fold by addition to pre-cooled 5mM sodium succinate in 0.9% saline (pH6.0) and filtered through a 5 μm filter. Diafiltration of the thiolated sugar was performed using 40 diafiltration volumes of pre-cooled 10mM sodium dihydrogen phosphate (pH 4.3). Aliquots of the activated thiolated sugar retentate were aspirated to determine sugar concentration and thiol content (Ellman) measurements.

Activation and purification of bromoacetylated carrier proteins

The free amino group of the carrier protein is bromoacetylated by reaction with a bromoacetylating agent, such as N-hydroxysuccinimide ester of bromoacetic acid (BAANS), bromoacetyl bromide, or another suitable reagent.

The carrier protein (pH 8.0. + -. 0.2 in 0.1M sodium phosphate) was first maintained at 8. + -. 3 ℃ around pH7 before activation. N-hydroxysuccinimide ester of bromoacetic acid (BAANS) was added to the protein solution as a stock solution of dimethyl sulfoxide (DMSO) (20mg/mL) at a ratio of 0.25-0.5BAANS: protein (w/w). The reaction was gently mixed for 30-60 minutes at 5. + -. 3 ℃. The resulting bromoacetylated (activated) protein is purified, for example, by ultrafiltration/diafiltration using a 10kDa MWCO membrane using 10mM phosphate (pH 7.0) buffer. After purification, the protein concentration of the bromoacetylated carrier protein was estimated by Lowry protein assay.

The extent of activation was determined by total bromide determination by ion exchange liquid chromatography (ion chromatography) coupled with suppressed conductivity detection. Bound bromide on the activated bromoacetylated protein is cleaved from the protein in the assay sample preparation and quantified along with any free bromide that may be present. Any remaining covalently bound bromide on the protein is released by heating the sample in basic 2-mercaptoethanol to convert it to ionic bromide.

₁₉₇Activation and purification of bromoacetylated CRM

CRM with 10mM phosphate buffered 0.9% NaCl pH 7(PBS)₁₉₇Diluted to 5mg/mL and made into 0.1M NaHCO using 1M stock solution₃The pH was 7.0. BAANS stock solution using 20mg/mL DMSO, in CRM₁₉₇BAANS 1:0.35(w: w) ratio BAANS was added. The reaction mixture was incubated at 3 ℃ to 11 ℃ for 30 minutes to 1 hour and then purified by ultrafiltration/diafiltration using a 10K MWCO membrane and 10mM sodium phosphate/0.9% NaCl (pH 7.0). Purified activated CRM as determined by Lowry assay₁₉₇To determine protein concentrationThen, the solution was diluted to 5mg/mL with PBS. Sucrose was added as a cryoprotectant to 5% wt/vol and the activated protein was frozen at-25 ℃ and stored until conjugation was required.

CRM₁₉₇The bromoacetylation of the lysine residues of (a) is very consistent, making 15-25 lysines from 39 lysines available for activation. The reaction produces high yields of activated protein.

Conjugation of activated thiolated saccharides to bromoacetylated carrier proteins

The reaction vessel was pre-cooled to 5 ℃ before the conjugation reaction was started. The bromoacetylated carrier protein and activated thiolated sugar are then added and mixed with agitation at 150-200 rpm. The sugar/protein input ratio was 0.9. + -. 0.1. The reaction pH was adjusted to 8.0. + -. 0.1 with 1M NaOH solution. The conjugation reaction was allowed to proceed for 20. + -.2 hours at 5 ℃.

Capping of residual reactive functional groups

Unreacted bromoacetylated residues on the carrier protein were quenched by reaction with 2 molar equivalents of N-acetyl-L-cysteine as capping reagent at 5 ℃ for 3 hours. The residual free thiol groups were capped with 4 molar equivalents of Iodoacetamide (IAA) for 20 hours at 5 ℃.

Purification of eTEC-linked glycoconjugates

The conjugation reaction (IAA capped) mixture was filtered through a 0.45 μm filter. Ultrafiltration/diafiltration of the glycoconjugate was performed against 5mM succinate-0.9% saline (pH 6.0). The glycoconjugate retentate was then filtered through a 0.2 μm filter. Aliquots of glycoconjugates were aspirated for assay. The remaining glycoconjugates were stored at 5 ℃.

Example 2 preparation of Pn-33F eTEC conjugates

Activation method

Pn33F activation of polysaccharides

Pn-33F polysaccharide was complexed with 500mM 1,2, 4-triazole (in WFI) to obtain 10 grams of triazole per gram of polysaccharide. The mixture was shell frozen in a dry ice-ethanol bath and then lyophilized to dryness. The lyophilized 33F polysaccharide was reconstituted in anhydrous dimethyl sulfoxide (DMSO). The moisture content of the lyophilized 33F/DMSO solution was determined by Karl Fischer (KF) analysis. The moisture content was adjusted by adding WFI to the 33F/DMSO solution to reach a moisture content of 0.2%.

To initiate activation, a 100mg/mL DMSO solution of 1, 1' -carbonyl-bis-1, 2, 4-triazole (CDT) was freshly prepared. Pn33F polysaccharide was activated with varying amounts of CDT prior to the thiolation step. CDT activation was performed at 23. + -. 2 ℃ for 1 hour. The level of activation was determined by HPLC (A220/A205). A 100mM sodium tetraborate solution (pH9.0) was added to quench any residual CDT in the activation reaction solution. Calculations were performed to determine the amount of tetraborate added and to bring the final moisture content to a total moisture content of 1.2%. The reaction was allowed to proceed at 23. + -. 2 ℃ for 1 hour.

Thiolation of activated Pn-33F polysaccharides

Cystamine-hydrochloride was freshly prepared in anhydrous DMSO and 1 molar equivalent of cystamine dihydrochloride was added to the activated polysaccharide reaction solution. The reaction was allowed to proceed at 23. + -. 2 ℃ for 21. + -. 2 hours. The thiolated sugar solution was diluted 10-fold by addition to pre-cooled 5mM sodium succinate in 0.9% saline (pH 6.0). The diluted reaction solution was filtered through a 5 μm filter. Diafiltration of thiolated Pn-33F polysaccharide using water for injection (WFI) was performed using 100K MWCO ultrafiltration membrane cartridges.

The thiolation level of the activated Pn-33F polysaccharide as a function of the molar equivalent of CDT is shown in FIG. 8.

Reduction and purification of activated thiolated Pn-33F polysaccharides

To the retentate was added a solution of tris (2-carboxyethyl) phosphine (TCEP) (5 molar equivalents) after dilution by 10% volume of 0.1M sodium phosphate buffer (pH 6.0). This reduction was allowed to proceed at 23. + -. 2 ℃ for 2. + -. 1 hours. Diafiltration of thiolated 33F polysaccharide was performed using 100KMWCO ultrafiltration membrane cartridges. Diafiltration was performed against pre-cooled 10mM sodium phosphate (pH 4.3). The thiolated 33F polysaccharide retentate was absorbed for both sugar concentration and thiol (Ellman) assays.

Alternative reduction and purification of activated thiolated Pn-33F polysaccharides

As an alternative to the purification operation described above, the 33F activated thiolated sugar was also purified as follows.

To the thiolated sugar reaction mixture was added a solution of tris (2-carboxyethyl) phosphine (TCEP) (5 molar equivalents) and allowed to proceed at 23 ± 2 ℃ for 3 ± 1 hour. The reaction mixture was then diluted 5-fold by addition to pre-cooled 5mM sodium succinate in 0.9% saline (pH6.0) and filtered through a 5 μm filter. Diafiltration of thiolated sugars was performed using 40 diafiltration volumes of pre-cooled 10mM sodium dihydrogen phosphate (pH4.3) and 100K MWCO ultrafiltration membrane cartridges. The thiolated 33F polysaccharide retentate was absorbed for both sugar concentration and thiol (Ellman) assays. A flow chart of the activation method is provided in fig. 7 (a).

Conjugation process

₁₉₇Conjugation of thiolated Pn33F polysaccharide to Bromoacetylated CRM

CRM by bromoacetylation as described in example 1₁₉₇The carrier protein is activated separately and then reacted with the activated Pn-33F polysaccharide for the conjugation reaction. The reaction vessel was pre-cooled to 5 ℃ before the conjugation reaction was started. Acetylating bromine into CRM₁₉₇And the thiolated 33F polysaccharide are mixed together in the reaction vessel at an agitation speed of 150-200 rpm. The sugar/protein input ratio was 0.9. + -. 0.1. The reaction pH was adjusted to 8.0-9.0. The conjugation reaction was allowed to proceed for 20. + -.2 hours at 5 ℃.

₁₉₇Bromoacetylated CRM and capping of reactive groups on thiolated Pn33F polysaccharide

CRM by₁₉₇Capping of unreacted bromoacetylated residues on proteins: with 2 molar equivalents of N-acetyl-L-cysteine at 5 ℃ for 3 hoursAny residual free thiol groups of the thiolated 33F-polysaccharide were then capped with 4 molar equivalents of Iodoacetamide (IAA) at 5 ℃ for 20 hours.

Purification of eTEC-linked Pn-33F glycoconjugates

The conjugation solution was filtered through a 0.45 μm or 5 μm filter. Diafiltration of the 33F glycoconjugate was performed using 300K MWCO ultrafiltration membrane cartridges. Diafiltration was performed against 5mM succinate-0.9% saline (pH 6.0). The Pn-33F glycoconjugate 300K retentate was then filtered through a 0.22 μm filter and stored at 5 ℃.

A flow chart of the conjugation process is provided in fig. 7 (B).

Results

The reaction parameters and characterization data for several lots of Pn-33F eTEC glycoconjugates are shown in Table 2. CDT activation-thiolation with cystamine dihydrochloride produced glycoconjugates with 63% -90% saccharide yields and < 1% -13% free saccharide.

TABLE 2 Experimental parameters and characterization data for Pn33F eTEC conjugates

₁₉₇OPA titres of glycoconjugates of Pn-33F eTEC with CRM

Pn-33F OPA titers were determined in mice under standard conditions. OPA titers (GMT with 95% CI) at 4 and 7 weeks are shown in table 3, confirming that serotype 33F Pn glycoconjugate elicits OPA titers in the murine immunogenicity model.

TABLE 3 Pn-33F OPA potency (GMT with 95% CI)

Example 3 preparation of Pn-22F eTEC conjugate

Activation method

Activation of Pn-22F polysaccharides

Pn-22F polysaccharide was complexed with 500mM 1,2, 4-triazole (in WFI) to obtain 10 grams of triazole per gram of polysaccharide. The mixture was shell frozen in a dry ice-ethanol bath and then lyophilized to dryness. The lyophilized 22F polysaccharide was reconstituted in anhydrous dimethyl sulfoxide (DMSO). The moisture content of the lyophilized 22F/DMSO solution was determined by Karl Fischer (KF) analysis. The moisture content was adjusted by adding WFI to the Pn-22F/DMSO solution to achieve a moisture content of 0.2%.

To initiate activation, a 100mg/mL DMSO solution of 1, 1' -carbonyl-bis-1, 2, 4-triazole (CDT) was freshly prepared. Pn-22F polysaccharide was activated with varying amounts of CDT and subsequently thiolated with 1 molar equivalent of cystamine dihydrochloride. CDT activation was performed at 23. + -. 2 ℃ for 1 hour. The level of activation was determined by HPLC (A220/A205). A100 mM sodium tetraborate solution (pH9.0) was added to quench any residual CDT in the activation reaction solution. Calculations were performed to determine the amount of tetraborate added and to bring the final moisture content to a total moisture content of 1.2%. The reaction was allowed to proceed at 23. + -. 2 ℃ for 1 hour.

Thiolation of activated Pn-22F polysaccharides

Cystamine-dihydrochloride was freshly prepared in anhydrous DMSO and added to the reaction solution. The reaction was allowed to proceed at 23. + -. 2 ℃ for 21. + -. 2 hours. The thiolated sugar solution was diluted 10-fold by addition to pre-cooled 5mM sodium succinate in 0.9% saline (pH 6.0). The diluted reaction solution was filtered through a 5 μm filter. Diafiltration of thiolated Pn-22F polysaccharide using water for injection (WFI) was performed using 100K MWCO ultrafiltration membrane cartridges.

Reduction and purification of activated thiolated Pn-22F polysaccharide

To the retentate was added a solution of tris (2-carboxyethyl) phosphine (TCEP) (5-10 molar equivalents) after dilution by 10% volume of 0.1M sodium phosphate buffer (pH 6.0). This reduction was allowed to proceed at 23. + -. 2 ℃ for 2. + -. 1 hours. Diafiltration of thiolated 22F polysaccharide was performed using 100KMWCO ultrafiltration membrane cartridges. Diafiltration was performed against pre-cooled 10mM sodium phosphate (pH 4.3). The thiolated 22F polysaccharide retentate was absorbed for both sugar concentration and thiol (Ellman) measurements.

Conjugation, capping and purification of Pn-22F eTEC glycoconjugates

Activated thiolated Pn22F polysaccharide with activated CRM according to the method described in example 2₁₉₇Conjugation, capping and purification of Pn-22F eTEC glycoconjugate.

Results

Representative Pn-22F eTEC and CRM₁₉₇The characterization and method data for glycoconjugates of (a) are provided in table 4.

TABLE 4 Experimental parameters and characterization data for Pn-22F eTEC conjugates

₁₉₇Example 4 preparation of Pn-10A eTEC conjugates bound to CRM

Preparation of Pn-10A eTEC glycoconjugates

Preparation according to the method described in example 2 comprising conjugation to CRM via eTEC spacer₁₉₇A glycoconjugate of streptococcus pneumoniae capsular polysaccharide serotype 10A (Pn-10A).

Characterization of Pn-10A eTEC glycoconjugates

Representative Pn-10A eTEC with CRM₁₉₇The characterization and method data for glycoconjugates of (a) are provided in table 5.

TABLE 5 Experimental parameters and characterization data for Pn-10A glycoconjugates

Pn-10A OPA potency

Determination of binding to CRM in mice under standard conditions₁₉₇The OPA titer of Pn-10A eTEC conjugate. OPA titers as a function of dose are shown in table 6. The OPA titer of the conjugate was significantly higher than that of the unconjugated serotype 10A polysaccharide.

TABLE 6 Pn-10A OPA potency (GMT with 95% CI)

10A Pn variants	0.001μg	0.01μg	0.1μg
				Pn-10A eTEC conjugates	691(389,1227)	1208(657,2220)	3054(1897,4918)
Unconjugated PS			602(193,1882)

Example 5 binding to CRM₁₉₇Preparation of Pn-11A eTEC conjugates of (A)

Preparation of Pn-11A eTEC glycoconjugates

Preparation according to the method described in example 2 comprising conjugation to CRM via eTEC spacer₁₉₇A glycoconjugate of streptococcus pneumoniae capsular polysaccharide serotype 11A (Pn-11A).

Characterization of Pn-11A eTEC glycoconjugates

Representative Pn-11A eTEC with CRM₁₉₇The characterization and method data for glycoconjugates of (a) are provided in table 7.

TABLE 7 Experimental parameters and characterization data for Pn-11A glycoconjugates

Pn-11A OPA potency

Determination of binding to CRM in mice under standard conditions₁₉₇The OPA potency of Pn-11A eTEC conjugate of (a). OPA titers as a function of dose are shown in table 8.

TABLE 8 Pn-11A OPA potency (GMT with 95% CI)

11A Pn variants	0.001μg	0.01μg	0.1μg
				Pn-11A eTEC conjugates	206(166,256)	906(624,1316)	5019(3648,6904)

Example 6 binding to CRM₁₉₇Preparation of Pn-33F RAC/aqueous conjugates of (A)

Preparation of Pn-33F RAC/aqueous glycoconjugates

Pn-33F glycoconjugates are prepared using reductive amination (RAC/aqueous) in an aqueous phase that has been successfully used to produce streptococcus pneumoniae conjugate vaccines (see, e.g., WO 2006/110381). This method comprises two steps. The first step is oxidation of the polysaccharide to form aldehyde functionality from the vicinal diol. The second step is conjugation of the activated polysaccharide to CRM₁₉₇Lysine (Lys) residue of (a).

Briefly, frozen polysaccharides were thawed and purified by adding varying amounts of sodium periodate (NaIO)₄) The oxidation was carried out in sodium phosphate buffer at pH 6.0. Concentration and diafiltration of the activated polysaccharide was performed and the purified activated polysaccharide was stored at 4 ℃. Reacting the activated polysaccharide with CRM₁₉₇And (4) protein compounding. Thoroughly mixing polysaccharide and CRM₁₉₇The bottles were then placed in a dry ice/ethanol bath, followed by polysaccharide/CRM₁₉₇The mixture was lyophilized. The lyophilized mixture was reconstituted in 0.1M sodium phosphate buffer. The conjugation reaction was initiated by adding 1.5 molar equivalents of sodium cyanoborohydride and incubating at 23 ℃ for 20hr and at 37 ℃ for another 44 hr. The reaction was diluted with 1 × volume of 0.9% brine and capped with 2 molar equivalents of sodium borohydride at 23 ℃ for 3 hr. The reaction mixture was diluted with 1 × volume of 0.9% brine, then filtered through a 0.45 μm filter, followed by purification. Concentration and diafiltration of the conjugate was performed using 100KMWCO ultrafiltration membrane cartridges.

Several conjugates were obtained using the above method by varying different parameters (e.g. pH, reaction temperature and concentration of polysaccharide).

Typical polysaccharide yields for these conjugates are about 50% and free sugars 15%, with conjugate molecular weights in the range of 2000-3500 kDa.

However, the native serotype 33F polysaccharide bears O-acetyl groups at C2 of its 5-galactofuranosyl residue, and it was found that about 80% of the acetyl functional groups were removed using reductive amination in the aqueous phase throughout the conjugation process. It was observed that the O-acetyl group on the five-membered ring structure (5-galactofuranoside) could migrate and could be easily removed using reductive amination chemistry in the aqueous phase.

Evaluation of Pn-33F RAC/aqueous glycoconjugate stability

Aliquots of representative RAC/aqueous conjugates prepared by the methods described above were dispensed into polypropylene tubes. The tubes were stored at 25 ℃ or 37 ℃ and stability was monitored for up to 3.5 months. At each stability time point, the% free sugar level was assessed. Stability data at both temperatures are summarized in table 9. As shown in table 9, the% free sugar levels increased significantly at 25 ℃ and 37 ℃. The increase in% free sugar levels during storage is a potential indicator of polysaccharide degradation occurring in the conjugate.

Table 9: stability data for RAC/aqueous conjugates at 25 ℃ and 37 ℃

wk is week; and M is month.

Although serotype 33F polysaccharide was successfully activated by reaction with sodium periodate and subsequently conjugated to CRM using aqueous reductive amination chemistry₁₉₇However, the combination of the% free saccharide stability results under accelerated conditions with the inability to retain the acetyl functionality (a key polysaccharide epitope for immunogenicity) during conjugation indicates that R isThe AC/aqueous approach is not the optimal approach for serotype 33F conjugation.

Example 7 binding to CRM₁₉₇Preparation of Pn-33F RAC/DMSO conjugate

Preparation of Pn-33F RAC/DMSO glycoconjugates

Conjugation by reductive amination in DMSO (RAC/DMSO) generally has a significantly lower chance of de-O-acetylation relative to the RAC/aqueous approach. In view of the challenges associated with the retention of O-acetyl functionality using the RAC/aqueous approach described in example 6, an alternative approach to using RAC/DMSO solvents, which has been successfully used to generate streptococcus pneumoniae conjugate vaccines, was evaluated (see, e.g., WO 2006/110381).

The activated polysaccharide was complexed with sucrose (50% w/v in WFI) using a ratio of 25 grams of sucrose per gram of activated polysaccharide. The components were mixed well and then shell frozen in a dry ice/ethanol bath. The shell-shaped frozen vials of the composite mixture were then lyophilized to dryness.

The lyophilized activated polysaccharide was reconstituted in dimethyl sulfoxide (DMSO). Addition of DMSO to lyophilized CRM₁₉₇For rehabilitation. Combining reconstituted activated polysaccharide with reconstituted CRM₁₉₇Combined in a reaction vessel. By mixing NaCNBH₃Addition to the reaction mixture to initiate conjugation. The reaction was incubated at 23 ℃ for 20 hr. By adding NaBH₄Termination of the conjugation (capping) reaction was achieved and the reaction was continued for an additional 3 hr. The reaction mixture was diluted with 4 volumes of 5mM succinate-0.9% saline pH6.0 buffer, then filtered through a 5 μm filter, followed by purification. Concentration and diafiltration of the conjugate was performed using a 100K MWCO membrane. Diafiltration was performed against 40 diafiltration volumes of 5mM succinate-0.9% saline pH6.0 buffer. The retentate was filtered through 0.45 μm and 0.22 μm filters and analyzed.

Several conjugates were obtained using the above method by varying different parameters (e.g. sugar-protein input ratio, reaction concentration, molar equivalent of sodium cyanoborohydride and water content). All data generated from conjugates prepared by the RAC/DMSO method proved superior to the RAC/aqueous method and allowed the preparation of conjugates with good conjugation yields, low% free sugar (< 5%) and higher degree of conjugation (conjugated lysine). In addition, more than 80% of the acetyl functionality can be retained throughout the RAC/DMSO conjugation process.

Evaluation of Pn-33F RAC/DMSO glycoconjugate stability

Aliquots of representative RAC/DMSO conjugates prepared by the methods described above were dispensed into polypropylene tubes, which were stored at 4 ℃ or 25 ℃ and the stability of the free sugars was monitored for 3 months. As shown in table 10, the samples stored at 4 ℃ showed a 4.8% increase in free sugar within 3 months. However, the samples stored at 25 ℃ showed a 15.4% increase in% free sugar within 3 months. The increase in% free saccharide in the RAC conjugate was attributed to degradation of the conjugate, especially at 25 ℃.

TABLE 10 stability results of RAC/DMSO conjugates at 4 ℃ and 25 ℃

wk is week; and M is month.

The stability of another batch of RAC/DMSO conjugate was also investigated at 4 deg.C, 25 deg.C and 37 deg.C. Aliquots were dispensed into polypropylene tubes and monitored for potential trends in% free sugar. As shown in table 11, the samples stored at 4 ℃ showed a 4.7% increase in free sugar% over 2 months. The increase in free sugar at 25 ℃ and 37 ℃ was significantly higher, indicating potential degradation of the conjugate.

TABLE 11 stability results of RAC/DMSO conjugates at 4 deg.C, 25 deg.C and 37 deg.C

wk is week; and M is month.

Even though the conjugate generated by the RAC/DMSO method retained the O-acetyl group, an increase in% free sugar and the potential instability described above was observed using this approach, especially at 25 ℃. In view of this observation of the potential instability of RAC/DMSO conjugates, RAC/DMSO was not considered the most suitable for serotype 33F conjugation and an alternative chemical route was developed to generate more stable conjugates (eTEC conjugates).

Example 8 preparation of other Pn-33F eTEC conjugates

Other Pn-33F eTEC conjugates were generated using the method described in example 2. The reaction parameters and characterization data for these other batches of Pn-33FeTEC glycoconjugates are shown in table 12.

TABLE 12 Experimental parameters and characterization data for other Pn33F eTEC conjugates

LOQ is the limit of quantitation.

As shown above and in table 12, several Pn33F conjugates were obtained using the eTEC conjugation described above. eTEC chemistry allows the preparation of conjugates with high yield, low% free sugar and high degree of conjugation (conjugated lysine). In addition, more than 80% of the acetyl functional groups can be retained using the eTEC conjugation method.

Example 9 evaluation of Pn-33F eTEC glycoconjugate stability: tendency to free sugar%

Aliquots of conjugate batch 33F-2B (see Table 2) were dispensed into polypropylene tubes and stored at 4 deg.C, 25 deg.C and 37 deg.C, respectively, and the trend for% free sugar was monitored. Data (% free sugar) are shown in table 13. As shown in this table, the% free sugar did not change significantly.

TABLE 13% stability of Pn-33F eTEC saccharide conjugates at 4 deg.C, 25 deg.C and 37 deg.C for free saccharide

wk is week; and M is month.

Another batch of conjugate (batches 33F-3C) was also tested for accelerated stability at 37 ℃ for up to 1 month. As shown in table 14, the% free sugar did not change significantly for up to 1 month at 37 ℃.

TABLE 14% stability of Pn-33F eTEC saccharide conjugates at 37 ℃ in free saccharide

To further confirm the stability of the eTEC conjugates, potential trends in% free sugar stored at 4 ℃ for other conjugate batches (33F-3C and 33F-5E (see tables 2 and 12)) were monitored for up to about 1 year. As shown in table 15, the% free sugar levels of the conjugates stored at 4 ℃ did not change significantly over an extended period of up to about 1 year.

TABLE 15% stability results for Pn-33F eTEC saccharide conjugates at 4 deg.C for free saccharide

M is equal to month

The serotype 33F conjugate generated by 33F eTEC chemistry proved significantly more stable than RAC/aqueous and RAC/DMSO conjugates, with no significant degradation, as monitored by the trend of free sugars at each temperature (real-time and accelerated).

All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Although the present invention has been described in considerable detail by way of illustration and example for purposes of clarity of understanding, certain changes and modifications may be practiced within the scope of the appended claims.

Claims

1. A method of preparing a glycoconjugate comprising a saccharide conjugated to a carrier protein through a (2- ((2-oxoethyl) thio) ethyl) carbamate (eTEC) spacer, comprising the steps of:

a) reacting a saccharide with 1,1 '-carbonyl-bis- (1,2, 4-triazole) (CDT) or 1, 1' -Carbonyldiimidazole (CDI) in an organic solvent to produce an activated saccharide;

b) reacting the activated saccharide with cystamine or cysteamine or a salt thereof to produce a thiolated saccharide;

c) reacting the thiolated saccharide with a reducing agent to produce an activated thiolated saccharide comprising one or more free sulfhydryl residues;

d) reacting the activated thiolated saccharide with an activated carrier protein comprising one or more α -haloacetamido groups to produce a thiolated saccharide-carrier protein conjugate, and

e) reacting the thiolated saccharide-carrier protein conjugate with (i) a first capping reagent capable of capping unconjugated α -haloacetamido groups of the activated carrier protein and/or (ii) a second capping reagent capable of capping unconjugated free thiol residues;

thereby producing an eTEC-linked glycoconjugate,

wherein the saccharide is a bacterial capsular polysaccharide.

2. The method of claim 1, wherein the capping step e) comprises reacting the thiolated saccharide-carrier protein conjugate with (i) N-acetyl-L-cysteine as a first capping reagent and/or (ii) iodoacetamide as a second capping reagent.

3. The method of claim 1, further comprising the step of complexing the saccharide with triazole or imidazole by reaction to provide a complexed saccharide, wherein the complexed saccharide is shell-frozen, lyophilized and reconstituted in an organic solvent prior to step a).

4. The process of claim 1, further comprising purifying the thiolated polysaccharide produced in step c), wherein the purification step comprises diafiltration.

5. The method of claim 1, wherein the reducing agent in step c) is tris (2-carboxyethyl) phosphine (TCEP), Dithiothreitol (DTT), or mercaptoethanol.

6. The method of claim 1, wherein the carrier protein is activated with an activated bromoacetic acid derivative.

7. The process of claim 6, wherein the bromoacetic acid derivative is N-hydroxysuccinimide ester of bromoacetic acid (BAANS).

8. The method of claim 1, wherein the method further comprises purifying the eTEC-linked glycoconjugate by diafiltration.

9. The process according to claim 1, wherein the organic solvent in step a) is a polar aprotic solvent selected from dimethyl sulfoxide (DMSO), Dimethylformamide (DMF), Dimethylacetamide (DMA), N-methyl-2-pyrrolidone (NMP), acetonitrile, 1, 3-dimethyl-3, 4,5, 6-tetrahydro-2 (1H) -pyrimidinone (DMPU) and Hexamethylphosphoramide (HMPA) or mixtures thereof.

10. The method according to claim 1, wherein the ratio of sugar to carrier protein (w/w) is 0.2-4.

11. The method according to claim 10, wherein the ratio of saccharide to carrier protein (w/w) is 0.4-1.7.

12. The method of any one of claims 1-11, wherein the saccharide is a capsular polysaccharide derived from streptococcus pneumoniae (s.

13. The method of claim 12, wherein the capsular polysaccharide is selected from pneumococcal (Pn) serotype 1,3, 4,5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F capsular polysaccharides.

14. The method of claim 13, wherein the capsular polysaccharide is a Pn-serotype 33F capsular polysaccharide.

15. The method of claim 13, wherein the capsular polysaccharide is a Pn-serotype 22F capsular polysaccharide.

16. The method of claim 13, wherein the capsular polysaccharide is a Pn-serotype 10A capsular polysaccharide.

17. The method of claim 13, wherein the capsular polysaccharide is a Pn-serotype 11A capsular polysaccharide.

18. The method according to any one of claims 1-11, wherein the saccharide is a capsular polysaccharide derived from neisseria meningitidis (n.meningitis).

19. The glycoconjugate of claim 18 wherein the capsular polysaccharide is selected from meningococcal (Mn) -serotype A, C, W135 and Y capsular polysaccharide.

20. The method of any one of claims 1-11, wherein the carrier protein is CRM₁₉₇。

21. A glycoconjugate produced by the method of any one of claims 1-20.

22. An immunogenic composition comprising the glycoconjugate of claim 21 and a pharmaceutically acceptable excipient, carrier or diluent.

23. A glycoconjugate comprising a bacterial capsular polysaccharide conjugated to a carrier protein through an eTEC spacer, wherein the polysaccharide is covalently linked to the eTEC spacer through a carbamate linkage, and wherein the carrier protein is covalently linked to the eTEC spacer through an amide linkage.

24. The glycoconjugate of claim 23, wherein the capsular polysaccharide is derived from streptococcus pneumoniae.

25. The glycoconjugate of claim 24 wherein the capsular polysaccharide is selected from Pn-serotype 1,3, 4,5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F capsular polysaccharides.

26. The glycoconjugate of claim 25, wherein the capsular polysaccharide is a Pn-serotype 33F capsular polysaccharide.

27. The glycoconjugate of claim 25, wherein the capsular polysaccharide is a Pn-serotype 22F capsular polysaccharide.

28. The glycoconjugate of claim 25, wherein the capsular polysaccharide is a Pn-serotype 10A capsular polysaccharide.

29. The glycoconjugate of claim 25, wherein the capsular polysaccharide is a Pn-serotype 11A capsular polysaccharide.

30. The glycoconjugate of claim 23, wherein the capsular polysaccharide is derived from neisseria meningitidis.

31. The glycoconjugate of claim 30 wherein the capsular polysaccharide is selected from Mn-serotype A, C, W135 and Y capsular polysaccharide.

32. The glycoconjugate of any one of claims 23-31 wherein the polysaccharide has a molecular weight of 10kDa-2,000 kDa.

33. The glycoconjugate of claim 32 wherein the polysaccharide has a molecular weight of 50kDa-2,000 kDa.

34. The glycoconjugate of any one of claims 23-31 wherein the glycoconjugate has a molecular weight of 50kDa-20,000 kDa.

35. The glycoconjugate of claim 34 wherein the glycoconjugate has a molecular weight of 500kDa-10,000 kDa.

36. The glycoconjugate of any one of claims 23-31 wherein the polysaccharide has a degree of O-acetylation of 75-100%.

37. The glycoconjugate of any one of claims 23-31 wherein the carrier protein is CRM₁₉₇。

38. The glycoconjugate of claim 37, wherein the CRM is₁₉₇Comprising 2-20 lysine residues covalently linked to the polysaccharide through an eTEC spacer.

39. The glycoconjugate of claim 37, wherein the CRM is₁₉₇Comprising 4-16 lysine residues covalently linked to the polysaccharide through an eTEC spacer.

40. The glycoconjugate of any one of claims 23-31 wherein the ratio of saccharide to carrier protein (w/w) is 0.2-4.

41. The glycoconjugate of claim 40 wherein the saccharide to carrier protein ratio (w/w) is 0.4-1.7.

42. The glycoconjugate of claim 37, wherein at least one CRM occurs at every 25 saccharide repeat units of the saccharide₁₉₇And a sugar.

43. The glycoconjugate of claim 37, wherein at least one CRM occurs at every 15 saccharide repeat units of the saccharide₁₉₇And a sugar.

44. The glycoconjugate of claim 37, wherein at least one CRM occurs at every 10 saccharide repeat units of the saccharide₁₉₇And a sugar.

45. The glycoconjugate of claim 37, wherein at least one CRM occurs at every 4 saccharide repeat units of the saccharide₁₉₇And a sugar.

46. The glycoconjugate of any one of claims 23-31 comprising less than 15% free saccharide relative to the total amount of saccharide.

47. The glycoconjugate of any one of claims 23-31 having a molecular size distribution (K) at ≤ 0.3 ≥ 35 ≥_d)。

48. An immunogenic composition comprising the glycoconjugate of any one of claims 23-47 and a pharmaceutically acceptable excipient, carrier or diluent.

49. The immunogenic composition of claim 48, further comprising an additional antigen.

50. The immunogenic composition of claim 49, wherein the additional antigen comprises a protein antigen or a glycoconjugate derived from capsular polysaccharide of Streptococcus pneumoniae.

51. The immunogenic composition of claim 50, wherein the additional antigen comprises a glycoconjugate of a capsular polysaccharide selected from Pn-serotype 1,3, 4,5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F capsular polysaccharides.

52. The immunogenic composition of claim 49, wherein the additional antigen comprises a protein antigen or glycoconjugate derived from capsular polysaccharide of Neisseria meningitidis.

53. The immunogenic composition according to claim 52, wherein the additional antigen comprises a glycoconjugate of a capsular polysaccharide selected from serotype A, C, W135 and Y capsular polysaccharide.

54. The immunogenic composition of any one of claims 48-53, further comprising an adjuvant.

55. The immunogenic composition of claim 54, wherein the adjuvant is an aluminum-based adjuvant.

56. The immunogenic composition of claim 55, wherein the aluminum-based adjuvant is selected from the group consisting of aluminum phosphate, aluminum sulfate, and aluminum hydroxide.

57. Use of the immunogenic composition of any one of claims 48-56 in the manufacture of a medicament for preventing, treating, or ameliorating a bacterial infection, disease, or disorder in a subject.

58. The use of claim 57, wherein the infection, disease or disorder is associated with Streptococcus pneumoniae bacteria, and the glycoconjugate comprises a capsular polysaccharide selected from Pn-serotype 1,3, 4,5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F capsular polysaccharides.

59. The use of claim 57, wherein the infection, disease or disorder is associated with Neisseria meningitidis bacteria and the glycoconjugate comprises a capsular polysaccharide selected from serotype A, C, W135 and Y capsular polysaccharides.

60. Use of the immunogenic composition of any one of claims 48-56 in the manufacture of a medicament for inducing a protective immune response in an individual.

61. The use according to claim 60, wherein the bacterial capsular polysaccharide is a capsular polysaccharide selected from Pn-serotype 1,3, 4,5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F capsular polysaccharides.

62. The use according to claim 60, wherein the bacterial capsular polysaccharide is a capsular polysaccharide selected from serotype A, C, W135 and Y capsular polysaccharides.