WO2004041310A1 - Preparation de conjugues de dendrimere et de glucide chimiquement bien definis - Google Patents

Preparation de conjugues de dendrimere et de glucide chimiquement bien definis Download PDF

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Publication number
WO2004041310A1
WO2004041310A1 PCT/DK2003/000766 DK0300766W WO2004041310A1 WO 2004041310 A1 WO2004041310 A1 WO 2004041310A1 DK 0300766 W DK0300766 W DK 0300766W WO 2004041310 A1 WO2004041310 A1 WO 2004041310A1
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conjugate
dendrimer
group
carbohydrate
conjugate according
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PCT/DK2003/000766
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English (en)
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Peter Heegaard
Ulrik Boas
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Danmarks Fødevareforskning
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Priority to AU2003275954A priority Critical patent/AU2003275954A1/en
Publication of WO2004041310A1 publication Critical patent/WO2004041310A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6903—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being semi-solid, e.g. an ointment, a gel, a hydrogel or a solidifying gel
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6949—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

  • the present invention concerns a method which allows fast and efficient synthesis of dendrimer conjugates having a well-defined chemical structure comprising one or more carbohydrate moieties and one or more immunomodulating substances coupled to a dendrimer.
  • the method comprises a first step in which the carbohydrate is coupled to the dendrimer in a chemoselective manner wherein the molar ratio between the carbohydrate and the dendrimer is predetermined and where the dendrimer is coupled to a specific region of the carbohydrate.
  • the conjugate between the carbohydrate and the dendrimer is reacted with an immunomodulating substance in a further chemoselective process, which may employ a weak electrophile that will react selectively with the accessible functional groups on the dendrimer surface, excluding the reaction with the carbohydrate hydroxyl groups and other weak nucleophilic groups present in the carbohydrate.
  • an immunomodulating substance in a further chemoselective process, which may employ a weak electrophile that will react selectively with the accessible functional groups on the dendrimer surface, excluding the reaction with the carbohydrate hydroxyl groups and other weak nucleophilic groups present in the carbohydrate.
  • the method comprises the synthesis of carbohydrate dendrimer conjugates (CD conjugates) comprising a combination of one or more same or different carbohydrates and one or more same or different immunomodulating substances coupled to a dendrimer.
  • the carbohydrate is a carbohydrate antigen originating from microorganisms such as e.g. bacteria and the immunomodulating substance is a substance such as e.g. an immunostimulating peptide, lipid, DNA fragment or chemical entity, including haptens.
  • the present invention also relates to novel CD conjugates.
  • the invention relates to the use of such CD conjugates as immunogens for the production of antibodies against the carbohydrate moiety of the conjugate. Further uses include uses as vaccine immunogens for the prophylactic protection and therapeutic treatment of humans and animals against infection by bacteria, viruses, fungi and other diseases such as cancer. The invention also discloses the use of CD conjugates in libraries and diagnostic assays.
  • Carbohydrate antigens are ubiquitous and biologically important, e.g. during infection processes.
  • bacterial surfaces present themselves to the surroundings under a blanket of carbohydrates composed of capsular polysaccharides, lipooligosaccharides and lipopolysaccharides.
  • These surface carbohydrates are of major importance for the phenotypical appearance of the bacterium, as well as for the interactions between bacteria and environments and hosts, being involved in adhesion to host tissues and to other environmental surfaces, in triggering host immune responses (mediated primarily by lipid-bound carbohydrate (lipopolysaccharide)) and in protecting against opsonisation and degradation by the host immune system.
  • Capsular polysaccharides are important determinants for bacterial virulence as non-encapsulated mutant bacteria are killed very efficiently and quickly by the host (see for example Rosendal & Maclnnes, 1990).
  • An indirect indication of the importance of these compounds is the presence of a broadly-reacting glycolipid-specific receptor (CD1 ) on antigen-presenting cells directed against bacterial carbohydrate structures, being part of the early, non-specific defence response of mammalian organisms (the "danger signal” hypothesis (Galluci & Matzinger, 2001)).
  • carbohydrates are classified as T- cell independent (Tl) immunogens, such immunogens being characterised by their lack of ability to induce immunological memory, lack of immunoglobulin class switching from IgM (low-affinity first line of defence antibodies) to IgG (high-affinity, later antibodies) and very low immunogenicity in neonates and young infants where the ability to mount Tl immune responses is not yet acquired (Mond et al., 1995).
  • Tl T- cell independent
  • the manufacture of efficient carbohydrate immunogens is thus a topic of great interest in the development of e.g. carbohydrate-based vaccines (Lindberg, 1999) and in diagnostic methods based on carbohydrate-specific antibodies.
  • the traditional strategy for producing carbohydrate immunogens for vaccines and for the production of diagnostic antibodies in laboratory rodents employs conjugation of purified carbohydrate antigens to macromolecular carriers, traditionally a naturally-derived protein, the latter functioning as an immunomodulator, and including e.g. ovalbumin, albumin, tetanus toxoid, pertussis toxoid, flagellin etc. (Adlin & Wriston, 1981).
  • the composition of this type of glycoconjugate is not well defined.
  • the conjugation between the immunomodulating carrier protein and the carbohydrate is hard to perform in a well- defined manner because of the large number of non-equivalent functional groups on the surface of the protein and the carbohydrate.
  • Standard conjugation methods used for joining carbohydrate and carrier are often not chemoselective, typically resulting in a molecular 'blend' of carbohydrate-carrier molecules, which sometimes has the desired immunogenic properties. It is often a matter of chance if a certain carbohydrate-carrier construct has a desirable immunogenicity and if so, no structural clues are evident as to what is controlling the immunogenicity (Jennings 1992, Verheul et al. 1989, Toyokuni et al., 1994). Due to the undefined structure of these types of conjugates, quality control by modern analytical methods as e.g. nuclear magnetic resonance (NMR) and high-pressure liquid chromatography (HPLC) is also not possible.
  • NMR nuclear magnetic resonance
  • HPLC high-pressure liquid chromatography
  • the naturally derived carrier-proteins as well as the carbohydrates are prepared by laborious methods not easily amenable to large-scale production. Additionally, the conjugation methods as a rule need to be optimised for each type of carbohydrate-carrier pair (Adlin & Wriston, 1981).
  • WO98/26662 provides evidence that dendrimer-coupled carbohydrates may be used in treating bacterial and viral diseases.
  • Lo-Man et al. (2001 ) describe a dendritic (Tn) 3 -CD4-stimulatory peptide substance that induces antibodies reactive with Tn-carbohydrate-structures associated with certain carcinoma carbohydrates. This substance can be used for both prophylactic and therapeutic immunization in a mouse model.
  • This paper illustrates how to induce antibodies against otherwise nonimmunogenic carbohydrate structures, but the method for doing this is different from that of the present invention; firstly the immunogen of Lo- Man et al. contains T-cell stimulatory moieties inside the structure and is not constructed by a modular approach. Secondly the structure is fully decorated by carbohydrate immunogen entities and is claimed to achieve the desired immunogenicity primarily due to the multimeric presentation of the carbohydrate antigen.
  • carbohydrate dendrimer conjugates of the present invention containing one or few carbohydrate antigens, are surprisingly very immunogenic, and can be constructed by a modular approach that allows a much more general use of CD conjugates for rendering carbohydrates immunogenic.
  • cyclodextrin polymers but does not specifically mention cyclodextrin dendrimers. If such cyclodextrin dend mers were to be prepared according to this invention and used for surface coupling of e.g. antigens, the result would be fully decorated cyclodextrins.
  • the focus of the invention is to utilise the ability of cyclodextrin to function as carriers of hydrophobic substances (drugs) inside their structure, combined with the surface attachment of biorecognition molecules for targeting of the cyclodextrin- drug complex (drug therapy, drug delivery). Furthermore, these beneficial properties of the cyclodextrin carrier system are enhanced by controlled polymerization of the cyclodextrin molecule by chemical cross-linking.
  • Bundle et al (2001) describe fully carbohydrate-decorated dendrimers that present carbohydrate epitopes in clusters, suitable for inhibiting the attachment of carbohydrate- specific bacterial toxins to its natural receptors.
  • the compounds of the invention of Bundle et al. (2001 ) do not utilise a modular aspect as opposed to the CD conjugates of the present invention.
  • the carbohydrate immunogens should be easy to analyse by chemical analysis, by HPLC and mass spectrometry and by use of 'fingerprint' regions in e.g. IR or NMR analysis.
  • the method should be easy to scale up and reaction conditions should be as environmentally friendly as possible.
  • the present invention concerns a method allowing fast and efficient synthesis of CD conjugates having a well-defined chemical structure comprising one or more carbohydrate moieties and one or more immunomodulating substances coupled to a dendrimer.
  • the invention relates to a chemoselective method of synthesising a CD conjugate, having the structure:
  • A is a functional, multivalent dendrimer or a conjugate of two or more functional, multivalent dendrimers
  • E' is a residue of a carbohydrate E or a derivative or a fragment thereof
  • L' is a residue of an immunomodulating substance L
  • m is an integer equal to or larger than 1
  • n is an integer equal to or larger than 1 , if more than one E' is present it could be the same or different, if more than one L' is present it could be the same or different,
  • (E') m -A contains A coupled to E' via the chemoselective group on E, wherein the molar ratio between E' and A is pre-determined, iii) identifying and/or introducing a chemoselective group on L, iv) binding L to (E') m -A in a chemoselective manner such that over 75% of the product (E') m -A-(L') n contains A coupled to L' via the chemoselective group on L, wherein the molar ratio between A' and L is pre-determined,
  • the method comprises 1) identifying and/or introducing a chemoselective group on the carbohydrate and 2) binding the carbohydrate to the dendrimer in a chemoselective manner such that over 75% of the product (E') m -A contains A coupled to E' via the chemoselective group on E, wherein the molar ratio between E' and A is pre-determined.
  • E' product
  • m -A contains A coupled to E' via the chemoselective group on E, wherein the molar ratio between E' and A is pre-determined.
  • the method further comprises 3) identifying and/or introducing a chemoselective group on the immunomodulating substance and 4) binding the immunomodulating substance to the CD conjugate containing only carbohydrate and dendrimer in a chemoselective manner, normally employing a weak electrophile that will react selectively with the large number of accessible functional groups on the dendrimer surface, excluding the reaction with the carbohydrate hydroxyl groups and other weak nucleophilic groups present in the carbohydrate.
  • a final CD conjugate is obtained wherein the molar ratio between the CD conjugate containing only carbohydrate and dendrimer and the immunomodulating substance is predetermined and wherein the numbers of carbohydrate moieties and immunomodulating substances conjugated directly to the dendrimer are independent of each other. Furthermore, over 75% of the product (E , ) m -A-(L') n contains A coupled to L' via the chemoselective group on L.
  • the method comprises the synthesis of CD conjugates comprising a combination of one or more same or different carbohydrates or derivatives or fragments thereof, such as e.g. carbohydrates originating from bacterial, viral or fungal antigens or cancer antigens, and one or more same or different immunomodulating substances.
  • the present invention also relates method for synthesising an intermediate compound with the formula
  • E', A and m are as defined as above, wherein A has at least one surface group that is not connected to an E' group
  • n is 1 , 2 or 3; more specifically m is 1.
  • the integer n may take values from 1 to 100 inclusive, such as e.g. from 1 to 70 inclusive, from 1 to 50 inclusive, from 3-20 inclusive such as e.g. 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15. Throughout the specification the following definitions and abbreviations have been used.
  • a dendrimer is defined as a molecule with a structure that extends from one or more core points through multiple generations of successive layers, with each layer having one or more branching points, to end in surface groups. They can be globular (spherical) or tree- shaped.
  • a DAB dendrimer is defined as a dendrimer consisting of poly(propyleneimine) layers built on a diaminobutane core unit. DAB dendrimers are commercially available and have well- studied physical and chemical properties.
  • a PAMAM dendrimer is defined as a dendrimer consisting of poly(amidoamine) layers built on a ethylenediamine core unit. PAMAM dendrimers are commercially available and have well-studied physical and chemical properties.
  • a conjugate is defined as an association of two or more compounds which are bound together to form a new compound. Bonding occurs between the compounds so that the structure of the conjugate can be determined chemically or spectroscopically.
  • carbohydrate includes monosaccharides, oligosaccharides and polysaccharides as well as substances derived from monosaccharides by oxidation of one or more terminal groups to carboxylic acids, by oxidation of vicinal hydroxyl groups to carbonyl groups or by replacement of one or more hydroxyl group(s) by a hydrogen atom, an amino group, thiol group or similar groups. It also includes derivatives or fragments of these compounds.
  • a carbohydrate which originates from a bacterial source is one which has a structure related to the native bacterial carbohydrate. This includes the complete carbohydrate, variants, modifications, synthetic analogues and fragments thereof.
  • An immunomodulating substance is a substance capable of influencing the magnitude and/or type of immune response against an antigen mounted by an animal being treated with the immunomodulating substance together with a compound containing the antigen.
  • examples of such substances include cytokines and cytokine fragments, certain lipids, peptides representing promiscous T-cell epitopes, microbially derived peptides and glycopeptides and oligodeoxynucleotides of the GpC-type.
  • a hapten is a small molecule (e.g. below 2000 Daltons) which can act as an antigenic epitope and/or contains an antigenic determinant but is incapable by itself of eliciting an immune response unless bound to another molecule.
  • haptens are substituted aromatic groups (e.g. substituted benzene or naphthalene groups such as dinitrophenols and dinitrobenzenes), adamantyl, biotin, digoxigenin, steroids, phosphorylcholine and dextran.
  • substituted aromatic groups e.g. substituted benzene or naphthalene groups such as dinitrophenols and dinitrobenzenes
  • adamantyl e.g. substituted benzene or naphthalene groups such as dinitrophenols and dinitrobenzenes
  • biotin e.g. substituted benzene or naphthalene groups such as dinitrophenols and dinitrobenzenes
  • adamantyl e.g. substituted benzene or naphthalene groups such as dinitrophenols and dinitrobenzenes
  • biotin e.g. substituted benzene or naphthalene groups such as dinitrophenol
  • a short alkyl chain is an alkyl chain which has a low relative molecular mass and contains a low number of carbon atoms (e.g. between 1 and 8 carbon atoms).
  • Chemoselectivity is defined as the preferential reaction of a chemical reagent with one of two or more different functional groups.
  • a reagent has a high chemoselectivity if reaction occurs with only a limited number of different functional groups.
  • chemoselectivity is achieved through the use of functional groups which show different reactivity towards one type of functionality over another.
  • the dendrimer is selective in which part of the carbohydrate it binds to, and the immunomodulating substance is selective in its binding to the dendrimer functional groups as opposed to the carbohydrate functional groups.
  • the use of chemoselective processes removes the need for protecting groups in a synthesis, thus avoiding many time-consuming protection and deprotection steps.
  • chemoselective processes allows the ratio of carbohydrate : immunomodulating substance bound to the dendrimer to be pre-determined. If a reaction occurs with a chemoselectivity of a certain percent (e.g. 90%), it means that the stated percentage of reagents react in the prescribed manner and the remaining percent (e.g. 10%) react otherwise. Hence, the higher the percent selectivity, the more preferred the reaction is over other reactions. Reactions with a chemoselectivity of 50% show no preference for either of two or more possible reaction routes.
  • a chemoselective group is an atom or group of atoms which reacts preferentially with one functional group over one or more alternative functional groups.
  • a pre-determined ratio is defined as a ratio which has been decided before a process takes place, and which is reflected in the product of said process. More specifically, the ratio of carbohydrate:dendrimer can be chosen before the reaction to give a CD conjugate containing said ratio of carbohydrate:dendrimer. The fact that the ratio is pre-determined and carries through to the product allows increased control over the stoichiometry of the product and decreases the need for subsequent analysis of the product to determine the number of moieties bound to the dendrimer. The ratio can be tailored according to the immunomodulating requirements of the CD conjugate.
  • Stoichiometry is defined as the ratio between the amounts of substance that react together in a particular chemical reaction.
  • a solid phase support is an insoluble polymer containing functional groups which are bound (usually via a linker entity) to a supported molecule. Use of such a support allows the supported molecule to participate in solution phase chemistry, and isolation and purification of the product is made easier due to the insolubility of the solid phase support. Supported molecules can be cleaved from the solid phase support after reaction. A large number of different compounds can be readily synthesised in this manner, often with only a small variation in structure or properties.
  • Solid phase supports are widely used in combinatorial chemistry and for the construction of compound libraries. Examples of commonly used solid phase supports are functionalised PEGA, functionalised tentagel and functionalised polystyrene.
  • a spacer is a non-cleavable short chain of atoms (e.g. alkyl chain), which connects two entities (two functional groups) together in a conjugate.
  • the spacer serves as a link between the two entities and at the same time keeps the entities separated from each other.
  • a linker is defined as a bifunctional reagent containing an anchor group to the polymer phase and an anchor group to the substrate.
  • the anchor groups are connected to each other through a spacer.
  • the anchor moieties of a linker are joined to the polymer phase and substrate in a selective manner and also cleaved in a selective way.
  • a linker is used to join a dendrimer or other fragment of a CD conjugate to a solid phase support.
  • a traceless linker is a class of linker entity. After cleavage of a molecule from a traceless linker, it is impossible to identify the point on the molecule at which the traceless linker was joined to said molecule. In this application, cleavage of a BAL linker releases an amide which is indistinguishable from other amides on the dendrimer.
  • a library is defined as a selection of chemical compounds which are catalogued together for a particular purpose. Such compounds often contain similar structural or chemical features or may be derived from a common synthetic route. In this application, libraries of CD conjugates can be assembled and used to map immunogenic determinants.
  • An antigen is a substance which is reactive with a specific antibody or T-cell.
  • An immunogen is an antigen capable of inducing an immune response, including antibody and T-cell responses.
  • High-throughput screening is a method by which a large number of chemical compounds can be analysed simultaneously. It often involves automated or semi- automated techniques. HTS is often combined with the techniques involved in chemical libraries and solid-phase synthesis in order to narrow a wide range of test compounds to those which show activity in a particular test.
  • An adjuvant is a substance added to a vaccine to improve the immune response.
  • a chemical entity is defined as a low molecular weight compound which exhibits immunomodulating behaviour when displayed multivalently on a dendrimer surface, such as e.g. a hapten.
  • Chemical entities particularly include compounds which have a molecular weight below 20kD and contain suitable functional groups allowing the chemoselective coupling to the dendrimer functional groups. Included in the group of small molecular weight compounds are also aromatic substances containing a suitable chemoselective functional group, typically a weak electrophile.
  • TFA trifluoroacetic acid TFFH tetramethylfluoroformamidinium hexafluorophosphate
  • the method according to the present invention allows fast and efficient synthesis of a CD conjugate having a well-defined chemical structure in a reproducible manner and by the use of easily accessible (e.g. commercially available) building blocks.
  • the chemoselective method comprises the steps of:
  • the percentage selectivities as described in steps ii and iv of this method may be over 80%, such as e.g. over 90%, over 95%, over 98%, over 99%.
  • the present invention relates to a method, wherein the steps are carried out in two or more step process in a one-, two- or multi pot synthesis. In a specific embodiment, the steps are carried out in a one-pot synthesis.
  • the first step of the method concerns the chemoselective coupling of the carbohydrate moiety to a specific number of free surface groups on the dendrimer.
  • the coupling of the carbohydrate moiety to the surface groups on the dendrimer occurs in such a way that only selected functional groups present on the carbohydrate react with the dendrimer and others do not. That is, only the specific parts (the chemoselective groups) of the carbohydrate are connected to the dendrimer.
  • the main product will contain one molecule of carbohydrate moiety conjugated to one molecule of dendrimer.
  • the choice of a 1 :1 ratio ensures a large molar excess of dendrimer amino groups compared to carbohydrate functional groups.
  • the surface amino groups of the dendrimer are all equivalent, all of the 1 :1 adducts obtained will be identical.
  • the method of the invention comprises conjugation of a carbohydrate to a dendrimer.
  • the carbohydrate must contain a chemoselective group in order to be coupled chemoselectively to the dendrimer.
  • the carbohydrate contains a chemoselective group in the form of a carbonyl group, the reducing end hemiacetal (or hemiketal) group, a carboxylic acid group, or other such specific functional groups of the carbohydrate, but in the event that it does not contain such a chemoselective group, one must be introduced before conjugation.
  • One skilled in the art will have the knowledge to introduce or identify an appropriate chemoselective group.
  • the chemoselective coupling between carbohydrate moiety E and the dendrimer A can be achieved through reductive amination between a reducing end of E and a dendrimer surface amino group to form a secondary glucosamine bond.
  • the chemoselective coupling between the carbohydrate moiety E and the dendrimer A can be achieved through oxidation of a reducing end of E to a carboxylic acid followed by chemoselective amide bond formation mediated by amide coupling reagents.
  • the chemoselective coupling between the carbohydrate moiety E and the dendrimer A can be achieved through oxidative coupling of a reducing end of E with a dendrimer surface amino group to yield a amide link between E and A.
  • the coupling of E to A can take place through either the keto-functionality or the acid functionality by reductive amination or amide bond formation, respectively.
  • the final step of the conjugation process of the method concerns the coupling of the immunomodulating substance L to a specific number of free surface groups on the CD conjugate containing only carbohydrate and dendrimer.
  • the coupling of the immunomodulating substance to the surface groups on the dendrimer occurs in such a way that the functional groups present on the carbohydrate (hydroxyl groups, amide such as acetamide, phosphate etc.) do not react with the immunomodulating substance.
  • the dendrimer is modified with the immunomodulating substance.
  • the stoichiometry of the reactants in the second step is such that the immunomodulating substance is coupled to one or more of the remaining available functional groups on the dendrimer part of the carbohydrate dendrimer.
  • This is typically achieved by employing a large surplus of the immunomodulating substance (e.g. the immunomodulating substance) in the reaction. This is possible because the chemoselectivity of the reaction allows the majority of the reaction between the immunomodulating substance and the dendrimer amino groups, while a minor reaction occurs between the immunomodulating substance and the carbohydrate functional groups.
  • the method of the invention comprises conjugation of the CD conjugate containing only carbohydrate and dendrimer with an immunomodulating substance.
  • the immunomodulating substance must contain a chemoselective group in order to be coupled chemoselectively. In the event that the immunomodulating substance per se does not contain such a chemoselective group, it must be introduced in the immunomodulating substance before conjugation.
  • the final step can be achieved by the introduction or identification of a chemoselective group on the immunomodulating substance, which is an electrophilic moiety, such as e.g. a weak electrophile.
  • a chemoselective group on the immunomodulating substance which is an electrophilic moiety, such as e.g. a weak electrophile.
  • the present invention relates to a method in which the chemoselective coupling between L and A can be achieved by the use of an electrophilic moiety on L.
  • the chemoselectivity of the method according to the invention lies in the choice of electrophilic moiety.
  • the electrophilic moiety required depends on the nature of the other functional groups present, especially those present on the carbohydrate.
  • An electrophilic moiety will be chosen which reacts only with the dendrimer, and not the carbohydrate. With knowledge of the functional groups present, a person skilled in the art will be able to select a suitable electrophilic moiety to achieve the desired chemoselectivity.
  • dendrimer surface amino groups can be substituted with immunomodulating substances to furnish the complete CD conjugate.
  • the weak electrophiles described above are suitable in the case where the dendrimer is amino-terminated and the carbohydrate contains free (unprotected) hydroxy groups.
  • the electrophilic moiety may be an active ester, a carboxylic acid (in the presence of an amide coupling reagent), or an isocyanate or isothiocyanate.
  • the immunomodulating substance has a suitable amino functional group
  • the immunomodulating substance has a suitable alcohol functional group
  • the immunomodulating substance has a suitable thiol functional group
  • Such a chemoselective group can be introduced to the immunomodulating substance in an aqueous or organic solvent or mixtures thereof.
  • solvents are water, aqueous media including aqueous buffers, alcohols, dichloromethane, ethers, N- methylpyrrolidone, N,N-dimethylformamide, acetonitrile, sulfolane, dimethyl sulfoxide, tetrahydrofuran and carbon disuifide.
  • the electrophilic moiety is introduced to L in an aqueous or organic solvent or mixtures thereof.
  • the electrophilic moiety is introduced to L in a solvent selected from the group comprising: water, alcohols, dichloromethane, ethers, A/-methylpyrrolidone, NJV-dimethylformamide, acetonitrile, sulfolanes, dimethylsulfoxide, tetrahydrofuran, carbon disuifide and mixtures thereof.
  • a solvent selected from the group comprising: water, alcohols, dichloromethane, ethers, A/-methylpyrrolidone, NJV-dimethylformamide, acetonitrile, sulfolanes, dimethylsulfoxide, tetrahydrofuran, carbon disuifide and mixtures thereof.
  • the method can be performed by solution phase chemistry or by solid phase techniques.
  • the present invention relates to a method for synthesising CD conjugates as described, wherein the carbohydrate moiety (E) to be coupled to the dendrimer can take a number of forms and can originate from a variety of sources, such as e.g. bacterial, viral or fungal antigens, neoplastic, tumour or cancer antigens and non-pathogenic, naturally occurring carbohydrates being parts of carbohydrate-lectin pairs.
  • sources such as e.g. bacterial, viral or fungal antigens, neoplastic, tumour or cancer antigens and non-pathogenic, naturally occurring carbohydrates being parts of carbohydrate-lectin pairs.
  • E originates from bacterial antigens.
  • CD conjugates comprise structures containing LPS-derived oligo- or polysaccharides devoid of lipid, especially such polysaccharides derived from bacterial LPS by acid hydrolysis or by alkaline or enzymatic delipidation and such oligosaccharides derived from bacterial LPS by controlled hydroysis or by the action of endoglycosidases such as phage-derived endoglycosidases.
  • the capsular polysaccharide from Salmonella typhi or smaller derivatives thereof is included in a CD conjugate and said CD conjugate is then used as a component in said vaccine.
  • CD conjugates containing capsular carbohydrates from one or several types of Streptococcus pneumoniae can be used in a vaccine for protection against lung and other infections.
  • the vaccines containing CD conjugates mentioned above may also be used for the production of antibodies for passive vaccination or therapy to counteract the corresponding infections.
  • CD conjugates containing capsular or lipopolysaccharide from the bacteria listed are useful for production of antibodies in laboratory rodents for use as diagnostic and research tools. Diagnostic uses comprise both the use of the antibodies produced for typing of bacteria at the serotype-level and the use of such antibodies for serodiagnosis (determination of specific antibodies) and research purposes.
  • CD conjugate More than one type of CD conjugate may be used in a vaccine, in order to induce protection against more than one type of bacterial infection. On the other hand it will also in some instances be possible to obtain protection against several types of bacteria by vaccination with a CD conjugate containing a cross-protective carbohydrate antigen. Also, vaccines containing CD conjugate as a component may also contain other types of inmunogens including protein antigens e.g. detoxified bacterial toxins.
  • E originates from bacterial antigens, wherein the bacteria is selected from the group comprising Haemophilus sp, Eschericia coli ssp, Salmonella sp, Klebsiella sp, Bordetella sp, Pseudomonas sp, Chlamydia sp, Neisseria sp., Vibrio cholerae, Shigella sp, Proteas sp, Brucella sp, Streptobacillus sp, Yersinia sp., Streptococcus sp., Staphylococcus sp., Legionella pneumophila, Campylobactersp., Actinobacillus sp., Mannheima sp., Pasteurella sp., Citrobacter sp. and Helicobacter sp.
  • the bacteria is selected from the group comprising Haemophilus sp, Eschericia coli
  • Carbohydrates of the present invention additionally include those originating from bacterial antigens wherein the bacteria is selected from the group comprising Haemophilus influenzae, Salmonella typhi, Salmonella paratyphi, Salmonella enterica sspp, Klebsiella pneumoniae, Bordetella pertussis, Pseudomonas aeruginosa, Chlamydia psitacci, Neisseria meningitidis, Neisseria gonorrhea, Shigella flexneri, Streptococcus pneumoniae, Staphylococcus aureus and Shigella dysenteria.
  • the bacteria is selected from the group comprising Haemophilus influenzae, Salmonella typhi, Salmonella paratyphi, Salmonella enterica sspp, Klebsiella pneumoniae, Bordetella pertussis, Pseudomonas aeruginosa, Chlamydia psitacci, Neisseria
  • E originates from bacterial antigens, wherein the bacteria are selected from the group consisting of enterobacteria and respiratory bacteria.
  • E originates from enterobacteriae selected from the group comprising Escherichia coli, Salmonella enterica and all serotypes thereof, especially Salmonella Typhimurium, Salmonella Infantis, Salmonella Choleraesuis, Salmonella Enteritidis, Salmonella Manhattan, Salmonella Thomson, and Salmonella Dublin, Escherichia coli sspp including 0157, and edema-disease causing Escherichia coli of the 0139 type, Yersinia enterocolitica, and Campylobacterjejuni.
  • E originates from respiratory bacteria selected from the group comprising the HA(M)P group of bacteria, especially Actinobacillus pleuropneumoniae of the serotypes 1 ,2,5,6,7,8, and 12, Haemophilus somnus, Mannheimia haemolytica (previously known as Pasteurella haemolytica), Pasteurella multocida, Haemophilus parasuis, Mannheimia sp., Citrobacter sp. and Helicobacter sp.
  • HA(M)P group of bacteria especially Actinobacillus pleuropneumoniae of the serotypes 1 ,2,5,6,7,8, and 12, Haemophilus somnus, Mannheimia haemolytica (previously known as Pasteurella haemolytica), Pasteurella multocida, Haemophilus parasuis, Mannheimia sp., Citrobacter sp. and Helicobacter sp.
  • the carbohydrate moiety E is a capsular polysaccharide-derived or lipopolysaccharide-derived carbohydrate or a derivative or fragment thereof.
  • the present invention also relates to a method wherein the carbohydrate E originates from viral antigens, especially those related to HIV, hepatitis virus or influenza virus.
  • the carbohydrate E originates from the Tn-carbohydrate found in the glycan moieties of gp120 of HIV.
  • the method of the present invention also relates to the use of carbohydrates originating from fungal antigens, such as e.g. mannans or carbohydrates from Saccharomyces spp.
  • the carbohydrate E originates from neoplastic, tumour or cancer antigens.
  • E originates from the group comprising cancer-related glycopeptides Tn ( ⁇ -GalNac-Ser/Thr) and Thomsen Friedenreich (T) ( ⁇ -Gal(1-3)- ⁇ GalNAc-Ser/Thr) antigens and their sialylated derivatives, glycolipids such as, e.g. acidic glycolipids as gangliosides GD2, GD3 and GM3 and neutral glycolipids such as, e.g. Lewis y (Le y ) and Globo H antigens, and their sialylated derivatives.
  • the present invention also relates a method wherein the carbohydrate E originates from non-pathogenic naturally occurring carbohydrates being parts of carbohydrate-lectin pairs, such as e.g. galactose, mannose, glucose, and their N-acetylated derivatives, and sialic acid.
  • carbohydrate-lectin pairs function as recognition molecules between cells of the immune system and tissue cells and between serum glycoproteins or blood cells and liver cell-localised lectins, in particular galactose being a ligand for the liver cell-localised asialoglycoprotein receptor lectin.
  • the carbohydrate E originates from CD-1 antigen-binding substances.
  • the carbohydrate E originates from a CD-1 antigen-binding glycolipid.
  • the CD1 antigen is present on professional antigen- presenting cells such as Langerhans cells and dendritic cells, and is specialised in presenting to T-cells a variety of antigenic structures consisting of lipid and glycolipids. By presenting those antigens to the T-cells a T-cell dependent, peptide-independent immune response is initiated.
  • the carbohydrate has a molecular weight between 10 kD and 150 kD.
  • the carbohydrate E has a molecular weight between 100 D and 2000 D, such as e.g. between 200 D and 2000 D.
  • the present invention also relates to a method, wherein the carbohydrate E is naturally occurring or synthetic or semi-synthetic.
  • interesting carbohydrates include mycobacterial glycolipids such as lipoarabinomannans and phosphoinositide mannosides and lipooligosaccharides of the bacteria mentioned, including Neisseria sp., Haemophilus sp., Bordetella sp., Branhamella sp., Campylobacter jejuni, and Campylobacter coli and parasitic GPI (glycosylphosphatidylinositol)-structures such as glycolipids which may act as a toxic principle in such pathogens e.g. in Plasmodium falciparum (Schofield, et al., 2002).
  • mycobacterial glycolipids such as lipoarabinomannans and phosphoinositide mannosides and lipooligosaccharides of the bacteria mentioned, including Neisseria sp., Haemophilus sp., Bordetella sp., Branhamella sp., Campylobacter jejuni, and Campyl
  • Suitable carbohydrates may be further derived to make them suited for inclusion in a CD conjugate obtained according to the invention.
  • derivatisation may comprise cleaving off the lipid portion of lipopolysaccharide, and fragmenting polymeric carbohydrate by controlled hydrolysis by chemical treatment, enzyme treatment, phage treatment or otherwise.
  • Capsular polysaccharides may contain lipid that can be removed as known in the art (se e.g. US5314811). Lipids can also be removed by certain naturally occurring enzymes (lipases).
  • lipases can also be removed by certain naturally occurring enzymes (lipases).
  • the immunomodulating substance L can be selected from a group consisting of: immunostimulating peptides, an antigen-presenting cell-derived peptide, cytokines or fragments thereof, immunostimulatory lipids, DNA fragments and chemical entities including haptens.
  • the immunomodulating substance L should contain a chemoselective group, which can be present on the immunomodulating substance or introduced to the immunomodulating substance.
  • the present invention relates to a method for synthesising CD conjugates, wherein a chemoselective group is introduced to the immunomodulating substance L.
  • the immunomodulating substance L is an immunostimulating peptide.
  • L is selected from a group comprising peptides representing T-cell epitopes, muramyldipeptides and muramyldipeptide variants, Tuftsin peptides and analogues thereof, peptides derived from complement factors, antigen-presenting cell derived T-cell binding peptides and interleukins and other relevant cytokines and fragments thereof.
  • the immunomodulating substance L is a CD4 T-cell stimulating peptide, such as a peptide containing e.g. the 103-1 5 sequence of VP1 protein from poliovirus type 1 (KFLAVWKITYKDT), the 830-844 sequence of tetanus toxoid (QYIKANSKFIGITEL), 947-967 sequence of the tetanus toxoid (FNNFTVSFWLRVPKVSASHLE), and the 1273-1284 sequence of tetanus toxoid (GQIGNDPNRDIL)
  • a CD4 T-cell stimulating peptide such as a peptide containing e.g. the 103-1 5 sequence of VP1 protein from poliovirus type 1 (KFLAVWKITYKDT), the 830-844 sequence of tetanus toxoid (QYIKANSKFIGITEL), 947-967 sequence of the tetanus toxoid (
  • promiscuous T-cell epitope peptides include Mycobacterium tuberculosis 38 kD antigen 350-369.
  • the active immunostimulating peptides are antigen-presenting cell derived T-cell binding peptides (Zimmerman, D.H., et al, 1996; Rosenthal, K.S., et al., 1999), such as e.g. DQTQDTEGGC (MHC-I peptide), NGQEEKAGVVSTGLIGGC (MHC-II peptide), or DLLKNGERIEKVEGGC (beta-2- microglobulin).
  • DQTQDTEGGC MHC-I peptide
  • NGQEEKAGVVSTGLIGGC MHC-II peptide
  • DLLKNGERIEKVEGGC beta-2- microglobulin
  • immunomodulating compound is a cytokine or fragment thereof, such as e.g. interferon gamma, tumor necrosis factor alpha, interleukin 6 or interleukin 1.
  • Tuftsin peptides having the sequence TKPR n (Friedkin & Najjar, 1989), where n is 1-10, such as e.g. 1-4 and 1 , analogues of the Tuftsin peptide and the interleukin-1 derived immunomodulatory nonapeptide VQGEESNDK (Antoni, et al., 1986) or larger IL-1 peptides including VQGEESNDKGGC and other immunomodulatory fragments of interleukin 1 and of other relevant cytokines, including interferon gamma, tumor necrosis factor alpha and interleukin 6. Also included as relevant moieties are peptides derived from complement factors.
  • peptides include muramyldipeptides and muramyldipeptide variants and derivatives, including the peptide part of muramyldipeptide and adamantyl- muramyldipeptide (Becker et al. 2001), and peptides originating from complement factor C3d.
  • Adamantyl MDP-derivatives are MDP derivatives in which the carbohydrate has been substituted with adamantyl bound to the glutamic acid side chain carboxylic function. MDP analogues consisting of other carbohydrate mimicking groups may also be useful.
  • the immunomodulating peptide may be comprised of a library of fragments from one or more specific proteins (protease digests) which are then coupled to the CD conjugate containing only carbohydrate and dendrimer.
  • the immunomodulating substance L of the present invention comprises immunostimulatory lipids.
  • Suitable lipids for inclusion in a CD conjugate according to the invention are straight chain fatty acids which may be saturated or unsaturated and which contain between 2 and 28 carbon atoms, such as e.g. between 4 and 10 carbon atoms.
  • Another group of immunomodulatory lipids include substances in which the immunomodulating substance L is coupled to an aromatic substituent through the straight chain of the fatty acid.
  • the immunostimulating lipid is dimethyldioctadecyl- ammoniumbromide (DDA, Snippe and Kraaijeveld, 1989) or the palmitoyl-Cys((RS)-2,3- di(palmitoyloxy)-propyl)-OH structure described by Wiesmueller et al. (1992).
  • DDA dimethyldioctadecyl- ammoniumbromide
  • RS palmitoyl-Cys((RS)-2,3- di(palmitoyloxy)-propyl)-OH structure described by Wiesmueller et al. (1992).
  • the CD1 antigen of professional antigen-presenting cells such as Langerhans cells and dendritic cells is specialised in presenting a variety of lipid and glycolipid structures to T- cells, thus initiating a T-cell dependent, peptide-independent immune response against certain glycolipids.
  • the CD1 antigen is not polymorphic and the binding of lipids and glycolipids is not restricted in the sense of traditional antigen presenting molecules but is instead focused on pathogen-related lipid and glycolipid structures having amphipathic properties, examples of which include mycobacteria-derived lipids.
  • certain autologous glycolipids can also be bound and CD1 can by itself activate certain T-cell subsets (Sugita et al., 1998; Prigozy et al., 2001).
  • Specific examples of such lipids include mycolic acids and glucose monomycolate of mycobacteria and other genera, lipoarabinomannans which are glycolipids also found in mycobacteria and a number of other mycobacterial lipids, phospholipids and glycolipids.
  • certain glycosphingolipids are bound, as e.g. alpha-galactosylceramide and galactose alpha 1-6 galactoseceramide.
  • the immunomodulating substance L corresponds to the lipid part of a CD-1 antigen-binding glycolipid.
  • a particular interesting immunomodulating substance L comprises a fragment of DNA which modulates the immune response.
  • L is unmethylated CpG-containing oligodeoxynucleotides (Krieg et al., 1995) containing the sequence (5')PuPuCpGPyPy(3'), wherein Pu is a purine base and Py is a pyrimidine base.
  • Such nucleotides have been shown to be more efficient immunomodulators when physically coupled to the antigen (see for example Maurer et al., 2002).
  • An alternative form of immunomodulating substance includes any other immunoactivating substance, in particular such substances having molecular weights below 20 kD and containing suitable functional groups allowing the chemoselective coupling to the dendrimer functional groups. Included in this group of small molecular weight compounds are also aromatic substances containing a suitable chemoselective functional group, typically a weak electrophile. Haptens are also included in this classification. Thus one embodiment of the present invention relates to a method wherein the immunomodulating substance is a chemical entity or a hapten.
  • the immunomodulating substance L is a short alkyl chain substituent, optionally comprising of an aromatic residue, such as e.g. benzene, naphthalene or substituted aromatic residues.
  • relevant immunomodulating substances also include substances that rather than just activating the immune system direct the immune response towards a desired type of reaction as e.g. a TH1 type response instead of a TH2 type response and vice versa.
  • a desired type of reaction e.g. a TH1 type response instead of a TH2 type response and vice versa.
  • such an immunomodulating substance is coupled together with a second immunomodulating substance resulting in a CD conjugate containing both types of modulators.
  • the present invention relates to a method for synthesising CD conjugates as described, wherein the multivalent functional dendrimer (A) of the CD conjugate has a dendritic structure that extends from one or more core points through multiple generations of successive layers with each layer having one or more branching points to end in surface groups.
  • A is a dendrimer represented by the formula:
  • X is a multifunctional segment having one or more branching points
  • Y is a linker or spacer group which may be branched or linear
  • Z is a surface group and a and b are integers such that each linker group Y terminates in a surface group Z
  • the dendrimer is globular or tree shaped.
  • the generation numbers of the dendrimer range from 0 to 20, such as e.g. from 1 to 10 or from 2 to 6.
  • the medium generation dendrimers e.g. 2 nd and 3 rd generation dendrimers
  • lower generation dendrimers give more space between the surface functionalities, giving enhanced possibilities for interaction with antibodies (and hence better immune recognition).
  • the dendrimer is a diaminobutane dendrimer (DAB) or a polyamidoamine dendrimer (PAMAM).
  • DAB diaminobutane dendrimer
  • PAMAM polyamidoamine dendrimer
  • Specific CD conjugates of the invention are built from simple and chemically unambiguous 2 nd and 3 rd generation dendrimers, preferably DAB or PAMAM dendrimers. Such dendrimers are commercially available, and all present a high density of equally reactive and accessible functional groups on their surface.
  • the molecular masses of the unmodified dendrimers lie from 50 to 30000 such as e.g. from 100 to 20000 or from 300 to 15000.
  • the dendrimer is a DAB dendrimer
  • the molecular mass of the unmodified DAB dendrimer lies from 50 to 10000, such as e.g. from 100 to 8000 or from 300 to 7200.
  • the dendrimer is a PAMAM dendrimer
  • the molecular mass of the unmodified PAMAM dendrimer lies from 200 to 20000, such as e.g. from 300 to 10000 or from 500 to 15000.
  • the DAB dendrimer has interior, free amines as opposed to the
  • PAMAM dendrimer and that this could be an advantage for an interaction with negatively charged cells.
  • the positively-charged amines are also advantageous in enhancing the solubility of the dendrimer in aqueous media.
  • the number of surface groups on the dendrimer lies between 2 and 256 such as e.g. between 2 and 64 or between 4 and 32 or between 8 and 32, such as e.g. 4, 8, 16, 32 or 64.
  • the surface groups of the dendrimer are amine functionalities.
  • the dendrimer A of the CD conjugate contains two or more multivalent, functional dendrimers as described above which are connected together within the CD conjugate.
  • the present invention relates to a method wherein the steps are carried out while at least one of the components E, A and L are in solution phase.
  • the solvent is aqueous or polar organic or mixtures thereof.
  • the solvent is selected from the group comprising: water, aqueous media including aqueous buffers, alcohols, ⁇ /. ⁇ /-dimethylformamide, ⁇ /-methylpyrrolidone, acetonitrile, dimethylsulfoxide, sulfolane and mixtures thereof.
  • the solvent is water or aqueous media including aqueous buffers.
  • Scheme 1 illustrates a general method for synthesis of a CD conjugate in solution phase employing a dendrimer containing amino groups as functional surface groups.
  • the present invention is not limited to such specific surface groups, however, in the case of surface groups other than amino, a person skilled in the art will know how to perform other kinds of chemoselective processes.
  • Scheme 1 Part A shows one embodiment of the synthesis of CD conjugates in a two-pot process.
  • a carbohydrate moiety containing a carbonyl group in this case an aldehyde group
  • a carbohydrate moiety containing a carbonyl group is coupled to the dendrimer surface amino group using reductive amination mediated by any suitable reagent or reagents known for use in such a reaction (e.g. NaBH 3 CN, NaBH 4 or NaB(OR) 3 H, where R is alkyl or acyl) to form a secondary glucosamine bond between the carbohydrate and the dendrimer.
  • This coupling process occurs selectively between a carbonyl group of the carbohydrate moiety and a dendrimer surface amino group, and other functional groups in the carbohydrate (e.g. alcohol, amide such as acetamide, phosphate etc.) are untouched.
  • other functional groups in the carbohydrate e.g. alcohol, amide such as acetamide, phosphate etc.
  • the second step illustrated in Scheme 1 ; Part A shows the coupling of the immunomodulating substance to a specific number of free surface amino groups on the carbohydrate dendrimer. This occurs in such a way that the functional groups present on the carbohydrate (hydroxyl groups, amide such as acetamide, phosphate etc.) do not react with the immunomodulating substance. That is, only the dendrimer is modified with the immunomodulating substance.
  • a CD conjugate having a predetermined amount of carbohydrate and predetermined amount of immunomodulating substance coupled to the dendrimer.
  • the CD conjugate is isolated and, if necessary, characterised.
  • the CD conjugate containing only carbohydrate and dendrimer thus formed is not isolated, but an immunomodulating substance provided with a chemoselective group (a weak electrophile as described above) is added to the reaction mixture. This reacts selectively with dendrimer surface amino groups as before, to produce the CD conjugate in one pot.
  • a chemoselective group a weak electrophile as described above
  • This route simplifies purification and obviates the need for isolation and purification of the intermediate.
  • Scheme 1 Part C describes an alternative embodiment of the invention. This route employs a carboxylic acid group which may be present on the carbohydrate, or introduced by oxidation of an aldehyde group.
  • a carbohydrate containing a carboxylic acid can be coupled to a dendrimer surface amino group via chemoselective amide bond formation mediated by various amide coupling agents or combinations thereof known to a person skilled in the art e.g. DCC/HOBt; BOP; PyBOP; HBTU; TBTU; BOP-CI; BOP-Br; HATU efc.
  • the hydroxyl groups present on the carbohydrate will not react under the above- mentioned conditions.
  • the product of this first step is a CD conjugate containing only carbohydrate and dendrimer, where the two components are joined through an amide bond.
  • the second step of Scheme 1 , Part C involves coupling of an immunomodulating substance to the dendrimer in a chemoselective manner such that only the free amine groups of the dendrimer react. This produces the required CD conjugate which is finally isolated and purified.
  • Part C can be carried out according to a one- or two-pot method as in Parts A and B.
  • Scheme 1 Part D describes an alternative method for synthesis of a CD conjugate.
  • a carbohydrate containing a carbonyl group can be coupled to a dendrimer surface amino group via chemoselective oxidative coupling mediated by various oxidative coupling agents or combinations thereof known to a person skilled in the art, such as e.g. iodine or dimethylsulfoxide.
  • the product of this first step is again a CD conjugate containing only carbohydrate and dendrimer where the two components are joined through an amide bond.
  • the second step of Scheme 1 , Part D involves coupling of an immunomodulating substance to the dendrimer in a chemoselective manner such that only the free amine groups of the dendrimer react (as in Part C). This produces the required CD conjugate which is isolated and purified.
  • the process in Scheme 1 , Part D can be carried out according to a one- or two-pot method as in Parts A and B.
  • Scheme 1 , Part E describes the case wherein the carbohydrate contains a KDO residue.
  • a carbohydrate can be coupled to a dendrimer surface amino group via chemoselective amide bond formation mediated by various amide coupling agents or combinations thereof known to a person skilled in the art e.g. DCC/HOBt; BOP; PyBOP; HBTU; TBTU; BOP-CI; BOP-Br; HATU etc.
  • the KDO residue can be coupled by reductive amination between a carbonyl group and a dendrimer surface amino group.
  • the CD conjugate containing only carbohydrate and dendrimer thus formed is reacted in a chemoselective second step with an immunomodulating substance which contains a weak electrophile as described above (Part C).
  • Part C an immunomodulating substance which contains a weak electrophile as described above.
  • This provides the desired CD conjugate containing both immunomodulating substance and carbohydrate in a predetermined ratio.
  • the steps described in Scheme 1 , Part E can be carried out in a one- or two-pot process.
  • CD conjugates can be performed by solid phase techniques.
  • one or more of the components dendrimer, carbohydrate, immunomodulating substance
  • a linker entity This method of synthesis allows straightforward variation of one or more component(s) and facilitates the recovery and purification of the product.
  • the present invention also relates to a method wherein the steps are carried out while the dendrimer A is grafted to a solid phase support through a linker entity.
  • the solid phase support is amino functionalised-PEGA, amino functionalised-tentagel or amino functionalised polystyrene.
  • the linker entity is a linker which upon acidolytic cleavage with e.g. trifluoroacetic acid releases free amines. In this way, a CD conjugate containing one unmodified dendrimer surface amine can be synthesised.
  • Specific linkers according to the invention are the chlorotrityl-chloride linker or other linkers having halobenzyl or general haloarenyl core structures.
  • the linker entity is a traceless linker.
  • the linker entity is the backbone amide linker ortho-BAL (Boas et al. 2002) or para-BAL (Jensen et al.
  • the CD conjugate (E) m -A-(L) n can be cleaved from the solid phase support with an acid, such as, e.g. 1-95% v/v trifluoracetic acid.
  • the present invention relates to a method wherein the solvent for the solid phase synthesis is aqueous or polar organic or mixtures thereof.
  • the solvent is selected from the group comprising: water, aqueous media including aqueous buffers, alcohols, N/V-dimethylformamide, ⁇ /-methylpyrrolidone, acetonitrile, dimethylsulfoxide, sulfolane and mixtures thereof.
  • the solvent is water or aqueous media including aqueous buffers.
  • Scheme 2 illustrates an example of the synthesis of CD conjugates using solid phase synthesis.
  • CD conjugates having one free primary surface amine can be synthesized and cleaved off.
  • Such CD conjugates having one free amine functionality can be further modified with e.g. a different chemical entity or immunomodulating substance, reporter molecule or a solubilising group in cases where the solubility of the CD conjugate is hampered by hydrophobicity.
  • Scheme 2 shows an alternative route for the synthesis of CD conjugates using solid phase methods.
  • the carbohydrate is coupled to the dendrimer to form an amide bond using the methods described in Scheme 1 , Parts C and D, above.
  • the resulting resin-bound dendrimer is further reacted chemoselectively with an immunomodulating substance containing a weak electrophile to furnish the desired CD conjugate bound to a solid phase support.
  • the product can be cleaved from the solid phase support through a method appropriate to the linker entity.
  • Scheme 3 illustrates an example of a solid phase synthesis of CD conjugates on a traceless BAL linker.
  • a linker entity is bound to a solid phase support so that all functional groups of the solid phase support are bound to a linker entity.
  • the other end of the linker entity is then coupled to a dendrimer surface functional group.
  • the functional surface groups are amino groups the coupling takes place through a reductive amination reaction mediated by any reagent or reagents known for use in such a reaction (e.g. NaBH 3 CN, NaBH 4 or NaB(OR) 3 H, where R is alkyl or acyl).
  • the product of this reaction is a number of dendrimer molecules bound to a solid phase support through a linker entity.
  • Scheme 4 shows suitable methods for the synthesis of immunomodulating substances containing chemoselective groups in the form of weak electrophiles. These immunomodulating substances are used in the synthesis of CD conjugates as described in Schemes 1 , 2 and 3.
  • agen e.g. DCC; EDC; DIPCDI; BOP; PyBOP; BOP-CI; BOP-Br; PyBrOP; HBTU; TBTU; TFFH;HATU; Haloformamidinium; Haloimidazolidinium- either in solution or attached to a resin.
  • agen e.g. DCC; EDC; DIPCDI; BOP; PyBOP; BOP-CI; BOP-Br; PyBrOP; HBTU; TBTU; TFFH;HATU; Haloformamidinium; Haloimidazolidinium- either in solution or attached to a resin.
  • the present invention also relates to a new type of chemically well-defined carbohydrate conjugate.
  • CD conjugates are easily prepared with a high degree of purity through the methods disclosed herein.
  • the CD conjugates of the invention display well-defined chemical structure and properties, as they are prepared from symmetrical dendrimers using chemoselective reactions in a few synthetic steps.
  • CD conjugates are very versatile and can comprise specific carbohydrates selected from a large number of different varieties, including simple oligosaccharides (for example cancer-related antigens, glycolipid determinants) and polysaccharides (for example bacterial lipopolysaccharide carbohydrates, bacterial capsular carbohydrates).
  • CD conjugates can comprise the two components in any desired ratio, as governed by the stoichiometry used for the chemoselective coupling reactions as described herein.
  • the present invention also relates to CD conjugates having the structure:
  • A is a functional, multivalent dendrimer or a conjugate of two or more functional, multivalent dendrimers
  • E' is a residue of a carbohydrate E or a derivative or a fragment thereof
  • L' is a residue of an immunomodulating substance L
  • m is 1
  • n is an integer equal to or larger than 1 , if more than one L' is present it could be the same or different,
  • CD conjugates having the structure:
  • A is a functional, multivalent dendrimer or a conjugate of two or more functional, multivalent dendrimers E' is a residue of a carbohydrate E or a derivative or a fragment thereof,
  • L' is a residue of an immunomodulating substance L
  • m is 1 , 2 or 3
  • n is an integer equal to or larger than 1 , if more than one E' is present it could be the same or different, if more than one L' is present it could be the same or different,
  • m may more specifically be 1 or 2, or most specifically 1.
  • the invention further relates to an intermediate compound with the formula
  • m is 1 , 2 or 3 and wherein A has at least one surface group that is not connected to an E' group.
  • m may more specifically be 1 or 2, or most specifically 1.
  • CD conjugates of the present invention is the case in which all functional groups on the CD conjugate are not occupied by either carbohydrate or immunomodulating substance. Free amino functionalities at the dendrimer surface could be used for coupling a third kind of molecule to the dendrimer such as e.g. another type of immunomodulating substance, or another CD conjugate.
  • n may be an integer from 1 to 100 inclusive, such as e.g. from 1 to 70 inclusive, from 1 to 50 inclusive, from 3-20 inclusive such as e.g. 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15.
  • CD conjugates in which immunomodulating substances are bound to the CD conjugate also called carbohydrate dendrimer immunogens (GDIs)
  • GDIs carbohydrate dendrimer immunogens
  • This kind of CD conjugate can be used for efficient immunization against carbohydrate antigens.
  • the basic inventive concept of a GDI conjugate is that a suitable and optimal immunogenicity of a carbohydrate is achieved by linking one or a few carbohydrate molecules together with a high number of immunomodulating molecules, dispensing the need for a highly repetitive carbohydrate structure.
  • the present invention does not rely on the use of dendrimers for achieving a so-called “cluster effect" of the carbohydrate moiety; this has been exploited in other structures ⁇ Lo-Man et al., 2001, Bundle et al. 2001).
  • the GDIs of the present invention somewhat surprisingly achieve biological activity (immunogenicity) by presenting one or very few carbohydrate entities together with an immunogenic substance that is present in the GDI compound in multiple copies.
  • the manufacture of such composite compounds is achieved by the modular, chemical approach described in the examples below and the result is fully defined and characterizable compounds.
  • Such GDI conjugates conform to the principles behind the structure of highly immunogenic glycoconjugates as outlined above. They can therefore be used to achieve a high immunogenicity for carbohydrates that have until now been considered non-immunogenic, allowing their use in vaccines aimed at establishing a desirable immunological response against the carbohydrate.
  • carbohydrate antigens that are useful for vaccination against human and veterinary diseases, including bacterial, viral and parasitic infections and certain types of cancers, including carcinoma and melanoma, as well as for immunization for other purposes, for example for the production of antibodies that are used in various diagnostic assays is significantly increased by the present invention.
  • CD conjugates The chemoselectivity involved in the construction of CD conjugates allows their immunological response to be tailored to suit the requirements of the application, for example by combining a number of different carbohydrates or immunomodulating entities in one CD conjugate.
  • CD conjugates used are not only limited to the novel CD conjugates disclosed herein, but also include those CD conjugates which are produced via the novel method of this invention.
  • CD conjugates An important field of application for CD conjugates is for immunizations leading to swift and high-titered antibody responses dominated by IgG or an equivalent type of antibodies and characterised by the development of immunological memory.
  • the present invention relates to CD conjugates synthesized via the disclosed methods which are used in the production of antibodies.
  • a particular embodiment of the invention is the case wherein CD conjugates are used in the production of antibodies against the carbohydrate E of the CD conjugate.
  • a method according to the present invention for the production of antibodies against the CD conjugate and/or the carbohydrate E comprises immunizing an animal with a CD conjugate as defined.
  • suitable animals for use in such an immunization are selected from the group comprising: mice, rats, rabbits, sheep, non-human primates and poultry.
  • the method according to the invention additionally relates to the production of monoclonal and/or polyclonal antibodies.
  • a suitable use of antibodies of the present invention is their use in diagnostic assays and high throughput screening.
  • An embodiment of the present invention is the use of CD conjugates as targeting compounds. This is achieved by coupling a predetermined number of carbohydrate moieties on a suitable dendrimer followed by the coupling of a suitable number of molecules of a drug that is intended for delivery in a target cell or target tissue, said carbohydrate being the ligand of a lectin residing in said target cell or target tissue. In a targeting dendrimer of this composition the number of carbohydrate moieties is greater than 1.
  • Additional therapeutic uses of CD conjugates according to the invention comprise the treatment of disease processes in which pathological cells, expressing carbohydrate binding receptors can be targeted by a CD conjugate containing the specific carbohydrate ligand in addition to a suitable drug.
  • Said pathological cells can be cells of the patient (e.g. cancerous cells or virally infected cells) or cells of a pathological agent as e.g. a bacterium.
  • the drug to be used should be a host cell cytotoxic drug while in the latter case it should be an antimicrobial drug as known by anyone skilled in the art.
  • Some examples of such useful antimicrobial drugs are listed in Goers et al. (US4867973).
  • the CD conjugates of the present invention are to be used in medicine.
  • a particular embodiment of such a use is the inhibition of bacterial adhesion, the inhibition of toxin action, such as e.g. glycosphingolipid-specific VT2 toxins and other such bacterial toxins with binding activities toward cell-surface carbohydrates of the host or inhibition of carbohydrate-mediated virus entry into host cells.
  • CD conjugates include their application for treating bacterial and viral diseases in which binding of a disease-causing component to a receptor can be blocked. Examples of this include inhibition of bacterial toxins binding to cell surface glycoconjugates, as for example the binding of glycosphingolipid-specific verotoxins to its cell surface receptor. Additional important bacterial toxins that may be the target of inhibition by specific CD conjugates of the present invention include cholera toxin, heat labile toxins of £. coli and Campylobacter jejuni (these are all specific for the carbohydrate part of the ganglioside GM1).
  • CD conjugates can be used to counteract carbohydrate-mediated binding (adherence) of bacteria or viruses to epithelial surfaces through surface glycoconjugates either present on the pathogen's surface or on the surface of host cells.
  • bacteria to host cell surface carbohydrates include Pseudomonas aeruginosa (asialo-GM1) and Helicobacter pylori (various gangliosides) while Pneumocystis carinii and Actinobacillus pleuropneumoniae are examples of bacteria adhering through the binding of bacterial cell surface carbohydrates to host cell carbohydrate receptors.
  • Examples of viral adherence mediated by carbohydrate ligands include HIV binding host galactosylated ceramides and rotavirus being bound by host receptors with specificity for certain ceramides.
  • a method for treating and/or preventing bacterial diseases comprising the administration to an animal an effective amount of a CD conjugate according to the method of the invention.
  • diseases include infection with bacteria described herein, viral diseases such as infection with HIV, hepatitis or influenza, fungal diseases and certain types of cancer such as carcinomas and melanomas.
  • a method for treating and/or preventing bacterial diseases comprising the administration to an animal an effective amount of an antibody obtained through the method described herein.
  • diseases include infection with bacteria described herein, viral diseases such as infection with HIV, hepatitis or influenza, fungal diseases and certain types of cancer such as carcinomas and melanomas.
  • Antibodies against cancer-associated carbohydrate structures are relevant for diagnosis and prognosis as well as for treatment as the result of active specific immunotherapy using the relevant CD conjugates of the invention for immunization.
  • the present invention also relates to pharmaceutical compositions comprising a CD conjugate as described herein.
  • the pharmaceutical composition comprises an antibody according to the invention.
  • Such compositions also comprise compounds appropriate for such a composition to facilitate delivery of said CD conjugate or antibody against a CD conjugate to a subject (human or non-human animal).
  • the use of bacterial carbohydrates for vaccines and for diagnostics is highly beneficial due to the extraordinarily specificity and biological importance of these molecules; however, their use has been hampered until now by the inadequacies of current methods of preparing carbohydrate immunogens.
  • the CD conjugates of the present invention are well suited for such an application. They are easily prepared by generally applicable methods, chemically well defined and fully analysable as well as having the desired immunological properties, including the ability to provoke the establishment of a high-titered immune response with immunological memory.
  • Also very useful for therapy are antibodies raised against carbohydrate structures specific for cancerous or otherwise pathological cell types, provided by immunization of a suitable laboratory animal or by immunization of the patient with a CD conjugate containing the relevant carbohydrate epitope.
  • CD conjugates which are produced through the method described herein are their use in vaccines.
  • the present invention relates to a vaccine composition comprising CD conjugates as described herein.
  • CD conjugates can be used for immunizations either alone or in combination with adjuvants in a way that is well known to anyone skilled in the art.
  • An adjuvant is a substance which augments or optimises specific immune response to antigens. Adjuvants should be efficient and safe.
  • Alum (aiuminiumhydroxide or aluminiumphosphate) is the only adjuvant licensed for use in humans.
  • adjuvants which may be used in immunisation include: Alum, MDP and MDP analogs (for example threonyl MDP, MDP-L-alanyl cholesterol), oil emulsions (Freund's adjuvant, pluronic block polymers, SAF, MF59, Emulsigen), lipid A derivatives (monophosphoryl lipid A), liposomes, surface active agents (saponins, quil A, Iscoms), cytokines and cytokine fragments (IL-2, interferon-gamma, IL-12, IL-1 nonapeptide) and immunostimulatory unmethylated CpG-containing oligodeoxynucleotides.
  • Alum threonyl MDP, MDP-L-alanyl cholesterol
  • oil emulsions Red's adjuvant, pluronic block polymers, SAF, MF59, Emulsigen
  • lipid A derivatives monophosphoryl lipid A
  • liposomes
  • the present invention describes a vaccine composition as defined above further comprising one or more adjuvants such as e.g. Freunds adjuvant, Alum, MDP and MDP analogs, oil emulsions, lipid A derivatives, liposomes or surface active agents such as e.g. saponins, quil A or iscoms.
  • adjuvants such as e.g. Freunds adjuvant, Alum, MDP and MDP analogs, oil emulsions, lipid A derivatives, liposomes or surface active agents such as e.g. saponins, quil A or iscoms.
  • compositions as described herein which further comprise one or more adjuvants including microcarriers such as for example polylactide-co-glycolide microparticles.
  • microcarriers such as for example polylactide-co-glycolide microparticles.
  • Such microcarriers may be combined with other adjuvants as for example MF59 or lipophilic muramyldipeptides.
  • the invention also concerns a vaccine composition
  • a vaccine composition comprising one or more additional immunoactivating substances, such as e.g. cytokines and cytokine fragments, unmethylated CpG -nucleotides or muramyldipeptides.
  • additional immunoactivating substances such as e.g. cytokines and cytokine fragments, unmethylated CpG -nucleotides or muramyldipeptides.
  • the present invention also discloses a vaccine composition comprising antibodies, additionally comprising an immunoglobulin fraction or a hyperimmune serum.
  • the invention also comprises vaccine formulations containing the CD conjugates of the invention, said formulations containing an effective amount of the CD conjugate in a pharmaceutically acceptable vehicle in liquid or emulsion form in the presence of an adjuvant. Equally suitable are such vaccines not containing an adjuvant.
  • Such vaccines are used by administering the vaccine by any of the usual routes used for vaccination such as e.g. parental or mucosal administration.
  • the vaccines of the present invention are to be used in the vaccination of animals, such as e.g. fish, pigs, sheep, humans, non-human primates and cattle.
  • the present invention relates to libraries comprising two or more CD conjugates as described above.
  • the libraries comprise conjugates representing combinations of different types of carbohydrates and different types of immunomodulating substances.
  • the CD conjugates which make up the library are synthesised via solid-phase synthesis.
  • the invention further relates to the use of said libraries for mapping of immunogenic determinants. Additionally, the libraries of the present invention can be used in high- throughput screening.
  • CD conjugates will be the mapping of immunogenic determinants using solid-phase coupled CD conjugate libraries containing different combinations of different types of carbohydrate and immunomodulating substance. This may be carried out in combination with cellular assays for immunogenicity. Libraries of CD conjugates may also be used in high-throughput screening studies in order to rapidly narrow a large range of potentially useful CD conjugates to those which have shown positive results in the required investigation. Other Uses
  • the CD conjugates of the present invention are to be used in diagnostic assays. Additionally disclosed is the use of such conjugates in high-throughput screening.
  • the CD conjugate comprises a reporter molecule
  • the CD conjugate is to be used in assays for the detection of antibodies against the carbohydrate moiety E.
  • Example 1 Preactivation of immunomodulating substances: (see scheme 4)
  • Phage degradation of PS is performed by a modification of the procedure published by Svenson & Lindberg (1978).
  • LPS (20 mg) is added to a dialysis bag (Slide a Lyzer MWCO 3500 Da) with purified bacteriophage p22c2 (containing endo-rhamnosidase activity), previously dialysed against 5 mM ammonium carbonate buffer (pH 7.1).
  • the dialysis bag is immersed in 200 ml of the same ammonium carbonate buffer and dialysed for 4 days at 37°C, to allow phage mediated hydrolysis of rhamnose 1-3 galactose ⁇ -linkage.
  • the oligosaccharide containing dialysis fluids is renewed and dialysis continues for an additional 40 h.
  • the combined oligosaccharide containing dialysis buffers are freeze- dried, removing the ammonium carbonate.
  • the crude oligosaccharide is obtained as a white crystalline solid and analysed by MS (MALDI-TOF): calc. C 52 H 8 6O 37 : 1302.5, found: 1325.7 (M+Na + ); 1283.7 (M+Na + -Ac); 1241.7 (M+Na + -2Ac).
  • the resin is washed with ethanol (or NMP), and 20 equiv of immunomodulating substance -isothiocyanate (or active ester) is added and the resin is shaken for 16 h. After wash of the resin with ethanol (NMP), ninhydrin test is negative. The CD conjugate is released from the resin upon treatment with 1-95%v/v trifluoroacetic acid for 1 h. Reprecipitation from either acetone or diethyl ether.
  • the BAL-modified resin is now ready for further solid-phase synthesis.
  • 2 Equiv amino-terminated dendrimer (2'nd or 3'rd generation DAB or PAMAM) is added to 1 equiv BAL- modified resin in 96 % ethanol (or NMP), 10 equiv NaBH 3 CN is added and the suspension is shaken 16 h at r.t.. Ninhydrin test shows positive.
  • the resin is washed with ethanol (or NMP), and 1.05 equiv purified p-22 phage degraded O-antigen (octa- saccharide fraction) is added to 1 equiv of dendrimer-modified resin.
  • the CD conjugate containing only carbohydrate and dendrimer is dissolved in 50% ethanol and excess of immunomodulating substance isothiocyanate or active ester (60 equiv) is added and the mixture is stirred overnight at r.t.
  • the carbohydrate dendrimer conjugate (CD conjugate) is precipitated by addition of 2 vol of acetone, reprecipitation from acetone.
  • the CD conjugate is redissolved in water and freeze-dried. Analysis by 1 H 13 C NMR, HPLC-MS, elemental analysis and melting point.
  • the carbohydrate dendrimer conjugate (CD conjugate) is precipitated by addition of 2 vol of acetone, reprecipitation from acetone- water.
  • the CD conjugate is redissolved in water and freeze-dried. Analysis by 1 H 13C NMR, HPLC-MS, elemental analysis and melting point.
  • the CD conjugate containing only carbohydrate and dendrimer is dissolved in 50% ethanol and excess of immunomodulating substance isothiocyanate (60 equiv) is added and the mixture is stirred overnight at r.t.
  • the carbohydrate dendrimer conjugate (CD conjugate) is precipitated by addition of 2 vol of acetone, reprecipitation from acetone.
  • the CD conjugate is redissolved in water and freeze-dried. Analysis by 1 H 13C NMR, HPLC- MS, elemental analysis and melting point.
  • the carbohydrate dendrimer conjugate (CD conjugate) is precipitated by addition of 2 vol of acetone, reprecipitation from acetone-water.
  • the CD conjugate is redissolved in water and freeze-dried. Analysis by 1 H-NMR, 13 C-NMR, HPLC- MS, elemental analysis and melting point.
  • Example 3 CD conjugate formation from delipidated full O-antigen (from Gram Negative lipopolysaccharide).
  • This conjugate is reprecipitated from acetone-water and redissolved in water and freeze-dried.
  • the CD conjugate containing only carbohydrate and dendrimer is dissolved in 50% ethanol and immunomodulating substance isothiocyanate or active ester (50-60 equiv) is added. The mixture is stirred 16 h at r.t.
  • the CD conjugate is precipitated by addition of acetone (1 vol).
  • the CD conjugate is redissolved in water and freeze-dried.
  • the resin is washed with ethanol, and 20 equiv of immunomodulating substance-isothiocyanate (or active ester) is added and the resin is shaken for 16 h. After wash of the resin with ethanol, ninhydrin test shows negative. The CD conjugate is released from the resin upon treatment with 1-95% trifluoroacetic acid for 1 h. Reprecipitation from either acetone or diethyl ether.
  • Example 4 CD conjugate formation from Tn-antigen and Thomson-Friedenheim antigen.
  • Tn-dendrimer conjugate is precipitated by addition of acetone (2vol), filtered off and redissolved in water followed by freeze-drying.
  • the Tn-dendrimer conjugate is analysed by NMR, HPLC-MS and elemental analysis.
  • the Tn-antigen orThomson-Friedenreich antigen conjugate is dissolved in 50% aqueous ethanol, 60 equiv of immunomodulating substance isothiocyanate or active ester is added and the mixture is stirred 2 days at r.t.
  • the conjugate is precipitated by addition of acetone (2 vol).
  • the conjugate is filtered off and the N-boc protection is removed by 5 equiv HCI in methanol (made by AcCI in methanol) and boiling for 5 min, precipitation by diethyl ether.
  • the CD conjugate is analysed by NMR, HPLC-MS and elemental analysis.
  • the conjugate is filtered off and the N-boc protection is removed by 5 equiv HCI in methanol (made by acetylchlorid in methanol) and boiling for 5 min, precipitation by diethyl ether.
  • the CD conjugate is analysed by NMR, HPLC-MS and elemental analysis.
  • the resin is washed with ethanol (or NMP), 20 equiv of immunomodulating substance-isothiocyanate (or active ester) is added and the resin is shaken for 48 h. After wash of the resin with ethanol (NMP), ninhydrin test is negative. The CD conjugate is released from the resin upon treatment with 1-95%v/v trifluoroacetic acid for 1 h. Reprecipitation from either acetone or diethyl ether.
  • the BAL-modified resin is now ready for further solid-phase synthesis.
  • 2 Equiv amino- terminated dendrimer (2'nd or 3'rd generation DAB or PAMAM) is added to 1 equiv BAL- modified resin in 96 % ethanol (or NMP), 10 equiv NaBH 3 CN is added and the suspension is shaken 16 h at r.t.. Ninhydrin test shows positive.
  • the resin is washed with ethanol (or NMP), and 1.05 equiv of preactivated (see) N-Boc protected Tn-antigen or Thomson-Friedenreich antigen is added to 1 equiv of dendrimer modified resin and the mixture is shaken for 16 h.
  • the resin is washed with ethanol (or NMP), and 20 equiv of immunomodulating substance-isothiocyanate (or active ester) is added and the resin is shaken for 48 h. After wash of the resin with ethanol (NMP), ninhydrin test is negative. The CD conjugate is released from the resin upon treatment with 1-95% trifluoroacetic acid for 1 h. Reprecipitation from either acetone or diethyl ether.
  • Example 5 The manufacture of CDI conjugates containing a large carbohydrate in a defined ratio to an immunomodulating substance and test of immunogenicity.
  • the polysaccharide part (20-100 kD) of a bacterial lipopolysaccharide from Salmonella Typhimurium, in another example from Escherichia coli and in yet another example from Actinobacillus pleuropneumoniae serotype 12 will be coupled through its carboxylate groups to a dendrimer as detailed in Example 2.6.
  • the immunogenicity of the CDI conjugate is tested by immunization of mice. This is done after the following procedure: The freeze-dried CDI conjugate is dissolved in sterile PBS at a concentration in the range of 0.1 to 5 mg/ml and preferably at least 1 mg/ml.
  • Preferable immunization doses are in the range of 1 to 500 ⁇ g CDI, preferably 5-100 ⁇ g at a concentration of between 0.1 to 5 mg/ml.
  • the CDI/PBS solution is then used for immunization of young (6-8 weeks) BALB/c mice using one or more of the following procedures in separate immunization experiments, using at least 4 mice in each group:
  • Immunization is performed 3 times with 14 days interval and blood samples are collected by tail bleeding before the first immunization (0 sample) and 10 days after the last immunization. Serum is prepared from the blood samples by clotting and analysed for reactivity against the carbohydrate antigen being presented in the CDI conjugate. The analysis is performed by indirect ELISA using the carbohydrate antigen conjugated to a suitable carrier protein or the bacterial lipopolysaccharide containing the carbohydrate antigen, for coating in a concentration of 1-50 ug/ml in PBS on Maxisorp microtitre plates (carrier protein conjugated carbohydrate antigen) or on Polysorp microtitre plates (lipopolysaccharides) (both types of plates are from Nunc).
  • the CDI itself may be used for coating in the same concentration in PBS.
  • a CDI or a protein- conjugate or an LPS comprising a structurally related nonsense carbohydrate should be used as a negative control coating antigen.
  • Coating is performed overnight at 4°C and followed by wash in PBS + 0.05 v/v%Tween 20 (PBS-T) and blocking in PBS-T-BSA (PBS-T + 0.1 w/v % BSA (Sigma A2153) for 1 hour.
  • mice sera is performed, using sera at a dilution of 1/100 and 1/1000 in PBS-T-BSA, incubating each dilution in duplicate and performing the incubation for 1 hour at room temperature. Then the plate is washed four times with PBS-T and incubated with peroxidase-conjugated rabbit anti mouse immunoglobulins (DAKO P260) at 1/1000 for 1 hour, followed by wash as before. Finally, the plate is developed by the peroxidase- orthophenyldiamine procedure and read at 490 nm using 650 nm for correction. For determination of titres, antisera are first tested in this ELISA as 2-fold dilution series from 1/1000 to 1/128000.
  • DAKO P260 peroxidase-conjugated rabbit anti mouse immunoglobulins
  • the end point is defined as the background OD reading (background OD being the mean OD of at least 4 wells in which serum has been replaced by the dilution buffer) + 2 standard deviations. If this end point is not reached in this first titration, further dilutions are performed in a new test until it is possible to determine the titre, defined as the highest dilution of the serum leading to an OD reading above the end point OD.
  • mice immunoglobulin type and isotypes sera are tested 1/1000 in an ELISA as above, but instead of DAKO P269 as the secondary antibody using biotinylated isotyping antibodies from Zymed according to the manufacturer's instructions followed by peroxidase-conjugated streptavidin from DAKO (P397) diluted 1/1000.
  • a competitive ELISA is performed. This is essentially done as the indirect ELISA described above but the antisera are mixed 1+1 in the well with a titration series of the free (non-coupled) carbohydrate antigen in question being titrated from 1 mg/ml and downwards in 2-fold steps.
  • a fixed dilution of the antiserum is used corresponding to an OD reading around 80% of the maximum OD-reading (as derived from a titration in the indirect ELISA decribed above) when mixed 1+1 with the competition antigen in the well. From the resulting curve it is possible to calculate the affinity of the antiserum with the free carbohydrate antigen, taking the free carbohydrate antigen concentration in mg/ml corresponding to half maximum OD. This concentration will correspond to the affinity constant of the antiserum-carbohydrate antigen system. From this, a mean affinity constant expressed in M "1 can be derived by using the mean molecular weight of the carbohydrate antigen in question.
  • Example 6 A diagnostic assay based on antibodies produced against CDIs specifying a specific, bacterial carbohydrate antigen.
  • antibodies against CDI are produced by the method described in example 5. The antibodies are then used to develop a diagnostic assay for infection with a specific bacterium carrying the antigenic carbohydrate on its surface.
  • the diagnostic antibody is produced as a monoclonal antibody allowing a detection of antibodies in the serum samples of the animal being analysed to become even more specific and more sensitive.
  • This principle can be applied on e.g. infection with Salmonella Typhimurium in swine using the lipopolysaccharide-derived carbohydrate antigen obtained by acid hydrolysis and obtained by acid hydrolysis and phage-degradation.
  • the principle can also be applied on diagnosis of infection with Actinobacillus pleuropneumoniae serotype 12 in swine using a carbohydrate mimic of the capsular polysaccharide antigen from this serotype in CDIs used for immunization.
  • Example 7 A diagnostic assay based on the use of CDIs specifying a specific, bacterial carbohydrate antigen.
  • CDIs may also be used directly as coating antigens in ELISA, provided that they are synthesised to become "good coaters", e.g. by supplying a suitably hydrophobic group as L. Also, by varying the ratio between E and L in order to introduce more carbohydrate units and less hydrophobic units in the CDI a highly efficient and specific coating antigen can be tailored.
  • the serum sample to be analysed is simply applied onto the coated plate and incubated whereafter the plate is washed and probed with peroxidase-conjugated antibodies against immunoglobulins of the species in question.
  • the presence of specific antibodies in the serum sample is revealed by an increased OD value in that sample.
  • This type of assay is expected to be highly specific and with a very high sensitivity due to the highly efficient, multimeric presentation of the carbohydrate antigens.
  • the assay is used to demonstrate the same infections as mentioned in Example 6.
  • Example 8 A CDI-based solid-phase screening assay for definition and characterization of immunostimulating compounds.
  • CDI conjugates with good immunostimulating properties will be able to stimulate unprimed CD1 expressing cells in addition to monocytes in culture. It is furthermore envisaged that the immunoactivator type, its type of binding to the dendrimer and the number of immunoactivators present on the dendrimer surface will all affect the immunogenicity of the CDI conjugate. It is, however a benefit inherent in the present invention that 1) CDI conjugates are made from a small number of modules and that 2) CDI conjugates can easily be prepared by solid-phase synthesis and this can be exploited to prepare solid phase coupled addressable combinatorial libraries of small-scale CDI conjugates that can be tested in assays for CD1 -binding and for monocyte activation.
  • Highly active compounds can then be identified and prepared in larger amounts by solid- phase or solution phase synthesis and used for immunizations of live animals as described above. This will provide a method for determining factors influencing the immunogenicity of CDIs compared to the in vitro immunostimulatory capacity of such compounds.
  • the method can be applied on e.g. a medium-sized carbohydrate included in a CDI conjugate combined with a range of different immunostimulating substances in various numbers and attached by various spacers, each variant being traceable to a specific part of the solid phase beads used for synthesis.
  • a suitable medium-sized carbohydrate is a phage-degraded Salmonella Typhimurium tetra- or octameric oligosaccharide, prepared as described in Example 2.1.
  • Suitable in vitro screening assays include the binding of free mouse CD1 molecules to solid-phase bound CDIs as detected by anti-CD1 antibodies and also include a mononuclear cell preparation which, after contact with solid-phase bound CDI is analysed for proliferation by a suitable assay including the MTT assay and the IL-2 assay. Furthermore this cell preparation can be obtained from already primed animals (eg. animals having been infected with the corresponding bacterium) to analyse the response of primed animals to the CDI variants present in the library.
  • already primed animals eg. animals having been infected with the corresponding bacterium
  • a group of pigs are immunized with an Ap12 CDI conjugate and then challenged with Ap12 either by aerosol or by intranasal installation.
  • Another group of pigs will be challenged in the same way but without being treated with Ap12 CDI conjugate first; instead they will receive pig serum from a group of pigs being immunised with Ap12 CDI conjugate (passive serum treatment).
  • Finally a third group of pigs will receive Ap12 challenge together with Ap12 CDI conjugate by simultaneous inoculation through the same route as the Ap12 challenge or through another route. All groups will be compared to a non-treated Ap12 challenged group with respect to development of clinical signs and pathology. It is expected that the CDIs will protect the pigs against disease and will clear the pigs of the infection.
  • Verheul, A. F. M. Versteeg, A. A.., De Reuver, M. J., Jansze, M., Snippe, H.(1989), Infect.

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Abstract

L'invention concerne un méthode de synthèse de conjugués de dendrimère présentant une structure chimique bien définie, comprenant une ou plusieurs fractions glucidiques et une ou plusieurs substances d'immunomodulation reliées à un dendrimère. D'abord, le glucide est relié au dendrimère de manière chimiosélective. Puis, la substance d'immunomodulation est également reliée au dendrimère de manière chimiosélective, pour donner un conjugué de dendrimère présentant une structure et une connectivité bien définie, et contenant un rapport précis et prédéterminé de glucide par rapport à la substance d'immunomodulation. L'invention concerne également de nouveaux conjugués de dendrimère et leur utilisation dans une vaccination, dans une production d'anticorps, dans un criblage à rendement élevé, dans des bibliothèques et dans des analyses diagnostiques.
PCT/DK2003/000766 2002-11-08 2003-11-07 Preparation de conjugues de dendrimere et de glucide chimiquement bien definis WO2004041310A1 (fr)

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