[go: up one dir, main page]
More Web Proxy on the site http://driver.im/

WO2011036560A2 - Glycoconjugate compositions and methods for treatment of hiv - Google Patents

Glycoconjugate compositions and methods for treatment of hiv Download PDF

Info

Publication number
WO2011036560A2
WO2011036560A2 PCT/IB2010/002541 IB2010002541W WO2011036560A2 WO 2011036560 A2 WO2011036560 A2 WO 2011036560A2 IB 2010002541 W IB2010002541 W IB 2010002541W WO 2011036560 A2 WO2011036560 A2 WO 2011036560A2
Authority
WO
WIPO (PCT)
Prior art keywords
glycoconjugate
composition according
hiv
composition
carrier
Prior art date
Application number
PCT/IB2010/002541
Other languages
French (fr)
Other versions
WO2011036560A3 (en
Inventor
Paolo Costantino
Anna Kabanova
Original Assignee
Novartis Ag
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novartis Ag filed Critical Novartis Ag
Publication of WO2011036560A2 publication Critical patent/WO2011036560A2/en
Publication of WO2011036560A3 publication Critical patent/WO2011036560A3/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/595—Polyamides, e.g. nylon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV

Definitions

  • HIV human immunodeficiency virus
  • Env HIV-1 envelope protein
  • the broadly neutralizing monoclonal antibody 2G12 binds a cluster of high-mannose-type oligosaccharides on the gp120 subunit of Env, revealing a conserved epitope on the glycan shield [2].
  • the sugar multivalency was demonstrated to be the key to higher 2G12 affinity [3].
  • Dendrimers are polymers of spherical or other three-dimensional shapes that have precisely defined compositions and that possess a precisely defined molecular weight. Dendrimers can be synthesized as water-soluble macromolecules through appropriate selection of internal and external moieties, See, U.S. Pat. Nos. 4,507,466 and 4, 568,737, incorporated by reference herein. The first well-defined, symmetrical, dendrimer family were the polyamidoamine (PAMAM) dendrimers, which are manufactured by the Dow Chemical Company.
  • PAMAM polyamidoamine
  • Figure 1 illustrates total IgG antibody titres specific against an9 in ELISA assay performed on microplates coated with human serum albumine (HSA) conjugate of Man9. Each dot represents single rabbit sera. Horizontal bars indicate GMT meaning of group.
  • HSA human serum albumine
  • Figure 2 illustrates inhibition of anti-Man9PAMAM4-CRM sera in ELISA assay with Man4-PAMAM4-CRM plate coating.
  • a composition having a glycoconjugate of at least 2 sugar moieties bound to a multivalent support and wherein said composition binds neutralizing antibodies greater than monovalent oligosaccharides.
  • Such glycoconjugates can be further bound to carriers.
  • the glycoconjugates are useful in vaccines and as antigens which bind to antibodies.
  • the present invention relates to glycoconjugates having sugar moieties bound to a multivalent support for the treatment of HIV.
  • glycoconjugates are provided which comprise oligosaccharides composed of synthetic high mannose sugars bound to dendrimer scaffolds, such as flexible polyamidoamine (PAMAM).
  • PAMAM flexible polyamidoamine
  • the invention also encompasses, glycoconjugates or oligosaccharides bound directly to carriers. The invention thus provides glyconconjugates as vaccine antigens which bind to neutralizing antibodies.
  • Oletaccharides refer to a saccharide polymer containing a number, for example two or more, of component sugars.
  • Man4, Man6, Man9, Man(X) refer to a polymer of mannose sugars linked together by the number of sugars, i.e., 4, 6, or 9 manrioses respectively. (X) refers to the number of sugars in the polymer.
  • “Sugar” or “sugar moiety” or “saccharide” refer to simple sugars, saccharide, saccharide residues, or derivatives thereof.
  • Cluster refers to a linear or branched polymers of sugars.
  • Glycoconjugate refers to a oligosaccharide or oligosaccharide cluster linked to or associated with carrier protein.
  • Oligosaccharides included in the present invention are selected from the group monosaccharide, disaccharide, trisaccharide, and polymers of sugar moieties including 4, 6, 8, 9, 10 units.
  • Suitable sugar moieties include at least one hexose residue, such as allose, altrose, glucose, mannose, gulose, idose, galactose, talose and combinations thereof.
  • a saccharide residue is a pentose, tetrose or triose.
  • a sugar moeity may contain a combination of hexose, pentose, tetrose and triose residues.
  • a sugar moiety may contain a 7-, 8-, 9, 10-, 11-, 12- or more carbon sugar, such as sialic acid.
  • a sugar moiety also encompasses steroisomers or anomer forms, combinations thereof, acid derivatives thereof, and glycosyl residues thereof.
  • the present invention relates to a glycoconjugate comprising oligosaccharides linked to or associated with a multivalent scaffold.
  • the number of sugar moieties which may be linked or associated with a multivalent scaffold is limited only by scaffold valency, the amount of active groups desired or presented and cross-reactivity to 2G12.
  • Preferably the number of sugar moieties that form a cluster most closely resembles glycosylation patterns on envelope regions of HIV.
  • a preferred glycoconjugate comprises a cluster of 4, 6, 8, 9, 10 or more sugar moieties conjugated to a multivalent support.
  • Preferred glycoconjugate includes Man6, Man9, Man4PA AM4, Man4PAMAM8, Man9PAMAM4, Man9PAMAM8.
  • a multivalent support may also be protein-based, including single amino acid polymers such as polylysine, polyarginine and polyhistidine, gelatin, collagen, complex natural ' protein carriers such as albumins and globulins; and including recombinant versions and combinations of these.
  • a multivalent support may also be carbohydrate- based, including for instance polymers such as carrageen, alginates, pectins and celluloses.
  • a multivalent support includes those that are lipid-based, such as cationic lipids of various compositions, liposome compositions of various types as described for instance in R. R. C. New, Liposomes: A Practical Approach, Oxford University Press.
  • a preferred multivalent support includes a dendrimer upon which monosaccharides and oligosaccharides are bound or associated.
  • Dendrimer creation is generally characterized by multistage synthesis of generational products having an organized branched structure. Preparation and synthesis of dendrimers is well known and is described by way of example in 4507466, 4558120, 4568737 and 4587329. Briefly, a dendrimer is synthesized from monomers added in stepwise fashion, such that each synthetic step produces a generation which is added to in a subsequent step. Each generation of dendrimers has an increasing number of terminals available for conjugation to a functional group.
  • a first generation dendrimer may have 4 sites available for functional group conjugation, a second generation 8, etc.
  • the number of functional groups conjugated to the sites available for attachment on the dendrimer may be equal to or less than the maximum number of sites available on the dendrimer.
  • Various types of dendrimers are known in the art including polyamidoamine (PAMAM) and poly (propylene imine) dendrimers.
  • Additional dendrimer compositions may be used as a multivalent support in an inventive composition include L-lysine and N,N'-bis(acrylamido)acetic acid dendrimers.
  • Dendrimer compositions, in the context of the present invention are advantageous due to their structural stability and ability to mimic the glycosylated regions of antigenic epitopes on HIV.
  • Preferred glycoconjugates include Man4PAMAM4, an4PAMAM8, Man9PAMAM4, Man9PAMAM8.
  • the glycoconjugates of the invention have a relative binding efficiency to 2G12 and HIV gp120 proteins similar to 2G12, as well as a 10 to 20- fold enhancement of capacity to inhibit gp120-2G 2 interaction in in vitro assays compared to monovalent sugars.
  • Man4 and an9 glycoconjugates showed cross-reactivity with 2G12 in ELISA assay.
  • the glycoconjugates of the invention induce antibodies having dissociation constant values of 10 "6 , W 7 , 10 10 "9 ,10 - 10 , 10 "11 , 10 2 , 10 "13 , 10 “14 , less than 10 "6 , preferably less than 10 "8 , preferably less than 10 "10 .
  • Conjugation of sugar to functional groups on multivalent supports utilizes techniques known in the art.
  • an amino group of synthetic oligosaccharide or of a linker bound to a synthetic oligosaccharide, activated with N-disuccinimidyl adipate is linked to an amine on a scaffold or carrier protein.
  • An exemplary method for conjugation is described in the Examples herein.
  • other conjugation chemistries can be used.
  • a free amino group on sugar or on a linker bound to a sugar it can be activated with squarate linker or other linker with ester groups. Further this activated amino group may be conjugated to an amino group on a protein or scaffold.
  • a carboxylic ester is present on a synthetic oligosaccharide or on a linker bound to it
  • treatment with a diamine can generate a free amino group which can be activated as described above.
  • activation can be performed by EDAC-NHS or any other chemistry capable of activating carboxylic groups.
  • the ratio of sugar or oligosaccharide to carrier protein (w/w) in a glycoconjugate may be determined calculating the ratio of carbohydrate to protein concentration or directly by mass analysis (ESI, MALDI).
  • the amount of protein is determined using a BCA or Lowry assay (for example Lowry et al (1951) J. Biol. Chem. 193, 265-275 or Peterson et al Analytical Biochemistry 100, 201-220 (1979)), and the amount of saccharide is determined using HPAEC-PAD analysis.
  • the conjugation method may also rely on activation of the saccharide with 1- cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP) to form a cyanate ester.
  • CDAP 1- cyano-4-dimethylamino pyridinium tetrafluoroborate
  • the activated saccharide may thus be coupled directly or via a spacer (linker) group to an amino group on the carrier protein.
  • the spacer could be cystamine or cysteamine to give a thiolated polysaccharide which could be coupled to the carrier via a thioether linkage obtained after reaction with a maleimide-activated carrier protein (for example using GMBS) or a holoacetylated carrier protein (for example using iodoacetimide or N-succinimidyl bromoacetatebromoacetate).
  • a maleimide-activated carrier protein for example using GMBS
  • a holoacetylated carrier protein for example using iodoacetimide or N-succinimidyl bromoacetatebromoacetate.
  • the cyanate ester obtained by CDAP chemistry
  • the amino-derivatised saccharide is conjugated to the carrier protein using carbodiimide (e.g. EDAC) chemistry via a carboxyl group on the protein carrier.
  • carbodiimide e.g. EDAC
  • the glycoconjugates of the inventions are bound to a carrier molecule.
  • the oligosaccharides may or may not be proximately associated (such as by conjugation) to the carrier molecule.
  • glyconconjugates are composed of oligosaccharides associated with multivalent structures which are further bound to carriers.
  • oligosaccharides are associated with or linked to carriers directly.
  • Preferred glycoconjugates of the invention comprise oligosaccharides bound to PAMAM and further conjugated to CRM197.
  • monovalent sugars are bound to a carrier having a glycosylation degree with a medium value of 41.5% carbohydrate/protein wt/wt %.
  • the inventors have found enhanced antigenic properties in glycoconjugates of the invention to inhibit gp120 and 2G12 interaction or reactivity with gp120 in comparison to monovalent sugars.
  • Carriers are known in the art. Plotkin, Vaccines 3rd Ed. Philadelphia, WB Saunders Co. (1999).
  • Bacterial carriers i.e., carriers derived from bacteria
  • Bacterial carriers include, but are not limited to, cholera toxin B subunit (CTB); diphtheria toxin mutant (CRM197); diphtheria toxoid; group B streptococcus alpha C protein; meningococcal outer membrane protein (OMPC); tetanus toxoid; outer membrane protein of nontypeable Haemophilus influenza (such as P6); recombinant class 3 porin (rPorB) of group B meningococci; heat-killed Brucella abortus; heat-killed Listeria monocytogenes; and Pseudomonas aeruginosa recombinant exoprotein A; N19 recombinant protein.
  • Another carrier is keyhole limpet hemo
  • carriers also encompass virus-like particles produced recombinantly in a eukaryotic cell line.
  • Virus-like particles could be of any viral nature providing sufficient immunogenicity to said glycoconjugate.
  • Virus-like particles encompassed by the invention include recombinant alphavirus, adenovirus, poxvirus, canaryvirus.
  • Alphavirus particles, methods of making and using are described in detail in 5843723, 5789245, 6342372, 5814482, 6015694, 63762366451592 and 753 180.
  • Methods for determining if glycoconjugates encompassed by the present invention can be performed by common assays for evaluating HIV antibodies and further provided below.
  • the formation of an antibody-antigen complex can be assayed using a number of well-defined diagnostic assays including conventional immunoassay formats to detect and/or quantitate antigen-specific antibodies.
  • diagnostic assays include, for example, enzyme immunoassays, e.g., ELISA, cell-based assays, flow cytometry, radioimmunoassays, and immunohistochemical staining.
  • Numerous competitive and non-competitive protein binding assays are known in the art and many are commercially available.
  • Representative assays include, for example, various binding assays with chemokine receptors (CCR5 or CXCR4), gp41 , characterized domains of these polypeptides, and competitive binding assays with characterized HIV-1 binding antibodies.
  • the glycoconjugates of the invention can also be employed to generate antibodies that recognize antigenic oligosaccharides mimicking the glycosylation pattern of HIV envelope polypeptides.
  • the method comprises administering to a subject or nonhuman animal an immunogenic composition comprising the glycoconjugate described herein.
  • immunogenic compositions of the invention can be administered to the subject by any suitable route of administration. Accordingly, in one embodiment, an immunogenic composition is administered to a subject to generate antibodies that recognize the heterologous HIV-1 neutralizing epitope.
  • Such antibodies find use in HIV research.
  • the nonhuman subjects employed in this embodiment is one typically employed for antibody production. Mammals, such as, rodents, rabbits, goats, sheep, etc., are preferred.
  • the antibodies generated can be either polyclonal or monoclonal antibodies.
  • Polyclonal antibodies are raised by injecting (e.g. subcutaneous or intramuscular injection) antigenic polypeptides into a suitable animal (e.g., a mouse or a rabbit). The antibodies are then obtained from blood samples taken from the animal.
  • the techniques used to produce polyclonal antibodies are extensively described in the literature.
  • Polyclonal antibodies produced by the subjects can be further purified, for example, by binding to and elution from a matrix that is bound with the polypeptide against which the antibodies were raised.
  • Monoclonal antibodies can also be generated using techniques known in the art.
  • the present invention provides a method for prophylactic or therapeutic treatment of an HIV or other viral infection in a human or non-human animal, which comprises administering to said human or animal an effective amount of a glycoconjugate as broadly described above.
  • An "effective amount” is defined herein as an amount of a biologically active agent that produces an intended biological activity.
  • viral infections for which glycoconjugates of the invention are useful in effecting treatment include, for example infections by HIV1 and H I V2 and other enveloped viruses including flaviviruses such as Hepatitis B and Hepatitis C, Bovine Viral Diarrhoea Virus, Human Influenza Virus A and B, Rhinovirus, Human Parainfluenza Virus, Respiratory Syncytial Virus (RSV), Varicella Zoster Virus (VZV), Human Cytomegalovirus (GMV), Epstein Barr Virus (EBV), Human Papilloma Virus (HPV), Adenovirus-8, Herpes Simplex Virus (HSV) type 1 and 2, Measles Virus, and Vesicular Stomatitis Virus (VSV).
  • flaviviruses such as Hepatitis B and Hepatitis C, Bovine Viral Diarrhoea Virus, Human Influenza Virus A and B, Rhinovirus, Human Parainfluenza Virus, Respir
  • a method of antiviral treatment is including the step of administering an effective amount of a glycoconjugate to an individual human or non- human animal in need of a therapeutic or prophylactic anti-viral treatment.
  • a glycoconjugate is administered to an individual human or non-human animal at risk of retroviral infection or in need of treatment for retrovirus infection.
  • a glycoconjugate is administered to an individual human or non-human animal at risk of HIV infection or in need of treatment for HIV infection.
  • glycoconjungates of the present invention are administered in combination with another composition, such as a protease inhibitor, nucleoside analog, virucidal agent, adjuvant or antibody, anti-viral nucleic acid such as an anti-sense construct or SIRNA, immune modulator, zinc finger inhibitor, integrase inhibitor or TAT inhibitor.
  • another composition such as a protease inhibitor, nucleoside analog, virucidal agent, adjuvant or antibody, anti-viral nucleic acid such as an anti-sense construct or SIRNA, immune modulator, zinc finger inhibitor, integrase inhibitor or TAT inhibitor.
  • the present invention provides a vaccine, wherein the host produces antibodies and/or CTL responses against the glycoconjugates of the invention, which responses then preferably serve to neutralize HIV viruses by, for example, inhibiting further HIV infection or generating neutralizing antibodies to glycoconjugates.
  • Administration of the glycoconjugates of the invention as a prophylactic vaccine would comprise administering to a subject a concentration of glycoconjugates effective in raising an immune response which is sufficient to elicit antibody and/or CTL responses to antigenic regions such as HIV Env, Gag, and Pol, and/or neutralize HIV, by, for example, inhibiting HIV ability to infect cells.
  • concentration will depend upon the specific compound to be administered, but may be determined by using standard techniques for assaying the development of an immune response which are well known to those of ordinary skill in the art.
  • the glyconconjugates of the invention may be formulated with a suitable adjuvant in order to enhance the immunological response.
  • suitable adjuvants may include, but are not limited to mineral gels such as aluminum hydroxide; surface active substances such as lysolecithin, pluronic polyols, polyanions; other peptides; oil emulsions; and potentially useful human adjuvants such as BCG and Corynebacterium parvum.
  • Adjuvants suitable for co-administration in accordance with the present invention should be those that are potentially safe, well tolerated and effective in people including QS-21 , Detox-PC, MPL-SE, MoGM-CSF, TiterMax-G, CRL-1005, GERBU, TERamide, PSC97B, Adjumer, PG-026, GSK-1 , GcMAF, B-alethine, MPC-026, Adjuvax, CpG ODN, Betafectin, Alum, and MF59 (see Kim et al., 2000, Vaccine, 18: 597 and references therein).
  • a variety of administration routes are available. The particular mode selected will depend, of course, upon the particular condition being treated and the dosage required for therapeutic efficacy.
  • the methods of this invention may be practised using any mode of administration that is medically acceptable, meaning any mode that produces therapeutic levels of the active component of the invention without causing clinically unacceptable adverse effects.
  • modes of administration include oral, rectal, topical, nasal, transdermal or parenteral (e.g. subcutaneous, intramuscular and intravenous) routes, such as by injection.
  • Formulations for oral administration include discrete units such as capsules, tablets, lozenges and the like.
  • Other routes include intrathecal administration directly into spinal fluid, direct introduction such as by various catheter and balloon angioplasty devices well known to those of ordinary skill in the art, and intraparenchymal injection into targeted areas.
  • a pharmaceutical composition according to the invention includes an inventive compound as described herein, and may further contain a pharmaceutically acceptable carrier or excipient formulation.
  • pharmaceutically acceptable is intended to mean a material that is not biologically or otherwise undesirable, which can be administered to an individual along with an inventive compound without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • a pharmaceutically acceptable carrier or excipient formulation may include sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile administrable solutions or dispersions.
  • aqueous and non-aqueous carriers, diluents, solvents or vehicles examples include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate.
  • a coating such as lecithin
  • surfactants for example, water, alcohol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like
  • suitable mixtures thereof examples include vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
  • a pharmaceutical composition according to the invention may include other pharmaceutical agents.
  • the general content of a pharmaceutically acceptable carrier or excipient formulation will depend on the form in which the pharmaceutical composition containing an inventive compound is given.
  • a pharmaceutical composition containing an inventive compound is delivered in solid, semi-solid or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, or suspensions, preferably in unit dosage form suitable for single administration of a precise dosage.
  • the glyconconjugate may be administered orally, parenterally (for example, intravenously), by intramuscular injection, by intraperitoneal injection, or transdermally.
  • fine powders or granules may contain diluting, dispersing, and/or surface active agents, and may be presented in water or in a syrup, in capsules or sachets in the dry state or in a nonaqueous solution or suspension wherein suspending agents may be included, in tablets wherein binders and lubricants may be included, or in a suspension in a liquid or gel. Tablets and granules are preferred oral administration forms, and these may be coated.
  • Parenteral administration is generally by injection. Injectables can be prepared in conventional forms, either liquid solutions or suspensions, solid forms suitable for solution or prior to injection, or as suspension in liquid prior to injection or as emulsions.
  • an excipient formulation may include conventional nontoxic solid carriers or fillers include, for example, pharmaceutical grades of cellulose, glucose, lactose, magnesium carbonate, magnesium stearate, mannitol, sodium saccharine, silicic acid, starches, sucrose and talc.
  • Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving or dispersing the glycoconjugate compound with optional pharmaceutical adjuvants in an excipient or inert diluent to thereby form a solution or suspension.
  • Liquid dosage forms include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs.
  • a liquid dosage form may contain such excipients or inert diluents as water or other solvents, solubilizing agents and emulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1 ,3-butyleneglycol, dimethylformamide, oils, in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan or mixtures of these substances, and the like.
  • solubilizing agents and emulsifiers as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
  • Suspensions in addition to the glycoconjugate compounds, may contain suspending agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.
  • suspending agents as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.
  • An excipient formulation may contain further inert customary ingredients, such as binders, as for example, carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone, sucrose, and acacia; humectants, as for example, glycerol; disintegrating agents, as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; solution retarders, as for example paraffin; absorption accelerators, as for example, quaternary ammonium compounds; wetting agents, as for example, cetyl alcohol, and glycerol monostearate; adsorbents, as for example, kaolin and bentonite; and lubricants illustratively including talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof.
  • binders as for example, carboxymethylcellulose, alignates
  • the excipient formulation can also include adjuvants, such as emulsifying agents, sweetening, flavoring, and perfuming agents.
  • adjuvants such as emulsifying agents, sweetening, flavoring, and perfuming agents.
  • time release preparations or intravenous preparations are exemplary effective dosage formulations.
  • release control components include: carbomer, alpha-starch, polyacrylamides, polysaccharides, polyvinylpyrrolidone; natural gums such as gum arabic; clays; lipophilic gelling agents; fatty acid metal salts such as aluminum stearates; hydrophobic silica; celluloses such as various hydroxyalkylcelluloses; polyethylene glycol; and combinations thereof.
  • the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as pH buffering agents, for example, sodium acetate, sodium citrate, dicalcium phosphate or triethanolamine oleate. Contamination by microorganisms can be prevented or inhibited by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride, and the like. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see A.
  • pH buffering agents for example, sodium acetate, sodium citrate, dicalcium phosphate or triethanolamine oleate. Contamination by microorganisms can be prevented or inhibited by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include iso
  • the exact amount of the pharmaceutical composition required will vary from subject to subject, depending on the age, weight and general condition of the subject, the condition being treated, the particular compound used, its mode of administration, and the like.
  • ., Cancer Chemother. Rep., 50:219-244, (1966) can be used to determine the maximum tolerated dose of an inventive compound for a human subject.
  • An appropriate amount may be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.
  • Synthetic high mannose oligosaccharides are available from Ancora Farmaceuticals (MA, USA).
  • CRMi 97 is internally produced in Novartis V&D, Siena, Italy (Research Disclosure 453077 Jan 2002).
  • the 2G12 antibody and HIV gp140 UG37 (clade A strain 92/UG/037, a.a. 32-662, NCBI protein database No. AAC97548, catalog no. ENV001) are purchased from Polymun Scientific (Vienna, Austria).
  • HIV gp 120 Bal (a.a.32- 518, Geneban No. M68893, catalog no. IT-001-002p), gp120 R2 (a.a.41-520, Geneban No.
  • AF128126, catalog no. IT-001-0029p), gp120 JRFL (a.a.34-518, Geneban No. U63632, catalog no. IT-001-0024p) are available from Immune Technology Corp. (New York, US). Polymun and Immune Technology recombinant proteins are expressed in CHO and 293T cells, correspondingly. Biotinylated Galantus nivalis lectin (GNL) is available from Vector Laboratories (CA, US).
  • Isocratic separations are performed using a 30-min 16 m NaOH followed by a 5-min 500 mM NaOH regeneration step and 15-min re-equilibration, set to a flow rate of 1.0 ml min "1 .
  • Monosaccharide peaks are detected directly by using quadruple-potential waveform pulsed amperometry on a gold working electrode and an Ag/AgCI reference electrode.
  • Raw data are elaborated on a Chromeleon 6.8 chromatography software (Dionex) with application of 0.5-10 g/ml mannose calibration curve. Rapid hexose quantification is achieved by Phenol-H 2 S04 method [24]. Protein concentration is determined by Micro BCA kit (Thermo Fisher Scientific).
  • PAMAM4 cluster formation is analyzed by direct sample injection in Micromass Q- Tof Micro system (Waters MS Technologies, UK).
  • PAMAM8 cluster formation is analyzed on Q-tof Micro system paired to UPLC system (ACQUITY UPLC System, Waters, UK).
  • the samples are diluted 1 :200 or less in 0.1 % formic acid, 1 :1 vol acetonitrile:water.
  • Chromatographic separations are performed on 2.1 mm i d. x 50 mm ACQUITY BEH C18 1.7 im column (Waters Corp., USA).
  • TOF MS analysis is performed operating in positive ion mode (ESI).
  • the nebulization gas is set to 800 LJh at a temperature of 250°C, the cone gas set to 50 L/h and the source temperature set to 100°C.
  • the capillary and cone voltages are 3500 V and 30 V, respectively.
  • the Q-Tof Micro is operated with a collision energy of 5 V.
  • the data acquisition rate is set to 0.1 s with a 0.1 s inter-scan delay.
  • the raw data are analyzed by the Micromass Marker-Lynx applications manager Version 1.0 (Waters, UK).
  • Oligosaccharide pellet is vaccum dried and weighed (9-10 mg, 90-100%). Oligosaccharide is coupled to PAMAM with stoichiometry of 8:1 mol CHO:PAMAM4 and 20:1 mol CHO:PAMAM8. Reaction in DMSO with 20 ⁇ /ml thiethylamine ias stirred overnight at RT. Cluster formation is monitored by ESI Q-TOF MS analysis. No signal of unreacted PAMAM is observed in any of the final reaction mixtures.
  • Reaction sample is lyophilized overnight and then dissolved in water. Excess of unreacted oligosaccharide is removed by hydrophobic interaction C4 column (0.5 ml resin, Bioselect, Grace Vydac) in a stepwise gradient of methanol (0-80% in water). Column is activated with methanol and then preconditioned with water. Fractions are analyzed by TLC and TOF-MS. Target fractions are dried to eliminate methanol. Carbohydrate yields for cluster formation and purification are 36.3% for Man4PAMAM4; 16.2% for Man9PAMAM4; 18% for Man4PAMAM8; 14.7% for Man9PAMAM8.
  • 2 Mg/ml 2G12 is preincubated with appropriate inhibitor for 15 minutes at 37°C before the start of analysis.
  • Analyte is injected at 10 ⁇ /min for 4 min, followed by 6 min dissociation and 30 sec of regeneration with 10 mM glycine, 3M NaCI pH 2.0. Similar conditions are used for analysis with 2G12 chips that had 490 RU and 4400 RU of immobilization.
  • oligosaccharides or oligosaccharide clusters are activated with disuccinimidyl adipate and purified according to the procedure mentioned above. Before activation Boc group of cluster is eliminated by stirring in 20% TFA for two hours. Conjugation stoichiometry of 30:1 or 40:1 CHO/cluster : protein mol is applied. Conjugation is done in 10-20 mg/ml protein solution in 200 mM NaPi pH 7,0 cluster. The mixture is incubated overnight at 37°C, and reaction outcome is verified by SDS-PAGE analysis.
  • Conjugates are purified from excess of unconjugated carbohydrate using ultrafiltration spin columns with 30 kDa or 50 kDa cut-off (Vivaspin, Sartorius). Glycosylation degree is calculated on the basis of protein and carbohydrate concentration.
  • Man9 is conjugated to HSA following similar procedures.
  • ELISA - 96-well Maxisorp plates (Nunc, Thermo Fisher Scientific) are coated with 100 ⁇ /well of PBS-diluted glycoproteins with 2 pg/ml coating concentration. Plates are incubated overnight at +4°C, then washed three times with TPBS (PBS with 0,05% Tween 20, pH 7.4) and blocked with 100 ⁇ well of 2% BSA (Sigma-Aldrich) for 1 hour at 37°C. Subsequently each incubation step is followed by triple TPBS wash. 200 ⁇ of prediluted 2G12 or lectin are transferred into coated-blocked plates (200 ⁇ ) and serially two-fold diluted followed by 2h incubation at 37°C.
  • TPBS PBS with 0,05% Tween 20, pH 7.4
  • BSA Sigma-Aldrich
  • Serum samples are initially diluted 1 :50-1 :1000 in 2 % BSA in TPBS. Then 100 ⁇ well of 1 :10000-1 :20000 diluted appropriate alkaline phosphatase-conjugated secondary antibody or streptavidin-alkaline phosphatase conjugate (Sigma-Aldrich) is added for 1 h at 37°C. 100 ⁇ /well of 1 mg/ml pNPP disodium hexahydrate (Sigma Aldrich) in 1 M diethanolamine (pH 9.8) is distributed onto plates to visualize the amount of bound alkaline phosphatase. After 30 minutes of development at RT plates are read with a microplate spectrophotometer at 405 nm. Antibody titres are those dilutions that give an optical density (OD) higher than triple average OD obtained for the mock-immunized rabbit sera.
  • OD optical density
  • One prime and two boosting doses of 20 pg carbohydrate antigen were administered i.m.
  • Control animals were immunized with MF-59 in PBS.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Chemical & Material Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Epidemiology (AREA)
  • Molecular Biology (AREA)
  • Virology (AREA)
  • AIDS & HIV (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Immunology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

Compounds are provided which are glycoconjugate compositions having oligosaccharides bound to multivalent structures for use in vaccines and binding to antibodies.

Description

Glycoconjugate compositions and methods for treatment of HIV
Background of the Invention
[001] The human immunodeficiency virus (HIV) is a very complex virus and presents many different potential targets for therapeutic intervention. Heavily glycosylated regions, highly variable loops, hidden co-receptor binding site(s), buried fusion peptides, and unfaithful replication of reverse transcriptase have all contributed to the inability to produce an effective vaccine to date. HIV-1 envelope protein (Env) contains numerous N-linked carbohydrates that shield conserved peptide epitopes and promote trans-infection of dendritic cells via binding to cell surface lectins [1]. The broadly neutralizing monoclonal antibody 2G12 binds a cluster of high-mannose-type oligosaccharides on the gp120 subunit of Env, revealing a conserved epitope on the glycan shield [2]. The sugar multivalency was demonstrated to be the key to higher 2G12 affinity [3]. These features make high mannose oligosaccharide epitope a viable target for vaccine design.
[002] Antiviral agents comprised of polysaccharides bound to mamcromolecular compounds such as dendrimers have been described in . US6190650 and US2004/0180852. Dendrimers are polymers of spherical or other three-dimensional shapes that have precisely defined compositions and that possess a precisely defined molecular weight. Dendrimers can be synthesized as water-soluble macromolecules through appropriate selection of internal and external moieties, See, U.S. Pat. Nos. 4,507,466 and 4, 568,737, incorporated by reference herein. The first well-defined, symmetrical, dendrimer family were the polyamidoamine (PAMAM) dendrimers, which are manufactured by the Dow Chemical Company.
[003] Currently, no synthetic glycoconjugates have been shown to elicit effective neutralizing antibodies capable of cross-reacting with HIV antigens. There remains a great need for further development of compounds in this area.
Brief Description of the Drawings
[004] Figure 1 illustrates total IgG antibody titres specific against an9 in ELISA assay performed on microplates coated with human serum albumine (HSA) conjugate of Man9. Each dot represents single rabbit sera. Horizontal bars indicate GMT meaning of group.
[005] Figure 2 illustrates inhibition of anti-Man9PAMAM4-CRM sera in ELISA assay with Man4-PAMAM4-CRM plate coating.
Summary of the Invention
[006] According to the present invention, a composition is provided having a glycoconjugate of at least 2 sugar moieties bound to a multivalent support and wherein said composition binds neutralizing antibodies greater than monovalent oligosaccharides. Such glycoconjugates can be further bound to carriers. The glycoconjugates are useful in vaccines and as antigens which bind to antibodies.
Detailed Description of the Invention
[007] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry and nucleic acid chemistry and hybridization described below are those well known and commonly employed in the art. Standard techniques are used for nucleic acid and peptide synthesis. Generally, enzymatic reactions and purification steps are performed according to the manufacturer's specifications. The techniques and procedures are generally performed according to conventional methods in the art and various general references, see, generally, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., which is incorporated herein by reference), which are provided throughout this document. The nomenclature used herein and the laboratory procedures in analytical chemistry, and organic synthetic described below are those well known and commonly employed in the art. Standard techniques, or modifications thereof, are used for chemical syntheses and chemical analyses.
All references cited herein are incorporated by reference in their entirety.
[008] The present invention relates to glycoconjugates having sugar moieties bound to a multivalent support for the treatment of HIV. Specifically, glycoconjugates are provided which comprise oligosaccharides composed of synthetic high mannose sugars bound to dendrimer scaffolds, such as flexible polyamidoamine (PAMAM). The invention also encompasses, glycoconjugates or oligosaccharides bound directly to carriers. The invention thus provides glyconconjugates as vaccine antigens which bind to neutralizing antibodies.
[009] "Monovalent" are used herein to refer to sugars or oligosaccharides which are not bound to a scaffold and hereby are referred to as the "non-clustered" form.
[0010] "Oligosaccharides" refer to a saccharide polymer containing a number, for example two or more, of component sugars.
[0011] "Man4, Man6, Man9, Man(X)" refer to a polymer of mannose sugars linked together by the number of sugars, i.e., 4, 6, or 9 manrioses respectively. (X) refers to the number of sugars in the polymer.
[0012] "Sugar" or "sugar moiety" or "saccharide" refer to simple sugars, saccharide, saccharide residues, or derivatives thereof.
[0013] "Cluster" refers to a linear or branched polymers of sugars.
[0014] "Glycoconjugate" refers to a oligosaccharide or oligosaccharide cluster linked to or associated with carrier protein.
[0015] Oligosaccharides included in the present invention are selected from the group monosaccharide, disaccharide, trisaccharide, and polymers of sugar moieties including 4, 6, 8, 9, 10 units. Suitable sugar moieties include at least one hexose residue, such as allose, altrose, glucose, mannose, gulose, idose, galactose, talose and combinations thereof. In some aspects, a saccharide residue is a pentose, tetrose or triose. A sugar moeity may contain a combination of hexose, pentose, tetrose and triose residues. Further, in some aspects, a sugar moiety may contain a 7-, 8-, 9, 10-, 11-, 12- or more carbon sugar, such as sialic acid. A sugar moiety also encompasses steroisomers or anomer forms, combinations thereof, acid derivatives thereof, and glycosyl residues thereof.
[0016] In another aspect, the present invention relates to a glycoconjugate comprising oligosaccharides linked to or associated with a multivalent scaffold. The number of sugar moieties which may be linked or associated with a multivalent scaffold is limited only by scaffold valency, the amount of active groups desired or presented and cross-reactivity to 2G12. Preferably the number of sugar moieties that form a cluster most closely resembles glycosylation patterns on envelope regions of HIV. A preferred glycoconjugate comprises a cluster of 4, 6, 8, 9, 10 or more sugar moieties conjugated to a multivalent support. Preferred glycoconjugate includes Man6, Man9, Man4PA AM4, Man4PAMAM8, Man9PAMAM4, Man9PAMAM8.
[0017] A multivalent support may also be protein-based, including single amino acid polymers such as polylysine, polyarginine and polyhistidine, gelatin, collagen, complex natural' protein carriers such as albumins and globulins; and including recombinant versions and combinations of these. A multivalent support may also be carbohydrate- based, including for instance polymers such as carrageen, alginates, pectins and celluloses. In addition, a multivalent support includes those that are lipid-based, such as cationic lipids of various compositions, liposome compositions of various types as described for instance in R. R. C. New, Liposomes: A Practical Approach, Oxford University Press.
[0018] A preferred multivalent support includes a dendrimer upon which monosaccharides and oligosaccharides are bound or associated. Dendrimer creation is generally characterized by multistage synthesis of generational products having an organized branched structure. Preparation and synthesis of dendrimers is well known and is described by way of example in 4507466, 4558120, 4568737 and 4587329. Briefly, a dendrimer is synthesized from monomers added in stepwise fashion, such that each synthetic step produces a generation which is added to in a subsequent step. Each generation of dendrimers has an increasing number of terminals available for conjugation to a functional group. Thus, for example, a first generation dendrimer may have 4 sites available for functional group conjugation, a second generation 8, etc. Depending on factors such as steric hindrance, the number of functional groups conjugated to the sites available for attachment on the dendrimer may be equal to or less than the maximum number of sites available on the dendrimer. Various types of dendrimers are known in the art including polyamidoamine (PAMAM) and poly (propylene imine) dendrimers. Additional dendrimer compositions may be used as a multivalent support in an inventive composition include L-lysine and N,N'-bis(acrylamido)acetic acid dendrimers. Dendrimer compositions, in the context of the present invention are advantageous due to their structural stability and ability to mimic the glycosylated regions of antigenic epitopes on HIV.
[0019] Preferred glycoconjugates include Man4PAMAM4, an4PAMAM8, Man9PAMAM4, Man9PAMAM8. The glycoconjugates of the invention have a relative binding efficiency to 2G12 and HIV gp120 proteins similar to 2G12, as well as a 10 to 20- fold enhancement of capacity to inhibit gp120-2G 2 interaction in in vitro assays compared to monovalent sugars. Man4 and an9 glycoconjugates showed cross-reactivity with 2G12 in ELISA assay. Surprisingly, the glycoconjugates of the invention induce antibodies having dissociation constant values of 10"6, W7, 10 10"9,10 -10, 10"11, 10 2, 10"13, 10"14, less than 10"6, preferably less than 10"8, preferably less than 10"10.
[0020] Conjugation of sugar to functional groups on multivalent supports utilizes techniques known in the art. In one aspect, an amino group of synthetic oligosaccharide or of a linker bound to a synthetic oligosaccharide, activated with N-disuccinimidyl adipate, is linked to an amine on a scaffold or carrier protein. An exemplary method for conjugation is described in the Examples herein. Optionally, other conjugation chemistries can be used. Where a free amino group on sugar or on a linker bound to a sugar is present, it can be activated with squarate linker or other linker with ester groups. Further this activated amino group may be conjugated to an amino group on a protein or scaffold.
Where a carboxylic ester is present on a synthetic oligosaccharide or on a linker bound to it, treatment with a diamine (hydrazine, ethylenediamine) can generate a free amino group which can be activated as described above. Where a carboxylic group is present on an oligosaccharide or on a linker bound to it, activation can be performed by EDAC-NHS or any other chemistry capable of activating carboxylic groups. Where a thiol group is present on an oligosaccharide or on a linker bound to it, it can linked to a C=C (such as allyl, or maleidimide) present in a previously activated protein or a scaffold. Alternatively, an oligosaccharide functionalized with a C=C (such as allyl, or maleidimide) can be linked to thio groups in a protein or a scaffold,
[0021] The ratio of sugar or oligosaccharide to carrier protein (w/w) in a glycoconjugate may be determined calculating the ratio of carbohydrate to protein concentration or directly by mass analysis (ESI, MALDI). The amount of protein is determined using a BCA or Lowry assay (for example Lowry et al (1951) J. Biol. Chem. 193, 265-275 or Peterson et al Analytical Biochemistry 100, 201-220 (1979)), and the amount of saccharide is determined using HPAEC-PAD analysis.
[0022] The conjugation method may also rely on activation of the saccharide with 1- cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP) to form a cyanate ester. The activated saccharide may thus be coupled directly or via a spacer (linker) group to an amino group on the carrier protein. For example, the spacer could be cystamine or cysteamine to give a thiolated polysaccharide which could be coupled to the carrier via a thioether linkage obtained after reaction with a maleimide-activated carrier protein (for example using GMBS) or a holoacetylated carrier protein (for example using iodoacetimide or N-succinimidyl bromoacetatebromoacetate). Optionally, the cyanate ester (obtained by CDAP chemistry) is coupled with hexane diamine or ADH and the amino-derivatised saccharide is conjugated to the carrier protein using carbodiimide (e.g. EDAC) chemistry via a carboxyl group on the protein carrier. Such conjugates are described in PCT published application WO 93/15760 Uniformed Services University and WO 95/08348 and WO 96/29094.
[0023] In a further aspect, the glycoconjugates of the inventions are bound to a carrier molecule. In some aspects comprising a carrier molecule, the oligosaccharides may or may not be proximately associated (such as by conjugation) to the carrier molecule. In one aspect glyconconjugates are composed of oligosaccharides associated with multivalent structures which are further bound to carriers. In another aspect oligosaccharides are associated with or linked to carriers directly. Preferred glycoconjugates of the invention comprise oligosaccharides bound to PAMAM and further conjugated to CRM197. In one aspect, monovalent sugars are bound to a carrier having a glycosylation degree with a medium value of 41.5% carbohydrate/protein wt/wt %. Surprisingly, the inventors have found enhanced antigenic properties in glycoconjugates of the invention to inhibit gp120 and 2G12 interaction or reactivity with gp120 in comparison to monovalent sugars.
[0024] Carriers are known in the art. Plotkin, Vaccines 3rd Ed. Philadelphia, WB Saunders Co. (1999). Bacterial carriers (i.e., carriers derived from bacteria) include, but are not limited to, cholera toxin B subunit (CTB); diphtheria toxin mutant (CRM197); diphtheria toxoid; group B streptococcus alpha C protein; meningococcal outer membrane protein (OMPC); tetanus toxoid; outer membrane protein of nontypeable Haemophilus influenza (such as P6); recombinant class 3 porin (rPorB) of group B meningococci; heat-killed Brucella abortus; heat-killed Listeria monocytogenes; and Pseudomonas aeruginosa recombinant exoprotein A; N19 recombinant protein. Another carrier is keyhole limpet hemocyanin (KLH). Examples of viral-derived carriers are known in the art and include hepatitis b surface antigen (HBsAg) particles and hepatitis b core antigen (HBcAg).
[0025] In another aspect, carriers also encompass virus-like particles produced recombinantly in a eukaryotic cell line. Virus-like particles could be of any viral nature providing sufficient immunogenicity to said glycoconjugate. Virus-like particles encompassed by the invention include recombinant alphavirus, adenovirus, poxvirus, canaryvirus. Alphavirus particles, methods of making and using are described in detail in 5843723, 5789245, 6342372, 5814482, 6015694, 63762366451592 and 753 180.
[0026] Methods for determining if glycoconjugates encompassed by the present invention can be performed by common assays for evaluating HIV antibodies and further provided below. The formation of an antibody-antigen complex can be assayed using a number of well-defined diagnostic assays including conventional immunoassay formats to detect and/or quantitate antigen-specific antibodies. Such assays include, for example, enzyme immunoassays, e.g., ELISA, cell-based assays, flow cytometry, radioimmunoassays, and immunohistochemical staining. Numerous competitive and non-competitive protein binding assays are known in the art and many are commercially available. Representative assays include, for example, various binding assays with chemokine receptors (CCR5 or CXCR4), gp41 , characterized domains of these polypeptides, and competitive binding assays with characterized HIV-1 binding antibodies.
[0027] The glycoconjugates of the invention can also be employed to generate antibodies that recognize antigenic oligosaccharides mimicking the glycosylation pattern of HIV envelope polypeptides. The method comprises administering to a subject or nonhuman animal an immunogenic composition comprising the glycoconjugate described herein. As outlined in detail below, immunogenic compositions of the invention can be administered to the subject by any suitable route of administration. Accordingly, in one embodiment, an immunogenic composition is administered to a subject to generate antibodies that recognize the heterologous HIV-1 neutralizing epitope. Such antibodies find use in HIV research. Generally, the nonhuman subjects employed in this embodiment is one typically employed for antibody production. Mammals, such as, rodents, rabbits, goats, sheep, etc., are preferred.
[0028] The antibodies generated can be either polyclonal or monoclonal antibodies. Polyclonal antibodies are raised by injecting (e.g. subcutaneous or intramuscular injection) antigenic polypeptides into a suitable animal (e.g., a mouse or a rabbit). The antibodies are then obtained from blood samples taken from the animal. The techniques used to produce polyclonal antibodies are extensively described in the literature. Polyclonal antibodies produced by the subjects can be further purified, for example, by binding to and elution from a matrix that is bound with the polypeptide against which the antibodies were raised. Those of skill in the art will know of various standard techniques for purification and/or concentration of polyclonal, as well as monoclonal, antibodies. Monoclonal antibodies can also be generated using techniques known in the art.
[0029] In another aspect, the present invention provides a method for prophylactic or therapeutic treatment of an HIV or other viral infection in a human or non-human animal, which comprises administering to said human or animal an effective amount of a glycoconjugate as broadly described above. An "effective amount" is defined herein as an amount of a biologically active agent that produces an intended biological activity. Other viral infections for which glycoconjugates of the invention are useful in effecting treatment include, for example infections by HIV1 and H I V2 and other enveloped viruses including flaviviruses such as Hepatitis B and Hepatitis C, Bovine Viral Diarrhoea Virus, Human Influenza Virus A and B, Rhinovirus, Human Parainfluenza Virus, Respiratory Syncytial Virus (RSV), Varicella Zoster Virus (VZV), Human Cytomegalovirus (GMV), Epstein Barr Virus (EBV), Human Papilloma Virus (HPV), Adenovirus-8, Herpes Simplex Virus (HSV) type 1 and 2, Measles Virus, and Vesicular Stomatitis Virus (VSV).
[0030] In another aspect, a method of antiviral treatment is including the step of administering an effective amount of a glycoconjugate to an individual human or non- human animal in need of a therapeutic or prophylactic anti-viral treatment. In particular, a glycoconjugate is administered to an individual human or non-human animal at risk of retroviral infection or in need of treatment for retrovirus infection. In a preferred aspect, a glycoconjugate is administered to an individual human or non-human animal at risk of HIV infection or in need of treatment for HIV infection.
[0031] Additionally, the glycoconjungates of the present invention are administered in combination with another composition, such as a protease inhibitor, nucleoside analog, virucidal agent, adjuvant or antibody, anti-viral nucleic acid such as an anti-sense construct or SIRNA, immune modulator, zinc finger inhibitor, integrase inhibitor or TAT inhibitor.
[0032] Accordingly, the present invention provides a vaccine, wherein the host produces antibodies and/or CTL responses against the glycoconjugates of the invention, which responses then preferably serve to neutralize HIV viruses by, for example, inhibiting further HIV infection or generating neutralizing antibodies to glycoconjugates. Administration of the glycoconjugates of the invention as a prophylactic vaccine, therefore, would comprise administering to a subject a concentration of glycoconjugates effective in raising an immune response which is sufficient to elicit antibody and/or CTL responses to antigenic regions such as HIV Env, Gag, and Pol, and/or neutralize HIV, by, for example, inhibiting HIV ability to infect cells. The exact concentration will depend upon the specific compound to be administered, but may be determined by using standard techniques for assaying the development of an immune response which are well known to those of ordinary skill in the art.
[0033] The glyconconjugates of the invention may be formulated with a suitable adjuvant in order to enhance the immunological response. Such adjuvants may include, but are not limited to mineral gels such as aluminum hydroxide; surface active substances such as lysolecithin, pluronic polyols, polyanions; other peptides; oil emulsions; and potentially useful human adjuvants such as BCG and Corynebacterium parvum.
[0034] Adjuvants suitable for co-administration in accordance with the present invention should be those that are potentially safe, well tolerated and effective in people including QS-21 , Detox-PC, MPL-SE, MoGM-CSF, TiterMax-G, CRL-1005, GERBU, TERamide, PSC97B, Adjumer, PG-026, GSK-1 , GcMAF, B-alethine, MPC-026, Adjuvax, CpG ODN, Betafectin, Alum, and MF59 (see Kim et al., 2000, Vaccine, 18: 597 and references therein).
[0035] A variety of administration routes are available. The particular mode selected will depend, of course, upon the particular condition being treated and the dosage required for therapeutic efficacy. The methods of this invention, generally speaking, may be practised using any mode of administration that is medically acceptable, meaning any mode that produces therapeutic levels of the active component of the invention without causing clinically unacceptable adverse effects. Such modes of administration include oral, rectal, topical, nasal, transdermal or parenteral (e.g. subcutaneous, intramuscular and intravenous) routes, such as by injection. Formulations for oral administration include discrete units such as capsules, tablets, lozenges and the like. Other routes include intrathecal administration directly into spinal fluid, direct introduction such as by various catheter and balloon angioplasty devices well known to those of ordinary skill in the art, and intraparenchymal injection into targeted areas.
[0036] In another aspect the glycoconjugates of the present invention are provided as a pharmaceutical composition. A pharmaceutical composition according to the invention includes an inventive compound as described herein, and may further contain a pharmaceutically acceptable carrier or excipient formulation. The term "pharmaceutically acceptable" is intended to mean a material that is not biologically or otherwise undesirable, which can be administered to an individual along with an inventive compound without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. A pharmaceutically acceptable carrier or excipient formulation may include sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile administrable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
[0037] Additionally, a pharmaceutical composition according to the invention may include other pharmaceutical agents. The general content of a pharmaceutically acceptable carrier or excipient formulation will depend on the form in which the pharmaceutical composition containing an inventive compound is given. For instance, depending on the intended mode of administration, a pharmaceutical composition containing an inventive compound is delivered in solid, semi-solid or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, or suspensions, preferably in unit dosage form suitable for single administration of a precise dosage.
[0038] The glyconconjugate may be administered orally, parenterally (for example, intravenously), by intramuscular injection, by intraperitoneal injection, or transdermally. For oral administration, fine powders or granules may contain diluting, dispersing, and/or surface active agents, and may be presented in water or in a syrup, in capsules or sachets in the dry state or in a nonaqueous solution or suspension wherein suspending agents may be included, in tablets wherein binders and lubricants may be included, or in a suspension in a liquid or gel. Tablets and granules are preferred oral administration forms, and these may be coated. Parenteral administration is generally by injection. Injectables can be prepared in conventional forms, either liquid solutions or suspensions, solid forms suitable for solution or prior to injection, or as suspension in liquid prior to injection or as emulsions.
[0039] For solid compositions, an excipient formulation may include conventional nontoxic solid carriers or fillers include, for example, pharmaceutical grades of cellulose, glucose, lactose, magnesium carbonate, magnesium stearate, mannitol, sodium saccharine, silicic acid, starches, sucrose and talc.
[0040] Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving or dispersing the glycoconjugate compound with optional pharmaceutical adjuvants in an excipient or inert diluent to thereby form a solution or suspension. Liquid dosage forms include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. Thus, in addition to the active compounds, a liquid dosage form may contain such excipients or inert diluents as water or other solvents, solubilizing agents and emulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1 ,3-butyleneglycol, dimethylformamide, oils, in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan or mixtures of these substances, and the like.
[0041] Suspensions, in addition to the glycoconjugate compounds, may contain suspending agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.
[0042] An excipient formulation may contain further inert customary ingredients, such as binders, as for example, carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone, sucrose, and acacia; humectants, as for example, glycerol; disintegrating agents, as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; solution retarders, as for example paraffin; absorption accelerators, as for example, quaternary ammonium compounds; wetting agents, as for example, cetyl alcohol, and glycerol monostearate; adsorbents, as for example, kaolin and bentonite; and lubricants illustratively including talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. Besides such ingredients, the excipient formulation can also include adjuvants, such as emulsifying agents, sweetening, flavoring, and perfuming agents. [0043] Where continuous pharmaceutical composition delivery is required, time release preparations or intravenous preparations are exemplary effective dosage formulations. Illustrative examples of release control components include: carbomer, alpha-starch, polyacrylamides, polysaccharides, polyvinylpyrrolidone; natural gums such as gum arabic; clays; lipophilic gelling agents; fatty acid metal salts such as aluminum stearates; hydrophobic silica; celluloses such as various hydroxyalkylcelluloses; polyethylene glycol; and combinations thereof.
[0044] If desired, the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as pH buffering agents, for example, sodium acetate, sodium citrate, dicalcium phosphate or triethanolamine oleate. Contamination by microorganisms can be prevented or inhibited by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride, and the like. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see A. Gennaro, Remington: The Science and Practice of Pharmacy, 2000, Lippincott, Williams & Wilkins; Goodman and Gilman's The Pharmacological Basis of Therapeutics by Hardman and Limbird, 9th Ed., 1996, McGraw-Hill, New York and in The Merck Index: an encyclopedia of chemicals, drugs, and bio!ogicals, 12th Edition, 1996, Merck & Co., Whitehouse Station, N.J.
[0045] The exact amount of the pharmaceutical composition required will vary from subject to subject, depending on the age, weight and general condition of the subject, the condition being treated, the particular compound used, its mode of administration, and the like. For example, formula of Freireich et a|., Cancer Chemother. Rep., 50:219-244, (1966) can be used to determine the maximum tolerated dose of an inventive compound for a human subject. An appropriate amount may be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.
EXAMPLES
[0046] Synthetic high mannose oligosaccharides are available from Ancora Farmaceuticals (MA, USA). CRMi97 is internally produced in Novartis V&D, Siena, Italy (Research Disclosure 453077 Jan 2002). The 2G12 antibody and HIV gp140 UG37 (clade A strain 92/UG/037, a.a. 32-662, NCBI protein database No. AAC97548, catalog no. ENV001) are purchased from Polymun Scientific (Vienna, Austria). HIV gp 120 Bal (a.a.32- 518, Geneban No. M68893, catalog no. IT-001-002p), gp120 R2 (a.a.41-520, Geneban No. AF128126, catalog no. IT-001-0029p), gp120 JRFL (a.a.34-518, Geneban No. U63632, catalog no. IT-001-0024p) are available from Immune Technology Corp. (New York, US). Polymun and Immune Technology recombinant proteins are expressed in CHO and 293T cells, correspondingly. Biotinylated Galantus nivalis lectin (GNL) is available from Vector Laboratories (CA, US).
[0047] Analytical methods. Total saccharide concentration is determined by HPAEC-PAD analysis (ICS-3000 Dionex system). Briefly, oligomannose carbohydrate preparation is hydrolyzed in 2 M trifluoracetic acid for 2h at 100°C, dried and then redissolved in water. 20 μΙ of sample are injected into CarboPac PA1 analytical column (250 mm x 4 mm i.d., Dionex) with CarboPac PA1 guard column (50 mm x 4 mm i.d., Dionex). Isocratic separations are performed using a 30-min 16 m NaOH followed by a 5-min 500 mM NaOH regeneration step and 15-min re-equilibration, set to a flow rate of 1.0 ml min"1. Monosaccharide peaks are detected directly by using quadruple-potential waveform pulsed amperometry on a gold working electrode and an Ag/AgCI reference electrode. Raw data are elaborated on a Chromeleon 6.8 chromatography software (Dionex) with application of 0.5-10 g/ml mannose calibration curve. Rapid hexose quantification is achieved by Phenol-H2S04 method [24]. Protein concentration is determined by Micro BCA kit (Thermo Fisher Scientific).
Example 1
ESI Q-TOF MS analysis
[0048] PAMAM4 cluster formation is analyzed by direct sample injection in Micromass Q- Tof Micro system (Waters MS Technologies, UK). PAMAM8 cluster formation is analyzed on Q-tof Micro system paired to UPLC system (ACQUITY UPLC System, Waters, UK). The samples are diluted 1 :200 or less in 0.1 % formic acid, 1 :1 vol acetonitrile:water. Chromatographic separations are performed on 2.1 mm i d. x 50 mm ACQUITY BEH C18 1.7 im column (Waters Corp., USA). Elution is performed with a linear gradient of 2-50% B for 8 min, then 50%-100% B for 1.5 min, reconditioned 2% B for 2 min each cycle, where A = water with 0.1% formic acid and B = acetonitrile with 0,1 % formic acid. Each cycle duration is 13 min at a flow rate 0.4 ml/min. 10 μΙ aliquots of sample are loaded.
[0049] TOF MS analysis is performed operating in positive ion mode (ESI). The nebulization gas is set to 800 LJh at a temperature of 250°C, the cone gas set to 50 L/h and the source temperature set to 100°C. The capillary and cone voltages are 3500 V and 30 V, respectively. The Q-Tof Micro is operated with a collision energy of 5 V. The data acquisition rate is set to 0.1 s with a 0.1 s inter-scan delay. The raw data are analyzed by the Micromass Marker-Lynx applications manager Version 1.0 (Waters, UK).
PAMAM cluster synthesis and purification
[0050] In a typical experiment synthetic oligosaccharide (10 mg, 20 mmol) ias treated with disuccinimidyl adipate (200 mmol) in 0.3 ml DMSO containing 20 g/ml triethylamine. After 2 hours of vigorous stirring activation of sugar is checked by TLC. TLC ias performed on aluminium plates coated with silica gel 60 A F25 (Merck) with detection by charring with 10% ethanolic H2S04. Excess linker is removed by oligosaccharide precipitation in 9:1 vol etylacetate : DMSO and centrifugation for 3 minutes at 10.000 xg. Pellet is washed two times with 1 ml of ethylacetate. Oligosaccharide pellet is vaccum dried and weighed (9-10 mg, 90-100%). Oligosaccharide is coupled to PAMAM with stoichiometry of 8:1 mol CHO:PAMAM4 and 20:1 mol CHO:PAMAM8. Reaction in DMSO with 20 μΙ/ml thiethylamine ias stirred overnight at RT. Cluster formation is monitored by ESI Q-TOF MS analysis. No signal of unreacted PAMAM is observed in any of the final reaction mixtures.
[0051] Reaction sample is lyophilized overnight and then dissolved in water. Excess of unreacted oligosaccharide is removed by hydrophobic interaction C4 column (0.5 ml resin, Bioselect, Grace Vydac) in a stepwise gradient of methanol (0-80% in water). Column is activated with methanol and then preconditioned with water. Fractions are analyzed by TLC and TOF-MS. Target fractions are dried to eliminate methanol. Carbohydrate yields for cluster formation and purification are 36.3% for Man4PAMAM4; 16.2% for Man9PAMAM4; 18% for Man4PAMAM8; 14.7% for Man9PAMAM8. [0052] Man4-PAMAM4-Boc ESI MS m/z (CieiHazoN^Oioo): calculated 4346.0508; found: 1450.3772 [M+3H]3+, 1458.3843 [M+3H+Na]3\ 1396.3909 [M+3H-mannose]3+, 4347.1250 [M+H]+, 4369.0664 [M+H+Na]+.
[0053] Man9-PAMAM4-Boc ESI MS m/z (C301H520N18O200): calculated 7587.1073; found: 2477.8005 [M+3H-mannose]3+, 2423.3994 [M+3H-2 mannoses]3+, 7610.3169 [M+H+Na]+, 7448.6763 [M+H+Na-mannose]+, 7287.0806 [M+H+Na-2 mannosesf.
[0054] Man4-PAMAM8-Boc ESI MS m/z (C365H644N38O200): calculated 8760,1391 ; found: 2923.4800 [M+3H]3+, 2869.3137 [M+3H-mannose]3+, 2192.8472 [M+4H] +, 2152.1152 [M+4H-mannose]4+, 1754.3076 [M+5H]5+, 1721.9094 [M+5H-mannoser, 8761.5898 [M+H]+, 8599.8057 [M+H-mannose]+.
[0055] Man9-PAMAM8-Boc ESI MS m/z (C605H1044N38O400): calculated 15242,2520; found 3051.5200 [M+5H]5+, 3019.1033 [M+5H-mannoser, 2543.2612 [M+6H]6+, 2516.0520 [M+6H-mannose]6+, 15241.5830 [M+H]+, 15081.1543 [M+H-mannose]+, 14920.1973 [M+H-2 mannoses]+, 14758.4053 [M+H-3 mannoses]+.
Example 2
Surface Plasmon resonance experiments: inhibition of 2G12-gp120 interaction by glycoconjugates.
[0056] The experiments are carried out with a BiaCore 3000 system in a HBS-EP buffer (10 mM HEPES, 150 mM NaCI, 0,005% surfactant p20, pH 7,4). Two flow cells of a CM5 chip (GE Healthcare) are activated with an EDC/NHS mixture; 10 ug/ml gp140 UG37 in sodium acetate pH 4.5 is injected over the channel two; both are then blocked with 1.0 M ethanolamine. Final immobilization level is 6800 RU. 2G12 solution with and without inhibitors (glycoconjugates) is injected over both channels, and a binding profile is obtained by subtraction of the reference signal in channel one from the gp 140 UG37 in channel two. 2 Mg/ml 2G12 is preincubated with appropriate inhibitor for 15 minutes at 37°C before the start of analysis. Analyte is injected at 10 μΙ/min for 4 min, followed by 6 min dissociation and 30 sec of regeneration with 10 mM glycine, 3M NaCI pH 2.0. Similar conditions are used for analysis with 2G12 chips that had 490 RU and 4400 RU of immobilization.
Example 3
Conjugation of oligosaccharides and clusters to CRM197
[0057] For conjugation, monovalent oligosaccharides or oligosaccharide clusters are activated with disuccinimidyl adipate and purified according to the procedure mentioned above. Before activation Boc group of cluster is eliminated by stirring in 20% TFA for two hours. Conjugation stoichiometry of 30:1 or 40:1 CHO/cluster : protein mol is applied. Conjugation is done in 10-20 mg/ml protein solution in 200 mM NaPi pH 7,0 cluster. The mixture is incubated overnight at 37°C, and reaction outcome is verified by SDS-PAGE analysis. Conjugates are purified from excess of unconjugated carbohydrate using ultrafiltration spin columns with 30 kDa or 50 kDa cut-off (Vivaspin, Sartorius). Glycosylation degree is calculated on the basis of protein and carbohydrate concentration. For ELISA purposes Man9 is conjugated to HSA following similar procedures.
[0058] ELISA - 96-well Maxisorp plates (Nunc, Thermo Fisher Scientific) are coated with 100 μΙ/well of PBS-diluted glycoproteins with 2 pg/ml coating concentration. Plates are incubated overnight at +4°C, then washed three times with TPBS (PBS with 0,05% Tween 20, pH 7.4) and blocked with 100 μΙ well of 2% BSA (Sigma-Aldrich) for 1 hour at 37°C. Subsequently each incubation step is followed by triple TPBS wash. 200 μΙ of prediluted 2G12 or lectin are transferred into coated-blocked plates (200 μΙ) and serially two-fold diluted followed by 2h incubation at 37°C. Serum samples are initially diluted 1 :50-1 :1000 in 2 % BSA in TPBS. Then 100 μΙ well of 1 :10000-1 :20000 diluted appropriate alkaline phosphatase-conjugated secondary antibody or streptavidin-alkaline phosphatase conjugate (Sigma-Aldrich) is added for 1 h at 37°C. 100 μΙ/well of 1 mg/ml pNPP disodium hexahydrate (Sigma Aldrich) in 1 M diethanolamine (pH 9.8) is distributed onto plates to visualize the amount of bound alkaline phosphatase. After 30 minutes of development at RT plates are read with a microplate spectrophotometer at 405 nm. Antibody titres are those dilutions that give an optical density (OD) higher than triple average OD obtained for the mock-immunized rabbit sera.
Example 4
[0059] Rabbit immunization
Animal experimental guidelines set forth by the Novartis Animal Care Department were followed in the conduct of all animal studies. Groups of 2-4 female white Zealand rabbits (2 kg weight) were immunized on days 1 , 21 arid 35 with 20 pg of carbohydrate antigen or PBS (250 μΙ final volume). Antigens were formulated with MF59 (250 μί). Antigens were delivered i.m. into both quadriceps. Sera were collected on days 20, 34 and 42.
[0060] Groups of rabbits (n=2 or 4) were immunized with glyeoconjugates formulated with oil-emulsion MF59 adjuvant that recently was shown to be effective for intramuscular immunization [Burke B. et al., 2009; Seubert A. et al. 2008]. One prime and two boosting doses of 20 pg carbohydrate antigen were administered i.m. Control animals were immunized with MF-59 in PBS. We assessed antibody response by ELISA recognition of Man9 conjugated to HSA via squarate linker. This ELISA was aimed to reveal mannose- specific antibodies but not anti-CRM197 or anti-linker antibodies. All glycoconjugate antigens induced high Man9-specific IgG titer meanwhile mock-immunized rabbits did not show any. PAMAM scaffold didn't seem to be immunogenic since inhibition ELISA showed that addition of PAMAM4 was not able to reduce the binding signal of anti-Man9PAMAM4- CRM serum on the plates coated with Man4-PAMAM4-CRM.

Claims

1. A composition comprising a glycoconjugate comprising at least 2 sugar moieties bound to a multivalent support and wherein said composition binds neutralizing antibodies more effectively than monovalent oligosaccharides.
2. The composition according to claim 1 wherein said multivalent support is selected from the group consisting of single amino acid polymers, gelatin, collagen, albumin, globulins, pectins, cellulose, polyaminoamides (PAMAM), propylene imine, L-lysine and N,N'-bis(acrylamido)acetic acid dendrimers.
3. The composition according to claim 1 wherein said dendrimer is further bound to a carrier.
4. The composition according to claim 3 wherein said carrier is selected from the group consisting of bacterial carriers, keyhole limpet hemocyanin, hepatitis surface antigens, hepatitis core antigens and virus-like particles.
5. The composition according to claim 3, wherein said carrier is CRM 197.
6. A glycoconjugate comprising synthetic oligomannose bound to a carrier for - treatment of HIV.
7. The composition according to claim 6 wherein said synthetic mannose is selected from the group consisting of Man4, Man6 and Man9.
8. The composition according to claim 7 wherein said glycoconjugate is bound to CRM197.
9. The composition according to claim 1 wherein said glycoconjugate binds to antibodies with a Kd of less than 10"6.
10. A pharmaceutical composition comprising an oligomannose glycoconjugate and a pharmaceutically acceptable carrier or diluent.
11. A method for treating a subject for viral infection in need thereof, comprising administering to said patient a therapeutically effective amount of an oligomannose glycoconjugate.
12. A method for screening HIV neutralizing antibodies in a sample comprising introducing a composition according to claim 1 to said sample under conditions suitable for binding said composition to HIV antigens, such that interaction of antibodies with the glycoconjugate is produced.
13. The method according to claim 12 wherein said HIV antigens are carbohydrate antigens.
14. The method according to claim 12 wherein said neutralizing antibodies have a greater binding affinity than 2G12.
15. A vaccine composition comprising the glycoconjugate of claim 1 and pharmaceutically acceptable solution in an effective amount
16. The vaccine composition according to claim 15 further comprising administering adjuvant.
PCT/IB2010/002541 2009-09-23 2010-09-21 Glycoconjugate compositions and methods for treatment of hiv WO2011036560A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US27732609P 2009-09-23 2009-09-23
US61/277,326 2009-09-23

Publications (2)

Publication Number Publication Date
WO2011036560A2 true WO2011036560A2 (en) 2011-03-31
WO2011036560A3 WO2011036560A3 (en) 2011-06-16

Family

ID=43598351

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2010/002541 WO2011036560A2 (en) 2009-09-23 2010-09-21 Glycoconjugate compositions and methods for treatment of hiv

Country Status (1)

Country Link
WO (1) WO2011036560A2 (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012158701A1 (en) 2011-05-18 2012-11-22 Matrivax Research & Development Corporation Protein matrix vaccine compositions including polycations
WO2012156750A1 (en) * 2011-05-19 2012-11-22 Isis Innovation Limited Hiv vaccine
CN102807621A (en) * 2011-06-01 2012-12-05 厦门大学 Fusion protein containing diphtheria toxin non-toxic mutant cross-reacting material 197 (CRM197) or segment thereof

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4507466A (en) 1983-01-07 1985-03-26 The Dow Chemical Corporation Dense star polymers having core, core branches, terminal groups
US4568737A (en) 1983-01-07 1986-02-04 The Dow Chemical Company Dense star polymers and dendrimers
WO1993015760A1 (en) 1992-02-11 1993-08-19 U.S. Government, As Represented By The Secretary Of The Army Dual carrier immunogenic construct
WO1995008348A1 (en) 1993-09-22 1995-03-30 Henry M. Jackson Foundation For The Advancement Of Military Medicine Method of activating soluble carbohydrate using novel cyanylating reagents for the production of immunogenic constructs
WO1996029094A1 (en) 1995-03-22 1996-09-26 Andrew Lees Producing immunogenic constructs using soluble carbohydrates activated via organic cyanylating reagents
US6190650B1 (en) 1994-06-15 2001-02-20 Biomolecular Research Institute Ltd. Antiviral dendrimers
US20040180852A1 (en) 2003-03-10 2004-09-16 Cara-Lynne Schengrund Use of multivalent glycodendrimers to inhibit the activity of human immunodeficiency virus

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7955605B2 (en) * 2005-04-08 2011-06-07 Wyeth Llc Multivalent pneumococcal polysaccharide-protein conjugate composition
TWI432448B (en) * 2006-09-14 2014-04-01 Univ Duke Vaccine

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4507466A (en) 1983-01-07 1985-03-26 The Dow Chemical Corporation Dense star polymers having core, core branches, terminal groups
US4568737A (en) 1983-01-07 1986-02-04 The Dow Chemical Company Dense star polymers and dendrimers
WO1993015760A1 (en) 1992-02-11 1993-08-19 U.S. Government, As Represented By The Secretary Of The Army Dual carrier immunogenic construct
WO1995008348A1 (en) 1993-09-22 1995-03-30 Henry M. Jackson Foundation For The Advancement Of Military Medicine Method of activating soluble carbohydrate using novel cyanylating reagents for the production of immunogenic constructs
US6190650B1 (en) 1994-06-15 2001-02-20 Biomolecular Research Institute Ltd. Antiviral dendrimers
WO1996029094A1 (en) 1995-03-22 1996-09-26 Andrew Lees Producing immunogenic constructs using soluble carbohydrates activated via organic cyanylating reagents
US20040180852A1 (en) 2003-03-10 2004-09-16 Cara-Lynne Schengrund Use of multivalent glycodendrimers to inhibit the activity of human immunodeficiency virus

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
"R. R. C. New, Liposomes: A Practical Approach", OXFORD UNIVERSITY PRESS
"The Merck Index: an encyclopedia of chemicals, drugs, and biologicals", 1996, MERCK & CO.
A. GENNARO: "Remington: The Science and Practice of Pharmacy", 2000, WILLIAMS & WILKINS
FREIREICH ET AL., CANCER CHEMOTHER. REP., vol. 50, 1966, pages 219 - 244
GOODMAN; GILMAN'S: "The Pharmacological Basis of Therapeutics", 1996, MCGRAW-HILL
KIM ET AL., VACCINE, vol. 18, 2000, pages 597
LOWRY ET AL., J. BIOL. CHEM., vol. 193, 1951, pages 265 - 275
PETERSON ET AL., ANALYTICAL BIOCHEMISTRY, vol. 100, 1979, pages 201 - 220
PLOTKIN: "Vaccines", 1999, WB SAUNDERS CO.
SAMBROOK ET AL.: "MOLECULAR CLONING: A LABORATORY MANUAL", 1989, COLD SPRING HARBOR LABORATORY PRESS

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2709656A4 (en) * 2011-05-18 2015-01-14 Matrivax Inc Protein matrix vaccine compositions including polycations
AU2012255878B2 (en) * 2011-05-18 2016-05-12 Matrivax Research & Development Corporation Protein matrix vaccine compositions including polycations
EP3524265A1 (en) * 2011-05-18 2019-08-14 Matrivax, Inc. Protein matrix vaccine compositions including polycations
EP2709656A1 (en) * 2011-05-18 2014-03-26 Matrivax, Inc. Protein matrix vaccine compositions including polycations
KR20140045416A (en) * 2011-05-18 2014-04-16 매트리백스 인코포레이티드 Protein matrix vaccine compositions including polycations
JP2014513726A (en) * 2011-05-18 2014-06-05 マトリヴァックス，インコーポレーテッド Protein matrix vaccine composition comprising a polycation
US9724401B2 (en) 2011-05-18 2017-08-08 Matrivax, Inc. Protein matrix vaccine compositions including polycations
KR101670340B1 (en) * 2011-05-18 2016-10-28 매트리백스 인코포레이티드 Protein matrix vaccine compositions including polycations
WO2012158701A1 (en) 2011-05-18 2012-11-22 Matrivax Research & Development Corporation Protein matrix vaccine compositions including polycations
WO2012156750A1 (en) * 2011-05-19 2012-11-22 Isis Innovation Limited Hiv vaccine
CN102807621B (en) * 2011-06-01 2016-04-13 厦门大学 Comprise the fusion rotein of diphtheria toxin non-toxic mutant CRM197 or its fragment
US9512185B2 (en) 2011-06-01 2016-12-06 Xiamen University Fusion protein comprising diphtheria toxin non-toxic mutant CRM197 or fragment thereof
US9757450B2 (en) 2011-06-01 2017-09-12 Xiamen University Fusion protein comprising diphtheria toxin non-toxic mutant CRM197 or fragment thereof
US9764028B2 (en) 2011-06-01 2017-09-19 Xiamen University Fusion protein comprising diphtheria toxin non-toxic mutant CRM197 or fragment thereof
CN102807621A (en) * 2011-06-01 2012-12-05 厦门大学 Fusion protein containing diphtheria toxin non-toxic mutant cross-reacting material 197 (CRM197) or segment thereof

Also Published As

Publication number Publication date
WO2011036560A3 (en) 2011-06-16

Similar Documents

Publication Publication Date Title
AU2003282821B2 (en) Carbohydrate-based synthetic vaccines for HIV
Agadjanyan et al. Peptide mimicry of carbohydrate epitopes on human immunodeficiency virus
Chabre et al. Recent trends in glycodendrimer syntheses and applications
Ni et al. Toward a carbohydrate-based HIV-1 vaccine: synthesis and immunological studies of oligomannose-containing glycoconjugates
Bhatia et al. Multivalent glycoconjugates as vaccines and potential drug candidates
JP4754689B2 (en) Bacterial meningitis vaccine with multiple oligosaccharide glycoconjugates
Wang et al. Novel template-assembled oligosaccharide clusters as epitope mimics for HIV-neutralizing antibody 2G12. Design, synthesis, and antibody binding study
US4950480A (en) Enhancement of antigen immunogenicity
Niederhafner et al. Glycopeptide dendrimers, Part III—a review: use of glycopeptide dendrimers in immunotherapy and diagnosis of cancer and viral diseases
WO2007079448A2 (en) Three component carbohydrate vaccine
Pifferi et al. Cyclopeptide scaffolds in carbohydrate-based synthetic vaccines
US10973910B1 (en) Neoglycoconjugates as vaccines and therapeutic tools
US20060229432A1 (en) Gp120 specific antigens and uses thereof
Kabanova et al. Preparation, characterization and immunogenicity of HIV-1 related high-mannose oligosaccharides-CRM 197 glycoconjugates
WO2006017180A2 (en) Glycopeptide dimers and uses thereof
WO2011036560A2 (en) Glycoconjugate compositions and methods for treatment of hiv
WO2007147823A1 (en) Method for the preparation of specific antibodies against saccharidic antigens
CN113274489B (en) Chitin oligosaccharide vaccine for preventing fungal infection and preparation method thereof
WO2012156750A1 (en) Hiv vaccine
Yin et al. Strategies for Synthesizing and Enhancing the Immune Response of Cancer Vaccines Based on MUC1 Glycopeptide Antigens
Liu et al. Synthesis and immunological evaluation of pentamannose-based HIV-1 vaccine candidates
US20080267989A1 (en) Hiv Gp-41-Membrane Proximal Region Arrayed On Hepatitis B Surface Antigen Particles as Novel Antigens
US20080181913A1 (en) Sugar immunogens
US7534442B2 (en) Immunogenic compositions comprising covalently bound polysaccharides, antigen, and bacterial toxoid
US20150024001A1 (en) Molecular mimic mucosal aids vaccine

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 10781999

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 10781999

Country of ref document: EP

Kind code of ref document: A2