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WO2011127989A1 - Vaccin à base conjugué de nicotine - Google Patents

Vaccin à base conjugué de nicotine Download PDF

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Publication number
WO2011127989A1
WO2011127989A1 PCT/EP2010/058328 EP2010058328W WO2011127989A1 WO 2011127989 A1 WO2011127989 A1 WO 2011127989A1 EP 2010058328 W EP2010058328 W EP 2010058328W WO 2011127989 A1 WO2011127989 A1 WO 2011127989A1
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WIPO (PCT)
Prior art keywords
nicotine
composition according
oil
immunoconjugate
water emulsion
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English (en)
Inventor
Ali Alloueche
Jean Paul Prieels
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GlaxoSmithKline Biologicals SA
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GlaxoSmithKline Biologicals SA
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/16—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from plants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0013—Therapeutic immunisation against small organic molecules, e.g. cocaine, nicotine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/30—Drugs for disorders of the nervous system for treating abuse or dependence
    • A61P25/32—Alcohol-abuse
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/30—Drugs for disorders of the nervous system for treating abuse or dependence
    • A61P25/34—Tobacco-abuse
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to adjuvanted nicotine immunoconjugates and their use in the treatment or prevention of nicotine addiction.
  • the invention also relates to methods of producing such adjuvanted immunoconjugates.
  • Nicotine ((S)-(-)-l-methyl-2-(3-pyridyl)pyrrolidine) is an alkaloid derived from the tobacco leaf.
  • the nicotine molecule is formed of an aromatic six-membered ring (pyridine) and an aliphatic five-membered ring (pyrrolidine) linked by a single bond.
  • the pyrrolidine ring is N-methylated and linked through its carbon -2 to the carbon -3 of pyridine.
  • the carbon -2 is chiral and there is virtually free rotation around the single bond linking the two rings.
  • the absolute configuration of carbon -2 is S, and so the natural enantiomer of nicotine is (5 , )-(-)-nicotine.
  • Nicotine is widely available in many different forms, such as cigarettes, cigars, pipe tobacco and chewing tobacco, and is highly addictive. Due to significant adverse health effects of tobacco use, in particular smoking, users often try to quit. However, the poor rates of success in those who try to quit indicate that there is a strong need for additional therapies for nicotine addiction.
  • Nicotine conjugate vaccines induce anti-nicotine antibodies that, upon exposure following smoking, bind to nicotine in the blood. Nicotine itself is a small molecule that readily crosses the blood-brain barrier but nicotine bound to an antibody cannot enter the brain. This mechanism of action has been largely demonstrated in mouse and rat animal models, and was correlated with an IgG dose-dependent activity attenuation. As a result, nicotine sequestered within the blood-stream cannot mediate its addictive effects on the CNS.
  • the invention provides an immunogenic composition comprising:
  • an oil-in-water emulsion adjuvant which comprises a metabolisable oil and an emulsifying agent.
  • the immunogenic composition may be used to induce a specific anti-nicotine immune response in a subject, and so may be used in the treatment or prevention of nicotine addiction.
  • the invention further provides:
  • an immunogenic composition of the invention for use in the prevention or treatment of nicotine addiction
  • an immunogenic composition of the invention for the manufacture of a medicament for the prevention or treatment of nicotine addiction
  • a method of preventing or treating nicotine addiction comprises administering an effective amount of an immunogenic composition of the invention to an individual in need thereof;
  • Figures 3 and 4 show that antibodies generated in mice immunised with NicA4-CRM + AS03 are poorly cross-reactive with (bind nicotine much better than) cotinine.
  • the results obtained for nicotine are shown as ⁇ and results for cotinine as ⁇ .
  • the present invention provides an immunogenic composition which may be used in the prevention or treatment of nicotine addiction.
  • immunogenic composition refers to a composition that comprises an immunogenic component capable of provoking an immune response in an individual, such as a human.
  • the invention provides an immunogenic composition comprising a nicotine immunoconjugate and an oil-in-water emulsion adjuvant which comprises a metabolisable oil and an emulsifying agent.
  • the immunogenic composition of the invention is a vaccine, i.e. the immunogenic composition can be administered to a subject to raise an immune response against nicotine that is capable of preventing or treating nicotine addiction in the subject.
  • the term "vaccine” as used herein refers to both therapeutic vaccines (for the treatment of the condition) as well as prophylactic vaccines (for prevention of the condition).
  • the nicotine immunoconjugate used in the present invention consists of a nicotine hapten and a carrier moiety, which may be connected via a linker molecule.
  • the nicotine hapten is capable of eliciting a specific anti-nicotine antibody response when attached to the carrier moiety, and so is the specificity-determining portion of the immunoconjugate.
  • the nicotine hapten will typically mimic the structure of the native nicotine molecule, although it need not be directly derived or synthesized from nicotine. Any nicotine hapten that is capable of eliciting a specific anti-nicotine antibody response when linked to a carrier moiety may be used in the present invention.
  • the term "nicotine hapten" is used to include both the specificity-determining portion (i.e. the nicotine-like structure) and the linker molecule used to attach it to the carrier, if present.
  • the nicotine hapten is not inherently immunogenic and requires coupling to a carrier moiety to elicit an antibody response.
  • carrier moiety refers to a conjugation partner that can enable the nicotine hapten to induce a specific anti- nicotine antibody response when administered to a subject.
  • Typical carrier moieties include proteins, peptides, virus-like particles (VLPs) and artificial vehicles such as microspheres.
  • Proteins that are suitable for use as carriers include, but are not limited to, tetanus toxoid (TT), diphtheria toxoid (DT), diphtheria toxin mutant CRM197, Pseudomonas exoprotein A (ExoA), cholera toxin B (CTB), pertussis toxin, outer membrane protein complex (OMPC) from Neisseria meningitidis, the B subunit of heat-labile E. coli, keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA).
  • TT tetanus toxoid
  • DT diphtheria toxoid
  • CTB cholera toxin B
  • OMPC outer membrane protein complex
  • Neisseria meningitidis the B subunit of heat-labile E. coli
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • the nicotine hapten may be covalently or non-covalently linked to the carrier moiety using any suitable method such as, but not limited to, covalent bond formation via amide, carbamate, urea, hydrazone, semicarbazone, thioether, triazole (Click chemistry) or disulfide bond formation.
  • a linker molecule may be used in order to displace the nicotine hapten a certain distance from the carrier molecule to improve its immunogenicity. The linking may take place at any position on the nicotine molecule.
  • nicotine haptens and immunoconjugates are already known in the art and may be used in the method of the invention. See, for example, the nicotine haptens and immunoconjugates disclosed in the following documents, which are incorporated by reference herein in their entirety: W098/14216, US5876727
  • the nicotine hapten is represented by formula (I): — Z
  • Z is NH 2 , COOH, CHO or SH and the -(CH 2 ) complicat-Z moiety can be bonded to the 3', 4' or 5' position of nicotine.
  • the Z moiety is capable of binding to the carrier moiety, directly or via an additional linker molecule. It will be apparent to a person skilled in the art that when the Z moiety is covalently attached to a linker molecule or directly to a carrier it will be modified by virtue of the covalent linking. For example, when covalently linked to a linker molecule or carrier the Z moiety may be represented by NH-, COO-, CO- or S- in the final structure. In a particular embodiment, Z is NH 2 and the -(CH 2 ) n -Z moiety is bonded to the 3' position of nicotine
  • the nicotine immunoconjugate is of formul
  • m is 1 to 2500; n is 0 to 12; y is 1 to 12; X is selected from the group consisting of NH-CO, CO-NH, CO-NH-NH, NH-NH-CO, NH-CO-NH, CO-NH-NH- CO, and S-S; Y is selected from the group consisting of NH-CO, CO-NH, CO-NH- NH, NH-NH-CO, NH-CO-NH, CO-NH-NH-CO, and S-S; and the -(CH 2 )n-X- (CH 2 )y-Y- moiety is bonded to the 3', 4' or 5' position of nicotine.
  • X is NH-CO
  • Y is CO-NH and the -(CH 2 )n-X-(CH 2 )y- Y- moiety is bonded to the 3' position of nicotine .
  • the terminal atom or atom group of the Y moiety is derived from the carrier molecule.
  • the terminal NH group in the Y moiety of formula (II) may be derived from an amino acid in a carrier protein, for example a lysine residue.
  • nicotine immunoconjugates Two examples of nicotine immunoconjugates that may be used in the present invention are:
  • R is a carrier moiety
  • the nicotine immunoconjugate is not AMI (ether link, 6 carbon linker) in conjugation to carrier proteins keyhole limpet hemocyanin (KLH), tetanus toxoid (TT) or diphtheria toxin cross-reactive mutant 197 (CRM) as disclosed in Moreno et ah Molecular Pharmaceutics; Vol 7, NO. 2, 431-441.
  • AMI ether link, 6 carbon linker
  • Oil-in-water emulsion adjuvant oil-in-water emulsion adjuvant
  • the present invention is based on the finding that when nicotine
  • immunoconjugates are adjuvanted with an oil-in-water emulsion, they induce significantly higher titer nicotine- specific antibody responses than the same immunoconjugates adjuvanted with alum or alum combined with 3D-MPL (3-de-O- acylated monophosphoryl lipid A).
  • the improved immune response obtained using an oil-in-water adjuvant may mean that fewer doses of the nicotine immunoconjugate are required in order to have the same therapeutic effect as a non-adjuvanted nicotine immunoconjugate or with an aluminium-based adjuvant and/or that the therapeutic effect is longer lasting.
  • the oil phase of the emulsion system has to comprise a metabolisable oil.
  • metabolisable is well known in the art, and can be defined as 'being capable of being transformed by metabolism' (Dorland's Illustrated Medical Dictionary, W.B. Sanders Company, 25th edition (1974)).
  • the oil may be any vegetable oil, fish oil, animal oil or synthetic oil, which is not toxic to the recipient, and which is capable of being transformed by metabolism. Nuts, seeds, and grains are common sources of vegetable oils. Synthetic oils may also be used and can include commercially available oils such as NEOBEE® and others.
  • a suitable metabolisable oil is squalene (2,6, 10,15, 19,23-Hexamethyl- 2,6,10,14,18,22-tetracosahexaene), an unsaturated oil which is found in large quantities in shark-liver oil, and in lower quantities in olive oil, wheat germ oil, rice bran oil and yeast.
  • Squalene is a metabolisable oil by virtue of the fact that it is an intermediate in the biosynthesis of cholesterol.
  • the metabolisable oil is typically present in an amount of 0.5% to 10% (v/v) of the total volume of the immunogenic composition.
  • the oil- in- water emulsion adjuvant further comprises an emulsifying agent.
  • a suitable emulsifying agent is polyoxyethylene sorbitan monooleate (Tween 80TM).
  • the emulsifying agent is typically present in the adjuvant composition in an amount of 0.125 to 4% (v/v) of the total volume of the immunogenic composition.
  • the oil- in- water emulsion of the present invention may further comprise a tocol.
  • Tocols are well known in the art and are described in EP0382271.
  • a suitable tocol is alpha-tocopherol or a derivative thereof such as alpha-tocopherol succinate (also known as vitamin E succinate).
  • the tocol is typically present in the adjuvant composition in an amount of 0.25% to 10% (v/v) of the total volume of the immunogenic composition.
  • the oil and emulsifier should be in an aqueous carrier.
  • the aqueous carrier may be, for example, phosphate buffered saline (PBS).
  • the oil-in-water emulsion adjuvant comprises squalene, polyoxyethylene sorbitan monooleate (Tween 80TM) and alpha- tocopherol.
  • the oil-in-water emulsion adjuvant will comprise from 2 to 10% squalene, from 0.3 to 3% polyoxyethylene sorbitan monooleate and from 2 to 10% alpha-tocopherol (percentages are of the total volume of the immunogenic composition), and may be produced according to the procedure described in WO
  • the amount of squalene may be equal to or less than the amount of alpha- tocopherol as this provides a more stable emulsion.
  • the oil-in-water emulsion may also contain polyoxyethylene sorbitan trioleate (Span 85) and/or Lecithin, for example at a level of 1% of the total volume of the immunogenic composition.
  • the method comprises mixing the oil phase (which may comprise a tocol) with a surfactant such as a PBS/TWEEN80TM solution, followed by homogenisation using a homogenizer.
  • a surfactant such as a PBS/TWEEN80TM solution
  • a method comprising passing the mixture twice through a syringe needle would be suitable for homogenising small volumes of liquid.
  • the emulsification process in microfluidiser MHOS Micro fluidics machine, maximum of 50 passes, for a period of 2 minutes at maximum pressure input of 6 bar (output pressure of about 850 bar)
  • the adaptation could be achieved by routine experimentation comprising the measurement of the resultant emulsion until a preparation was achieved with oil droplets of the required diameter.
  • the oil- in- water emulsion systems of the present invention may have a small oil droplet size in the sub-micron range.
  • the droplet sizes will be in the range of 120 to 750 nm, for example sizes from 120 to 600 nm in diameter.
  • the oil-in water emulsion may contain oil droplets of which at least 70% by intensity are less than 500 nm in diameter, at least 80% by intensity are less than 300 nm in diameter, or at least 90% by intensity are in the range of 120 to 200 nm in diameter.
  • the oil droplet size (i.e. diameter) according to the present invention is given by intensity.
  • Intensity is measured by use of a sizing instrument, suitably by dynamic light scattering such as the Malvern Zetasizer 4000 or the Malvern Zetasizer 3000HS.
  • a first possibility is to determine the z average diameter ZAD by dynamic light scattering (PCS-Photon correlation spectroscopy); this method additionally gives the polydispersity index (PDI), and both the ZAD and PDI are calculated with the cumulants algorithm. These values do not require the knowledge of the particle refractive index.
  • a second means is to calculate the diameter of the oil droplet by determining the whole particle size distribution by another algorithm, either the Contin, or NNLS, or the automatic "Malvern" one (the default algorithm provided for by the sizing instrument).
  • Another algorithm either the Contin, or NNLS, or the automatic "Malvern" one (the default algorithm provided for by the sizing instrument).
  • nicotine-specific antibodies are those that bind specifically to the nicotine molecule. Specific binding is distinguished from non-specific binding (e.g., binding to surfaces such as ELISA wells or blotting membranes). Nicotine- specific antibodies bind to nicotine with high affinity, compared to nonspecific binding. For nicotine vaccines to be effective, the antibodies generated to nicotine after immunization must able to bind nicotine as it moves from the lungs into the blood, in order to prevent nicotine movement into the brain. Cotinine is the major by-product of nicotine metabolism and, because of its long half- life, is present at much higher levels in the blood of smokers than nicotine (approximately 10-fold higher). If the antibodies generated by a vaccine bind to cotinine to a significant degree, the effectiveness of the antibodies for preventing movement of nicotine into the brain may be compromised.
  • a "nicotine specific antibody” is one that binds more avidly to nicotine than to cotinine.
  • Substance dependence is a state in which there is a compulsive or chronic need for a drug or other substance (Dorlands Illustrated Medical Dictionary, 30 th Edition, Saunders). Substance dependence is a compulsive use of a substance despite significant problems resulting from such use ⁇ Id.). Substance dependence may include physiological as well as psychological dependence.
  • Nicotine addiction can thus be defined as a dependence on the drug nicotine. Nicotine addiction may also be termed "nicotine use disorder" (see Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revision (DSM-IV-TR)). The DSM-IV-TR provides criteria for the diagnosis of nicotine use disorder.
  • questionnaires as the Fagerstrom Test for Nicotine Dependence (FTND) are also available to aid health care workers in determining the level of a subject's nicotine dependence.
  • treatment of nicotine addiction does not imply 100% success rate, i.e., does not imply that every subject treated will quit tobacco use indefinitely.
  • a successful treatment for nicotine addiction is one that results in a higher percentage of subjects quitting or reducing tobacco use during or immediately after treatment, compared to a control group (subjects not receiving treatment for nicotine addiction).
  • the effect of nicotine addiction treatment may be assessed by various parameters, such as decrease in intensity of tobacco use, cessation of tobacco use, the total length of time over which the decrease or cessation persists, or the number of subjects who maintain cessation of tobacco use over time.
  • a preventive treatment for nicotine addiction is one that reduces the number of subjects who become addicted to nicotine, compared to the number of subjects who become addicted in a control group not receiving the treatment.
  • the amount of the nicotine immunoconjugate of the present invention present in each dose of immunogenic composition is selected as an amount which induces the desired immune response without significant, adverse side effects. Such amount will vary depending upon which specific immunoconjugate is employed and the type and amount of oil- in- water adjuvant used. An optimal amount for a particular
  • immunoconjugate may be ascertained by standard studies involving observation of antibody titres and other responses in subjects. Generally, it is expected that each human dose will comprise l-1000 ⁇ g of immunoconjugate, for example 20-800 ⁇ g, or 50-600 ⁇ g, or 100-500 ⁇ g. A typical human dose may contain approximately 20, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550 or 600 ⁇ g of immunoconjugate.
  • human dose is used to mean a dose which is in a volume suitable for human use. Generally this is between 0.3 and 1.5 ml. In one embodiment, a human dose is 0.5 ml.
  • boosts Following an initial vaccination, subjects typically receive a boost after a certain interval of time. Additional boosts may also be given, for example a total of 2, 3, 4 or 5 boost vaccinations.
  • the initial and boost vaccinations may be given at 1, 2, 3, 4, 5 or 6 day intervals, at 1, 2, 3 or 4 week intervals, at 1, 2, 3, 4, 5 or 6 month intervals, at 1 year intervals, or at any combination of these time intervals.
  • the initial vaccination may consist of the
  • the immunogenic composition of the invention may contain only the nicotine immunoconjugate as an active component, or the nicotine immunoconjugate in combination with a different adjuvant, such as alum.
  • the immunogenic compositions of the invention may be provided by any of a variety of routes such as oral, topical, subcutaneous, mucosal, intra veneous, intramuscular, intranasal, sublingual, intradermal and via suppository.
  • the immunogenic composition will be administered via intramuscular injection or transdermally, for example using a patch.
  • the initial vaccination is given by one route of administration (for example by intramuscular injection) whereas the boosting composition is given by a different route of administration (for example using a transdermal patch).
  • the nicotine immunoconjugate and oil- in- water emulsion adjuvant may be co-administered separately.
  • the immunoconjugate and oil- in- water emulsion adjuvant may be administered simultaneously or within a short period of time (such as within 2, 5, 10 or 15 minutes) by the same or different routes of administration as discussed herein.
  • the immunogenic composition may be prophylactic or therapeutic.
  • the invention described herein is primarily but not exclusively concerned with therapeutic use of adjuvanted nicotine immunoconjugates for the treatment of nicotine addiction.
  • Vaccine preparation is generally described in Pharmaceutical Biotechnology, Vol.61 Vaccine Design - the subunit and adjuvant approach, edited by Powell and Newman, Plenum Press New York, 1995. New Trends and Developments in Vaccines, edited by Voller et al., University Park Press, Baltimore, Maryland, U.S.A. 1978.
  • the immunogenic compositions of the invention comprise certain components as laid out above.
  • the immunogenic composition consists essentially of, or consists of, said components.
  • NicA4 hapten was dissolved in dimethylformamide (DMF) to a concentration of 30-40 mg/mL.
  • DMF dimethylformamide
  • HBTU 4-Benzotriazole-N,N,N',N'-tetramethyl-uronium- hexafluoro -phosphate
  • the mixture was incubated at room temperature for 30 minutes with shaking.
  • the CRM-197 was diulted in 0.2M phosphate buffer, pH 7.2 to a concentration of 2 mg/mL.
  • the CRM-197 solution was diluted to 3 mg/ml. After the resin had settled (or had been briefly centrifuged) the supernatant was drawn off and added to the CRM-197 solution. The mixture was allowed to react at room temperature for 20 - 24 hours or until RP-HPLC showed no hapten-SNHS ester remaining. Purification of the conjugate was carried out by either ultrafiltration or small scale dialysis. Hapten: carrier ratio
  • the nicotine immunoconjugates were analyzed for hapten:carrier ratio with the following results: NicA6-CRM (Lot #1525-161, 17 haptens / carrier, 4276 ⁇ g/mL) NicA4-CRM (Lot #1520-173, 14 haptens / carrier, 3155 ⁇ g/mL).
  • the oil- in- water emulsion used in the subsequent examples is composed of an organic phase made of two oils (alpha-tocopherol and squalene), and an aqueous phase of phosphate buffered saline (PBS) containing polyoxyethylene sorbitan monooleate (Tween 80TM) as emulsifying agent.
  • PBS phosphate buffered saline
  • Tween 80TM polyoxyethylene sorbitan monooleate
  • the oil- in- water emulsion adjuvant formulations used in the subsequent examples were made comprising the following oil- in- water emulsion component (final concentrations given): 2.5% squalene (v/v), 2.5% alpha-tocopherol (v/v), 0.9% polyoxyethylene sorbitan monooleate (v/v). This emulsion was prepared as follows as a two-fold concentrate.
  • the emulsion is made by mixing under strong agitation an oil phase composed of hydrophobic components (a-tocopherol and squalene) and an aqueous phase containing the water soluble components (polyoxyethylene sorbitan monooleate and PBS mod (modified), pH 6.8). While stirring, the oil phase (1/10 total volume) is transferred to the aqueous phase (9/10 total volume), and the mixture is stirred for 15 minutes at room temperature. The resulting mixture is then subjected to shear, impact and cavitation forces in the interaction chamber of a micro fluidizer (15000 PSI - 8 cycles) to produce submicron droplets (distribution between 100 and 200 nm). The resulting pH is between 6.8 + 0.1.
  • the emulsion is then sterilised by filtration through a 0.22 ⁇ membrane and the sterile bulk emulsion is stored refrigerated in Cupac containers at 2 to 8 °C.
  • Sterile inert gas nitrogen or argon
  • Each immunoconjugate (NicA4-CRM and NicA6-CRM) was combined with one of three adjuvants: alum (alhydrogel aluminum hydroxide batch 4095) at 50 ⁇ g A1+++ per dose; AS03 (oil- in- water emulsion) at 25 ⁇ per dose; or AS04 (alum and 3D-MPL) at 50 ⁇ g A1+++ combined with 5 ⁇ g 3D-MPL per dose.
  • the AS04- containing preparations were made as follows: 1 or 10 ⁇ g of immunoconjugate was adsorbed to 30 ⁇ g of alum.
  • a preparation of 3D-MPL adsorbed to alum was added to make a final dosage of 1 or 10 ⁇ g immunoconjugate, 50 ⁇ g alum and 5 ⁇ g 3D-MPL in 50 ⁇ ⁇ .
  • mice C57B1/6 or BALB/c female mice obtained from The Jackson Laboratory, Bar Harbor, Maine were fed ad libitum, housed 13 mice per cage, and allowed free access to sterile water and food. The mice were 13 weeks old when the primary vaccination was administered.
  • the adjuvanted nicotine immunoconjugates were administered at 10 ⁇ g/dose or ⁇ g/dose (see Table 1).
  • Sufficient antigen/alum solution was prepared for both the primary and secondary injections in the case of AS04 solutions.
  • Diluent was 10 mM sodium phosphate buffer, pH 7.2. Two 50 ⁇ injections were performed, 14 days apart via the intramuscular route in alternating (right for primary, left for secondary) biceps femoris muscles. Blood was collected on day 21 via cardiac draw on anesthetized animals. Blood was allowed to clot, and serum was collected and used in the following analyses to determine anti-nicotine antibody concentration and affinity.
  • the quantity of nicotine-binding IgG in the sera of mice immunized with the conjugate vaccines was determined by extrapolation from a standard curve generated with a monoclonal antibody (mAb 9D9) specific for nicotine. Serum from each of the thirteen C57bl/6 mice in each of the twelve groups shown in Table 1 was tested, as was serum from each of the thirteen Balb/6 mice in each of the groups.
  • the antibodies generated to nicotine after immunization must able to bind nicotine as it moves from the lungs into the blood in order to prevent its movement into the brain.
  • Cotinine is the major by-product of nicotine metabolism and, because of its long half- life, is present at much higher levels in the blood of smokers than nicotine ( ⁇ 10-fold higher). If the antibodies generated by nicotine vaccines bind to cotinine to a significant degree, it is possible that the effectiveness of the antibodies for preventing movement of nicotine into the brain will be compromised. For this reason, it is important to determine the level to which nicotine- specific antibodies generated by candidate vaccines cross-react with cotinine.
  • Radioimmunoassay was also used to evaluate the level of antibody cross- reactivity between nicotine and cotinine. Because there is no pre-binding of the antibodies to nicotine tethered to a carrier in this assay, any antibodies induced by the vaccine that bind to determinants on the tether become irrelevant. Thus, the RIA assay allows a more "pure” evaluation of the nicotine binding ability of the antibodies. In this system, radiolabeled (tritium) "hot” nicotine was bound to the antibodies in pooled sera from C57B1/6 mice immunized with NicA4-CRM plus AS03. The amount of free "cold” nicotine or cotinine required to prevent binding of the "hot” nicotine was determined.
  • nicotine immunoconjugate vaccines consisting of nicotine haptens coupled to CRM and adjuvanted with AS03 (oil- in- water emulsion) induce significantly higher titre nicotine- specific antibody responses than the same vaccines adjuvanted with alum or AS04 (alum and 3D-MPL).
  • the antibodies induced by the vaccines have moderate affinity for nicotine and appear to be nicotine- specific, since cotinine, the major by-product of nicotine metabolism, is poorly cross-reactive.

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Abstract

L'invention concerne une composition immunogène comprenant un immunoconjugué de nicotine et un adjuvant d'émulsion huile-dans-l'eau et un procédé de fabrication de telles compositions. L'invention concerne également l'utilisation de tels immunoconjugués de nicotine avec adjuvant comme médicaments et dans le traitement ou la prévention de la dépendance à la nicotine.
PCT/EP2010/058328 2010-04-15 2010-06-14 Vaccin à base conjugué de nicotine Ceased WO2011127989A1 (fr)

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US20150056251A1 (en) * 2012-02-28 2015-02-26 Cornell University Aav-directed persistent expression of an anti-nicotine antibody gene for smoking cessation
US20150297739A1 (en) * 2012-04-13 2015-10-22 Cornell University Development of a highly efficient second generation nicotine-conjugate vaccine to treat nicotine addiction

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CN115583992A (zh) * 2022-11-02 2023-01-10 江南大学 一种氧化还原响应的载体蛋白及其在制备疫苗中的应用

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150056251A1 (en) * 2012-02-28 2015-02-26 Cornell University Aav-directed persistent expression of an anti-nicotine antibody gene for smoking cessation
US10093947B2 (en) * 2012-02-28 2018-10-09 Cornell University AAV-directed persistent expression of an anti-nicotine antibody gene for smoking cessation
US20150297739A1 (en) * 2012-04-13 2015-10-22 Cornell University Development of a highly efficient second generation nicotine-conjugate vaccine to treat nicotine addiction
US10004811B2 (en) * 2012-04-13 2018-06-26 Cornell University Development of a highly efficient second generation nicotine-conjugate vaccine to treat nicotine addiction

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