EP1363664A2 - Agents de vaccin synthetiques - Google Patents

Agents de vaccin synthetiques

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Publication number
EP1363664A2
EP1363664A2 EP02700174A EP02700174A EP1363664A2 EP 1363664 A2 EP1363664 A2 EP 1363664A2 EP 02700174 A EP02700174 A EP 02700174A EP 02700174 A EP02700174 A EP 02700174A EP 1363664 A2 EP1363664 A2 EP 1363664A2
Authority
EP
European Patent Office
Prior art keywords
immunogen
polyhydroxypolymer
poly
epitope
antigen
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP02700174A
Other languages
German (de)
English (en)
Inventor
Klaus Gregorius c/o M & E Biotech A/S NIELSEN
Peter c/o Pharmexa A/S KOEFOED
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Affitech AS
Original Assignee
Affitech AS
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
Priority claimed from PCT/DK2001/000113 external-priority patent/WO2001062284A2/fr
Priority claimed from US09/785,215 external-priority patent/US7135181B2/en
Application filed by Affitech AS filed Critical Affitech AS
Publication of EP1363664A2 publication Critical patent/EP1363664A2/fr
Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0007—Nervous system antigens; Prions
    • 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
    • A61K39/385—Haptens or antigens, bound to carriers
    • 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
    • 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
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • 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
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • 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
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • 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]
    • 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
    • A61K2039/6068—Other bacterial proteins, e.g. OMP
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6087—Polysaccharides; Lipopolysaccharides [LPS]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6093—Synthetic polymers, e.g. polyethyleneglycol [PEG], Polymers or copolymers of (D) glutamate and (D) lysine
    • 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
    • A61K2039/64—Medicinal preparations containing antigens or antibodies characterised by the architecture of the carrier-antigen complex, e.g. repetition of carrier-antigen units
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention provides for novel vaccine agents, which have a controllable distribution of different, well-defined peptides and which allows for detachment of these peptides from the agent by means of peptidase cleavage.
  • Agents for immunizing or vaccinating animals include a large variety of different solutions to various problems involved in immunization .
  • One known way of achieving coupling of peptides to an immuno- logically inert carrier is to activate a suitable polyhydroxypolymer with tresyl (trifluoroethylsulphonyl) groups or other suitable activation groups such as maleimido, p-Nitrophenyl cloroformate (for activation of OH groups and formation of a peptide bond between peptide and polyhydroxypolymer) , and to- syl (p-toluenesulfonyl) . It is e.g.
  • Molecules can be obtained that comprise a T helper epitope and molecules representing or including B-cell epitopes linked co- valently to a non-immunogenic polymer molecule acting as a vehicle, e.g. a multivalent activated poly-hydroxypolymer - this type of molecule functions as a vaccine molecule that only contains the immunologically relevant parts of antigens.
  • Promiscuous or so-called universal T-helper epitopes can be used if e.g.
  • the target for the vaccine is a hapten (such as a self-antigen) or an antigen that otherwise could be rendered more immunogenic by adding further T H epitopes.
  • elements that enhance the immunological response could be also co-coupled to the vehicle and thereby act as an adjuvant.
  • Such elements could be mannose, tuftsin, muramyl dipeptide, CpG motifs etc, cf. below. In that case, subsequent adjuvant formu- lation of the vaccine product might be unnecessary and the product could be administered in pure water or saline.
  • CTL cytotoxic T cell
  • At least one first antigenic determinant that includes at least one B-cell epitope and/or at least one CTL epitope
  • T H epitope T helper cell epitope
  • Another aspect of the invention relates to an immunogenic com- position comprising the immunogen of the invention.
  • a third aspect of the invention relates to a method of immunizing/vaccinating an animal against an antigen of choice by administering the immunogen or the immunogenic composition of the invention, where the immunogen and the antigen of choice shares the same at least one first antigenic determinant.
  • a fourth aspect of the invention relates to a method for preparing and selecting useful immunogens of the invention.
  • the present disclosure focuses on the use of antigenic peptide determinants - in fact it is neces- sary that the 2 nd antigenic determinant is, or at least contains, an amino acid sequence that constitutes an MHC Class II binding peptide (a T H epitope) .
  • the 1 st determinant includes B-cell epitopes, it need not necessarily be a peptide - it will be understood by the skilled reader that all disclo- sures relating to the 1 st antigenic determinant may relate to other B-cell epitope-containing molecular entities, unless the present disclosure specifically discusses characteristics of 1 st peptide determinants that would not be applicable to other antigenic determinants (for instance in the event the 1 st de- terminant is a CTL epitope) .
  • Fig. 1 Schematic drawing of the synthesis of immunogens of the invention, exemplified by peptide-coupling to TAD.
  • a mixture of peptides A and B is brought in contact (essen- tially as described in Example 2) with a tresyl-activated, water-soluble dextran.
  • Peptides A and B react with the activation groups on the TAD, and after purification the immunogen is provided.
  • T-lymphocyte and "T-cell” will be used inter- changeably for lymphocytes of thymic origin that are responsible for various cell mediated immune responses as well as for helper activity in antigen specific immune responses.
  • B-lymphocyte and “B-cell” will be used interchangeably for antibody-producing lymphocytes.
  • cytotoxic T-cell and the term
  • CTL are also used interchangeably for lymphocytes that induce cell killing in response to recognition of peptide sequences bound to MHC Class I molecules on the surface of the cells .
  • An immunogen is herein meant to designate a single molecule that includes one or more antigenic determinants of an antigen of interest and one or more T H epitopes that are recognized by the animal to be immunized.
  • Antigenic determinant is herein designating a molecule or a part of a molecule that is specifically recognised by certain clones of lymphocytes.
  • An antigenic determinant may e.g. be a B-cell epitope and thus recognized by B-cells and their corresponding antibodies.
  • a B-cell epitope is characterized by its 3D shape and in essence any part of a molecule that can fit into the antigen binding site of an antibody may constitute an antigenic determinant of that molecule.
  • Other antigenic determinants are CTL and T H epitopes - these are always peptides or contains peptides and they are characterized by their linear structure.
  • peptide is in the present context intended to mean both short peptides of from 2 to 10 amino acid residues, oli- gopeptides of from 11 to 100 amino acid residues, and polypep- tides of more than 100 amino acid residues. Furthermore, the term is also intended to include proteins, i.e. functional biomolecules comprising at least one polypeptide.
  • the peptide may be glycosylated and/or lipidated and/or comprise prosthetic groups.
  • sequence means any consecutive stretch of at least 3 amino acids or, when relevant, of at least 3 nucleo- tides, derived directly from a naturally occurring amino acid sequence or nucleic acid sequence, respectively.
  • animal is in the present context in general intended to denote an animal species (preferably mammalian) , such as Homo sapiens, Canis domesticus, etc. and not just one single animal. However, the term also denotes a population of such an animal species, since it is important that the indi- viduals immunized according to the invention all harbour substantially the same antigen allowing for immunization of the animals with the same immunogen (s) . If, for instance, genetic variants of the antigen exists in different human populations it may be necessary to use different immunogens in these different populations in order to be able to break the tolerance towards the antigen in each population. It will be clear to the skilled person that an animal in the present context is a living being which has an immune system. It is preferred that the animal is a vertebrate, such as a mammal.
  • effecting presentation ... to the immune system is intended to denote that the animal's immune system is subjected to an immunogenic challenge in a controlled manner.
  • challenge of the immune system can be by vaccination with peptide-contain- ing immunogens of the invention.
  • immune competent cells in the animal are confronted with the antigen in an immunologically effective manner, whereas the precise mode of achieving this result is of less importance to the inventive idea underlying the present invention.
  • immunogenically effective amount has its usual meaning in the art, i.e. an amount of an immunogen which is capable of inducing an immune response which significantly en- gages agents (e.g. pathogenic agents) which share immunologi- cal features with the immunogen.
  • a “foreign T-cell epitope” is a peptide which is able to bind to an MHC molecule and which stimulates T-cells in an animal species .
  • Preferred foreign T-cell epitopes in the invention are "promiscuous” (or “universal” or “broad-range”) epitopes, i.e. epitopes which bind to a substantial fraction of a particular class of MHC molecules in an animal species or population. Only a very limited number of such promiscuous T-cell epitopes are known, and they will be discussed in detail below.
  • a "foreign T helper lymphocyte epitope" (a foreign T H epitope) is a foreign T cell epitope which binds an MHC Class II molecule and can be presented on the surface of an antigen presenting cell (APC) bound to the MHC Class II molecule.
  • APC antigen presenting cell
  • MHC Class II binding amino acid sequence that is heterolo- gous to an antigen is therefore an MHC Class II binding peptide that does not exist in the multimeric protein in question. Such a peptide will, if it is also truly foreign to the animal species harbouring the multimeric protein, be a foreign T H epitope.
  • a "functional part" of a (bio) molecule is in the present context intended to mean the part of the molecule which is responsible for at least one of the biochemical or physiological effects exerted by the molecule. It is well-known in the art that many enzymes and other effector molecules have an active site which is responsible for the effects exerted by the molecule in question. Other parts of the molecule may serve a stabilizing or solubility enhancing purpose and can therefore be left out if these purposes are not of relevance in the context of a certain embodiment of the present invention.
  • adjuvant has its usual meaning in the art of vaccine technology, i.e. a substance or a composition of matter which is 1) not in itself capable of mounting a specific immune response against the immunogen of the vaccine, but which is 2) nevertheless capable of enhancing the immune response against the immunogen.
  • vaccination with the adjuvant alone does not provide an immune response against the immunogen
  • vaccination with the immunogen may or may not give rise to an immune response against the immunogen, but the combination of vaccination with immunogen and adjuvant induces an immune response against the immunogen which is stronger than that induced by the immunogen alone.
  • Targeting of a molecule is in the present context intended to denote the situation where a molecule upon introduction in the animal will appear preferentially in certain tissue (s) or will be preferentially associated with certain cells or cell types.
  • the effect can be accomplished in a number of ways including formulation of the immunogen in a composition facilitating targeting or by introduction in the immunogen of groups which facilitates targeting.
  • Stimulation of the immune system means that a substance or composition of matter exhibits a general, non-specific immu- nostimulatory effect.
  • a number of adjuvants and putative adju- vants (such as certain cytokines) share the ability to stimulate the immune system.
  • the result of using an immunostimula- ting agent is an increased "alertness" of the immune system meaning that simultaneous or subsequent immunization with an immunogen induces a significantly more effective immune re- sponse compared to isolated use of the immunogen.
  • polyhydroxypolymer carrier is intended to denote the part of the immunogen that carries the amino acid sequences.
  • the polyhydroxypolymer carrier has its outer limits where amino acid sequences can be cleaved of by a peptidase, e.g. in an antigen presenting cell that is processing the immunogen.
  • the polyhydroxypolymer carrier can be the polyhydroxypolymer with an activation group, where the bond between the activation group and the amino acid sequence is cleavable by a peptidase in an APC, or the polyhy- droxypolymer carrier can be a polyhydroxypolymer with activa- tion group and e.g. a linker such as a single L-amino acid or a number of D-amino acids, where the last part of the linker can bond to the amino acid sequences and be cleaved by a peptidase in an APC.
  • a linker such as a single L-amino acid or a
  • polysaccharide is intended to be used with its normal meaning, i.e. "a combination of nine or more monosaccha- rides, linked together by glycosidic bonds", cf. Hawley's Condensed Chemical Dictionary, 11 th ed., Sax and Lewis, eds . , Van Nostrand Reinhold Co., New York, 1987.
  • Examples of such poly- saccharides are dextran (e.g. Dextran 40, Dextran 70, Dextran 75) , agarose, cellulose and starch.
  • the rationale behind the present invention is that the provision of synthetic immunogens (especially peptide-containing immunogens) provides a greater deal of control over the composition of the end-product.
  • synthetic immunogens especially peptide-containing immunogens
  • purification and characterization is always a major task.
  • epitopes epitopes
  • epitopes both B-cell epitopes and CTL epitopes
  • a known protein may be "scanned" for epitopes by preparing overlapping truncates thereof that are reacted with monoclonal antibodies raised against the protein. The fragments that react with the antibody constitute local epitopes.
  • suitable technologies include those described in Irving MB et al . , Curr Opin Che Biol 2001 5(3): 314-24, Parker CE and Tomer KB, Methods Mol Biol 2000;146:185-201, and Nelson PN et al . , Mol Pathol 2000; 53 (3) : 111-7.
  • CTL epitopes may be predicted via the methods described in Rothbard et al . EMBO J. 7:93-100 (1988) and in de Groot MS et al . , Vaccine 2001;19(31) .4385-95 or otherwise identified via the technologies described in Rammensee H-G. et al . (1995), Immunogenetics 41: 178-228, Schirle M et al . Eur J Immunol 2000; 30 (18) : 2216- 2225.
  • either or both of the at least one first and second antigenic determinants of the immunogen are peptides, i.e. that they are constituted by amino acid sequences - since the 1 st and 2 nd amino acid sequences are normally attached separately to the activated polyhydroxypolymer carrier, their respective lengths can be kept at a minimum, thus facilitating the synthesis steps for each peptide species.
  • the length of such amino sequences are contemplated to range from about 4 amino acids to about 100 (even though there is no upper limit) , but preferably the upper limit will not exceed 80 amino acids, and it is preferred that the upper limit does not exceed 60 amino acids.
  • peptidic antigenic determinants are thus in the range between about 4 to about 50 amino acids. Even lower upper limits are envisaged, such as at most 40 , at most 30 and at most 25 amino acids. It is especially preferred that a peptidic antigenic determinant is constituted by an amino acid sequence of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids. It is also contemplated to utilise e.g. recombinantly produced polypeptides as the molecule constituting the 1 st antigenic determinant and using synthetic peptides as the 2 nd antigenic determinant. This would e.g.
  • the APCs In order to obtain processing and presentation of peptides of the immunogen by antigen presenting cells (APCs) , the APCs must be able to cleave off the peptide antigenic determinant from the polyhydroxypolymer carrier. This is most conveniently achieved by having such a peptide antigenic determinant coupled to the carrier via a bond that can be cleaved by a peptidase, such as the peptidases that are active in the APCs' processing of peptide sequences. This can e.g. be achieved by having the determinants coupled to the carrier by means of a peptide bond or an amide bond.
  • the antigenic determinant is bound to the carrier, where the antigenic determinant provides for the nitrogen atom of the amide or peptide bond - for peptidic antigenic determinants, this has the consequence that the peptide has a free carboxy termi- nus, an effect obtained when using a tresyl group as the activation group.
  • the 1 st and 2 nd antigenic determinants will be derived from different molecules, i.e. they are not found in the same molecule in nature. Further, it is normally also the case that they are not derived from the same species. And, even though it is preferred that the 1 st and 2 nd antigenic determi- nants are coupled independently to the activated polyhydroxypolymer it can be envisaged that they are e.g. fused to each other and coupled to the carrier at the terminus of the fusion product - this will be feasible when both antigenic determi- nants are relatively small (such as when the 1 st antigenic determinant is a CTL epitope and the 2 nd is a pure T H epitope) .
  • the 2 nd antigenic determinant preferably includes an immunodommant T H epitope, that is, epitope which in the vaccinated individual gives rise to a significant immune response, but it is a well-known fact that a T H epitope which is immunodommant in one individual is not necessarily immunodommant in another individual of the same species, even though it may be capable of binding MHC Class II molecules in the latter individual. However, normally it will suffice to utilise a peptide that strongly binds to an MHC class II molecule.
  • the T H epitope being part of the 2 nd antigenic determinant is preferably a promiscuous T H epitope.
  • the promiscuous epitope can according to the invention be a naturally occurring human TH epitope such as epitopes from tetanus toxoid (e.g. the P2 and P30 epitopes, cf. SEQ ID NOs : 1 and 2, respectively), diphtheria toxoid, Influenza virus hemagluttinin (HA), and P. falciparum CS antigen.
  • tetanus toxoid e.g. the P2 and P30 epitopes, cf. SEQ ID NOs : 1 and 2, respectively
  • diphtheria toxoid e.g. the Influenza virus hemagluttinin (HA), and P. falciparum CS antigen.
  • T-cell epitopes Over the years a number of other promiscuous T-cell epitopes have been identified. Especially peptides capable of binding a large proportion of HLA-DR molecules encoded by the different HLA-DR alleles have been identified and these are all possible T-cell epitopes to be introduced in modified CEA used according to the present invention. Cf. also the epitopes discussed in the following references which are hereby all incorporated by reference herein: WO 98/23635 (Frazer IH et al . , assigned to The University of Queensland); Southwood S et. al, 1998, J. Immunol. 160: 3363-3373; Sinigaglia F et al .
  • the epitope can be any artificial T-cell epitope which is capable of binding a large proportion of haplo- types.
  • the pan DR epitope peptides PADRE
  • the most effective PADRE peptides disclosed in these papers carry D-amino acids in the C- and N-termini in order to improve stability when administered.
  • the present invention primarily aims at incorporating the relevant epitopes as part of the immunogen which should then subsequently be broken down enzymatically inside the ly- sosomal compartment of APCs to allow subsequent presentation in the context of an MHC-II molecule and therefore it is not expedient to incorporate D-amino acids in the epitopes used in the present invention.
  • PADRE peptide is the one having the amino acid sequence AKFVAAWTLKAAA (SEQ ID NO: 3) or an immu- nologically effective subsequence thereof.
  • This, and other epitopes having the same lack of MHC restriction are preferred T-cell epitopes that should be present in the modified CEA used in the inventive method.
  • Such super-promiscuous epitopes will allow for the simplest embodiments of the invention wherein only one single modified CEA is presented to the vac- cinated animal' s immune system.
  • the immunogen of the invention may also contain other moieties that provide for desirable features of the immunogen.
  • the immunogen may further comprise at least one moiety coupled to the polyhydroxypolymer, said at least one moiety being selected from the group consisting of an immune stimulating moiety, a targeting moiety, or a presentation enhancing moiety - these are also discussed in detail below in connection with adjuvants. If such a moiety is a peptide it may be coupled to the activated polyhydroxypolymer in the same step as the coupling of the 1 st and 2 nd antigenic determinants.
  • the immunogen of the invention can also include the introduction of a moiety which targets the immunogen to an APC or a B-lymphocyte.
  • the first moiety can be a specific binding partner for a B-lymphocyte specific surface antigen or for an APC specific surface antigen.
  • the moiety can be a carbohydrate for which there is a receptor on the B-lymphocyte or the APC (e.g. mannan or mannose).
  • the second moiety can be a hapten.
  • an antibody fragment which specifically recognizes a surface molecule on APCs or lymphocytes can be used as a first moiety (the surface molecule can e.g.
  • CD40 ligand, antibodies against CD40, or variants thereof which bind CD40 will target the modified CEA to dendritic cells.
  • CD40 binding molecules as the first moiety (or as adjuvants, cf . below) will enhance the CTL response considerably; in fact, the use of such CD40 bin- ding molecules as adjuvants and "first moieties" in the meaning of the present invention is believed to be inventive in its own right.
  • an immune system stimulating moiety As an alternative or supplement to targeting the immunogen to a certain cell type in order to achieve an enhanced immune re- sponse, it is possible to increase the level of responsiveness of the immune system by including the an immune system stimulating moiety.
  • Typical examples of such moieties are cyto- kines, heat-shock proteins, and hormones, as well as effective parts thereof.
  • Suitable cytokines to be used according to the invention are those which will normally also function as adjuvants in a vac- cine composition, e.g. interferon ⁇ (IFN- ⁇ ) , Flt3 ligand (Flt3L) , interleukin 1 (IL-1) , interleukin 2 (IL-2) , interleu- kin 4 (IL-4), interleukin 6 (IL-6) , interleukin 12 (IL-12) , interleukin 13 (IL-13) , interleukin 15 (IL-15) , and granulo- cyte-macrophage colony stimulating factor (GM-CSF) ; alternatively, the functional part of the cytokine molecule may suffice as the second moiety.
  • cy- tokines as adjuvant substances, cf . the discussion below.
  • the immune stimulating moiety can be a toxin, such as listeriolycin (LLO) , lipid A and heat-labile entero- toxin.
  • LLO listeriolycin
  • lipid A lipid A
  • heat-labile entero- toxin lipid A
  • mycobacterial derivatives such as MDP (muramyl dipeptide) , CFA (complete Freund' s adjuvant) and the trehalose diesters TDM and TDE are interesting possibilities.
  • suitable heat shock proteins used as the immune stimulating moiety can be HSP70, HSP90, HSC70 (a heat shock cognate) , GRP94, and calreticulin (CRT) .
  • CpG motifs and the immune stimulating peptide tuftsin are other possibilities.
  • the possibility of introducing a presentation enhancing moiety is an important embodiment of the invention.
  • the art has shown several examples of this principle.
  • the palmitoyl lipidation anchor in the Borrelia burgdorferi protein OspA can be utilised so as to provide self-adjuvating polypeptides (cf. e.g. WO 96/40718).
  • the lipidated proteins form up micelle-like structures with a core consisting of the lipidation anchor parts of the polypeptides and the remaining parts of the molecule protruding therefrom, resulting in multiple presentations of the antigenic determinants.
  • this and related ap- proaches using different lipidation anchors (e.g.
  • lipidation anchor in a recombinantly produced protein is fairly straightforward and merely requires use of e.g. a naturally occurring signal sequence as a fusion partner for a component of the immunogen.
  • Another possibility is use of the C3d fragment of complement factor C3 or C3 itself (cf. Dempsey et al., 1996, Science 271, 348-350 and Lou & Kohler, 1998, Nature Biotechnology 16, 458-462) .
  • the polyhydroxypolymer carrier is the polyhydroxypolymer carrier
  • polyhydroxypolymer is generally intended to have the same meaning as in WO 00/05316, i.e. the polyhydroxypolymer can have exactly the same characteristics as is specifically taught in that application.
  • the polyhydroxypoly- mer can be water soluble or insoluble (thus requiring different synthesis steps during preparation of the immunogen) .
  • the coupling of peptides to a water-soluble activated polyhydroxypolymer is chemically simpler than the synthesis applica- ble for an insoluble polymer.
  • induction of CTLs will be facilitated by using water-soluble carriers in the invention since such immunogens would be likely to be taken up via pinocytosis.
  • the insoluble polymer can, when grinded, form a particulate compo- sition that is believed to be well-suited for inducing T-cell help due to it being predominantly taken up by APCs and since it results in local concentration of antigen. It is therefore also within the scope of the present invention to utilise vaccination strategies where both water-soluble and water-insolu- ble immunogens are utilised.
  • the polyhydroxypolymer can be selected from naturally occur- ring polyhydroxy compounds and synthetic polyhydroxy compounds .
  • polyhydroxypolymers are polysaccharides selected from acetan, amylopectin, gum agar-agar, agarose, alginates, gum Arabic, carregeenan, cellulose, cyclodextrins, dextran, furcellaran, galactomannan, gelatin, ghatti, glucan, glycogen, guar, karaya, konjac/A, locust bean gum, annan, pectin, psyllium, pullulan, starch, tamarine, tragacanth, xan- than, xylan, and xyloglucan. Dextran is especially preferred.
  • the polyhydroxypolymer can also be selected from highly branched poly (ethyleneimine) (PEI), tetrathienylene vi- nylene, Kevlar (long chains of poly-paraphenyl terephtala- mide) , Poly (urethanes) , Poly (siloxanes) , polydimethylsiloxane, silicone, Poly (methyl methacrylate) (PMMA) , Poly(vinyl alcohol), Poly (vinyl pyrrolidone) , Poly (2-hydroxymethylmethacry- late), Poly(N-vinyl pyrrolidone), Poly (vinyl alcohol),
  • PEI poly (ethyleneimine)
  • tetrathienylene vi- nylene Kevlar (long chains of poly-paraphenyl terephtala- mide)
  • Poly (urethanes) Poly (siloxanes) , polydimethylsiloxane, silicone, Poly (methyl methacryl
  • the (weight) average molecular weight of the polyhydroxypoly- mer in question is at least 500, typically at least 1,000, such as at least 2,000, preferably in the range of 2,500-2,000,000, more preferably in the range of 3,000-1,000,000, in particular in the range of 5,000- 500,000. It is believed that polyhydroxypolymers having an av- erage molecular weight in the range of 10,000-200,000 are particularly advantageous.
  • a water-soluble polyhydroxypolymer When using a water-soluble polyhydroxypolymer, it is preferably water soluble to an extent of at least 10 mg/ l, preferably at least 25 mg/ml, such as at least 50 mg/ml, in particular at least 100 mg/ml, such as at least 150 mg/ml at room temperature. It is known that dextran, even when activated as described herein, fulfils the requirements with respect to water solubility.
  • the ra- tio between C (carbon atoms) and OH groups (hydroxy groups) of the unactivated polyhydroxypolymers is in the range of 1.3 to 2.5, such as 1.5-2.3, preferably 1.6-2.1, in particular 1.85-2.05.
  • a C/OH ratio of the unactivated polyhydroxypolymer represents a highly advantageous level of hydrophilicit .
  • Polyvinylalcohol and polysaccharides are examples of polyhydroxypolymers which fulfil this requirement. It is believed that the above-mentioned ratio should be roughly the same for the activated polyhydroxypolymer as the activation ratio should be rather low.
  • the polyhydroxypolymers carry functional groups (activation groups) , which facilitates the anchoring of peptides to the carrier.
  • a wide range of applicable functional groups are known in the art, e.g. tresyl (trifluoroethylsul- phonyl) , maleimido, p-nitrophenyl cloroformate, cyanogenbro- mide, tosyl (p-toluenesulfonyl) , triflyl (trifluoromethanesul- fonyl) , pentafluorobenzenesulfonyl, and vinyl sulphone groups.
  • Preferred examples of functional groups within the present invention are tresyl, maleimido, tosyl, triflyl, pentafluorobenzenesulfonyl, p-nitrophenyl cloroformate, and vinylsulphone groups, among which tresyl, maleimido, and tosyl groups are particularly relevant.
  • Tresyl activated polyhydroxypolymers can be prepared using tresyl chloride as described for activation of dextran in Example 1 in WO 00/05316 or as described in Gregorius et al . , J. Immunol. Meth. 181 (1995)' 65-73.
  • Maleimido activated polyhydroxypolymers can be prepared using p-maleimidophenyl isocyanate as described for activation of dextran in Example 3 of WO 00/05316.
  • maleimido groups could be introduced to a polyhydroxypolymer, such as dextran, by derivatisation of a tresyl activated polyhydroxypolymer (such as tresyl activated dextran (TAD)) with a dia- mine compound (generally H 2 N-C n H 2n -NH 2 , where n is 1-20, preferably 1-8), e.g.
  • succinimidyl 4- N-maleimidomethyl) cyclohexane-1-car- boxylate (SMCC)
  • sulfo-succinimidyl 4- N-maleimidomethyl) - cyclohexane-1-carboxylate (sulfo-SMCC)
  • succinimidyl 4- p- maleimidophenyl) butyrate (SMPB)
  • sulfo-succinimidyl 4- p- maleimidophenyl) butyrate
  • sulfo-SMPB N- ⁇ -maleimidobutyryloxy- succinimide ester
  • GMBS N- ⁇ -maleimidobutyryloxy-sulfosuc- cinimide ester
  • maleimide activated polyhydroxypolymers Although the different reagents and routes for activation formally results in slightly different malei- ide activated products with respect to the linkage between the maleimide functionality and the remainder of the parent hydroxy group on which activation is performed, all and every are considered as "maleimide activated polyhydroxypolymers".
  • Tosyl activated polyhydroxypolymers can be prepared using tosyl chloride as described for activation of dextran in Example 2 in WO 00/05316.
  • Triflyl and pentafluorobenzenesulfonyl activated polyhydroxypolymers are prepared as the tosyl or tresyl activated analogues, e.g. by using the corresponding acid chlorides.
  • Cyanogenbromide activated polyhydroxypolymer can be prepared by reacting the polyhydroxypolymer with cyanogenbromide using conventional methods.
  • the resulting functional groups are normally cyanate esters with two hydroxy groups of the polyhy- droxypolymer .
  • the degree of activation can be expressed as the ratio between the free hydroxy groups and the activation groups (i.e. func- tionalised hydroxy groups) . It is believed that a ratio between the free hydroxy groups of the polyhydroxypolymer and the activation groups should be between 250:1 and 4:1 in order to obtain an advantageous balance between the hydrophilicity and the reactivity of the polyhydroxypolymer. Preferably the ratio is between 100:1 and 6:1, more preferably between 60:1 and 8:1, in particular between 40:1 and 10:1.
  • Especially interesting activated polyhydroxypolymers for use in the method for producing the generally applicable immunogen according to the invention are tresyl, tosyl and maleimido activated polysaccharides, especially tresyl activated dextran (TAD) , tosyl activated dextran (TosAD) , and maleimido activated dextran (MAD) .
  • TAD tresyl activated dextran
  • TosAD tosyl activated dextran
  • MAD maleimido activated dextran
  • the polyhydroxypolymer carrier may be substantially free of amino acid residues, necessitating that the activation group provides for part of a peptidase cleavable bond since the an- tigenic determinant would not otherwise be able to enter the antigen processing steps of the APCs metabolism.
  • the carrier may also simply include a spacer including at least one L-amino acid. Nevertheless, the at least first and at least second antigenic determinants are normally bound to the activated version of the polyhydroxypolymer via a nitrogen, preferably at the N-terminus of an amino acid sequence .
  • the ac- tivated polyhydroxypolymer must normally contain at least one amino acid group if the bond between an amino acid and the activation group cannot be broken by the APCs biochemical machinery.
  • the at least one amino acid group (s) then serves as spacer (s) between the activation group and the antigenic pep- tide determinant, with the consequence that the bond between the carrier's amino acid(s) and the peptide determinant may be cleaved, leaving the peptide free for attack by peptidases of the APCs processing pathway.
  • the binding between an activation group and a peptide determinant need not be sen- sitive to peptidase cleavage - it may equally well be sensitive to other physicochemical conditions that are found in the APC.
  • the important goal to achieve is that the antigenic determinant is liberated inside the APC, thus allowing for binding to an MHC molecule and subsequent presentation to lymphocytes .
  • the activated polyhydroxy polymer is used to combine the antigenic determinants containing the B-cell epitope and the T-helper epitopes it can, as mentioned above be performed as a solid phase synthesis and the final product can be harvested and purified by wash and filtration.
  • the elements to be coupled to an activated polyhydroxypolymer peptides, tags etc
  • the pH can be raised to pH 9-10 to start the reaction of the primary amino groups on the peptides and tags to the tresyl groups on the polyhydroxy polymer.
  • the gel is grinded to form particles of suitable size for immunization.
  • the ratio of B-cell epitopes and T-helper epitopes (P2, P30, PADRE or other suitable epitopes) in the final product can be varied by varying the concentration of these molecules in the synthesis step.
  • the immunogenic molecule can be tagged with e.g. mannose, tuftsin, CpG-motifs or other immune stimulating substances (described herein) by adding these, if necessary by using e.g. aminated derivatives of the substances, to the carbonate buffer in the synthesis step.
  • the formulation of the immunogen follows the principles generally acknowledged in the art.
  • peptide vaccines and immunizing compositions since peptides constitute the most preferred embodiments of the above-mentioned 1 st and 2 nd antigenic determinants.
  • the skilled reader will, though, appreciate that the general principles given below also will be applicable for most, if not all, vaccine strategies that include an immunogen of the invention.
  • vaccines and immunizing agents that contain peptide sequences as active ingredients is generally well un- derstood in the art, as exemplified by U.S. Patents 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792; and 4,578,770, all incorporated herein by reference.
  • such vaccines are prepared as injectables either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared.
  • the preparation may also be emulsified.
  • the active immunogenic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient.
  • Suitable excipients are, for example, water, saline, dextrose, gly- cerol, ethanol, or the like, and combinations thereof.
  • the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants which enhance the effectiveness of the vaccines; cf. the detailed discussion of adjuvants be- low.
  • the vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously, intracutane- ously, or intramuscularly.
  • Additional formulations which are suitable for other modes of administration include supposito- ries and, in some cases, oral, buccal, sublinqual, intraperi- toneal, intravaginal, anal, epidural, spinal, and intracranial formulations.
  • suppositories traditional binders and carriers may include, for example, polyalkalene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1-2% .
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These com- positions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10-95% of active ingredient, preferably 25-70%.
  • cholera toxin is an interesting formulation partner (and also a possible conjugation partner) .
  • the immunogens may be formulated into the vaccine as neutral or salt forms.
  • Pharmaceutically acceptable salts include acid addition salts (e.g. formed with the free amino groups of a peptide constituting part of an immunogen of the invention) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, his- tidine, procaine, and the like.
  • the vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeuti- cally effective and immunogenic.
  • the quantity to be administered depends on the subject to be treated, including, e.g., the capacity of the individual's immune system to mount an im- mune response, and the degree of protection desired.
  • Suitable dosage ranges are of the order of several hundred micrograms active ingredient per vaccination with a preferred range from about 0.1 ⁇ g to 2,000 ⁇ g (even though higher amounts in the 1- 10 mg range are contemplated) , such as in the range from about 0.5 ⁇ g to 1,000 ⁇ g, preferably in the range from 1 ⁇ g to 500 ⁇ g and especially in the range from about 10 ⁇ g to 100 ⁇ g.
  • Suitable regimens for initial administration and booster shots are also variable but are typified by an initial administration followed by subsequent inoculations or other administra- tions.
  • the manner of application may be varied widely. Any of the conventional methods for administration of a vaccine are applicable. These include oral application on a solid physiologically acceptable base or in a physiologically acceptable dispersion, parenterally, by injection or the like.
  • the dosage of the vaccine will depend on the route of administration and will vary according to the age of the person to be vaccinated and the formulation of the antigen.
  • the immunogens of the vaccine are sufficiently immuno- genic in a vaccine, but for some of the others the immune response will be enhanced if the vaccine further comprises an adjuvant substance.
  • an adjuvant which can be demonstrated to facilitate breaking of the autotolerance to self-protiens and other haptens that are not merely non-immu- nogenic due to their small size; however, in cases where the antigenic determinants coupled to the polyhydroxy polymer are both sufficiently large and sufficiently immunogenic, the adjuvant need not be incorporated.
  • Non-limiting examples of suitable adjuvants are selected from the group consisting of an immune targeting adjuvant; an im- mune modulating adjuvant such as a toxin, a cytokine, and a mycobacterial derivative; an oil formulation; a polymer; a micelle forming adjuvant; a saponin; an immunostimulating complex matrix (ISCOM matrix) ; a particle; DDA; aluminium adjuvants; DNA adjuvants; ⁇ -inulin; and an encapsulating adjuvant.
  • an immune targeting adjuvant an im- mune modulating adjuvant such as a toxin, a cytokine, and a mycobacterial derivative
  • an oil formulation a polymer; a micelle forming adjuvant; a saponin; an immunostimulating complex matrix (ISCOM matrix) ; a particle; DDA; aluminium adjuvants; DNA adjuvants; ⁇ -inulin; and an encapsulating adjuvant.
  • adjuvants include use of agents such as aluminium hydroxide or phosphate (alum), commonly used as 0.05 to 0.1 percent solution in buffered saline, admixture with synthetic polymers of sugars (e.g. Carbopol®) used as 0.25 percent solution, aggregation of protein in the vaccine by heat treatment with temperatures ranging between 70° to 101°C for 30 second to 2 minute periods respectively and also aggregation by means of cross-linking agents are possible. Aggrega- tion by reactivation with pepsin treated antibodies (Fab fragments) to albumin, mixture with bacterial cells such as C.
  • agents such as aluminium hydroxide or phosphate (alum), commonly used as 0.05 to 0.1 percent solution in buffered saline, admixture with synthetic polymers of sugars (e.g. Carbopol®) used as 0.25 percent solution, aggregation of protein in the vaccine by heat treatment with temperatures ranging between 70° to 101°C for 30 second to 2 minute periods respectively and also
  • parvum or endotoxins or lipopolysaccharide components of gram- negative bacteria emulsion in physiologically acceptable oil vehicles such as mannide mono-oleate (Aracel A) or emulsion with 20 percent solution of a perfluorocarbon (Fluosol-DA) used as a block substitute may also be employed. Admixture with oils such as squalene and IFA is also preferred.
  • DDA dimethyldioctadecylammonium bromide
  • DNA and ⁇ -inulin are interesting candidates for an adjuvant as is DNA and ⁇ -inulin, but also Freund's complete and incomplete adjuvants as well as quillaja saponins such as QuilA and QS21 are interesting as is RIBI.
  • Further possibilities are monophos- phoryl lipid A (MPL) , the above mentioned C3 and C3d, and u- ramyl dipeptide (MDP) .
  • MPL monophos- phoryl lipid A
  • C3 and C3d the above mentioned C3 and C3d
  • MDP u- ramyl dipeptide
  • Liposome formulations are also known to confer adjuvant ef- fects, and therefore liposome adjuvants are preferred according to the invention.
  • immunostimulating complex matrix type (ISCOM® matrix) adjuvants are preferred choices according to the invention, especially since it has been shown that this type of adjuvants are capable of up-regulating MHC Class II expression by APCs.
  • An ISCOM® matrix consists of (optionally fractionated) sapo- nins (triterpenoids) from Quillaja saponaria, cholesterol, and phospholipid.
  • the resulting particulate formulation is what is known as an ISCOM particle where the saponin constitutes 60-70% w/w, the cholesterol and phospholipid 10-15% w/w, and the protein 10-15% w/w. Details relating to composition and use of immunostimulating complexes can e.g.
  • Another highly interesting (and thus, preferred) possibility of achieving adjuvant effect is to employ the technique described in Gosselin et al . , 1992 (which is hereby incorporated by reference herein) .
  • the presentation of a relevant antigen such as an antigen of the present invention can be enhanced by conjugating the antigen to antibodies (or antigen binding antibody fragments) against the Fc ⁇ receptors on mono- cytes/macrophages .
  • Especially conjugates between antigen and anti-Fc ⁇ RI have been demonstrated to enhance immunogenicity for the purposes of vaccination.
  • Suitable mycobacterial derivatives are selected from the group consisting of muramyl dipeptide, complete Freund's adjuvant, RIBI, and a diester of trehalose such as TDM and TDE.
  • Suitable immune targeting adjuvants are selected from the group consisting of CD40 ligand and CD40 antibodies or specifically binding fragments thereof (cf. the discussion above), mannose, a Fab fragment, and CTLA-4.
  • Suitable polymer adjuvants are selected from the group consisting of a carbohydrate such as dextran, PEG, starch, man- nan, and mannose; a plastic polymer; and latex such as latex beads .
  • VLN virtual lymph node
  • the VLN (a thin tubular device) mimics the structure and function of a lymph node. Insertion of a VLN under the skin creates a site of sterile inflammation with an upsurge of cytokines and chemokines. T- and B-cells as well as APCs rapidly respond to the danger signals, home to the in- flamed site and accumulate inside the porous matrix of the VLN.
  • the vaccine of the invention should be administered at least once a year, such as at least 1, 2, 3, 4, 5, 6, and 12 times a year. More specifically, 1-12 times per year is expected, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 times a year to an individual in need thereof. It has previously been shown that the memory immunity induced by the use of the preferred autovaccines according to the invention is not permanent, and therefor the immune system needs to be periodically challenged with the analogues.
  • the vaccine according to the invention may comprise several different immunogens with differences in the choice of antigenic determinants in order to increase the immune response, cf. also the discussion above concerning the choice of foreign T-cell epitope introductions .
  • the vaccine may comprise two or more immunogens, where all of the immunogens are as de- fined above.
  • the vaccine may consequently comprise 3-20 different immunogens of the invention, such as 3-10 immunogens.
  • analogues normally the number of analogues will be sought kept to a minimum such as 1 or 2 analogues, especially because the immunogens may be tailored to include both the necessary 1 st antigenic determinants and the necessary 2 nd antigenic determinants in order to target as large a fraction of the vaccinated population.
  • an advantage of the immunogens of the invention is that they can be tailored to provide the optimum ratio between 1 st and 2 nd antigenic determinants.
  • This also allows for the provision of a method for selection and preparation of useful immunogens of the invention.
  • the method entails preparation of at least two different immunogens of the invention where each different immunogen has a characteristic ratio between the 1 st and 2 nd antigenic determinants.
  • These immunogens are then formulated as immunizing agents (cf. example 3) and their respective capabilities of inducing a specific immune response against the 1 st antigenic determinant are determined.
  • the most effective immunogen (s) is/are selected and used for further development.
  • the 1 st and 2 nd antigenic determinants as well as possible further moieties are coupled to the activated polyhydroxypolymer essentially as described in the present examples. And since the ratio of incorporated molecules in the immunogen is de- pendent on the relative concentrations of the molecules in the incubation mixture, it is unproblematic to prepare a large number of different immunogens merely be adjusting the relative concentrations of the molecules .
  • a traditional conjugate vaccine consists of an antigen or hapten (e.g. in the form of a polypeptide antigen or hapten) coupled covalently to a carrier, such as a carrier protein.
  • the antigen/hapten contains the B-cell or CTL epitope (s) and the carrier protein provides T H epitopes.
  • the carrier protein will normally be irrelevant as a source for T H epitopes, since only a minor part of the total sequence contains the relevant T H epitopes.
  • Such epitopes can be defined and synthesized as peptides of e.g. 9-15 amino a- cids .
  • a vaccine molecule that only contains the relevant parts of the antigen and the traditional carrier can be obtained. It is further possible to provide an immunogen that contains an optimized compo- sition of B-cell epitopes, CTL epitopes, and T H epitopes.
  • Polyhydroxypolymers such as dextran, starch, agarose etc. can be activated with 2, 2, 2-trifluoroethanesulfonyl chloride (tresyl chloride) , either by means of a homogenous synthesis (dextran) dissolved in N-methylpyrrolidinone (NMP) or by means of a heterogeneous synthesis (starch, agarose, cross-linked dextran) in e.g. acetone.
  • tresyl chloride 2, 2, 2-trifluoroethanesulfonyl chloride
  • NMP dry N-methyl pyrrolidinone
  • the flask is placed into the oil bath agaom, still with magnetic stirring, and tresyl chloride (2.764 ml, 25 mmol) is added drop- wise. After 15 min, dry pyridine (anhydrous, 2.020 ml, 25 mmol) is added drop-wise. The flask is removed from the oil bath and stirred for 1 hour at room temperature.
  • the product Tesyl Activated Dextran, TAD
  • the supernatant is decanted and the precipitate is harvested in 50 ml polypropylene tubes in a centrifuge at 2000 rp . The precipitate is dissolved in 50 ml 0.5% acetic acid, dialyzed 2 times against 5000 ml 0.5% acetic acid and freeze- dried.
  • TAD can be stored as a freeze-dried powder at -20°C.
  • an insoluble poly-hydroxypolymer such as aga- rose or cross-linked dextran can be tresyl activated by making a suspension of the poly-hydroxypolymer in e.g. acetone and perform the synthesis as a solid phase synthesis.
  • the activated poly-hydroxypolymer can be harvested by filtration. Suitable methods are reported in e.g. Nilsson K and Mosbach K (1987), Methods in Enzymology 135, p. 67, and in Hermansson GT et al . (1992), in "Immobilized Affinity Ligand Techniques", Academic Press, Inc., p. 87.
  • the peptides are prepared by means of a standard Fmoc strategy, where the conventional Fmoc-Lys (Boc) -OH has been substituted with Fmoc- Lys(Dde)-OH (obtained from Novabiochem , cat. no. 04-12-1121), i.e. the ⁇ -amino group in lysine is protected with Dde instead of Boc.
  • the pH value is measured and adjusted to 9.6 using 1 M HC1. After 2.5 hours at room temperature, hydrazine from an 80% solution is added to a final hydrazine concentration of 8% and the solution is incubated for another 30 min. at room tempera- ture and freeze-dried immediately thereafter. The freeze-dried product is dissolved in H 2 0 and dialysed extensively against H 2 0 before the final freeze-drying.
  • the ratio between B-cell epitopes (A ⁇ ) and T-helper epitopes (P2 and P30) in the final product is varied using different concentrations of these peptides in the synthesis step.
  • the final product can be tagged with e.g. mannose (so as to target the conjugate to APCs) by adding aminated mannose to the carbonate buffer in the synthesis step. If an insoluble activated poly-hydroxypolymer is used to combine the peptides containing the B-cell epitope and the T-helper epitopes, the coupling to the polymer can be performed as a traditional solid phase synthesis and the final product is harvested and purified by wash and filtration.
  • the presently described approach for preparing a peptide based vaccine may be applied to any other polypeptide antigen where it would be convenient to prepare a purely synthetic peptide vaccine and where the polypeptide antigen in question provides sufficient immunogenicity in one single peptide:
  • TAD (10 mg) is dissolved in 100 ⁇ l H 2 0 and 1000 ⁇ l carbonate buffer, pH 9.6, containing 1-5 mg peptide A (any immunogenic peptide of interest) , 1-5 mg P2 (diphtheria toxoid P2 epitope) and 1-5 mg P30 (diphtheria toxoid P30 epitope) is added.
  • the pH value is measured and adjusted to 9.6 using 0.1 M HC1. After 2.5 hours at room temperature the solution is freeze dried immediately hereafter.
  • the freeze-dried product is dissolved in H 2 0 and dialysed extensively against H 2 0 or desalted on a gelfiltration column before the final freeze-drying.
  • Such immunogens have been utilised by the inventors with the short 8 amino acid C-terminal fragment of the Borrelia burgdorferi protein OspC as "peptide A” and a diptheria toxoid epitope (P2 or P30) as peptide B.
  • the results of immunization studies with this antigen revealed that only the immunogen of the invention including the OspC fragment and a foreign dipth- eria epitope matching the MHC haplotype of the vaccinated mice were capable of inducing antibodies reactive with OspC in these mice.
  • the immunogen of the invention is injected into a suitable animal species such as mouse, rat, guinea pig, rabbit, or monkey.
  • the immunogens can be mixed with a suitable adjuvant such as for example Freund's Adjuvant, ISA-51, aluminum-based adjuvants (aluminium phosphate or aluminium hydroxide, e.g. from Danfoss), Calcium Phosphate, QS21 (Antigenics), MF59 (Chiron Corp.), and Ribi (Glaxo SmithKline) .
  • a suitable adjuvant such as for example Freund's Adjuvant, ISA-51, aluminum-based adjuvants (aluminium phosphate or aluminium hydroxide, e.g. from Danfoss), Calcium Phosphate, QS21 (Antigenics), MF59 (Chiron Corp.), and Ribi (Glaxo SmithKline) .
  • Protein vaccines are usually administered 3-5 times, for example at weeks 0, 3, ⁇ , 9, 12.
  • Sera from vaccinated animals can be tested for specific antibodies by ELISA.
  • 96-well Maxisorb plates e.g obtained from Nunc, Life Technologies, Taastrup, Denmark
  • a suitable volume e.g. 50 ul
  • This is done in a suitable buffer such as carbonate buffer pH 9.6 in a suitable concentration giving a final content of e.g. 1 ug/well.
  • the plates are incubated, e.g. for 1 hour, washed in washing buffer, e.g. PBS + 0.5M NaCl + 1% Triton X-100 and then blocked for e.g.
  • dilution buffer 1 hour in dilution buffer that could e.g. be washing buffer plus 1% BSA.
  • Standards and diluted serum samples can be added in duplicate and incubated in the plates, e.g. for 30 minutes.
  • a dilution of secondary antibody e.g. HRP conju- gated rabbit-anti-mouse IgG e.g. from DAKO, Glostrup, Denmark
  • HRP conju- gated rabbit-anti-mouse IgG e.g. from DAKO, Glostrup, Denmark
  • the plates can then be washed in washing buffer, and a chromogenic substrate, e.g. OPD substrate (e.g. from Sigma-Aldrich, Vallensbask Strand, Denmark) can be added.
  • OPD substrate e.g. from Sigma-Aldrich, Vallensbask Strand, Denmark
  • the reaction can be stopped with e.g 2N H 2 S0 and the optical density be measured in e.g. a Dynex MRX ELISA plate reader at 490nm.
  • Serum antibody concentrations can be calculated e.g. by relating the optical densities of the samples to a standard curve.
  • these antibodies can be used to generate a standard curve, but in the event such an antibody should not be available, it can be prepared by methods known in the art.
  • the ELISA can be modified for different purposes.
  • ELISA can be used to monitor whether the antisera contains reactivities that can displace e.g. a biotin-labeled, therapeu- tically relevant antibody from binding to plate-coated antigen or 1 st antigenic determinant. Specialized ELISA kits can be used to determine the isotype subclass distribution in anti- sera etc.
  • CTL epitope containing vaccines can induce an antigen-specific CTL re- sponse.
  • the CTL epitopes used in the immunogen can be used to pulse APCs in e.g. typical cytotoxic T cell assays.
  • Antigen- expressing target cells e.g. MHC class I expressing cell lines transfected with a gene encoding the antigen of supplements
  • CTLs cytotoxic T lym- phocytes
  • Mice will be vaccinated with the inventive immunogens using an appropriate vaccination scheme, cf. above. As positive control, mice could be immunized with e.g.
  • splenocytes from immunized mice can be re-stimulated e.g. with mytomicin C-treated (e.g. 50 ⁇ g/ml, 20 min at 37 °C) syngeneic splenocytes loaded with the peptides.
  • mytomicin C-treated e.g. 50 ⁇ g/ml, 20 min at 37 °C
  • syngeneic splenocytes loaded with the peptides.
  • a suitable number e.g. lOOxlO 6
  • pep- tide-loaded syngeneic splenocytes can be mixed with a suitable number (e.g.
  • splenocytes from vaccinated mice in and incubated at 37 °C for e.g. 7 days.
  • the cytotoxic activity of the effector cells can be monitored e.g. in a chromium release assay.
  • a suitable number (e.g. 5x10 s ) of target cells is labeled with 51 Chromium (e.g. 200 mCi) , if necessary loaded with antigen-derived peptide, and used as targets in a 51 Chromium release assay.
  • Cell lines transfected with the relevant antigen can also be used as targets for CTLs .
  • Such CTL assays can e.g. be performed in normal wild type mice, transgenic mice and/or mice transgenic for hu- man HLA class I molecules (e.g. HHD mice) using suitable compatible target cells and cells for restimulation.
  • the ability of a vaccine of the invention to induce antigen- specific CTL responses can also be measured using other assay methods including antigen-specific tetramer stainings and Elispot assays; all such methods are well-known to the person skilled in immunology.

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Abstract

L'invention concerne de nouveaux immunogènes constitués d'un squelette polyhydroxypolymère activé auquel sont fixés 2 déterminants antigènes séparés. Le premier déterminant antigène comprend une cellule B ou un épitope CTL et le second déterminant antigène un épitope T auxiliaire. Dans des modes de réalisation préférés, les déterminants antigènes sont dérivés de molécules et d'espèces différentes. Des immunogènes exemplaires selon l'invention sont constitués d'un squelette dextrane linéaire activé par trésyl auquel sont couplés une celle B ou des épitopes CTL d'un antigène, ainsi que des épitopes T auxiliaires universels. L'invention concerne également des compositions immunogènes comprenant lesdits immunogènes, ainsi que des procédés d'immunisation et un procédé d'identification d'immunogènes appropriés.
EP02700174A 2001-02-19 2002-02-19 Agents de vaccin synthetiques Withdrawn EP1363664A2 (fr)

Applications Claiming Priority (9)

Application Number Priority Date Filing Date Title
WOPCT/DK01/00113 2001-02-19
PCT/DK2001/000113 WO2001062284A2 (fr) 2000-02-21 2001-02-19 Nouvelle methode de regulation negative d'amyloide
US09/785,215 US7135181B2 (en) 2000-02-21 2001-02-20 Method for down-regulation of amyloid
US785215 2001-02-20
DK200101231 2001-08-20
DKPA200101231 2001-08-20
US33754301P 2001-10-22 2001-10-22
US337543P 2001-10-22
PCT/DK2002/000112 WO2002066056A2 (fr) 2001-02-19 2002-02-19 Agents de vaccin synthetiques

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EP1363664A2 true EP1363664A2 (fr) 2003-11-26

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EP (1) EP1363664A2 (fr)
JP (1) JP2004529881A (fr)
CN (1) CN100562338C (fr)
AU (1) AU2002233166B2 (fr)
CA (1) CA2440197A1 (fr)
IL (1) IL157475A0 (fr)
NO (1) NO335602B1 (fr)
NZ (1) NZ527720A (fr)
WO (1) WO2002066056A2 (fr)
ZA (1) ZA200400895B (fr)

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EP1539232A1 (fr) * 2002-09-12 2005-06-15 Pharmexa A/S Immunisation contre la ghreline autologue
WO2005037190A2 (fr) * 2003-09-03 2005-04-28 Dendritherapeutics, Inc. Vaccins multiplex
EP1682171A4 (fr) * 2003-11-07 2008-02-27 Univ Rochester Compositions et procedes de traitement de maladies nerveuses
US20120231044A1 (en) * 2011-03-11 2012-09-13 Flow Pharma, Inc. Vaccine formulation of mannose coated peptide particles
CN103665113A (zh) * 2012-09-14 2014-03-26 深圳市安群生物工程有限公司 人Aβ42抗原决定簇多肽、抗原、抗体、用途及试剂盒
RU2635517C1 (ru) * 2016-09-14 2017-11-13 Закрытое акционерное общество "Институт фармацевтических технологий" (ЗАО "ИФТ") Синтетический иммуноген для защиты и лечения от зависимости от психоактивных веществ
CN112165956A (zh) * 2018-04-10 2021-01-01 Ac免疫有限公司 抗Aβ治疗性疫苗

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DE69333107T2 (de) * 1992-02-11 2004-01-29 Jackson H M Found Military Med Dualer träger für immunogene konstrukte
AU4377793A (en) * 1992-05-20 1993-12-13 Johns Hopkins University, The Alternative receptor therapy
DE69918146T2 (de) * 1998-10-05 2005-07-07 Pharmexa A/S Verfahren zur therapeutischen impfung

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See references of WO02066056A2 *

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WO2002066056A8 (fr) 2004-04-29
CN100562338C (zh) 2009-11-25
JP2004529881A (ja) 2004-09-30
CN1893970A (zh) 2007-01-10
ZA200400895B (en) 2005-05-03
NZ527720A (en) 2005-03-24
WO2002066056A3 (fr) 2003-01-03
AU2002233166B2 (en) 2006-06-29
IL157475A0 (en) 2004-03-28
NO335602B1 (no) 2015-01-12
WO2002066056A2 (fr) 2002-08-29
NO20040431L (no) 2004-04-16
CA2440197A1 (fr) 2002-08-29

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