EP0961793A1 - Polymeres dans lesquels sont incorpores des peptides - Google Patents

Polymeres dans lesquels sont incorpores des peptides

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Publication number
EP0961793A1
EP0961793A1 EP98901884A EP98901884A EP0961793A1 EP 0961793 A1 EP0961793 A1 EP 0961793A1 EP 98901884 A EP98901884 A EP 98901884A EP 98901884 A EP98901884 A EP 98901884A EP 0961793 A1 EP0961793 A1 EP 0961793A1
Authority
EP
European Patent Office
Prior art keywords
polymer
peptide
peptides
cooh
different
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Withdrawn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
EP98901884A
Other languages
German (de)
English (en)
Other versions
EP0961793A4 (fr
Inventor
David Charles Jackson
Neil Martin O'brien-Simpson
Lorena Elizabeth Brown
Wieguang Zeng
Nicholas Jon Ede
Evelyn Rosemary Brandt
Michael Francis Good
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.)
Commonwealth Scientific and Industrial Research Organization CSIRO
Walter and Eliza Hall Institute of Medical Research
CSL Ltd
University of Melbourne
QIMR Berghofer Medical Research Institute
Original Assignee
Commonwealth Scientific and Industrial Research Organization CSIRO
Walter and Eliza Hall Institute of Medical Research
Queensland Institute of Medical Research QIMR
CSL Ltd
University of Melbourne
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Filing date
Publication date
Priority claimed from AUPO5071A external-priority patent/AUPO507197A0/en
Application filed by Commonwealth Scientific and Industrial Research Organization CSIRO, Walter and Eliza Hall Institute of Medical Research, Queensland Institute of Medical Research QIMR, CSL Ltd, University of Melbourne filed Critical Commonwealth Scientific and Industrial Research Organization CSIRO
Publication of EP0961793A1 publication Critical patent/EP0961793A1/fr
Publication of EP0961793A4 publication Critical patent/EP0961793A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1018—Orthomyxoviridae, e.g. influenza virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1275—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Streptococcus (G)
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F246/00—Copolymers in which the nature of only the monomers in minority is defined
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to polymers incorporating peptides.
  • the polymers may be used for raising an immune response or in delivery of the peptides or as a diagnostic tool.
  • Synthetic peptides are widely used to generate site-specific antibodies, a fact which has stimulated considerable interest in evaluating their use as vaccine candidates.
  • the advantages of this approach include safety, as there is no requirement for infectious material, and the ability to chemically define the product.
  • synthetic peptide-based vaccines there are currently major problems limiting their exploitation. Many of these limitations center around the small size, low copy number and low immunogenicity of peptide-based immunogens.
  • T- and B-cell epitopes defined in a single host of a particular MHC type may be inadequate for eliciting immunity in outbred populations and because many diseases are ca ised by organisms where the target antigens are polymorphic, this restriction in the number of different epitopes that can be incorporated is an important consideration.
  • the Birr et al. polymeric vaccine as an immunogen, only had limited effectiveness in antibody response in providing specific antibody titres in vaccination trials which were intermediate between a higher titre obtained by a first vaccine derived from ⁇ -terminal conjugation of each of the same seven epitopes with ⁇ -palmitoyl-S-[2,3 bis-(palmitoyloxy)-(2RS)- propyl]-(R) cysteinyl-serine as discussed in Jung et. al., 1985, Int. Ed. Engl. 24 872 and a lower titre obtained by another vaccine which was produced by recombinant co-expression in E.
  • the approach developed by the present inventors is also "modular" and permits peptides to be synthesised, purified and then assembled into polymers.
  • very large (> 600,000 Dalton) molecular species can be assembled with virtually any number of the same or different epitopes.
  • the orientation of a peptide within a polymer may affect the resulting immune response as described, for example, in Silversides et al. (A synthetic luteinizing hormone releasing hormone vaccine. I. Conjugation and specificity trials in BALB/c mice. Silversides-DW; Allen-AF; Misra-V;
  • lysine derivatives which have different types of amino protecting groups, for example, 9-fluorenylmethoxycarbonyl (Fmoc), Mtt (4-methyltrityl) and Dde (4,4-dimethyl-2,6-dioxocyclohex-l-ylidene ethyl), allows orthogonal chemistries to be used to selectively expose a particular amino group by removal of its protecting gimip such that the acryloyl group can be introduced at a specified point or points within the peptide sequence.
  • Fmoc 9-fluorenylmethoxycarbonyl
  • Mtt 4-methyltrityl
  • Dde 4,4-dimethyl-2,6-dioxocyclohex-l-ylidene ethyl
  • the peptides can be polymerised by free radical initiation of chain elongation in much the same way that acrylamide is lmitinely polymerised into polyacrylamide gels. In this way peptides are assembled into polymers in which the peptides form side chains pendant from an alkane backbone (Fig. 1).
  • the method allows purification of the individual determinants, avoids errors inherent in long sequential syntheses and, in contrast to an approach described by Eckstein .17 has the advantage that protected peptide fragments are not ised thereby avoiding sohibility and purification problems.
  • multiple copies of many different peptide epitopes can be incorporated into a single polymeric structure to allow utilisation of the range of T cell epitopes required for outbred populations, in conjunction with epitopes representing different pathogenic serodemes. This approach may prove to be a significant advance in synthetic vaccine technology.
  • the present invention consists in a polymer comprising polymerised units of
  • R ⁇ is a peptide, each R ⁇ being the same or different;
  • R 2 is NH2
  • Y is H, COOH, CO-NH 2 ;
  • W is H, CH 3) CH 2 CO-NH 2 , CH 2 COOH, CH 2 OH, CH(CH 3 )OH,
  • Z is H, (CH 2 ) h QT, in which h is 0-10, Q is O, NH, CH 2 and T is a lipid, labelling molecule, targeting molecule or functional group;
  • R 3 is CH 3 , H, NH 2 , OH, CN or halogen, each R 3 being the same or different;
  • R 4 is CH 3 , H, NH 2 , OH, CN or halogen, each R 4 being the same or different; and the ratio of (1):(2) being in the range of about 1:1 to about
  • R ⁇ is a peptide, each Rj being the same or different;
  • R 4 is CH 3 , H, NH 2 , OH, CN or halogen, each R 4 being the same or different.
  • Y is H, COOH, CO-NH 2 ;
  • W is H, CH 3 , CH 2 CO-NH 2 , CH 2 COOH, CH 2 OH, CH(CH 3 )OH,
  • Z is H, (CH 2 ) h QT, in which h is 0-10, Q is O, NH, CH 2 and T is a lipid, labelling molecule, targeting molecule or functional group;
  • R 3 is CH 3 , H, NH 2 , OH, CN or halogen, each R 3 being the same or different;
  • R 4 is CH 3 , H, NH 2 , OH, CN or halogen, each R 4 being the same or different; and (ii) polymerising the monomers in the presence of a free radical initiator.
  • R t is a peptide, each R ⁇ being the same or different, at least one of R being the peptide epitope;
  • R 2 is NH2
  • Y is H. COOH, CO-NH 2 ;
  • W is H, CH 3 , CH 2 CO-NH 2 , CH 2 COOH, CH 2 OH, CH(CH 3 )OH,
  • Z is H, (CH 2 ) h QT, in which h is 0-10, Q is O, NH, CH 2 and T is a lipid, labelling molecule, targeting molecule or functional group;
  • R 3 is CH 3 , H, NH 2 , OH, CN or halogen, each R 3 being the same or different;
  • R 4 is CH 3 , H, NH 2 , OH, CN or halogen, each R 4 being the same or different; and the ratio of (1):(2) being in the range of about 1:1 to about 1:1000.
  • the polymer will typically be random, however, it may be block or alternating.
  • the polymer may also be cross-linked. This may be achieved any suitable cross-linking agent such as bisacrylamide.
  • the polymer will normally include a plurality of R ⁇ groups. It is preferred that the peptides R, are epitopes and that this plurality of R ⁇ groups provides a mixture of T cell and/or B cell epitopes. It will be recognised that the second of the elements in the polymer basically acts as a spacer between R ⁇ groups.
  • R 2 may include a lipid component or may include a group which will direct the polymer to a particular target.
  • R 2 may include lipids such as palmitic acids or
  • N ⁇ -Palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-L-cysteine is one way in which self-adjuvanting properties may be added to the polymer. It is also possible to include molecules with labelling functions in R 2 eg fluorescamine, metal ion chelating molecules for chelating radioactive metal ions etc. Molecules with targeting properties may also be included in R 2 . For example folic acid to target cancer cells. As will also be readily appreciated that molecules which modify the physiochemical properties, such as solubility, viscosity etc, of the polymer may be included in R 2 .
  • a spacer group X is present, however, in others others its presence is optional. It is presently preferred that the polymer includes this spacer so that the peptide R ⁇ is spaced away from the polymer backbone. It is preferred that spacer group X includes an enzymaticly cleavable site. This is particularly preferred where R t are peptide epitopes. Examples of cleavable sites include the peptides GLFG and NYLKY.
  • the ratio (1):(2) is in the range 1:10 to 1:50. While the bulk of the discussion and examples in this application relate to polymers in which R ⁇ is or are peptide epitopes it is to be understood that it is not essential that R x is or are peptide epitopes.
  • the present invention contemplates polymers in which R ⁇ is or are other biologically active peptides, such as hormones etc..
  • the polymers of the present invention may provide useful vehicles for the delivery of a range of biologically active peptides. In this arrangement by adjusting the spacer X it may be possible to produce polymers which release the peptide over prolonged periods.
  • the invention provides a method of preparation of a polymeric immunogen having a polymer backbone and a plurality of pendant peptides attached thereto. In general the method includes the steps of:-
  • the peptides in step (i) may be obtained from any suitable source which may include extraction of a natural protein, chemical cleavage of a natural synthetic or recombinant protein, recombinant expression of an oligopeptide or from a naturally occurring small protein. However, preferably each peptide is synthesised either manually or by using an automatic peptide synthesiser.
  • the peptides in step (i) may be synthesised using solution synthesis or solid phase synthesis as described, for example, in Chapter 9 entitled “Peptide Synthesis” by Atherton and Sheppard which is included in a publication entitled “Synthetic Vaccines” edited by Nicholson and published by Blackwell Scientific Publications.
  • a solid phase support is utilised which may be polystyrene gel beads wherein the polystyrene may be cross-linked with a small proportion of divinylbenzene (e.g. 1%) which is further swollen by lipophilic solvents such as dichloromethane or more polar solvents such as dimethylformamide (DMF).
  • the polystyrene may be functionalised with chloromethyl or aminomethyl groups.
  • cross-linked and functionalised polydimethyl-acrylamide gel is used which may be highly solvated and swollen by DMF and other polar aprotic solvents.
  • Other supports can be utilised based on polyethylene glycol which is usually grafted or otherwise attached to the surface of inert polystyrene beads.
  • use may be made of a variety of commercial solid supports or resins such as PAL-PEG, PAK-PEG, KA, KR or TGR.
  • each peptide may have additional moieties selected from carbohydrate, lipid, nucleotide or nucleoside as may be desired. Especially preferred are oligosaccharides, oligonucleotides or glycosidic groups.
  • step (ii) use may be made of an suitable acryloylating agent such as acryloyl chloride, cyanoacryloyl chloride, methacryloyl chloride or esters of acrylic acid or methacrylic acid.
  • an acryloylating agent such as acryloyl chloride, cyanoacryloyl chloride, methacryloyl chloride or esters of acrylic acid or methacrylic acid.
  • the peptide may be cleaved from the resin and the protecting groups removed by a suitable cleaving agent or sequential use of cleavage reagents.
  • a suitable cleaving agent or sequential use of cleavage reagents.
  • the cleaving agent does not contain a thiol group because the thiol group may react with double bonds.
  • the peptides may be purified in any suitable manner such as, for example, by reverse phase, ion exchange or size exclusion chromatography.
  • polymerisation of the acryloylated peptides may occur in any suitable manner.
  • a solution of the acryloylated peptides may be treated with a reagent capable of generating a very small concentration of free radicals.
  • a trace of hydrogen peroxide and a ferrous salt can be used or a trace of ammonium persulphate with N,N,N',N'-tetramethylenediamine.
  • the peptide entities are therefore pendant from a linear backbone.
  • Each peptide could terminate in either a carboxyl or carboxyamide group to mimic the natural situation of the particular epitope, i.e. whether the particular epitope forms the C-terminus of the protein or whether it represents an internal sequence of amino acids.
  • the distribution of the peptides along the polymer backbone may be controlled to some extent by controlling the stoichiometry of component peptides in the polymerisation mixture. Such an arrangement may even allow for the simulation of conformational determinants of the native protein where different peptides representing regions remote in sequence of the parent protein are attached to the backbone.
  • Co-polymerisation of the acryloyl peptides with different amounts of a suitably functionalised reagent such as N, N'-dimethylacrylamide or acryloylated amino acids, would allow for the pendant peptides to be separated to a greater or lesser extent from one another along the polymer backbone. This arrangement may prove necessary in some cases where steric hindrance or adventitious interactions between neighbouring peptides occurs. It could also increase or decrease solubility of the resulting polymeric peptide as desired.
  • this may occur by preparing the acryloyl derivative of a first peptide and the methacryloyl derivative of a second peptide and then co-polymerise the two.
  • the sequence order of the two peptides along the backbone of the resultant polymer could well be different (and possibly more ordered) than might be the case for the co-polymerisation of the acryloylated derivatives of the first peptide and the second peptide where a more random arrangement could be expected.
  • Small amounts of low molecular weight acrylic amides or esters, chosen for specific features, could also be added to the mixture of monomers prior to the polymerisation step.
  • additional monomers could include an acryloyl compound having a radioactive label attached, or an acryloyl derivative of a fluorescent or chemiluminescent reagent.
  • the resulting polymer would then contain, in chemical side chains distinct from the peptide epitopes themselves, the potential for subsequent easy detection of that polymer. This feature would be particularly advantageous at the research and development stage. Assembly of an appropriate combination of B- and T-cell epitopes should allow antibody and T-cell production in animals in a wide range of different histocompatibility types.
  • “Slow release” form of these peptide polymers may become available through preparation of the peptide acryloyl derivative and polymerisation in the presence of a cross-linking reagent such as bisacrylamide. These polymers may also express interesting biological activity per se by virtue of the fact that each polymer molecule would present a very high local concentration of the active monomeric unit. Numerous peptide hormones are now known including insulin, gastrin, oxytocin, vasopressin, adrenocorticotrophic hormone (ACTH), growth hormone, cholecystokinin, bombesin, substance P, relaxin, encephalin, angiotensin, somatostatin, bradykinin and so on.
  • a "poly-vasopressin” or analogue might be extremely efficient in blocking the natural vasopressin receptor on a cell surface.
  • Immobilisation of a biologically-active peptide on a polymeric support already has many known applications in research, for example in the isolation of receptor molecules, purification of antibodies and in immunochemical analysis.
  • Cross-linked polyacrylamides with specific peptide side chains, such as those described here, could serve similar functions.
  • Figure 1 Scheme for the preparation of synthetic peptide based polymers. Peptides are assembled in the normal way on solid phase supports and then acylated at the N- terminus with acryloyl chloride. Following removal of the peptide from the support and concommitant removal of the side chain protecting groups, the peptide epitopes are purified and polymerised by exposure to free radical.
  • FIG. 1 Analytical reverse phase HPLC chromatograms of crude H 2 N-P8 (A) and crude N-acryloyl P8 (B) using a Vydac C18 column (4.6 x 300 mm) installed in a Waters HPLC system. The chromatogram was developed at a flow rate of 1 mL min -1 using a gradient, 0-100% solvent B developed over 30mins.
  • FIG. 3 ⁇ NMR of N-acryloyl-GFGA.
  • the N-acryloylated peptide was cleaved from the resin using reagent B, purified by RP-HPLC and examined by J H NMR.
  • FIG. 4 Gel permeation chromatography of monomeric and polymeric peptides.
  • the column effluent was monitored at 280nm to detect tryptophan and tyrosine residues.
  • the arrows indicate the retention time of the molecular weight standards; thyroglobulin 669 kDa (A), bovine serum albumin 67 kDa (B) and ribonuclease 13.7 kDa (C).
  • Figure 5 Antigenic integrity of peptides and peptide polymers.
  • the binding of anti-P8 antisera to polymerised P8 ( ⁇ ), P8 monomer ( ⁇ ) or to polyacrylamide ( ⁇ ) and the binding of MAb 1/1 to polymerised P5 (•), P5 monomer (o) or to polyacrylamide (v) was examined by ELISA.
  • Figure 6 Ability of soluble monomeric and polymeric peptides to inhibit antigen-antibody binding. Dilutions of peptide, or peptide polymer were mixed with a constant amount of antibody and added to wells of a microtiter tray coated with peptide antigen. Binding of the antibody was measured by ELISA and the result expressed as the percentage of the antibody bound in the absence of inhibitor.
  • P2 monomer (O ), polymerised P2 ( ⁇ ) or polymerised P4 (0 ) were incubated with MAb 1/1 and binding assessed on P5 coated wells.
  • FIG. 8 Effect of free radicals on the antigenicity of synthetic peptides.
  • MAbs 1/1 and 2/1 were examined for their ability to bind to untreated peptide 306PKYVKQNTLKLATGMRNNPEKQT328 (•) and peptide exposed to various concentrations of ammonium persulphate.
  • the molar ratios of ammonium persulphate to peptide were; 0.1:1, (O); 1:1, (O); 10:1, ( ⁇ ); 100:1, (V); and 1000:1, ( ).
  • the level of binding of ⁇ MS to each of the different peptide preparations ( ⁇ ) is also shown.
  • Figure 9 Antigenic integrity of polymeric peptides.
  • Panel A Proliferation induced by a polymer of the following acryloylated peptide sequences: VYLKY-PKYVKQNTLKLA co-polymerised with acryloylserine (•), GFLG-PKYVKQNTLKLA co-polymerised with acryloylserine ( ⁇ ), PKYVKQNTLKLA co-polymerised with acrylamide ( ⁇ ), VYLKY-PKYVKQNTLKLA co-polymerised with acrylamide (A), GFLG-PKYVKQNTLKLA co-polymerised with acrylamide (T), monomeric peptide PKYVKQNTLKLATGMRNVPEKQT (D).
  • Panel B Proliferation induced by VYLKY-PKYVKQNTLKLA co-polymerised with acryloylglutamic acid (0), GFLG-PKYVKQNTLKLA co-polymerised with acryloylglutamic acid (0), PKYVKQNTLKLA co-polymerised with acrylamide ( ⁇ ), monomeric peptide PKYVKQNTLKLATGMRNVPEKQT p).
  • Figure 15 Cytotoxic T-lymphocyte activity of polymer-primed cells against virus and peptide-pulsed targets.
  • mice were immunised subcutaneously with polymers (containing lO ⁇ g of peptide) in CFA in the hind footpad or inoculated intranasally with infectious influenza virus A/Memphis/1/71 (lxlO 4'5 pfu). Lymph nodes of peptide-primed mice and spleen cells from virus infected mice were removed 7 and 21 days after inoculation and restimulated in vitro with either influenza virus-infected spleen cells or the CTL epitope, TYQRTRALV at a dose of lOO ⁇ g/mL for 5 days.
  • Panel A 5lCr released by CTL activity of cells restimulated in vitro with virus.
  • Panel B 5 lCr released by CTL activity of cells restimulated in vitro with TYQRTRALV peptide.
  • Figure 16. Antibody response induced by polymers of peptides at various doses and differing backbone compositions. BALB/c mice were inoculated either with acrylamide, or serine or glutamic acid-based co-polymers of the peptides GMRNVPEKQT and ALNNRFQIKGVELKS at doses of 5, 0.5 or 0.05 nmoles of peptide. Antisera were taken at day 30 after the primary inoculation and 12 days after the secondary inoculation and antibody titers determined by ELISA on plates coated with the peptide
  • PKYVKQNTLKLATGMRNVPEKQT Antibody titres are expressed as the titre obtained at an optical density of 0.25 units. Titers obtained following the primary (open symbols) and secondary (closed symbols) inoculations are indicated. The bar represents the mean titre for each group of antisera.
  • A Heteropolymer antisera to peptide ST156;
  • B Heteropolymer antisera to peptide M52;
  • C ST156 homopolymer antisera: recognition of peptide ST156;
  • D M52 homopolymer antisera: recognition of peptide M52.
  • Each line represent the result of one mouse.
  • FIG. 1 Antibodies raised against the complete heteropolymer. Each line represents the result of one mouse.
  • HBTU 0'Benzotriazole-N,N,N',N'-tetra methyl -uronium- hexafluorophosphate
  • HOBt 1-hydroxybenzotriazole
  • DIPEA diisopropylethylamine
  • DMF N,N-dimethylformamide
  • TFA trifluoroacetic acid
  • Fmoc 9-fluorenylmethoxycarbonyl
  • Peptides were synthesised manually or using an automatic peptide synthesiser (either a Novasyn Crystal, Novabiochem, U.K. or a 9050 Plus PepSynthesiser (Milligen, Millford, MA). Standard solid phase peptide synthesis protocols for Fmoc chemistry were used throughout. Peptides were assembled as the C-terminal carboxyl or carboxyamide form using Novasyn KA 100 or KR 100 resins (Calbiochem-Novabiochem, New South Wales, Australia) respectively. Coupling was accomplished with HBTU/HOBt activation using 4 equivalents of amino acids and 6 equivalents of DIPEA. The Fmoc group was removed by 20% piperidine in DMF or 2.5% DBU in DMF.
  • an automatic peptide synthesiser either a Novasyn Crystal, Novabiochem, U.K. or a 9050 Plus PepSynthesiser (Milligen, Millford, MA). Standard solid phase peptide synthesis protocols for Fmoc chemistry
  • Cleavage of peptides from the resin support was performed using reagent B (88% TFA, 5% phenol, 5% water, 2% TIPS) for 2 or 4 hours depending on the arginine content of the peptide. After cleavage the resin was removed by filtration and the filtrate concentrated to approximately 1 mL under a stream of nitrogen. After the peptide products were precipitated in cold ether, they were centrifuged and washed 3 times. The peptide precipitate was then dissolved in 5 to 10 mL of water containing 0.1%v/v TFA and insoluble residue removed by centrifugation. N-acryloylation of peptides.
  • Resins bearing peptides were swollen in a minimum amount of anhydrous, de-aerated DMF and acryloylated under nitrogen. After cooling on ice, a 20-fold molar excess of DIPEA in 0.5 ml DMF and a 10-fold molar excess of acryloyl chloride in 0.5 ml DMF were added to the resin. The mixture was stirred for 1 hour on ice and then for a further 1 hour at room temperature. The progress of acryloylation was monitored by the trinitrobenezene sulphonic acid (TNBSA) test. When a negative TNBSA test was returned the resin was washed (5 x in DMF, 3 x in DCM and 3 x in diethyl ether). The resin was than dried under vacuum
  • Peptides P4 and P6 were assembled with Fmoc-Lys(Mtt)-OH (Calbiochem-Novabiochem, New South Wales, Australia) at their C-terminus and Boc-Pro-OH and Boc-Asp(OtBu)-OH at the N-terminus of P4 and P6 respectively.
  • the Mtt group was removed with 1% TFA containing 5% TIPS in DCM and Fmoc-Ahx then coupled to the free ⁇ -amino group using HBTU activation.
  • the Fmoc group was removed from the Ahx with 2.5% DBU in DMF and the exposed amino group acryloylated as above. Purification of Peptide Monomers.
  • Chromatograms were developed using solvent A and solvent B at a flow rate of 1 mL min" 1 and a 0-100% linear gradient of solvent B formed over 30 mins. Material eluted from columns was detected by determining the absorbance at 214nm or 280nm.
  • Peptide polymers were isolated by gel permeation chromatography (GPC) using a column (1.6x60 cm) of Superdex 200 installed in a FPLC system. Chromatography was carried out at a flow rate of 3 mL min -1 in 50mM NH 4 HCO 3 . All polymers eluted in the void volume. Peptide polymers isolated in this way were then lyophilised.
  • GPC gel permeation chromatography
  • Amino acid composition of the peptide monomers and polymers was confirmed by amino acid analysis of purified material. Peptide material was hydrolyzed (0.001% w/v phenol in 6N HCl for 24 hours at 110°C) and the hydrolysate derivatised with Fmoc-Cl. Amino acid analysis was carried out using a GBC Aminomate system using fluorometric detection. Enzyme-linked immunosorbent assay (ELISA).
  • ELISAs were performed as described 23 using a solution (5 ⁇ g mL "1 ) of peptide or peptide polymer to coat wells of flat-bottomed polyvinyl microtitre plates (Microtiter, Dynatech Laboratories, VA., U.S.A.). Bound antibody was then detected by incubation with horseradish peroxidase- conjugated (HRPO) rabbit immunoglobulin (Ig) directed against mouse Ig (DAKO, Denmark) or HRPO donkey Ig directed to sheep Ig (DAKO, Denmark) for 1.5h.
  • HRPO horseradish peroxidase- conjugated
  • Inhibition ELISAs were carried out using a 1 / 150 o dilution of MAb 1/1 or anti-P8 antiserum incubated with known concentrations of inhibitor (soluble peptide or polymer) for 2 hours and then transferred to flat- bottomed polyvinyl microtiter plates coated with P5 or P8. Following overnight incubation, the plates were washed and the ELISA developed as above.
  • MAb 1/1 The preparation and properties of monoclonal antibody MAb 1/1 has been described elsewhere 19 .
  • MAb 1/1 was raised against the synthetic peptide representing the C-terminal 24 residues ( 305 CPKYVKQNTLKLATGMRNVPEKQT 328 ) of the heavy chain [UA_] of the hemagglutinin of influenza virus A/Memphis/l/71 (H3) and is specific for the B cell determinant RNVPEKQT 20 .
  • Hyperimmune serum (HIS) was raised in sheep to luteinising hormone releasing hormone (LH-RH; P8). Synthesis of acryloyl peptides.
  • Reagent B 36 was used for the cleavage of N-acryloyl peptides from the resin support and side chain deprotection because the use of reagent R (peptide NH2-Gly-Phe-Gly- Ala, when acryloylated with acryloyl chloride and then cleaved with reagent R (TFA : thioanisole : anisole : ethane dithiol 90:5:3:2) gave an m z value 124 Daltons greater than expected by FAB mass spectrometry, a result consistent with thioanisole addition to the double bond) resulted in the addition of thioanisole to the double bond.
  • peptides P4 and P6 have been synthesised with the acryloyl group at their C-terminus. This was achieved by the acryloylation of the ⁇ -amino group of lysine which was introduced at the C-terminal position as Fmoc-Lys-(Mtt)-COOH.
  • the ability to synthesise N- or C-terminally acryloylated peptides allows for opposite orientations of a peptide within a polymer. Polymerisation of synthetic peptides.
  • the polymerisation reaction was routinely carried out in degassed 6M guanidine-HCl (GuHCl) and 2mM EDTA in 0.5M Tris (pH 8.3) although solvents such as 1.5M Tris-HCl (pH 8.8) and 8M urea were also successfully employed.
  • a 50-fold molar excess of acrylamide over the amount of acryloylated peptide was introduced to allow stretches of polyacrylamide to be interspersed between the various acryloyl peptide units with the aim of minimising steric interactions between the peptide chains and to maximise water solubility of the overall polymer. This was not always necessary because peptides P2, P9, P10 and P13 incorporating Ahx were polymerised successfully in the absence of added acrylamide. Polymerisation was initiated by addition of ammonium persulphate and
  • a polymer of P4 (which does not contain epitopes recognized by MAb 1/1 or anti-P8 antiserum) did not inhibit the binding of these antibodies to their relevant peptides indicating that increased inhibition caused by the peptide polymers is a consequence of the presentation of multiple copies of each peptide determinant.
  • the results in figures 5 and 6 demonstrate not only that polymerised peptides retain antigenic integrity but are more antigenic than the corresponding peptide monomer, presumably because multiple copies of the same antigenic determinant allows high avidity interaction with antibody.
  • Free radical polymerisation does not affect the antigenic integrity of peptides and peptide polymers are more antigenic than monomeric peptides.
  • An advantage of this approach is that any number of the same or different acryloyl peptides can be assembled into a polymer with the expectation that the overall antigenic activity of the construct will be largely determined by the nature of the peptide units which are pendant from a hydrocarbon chain.
  • polymerising a mixture of B-cell and T-cell peptide epitopes it will be possible to assemble a construct in which all or most of the important epitopes of a pathogen or of several pathogens are represented. This is particularly important for those organisms such as the malaria parasite, HIV and influenza virus and group A streptococci which occur as serologically diverse strains.
  • N,N'-dimethylformamide (DMF), piperidine, trifluoracetic acid (TFA), 0'Benzotriazole-N,N,N',N'-tetra methyl-uronium-hexafluorophosphate (HBTU), l-hydroxybenzotriazole (HOBt) and diisopropylethylamine (DIPEA) were obtained from Auspep Pty Ltd (Melbourne Australia).
  • Phenol, triisopropylsilane and acryloyl chloride were from Aldrich (Milwaulkee, WI) and trinitrobenzylsulphonic acid (TNBSA) from Fluka (Switzerland); l,8-diazabicyclo-[5.4.0]undec-7-ene (DBU) was obtained from Sigma and acrylamide, ammonium persulphate and
  • TEMED N,N,N',N'-tetramethylethylenediamine
  • Peptides were either synthesised manually or using an automatic peptide synthesiser (either a Novasyn Crystal, Novabiochem UK or a 9050 Plus, Milligen). Fmoc chemistry was used throughout and peptides were assembled as the C-terminal carboxyl or carboxamide form. Fmoc amino acids were either incorporated into the peptide chain as the activated
  • Peptides were cleaved from the resin support and the side chain protecting groups removed using reagent B (TFA, phenol, water and triisopropylsilane in the ratio 88:5 :5:2) at room temperature under nitrogen for either 2 or 4 hours, depending on the arginine content of the peptide.
  • reagent B TAA, phenol, water and triisopropylsilane in the ratio 88:5 :5:2:2
  • PepRPC column (1.6 x 10cm) installed in an FPLC system (Amrad/Pharmacia Pty. Ltd) and the chromatogram developed at a flow rate of 4ml/min using 0.1% TFA in H z O and 0.1% TFA in CH3CN as the limit solvent.
  • Analytical HPLC was carried out using a Vydac C4 column (4.6 x 300 mm) installed in a Waters HPLC system and developed at a flow rate of lml/min using similar solvents.
  • N-acryloylated peptides were carried out under nitrogen in degassed 0.5M Tris (pH 8.3) containing 6M guanidine-HCl and 2mM EDTA. Assembly of polymers of peptides was typically carried out with 3 ⁇ moles of N-acryloyl-peptide(s) and a 50-fold molar excess of acrylamide. Polymerisation was initiated by addition of ammonium persulphate (2% of the total number of moles of acrylamide) and TEMED (5 ⁇ l of a 1.32 ⁇ M solution) and the reaction mixture allowed to stand for 18 hrs at room temperature. Peptide polymers were isolated using a column (1.6 x 60cm) of
  • HiLoad Superdex 200 prep grade (Amrad/Pharmacia Pty. Ltd.) installed in an FPLC system and the chromatogram developed at a flow rate of 3ml/min using 50mM ammonium bicarbonate.
  • Molecular weight estimations were carried out using a Waters HPLC system with a Superose 6 HR column (1 x 30 cm) and developed at a flow rate of 0.5 ml/min in 50mM ammonium bicarbonate.
  • MAbs 1/1 and 2/1 have been described elsewhere . These MAbs were raised against the synthetic peptide 305 CPKYVKQNTLKLATGMRNVPEKQT 328 representing the C-terminal 24 residues of the heavy chain (HA ) of the hemagglutinin of influenza virus A/Memphis/1/71.
  • MAb 1/1 is specific for the B cell determinant RNVPEKQT (B and requires 32Z N, 325 E and 327 Q for binding, whereas MAb 2/1 recognises the B cell determinant LKLAT (B 2 ) and has an absolute requirement for each of these five residues . T Cells.
  • T cell clone 12V1 was raised in mice immunized with purified hemagglutinin light chain (HA 2 ) and proliferates in response to the peptide ALNNRFQIKGVELKS derived from HA 2 . The derivation, properties and maintenance of this clone have been described elsewhere 21 . Prior to use, the cultured cells were passaged over Isopaque-Ficoll gradients 22 ' 23 .
  • Lymph node T cells were derived from pooled inguinal and popliteal lymph node cell suspensions of mice primed seven days previously with peptide. T cells were enriched by passage through nylon wool columns . Immunization Protocols. Female Balb/c mice, 6 to 8 weeks of age, were used. For antibody studies, animals were inoculated i.p. with synthetic peptide in monomeric or polymeric form and emulsified in complete Freund's adjuvant (CFA). Mice received 5 or 0.5 nmoles of monomeric peptide or the equivalent number of moles of determinant within a polymer. For T-cell studies, mice were inoculated s.c. in the hind footpads with a total of 10 nmoles of T-cell determinant in CFA. Antibody Binding Assays.
  • Enzyme-linked immunosorbent assays were performed as described 23 using microtitre plates coated with a solution of 5 ⁇ g/ml peptide or polymer antigen. Bound antibody was detected by incubation with horseradish peroxidase-conjugated rabbit immunoglobulin (Ig) directed against mouse Ig (DAKO, Denmark) for 1.5h followed, after washing, with substrate (0.2mM 2,2'-azino-bis 3-ethylbenzthiazoline-sulphonic acid in 50mM citric acid pH4.0 containing 0.004% v/v hydrogen peroxide). The absorbance at a wavelength of 405 nm was determined using a Labsystems Multiscan Multisoft microplate reader (Pathtech Diagnostics Pty.
  • rabbit antisera to mouse IgM, IgGl, IgG2a, IgG2b, IgG3 or IgA was added and allowed to bind for 4h. Plates were again washed and a 1/400 dilution of horseradish peroxidase-conjugated swine anti-(rabbit Ig) was added. After 1.5h, the trays were again washed, substrate added and the absorbance determined as above. T Cell Proliferation Assays.
  • T cells were cultured (at a concentration of 3 x 10 cells/well for lymph node T cells and 1 x 10 4 /well for the T-cell clone) in 96-well microtitre trays (Nunc, Denmark) in the presence of syngenic ⁇ -irradiated (2,200 R, Co source) spleen cells as a source of antigen presenting cells, together with antigen in a total volume of 250 ⁇ l 22 ' 23 .
  • the culture medium was RPMI 1640 supplemented with 10% heat inactivated (56°C, 30min) fetal calf serum, 2mM glutamine, 2mM sodium pyruvate, O.lmM 2-mercaptoethanol, 30 ⁇ g/ml gentamicin, 100 I.U./ml penicillin and lOO ⁇ g/ml streptomycin.
  • Cultures were incubated for 4 days at 37°C in an atmosphere of 5% C0 2 with l ⁇ Ci/well 3 H-thymidine present during the final 18hr. Cells were then harvested onto glass fibre filters and incorporation of 3 H- thymidine was measured on a Hewlett Packard Matrix 9600 direct ⁇ -counter.
  • This peptide contains two B-cell determinants encompassed by the sequences GMRNVPEKQT (B and TLKLATG (B 2 ) and a helper T-cell determinant KYVKQNTLKL that overlaps with B 2 .
  • a second T cell determinant ALNNRNFQIKGVELKS (T) located elsewhere on the viral hemagglutinin within the light chain (HA 2 ) has been reported to be a more potent inducer of B cell help for the production of antibody directed to B_ than is the native overlapping helper determinant 26 .
  • Polymers of T alone or in combination with B 1 ⁇ B 2 or B ⁇ together with B 2 were prepared using the scheme shown in Figure 1.
  • Peptides were synthesised with an Ahx group at the N-terminus followed by acylation with the acryloyl group. Polymerisation of N-acryloylated peptides was then carried out in guanidine-HCl to aid solubility. A 50-fold molar excess of acrylamide was also added to act as a spacer along the polymer backbone. The reaction is comparable to the formation of polyacrylamide gels in the absence of crosslinking reagent.
  • Figure 7 shows the elution profile of poly (T + B ⁇ + B 2 ) from a column (1 x 30 cm) of Superose 6 HR. This profile was representative of that obtained with each of the different polymeric immunogens. The polymer eluted in the void volume of the column, a position quite distinct from that of monomeric peptides. In all cases, very little non-polymerised peptide remained after the polymerisation process. Antigenicity of Peptides Exposed to Free Radicals.
  • the polymerisation procedure utilises free radicals which are potentially capable of damaging functional groups of proteins.
  • monomers of peptide 306-328, lacking the acryloyl group were exposed to various concentrations of ammonium persulphate in the presence of acrylamide.
  • the peptide was then separated from the acrylamide polymer and used to coat wells for an ELISA assay. The ability of these treated peptides to bind MAbs 1/1 and 2/1, which have specificity for B 1 and B 2 respectively, is shown in Fig. 8.
  • the T cells recognised the T monomer and poly (T + B 2 ) with equal efficiency, and poly (T + B ⁇ + B 2 ) to a slightly less extent.
  • Poly (T + B was the least well recognised. Immunogenicity of the Polymeric Immunogens.
  • the relative immunogenic properties of monomeric and polymeric immunogens were assessed by comparing the antibody levels elicited by monomeric peptide 306-328 and a polymer of the peptide 306-319 which contains the B 2 determinant and the overlapping T cell determinant, KYVKQNTLKL 27, 28 . These two immunogens were tested in mice at a dose of 5 nmoles of peptide epitope and sera were taken during the primary and secondary responses. At this dose the monomeric peptide gave a very weak primary antibody response which rose some 10-fold following the second inoculation. In contrast, the polymeric immunogen gave a substantial primary response (approximately 30-fold higher mean titre than the monomer) and this was increased a further 10-fold after a second dose to give an overall 30-fold enhancement of antibody production.
  • the multi-component polymers based on the T sequence together with B ⁇ or B 2 or both, were also inoculated into mice and the antibody response determined. Doses of O. ⁇ nmoles and ⁇ nmoles elicited similar levels of antibody and the data obtained for the O. ⁇ nmole dose only is reported (Fig. 11B). Not unexpectedly, the individual monomeric Jz or B 2 determinants yielded no antibody when inoculated into mice, but the same determinants when co-polymerised with the T sequence were potent immunogens.
  • the levels of antibodies elicited in response to a single dose of polymer (panel B) were comparable or higher than those achieved after two doses of peptide 306-328 monomer (panel A) and those obtained after two doses of the polymers were 50 to 100-fold greater than with the peptide 306-328.
  • the poly (T + B t ) construct elicited the highest levels of antibody, higher on average than the titres of MAbs in the control ascitic fluid preparations. Specificity of Antibody to the Polymeric Immunogen containing two B-cell Determinants.
  • Certain polyvalent immunogens can trigger B cells in a T-independent manner and this interaction results in antibody of a restricted isotype profile with the dominant subclass being IgM.
  • the isotype profiles of serum antibody produced in response to poly (T + B , poly (T + B 2 ) or poly (T + Bj + B 2 ) were therefore examined to determine whether these polymers were restricted in the isotypes of antibody that they could elicit.
  • Figure 13 shows that a range of different antibody isotypes are produced in response to each of the three polymers.
  • Multicomponent polymers of a T-cell determinant sequence together with the B and/or B 2 determinants were successfully constructed and shown to be antigenically intact.
  • Results from subsequent experiments in which a polymer was assembled from the co-polymerisation of nine serologically distinct peptides demonstrated recognition of the polymer by antisera raised to each of the individual epitopes. These findings raise the strong possibility that such polymers could be used diagnostically.
  • the present study also shows that the polymers are strong immunogens and in the case of the polymer containing two different B-cell determinants, antibody was elicited against each one.
  • a great advantage of the approach described here is that the component synthetic peptide epitopes are purified prior to their incorporation into the immunogen. Furthermore, because the component peptides are individually assembled, other molecular properties such as intra-peptide disulphide loops can be incorporated to mimic similar structures in the native protein.
  • the ability to assemble multiple copies of the same or different peptides using this technology not only provides a way of addressing the polymorphism of some pathogens but also that associated with the MHC antigen system in the recognition of T-cell epitopes. In this way the assembly of appropriate combinations of B- and T-cell epitopes into polymeric supports should allow antibody production in animals of a wide range of different histocompatibility types.
  • the reaction mixture was washed 1 x water, 1 x 5% aqueous NaHC0 3 , 1 x water, 2 x 10% aqueous citric acid, 1 x water and 1 x brine and then dried overnight with Na 2 S0 4 .
  • the DCM was removed by rotary evaporation ( ⁇ 30°C) to a clear oil (99% yield) and the product was pure by TLC (chloroform:methanol 95:5).
  • the amino acid side chain and carboxyl protecting groups was removed with TFA:water (95:5) cleavage mixture for two hours, under N 2 , no light.
  • the TFA was removed under a stream of nitrogen and the product rotary evaporated ( ⁇ 30°C) to a clear oil.
  • the acryloyl amino acid was dissolved in 6M guanidine-HCl + 2mM EDTA in 0.5M Tris (GuHCl) and the pH adjusted to 8.3 with 4M Tris. A working stock solution was made containing 0.15 mg/ ⁇ l of an acryloyl amino acid. Each acryloyl amino acid had the expected mass as indicated by FAB mass spectra.
  • mice Female BALB/c mice, 6 to 8 weeks old, were used and were inoculated with at the following doses 5, 0.5, 0.05 nmoles of peptide (contained within the polymer) of either poly(serine) ALN+C10, poly(glutamic acid) ALN+ClO or poly(acrylamide) ALN+C10 emulsified in complete Freund's adjuvant (CFA).
  • Mice received a second dose on day 30. , All animals were bled from the retro-orbital plexus on day 30 after the primary peptide polymer inoculation and day 12 after the secondary peptide polymer inoculation, the sera was stored at -20°C until required.
  • mice were inoculated s.c. in the hind footpads (50 ⁇ l per footpad) with a total of 10 ⁇ g (7.8 nmoles) of the CTL determinant in monomeric or polymeric form. Mice were inoculated intranasally with infectious influenza virus A/Memphis/1/71 at lxlO 4 - 5 pfu in 50 ⁇ l.
  • Antibody titres were expressed as the reciprocal of the antibody dilution giving an absorbance of 0.25, which corresponds to at least five times the background value.
  • T-cell medium consisted of RPMI-1640 (CSL, Ltd. Parkville, Australia) supplemented with 10% heat inactivated (56°C, 30min) foetal calf serum(vol/vol), 2mM glutamine, 2mM sodium pyruvate, O.lmM
  • 2-mercaptoethanol 30 ⁇ g/ml gentamicin, 100 I.U./ml penicillin and lOO ⁇ g/ml streptomycin.
  • T Cell proliferation assay T-cell clone 4.51 was used and was raised in mice immunised with purified hemagglutinin heavy chain (HAi) and proliferates in response to the peptide KYVKQNTLKL derived from HAi. The derivation, properties and maintenance of this clone have been described elsewhere 28 . Prior to use , the cultured cells were passaged over Isoplaque-Ficoll gradients 2 ' 23 .
  • Proliferation assays were set up in 96-well flat-bottom microtitre trays (Nunc, Denmark) containing peptide antigen in monomeric or polymeric form (40 ⁇ M in the first well) with 1 x 10 4 T-cells (clone 4.51) in the presence of 3 x 10 5 (lOO ⁇ l) ⁇ -irradiated (2,200 rad, 60 Co source) normal BALB/c spleen cells, as a source of antigen presenting cell in a total volume of 250 ⁇ l 22 ' 23 . Cultures were incubated for 4 days at 37°C in an atmosphere of 5% C0 2 with l ⁇ Ci well" 1 3 H-thymidine present during the final 18hr. Cells were then harvested onto glass fibre filters and incorporation of 3 H-thymidine was measured on a Hewlett Packard Matrix 9600 direct ⁇ -counter.
  • Secondary effector cells were prepared from (i) popliteal and inguinal lymph node cell suspensions from BALB/c mice inoculated 7 days previously with peptide monomer or polymer (lO ⁇ g of peptide), (ii) spleen cells, depleted of erythrocytes by treatment with tris-buffered ammonium chloride (ATC; 0.15M NH4CI in 17 mM tris-HCl at pH 7.4) from mice primed at least 21 days previously with infectious virus.
  • ATC tris-buffered ammonium chloride
  • the effector cell populations were incubated (3xl0 7 cells/flask) in 25ml of T-cell medium containing lxlO 7 virus infected or peptide pulsed spleen cells as a source of antigen presenting cells.
  • the virus infected spleen cells had been preincubated at 37°C for 30 mins with 1000-5000 HAU of infectious virus in lml of serum free RPMI and washed once prior to use, the peptide pulsed cells were incubated with lOO ⁇ g of the CTL peptide determinant (monomer) in lml of serum free RPMI at 37°C for 2 hours. Cultures were incubated for 5 days at 37°C in an atmosphere of 5% C0 2 , after which the cells were washed and used in the CTL assay.
  • P815 mastocytoma cells were used .as target cells in the CTL assay, virus infected and uninfected targets were prepared by incubating 2X10 6 P815 cells in 500 ⁇ l of infectious virus solution (1000-5000 HAU mL- 1 ) or serum free RPMI, respectively at 37°C. After 2 hours the cells washed and resuspended in 200 ⁇ l of TCM containing 200 ⁇ Ci 51 Cr (Amersham, Australia). After 2 hours the cells were washed 3 times and their concentration adjusted to 1 x 10 5 cells ml -1 .
  • enzymatic cleavage sequences Two enzymatic cleavage sequences were chosen and synthesised onto the N-terminus of peptide PKY which was then acryloylated as previously described.
  • the sequences a tetramer (GLFG) and a pentamer (VYLKY) present the cleavage site of cathepsin B 38 and the cleavage motif described by van Noort and van der Drift 39 .
  • Serine, glutamic acid and acrylamide based polymers were assembled containing PKY either with or without the enzymatic cleavage sequences inserted between the peptide and the acryloyl -Ahx- group. Each of these polymers were used to assess their ability to stimulate clone 4.51 in a proliferation assay.
  • those polymers which contained either the cathepsin B or the cathepsin D cleavage sequences induced a proliferation response.
  • the incorporation- of the cathepsin D sequence induced higher proliferation as compared to the cathepsin B sequence for each type of polymer.
  • Poly(glutamic acid) based polymers elicited a response 35% to 50% higher proliferative response than poly(serine) based polymers at 0.1 ⁇ mole dilution.
  • the amino acid based polymer induced a much higher proliferative response than poly (acrylamide) based polymers.
  • the CTL epitope 147 TYQRTRALV 155 from the nucleoprotein of influenza virus was acryloylated and polymerised with acrylamide, acryloyl-serine and acryloyl-glutamic acid.
  • Four lysines were synthesised onto the N-terminus of the CTL epitope to act as a spacer between the polymer backbone and the peptide, this peptide was also acryloylated and polymerised with the same co- monomers.
  • the CTL peptide polymers were used to prime BALB/c mice and T-cells were isolated from the inguinal and popliteal lymph nodes after 7 days and were restimulated in vitro with virus or CTL peptide. The restimulated T-cells were then used in a CTL assay to evaluated their ability to kill virus infected or uninfected target cells.
  • Figure 15 illustrates that amino acid based polymers containing the CTL or K 4 -CTL determinant are able to prime for a CTL response, whereas the CTL peptide polymerised with acrylamide did not.
  • the CTL response induced by the poly(amino acid/s) CTL is comparable to that induced by the CTL peptide alone and similar to that induced by infectious virus (panel A).
  • the response induced by restimulating the poly(amino acid) CTL T-cells with CTL peptide is weaker than T-cells derived from mice exposed to infectious virus (panel B) they are still able to kill viral targets.
  • T-cells primed with peptide polymer and then restimulated with peptide are able to kill virus infected targets.
  • a surprising result is that acryloyl-Ahx-CTL did not induce a CTL response whereas CTL monomer and acryloyl- AI1X-K 4 CTL did. This may be a reflection of antigen processing events which are able to cleave the acryloyl-Ahx-K 4 from the CTL epitope but not acryloyl-Ahx, which in turn would affect the peptides ability to stimulate a CTL response.
  • ALN and CIO were polymerised with acryloyl-serine, acryloyl-glutamic acid or acrylamide, emulsified with CFA and inoculated into BALB/c mice at 5, 0.5 and 0.05 nmole peptide doses.
  • the primary and secondary antibody response was determined by ELISA ( Figure 16). All of the polymers were able to induce comparably high antibody litres at 5 and 0.5 nmole peptide dose.
  • serine based polymers elicited an equally high antibody titre. Section 4
  • the N-terminal amino acid can be added as the BOC-amino acid derivative to allow final exposure of an amino group at the N-terminus if needed but to prevent its acryloylation.
  • the Mtt protecting group at the C-terminus of the peptide was then removed by treating 3 times with a 1% solution of TFA in dichlorome thane containing 5% triisopropylsilane for 3 minutes at which point additional spacers can then be added to the amino group exposed by this treatment and the acryloyl group finally attached.
  • Homopolymers containing multiple copies of one peptide stitched onto the polymeric backbone were synthesised. Homopolymers for the N- terminal peptides ST156(1-19) and M52(l-19) were constructed. For both peptides, a separate construct was made in. which multiple copies of each peptide were attached to the backbone by both the C-terminal end and the N-terminal end. In addition, one heteropolymer containing both the peptides ST156(1-19) and M52(l-19), attached by the C-terminal end onto the same polymeric backbone were constructed.
  • mice were tested for immunogenicity by immunising B10.BR mice. Mice were given an initial immunisation of construct (30 ⁇ g/mouse) emulsified in complete Freunds adjuvant (CFA) and boosted with the construct in PBS only at 21, 31, 41 and 51 days. Mice were bled at 9, 19, 29, 49 and 59 days after the initial immunisation. At each time point the sera were tested for peptide-specific antibodies using ELISAs. Antisera were also tested for opsonic activity using the indirect bactericidal assay.
  • CFA complete Freunds adjuvant
  • Table 3 summaries the results from sera obtained 59 days after initial immunisation. Antisera from both the ST156(1-19) homopolymer and M52(l-19) homopolymer in which peptides are attached by the N-terminus to the backbone, did not recognise the respective individual peptides with the exception of mouse 2 immunised with the M52(l-19) homopolymer, nor were these sera opsonic for their respective group A streptococci (GAS) strains. However, when peptides are attached by the C-terminal end to the backbone, all mice raised significantly high levels of antibody to their respective peptides (Table 3; Figure 17).
  • mice were immunised with a M52/ST156 heteropolymer, that is both peptides were attached by the C-terminus onto the same backbone.
  • Figure 17 shows that sera from mice immunised with the heteropolymer were able to recognise both individual peptides with titres greater than 40000 by 69 days post initial immunisation.

Abstract

La présente invention concerne des polymères dans lesquels sont incorporés des peptides. Une première réalisation prévoit des polymères comprenant des unités polymérisées de (1) CH2=CR4-CO-X-R1 et de (2) CH2=CR3-CO-R2, et éventuellement d'un ou plusieurs autres monomères. Une seconde réalisation de la présente invention prévoit des polymères provenant de CH2=CR4-CO-X-R1 et éventuellement un ou plusieurs autres monomères. Lorsque X est une séquence d'espacement d'une longueur équivalente à 1 à 30 liaisons C-C simples, R1 est un peptide, chacun des R1 étant identique ou différent, -COR2 étant un ester un amide et/ou leurs dérivés tels que décrits ici. L'invention concerne également des procédés de production de polymères ainsi que des procédés d'induction de réponse immunitaire par utilisation de ces polymères.
EP98901884A 1997-02-11 1998-02-10 Polymeres dans lesquels sont incorpores des peptides Withdrawn EP0961793A4 (fr)

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AUPO5071A AUPO507197A0 (en) 1997-02-11 1997-02-11 Peptide polymers
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CA002217321A CA2217321A1 (fr) 1997-02-11 1997-10-03 Peptides utilises comportant des polymeres
PCT/AU1998/000076 WO1998034968A1 (fr) 1997-02-11 1998-02-10 Polymeres dans lesquels sont incorpores des peptides

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GB9930591D0 (en) * 1999-12-23 2000-02-16 Univ London Component for vaccine
GB9930585D0 (en) * 1999-12-23 2000-02-16 Univ London Component for vaccine
WO2002038596A1 (fr) * 2000-11-08 2002-05-16 Commonwealth Scientific And Industrial Research Organisation Procede servant a identifier des composes antibacteriens
FR2828648A1 (fr) * 2001-08-16 2003-02-21 Adam Bouaziz Constitution d'un vaccin extractible a partir d'un insert immunogene.
US7255867B2 (en) * 2002-11-15 2007-08-14 Id Biomedical Corporation Of Quebec Vaccine
US20110262473A1 (en) * 2008-07-07 2011-10-27 The University Of Melbourne Synthetic vaccine component
ES2333511B1 (es) * 2009-06-15 2011-01-24 Universitat De Girona Derivados polimerizables de peptidos lineales antimicrobianos.
KR102003904B1 (ko) * 2016-08-17 2019-07-25 연세대학교 산학협력단 안정화된 알파-헬릭스 이차 구조를 갖는 펩타이드-고분자 결합체 제조방법 및 이를 이용해 제조된 펩타이드-고분자 결합체

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JP2001513120A (ja) 2001-08-28
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