JP2001513120A - Polymer containing peptide - Google Patents

Abstract

(57) Abstract The present invention provides a polymer-bound peptide comprising a peptide. In a first embodiment, the polymer comprises (1) CH ₂ CRCR ₄ —CO—XR ₁ and (2) CH ₂ CRCR ₃ —CO—R ₂ , and optionally one or more other monomers. Contains polymerized units. In a second embodiment, the present invention provides a _{CH 2 = CR 4 -CO-XR} 1 and polymer formed from any one or more other monomers. Here, X is a spacer having a length equivalent to 1 to 30 CC single bonds, R ₁ is a peptide, each R ₁ is the same or different, and —COR ₂ is an ester or amide. And derivatives thereof as defined herein. The invention further relates to a method for producing said polymer and a method for inducing an immune response using said polymer.

Description

DETAILED DESCRIPTION OF THE INVENTION                          Polymer containing peptide   The present invention relates to a polymer comprising a peptide. This polymer produces an immune response Used for the delivery of peptides, or as a diagnostic tool You can be.   Synthetic peptides are widely used to generate site-specific antibodies, The fact that they have stimulated considerable interest in their assessment as potential You. The advantages of this approach include security. Because infectious substances , And the ability to chemically define the product. Synthetic pep There are several potential advantages in the development of tide-based vaccines However, there are serious problems that generally limit their development. Many of these restrictions are Small size, low copy number and peptide-based immunogen Focuses on poor immunogenicity. These problems have traditionally been Binding of tope to carrier protein (this is a synthetic peptide B cell epithelium) To provide T cell help to the immune system and to provide a multivalent representation of the epitope to the immune system. Work). This method generally involves the use of a peptide as well as a carrier. Before exposing the individual to the carrier protein, while inducing any antibodies specific to The suppression of the B cell response to the peptide may occur.^1,2And also this bond Manipulation can have a deleterious effect on the integrity of peptide determinants^Three.   Total synthetic approach in which peptide epitopes are included in tandem in a linear sequence^4-8 Are also described. This approach depends on the length of the immunogen that can be synthesized Of unrelated immunogenic unrelated sequences at the junction of restricted and distinct determinants It is also limited by the possibility of introduction. To overcome these problems, Tam and And collaborators^9-11Assembled multiple peptides on a branched oligolysine carrier And the present inventors and others^12,13Is not suitable for exposure to trifluoroacetic acid. Peptide is synthesized on a cross-linked acrylamide resin A long single-chain polyamide to which multiple copies of the amide were attached was obtained.   However, for each of these synthetic approaches, the available component pairs The purity of the peptide is limited. Purification of vaccines based on oligomeric synthetic peptides The question of the degree is Rose¹⁴And Tam^15,16Concise chemical ligation described by Has been addressed in several studies, such as the research approach. Also, these The approach also involves, in principle, coupling various purified peptides onto a template carrier. Provides greater flexibility, but is built into these structures The number of different peptide epitopes that can be rare is still limited. Specific MHC T cell and B cell epitopes defined in a single host of the type May be insufficient to elicit immunity in an outbred population Many diseases are caused by organisms whose target antigen is polymorphic. As such, such limitations in the number of different epitopes that can be incorporated are important considerations It is a matter to be done.   As further background to the present invention, Birr et al., PEPTIDES-Chemistry and Biology ( The first of June 16-21, 1991 in Cambridge, Mass., USA Proceedings of the 12th American Peptide Symposium, John A. Smith and Jean E. Riv ier)) "Anti-FeLV Synthetic Peptide Vaccine Development" May also be referred to. In this paper, we include the seven best FeLV epitopes. The construction of synthetic vaccines, including polymer vaccines, has been discussed, N-terminal acryloylation of each epitope, and macromolecules of all seven epitopes But with polyamide copolymerization to a water soluble "brush" type antigen. Epitope Nick et al., 1981, Proc. Acad. Sci. USA 78: 3824. But Specific conditions for polymerization performed in connection with the production of synthetic polymer vaccines. Are not described, and therefore the disclosure in Birr et al. No practicable disclosure could be constructed.   Birr et al.'S polymer vaccine has limited efficacy in antibody response as immunogen Vaccinated in Jung et al., 1985, Int. Ed. Engl. 24: 872 Each of the same seven epitopes and N-palmitoyl-S- [ 2,3-bis- (palmitoyloxy)-(2RS) -propyl]-(R) cysteinyl-serine High titer obtained by the first vaccine derived from N-terminal ligation of Clarke Et al., 1987, Nature 330: 381, the C-terminal of β-galactosidase. Another vaccine produced by recombinant co-expression in E. coli as an end fusion product Specific antibody titers were obtained that were intermediate between the low titers obtained by.   Birr et al. Also noted that the polymer vaccine was compared to the first vaccine. In particular, the effectiveness as a vaccine is substantially less than 30%. And such limited effectiveness would not be commercially acceptable.   N-terminal acryloylation of each epitope followed by polyamide copolymerization The polymer vaccine reported in Birr et al. Is clearly not effective This means that the position of each peptide and polymer is applicable to the polymerization process. As a result of the fact that physical constraints were not taken into account in the polymerization process It is thought to get.   Approval developed by the present inventors, such as chemical ligation operation Chi is also "modular", synthesizing and purifying the peptide, and then It is possible to assemble into a polymer. However, in this case, very Large (> 600,000 daltons) species can contain virtually any number of identical or different It can be assembled with a pitope. This technique is based on certain aspects of the peptide sequence. Depending on the acylation of the amino group in the Royl (CH_Two= CH-CO-) group still attached to the solid support. Polymer The orientation of the peptide in the gel is determined, for example, by the method of Silversides et al. ormone releasing hormone vaccine, I. Conjugation and specificity trials in BALB / c mice 、 Silversides-DW; Allen-AF; Misra-V; Qualtiere-L; Mapletoft-RJ Murphy-BD. J-Reprod-Immunol. August 1988; 13 (3): 249-51). Can affect the immune response obtained. Therefore, in some cases Is attached to the polymer backbone at some other point in the sequence other than its C-terminal or N-terminal. It may be necessary to have a combined peptide epitope. For example, 9-fluo Renylmethoxycarbonyl (Fmoc), Mtt (4-methyltrityl) and Dde (4,4- Dimethyl-2,6-dioxocyclohex-1-ylideneethyl) The effectiveness of lysine derivatives with a protecting group is that the acryloyl group Orthogonal chemistry so that it can be introduced at one or more specific points l chemistries) to selectively remove specific amino groups by removing their protecting groups. Allows for exposure. After cleavage, deprotection, and purification, the peptide Chain elongation in the same way that luamide is routinely polymerized onto polyacrylamide gels. It can be polymerized by a free radical initiator. In this method, the peptide is Peptides assemble into polymers forming side-chain pendants from the alkane backbone (Figure 1). This method allows for the purification of individual determinants, Errors inherent in continuous synthesis of Eckstein¹⁷Approach described by In contrast, the use of protected peptide fragments avoids solubility and purification problems It has the advantage of being done. In addition, many different peptide epitopes Copies can be incorporated into a single polymer structure, and different pathogenic serum dome (s erodemes) and T-cell epitopes required for outcrossing populations Ranges are available. This approach represents a significant advance in synthetic vaccine technology. You will see that it is a step.   Therefore, in the first aspect, the present invention provides (1) CH_Two= CR_Four-CO-X-R₁And And (2) CH_Two= CR_Three-CO-R_Two [Wherein X is absent or a space having a length equivalent to 1 to 30 CC single bond] Sir,   R₁Is a peptide and each R₁Are the same or different,   R_TwoIs NH_Two,   Ｕwhere U is O or NH,     f is an integer of 0 to 17,     Y is H, COOH, CO-NH_TwoAnd     W is H, CH_Three, CH_TwoCO-NH_Two, CH_TwoCOOH, CH_TwoOH, CH (CH_Three) OH, CH_TwoSH, CH_TwoCH_TwoCOOH , CH_TwoCH_TwoCO-NH_Two, CH_TwoCH (CH_Three)_Two, CH (CH_Three) CH_TwoCH_Three, (CH_Two)_FourNH_Two, CH_TwoCH_TwoSCH_Three, CH (CH_Three )_Two,And     Z is H, (CH_Two)_hQT (where h is 0-10, Q is O, NH, CH_TwoAnd T is a lipid, a labeling molecule, a target molecule or a functional group). And   R_ThreeIs CH_Three, H, NH_Two, OH, CN or halogen, each R_ThreeAre the same or different Things,   R_FourIs CH_Three, H, NH_Two, OH, CN or halogen, each R_FourAre the same or different And   The ratio of (1) :( 2) ranges from about 1: 1 to about 1: 1000] And optionally a polymer comprising polymerized units of one or more other monomers.   In a second aspect, the present invention provides_Two= CR_Four-CO-X-R₁ (Wherein X is a spacer having a length equivalent to 1 to 30 CC single bonds,   R₁Is a peptide and each R₁Are the same or different,   R_FourIs CH_Three, H, NH_Two, OH, CN or halogen, each R_FourAre the same or different Is) And optionally a polymer formed from one or more other monomers.   In a third aspect, the present invention provides a method comprising: (i) monomer (1) CH_Two= CR_Four-CO-X-R₁You And (2) CH_Two= CR_Three-CO-R_Two [Wherein X is absent or a space having a length equivalent to 1 to 30 CC single bond] Sir,   R₁Is a peptide and each R₁Are the same or different,   R_TwoIs NH_Two,  Ｕwhere U is O or NH,     f is an integer of 0 to 17,     Y is H, COOH, CO-NH_TwoAnd     W is H, CH_Three, CH_TwoCO-NH_Two, CH_TwoCOOH, CH_TwoOH, CH (CH_Three) OH, CH_TwoSH, CH_TwoCH_TwoCOOH , CH_TwoCH_TwoCO-NH_Two, CH_TwoCH (CH_Three)_Two, CH (CH_Three) CH_TwoCH_Three, (CH_Two)_FourNH_Two, CH_TwoCH_TwoSCH_Three, CH (CH_Three )_Two, And     Z is H, (CH_Two)_hQT (where h is 0-10, Q is O, NH, CH_TwoAnd T is a lipid, a labeling molecule, a target molecule or a functional group). And   R_ThreeIs CH_Three, H, NH_Two, OH, CN or halogen, each R_ThreeAre the same or different Things,   R_FourIs CH_Three, H, NH_Two, OH, CN or halogen, each R_FourAre the same or different Is) And optionally preparing one or more other monomers; and (Ii) polymerizing the monomer in the presence of a free radical initiator Having a polymer main chain and a plurality of pendant peptides bonded to the main chain. In the preparation of peptide polymers.   In a fourth aspect, the present invention provides an acceptable carrier, and (1) CH_Two= CR_Four- CO-X-R₁And (2) CH_Two= CR_Three-CO-R_Two [Wherein X is absent or a space having a length equivalent to 1 to 30 CC single bond] Sir,   R₁Is a peptide and each R₁Are the same or different and R₁At least one of One is a peptide epitope,   R_TwoIs NH_Two,   Ｕwhere U is O or NH,     f is an integer of 0 to 17,     Y is H, COOH, CO-NH_TwoAnd     W is H, CH_Three, CH_TwoCO-NH_Two, CH_TwoCOOH, CH_TwoOH, CH (CH_Three) OH, CH_TwoSH, CH_TwoCH_TwoCOOH , CH_TwoCH_TwoCO-NH_Two, CH_TwoCH (CH_Three)_Two, CH (CH_Three) CH_TwoCH_Three, (CH_Two)_FourNH_Two, CH_TwoCH_TwoSCH_Three, CH (CH_Three )_Two, And     Z is H, (CH_Two)_hQT (where h is 0-10, Q is O, NH, CH_TwoAnd T is a lipid, a labeling molecule, a target molecule or a functional group). And   R_ThreeIs CH_Three, H, NH_Two, OH, CN or halogen, each R_ThreeAre the same or different And   R_FourIs CH_Three, H, NH_Two, OH, CN or halogen, each R_FourAre the same or different And   The ratio of (1) :( 2) ranges from about 1: 1 to about 1: 1000] And optionally a polymer comprising polymerized units of one or more other monomers Immunizing a peptide epitope in an animal, comprising administering to the animal Lies in the way to generate a response.   Polymers are typically random, however, in blocks or alternating possible.   Also, the polymer may be cross-linked. This is for bisacrylamide It can be obtained with any suitable crosslinking agent. The crosslinking agent has the general formula CH_Two= CH-CO-R_Five-CO-CH = C H_TwoR where R_FiveIs the amino acid sequence NH (CH_Two)_TwoSS (CH_Two)_TwoNH or NH (CH_Two)_kNH (k is 1 to 10).   As will be readily appreciated, polymers typically have more than one R₁including. Peptide R₁Is Epitopes, this multiple R₁The group is a T cell and / or B cell epitope Preferably, it provides a mixture.   The second element in the polymer is essentially R₁Acts as a spacer between groups Is recognized. This spacer is made of acrylamide (R_Two= NH_Two) However, the spacer may be an acryloylated amino acid, especially serine or glutamate. It is preferably formed from an acid. Also, different spacers are Can be used (R_TwoWill be understood). In other embodiments In addition, R_TwoCan contain lipid components or can be used May be included. For example, R_TwoIs palmitic acid or Nα-palmi Toyl-S- [2,3-bis (palmitoyloxy)-(2RS) -propyl] -L-cysteine Such lipids may be included. As will be appreciated, this is a self-adjuvant It is one way in which self-adjuvanting properties can be added. Also examples For example, fluorescamine, a metal ion chelator for chelating radioactive metal ions R such as molecule_TwoMay contain a molecule having a labeling function. Target Molecules with cut-off properties, e.g._Two Can be included. Molecules that alter physicochemical properties such as polymer solubility and viscosity R_TwoWill be readily recognized.   In one aspect, the spacer group X is present, but its presence is optional. . At present, the polymer is a peptide R₁Are spaced from the polymer backbone It is preferable to include such a spacer. Spacer group X can be cleaved enzymatically It preferably contains a functional site. This is R₁Is a peptide epitope Particularly preferred. Examples of cleavable sites include the peptides GLFG and VYLKY. No.   The ratio of (1) :( 2) is preferably in the range of 1:10 to 1:50.   Most of the discussions and examples in this application are based on one or more R₁Is Peptide For polymers that are pitopes, but one or more R₁Is a peptide It will be appreciated that it is not essential that it be an epitope. The present invention Is one or more R₁Is a biologically active peptide such as a hormone It is intended to be a mer. For example, the polymers of the present invention have a range of biological activities. A vehicle useful for delivery of the peptide may be provided. To adjust the spacer X Such an arrangement releases the peptide for an extended period of time It may be possible to produce a polymer. R_TwoTo give a crosslinkable part to , It is also possible to crosslink a plurality of the polymers of the present invention. This is R₁Late It may be advantageous in providing an extended release.   As will be appreciated, the present invention relates to a polymer backbone and a plurality of pairs attached to the backbone. A method for preparing a polymer immunogen having a dant peptide is provided. In general, The method of the present invention comprises: (I) optionally adding a lateral spacer to each peptide, (Ii) acryloylating each peptide with an acryloylating agent, and (Iii) each acryloylated peptide, and optionally a linear spacer, Polymerizing in the presence of a radical initiator to prepare the required polymer immunogen. The linear spacers separate the peptide sequences from each other along the polymer backbone. Wherein the side spacers distance each peptide from the polymer backbone. And place them.   The peptide of step (i) may be a natural protein extracted, natural, synthetic or recombinant. Chemical cleavage of proteins, oligopeptides or naturally occurring small proteins It can be obtained from any suitable source that can include recombinant expression. However, preferred Alternatively, each peptide can be synthesized manually or synthesized using an automated peptide synthesizer. Is done.   The peptide of step (i) was edited, for example, by Nicholson and Title "Synthetic Vaccines" published by cientific Publications Entitled "Peptide Synthesis" by Atherton and Shepard Can be synthesized using solution synthesis or solid phase synthesis, as described in Chapter 9 of the Name . Preferably, a solid support, which can be polystyrene gel beads, is utilized, wherein The polystyrene can be cross-linked with a small amount of divinylbenzene (eg, 1%). Lipophilic solvents such as dichloromethane or dimethylformamide ( Further swollen with more polar solvents such as DMF). Polystyrene is It can be functionalized with a chloromethyl or aminomethyl group. On the other hand, highly solvated Crosslinked, which can be swollen with DMF and other polar aprotic solvents, A functionalized polydimethyl-acrylamide gel is used. Other carriers Are usually grafted or bonded to the surface of inert polystyrene beads. It can be utilized on the basis of polyethylene glycol. In a preferred embodiment, PAL- Various commercially available solid carriers or resins such as PEG, PAK-PEG, KA, KR or TGR can be used. Can be used.   In solid-phase synthesis, mask the reactivity of α-amino groups and side chain functional groups A removable protecting group is used.   In addition, each peptide may optionally contain a carbohydrate, lipid, nucleotide or nucleotide. It will also be appreciated from the foregoing that it may have additional components selected from oside. Would. Particularly preferred are oligosaccharides, oligonucleotides, or glycoside groups. is there.   Regarding step (ii), acryloyl chloride, cyanoacryloyl chloride, Suitable acrylates such as acryloyl or esters of acrylic or methacrylic acid Chryloylating agents can be used.   After step (ii), the peptide can be cleaved from the resin and the protecting group is It can be removed by continuous use of the appropriate cleavage agent or cleavage reagent. Preferably off The blocking agent does not contain a thiol group. Because the thiol group reacts with the double bond Because you get it.   After removal of the resin, the peptide is recovered, for example, by reversed phase, ion exchange or It can be purified by any suitable method, such as by chromatography.   With respect to step (iii), the polymerization of the acryloylated peptide may be performed in any suitable manner. Can occur. Solutions of acryloylated peptides are free of very low concentrations of free radicals. Can be treated with a reagent capable of generating In aqueous solutions, traces of hydrogen peroxide and Trace persulfuric acid containing ferrous salts and ferrous salts or N, N, N ', N'-tetramethylenediamine Ammonium can be used. Therefore, the peptide is a vendor from a linear backbone. Be an event. Each peptide has either a carboxyl or carboxamide group In the natural state of the particular epitope, i.e., Either form the C-terminus of the protein or show the internal sequence of amino acids Can be imitated.   In the case of copolymerization of two or more acryloyl peptides, the peptide along the polymer backbone The distribution of the compounds is controlled by adjusting the stoichiometry of the component peptides in the polymerization mixture. To some extent. Such an arrangement allows for the distribution of the parent protein. If different remote peptide presentation regions are attached to the backbone in a Even simulations of protein conformational determinants may be possible.   Various, such as N, N′-dimethylacrylamide or acryloylated amino acids Copolymerization of an acryloyl peptide with an amount of an appropriately functionalized reagent Dant peptides are separated from each other to a greater degree or better along the polymer backbone. Can be separated to a smaller extent. This arrangement consists of two adjacent pep Required in some cases where steric hindrance or accidental interaction between tides occurs You will see that Also, the solubility of the resulting polymer peptide is desired Can be increased or decreased.   For the polymerization of various peptides, this is the acryloyl derivation of the first peptide. And a methacryloyl derivative of the second peptide and the two peptides are then prepared. It can be caused by polymerization. Two peptides along the main chain of the resulting polymer The sequence order of the first and second peptides can be expected to be more random sequences. Is significantly different from the copolymerization of acryloylated derivatives of the peptide (And could be more regular).   Small amounts of low molecular weight acrylamides or esters selected for specific characteristics Can also be added to the monomer mixture before the polymerization step. Such additional monomers Acryloyl compounds with a radiolabel attached, or fluorescent Or an acryloyl derivative of a chemiluminescent reagent. Then the resulting poly Is a polymer in a separate chemical side chain from the peptide epitope itself. It will include the possibility for subsequent easy detection. This feature is Particularly convenient during the development phase.   The assembly of appropriate combinations of B-cell and T-cell epitopes Allows the production of antibodies and T cells in a wide variety of histocompatible types. This This can be any vaccine administered to an outbred population, such as a human or its human livestock. Very important in chin design. Carefully selected for polymerization mixture Assembly of peptides and lipid-containing materials as linear or side spacers Eliminate the need for adjuvants by incorporating other reagents such as Is also possible.   "Slow release" forms of these peptide polymers are derived from peptide acryloyl derivatives Obtained by the preparation and polymerization in the presence of a cross-linking reagent such as bisacrylamide It can be possible. In addition, these polymers have a very high local The fact that it exhibits an effective concentration of active monomer units, May also be shown. Currently insulin, gastrin, oxytocin, vasopressin , Adrenocorticotropic hormone (ACTH), growth hormone, cholecystokinin, bon Besin, substance P, relaxin, ensephalin, angiotensin, Many peptide hormones are known, such as matostatin, bradykinin. this These molecules and many variants are particularly useful for agonist and antagonist analogs. Investigation, and further investigation of modified peptides with improved bioselectivity and stability Has been synthesized and tested. For example, "poly-vasopressin" or Analogs are extremely effective at blocking cell surface natural vasopressin receptors. Can be efficient.   Also, high molecular weight polymers, especially those polymers make it insoluble Hormone or other pharmacological effects in experimental animals may be Restriction to a particular site or organ may also be feasible.   Immobilization of a bioactive peptide on a polymeric carrier has been studied, e.g., for the receptor molecule. There are already many known uses in isolation, antibody purification, and immunochemical analysis. Have. Polyacrylic cross-linked with specific peptide side chains as described herein Luamide provides a similar function.   In order that the nature of the invention may be more clearly understood, its preferred embodiments are described in the following non-limiting examples. A non-limiting example is described. Description of the drawings   Figure 1: Scheme for the production of polymers based on synthetic peptides. Peptide Assembled in the usual way on a solid support, then with acryloyl chloride N-terminal acylation. The peptide is removed from this support, while simultaneously protecting the side chain After removal, the peptide epitope is purified and exposed to free radicals. Polymerizes.   Figure 2: A Vydac C18 column (4.6 x 300 mm) attached to a Waters HPLC system Unpurified H used_TwoAnalysis of N-P8 [A] and unpurified N-acryloyl P8 [B] Chromatogram. The chromatogram is a 0-100% solution that evolves over 30 minutes. It was developed using a gradient of medium B at a flow rate of 1 mL / min.   Figure 3: N-Acryloyl-GFGA¹1 H NMR. Use reagent B for N-acryloylated peptide And cleaved from the resin, purified by RP-HPLC, and then¹Measured by 1 H NMR. A Characteristic chemical shift of the cryloyl group, It is shown.   Figure 4: Gel permeation chromatography of monomeric and polymeric peptides. CH_Two= CHCO-P6 [―] and NH_Two-P6 […] was added to the polymerization protocol, Introduced into ram [1x30cm], 50mM NH_FourHCO_ThreeMedium, chromatogram at 0.5 mL / min flow rate Was expanded. Monitor the column effluent at 280 nm and tryptophan and chitin. Rosin residues were detected. Arrow indicates retention time of molecular weight standard; thyroglobulin 66 9 kDa [A], bovine serum albumin 67 kDa [B] and ribonuclease 13.7 kDa [C].   Figure 5: Antigen binding of peptides and peptide polymers. Polymerized P8 of anti-P8 antiserum ■], binding to P8 monomer [□] or polyacrylamide [△], and MAb1 / 1 Binds to polymerized P5 [●], P5 monomer [○] or polyacrylamide [▽] by ELIS Measured by A.   Figure 6: The ability of soluble monomers and polymer peptides to inhibit antigen-antibody binding. Pepti A dilution of the peptide or peptide polymer is mixed with a fixed amount of antibody and covered with peptide antigen. Added to the wells of a covered microtiter plate. Antibody binding is measured by ELISA And the results are expressed as the percentage of antibody bound in the absence of inhibitor. Was. [A] The P2 monomer [◇], polymerized P2 [△], or polymerized P4 [○] are mixed with MAb1 / 1 Binding was assessed in wells cubated and coated with P5. [B] P8 monomer [◇], heavy P8 [△] or polymerized P4 [●] is incubated with anti-P8 antiserum and coated with P8 Was evaluated on the wells.   Figure 7: From a Superose6 column [1x30cm] attached to a Waters HPLC system Elution profile of poly [T + B1 + B2]. Chromatogram is 50 mM ammonium bicarbonate The medium was developed at a flow rate of 0.5 ml / min. The polymer is the void volume of the column [approximately 7.5 ml] And the monomeric peptide eluted at the position indicated by the arrow. Column inspection Quantitative curves were generated using the indicated standard proteins.   Figure 8: Effect of free radicals on the antigenicity of synthetic peptides. MAbs 1/1 and 2 / 1 with their untreated peptides 306PKYVKQNTLKLATGMRNVPEKQT328 [●] and various The ability to bind peptides exposed to concentrations of ammonium persulfate was measured. The molar ratio of ammonium persulfate to peptide was 0.1: 1, [◇], 1: 1, [○], 1 The level of binding to each of the preparations [■] is also shown.   Figure 9: Antigen binding of polymer peptide. Peptide 306 PK for MAb 1/1 and MAb 2/1 Binding with YVKQNTLKLATGMRNVPEKQT328 [●], and poly T [□], poly T + B1 [◇], Pepti contained in the polymer of T + B2 [○] and poly T + B1 + B2 [△] Binding to the epitope was measured by ELISA. In addition, various types of normal mouse serum Was also measured [ペ プ チ ド].   Figure 10: Antigen binding of peptide polymer as measured by T cell proliferative activity. mono For mer peptide T [□], poly T + B1 [◇], poly T + B2 [●] or poly T + B1 + B2 [△] Proliferation of lymphocyte T cells [A] or T cell clone 12V1 [B] Answer.   Figure 11: Comparison of immunoreactivity of monomer and polymer peptides. Peptide monomer In response to primary and secondary antibody responses to [open symbols] and peptide polymer [black symbols] The titer of the prepared antiserum was measured by ELISA. Each symbol is an individual animal , And the average antibody titer of each group is indicated by a solid line.   Figure 12: Specificity of generated antisera for monomeric and polymeric peptides. one Antiserum elicited by poly T + B1 + B2 in secondary [△] and secondary [▲] antibody responses The ability of the to bind to analog A and analog B was measured in an ELISA assay. MAb  Binding to analogs of 1/1 [MA], MAb 2/1 [●] and NMS [■] is also shown.   FIG. 13: Profile of isotype antibody elicited by polymer peptide. Poly T + Primary [white bar] and secondary [black bar] for B1, poly T + B2, and poly T + B1 + B2 ] The isotype immunoglobulin obtained in the antibody response was analyzed using isoform-specific reagents. Measured by LISA.   Figure 14: Incorporation of various cathepsin-sensitive sequences and serine, glutamic acid, etc. Or PKYVKQNT helper epitope copolymerized with either acrylamide Expansion of T cell clone 4.51 in response to LKLA. Panel A: The following acryloylated pep Pide sequence: VYLKY-PKYVKQNTLKLA [●] copolymerized with acryloylserine, acrylo GFLG-PKYVKQNTLKLA [◆] copolymerized with ilserin, copolymerized with acrylamide PKYVKQNTLKLA [▲], GFLG- copolymerized with acrylamide PKYVKQNTLKLA [▼], monomer peptide Proliferation induced by the polymer of PKYVKQNTLKLATGMRNVPEKQT [□]. Panel B: Access VYLKY-PKYVKQNYLKLA [○] copolymerized with Liloylglutamic acid, acryloyl Copolymerized with glutamic acid GFLG-PKYVKQNTLKLA [◇], copolymerized with acrylamide Proliferation induced by PKYVKQNTLKLATGMRNVPEKQT [□].   Figure 15: Virus and peptide-pulsed markers of polymer-sensitized cells T-lymphocyte activity on target. BALB / c mice have polyhedral By subcutaneous inoculation (containing 10 μg of peptide) Fluenza virus A / Memphis / 1/71 [1x10^4.5pfu] was intranasally inoculated. After inoculation On days 7 and 21, the lymph nodes and spleen cells of the peptide-sensitized mice were sensitized to the virus. Removed from stained mice and infected with influenza virus in vitro Splenocytes or CTL epitope TYQRTRALV at 100 μg / mL for 5 days Restimulation was used in volume. Next, [■] virus and [◆] acryloylserine Acryloyl TYQRTRALV combined, [●] Acryloyl KKKTYQRTR copolymerized with serine ALV, [▲] Acryloyl TYQRTRALV copolymerized with acryloylglutamic acid, [▼] Acryloyl KKKTYQRTRALV copolymerized with acryloylglutamic acid, or [*] Fine cells obtained from animals sensitized with acryloyl TYQRTRALV copolymerized with acrylamide. The CTL activity of the vesicles can be measured by virus-infected target (black symbol) or non-infected target (white symbol) In a 51Cr release assay using Panel A: Virus in vitro 51Cr released by CTL activity of restimulated cells. Panel B: TYQR in vitro 51Cr released by CTL activity of cells restimulated with TRALV peptide.   Figure 16: Caused by different doses of peptide polymer and different backbone compositions Antibody response. BALB / c mice are acrylamide, or serine or glue Copolymers of the tamnic acid based peptides GMRNVPEKQT and ALNNRFQIKGVELKS Some were inoculated at a dose of 5, 0.5 or 0.05 nanomolar peptide. 30 after primary inoculation Day and 12 days after the second inoculation, antiserum was collected and the peptide PKYVKQNTLKLATGMRNVPEKQT Antibody titers were determined by ELISA on plates coated with. Antibody titer is 0.25 single It is expressed as the titer obtained at the optical density of the order. once The titers obtained after inoculation [open symbols] and secondary inoculation [black symbols] are shown. Bars in each group Represents the average titer of antiserum.   Figure 17: Immunogenicity of ST156 / M52 heteropolymer. [A] Hete against peptide ST156 [B] Heteropolymer antiserum against peptide M52, [C] ST156 Mopolymer antiserum: Recognition of peptide ST156, [D] M52 homopolymer antiserum: Pepti Recognition of de M52. Each row represents the results for one mouse. Day 0 of first immunization, one Day 20 of secondary antigen stimulation, Day 30 of secondary antigen stimulation.   Figure 18: Antibodies generated against fully heteropolymer. Each row contains the results for one mouse. Represents a fruit. Example Section 1 Materials and methods   Unless otherwise noted, chemicals are of peptide synthesis grade or Or the equivalent. O'benzotriazole-N, N, N ', N'-tetramethyl Ru-uronium-hexafluorophosphate (HBTU), 1-hydroxybenzotria Zole (HOBt), diisopropylethylamine (DIPEA), N, N-dimethylformamide (DMF), piperidine, trifluoroacetic acid (TFA), and 9-fluorenylmethoxy Amino acids protected with carbonyl (Fmoc) were obtained from Auspep Pty (Melbourne, Australia) Obtained from. Thioanisole, anisole, ethanedithiol (EDT), torii Sopropylsilane (TIPS) and acryloyl chloride are available from Aldrich (New South Wales , Australia). 1,8-Diazabicyclic- [5.4.0] undec-7-ene (DBU) Acrylamide, obtained from Sigma Chemical Company (New South Wales, Australia) Ammonium persulfate, TEMED and Tris (hydroxymethyl) aminomethane (Tris ) Was obtained from BioRad (Richmond, CA). Phenol, dichloromethane (DCM) and And diethyl ether were obtained from BDH (Poole, UK). Solid phase peptide synthesis   Peptides can be prepared manually or on an automated peptide synthesizer (Novasyn Crystal, Novabioc). hem, U.K, or 9050 Plus PepSynthesiser (Milligan, Millford, MA ). Standard solid phase peptide synthesis protocol for Fmoc chemistry Was used throughout. Novasyn KA 100 or KR 100 resin respectively (Calbiochem- Novabiochem, New South Wales, Australia) using C-terminal carboxy Incorporated as xyl or carboxamide form. 4 equivalents of amino acids and 6 Coupling was performed by activation of HBTU / HOBt using an equivalent amount of DIPEA. 2 in DMF Fmoc groups were removed with 0% piperidine or 2.5% DBU in DMF. Peptide resin support Cleavage from reagent B (88) for 2 or 4 hours depending on the arginine content of the peptide. % TFA, 5% phenol, 5% water, 2% TIPS). After cutting, resin by filtration Was removed and the filtrate was concentrated to approximately 1 mL under a stream of nitrogen. Cool the peptide product After precipitating in the water, they were centrifuged and washed three times. Then pepti Dissolve the precipitate in 5-10 mL of water containing 0.1% v / v TFA and centrifuge the insoluble residue. Therefore, it was removed. N-acryloylation of peptides   The resin holding the peptide is swollen in a minimum amount of degassed anhydrous DMF and nitrogen Acryloylated below. After cooling on ice, add this resin to 20x in 0.5 ml DMF Add a molar excess of DIPEA and a 10-fold molar excess of acryloyl chloride in 0.5 ml DMF. Added. The mixture was stirred on ice for 1 hour and then at room temperature for another hour. Aku The progress of reloylation is monitored by the trinitrobenzenesulfonic acid (TNBSA) test. Taling. The resin was washed when the TNBSA test returned negative (5 times in DMF, DC M 3 times and diethyl ether 3 times). The resin was then vacuum dried .   Peptides P4 and P6 have Fmoc-Lys (Mtt) -OH (Calbioc hem-Novabiochem, New South Wales, Australia) and also at the N-terminus of P4 and P6. Boc-Pro-OH and Boc-Asp (OtBu) -OH were coupled. After peptide binding, the Mtt group is changed to D Removed using 1% TFA containing 5% TIPS in CM, then Fmo using HBTU activation c-Ahx was coupled to a free ε-amino group. Fmoc group using 2.5% DBU in DMF Was removed from Ahx and the exposed amino groups were acryloylated as described above. Purification of peptide monomers   Purification of the synthesized peptide was performed using Fast Protein Liquid Chromatography (FPLC) system. Pharma attached in the tem (Amrad-Pharmacia Pty. Ltd., Victoria, Australia) This was performed using a cia C18 Pep RPC column (1.6 × 10 cm). Underwater as restriction buffer With 0.1% TFA (solvent A) and 0.1% TFA in acetonitrile (solvent B) at a flow rate of 4 mL / min Developed the chromatogram. All peptides are formed over 40 minutes Eluted with a linear gradient of 10-30% solvent B. Installed in Waters HPLC system Analytical HPLC was performed using a Vydac C18 column (4.6 × 300 mm). Solvent A and solvent B Chromatogram is developed at a flow rate of 1 mL / min using a 0 to 100% linear gradient of solvent B for 30 minutes. Was formed. Analyze the material eluted from the column at 214 nm or 280 nm. It was detected by measuring.   N-acryloyl peptide¹1 H NMR spectra were obtained on a Bruker-AM300 (in DMSO-d6). Recording was performed at 300 MHz on a spectrometer. FAB mass spectrum is the first ion energy of FAB The JOEL LMS-DX300 spectrometer was operated at an acceleration voltage of 3 keV using 6 keV and an emission current of 20 A. Recorded using. Xenon was used as the shock gas. Scan area is 100-150 It was 0 m / z. Copolymerization of acryloyl peptide with acrylamide   The general conditions for all polymerizations are as follows: 1:50 molar ratio of acryloy Peptide and comonomer acrylamide (5% w / v final concentration) with 2 mM EDTA and And mixed in degassed 6M guanidine-HCl (GuHCl) in 0.5M Tris (pH 8.3). Existence Ammonium persulfate at a concentration of 2% of the molar concentration of acrylamide present, and 5 μl The polymerization was initiated by adding a 20% v / v solution of TEMED. At room temperature under nitrogen The polymerization was allowed to proceed for 18 hours. Purification and partial characterization of peptide polymers   Gel immersion using a Superdex 200 column (1.6 x 60 cm) mounted in an FPLC system The peptide polymer was isolated by permeation chromatography (GPC). Chromatog Raffy is 50mM NH_FourHCO_ThreeMedium and at a flow rate of 3 mL / min. All polymers have void volume Eluted in. The peptide polymer thus isolated is then freeze-dried. Dried.   Amino acid composition of peptide monomer and polymer Confirmed by. Hydrolyze the peptide material (0.001% in 6N HCl for 24 hours at 110 ° C. w / v phenol), and the hydrolyzate was derivatized with Fmoc-Cl. G with fluorometric detection Amino acid analysis was performed using the BC Aminomate system. Enzyme-linked immunosorbent assay (ELISA)   As described^{twenty three}, Flat bottom polyvinyl made microtiter plate (Microtiter , Dynatech Laboratories, VA., U.S.A.) Alternatively, ELISA was performed using a peptide polymer solution (5 μg / mL). Then bound anti The body was transferred to horseradish peroxidase-conjugated (HRPO) rabbits directed against mouse Ig. HRPO directed against goat immunoglobulin (Ig) (DAKO, Denmark) or sheep Ig Detection by incubation for 1.5 hours with donkey Ig (DAKO, Denmark) did. After washing, the substrate (0.2 in 50 mM citric acid pH 4.0 containing 0.004% v / v hydrogen peroxide). mM 2,2'-azino-bis 3-ethylbenzothiazoline-sulfonic acid). After that, the optical density (O.D.) at 405 nm was measured using Titertek Multiskan MC (Flow Laboratories , Melbourne, Australia).   1/1500 dilution of MAb 1/1 or a known concentration of inhibitor (soluble peptide or 2 hour inhibition ELISA using anti-PS antiserum incubated with polymer) And then a flat-bottom polyvinyl microtiter plate coated with P5 or P8 Moved to After overnight incubation, wash the plates and perform ELIS as described above. Expanded A. antibody   Preparation and properties of monoclonal antibody MAb 1/1 are elsewhere¹⁹It is described in. MAb  1/1 is the hemagglutinin heavy chain (HA) of influenza virus A / Memphis / 1/71 (H3)₁ ) C-terminal 24 residues (³⁰⁵CPKYVKQNTLKLATGMRNVPEKQT³²⁸) Is made for the synthetic peptide Cell determinant RNVPEKQT²⁰Is specific to Hyperimmune serum (HIS) is luteal in sheep Made against adult hormone releasing hormone (LH-RH; P8). Synthesis of acryloyl peptide   Peptide sequences varying in residue length from 4 to 23 (Table 1) were analyzed using Fmoc chemistry. Assembled by solid phase peptide synthesis. Generally, 6-aminohexanoic acid (Ahx) Introduced as the final "residue" and then acylated at the N-terminus by acryloylation did. Thus, Ahx is a spacer that separates the acryloyl group from the peptide. Function as Table 1: Amino acid sequences of the peptides used ^a  One letter notation^b   All but P2, P5 and P7 synthesized with C-terminal free-COOH The peptides were assembled as carboxamides.^c   Luteinizing hormone-releasing hormone   Three techniques for acryloylating resin-bound peptides were evaluated . Symmetric anhydride pathway¹⁷Or O-benzotriazole-N, N, N ', N'-tetramethyl- Uronium-hexafluorophosphate and N-hydroxybenzotriazolate Two methods involving the coupling of acrylic acid to the N-terminus by either The method yielded only low yields of pure N-acryloyl peptide. But However, consistent high yields of pure N-acryloyl peptide are Obtained by acylating the amino group with acryloyl chloride (Scheme 1 ).   As can be seen from FIG. 2, the acryloylation of the peptide resulted in reversed-phase chromatography. Retention compared to non-acryloylated peptide when measured in fee A long material in between is obtained. By using reagent R (peptide NH_Two-Gly-Phe-Gly-Ala is , Acryloyl chloride with acryloyl chloride, then the reagent R (TFA: thioanisole: Anisole: FAB mass spectrum when cut with ethanedithiol 90: 5: 3: 2) The m / z value is 124 daltons greater than predicted by the rhometry, and the result is Thioanisole is added to the double bond). Reagent B³⁶Cleavage of the N-acryloyl peptide from the resin support And for side chain deprotection. Unpurified acryloylated peptide was obtained by reversed-phase chromatography. Purification by luffy, which provided a putative amino acid analysis. Acryloyl Typical of the group¹H NMR resonances were obtained with various N-acryloylated peptides . Shows a typical spectrum for the model peptide N-acryloyl GFGA (P16) (Figure 3).   Most peptides are acryloylated at the N-terminus, whereas peptides P4 and P6 It was synthesized to have an acryloyl group at their C-terminus. This means that the C-terminal position For acryloylation of ε-amino group of lysine introduced as Fmoc-Lys- (Mtt) -COOH Thus achieved. Synthesize N- or C-terminally acryloylated peptides The ability takes into account the reversed peptide within the polymer. Polymerization of synthetic peptides   A simple prototype similar to that used in the routine preparation of polyacrylamide gels Cole (Scheme 1) was converted to a linear polyacrylamide holding a pendant peptide. Applied to assemble the code. From this method, high molecular weight water-soluble peptide poly You get the mer. Polymerization of short (tetra or penta) peptides follows a similar methodology Has been reported^17,34, In those studies disadvantageous to biological activity Potentially high temperatures are used, which can lead to potential solubility problems Side-chain protecting groups remain in place during the polymerization process. Polyacrylic acid that retains sialic acid groups Mid's assembly has also been reported³⁵.   Solvents such as 1.5 M Tris-HCl (pH 8.8) and 8 M urea have also been used successfully, The coupling reaction is usually 6 M guanidine-HCl (GuHCl) and 2 mM EDTA in 0.5 M Tris degassed. (pH 8.3). Minimize steric interactions between peptide chains and reduce Stretch polyacrylamide to maximize the water solubility of the 50 times the amount of acryloylated peptide to intersperse between loyl peptide units A molar excess of acrylamide was introduced. Peptides P2, P9, P10 and Ahx And P13 polymerized well without the addition of acrylamide. It was not always necessary. Ammonium persulfate and N, N, N ', N'-tetra The polymerization was initiated by the addition of methylethylenediamine (TEMED) and the reaction mixture was_Two It was left at room temperature for 18 hours. Analysis of the polymerization process over time showed that some peptides Peptide polymer with evidence that it is incorporated into the polymer within minutes. Uptake was shown to be rapid. FIG._Two-CH instead of P8_Two = Indicates high molecular weight peptide-containing substance formed when CHCO-P8 was subjected to polymerization conditions ing. Each test peptide polymer is approximately 4 × 10⁷Da exclusion Eluted into the void volume of a Superose 6 column (1 × 30 cm) having a molecular weight. Polymer According to the amino acid analysis of the acryloylated monomer peptide, 80-97% It was shown to be incorporated during the limer. This data also shows that different peptides It may be incorporated into the same polymer structure, and when copolymerized in equimolar amounts, These ratios are almost equal (Table 2). This technology uses the following peptides Successfully applied to the copolymerization of combinations of: P1 + P3, P2 + P3, P1 + P2 + P3, P1 + P4, P 2 + P4, P1 + P2 + P4, P8 + P11, P9 + P11, P9 + P10, P9 + P10 + P11, P1 + P3 + P14 + P15, P1 + P1 4 and P1 + P15. Table 2: Amino acid composition of peptide polymer ^a  One letter notation^b   Predicted stoichiometric incorporation of each peptide into the polymer Antigenicity of synthetic peptide polymers   Concerns over free radical-induced polymerization of unprotected peptide monomers What they gain is that their side chain function is altered to affect their antigenicity and immunogenicity That is to exert. To examine this possibility, both groups have free functional groups. -Polymer P8 and P2 containing side chains that are susceptible to radicals Antiserum directed to P8 or a P2-specific Probed with noclonal antibody (MAb 1/1)^19,20. The results (Fig. 5) Demonstrated that antibodies can bind efficiently to determinants in the polymer. Which makes the epitope antigenic despite exposure to free radicals Indicates that the symbol remains as it is.   The advantage of polymerizing synthetic peptides is the multiplexing of peptides present in a single molecule. Is that the copy will confer increased antigenicity to the peptide monomer³⁷ . To assess this, the ability of antigen to bind to microtiter plates Using an inhibition ELISA that is independent of any differences in The antigenic properties of the mer peptides were compared. P5 coating of MAb 1/1 using P2 and polymerized P2 Inhibits binding to microtiter plates, and also reduces P8 monomer and polymerized P8 Was used to inhibit the binding of anti-P8 antiserum to P8 coated plates. As can be seen from FIG. In addition, polymer peptides have higher antigenicity than peptide monomers. P4 polymer (this It does not contain the epitope recognized by MAb 1/1 or anti-P8 antiserum) These antibodies do not inhibit the binding of them to the relevant peptides, which The enhanced inhibition caused by the tide polymer is due to the multiple cores of each peptide determinant. Indicates that the result is present. The results in FIGS. Not only does the synthesized peptide retain antigen binding, Demonstrate that they are more antigenic than the This is because multiple copies of the determinant allow for a very strong interaction with the antibody. Conclusion   The synthesis and polymerization of unprotected acryloyl peptides are To provide a practical genetic method for the synthesis of Using acryloyl chloride However, the N-terminus of the protected peptide attached to the resin support is derivatized and then Cleavage and deprotection with reagent B yields high yields of acryloyl peptide Is obtained. 6-aminohexyl as a spacer between the peptide and the acryloyl group Due to the interposition of sanic acid, certain types of paper can be used even in the absence of acrylamide. Polymerization of the peptide becomes possible. Free radical polymerization affects antigen binding of peptides And the peptide polymer is more antigenic than the monomeric peptide. This app The advantage of roach is that any number of identical or different acryloyl peptides can be constructed The antigenic activity of the whole product depends on the nature of the peptide unit hanging from the hydrocarbon chain. Can be incorporated into one polymer with the expectation that it will be largely determined You. By polymerizing a mixture of B cell and T cell peptide epitopes, All or most of the important epitopes of one or several pathogens Can be assembled. This is the malaria parasite , HIV and influenza viruses and serologically distinct strains It is particularly important for organisms such as streptococci A, which do so. Section 2 Materials and methods chemicals   Unless otherwise noted, chemicals are of analytical grade or the same. And so on. N, N'-dimethylformamide (DMF), piperidine, trifluoro Acetic acid (TFA), O'benzotriazole-N, N, N ', N'-tetramethyl-uronium-hex Safluorophosphate (HBTU), 1-hydroxybenzotriazole (HOBt) and Diisopropylethylamine (DIPEA) from Anspep Pty (Melbourne Australia) obtained. Phenol, triisopropylsilane and acryloyl chloride are Aldr From ich (Milwaulkee, WI), trinitrobenzylsulfonic acid (TNBSA) was obtained from Fluka (Sw 1,8-diazabicyclic- [5.4.0] undec-7-ene (DBU) from Sigma And acrylamide, ammonium persulfate, and N, N, N ', N',-tetrame Tylethylenediamine (TEMED) is available from BioRad (Bio-Rad Laboratories Pty., Ltd., A ustralia) and dichloromethane (DCM) and diethyl ether are Obtained from BDH (Poole, UK). Peptide synthesis   Peptides can be prepared manually or on an automated peptide synthesizer (Novasyn Crystal, Novabioc). hem, U.K, or 9050 Plus Milligan). Fmoc chemistry And use the peptide as the C-terminal carboxyl or arboxamide type. Assembled. Activated O-pentafluorophenyl ester or HBTU and And HOBT are present in equimolar amounts and DIPEA is present in a 1.5-fold molar excess. Fmoc amino acids were incorporated into the peptide chain either by using an acid. amino acid After incorporation of the residues, Fmoc using piperidine (20% in DMF) or DBU (2.5% in DMF) The group was removed. Fmoc-6-aminohexanoic acid (Ahx) using HBTU, HOBT and DIPEA Manually coupled to the N-terminus of the peptide. Ahx group is a polymer-based peptide It served the function of keeping a certain distance from the chain. All coupling reactions are performed manually. Yes, TNBSA test¹⁸Was monitored using   Acryloylation of the N-terminal of the protected peptide bound to the resin is performed by degassing at 0 ° C under nitrogen. Performed manually in anhydrous DMF. 20-fold molar excess of DIPEA (in 0.5 ml DMF) and 10-fold A molar excess of acryloyl chloride (in 0.5 ml DMF) was added and the reaction was left on ice for 1 hour. Then mixed at room temperature for another hour. The progress of acryloylation is determined using the TNBSA test Monitoring. At the end of peptide integration, the peptide is bound The support was washed sequentially with DMF, DCM and diethyl ether and then dried in vacuo.   The peptide is cleaved from the resin support and the arginine content of the peptide at room temperature under nitrogen. For 2 or 4 hours depending on the side chain protecting group of reagent B (TFA, 88: 5: 5: 2, (Ethanol, water and triisopropylsilane). Under a nitrogen stream, After partial evaporation of the peptide-containing filtrate, the crude peptide was precipitated in cold ether. And then washed three times. Purification of peptides   PepRPC column (1.6 × 1) mounted in FPLC system (Amrad / Pharmacia Pty. Ltd.) (0 cm) by reverse phase chromatography and purified with water as the limiting solvent. 0.1% TFA and CH in_ThreeChromatogram was developed using 0.1% TFA in CN at a flow rate of 4 ml / min. Opened. Analytical HPLC was performed on a Vydac C4 column (4.6x) mounted in a Waters HPLC system. 300 mm), and developed using the same solvent at a flow rate of 1 ml / min.   The authenticity of the purified peptide can be confirmed by analytical RF-HPLC and amino acid analysis. And the presence of the -ene group¹Monitored by 1 H NMR. Bruker-AM30 A spectrum was obtained at 300 MHz on a 0 spectrometer. Polymerization of N-acryloylated peptides   Degassed 0.5 MTris containing 6 M guanidine-HCl and 2 mM EDTA under nitrogen (pH In 8.3), polymerization of the N-acryloylated peptide was carried out. Typically, 3 micromo N-acryloyl peptide and a 50-fold molar excess of acrylamide Polymer incorporation was performed. Ammonium persulfate (total moles of acrylamide 2%) and TEMED (5 μl of a 1.32 μM solution) to initiate the polymerization. Was left at room temperature for 18 hours.   HiLoad Superdex 200 prepgrade (Amrad / Pharm acia Pty. The peptide polymer was isolated using a column (1.6 x 60 cm) of The chromatogram was developed using ammonium bicarbonate at a flow rate of 3 ml / min. Molecular weight Was evaluated on a Superose 6 HR column (1 x 30 cm) using a Waters HPLC system and 50 The chromatogram was developed in mM ammonium bicarbonate at a flow rate of 0.5 ml / min. Monoclonal antibody   Other sources for preparation and characterization of monoclonal antibodies (MAbs) 1/1 and 2/1¹⁹To Has been described. These MAbs are linked to the influenza virus A / Memphis / 1/71. Magglutinin heavy chain (HA₁) Synthetic peptide representing the C-terminal 24 residues³⁰⁵CPKYVKQNTLKLATGMR NVPEKQT³²⁸Prepared for MAb 1/1 is the B cell determinant RNVPEKQT (B₁Specific to Yes, to join³²²N,³²⁵E and³²⁷Requires Q, whereas MAb 2/1 has the B cell determinant LK LAT (B_Two) And there is an absolute need for each of these five residues. Do²⁰. T cells   T cell clone 12V1 contains purified hemagglutinin light chain (HA_TwoIn mice immunized with Generate and HA_TwoProliferates in response to the derived peptide ALNNRFQIKGVELKS. This clone Derivatization, properties and maintenance elsewhere^{twenty one}It is described in. Prior to use , Culture cells in Isopaque-Ficoll gradient^22,23Through.   Lymph node T cells were collected from pooled groin of mice previously sensitized with peptide for 7 days. And popliteal lymph node cell suspension. Made of nylon wool T cells were enriched by passing through a column^{twenty four}. Immunization protocol   6-8 week old female Balb / c mice were used. For antibody research, monomers or Is a polymer type, synthetic peptide emulsified in Freund's complete adjuvant (CFA). Animals were intraperitoneally inoculated with pide. Mice have 5 or 0.5 nanomolar monomeric peptides Or an equimolar determinant in the polymer. In CFA for T cell studies Were inoculated subcutaneously in the hind footpads of mice. Antibody binding assay   Microtiter tape coated with a solution of 5 μg / ml peptide or polymer antigen Using the rates, as described^{twenty three}And enzyme-linked immunosorbent assay (ELISA). Conclusion Antibodies were tested with horseradish peroxidase-conjugated rabbit immunized antibodies directed against mouse Ig. Incubate for 1.5 hours with Roblin (Ig) (DAKO, Denmark) (0.2 mM 2,2'-azino-bis in 50 mM citric acid containing 0.004% v / v hydrogen peroxide Incubate with ethylbenzthiazoline sulfonic acid, pH 4.0) Detected by 405nm absorbance at Labsystems Multiscan Multisoft Microphone Using a plate reader (Pathtech Diagnostics Pty. Ltd., Aust.). , Antibody dilution giving an absorbance of 0.25 corresponding to at least 5 times the background value The antibody titer was expressed as the reciprocal of the solution. Isotyping of mouse serum   Sera collected from mice that received two doses of the specific antigen were pooled and analyzed by ELISA. Tested the isoform profile of the antibody. Dilute antiserum to peptide³⁰⁶PKYVK QNTLKLATGMRNVPEKQT³²⁸Added to coated plates and incubated overnight. C After washing the wells, mouse IgM, IgG1, IgG2a, IgG2b, IgG3 or IgA (ICN Pharm Rabbit antiserum against aceuticals Inc., Costa Mesa, CA) was added and bound for 4 hours. I let it. The plate was washed again and horseradish peroxidase-conjugated pig anti- A 1/400 dilution of G. Ig) was added. After 1.5 hours, wash the tray again and add substrate The absorbance was measured as described above. T cell proliferation assay   Syngeneic γ-irradiation (2,20) as a source of antigen-presenting cells together with antigen in a total volume of 250 μl 0R,⁶⁰Co source) 96-well microtiter trays in the presence of spleen cells (Nunc, Denma rk) (3x10 cells for lymph node T cells)^FiveCells / well and T cells 1x10 for clone^Four/ Well concentration)^22,23. culture The culture medium was 10% heat-inactivated (56 ° C, 30 minutes) fetal bovine serum, 2 mM glutamine, 2 mM pill. Sodium borate, 0.1 mM 2-mercaptoethanol, 30 μg / ml gentamicin, RPMI 1640 supplemented with 100 I.U./ml penicillin and 100 μg / ml streptomycin there were. Culture 5% CO_Two4 days at 37 ° C in atmosphere, last 1 hour at 1μCi / well^Three Incubated in the presence of H-thymidine. The cells are then placed on a glass fiber filter. Collected and captured by^ThreeH-thymidine to Hewlett Packard Matrix 9600 Direct It was measured with a β-counter. result   Polymerization of N-acryloyl peptide. In this study, separate T-cell and B-cell A simple method to polymerize determinants into a single construct has been developed, and a powerful T cell antigen Efficient delivery of determinants into multimeric immunogens along with B-cell antigenic determinants Noh. To assess the potential of this technology, we set out influenza virus. Ils A / Memphis / 1/71 (H_Three(Subtype) Hemagglutinin heavy chain (HA₁) 24 residues C-terminal Well-identified residues remaining in peptides 306-328 representing the sequence^{twenty five}B cells and Antigenic determinants for helper T cells were used. This peptide has the sequence GMRNVPEKQT (B₁ ) And TLKLATG (B_TwoThe two B cell antigenic determinants contained in_TwoOverlap with Contains the helper T cell antigenic determinant KYVKQNTLKL. Light chain (HA_Two) A second T cell antigenic determinant ALNNRNFQIKGVELK located somewhere on glutinin S (T) is the original overlapping helper antigenic determinant²⁰B than is_TwoTurned to More likely to be an effective inducer of B cells to help produce antibodies Has been reported. T alone or B₁, B_TwoOr B₁With B_Two(B₁+ B_Two) And combined The loaded polymer was prepared using the scheme shown in FIG. Has Ahx group at N-terminus The peptide was synthesized and subsequently acylated with an acryloyl group. Then N-Acry Polymerization of the roylated peptide was performed in guanidine-HCl to aid solubility. Ma A 50-fold molar excess of acrylamide was also added to add spacers along the polymer backbone. Function. This reaction is performed on polyacrylamide gels in the absence of a crosslinker. Equivalent to generation. Amino acids in peptide polymers consisting of equimolar amounts of different peptides Acid analysis confirmed the presence of an approximately equimolar ratio of the relevant peptide. Poly (T + B₁) For T vs. B₁Was 1: 1.3. Poly (T + B_TwoIn), the ratio was 1: 1.2. Poly (T + B₁+ B_Two)) Is 1: 1.4: 1 Was .1   What are the chromatographic properties of the polymers produced using this protocol? Some were examined by gel permeation chromatography and FIG. 7 shows Superose 6 HR. (T + B) obtained from a column (1x30 cm)₁+ B_Two3) shows the elution profile. this Profiles represent what was obtained for each of the different polymer immunogens Was. The polymer elutes into the void volume of the column and is It was completely different from de. In all cases, after the polymerization step, Little peptide remained. Antigenicity of peptides exposed to free radicals   This polymerization method uses free radicals that can destroy protein functional groups. Use Selected for free radical-induced polymerization of the synthetic peptides described here Conditions determine whether the antigenic activity of the peptide epitope is deleterious To achieve this, the monomers of peptide 306-328 lacking an acryloyl group were converted to acrylamide Were exposed to various concentrations of ammonium persulfate. Acrylamide peptide And used to coat wells for ELISA assays. This Each of these treated peptides is B₁And B_TwoMAb 1/1 with specificity for And the ability to bind 2/1 is shown in FIG. These results indicate that each of the tested persulfates At concentration (varied persulfate to peptide ratio between 0.1: 1 and 1,000: 1) It has little or no effect on antigen activity. This weight The ratio of persulfate to peptide used in the synthesis method was 0.02: 1, well within this range. It is below. Antigen binding of synthetic polymer immunogens.   B-cell antigenic determinants in the polymer immunogen are directed against monomeric peptides 306-328. Microtiter with polymer to determine accessibility to engineered antibodies Cover the wells of the tray (micotitre tray) and MAb 1/1 or 2/1 by ELISA (FIG. 9). MAb 1/1 is similar to monomeric peptide Also exhibit similar binding properties and B₁Efficiently binds to polymers containing antigenic determinants be able to. MAb 2/1 also B_TwoOften used with polymers containing antigenic determinants Joined.   The binding of T cell antigenic determinants within the polymer immunogen was also tested. FIG. Polymer is T-array ALNNRFQIKGVELKS^{twenty one}Specific T cell clone 12V1 (Figure 10A) Shows the ability to stimulate proliferation in vitro. This system^26,27Typically observed in Although high doses of antigen suppression were apparent for each antigen, peak growth By comparing the doses required for the answer, the polymer is a monomeric T cell antigenic determinant peptide. Demonstrated a greatly reduced ability to stimulate related clones. this child This means that antigenic determinants for presentation to clones are easily processed from the polymer. Means that it is not shingled. However, in terms of recognition efficiency, Large differences such as were made for monomeric T cell epitope peptides Nothing was observed in the polyclonal population of lymph node T cells (FIG. 10B). in this case In T cells, T cells recognize T monomers and produce poly (T + B_Two) Is equivalent efficiency, Re (T + B₁+ B_Two) Was slightly lower. Poly (T + B₁) Recognition was the lowest. Immunogenicity of polymer immunogens   B_TwoAntigenic determinants and overlapping T cell determinants, KYVKQNTLKL^27,28Monomers containing Antibodies raised by polymers of peptides 306-328 and peptides 306-319 By comparing levels, the relative immunity of monomeric and polymeric immunogens The original properties were evaluated. These two immunogens were injected in mice at a dose of 5 nanomolar. Serum was collected during the primary and secondary responses, testing for peptide epitopes. This dose The monomeric peptide shows only a very weak primary antibody response, which is It has increased several tens of times. In contrast, polymer immunogens produce significant primary responses. (Average titer approximately 30 times higher than monomer) Increased antibody production 30-fold.   Also, B₁Or B_TwoMulticomponent polymers based on T sequences used with or both Mice were also inoculated and antibody responses were measured. 0.5 and 5 nanomolar doses Elicited antibodies to similar levels and only reports data obtained at 0.5 nmole dose (FIG. 11B). Not surprisingly, the individual monomer B₁Or B_TwoAntigenic determinant When a mouse is inoculated with T, no antibody is generated, but the same antigenic determinant is shared with the T sequence. When polymerized, it became a strong immunogen. In response to a single dose of polymer The level of antibody elicited (panel B) was the peptide 306-328 monomer (panel A). Equal to or higher than that achieved after two doses, two doses of polymer Obtained after administration is 50 to 100 times higher than with peptide 306-328 won. Among the three polymer immunogens containing T sequences, poly (T + B₁) Elicited antibodies at higher levels and averaged higher than MAb titers in control ascites preparations. won. Antibody specificity for polymeric immunogens containing two B cell antigenic determinants   In FIG. 11, poly (T + B₁+ B_Two) The level of antibodies obtained with the immunogen is poly (T + B₁) Re (T + B_Two) Did not reach the sum of the antibodies obtained. B cell antigenic determinants If the ratio of different T cell antigenic determinants affects the total amount of antibody produced, Is not surprising. However, poly (T + B₁+ B_Two) Immunogen is poly (T + B_Two) Immunity Both B cell antigenic determinants were poly (T + B₁+ B_Two) Whether one is recognized within, or one becomes immunocompetent at the expense of the other The question was raised as to whether or not it was. To investigate this, Re (T + B₁+ B_TwoPeptides called analog A and analog B by ELISA Tested against. Where analogs A and B are analogous to peptides 306-328 Have a similar sequence but only one of the two B cell antigenic determinants of this peptide , Each B₁And B_TwoContains¹⁹. FIG. 12 shows poly (T + B₁+ B_Two) Serum collected during the secondary and secondary responses can bind to both of these analogous peptides Which means that B₁And B_TwoAntibodies specific to both are multicomponent polymers Means that it was triggered by Antibody isotype elicited by polymer immunogen   Certain multivalent immunogens can induce B cells independently of T, and this interaction results in Ig Limited isotype profile antibodies with a predominant subclass of M Occurs. Therefore poly (T + B₁), Poly (T + B_Two) Or poly (T + B₁+ B_TwoIn response to The isotype profiles of the serum antibodies produced were tested and these polymers were We examined whether they were restricted in the isotype of the raised antibodies. Figure 13 Response of a range of different antibody isotypes to each of the three polymers It shows that it is produced in. Consideration   Defined antibodies, rather than crude antibody preparations, form third generation vaccines And to all individuals against the various serotypes of a particular pathogen. The difficulty associated with eliciting an immune response in A mid-range approach will be required. Molecular immunological techniques can cause disease Enables identification of many epitopes involved in the immune response to microbes I've been. The best of the techniques used to identify these epitopes What is being described is peptide synthesis, and we have now found these epitopes useful. Developing methods to deliver to the immune system to elicit an immune response is at a necessary stage . Thus, the inventors have developed polyvalent homo and heteropolymers of B and T cell epitopes. We have approached this problem by developing an embedded method.   In the method we describe, very large elutions elute into the void volume of the column. A polymer is obtained, and the excluded molecular weight of its spherical structure is about 4 × 10⁷Dalton. This It is not clear if such large structures are required to enhance immunogenicity However, in theory, the uptake of such polymers by antigen-presenting cells could The presentation of multiple copies of the antigenic determinant per cell will be possible. These "go The `` synthetic protein '' form also relates to antigenic determinants as compared to the free single peptide. Would give better stability of the serum. Polymerization protocol for chain transfer agent The size of the polymer by incorporating it into the It is possible to study the influence of size on immunogenicity.   The multi-component polymer of the T-cell antigenic determinant sequence is B₁And / or B_TwoAntigenic determinants of And was shown to be complete as an antigen. Serologically different The results of a subsequent experiment to construct a polymer by copolymerization of nine peptides As a result, polymer recognition by antisera generated for each individual epitope Was proved. From these findings, such polymers can be used diagnostically The strong possibility that it may be raised. The present invention also provides that polymers are strongly exempt If it is an epidermis and the polymer contains two different B-cell antigenic determinants, The body shows that each one was provoked.   A great advantage of the approach described herein is that the component synthetic peptide epitopes Is purified before being incorporated into the immunogen. In addition, its ingredients Since the tides are incorporated individually, they can be used as disulfide loops in peptides. Can incorporate other molecular properties to mimic similar structures in native proteins It is. Use this technology to incorporate multiple copies of the same or different peptides Not only provides a way to elucidate the polymorphism of some pathogens, It also relates to the MHC antigen system in recognition of T cell epitopes. This way In the association of the appropriate combination of B and T cell epitopes to the polymeric support This should allow the production of a wide range of different histocompatible antibodies in animals. is there. This is relevant for outbred populations, such as humans or human domestic animals. It is very important in the design of any given vaccine. If adjuvant If PAM is found to be necessary, PAM_ThreeCys²⁹A new generation of substances such as Can be incorporated into rimmers. Incorporation of hydrophobic sequences into polymerized synthetic peptides By making them liposomal ISCOMS^30-33Medium or protein antigen successfully Fixes in other amphiphilic substances such as DOTAP, which have been proven to help Will be possible.   Copolymerization of acryloyl peptide with other reagents results in peptide in the polymer Separation of epitopes from each other is possible, but also separation of epitopes from the main chain This allows minimization of steric limitations in the polymerization process. In this test In order to keep the space between the peptide and the main chain, the inventors thought that the peptide and acryloyl Ahx was used between the group. B, the shorter of the two B cell antigen-determining peptides_Two Has a slightly reduced ability to interact with MAbs when it is in the polymerized form. This is Probably the problem of accessibility, resulting in poly (T + B_Two) Is poly (T + B₁)Than A weak immunogen, but possibly longer spacer sequences It may be overcome by using it. Acrylamide is a peptide Used for spacing. However, acrylamide is converted to α-amino group Can be replaced with roylated amino acids or other spacing groups. You. The inventors constructed the polymer using acryloyl-lysine as a spacer. Have been built, but also to provide appropriate physical properties in the polymer backbone. Other amino acids that are aqueous, polar, charged or even hydrophobic may be used. U. Lysine is persistent when acryloylated at both α- and ε-amino groups. It provides cross-linking that allows the formation of gels with excellent sustained release properties. These methods Distance separating the peptide from the polymer backbone, or between the peptides on the backbone And the charge characteristics of the polymer can be adjusted to taste. these Approach avoids steric hindrance or accidental interaction between adjacent peptides If necessary, or as two or more separate Useful when it may be advantageous to increase the chances of the peptides interacting with each other It is. Polymerization of other reagents containing radioactive, fluorescent or chemiluminescent reagents To provide a tracer group on a side chain separate from the peptide. Protease resistance between the peptide epitope and the backbone or protease The incorporation of the sensitive sequence allows for controlled release of the epitope. Section 3 Synthesis of acryloyl amino acid   The amino acids H-lysine (Boc) -OtBu, H-glutamic acid (OtBu) -OtBu and H-serine (tB u) -OtBu (Calbiochem-Novabiochem, New South Wales, Australia) under the following conditions Acryloylated using: DCM 10 containing 7.2 mmol (2.4 molar equivalents) of DIPEA Acryloyl chloride (DCM 10 mL) 3.6 mmol (1.2 mol equivalent) Amount) was added dropwise at −10 ° C. under nitrogen. 1 hour at -10 ° C with vigorous stirring For another hour at room temperature. Monitor the reaction with the Kaiset test, This indicated that the reaction was complete after 2 hours. Reaction mixture 1x water, 1x5% NaHCO_ThreeWash with aqueous solution, 1x water, 2x10% citric acid aqueous solution, 1x water, and 1x brine, Evening Na_TwoSO_FourAnd dried. After filtration, remove DCM with a rotary evaporator (<30 ° C). Upon leaving, a clear oil was obtained (99% yield). TLC (chloroform: methanol The product was pure according to Le 95: 5). Amino acid side chains and carboxyls The protecting groups were removed with TFA-water (95: 5) cleavage mixture for 2 hours under nitrogen and protected from light. Rotary -When TFA is removed in an evaporator (<30 ° C) under a stream of nitrogen, the product is clear. An oil was obtained (99% yield). Acryloyl amino acid is converted to 6M guanidine-HCl + 0.5 Dissolve in 2 mM EDTA in M Tris (GuHCl) and adjust pH with 4 M Tris. Adjusted to 8.3. Make a stock solution containing 0.15mg / μl acryloyl amino acid did. Each acryloyl amino acid has a predicted Had a mass. Polymerization of acryloyl peptide with acryloyl amino acid   Polymerization was performed as described above. Acryloyl amino acids are peptide polymers Acrylamide as a comonomer for the formation of 50: 1 (acryloyl amino acid: (Cryloyl peptide / peptides). Peptide polymer -Based amino acids are prepared and converted to peptide polymer-based acrylamide. It was then purified and lyophilized as described above. By amino acid analysis, Confirm the presence of peptide in the polymer and the ratio of peptide to amino acid did. Immunization protocol   Using 6-8 week old BALB / c female mice, complete Freund's adjuvant (CFA) Poly (serine) ALN + C10, poly (glutamic acid) ALN + C10, or (Acrylamide) Any peptide of ALN + C10 (included in the polymer) The following doses were inoculated at 5, 0.5, 0.05 nanomolar. Mice received a second dose on day 30 I did. All animals were treated 30 days after the first peptide polymer inoculation and a second pep Blood is collected from the retro-orbital plexus 12 days after inoculation of the tide polymer, and the serum is -2 until needed. Stored at 0 ° C.   Monomeric or polymeric forms in BALB / c mice for cytotoxic T cell (CTL) studies 10 μg (7.8 nmoles) of CTL antigen determinants subcutaneously in the hind footpad (50 μl per footpad) Inoculated. Infectious influenza virus A / Memphis / 1/71 in mice 1x1 in 50 μl 0^4.5It was inoculated intranasally with pfu. Antibody binding assay   ELISA assays were performed as described above. Antibody titer shows absorbance of 0.25 Expressed as the reciprocal of antibody dilution, which is at least five times background Is equivalent to Cell culture medium   T cell culture medium was 10% heat-inactivated (56 ° C, 30 minutes) fetal bovine serum (vol / vol), 2 mM glutamate. , 2 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, 30 μg / ml gene Supplemented with tamycin, 100 I.U./ml penicillin and 100 μg / ml streptomycin. And RPMI-1640 (CSL, Ltd. Parkville, Australia). T cell proliferation assay   T cell clone 4.51 was used, which was purified hemagglutinin heavy chain (HA₁) HA₁In response to the peptide KYVKQNTLKL derived from Multiply. The induction, characterization and maintenance of this clone is described elsewhere²⁰Have been. Use Prior to use, culture the cells in an Isoplaque-Ficoll gradient.^22,23Subculture.   Proliferation assay is 1x10^FourMonomer or polymer with T cells (clone 4.51) 96-well flat-bottom microtiter containing a peptide antigen (40 μM in the first well) In a tray (Nunc, Denmark), a total volume of 250 μl of 3 × 10^Five(1 00μl) gamma irradiation (2,200rad,⁶⁰(Co source) Set in the presence of normal BALB / c spleen cells^{22,2 Three} . Culture 5% CO_Two1μCi well for 4 days at 37 ° C in the atmosphere for the last 18 hours^{-1 Three}H- Incubated in the presence of thymidine. The cells are then filtered on a glass fiber filter. Collected,^ThreeIncorporate H-thymidine into Hewlett Packard Matrix 9600 direct β-cow Measured at the center. Cytotoxic T cell assay   The second effector cell is (i) a peptide monomer or polymer (peptide 10 μl). g) Lymph node cell suspension in popliteal and inguinal regions of BALB / c mice 7 days previously inoculated with g) (Ii) Tris-buffered ammonium chloride (ATC: 17 mM tris at pH 7.4) 0.15M NH in s-HCl_FourCl), the red blood cells were reduced by the pre-transmissible virus. Prepared from sensitized, spleen cells of mice for at least 21 days. Effector cells Group, 1x10⁷Peptide-pulsed spleen cells as a source of infectious virus or antigen presenting cells Incubate in 25 ml of T cell medium containing⁷Cells / flask). Wooly Ruth-infected spleen cells were inoculated with infectious virus 1000-5000 HAU in 1 ml At 37 ° C. for 30 minutes and washed once prior to use. Pep Tide pulsed cells in 1 ml of serum-free RPMI, CTL peptide antigenic determinant (monomer) 1 Incubated for 2 hours at 37 ° C. with 00 μg. Culture 5% CO_TwoAtmosphere, 37 ℃ For 5 days, then wash the cells and Used for assay.   P815 mast cell tumor cells were used as target cells in the CTL assay, And uninfected targets with infectious virus solution (1000-5000 HAU mL)^-1) Or serum 2x10 at 37 ° C in 500 μl of Ree RPMI^ThreeBy incubating P815 cells Prepared. After 2 hours, the cells are washed and 200 μCi⁵¹Cr (Amersham, Australia) Resuspended in 200 μl containing TCM. After 2 hours, wash the cells three times, Concentration of 1x10^FiveCells / ml¹Was adjusted to 100 microliter aliquot (1x10^FourFine Target cells (infected and uninfected) in 96-well U-bottom tissue culture media 100 μl of effector cells in addition to the following effector: target cell ratio; Added at 1: 50: 1, 25: 1, 12: 1, 6: 1, 3: 1, 1.5: 1 and 0: 1. Centrifuge the cells Pellet slowly at 600xg for 30 seconds, 5% CO_Two4 hours in at 37 ℃ in atmosphere Cuvette, then take 100 μl of the supernatant and evaluate the radioactivity using a γ-counter. Valued. Unique for each effector⁵¹Cr release percentage: target ratio is calculated by the formula: Indicated by Of target cells incubated in medium alone⁵¹Maximum spontaneous release of Cr They ranged from 1-10% of the total possible. Data are the average of values obtained from three cultures. Is represented by result Proliferation of T cell clone 4.51 on polymerized PKY containing an enzyme cleavage sequence.   Two enzymatic cleavage sequences were selected and acryloylated Peptides as described above. Synthesized onto the N-terminus of tide PKY. Its sequence tetramer (GLFG) and pentamer ( VYLKY) for van Noort and van der Drift³⁹Cathepsin B described by³⁶Off Represents the cleavage site and cleavage motif. Inserted between peptide and acryloyl-Ahx-group Serine, glutamine containing PKY with or without inserted enzyme cleavage sequence A polymer based on acid and acrylamide was constructed. In a proliferation assay, Each of these polymers was used to evaluate their ability to stimulate clone 4.51.   As can be seen in FIG. 14, either cathepsin B or cathepsin D cleavage sequence was included. These polymers induced a proliferative response. Cathepsin D for each type of polymer Sequence integration induced higher proliferation compared to the cathepsin B sequence. 0.1 microphone At romole dilution, a polymer based on poly (glutamic acid) is converted to poly (serine) It induced 35% to 50% higher proliferative response than the based polymer. Amino acid based Remer has a much higher proliferative response than poly (acrylamide) based polymers Induced. The ability of peptide polymers to induce cytotoxic T lymphocyte responses   CTL epitopes derived from influenza virus nucleoprotein¹⁴⁷TYQRTRALV¹⁵⁵ To acryloyl, acrylamide, acryloyl-serine and acryloyl Polymerized with ru-glutamic acid. With a spacer between the polymer backbone and the peptide In order to function properly, four lysines were synthesized at the N-terminus of the CTL epitope, The tide was also acryloylated and polymerized with the same comonomer. CTL peptide BALB / c mice were sensitized using a limmer, and after 7 days, T cells were transferred to the groin and popliteal phosphorus. It was isolated from the rat node and restimulated in vitro with virus or CTL peptide. Next The restimulated T cells can then be used in CTL assays to target virus-infected target cells or non- The ability to kill infected target cells was evaluated.   Figure 15 shows CTL or K_FourPolymer based on amino acid containing -CTL antigenic determinant is C While sensitizing to the TL response, CTL peptides polymerized with acrylamide It was not. Induced by poly (amino acids / amino acids) CTL The induced CTL response was comparable to that induced by the CTL peptide alone, indicating that Equivalent to that induced by the virus (Panel A). Poly (A) Responses induced by restimulating (amino acid) CTL T cells are Weaker than T cells from exposed mice (Panel B), but still they are virus Can kill a target. In this way, sensitization with peptide polymer T cells restimulated with peptides can kill viral infection targets. Surprise A good result is that acryloyl-Ahx-CTL does not induce a CTL response, while Mer and Acryloyl-Ahx-K_FourThat CTL was induced. Was. This means that acryloyl-Ahx-K but not acryloyl-Ahx_FourCut off Effect of the consequences of antigen processing, which in turn stimulates the CTL response It will affect the ability of the peptide. Poly (serine) ALN + C10, poly (glutamic acid) ALN + C10 Comparison of Li (acrylamide) ALN + C10   Replace ALN and C10 with acryloyl-serine, acryloyl-glutamic acid, or Polymerized with acrylamide, emulsified with CFA, 5, 0.5 and 0.05 nanomolar peptide BALB / c mice were inoculated at the dose. First and second antibody responses were measured by ELISA (FIG. 16). All polymers compared at 5 and 0.5 nanomolar peptide doses An antibody titer as high as possible could be induced. Weaker at 0.05 nanomolar dose Unlike polymers based on glutamic acid or acrylamide that elicited a response , Serine-based polymers elicited equally high antibody titers. Section 4 Antigenic and immunogenic properties of polymers synthesized by polyacryloylation   To assess the effect of peptide orientation within the polymer, the streptococcal M protein Multiple peptide epitopes derived from a protein with an acryloyl group at the C-terminus . This is because Fmoc Lys (Mtt) -COOH (Calbiochem-Novabiochem, New So uth Wales, Australia). Add the terminating (N-terminal) amino acid by synthesis After removing the Fmoc group, the exposed N-terminal amino group was replaced with N-acetylimidazolone. And treated with acetic anhydride. Alternatively, the acryloyl In addition to inhibiting the hydrolysis, if necessary, to finally expose the amino group at the N-terminus An N-terminal amino acid may be added as a BOC-amino acid derivative. 5% triisopropyl Treated three times for 3 minutes with a 1% solution of TFA in dichloromethane containing silane. The Mtt protecting group at the peptide C-terminus was removed, at which point an additional spacer was added to this Treatment can add to exposed amino groups and ultimately bound acryloyl groups. You. Method   Synthesize homopolymers containing multiple copies of one peptide attached on the polymer backbone did. Construct homopolymer for N-terminal peptides ST156 (1-19) and M52 (1-19) I built it. Separate constructs were made for both peptides, where each peptide was multiplexed. Copies are attached to the backbone by both the C-terminus and the N-terminus. In addition, C-terminal Both peptides ST156 (1-19) and M52 (1-19) linked on the same polymerized main chain at One containing heteropolymer was constructed.   Immunization of B10BR mice will test the immunogenicity of these polymers. did. Mice were treated with a construct (30 μg / mouse) emulsified in Freund's complete adjuvant (CFA). S) received the primary immunization and boosted only at 21, 31, 41 and 51 days with the construct in PBS Stimulation was performed. After the primary immunization, mice were bled on days 9, 19, 29, -49 and -59 . At each time point, the serum is tested for peptide-specific antibodies using ELISA. did. Antisera were also tested for opsonic activity using an indirect bactericidal assay . result Homopolymer   Antibodies raised against ST156 (1-19) homopolymer and M52 (1-19) homopolymer Serum has a peptide attached to the backbone at both the C-terminus and the N-terminus, Test in indirect ELISA to determine if peptide antigens of such constructs are found did. Table 3 summarizes the results obtained 59 days after the primary immunization. Peptide ST156 (1-19) and M52 (1-19) homopolymers that bind to the main chain at the N-terminus Antisera from both, except for mouse 2 immunized with the M52 (1-19) homopolymer, Did not recognize each individual peptide or their respective streptococcal group A ( For the GAS) strain, these sera did not contain opsonin. But However, if the peptides are attached to the backbone at the C-terminus, all mice will have their own Peptide antibodies were produced at significantly higher levels (Table 3; FIG. 17). Table 3; Immunogenicity of ST156 (1-19) and M52 (1-19) homopolymersHeteropolymer   Mice were immunized with the M52 / ST156 heteropolymer, ie both peptides were identical Linked at the C-terminus on the backbone. FIG. 17 shows a mouse derived from a heteropolymer immunized mouse. Sera were separated by two separate titers greater than 40,000 by day 69 after primary immunization. This indicates that the peptide could be recognized.   A heteropolymer having eight N-terminal peptides attached to the main chain at the N-terminus was used Studies did not show a significant increase in antibody levels to individual peptides ( Table 4). However, significant levels of antibodies against fully heteropolymers (Table 4; FIG. 18). From the competitive ELISA, the peptide was attached to the backbone at the N-terminus. Various homopolymers (described above) and hetero Antisera raised against the polymer were shown to be uninhibited. Especially individual The antiserum raised against the peptide alone reacted with the heterozygote in the ELISA. Returning to the rimer, the heteropolymer recognizes the peptide-specific antibody, Suggests that the heteropolymer is still antigenic (Table 5). Table 4: Immunity of antisera raised against heteropolymer consisting of 8 peptides Originality Table 5: Cross-reactivity of peptide-specific antisera to heteropolymers   Specific implementations may be used without departing from the spirit or scope of the broadly described invention. Many variations and / or modifications as set forth in the It will be appreciated by those skilled in the art that Therefore, this embodiment is not In all respects, this is merely illustrative and is not to be construed as limiting.

[Procedure of Amendment] Article 184-8, Paragraph 1 of the Patent Act [Submission Date] April 28, 1999 (1999.4.28) [Correction contents]                                The scope of the claims   1. (1) CH_Two= CR_Four-CO-X-R₁And (2) CH_Two= CR_Three-CO-R_Two [Wherein X is absent or a space having a length equivalent to 1 to 30 CC single bond] Sir,   R₁Are epitopes, each R₁Are the same or different,   R_TwoIs NH_Two,   Ｕwhere U is O or NH,     f is an integer of 0 to 17,     Y is H, COOH, CO-NH_TwoAnd     W is H, CH_Three, CH_TwoCO-NH_Two, CH_TwoCOOH, CH_TwoOH, CH (CH_Three) OH, CH_TwoSH, CH_TwoCH_TwoCOOH , CH_TwoCH_TwoCO-NH_Two, CH_TwoCH (CH_Three)_Two, CH (CH_Three) CH_TwoCH_Three, (CH_Two)_FourNH_Two, CH_TwoCH_TwoSCH_Three, CH (CH_Three )_Two, And     Z is H, (CH_Two)_hQT (where h is 0-10, Q is O, NH, CH_TwoAnd T is a lipid, a labeling molecule, a target molecule or a functional group). And   R_ThreeIs CH_Three, H, NH_Two, OH, CN or halogen, each R_ThreeAre the same or different And   R_FourIs CH_Three, H, NH_Two, OH, CN or halogen, each R_FourAre the same or different And   The ratio of (1) :( 2) ranges from about 1: 1 to about 1: 1000] And optionally a polymer comprising polymerized units of one or more other monomers.   2. Multiple different R₁The polymer of claim 1 having a group.   3. Individual R₁T-cells wherein the group comprises a mixture of T-cell epitopes 2. The polymer of claim 1, which is an epitope.   4. Individual R₁B-cells wherein the group comprises a mixture of B-cell epitopes 2. The polymer of claim 1, which is an epitope.   5. Individual R₁Group so that the polymer comprises a mixture of B and T cell epitopes 2. The polymer of claim 1, which is a unique T cell or B cell epitope.   6. When the spacer group X is R₁Are spaced from the polymer backbone 2. The polymer of claim 1, which is present.   7. 2. The polymer of claim 1, wherein X comprises a site that is enzymatically cleavable.   8. The polymer of claim 7, wherein X comprises the amino acid sequence GLFG or VYLKY.   9. f = 0, U = NH, Z = H, W = CH_TwoOH, and Y = COOH or CO-NH_TwoIn The polymer of claim 1, wherein   10. f = 0, U = NH_Two, Z = H, W = CH_TwoCH_TwoCOOH, and Y = COOH or CO- NH_TwoThe polymer of claim 1, wherein   11. 2. The method of claim 1, wherein the ratio of (1) :( 2) ranges from about 1:10 to about 1:50. The listed polymer.   12. 9. The polymer of claim 8, wherein X comprises the amino acid sequence GLFG or VYLKY. .   13. The polymer of claim 1, wherein the polymer is crosslinked.   14． (I) Monomer (1) CH_Two= CR_Four-CO-X-R₁And (2) CH_Two= CR_Three-CO-R_Two [Wherein X is absent or a space having a length equivalent to 1 to 30 CC single bond] Sir,   R₁Are epitopes, each R₁Are the same or different,   R_TwoIs NH_Two,   Ｕwhere U is O or NH,     f is an integer of 0 to 17,     Y is H, COOH, CO-NH_TwoAnd     W is H, CH_Three, CH_TwoCO-NH_Two, CH_TwoCOOH, CH_TwoOH, CH (CH_Three) OH, CH_TwoSH, CH_TwoCH_TwoCOOH , CH_TwoCH_TwoCO-NH_Two, CH_TwoCH (CH_Three)_Two, CH (CH_Three) CH_TwoCH_Three, (CH_Two)_FourNH_Two, CH_TwoCH_TwoSCH_Three, CH (CH_Three )_Two, And     Z is H, (CH_Two)_hQT (where h is 0-10, Q is O, NH, CH_TwoAnd T is a lipid, a labeling molecule, a target molecule or a functional group). And   R_ThreeIs CH_Three, H, NH_Two, OH, CN or halogen, each R_ThreeAre the same or different Things,   R_FourIs CH_Three, H, NH_Two, OH, CN or halogen, each R_FourAre the same or different Is) And optionally preparing one or more other monomers; and (Ii) polymerizing the monomer in the presence of a free radical initiator Having a polymer main chain and a plurality of pendant peptides bonded to the main chain. A method for preparing a peptide polymer.   15. Acceptable carrier, and (1) CH_Two= CR_Four-CO-X-R₁And (2) CH_Two= CR_Three -CO-R_Two [Wherein X is absent or a space having a length equivalent to 1 to 30 CC single bond] Sir,   R₁Are epitopes and each R₁Are the same or different and R₁At least One is a peptide epitope,   R_TwoIs NH_Two,   Ｕwhere U is O or NH,     f is an integer of 0 to 17,     Y is H, COOH, CO-NH_TwoAnd     W is H, CH_Three, CH_TwoCO-NH_Two, CH_TwoCOOH, CH_TwoOH, CH (CH_Three) OH, CH_TwoSH, CH_TwoCH_TwoCOOH , CH_TwoCH_TwoCO-NH_Two, CH_TwoCH (CH_Three)_Two, CH (CH_Three) CH_TwoCH_Three, (CH_Two)_FourNH_Two, CH_TwoCH_TwoSCH_Three, CH (CH_Three )_Two,And     Z is H, (CH_Two)_hQT (where h is 0-10, Q is O, NH, CH_TwoAnd T is a lipid, a labeling molecule, a target molecule or a functional group). And   R_ThreeIs CH_Three, H, NH_Two, OH, CN or halogen, each R_ThreeAre the same or different Things,   R_FourIs CH_Three, H, NH_Two, OH, CN or halogen, each R_FourAre the same or different And   The ratio of (1) :( 2) ranges from about 1: 1 to about 1: 1000] And optionally a polymer comprising polymerized units of one or more other monomers Immunizing a peptide epitope in an animal, comprising administering to the animal How to generate a response.   16. When the spacer group X is R₁Are spaced from the polymer backbone 16. The method of claim 15, wherein   17． 16. The method of claim 15, wherein X comprises an enzymatically cleavable site.   18. 18. The method according to claim 17, wherein X comprises the amino acid sequence GLFG or VYLKY.   19. f = 0, U = NH, Z = H, W = CH_TwoOH, and Y = COOH or CO-NH_Twoso 16. The method of claim 15, wherein there is.   20. f = 0, U = NH, Z = H, W = CH_TwoCH_TwoCOOH, and Y = COOH or CO-N H_TwoThe method of claim 15, wherein   21. 16. The method of claim 15, wherein the ratio of (1) :( 2) ranges from about 1:10 to about 1:50. The described method.   22. 16. The method of claim 15, wherein the polymer is cross-linked.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 39/385 A61K 39/385 47/48 47/48 A61P 31/04 A61P 31/04 31/16 31 / 16 C07K 17/08 C07K 17/08 C08F 220/26 C08F 220/26 220/34 220/34 222/32 222/32 (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), AU, JP (71) Applicant Commonwealth Scientific and Industrial Research Organization Australia 2601 Australian Capital Territory, Campbell , Limestone Avenue (without address) (71) Applicant The University 3052 Victoria, Parkville, Royal Parade, Loyal Melbourne Hospital, Australia 3052 Victoria, Parkville, Victoria (No address) (71) (71) Applicant CSL Limited Australia 3052 Victoria, Victoria, Poplar Road 45 (72) Inventor Jackson, David, Charles Australia 3104 Victoria, North Balwyn, Woodville Street 74 (72) Inventor E Brian-Simpson, Nail, Martin 3056 Victoria, Victoria, Brunswick, South Audrey Street 7/10 (72) Inventor Brown, Lorena, Rezabeth Australia 3104 Woodville Street, North Balwyn, Victoria 74, (72) Inventor Zen, Wiegan Australia 3051 Victoria, North Melbourne, Highness Street 6/11 (72) Inventor Ed, Nicholas, John Australia 3145 Melrose Avenue, East Malvern, Victoria 10 (72) Inventor Brand, Evelyn, Rosemary Australia 4030 Queensland, Windsor Sixth Avenue 11 (72) Inventor Good, Michael, Frances Australia 4061 Queensland Do Governorate, The Gap, Weemara Street 46

Claims (1)

[Claims]   1. (1) CH_Two= CR_Four-CO-X-R₁And (2) CH_Two= CR_Three-CO-R_Two [Wherein X is absent or a space having a length equivalent to 1 to 30 CC single bond] Sir,   R₁Is a peptide and each R₁Are the same or different,   R_TwoIs NH_Two,   Ｕwhere U is O or NH,     f is an integer of 0 to 17,     Y is H, COOH, CO-NH_TwoAnd     W is H, CH_Three, CH_TwoCO-NH_Two, CH_TwoCOOH, CH_TwoOH, CH (CH_Three) OH, CH_TwoSH, CH_TwoCH_TwoCOOH , CH_TwoCH_TwoCO-NH_Two, CH_TwoCH (CH_Three)_Two, CH (CH_Three) CH_TwoCH_Three, (CH_Two)_FourNH_Two, CH_TwoCH_TwoSCH_Three, CH (CH_Three )_Two, And     Z is H, (CH_Two)_hQT (where h is 0-10, Q is O, NH, CH_TwoAnd T is a lipid, a labeling molecule, a target molecule or a functional group). And   R_ThreeIs CH_Three, H, NH_Two, OH, CN or halogen, each R_ThreeAre the same or different Things,   R_FourIs CH_Three, H, NH_Two, OH, CN or halogen, each R_FourAre the same or different And   The ratio of (1) :( 2) ranges from about 1: 1 to about 1: 1000] And optionally a polymer comprising polymerized units of one or more other monomers.   2. Multiple different R₁The polymer of claim 1 having a group.   3. R₁3. The polymer of claim 2, wherein is an epitope.   4. Individual R₁T-cells wherein the group comprises a mixture of T-cell epitopes 2. The polymer of claim 1, which is an epitope.   5. Individual R₁B-cells wherein the group comprises a mixture of B-cell epitopes 2. The polymer of claim 1, which is an epitope.   6. Individual R₁Group so that the polymer comprises a mixture of B and T cell epitopes 2. The polymer of claim 1, which is a unique T cell or B cell epitope.   7. When the spacer group X is R₁Are spaced from the polymer backbone 2. The polymer of claim 1, which is present.   8. 2. The polymer of claim 1, wherein X comprises a site that is enzymatically cleavable.   9. 9. The polymer according to claim 8, wherein X comprises the amino acid sequence GLFG or VYLKY.   10. f = 0, U = NH_Two, Z = H, W = CH_TwoOH, and Y = COOH or CO-NH_Twoso The polymer of claim 1, wherein:   11. f = 0, U = NH_Two, Z = H, W = CH_TwoCH_TwoCOOH, and Y = COOH or CO- NH_TwoThe polymer of claim 1, wherein   12. 2. The method of claim 1, wherein the ratio of (1) :( 2) ranges from about 1:10 to about 1:50. The listed polymer.   13. CH_Two= CR_Four-CO-X-R₁ (Wherein X is a spacer having a length equivalent to 1 to 30 CC single bonds,   R₁Is a peptide and each R₁Are the same or different,   R_FourIs CH_Three, H, NH_Two, OH, CN or halogen, each R_FourAre the same or different Is) And optionally a polymer formed from one or more other monomers.   14． Multiple different R₁14. The polymer of claim 13, wherein the group is present.   15. R₁15. The polymer of claim 14, wherein is an epitope.   16. Individual R₁The group is a T-cell such that the polymer comprises a mixture of T-cell epitopes. 14. The polymer of claim 13, which is a vesicle epitope.   17． Individual R₁The group is a B-cell such that the polymer comprises a mixture of B-cell epitopes. 14. The polymer of claim 13, which is a vesicle epitope.   18. Individual R₁Group if the polymer contains a mixture of B and T cell epitopes 14. The polymer of claim 13, which is a T cell or B cell epitope.   19. 14. The polymer of claim 13, wherein X comprises an enzymatically cleavable site.   20. 9. The polymer of claim 8, wherein X comprises the amino acid sequence GLFG or VYLKY. .   21. 14. The polymer of claim 1 or claim 13, wherein the polymer is crosslinked. -   22. (I) Monomer (1) CH_Two= CR_Four-CO-X-R₁And (2) CH_Two= CR_Three-CO-R_Two [Wherein X is absent or a space having a length equivalent to 1 to 30 CC single bond] Sir,   R₁Is a peptide and each R₁Are the same or different,   R_TwoIs NH_Two,   Ｕwhere U is O or NH,     f is an integer of 0 to 17,     Y is H, COOH, CO-NH_TwoAnd     W is H, CH_Three, CH_TwoCO-NH_Two, CH_TwoCOOH, CH_TwoOH, CH (CH_Three) OH, CH_TwoSH, CH_TwoCH_TwoCOOH , CH_TwoCH_TwoCO-NH_Two, CH_TwoCH (CH_Three)_Two, CH (CH_Three) CH_TwoCH_Three, (CH_Two)_FourNH_Two, CH_TwoCH_TwoSCH_Three, CH (CH_Three )_Two,And     Z is H, (CH_Two)_hQT (where h is 0-10, Q is O, NH, CH_TwoAnd T is a lipid, a labeling molecule, a target molecule or a functional group). And   R_ThreeIs CH_Three, H, NH_Two, OH, CN or halogen, each R_ThreeAre the same or different Things,   R_FourIs CH_Three, H, NH_Two, OH, CN or halogen, each R_FourAre the same or different Is) And optionally preparing one or more other monomers; and (Ii) polymerizing the monomer in the presence of a free radical initiator Having a polymer main chain and a plurality of pendant peptides bonded to the main chain. A method for preparing a peptide polymer.   23. Acceptable carrier, and (1) CH_Two= CR_Four-CO-X-R₁And (2) CH_Two= CR_Three -CO-R_Two [Wherein X is absent or a space having a length equivalent to 1 to 30 CC single bond] Sir,   R₁Is a peptide and each R₁Are the same or different and R₁At least one of One is a peptide epitope,   R_TwoIs NH_Two,  Ｕwhere U is O or NH,     f is an integer of 0 to 17,     Y is H, COOH, CO-NH_TwoAnd     W is H, CH_Three, CH_TwoCO-NH_Two, CH_TwoCOOH, CH_TwoOH, CH (CH_Three) OH, CH_TwoSH, CH_TwoCH_TwoCOOH , CH_TwoCH_TwoCO-NH_Two, CH_TwoCH (CH_Three)_Two, CH (CH_Three) CH_TwoCH_Three, (CH_Two)_FourNH_Two, CH_TwoCH_TwoSCH_Three, CH (CH_Three )_Two, And     Z is H, (CH_Two)_hQT (where h is 0-10, Q is O, NH, CH_TwoAnd T is a lipid, a labeling molecule, a target molecule or a functional group). And   R_ThreeIs CH_Three, H, NH_Two, OH, CN or halogen, each R_ThreeAre the same or different Things,   R_FourIs CH_Three, H, NH_Two, OH, CN or halogen, each R_FourAre the same or different Thing, and   The ratio of (1) :( 2) ranges from about 1: 1 to about 1: 1000] And optionally a polymer comprising polymerized units of one or more other monomers Immunizing a peptide epitope in an animal, comprising administering to the animal How to generate a response.   24. When the spacer group X is R₁Are spaced from the polymer backbone 24. The method of claim 23, wherein   25. 24. The method of claim 23, wherein X comprises an enzymatically cleavable site.   26. 26. The method of claim 25, wherein X comprises the amino acid sequence GLFG or VYLKY.   27. f = 0, U = NH_Two, Z = H, W = CH_TwoOH, and Y = COOH or CO-NH_Two 24. The method of claim 23, wherein   28. f = 0, U = NH_Two, Z = H, W = CH_TwoCH_TwoCOOH, and Y = COOH or CO- NH_Two24. The method of claim 23, wherein   29. 24. The method of claim 23, wherein the ratio of (1) :( 2) ranges from about 1:10 to about 1:50. The described method.   30. 24. The method of claim 23, wherein the polymer is crosslinked.