CN101123990A - Nanoparticles comprising antigens and adjuvants and immunogenic structure - Google Patents

Abstract

Nanoparticles comprising adjuvants and antigens, such as tumour and pathogen antigens, are disclosed and their use in a range of applications such as for the treatment of cancer and infectious diseases. Immunogenic structures based on nanoparticles or antibodies with carbohydrate ligands, and their use for therapeutic and prophylactic purposes, and for the isolation and detection of antibodies directed against the carbohydrate structures.

Description

The nano-particle that comprises antigen and adjuvant, and immunogenic structure

Invention field

The present invention relates to nano-particle, relate more particularly to comprise adjuvant and antigenic nano-particle, antigen such as tumor and pathogen antigen, and their purposes in a series of application.The invention further relates to the immunogenic structure that has the saccharide part, and they are used for the treatment of and prevent the purposes of purpose, with the purposes that is used to separate and detect the antibody of this carbohydrate structure of antagonism.

Background of invention

Lack immunogenicity when saccharide and peptide antigen direct injection are given the patient and hindered their application in vaccine greatly.Usually ignored when this antigen is injected separately, removed rapidly and not induction of immunity reaction by antigen-presenting cell (APCs).

In most of the cases, this antigen is combined with adjuvant be still essential.Adjuvant can be simple delivery system such as liposome, and it is slowly removed antigen and it more may be arrived and be accepted by APCs.Yet this is not in fact very effective and need combines with the reagent of stimulating immune system usually, as the bacterial product of stimulating cytokine formation.Cytokine itself also can give jointly.Many toxicity in these products too greatly or too tool are experimental and can not be applied to the mankind, and the most effective adjuvant also is not approved for the mankind.The effectiveness that can be used for human most of adjuvants is limited.It is a persistent challenges that searching is suitable for the human effective adjuvant that uses.

The sugar antigen immunogenicity especially a little less than because they only can stimulate the B cell effect, can not stimulate t cell responses.This reaches by saccharide is combined with protein carrier usually.Yet for the enhance immunity reaction, it is essential to use adjuvant to be still.

Sugar antigen is the potential target that is used for anticancer immunotherapy, because but they are exposed to tumor cell surface are hidden on the normal cell.Many antibacterials and other pathogen are also distinguished because of sugar antigen, if the saccharide immunogenicity be not so a little less than, it is the good target that is used for vaccine.Therefore the immunogenicity of improving sugar antigen will be applied to multiple treatment field.

Cancerous cell usually with anomalous mode by glycosylation, be the feature that they and normal cell are made a distinction.(Glycoconjugate J.(1997)，14：569；Adv.Cancer Res.(1989)，52：257；Cancer Res.(1996)，56：5309)。In most of the cases, unusual glycosylation is present in cell surface with glycoprotein and glycolipid form.Therefore the carbohydrate structure of these changes can be known as tumor associated antigen (TAA), and it usually is not present on the normal cell.Under many circumstances, cell does not demonstrate glycosylation of the same race, that is, exist on the cell surface complex plycan chain the different sugar form (Annu.Rev.Biochem. (1988), 57:785).In the discovery of the tumor associated antigen that great majority change with subsequently in the process of sign, studies show that they have important function for cancerous cell.For example, tumor associated antigen can make degenerated cell demonstrate malignant phenotype's characteristic attributes, increases as adhesive capacity, and described sticking in the foundation transfer plays an important role.Yet this antigen also can be expressed in normal cell in some stage, and they are responsible for the normal function of these cells there.Therefore, tumor associated antigen is the structure of mainly being presented by tumor cell, and on cell membrane or in the film, this allows they and non-malignant tissue to distinguish usually.Tumor associated antigen can be for example polypeptide, particularly glycosylated protein, or the polypeptide of glycosylation form.Can represent other structures of tumor associated antigen to comprise glycolipid, for example ganglioside such as GM2.This tumor associated antigen can show as the change of cell membrane lipid constituents, and it may be the feature of cancerous cell.

Tumor associated antigen comprises following example.

N-CAM (nerve cell adhesion molecule), it is usually expressed on the tumor of nerve origin and causes homophilic adhesion (J.Cell Biol.118 (1992), 937).

Lewis Y sugar antigen, it is present on the tumor of most of epithelium genesis, but it also plays an important role in the fetal development of epithelial tissue.Shown that expression and the prognosis mala of this antigen in pulmonary carcinoma has tight association, because Lewis Y positive cancer cell obviously has higher metastatic potential (N.Engl.J.Med.327 (1992), 14).

CEA (carcinoembryonic antigen), it usually is present in the gastrointestinal epithelial tumor, and has been accredited as from viscoelastic element (Cell 57 (1989), 327).

Ep-CAM (epithelial cell adhesion molecule), it is expressed on the tumor of nearly all epithelium genesis, but it also is present on many normal epithelial.It has been characterized as being from viscoelastic element (self-adhesion molecule) and therefore can be classified as full epithelium (pan-epithelial) Adhesion Antigen (J.Cell Biol.125 (1994), 437).

More examples of tumor associated antigen are Sialyl Tn saccharides, Lewis antigen (Lewis-x, Lewis-b, Lewis y-structure), Globo H saccharide, ganglioside such as GD2/GD3/GM2, prostate specific antigen (PSA), CA 125, CA 19-9, CA 15-3, TAG-72, EGF receptor, Her2/Neu receptor, p97, CD20 and CD21.Resisting all these antigenic monoclonal antibodies can obtain.The example of tumor associated antigen is at DeVita etal. (Eds., " Biological Therapy of Cancer ", 2.Edition, Chapter3:Biology of Tumor Antigens, Lippincott Company, ISBN0-397-51416-6 (1995), (Elektrophoresis (1999), 20:362; Curr.Pharmaceutical Design (2000), 6:485, Neoplasma (1996), 43:285)) the middle description.

The treatment for cancer method has multiple, yet the success rate of therapeutic scheme is still waited to improve at present.Except that operation and chemotherapy, also known immunotherapy.

In passive immunization therapy, monoclonal antibody (MAbs) gives the patient with suitable amount whole body, directly combines with target.The purpose of treatment is to form immune complex, by a series of immunoreation, has the cell or biological elimination of this target.Therapeutical effect depends on the concentration of MAbs in the circulation and their biological half-life, and its biological half-life is quite short usually.Therefore repeat administration is essential in suitable a period of time.If use xenogenesis MAbs, as murine antibody, untoward reaction may take place, may cause anaphylactic shock.Because this shortcoming, this immunotherapy only has been used limited a period of time.

The active immunity mode activates patient's immune system with distinct methods.After being similar to the antigen of concrete target, patient's body fluid and T cell-specific immunoreation induced defence mechanism are struggled with this target in the body.Vaccine antigens various types of and anti-various various disease are well known in the art.For example, using the vaccination resistance of hepatitis B that contains hbs antigen is that everybody knows.Shown that the high dose scope antigen and the low dose of inoculation that are used to inoculate can provide sufficient seroconversion rate (Parish D.C.et al., 1991, SouthernMedical Journal, 84,426-430; Goudeau A.etal., 1984, TheLancet, 10,1091-1092).

Also known mannan-mucin fused protein and can be used to produce cytotoxic T cell.Shown to rely on the fusion rotein dosage give mice, almost only inducing cell immunity (low dosage), only induce humoral immunization (high dose) (Pietersz G.A.et al., 1998, Cancer Immunol.Immunother., 45,321-326).

For active immunity, antigen is present in the immunogenic formulation usually so that vaccine to be provided.Simulate the antigen of this target and this target or its segmental one-level or secondary sequence and have similarity.Yet the tertiary structure of mimic epitope (mimotope) or mimic epitope antigen and this target has similarity.

Although developed many products that are used for the treatment of cancer, still being starved of provides and the known substance material that improves of ratio characteristic mutually.Particularly, aspect vaccination, need high immunogenicity, be easy to repetition and fruitful, still do not cause the product of serious side effects.

WO 02/32404 (Consejo Superior de InvestigacionesScientificas) discloses the nano-particle that is formed by metal or semiconductor atom, and the part and the nano-particle core that wherein comprise saccharide are covalently bound.These nano-particle are used to regulate the interaction of saccharide mediation and are solvable and nontoxic.Require the PCT application of the priority of GB-A-0313259.4 (ConsejoSuperior de Investigaciones Scientificas and Midatech Limited) to disclose magnetic nanoparticle, it has the core that comprises inertia and magnetic metal atom, and this core and part are covalently bound.GB application 0411537.4 (ConsejoSuperior de Investigaciones Scientificas and Midatech Limited) discloses nano-particle, and it comprises and the bonded magnetic nanoparticle of RNA part, particularly siRNA part.

Summary of the invention

The wide in range immunogenic structure that comprises adjuvant and antigenic nano-particle and have the saccharide part that relates to of the present invention.

In one aspect, the invention provides the immunogenic structure of the improvement that can be used for the treatment of various diseases, particularly in cancer prevention and treatment, be used to inoculate purpose.

Therefore, the invention provides immunogenic structure, it is by forming with covalently bound core element of saccharide part and the auxiliary peptide part of at least one T cell.

On the other hand, the invention provides immunogenic structure, it is by forming with the covalently bound core element of a plurality of saccharide parts, and wherein the saccharide part comprises at least one new epi-position (neoepitope) structure.

Core element can be antibody or derivatives thereof or fragment.Alternatively, the nano-particle that core element also can be made up of metal or semiconductor atom core, as further described herein.For example, metallic core can comprise Au, Ag, Cu, Pd or Al.WO 02/32404 and Crespo et al, Physical Review Letters, 93 (8): 87204-14, describe nano-particle and preparation thereof in detail in 2004.According to immunogenic structure of the present invention can be any saccharide part.For example, in preferred embodiments, it can demonstrate structure or functional similarity with the tumor associated antigen based on saccharide, as Lewis antigen, sialic acid or non-sialylated, or Sialyl Tn or non-sialylated Tn or even any tumor associated antigen of partly discussing of above-mentioned background.

Single saccharide part may also comprise new epi-position structure.New epi-position can be formed by the antigenic glycosylation of cell surface protein.Carbohydrate structure seldom is positioned on the tumor cell as individual molecule, but mainly with bunch existing of forming by a plurality of carbohydrate structures.These bunches can form new epi-position, and wherein single part is the destruction of mediate antibody combination and tumor cell not, but the result of saccharide bunch identification is effective immunoreation.

Can design and simulate the saccharide that exists on the tumor cell bunch by using a plurality of saccharide parts so that presenting highdensity a large amount of part.These parts can closely be connected with core element and imitate bunch, i.e. the structure that presents on the tumor cell.

For example, antibody can combine with unusual glycosylation structure on the tumor cell, but can not combine with the single saccharide part that presents on the surface texture.Therefore optional and unusual glycosylated other the antigenic inventive combination of saccharide part that derives from these glycoproteins becomes and may and can produce new epi-position structure.

By carrying out immunization therapy with unusual glycosylation target, the almost whole tumour-specific receptors that turn to feature with this unusual glycosyl can be blocked.In the middle of them be, for example whole receptors of EGF-receptor family, CD55 (791Tgp72/DAF-decay accelerating factor) receptor, TfR and P-glycoprotein.

Also have been found that the several receptors bind of the unusual glycosylated immunogenic structure of antagonism, so the signal cascade of inducing cell growth can be blocked effectively with functional mode and EGF receptor family.Therefore stop respectively or reduced combining of somatomedin and receptor.This treatment is compared more special with the immunotherapy of the antibody that uses anti-EGF receptor extracellular protein part, because do not find rare tumor associated sugars class formation on the normal cell EGF receptor.On the other hand, this treatment is more general, can be blocked simultaneously because have identical unusual glycosylated not isoacceptor.

By using the unusual glycosylated immunotherapy of antagonism, also may stop or reduce EGF or transfer albumen that the mitogenesis of cancerous cell is stimulated.The specificity of antibody and the relevant glycosylated cancerous cell of tumor is in conjunction with the interaction of having blocked growth factor receptors and its physiology part and suppress via the signal transduction of these receptors and therefore suppress cell development.Simultaneously, this antibody can be attacked tumor cell by the effect specificity in its body fluid and the cell immune system.According to the present invention, express respectively EGF receptor or EGF receptor family receptor tumor cell by specificity in conjunction with and can be cleaved or stunt disconnected.

The immunotherapy that will carry out with the target of new epi-position is improved, and it does not influence the epithelial cell of normal structure, and only influences tumor cell.

The example of this new epi-position is the epi-position that is formed by EpCAM albumen or Her-2/neu receptor and Lewis Y saccharide or suitably sialylated glycoprotein glycosylation.When producing and preparation during to the antibody of these new epitope specificities, preferably they not with deglycosylated protein binding, the glycosyl preface combination on the also not different albumen with structure.Preferred these antibody of suggestion are as the monoclonal antibody of passive immunization therapy, so that avoid non-specific interaction and side effect.The evaluation of new epi-position also can become the exploitation basis of antigen inoculation, promptly only presents the immunogen with this epi-position.This epi-position or epitope mimic thing can easily produce from suitable peptide library or by the anti-idiotype antibody technology, or can be used as natural antigenic derivant, for example fragment of existing.According to the new epi-position of selecting, can obtain just to have the antigen preparation of this new epi-position or its analogies, analogies are anti-idiotype antibody, mimic epitope for example.This antigen preparation is the valuable active substance that is used for cancer patient's active immunity, or they also can be used as diagnostic preparation.

According to immunogenic structure of the present invention can be to derive from for example auxiliary peptide part of T cell of tetanus toxoid, diphtheria toxoid or keyhole limpet hemocyanin of toxoid.This part can be a different length, for example 5 to 50 aminoacid of length, perhaps 5 to 30 aminoacid or 5 to 20 aminoacid.

Use the immunogenic peptide that is connected with immunogenic structure, when individuality is given in injection, can increase immunogenicity.For example, aminoacid sequence can be FKLQTMVKLFNRIKNNVA (SEQ IDNo.1).

The present invention also comprises the compositions that comprises one or more immunogenic structures, is used to prepare the medicine of prevention or treatment cancer.This treatment can be the active or the passive immunization therapy of following further discussion.In addition, immunogenic structure disclosed by the invention is used to separate the application that is suitable for detecting with the antibody of separating tumor cell and also can implements.

For term antibody, its derivant or fragment, be interpreted as all types of antibody, especially monospecific and polyspecific monoclonal antibody, or also has the antibody for preparing on chemistry, biochemistry or the molecular biology, or have certain specific polyclonal antibody, for example a fraction of immune serum or immune serum.

Antibody according to the invention use is preferably natural, promptly has the antibody of functional activity.Preferred this antibody does not have labelling or other detectable of connection, so that do not damage its function.Natural antibody has the character of the antibody that exists naturally among the patient.Natural antibody is different tetramer glycoprotein, and it is made up of with two identical heavy chains two identical light chains.

Yet also can use antibody derivatives, preferably it is selected from antibody fragment, conjugate, congener or derivant, or has the complex of other effector effect.In any case the preferred antibody derivant contains the segmental at least a portion of Fab, preferably together with the part of F (ab ') 2 segmental at least a portion and/or hinge region and/or the Fc part of λ or κ antibody.

In addition, can also use the single-chain antibody derivant, as so-called single-chain antibody according to the present invention.Antibody used according to the invention is immunoglobulin class preferably, as IgG, IgE, IgM, IgA or IgD.

The nano-particle of term metal or semiconductor atom bunch comprises the substrate that is suitable for as fixed ligands or a plurality of parts, and this part comprises carbohydrate group.Suitable nano-particle is described in for example WO02/32404 and Crespo R.et al. (Physical Review Letters, 2004,93,8, pp 87204-1-4), and further discusses below.Using under the situation of gold bunch, can use 50 to 500 gold atoms that core diameter in the nanometer range is provided usually.For example, core diameter can be between 50 to 500nm, 50 to 250nm, 50 to 150nm.

Part and granular core are covalently bound.The scheme of implementing this step is well known in the art, and covalent bond can for example be undertaken by sulfydryl.

The saccharide part also can link together with antibody.In order to obtain having the antibody of a plurality of bonded parts, can use the branch joint that contains the high density active group.These joints can be in conjunction with a plurality of saccharide parts, and this part can be used as clustering architecture then and is positioned on the antibody.

Can be used to prepare prevention and treat the medicine that disease resembles cancer according to immunogenic structure of the present invention.This pharmaceutical preparation can be used for immunotherapy.

On the other hand, the present invention relates to nano-particle, it has the core that comprises metal and/or semiconductor atom, and this core is connected with the antigen part.This part is saccharide or peptide antigen typically.This nano-particle can be used for delivery of antigens and is applied to various application, particularly is used in the treatment application as vaccine.In preferred embodiments, nano-particle can also be connected with adjuvant, for example the T secondary stimulus peptide or the saccharide of stimulating innate immunity system.

This delivery system has several advantages that are better than existing method.This nano-particle itself can be by stoping antigenic destruction or removing and improving antigenic immunoreation by the antigen that particle form is provided.

Using under the situation of other adjuvant, the present invention allow to use single delivery vector with delivery of antigens and adjuvant the two, or multiple antigen or adjuvant.

This nanoparticle size is little, is absorbed by cell and allows antigen to be presented on the cell surface to being enough to for a short time.Under the situation that the auxiliary peptide of T is also puted together with nano-particle, the auxiliary peptide of T also may be presented.

Therefore, on the other hand, the invention provides nano-particle, it comprises the core that comprises metal and/or semiconductor atom, and wherein core and a plurality of part are covalently bound, and part comprises the antigen part.In preferred embodiments, part also comprises adjuvant.

This antigen can be for example peptide or saccharide.In preferred embodiments, antigen is tumor specific antigen.Preferred saccharide tumor antigen comprises Sialyl Tn (STn), SialylLewis ^a(Le ^a), Sialyl Lewis ^x(Le ^x) or Sialyl Lewis ^y(Le ^y) and non-sialylated form.In a further preferred embodiment, this antigen is pathogen specific antigen, as antibacterial, virus or parasite antigen.For example, can use HIV antigen Man alpha1-2Man or parasite antigen Gal alpha 1-3 Gal.

Adjuvant can stimulating innate immunity the cell of reaction and/or the cell of adaptive immunity reaction, as T cell, especially t helper cell.This adjuvant can be saccharide part or peptide moiety.Preferred saccharide partly comprises glucose, mannose, fucose and/or N-acetyl-glucosamine.Preferred peptide moiety comprises the peptide that activates t helper cell, as from bacteriotoxic immunogenic peptide.Particularly preferred peptide moiety comprises aminoacid sequence FKLQTMVKLFNRIKNNVA (SEQ IDNo.1).

Preferably, nano-particle of the present invention is water miscible.In preferred embodiments, nano-particle of the present invention has average diameter in 0.5 to 10nm core, and more preferably 1 to 2.5nm.Preferably, the nano-particle that comprises their part has 10 to 30nm average diameter.

Except that antigen and adjuvant, nano-particle can comprise the part of one or more types.For example, other part or ligand groups or domain can comprise one or more peptides, protein structure domain, nucleic acid molecules, lipid group, carbohydrate group, any organic or anion or cation group.Carbohydrate group can be polysaccharide, oligosaccharide or monosaccharide group.Preferred part comprises glycoconjugate, therefore forms sugared nano-particle.Exist under the situation of nucleic acid molecules, nucleic acid molecules can comprise list or double-stranded DNA or RNA.In particularly preferred embodiments, this nano-particle comprises film transposition signal, and cell membrane is passed through in auxiliary their infiltrations.

This granule can have the part of an above kind that is fixed thereon, and for example 2,3,4,5,10,20 or 100 kind of different part.Alternatively or additionally, a plurality of dissimilar nano-particle can use together.In preferred embodiments, the par of the total part that links to each other with particulate single metal core is at least one part, more preferably 50 parts and most preferably 60 parts.

This nano-particle can also comprise labelling, as fluorophor, radionuclide, magnetic mark, dyestuff, NMR active atomic or the atom that uses surface plasma body resonant vibration to detect.Preferred magnetic mark comprises the paramagnetic group, comprises Mn ^{+ 2}, Gd ^{+ 3}, Eu ^{+ 2}, Cu ^{+ 2}, V ^{+ 2}, Co ^{+ 2}, Ni ^{+ 2}, Fe ^{+ 2}, Fe ^{+ 3}Or lanthanide series ^{+ 3}Preferred NMR active atomic comprises Mn ^{+ 2}, Gd ^{+ 3}, Eu ^{+ 2}, Cu ^{+ 2}, V ^{+ 2}, Co ^{+ 2}, Ni ^{+ 2}, Fe ^{+ 2}, Fe ^{+ 3}Or lanthanide series ^{+ 3}

The core of nano-particle can be a metallic core.Preferably, this metallic core comprises Au, Ag or Cu, for example is selected from the alloy of Au/Ag, Au/Cu, Au/Ag/Cu, Au/Pt, Au/Pd, Au/Ag/Cu/Pd, Au/Fe, Au/Cu, Au/Gd, Au/Fe/Cu, Au/Fe/Gd or Au/Fe/Cu/Gd.

In some embodiments, the core of nano-particle is a magnetic.Preferred magnetic nanoparticle core can comprise inertia (passive) metallic atom and magnetic metal atom, and its ratio in core is about 5: 0.1 to about 2: 5.This inert metal can be that for example gold, platinum, silver or copper and magnetic metal are ferrum or cobalt.

In yet another aspect, the invention provides the compositions of the colony that comprises one or more nano-particle as described herein.In some embodiments, the colony of nano-particle can have the identical or different part of the different densities that is connected with core.In some cases, may need to seal nano-particle, make a plurality of nano-particle can be delivered to target site.Suitable wrapper technology is well known to those skilled in the art.The colony that seals of nano-particle can be that how dissimilar one, two, three are perhaps.In preferred embodiments, said composition comprises nano-particle and drug acceptable carrier.

Many-sided, the invention provides the method for preparation nano-particle as described herein.Easily, this method comprises puts together part and nano-particle core, by part being derived joint and deutero-part being included in the reactant mixture of synthesis of nano granular core.Assemble in the nano-particle process the oneself, the nano-particle core is connected with part by joint.This joint can comprise sulfydryl, alkyl, glycol-based or peptide group.The joint group of example is by general formula HO-(CH ₂) _n-S-S-(CH ₂) _m-OH represents that wherein n and m are 1 to 5 independently.When having synthesized nano-particle, division-S-S-joint to be forming the joint of two sulfur-bearings, its each allow by-the S-base is covalently bound with the nano-particle core.In preferred embodiments, the joint group comprises C2, C 3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13 or C15 alkyl and/or C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13 or C15 glycol-based.This joint can be a hybrid juction, for example hexaethylene glycol-C11 alkyl.

Different joints can be controlled peptide and be released or keep being connected with nano-particle.For example, under the situation of BMIX described herein and BCll peptide, preceding two residues of this peptide are FK, and it is the cleavage site of cathepsin.If, can discharging the auxiliary peptide LQTMVKLFNRIKNNVA (SEQ ID NO:2) of T apart from nano-particle (use sept) enough far away, this is used for processing in the cell.

In one embodiment, can use the synthetic nano-particle of the scheme of at first describing among the WO 02/32404, wherein use the disulphide joint that part is derived, institute's derived ligand and HAuCl with the core that comprises gold atom ₄(tetra chlorauric acid) reacts in the presence of Reducing agent and prepares nano-particle.In this method, the part that is in the disulphide protection in methanol or the water can add in the tetra chlorauric acid aqueous solution.Preferred Reducing agent is a sodium borohydride.This of this method and other features are described in WO 02/32404.

In yet another aspect, the present invention also provides nano-particle described herein to be used for the prevention or the treatment that takes stopgap measures.Especially, this nano-particle can be used as vaccine.

In one aspect, the invention provides the nano-particle that limits above and be used to prepare the purposes of treatment by the medicine that gives the disease that this nano-particle improves.For example, nano-particle described herein or their derivant can be formulated as pharmaceutical composition, and give the patient with various forms, especially treat by giving the disease that antigen improves.

In one embodiment, the invention provides the purposes of nano-particle of the present invention in the medicine of preparation treatment cancer.Cancer can be for example colon, pancreas, intestinal, lung, lining, ovary or bladder cancer.

The purposes of nano-particle of the present invention in the medicine of preparation treatment infectious disease also is provided.The pathogen that causes this disease can be virus, antibacterial or parasite.

The example of treatable concrete purposes has below been described according to the present invention, and nano-particle other application in vitro and in vivo.

Describe embodiment of the present invention for example referring now to accompanying drawing, this embodiment is nonrestrictive.

The accompanying drawing summary

Fig. 1 has shown part Glc (A), Stn (B) and Le ^y(C).

Fig. 2 has shown the structure of the auxiliary peptide part BC11 of T.

Fig. 3 has shown the structure of the auxiliary peptide part BMIX of T.

Fig. 4 A shown for the starting mixt of BC11 I nano-particle (top) and the NMR spectrum of the nano-particle that produces (following).

Fig. 4 B shown for the starting mixt of BC11 II nano-particle (top) and the NMR spectrum of the nano-particle that produces (following).

Fig. 5 A has shown for the transmission electron microscope photo (left side) of BC11 I nano-particle and particle size distribution rectangular histogram (right side).

Fig. 5 B has shown for the transmission electron microscope photo (left side) of BC11 II nano-particle and particle size distribution rectangular histogram (right side).

Fig. 6 has shown the sketch map of the BC11 I nano-particle of inferring.

Fig. 7 has shown the anti-HSA-Le from the mice of inoculation BC11 I (circular and square) or BC11 II (triangle) ^yThe serum titer of IgG.Be shown as one filled squares from the control serum of not inoculating mice.Arrow is represented inoculation time.

Fig. 8 has shown the anti-HSA-Le from the mice of inoculation BC11 III (triangle) or BC11 IV (square and circular) ^yThe serum titer of IgG.Be shown as open squares from the control serum of not inoculating mice.Arrow is represented inoculation time.

Fig. 9 shows the anti-HSA-Le from the mice of inoculation BC11 II companion tetanus toxin sensitization ^yThe serum titer of IgG.Arrow is represented inoculation time.

Detailed Description Of The Invention

Nano particle

Nano particle is granule, for example metal or semiconductor atom bunch, can be as the substrate of fixed ligands.

Nano particle of the present invention dissolves in most of organic solvents, especially water. This can be used in their purification and mean that importantly they can use the part that is fixed in particle surface to present in solution. The soluble fact of nano particle has the advantage of presenting nature conformation part. Use for treatment, this nano particle is nontoxic, solvable and be stable under physiological condition.

Preferably, nano particle has the core of average diameter 0.5 to 50nm, and more preferably 0.5 to 10nm, and more preferably 0.5 to 5nm, and more preferably 0.5 to 3nm and also more preferably 0.5 to 2.5nm. Except core, when also considering part, the overall average diameter of preferred particulates is 5.0 to 100nm, and more preferably 5 to 50nm and most preferably 10 to 30nm. Can use well known commercial measurement average diameter, such as transmission electron microscopy.

Core material can be metal or semiconductor and can be comprised of the atom of an above type. Preferably, core material is the metal that is selected from Au, Fe or Cu. The nano particle core can also be formed by alloy, comprises Au/Fe, Au/Cu, Au/Gd, Au/Fe/Cu, Au/Fe/Gd and Au/Fe/Cu/Gd, and can be used for the present invention. Preferred core material is Au and Fe, and most preferred material is Au. The nano particle core preferably includes about 100 to 500 atoms (for example gold atom) so that the core diameter of nanometer range to be provided. Other useful especially core materials are with to have one or more of NMR activity atom doped, and this allows to use NMR to detect in vitro and in vivo nano particle. The NMR active atomic comprises Mn⁺²、Gd ⁺³、Eu ⁺²、Cu ⁺²、V ⁺²、Co ⁺²、Ni ⁺²、 Fe ⁺²、Fe ⁺³Or lanthanide series⁺³Or other local quantum dot of describing of the application.

The nano particle core that comprises semiconductor atom can be detected, because the semiconductor crystal of nanoscale can be taken on quantum dot, that is to say that they can absorption optical, thus with the electron excitation in the material to high-energy level more, the characteristic frequency with material discharges photon subsequently. The example of semiconductive core material is cadmium selenide, cadmium sulfide, cadmium telluride. Also comprise zinc compound such as zinc sulphide.

In some embodiments, the core of nano particle can be magnetic and comprise the magnetic metal atom, optional and the former sub-portfolio of inert metal. For example inert metal can be gold, platinum, silver or copper, and magnetic metal can be iron or gadolinium. In preferred embodiments, inert metal is gold, and magnetic metal is iron. In this case, easily, the ratio of inert metal atom and magnetic metal atom is about 5: 0.1 to about 2: 5 in the core. More preferably, this ratio is about 5: 0.1 to about 5: 1. As used herein, term " inert metal " refers to not show on magnetic and the chemical property to oxidation-stabilized metal. Inert metal can be diamagnetic or superparamagnetism. Preferably, this nano particle is superparamagnetism.

Has the example of nano particle of the core that comprises paramagnetic metal for comprising Mn⁺²、Gd ⁺³、 Eu ⁺²、Cu ⁺²、V ⁺²、Co ⁺²、Ni ⁺²、Fe ⁺²、Fe ⁺³Or lanthanide series⁺³Those.

Other magnetic nanoparticles can be comprised of material such as MnFe (spinel brick) or CoFe (Conjugate ferrite), can form nano particle (magnetic fluid adds or do not add the other core material that is defined as above). Biotechnol.Prog., 19:1095-100 (2003), J. Am.Chem.Soc.125:9828-33 (2003) has provided the example for the preparation of the self-assembly connection chemistry of this nano particle among the J.Colloid Interface Sci. 255:293-8 (2002).

In some embodiments, nano particle of the present invention or its part comprise detectable label. This mark can be element or the part of nano particle core. This mark can be detectable, because the inherent characteristic of that element of nano particle, or is connected, puts together or unite with detectable other parts. The preferred embodiment of mark comprises fluorophor, radionuclide, magnetic mark or dyestuff. Fluorophor comprises fluorescein, rhodamine or tetramethyl rhodamine, Texas-Red, Cy3, Cy5 etc., and can excite and use the utilizing emitted light of raman scattering spectrum to detect (Y.C.Cao by fluorescently-labeled, R.Jin, C.A.Mirkin, Science 2002,297:1536-1539).

In some embodiments, this nano particle comprises the radionuclide for detection of nano particle, and it uses the radioactivity of radionuclide radiation, for example by use PET, SPECT, or is used for the treatment of, and namely kills target cell. Normally used can being changed easily in this area and the example of the radionuclide that uses in the present invention comprises^99mTc, it exists with the various states of oxidation, although the most stable be TcO^4-； ³²P or³³P； ⁵⁷Co； ⁵⁹Fe； ⁶⁷Cu, it is usually as Cu²⁺Salt;⁶⁷Ga, it uses Ga usually³⁺Salt, for example gallium citrate;⁶⁸Ge； ⁸²Sr； ⁹⁹Mo； ¹⁰³Pd； ¹¹¹In, it is typically used as In³⁺Salt;¹²⁵I or¹³¹I, it is typically used as sodium iodide;¹³⁷Cs； ¹⁵³Gd； ¹⁵³Sm； ¹⁵⁸Au； ¹⁸⁶Re; Be typically used as Tl⁺Salt is such as thallium chloride²⁰¹Tl； ³⁹Y ³⁺； ⁷¹Lu ³⁺With²⁴Cr ²⁺ Radionuclide serves as a mark and the universal use of tracer is well known in the art and can is changed easily and be used for aspect of the present invention by those skilled in the art. Can extremely easily use radionuclide, by mixing the nano particle core or they being comprised the mark that exists as with the ligand moiety that is fixed on the nano particle.

In addition or alternatively, nano particle of the present invention, or they and the interactional result of other kinds, can with a lot of utilizations well known in the art as noted above the mark of being combined with nano particle or be detected by the character of utilizing them. These methods that detect nano particle can be from detecting the aggregation that produces when nano particle when another kind is combined, for example by simply visually observing or using complicated technology such as transmission electron microscopy (TEM) or atomic force microscopy (AFM) with the developing nano particle by using light scattering (containing the transmission of the solution of nano particle), arriving. Other method that detects metallic particles is to use plasma resonance, and it is the metal surface electron excitation, is usually caused by light radiation. Surface plasma body resonant vibration (SPR) phenomenon is present in the interface of metal (such as Ag or Au) and dielectric material such as air or water. When analyte and the ligand binding that is fixed in nano grain surface have changed interfacial refractive index, SPR just changes. More advantages of SPR are that it can be used for monitoring real-time interaction. As mentioned above, if nano particle comprises or with have the atom doped of NMR activity, so this technology be used in external or body in detect this particle, use technology well known in the art. Use can detect nano particle based on the system that uses nano particle to promote the quantitative signal of silver reduction (I) to amplify. If nano particle comprises part as fluorescence probe, can use fluorescence spectrum. And the tagging of carbohydrate can be used for promoting their detection.

This part can comprise the inertia carbohydrate content (for example glucose) that allows antigen and support density in the final construct of arbitrarily control.

Antigen

Antigen is a kind of molecule, and it is by the cell of adaptive immune system, i.e. T cell or B cell or the two specific recognition simultaneously. Antigen comprises albumen, carbohydrate, nucleic acid and even little molecule such as toxin. In a preferred embodiment of the invention, antigen is tumour specific antigen, especially peptide or carbohydrate tumour specific antigen. In other preferred embodiments, antigen is the antigen of finding on the pathogen, such as virus, bacterium or parasite.

The example of carbohydrate tumour specific antigen comprises the sialylated and non-sialylated Lewis structure that the sugar chain by glycoprotein on the tumor cell surface and glycolipid carries. These antigens are usually crossed the adhesion of expressing and seem to participate in tumour cell and endothelium in tumour. For example, Sialyl Lewis^aBe responsible for adhering to people's colon, pancreas and stomach cancer cell, and Sialyl Lewis^xBe responsible for the combination of lung, liver and ovarian cancer cell. Sialyl Le^aIn colorectum, liver and cancer of the stomach, excessively express (for summary, referring to Ugorski and Laskowska (2002), Acta Biochimican Polonica, 49,303-311).

Adjuvant

Adjuvant is the immunoreactive reagent that improves antigen. Adjuvant can improve antibody response by the cell that stimulates the adaptive immunity reaction, and/or can act on by non-specific increase innate immune system is active. Usually, the antigen that improves antibody response is by being exposed to lymphocytic suitable position concentrated antigen or accomplishing this point by the generation of stimulating cytokine in that antigen more. When the vaccine delivery platform, nano particle oneself can serve as adjuvant, by the antigen of particle form is provided, so that they are absorbed easilier by antigen presenting cell, such as macrophage. Yet, use otherwise other reagent of raising immune system of puting together with nano particle, this effect can strengthen greatly.

The adjuvant that increases the innate immune system activity comprises the carbohydrate part, such as wood sugar, fucose, mannose and N-acetyl-amino glucose. These work in several modes. They can be combined in the secretion molecule that circulates in blood and the lymph, and it triggers the complement component division and causes complement fixation. They also can resemble macrophage in conjunction with the surface receptor on the phagocyte, and such as CD2-6 (MMR), it stimulates phagocytosis and cell endocytic.

This adjuvant also can work in stimulating the adaptive immunity reaction.

They can cause in conjunction with startup the cell surface receptor of the signal of effector molecule (cell factor) release. For example, the combination of To11 sample acceptor causes their secrete cytokines on carbohydrate and the surface of dendritic cells, comprises interleukin 6 (IL-6), and it hinders regulatory T cells depression effect T cell to the ability of replying of antigen.

The B cell also has To11 sample acceptor. When bind receptor, it improves the B cell to the reaction of antigen.

Other adjuvants directly stimulate the cell of adaptive immunity reaction. For example, can use the peptide that stimulates helper cell (HTL) reaction, to strengthen the ctl response to antigenic peptides. This peptide can be for example from the high immunogenicity antigen of tetanus toxoid, and it produces non-specific htl response. Propagation, survival and the effector function of the HTL enhanced CT L of activation.

This peptide is particularly useful in the immune response that improves sugar antigen. Although sugar antigen can be by the combination of carbohydrate specific b cells and internalization, they can not activate the HTL that is only activated by peptide. Yet nano particle of the present invention can evoke B cell and htl response, because they all are combined with sugar antigen and HTL activated peptide. This provides the immune response that greatly improves.

Administration and treatment

Nanoparticulate compositions of the present invention can give the patient by a lot of different approach, comprises intestines or parenteral route. Parenteral comprises by following administration: intravenous, skin or subcutaneous, nose, intramuscular, intraocular, thoroughly in epithelium, the peritonaeum and local (comprise in corium, eyes, rectum, the nose, suction and aerosol) and rectum whole body approach.

For example send the rifle ballistic by injection or use and implement administration, send rifle and accelerate them via the outer field transdermal route of epidermis. Then nano particle can be absorbed by for example dendritic cells, and dendritic cells are ripe by the lymphatic system migration, cause the inoculation of regulating immune response and anti-this antigen. Nano particle can also be sent with aerosol. Small-sized nano particle may be done like this.

Compact nano particle of the present invention is the great advantages that is delivered to cell and tissue, because they can be by Cell uptake, even when itself and target-seeking or treatment minute sub-connection.

Nano particle of the present invention can be configured to pharmaceutical composition, can be solid or forms of liquid compositions. This composition will comprise the carrier of some types usually, for example solid carrier such as gelatin or adjuvant or inert diluent, or liquid-carrier such as water, oil, animal or plant oil, mineral oil or artificial oil. Can comprise saline, or dihydroxylic alcohols such as ethylene glycol, propane diols or polyethylene glycol. This composition and preparation contain at least this compound of 0.1wt% usually.

For intravenous, skin or hypodermic injection, or in the diseased region injection, active component will be parenteral acceptable aqueous solution form, and this solution is pyrogen-free and has suitable pH, isotonicity and stability. Those skilled in the relevant art can prepare suitable solution certainly, use for example solution of this compound or derivatives thereof, for example in physiological saline, with the dispersion liquid of glycerine, liquid macrogol or oils preparation. Except one or more compounds, optional and other active components make up, and said composition can comprise one or more medicines can accept excipient, carrier, buffer, stabilizing agent, isotonic agent, anticorrisive agent or antioxidant or other materials well known to those skilled in the art. This material should be nontoxic and the effect that can not hinder active component. The definite character of carrier or other materials can depend on method of administration, for example oral or parenteral.

Composition of liquid medicine typically is formulated as has about pH of 3.0 to 9.0, more preferably about 4.5 to 8.5, and also more preferably about 5.0 to 8.0. The pH of composition can keep with buffer solution, and such as acetate, citrate, phosphate, succinate, Tris or histidine, buffer solution typically uses with the scope of about 1mM to 50mM. Perhaps the pH of composition can be by regulating with physilogically acceptable acid or alkali.

Generally include anticorrisive agent in the pharmaceutical composition to hinder growth of microorganism, prolong the shelf life of composition and allow multi-purpose package. The example of anticorrisive agent comprises carbolic acid, metacresol, phenmethylol, P-hydroxybenzoic acid and its ester, methylparoban, propylparaben, benzalkonium chloride and benzethonium chloride. Typically anticorrisive agent uses with the scope of about 0.1 to 1.0% (w/v).

Preferably, pharmaceutical composition gives individuality with prevention effective dose or treatment effective dose (depending on the circumstances, although prevention may be considered to treatment), and this is enough to show and is beneficial to individuality. Typically, this provides generation and benefits effectively activity of individual treatment. Give the actual amount of compound, and injection speed and time course, will depend on the sanatory character of institute and seriousness. The requirement for the treatment of is the thing in general doctor and other doctor scopes of offical duty as determining dosage etc., typically considers other known factors of situation, site of delivery, medication and professional of disorder to be treated, patient. Handbook of Pharmaceutical Additives, 2nd Edition (eds.M.Ash and I.Ash), 2001 (Synapse Information Resources, Inc., Endicott, New York, USA); Remington ' s Pharmaceutical Sciences, 20th Edition, 2000, pub.Lippincott, Williams ﹠ Wilkins; And Handbook of Pharmaceutical Excipients, 2nd edition can find the example of above-mentioned technology and scheme in 1994. For example, preferred composition is with about 0.01 to the 100mg reactive compound of per kilogram of body weight, and more preferably the dosage of about 0.5 to 10mg/kg body weight gives the patient.

In the very clear situation that relates to oncotherapy, treatment comprises by the doctor implements ameliorate tumor to any means of patient's effect. Therefore, although the fully alleviation of tumour is expectation target, effectively treatment also comprises and can reach that tumor section is alleviated and the tumor growth rate that slows down comprises any means of transfer. This means can effectively prolong and/or improve the quality of living and palliate a disease symptom.

Immunotherapy

Composition of the present invention such as immunogenic structure, can be used for prevention and treatment disease, resembles cancer, and more specifically to immunotherapy.

In the present invention, term " inoculation " meaning is active immunity, exactly owing to give a small amount of antigen induction specific immune response, for example via approach in subcutaneous, the corium, in the intramuscular, oral or nose, the individual identification that this antigen is vaccinated is external and therefore has immunogenicity in suitable preparation. Therefore in order to set up the specific immune response of anti-this antigen, this antigen is used as immune " triggering son ".

According to the present invention, inoculation can be treatment or preventative, as all antimicrobial vaccines. For example, do not suffer from cancered individual inoculation by giving, perhaps may reach the preventive protection of anticancer outburst. The example that may apply this premunitive individuality is the individuality that develops into the risk increase of cancer, although this application is not limited to this individuality. The patient who is in risk of cancer may have the tumour (primary tumo(u)r or transfer) of development, or demonstrates cancer susceptibility.

For the active immunity according to cancer patient of the present invention, immunogenic structure typically is configured to vaccine. Preferably, this pharmaceutical preparation contains drug acceptable carrier, and described carrier for example may further include auxiliary substance, buffer, salt and/or anticorrisive agent. Pharmaceutical preparation can for example be used for prevention and treatment cancer patient's cancer related disorders, forms as shifting. When doing like this, antigen presenting cell in vivo or stripped specificity regulate so that produce the immune response of anti-TAA.

For the active immunity with specific antigen or antigen combination, usually use bacterin preparation, it contains, and immunogene-it is natural TAA or its epi-position, analogies or new epitope mimic thing, or immunogenic antibodies-mainly with low concentration, for example from the immunogenicity amount of 0.01 μ g to 10mg scope, yet dosage range can increase to 100 to 500mg scope. Immunogenicity according to antigen inoculation, this immunogenicity is for example determined by the sequence of external kind or by derivatization, or also respectively according to the auxiliary substance or the adjuvant that use, for example can select in 0.01 μ g to the 1mg scope the suitable immunogenicity dosage of preferred 100 μ g to 500 μ g. Yet, will be delivered to the antigen inoculation that biological bank vaccine can also contain much higher amount in the extended period, for example at least more than the 1mg to 100mg.

Concentration will depend on the liquid that gives or the amount of suspension vaccine. Vaccine provides with instant syringe or ampoule form usually, has the volume of 0.01 to 1ml scope, and preferred 0.1 to 0.75ml. The antigen inoculation of kit composition of the present invention preferably is present in and is suitable in subcutaneous, intramuscular and the corium or in the drug acceptable carrier of cutaneous penetration. Also can by the mucus administration, for example pass through in the nose or per oral inoculation. If use solid matter as the auxiliary agent of bacterin preparation, for example will contain respectively adsorbate or the suspended mixture of the vaccine antigen of auxiliary agent. In particular embodiments, vaccine exists with the aqueous vaccine in solution or the aqueous solvent.

Preferably, the inoculation unit of tumor vaccine provides in instant syringe or ampoule. The stabilization formulations of vaccine can advantageously be sold with the instant form. Although needn't require anticorrisive agent, such as the content of other anticorrisive agents of thimerosal or tolerable property improvement, it can so that preparation provide in cryogenic temperature to the storage temperature stability inferior of room temperature form more of a specified duration. Yet can also provide and can melt respectively when needed or reconstruct with freezing or lyophilized form according to vaccine of the present invention.

Verified by using adjuvant to be suitable for increasing the immunogenicity of antibody used according to the invention. For this purpose, use solid matter or aqueous vaccine adjuvant, for example aluminium hydroxide (Alu-Gel) or aluminum phosphate, growth factor, lymphokine, cell factor such as IL-2, IL-12, GM-CSF, IFN-γ or complement factor such as C3d, other Liposomal formulation or have the preparation of other antigen, immune system has produced strong immune response to this antigen, such as tetanus toxoid, bacteriotoxin, such as the derivative of PE and lipid A and lipopolysaccharides.

In the covalently bound situation of toxoid peptide and core texture, the needs of adjuvant can reduce or cancel.

Embodiment

Embodiment 1

Load sugar antigen, T assistant carrier and the preparation and the sign of nano-particle that is connected in the glucose of gold surface are described below.

Use part Glc, STn and Le ^y(Fig. 1).Select C ₂The aliphatic sept is connected glucose residue with gold surface, and C ₅The aliphatic joint is used for two antigenic connections.

By the amino terminal group will be from tetanus toxoid mix T cell peptide epitopes (FKLQTMVKLFNRIKNNVA) and C ₁₁The aliphatic sept connects and the auxiliary peptide part BC11 (Fig. 2) of preparation T.By the amino terminal group with identical tetanus toxoid T cell peptide epitopes with by hexaethylene glycol and C ₁₁The hybrid juction that the aliphatic sept is formed connects and the auxiliary peptide part BMIX (Fig. 3) of preparation T.

For the preparation of sugared nano-particle, Glc, STn, Le ^yBe dissolved in the deuterate methanol with desired proportion with BC11 or BMIX and write down these solution at 500MHz ¹H NMR spectrum.The spectrum of these mixture allows clearly to identify that signal that belongs to separate constituent and the intensity that confirms these signals are equivalent to according to the desired intensity of the ratio of different ligands in the original solution (Fig. 4).After the methanol dilution, this mixture of processing as described below repeats this granule of purification to obtain corresponding sugared nano-particle by centrifugal filtration.These constructs are in the deuterate heavy water ¹H NMR spectrum (Fig. 4) shows among the GNPs that obtains under the experiment condition of setting up before used has kept original part ratio.Supernatant ¹H NMR spectrum also confirms described part ratio.

According to said procedure, prepare following sugared nano-particle: BC11 I (Glc: STn: BC1128: 1: 1), BC11 II (Glc: STn: BC11 20: 9: 1), BC11 III (Glc: STn: Le ^y: 1), BC11 IV (Glc: STn: Le BC11 18: 10: 1: ^y: BC1118: 1: 10: 1), BMIX I (Glc: STn: BMIX 28: 1: 1), BMIX II (Glc: STn: BMIX20: 9: 1), BMIX III (Glc: STn: Le ^y: 1), BMIX IV (Glc: STn: Le BMIX 18: 10: 1: ^y: BMIX 18: 1: 10: 1) BMIX V (Glc: Le ^y: BMIX 28: 1: 1) and BMIXVI (Glc: Le ^y: BMIX 20: 9: 1).

Use transmission electron microscopy (TEM) to measure the average diameter of these constructs, be respectively 2.25nm, 1.45nm, 2.05nm and 1.81nm and be respectively 1.80nm, 1.55nm, 2.19nm, 1.77nm, 1.64nm and 1.79nm for BC11 I, BC11 II, BC11 III and BC11IV for BMIX I, BMIX II, BMIX III, BMIX IV, BMIX V and BMIX VI.From these average diameters, quantity, the chain that is connected with gold and the approximate molecular weight of GNPs of gold atom in estimating bunch. ^[3]These values have below been provided.

Prepared and characterized several other sugared nano-particle (1-4) (data not shown).The component of these constructs is as follows: 1 (STn, 100%), 2 (Le ^y100%) 3 (Glc: STn: HS (CH, ₂) ₁₀COOH joint 20: 1: 1), 4 (Glc: STn: HS (CH ₂) ₁₁O (CH ₂CH ₂O) ₆CH ₂COOH joint 28: 1: 1).

Experimental section

HAuCl ₄And NaBH ₄Available from Aldrich Chemical Company.For all experiments and solution, use nanometer pure water (Nanopure water) (18.1m Ω).

The preparation of peptide BC11-Au-antigenic carbohydrates nano-particle

a)BC11 I(Glc∶STn∶BC11 28∶1∶1).

Peptide BC11 (3.1mg, 1.31 μ mol) is dissolved in CF ₃COOD (100 μ L) flows down concentrated this solution until the formation of observing oil at argon.Add Glc (8.8mg, 36.7 μ mol) and STn (0.8mg, 1.31 μ mol) then, and mixture is dissolved in CD ₃Among the OD (500 μ L). ¹Ratio is 28: 1: 1 between the signal of H-NMR spectrum demonstration Glc, STn and BC11.

Dilute this solution and add trifluoroacetic acid with MeOH (2.8mL) pH value is transferred to 1.Add HAuCl ₄Aqueous solution (286 μ L, 0.025M).Then, add 1N NaBH with several parts ₄Aqueous solution (157 μ L), the companion shakes fast.The black suspension that forms is shaken other 2h and is passed through decant separation of methanol layer ^[4]Black solid water-soluble (700 μ L) is also used centrifugal filtration (AMICON MW10000,30min, 14000rpm) purification.This process repeats twice, until nano-particle not saliferous and initiation material.Residue in the AMICON filter be dissolved in the 500 μ L water and lyophilizing so that 1.2mg BC11 I to be provided nano-particle.TEM: average diameter 2.25nm, 309 gold atoms, 92 chains, MW=90586.

b)BC11 II(Glc∶STn∶BC11 20∶9∶1).

Peptide BC11 (4.0mg, 1.7 μ mol) is dissolved in CF ₃COOD (100 μ L) flows down concentrated this solution until the formation of observing oil at argon.Add Glc (8.1mg, 33.9 μ mol) and STn (9.3mg, 15.2 μ mol) then, and mixture is dissolved in CD ₃Among the OD (500 μ L). ¹Ratio is 20: 9: 1 between the signal of H-NMR spectrum demonstration Glc, STn and BC11.

Dilute this solution and add trifluoroacetic acid with MeOH (3.7mL) pH value is transferred to 1.Add HAuCl ₄Aqueous solution (368 μ L, 0.025M).Then, add 1N NaBH with several parts ₄Aqueous solution (202 μ L), the companion shakes fast.The black suspension that forms is shaken other 2h and is passed through decant separation of methanol layer ^[4]Black solid water-soluble (500 μ L) is also used centrifugal filtration (AMICON MW10000,30min, 14000rpm) purification.This process repeats twice, until nano-particle not saliferous and initiation material.Residue in the AMICON filter is dissolved in the 500 μ L water and lyophilizing so that 1.8mg BC11 II to be provided nano-particle.TEM: average diameter 1.45nm, 116 gold atoms, 53 chains, MW=45358.

c)BC11 III(Glc∶STn∶Le ^y∶BC11 18∶10∶1∶1).

Peptide BC11 (2.8mg, 1.2 μ mol) is dissolved in CF ₃COOD (100 μ L) flows down concentrated this solution until the formation of observing oil at argon.Add Glc (5.1mg, 21.3 μ mol), Le then ^y(0.9mg, 1.2 μ mol) and STn (7.3mg, 11.8 μ mol), and mixture is dissolved in CD ₃Among the OD (500 μ L). ¹The H-NMR spectrum shows Glc, STn, Le ^yAnd ratio is 18: 10: 1 between the signal of BC11: 1.

Dilute this solution and add trifluoroacetic acid with MeOH (2.4mL) pH value is transferred to 1.Add HAuCl ₄Aqueous solution (256 μ L, 0.025M).Then, add 1N NaBH with several parts ₄Aqueous solution (142 μ L), the companion shakes fast.The black suspension that forms is shaken other 2h and is passed through decant separation of methanol layer ^[4]Black solid water-soluble (500 μ L) is also used centrifugal filtration (AMICON MW10000,30min, 14000rpm) purification.This process repeats twice, until nano-particle not saliferous and initiation material.Residue in the AMICON filter is dissolved in the 500 μ L water and lyophilizing so that 0.5mg BC11 III to be provided nano-particle.TEM: average diameter 2.05nm, 225 gold atoms, 71 chains, MW=76661.

d)BC11 IV(Glc∶STn∶Le ^y∶BC11 18∶1∶10∶1).

Peptide BC11 (2.7mg, 1.1 μ mol) is dissolved in CF ₃COOD (100 μ L) flows down concentrated this solution until the formation of observing oil at argon.Add Glc (4.9mg, 20.6 μ mol), Le then ^y(8.8mg, 11.4 μ mol) and STn (0.7mg, 1.1 μ mol), and mixture is dissolved in CD ₃Among the OD (500 μ L). ¹The H-NMR spectrum shows G1c, STn, Le ^yAnd ratio is 18: 1: 10 between the signal of BC11: 1.

Dilute this solution and add trifluoroacetic acid with MeOH (2.3mL) pH value is transferred to 1.Add HAuCl ₄Aqueous solution (248 μ L, 0.025M).Then, add 1N NaBH with several parts ₄Aqueous solution (137 μ L), the companion shakes fast.The black suspension that forms is shaken other 2h and is passed through decant separation of methanol layer ^[4]Black solid water-soluble (500 μ L) is also used centrifugal filtration (AMICON MW10000,30min, 14000rpm) purification.This process repeats twice, until nano-particle not saliferous and initiation material.Residue in the AMICON filter is dissolved in the 500 μ L water and lyophilizing so that 1.2mg BC11 IV to be provided nano-particle.TEM: average diameter 1.81nm, 201 gold atoms, 71 chains, MW=75409.

The preparation of peptide BMIX-Au-antigenic carbohydrates nano-particle

a)BMIX I(Glc∶STn∶BMIX 28∶1∶1).

Peptide BMIX (3.5mg, 1.31 μ mol) is dissolved in CF ₃COOD (100 μ L) flows down concentrated this solution until the formation of observing oil at argon.Add Glc (8.8mg, 36.6 μ mol) and STn (0.8mg, 1.31 μ mol) then, and mixture is dissolved in CD ₃Among the OD (500 μ L). ¹Ratio is 28: 1: 1 between the signal of H-NMR spectrum demonstration Glc, STn and BMIX.

Dilute this solution and add trifluoroacetic acid with MeOH (2.7mL, cumulative volume 3.2mL) pH value is transferred to 1.Add HAuCl ₄Aqueous solution (314 μ L, 0.025M).Then, add 1N NaBH with several parts ₄Aqueous solution (157 μ L), the companion shakes fast.The black suspension that forms is shaken other 2h and is passed through decant separation of methanol layer ^[2]Black solid water-soluble (700 μ L) is also used centrifugal filtration (AMICON MW 10000,30min, 14000rpm) purification.This process repeats twice, until nano-particle not saliferous and initiation material.Residue in the AMICON filter is dissolved in the 500 μ L water and lyophilizing so that 1.0mg BMIX I to be provided nano-particle.TEM: average diameter 1.80nm, 201 gold atoms, 71 chains, MW=63300.

b)BMIX II(Glc∶STn∶BMIX 20∶9∶1).

Peptide BMIX (3.5mg, 1.31 μ mol) is dissolved in CF ₃COOD (100 μ L) flows down concentrated this solution until the formation of observing oil at argon.Add Glc (6.3mg, 26.2 μ mol) and STn (7.2mg, 11.7 μ mol) then, and mixture is dissolved in CD ₃Among the OD (500 μ L). ¹Ratio is 20: 9: 1 between the signal of H-NMR spectrum demonstration Glc, STn and BMIX.

Dilute this solution and add trifluoroacetic acid with MeOH (2.7mL, cumulative volume 3.2mL) pH value is transferred to 1.Add HAuCl ₄Aqueous solution (314 μ L, 0.025M).Then, add 1N NaBH with several parts ₄Aqueous solution (157 μ L), the companion shakes fast.The black suspension that forms is shaken other 2h.Here can not be by decant separation of methanol layer.Then volume is reduced to 1mL, add water (700 μ L) and use centrifugal filter (AMICON MW 10000,30min, 14000rpm) purification.This process repeats twice, until nano-particle not saliferous and initiation material.Residue in the AMICON filter is dissolved in the 500 μ L water and lyophilizing so that 2.5mg BMIX II to be provided nano-particle.

TEM: average diameter 1.55nm, 140 gold atoms, 53 chains, MW=50567.

c)BMIX III(Glc∶STn∶Le ^y∶BMIX 18∶10∶1∶1).

Peptide BMIX (3.9mg, 1.46 μ mol) is dissolved in CF ₃COOD (100 μ L) flows down concentrated this solution until the formation of observing oil at argon.Add Glc (6.3mg, 26.2 μ mol), Le then ^y(1.1mg, 1.46 μ mol) and STn (9.0mg, 14.6 μ mol), and mixture is dissolved in CD ₃Among the OD (500 μ L). ¹The H-NMR spectrum shows Glc, STn, Le ^yAnd ratio is 18: 10: 1 between the signal of BMIX: 1.

Dilute this solution with MeOH (3.2mL, cumulative volume 3.7mL).Add HAuCl ₄Aqueous solution (350 μ L, 0.025M).Then, add 1N NaBH with several parts ₄Aqueous solution (193 μ L), the companion shakes fast.The black suspension that forms is shaken other 2h and is passed through decant separation of methanol layer ^[2]Black solid water-soluble (500 μ L) is also used centrifugal filter (AMICON MW 10000,30min, 14000rpm) purification.This process repeats twice, until nano-particle not saliferous and initiation material.Residue in the AMICON filter is dissolved in the 500 μ L water and lyophilizing so that 2.8mg BMIXIII to be provided nano-particle.

TEM: average diameter 2.19nm, 309 gold atoms, 92 chains, MW=103569.

d)BMIX IV(Glc∶STn∶Le ^y∶BMIX 18∶1∶10∶1).

Peptide BMIX (3.7mg, 1.38 μ mol) is dissolved in CF ₃COOD (100 μ L) flows down concentrated this solution until the formation of observing oil at argon.Add Glc (6.0mg, 24.8 μ mol), Le then ^y(10.7mg, 13.8 μ mol) and STn (0.85mg, 1.38 μ mol), and mixture is dissolved in CD ₃Among the OD (500 μ L). ¹The H-NMR spectrum shows Glc, STn, Le ^yAnd ratio is 18: 1: 10 between the signal of BMIX: 1.

Dilute this solution and add trifluoroacetic acid with MeOH (3.0mL, cumulative volume 3.5mL) pH value is transferred to 1.Add HAuCl ₄Aqueous solution (330 μ L, 0.025M).Then, add 1N NaBH with several parts ₄Aqueous solution (182 μ L), the companion shakes fast.The black suspension that forms is shaken other 2h and is passed through decant separation of methanol layer ^[2]Black solid water-soluble (500 μ L) is also used centrifugal filter (AMICON MW 10000,30min, 14000rpm) purification.This process repeats twice, until nano-particle not saliferous and initiation material.Residue in the AMICON filter is dissolved in the 500 μ L water and lyophilizing so that 1.8mg BMIX IV to be provided nano-particle.

TEM: average diameter 1.77nm, 201 gold atoms, 71 chains, MW=75934.

e)BMIX V{Glc∶Le ^y∶BMIX 28∶1∶1).

Peptide BMIX (3.7mg, 1.4 μ mol) is dissolved in CF ₃COOD (100 μ L) flows down concentrated this solution until the formation of observing oil at argon.Add Glc (9.4mg, 39.1 μ mol) and Le then ^y(1.1mg, 1.4 μ mol), and mixture is dissolved in CD ₃Among the OD (500 μ L). ¹The H-NMR spectrum shows Glc, Le ^yAnd ratio is 28: 1: 1 between the signal of BMIX.

Dilute this solution and add trifluoroacetic acid with MeOH (3.0mL, cumulative volume 3.5mL) pH value is transferred to 1.Add HAuCl ₄Aqueous solution (331 μ L, 0.025M).Then, add 1N NaBH with several parts ₄Aqueous solution (182 μ L), the companion shakes fast.The black suspension that forms is shaken other 2h and is passed through decant separation of methanol layer ^[2]Black solid water-soluble (700 μ L) is also used centrifugal filter (AMICON MW 10000,30min, 14000rpm) purification.This process repeats twice, until nano-particle not saliferous and initiation material.Residue in the AMICON filter is dissolved in the 500 μ L water and lyophilizing so that 0.7mg BMIX V to be provided nano-particle.

TEM: average diameter 1.64nm, 140 gold atoms, 53 chains, MW=45568.

f)BMIX VI(Glc∶Le ^y∶BMIX 20∶9∶1).

Peptide BMIX (3.5mg, 1.31 μ mol) is dissolved in CF ₃COOD (100 μ L) flows down concentrated this solution until the formation of observing oil at argon.Add Glc (6.3mg, 26.2 μ mol) hLe then ^y(9.2mg, 11.8 μ mol), and mixture is dissolved in CD ₃Among the OD (500 μ L). ¹The H-NMR spectrum shows Glc, Le ^yAnd ratio is 20: 9: 1 between the signal of BMIX.

Dilute this solution and add trifluoroacetic acid with MeOH (2.7mL, cumulative volume 3.2mL) pH value is transferred to 1.Add HAuCl ₄Aqueous solution (314 μ L, 0.025M).Then, add 1N NaBH with several parts ₄Aqueous solution (157 μ L), the companion shakes fast.The black suspension that forms is shaken other 2h.Here can not be by decant separation of methanol layer.Then volume is reduced to 1mL, add water (700 μ L) and use centrifugal filter (AMICON MW 10000,30min, 14000rpm) purification.This process repeats twice, until nano-particle not saliferous and initiation material.Residue in the AMI CON filter is dissolved in the 500 μ L water and lyophilizing so that 1.8mg BMIX VI to be provided nano-particle.

TEM: average diameter 1.79nm, 201 gold atoms, 71 chains, MW=73864.

The purposes of the nano-particle induction of immunity reaction of conjugated antigen

With nano-particle BC11 I, II, III and IV inoculation mice and monitoring immunoreation to conjugated antigen.30 μ g nano-particle are injected in 200 μ l adjuvants (Sigma M-6536-MPL+TDM).Gave four injections of 2 * 100 μ l at the 0th, 28,40 and 157 day.First three time is subcutaneous to be given and injects intraperitoneal for the last time to give.39th, blood sampling in 48 and 67 days and measure anti-HSA-Le ^yIgG titre (Fig. 7 and 8).See among BC11 I/II and the BC11 III/IV big difference is arranged between the result.The rising of the titre of anti-Ley is owing to use the nonspecific effect of adjuvant among the BC11 I/II.When repeating immunity, titre does not increase but begins and reduces.On the contrary, use BC11 III/IV, titre increases with booster immunization, proves real immunization.

BC11 II is used for the inoculation of tetanus toxoid sensitization.At the 0th day, give the 2.35IU tetanus toxoid of animal subcutaneous injection from Aventis Pasteur MSD-Diftavax (2 bottles are mixed with common 3.4ml, and administration 100 μ l) with 0.9% saline.At the 14th day, 2 * 100 μ l that contain 50 μ g nano-particle subcutaneous injection and at the 34th day in adjuvant (Sigma M-6536) contained 2 * 100 μ l peritoneal injection in adjuvant of 50 μ g nano-particle.Blood sampling in the 33rd and 44 day.Fig. 9 has shown the result (different animals with different symbolic representations) of 5 mices.First arrow is represented tetanus toxoid sensitization, and second arrow represented to represent to use the nano-particle peritoneal injection with nano-particle subcutaneous injection and the 3rd arrow.

Embodiment 2

Gold nano grain is as immunogenic structure

The preparation for preparing gold nano grain according to WO 02/32404 described technology.Preparing ratio with the peptide sequence FKLQTMVKLFNRIKNNVA of various density is alpha-Sialyl-Tn: Lewis y=30: 3 with the different constructs of 3: 30 gold nano grain.Remaining space can seal with Glc-C2.Alternatively, joint also can be sequence FKFQILYNSIMG.

The ratio of Sialyl-Tn or Lewisy or two splice combinations also can use the technology according to WO02/32404 to increase.For example, can easily be connected up to hundreds of carbohydrate groups with core element.The ratio of different ligands can easily change.Alternatively, also can be and single Sialyl Tn of core element covalent bond or Lewis y saccharide.

Antibody is as immunogenic structure

The coupling of SialylTn saccharide and HE2

SialylTn-O (CH ₂) ₃NH (CH ₂) ₄COO-pNp and HE2 coupling.In order to increase the quantity of SialylTn saccharide part, can use branch joint well known in the art with the saccharide ligand coupling to antibody.By SEC, LDS-PAGE, Western blotting and different ELISA analysis of experiments end-products.

Experimental section

Material and method

HE2 Panorex，10mg/ml，Lotl70901

SialylTn-O (CH ₂) ₃NH (CH ₂) ₄COO-pNp, 2 * 5mg, Fa.LectinityDMF (N, and dinethylformamide (anhydrous, Merck)

Coupling buffer: 0.1M Na ₂HPO ₄+ 0.15M NaCl (pH=8)

Preparation buffer: NaCl 0.86%+1mM Na ₂HPO ₄(pH=6.0).

Step

1. use the Slide-A-Lyzer dialysis cassette, with 100mg HE2 (V=I0ml; Concentration: 10mg/ml) dialysed 20 hours at 4 ℃ facing to 2 * 700ml coupling buffer, until～the 10ml volume, according to the about 10mg/ml of the concentration of SEC.

2. with 2 * 100 μ l DMF (100 μ l/ bottle) dissolving, 2 * 5mg SialylTn-O (CH ₂) ₃NH (CH ₂) ₄COO-pNp.

(3.SialylTn in DMF) solution is added into～10ml (～100mg) icy HE2 (in coupling buffer).

4. with 100 μ l DMF (being transferred to bottle 2 from bottle 1) rinsing SialylTn-bottles, it also is added into reactant mixture.

5. reactant mixture is in+4 ℃ of rotations spend the night (28 hours).With SEC observing response kinetics (referring to 5.3.1 and 6.3.1).

6. use the Slide-A-Lyzer dialysis cassette, with HE2-SialylTn (10ml ,～whole solution 10mg/ml) facing to 2 * 800ml preparation buffer 4 ℃ of dialysis 20 hours.

Analyze

Size exclusion chromatography

On ZORBAX GF-250 post, in the Dionex system, roll over (SEC) quantitatively concentration of HE2-SialylTn by the size exclusion layer.With gel filtration standard (Fa.BioRAD) test HPLC system.HE2 is as the quantitative reference standard of HE2-SialylTn.The efficient of the coupling reaction of retention time (increasing relevant with molecular weight) shortening and SialylTn and HE2 is relevant.The data of receiving show that coupling efficiency is with reaching capacity at the little the reaction time of 23-27 and increase.

LDS-PAGE (lithium dodecyl sulfate PAGE)

Use Bis-Tris-Gel (4-12%) " SilverXpress ^TMThe LDS-PAGE of-Stain: referring to " NuPAGE Bis-Tris-Gel " pamphlet is described, the 13rd page.The result is shown in Figure 7.

Swimming lane	Sample	Concentration	Volume [μ l]	Preparation
					1	Mark 12MW standard	-	10	Do not have
2	HE2 dialyses in coupling buffer	20μg/ml	10	Referring to SOP
					3	HE2 dialyses in coupling buffer	10μg/ml	10	Referring to SOP
4	HE2 dialyses in coupling buffer	50μg/ml	10	Referring to SOP
					5	HE2 dialyses in coupling buffer	2.5μg/ml	10	Referring to SOP
6	HE2SiaTn dialyses in the preparation buffer	20μg/ml	10	Referring to SOP
					7	HE2SiaTn dialyses in the preparation buffer	10μg/ml	10	Referring to SOP
8	HE2SiaTn dialyses in the preparation buffer	5μg/ml	10	Referring to SOP
					9	HE2SiaTn dialyses in the preparation buffer	2.5μg/ml	10	Referring to SOP
10	Mark 12MW standard	-	10	Do not have

Western blotting

Use the Western blotting of rabbit x mice IgG2a

Step:

1. the LDS-Gel that contains Bis-Tris-Gel (4-12%)

2. protein shifts: (use Immobilon transfer membrane PVDF 0.45 μ m, Fa.Millipore) referring to NuPAGE Bis-Tris-Gel explanation pamphlet 14-20 page or leaf

3. film colour developing

Material:

Put together: rabbit x mice IgG2a-HRP, #61-0220, Fa.Zymed

Dyeing liquor 1: the 5ml MetOH that contains 15mg HRP-Color Reagent (Fa.BioRAD)

Dyeing liquor 2: contain 15 μ l 30%H ₂O ₂25ml PBS def.1x

Step:

With closing membrane under the PBS room temperature that contains 3% defatted milk powder 1 hour.

Wash film with PBS.

At room temperature hatched 1 hour with conjugate (being diluted among the PBS at 1: 1000).

Wash film with PBS.

Stop with dyeing liquor 1+2 colour developing and water.

Western blotting with anti-SialylTn CD175s (IgG type)/rat x mice IgG1-HRP.

Step:

1. the LDS-Gel that contains Bis-Tris-Gel (4-12%)

2. protein shifts: (use Immobilon transfer membrane PVDF 0.45 μ m, Fa.Millipore) referring to NuPAGE Bis-Tris-Gel explanation pamphlet 14-20 page or leaf

3. film colour developing

Material:

The 2nd Ab: anti-SialylTn CD175s (IgG type), 90 μ g/ml, Fa.DAKO, Code.No.M0899, Lot.089 (601).

Put together: rat x mice IgG1-HRP, Fa.Becton Dickinson, Mat.No.559626, Batch:37205.

3% defatted milk powder is in PBS deflx.

Dyeing liquor 1: the 5mlMetOH that contains 15mg HRP-Color Reagent (Fa.BioRAD).

Dyeing liquor 2: contain 15 μ l 30%H ₂O ₂25ml PBS.

Step:

Descended closing membrane 1 hour with the PBS room temperature (RT) that contains 3% defatted milk powder.

Wash film with PBS

With the 2nd Ab (concentration 10 μ g/ml) V=5ml, hatched under the room temperature 1 hour.

Wash film with PBS.

With hatched 1 hour under conjugate (being diluted among the PBS at 1: the 1000) room temperature.

Wash film with PBS.

Stop with dyeing liquor 1+2 colour developing and water.

After the SialylTn coupling, Western blotting also confirms the increase of HE2 heavy chain of antibody molecular weight with the anti-mice IgG2a-HRP of rabbit dyeing.

In order to show how many (HE2's) antiidiotypes is retained in the coupling product in conjunction with active, Implementation criteria ELISA

Fixed IGN111 captures antiidiotype HE2, is detected by anti-mice IgG2a-HRP.Show HE2 reactive high 2-3 times than HE2-SialylTn, bonded moderate loss only takes place after showing coupling in this.

Implement other standard ELISA, by mouse anti SialylTn-antibody test SialylTn.Wherein initiation material HE2 and coupling product HE2-SialylTn are fixed.For detection, anti-SialylTn (mice IgG)/rat anti-mouse IgG1-HRP is used for the detection of SialylTn.

The result shows that the HE2-SialylTn product carries SialylTn really, and is opposite with HE2 before the coupling.

Conclusion

SialylTn success and HE2 antibody coupling are together.Coupling reaction kinetics time lengthening reaches capacity after about 24 hours.SialylTn mainly is coupled at the HE2 heavy chain of antibody, and light chain only the part and the SialylTn coupling.HE2-SialylTn coupling product has kept the idiotype specificity of most of HE2, and the SialylTn part of this neoglycoprotein is discerned by the SialylTn specific antibody.Level of endotoxin is lower than detectable limit.

List of references

The list of references that this paper mentions all is incorporated herein by reference clearly with its integral body.

[1]J.M.de Ia Fuente，A.G.Barrientos，T.C.Rojas，J.Canada，A.Fernandez，S.Penades，Angew.Chem.Int.Ed.，2001，40，2257.

[2]A.G.Barrientos，J.M.de Ia Fuente，T.C.Rojas，A.Fernandez，S.Penades，Chem.Eur.J.，2003，9，1909.

[3]M.J.Hostetler，J.E.Wingate，C.Z.Zhong，J.E.Harris，R.W.Vachet，M.R.Clark，J.D.Londono，S.J.Green，J.J.Stokes，G.D.Wignall，G.L.Clish，M.D.Porter，N.D.Evans，R.W.Murray，Langmuir，1998，14，17.

[4]This methanolic layer was concentrated under reducedpressure.The ¹H-NMR spectrum of the residue showed the sameinitial ratio，approximately，between Glc，STn，Le ^y and BC11signals.

Claims (69)

1. nano-particle, it comprises the core that comprises metal and/or semiconductor atom, and wherein this core and a plurality of part are covalently bound, and this part comprises at least a antigen and at least a adjuvant.

2. the nano-particle of claim 1, wherein at least a adjuvant stimulating innate immunity reaction.

3. the nano-particle of claim 1 or claim 2, wherein at least a adjuvant stimulates t cell responses.

4. the nano-particle of claim 3, wherein t cell responses comprises the t helper cell reaction.

5. the nano-particle of claim 1 or claim 2, comprising is the adjuvant of saccharide part.

6. the nano-particle of claim 5, wherein saccharide partly comprises glucose, mannose, fucose and/or N-acetyl-glucosamine.

7. the nano-particle of above-mentioned each claim, comprising is the adjuvant of peptide moiety.

8. the nano-particle of claim 3 or claim 4, wherein this peptide moiety is the peptide of activation t helper cell.

9. the nano-particle of claim 7 or claim 8, wherein this peptide moiety comprises protease cutting site.

10. the nano-particle of claim 9, wherein this peptide comprises aminoacid sequence FKLQTMVKLFNRIKNNVA.

11. the nano-particle of aforementioned each claim, wherein antigen is tumor specific antigen.

12. the nano-particle of claim 11, wherein antigen is sugar antigen.

13. the nano-particle of claim 12, wherein antigen is sialylated.

14. the nano-particle of claim 12, wherein antigen is sialylated.

15. the nano-particle of claim 14, wherein antigen is sialyl Tn, sialylLewis ^a, sialyl Lewis ^xOr sialyl Lewis ^y

16. the nano-particle of aforementioned each claim, wherein antigen is pathogen specific antigen.

17. the nano-particle of claim 16, wherein pathogen is antibacterial, virus or parasite.

18. the nano-particle of aforementioned each claim, wherein at least a part is connected with nano-particle by the joint group.

19. the nano-particle of claim 18, its center tap group comprises sulfydryl, alkyl, dihydroxylic alcohols group or peptide group.

20. the nano-particle of claim 19, its center tap group comprises C2-C15 alkyl and/or C2-C15 dihydroxylic alcohols.

21. the nano-particle of claim 20, its center tap group are C2-C15 alkyl or hexaethylene glycol-C11 alkyl.

22. the nano-particle of aforementioned each claim, wherein nano-particle comprises labelling.

23. the nano-particle of claim 22, wherein labelling is the atom that fluorophor, radionuclide, magnetic mark, dyestuff, NMR active atomic or use surface plasma body resonant vibration can detect.

24. the nano-particle of claim 23, wherein magnetic mark is the paramagnetic group, comprises Mn ^{+ 2}, Gd ^{+ 2}, Eu ^{+ 2}, Cu ^{+ 2}, V ^{+ 2}, Co ^{+ 2}, Ni ^{+ 2}, Fe ^{+ 2}, Fe ^{+ 2}Or lanthanide series ^{+ 3}

25. the nano-particle of claim 23, wherein the NMR active atomic is Mn ^{+ 2}, Gd ^{+ 3}, Eu ^{+ 2}, Cu ^{+ 2}, V ^{+ 2}, Co ^{+ 2}, Ni ^{+ 2}, Fe ^{+ 2}, Fe ^{+ 3}Or lanthanide series ^{+ 3}

26. the nano-particle of aforementioned each claim, wherein nano-particle is water miscible.

27. the nano-particle of aforementioned each claim, wherein the core of nano-particle has 0.5 to 10nm average diameter.

28. the nano-particle of aforementioned each claim, wherein the core of nano-particle has 1 to 2.5nm average diameter.

29. the nano-particle of aforementioned each claim, the nano-particle that wherein comprises its part has 10 to 30nm average diameter.

30. the nano-particle of aforementioned each claim, wherein core is a metallic core.

31. the nano-particle of claim 30, wherein metallic core comprises Au, Ag or Cu.

32. the nano-particle of claim 30 or claim 31, wherein metallic core is the alloy that is selected from Au/Ag, Au/Cu, Au/Ag/Cu, Au/Pt, Au/Pd, Au/Ag/Cu/Pd, Au/Fe, Au/Cu, Au/Gd, Au/Fe/Cu, Au/Fe/Gd or Au/Fe/Cu/Gd.

33. each nano-particle of claim 30 to 32, wherein the core of nano-particle is a magnetic.

34. the nano-particle of claim 33, wherein nano-particle comprises inert metal atom and magnetic metal atom in core, and its ratio is about 5: 0.1 to about 2: 5.

35. the nano-particle of claim 34, wherein inert metal is gold, platinum, silver or copper, and magnetic metal is ferrum or cobalt.

36. each nano-particle of claim 1 to 29, wherein core comprises semiconductor atom.

37. the nano-particle of claim 36, wherein semiconductor atom can serve as quantum dot.

38. the nano-particle of aforementioned each claim, wherein part also comprises peptide, protein structure domain, nucleic acid fragment, glycolipid or glycoprotein.

39. the nano-particle of aforementioned each claim, wherein part comprises DNA or RNA.

40. compositions comprises the colony of the nano-particle of one or more aforementioned each claim.

41., further comprise drug acceptable carrier according to the compositions of claim 40.

42. immunogenic structure is by assisting the covalently bound core element of peptide part to form with saccharide part and at least a T cell.

43. immunogenic structure, by forming with the covalently bound core element of a plurality of saccharide parts, wherein the saccharide part comprises at least a new epi-position structure.

44. according to the immunogenic structure of claim 42 or claim 43, wherein core element is selected from the nano-particle of being made up of metal or semiconductor atom core and antibody or derivatives thereof or fragment.

45. according to each carrier molecule of claim 42 to 44, wherein metallic core comprises Au, Ag, Cu, Pd or Al.

46., comprise new epi-position structure according to the immunogenic structure of claim 42.

47. according to each immunogenic structure of claim 42 to 46, wherein the saccharide part is Lewis antigen or Sialyl Tn.

48. according to the immunogenic structure of claim 47, wherein Lewis antigen is sialylated.

49. according to the immunogenic structure of claim 47, wherein Lewis antigen is sialylated.

50. according to each immunogenic structure of claim 42 to 49, wherein the auxiliary peptide part of T cell derives from the toxoid that is selected from tetanus toxoid, diphtheria toxoid or keyhole limpet hemocyanin.

51. according to each immunogenic structure of claim 42 to 50, wherein the auxiliary peptide part of T cell has aminoacid sequence FKLQTMVKLFNRIKNNVA.

52. pharmaceutical composition comprises one or more according to each immunogenic structure of claim 42 to 51.

53., contain at least a vaccine adjuvant according to the pharmaceutical composition of claim 52.

54. the compositions according to claim 52 or claim 53 is used to prepare the purposes of immunization therapy with medicine.

55. the purposes of claim 54, its Chinese medicine are used for prevention or treatment cancer.

56. be used to separate the purposes that is suitable for detecting with the antibody of separating tumor cell according to each immunogenic structure of claim 42 to 51.

57. one kind prepares each the method for nano-particle of claim 1 to 39 by the core of at least a antigen and at least a adjuvant and nano-particle is puted together, this method comprises: with the joint antigen of deriving; With the joint adjuvant of deriving; And, make that in nano-particle self-assembly process, the nano-particle core is connected by joint with adjuvant with antigen with the reactant reaction of the core of the deutero-antigen of joint and adjuvant and preparation nano-particle.

58. the method for claim 57, its center tap group comprises sulfydryl, alkyl, dihydroxylic alcohols group or peptide group.

59. the method for claim 57, its center tap group comprises C2-C15 alkyl and/or C2-C15 dihydroxylic alcohols.

60. the method for claim 57, its center tap group are C2-C15 alkyl or hexaethylene glycol-C11 alkyl.

61. each method of claim 57 to 60, wherein reactant mixture comprises the antigen of deriving, the salt of the adjuvant of deriving, metal and/or semiconductor atom and Reducing agent be with the preparation nano-particle.

62. with each the obtainable nano-particle of method of claim 57 to 61.

63. each nano-particle of claim 1 to 39, or the compositions of claim 40 or claim 41 are used for preventative or the treatment of taking stopgap measures property.

64. each nano-particle of claim 1 to 39, or the compositions of claim 40 or claim 41 are as vaccine.

65. each nano-particle of claim 11 to 15 is used to prepare the purposes of treatment cancer with medicine.

66. the purposes of claim 65, wherein cancer is colon, pancreas, intestinal, lung, liver, ovary or bladder cancer.

67. the nano-particle of claim 16 or claim 17 is used to prepare the purposes of treatment infectious disease with medicine.

68. the purposes of claim 67, wherein disease is malaria or tuberculosis.

69. each purposes of claim 65 to 68, wherein nano-particle comprises film transposition signal, so that they can the permeation cell film.

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