CN101426533A - Immunostimulatory compositions and methods - Google Patents

Abstract

The invention provides conjugates comprising immunity co-stimulatory polypeptide and antigen or infection dose. The conjugate is used for immunity response for generating or enhancing antigen or infection dose. The invention provides immunity cell for modifying conjugates for generating or enhancing immunity response of antigen or infection dose. The invention also provides immunity stimulating part comprising immunity co-stimulatory polypeptide for stimulating immunity response, and method in the treatment of immunity or prevention of infection.

Description

Immunostimulatory compositions and method

The cross reference of related application

The application requires the rights and interests of the applying date of following U.S. provisional application according to 35 U.S.C. § 199 (e): 60/748,177 (December in 2005 application on the 8th); 60/771,179 (application on February 6th, 2006); 60/799,643 (application on May 12nd, 2006); With 60/863,173 (application on October 27th, 2006).At this aforesaid each piece application is incorporated herein by reference with its integral body.

Invention field

The present invention generally relates to and produces or method that enhance immunity is replied, comprises the antigenic immunne response of antagonism, relates to the compositions that is used to realize this method, and relates to the modification immunocyte that is used for this method.

Background of invention

Having described at the immunne response of strengthening the host comes processing feature to be to lack immunity or the method by soluble disease of the bigger immunne response of aggressiveness and disease.Exemplary disease that this method is favourable therein or disease comprise cancer, influenza and human immunodeficiency virus (HIV).

Usually use the cancer that relates to surgical operation, chemotherapy and radiation to handle, but these methods lack tumour-specific, cause disadvantageous side effect and not too satisfied clinical response.Therefore, strengthen the immunne response of cancer at replying of targeted cancerous cells by specificity and will present the clear superiority that surpasses traditional cancer therapy the method that normal cell is not showing adverse effect.

Have consensus: immunologic surveillance plays effect in the prevention of tumor with in eradicating, and the cell-mediated acquired immunity of T also plays effect in this process.Referring to, for example, Pardoll, Nat.Rev.Immunol.2002,2:227-38; Rosenberg, Nature 2001,411:380-84; Finn, OJ.Nat.Rev.Immunol.2003,3:630-41.The cell-mediated immunity of T is also worked in various immunotherapy methods, demonstrates effect before clinical and in the limited clinical setting.Referring to, for example, Pardoll, above; Finn, above; Antonia etc., Curr.Opin.Immunol 2004,16:130-6.Come target tumor by immune system, because their expressing tumor related antigens (TAA), it is autologous protein sudden change or super/unconventionality expression, or is derived from the protein of oncogenic virus.Referring to, for example, Finn, above; Antonio, above.Under physiological condition, (DC) selects tumor antigen by dendritic cell, is carried into peripheral lymphoid organs, and submission makes its activation and is divided into effector lymphocyte (T to natural T cell under immune condition _Eff).These cells are transported to tumor sites and produce the antitumor that is used for the tumor elimination and reply then.Referring to, for example, Spiotto etc., Immunity.2002; 17:737-47; Ochsenbein etc., Nature 2001; 411:1058-64; Yu etc., Nat.Immunol.2004; 5:141-9).

The productivity t cell response needs three different signals: signal 1,2 and 3.Produced signal 1 by TXi Baoshouti (TCR) and special antigen presenting cell (APC) the compatible complex of lip-deep main tissue (MHC) interaction of molecules.Mediated signal 2 by a series of costimulatory moleculeses, and be crucial for the immunne response that continues.By activated lymphocyte and the elaborate cytokine of APC (as macrophage and the DC) signal 3 of having transduceed, and be important for keeping of effect immunne response.

Tumor has produced various mechanism and has evaded immune surveillance.These mechanism comprise: (i) lack signal 1, invalid displaying by the MHC/ tumor antigen biomolecule complex on the tumor cell, the expression of the shortage of this signal transduction or MHC analog MIC causes, has suppressed to express the NKT (NK cell) that NKG2 suppresses receptor; (ii) lack signal 2, cause by the expression that lacks costimulatory molecules on the tumor cell or suppress molecule altogether; The (iii) secretion by anti-inflammatory molecular, the inducing of anergy in the tumor response T cell is by the T of apoptosis _EffThe physics elimination of cell or the CD4 of natural generation ⁺CD25 ⁺FoxP3 ⁺T regulates and control (T _Reg) cell the mediation of inductive tumor immunne response inhibition and (iv) regulate by the immunity of tumor stroma.The evidence of accumulation has shown many can the operation simultaneously in these mechanism in having patient's body of big tumor load.

Owing to can prevent the prospect of immune specificity, safety, effect and the longterm memory of cancer return, comprise that the antigenic cancer vaccine from the target cancer has caused special concern.In case determined that immune system plays an important role and can regulate and eradicated existing tumor in the animal model in the individual antagonism of protection cancer, dropped into sizable effort and researched and developed the treatment vaccine.Referring to, for example, Berzofsky etc., J.Clin.Invest 2004,113:1515-25; Platsoucas etc., Antivcancer Res.2003; 23,1969-96; Finn, above.Present vaccine strategy comprises the specificity T AA of use in conjunction with non-specific or specificity adjuvant, complete tumor cell lysate, genetic modification is expressed the tumor cell of costimulatory molecules, cytokine and/or chemotactic factor, with the tumor antigen pulse or with the DC of tumor RNA or DNA transfection, and the carrier of the various molecules of immunization stimulus of the various codings of injection in the tumor.The effective effect of these methods comes from uses one or several immune evasion strategy to advance tumor to control the ability of immunne response, or owing to being the invalid submission of immunosuppressant mechanism, TAA or lacking effective DC, T _EffThe activation of cell and NK cell.

Summary of the invention

The invention provides immunostimulatory compositions and method.

According to an embodiment, the invention provides and comprise that (a) first conjugate and (b) combination of second conjugate, first conjugate comprise that (i) comprises that first immunity stimulates the conjugate member of polypeptide altogether and (ii) comprises conjugate member in conjunction with first right member; Second conjugate comprises that (i) comprises the first antigenic conjugate member and (ii) comprise conjugate member in conjunction with second right member.In one embodiment, can comprise avidin or Succ-PEG-DSPE in conjunction with first right member, and can comprise biotin in conjunction with second right member.In another embodiment, first conjugate can comprise and contains that first immunity stimulates polypeptide altogether and in conjunction with first right member fused polypeptide.In a specific embodiment, first immunity stimulates polypeptide to be selected from 4-1 BBL, CD86, ICOSL, PD-L1, PD-L2, B7-H3, B7-H4, OX40L, CD27L, CD30L, LIGHT, BAFF, APRIL, CD80 and CD40L altogether.

In a specific embodiment, first antigen is relevant with infectious agent, as people or bird flu or human immunodeficiency virus.In another specific embodiment, first antigen is tumor associated antigen.

In one embodiment, compositions further comprises the 3rd conjugate, and it comprises that (i) comprises that second immunity stimulates polypeptide altogether and in conjunction with first right member conjugate member with (ii) comprise second antigen and in conjunction with second right member conjugate member.In this embodiment, second immunity stimulates the polypeptide and first immunity to stimulate polypeptide identical or different altogether altogether; Second antigen and first antigen are identical or different; First of the 3rd conjugate and second combination combine the member identical or different to first of member and first and second conjugates with second.In addition, the first conjugate member can come in conjunction with the second conjugate member the combination between the member by first and second combination.

In another embodiment, second conjugate of combination comprises that (i) comprises the conjugate member of infectious agent and (ii) comprise conjugate member in conjunction with second right member.

According to another aspect of the present invention, provide to produce or strengthen the method for immunne response that the tumor of first tumor associated antigen is expressed in antagonism, described method comprises that the material with following has the patient of tumor: comprise that (a) (i) comprises that first immunity stimulates the conjugate member of polypeptide altogether and (ii) comprises first conjugate in conjunction with first right member conjugate member; With comprise that (i) comprises the conjugate member of first tumor associated antigen and (ii) comprise second conjugate in conjunction with second right member conjugate member; Or the immunocyte of (b) having handled at external use first and second conjugates.

In one embodiment, give the patient, separate or the while, comprise a part as single compositions with first and second conjugates.

In another embodiment, give the patient with the immunocyte of handling at external use first and second conjugates.In a specific embodiment, immunocyte comprises that being used for immunity stimulates the receptor of polypeptide altogether, and wherein stimulate the combination between polypeptide and the receptor that first conjugate is puted together immunocyte altogether, the combination between the member is puted together immunocyte with second conjugate by first and second combination by immunity.

In one embodiment, this method further comprises and gives the 3rd conjugate, and it comprises that (i) comprises that second immunity stimulates polypeptide altogether and in conjunction with first right member conjugate member with (ii) comprise second tumor associated antigen and in conjunction with second right member conjugate member.In this embodiment, second immunity stimulates the polypeptide and first immunity to stimulate polypeptide identical or different altogether altogether; Second antigen and first antigen are identical or different; First of the 3rd conjugate and second combination combine the member identical or different to first of member and first and second conjugates with second.In addition, the first conjugate member can come in conjunction with the second conjugate member the combination between the member by first and second combination.

According to another aspect of the present invention, provide and modified immunocyte and produce or strengthen method the immunne response of the tumor of expressing tumor associated antigen or infectious agent, comprise stimulating immunocyte contact (a) first conjugate of polypeptide receptor and (b) second conjugate altogether that first conjugate comprises that (i) comprises that first immunity stimulates the conjugate member of polypeptide altogether and (ii) comprises conjugate member in conjunction with first right member with expressing first immunity; Second conjugate comprises that (i) comprises the conjugate member of antigen relevant with tumor or infectious agent or infectious agent and (ii) comprise conjugate member in conjunction with second right member.Can stimulate the combination between polypeptide and the receptor that first conjugate is puted together immunocyte altogether by immunity, and can to the combination between the member second conjugate be puted together immunocyte by first and second combination.

In one embodiment, immunocyte is the T cell, as CD4+ cell or CD8+ cell, or neutrophil cell, natural killer cell, mononuclear cell or dendritic cell.

In another embodiment, immunocyte comprises that first immunity stimulates the receptor of polypeptide altogether, and this method further comprises immunocyte contacted the 3rd conjugate, the 3rd conjugate comprise (i) comprise second immunity stimulate altogether polypeptide with in conjunction with first right member conjugate member with (ii) comprise second antigen relevant or infectious agent and in conjunction with second right member conjugate member with tumor or infectious agent.In this embodiment, second immunity stimulates the polypeptide and first immunity to stimulate polypeptide identical or different altogether altogether; Second antigen, if it is exist, identical or different with first antigen (if existence); First of the 3rd conjugate and second combination combine the member identical or different to first of member and first and second conjugates with second.In addition, the first conjugate member can come in conjunction with the second conjugate member the combination between the member by first and second combination.

According to another aspect of the present invention, provide and expressed the modification immunocyte that first immunity stimulates polypeptide receptor altogether, wherein use (a) first conjugate and (b) immunocyte of the second conjugate modification, first conjugate comprises that (i) comprises that first immunity stimulates the conjugate member of polypeptide altogether and (ii) comprises conjugate member in conjunction with first right member, second conjugate comprises that (i) comprises the conjugate member of first antigen or infectious agent and (ii) comprise conjugate member in conjunction with second right member, wherein first conjugate stimulates the combination between polypeptide and the receptor to put together immunocyte by immunity altogether, and second conjugate is puted together immunocyte by first and second combination to the combination between the member.In one embodiment, immunocyte is the T cell, as CD4+ cell or CD8+ cell, or neutrophil cell, natural killer cell, mononuclear cell or dendritic cell.

According to another aspect of the present invention, provide and induced or strengthen method the immunne response of anti-infective, described method comprises that following material is suffered from infectious agent infects or be in patient in the infectious agent infection risk: (a) first conjugate and (b) second conjugate, first conjugate comprises that (i) comprises that first immunity stimulates the conjugate member of polypeptide altogether and (ii) comprises conjugate member in conjunction with first right member, and second conjugate comprises that (i) comprises first antigen relevant with infectious agent or comprise the conjugate member of infectious agent and (ii) comprise conjugate member in conjunction with second right member.In one embodiment, give in first and second conjugates at least one by direct injection to infection site.

In a specific embodiment, infection is people or bird flu, and first antigen is selected from H, N, M1, M2e, NS1, NS2 (NEP), NP, PA, PB1 and PB2.In another specific embodiment, infection is HIV, and first antigen is selected from the HIV antigen that is made of Gag albumen, Pol, Vif, Vpr, Rev, Vpu, envelope antigen decision position, Tat and Nef.

In one embodiment, this method further comprises and gives the 3rd conjugate, it comprise (i) comprise second immunity stimulate altogether polypeptide with in conjunction with first right member conjugate member with (ii) comprise second antigen relevant or infectious agent and in conjunction with second right member conjugate member, wherein second immunity stimulates the polypeptide and first immunity to stimulate polypeptide identical or different altogether altogether with infectious agent; Second antigen, if it is exist, identical or different with first antigen (if existence); First of the 3rd conjugate and second combination combine the member identical or different to first and second of member and first and second conjugates, and the first conjugate member by first and second combination to the combination between the member in conjunction with the second conjugate member.

According to another aspect of the present invention, provide and comprised that immunity stimulates the conjugate of polypeptide and avidin or Succ-PEG-DSPE altogether.

According to another aspect of the present invention, provide the method for induced animal immunostimulatory response, comprised and to comprise that immunity stimulates the conjugate of polypeptide and avidin or Succ-PEG-DSPE to give animal altogether.In some embodiments, this method further comprises and gives animal with antigen.

The accompanying drawing summary

Figure 1A and 1B have listed the nucleotide sequence (SEQ ID NO:1) and the aminoacid sequence (SEQ ID NO:2) of the fusion rotein that comprises core Succ-PEG-DSPE and Mus LIGHT albumen ectodomain separately.Underscore is core Succ-PEG-DSPE sequence among Figure 1B.

Fig. 2 A and 2B have listed the nucleotide sequence (SEQ ID NO:3) and the aminoacid sequence (SEQID NO:4) of the fusion rotein that comprises people CD80 ectodomain and core Succ-PEG-DSPE separately.Underscore is core Succ-PEG-DSPE sequence among Fig. 2 B.

Fig. 3 A and 3B have listed the nucleotide sequence (SEQ ID NO:5) and the aminoacid sequence (SEQ ID NO:6) of the fusion rotein that comprises Mus 4-1BBL ectodomain and core Succ-PEG-DSPE separately.Underscore is core Succ-PEG-DSPE sequence among Fig. 3 B.

Fig. 4 A and 4B have listed the nucleotide sequence (SEQ ID NO:7) and the aminoacid sequence (SEQ ID NO:8) of the fusion rotein that comprises core Succ-PEG-DSPE and people 4-1BBL ectodomain separately.Underscore is core Succ-PEG-DSPE sequence among Fig. 4 B.

Fig. 5 A and 5B have listed the nucleotide sequence (SEQ ID NO:9) and the aminoacid sequence (SEQID NO:10) of the fusion rotein that comprises core Succ-PEG-DSPE and people CD86 ectodomain separately.Underscore is core Succ-PEG-DSPE sequence among Fig. 5 B.

Fig. 6 A, 6B and 6C have listed the aminoacid sequence of HPV16E6 (SEQ ID NO:11), HPV 16 E6 variants (SEQ ID NO:12) and HV 16 E7 (SEQ ID NO:13).

Fig. 7 A ﹠amp; 7B has listed the nucleotide and aminoacid sequence (the SEQ ID NO:14﹠amp of used CSA-human CD 40 L among the embodiment; 15).

Fig. 8 has shown the result of the blended lymphocyte reaction of allos, use the conduct of natural B LAB/c lymphocyte to reply thing, and the splenocyte that allos C57BL/6 shone is as stimulus object.Culture shown in giving replenishes 1 μ g/ml CSA-41 BBL fusion rotein.

Fig. 9 has shown the result of ex vivo T cell proliferation, wherein with solubility anti--CD8 that CD3 monoclonal antibody (0.5 μ g/ml) and the splenocyte stimulation of shining are selected from the C57B/6 mice ⁺The T cell is in the existence of CSA-41BBL fusion rotein (0.5 μ g/ml), contrast CSA albumen (0.19 μ g/ml) or anti-4-1BB monoclonal antibody clone 3H3 (5 μ g/ml) or not.

Figure 10 has shown with CFSE labelling 1,000,000 OT-I CD8 ⁺The T cell and be transferred to biotinylated OVA (10 μ g/ injection) and with the blended CSA-4-1 BBL of biotinylated OVA fusion rotein (1 μ g/ injection) (41BBL+OVA) or when comprising in the B6.SJL mice of conjugate immunity of biotinylated OVA and CSA-4-1BBL, the proliferative of antigenic specificity CD8+T cell is replied.Last group ( ^*) shown and used and the replying of CSA-4-1 BBL that 5 μ g of mol level such as 4-1BBL put together 10 μ g biotinylated OVA.

Figure 11 A is a rectangular histogram, has shown PE+ the be untreated cell (filling gray zone) of DC, the DC (dotted line) that handles with biotinylation PE and with the DC (solid line) of biotinylated PE//CSA-4-1BBL conjugate processing.Figure 11 B has shown the average fluorescent strength (MFI) of the PE of the DC that accepts each processing.

Figure 12 A has shown the result of flow cytometry, carries out that flow cytometry analysis is untreated (grey black color) or the CD86 of the DC that handles with CSA-41BBL (solid line) or LPS (dotted line) in the presence of GM-CSF and the level of II class MHC.Figure 12 B has shown the average fluorescent strength of CD86 and II class MHC.

Figure 13 has shown the average fluorescent strength that CD40, CD86 and II class MHC express on the DC cell of the animal of handling with biotinylated OVA/CSA-4-1 BBL of handling from natural, biotinylated OVA/CSA.

Figure 14 A has shown the result of co-culture experiments, wherein selects CD4 from spleen and the periphery lymph node of natural B ALB/c mice ⁺CD25 ^-(single positive, SP) and CD4 ⁺CD25 ⁺(two positives, DP) T cell, and single culture 3 days, or replenish splenocyte shine, anti-cd 3 antibodies (0.5 μ g/ml) and shown in the proteic culture of contrast CSA of the 4-1 BBL of concentration (μ g/ml) or equimolar amounts, with 1:1 ratio cultivation 3 days.Shown the result of CFSE test among Figure 14 B, wherein will under condition described in Figure 14 A, be used for suppressing test, except using the 4-1 BBL of 0.5 μ g/ml with the SP T cell of CFSE labelling.The percentage ratio that has shown each histogrammic somatoblast.

Figure 15 has shown the result of ex vivo T cell proliferation, wherein selects CD4 from spleen and the periphery lymph node of natural B ALB/c mice ⁺CD25 ^-(single positive, SP) and CD4 ⁺CD25 ⁺(two positives, DP) T cell, and single culture, or in the presence of 0.5 μ g/ml anti-cd 3 antibodies and the splenocyte that shine with the cultivation of 1:1 ratio, use or 4-1BBL that need not 1 μ g/ml.

Figure 16 has shown the structure of CSA-hCD40L and CSA-mCD40L construct.Haircut is represented primer (a, b, c, d) and is used to clone the direction of purpose.

Figure 17 has shown flow cytometry, proved combining of CSA-mCD40L and CSA-hCD40L and CD40 receptor, wherein hatch people THP-1 and Mus A20 cell line with CSA-mCD40L or CSA-hCD40L, with the anti--Succ-PEG-DSPE antibody staining of FITC labelling, and in flow cytometry, analyze.Figure (a) and (b) shown combining of CSA-mCD40L and people and mouse cell line separately, and scheme (c) and (d) shown combining of CSA-hCD40L and people and mouse cell line separately.

Figure 18 has shown flow cytometry, proved with the II class HLA on the macrophage of CSA-hCD40L stimulation and the rise of costimulatory molecules, wherein people THP-1 cell line is stimulated 48 hours (fine line), and in flow cytometry, use the antibody of II class HLA (Figure 18 A) and CD80 (Figure 18 B) to analyze with 100ng/mlCSA-hCD40L.The cell that to hatch with CSA albumen (solid rectangular histogram) and express film in conjunction with the Chinese hamster ovary celI transfectant of Mus CD40L (heavy line) respectively as feminine gender and positive control.

Figure 19 has shown flow cytometry, proved the phenotype sudden change of the Mus DC that stimulates with CSA-mCD40L, wherein use the CSA-mCD40L (hollow rectangular histogram) of various concentration to stimulate the various time periods of jejune DC of bone marrow derived, and use and analyze for the antibody of CD80 (Figure 19 A) and CD86 (Figure 19 B) expression specificity.Stay not stimulated cells (fine line) or with CSA albumen stimulated cells (solid rectangular histogram) with comparing.The data that per 106 cells stimulated with 200ng albumen 48 hours have been shown.

Figure 20 has shown the person monocytic cell's secrete cytokines that stimulates by with CSA-CD40L, wherein use 1 μ g/ml rhsCD40L+ reinforcing agent (Figure 20 A) and 100ng/ml CSA-hCD40L (Figure 20 B) or 250ng/ml CSA-mCD40L (Figure 20 C) or CSA to stimulate the person monocytic cell 18 hours of preliminary eluting, and IL-1 β and IL-6 by the elisa assay supernatant.Result when Figure 20 D has shown the mouse macrophage cell line of coming expressing human CD40 (CD40KO) with CSA or CSA-hCD40L (1 μ g/ml) stimulation heredity improvement; extract RNA and protect test to analyze by RNAse; use RiboQuant multiprobe RNAse protection system, use template mck-3b.Shown the protection probe that is used for IL-6, L32 and GAPDH.Rectangular histogram is represented the ribbon density with the IL-6 after the house-keeping gene L32 standardization.

Figure 21 has shown the stimulation that the iNOS in the macrophage that stimulates with CSA-hCD40L expresses, wherein prepare transfection and be used for the Mus CD40KO macrophage system 24 hours of expressing human CD40 with IFN-γ, use commercial rhsCD40L of 1 μ g/ml and reinforcing agent or 300ng/mlCSA-hCD40L or CSA albumen irritation cell 24 hours then, use anti-iNOS antibody by the western blot analysis cell lysate.Rectangular histogram is represented the density of iNOS band.

Figure 22 has shown the dosage at 12.5 and 25 μ g, compares with LPS, and the adjuvant influence uses 50 μ gOVA as antigen in the strong body of CSA-4-1BBL.Reported the result according to killing percentage ratio in the body.

Figure 23 shown inoculation (i) PBS (◆, n=20); (ii) 50 μ g P1+12.5 μ g CSA (■, n=6); (iii) 25 μ g CSA-4-1BBL (▲, n=10); (iv) 50 μ g P1+25 μ gCSA-4-1BBL (Δ, n=13) or (v) (is n=7) to the effect of existing cervix neoplasms for 50 μ g P1+10 μ g CpG.The compositions of inoculation P1 and CSA-4-1BBL has caused showing the survival rate that improves, and inoculation P1 or CSA-4-1 BBL provide quite successful immunization therapy.

Figure 24 has shown and has inoculated the result of biotinylated OVA/CSA-4-1BBL conjugate in preventing tumor growth.The mice that has shown inoculation OVA (Δ), biotinylated OVA/CSA-4-1BBL conjugate (▲) and control mice (●) does not have tumor survival, and the mice that inoculates biotinylated OVA/CSA-4-1BBL conjugate demonstrates 100% survival.

Figure 25 demonstrates the flow cytometry of the painted cell of CFSE, proved with antigen separately or antigen and LPS compare, 4-1BBL can reply intravital antigenic specificity CTL the higher level that is enhanced to.Percentage ratio cracking as the CFSE peak of peptide pulse on the angle of each figure comes ecbatic, and is normalized to comparing with reference to the CFSE ebb of natural animal.

Figure 26 has shown the flow cytometry data, has proved that 4-1BBL stimulates altogether to have improved intravital antigen presentation.

Figure 27 has shown the flow cytometry data, has proved that 4-1BBL stimulates altogether to have improved dendritic cell antigen absorption in vivo.

Detailed Description Of The Invention

The invention provides for generation of or strengthen the method and composition of immune response, comprise the immune response of resisting antigen, antigen such as TAA or the antigen relevant with infectious agent, infectious agent such as people and bird flu or HIV. The present invention also provides for generation of or has strengthened modification immunocyte to the immune response of antigen. The present invention also provides immunotherapy method, comprises the cancer immunotherapy method, as reduces the method for tumor size and the method for inhibition tumor cell growth, and the method for the treatment of or infection prevention.

The productivity Acquired immune response needs collaborative between interior natural T effect (" the Teff ") cell of the systematism structure of less important lymphoid organ and the APC and interacts timely. This interaction has promoted the mutual activation of Teff cell and APC, causes various cell surface parts and acceptor and to starting, keep the expression of replying important soluble protein with long-term memory. As mentioned above, at least three signals ( signal 1,2 and 3) relate to the activation of initial natural T cell. Hinted that several co-stimulators have stimulated one or more in these signals.

The present invention relates to one or more immunity stimulates polypeptide and one or more antigens relevant with tumour or infectious agent with the purposes of antigen submission to the method for immunocyte altogether, so that induce effectively the immune response to antitumor or infectious agent. Perhaps, can substitute relative antigen and use infectious agent. Although do not wish to be bound by any theory, think that the present invention has obtained favourable result by promoting antigen submission and activate immunity to reply. In another interchangeable embodiment, the invention provides and comprise that immunity stimulates the immunostimulation part of polypeptide altogether, be used for immune stimulatory and reply.

For purposes of this application, following term has these definition:

As used in this, " one " or " a kind of " meaning is one or more, unless specially point out only to represent one.

" give " as used in this suitable method that comprises that all provide material to the patient. That approach commonly used comprises is oral, the hypogloeeis, through mucous membrane, in skin, rectum, vagina, subcutaneous, intramuscular, intravenous, artery, in the sheath, by conduit, by implanting etc. In some embodiments, composition is approached or directly gives tumour, as by directly being injected in the tumour or being injected to blood, when being neoplastic hematologic disorder such as tumour.

" antigen " without limits as used in this. Antigen comprises the molecule of protein, lipid, sugar, nucleic acid, chemical part and other induce immune response. Antigen comprises protein, and it can be to modify or unmodified, as by glycosylation or methylate to modify, for example be cyclisation or in conjunction with lipid. Comprise it being the antigen of an infectious agent part with the antigen of infectious agent or disease association, such as envelope protein, capsid protein, surface protein, toxin, cell membrane, antigenicity lipid etc. Other antigen can only be expressed in the presence of the host. In some embodiments, other suitable antigen can comprise host's antigen, comprises inducing, modifying or as infection or disease marker and other those of overexpression. The antigen that is derived from infectious agent, infection, illness or disease that all are such or be applicable among the present invention with the antigen of infectious agent, infection, illness or disease association. What also be suitable for use as " antigen " according to the present invention is the peptide that comprises the full-length proteins antigenic portions, as comprises the peptide of a part of the protein of induce immune response, such as the immunogenicity epitope. For example, suitable antigen can comprise the peptide of synthetic induce immune response.

" in conjunction with to " refers to by interactional two molecules of any molecular force, and molecular force comprises, for example, ion, covalency, hydrophobic, Van der Waals and hydrogen bond are so that this combination is to having the characteristic of mutual specific binding. The meaning of specific binding is less than under the condition in conjunction with another molecule, mutually combines in conjunction with the member is presented. Biotin-avidin, hormone-acceptor, receptor-ligand, enzyme-substrate, IgG-albumin A, Ag-Ab etc. in conjunction with right example.

" immunity stimulates polypeptide altogether " meaning is to improve the polypeptide of the immune response of individual enantiopathy substance (comprising infectious agent) or tumour.

" immunocyte " comprises any cell of the generation, adjusting or the effect that relate to acquired or innate immune system as used in this. Immunocyte comprises the T cell, such as CD4+ cell, CD8+ cell and various other T cell subset, B cell, NK, macrophage, monocyte and dendritic cells, and neutrophil cell.

" patient " comprises any vertebrate as used in this, comprises horse, sheep, he-goat, ox, pig, birds, dog, cat and primate species. In one embodiment, the patient is the people. Those skilled in the art will recognize that specific co-stimulators, signaling molecule, cell marking, cell type, infectious agent etc., when discussing about species, in different species, have corresponding analog, and the present invention includes such analog and the purposes in corresponding and relative species thereof.

" tumour " comprises entity and non-entity tumor (such as lymthoma) as used in this; And be developed to carcinous, different tumour stage pernicious and metastatic tumo(u)r from precancerous lesion and benign tumour.

Briefly, the invention provides use (a) the first conjugate and (b) the second conjugate produce or strengthen the method for the immune response of antagonism the first antigen, the first conjugate comprises that (i) comprises that the first immunity stimulates the conjugate member of polypeptide and the conjugate member who (ii) comprises in conjunction with first right member altogether, and the second conjugate comprises that (i) comprises the conjugate member of the first antigen and the conjugate member who (ii) comprises in conjunction with second right member. Antigen is TAA or the antigen relevant with infectious agent, or infectious agent self. Can directly give the patient with the conjugate that comprises antigen or infectious agent, maybe can be used for processing immunocyte, then give the patient with it. The method of the immunocyte that the immunocyte that the present invention also provides the composition that comprises conjugate, processed with conjugate and preparation were processed.

The present invention also provides and has comprised that immunity stimulates the immunostimulation part of polypeptide altogether, as comprises that immunity stimulates conjugate or the fusion of polypeptide and avidin or streptavidin altogether. The present invention also provides the method for induced animal immunostimulatory response, comprises partly giving animal with immunostimulation. In some embodiments, also give animal with antigen. The composition that comprises this part also is provided.

According to an aspect of the present invention, immunity as the immunocyte that comprises selective one or more types of target stimulates the part of the conjugate of polypeptide that antigen or infectious agent are presented to immunocyte altogether, and immunity as described below stimulates any of polypeptide altogether. Therefore, according to an embodiment, the invention provides and comprise that immunity stimulates the polypeptide antigen relevant with tumour or infectious agent or the conjugate of infectious agent altogether. Can antigen or infectious agent and immunity be stimulated conjugation of polypeptides altogether by any mode, comprise by covalent bond, directly or by attachment or by in conjunction with to the member.

In one embodiment, by in conjunction with right binding interactions antigen or infectious agent and immunity being stimulated conjugation of polypeptides altogether. According to this embodiment, each antigen (or infectious agent) and immunity are stimulated altogether polypeptide and puts together in conjunction with right member, and stimulate altogether polypeptide chain to be connected together the antigen in the conjugate (or infectious agent) and immunity in conjunction with the binding interactions to the member, stimulate altogether polypeptide-in conjunction with first right member such as immunity:: in conjunction with right second member-antigen (or infectious agent) conjugate.

According to this embodiment, the invention provides and comprise (a) first conjugate and (b) combination of the second conjugate, the first conjugate comprises that (i) comprises that the first immunity stimulates the conjugate member of polypeptide and the conjugate member who (ii) comprises in conjunction with first right member altogether, and the second conjugate comprises that (i) comprises the conjugate member of first antigen (or infectious agent self) relevant with tumour or infectious agent and the conjugate member who (ii) comprises in conjunction with second right member.

In another embodiment, combination comprises the 3rd conjugate, it comprises that conjugate member that (i) comprise that the second immunity stimulates polypeptide altogether and (ii) comprise the conjugate member of second antigen (or infectious agent self) relevant with tumour or infectious agent, wherein the immunity of the 3rd conjugate stimulates the immunity of polypeptide and the first conjugate to stimulate altogether polypeptide identical or different altogether, and the second antigen and the first antigen are identical or different. In the particular aspects of this embodiment, by in conjunction with the combination that member and each immunity is stimulated altogether between polypeptide and the second antigen, immunely stimulate altogether the second antigen of polypeptide and the 3rd conjugate to combine. According to this embodiment, the combination of the 3rd conjugate is identical or different to the member to first and second combination of member and the first and second conjugates.

First, second can be provided in the compositions of separating and choose the 3rd conjugate wantonly.Perhaps, can in single compositions, provide first and second conjugates, and the 3rd conjugate is provided in the compositions of separating.In another interchangeable embodiment, in single compositions, provide first, second and the 3rd conjugate.In another interchangeable embodiment, in a compositions, provide first conjugate, and second and the 3rd conjugate are provided in another compositions.

Each compositions is optional can to comprise acceptable carrier on the materia medica.Acceptable carrier is can be as the material of compositions medium on the materia medica, because in the administration scope, this material is inert or medically is being acceptable in addition, and with activating agent be compatible.Carrier can be accepted on the materia medica and conventional medicine additive well known in the art can be contained.

Immunity stimulates polypeptide altogether

The immunity costimulatory molecules relates to the natural interaction between natural T cell and the antigen presenting cell, its cause mutual activation and promote various cell surface parts and receptor expression and cause immunne response startup, keep the soluble protein with longterm memory.As mentioned above, the initial activation of at least three natural T cells of signal demand.Interaction between the nominal peptide that presents by TXi Baoshouti (TCR) and special APC (as dendritic cell (DC)) the compatible complex of lip-deep main tissue (MHC) molecule has produced signal 1.By by several different numerator mediated signals 2, and be crucial for the immunne response that continues.By activated T cells and the elaborate cytokine transduction signal 3 of APC, and be important for keeping of effect immunne response.

Identified the panimmunity costimulatory molecules.The exemplary immunization costimulatory molecules (polypeptide) useful according to the present invention comprises, but be not limited to LIGHT, CD80 (B7-1), CD86 (B7-2), ICOS, ICOSL (comprises B7h, B7-H2, B7RP-1, GL-50 and LICOS), CD94 (KP43), CD40L (CD154), ICAM-I (CD54), ICAM-2, ICAM-3, SLAM (CD150), HAS (CD24), 4-1BB (CDw137), 4-1BBL (CDw137L), OX40L, CD28, CD40 (BP50), CD25 (IL-2R α), lymphotoxin (LT α or LT β), TNF, Fas-L, GITR (activation can be induced TNRF), the GITR part, CD1 1a (α _LIntegrin), CD11b (α _MIntegrin), L-selectin (CD62L), CD69 (very early stage activation antigen), CD70 (CD27L), PD-L1, PD-L2, B7-H3, B7-H4, OX40L, 4-1BBL, CD27L, CD30L, LIGHT, BAFF and APRIL. Referring to, For example,Watts ﹠amp; DeBenedette, 1999, Curr.Opin.Immunol., 11:286-93.

Be " total length " unless refer in particular to, stimulate polypeptide to comprise full-length polypeptide altogether with reference to immunity and present the immunity fragment or the part of stimulatory function altogether, include but not limited at this at this, below those fragments and the part specially identified.Therefore, for example, mean fragment or the polypeptide partly that comprises the total length 4-1 BBL that presents the common stimulatory function of immunity with reference to the 4-1BBL polypeptide, as ectodomain or the total length 4-1 BBL albumen of 4-1BBL.In one embodiment, immunity stimulates polypeptide not comprise the membrane spaning domain of immune costimulatory molecules altogether.In one embodiment, the immune ectodomain that stimulates polypeptide to comprise immune costimulatory molecules altogether, or its receptors bind fragment.

Representational nucleotide sequence and coded immunity stimulate the example of polypeptide to comprise GenBank accession number No.AB029155 (Mus LIGHT) altogether; NM_172014 (people TNFSF14mRNA transcript variant 2); NM_003807 (people TNFSF14 mRNA transcript variant 1); NM_005191 (people CD80 mRNA); NM_009855 (Mus CD80 mRNA); NM_214087 (pig CD80 mRNA); NM_009404 (Mus Tnfsf9 mRNA); NM_003811 (people TNFSF9 mRNA); NM_181384 (Rattus norvegicus TnfsfP mRNA); BAA88559 (Mus LIGHT albumen); Q9QYH9 (Mus TNFSF 14 embrane-associated proteins and soluble protein); AAH18058 (people TNFSF14 albumen); NP_005182 (people CD80 albumen); NP_033985 (Mus CD80 albumen); NP_037058 (Rattus norvegicus CD80 albumen); NP_003802 (people TNFSF9 albumen); NP_033430 (Mus TNFSF9 albumen); NP_852049 (Rattus norvegicus TNFSF9 albumen); NM_012967 (Rattus norvegicus ICAM-1mRNA); X69711 (H-ICAM-1 mRNA); X52264 (Mus ICAM-I mRNA); X69819 (people ICAM-3 mRNA); AF296283 (Mus ICAM-4 mRNA); NM_021181 (people SLAMF7 mRNA); NM_033438 (people SLAMF9 mRNA); NM_029612 (Mus SLAMF9 mRNA); NM_144539 (Mus SLAMF7 mRNA); L13944 (Mus CD18 gene); X53586 (human beta 2 integrin alpha 6 mRNA); X68742 (human beta 2 integrin alpha mRNA); J04145 (people has a liking for medium-sized grain cell adhesion receptor α-M Subunit mRNA); AJ246000 (human leukocyte adhesion receptor, L-selectin mRNA); AY367061 (people L-selectin mRNA, part cds); Y13636 (Mus CD70 mRNA); NM_001252 (people TNFSF7 mRNA); BC000725 (people TNFSF7 mRNA (cDNA clone MGC:1597 IMAGE:3506629), complete cds); X69397 (people CD24 gene and complete CDS); NM_013230 (people CD24 mRNA); NM_012752 (Rattus norvegicus CD24 mRNA); Y00137 (Mus tumor necrosis factor-β (lymphotoxin) gene); X02911 (human tumour necrosis factor-β (lymphotoxin) gene); D00102 (human lymphotoxin mRNA, complete CDS); X01393 (human lymphotoxin mRNA); And A06316 (those shown in (human lymphotoxin mRNA).Can find coding identical or other immunely stimulate the nucleotide sequence of polypeptide altogether and/or stimulate amino acid sequence of polypeptide altogether, for example, by the search obtainable GenBank data base of the public (for example, the ncbi.nlm.nih.gov on World Wide Web can obtain).

As if the interaction between CD28 and the CD80/CD86 play an important role in the transduction of signal 2.Referring to, for example, Harding ﹠amp; Allison, J.Exp.Med.1993,177:1791-96; Ramarathinam etc., J.Exp.Med.1994,179:1205-14; Townsend﹠amp; Allison, Science 1993,259:368-70; Gause etc., J.Immunol.1997,159:1055-58.CD80 does not express on immobilized B cell usually, but on peripheral blood lymphocytes and DC with low expression level; Yet after activation, the CD80 on these two kinds of cells and macrophage and other APC expresses and obtains raising.Referring to, for example, Lenschow etc., Ann.Rev.Immunol.1996,14:233-58; Freeman etc., J.Immunol 1989,143:2714-22.On the contrary, CD86 obtains constitutive expression on peripheral blood lymphocytes and DC, and is raised faster on the B cell.Referring to, for example, Lenschow etc., above, Inaba etc., J.Exp.Med.1994,180:1849-60.The interaction of the MHC/ peptide complexes on TCR and the APC makes CD80/86 molecule and CD28 strengthen and cause the tyrosine phosphorylation of lipid kinase phosphatidyl-inositol 3-kinase simultaneously, and it suppresses the inducing of a series of IL-2 of causing gene expressions, cell proliferation subsequently and is divided into incident in the complicated born of the same parents of effector function.Referring to, for example, Slavik etc., Immunol.Res.1999,19:1-24; Azuma etc., Nature 1993,366:76-79; Allison ﹠amp; Krummel, Science1995,270:932-33.

Signal 2 can further increase generation property immunne response by regulating anti-apoptotic genes expression (as Bal-xL) to prevent cell death.Referring to, for example, Radvanyi etc., J.Immunol.1996,156:1788-98; Boise etc., Immunity 1995,3:87-98; Boise ﹠amp; Thompson, Science 1996,274:67-68.After the initial period of immune activation, lip-deep multiple other receptor-ligand of T cell and APC raises obtaining.These " accessory " receptor/ligand are right, as 4-1 BBL/4-1 BB, play an important role in the generation of keeping after initial activation the, immune body inner equilibrium and immunological memory.Referring to, for example, Yu etc., Nat.Immunol.2004,5:141-49; Armitage etc., Nature 1992,357,80-82; Zhai etc., J.Clin.Invest.1998,102:1142-51; Bourgeois etc., Science 2002,297:2060-63; Kikuchi etc., Nat.Med.2000,6:1154-59.

1.?4-1?BBL

In a particular of the present invention, it is 4-1 BBL polypeptide that immunity stimulates polypeptide altogether.4-1 BBL (being also referred to as 4-BB-L, 4-BB part, TNFSF9, ILA part) is the II type albumen of expressing on activated B cell, macrophage and DC in two to three days after the activation.Referring to, for example, Alderson etc., Eur.J.Immunol.1994,24:2219-27; Goodwin etc., Eur.J.Immunol.1993,23:2631-41; Pollok etc., Eur.J.Immunol.1994,24:367-74; DeBenedette etc., J.Immunol.1997,158:551-59.Its receptor, 4-1 BB (CD137) is at activated CD4 ⁺And CD8 ⁺On the T cell surface, on natural killer cell, mononuclear cell and immobilized DC, obtain expressing.Referring to, for example, Pollock, above; Wilcox etc., J.Immunol.2002,169:4230-36; Futagawa etc., Int.Immunol.2002,14:275-86; Pollok etc., J.Immunol.1993,150:771-81.

4-1 BB/4-1 BBL interacts and also signal 2 is transferred to CD8 in CD28-independence mode ⁺The T cell, and stimulate them to produce cytokine, enlarge and obtain effector function.Referring to, for example, Cannons etc., J.Immunol.2001,167:1313-24; Hurtado etc., J.Immunol.1995,155:3360-67.Kim ﹠amp; Broxmeyer, J.Hematother.Stem Cell Res.2001,10:441-49; Saoulli etc., J.Exp.Med.1998,187:1849-62; Shuford etc., J.Exp.Med.1997,186:47-55; Tan etc., J.Immunol.1999,163:4859-68; Vinay ﹠amp; Kwon, Semin.Immunol.1998,10:481-89.4-1 BB/4-1 BBL interact for the synthetic of the activation of mononuclear cell and DC, cytokine and with the communication for information of NK cell also be important.Referring to, for example, Futagawa etc., above; Wilcox etc., J.Clin.Invest 2002,109:651-9.Similarly, except the downward modulation of going up mediation cyclin-dependant kinase inhibitors p27kip1 by cyclinD2 and E promotes the effect that T cells with antigenic specificity enlarges, the 4-1BB signal plays effect in the T cell survival, because it has prevented the cell death of activation-inducing by the foundation of going up the memory of mediation permanent immunity of anti-apoptosis Bcl-xL and Bcl-1.Referring to, for example, Takahashi etc., J.Immunol.1999,162:5037-40; Hurtado etc., J.Immunol.1997,158:2600-09; Kim etc., Eur.J.Immunol.1998,28:881-90.Also demonstrate the 4-1BB/4-1BBL interaction and optionally promote 1 cytokines, as IL-2, IFN-γ and TNF-α, show that 4-1BB is the specific costimulatory molecules of 1 type effector T cell, it plays effect in tumor is eradicated.

Shown T recently _RegCell constitutive expression 4-1BB receptor and the signal transduction by 4-1 BB receptor have suppressed these cell inhibiting functions.Referring to, for example, Choi etc., above; Morris etc., above, and following embodiment.This is important, because T _RegCell plays an important role in immune tumor is escaped.Several clinical researches have proved T _RegExist directly related between the number of cell and the tumour progression.Curiel etc., Nat.Med.2004,10:942-49.In fact, T in the animal model _RegThe elimination of cell causes big tumor to be eradicated, and the positive evidence of they main effects in tumour progression is provided.Yu etc., J.Exp.Med.2005,201:779-91.Similarly, infectious agent as HIV, can be used T _RegCell is used for immune evasion.

Although do not wish to be bound by theory, thinking according to the present invention stimulates the 4-1BB homoreceptor of polypeptide on can activated T cell with 4-1BBL altogether as immunity, forms several important immunostimulations.An effect is that signal 2 is transferred to CD8 in CD28-independence mode ⁺The T cell, this stimulates the T cell to produce cytokine, enlarge and obtain effector function.Interactional another effect of 4-1 BB/4-1 BBL is activated mononuclear cell and DC, and it causes the synthetic of cytokine and discharges.Another effect of 4-1 BB signal is to promote the T cell survival and set up the permanent immunity memory by the cell death (AICD) that prevents activation-inducing.Interactional another effect of 4-1 BB/4-1 BBL is optionally to produce 1 cytokines from T cell, DC and macrophage, and as IL-2, IFN-γ and TNF-α, it acts on eradicates 1 important type effector T cell to tumor.In addition, as explained above, 4-1 BB/4-1 BBL interacts can suppress T _RegCell inhibiting agent function.Therefore, for example, 4-1 BBL antigen conjugate can promote antigen presentation specifically in conjunction with the DC that expresses 4-1 BB receptor, activates DC, is used to produce elementary t cell response, directly acts on activated T cells and (comprises T _RegCell) and the NK cell strengthen resisting the antigenic T of replying _RegCell.

4-1 BBL contains 254 aminoacid (26624Da).Referring to Alderson etc., Eur JImmunol.1994 JIUYUE; 24 (9): 2219-27.Can in the Swiss-Prot data base, find whole aminoacid sequences of people 4-1 BBL according to accession number no.P41273.4-1 BBL is an II type glycoprotein, forms potential Cytoplasm domain by residue 1-28, and residue 29-49 forms the membrane spaning domain of single prediction, and residue 50-254 forms potential ectodomain, and residue 35-41 represents to gather-and the Leu chain.Can in GenBank accession number no.NM_003811, find the nucleotide sequence of coding people 4-1 BBL.

As mentioned above, activate back 2-3 days,, comprise that activated B cell, macrophage and DC express 4-1 BBL by activated antigen presenting cell.4-1 BB is the receptor of 4-1 BBL, at activated CD4 ⁺And CD8 ⁺On the T cell surface, on natural killer cell, mononuclear cell and immobilized DC, obtain expressing.Can connect in conjunction with to the member or be expressed as the fusion rotein that contains in conjunction with to the member in conjunction with the residue 50-254 of the 4-1 BBL of its homoreceptor 4-1 BB or its fragment, use according to the present invention.For example, Fig. 3 A and 3B have listed nucleotide and the aminoacid sequence (SEQ ID NO:5 and 6) of CSA-Mus 4-1 BBL.Fig. 4 and B have shown the nucleotide and the aminoacid sequence (SEQ ID NO:7 and 8) of the fusion rotein that comprises people 4-1 BBL ectodomain and core Succ-PEG-DSPE.

2.?CD80?&?CD86

CD80 (be also referred to as B7.1, CD28LG, LAB7) and CD86 (be also referred to as B7.2, CD28LG2 is that example stimulates polypeptide altogether LAB72), and the both is in conjunction with the CD28/CTLA4 co-receptor of T cellular expression.CD80 contains 288 aminoacid (33048Da).Referring to Freeman etc., J.Immunol.143 (8), 2714-2722 (1989).Can in the Swiss-Prot data base, find whole aminoacid sequences of people CD80 according to accession number no.P33681.CD80 is an I type glycoprotein, and 1-34 forms secretion signal by residue, and residue 35-242 forms potential ectodomain, and residue 243-263 forms potential membrane spaning domain, and residue 264-288 forms potential Cytoplasm domain.Therefore, there is not the ripe CD80 of secretory signal sequence to divide subrepresentation aminoacid 35-288.Can in GenBank accession number no.NM_005191, find the nucleotide sequence of coding people CD80.

Can connect in conjunction with to the member or be expressed as the fusion rotein that contains in conjunction with to the member in conjunction with the residue 35-242 of the CD80 of its homoreceptor CD28 or its fragment, use according to the present invention.For example, Fig. 2 A and 2B have listed the nucleotide (SEQ ID NO:3) and the aminoacid sequence (SEQ ID NO:4) of the chimeric protein that comprises people CD80 (B7.1) ectodomain and core Succ-PEG-DSPE.

CD86 (B7.2) contains 329 aminoacid (37696Da).Referring to Freeman etc., Science 262 (5135), 909-911 (1993).Can in the Swiss-Prot data base, find whole aminoacid sequences of people CD86 according to accession number no.P42081.CD86 is an I type glycoprotein, and 1-23 forms secretion signal by residue, and residue 24-247 forms potential ectodomain, and residue 248-268 forms potential membrane spaning domain, and residue 269-329 forms potential Cytoplasm domain.Therefore, there is not the ripe B7.2 of secretory signal sequence to divide subrepresentation aminoacid 24-329.Can in GenBank accession number no.NM_175862, find the nucleotide sequence of coding people CD86.

Can connect in conjunction with to the member or be expressed as the fusion rotein that contains in conjunction with to the member in conjunction with the residue 24-247 of the CD86 of its homoreceptor CD28 or its fragment, use according to the present invention.For example, Fig. 5 A and 5B have listed the nucleotide (SEQ ID NO:9) and the aminoacid sequence (SEQ ID NO:10) of the chimeric protein that comprises people CD86 (B7.2) ectodomain and core Succ-PEG-DSPE.

CD86 does not express on immobilized B cell usually, but goes up with low expression level in peripheral blood lymphocytes (PBC) and DC; Yet after activation, the expression on B cell and other APC such as macrophage and the DC obtains raising.On the contrary, CD86 obtains constitutive expression on PBC and DC, and is raised faster on the B cell.The interaction of the MHC/ peptide complexes on TXi Baoshouti (TCR) and the APC strengthens CD80/86 and CD28 on the T cell simultaneously, this causes the tyrosine phosphorylation of lipid kinase phosphatidyl-inositol 3-kinase, and it suppresses the inducing of a series of IL-2 of causing gene expressions, cell proliferation subsequently and is divided into incident in the complicated born of the same parents of effector function.Signal 2 can further increase generation property immunne response by regulating anti-apoptotic genes expression (as Bal-xL) to prevent cell death.

3.?L1GHT

After the initial period of immune activation, " accessory " receptor/ligand is right, as 4-1BBL/4-1BB and LIGHT/HVEM, obtains raising on the surface of T cell and APC.These receptor/ligand are to relating to the generation of keeping after the initial activation incident, immune body inner equilibrium and immunological memory.

LIGHT polypeptide (being also referred to as TNFS14, HVEM-L, LTg, TR2) is and the homologous TNF superfamily member of lymphotoxin.Referring to Mauri etc., Immunity 8 (1), 21-30 (1998).Can in the Swiss-Prot data base, find whole aminoacid sequences of people LIGHT according to accession number no.O43557.LIGHT contains 240 aminoacid (26351Da) and is II type glycoprotein, forms potential Cytoplasm domain by residue 1-37, and residue 38-58 forms the membrane spaning domain of single prediction, and residue 59-240 forms potential ectodomain.The division site relates to residue 82-83.Can in GenBank accession number no.NM_172014, find the nucleotide sequence of coding people LIGHT.

Can connect in conjunction with to the member or be expressed as the fusant that contains in conjunction with to the member in conjunction with the residue 59-240 of the LIGHT of its homoreceptor HVEM, LT β R or TR6 or its fragment, use according to the present invention.For example, Figure 1A and 1B have listed the nucleotide (SEQ ID NO:1) and the aminoacid sequence (SEQ ID NO:2) of the chimeric protein that comprises core Succ-PEG-DSPE and Mus LIGHT ectodomain.

LIGHT mainly expresses on activated T cells, NK cell and immature dendritic cell, and is used for regulating the various aspects of immunne response.Synthesize LIGHT as embrane-associated protein, but its cell surface expression is subjected to the adjusting of several translations back mechanism.In a few minutes of expressing, separate LIGHT and accumulate (obform body 1 from cell surface as shla molecule by matrix metalloproteinase; General expression residue 83-240; Swiss-Prot O43557-1).Cell surface Cytoplasm fragment is represented obform body 2 (Swiss-Prot O43557-2).In addition, various cell types are stored LIGHT and are secreted out by various biostimulations when activating in vesicle.Although the effect of the soluble form of LIGHT is not characterized fully, can be used as negative feedback loop, by competing the function that suppresses film combining form with HVEM and LT β R.

LIGHT and three different acceptor interactions: the herpesvirus on (1) T cell enters medium (HVEM), (2) LT β R, its mainly express on epithelial cell and the stromal cell and (3) various cells on solubility inveigle receptor.LIGHT is given in these interactions different functions.Take place relevant with the interaction of LT β R on the stromal cell and generation, lymph node (LN) organ of various cytokine/chemotactic factors with the recovery of secondary lymph structure.On the other hand, the interaction of the HVEM receptor on LIGHT and the lymphocyte causes the activation and the generation of cytokine, is mainly IFN-γ and GM-CSF.As if about this point, the LIGHT/HVEM axle transmits with the Th1 type and replys the relevant costimulatory signal of activation, this plays pivotal role in oncolysis.

LIGHT takes place and the Th1 type is replied in the generation and worked at lymphatic organ. Referring to, for example, Yang etc., 2002, J.Biol.Regul.Homeost.Agents, 16:206-10; Schneider etc., 2004, Immunol.Rev., 202:49-66.

Demonstrated the effect of LIGHT in the different tumor models in vitro and in vivo.Reported that the chronic lymphocytic leukemia cells of transduceing by the type simple herpesvirus amplicon of expressing LIGHT can strengthen the T cell proliferation in the mixed lymphocyte reaction.Shown that the LIGHT overexpression on the MDA-MB-231 human breast cancer cell suppresses tumor growth.The ICAM-1 of LIGHT transfection to different these cells of cancerous cell line moderate stimulation expresses.Think that the existence of ICAM-1 is useful, because its effective conducted signal, in tumor cell, to produce anti-tumor activity.Except t cell activation, another critical function of LIGHT is that this plays an important role in the generation of secondary lymph structure by the ability of LT β R transduction signal, by chemokine expression induce and stromal cell in adhesion molecule mediate.The expression of CCL21 is regulated in the interaction of LT β R on LIGHT and the stromal cell, and this controls natural T cell and gets back to lymphoid tissue.

An advantage of wherein the tumor cell through engineering approaches being showed embodiment of the present invention of LIGHT is that LIGHT stimulates the lymphatic organ generation and supports the Th1 type to reply the ability of generation.Another advantage is that the LIGHT stimulation further enlarges the ability that these are replied to antineoplastic immunne response and activation tumor stroma.

Substrate prevents that as physical barriers lymphocyte from infiltrating in the tumor locus.Substrate has also suppressed the lymphocyte activator in the tumor microenvironment.This may be that as TGF-β and IL-10, these are by substrate fibroblast and the synthetic justacrine of tumor cell owing to lack the costimulatory signal that t cell activation needs and/or the existence of various immunosuppressant solubility media.Substrate promotes immune ignorant by tumor cell being limited to tumor locus, therefore prevents that they are transferred to zonal lymph node.

The tumor stroma cell is also expressed various immunity receptors, as LT β R, its exploitation can be used for the enhancing of antineoplastic immune according to the present invention.

4.?OX40L

OX40L is expressed by dendritic cell and other APC, and in conjunction with the OC40 that is present on the activated T cells.OX40L contains 183 aminoacid (21950Da).Referring to Miura etc., Mol.Cell.Biol.11:1313-1325 (1991).Can in the Swiss-Prot data base, find whole aminoacid sequences of OX40L according to accession number no.P23510.OX40L is an II type glycoprotein, and residue 1-23 is the Cytoplasm domain, and residue 24-50 is a membrane spaning domain, and residue 51-183 is an ectodomain.The nucleotides sequence of OX40L is classified 3510bp as, and coded sequence is 157-708 (referring to GenBank accession number no.NM_003326.2).Can connect in conjunction with to the member or be expressed as the C-end fusant that contains in conjunction with to the member in conjunction with the residue 51-183 of the OX40L of its homoreceptor OX40 or its fragment, use according to the present invention.

5.?CD40L

CD40L is expressed by activated T cells, and exists as the outer soluble form of born of the same parents, and it is derived from form membrane by the Proteolytic enzyme process.CD40L (being also referred to as TNFSF5) contains 261 aminoacid (29350Da).Referring to Villinger etc., Immumogenetics 53:315-328 (2001).Can find whole aminoacid sequences of CD40L according to accession number no.Q9BDN3.CD40L is an II type glycoprotein, and residue 1-22 is the Cytoplasm domain, and residue 23-43 is a membrane spaning domain, and residue 44-261 is an ectodomain.The nucleotides sequence of CD40L is classified 1834bp as, and coded sequence is 73-858 (referring to GenBank accession number no.NM_000074).Can connect in conjunction with to the member or be expressed as the N-end fusant that contains in conjunction with to the member in conjunction with the residue 44-261 of the CD40L of its homoreceptor CD40 or its fragment, use according to the present invention.

6.?PD-L1

PD-L1 obtains expressing on activated T and B cell, dendritic cell, keratinocyte and mononuclear cell.PD-L1 (is also referred to as B7-H; B7H1; PDL1; PDCD1L1) contain 290 aminoacid (33275Da).Referring to, Dong etc., Nat.Med.5:1365-1369 (1999).Can in the Swiss-Prot data base, find whole aminoacid sequences of PD-L1 according to accession number no.Q9NZQ7.PD-L1 contains 290 aminoacid, represents signal sequence at 18 aminoacid of N end.Ectodomain is positioned at amino acid/11 9-238, and membrane spaning domain is positioned at residue 239-259, and the Cytoplasm domain is positioned at residue 260-290.The nucleotide sequence of PD-L1 (1553bp) can obtain (referring to GenBank accession number no.NM_014143) (coded sequence is 53-925) in public data base.By interchangeable montage, there is the obform body of PD-L1.Can connect in conjunction with to the member or be expressed as the N-end fusant that contains in conjunction with to the member in conjunction with the ectodomain of the PD-L1 of its homoreceptor PDCD1 or its fragment, use according to the present invention.

7.?GL50

GL50 obform body 1 obtains wide expression (brain, heart, kidney, liver, lung, pancreas, Placenta Hominis, skeletal muscle, bone marrow, colon, ovary, prostate, testis, lymph node, lymphocyte, spleen, thymus and tonsil); GL50 obform body 2 (swissprot O75144) is expressed in lymph node, lymphocyte and spleen and on activated mononuclear cell and dendritic cell.GL50 (is also referred to as B7-H2; B7H2; B7RP-1; B7RP1; ICOS-L; ICOSLG; KIAA0653; And LICOS) contains 290 aminoacid (33275Da).Referring to, Wang etc., Blood96:2808-2813 (2000).Can in the Swiss-Prot data base, find whole aminoacid sequences of GL50 according to accession number no.O75144.GL50 contains 302 aminoacid, and 18 aminoacid of N end are represented signal sequence.Ectodomain is positioned at amino acid/11 9-256, and membrane spaning domain is positioned at residue 257-277 and the Cytoplasm domain is positioned at residue 278-302.The nucleotide of GL50 (3239bp) can obtain (referring to GenBank accession number no.NM_015259) (encode fragment is represented 135-1043) in public data base.By interchangeable montage, there is the obform body of GL50.Can connect in conjunction with to the member or be expressed as the N-end fusant that contains in conjunction with to the member in conjunction with the ectodomain of the GL50 of its homoreceptor ICOS or its fragment, use according to the present invention.

Table 1 has been summarized various exemplary costimulatory moleculeses and receptor thereof and has been comprised the embodiment of co-receptor part to conjugate.

Table 1

Construct title and direction	Receptor	Expression of receptor
			CD80-CSA	CD28	Constitutive expression on almost all people CD4 T cell and about 50% the cd8 t cell
GL50-CSA	ICOS	On immobilized T cell, can detect at activated CD4 +T and CD8 +Obtain on T cell and the NK cell raising
			PD-L1-CSA	PD-1	At CD4 +And CD8 +Low-level upward expression on the inducible expression NK-T cell on T cell, B cell and the mononuclear cell
CSA-CD40L	CD40	Constitutive expression on B cell, mononuclear cell, DC, endotheliocyte and the epithelial cell
			CSA-4-1BBL	CD137	NK cell DC subclass (low), person monocytic cell, folliculus DC, CD4 that activated T cells (48h peak value, 96h descends) and cytokine are handled +CD25 +Regulate constitutive expression on the T cell
CSA-OX40L	OX40	Activated CD4 (preferably) and CD8 (intensive antigen is replied) T cell (48h peak value, 96h descends)
			CSA-LIGHT	HVEM	Immobilized T cell, mononuclear cell and immature DC obtain downward modulation when going up constitutive expression t cell activation and DC maturation

Can use other immunity to stimulate polypeptide altogether according to the present invention.For example, US2003/0219419 (its content all being incorporated herein by reference at this) has described IL-2-CSA fusion rotein and the CSA-CD40L fusion rotein that can be used among the present invention.In a word, according to the present invention useful exemplary immunization to stimulate polypeptide to comprise altogether following.

Table 2:B7 and CD28 family member

Part	Receptor
		CD80(B7.1)	CD28，CTA-4(CD152)
CD86(B7.2)	CD28，CTA-4
		ICOSL(B7h，B7-H2，B7RP-1，GL50，LICOS)	ICOS(AILIM)
PD-L1(B7-H1)	PD-1
		PD-L2(B7-DC)	PD-1
B7-H3	Unknown
		B7-H4(B7x；B7S1)	Unknown (BTLA?)
Unknown (HVEM *)	BTLA

^*Be TNF member

Table 3:TNF family member

Part	Receptor
		OX40L	OX40(CD134)
4-1?BBL	4-1?BB(CD137)
		CD40L(CD154)	CD40
CD27L(CD70)	CD27
		CD30L	CD30
LIGHT	HVEM、LTβR、DcR3
		GITRL	GITR
BAFF(BLyS)**	BAFF-R、TACI、BCMA
		APRIL**	TACI、BCMA

^*These are that the B cell is relevant

Table 4A:B7 family member's the nucleotide and/or the list of references of aminoacid sequence

Part (people)	List of references
		CD80 (B7.1)	Freeman etc., J.Immunol.143:2714-2722 (1989).
CD86 (B7.2)	Freeman etc., Science 262:909-911 (1993).
		ICOSL	Wang etc., Blood 96:2808-2813 (2000).Yoshinaga etc., Int.Immunol.12:1439-1447 (2000).
PD-L1	Dong etc., Nat.Med.5:1365-1369 (1999).Freeman etc., J.Exp.Med.192:1027-1034 (2000)
		PD-L2	Tseng etc., J.Exp.Med.193:839-846 (2001) Latchman etc., Nat.Immunol.2:261-268 (2001)
B7-H3	Steinberger etc., (in JIUYUE, 2003) is committed to the EMBL/GenBank/DDBJ data base.Mingyi etc., J.Immunol 168:6294-6297 (2002).
		B7-H4 (B7x; B7S1)	Zang etc., Proc.Natl.Acad.Sci.U.S.A.100:10388-92 (2003).Sica etc., (in April, 2003) is committed to the EMBL/GenBank/DDBJ data base.

Table 4B:TNF family member's the nucleotide and/or the list of references of aminoacid sequence

Part	List of references
		OX40L	Baum etc., Circ.Shock 44:30-34 (1994).Miura etc., Mol.Cell.Biol.11:1313-1325 (1991).Godfrey etc., J.Exp.Med.180:757-762 (1994).
4-1BBL	Alderson etc., Eur.J.Immunol.24:2219-2227 (1994).
		CD40L	Graf etc., Eur.J.Immunol.22:3191-3194 (1992).Hollenbaugh etc., EMBO are (1992) J.11:4313-4321.
CD27L (CD70)	Goodwin etc., Cell 73:447-456 (1993).
		CD30L	Smith etc., Cell 73:1349-1360 (1993).
LIGHT	Mauri etc., Immunity 8:21-30 (1998).
		GITRL	Gurney etc., Curr.Biol.9:215-218 (1999).
BLyS	Moore etc., Science 285:260-263 (1999).
		APRIL	Hahne etc., J.Exp.Med.188:1185-1190 (1998).

Kang Yuan ﹠amp; Infectious agent

Method and composition of the present invention is used to produce or strengthens the immunne response of any antigen of antagonism or infectious agent, comprises antagonism TAA, antigen and the infectious agent self relevant with infectious agent.According to the present invention, antigen that will be relevant with target tumor or infectious agent (or infectious agent self) is presented in immunocyte, and therefore generation or enhance immunity are replied.

1.TAA

In one embodiment, antigen is TAA, and the invention provides effective generation or strengthen the cancer immunotherapy method of patient to the antineoplastic immunne response.According to this embodiment, the invention provides method that reduces the tumor size and the method that suppresses growth of tumour cell.

The present invention resists useful representative tumor cell and includes, but not limited to cancer, and it can be derived from the various organs any, comprises lung, liver, breast, bladder, stomach, colon, pancreas, skin etc.Cancer can comprise the adenocarcinoma that results from organ or the body of gland and come from the squamous cell carcinoma of squamous epithelial cancer.Treatable other cancer comprises sarcoma, as osteosarcoma or osteogenic sarcoma (bone), chondrosarcoma (cartilage), leiomyosarcoma (smooth muscle), rhabdomyosarcoma (skeletal muscle), mesotheliosarcoma or mesothelioma (film inner layer of body cavity), fibrosarcoma (fibrous tissue), angiosarcoma or hemangioendothelioma (blood vessel), liposarcoma (fatty tissue), glioma or astrocytoma (the neural connective tissue of finding in the brain), myxosarcoma (original embryo's connective tissue), esenchymous or mixing mesoderm tumor (blended connective tissue type).Except myeloma, also be easy to treat leukemia and lymphoma.

Identified the multiple TAA relevant with the specific tumors type.These comprise robot end enzyme reverse transcriptase (hTERT), survivin, MAGE-1, MAGE-3, human chorionic gonadotropin, carcinoembryonic antigen, alpha fetal protein, cancer of pancreas embryonal antigen, MUC-1, CA125, CA15-3, CA19-9, CA549, CA195, prostate specific antigen; Prostate specific membrane antigen, Her2/neu, gp-100, sudden change K-ras albumen, sudden change p53, the epithelial growth factor receptor that blocks, chimeric protein ^P210BCR-ABL; Human papillomavirus's the E7 albumen and the EBNA3 albumen of Epstein-Barr virus.In can these antigens used according to the invention any, its antigen fragment and antigen and/or segmental mixture produce or strengthen patient's anti-tumor immune response.Table 5 has been listed example T AA and the disease relevant with these TAA.

Table 5.

Antigen	Disease
		CTAGE-1 and variant	Cutaneous T cell lymphoma
BLA or glycosyl sphingolipid (P kAntigen)	Burkitt ' s lymphoma
		The membrane antigen that the human T-cell leukemia virus is correlated with (HTLV-MA)	Adult T-cell leukemia's lymphoma (ATL)
Thymocyte cell surface antigen JL1	Most acute leukemia
		The relevant antigen (ATLA) of human reverse transcript virus that the adult T cell leukemia is correlated with	The adult T cell leukemia
Epstein-Barr virus (EPV) antigen	Burkitt ' s lymphoma, the hodgkin's disease
		Between modification lymphoma kinases (ALK)	Modification large celllymphoma between CD30+
Fusion rotein (NPM/ALK and variant)	(ALCL)
		Common acute lymphoblast leukemia antigen (CALLA)	Most of acute lymphoblast leukemia
Immunoglobulin Id; II type glycoprotein (for example, HM1.24; KW-2, KW-4, KW-5, KW-12); Carcinoembryonic antigen immaturity laminin receptor albumen (OFA-iLRP); EBV albumen (for example, LMP2A)	The lymphocytic hyperplasia disease

For example, can be at Novellino etc., " A listing of human tumor antigensrecognized by T cells:March 2004 update " (by the tabulation of the human tumor antigen of T cell recognition: upgrade in March, 2004) Cancer Immunology and Immunotherapy, find other people TAA among the 54:187-207 (2005), be introduced into as a reference at this by the T cell recognition.Be known in the art and comprise within the scope of the present invention corresponding to the animal correllaries of these diseases and corresponding to many animal TAA of other Animal diseases.

In one embodiment of the invention, TAA is selected from robot end enzyme reverse transcriptase (hTERT) and survivin, as TAA.Express hTERT in 85% human cancer, although its expression is limited in the normal structure.Referring to, for example, Vonderheide etc., Immunity 1999,10:673-79.Similarly, survivin has been accredited as inhibitors of apoptosis, does not exist in normal structure, but obtains expressing in most of tumor type, comprises in lung, colon, pancreas, prostate and the mastocarcinoma.Referring to, for example, Ambrosini etc., Nat.Med.1997,3:917-21.Because these TAA obtain expressing in most of cancer types, and seldom or not exist in normal structure, be attractive antigen therefore for the use in the cancer immunotherapy method according to the present invention.

In another embodiment of the invention, TAA is relevant with cervical cancer.The whole world probably has 500,000 women to produce cervical cancer and be the second largest inducement of woman cancer death every year.The genitals viral infection that cervical cancer and human papillomavirus cause is directly related and be global health problem.In general half cervical cancer, 16 type HPV have been found especially.Genitals 16 and 18 type HPV, and 31,33,35,45,51 and 56 not too common types also relate to the nosetiology of cervical cancer and other anogenital cancer.The HPV type of finding in the cancerous cell has activity of conversion in vitro study, and viral transforming protein, and E6 and E7 (being also referred to as " in early days " albumen) obtain constitutive expression in cervical cancer tumer line and HPV-associated cancer.Known E6 and E7 are separately in conjunction with tumor inhibitor p53 and retinoblastoma (Rb).In the relevant vicious transformation of HPV, late gene (L1 and L2) and some early genes (E1 and E2) are lost usually, stay E6 and E7 as unique open reading frame of finding usually in the cancer.The expression of E6 and E7 has surpassed usually the adjusting by the cell proliferation of the mediation of albumen such as p53 and Rb probably, it is grown uncontrollably and the potential of vicious transformation is provided.

Therefore, according to a particular of the present invention, TAA is one or more among E6 and the E7.Use according to E6 of the present invention and E7 has given several advantages.At first, E6 and E7 obtain constitutive expression in most of cervical cancer.Secondly, although most of tumor antigen is derived from the autologous protein of normal protein or sudden change, E6 and E7 are exogenous virus albumen completely, and have more antigenic peptides or epitope than mutain.The 3rd, E6 and E7 the malignant phenotype induce and keep in play an important role, and if do not have functional E6 and E7, these cells will no longer be carcinogenic.

E6 from different plant species (for example, people, cattle) is known in the art with aminoacid sequence with being used for different papillomavirus types (for example, HPV 16 and 18) with the proteic nucleotide of E7.Referring to, for example, the HPV sequence library of http://www.stdgen.lanl.gov/stdgen/virus/hpv/index.html..The aminoacid of HPV 16 E6, HPV 16 E6 variants and E7 is listed among Fig. 6 A (SEQ ID NO:11), 6B (SEQ ID NO:12) and the 6C (SEQ ID NO:13) separately.

2. infectious agent

The present invention resists useful representative infectious agent and includes, but not limited to any virus, antibacterial, fungus or protozoacide.Table 6 has been listed the example of infectious agent.

Table 6

People and bird flu, HIV, hepatitis C, pulmonary tuberculosis, west Nile virus, cryptococcus (meningitis), herpes, chlamydia and anthrax are representational infectious agent.Can use any antigen relevant according to the present invention with infectious agent.

According to an embodiment, infectious agent self is used for according to conjugate of the present invention.According to this embodiment, use to comprise that infectious agent is as virus with in conjunction with the conjugate to the member.Any infectious agent be can use,, people and bird flu virus or HIV comprised as virus, or any other virus.Infectious agent improvement or attenuation can be reduced or eliminate its infectivity.

Only for illustrative purposes, with reference to influenza this aspect of the present invention has been described in more detail.Influenza is the Contagious disease that is caused by influenza virus, causes the symptom in nose, throat and the lung usually, and heating, headache, fatigue and headache.Also caused complication, as the chronic disease of pneumonia, bronchitis or hole and ear infections or deterioration.Influenza virus is divided into A, B or C type.The bacterial strain that belongs to A and Type B circulates in colony and is relevant with most people's influenza case.A type influenza causes the most public health problem that can't keep out in the crowd.

According to two proteic composition with A type influenza virus subclassification; Hemagglutinin (H), promote virus in conjunction with and enter the albumen of target cell, and neuraminidase (N), the virion that relates to new formation discharges from infected cell and propagates by health.14 hemagglutinin hypotypes (H1-H15) and 9 neuraminidase hypotypes (N1-N9) have been identified.Flu outbreak big among the crowd is caused by single hemagglutinin hypotype (H1, H2 and H3) and two neuraminidase hypotypes (N1 and N2).For example, the hemagglutinin of influenza virus in 1918 is H1, and its neuraminidase is N1, therefore is called the H1N1 hypotype.Other outburst comprises H2N2 hypotype in 1975, the H3N2 hypotype of nineteen sixty-eight and recently in Southeast Asia, China and the present H5N1 that in the birds and the mankind, breaks out in Europe and the Middle East.

Influenza A virus is reaction or the sudden change of antigen site or the mechanism of change by relating to hemagglutinin and neuraminidase often, or hemagglutinin or neuraminidase hypotype are substituted by the burst of another hypotype and develop.These mechanism form new virus subtype and make influenza virus escape immune defence and propagation.The annual antigenic variant that influenza A virus all can occur, and, need up-to-date bacterin preparation based on the international supervision of the ongoing influenza virus of World Health Organization (WHO).Because the phenomenon that this new influenza virus sub-strain constantly occurs, as H5N1 in recent years, bigger flu outbreak will take place in expection.In specific specious bioterrorism situation, the virus of laboratory generation is carried out similar design realize that antigen changes, and therefore cause the outburst of the host defense that escape has been set up.

Conjugate of the present invention can be used for influenza vaccines, influenza vaccines produce easily and can make fast, can need change and upgrade its antigenic component based on current health, and without any difficulty, it is targeting virus mechanism and infected cell optionally, and for therapeutic effect, gives after can injecting, and give before the injection, be used for prevention.

Therefore, according to an embodiment, with the antigenic component of influenza antigens (or its antigen fragment) as conjugate of the present invention.For example, antigen can comprise one or more H1 and N1 (all being highly immunogenic) and/or one or more nucleoprotein (NP) and stromatin 1 (MP1) and/or stromatin 2 (MP2) (all being the structural protein of high conservative).According to the present invention,, also can be used as antigen from the albumen of epidemic strain such as H5.Although do not wish to receive the constraint of any theory, intracellular protein such as NP and MP2 can provide more general vaccine, do not change because they present almost, and therefore prevent the xenogenesis viral infection, and do not need annual the adjustment.For example, NP presents in influenza A separator〉90% protein sequence homology, and contain dominant cytotoxic T cell target antigen decision position.Other useful influenza antigens comprises PA, PB1 and PB2 (RNA polymerase subunit) and NS1 and NS2 (interferon response inhibitor and the output of RNP nuclear) among the present invention.See also Brown, 2000, Biomed.Pharmacother.54:196-209; Steinhauer etc., 2002,36:305-32; De Jong etc., 2000,40:218-28; Alexander, Vet.Microbiol.74:3-13.

Therefore, according to an embodiment, with the antigenic component of A type influenza hemagglutinin protein (or its antigen fragment) as conjugate of the present invention.At present contain H obtains influenza as the influenza vaccines of main component prevention by subcutaneous injection.For example, be main circulation H albumen from influenza virus A/PuertoRico/8/34 (PR8) H1 (N1H1), and characterized fully, and can use according to the present invention.In another embodiment, with the antigenic component of A type neuraminidase influenza albumen (or its antigen fragment) as conjugate.To comprise that the compositions that contains the proteic conjugate of H-or contain the proteic conjugate of N-is as the vaccine to influenza.(for example, present influenza vaccines comprise the H albumen as main component, and demonstrated to induce enough immunity to prevent the popular of homology virus).Perhaps, comprise the proteic conjugate of H and comprise the proteic conjugate of N, or antigenic arbitrary composition, will be favourable as variable and compositions conservative antigen.Realize this by giving two or more compositionss, each compositions comprises and contains single antigenic conjugate, or provides two or more conjugates (and therefore two or more antigens are provided) to realize this in single compositions.In one embodiment, need select the antigenic component of conjugate based on present publilc health.

In one embodiment, conjugate comprises that 4-1 BBL stimulates polypeptide altogether as immunity.Be not bound by any theory although do not wish, think this is puted together when giving the patient in the object, to come in conjunction with DC by the 4-1 BBL on the DC and the interaction between the 4-1 BB, make vaccine internalization and influenza antigens submission on the surface of DC and the activation of DC and maturation.The activation of DC causes with the interaction of CD8 and cd4 t cell subsequently and activates these cells.Then, activated CD4 T cell and B cell effect, and make them activate and be divided into the cell of secretory antibody, cause humoral response.The activation of CD8 T cell also causes the differentiation and the propagation of more CD8 T cell, and it is used for killing the cell that is infected by the virus.Conjugate can also be expressed 4-1 BB receptor, and further enlarge signal directly in conjunction with activated CD8 and CD4 T cell.In addition, conjugate (by 4-1 BBL) can be directly in conjunction with activated NKT (NK) cell, and it also is used for killing the cell that is infected by the virus, and these all cause stronger immunne response.Therefore, conjugate will produce cell and humoral response, support its effect in therapeutic and preventative vaccine.

Those skilled in the art can select to be used as the antigenic component of the conjugate that resists other infectious agent vaccine with similar method, based on the antigen relevant with these infectious agent.For example, the antigen relevant with HIV comprises HIV peplos gp120 epitope (for example, variable loop is as V3), or other HIV albumen, as Gag albumen (Pr55 ^Gag, substrate p17, capsid p24, nucleocapsid p7), p5; Pol (polymerase), Vif (viral communication power factor p23); Vpr (viral protein R p15); Rev (the regulator p19 of viral gene expression); Vpu (virus protein U); Env (gp160, gp120, gp41); Tat (transcription activator p14); With negative effector p24).Referring to, for example, Peters, 201.Vaccine 2:688-705; Michael, 2003, Clin.Med.3:269-72; Gandhi ﹠amp; Walker, 2002, Ann.Rev.Med.53:149-72; Haseltine, 1991, FASEB 5:2349-60.Other useful antigen comprises the capsular polysaccharide of b type hemophilus influenza (Haemophilius influenzae), the capsular polysaccharide of Neisseria meningitidis (Neisseriameningitidis), the capsular polysaccharide of streptococcus pneumoniae (Streptococcus pneumoniae), the surface antigen of hepatitis B in the vaccine, and the diphtheria extracellular toxin and the tetanus toxin of inactivation.With reference to aforesaid influenza antigens, use these antigen according to the present invention.

In conjunction with right

The example combination is to being biotin and Succ-PEG-DSPE (SA) or avidin.Can also use maintenance to biotin substantially in conjunction with active SA or avidin fragment, as separately at least 50% or how natural SA or avidin in conjunction with active.Such fragment comprises " core Succ-PEG-DSPE " (" CSA "), the clipped form of total length Succ-PEG-DSPE polypeptide, and it comprises Succ-PEG-DSPE residue 13-138,14-138,13-139 and 14-139.Referring to, for example, Pahler etc., J.Biol.Chem.1987.262:13933-37.Can also use and keep strong in conjunction with other Succ-PEG-DSPE of biotin and the clipped form of avidin.Referring to, for example, Sano etc., J Biol Chem.1995 November 24,270 (47): 28204-09 (having described core Succ-PEG-DSPE variant 16-133 and 14-138) (U.S. patent no.6.022,951).Can also use keep basic biotin in conjunction with the active or biotin that improves in conjunction with active Succ-PEG-DSPE mutant and Succ-PEG-DSPE core form.Referring to, Chilcoti etc., Proc Natl Acad Sci U S is Feb28 A.1995; 92 (5): 1754-8; Reznik etc., Nat Biotechnol.1996 August; 14 (8): 1007-11.For example, can use to have the immunogenic mutant of reduction, as remove the mutant of potential T cellular antigens decision position or lymphocyte antigen decision position sudden change by direct mutagenesis.Referring to Meyer etc., Protein Sci.200110:491-503.Equally, also can use keep basic biotin in conjunction with the active or biotin that improves in conjunction with the mutant of active avidin and the core form of avidin.Referring to, Hiller etc., J.Biochem. (1991) 278:573-85; Livnah etc., Proc Natl Acad Sci USA (0:5076-80 (1993).For convenience, in the description at the moment, term " avidin " and " Succ-PEG-DSPE " comprise that these are in conjunction with biotin binding fragment, mutant and core form to the member as used in this.Avidin and Succ-PEG-DSPE can obtain from commercial supplier.In addition, can find nucleotide sequence and the Succ-PEG-DSPE and the avidin aminoacid sequence of coding Succ-PEG-DSPE and avidin, for example, at GenBank accession number No.X65082; X03591; NM_205320; X05343; Z21611 and Z21554.

As used in this " biotin " comprise can mating surface such as cell surface (comprising tumor cell surface) contain biotin moiety, as NHS-biotin and EZ-Link ^TMSulfo-NHS-LC-biotin (Pierce).The albumino reaction form of such biotin can be buied.

In scope of the present invention, several advantages have been given in the interaction between biotin and binding partners avidin or the Succ-PEG-DSPE.For example, biotin is for Succ-PEG-DSPE (10 ¹³M ^-1) and avidin (10 ¹⁵M ^-1) have a very high affinity.In addition, Succ-PEG-DSPE and avidin are separately in conjunction with four polymerization polypeptide of four biotin molecules.Therefore, the immunity that comprises Succ-PEG-DSPE or avidin stimulates part to have the trend that forms tetramer and higher structure altogether.Therefore, they can crosslinked its corresponding immunocyte receptor, is used for the more efficient signal transduction, as the accumulation by receptor.

Those skilled in the art will recognize that and (for example to use other mechanism, other conjugation methods, for example use other coupling part or chemistry or the gene crosslinked) the high stage structure of immune costimulatory molecules is provided, as comprising the conjugate of dimer, trimer, tetramer and the high-order polymer of immune costimulatory molecules, it presents favourable characteristic equally.Such conjugate comprises within the scope of the invention.

Conjugate

Can form by means commonly known in the art and comprise that immunity stimulates polypeptide, antigen or infectious agent altogether and in conjunction with to member's conjugate.For example, can be with polypeptide/antigen/infectious agent with in conjunction with to the mutual covalent bond of member or directly put together mutually or put together mutually by junctional complex.According to an embodiment, polypeptide/antigen/infectious agent and combination are the compositions of fusion rotein to the member.Can prepare fusion rotein by in the various distinct methods known in the art any.For example, can chemosynthesis or use known recombinant nucleic acid technology to produce one or more composition polypeptide of fusion rotein.(as used in this, " nucleic acid " refers to RNA or DNA).For example, can use polymerase chain reaction (PCR) to obtain nucleotide sequence useful among the present invention.For example, various PCR method are described in PCR Primer:A Laboratory Manual (the PCR primer: laboratory manual), Dieffenbach 7 Dveksler edit, Cold Spring HarborLaboratory Press, 1995.

According to an embodiment, immunity stimulates polypeptide to pass through its C-end and the N-end combination that combines the member altogether.For example, immunity stimulates polypeptide to pass through the N-end of its C-end in conjunction with core Succ-PEG-DSPE (CSA) altogether.Therefore, the present invention includes the CD80-CSA fusion rotein, wherein CD80 is partly by the N-end of its C-end in conjunction with CSA.According to another embodiment, immunity stimulates polypeptide to pass through its N-end and the C-end combination that combines the member altogether.For example, immunity stimulates polypeptide to pass through the C-end of its N-end in conjunction with CSA altogether.For example, the present invention includes CSA-4-1 BBL, CSA-LIGHT, CSA-CD40L and CSA-OX40L fusion rotein, wherein CSA partly stimulates the N-end of polypeptide altogether by its C-end binding immunoassay.Immunity stimulate altogether polypeptide can be directly with in conjunction with to the member in conjunction with or by one or more coupling parts such as one or morely be connected polypeptide and combine combination to the member.

According to an embodiment, immunity is stimulated polypeptide, antigen or infectious agent biotinylation altogether.Can form biotinylated conjugate by methods known in the art, and in following embodiment illustrated.

For example, can use from Avidity, (Denver, biotin AviTag technology CO) produces biotinylated protein or infectious agent to Inc..Biotin AviTag is made of 15 amino acid peptides of uniqueness, and this peptide is by biotin ligase BirA identification, with the lysine residue in the biotin connection peptides sequence.Schatz，1993，Biotechnology，11：1138-43。Biotin AviTag can merge with any target protein in heredity, makes the labelling of protein receptor to biotin molecule.

A latent defect of biotin AviTag technology is the biotinylated probability of low degree, because the single unique lysine residue place biotinylated protein matter of system in labeled fragment.In order to overcome such problem, can use the labelled protein of the biotin ligase of purification at external modification purification.Because be the biotinylation that is undertaken by enzyme, reaction condition is comparatively gentle, and labelling is a high degree of specificity, and reaction is modified more effective than the protein chemistry that uses biotin derivative.Perhaps, can use Jordan etc., 2003, the method described in the Clin.Diag.Lab.Immunol.10:339-44 produces genetically engineered biotinylated protein.

It is useful to the fragment of member, antigen or infectious agent in the present invention that immunity stimulates polypeptide, combination altogether, as long as fragment keeps the activity of the total length part of reference.Therefore, for example, immunity stimulates polypeptide fragment should keep its immunity (for example to be total to stimulating activity altogether, keep its ability) in conjunction with its receptor or part, in conjunction with should keeping its ability in conjunction with its binding partners to member's fragment, and antigen or infectious agent fragment should keep it to induce the ability of antagonism with reference to the immunne response of total length antigen or infectious agent.Can keep active fragment by the conventional method screening of this area.More than listed the immune exemplary fragment that stimulates polypeptide altogether.

Conjugate can be comprising junctional complex in conjunction with member and immunity are stimulated between polypeptide, antigen or the infectious agent altogether, as the peptide junctional complex.Can select the length of junctional complex and form the activity (for example, stimulating polypeptide/antigen, infectious agent or combination altogether) that strengthens the arbitrary of conjugate or two function ends the member.Junctional complex is generally about 3 to about 15 amino acid longs, and more preferably from about 5 to about 10 amino acid longs, yet, shorter or longer junctional complex can be used, or junctional complex can be all distributed.In this, can use and be used for connecting the heavy chain of single-chain antibody and elasticity junctional complex (for example, (Gly of light chain ₄Ser) ₃).Referring to, Huston etc. (1988) Proc.Nat.Acad.Sci.USA, 85:5879-5883; U.S. patent No.5,091,513,5,132,405,4,956,778; 5,258,498 and 5,482,858.Other junctional complex is FENDAQAPKS or LQNDAQAPKS.The segmental one or more domains of immunoglobulin Fc (for example, CH1, CH2 and/or CH3) also can be used as junctional complex.Can also use chemical linkers.

Nucleic acid and polypeptide that modify, that change or sudden change also are useful in the present invention, as long as they keep the activity with reference to nucleic acid or polypeptide.For example, be applicable to nucleic acid of the present invention and peptide sequence and, that is, stimulate polypeptide altogether with the known immunity of coding or combine nucleotide sequence to have sequence homogeneity (comprising at least 80% sequence homogeneity) at least about 80% to the member with reference to nucleic acid or polypeptide.In some embodiments, nucleotide sequence or polypeptide and with reference to nucleic acid or polypeptide have at least about 85%, at least about 90%, at least about 95% or at least about 99% sequence homogeneity.

The present invention includes the nucleic acid of sequence change " silence ", identical aminoacid (promptly because their are encoded.The degenerate core acid sequence).The present invention comprises that also coding has the nucleic acid of the polypeptide of conservative amino acid replacement, and such polypeptide.Conservative amino acid replacement (for example, substituting a hydrophobic residue with different hydrophobic residue) is well known in the art and can for example waits by point mutation and realize.Can use receptors bind known in the art and/or biological screening method to confirm the suitability of given modification sequence, variant or mutant, as above about described those methods of fragment.

As used in this, the quantity of mated position in nucleic acid by measuring comparison or the peptide sequence, with the quantity of mated position divided by comparison nucleotide or aminoacid separately sum and multiply by 100 and calculate " % sequence homogeneity ".Identical nucleotide or amino acid whose position appear in the same position that paired position refers in aligned sequences.Comparison nucleotide or amino acid whose sum refer to nucleotide or the amino acid whose minimum number that second sequence of comparison needs, the comparison that does not comprise non-homogeneous sequence (for example, the comparison that forces), as those sequences (that is, coding immunity the stimulation altogether polypeptide or combination are to member's sequence) that can hold at the N-of target sequence or the C-end merges.Comparison nucleotide or amino acid whose sum can be corresponding to complete coded sequences, or can be corresponding to the fragment of full length sequence, as defined in this.

Can use Altschul etc. (1997, Nucleic Acids Res., 25:3389-3402) algorithm of Miao Shuing comes aligned sequences, because introduced in BLAST (the basic local comparison research tool) program, can obtain at the ncbi.nlm.hih.gov of World Wide Web.Can carry out blast search or compare the percentage ratio sequence homogeneity of measuring between nucleic acid molecules (" inquiry sequence ") and any other sequence or its fragment, use the Altschul algorithm.Can use BLASTN to compare and the homogeneity between the nucleotide sequence relatively, and use BLASTP to compare and the comparing amino acid sequence between homogeneity.When the nucleotide sequence of use blast program calculation code therapeutical peptide and the percentage ratio homogeneity between another sequence, can use the default parameter of program separately, comprise the default of breach punishment.

Can be by detecting nucleic acid of the present invention as DNA or rna blot analysis (that is hybridization), PCR or the such method of in situ hybridization analysis.Usually by the immunocytochemistry in the transfectional cell series or by sodium lauryl sulphate (SDS)-polyacrylamide gel electrophoresis follows Coomassie blue stain or western blot analysis detects polypeptide, use the antibody (monoclonal or polyclone) of specificity binding affinity with specific polypeptide.Many Sambrook etc. (1989 that are described in greater detail in these methods, Molecular Cloning:A Laboratory Manual (molecular cloning: laboratory manual), the 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

Coding immunity the stimulation altogether polypeptide and combination can operationally interconnect in construct member's nucleotide sequence, use conventional Protocols in Molecular Biology.Referring to, for example, MolecularCloning:A Laboratory Manual (molecular cloning: (Sambrook etc. laboratory manual), 2001, the 2nd edition, Cold Spring Harbor Laboratory Press) or Short Protocolsin Molecular Biology (molecular biological succinct experimental program) (Ausubel etc., 2002, the 5th edition, Current Protocols).Be applicable to construct in these methods be can buy and be that this area is used in everyday.Construct can comprise the sequence of expression needs, and as promoter sequence, regulating and controlling sequence as enhancer sequence and response sequence and/or derivable sequence, is regulated the expression of nucleotide sequence.As used in this, " be operably connected " and refer to (i) and place promoter and/or other regulating and controlling sequence with respect to nucleotide sequence in the mode that instructs or regulate expression of nucleic acid; And/or (ii) place the nucleic acid of coding immunity the stimulation altogether polypeptide and coding in conjunction with nucleic acid to the member, and make coded sequence " in frame ", that is, make the construct coding comprise that immunity stimulates polypeptide altogether and in conjunction with fusion rotein to the member.

Can be by methods known in the art with construct breeding or expression, in host cell, to produce polypeptide.As used in this, term " host " or " host cell " meaning are not only to comprise prokaryote, as escherichia coli, and comprise eukaryote, as yeast, insecticide, plant and animal cell.Zooblast comprises, for example, and COS cell and HeLa cell.Can use dna molecular (for example, construct) to transform or transfection host cell, use and well known to a person skilled in the art any technology, as calcium phosphate or lithium acetate precipitation, electroporation, fat transfection and microparticle bombardment.The host cell that contains carrier of the present invention can be used for as the breeding carrier, produces nucleic acid (for example, DNA or RNA), express the purpose that immunity stimulates polypeptide or its fragment or expressed fusion protein altogether, as mentioned above.

Tu1A ﹠amp; 1B, 2A ﹠amp; 2B, 3A ﹠amp; 3B, 4A ﹠amp; 4B, 5A ﹠amp; 5B and 7A ﹠amp; 7B has shown representational nucleotide sequence (SEQ ID NO.1,3,5,7,9﹠amp; 14), these sequences comprise that comprising the core Succ-PEG-DSPE stimulates the immunity of polypeptide to stimulate coded sequence partly altogether with immunity altogether, and the aminoacid sequence of corresponding encoded (SEQ ID NO.2,4,6,8,10; 15).

Immunization therapy

One embodiment of the invention provide the method that produces or strengthen the immunne response of antagonism first antigen or infectious agent, by with (a) first conjugate and (b) patient that needs of second conjugate, first conjugate comprises that (i) comprises that first immunity stimulates the conjugate member of polypeptide altogether and (ii) comprises conjugate member in conjunction with first right member, and second conjugate comprises that (i) comprises the conjugate member of first antigen or infectious agent and (ii) comprise conjugate member in conjunction with second right member.In interchangeable embodiment, handle immunocyte with first and second conjugates, give the patient then.As mentioned above, can use any immunity to stimulate polypeptide and any antigen (or infectious agent self) relevant altogether, can use any equally in conjunction with right with tumor or infectious agent.

The present invention includes and use chimeric costimulatory molecules, use or do not use and the puting together of target antigen.

Conjugate is directly being given in patient's the embodiment, can give first conjugate and second conjugate in order simultaneously or at different time.In one embodiment, before giving the patient, by conjugate being combined in conjunction with combination activity to the member.For example, can and in single compositions, give at external mixing first and second conjugates.In another embodiment, at first give first conjugate, then after being enough to make immunity stimulate the time of polypeptide binding immunoassay cell altogether, give second conjugate.For example, the time period can be one to several hours, and one was all or longer to a couple of days in one day.

Can be with the first or second conjugate whole body or topical administration patient, as passing through intravenous, intranasal, peritoneum or subcutaneous injection.In one embodiment, to tumor locus, as by injecting in the tumor, or be injected to the local infection position, come the one or more compositionss of topical by direct injection.In another embodiment, come the one or more compositionss of administration by different approach.For example, can the one or more compositionss of topical, and the one or more compositionss of whole body administration.

Being used for processing subsequent at conjugate directly gives to handle immunocyte in order simultaneously or in the different time with first and second conjugates in patient's the embodiment of immunocyte.In one embodiment, before being used to handle immunocyte, first and second conjugates are by combination combining in conjunction with activity to the member.For example, can be blended in first and second conjugates in the single compositions and be used for, as by immunocyte is contacted compositions at the extracorporeal treatment immunocyte.In another embodiment, handle immunocyte, handle with second conjugate after a period of time with first conjugate.This time period for example, is one to several hours, one day to several days, and a week or longer time.The immunocyte that to handle by above-mentioned any way gives the patient, comprises whole body or topical, as injecting in the tumor or being injected in the position of local infection.

According to an embodiment, this method further comprises and gives the 3rd conjugate or handle immunocyte with the 3rd conjugate.In one embodiment, the 3rd conjugate comprises conjugate member that (i) comprise that immunity stimulates polypeptide altogether and (ii) comprises second antigen relevant with tumor or infectious agent or the conjugate member of infectious agent self.The immunity of the 3rd conjugate stimulates polypeptide to stimulate polypeptide identical or different altogether with the immunity of first conjugate altogether, and second antigen can be identical or different with first antigen.In the particular aspects of this embodiment, immunity stimulates polypeptide altogether to stimulate polypeptide and second antigenic interaction to combine to the member with the immunity that links to each other separately by combination with second antigen altogether.According to this embodiment, first of the 3rd conjugate and second combination can combine the member identical or different with first and second of first and second conjugates to the member.

In one embodiment, first conjugate comprises that the immunity in conjunction with the composing type receptor stimulates polypeptide altogether, and as CD80, LIGHT and CD40L, and the 3rd conjugate comprises that the immunity of zygotic induction receptor stimulates polypeptide altogether, as 4-1 BBL and OX40L.

Following table 7 provides the tabulation of composing type and induction type costimulatory molecules.

Table 7

Composing type	Induction type
		CD80-CSA	CSA-4-1BBL
CSA-CD40L	CSA-OX40L
		CSA-LIGHT	PD-L1-CSA
	GL50-CSA

The effect that can test cancer immunotherapy by the reduction of measuring tumor cell proliferation and/or tumor size.The quantity of tumor cell is not immobilized, and reflects and stand fissional cell quantity and cell death quantity (for example, passing through apoptosis).Improve the individual propagation that can suppress cell to the immunne response of antitumor cell.Refer in the section preset time increase (external or in vivo) of (for example, several hours, several days, a few weeks or months) tumor cell quantity in the propagation of this used tumor cell.The reduction that can advance the speed by tumor cell, tumor cell lose fully or wherein any reduction of propagation measure tumor cell proliferation inhibition.The reduction of entity tumor size is the sign that tumor cell proliferation suppresses.

Stimulate the interaction of polypeptide (as 4-1 BBL) and its receptor altogether by immunity, by the ability with the selectively targeted DC of TAA is provided, the present invention has given the advantage that surpasses existing cancer vaccine.In addition, the invention provides and to give the patient and by the vaccine that DC absorbs in vivo by injection, make antigen presentation and activation, and do not need the separation of DC and ex vivo to handle or use gene therapy.

Can,, measure immunization therapy by measuring patient's infection load to anti-infectious effect as fever or swelling by measuring clinical endpoint.

Avidin/biotin according to the present invention has been given more superiority in conjunction with right use (or provide other mechanism of the high-order immunity costimulatory molecules): tetramer structure (or other polymer structure) is provided, this structure allows immunity, and costimulatory receptor is crosslinked altogether, be used for stronger replying, and allow a plurality of antigen molecules to be sent to DC.In one embodiment, in conjunction with first right member, promptly, the combination of first conjugate (comprising that first immunity stimulates polypeptide altogether) is avidin, Succ-PEG-DSPE or core Succ-PEG-DSPE to the member, and in conjunction with second right member, that is, the combination of second conjugate is a biotin to member's (comprising first antigen or infectious agent).In another embodiment, be biotin in conjunction with first right member, and be avidin, Succ-PEG-DSPE or core Succ-PEG-DSPE in conjunction with second right member.

Stimulate polypeptide can give more superiority altogether as immunity 4-1 BBL, because stimulate DC to show the T that sends as an envoy to 4-1 BBL _RegThe cell inhibiting function is invalid, T _RegCell plays main effect in immune tumor is escaped.Therefore, the conjugate that the present invention includes 4-1 BBL and TAA is sent to DC with TAA, is used for effective submission, activates DC, is used to produce various cytokines, and makes T _RegThe function of cell is invalid, strengthens the function of Teff and NK cell simultaneously, is used for tumor and eradicates.

The immunocyte of modifying

The present invention also provides the modification that can be used in above-mentioned immunotherapy method immunocyte, and preparation method thereof.According to this aspect of the invention, provide and modify immunocyte and produce or strengthen to the tumor of expressing the first antigen related antigen or to the method for the immunne response of infectious agent.This method comprises stimulates immunocyte contact (a) first conjugate of polypeptide receptor and (b) second conjugate altogether with expressing first immunity, and first conjugate comprises that (i) comprises that first immunity stimulates the conjugate member of polypeptide altogether and (ii) comprises conjugate member in conjunction with first right member; Second conjugate comprises that (i) comprises the conjugate member of antigen relevant with tumor or infectious agent or infectious agent and (ii) comprise conjugate member in conjunction with second right member.According to this method, stimulate the combination between polypeptide and the receptor that first conjugate and immunocyte are puted together altogether by immunity, by first and second combination second conjugate and immunocyte are puted together in the combination between the member.

Can immunocyte be contacted with first and second conjugates by any way, and can realize this in vivo or external.For example, method can comprise and first and second conjugates are contained immunocyte and contains tumor or infectious agent or be in the patient of containing in tumor or the infectious agent risk in the body.According to this method, can be basically simultaneously (in identical or the compositions of separating) or in order (in the compositions of separating) give first and second conjugates.Contain in the embodiment of tumor the patient, by injecting at least one that gives in first and second conjugates in the tumor.

Exemplary in vitro method can comprise immunocyte at external contact first and second conjugates, and as comprising the single compositions of first and second conjugates by contact, or contact comprises first and second compositions of first and second conjugates separately.When providing conjugate in single compositions, they can combine to the combination between the member by first and second combination that provides in the compositions.

In this aspect of the invention, can use any immunity to stimulate polypeptide, antigen or infectious agent altogether, and in conjunction with to the member, comprise above-mentioned each.

Can modify any immunocyte that expression first immunity stimulates polypeptide receptor altogether according to this method.In one embodiment, immunocyte is T cell or neutrophil cell.The example T cell comprises the CD4+ cell, CD8+ cell, natural killer cell, mononuclear cell and dendritic cell.

In the more embodiments of the present invention aspect this, immunocyte comprises that second immunity stimulates the receptor of polypeptide altogether, and this method comprises further immunocyte is contacted with the 3rd conjugate that the 3rd conjugate comprises that (i) comprises that first immunity stimulates the first conjugate member and the second conjugate member who (ii) comprises the antigen relevant with tumor or infectious agent (or infectious agent self) of polypeptide altogether.In this embodiment, second immunity stimulates polypeptide to stimulate polypeptide identical or different altogether with first immunity altogether, and second antigen, if exist, can be identical or different with first antigen (if existence).In another specific embodiment, the first and second conjugate members comprise further that separately first and second combination are to the member.According to this embodiment, first of the 3rd conjugate and second combination combine the member identical or different to first of member and first and second conjugates with second.In addition, the first conjugate member can combine with the second conjugate member the combination between the member by first and second combination.

As aforesaid first and second conjugates, the 3rd conjugate can contain that any immunity stimulates polypeptide, antigen or infectious agent altogether and in conjunction with to the member, comprise described herein arbitrarily those.

In related fields, the invention provides the immunocyte group who makes by this method.When contacting with other immunocyte, such immunocyte generation or enhancing are to the immunne response of tumor.

The present invention also provides and has expressed the modification immunocyte that first immunity stimulates polypeptide receptor altogether, wherein use (a) first conjugate and (b) immunocyte of the second conjugate modification, first conjugate comprises that (i) comprises that first immunity stimulates the conjugate member of polypeptide altogether and (ii) comprises conjugate member in conjunction with first right member; Second conjugate comprises that (i) comprises the conjugate member of first antigen or infectious agent and (ii) comprise conjugate member in conjunction with second right member.According to this embodiment, stimulate the combination between polypeptide and the receptor that first conjugate and immunocyte are puted together altogether by immunity, by first and second combination second conjugate and immunocyte are puted together in the combination between the member.

According to above-mentioned method, in this aspect of the invention in, can use any immunity to stimulate polypeptide, antigen or infectious agent altogether, and in conjunction with to the member, comprise above-mentioned each.

In addition, according to this method, can modify and express any immunocyte that first immunity stimulates polypeptide receptor altogether.In one embodiment, immunocyte is T cell or neutrophil cell.The example T cell comprises CD4+ cell, CD8+ cell, natural killer cell, mononuclear cell and dendritic cell.

The immunostimulation part

The present invention also provides the part of the immunostimulation with immunostimulatory activity.When individually dosed, or during as adjuvant conjugated antigen or other immunostimulant administration, immunostimulation partly is useful.For example, in vaccine, cancer immunotherapy and the treatment based on immune disorder, immunostimulation partly is useful.Immunostimulation partly can be formulated in and be applicable in the compositions that gives animal, and can need immunostimulating animal, as accept vaccine, cancer immunotherapy or stand animal based on the immune disorder treatment.

According to an embodiment, immunostimulation comprises that partly above-mentioned immunity stimulates any of polypeptide altogether, as 4-1 BBL, CD86, ICOSL, PD-L1, PD-L2, B7-H3, B7-H4, OX40L, CD27L, CD30L, LIGHT, BAFF, APRIL, CD80 and CD40L.In another embodiment, immunity stimulates polypeptide to be selected from 4-1BBL, CD86, ICOSL, PD-L1, PD-L2, B7-H3, B7-H4, OX40L, CD27L, CD30L, LIGHT, BAFF and APRIL altogether.In another embodiment, it is 4-1 BBL that immunity stimulates polypeptide altogether.

According to a particular aspects of this embodiment, the immunostimulation part further comprises Succ-PEG-DSPE or core Succ-PEG-DSPE.For example, the immunostimulation part can be conjugate or the fusion rotein that comprises immunostimulation polypeptide and core Succ-PEG-DSPE.

In another embodiment, the immunostimulation part stimulates polypeptide and Succ-PEG-DSPE or core Succ-PEG-DSPE to constitute by immunity basically altogether.According to this embodiment, immunostimulation part do not comprise, or do not put together or in addition in conjunction with any other immunostimulant, stimulate polypeptide or antigen altogether as another immunity.

The present invention also comprises the immunostimulation method, and comprising partly needs immunostimulating patient with immunostimulation.In an embodiment of this method, immunostimulation comprises that partly immunity stimulates polypeptide and Succ-PEG-DSPE or core Succ-PEG-DSPE altogether.In another embodiment, the immunostimulation part stimulates polypeptide and Succ-PEG-DSPE or core Succ-PEG-DSPE to constitute by immunity basically altogether.In further embodiment, this method further comprises and gives the patient with antigen, with the administration of immunostimulation part simultaneously or in order (before or after).At the same time in some embodiments of administration, administration immunostimulation part and antigen in single compositions, as comprise immunostimulation part and antigenic mixture.At the same time in other embodiment of administration, the compositions administration immunostimulation part and the antigen that are separating.In some embodiments, give antigen, as comprise antigen and in conjunction with to member's conjugate as the above-mentioned antigen conjugate that contains.In other embodiments, do not have as comprising that the conjugate in conjunction with to the member gives antigen.

Another embodiment of immunostimulation method partly constitutes by giving immunostimulation basically, and the immunostimulation part stimulates polypeptide and Succ-PEG-DSPE or core Succ-PEG-DSPE to constitute by immunity basically altogether.According to this embodiment, not giving other will partly put together with immunostimulation or other bonded immunostimulant, stimulate polypeptide or antigen altogether as another immunity.Therefore, for example, do not give biotinylated molecule, as biotinylated cell or comprise the protein conjugate of biotin.

Although do not wish to be bound by any theory, think that immunostimulation of the present invention has partly stimulated the interaction between cell surface immunity receptor and the part thereof, therefore promoted body fluid and cellullar immunologic response.The immunostimulation that comprises Succ-PEG-DSPE (or core Succ-PEG-DSPE) partly forms the saturated oligomer of the stable tetramer, it is bind receptor and stimulate B cell, mononuclear cell and dendritic cell effectively, is used to produce cytokine, chemotactic factor and rise molecules of immunization stimulus.

Following embodiment understands the present invention in more detail, and is not the scope that is used for limiting any aspect of the present invention.

Embodiment

Experimental technique

Animal.Inbreeding BALB/c (H-2 grows up ^d) and the C57B/6 mice available from JacksonLaboratories (Bar Harbor, Maine).(Germantown instructs NY) and according to NIH and to raise available from Taconics for TCR transgenic OT-I, DO11.10, C57BL/6.SJL animal.

Express the foundation of the A20 cell of OVA.The OVA construct is available from the Dr.Tom Mitchell of Lewis's Weir university and directly be cloned in the pcDNA3 carrier with BglII and EcoRI restriction (Invitrogen, San Diego, CA).Antibacterial transforms and after selecting on the ampicillin medium, several clones is accepted mini plasmid preparation and digest with BglII/EcoRI to identify positive colony.To contain the segmental clone of insertion then and be used for big plasmid preparation, and use Lipofectamine according to the explanation of manufacturer ^TMThe transfection of 2000 (Invitrogen) test kit is to the A20 cell.In the culture medium that contains G418 (Geneticin), select cell and use antibody or the T cell proliferation of Western blotting, antagonism OVA to test the expression of measuring OVA.

The foundation of TC-1 transplantation cervical cancer model.In the C57BL/6 mice, set up the TC-1 tumor model.By producing oncogenic TC-1 cell line, and be characterized by human cervical carcinoma's model with HPV-16 E6 and E7 and the elementary C57BL/6 mouse lung of activated ras oncogene cotransformation epithelial cell.The TC-1 cell forms tumor in homology C57BL/6 mice.In order to set up model, with 1 * 10 ⁵Individual tumor cell transplantation is to the right side of C57BL/6 mice, and the tumor growth of monitor animal.

Use expression and the purification of the reorganization 4-1BBL of insecticide DES expression system.According to Singh etc., the 2003, (Invitrogen of the use fruit bat DES expression system described in the Cancer Res.63:4067-73; Carlsbad CA) sets up stable conversion of expressing 4-1BBL.Shake in the bottle at the incubators that are set at 25 ℃ and 105rpm, at the fruit bat serum-free medium (Gibco that replenishes 1mM copper sulfate; Carlsbad induces the expression of recombinant proteins 72 hours of transformant in CA).By centrifugal collection culture supernatants and accept large scale purification, use the crosslinked fixed metal affinity of the agarose resin (BD-Talon of cobalt (II)-carboxymethyl asparagic acid salt, BDBiosciences) or Ni-NTA metal affinity resin (Qiagen), utilize the 6x-His-label to be engineered in the protein.

In brief, produce 10% alcoholic acid final concentration by dropwise adding 95% ethanol, precipitate the culture medium that contains 4-1 BBL, after 4 ℃ of night incubation, sedimentary 4-1BBL is dissolved in again in 1/10 the volume, uses binding buffer liquid (50mM sodium phosphate pH7.0; 500mM sodium chloride; 0.5% tween 20; 1% glycerol; The 5mM 2 mercapto ethanol).Use the binding buffer liquid of 5 * gel bed volume to come balance metal affinity resin, add the dissolved again protein solution that contains 4-1 BBL, and be rotated in incubated at room 45 minutes with putting upside down.With 50-100ml lavation buffer solution (50mM sodium phosphate pH7.0; 500mM sodium chloride) will wash 2 times in conjunction with the metal affinity resin of 4-1 BBL.Elution buffer (50mM sodium phosphate pH7.0 with 2 * gel bed volume; 500mM sodium chloride, the 150mM imidazoles) from the bonded 4-1 BBL of metal affinity resin elution.

Merge the 4-1 BBL eluate of purification and be loaded into Amicon Ultra ^TM(Millipore; Bedford Mass) in the centrifugal filter device, uses 30kD molecular weight mwco membrane.(2000 * g) 4 ℃ centrifugal 15 minutes at 3000rpm with centrifugal filter device.Aseptic PBS is added in the trapped substance, and with filter at 3000rpm (2000 * g) recentrifuge.Sucking-off concentrates/trapped substance of the 4-1BBL of desalination from centrifugal filter device, places the sterile cryogenic bottle, and is stored in the liquid nitrogen.Measure the purity of protein isolate by the SDS-polyacrylamide gel electrophoresis.Use BCA protein determination (Pierce) to measure protein concentration according to the explanation of manufacturer.

The expression of biotinylated OVA and purification.Extremely from Avidity, (Denver in pAN CO) and the pAC carrier, is used for expressing separately N-end and C-end AviTag-protein blend zoarium to Inc. with above-mentioned OVA construct sub-clone.Antibacterial transforms and after selecting on the ampicillin medium, several clones is accepted minimum plasmid preparation and digest with suitable restriction endonuclease to identify positive colony.To contain the segmental clone of insertion and be used for big plasmid preparation.Plasmid is used to transform the AVB100 escherichia coli, and this is the bacterial strain with stable integration birA ligase gene to the chromosome.Express with the L-arabinose induced protein, L-arabinose is used to have the high level expression of the OVA of biotin labeling thing.Use the expressed protein of AviTag antibody agarose purification.Use Western blotting to measure concentration, level of endotoxin and the biotinylation of purification OVA, and the Succ-PEG-DSPE that alkali phosphatase is puted together is used for surveying.If desired, use Detoxi-gel endotoxin removal test kit (Pierce) to remove endotoxin.Biotinylated OVA is puted together the CSA-4-1BBL fusion rotein, and use OT-I TCR transgenic cell to test in the propagation test in vivo, as described below.With the protein five equilibrium and at-70 ℃ until use.

The propagation test.For the proliferation in vivo test, from OT-I (OVA _257-264/ K ^b) TCR transgenic animal collection spleen and lymph-node cell.With 5 μ M CFSE (CF 5(6)-Carboxyfluorescein diacetate succinimide ester) labeled cell and by tail vein injection with the cell transfer of 1,000,000 CFSE-labellings to CD45.1 ⁺In the carcinogenic B6-SJL mice.After 24 hours, with 10 independent μ gOVA, with CSA or the blended OVA of CSA-4-1BBL or put together CSA or the OVA of CSA-4-1 BBL excites animal.Collect spleen and lymph-node cell after 3 days, and by the CD8 among the lymph node gate ⁺CD45.1 ^-(OT-1) propagation is measured in the CFSE dilution analysis in the cell mass.The cell of never accepting the more proteic animals collections of OVA is used for measuring parental population, is used for analyzing.

Followingly carry out in-vitro multiplication test: will be from the DO11.10 (OVA of the CFSE labelling of BALB/c mouse _323-339/ I-A ^d) the TCR transgenic cell is with the agent that responses, and resists the A20 transfectant of the expression OVA that shone with different ratios, continues 3 days.Collect culture and in flow cytometry, analyze propagation.

Flow cytometry.Use standardization program to carry out flow cytometry, at first titration target one is anti-and two anti-, uses optium concentration at flow cytometry then.Referring to, for example, Mhoyan etc., 1997, Transplantation 64:1665-70.The antibody of homotype coupling is as negative contrast.With sample at FACS Calibur or Vantage Sorter (Becton Dickinson; MountainView CA) goes up operation, and uses FlowJo software (TreeSoft) to analyze.

Immunization therapy.The following inoculation.In brief, with various mol ratios the CSA-4-1BBL fusion rotein is mixed with biotinylation OVA among the PBS, then by peritoneal injection to BALB/c mouse, be used for pre-inoculation, or be used for immunization therapy, the A20 alive (1 * 10 that is injected at side subcutaneous vaccination lethal dose ⁶) in the animal of cell.Contrast comprises the animal that not have inoculation or inoculates those of reference protein.In case detect, the use slide calliper rule are every other day measured tumor and are reported as the mean+/-standard error of longest diameter and perpendicular diameter.When the tumor size reaches about 20mm diameter, with Animal Anesthesia, to avoid uncomfortable.

Statistics.Use the effect of Kaplan-Meier curve estimation processing to tumor survival.Use the survival difference of logarithm level testing (Savage/Mantel Cox is generalized) between measuring not on the same group.Relate to from the program relatively of animal groups experimental data separately and equating with the variation of using F check (two groups) or Levene ' s check (many groups) detection.If change unequally, carry out logarithmic transformation.When comparing the sample mean of normal distribution, use Student ' s t check (two groups) or Newman-Keuls check (organize) more.When data do not have normal distribution, use Mann-Whitney U check (two groups) or Kruskal-Wallis check (many groups).Significant difference is defined as P＜0.05.

Embodiment 1:CSA-4-1 BBL has enlarged replying that isoantigen produces

As mentioned above, 4-1 BBL replys in the adjusting in acquired and innate immunity and plays an important role.4-1 BBL is used to activate CD4 as costimulatory molecules ⁺And CD8 ⁺T cell, NK cell and DC, and suppress T _RegThe cell inhibiting function.Therefore, this molecule can be used for treatment of cancer as the specific adjuvant that produces effective tumor response.

Because the existence of core Succ-PEG-DSPE part, CSA-4-1 BBL fusion rotein form four poly-/oligomerization structures, and are the molecules of solubility.Use isoantigen mixed lymphocyte reaction (MLR) to prove the immunostimulatory activity of CSA-4-1 BBL to t cell response, as described below.

In the existence of CSA-4-1 BBL or not, with of the reply agent of C57BL/6 mouse lymph nodal cell as the splenocyte that shone of antagonism BALB/c.At last 18 hours of cultivation stage, use [ ³H] thymidine labelling culture, and measure propagation.With compare, the culture that replenishes CSA-4-1BBL demonstrates effective proliferation activity (Fig. 8).

Embodiment 2: CSA-4-1 BBL has improved the T cell proliferation

In order to measure the relative activity of 4-1-BBL fusion rotein and the monoclonal antibody of antagonism 4-1 BB, in the propagation test, in the presence of the 4-1 of various content BBL fusion rotein and antibody, will be with the anti-CD 3 antibodies of time good concentration by the CD4 of flow cytometry sorting ⁺And CD8 ⁺T cell polyclone stimulates.Fusion rotein is to T cell proliferation the high 70-of specific activity antibody times (Fig. 9).

Because having demonstrated, this antibody A b in the animal model of cancer immunotherapy, has effective activity.Referring to, for example, Melero etc., 1998, Cell Immunol.190:167-72; Melero etc., 1997Nat.Med.3:682-85, these data show that the CSA-4-1BBL fusion rotein will be the useful component of cancer vaccine, as adjuvant or as the carrier that TAA is sent to DC.

Embodiment 3: biotinylated OVA/CSA-4-1BBL conjugate is to CD8 ⁺The effect of T cell

(Pierce Biotechnology, Rockford is IL) with ovalbumin peptide (OVA) biotinylation for the test kit that use can be buied.Biotinylated OVA is pre-mixed with different ratios with the CSA-4-1BBL fusion rotein external, is used to put together, and with 1,000,000 OT-1T cells at peritoneal injection to the natural C58BL/6.SJL animal of acquired transfer.Particularly, with 1,000,000 OT-I CD8 of CFSE labelling ⁺The T cell also is transferred in the B6.SJL mice, this mice with biotinylated ovalbumin (10 μ g/ injection) (" OVA ") and with the blended CSA-4-1BBL of biotinylated OVA (1 μ g/ injection) (" 41BBL+OVA ") or OVA-biotin/CSA-4-1 BBL (" 41BBL-OVA ") immunity of puting together.(Figure 10) last figure of Figure 10 (" 41 BBL-OVA ") has shown the replying of CSA-4-1 BBL of 5 μ g being puted together the biotinylated OVA of 10 μ g.In order to contrast, to use core Succ-PEG-DSPE (" SA ") with mol level such as CSA-4-1BBL.

As shown in Figure 10, compare with contrast " SA/OVA " conjugate (33.6%) or unconjugated single protein " 41 BBL+OVA " (35.5%), 4-1 BBL/OVA conjugate has produced effective (73.5%) propagation and has replied in the OT-1 cell.It is dose dependent that propagation is replied, because the CSA-4-1 BBL of 5 μ g dosage has produced higher replying (94.5%) than 1 μ g dosage (73.5%).

This embodiment has shown that CSA-4-1 BBL fusion rotein has improved antigenic specificity CD8 ⁺The propagation of T cell is replied, and shows that the biotinylated antigen construct of 4-1 BBL-CSA/ can successfully be sent to antigen special APC and activate these cells, is used to produce efficient immune.

Embodiment 4: CSA-4-1 BBL is sent to DC with antigen

This embodiment has proved that CSA-41BBL is sent to DC with antigen effectively.Biotinylated PE is as fluorescent antigen.Puted together 30 minutes with biotinylated PE (250ng) on ice with 250ng CSA-41 BBL.With biotinylated PE (250ng/ml) or biotinylated PE/CSA-41BBL conjugate with Jaw II dendritic cell (5 * 10 ⁵/ hole) cultivated 16 hours.Use the level of Flow cytometry PE.Figure 11 A is the block diagram that shows the PE+ cell.The Lycoperdon polymorphum Vitt filling part is represented untreated cell, and black dotted lines is represented the cell with biotinylation PE processing, and black line is represented the cell with the processing of biotinylation PE/CSA-41 BBL conjugate.Figure 11 B has shown the PE average fluorescent strength (MFI) of each processing, and has proved that the cell that conjugate was handled presents significantly higher replying.

Embodiment 5: CSA-4-1 BBL activates DC

This embodiment has proved that 4-1 BBL activates dendritic cell.Jaw II dendritic cell (5 * 10 ⁵/ hole) is untreated, or in the flat board of 24-hole, handling 48 hours in the presence of the 5ng/ml GM-CSF with 5 μ g/ml CSA-41BBL conjugates or 5 μ g/ml lipopolysaccharide (LPS).Use flow cytometry CD86 and II class MHC level, as shown in Figure 12 A.Right side Lycoperdon polymorphum Vitt filling part is represented the cell that homotype is handled, and the untreated cell of expression that the grey black color is filled, black line are represented the cell that CSA-4-1BBL handles, and dotted line is represented the cell that LPS handles.Figure 12 B has shown the average fluorescent strength (MFI) of CD86 and II class MHC, and has proved that the cell that CSA-4-1BBL handles presents significantly higher replying.

Embodiment 6: CSA-4-1 BBL is sent to antigen DC and activates DC in vivo

This embodiment has proved that CSA-4-1 BBL is sent to dendritic cell with biotinylated antigen and drives these cells and has activated in vivo.Biotinylated OVA contact CSA-41BBL is produced biotinylated OVA/CSA-4-1 BBL conjugate.With conjugate or the intravenous injection of biotinylated OVA/CSA conjugate to natural C57BL/6 mice.After 24 hours, with Animal Anesthesia and collect splenocyte.Use the dendritic cell in the flow cytometry CD11c+ cell mass to activate.The average fluorescent strength (MFI) that CD40, CD86 on the dendritic cell of the animal of handling with biotinylation OVA/CSA-41-BBL that mensuration is handled from natural, biotinylation OVA-SA and II class MHC express, as shown in figure 13.This figure has proved that the animal that biotinylated OVA/CSA-41-BBL handled presents significantly higher replying.

Embodiment 7: among the CSA-4-1 BBL and Treg cell inhibiting function

As mentioned above, the CD4 of natural generation ⁺CD25 ⁺FoxP3 ⁺4-1 BB receptor is expressed on Treg groups of cells molding ground, and therefore, replying 4-1 BBL stimulates.Following embodiment has proved the stimulating activity of 4-1BBL fusion rotein on the Treg cell.

Use flow sort separation of C D4 ⁺CD25 ^-Teff cell and CD4 ⁺CD25 ⁺The Treg cell is cultivated in the presence of homology cell that shone and anti-cd 3 antibodies with the 1:1 ratio.In order to distinguish CD4 ⁺CD25 ⁺(DP) to CD4 ⁺CD25 ^-(SP) propagation of T cell in co-culture experiments, (CFSE, Molecular Probes is OR) with CD4 with CF 5(6)-Carboxyfluorescein diacetate succinimide ester ⁺CD25 ^-Dyeing also is used for suppressing test.In brief, use the PBS washed cell, in 4ml2.5 μ M CFSE/1 * 10 ⁶(when more a spot of cell obtains labelling, keep this ratio) in the cell incubated at room 7 minutes.Then cell was hatched 1 minute in the peptide Ox blood serum of two volumes, and guarantee to remove all excessive CFSE 2 times with the PBS washing.Use flow cytometry to measure propagation.

The Treg cell is not replied anti-CD3 to stimulate, because they are anergies, but demonstrates the appropriateness propagation (Figure 15) of replying 4-1BBL.Especially, the Treg cell suppresses the propagation of Teff cell replys, and this is by adding a kind of effect that 4-1BBL can reverse.This is consistent with the data of using natural Treg cell, has wherein offset the effect of expansion cell inhibiting by the existence of CSA-4-1BBL.

These data acknowledgements the immunoregulation effect of 4-1 BBL, and be used for the purposes of cancer immunotherapy.For example, 4-1 BBL fusion rotein has been strengthened the Teff function, and has reduced Treg cell inhibiting function, is used for more strong anti-tumor immune response.

Embodiment 8: the dual function of 4-1 BBL

The effect of signal in regulating the Treg function of 4-1BB/4-1 BBL mediation is the theme of nearest two researchs, has opposite discovery.Although one studies have shown that the effect of 4-1 BB signal cancellation Treg cell inhibiting, Chol etc., 2004, J.Leukoc.Biol.75:785-91,4-1 BB signal mediation Treg propagation has then been reported in another research, to the not significantly effect of its inhibit feature, Zheng etc., 2004, J.Immonul.173:2428-34.In order to clarify this contradiction, use CSA-4-1 BBL fusion rotein to study the effect of 4-1BB signal in the Treg function.

Spleen and periphery lymph node sorting CD4 from natural B ALB/c mice ⁺CD25 ^-(single positive; SP) and CD4 ⁺CD25 ⁺(two positives; DP) T cell, and cultivated 3 days separately or with the ratio of 1:1.Culture is replenished the splenocyte that shone, anti-cd 3 antibodies (0.5 μ g/ml), the contrast CSA albumen of the concentration of 4-1BBL (μ g/ml) or equivalent is shown among Figure 14 A.In co-culture experiments, use the CD4 of fluidic cell sorting from natural B ALB/c mice purification ⁺CD25 ⁺Two positives (DP) T cell has significantly suppressed inductive single positive (SP) CD4 by antibody antagonism CD3 ⁺CD25 ^-The propagation of Teff cell is replied.Effectively and specifically reversed this inhibitory action by replenishing 1 μ g/mlCSA-4-1 BBL to culture, but the contrast CSA of mol level such as use not.

In order to test whether the viewed inhibition reverse that is caused by CSA-4-1 BBL fusion rotein is because the recovery that the propagation of SP cell is replied causes, with CFSE labelling SP cell, and at CSA-4-1 BBL (0.5 μ g/ml) or as being used for co-culture experiments in the presence of the CSA of reference protein.CSA-4-1 BBL is increased to 60% of DP cell with the propagation of SP from 44% and the CSA proteic 46% that contrasts, and has significantly reduced the propagation of SP T cell (16%), and this is significantly recovered (34%) by 4-1BBL, but the CSA reference protein does not have (17%).(Figure 14 B) these digital proofs 4-1 BBL fusion rotein reduced Treg cell inhibiting function.

Therefore, our worksheet understands that CSA-4-1 BBL fusion rotein presents two kinds of opposite activity to the Treg cell.On the one hand, it and anti-cd 3 antibodies and IL-2 synergism promote the Treg cell to enlarge.On the other hand, it blocks natural and activated Treg cell inhibiting function, is not only when Treg cells contacting 4-1 BBL fusion rotein, because be removed the recovery that has caused inhibit feature from culture medium.

This back of 4-1 BBL is a kind of to act on to use in the scope of Treg cell as the tumor of immune evasion mechanism and infection and has certain meaning.

Embodiment 9: antigen-4-1-BBL conjugate is expressed generation as the A20 transfectant of the OVA of soluble protein as the purposes (a) of cancer vaccine.

Experimental program according to manufacturer (Invitrogen) uses Lipofectamine ^TM2000 test kits with above-mentioned OVA construct transfection to the A20 cell.Select to select in the culture medium stable transfectant at G418,, and use Western blotting to test the expression of OVA unicellular level clone.The clone that will have the OVA expression of significant level breeds the DO11.10 CD4 of OVA peptide specific in the test as CFSE ⁺The stimulant of T cell is as described with reference to above Figure 10.In case it is male having confirmed to express for OVA, is injected in the right side of BALB/c mouse under the A20 cell skin with 1,000,000 work.Every other day the tumor of monitor animal produces and survival, and when diameter of tumor reaches the 20mm size, with Animal Anesthesia.The contrast of the animal of parent A20 cell as tumor growth will be inoculated.In the time of in being injected to the homology BALB/c mouse, A20 cell transfecting of expressing OVA will form tumor.

(b) use insecticide DES system to produce CSA-4-1 BBL fusion rotein.

Use DES expression system (Invitrogen) and our experimental program of foundation to prepare CSA-4-1 BBL albumen.Referring to, for example, Singh etc., 2003, Cancer Res.63:4067-73; Yolcu etc., 2002, Immunity 17:795-808.Use immobilization metal base affinity chromatograph purified fusion protein, utilize to be engineered to CSA-4-1 BBL fusion rotein single 6XHis label afterwards.With the protein desalination, by ultrafiltration and concentration, and by the SDS-PAGE purity assay.Use dihomocinchonine acid (BCA) test (Pierce) to measure the concentration of protein formulation, and use from Cambrex's

Chromogenic LAL terminal test detects endotoxic existence.

(c) biotinylation of OVA

(Molecular Probes, San Diego, experimental program CA) use DSB-X biotin labeling test kit with the activated no endotoxic chicken OVA of maleimide (Pierce) biotinylation according to manufacturer.In PBS, thoroughly after the dialysis, use Western blotting to measure concentration, level of endotoxin and the biotinylation of biotinylation OVA, and the Succ-PEG-DSPE that will put together alkali phosphatase is used for surveying.If desired, use Detoxi-gel endotoxin removal test kit (Pierce) to remove endotoxin.Biotinylated OVA is puted together the CSA-4-1BBL fusion rotein.With the protein conjugate five equilibrium, and be frozen in-80 ℃, until use.(d) biotinylated OVA/CSA-4-1BBL conjugate is as the purposes of cancer vaccine

CSA-4-1BBL is pre-mixed with different mol ratios in PBS with biotinylated OVA, as the 4-1BBL:OVA of 1:1,1:5,1:10,5:1 and 10:1, and with various dose (as 10,50 and 100 μ gOVA) with on every Wendesdays time interval by in peritoneal injection to the group BALB/c mouse.The biotinylation OVA of Succ-PEG-DSPE is puted together in injection, biotinylated OVA separately or the not biotinylated OVA animal of mixed C SA-4-1BBL with comparing.

Excite animal with the A20 tumor cell of 100 ten thousand work is subcutaneous on the right side, monitor every other day that tumor produces and survival, and when diameter of tumor reaches the 20mm size, with Animal Anesthesia.

To produce the effective antitumour immunne response with biotinylated OVA/CSA-4-1 BBL conjugate inoculation, and make prophylaxis of tumours grow.Also can produce with unconjugated 4-1 BBL and OVA inoculation and to reply, but any such replying will produce less than antigen/4-1 BBL conjugate.Only producing minimum replying with independent OVA or CSA-OVA inoculation, should be invalid in the prophylaxis of tumours growth therefore.

Embodiment 10: with the early stage/post incoulation of antigen-4-1BBL conjugate

As mentioned above, tumor is escaped immune system by the various mechanism that produce along with the tumor growth process.When also not setting up escape mechanism, inoculate animal simultaneously and prove that conjugate of the present invention is in the early stage effect of tumour progression by exciting with tumor.In case set up tumor and produced immune evasion mechanism fully, proved that by the inoculation animal conjugate of the present invention resists the effect of having set up tumor.

(a) in the early stage effect of tumour progression

A20 cell with 1,000,000 work excites BALB/c mouse on the right side, inoculates biotinylated OVA/CSA-4-1 BBL conjugate at intraperitoneal simultaneously.One all repeated inoculation antigen/4-1 BBL conjugate once around continuing, arrives this time, and the diameter of tumor in the control animal will reach the 10-15mm size.The animal of unsteered animal and inoculation CSA-OVA conjugate will be in contrast.

(b) effect of tumor has been set up in antagonism

On the right side with the A20 tumor cell of 100 ten thousand work of BALB/c mouse subcutaneous vaccination.The tumor of monitor animal produces, and when tumor reaches diameter 4-6mm size, inoculates biotinylated OVA/CSA-4-1 BBL conjugate.Inoculation experiments side's amine will be referred to peritoneal injection weekly at first, until mice with tumor or reach the size of diameter 20mm.

The tumor back animal of effectively eradicating its tumor with the A20 cell activation of 200 ten thousand work in 60 days that disappears, be used for testing memory response.

Although do not wish to be subjected to the restriction of any theory, when tumour progression gives in early days, in a single day compare with the administration of having set up tumor, antigen of the present invention/4-1 BBL conjugate has higher effect in the prophylaxis of tumours growth, this is because in various inhibition mechanism of lacking in early days of tumour progression process.However, because antigen is selectively targeted to DC's, is used for the effective antigens submission, the activation of DC, be used to produce danger signal (adjuvant effect) and downward modulation Treg cell inhibiting function, antigen/4-1 BBL conjugate demonstrates effect in eradicating the tumor of having set up.Except indirect action to DC, by in conjunction with the 4-1BB receptor that activates on T and the NK cell, cause their strong propagation, survival and memory T cell function, the duplicate injection vaccine is the booster immunization system further.

Embodiment 11: antigen-4-1 BBL conjugate is by the effect of onlooker's effect.

Following embodiment will prove that biotinylated OVA/CSA-4-1 BBL conjugate has produced the immunne response of the undetermined A20 tumor antigen of antagonism (rather than OVA), by onlooker's effect or epitope expansion.

With the A20 of BALB/c animal, at the A20 cell of left side inoculation parent unmodified at right side inoculation expression OVA.In case can touch out tumor, with the biotinylated OVA/CSA-4-1 BBL of animal inoculation conjugate.According to above-mentioned inoculation time table, but also can change as required to improve effect.The growth of two kinds of tumor types in the monitor animal.

Perhaps, after eradicate expressing the A20 tumor of OVA 60 days, excite animal at offside under with 200 ten thousand parent A20 cell skins, this animal has successfully been eradicated tumor (as above embodiment) behind the OVA/CSA-4-1 BBL conjugate of inoculating bioid.

Biotinylated OVA/CSA-4-1BBL conjugate vaccine will demonstrate the effect of antagonism shortage as the parent A20 tumor of the OVA of TAA.For example, effective immunne response of antagonism OVA will cause killing of tumor, the coming off of tumor antigen, and catching and submission by APC, the t cell response of the new one group of TAA that is used to create antagonism.Can be used for further promoting the elimination of parent's tumor by the onlooker of the antagonism A20-OVA tumor that produces.

Embodiment 12: use bacterial expression system to produce biotinylated antigen.

In some cases, advantageously in heredity, produce biotinylated antigen, as the antigenic component of vaccine of the present invention.In this, can use Avidity, Inc. (Denver, biotin AviTag technology CO).Biotin AviTag is made of 15 unique amino acid peptides, and this peptide is by biotin ligase BirA identification, and biotin enzyme connects the lysine residue in biotin and the peptide sequence.Biotin AviTag can merge in heredity to any target protein, makes and can use the biotin molecule labelled protein.

With the cDNA sub-clone of coding OVA to pAN and pAC carrier, to express N-end and C-end AviTag-albumen fusant separately.With the birA gene transformation AVB100 escherichia coli of stable integration to the chromosome, and induce, be used for the OVA that high level expression carries the biotin labeling thing with L-arabinose.Use the expressed albumen of AviTag antibody agarose purification.Use BCA test kit, Chromogenic LAL test kit and measure concentration, level of endotoxin and the biotinylation of purification OVA with the Western blotting that the Succ-PEG-DSPE of puting together alkali phosphatase is made probe.If desired, use Detoxi-gel endotoxin removal test kit (Pierce) to remove endotoxin.

As mentioned above, biotinylated OVA and CSA-4-1BBL are puted together.With the protein conjugate five equilibrium and-80 ℃ freezing, until use.

Embodiment 13: the purposes that comprises antigen/4-1 BBL conjugate of TERT and Survivin

The biotinylated antigen of more than giving an example/CSA-4-1 BBL conjugate and biotinylated OVA/CSA-4-1 BBL can be used for any vaccine situation with any antigen.In the scope of cancer vaccine, two kinds of general people TAA, terminal enzyme reverse transcriptase and survivin can be the favourable antigenic components of biotinylated antigen of the present invention/CSA-4-1 BBL conjugate.

Embodiment 14: E7/4-1 BBL conjugate

As mentioned above, comprise that human papillomavirus E7 antigen is useful as the conjugate of the present invention of antigenic component in to anti-cervical cancer.This embodiment relates to this particular of the present invention.

(a) generation of biotinylated HPV-16E7

With the antigenic component of biotinylated E7 as conjugate, this conjugate is used as the HPV vaccine according to the present invention.In one embodiment, total length E7 albumen provides the epitope of maximum quantity.The cDNA of RT-PCR clones coding total length HPV-16 E7 is passed through in use from total RNA of TC-1 cell.After confirming sequence, cDNA sub-clone in the frame with 6X-His label is used in DES system constitutive expression and secretion to pMIB/V5-His carrier (Invitrogen).Use the excretory albumen of metal affinity resin purification as mentioned above.Experimental program (Pierce) according to manufacturer uses EZ-Link Sulfo-NHS-LC-biotin at external E7 biotinylation with purification.In brief, the concentrated E7 of purification is cushioned exchange in the saline (PBS) of phosphate-buffered, and hatch 1 in room temperature with EZ-Link Sulfo-NHS-LC-biotin and disappear.(Spectrum Labs NJ) removes unconjugated biotin to use grossflow filtration.

(b) generation of E7/4-1 BBL conjugate

According to above-mentioned general procedure, use biotinylated E7 and CSA-4-1 BBL fusion rotein to produce the conjugate that comprises E7 and 4-1 BBL.In order to compare following generation E7/4-1 BBL fusion rotein.CDNA sub-clone in the frame with 6X-His label of coding E7 and 4-1 BBL is used in DES system constitutive expression and secretion to pMIB/V5-His carrier (Invitrogen), and with protein expression and purification, as mentioned above.

(c) combination of E7/4-1 BBL conjugate is active

The biotin combination and the 4-1BB receptor-binding activity of following mensuration E7/4-1 BBL conjugate.

For the biotin combination, use the CSA-4-1 BBL (100ng/10 among the PBS with TC-1 cell biological elementization and on ice ⁶Cell) hatches.With PBS cell is thoroughly washed, dye with the antibody of the anti-4-1 BBL of fluorochrome label, and use flow cytometry to analyze.The biotinylation cell of puting together CSA is with comparing.

In order to test combining of 4-1 BB receptor on conjugate or fusion rotein and the activated T cell, Con A Concanavalin (ConA) with 5 μ g/ml will be activated 36h from the splenocyte of C57BL/B6 mice, wash and will be used on ice with PBS and hatch with the conjugate or the fusion rotein of various concentration.Cell is thoroughly washed, and dye, and in flow cytometry, analyze with the antibody of 4-1BBL, core Succ-PEG-DSPE or the E7 of suitable fluorochrome label.

Use 1:4 ratio (CSA-4-1BBL:E7) to form conjugate and measure combining of CSA-4-1 BBL conjugate and biotinylation E7, follow the bonded Chemical Calculation of CSA-biotin, in sandwich ELISA, test conjugate then by first.In brief, the protein binding that will put together covers the 96-hole flat board of anti-E7 antibody, and washing resists-the Succ-PEG-DSPE antibody incubation with reactive then, measures the content of the E7/4-1 BBL complex of existence.After confirming the formation of conjugate, test their abilities, as mentioned above in conjunction with 4-1 BB receptor on the activated T cell.

Embodiment 15: by the inductive immunne response of E7/4-1 BBL conjugate

(a) optimization of dosage

The optimal dose that can following mensuration comprises the vaccine of E7/4-1BBL conjugate.Use the biotinylated E7 of 1,10 or 50 μ g, form the conjugate that comprises biotinylation E7 and CSA-4-1 BBL by E7 and CSA-4-1 BBL with two ratios (as the CSA-4-1 BBL:E7 of 1:4 and 1:8) mixed biologic elementization.Also used the not biotinylated E7 of contrast of a great deal of.(these E7 dosage be based on proved inoculate can effectively the create antagonism research of protective immune response of TC-1 cell of 50 μ g E7).By under various experimental programs, as described below those are measured the immunne response to vaccine, can measure optimal proportion and the antigenic optimum content of 4-1 BBL:E7, and regulate by experiment.

(b) tetramer analysis

For CD8 ⁺The expansion of T cell, tetramer dyeing can be measured efficacy of vaccines.Give the bacterin preparation among the above-mentioned PBS of C57BL/6 female mice peritoneal injection.The mice of injection PBS, CSA-4-1 BBL, the not biotinylated E7 of CSA-4-1 BBL+ or E7-4-1 BBL fusion rotein in contrast.After 10 days, intraperitoneal gives equal for the second time dosage, and inoculates back three days the last time, collects splenocyte, and uses the quantity of tetramer technology and the quantitative E7-specific C of flow cytometry D8+T cell.In brief, with FITC-anti--the CD8 antibody labeling is from the splenocyte of immune animal, and determines position (peptide 49-57 (RAHYNIVTF)) labelling I class MHC H-2D with the immunodominant antigen of E7 ^bThe PE-tetramer.(tetramer can be available from National Institutes of Health Tetramer Facility (Atlanta, GA)).Load the I class H-2D of Sendai virus nucleoprotein 324-332 peptide (FAPGNYPAL) ^bMolecule is as negative contrast.After the dyeing, come analysis of cells, with quantitative percentage ratio to the male CD8+T cell of tetramer by flow cytometry.

(c) IFN-γ analyzes in the born of the same parents

Vaccine-induced CD8+T cell is for IFN-γ (effect CD8 ⁺The signal cytokine of T cell) the feasible function that can measure the T cell of the sign of Biao Daing.Biotinylation E7/CSA-4-1 BBL conjugate vaccine (measuring as mentioned above) with female C57BL/6 peritoneal injection optimal dose, and after 10 days, collect from the splenocyte of immune animal, and with the TC-1 co-culture of cells of the irradiation of expressing E7 5 days, replenish Golgi transport inhibitors brefeldin A then and spend the night.Use the Ficoll gradient to collect living cells and also hatched one hour, follow anti--CD8 antibody staining with the FITC labelling with anti-Mus Fc γ receptor antibody (from the 2.4G2 at U.S. typical case's culture center).Then with cell fixation, infiltration, dye and pass through flow cytometry for the anti-IFN-gamma antibodies (Pharmingen) of PE labelling.With the cell of homotype antibody staining with comparing.Use by oneself PBS, E7-4-1 BBL fusion rotein or CSA-4-1 BBL+ not the splenocyte of biotinylation E7 immune animal with comparing.

(d) kill and reply

The following ability of having measured the TC-1 cell of vaccine-induced CD8+T lysis expression E7 molecule.To in the presence of 100 μ g/ml E7 albumen, cultivate altogether 5 days from the splenocyte that the animal of above-mentioned inoculation is collected.Culture is replenished the growth that 50U/ml external source IL-2 supports the CD8+T cell.Use the Ficoll gradient to collect great-hearted splenocyte and, with different effectors: object ratio (as 1:1,10:1,20:1,40:1 and 80:1) as the effector lymphocyte of antagonism TC-1 target cell in the JAM test.Referring to, for example, Singh etc., 2003, Cancer Res.63:4067-73.Because the tumor cell that causes by the CD8+T cell directly to kill cancer immunotherapy be important, the proof of effect will further be supported the effect of vaccine to anti-cervical cancer in this test.

(e) the CD4+T cell proliferation is replied

The following effect of biotinylated E7/CSA-4-1 BBL in inducing the CD4+T cell response of having measured.With the splenocyte of CFSE labelling, and under above-mentioned same culture conditions, cultivate altogether, except IL-2 not being added in the culture with reorganization E7 from immune animal.Not collecting cell on the same day in incubation is used the APC-CD4 antibody staining, and uses flow cytometry propagation.Do not use E7 albumen or use the proteic culture of OVA with comparing.Because there is general suggestion: the CD4+T cell response is important for CD8+T cell and B cell response, and the proof of effect has further been supported the effect of vaccine to anti-cervical cancer in this test.

(f) humoral response

Following mensuration is with handling the ability that produces humoral response in the biotinylated E7/CSA-4-1 BBL vaccine body.Collect from inoculating the blood of mice, and separation of serum and be used for screening and cover E7 proteic Maxisorb ELISA flat board (Nalgene Nunc International).With the goat of puting together horseradish peroxidase anti--Mus IgG and goat be anti--Mus IgM antibody test is anti--E7IgG and IgM.Contrast comprises the serum of collecting from inoculation PBS and reference protein such as above-mentioned those animal.

Biotinylated E7/CSA-4-1 BBL conjugate vaccine will produce effectively in these tests and reply.Inoculation E7-4-1 BBL fusion rotein has also produced replys, but expects that any such replying will be on a small scale.Inoculation CSA-4-1 BBL adds not biotinylated E7 and can produce and reply, but any such replying can be the same with conjugate vaccine not strong, antigen is random case because APC absorbs E7, can not to be sent to APC the same effective with the E7 targeting that obtains by conjugate.

Embodiment 16: the therapeutic efficiency of E7/4-1 BBL conjugate

In two kinds of different situations, measured the vaccine effect that tumor forms in prevention and elimination TC-1 transplantable tumor model that the present invention includes E7/4-1 BBL conjugate.First kind of situation relates to before tumor injection, inoculation when immune evasion mechanism does not also produce.Second kind of situation relates to the inoculation of setting up tumor that antagonism has produced immune evasion mechanism fully.Come induced tumor to form for C57BL/6 injected in mice TC-1 cell, and before the TC-1 injection and inoculate biotinylated E7/CSA-4-1 BBL conjugate afterwards.Measure immunne response as mentioned above.The tumor of every other day also monitoring mice produces and survival, and when tumor reaches diameter 20mm size, with Animal Anesthesia.

(a) E7/4-1 BBL conjugate resists the effect that tumor subsequently excites.

Following examples will prove with E7/4-1 BBL conjugate of the present invention immunity and produce the protective immunity that antitumor is excited.

Give the independent PBS of injection in the female C57BL/6 mouse peritoneum, independent CSA-4-1 BBL, with the not blended CSA-4-1 BBL of biotinylation E7, biotinylation E7/CSA-4-1 BBL conjugate of the present invention and E-7-4-1 BBL fusion rotein.Use optimized as mentioned above dosage.Collect the TC-1 cell, be resuspended among the aseptic PBS and be used for immune the last time back and inject fortnight.On the right side with 1 * 10 ⁵Excite mice under the individual TC-1 cell skin and observed 60 days.As confirming that conjugate is to E7 ⁺The contrast of tumour-specific excites one group of immune mice that crosses with the A20 cancerous cell.The tumor of every other day monitoring mice produces and survival, and when tumor reaches the 20mm diameter, with Animal Anesthesia.The first time tumor excite back 60 days with 1 * 10 ⁶Individual TC-1 cell excites the animal that does not produce tumor to test memory response once more.With the interval of fortnight, will sacrifice from every group mice, and collect their splenocyte.As mentioned above, use tetramer dyeing and cytokine dyeing, splenocyte is used to measure the CD8+T cell response.

(b) effect of the existing tumor of E7/4-1 BBL conjugate antagonism

The following tumor treatment effect that has proved that E7/4-1 BBL conjugate antagonism of the present invention has existed in advance.

With female C57BL/6 mice at right side subcutaneous injection TC-1 cell and when 100% mice has palpable tumor, inoculate.Give as mentioned above at intraperitoneal that the vaccine of optimization dosage reaches the 20mm diameter until the tumor size weekly, at this moment with mouse anesthesia.Measure growth of tumor rate and sickness rate, continue 60 days.In addition, measure long-term surviving and also continue 90 days.

To in two kinds of situations, produce the effective antitumour immunne response with biotinylation E7/CSA-4-1 BBL conjugate inoculation of the present invention, and make to prevent tumor growth and eradicate existing tumor.Inoculate unconjugated CSA-4-1 BBL and E7 and inoculation E7-4-1 BBL fusion rotein and also can produce antitumor and reply, but any such replying will be minimum, and prevent tumor growth or eradicating in the existing tumor to be likely invalid.

Embodiment 17: comprise the antigenic conjugate of influenza A

By produce the cDNA of target influenza proteins (for example, H1, N1, NP and/or MP2) from reverse transcriptase-polymerase chain reaction of influenza A RNA.The cDNA sub-clone to the pCSA carrier, and is transferred in the fruit bat insect cell, is used to set up stable transfectant.

Utilization is engineered to the 6X-His label in the albumen, uses metal affinity resin and grossflow filtration (already used method of ApoImmune and technology) from fruit bat culture medium purification secreted H1, N1, NP and MP2 albumen.Analyze the albumen of purification by gel electrophoresis, immunoblot assay, substance assistant laser desorpted/ionization massspectrum (MALDI-MS) and analytical ultracentrifugation.

CSA-4-1 BBL (making as mentioned above) is mixed with biotinylated H1, N1, NP or MP2 with the mol ratio of 1:4, form influenza A/4-1BBL conjugate.In brief, with CSA-4-1BBL biotinylated H1, N1, NP or MP2 were hatched one hour at 4 ℃.Hatch not biotinylated H1, N1, NP or MP2 with CSA-4-1BBL, be used as unconjugated contrast.Conjugate can be formulated in the compositions as vaccine.

Embodiment 18: to the inoculation of influenza A

(a) dosage optimization

Give the influenza A antigen/4-1 BBL conjugate of C57BL/6 mouse inoculation various dose, as mentioned above.Use the immunne response in the standard immunoassay technical measurement mice, these technology comprise the measurement of tetramer technology, cytokine dyeing, cytotoxicity test and humoral response, as mentioned above.Initial result is used for measuring the optimal dose regime of vaccine.

(b) inoculation and infection excite

The following preventative and therapeutic effect that in the mice that excites with influenza A, proves influenza A antigen/4-1 BBL conjugate vaccine.Before and after infecting, treat human influenza virus's infected animals, and measure virus titer and measure therapeutic efficiency with influenza A antigen/4-1 BBL conjugate vaccine.After infection, collected from the lung of inoculation and contrast infection animal in the 1st, 3,5,7 and 9 day.Measure weight loss every day, as the direct measurement of sickness rate.

In the experiment of another series, estimate pneumonopathy Neo-Confucianism and measure Pneumovirinae titer.For this purpose, after 1500 * g is centrifugal 15 minutes, collect viral supernatant, and be frozen in-80 ℃ until subsequently analysis with the lung homogenizing and with homogenate.To add from the diluent of the viral supernatant of lung in the U base plate of 96-hole, to 3 * 10 ⁴Mdck cell/hole continues 24 hours at 37 ℃, removes culture medium from the hole, and adds serum-free medium.After four days, identifying culture supernatants no longer behind the erythrocytic dilution factor of coagulation chicken, using the Reed-Munch of the standard curve of known viruse concentration and TCID to calculate and measure virus titer.

Embodiment 19: immunity stimulates the CD40L part altogether

Used in this embodiment person monocytic cell's property leukemia THP-1 and Mus A20B-cell lymphoma system available from U.S. typical case's culture center (ATCC, Rockville, MD, USA).At additional 10% hot deactivation peptide Ox blood serum (FBS; Valley Biomedical, Winchester, VA, USA), 12mM L-glutaminate, 100U/ml penicillin, 100 μ g/ml streptomycins (all from GIBCO) and 50 μ M 2 mercapto ethanol (Sigma, St.Louis, MO, USA) DMEM (GIBCO, Gaithersburg, MD, USA) the middle A20 cell of cultivating.In the RPMI that replenishes 5% FBS, 100U/ml penicillin and 0.1mM Hepes buffer (GIBCO), cultivate the THP-1 cell, at 37 ℃, at the 5%CO of humidity ₂In the incubator.Cell is grown in suspension, 37 ℃, 5%CO ₂

The CHO (CHO-mCD40L) of used Chinese hamster ovary (CHO) and stable Mus CD154 transfection is provided by Dr.Gail Bishop (Iowa university) among this embodiment, and be maintained at and contain 100mM Hepes, among the RPMI 1640 of 50 μ g/ml gentamycins and 5% FBS (GIBCO).

Is present from Dr.Laryy Wahl (NICDR) from the human peripheral blood mononuclear cell by the isolating elementary mononuclear cell of adverse current eluting.

According to described before, set up permanent macrophage system from CD40 knock-out mice (CD40KO cell line) with the Mus reorganization J2 retroviral infection medullary cell that contains v-myc and v-raf oncogene.Referring to, for example, Clemon-Miller etc., 2000, Immunobiol.202:477-92.

In order to produce the people CD40 of stable expression transfectant, at 600v, what 20 μ seconds and 2 pulses transformed J2 is electroporation with 10 μ gDNA.24h adds zeocin (100 μ g/ml) in the culture medium after the transfection, and with resistive bacterium colony dyeing, is used for the surface expression of CD40.Use FACS Vantage SE (Becton Dicknson, San Jose CA, the USA) cell of the high CD40 expression of sorting, and keep, be used for these research.

(a) clone and the expression of CSA-CD40L part

Use to separate genomic DNA from avidin streptomycete (Streptpmyces avidinii) as template and Auele Specific Primer (a among Figure 16 and b) gene of clones coding CSA in PCR.The first chain cDNA that total RNA is originated from use is as clone people in the PCR ectodomain of CD40L of template and CD40L-Auele Specific Primer (c among Figure 16 and d), and total RNA separates from the activated human peripheral lymphocyte of lectins (PHA).To clone Mus CD40L, use the total RNA that separates the personal activated mice spleen cell of concanavalin A (ConA) with the same way as of CSA-hCD40L.

Then in frame with CSA/CD40L gene sub-clone to the derivable carrier of pMT/BiP/V5-His CuSO4-, be used for expressing to fruit bat S2 expression system (DES; Invitrogen, San Diego, CA, USA).Experimental program (Invitrogen) according to manufacturer uses the calcium phosphate transfection test kit to come transfection fruit bat S2 cell with 20 μ g recombinant vectors.By setting up stable transfectant with 1 μ gpCoHygro carrier cotransfection and in the presence of 300 μ g/ml hygromycin, keeping.Use the copper sulfate of 500 μ M final concentrations to obtain Recombinant Protein Expression.Induce and collected culture supernatants in back 3 days, precipitate with 40% Ammonium persulfate., and carry out dialysis for PBS.

According to described before, use improved metal ion affinity chromatograph method purification of recombinant proteins.Referring to, for example, Lehr etc., 2000, Protein Expression Purif., 19:362-68.In brief, culture supernatant or sedimentary albumen are flowed (Pharmacia Biotech, Upsala, Pharmacia XK16 post Sweden), and with 50mM imidazoles eluting recombiant protein soon by packing chelating agarose.Use Bradford dyestuff associated methods or ELISA (R﹠amp; DSystems, Minneapolis, MN USA) measures protein concentration.

(b) characterize the CSA-CD40L part by Western blotting and ELISA

Use Quantikine CD40L immunoassay to detect and the quantitatively expression of CSA-hCD40L, it uses the specific polyclone Ab of CD40L that is covered in advance on the micro-flat board, according to the described (R﹠amp of the explanation of manufacturer; D Systems).For western blot analysis, at first by sodium dodecyl sulfate-polyacrylamide gel electrophoresis supernatant classification with CSA-hCD40L and CSA-mCD40L under natural and degeneration condition, use half-dried blotter (BioRad then, Hercules, CA USA) is transferred on the polyvinylidene difluoride film.At first hatch film in the buffer in blocking-up, then in the blocking-up buffer with the dilution goat of 1:1000 anti--SA Ab (Pierce, Rockford, IL, USA) in incubated at room 1 hour.Then film is thoroughly washed, and hatched 1 hour with the dilution anti-goat antibody of puting together horseradish peroxidase of 1:4000.At last, (ECL, AmershamBiosciences UK) use chemical luminous substrate to detect protein according to the explanation of manufacturer.

Under non-degeneration PAGE condition, transfectant is expressed the CSA-CD40L part that tetramer and high stage structure are stablized in high-caliber formation.Only at 100 ℃ but not under the degeneration condition after 60 ℃ of heating, just be dissociated into monomer.These digital proofs the immune partial C D40L polypeptide that stimulates altogether do not influence expression, correct folding and CSA existence as oligomer.

(c) receptors bind and activation test

CSA-CD40L (people or Mus) part or contrast CSA albumen with 200ng/ml are hatched positive Mus A20B cell lymphoma of 1,000,000 CD40 or human macrophage THP-1 cell 30 minutes at 4 ℃.After PBS washing several times, (CA USA) detects bonded albumen in flow cytometry for Vector Laboratories, Burlingame to use the anti-Succ-PEG-DSPE antibody of puting together FITC.CSA is detected non-specific binding as negative contrast.By cultivating 0.5 * 10 with 100ng/ml CSA-hCD40L or CSA ⁶Individual Chinese hamster ovary celI or with 0.5 * 10 of CD40L form membrane transfection ⁶Individual Chinese hamster ovary celI is cultivated the effect of CSA-CD40L stimulation to CD80 and II class MHC developed by molecule of measuring in 48 hours altogether.Then with cell washing, and with anti--CD80 (L307.4) that puts together FITC of saturated concentration and II class HLA (TU36) antibody (CA USA) dyes, and analyzes by flow cytometry for BD-PharMingen, San Diego.

CSA-mCD40L combines people and Mus CD40 receptor (Figure 17 A﹠amp; B, concealed wire), as measuring, and be shown among Figure 17 by flow cytometry.On the contrary, CSA-hCD40L only with people's cell on acceptor interaction, have with the Mus cell and to be minimal to undetectable combination the (Figure 17 C﹠amp; D, concealed wire), proved its species specificity.These interactions make CD40-specific because with CSA when the reference protein, do not have detectable combination (Figure 17, Lycoperdon polymorphum Vitt filling part).

The antibody that uses II class HLA (Figure 18 A) and CD80 (Figure 18 B) molecule the II class MHC on the CSA-hCD40L of all tests protein concentration detection THP-1 cell surface and up-regulated expression of CD80 molecule in flow cytometry, back 48 hours of stimulation in 100ng protein/5 * 10 ⁵Individual cell obtains maximum the rise.The CD40L (heavy line) more effective (55.8 MFI is to 35.5) of the film combining form that CSA-hCD40L (fine line) expresses on than Chinese hamster ovary celI in raising II class HLA molecule.On the contrary, to raise almost be suitable (33.6 MFI is to 36.2) to the CD80 of the CD40L by two kinds of forms.The expression of raising is that CSA-hCD40L is specific, has no significant effect the expression of CD80 and II class HLA molecule above background level because hatch (solid block diagram) with CSA is proteic.

(d) generation of the DC of bone marrow derived

From 6 to 8 weeks, the femurs of big mices flushed out bone marrow, and moved by suction and to be dispersed into individual cells, with ammonium chloride potassium (ACK) solution cracking erythrocyte.Use the mixture of the saturated supernatant of TIB105, TIB146 and clone RL-172 Hybridoma Cell Culture thing to exhaust the T and the B cell of single-cell suspension liquid then, continuing 30 minutes on ice.(culture supernatants is the present of the Dr.Tatiana Zorina of University of Pittsburgh, PA).With the rabbit complement cell was hatched 30 minutes at 37 ℃, and complete medium (RPMI 1640,2mM L-glutaminate, 100 μ g/ml penicillins and streptomycin, 10%FBS, 0.1mM non essential amino acid, 1mM Sodium Pyruvate, 1 μ g/ml indomethacin and 50 μ M N-methyl-L-arginine) (Sigma) in six hole flat boards with 10 ⁶Individual cell/ml concentration overnight incubation (37 ℃, 5%CO ₂).Move the cell that collection is not adhered to by the gentleness suction, and with 10 ⁵The concentration of individual cell/ml be resuspended to replenish reorganization Mus granulocyte-macrophage colony stimutaing factor (5ng/ml) and rmIL-4 (5ng/ml) (all from USBiological, Swampscott, MA is in complete medium USA).Cell (4ml/ hole) in six hole flat boards was cultivated 5 days.

At the 5th day, for the expression of cell surface MHC and costimulatory molecules, measure the type of the DC that exists in the culture, and with variable concentrations (0.1-0.5 μ g/10 ⁶Individual cell) CSA-mCD40L, independent culture medium or CSA are hatched.At different natural law collecting cells and use the monoclonal antibody (HL3) of the anti-CD11c of PE labelling and the mAb (all from PharMingen) of the CD80 (16-10A1) of FITC labelling and CD86 (GL1) analyzes the expression of ripe labelling.

With various concentration (0.1-0.5 μ g/10 ⁶Individual cell) the immaturity dendritic cell from the 5th day Os Mus marrow culture that Mus CSA-CD40L was hatched 48 hours demonstrate the CD80 of raising and expression (Figure 19 A﹠amp of CD86 costimulatory molecules; B), at 0.2 μ g/10 ⁶The concentration of individual cell is higher than the effect of CD80 up-regulated CD86 (doubly 3 pairs 2 times).The CSA-CD40L fusion rotein of higher concentration or longer incubation time section do not cause further rise (data not shown).This effect is that immunity stimulates part specific altogether, is minimal to undetectable change because the cell of hatching with CSA albumen has in the costimulatory molecules that surpasses background level is expressed.

(e) analysis of proinflammatory cytokine generation

Trimerization recombined human CD40L (rhsCD40L)+1 μ g/ml enhancer (the Alexis Bochemicals that coats the person monocytic cell in the 96-hole microtitration flat board and use 1 μ g/ml to buy, San Diego, CA USA) stimulates with 100ng/ml CSA-hCD40L, CSA-mCD40L or CSA.After hatching 18 hours, collect supernatant and test, all cytokines are used OptEIA by ELISA ^TMDevice (PharMinger).(CA USA) analyzes for Molecular Devices, Sunnyvale to use E-maxPrecision microtitration plate reader.

The person monocytic cell connects CSA-hCD40L and causes surpassing independent CSA5 people IL-1 β generation stimulation doubly, and this equals by the inductive level of rhsCD40L (Figure 20 A﹠amp; B).Similarly, the person monocytic cell's stimulation with CSA-mCD40L causes strong people IL-6 to stimulate (Figure 20 C﹠amp; D).Therefore, people and Mus CSA-CD40L fusion rotein can both stimulate the CD40 on the person monocytic cell to produce IL-1 β and IL-6.

(f) RNAse protection test

Carry out the synthetic analysis of cytokines mRNA by RNAse protection test.Coat cell in the flat board of 6-hole and use CSA-CD40L to stimulate 3 to 4 hours by CD40.Express the CHO transfectant of CD40L and rhsCD40L with comparing with enhancer.The described use Trizol of explanation (Invitrogen) according to manufacturer extracts RNA.RNA (5 μ g) is spent the night 55 ℃ of hybridization with radiolabeled probe, and probe originates from human cell factor/RNA template cover system, and mCK-3b (RiboQuant, BD-PharMingen, San Diego, CA, USA).The RNAse processing was carried out 45 minutes at 37 ℃, and 5% polyacrylamide gel (BioRad) in the shielded probe of purification after this, and the use tbe buffer liquid is collected by gel electrophoresis.With gel drying and be exposed to Kodak Biomax XL x-ray film (Eastman Kodak, Rochester, NY, USA).Use indigested probe to serve as a mark, on semilogarithmic paper, length of nucleotides is made standard curve with displacement.From figure, infer the characteristic of RNAse protection band in the sample then.

As shown in Figure 20 D, cause IL-6mRNA to surpass independent CSA 2.8-with the CSA-hCD40L stimulation and doubly improve.

Generally speaking, these tables of data person of good sense and Mus CSA-CD40L can induce the CD40 signal in mononuclear cell and the macrophage.

(g) iNOS in the macrophage of CSA-hCD40L stimulation IFN-γ initiation produces

The CD40 of the macrophage that IFN-γ causes connects the stimulation that causes nitric oxide to produce, and this plays pivotal role in the microbicidel of macrophage and cytotoxic activity.Derivable nitricoxide synthase (iNOS) belongs to from the synthetic nitric oxide production nitricoxide synthase of L-arginine catalysis family.Th1 and Th2 t helper cell can differently be regulated the arginine metabolism in the macrophage.The Th1 cell induction produces by the iNOS of macrophage, and Th2 cell induction macrophage produces arginase, and it is relevant with anti-inflammatory properties.Therefore, macrophage iNOS generation is the sign of Th1 type immunne response.

The following CSA-CD40L that proved partly stimulates the ability that iNOS produces in the mouse macrophage.With IFN-γ CD40KO-people CD40 cell was caused 24 hours, stimulate 24h with CSA-hCD40L, rhsCD40L or independent CSA subsequently.Analyze with the cell lysate standardization and by Western blotting, use anti-iNOS Ab.As proving among Figure 21, the stimulation that stimulates macrophage to cause iNOS to produce with CSA-hCD40L or rhsCD40L rather than CSA.Stimulate the iNOS that causes above 6 times of backgrounds to stimulate with the commercial rhsCD40L of 1 μ g/ml, and cause surpassing independent CSA9 iNOS stimulation doubly with the CSA-hCD40L stimulation of 300ng/ml.These data show that CSA-hCD40L is the effective stimulus agent that macrophage iNOS produces.

Embodiment 20: reply by killing in the inductive body of CSA-4-1BBL

With 50 μ g ovalbumins (OVA) as the CSA-4-1BBL of antigen and two dosage (12.5 μ g and 25 μ g respectively do for oneself) or LPS as adjuvant, the natural C57BL/6 mice of intravenous immunity.With natural animal with comparing.

After seven days, the target cell of the CFSE labelling that all mices acceptance are prepared as follows.To be divided into two groups from the splenocyte of natural C57BL/6 mice.With 0.25 μ M CFSE (CFSE ^Low) first group of labelling, with second group of 2.5 μ M CFSE labellings, use 2 μ g/ml OVA then _257-2641 hour (CFSE of peptide (SIINFEKL) pulse ^High).With the mixed of cell, with total 1 * 10 with 1: 1 ⁷Individual cell intravenous injection is to accepting in the animal.Collect spleen after 48 hours, and by flow cytometry CFSE fluorescence intensity.The results are shown among Figure 22, be expressed as the CFSE of peptide pulse ^HighThe peak with reference to CFSE ^LowThe percentage ratio cracking of comparing in the peak is normalized to natural animal.As shown in Figure 22, produced to kill in the effective body in target cell with OVA and CSA-4-1BBL immunity and replied, CSA-4-1BBL has proved the stronger adjuvant effect at the concentration ratio LPS of two tests.

Embodiment 21: stimulate altogether with 4-1BBL and to have improved dramatically in the mice, controlled the TC-1 tumor and induced the antitumor memory the proteic immunne response of HPV16 E7.

In inoculation experiments scheme based on the CD8+T cellular antigens decisions positions (having aminoacid sequence RAHYNIVTF) that give HPV16 E7 epitope P1, on the right side with 1 * 10 ⁵Excite natural B 6 mices under the TC-1 cell skin of individual work, TC-1 cytotostatic expressing human papillomavirus-16E7 albumen.Figure 23 has shown the survival of mice in this TC-1 tumor model.After 10 days, mice is accepted once one of following subcutaneous injection: (i) PBS (◆, n=20); (ii) 50 μ g P1+12.5 μ g CSA (■, n=6); (iii) 25 μ g CSA-4-1BBL (▲, n=10); (iv) 50 μ g P1+25 μ g CSA-4-1 BBL (Δ, n=13), or (v) 50 μ g P1+10 μ gCpG (, n=7).

As shown in Figure 23, obtained some successful immunization therapies, but obtained higher result (comprising the survival of raising) with P1 and CSA-4-1 BBL inoculation with P1 or CSA-4-1 BBL immunity.All animals of only accepting PBS have produced tumor.

Excite once more the 60th day (black arrow) animal survival.One week monitoring tumor growth 3 times.With independent P1 or CSA-4-1 BBL, or P1 and CpG compare, and gives the survival that CSA-4-1 BBL and P1 restriction has improved animal after tumor excites together.Importantly, the neither one surviving animals produces tumor in the P1+CSA-4-1 BBL group when exciting for the second time, has proved immunological memory.

Embodiment 22: inoculation OVA/CSA-4-1 BBL has prevented tumor growth

With 50 μ g OVA that put together 25 μ g CSA-4-1 BBL or the natural C57BL/6 mice of the biotinylated OVA immunity of 50 μ g.Staying some animals is untreated in contrast.After 7 days, with 1 * 10 ⁵Individual OVA expression-EG.7 tumor cell is subcutaneous on the right side to excite mice.Use slide calliper rule one week monitoring tumor growth three times.Result's (no tumor survival) is shown among Figure 24.As directed, the animal of all control animals and inoculation OVA has produced tumor, and all animals that inoculated biotinylated OVA/CSA-4-1 BBL do not produce tumor, have proved that inoculating biotinylated OVA/CSA-4-1 BBL causes 100% of thyoma tumor growth to prevent.

Embodiment 23: 4-1 BBL has improved body endoantigen specific CTL strongly and has replied

With (1) 50 μ g OVA, (ii) 50 μ g OVA and 25 μ g CSA-4-1BBL, (iii) 50 μ gOVA and 25 μ g anti--CD137 antibody or (iv) 50 μ g OVA and 25 μ g LPS give immunity in the natural C57BL/6 mouse vein.Natural animal is with comparing.After seven days, all mices are accepted the target cell of CFSE labelling.In brief, will be divided into two groups from the spleen of natural C57BL/6 mice.With first group's (CFSE is low) of 0.25 μ M CFSE labelling.With second group of 2.5 μ M CFSE labellings, use 2 μ g/ml OVA then _257-264SIINFEKL peptide pulse 1 hour (CFSE height).With the mixed of cell with 1:1, and with total 1 * 10 ⁷Individual cell intravenous injection is to accepting in the animal.Collect spleen after 48 hours, and, the results are shown among Figure 25 by flow cytometry CFSE fluorescence intensity.The results are shown on the angle of each figure,, be normalized to natural animal as the CFSE peak of peptide pulse and the percentage ratio cracking of comparing with reference to the CFSE ebb.This test has disclosed 4-1BBL and the CTL of antigenic specificity can have been replied and be increased to higher level (95%), and with independent antigen (OVA) (24.2%), or antigen and LPS (35%) compare, and causes killing of most of target cell.

Embodiment 24: 4-1BBL stimulate altogether improved body endoantigen submission to the CD8+T cell with (i) 10 μ g OVA, (ii) 10 μ g OVA and 5 μ g 4-1BBL are the immunity of natural B 6-SJL (CD45.1+) animal intravenous, or (iii) stay and be untreated.After 2 days, by intravenous injection, animals received 1 * 10 ⁶The OT-1 cell (CD45.2+) of CFSE labelling.Collect spleen after 3 days, and use the propagation of flow cytometry OT-1 cell, as shown in Figure 26.4-1BBL and the antigenic antigen presentation that improved together be to the CD8+T cell, as by most of OT-1 cell propagation proved.(for OVA+4-1BBL is 83.2%; For OVA is 13.7%; For untreated be 8.8%).

Embodiment 25: 4-1BBL stimulates the antigen absorption that has improved dendritic cell altogether

Give natural B ALB/c mouse subcutaneous injection 25 μ g OVA-FITC, 25 μ g OVA-FITC and 10 μ g CSA, or 25 μ g OVA-FITC and 25 μ g CSA-4-1BBL.After 3 hours, collect the inguinal lymph nodes of injection site.Use the cell among the flow cytometry FITC+CD11c+ group, measure fluorescently-labeled antigen renewal in the body, as seeing among Figure 27.As directed, 4-1 BBL signal has improved the antigen absorption of CD11c+DC, and contrast CSA albumen does not have effect.

* * * * *

Although enough describe in detail and for example understand the present invention, be used for those skilled in the art and prepare and use, various replacements, changes and improvements should be clearly, and do not break away from the spirit and scope of the present invention.In the expression that this embodiment that provides is a preferred embodiment, be not to plan to limit the scope of the invention.Those skilled in the art will produce change and other purposes wherein.These changes are included in the spirit of the present invention, and are limited by the scope of claim.

To the present invention disclosed herein form various substitute and change and do not depart from the scope of the present invention with spirit be that those skilled in the art are conspicuous.

All patents mentioned in the description and publication are the expressions of those skilled in the art's level.Be incorporated herein all patents and publication as a reference, to the same degree of specially and separately representing as every piece of independent publication to be incorporated herein by reference.

The present invention who is implemented in this illustrative description under the situation of this specially not disclosed any key element, restriction suitably can lacked.Therefore, for example, in this each situation, term " comprises ", " consisting essentially of " and in " comprising " any can be substituted by in other two terms any.Already used term and statement be unrestricted term as describing, do not use shown in such term and the statement eliminating and the intention of described any equivalent characteristics or its part, but think that various changes are possible in the desired scope of the invention.Therefore, although be to be understood that and disclose the present invention specifically by embodiment preferred and optional feature, the change of notion disclosed herein and change to rely on those skilled in the art, and think such change and changing in the scope of the present invention that claims limit.

Below listed other exemplary and claim:

Exemplary

1. combination, it comprises:

(a) first conjugate comprises that (i) comprises that first immunity stimulates the conjugate member of polypeptide altogether and (ii) comprises conjugate member in conjunction with first right member; With

(b) second conjugate comprises that (i) comprises the first antigenic conjugate member and (ii) comprise described conjugate member in conjunction with second right member.

2. the combination of embodiment 1 wherein saidly comprises avidin or Succ-PEG-DSPE in conjunction with right described first member, and describedly comprises biotin in conjunction with right described second member.

3. the combination of embodiment 2 wherein saidly comprises the core Succ-PEG-DSPE in conjunction with right described first member.

4. the combination of embodiment 1, wherein said first conjugate comprises fused polypeptide, this fused polypeptide comprises that described first immunity stimulates polypeptide and described in conjunction with right described first member altogether.

5. the combination of embodiment 1, wherein said first immunity stimulates polypeptide to be selected from 4-1 BBL, CD86, ICOSL, PD-L1, PD-L2, B7-H3, B7-H4, OX40L, CD27L, CD30L, LIGHT, BAFF, APRIL, CD80 and CD40L altogether.

6. it is 4-1 BBL that the combination of embodiment 5, wherein said first immunity stimulate polypeptide altogether.

7. the combination of embodiment 6, wherein said first conjugate comprises fused polypeptide, this fused polypeptide comprises the aminoacid sequence of SEQ ID NO:8.

8. the combination of embodiment 1, wherein said first antigen is relevant with infectious agent.

9. the combination of embodiment 8, wherein said infectious agent are selected from people or bird flu and human immunodeficiency virus.

10. the combination of embodiment 1, wherein said first antigen is tumor associated antigen.

11. the combination of embodiment 10, wherein said tumor associated antigen are selected from robot end enzyme reverse transcriptase, survivin, MAGE-1, MAGE-3, human chorionic gonadotropin, carcinoembryonic antigen, alpha fetal protein, cancer of pancreas embryonal antigen, MUC-1, CA125, CA15-3, CA19-9, CA549, CA195, prostate specific antigen; Prostate specific membrane antigen, Her2/neu, gp-100, sudden change K-ras albumen, sudden change p53, the epithelial growth factor receptor that blocks, chimeric protein ^P210BCR-ABL; HPV E6, HPV E7; Epstein-Barr virus EBNA3 albumen, and combination or fragment.

12. the combination of embodiment 1 wherein provides described first and second conjugates as the compositions of separating.

13. the combination of embodiment 1 wherein provides described first and second conjugates as single compositions.

14. the combination of embodiment 13, wherein said compositions comprises acceptable carrier on the materia medica, excipient or diluent.

15. the combination of embodiment 13 wherein, provides in described compositions, described first conjugate combines with described second conjugate the combination between the member by described first and second combination.

16. the combination of embodiment 1, wherein said first immunity stimulates polypeptide not comprise the membrane spaning domain of immune costimulatory molecules altogether.

17. the combination of embodiment 1, wherein said first immunity stimulate polypeptide to comprise the membrane spaning domain of immune costimulatory molecules or its receptor binding moiety altogether.

18. the combination of embodiment 1, further comprise the 3rd conjugate, comprise that (i) comprises that second immunity stimulates polypeptide altogether and in conjunction with first right member conjugate member with (ii) comprise second antigen and in conjunction with second right member conjugate member, wherein:

Described second immunity stimulates polypeptide and described first immunity to stimulate polypeptide identical or different altogether altogether; Described second antigen and described first antigen are identical or different; First of described the 3rd conjugate and second combination to member and described first and second conjugates described first with second combine to the member identical or different and

The described first conjugate member combines with described second conjugate the combination between the member by described first and second combination.

19. a combination comprises:

(a) first conjugate comprises that (i) comprises that first immunity stimulates the conjugate member of polypeptide altogether and (ii) comprises conjugate member in conjunction with first right member; With

(b) second conjugate comprises that (i) comprises the conjugate member of infectious agent and (ii) comprise described conjugate member in conjunction with second right member.

20. produce or strengthen the method for the immunne response of the tumor of resisting the expressing tumor related antigen, comprise the patient who following material is had described tumor:

(a) first conjugate, comprise that (i) comprises that first immunity stimulates the conjugate member of polypeptide altogether and (ii) comprises conjugate member in conjunction with first right member, with second conjugate, comprise that (i) comprises the conjugate member of described first tumor associated antigen and (ii) comprise described conjugate member in conjunction with second right member; Or

(b) immunocyte of having crossed in extracorporeal treatment with described first conjugate and second conjugate.

21. the method for embodiment 20 wherein gives described patient with described first and second conjugates.

22. the method for embodiment 21 wherein separately gives described first and second conjugates.

23. the method for embodiment 21 wherein gives described first and second conjugates simultaneously.

24. the method for embodiment 20 wherein provides described first and second conjugates in single compositions.

25. the method for embodiment 24 wherein, provides in described compositions, described first conjugate combines with described second conjugate the combination between the member by described first and second combination.

26. the method for embodiment 21 is wherein by injecting at least one that gives in described first and second conjugates in the tumor.

27. the method for embodiment 20, wherein said first tumor associated antigen is selected from robot end enzyme reverse transcriptase, survivin, MAGE-1, MAGE-3, human chorionic gonadotropin, carcinoembryonic antigen, alpha fetal protein, cancer of pancreas embryonal antigen, MUC-1, CA125, CA15-3, CA19-9, CA549, CA195, prostate specific antigen; Prostate specific membrane antigen, Her2/neu, gp-100, sudden change K-ras albumen, sudden change p53, the epithelial growth factor receptor that blocks, chimeric protein ^P210BCR-ABL; HPV E6, HPV E7; Epstein-Barr virus EBNA3 albumen, and combination or fragment.

28. the method for embodiment 20, further comprise and give the 3rd compositions, it comprises that (i) comprises that second immunity stimulates polypeptide altogether and in conjunction with first right member conjugate member with (ii) comprise second tumor associated antigen and in conjunction with second right member conjugate member, wherein:

Described second immunity stimulates polypeptide and described first immunity to stimulate polypeptide identical or different altogether altogether; Described second antigen and described first antigen are identical or different; First of described the 3rd conjugate and second combination to member and described first and second conjugates described first with second combine to the member identical or different and

The described first conjugate member combines with described second conjugate the combination between the member by described first and second combination.

29. the method for embodiment 28, wherein said second tumor associated antigen is selected from robot end enzyme reverse transcriptase, survivin, MAGE-1, MAGE-3, human chorionic gonadotropin, carcinoembryonic antigen, alpha fetal protein, cancer of pancreas embryonal antigen, MUC-1, CA125, CA15-3, CA19-9, CA549, CA195, prostate specific antigen; Prostate specific membrane antigen, Her2/neu, gp-100, sudden change K-ras albumen, sudden change p53, the epithelial growth factor receptor that blocks, chimeric protein ^P210BCR-ABL; HPV E6, HPV E7; Epstein-Barr virus EBNA3 albumen, and combination or fragment.

30. the method for embodiment 20 wherein saidly comprises avidin or Succ-PEG-DSPE in conjunction with right described first member, and describedly comprises biotin in conjunction with right described second member.

31. the method for embodiment 30 wherein saidly comprises the core Succ-PEG-DSPE in conjunction with right described first member.

32. the method for embodiment 20, wherein said first conjugate comprises fused polypeptide, and this fused polypeptide comprises that described first immunity stimulates polypeptide and described in conjunction with right described first member altogether.

33. the method for embodiment 20, wherein said first immunity stimulates polypeptide to be selected from 4-1BBL, CD86, ICOSL, PD-L1, PD-L2, B7-H3, B7-H4, OX40L, CD27L, CD30L, LIGHT, BAFF, APRIL, CD80 and CD40L altogether.

34. the method for embodiment 33, it is 4-1BBL that wherein said first immunity stimulates polypeptide altogether.

35. the method for embodiment 34, wherein said first conjugate comprises fused polypeptide, and this fused polypeptide comprises the aminoacid sequence of SEQ ID NO:8.

36. the method for embodiment 20, wherein said immunity stimulate polypeptide not comprise the membrane spaning domain of immune costimulatory molecules altogether.

37. the method for embodiment 20, wherein said immunity stimulate polypeptide to comprise the ectodomain of immune costimulatory molecules or its receptor binding moiety altogether.

38. the method for embodiment 20, wherein the immunocyte that will cross in extracorporeal treatment with described first and second conjugates gives described patient.

39. the method for embodiment 38, wherein said immunocyte comprises that described immunity stimulates the receptor of polypeptide altogether, and wherein said first conjugate stimulates the combination between polypeptide and the described receptor to put together with described immunocyte by described immunity altogether, and described second conjugate is puted together combination between the member and described immunocyte by described first and second combination.

40. the method for embodiment 38 is wherein handled described immunocyte simultaneously with described first and second conjugates.

41. the method for embodiment 38 is wherein used the described immunocyte of the described first and second conjugate separate processes.

Produce or strengthen 42. modify immunocyte, comprise stimulating the immunocyte contact of polypeptide receptor altogether expressing first immunity to the tumor of expressing tumor associated antigen or to the method for the immunne response of infectious agent:

(a) first conjugate comprises that (i) comprises that first immunity stimulates the conjugate member of polypeptide altogether and (ii) comprises conjugate member in conjunction with first right member; With

(b) second conjugate comprises that (i) comprises the conjugate member of infectious agent and (ii) comprise described conjugate member in conjunction with second right member,

Wherein said first conjugate stimulates the combination between polypeptide and the described receptor to put together with described immunocyte by described immunity altogether, and described second conjugate is puted together combination between the member and described immunocyte by described first and second combination.

43. the method for embodiment 42 wherein separately contacts described first conjugate with second conjugate.

44. the method for embodiment 42 wherein contacts described first and second conjugates simultaneously.

45. the method for embodiment 44 wherein provides described first and second conjugates in single compositions.

46. the method for embodiment 45 wherein, provides in described compositions, described first conjugate combines with described second conjugate the combination between the member by described first and second combination.

47. the method for embodiment 42 wherein saidly realizes described contact by the patient that described first and second conjugates are contained described immunocyte.

48. the method for embodiment 47, wherein said second conjugate comprises tumor associated antigen, and described patient further comprises described tumor, by injecting at least one that gives in described first and second conjugates in the tumor.

49. the method for embodiment 42, wherein said immunocyte are T cell or neutrophil cell.

50. the method for embodiment 49, wherein said T cell is selected from CD4+ cell, CD8+ cell, natural killer cell, mononuclear cell and dendritic cell.

51. the method for embodiment 42, wherein said second antigen comprises tumor associated antigen.

52. the method for embodiment 51, wherein said tumor associated antigen are selected from robot end enzyme reverse transcriptase, survivin, MAGE-1, MAGE-3, human chorionic gonadotropin, carcinoembryonic antigen, alpha fetal protein, cancer of pancreas embryonal antigen, MUC-1, CA125, CA15-3, CA19-9, CA549, CA195, prostate specific antigen; Prostate specific membrane antigen, Her2/neu, gp-100, sudden change K-ras albumen, sudden change p53, the epithelial growth factor receptor that blocks, chimeric protein ^P210BCR-ABL; HPV E6, HPV E7; Epstein-Barr virus EBNA3 albumen, and combination or fragment.

53. the method for embodiment 42, wherein said second conjugate comprises antigen relevant with infectious agent or infectious agent.

54. the method for embodiment 42, wherein said infectious agent is an antibacterial.

55. the method for embodiment 54, wherein said antibacterial is selected from conjunction with mycobacteria; Anthrax bacillus; Staphylococcus aureus.

56. the method for embodiment 42, wherein said infectious agent are virus.

57. the method for embodiment 56, wherein said virus is selected from adenovirus; Arenavirus; Calicivirus; Coronavirus; Filamentous virus; Banzi virus; Hepatovirus; Herpesvirus; Positive myxovirus; Human papillomavirus; Piconavirus; Poxvirus; Breathe lonely virus; Retrovirus; Rhabdovirus; And togavirus.

58. the method for embodiment 42, wherein said infectious agent is parasite.

59. the method for embodiment 58, wherein said parasite are selected from deformable body and Leishmania.

60. the method for embodiment 42, wherein said infectious agent is a fungus.

61. the method for embodiment 60, wherein said fungus is selected from aspergillus; Mycocandida; Globidium; Cryptococcus; Geotricha; Histoplasma capsulatum; Microsporidian; Pneumocystis.

62. the method for embodiment 47, wherein said patient is selected from horse, sheep, he-goat, cattle, pig, birds, Canis familiaris L., cat and primate species.

63. the method for embodiment 47, wherein said patient is the people.

64. the method for embodiment 42, wherein said immunocyte comprises that second immunity stimulates the receptor of polypeptide altogether, this method further comprises described immunocyte is contacted the 3rd conjugate, it comprise (i) comprise described second immunity stimulate altogether polypeptide with in conjunction with first right member conjugate member with (ii) comprise second antigen relevant or described infectious agent and described conjugate member in conjunction with second right member with described tumor or infectious agent, wherein:

Described second immunity stimulates polypeptide and described first immunity to stimulate polypeptide identical or different altogether altogether; Described second antigen, if it is exist, identical or different with described first antigen (if existence); First of described the 3rd conjugate and second combination to member and described first and second conjugates described first with second combine to the member identical or different and

The described first conjugate member combines with described second conjugate the combination between the member by described first and second combination.

65. the method for embodiment 42 wherein saidly comprises avidin or Succ-PEG-DSPE in conjunction with right described first member, and describedly comprises biotin in conjunction with right described second member.

66. the method for embodiment 65 wherein saidly comprises the core chain mycin in conjunction with right described first member.

67. the method for embodiment 42, wherein said first conjugate comprises fused polypeptide, and this fused polypeptide comprises that described first immunity stimulates polypeptide and described in conjunction with right described first member altogether.

68. the method for embodiment 42, wherein said first immunity stimulates polypeptide to be selected from 4-1BBL, CD86, ICOSL, PD-L1, PD-L2, B7-H3, B7-H4, OX40L, CD27L, CD30L, LIGHT, BAFF, APRIL, CD80 and CD40L altogether.

69. the method for embodiment 68, it is 4-1BBL that wherein said first immunity stimulates polypeptide altogether.

70. the method for embodiment 69, wherein said first conjugate comprises fused polypeptide, and this fused polypeptide comprises the aminoacid sequence of SEQ ID NO:8.

71. the method for embodiment 42, wherein said immunity stimulate polypeptide not comprise the membrane spaning domain of immune costimulatory molecules altogether.

72. the method for embodiment 42, wherein said immunity stimulate polypeptide to comprise the ectodomain of immune costimulatory molecules or its receptor binding moiety altogether.

73. the immunocyte group that the method by embodiment 42 makes, when wherein contacting other immunocytes, described immunocyte produces or strengthens immunne response to described tumor.

74. express the modification immunocyte that first immunity stimulates polypeptide receptor altogether, the immunocyte of wherein said modification comprises:

(a) first conjugate comprises that (i) comprises that described first immunity stimulates the conjugate member of polypeptide altogether and (ii) comprises conjugate member in conjunction with first right member; With

(b) second conjugate comprises that (i) comprises the conjugate member of first antigen or infectious agent and (ii) comprise described conjugate member in conjunction with second right member,

Wherein said first conjugate stimulates the combination between polypeptide and the described receptor to put together with described immunocyte by described immunity altogether, and described second conjugate is puted together combination between the member and described immunocyte by described first and second combination.

75. the immunocyte of embodiment 74, wherein said immunocyte is selected from T cell, neutrophil cell, natural killer cell, mononuclear cell and dendritic cell.

76. the immunocyte of embodiment 75, wherein said T cell is selected from CD4+ cell and CD8+ cell.

77. the immunocyte of embodiment 76 wherein saidly comprises avidin or Succ-PEG-DSPE in conjunction with right described first member, and describedly comprises biotin in conjunction with right described second member.

78. the immunocyte of embodiment 76 wherein saidly comprises the core Succ-PEG-DSPE in conjunction with right described first member.

79. the immunocyte of embodiment 74, wherein said first conjugate comprises fused polypeptide, and this fused polypeptide comprises that described first immunity stimulates polypeptide and described in conjunction with right described first member altogether.

80. the immunocyte of embodiment 74, wherein said first immunity stimulates polypeptide to be selected from 4-1BBL, CD86, ICOSL, PD-L1, PD-L2, B7-H3, B7-H4, OX40L, CD27L, CD30L, LIGHT, BAFF, APRIL, CD80 and CD40L altogether.

81. the immunocyte of embodiment 80, it is 4-1BBL that wherein said first immunity stimulates polypeptide altogether.

82. induce or strengthen method, comprise that following material is suffered from described infectious agent to be infected or be in described infectious agent and infect patient in the risk to the immunne response of anti-infective:

(a) first conjugate comprises that (i) comprises that described first immunity stimulates the conjugate member of polypeptide altogether and (ii) comprises conjugate member in conjunction with first right member; With

(b) second conjugate comprises that (i) comprises the antigen that first is relevant with described infectious agent or comprise the conjugate member of described infectious agent and (ii) comprise described conjugate member in conjunction with second right member.

83. the method for embodiment 82 wherein separately gives described first and second conjugates.

84. the method for embodiment 82 wherein gives described first and second conjugates simultaneously.

85. the method for embodiment 84 wherein provides described first and second conjugates in single compositions.

86. the method for embodiment 85 wherein, provides in described compositions, described first conjugate combines with described second conjugate the combination between the member by described first and second combination.

87. the method for embodiment 82 wherein gives in described first and second conjugates at least one by being selected from following approach: oral; The Sublingual; Through mucous membrane; Percutaneous; Rectum; Vagina; Subcutaneous; Intramuscular, intravenous; Intra-arterial; In the sheath; Pass through conduit; By implanting; Directly to tumor.

88. the method for embodiment 82, wherein said infectious agent is an antibacterial.

89. the method for embodiment 88, wherein said antibacterial is selected from conjunction with mycobacteria; Anthrax bacillus; Staphylococcus aureus.

90. the method for embodiment 82, wherein said infectious agent are virus.

91. the method for embodiment 90, wherein said virus is selected from adenovirus; Arenavirus; Calicivirus; Coronavirus; Filamentous virus; Banzi virus; Hepatovirus; Herpesvirus; Positive myxovirus; Human papillomavirus; Piconavirus; Poxvirus; Breathe lonely virus; Retrovirus; Rhabdovirus; And togavirus.

92. the method for embodiment 82, wherein said infectious agent is parasite.

93. the method for embodiment 92, wherein said parasite are selected from deformable body and Leishmania.

94. the method for embodiment 82, wherein said infectious agent is a fungus.

95. the method for embodiment 94, wherein said fungus is selected from aspergillus; Mycocandida; Globidium; Cryptococcus; Geotricha; Histoplasma capsulatum; Microsporidian; Pneumocystis.

96. the method for embodiment 82, wherein said patient is selected from horse, sheep, he-goat, cattle, pig, birds, Canis familiaris L., cat and primate species.

97. the method for embodiment 96, wherein said patient is the people.

98. the method for embodiment 82, wherein said infection are people or bird flu, and described antigen is selected from H, N, M1, M2e, NS1, NS2 (NEP), NP, PA, PB1 and PB2.

99. the method for embodiment 82, wherein said infection is HIV, and described first antigen is selected from the HIV antigen that is made of Gag albumen, Pol, Vif, Vpr, Rev, Vpu, envelope antigen decision position, Tat and Nef.

100. the method for embodiment 82, further comprise and give the 3rd conjugate, it comprise (i) comprise second immunity stimulate altogether polypeptide with in conjunction with first right member conjugate member with (ii) comprise second antigen relevant or described infectious agent and described conjugate member in conjunction with second right member with described infection, wherein:

Described second immunity stimulates polypeptide and described first immunity to stimulate polypeptide identical or different altogether altogether; Described second antigen, if it is exist, identical or different with described first antigen (if existence); First of described the 3rd conjugate and second combination to member and described first and second conjugates described first with second combine to the member identical or different and

The described first conjugate member combines with described second conjugate the combination between the member by described first and second combination.

101. the method for embodiment 100, wherein said infection are people or bird flu, and described second antigen is H, N, M1, M2e, NS1, NS2 (NEP), NP, PA, PB1 and PB2.

102. the method for embodiment 101, wherein said infection is HIV, and described second antigen is selected from the HIV antigen that is made of Gag albumen, Pol, Vif, Vpr, Rev, Vpu, envelope antigen decision position, Tat and Nef.

103. the method for embodiment 82 wherein saidly comprises avidin or Succ-PEG-DSPE in conjunction with right described first member, and describedly comprises biotin in conjunction with right described second member.

104. the method for embodiment 103 wherein saidly comprises the core Succ-PEG-DSPE in conjunction with right described first member.

105. the method for embodiment 82, wherein said first conjugate comprises fused polypeptide, and this fused polypeptide comprises that described first immunity stimulates polypeptide and described in conjunction with right described first member altogether.

106. the method for embodiment 82, wherein said first immunity stimulates polypeptide to be selected from 4-1BBL, CD86, ICOSL, PD-L1, PD-L2, B7-H3, B7-H4, OX40L, CD27L, CD30L, LIGHT, BAFF, APRIL, CD80 and CD40L altogether.

107. the method for embodiment 106, it is 4-1BBL that wherein said first immunity stimulates polypeptide altogether.

108. the method for embodiment 107, wherein said first conjugate comprises fused polypeptide, and this fused polypeptide comprises the aminoacid sequence of SEQ ID NO:8.

109. the method for embodiment 82, wherein said immunity stimulate polypeptide not comprise the membrane spaning domain of immune costimulatory molecules altogether.

110. the method for embodiment 82, wherein said immunity stimulate polypeptide to comprise the ectodomain of immune costimulatory molecules or its receptor binding moiety altogether.

111. the conjugate that stimulates polypeptide and avidin or Succ-PEG-DSPE to constitute altogether by immunity basically, wherein said immunity stimulates polypeptide to be selected from 4-1 BBL, CD86, ICOSL, PD-L1, PD-L2, B7-H3, B7-H4, OX40L, CD27L, CD30L, LIGHT, BAFF, APRIL, CD80 and CD40L altogether.

112. the conjugate of embodiment 111 comprises the core Succ-PEG-DSPE.

113. the conjugate of embodiment 111, wherein said immunity stimulate polypeptide to be selected from 4-1BBL, CD86, ICOSL, PD-L1, PD-L2, B7-H3, B7-H4, OX40L, CD27L, CD30L, LIGHT, BAFF and APRIL altogether.

114. the method for immunostimulatory response in the induced animal consists essentially of and gives animal with conjugate, this conjugate stimulates polypeptide and avidin or Succ-PEG-DSPE to constitute by immunity basically altogether.

115. the method for embodiment 114, wherein said conjugate comprises the core Succ-PEG-DSPE.

116. the method for embodiment 114, wherein said immunity stimulate polypeptide to be selected from 4-1BBL, CD86, ICOSL, PD-L1, PD-L2, B7-H3, B7-H4, OX40L, CD27L, CD30L, LIGHT, BAFF, APRIL, CD80 and CD40L altogether.

117. the method for embodiment 116, wherein said immunity stimulate polypeptide to be selected from 4-1BBL, CD86, ICOSL, PD-L1, PD-L2, B7-H3, B7-H4, OX40L, CD27L, CD30L, LIGHT, BAFF and APRIL altogether.

Stimulate the conjugate of polypeptide and avidin or Succ-PEG-DSPE altogether 118. comprise immunity, wherein said immunity stimulates polypeptide to be selected from 4-1 BBL, CD86, ICOSL, PD-L1, PD-L2, B7-H3, B7-H4, OX40L, CD27L, CD30L, LIGHT, BAFF, APRIL, CD80 and CD40L altogether.

119. the method for immunostimulatory response in the induced animal, comprise and give animal conjugate, this conjugate comprises that immunity stimulates polypeptide and avidin or Succ-PEG-DSPE altogether, and wherein said immunity stimulates polypeptide to be selected from 4-1 BBL, CD86, ICOSL, PD-L1, PD-L2, B7-H3, B7-H4, OX40L, CD27L, CD30L, LIGHT, BAFF, APRIL, CD80 and CD40L altogether.

120. the method for embodiment 119 further comprises giving animal with antigen.

121. the method for embodiment 120 is wherein as comprising described antigen and coming the described antigen of administration in conjunction with the conjugate to the member.

Sequence table

<110>UNIVERSITY?OF?LOUISVILLE?RESEARCH?FOUNDATION，INC.

＜120〉immunostimulatory compositions and method

<130>060592-0139

<140>PCT/US06/046662

<141>2006-12-07

<150>60/748,177

<151>2005-12-08

<150>60/771,179

<151>2006-02-06

<150>60/799,643

<151>2006-05-12

<150>60/863,173

<151>2006-10-27

<160>22

<170>PatentIn?Ver.3.3

<210>1

<211>1213

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic constructs

<400>1

<210>2

<211>307

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic fusion rotein

<400>2

<210>3

<211>1268

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic constructs

<400>3

<210>4

<211>368

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic fusion rotein

<400>4

<210>5

<211>1294

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic constructs

<400>5

<210>6

<211>358

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic fusion rotein

<400>6

<210>7

<211>1295

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic constructs

<400>7

<210>8

<211>357

<212>PRT

＜213〉artificial sequence

<220>

The description of artificial sequence: synthetic fusion rotein

<223>

<400>8

<210>9

<211>1334

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic constructs

<400>9

<210>10

<211>376

<212>PRT

＜213〉artificial sequence

<220>

The description of artificial sequence: synthetic fusion rotein

<223>

<400>10

<210>11

<211>158

<212>PRT

＜213〉human papillomavirus

<400>11

<210>12

<211>151

<212>PRT

＜213〉human papillomavirus

<400>12

<210>13

<211>98

<212>PRT

＜213〉human papillomavirus

<400>13

<210>14

<211>1303

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic constructs

<400>14

<210>15

<211>364

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic nuclear fusion albumen

<400>15

<210>16

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>16

<210>17

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>17

<210>18

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>18

<210>19

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic 6x His label

<400>19

<210>20

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>20

<210>21

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>21

<210>22

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>22

Claims (20)

1. combination, it comprises:

(a) first conjugate, it comprises that (i) comprises that first immunity stimulates the conjugate member of polypeptide altogether and (ii) comprises conjugate member in conjunction with first right member; With

(b) second conjugate, it comprises that (i) comprises the first antigenic conjugate member and (ii) comprise described conjugate member in conjunction with second right member.

2. the combination of claim 1 wherein saidly comprises avidin or Succ-PEG-DSPE in conjunction with right described first member, and describedly comprises biotin in conjunction with right described second member.

3. the combination of claim 1, wherein said first immunity stimulates polypeptide to be selected from 4-1BBL, CD86, ICOSL, PD-L1, PD-L2, B7-H3, B7-H4, OX40L, CD27L, CD30L, LIGHT, BAFF, APRIL, CD80 and CD40L altogether.

4. the combination of claim 1, wherein said first antigen is selected from antigen relevant with infectious agent and tumor associated antigen.

5. combination, it comprises:

(a) first conjugate, it comprises that (i) comprises that first immunity stimulates the conjugate member of polypeptide altogether and (ii) comprises conjugate member in conjunction with first right member; With

(b) second conjugate, it comprises that (i) comprises the conjugate member of antigen relevant with infectious agent or infectious agent and (ii) comprise described conjugate member in conjunction with second right member.

6. the combination of claim 5, wherein infectious agent is selected from antibacterial, virus and parasite.

7. produce or strengthen the method for the immunne response of resisting the tumor of expressing first tumor associated antigen, described method comprises the patient who following material is had described tumor:

(a) first conjugate, it comprises that (i) comprises that first immunity stimulates the conjugate member of polypeptide altogether and (ii) comprises conjugate member in conjunction with first right member, with second conjugate, it comprises that (i) comprises the conjugate member of described first tumor associated antigen and (ii) comprise described conjugate member in conjunction with second right member; Or

(b) immunocyte of having crossed in extracorporeal treatment with described first and second conjugates.

8. the method for claim 7 wherein saidly comprises avidin or Succ-PEG-DSPE in conjunction with right described first member, and describedly comprises biotin in conjunction with right described second member.

9. the method for claim 7, wherein said first immunity stimulates polypeptide to be selected from 4-1BBL, CD86, ICOSL, PD-L1, PD-L2, B7-H3, B7-H4, OX40L, CD27L, CD30L, LIGHT, BAFF, APRIL, CD80 and CD40L altogether.

10. the method for claim 7, wherein said patient is selected from horse, sheep, he-goat, cattle, pig, birds, Canis familiaris L., cat and primate species.

11. the method for claim 10, wherein said patient is the people.

12. modifying immunocyte produces or strengthens the tumor of expressing tumor associated antigen or to the method for the immunne response of infectious agent, described method comprises stimulates the immunocyte contact of polypeptide receptor altogether with expressing first immunity:

(a) first conjugate comprises that (i) comprises that described first immunity stimulates the conjugate member of polypeptide altogether and (ii) comprises conjugate member in conjunction with first right member; With

(b) second conjugate comprises that (i) comprises the conjugate member of antigen relevant with described tumor or infectious agent or described infectious agent and (ii) comprise described conjugate member in conjunction with second right member,

Wherein said first conjugate stimulates the combination between polypeptide and the described receptor to put together with described immunocyte by described immunity altogether, and described second conjugate is puted together combination between the member and described immunocyte by described first and second combination.

13. express the modification immunocyte that first immunity stimulates polypeptide receptor altogether, the immunocyte of wherein said modification comprises:

(a) first conjugate, it comprises that (i) comprises that described first immunity stimulates the conjugate member of polypeptide altogether and (ii) comprises conjugate member in conjunction with first right member; With

(b) second conjugate, it comprises that (i) comprises the conjugate member of first antigen or infectious agent and (ii) comprise described conjugate member in conjunction with second right member,

Wherein said first conjugate stimulates the combination between polypeptide and the described receptor to put together with described immunocyte by described immunity altogether, and described second conjugate is puted together combination between the member and described immunocyte by described first and second combination.

14. induce or strengthen method to the immunne response of anti-infective, described method comprises that following material is suffered from described infectious agent infects or be in patient in the described infectious agent infection risk:

(a) first conjugate, it comprises that (i) comprises that first immunity stimulates the conjugate member of polypeptide altogether and (ii) comprises conjugate member in conjunction with first right member; With

(b) second conjugate, it comprises that (i) comprises first antigen relevant with described infectious agent or comprise the conjugate member of described infectious agent and (ii) comprise described conjugate member in conjunction with second right member.

15. the method for claim 4, wherein said patient is selected from horse, sheep, he-goat, cattle, pig, birds, Canis familiaris L., cat and primate species.

16. the method for claim 15, wherein said patient is the people.

Stimulate the conjugate of polypeptide and avidin or Succ-PEG-DSPE altogether 17. comprise immunity, wherein said immunity stimulates polypeptide to be selected from 4-1BBL, CD86, ICOSL, PD-L1, PD-L2, B7-H3, B7-H4, OX40L, CD27L, CD30L, LIGHT, BAFF, APRIL, CD80 and CD40L altogether.

Give animal 18. the method for immunostimulatory response in the induced animal, described method comprise with conjugate, this conjugate comprises that immunity stimulates polypeptide and avidin or Succ-PEG-DSPE altogether.

19. the method for claim 18, wherein this method further comprises and gives animal with antigen.

20. the method for claim 18, wherein said animal is selected from horse, sheep, he-goat, cattle, pig, birds, Canis familiaris L., cat and primate species.

Images