CN101506266A - Vaccine delivery compositions and methods of use - Google Patents
Vaccine delivery compositions and methods of use Download PDFInfo
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- CN101506266A CN101506266A CNA2006800035269A CN200680003526A CN101506266A CN 101506266 A CN101506266 A CN 101506266A CN A2006800035269 A CNA2006800035269 A CN A2006800035269A CN 200680003526 A CN200680003526 A CN 200680003526A CN 101506266 A CN101506266 A CN 101506266A
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Abstract
The present invention provides synthetic vaccine delivery compositions based on polyester amide (PEA), polyester urethane (PEUR), and polyester urea (PEU) polymers for stimulating an immune response to a variety of pathogenic organisms and tumor cells in humans and other mammals. The vaccine delivery compositions are formulated as a liquid dispersion of polymer particles or molecules including class I or class II antigen peptides derived from organism or tumor cell proteins, which are taken up by antigen presenting cells of the mammal to induce an immune response in the mammal. Methods of inducing an immune response to the pathogenic organism or tumor cells in the invention compositions are also included.
Description
Related application
[0001] the application requires the provisional application series the 60/649th in submission on February 1st, 2005,60/689 of submission on June 8th, No. 289 1,003,60/742 of submission on December 2nd, 2005,188,60/748 of submission on December 7th, 2005,486,60/719 of submission on September 22nd, 2005,950,60/759,179 the right of priority of submitting to 13,60/687,570,2006 on the January of submitting on June 3rd, 2005 under 35 U.S.A. § 119 (e).
Invention field
[0002] relate generally to immunogenic composition of the present invention, and relate to allelic vaccine delivery compositions particularly in conjunction with MHC.
Technical background
[0003] although vaccine development and use obvious improvement has been arranged, the optional mode that strengthens the efficient of vaccine preparation and security is still under study for action.Subunit vaccine for example recombinant protein, synthetic peptide and polysaccharide-peptide conjugate just occurs as the candidate new vaccine.Yet the traditional vaccine of being made up of attenuated pathogens and full deactivation organism contains impurity and bacterium composition, and it can be as adjuvant, and this is a kind of activity that these subunit vaccines lack.Therefore, the efficient of the highly purified subunit vaccine of carrying with independent preparation will require to add effective adjuvant.
[0004] present, in the U.S., the adjuvant that is used for people's vaccine that the FDA that aluminum compound remains unique ratifies.Although they have the good safety record, they are weak relatively adjuvants and require the multiple dose administration scheme usually, to induce the antibody horizontal relevant with protective immunity.Therefore aluminum compound may not be the desirable adjuvant of inducing the protective immune response of subunit vaccine.Although among present many candidate's adjuvants were being studied, they had numerous defectives, the prerequisite that is included in the toxicity among the mankind and introduces antigenic pipoint technique.
[0005] in vaccine the antigenic use of peptide be based on handle in Mammals and other animal immune knowledge, especially major histocompatibility complex (major histocompatability complexes, MHC).The MHC molecule is synthetic and displaying by the most cells of body.The T cell of MHC and specialization type (for example cytotoxic T cell) is coordinated to work, with " non-self (nonself) " or the adventitious viruses albumen of removing body.Antigen receptor identification epi-position on the T cell, described epi-position is the mosaic of the part of binding peptide and α spiral, partly constituting of described α spiral is positioned at the lateral ditch of binding peptide.Produce after the peptide fragment by the cutting foreign protein, by MHC molecular presentation peptide fragment, this makes antigen restrictive cell toxicity T cell monitoring cell expressing " non-self " or adventitious viruses albumen.Functional T cell will show cytotoxic immune and reply after identification contains the antigenic MHC molecule of binding peptide of T cell-specific.
[0006] exogenous antigen is from those antigens outside the body cell.Example comprises bacterium, free virus, yeast, protozoon and toxin.These exogenous antigens enter antigen presenting cell (antigen-presenting cells) or APCs (scavenger cell, dendritic cell and bone-marrow-derived lymphocyte) by phagolysis.Microorganism is swallowed, and proteantigen is degraded to a series of peptides by proteolytic enzyme.These peptides finally are attached on the ditch in the MHC-II molecule and are transported to the surface of APC.Then, the T4 lymphocyte by they TXi Baoshouti (T-cell receptors, TCRs) and the CD4 molecule can identification polypeptide/MHC-II complex body.The peptide of presenting in II class MHC by APCs is that length is about 10 to about 30 amino acid, for example about 12 to about 24 amino acid (Marsh, S.G.E.et al. (2000) The HLA Facts Book, Academic Press, p.58-59).Activated T 4 lymphocytic effector functions comprise: by the microbiocidal activity of B cell generation antibody and scavenger cell, it is outside the born of the same parents or is engulfed microorganism and suffer mainly mechanism of destructive.
[0007] body is to destroy endogenous infected cell and tumour cell by cytotoxic T lymphocyte (cytotoxic T-lymphocytes) or CTLs to one of main defence of virus, intracellular bacteria and cancer.These CTLs are effector cells, derive from the T8 lymphocyte during the cell-mediated immunity.Yet in order to become CTLs, inmature T8 lymphocyte must become active state by the cytokine that APCs produces.Interaction between this APCs and the inmature T8 lymphocyte mainly occurs in lymphoglandula, lymphatic nodule and the spleen.This process relates to dendritic cell and scavenger cell, their swallow and degrade resistatess of infected cell, tumour cell and killed infected cell and tumour cell.It is believed that, in this way, endogenous antigen from diseased cells can enter APC, and proteolytic enzyme isolates into a series of peptides with peptase with protein there, length be about 8 to about 10 amino acid, may about 8 to about 11 amino acid or about 8 to about 12 amino acid.Have the MHC I quasi-molecule of binding peptide, it appears on the surface of APCs, can be had the inmature T8 lymphocyte identification of the TCRs that contains complementary shape and CD8 molecule now.This peptide epitopes recognition process of passing through TCR is used for the cell-mediated immunity function as lymphocytic first signal of the inmature T8 of activation.The signal cell can have the MHC-I molecule that contains up to 250,000 in conjunction with epi-position on its surface.
[0008] therefore, still exist in the art demand novel and improved vaccine delivering composition and their using method, wherein said vaccine delivery compositions utilizes peptide antigen but not deactivation pathogenic former, so that induce the immunne response at the pathogenic micro-organism of being determined by MHC I class and II class allelotrope in individuality.
Summary of the invention
[0009] the present invention is based on such prerequisite: in polymer chain, contain amino acid whose biodegradable polymers, some polyesteramide (poly (ester amide) for example, PEA), polyester urethane (poly (esterurethane), PEUR) and polyester-urea (poly (ester urea), (PEU)) polymkeric substance, thereby can be used to prepare complete synthetic, and the vaccine delivery compositions that is easy to produce, be used in people and other Mammals moderate stimulation immunne response to various pathogenic microbes.
[0010] therefore, in one embodiment, the invention provides vaccine delivery compositions, it comprises at least a MHC I class or the II class peptide antigen of significant quantity, described peptide antigen contains 5 to about 30 amino acid, be dispersed in biodegradable polymers molecule or the particle, described biodegradable polymers molecule or particle contain the amino acid that is coupled at least a type at least a non-amino acid moiety in each monomer.
[0011] in another embodiment; the invention provides vaccine delivery compositions; the quilt preparation is used for using with the form of the liquid dispersion of at least a MHC I class that is coupled to significant quantity or II class peptide antigenic polymer particle or molecule; described peptide antigen contains 5 to about 30 amino acid; and biodegradable PEA has the described structural formula of structural formula (I)
Formula (I)
Wherein the scope of n is between about 5 to about 150; R
1Be independently selected from α, ω-two (4-carboxyl phenoxy group)-(C
1-C
8) alkane, 3,3 '-(alkane two acyl dioxy bases) two styracins or 4,4 '-the residue, (C of (alkane two acyl dioxy bases) two styracins
2-C
20) alkylene hydrocarbon or (C
2-C
20) inferior alkene; R in an independent n monomer
3Be independently selected from hydrogen, (C
1-C
6) alkyl, (C
2-C
6) thiazolinyl, (C
2-C
6) alkynyl, (C
6-C
10) aryl (C
1-C
20) alkyl and-(CH
2)
2S (CH
3); And R
4Be independently selected from (C
2-C
20) alkylidene group, (C
2-C
20) alkenylene, (C
2-C
8) alkoxyl group, (C
2-C
20) alkylidene group, saturated or unsaturated therapeutic glycol residue, structural formula (II) 1,4:3, dicyclo part and their the combination, (C of the two anhydrous hexitols (dianhydrohexitol) of 6-
2-C
20) alkylidene group and (C
2-C
20) alkenylene;
Formula (II)
Perhaps the PEA polymkeric substance has the described chemical formula of structural formula (III):
Formula (III)
Wherein n is between about 5 to about 150, and m is between about 0.1 to 0.9, and p is between about 0.9 to 0.1; R wherein
1Be independently selected from α, ω-two (4-carboxyl phenoxy group)-(C
1-C
8) alkane, 3,3 '-(alkane two acyl dioxy bases) two styracins or 4,4 '-(alkane two acyl dioxy bases) two styracins, (C
2-C
20) alkylene hydrocarbon or (C
2-C
20) residue of inferior alkene; Each R
2Be hydrogen, (C independently
1-C
12) alkyl or (C
6-C
10) aryl or protecting group; R in an independent m monomer
3Be independently selected from hydrogen, (C
1-C
6) alkyl, (C
2-C
6) thiazolinyl, (C
2-C
6) alkynyl, (C
6-C
10) aryl (C
1-C
20) alkyl and-(CH
2)
2S (CH
3); And R
4Be independently selected from (C
2-C
20) alkylidene group, (C
2-C
20) alkenylene, (C
2-C
8) alkoxyl group, (C
2-C
20) alkylidene group, saturated or unsaturated therapeutic glycol residue, general formula (II) 1,4:3, the dicyclo part and their combination of the two anhydrous hexitols (dianhydrohexitol) of 6-.
[0012] in another embodiment, polymkeric substance is the PEUR polymkeric substance with the described chemical formula of structural formula (IV),
Formula (IV)
Wherein n is between about 5 to about 150; R wherein
3Be independently selected from hydrogen, (C
1-C
6) alkyl, (C
2-C
6) thiazolinyl, (C
2-C
6) alkynyl, (C
6-C
10) aryl (C
1-C
20) alkyl and-(CH
2)
2S (CH
3); R
4Be selected from (C
2-C
20) alkylidene group, (C
2-C
20) alkenylene or alkoxyl group, saturated or unsaturated therapeutic glycol residue, structural formula (II) 1,4:3, the dicyclo part and their combination of the two anhydrous hexitols (dianhydrohexitol) of 6-; And R
6Be independently selected from (C
2-C
20) alkylidene group, (C
2-C
20) alkenylene or alkoxyl group, general formula (II) 1,4:3, the dicyclo part and their combination of the two anhydrous hexitols (dianhydrohexitol) of 6-;
Perhaps the PEUR polymkeric substance has the described chemical formula of structure formula V:
Formula V
Wherein n is between about 5 to about 150, and m is between about 0.1 to about 0.9, and p is between about 0.9 to about 0.1; R
2Be independently selected from hydrogen, (C
6-C
10) aryl (C
1-C
20) alkyl or protecting group; R in an independent m monomer
3Be independently selected from hydrogen, (C
1-C
6) alkyl, (C
2-C
6) thiazolinyl, (C
2-C
6) alkynyl, (C
6-C
10) aryl (C
1-C
20) alkyl and-(CH
2)
2S (CH
3); R
4Be selected from (C
2-C
20) alkylidene group, (C
2-C
20) alkenylene or alkoxyl group, saturated or unsaturated therapeutic glycol residue, structural formula (II) 1,4:3, the dicyclo part and their combination of the two anhydrous hexitols (dianhydrohexitol) of 6-; And R
6Be independently selected from (C
2-C
20) alkylidene group, (C
2-C
20) alkenylene or alkoxyl group, general formula (II) 1,4:3, the dicyclo part of the two anhydrous hexitols (dianhydrohexitol) of 6-, the residue of the saturated or unsaturated therapeutic glycol of significant quantity and their combination.
[0013] still in another embodiment, polymkeric substance is biodegradable PEU polymkeric substance, and it has the described chemical formula of structural formula (VI):
Formula (VI),
Wherein n is between about 10 to about 150; R in an independent n monomer
3Be independently selected from hydrogen, (C
1-C
6) alkyl, (C
2-C
6) thiazolinyl, (C
2-C
6) alkynyl, (C
6-C
10) aryl (C
1-C
20) alkyl and-(CH
2)
2S (CH
3); R
4Be independently selected from (C
2-C
20) alkylidene group, (C
2-C
20) alkenylene, (C
2-C
8) alkoxyl group (C
2-C
20) residue of alkylidene group, saturated or unsaturated therapeutic glycol; Or structural formula (II) 1,4:3, the dicyclo part of the two anhydrous hexitols of 6-; Perhaps the PEU polymkeric substance has the described chemical formula of structural formula (VII)
Formula (VII),
Wherein m is between about 0.1 to about 1.0, and p is between about 0.9 to about 0.1, and n is between about 10 to about 150; Each R
2Be hydrogen, (C independently
1-C
12) alkyl or (C
6-C
10) aryl; R in an independent m monomer
3Be independently selected from hydrogen, (C
1-C
6) alkyl, (C
2-C
6) thiazolinyl, (C
2-C
6) alkynyl, (C
6-C
10) aryl (C
1-C
20) alkyl and-(CH
2)
2S (CH
3); Each R
4Be independently selected from (C
2-C
20) alkylidene group, (C
2-C
20) alkenylene, (C
2-C
8) alkoxyl group (C
2-C
20) alkylidene group, saturated or unsaturated therapeutic glycol residue, structural formula (II) 1,4:3, the dicyclo part and their combination of the two anhydrous hexitols of 6-.
[0014] still in another embodiment; the invention provides the method for induce immune response in Mammals; it is by carrying out described administration invention vaccine delivery compositions; the form of described composition is structural formula I and the particle of the described polymkeric substance of III-VII or the liquid dispersion of molecule, and described particle or molecule are coupled on the peptide antigen of the I class of significant quantity or II class.Said composition is absorbed by described mammiferous antigen presenting cell, with induce immune response in described Mammals.
[0015] in another embodiment; the invention provides and carry vaccine to mammiferous method; it is by carrying out described administration invention vaccine delivery compositions; the form of described composition is structural formula I and the particle of the described polymkeric substance of III-VII or the liquid dispersion of molecule, and described particle or molecule are coupled to I class or II class peptide antigen.Said composition is absorbed by described mammiferous antigen presenting cell, so that I class or II class peptide antigen are delivered to described Mammals.
Description of drawings
[0016] Fig. 1 is a synoptic diagram, and generation PEA, PEUR or PEU particle have been described, they have the promoting agent that is dispersed in wherein each type by dual and triple emulsifying steps described herein, for example peptide antigen.
[0017] Fig. 2 is a synoptic diagram, illustrates to contain the antigenic micelle of the present invention of dispersive peptide, as described herein.
[0018] Fig. 3 is preparation vaccine of the present invention and tests the schema of the method that human T-cell in the body of vaccine of the present invention is replied.
[0019] Fig. 4 A-B shows the figure of the T cell activation that the dendritic cell that is exposed to polymkeric substance-peptide conjugate is replied.Fig. 4 A shows the propagation of T cell during 96 hours, and wherein the PEA-peptide conjugate with respect to independent peptide or PEA, has stimulated remarkable propagation.Fig. 4 B shows the IL-2 secretion of T cell during 96 hours, and wherein, than independent peptide or PEA, PEA-peptide (formula III, Embodiment B 1) has stimulated significant IL-2 secretion.
Detailed Description Of The Invention
[0020] the present invention is based on such discovery: each monomer contains at least one amino acid whose biodegradable polymers can be for the production of the synthetic vaccine delivering composition that is used for subcutaneous or intramuscular injection or mucosa delivery, described composition be a large amount of repeatably, safety (not containing attenuated virus), stable, and can be lyophilized, so that transportation and storing. Because the architectural characteristic of employed polymer, described vaccine delivery compositions provides the antigen of high copy number and local density.
[0021] described polymer can be formulated into and have vaccine delivery compositions of different nature. In one embodiment, described polymer as the time meta-release type polymer depot work, when polymer depot in vivo biodegradation occured, this bank discharged peptide antigen and the antigen-polymer segments that is absorbed by APCs in the future and presented by MHC I class or II class allele. In other embodiments, described polymer works as the carrier that peptide antigen enters APC, and described peptide antigen is released, and presents in the born of the same parents carrying out. Described polymer may be really phagocytosis by induced polymer-antigen conjugates stimulate APC.
[0022] in another embodiment, the invention provides the method for induce immune response in mammal, it is undertaken by the invention vaccine delivery compositions to described administration effective dose, described composition is absorbed by described mammiferous antigen presenting cell, with induce immune response in described mammal.
[0023] except treating the mankind, vaccine delivery compositions of the present invention also is intended to for multiple mammality patient, for example the veterinary treatment of pet (such as cat, dog, rabbit and ferret), agricultural animal (such as pig, horse, donkey, milk cow and beef cattle) and horse racing.
[0024] directly carries or made according to size from polymer beads or polymer molecule that body interpolymer bank discharges; to be convenient to by antigen presenting cell (antigen presenting cells; APCs) absorb, and contain and be dispersed in the polymer beads or be coupled to peptide antigen in the functional group on the polymer molecule and optional adjuvant. APCs passes through MHC complex displayed polypeptide antigen, and is identified by T cell such as cytotoxic T cell, to produce and to promote to cause to be loaded with the endogenous immune response of the pathogenic cell disintegration of coupling antigen or comm on antigen. The polymer that is used in the vaccine delivery compositions of the present invention can be designed, and to be fit to the biodegradation rate of polymer depot, molecule and particle, causes the Continuous Contact of peptide antigen and antigen presenting cell within selected a period of time. For example, typically, polymer depot will be degraded being selected from about twenty four hours, about seven days, about 30 days or about 90 days or longer a period of time. Longer time span is particularly suitable for providing implantable vaccine delivery compositions, and it has eliminated the duplicate injection vaccine to obtain the needs of suitable immune response.
[0025] the present invention has utilized the conveying technology of biodegradable polymers mediation, to induce the immune response that various pathogen is comprised the pathogen of mucous membrane transmission. Described composition provides strong immune response, even also is like this when antigen itself is weak immunogenicity. Although the separate constituent of described vaccine delivery compositions and method described here are known, but unexpected and surprisingly, such combination will make antigen efficient be strengthened on the level that reaches when described composition is used alone, and the polymer that uses in the described vaccine delivery compositions of preparation will be eliminated the demand to extra adjuvant in some cases.
[0026] although the present invention is widely applicable for to be provided any above-mentioned former immune response of causing a disease, the present invention passes through reference stream Influenza Virus and HIV by illustrative explanation in this article.
[0027] method of the present invention provides cell-mediated immunity and/or humoral antibody to reply. Therefore, method of the present invention will can be used for any antigen, for described antigen, cellullar immunologic response and/or HI are expected, described antigen comprises the antigen that derives from virus, bacterium, fungi and parasitic disease substance, and it can induce antibody, T-helper activity and T-cell cytotoxic activity. Therefore, " immune response " used herein refers to produce antibody, T-helper activity or the T cell cytotoxic activity that is specific to used peptide antigen. This type of antigen includes but not limited to those antigens by the pathogenic original encoding of humans and animals, and can be corresponding to structural proteins or non-structural protein, polysaccharide-peptide conjugate or DNA.
[0028] for example, the present invention can be used for stimulating the immune response for various albumen from herpesviral family, comprise the albumen from herpes simplex virus (herpes simples virus, HSV) Class1 and 2, such as HSV-1 and HSV-2 glycoprotein gB, gD and gH; Antigen from varicellazoster virus (varicella zoster virus, VZV), Epstein-Barr virus (Epstein-Barr virus, EBV) and cytomegalovirus (cytomegalovirus, CMV) comprises CMV gB and gH; And from other herpes virus hominis's antigen, such as the antigen of HHV6 and HHV7. (referring to, Chee et al for example, Cytomegaloviruses (J.K.McDougall, ed., Springer-Verlag 1990) pp.125-169, about the summary of the encoding histone content (protein coding content) of cytomegalovirus; McGeoch et al., J.Gen.Virol. (1988) 69:1531-1574 is about the discussion of various HSV-1 encoding proteins; United States Patent (USP) 5,171,568 is about the discussion of HSV-1 and HSV-2gB and gD albumen and their gene of coding; Baer et al., Nature (1984) 310:207-211 is about differentiating albumen coded sequence in the EBV genome; And Davison and Scott, J.Gen.Virol. (1986) 67:1759-1816 is about the summary of VZV. )
[0029] from the antigen of hepatitis viruse family, comprise hepatitis A virus (hepatitis A virus, HAV), hepatitis type B virus (hepatitis B virus, HBV), HCV (hepatitis C virus, HCV), Hepatitis D virus (delta hepatitis virus, HDV), HEV (hepatitis E virus, HEV) and HGV RNA (hepatitis G virus, HGV), also can be used in the technology of describing in this article easily. As an example, the virus genome sequence of HCV is known, and the method that obtains simultaneously this sequence also is known. Referring to, for example No. 90/14436, international publication WO 89/04669, WO 90/11089 and WO. The some virus proteins of HCV genome encoding, comprise that E1 (being also referred to as E) and E2 (being also referred to as E2/NSI) and N end nucleocapsid protein (being called ＂ core (core) ＂) is (referring to Houghton et al., Hepatology (1991) 14:381-388, about the discussion of HCV albumen, comprise E1 and E2). Each of these albumen and their antigenicity fragment will can be used in this method. Similarly, from the sequence of δ-antigen of HDV be known (referring to, for example United States Patent (USP) the 5th, 378, No. 814), and this antigen also can be easily with in the method. In addition, derive from the antigen of HBV, for example cAg, surface antigen sAg and front surface sequence (presurface sequence (presurface sequences)) pre-S1 and pre-S2 (being called in the past pre-S) and above-mentioned combination, for example sAg/pre-S1, sAg/pre-S2, sAg/pre-S1/pre-S2 and pre-S1/pre-S2 will find useful in this article. Referring to, for example ＂ HBV Vaccines-from the laboratory to license:a case study ＂ is in Mackett, M.and Williamson, J.D., Humnan Vaccines and Vaccination is among the pp.159-176, about the discussion of HBV structure; And United States Patent (USP) the 4th, 722, No. 840, the 5th, 098, No. 704, the 5th, 324, No. 513, all be incorporated in this as a reference; Beames et al., J.Virol. (1995) 69:6833-6838, Birnbaum et al., J. Virol. (1990) 64:3319-3330; With Zhou et al., J.Virol. (1991) 65:5457-5464.
[0030] also will can be used in the method required for protection from other viral antigen; for example and nonrestrictive; protein from the member of following section: the member of Picornaviridae (Picornaviridae) (such as poliovirus etc.); Caliciviridae (Caliciviridae); Togaviridae (Togaviridae) (rubella virus for example; dengue fever virus etc.); flaviviridae (Flaviviridae); coronaviridae (Coronaviridae); Reoviridae (Reoviridae); birnavirus section (Birnaviridae); Rhabdoviridae (Rhabodoviridae) (such as rabies viruses etc.); filamentous form virus section (Filoviridae); paramyxovirus section (Paramyxoviridae) (mumps virus for example; measles virus; Respiratory Syncytial Virus(RSV) etc.); (the A for example of orthomyxovirus section (Orthomyxoviridae); B and C type influenza virus etc.); Bunyaviridae (Bunyaviddae); Arenaviridae (Arenaviridae); Retroviridae (Retroviradae) (HTLV-I for example; HTLV-II; HIV-1 (also is known as HTLV-III LAV; ARV; hTLR etc.)), include but not limited to from separated strain HIVIIIb、HIVs
F2、HIV
LAV、HIV
LAI、HIV
MN；HIV-1
CM235、
HIV-1
US4 HIV-2; Simian immunodeficiency virus (simian immunodeficiency virus (SIV)); Etc. antigen. In addition, antigen can also be from HPV (human papillomavirus, HPV) and tick-brone encephalitis virus. Referring to, Virology for example, 3rd Edition (W.K.Joklik ed.1988); Fundamental Virology, 2nd Edition (B.N.Fields and D.M.Knipe, eds.1991) is about the description of these and other virus.
[0031] more specifically, envelope protein from any above-mentioned HIV separated strain---member who comprises the various science of heredity hypotypes of HIV---be known and reported (referring to for example Myers et al., Los Alamos Database, Los Alamos National Laboratory, Los Alamos, N.Mex. (1992); Myers et al., Human Retroviruses and Aids, 1990, Los Alamos, N.Mex.:Los Alamos National Laboratory; And Modrow et al., J.Virol. (1987) 61:570-578 is about the comparison of the envelope protein sequence of various HIV separated strains), and will can be used in this method from any antigen of above-mentioned these separated strains. Particularly, synthetic peptide R15K (Nehete et al.Antiviral Res. (2002) 56:233-251)---derive from the V3 ring of gp120 and have sequence RIQRGPGRAFVTIGK (SEQ ID NO:1), will can be used in the present composition and the method. And, the present invention is equally applicable to derive from other one of any immunogenic protein of various HIV separated strains, comprise any such as gp160 and gp41 of all kinds envelope protein, gag antigen such as p24gag and p55gag, and the albumen that derives from the pol district. And, can be used to the various epi-positions from HIV albumen, the multi-epitope mixture of imagination polymer-peptide conjugate. For example, 6 of gp120 and gp41 conserved epitopes have shown and have reduced viral load and prevent from propagating in macaque/SHIV model: SVITQACSKVSFE (S13E) (SEQ ID NO:2), GTGPCTNVSTVQC (G13C) (SEQ ID NO:3), LWDQSLKPCVKLT (L13T) (SEQ ID NO:4), VYYGVPVWKEA (V11A) (SEQ ID NO:5), YLRDQQLLGIWG (V12G) (SEQ ID NO:6) and FLGFLGAAGSTMGAASLTLTVQARQ (F25Q) (SEQ ID NO:7) (Nehete et al. Vaccine (2001) 20:813-). In the present composition and method, the amino acid sequence of the antigen that is detected by the applicant is IFPGKRTIVAGQRGR (SEQ ID NO:8), and wherein all amino acid all are natural L-amino acid.
[0032] as explained above, the present invention general is influenza virus to another example of its useful virus especially. Particularly, the tunicle glycoprotein h A of influenza virus A and NA are to make us especially interested for producing immune response, as nucleoprotein. A lot of HA hypotypes of influenza virus A are identified (Kawaoka et al., Virology (1990) 12:759-767; Webster et al., ＂ Antigenic variation among type A influenza viruses, ＂ is and D.W.Kingsbury (ed.) p.127-168.In:P.Palese, Genetics of influenza viruses.Springer-Verlag, New York). Therefore, the albumen that derives from any these separated strains also can be used in the immunological technique described herein. Particularly, 13 conservative amino acid sequences of HA can be used in vaccine delivery compositions of the present invention and the method. In the employed H3 strain, this amino acid sequence is PRYVKQNTLKLAT (SEQ ID NO:9) in present bacterin preparation, and in the H5 strain, it mainly is PKYVKSNRLVLAT (SEQ ID NO:10).
[0033] method of describing in this article also can be utilized many bacterial antigens, for example derive from those antigens that cause diphtheria, cholera, pulmonary tuberculosis, lockjaw, pertussis, meningitic organism and other pathogeny organism, include but not limited to: meningococcus A, B and C, Type B haemophilus influenzae (Hemophilus influenza type B, HIB) and helicobacter pylori. The example of parasite antigen comprises those antigens that derive from the organism that causes malaria and Lyme disease.
[0034] and, method described herein provides instrument for treating various malignant cancer. For example, composition of the present invention can be used for starting for the HI of the special specific protein of the cancer of suspecting and cell-mediated immune response, for example oncogene, fetal antigen or the activation marker of activation. This type of tumour antigen comprises that various MAGEs' (melanoma associated antigen E (melanoma associated antigen E)) is any, comprise MAGE1,2,3, (Boon, the T.Scientific American (March 1993): 82-89) such as 4; Various tyrosinases any; MART1 (by the melanoma antigen of T cell recognition), the ras mutant; The p53 mutant; The p97 melanoma antigen; CEA (carcinomebryonic antigen (carcinoembryonic antigen)); Etc.. In the present composition and composition useful other melanoma peptide antigen comprise following these:
| Title | Antigen sequence | Protein |
| Mart1-27 | AAGIGILTV | MART1 |
| (SEQ ID NO：11) | ||
| Gp100-209 * | ITDQVPFSV (SEQ ID NO：12) | Melanocyte system specific antigen GP100 |
| Gp100-154 | KTWGQYWQV (SEQ ID NO：13) | Melanocyte system specific antigen GP100 |
| Gp100-280 | YLEPGPVTA (SEQ ID NO：14) | Melanocyte system specific antigen GP100 |
*GP100 is also referred to as the relevant ME20 antigen (melanoma-associated ME20 antigen) of melanoma.
[0035] evidently, the invention of this theme can be used to prevention or treatment various diseases.
[0036] the peptide antigen that is dispersed in the polymer of vaccine delivery compositions of the present invention can have any suitable length, but can mix by the identification of peptide restricted type T lymphocyte, have 8 to about 30 amino acid whose peptide antigen fragments. Particularly, described peptide antigen fragment by the identification of corresponding I class peptide restricted type cytotoxic T cell contains 8 to about 12 amino acid, and for example 9 to about 11 amino acid; And described peptide antigen fragment by the identification of corresponding II class peptide restricted type t helper cell contains 8 to about 30 amino acid, for example about 12 to about 24 amino acid.
[0037] although presenting (on the surface of APCs) by the peptide epitopes that is undertaken by the MHC molecule, the cell-mediated immunity of natural T works, but MHCs can also present the peptide adjuvant---particularly, sugar-peptide and fat-peptide, wherein peptide moiety is controlled by MHC, to T cell display sugar or fat part. This Consideration is correlated with in cancer vaccine is learned especially because several tumour is crossed expression sugar-derived protein or fat-derived protein, and these sugar-or fat-derived peptide fragment can be strong t cell epitope in some cases. And the fat in this type of t cell epitope can be sugar-fat.
[0038] present different from normal separately peptide, in these cases, the T cell recognition is subjected to sugar or the fat group on this peptide to prop up such degree that is fitted on, so that short synthetic peptide successfully is used as peptide antigen, described short synthetic peptide with high affinity in conjunction with MHCs, but be not derived from oncoprotein, however Tumor-assaciated sugar or fat molecule be synthesized ground covalently bound on it. Should be used for setting up the method for the artificial t cell epitope of natural tumor cell line, by Franco et aL, J.Exp.Med (2004) 199 (5): 707-716 adopts recently. Therefore, the synthetic peptide derivative, even intend peptide, can replace the peptide antigen in the vaccine delivery compositions of the present invention, with as high affinity MHC binding partner, be formed for to the platform of T presented by cells peptide branch (peptide branches) and non-peptide antigen.
[0039] therefore, term " peptide antigen (peptidic antigen) ", as used herein, refer to peptide, full peptide derivant (wholly peptide derivative) (for example branched peptide (branched peptide)) and the covalency of peptide different-(for example sugared-and fat-and glycolipid-) derivative. Also intention comprises that this type of is by the fragment of the material of specific antibodies or the combination of specific T lymphocyte specific.
[0040] utilize technology as known in the art, can antigenic synthetic peptide. Described peptide antigen can also comprise " peptide mimics (peptide mimetics) ". Peptide analogues is often used as the non-peptide biological activity agent with the character that is similar to the template peptide in pharmacy industry. The non-peptide compound of these types is called as " peptide mimics (peptide mimetics) " or " intending peptide (peptidomimetics) " Fauchere, J. (1986) Adv.Bioactive agent Res., 15:29; Veber and Freidinger (1985) TINS p.392; With Evans et al. (1987) J.Med.Chem., 30:1229; And usually under the help of computerization molecular modeling, be developed. Usually, intend peptide and structurally be similar to example polypeptide (that is, having the polypeptide of biochemical property or pharmacological activity), chosen wantonly with being selected from following key replacement :-CH but have one or more peptide bonds2NH-、-CH
2S-、CH
2—CH
2-,-CH=CH-(cis and trans) ,-COCH2-、-CH(OH)CH
2-and-CH2SO-, described replacement is undertaken by method as known in the art and that further describe in following list of references: Spatola, A.F.in ＂ Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, ＂ B.Weinstein, eds., Marcel Dekker, New York, p.267 (1983); Spatola, A.F., Vega Data (March 1983), Vol.1, Issue 3, ＂ Peptide Backbone Modifications ＂ (general summary); Morley, J.S., Trends.Pharm. Sci, (1980) pp.463-468 (general summary); Hudson, D.et al., Int.J.Pept.Prot.Res., (1979) 14:177-185 (CH2NH-，CH
2CH
2-)；Spatola，A.F.et al.，Life Sci，(1986)38：1243-1249
(-CH
2-S-); Harm, M.M., J.Chem.Soc.Perkin Trans I (1982) 307-314 (CH=CH-, cis and trans); Almquist, R.G.et al., J.Med.Chem., (1980) 23:2533 (--COCH2-)；
Jennings-Whie，C.et al.，Tetrahedron Lett.，(1982)23：2533(-COCH
2-)；Szelke，M.et
al.，European Appln.，EP 45665(1982)CA：97：39405(1982)(-CH(OH)CH
2-)；
Holladay，M.W.et al.，Tetrahedron Lett，(1983)24：4401-4404(--C(OH)CH
2-); And Hruby, V.J., Life Sci, (1982) 31:189-199 (CH2-S-). These type of polypeptide analogies can have significant advantage with respect to the polypeptide embodiment, comprise, such as: the pharmacological property of more economical production, larger chemical stability, enhancing (half-life, absorb, tire and effectiveness etc.), the specificity (for example broad-spectrum biological is learned active) that changes, the antigenicity that reduces and other.
[0041] in addition, the one or more amino acid whose replacement in peptide (for example, replacing L-lysine with D-Lys) can be used for producing more stable peptide and the peptide of anti-endogenous protease. Alternatively, synthetic peptide antigen for example is covalently bound to biodegradable polymers, can also by the preparation of D-amino acid, be called trans peptide (inverso peptides). When peptide during with the assembling of the rightabout of native sequence polypeptide, it is called as backward peptide (retro peptide). Generally speaking, the peptide from the preparation of D-amino acid is very stable for enzymatic hydrolysis. Many situations (US patent 6,261,569 B1 and the list of references wherein of the trans peptide of contrapositive or Partial Inverse (retro-inverso or partial retro-inverso peptides) preservation BA have been reported; B.Fromme et al., Endocrinology (2003) 144:3262-3269).
[0042] in adjuvant existence or non-existent situation, selected peptide antigen is combined with biodegradable polymers, is used for subsequently using mammalian object. Vaccine delivery compositions of the present invention can be produced, and is used for intravenous, mucous membrane, intramuscular or subcutaneous transmission. For example, useful polymer in method described herein includes but not limited to: PEA, PEUR and the PEU polymer described herein. These polymer can be produced with various molecular weight, and the suitable molecular weight that is applicable to given antigen can easily be determined by those of ordinary skills. Therefore, for example, suitable molecular weight will be about 5,000 to about 300,000 the order of magnitude, and for example about 5,000 to about 250,000 or about 75,000 to about 200,000 or about 100,000 to about 150,000.
[0043] in some embodiments, the continuation, protection and the peptide that obtain by polymer composition itself are passed to APCs, may be enough to provide adjuvanticity. In other embodiments, vaccine delivery compositions of the present invention can comprise adjuvant, adjuvant can produce by transmission, the stimulating cytokine that increases antigen and/or stimulator antigen is delivery cell, strengthens immune response, particularly cellullar immunologic response to Soluble protein antigen. Described adjuvant can be dispersed in the polymer substrate with peptide antigen, for example by described adjuvant is coupled to described antigen, thereby described adjuvant can be applied. Alternatively, adjuvant can be applied with vaccine delivery compositions of the present invention, for example in same composition or in several compositions that separating. For example, adjuvant can be applied before or after vaccine delivery compositions of the present invention. Still alternatively, adjuvant or adjuvant/peptide antigen can be arrived as described herein polymer by chemical bond, to carry simultaneously. This type of adjuvant includes, but are not limited to: (1) aluminium salt (alum), such as aluminium hydroxide, aluminum phosphate, aluminum sulfate etc.; (2) oil in water emulsion formulation (other specific immunity stimulant of with or without, such as muramyl peptide (muramyl peptides) or bacteria cell wall composition), for example, such as (a) MF59 (international publication number WO 90/14837), contain 5% squalene (Squalane), 0.5% Tween 80TMWith 0.5% Span 85, the optional MTP-PB that contains different amounts, it utilizes microfluidizer (microfluidizer) such as Model HOY microfluidizer (Microfluidics, Newton, Mass.) be formulated into submicron particles, (b) SAF contains 10% squalene, 0.4% Tween 80TM, 5% general stream Buddhist nun restrains block copolymer L121 (pluronic blocked polymer L121) and thr-MDP, itself or become the submicron emulsion by microjet, or be rotated vibration producing the greater particle size emulsion, and (c) RibiTMAdjunvant composition (RAS), (Ribi Immunochem, Hamilton, Mont.) contains 2% squalene, 0.2% Tween 80TM, and be selected from one or more following bacteria cell wall compositions: monophosphoryl lipid A (monophosphorylipid A) (MPL), two mycolate trehalose (trehalose dimycolate; TDM) and cell wall skeleton (cell wall skeleton; CWS), preferred MPL+CWS (DetoxTM); (3) saponin adjuvant is such as StimulonTM(Cambridge Bioscience, Worcester, Mass.) can be used, perhaps from particle such as the ISCOMs (immunostimulation complex (immunostimulating complexes)) of its generation; (4) complete Freund's adjuvant (Complete Freunds Adjuvant (CFA)) and incomplete Freund's adjuvant (Incomplete Freunds Adjuvant (IFA)); (5) cell factor is such as interleukin (IL-1, IL-2 etc.), macrophage colony stimulating factor (M-CSF), TNF (TNF) etc.; (6) bacterium ADP-ribosylation toxin cholera toxin (cholera toxin for example, CT), the daytime is coughed toxin (pertussis toxin, PT) or colibacillary heat-labile toxin (heat-labile toxin, LT) detoxification variant, especially LT-K63 is (wherein in the position 63, lysine replaces wild-type amino acid), LT-R72 is (wherein in the position 72, arginine replaces wild-type amino acid), CT-S 109 is (wherein in the position 109, serine replaces wild-type amino acid) and PT-K9/G129 is (wherein, in the position 9, lysine replaces wild-type amino acid; And in the position 129, glycine replaces wild-type amino acid) (referring to for example international publication WO93/13202 and WO92/19265); And (7) QS21, the saponin(e of purified form and 3D-MPL (3D-monophosphoryl lipid A) are (MPL), it is a kind of non-toxic derivative of lipopolysaccharides (lipopolysaccharide (LPS)), to strengthen cell and HI (Moore, et al., Vaccine.1999 Jun 4; 17 (20-21): 2517-27). As other material of immunostimulant, also can be used for strengthening the validity of described composition.
[0044] is suitable for being used in polymer in the practice of the present invention, has so that peptide antigen, adjuvant or antigen-adjuvant conjugate covalently bound functional group to described polymer easily. For example, can be easily and amino partial reaction with the polymer of carboxylic group, thereby by the amide group that produces, peptide is covalently bound to this polymer. As will describing herein, biodegradable polymers and polypeptide or adjuvant can contain the functional group of many complementations, and it can be used for peptide antigen and/or adjuvant covalently bound to biodegradable polymers.
[0045] passes through in injection site maintenance peptide antigen and optional adjuvant a period of time, be enough to make individual immunocyte and peptide antigen and the adjuvant of choosing wantonly to interact to affect immunologic process; Simultaneously during the biodegradation of polymer, slowly discharge particle or the polymer molecule that contains this type of preparation, thus the polymer in vaccine delivery compositions of the present invention in the endogenous immunologic process of implant site, play a part one positive. By slower biological degradation polyalcohol, fragile biological peptide antigen is protected, with half-life and the continuation that increases described antigen.
[0046] by stimulating phagolysis to polymkeric substance-antigen composition, polymkeric substance itself also can the antigen transmission is advanced play a part among the APCs positive.In addition, polymkeric substance disclosed herein (for example, having those of structural formula (I and III-VIII)), in case behind enzymatic degradation, provide the indispensable amino acid of bringing up cell, other degradation production can be able to metabolism with the approach of lipid acid and sugar metabolism simultaneously.The picked-up that has antigenic polymer is safe: but studies show that---APCs survival, function be normal, and metabolism/removing polymer degradation products.Therefore, these polymkeric substance and vaccine delivery compositions except injecting the wound that itself causes, in the injection site or whole body, all are non-inflammatory basically for object.And, initiatively absorbing at APCs under the situation of polymkeric substance, polymkeric substance can be used as antigenic adjuvant and works, and therefore, does not have the exclusive requirement of independent preparation being used adjuvant.
[0047] can be used for forming the biodegradable polymers of biocompatibility vaccine delivery compositions of the present invention, comprise that each monomer contains those polymkeric substance that at least one amino acid is coupled at least one non-amino acid moiety.As used herein, term " non-amino acid moiety (non-amino acid moiety) " comprises various chemical part, but gets rid of amino acid derivative particularly and intend peptide (peptidomimetics), as described herein.In addition, contain at least a polymer of amino acid and be not considered to include the amino acids fragment, comprise the polypeptide of natural generation, unless carrying out under the so special situation about describing.In one embodiment, non-amino acid (non-amino acid) is in monomer between two adjacent amino acids.In another embodiment, non-amino acid moiety (non-amino acid moiety) is hydrophobic.Polymkeric substance also can be a segmented copolymer.
[0048] being preferred in the present composition and the method is polyesteramide (polyester amides (PEAs)) and polyester urethane (polyester urethanes (PEURs)), it has the functional group of embedding on PEA or PEUR skeleton, and these embeddings functional group can with other chemical reaction, and cause mixing of extra functional group, with the functionality of further expansion PEA or PEUR.Therefore, usefulness this base polymer in the methods of the invention is easy to have with other chemical reaction of hydrophilic-structure, and is water-soluble to increase, and reacts with peptide antigen, adjuvant and other preparation, and unnecessary modification in advance.
[0049] in addition, the polymkeric substance that uses in vaccine delivery compositions of the present invention is when when detected, show no hydrolysis degraded in salt solution (PBS) medium; But in enzyme solution, for example among Quimotrase (chymotrypsin) or the CT, observe consistent corrosion behavior.
In one embodiment, polymkeric substance is the PEA with the described chemical structural formula of structural formula (I),
Formula (I)
Wherein n is between about 5 to about 150; R
1Be independently selected from α, ω-two (4-carboxyl phenoxy group)-(C
1-C
8) alkane, 3,3 '-(alkane two acyl dioxy bases) two styracins or 4,4 '-(alkane two acyl dioxy bases) two styracins, (C
2-C
20) alkylene hydrocarbon or (C
2-C
20) residue of inferior alkene; R in an independent n monomer
3Be independently selected from hydrogen, (C
1-C
6) alkyl, (C
2-C
6) thiazolinyl, (C
2-C
6) alkynyl, (C
6-C
10) aryl (C
1-C
20) alkyl and-(CH
2)
2S (CH
3); And R
4Be independently selected from (C
2-C
20) alkylidene group, (C
2-C
20) alkenylene, (C
2-C
8) alkoxyl group, (C
2-C
20) alkylidene group, saturated or undersaturated therapeutic glycol residue, structural formula (II) 1,4:3, dicyclo part and their the combination, (C of the two anhydrous hexitols (dianhydrohexitol) of 6-
2-C
20) alkylidene group and (C
2-C
20) alkenylene;
Formula (II)
Perhaps the PEA polymkeric substance has the described chemical formula of structural formula (III):
Formula (III)
Wherein n is between about 5 to about 150, and m is between about 0.1 to 0.9, and p is between about 0.9 to 0.1; R wherein
1Be independently selected from α, ω-two (4-carboxyl phenoxy group)-(C
1-C
8) alkane, 3,3 '-(alkane two acyl dioxy bases) two styracins or 4,4 '-(alkane two acyl dioxy bases) two styracins, (C
2-C
20) alkylene hydrocarbon or (C
2-C
20) residue of inferior alkene; Each R
2Be hydrogen, (C independently
1-C
12) alkyl or (C
6-C
10) aryl or protecting group; R in an independent m monomer
3Be independently selected from hydrogen, (C
1-C
6) alkyl, (C
2-C
6) thiazolinyl, (C
2-C
6) alkynyl, (C
6-C
10) aryl (C
1-C
20) alkyl and-(CH
2)
2S (CH
3); And R
4Be independently selected from (C
2-C
20) alkylidene group, (C
2-C
20) alkenylene, (C
2-C
8) alkoxyl group, (C
2-C
20) alkylidene group, saturated or unsaturated therapeutic glycol residue, general formula (II) 1,4:3, the dicyclo part and their combination of the two anhydrous hexitols (dianhydrohexitol) of 6-.
[0050] in another embodiment, polymkeric substance is the PEUR polymkeric substance, and it has the described chemical formula of structural formula (IV),
Formula (IV)
Wherein n is between about 5 to about 150; R wherein
3Be independently selected from hydrogen, (C
1-C
6) alkyl, (C
2-C
6) thiazolinyl, (C
2-C
6) alkynyl, (C
6-C
10) aryl (C
1-C
20) alkyl and-(CH
2)
2S (CH
3); R
4Be selected from (C
2-C
20) alkylidene group, (C
2-C
20) alkenylene or alkoxyl group, saturated or unsaturated therapeutic glycol residue, structural formula (II) 1,4:3, the dicyclo part and their combination of the two anhydrous hexitols (dianhydrohexitol) of 6-; And R
6Be independently selected from (C
2-C
20) alkylidene group, (C
2-C
20) alkenylene or alkoxyl group, general formula (II) 1,4:3, the dicyclo part and their combination of the two anhydrous hexitols (dianhydrohexitol) of 6-.
[0051] in another embodiment, polymkeric substance is the PEUR with the described chemical structure of universal architecture formula V:
Formula V
Wherein n is between about 5 to about 150, and m is between about 0.1 to about 0.9, and p is between about 0.9 to about 0.1; R
2Be independently selected from hydrogen, (C
6-C
10) aryl (C
1-C
20) alkyl or protecting group; R in an independent m monomer
3, be independently selected from hydrogen, (C
1-C
6) alkyl, (C
2-C
6) thiazolinyl, (C
2-C
6) alkynyl, (C
6-C
10) aryl (C
1-C
20) alkyl and-(CH
2)
2S (CH
3); R
4Be selected from (C
2-C
20) alkylidene group, (C
2-C
20) alkenylene or alkoxyl group, saturated or unsaturated therapeutic glycol residue, structural formula (II) 1,4:3, the dicyclo part and their combination of the two anhydrous hexitols (dianhydrohexitol) of 6-; And R
6Be independently selected from (C
2-C
20) alkylidene group, (C
2-C
20) alkenylene or alkoxyl group, general formula (II) 1,4:3, the dicyclo part of the two anhydrous hexitols (dianhydrohexitol) of 6-, the residue of the saturated or unsaturated therapeutic glycol of significant quantity and their combination.
[0052] still in another embodiment, polymkeric substance is biodegradable PEU polymkeric substance, and it has the described chemical formula of universal architecture formula (VI):
Formula (VI),
Wherein n is between about 10 to about 150; R in an independent n monomer
3, be independently selected from hydrogen, (C
1-C
6) alkyl, (C
2-C
6) thiazolinyl, (C
2-C
6) alkynyl, (C
6-C
10) aryl (C
1-C
20) alkyl and-(CH
2)
2S (CH
3); R
4Be independently selected from (C
2-C
20) alkylidene group, (C
2-C
20) alkenylene, (C
2-C
8) alkoxyl group (C
2-C
20) residue of alkylidene group, saturated or unsaturated therapeutic glycol; Or structural formula (II) 1,4:3, the dicyclo part of the two anhydrous hexitols of 6-.
[0053] still in another embodiment, polymkeric substance is the PEU with the described chemical formula of structural formula (VII)
Formula (VII),
Wherein m is between about 0.1 to about 1.0, and p is between about 0.9 to about 0.1, and n is between about 10 to about 150; Each R
2Be hydrogen, (C independently
1-C
12) alkyl or (C
6-C
10) aryl; R in an independent m monomer
3, be independently selected from hydrogen, (C
1-C
6) alkyl, (C
2-C
6) thiazolinyl, (C
2-C
6) alkynyl, (C
6-C
10) aryl (C
1-C
20) alkyl and-(CH
2)
2S (CH
3); Each R
4Be independently selected from (C
2-C
20) alkylidene group, (C
2-C
20) alkenylene, (C
2-C
8) alkoxyl group (C
2-C
20) alkylidene group, saturated or unsaturated therapeutic glycol residue, structural formula (II) 1,4:3, the dicyclo part and their combination of the two anhydrous hexitols of 6-.
[0054] for example, in an optional scheme, in being used for the PEA polymkeric substance of particle transport composition of the present invention, at least one R
1Be α, ω-two (4-carboxyl phenoxy group)-(C
1-C
8) alkane, 3,3 '-(alkane two acyl dioxy bases) two styracins or 4,4 '-residue of (alkane two acyl dioxy bases) two styracins, and R
4Be 1 of general formula (II), 4:3, the dicyclo part of the two anhydrous hexitols of 6-.In another optional scheme, the R in the PEA polymkeric substance
1Or α, ω-two (4-carboxyl phenoxy group)-(C
1-C
8) alkane, 3,3 '-(alkane two acyl dioxy bases) two styracins or 4,4 '-residue of (alkane two acyl dioxy bases) two styracins.In another possibility, the R in the PEA polymkeric substance
1Be α, ω-two (4-carboxyl phenoxy group) (C
1-C
8) alkane for example 1, two (the 4-carboxyl phenoxy group) propane (CPP), 3,3 of 3-'-(alkane two acyl dioxy bases) two styracins or 4,4 '-residue of (alkane two acyl dioxy bases) two styracins, and R
4Be 1 of general formula (II), 4:3, the dicyclo part of the two anhydrous hexitols of 6-, for example DAS.
[0055] in another embodiment, polymkeric substance is the PEUR with the described chemical formula of structural formula (IV),
Formula (IV)
Wherein n is between about 5 to about 150; R wherein
3Be independently selected from hydrogen, (C
1-C
6) alkyl, (C
2-C
6) thiazolinyl, (C
2-C
6) alkynyl, (C
6-C
10) aryl (C
1-C
20) alkyl and-(CH
2)
2S (CH
3); R
4Be selected from (C
2-C
20) alkylidene group, (C
2-C
20) alkenylene or alkoxyl group, saturated or unsaturated therapeutic glycol residue, structural formula (II) 1,4:3, the dicyclo part of the two anhydrous hexitols (dianhydrohexitol) of 6-; And R
6Be independently selected from (C
2-C
20) alkylidene group, (C
2-C
20) alkenylene or alkoxyl group, general formula (II) 1,4:3, the dicyclo part of the two anhydrous hexitols (dianhydrohexitol) of 6-, the residue of the saturated or unsaturated therapeutic glycol of significant quantity and their combination;
The PEUR polymkeric substance that perhaps has the described chemical formula of universal architecture formula V:
Formula V
Wherein n is between about 5 to about 150, and m is between about 0.1 to about 0.9, and p is between about 0.9 to about 0.1; R
2Be independently selected from hydrogen, (C
6-C
10) aryl (C
1-C
20) alkyl or protecting group; R in an independent m monomer
3Be independently selected from hydrogen, (C
1-C
6) alkyl, (C
2-C
6) thiazolinyl, (C
2-C
6) alkynyl, (C
6-C
10) aryl (C
1-C
20) alkyl and-(CH
2)
2S (CH
3); R
4Be selected from (C
2-C
20) alkylidene group, (C
2-C
20) alkenylene or alkoxyl group, structural formula (II) 1,4:3, the dicyclo part of the two anhydrous hexitols (dianhydrohexitol) of 6-; And R
6Be independently selected from (C
2-C
20) alkylidene group, (C
2-C
20) alkenylene or alkoxyl group, general formula (II) 1,4:3, the dicyclo part and their combination of the two anhydrous hexitols (dianhydrohexitol) of 6-.
[0056] in an optional scheme, in the PEUR polymkeric substance, at least one R
4Be 1,4:3, the dicyclo part of the two anhydrous hexitols (formula (II)) of 6-, for example 1,4:3, the two anhydrous Sorbitol Powders (1,4:3,6-dianhydrohexitol (DAS)) of 6-; Perhaps R
6Be 1,4:3, the dicyclo part of the two anhydrous hexitols of 6-, for example 1,4:3, the two anhydrous Sorbitol Powders (1,4:3,6-dianhydrohexitol (DAS)) of 6-.In another optional scheme, in the PEUR polymkeric substance, R
4And/or R
6Be 1,4:3, the dicyclo part of the two anhydrous hexitols of 6-, for example 1,4:3, the two anhydrous Sorbitol Powders (1,4:3,6-dianhydrohexitol (DAS)) of 6-.
[0057] still in another embodiment, the polymkeric substance in particle transport composition of the present invention is the PEU polymkeric substance, and it has the described chemical formula of universal architecture formula (VI):
Formula (VI),
Wherein n is between about 10 to about 150; R in an independent n monomer
3Be independently selected from hydrogen, (C
1-C
6) alkyl, (C
2-C
6) thiazolinyl, (C
2-C
6) alkynyl, (C
6-C
10) aryl (C
1-C
20) alkyl and-(CH
2)
2S (CH
3); R
4Be independently selected from (C
2-C
20) alkylidene group, (C
2-C
20) alkenylene, (C
2-C
8) alkoxyl group (C
2-C
20) residue of saturated or unsaturated therapeutic glycol of alkylidene group, significant quantity; Or structural formula (II) 1,4:3, the dicyclo part of the two anhydrous hexitols of 6-;
The PEU that perhaps has the described chemical formula of structural formula (VII)
Formula (VII),
Wherein m is between about 0.1 to about 1.0, and p is between about 0.9 to about 0.1, and n is between about 10 to about 150; Each R
2Be hydrogen, (C independently
1-C
12) alkyl or (C
6-C
10) aryl or other blocking group; R in an independent m monomer
3Be independently selected from hydrogen, (C
1-C
6) alkyl, (C
2-C
6) thiazolinyl, (C
2-C
6) alkynyl, (C
6-C
10) aryl (C
1-C
20) alkyl ,-(CH
2)
3-and-(CH
2)
2S (CH
3); R
4Be independently selected from (C
2-C
20) alkylidene group, (C
2-C
20) alkenylene, (C
2-C
8) alkoxyl group (C
2-C
20) residue of saturated or unsaturated therapeutic glycol of alkylidene group, significant quantity; Or structural formula (II) 1,4:3, the dicyclo part of the two anhydrous hexitols of 6-;
[0058] the suitable protecting group that is used for practice of the present invention comprises the tertiary butyl and other protecting group known in the art.1,4:3, the suitable dicyclo part of the two anhydrous hexitols of 6-can derive from sugar alcohol, for example D-sorbitol, D-N.F,USP MANNITOL and L-iditol.For example, 1,4:3, (Isosorbide DAS) is particularly suitable for as 1 the two anhydrous Sorbitol Powders of 6-, 4:3, the dicyclo part of the two anhydrous hexitols of 6-.
[0059] these PEU polymkeric substance can be prepared to high-molecular weight polymer, are used to prepare vaccine delivery compositions of the present invention, to carry on the various pharmacopedics and biologically activated preparation to people and other Mammals.PEUs of the present invention mixes the ester group that hydrolysis can cut and contains the non-toxicity of a-amino acid, the monomer of natural generation in polymer chain.The ultimate biodegradation product of PEUs will be a-amino acid (no matter be biological or be not biological), glycol and CO
2Than PEAs and PEURs, PEUs of the present invention is lenticular or semi-crystalline shape, and have favourable machinery, chemistry with biological degradation character, this allows preparation is complete synthetic, thereby be easy to produce: lenticular and semi-crystalline shaped polymer particle, as nano particle.
[0060] for example, the PEU polymkeric substance that uses in vaccine delivery compositions of the present invention has high mechanical strength, and the surface erosion of PEU polymkeric substance can be subjected to the catalysis of the enzyme that exists under physiological condition, as the catalysis of lytic enzyme institute.
[0061] in a possibility, in the PEU polymkeric substance, at least one R
1Be 1,4:3, the dicyclo part of the two anhydrous hexitols of 6-, for example 1,4:3, the two anhydrous Sorbitol Powders (1,4:3,6-dianhydrohexitol (DAS)) of 6-.
[0062] is used for the suitable protecting group of practice of the present invention, comprises the tertiary butyl and other protecting group known in the art.Suitable 1,4:3, the two anhydrous hexitol dicyclos parts of 6-can derive from sugar alcohol, for example D-sorbitol, D-N.F,USP MANNITOL and L-iditol.For example, two anhydrous Sorbitol Powders are particularly suitable for as 1,4:3, the dicyclo part of the two anhydrous hexitols of 6-.
[0063] in a selection, a plurality of R at least one n monomer
3Be CH
2Ph, and the a-amino acid that uses in synthetic is the L-phenylalanine.A plurality of R in monomer
3Be-CH
2-CH (CHB)
2Possibility in, polymkeric substance contains a-amino acid, leucine.By changing a plurality of R
3, other a-amino acid also can be used, and for example glycine (is worked as R
3Be-during H), proline(Pro) (works as R
3When being ethylidene acid amides (ethylene amide)); L-Ala (is worked as R
3Be-CH
3The time), Xie Ansuan (works as R
3Be-CH (CH
3)
2The time), Isoleucine (works as R
3Be-CH (CH
3)-CH
2-CH
3The time), phenylalanine (works as R
3Be-CH
2-C
6H
5The time); Methionin (is worked as R
3Be-(CH
2)
4-NH
2The time); Perhaps methionine(Met) (is worked as R
3Be-CH
2S (CH
3) time).
[0064] in another embodiment, wherein polymkeric substance is PEA, PEUR or the PEU of formula I or III-VII, at least one R
3Further can be-(CH
2)
3-; And described at least one R
3Cyclisation is to form the described chemical structure of structural formula XVIII
Formula (XVIII).
[0065] works as R
3Be-(CH
2)
3, the a-amino acid that is similar to tetramethyleneimine-2-carboxylic acid (proline(Pro)) is used.
[0066] PEAs, PEURs and PEUs are biodegradable polymers, and they are mainly by the enzymatic action biological degradation, discharge dispersive peptide antigen and optional adjuvant with in time past.Because the structural performance of employed polymkeric substance, vaccine delivery compositions of the present invention provide the steady load of peptide antigen with the adjuvant of choosing wantonly, keep their three-dimensional structure simultaneously, thus retains biological activity.
[0067] as used herein, term " amino acid " and " a-amino acid " are meant the compound that contains amino, carboxyl and side chain R group, and for example side chain R group is R defined herein
3Group.As used herein, term " biological a-amino acid (biological α-amino acid) " is meant the amino acid (one or more) that is used in synthesizing, and is selected from phenylalanine, leucine, glycine, L-Ala, Xie Ansuan, Isoleucine, methionine(Met), proline(Pro) or their mixture.
[0068] in PEA, the PEUR and PEU polymkeric substance that are used for the present invention's practice, a plurality of different a-amino acids can be used in the single polymer molecule.Each repeating unit of these polymkeric substance can comprise at least two different amino acid, and single polymer molecule can contain a plurality of different a-amino acids in this polymer molecule, and this depends on this bulk of molecule.In a selection, at least one in the preparation of polymkeric substance of the present invention in the employed a-amino acid is biological a-amino acid.
[0069] for example, work as R
3Be CH
2Ph, employed biological a-amino acid is the L-phenylalanine in synthetic.At R
3Be CH
2-CH (CH
3)
2Selection in, polymkeric substance contains this biology a-amino acid, i.e. leucine.By changing the R in the comonomer as described herein
3, also can use other biological a-amino acid, for example glycine (is worked as R
3When being H), L-Ala (works as R
3Be CH
3The time), Xie Ansuan (works as R
3Be CH (CH
3)
2The time), Isoleucine (works as R
3Be CH (CH
3)-CH
2-CH
3The time), phenylalanine (works as R
3Be CH
2-C
6H
5The time) or methionine(Met) (work as R
3Be-CH
2S (CH
3) time) and their mixture.Work as R
3Be-(CH
2)
3In-time, as in pyrrolidine 2 carboxylic acid (proline(Pro)), biological a-amino acid can be used.In another optional embodiment, all various a-amino acids that are included in the vaccine delivery compositions of the present invention are biological a-amino acids, as described herein.
[0070] polymer molecule also can make peptide antigen and its coupling by joint, and peptide antigen is incorporated in the linking agent between the molecule.For example, in one embodiment, polymkeric substance is comprised in the have structural formula polymkeric substance-antigen conjugates of (VIII):
Formula (VIII)
Wherein n, m, p, R
1, R
3And R
4For as mentioned above, R
5Be selected from-O-,-S-and-NR
8-, R wherein
8Be H or (C
1-C
8) alkyl; And, R
7Be peptide antigen.
[0071] in another embodiment, the polymkeric substance of the structural formula of two molecules (IX) can be crosslinked, to provide-R
5-R
7-R
5-conjugate.In another embodiment, as shown in structural formula IX below, two portions of the single polymer molecule of peptide antigen and structural formula IV pass through-R
5-R
7-R
5-conjugate is covalently bound, and R
5Be independently selected from-O-,-S-and-NR
8-, R wherein
8Be H or (C
1-C
8) alkyl; And R
7Be peptide antigen.
Formula (IX)
[0072] still alternatively, as shown in structural formula (X) below, joint-X-Y-can be inserted into the R in the molecule of structural formula (VIII)
5With peptide antigen R
7Between, wherein X is selected from (C
1-C
18) alkylidene group, substituted alkylene, (C
3-C
8) encircle alkylidene group, substituted ring alkylidene group, contain 1-3 the first heterocyclic system of heteroatomic 5-6, substituted heterocycle, (C that is selected from O, N and S
2-C
18) thiazolinyl, substituted alkenyl, alkynyl, substituted alkynyl, C
6And C
10Aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkylaryl, substituted alkyl aryl, aromatic yl polysulfide yl, substituted aryl alkynyl, aryl alkenyl, substituted aryl thiazolinyl, aromatic yl polysulfide yl, substituted aryl alkynyl, wherein substituting group is selected from H, F, Cl, Br, I, (C
1-C
6) alkyl ,-CN ,-NO
2,-OH ,-O (C
1-C
4) alkyl ,-S (C
1-C
6) alkyl ,-S[(=O) (C
1-C
6) alkyl)] ,-S[(O
2) (C
1-C
6) alkyl] ,-C[(=O) (C
1-C
6) alkyl], CF
3,-O[(CO)-(C
1-C
6) alkyl)] ,-S (O
2) [N (R
9R
10) ,-NH[(C=O) (C
1-C
6) alkyl] ,-NH (C=O) N (R
9R
10) ,-N (R
9R
10); R wherein
9And R
10Be H or (C independently
1-C
6) alkyl; And Y be selected from-O-,-S-,-S-S-,-S (O)-,-S (O
2)-,-NR
8-,-C (=O)-,-OC (=O)-,-C (=O) O-,-OC (=O) NH-,-NR
8C (=O)-,-C (=O) NR
8-,-NR
8C (=O) NR
8-,-NR
8C (=O) NR
8-and-NR
8C (=S) NR
8-.
Formula (X)
[0073] in another embodiment, the antigenic two portions of single peptide pass through-R
5-R
7-Y-X-R
5-bridge (formula XI) is covalently bound with biologically active agent:
Formula (XI)
Wherein, X is selected from (C
1-C
18) alkylidene group, substituted alkylene, (C
3-C
8) encircle alkylidene group, substituted ring alkylidene group, contain 1-3 the first heterocyclic system of heteroatomic 5-6, substituted heterocycle, (C that is selected from O, N and S
2-C
18) thiazolinyl, substituted alkenyl, alkynyl, substituted alkynyl, (C
6-C
10) aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkylaryl, substituted alkyl aryl, aromatic yl polysulfide yl, substituted aryl alkynyl, aryl alkenyl, substituted aryl thiazolinyl, aromatic yl polysulfide yl, substituted aryl alkynyl, wherein substituting group is selected from H, F, Cl, Br, I, (C
1-C
6) alkyl ,-CN ,-NO
2,-OH ,-O (C
1-C
6) alkyl ,-S (C
1-C
6) alkyl ,-S[(=O) (C
1-C
6) alkyl)] ,-S[(O
2) (C
1-C
6) alkyl] ,-C[(=O) (C
1-C
6) alkyl], CF
3,-O[(CO)-(C
1-C
6) alkyl)] ,-S (O
2) [N (R
9R
10) ,-NH[(C=O) (C
1-C
6) alkyl] ,-NH (C=O) N (R
9R
10); R wherein
9And R
10Be H or (C independently
1-C
6) alkyl and-N (R
11R
12), R wherein
11And R
12Be independently selected from (C
2-C
20) alkylidene group and (C
2-C
20) alkenylene.
[0074] in another embodiment, vaccine delivery compositions contains four molecules of polymkeric substance, just only has two to omit R in four molecules
7, and be crosslinked, so that single-R is provided
5-X-R
5-conjugate.
When [0075] mentioning the structural formula of this paper, term " aryl (aryl) " is used to refer to phenyl (phenyl radical) or has the monolateral fused bicyclic carbocyclic group group of about 9 to 10 annular atomses, and wherein at least one ring is an aromaticity.In some embodiments, one or more annular atomses can replace with one or more nitros, cyano group, halogen, trifluoromethyl or trifluoromethoxy.The example of aryl includes, but not limited to phenyl, naphthyl and nitrophenyl.
When [0076] mentioning the structural formula of this paper, term " alkenylene (alkenylene) " is used in reference to divalence side chain or the unbranched hydrocarbon chain that contains at least one unsaturated link(age) in main chain or side chain.
[0077] as used herein, " therapeutic glycol (therapeutic diol) " is meant any glycol molecules of synthetic generation or natural generation (for example, interior seedbed takes place); When being applied to Mammals, it influences the biological procedures of mammalian subject, as the biological procedures in the human body with therapeutic or appeasing property mode.
[0078] as used herein, term " residue of therapeutic glycol (residue of a therapeutic diol) " is meant the part of therapeutic glycol, and so the place is described, and this part does not comprise two oh groups of described glycol.The corresponding treatment glycol that contains its " residue " was used in synthesizing of polymer composition.By biological degradation in a controlled manner after polymer backbone discharges, the residue of therapeutic glycol (or under similar pH condition, aqueous medium conditioned disjunction conditions of similarity) in vivo is reproduced into corresponding diol, described controlled way depends on the PEA, the PEUR that are selected to prepare composition or the character of PEU polymkeric substance, and this character is known and as described herein in this area.
[0079] owing to be used in PEA, PEUR in the present composition and the diversity of PEU polymkeric substance, the amount of mixing the therapeutic glycol of polymer backbone can be controlled by the ratio that changes constitutional unit.For example, depend on the composition of PEA, can be implemented up to the 17 β estradiol contents of 40% w/w.Three kinds of different regular, linear PEA with 17 different β estradiol content ratios, set forth in the diagram 1 below:
＂ homopolymerization ＂-two-leucine-estradiol-hexanodioic acid (estradiol on the 40%w/w-polymkeric substance)
Multipolymer: Leu (ED)
3Lys (OEt) Adip
4Estradiol content with 38%w/w
Diagram 1
[0080] similarly, the capacity value of therapeutic glycol in PEUR and PEU polymkeric substance can pass through the amount change of two or more structural units of change polymkeric substance.
[0081] in addition, synthetic steroid radical glycol based on testosterone or cholesterol, 4-androstene-3 for example, 17 glycol (4-rostenediol), 5-androstene-3,17 glycol (5-rostenediol), 19-remove first 5-androstene-3,17 glycol (19-norandroslenediol) are suitable for mixing in the skeleton according to PEA of the present invention and PEUR polymkeric substance.And the therapeutic diol compound that is suitable for being used in the preparation of vaccine delivery compositions of the present invention comprises, for example ammonia hydroxyl butyl kanamycin A (amikacin); Amphotericin B; Apicycline (apicycline); Apramycin; Arbelkacin (arbekacin); Azidoamphenicol (azidamfenicol); Bambermycin (one or more); Fourth glycosides bacterium; Magnamycin A; Cefpiramide (cefpiramide); Paraxin; Uromycin; Clindamycin; Clomocycline; Deoxidation duomycin; Thymol sulfone (diathymosulfone); Dibekacin, dihydrostreptomycin; Dirithromycin; Vibravenos; Erythromycin; Fortimicin (one or more); Gentamicin (one or more); Glucosulfone (glucosulfone) Solasulfone (solasulfone); Guanidine first mycin; Isepamicin (isepamicin); Josamycin; Kantlex (one or more); Leucomycin (one or more); Lincomycin; Lucensomyein; Rely amine first tsiklomitsin; The first Uromycin; Methacycline; Micronomicin; Mydecamycin (one or more); MINOCYCLINE HCL; Mupirocin (mupirocin); Natamycin; Xin Meisu; Anti-for Mi Xing; Romicil; Terramycin; Paromycin; Pipacycline (pipacycline); Podophyllinic acid 2 ethylhydrazide; Primycin; Ribostamycin; Rifamide; Rifampin; Rifamycin Sodium; Rifapentine; Rifaximin; Spontycine; Rokitamycin (rokitamycin); Rolitetracycline; Rosaramicin (rasaramycin); Roxithromycin; Orsomycin (sancycline); Sisomycin; Spectinomycin; Spiramycin Base; Streptomycin sulphate; Trip wall rhzomorph; Tsiklomitsin; Thiamphenicol; Theiostrepton; Tobramycin; The holder miramycin; Tuberactinomycin; Vancomycin; Candicidin (one or more); Chlorphenesin (chlorphenesin); Dermastatin (one or more); Filipin; Fungichromin; Kantlex (one or more); Leucomycin (one or more); Lincomycin; Lucensomyein (lvcensomycin); Lymecycline; The first Uromycin; Methacycline; Micronomicin; Mydecamycin (one or more); MINOCYCLINE HCL; Mupirocin (mupirocin); Natamycin; Xin Meisu; Anti-for Mi Xing; Romicil; Terramycin; Paromycin; Pipacycline (pipacycline); Podophyllinic acid 2 ethylhydrazide; Primycin (priycin); Ribostamycin; Rifamide; Rifampin; Rifamycin Sodium; Rifapentine; Rifaximin; Spontycine; Rokitamycin (rokitamycin); Rolitetracycline; Rosaramicin; Roxithromycin; Orsomycin (sancycline); Sisomycin; Spectinomycin; Spiramycin Base; Streptomycin sulphate (strepton); Otbramycin; The holder miramycin; Tuberactinomycin; Vancomycin; Candicidin (one or more); Chlorphenesin (chlorphenesin); Dermastatin (one or more); Filipin; Fungichromin; First three rhzomorphs; Nystatin (mystatin); Oligomycin (one or more); Erimycin A; Tubercidin; The 6-azauridine; Aclacinomycin (one or more); Ancitabine (ancitabine); The ammonia aspergillin; Azacitadine; Bleomycin (one or more) carubicin; Carzinophillin A; Chlorine assistant star (chlorozotocin); Chromomcin (s); Doxifluridine (doxifiuridine); Enocitabine (enocitabine); Pidorubicin (epirubicin); Gemcitabine (gemcitabine); Mannomustin (mannomustine); Menogaril (menogaril); Atorvasi Pravastatin (pravastatin); Clarithromycin; Leuproline; Taxol (paclitaxel); Mitobronitol; Mitolactol; Mopidamol (mopidamol); U-15167; Olivomycine (one or more); Peplomycin (peplomycin); Perarubicin (pirarubicin); Prednimustine (prednimustine); Tetracycline (puromycin); Ranomustine (ranimustine); Tubercidin (tubercidin); Vinesine; Zorubicin (zorubicin); Cumetharol (coumetarol); Temparin (dicoumarol); Biscoumacetate (ethyl biscoumacetate); Ethylidine temparin (ethylidine dicoumarol); Iloprost (iloprost); Taprostene (taprostene); Tioclomarol (tioclomarol); Therafectin (amiprilose); Romurtide (romurtide); Sirolimus (sirolimus) (rapamycin (rapamycin)); Tacrolimus (tacrolimus); Saligenol; Bromosaligenin (bromosaligenin); Ditazol (ditazol); Fepradinol (fepradinol); 2, the 5-protocatechuic acid; Glacamethacin; Olsalazine (olsalazine); S-adenosylmethionine; Azythromycin (azithromycin); Salmeterol (salmeterol); Budesonide (budesonide); Albuteal; That dimension of indenes ground (indinavir); Fluvastatin (fluvastatin); U-9889 (streptozocin); Zorubicin (doxorubicin); Daunorubicin (daunorubicin); Plicamycin (plicamycin); Darubicin (idarubicin); Pentostatin (pentostatin); Rice holder anthracene a kind of jade (metoxantrone); Cytosine arabinoside (cytarabine); Fludarabine phosphate (fludarabine phosphate); Fluridine (floxuridine); Cladriine; Xeloda (capecitabien); Docetaxel (docetaxel); Etoposide (etoposide); Hycamtin (topotecan); Vinealeucoblastine(VLB) (vinblastine); Teniposide (teniposide), and similar compound.The therapeutic glycol can be selected from saturated or unsaturated diol.
[0082] molecular weight of this paper and polymolecularity are by gel permeation chromatography (gel permeationchromatography (GPC)), use polyethylene standard product and mensuration.More specifically, number-average molecular weight and weight-average molecular weight (M have been measured
nAnd M
w), for example, use Model 510 gel permeation chromatographs (Water Associates, Inc., Milford, MA), it is equipped with high pressure liquid chromatography pump (high-pressure liquid chromatographicpump), Waters 486 UV detectors and Waters 2410 differential refractive index detectors.Use tetrahydrofuran (THF) (THF), N, dinethylformamide (DMF) or N,N-dimethylacetamide (DMAc) are as elutriant (1.0mL/min).Polystyrene or polymethylmethacrylate standard substance with narrow molecular weight distributions are used for calibration.
[0083] as used herein, term " amino acid " and " a-amino acid " are meant and contain amino, carboxyl and side chain R group R for example defined herein
3The compound of group.As used herein, term " biological a-amino acid (biological α-amino acid) " is meant the amino acid (one or more) that is used in synthesizing, and is selected from phenylalanine, leucine, glycine, L-Ala, Xie Ansuan, Isoleucine, methionine(Met) or their mixture.
[0084] preparation contains the structural formula (I) of a-amino acid and the method for polymkeric substance (III-VII) in general formula, knows in this area.For example, for the embodiment of the polymkeric substance of structural formula (I), wherein R
4Be merged in the a-amino acid, synthetic for polymkeric substance, have side chain R
3A-amino acid can be converted to two-α by esterification, ω-diamines for example can be by having side chain R
3A-amino acid and glycol HO-R
4The condensation of-OH.Therefore, have reactive α, the diester monomer of omega-amino-group is formed.Then, two-α, ω-diamines enters the polycondensation with diacid such as sebacic acid or its pair Acibenzolar or its two-acyl chlorides, to obtain to have the final polymkeric substance (PEA) of ester bond and amido linkage.Alternatively, for PEUR, use two carbonic acid ester derivatives (formula XII) to replace diacid, wherein R
6As defined above, and R
13Independent of choosing (C wantonly with the one or more replacements in nitro, cyano group, halogen, trifluoromethyl or the trifluoromethoxy
6-C
10) aryl.
Formula (XII)
[0085] more specifically, as being described the synthesizing of unsaturated polyester (carboxylic acid amide esters) class (unsaturated poly (ester-amide) s (UPEAs)) of the biodegradable polymers of structure (I) as mentioned above, wherein
And/or (b) R
4Be-CH
2-CH=CH-CH
2-.There is (a) and (b) under the non-existent situation, (I) R in
4Be-C
4H
8-or-C
6H
12-.There is not and under the situation about (b) existing (I) R at (a)
1Be-C
4H
8-or-C
8H
16-.
[0086] UPEAs can prepare by following solution polycondensation, or: two tosilate of (1) two (a-amino acid) diester---at R
4In comprise at least one two key---with the solution polycondensation of two p-nitrophenyl esters of saturated dicarboxylic acid, or two tosilate of (2) two (a-amino acid) diester---at R
4In do not contain two keys---with the solution polycondensation of the dinitrobenzene phenyl ester of unsaturated dicarboxylic acid, or two tosilate of (3) two (a-amino acid) diester---at R
4In comprise at least one two key---with the solution polycondensation of the dinitrobenzene phenyl ester of unsaturated dicarboxylic acid.
[0087] salt of known tosic acid is used for the synthetic polymkeric substance that contains amino-acid residue.Substitute free alkali with arylsulphonate, reason is that the arylsulphonate of two (a-amino acid) diester easily carries out purifying by recrystallization, and to make amino group in entire operation be the inert ammonium tosylate.In polycondensation, nucleophilic amino appears easily by the adding of organic bases such as triethylamine, so polymer product obtains with high yield.
[0088] two p-nitrophenyl esters of unsaturated dicarboxylic acid can be synthetic from p-NP and unsaturated dicarboxylic acid muriate (unsaturated dicarboxylic acid chloride), for example by triethylamine and p-NP are dissolved in the acetone, under-78 ℃, follow stirring dropwise to add the unsaturated dicarboxylic acid muriate, and pour in the water, be settled out product.The suitable chloride of acid (acid chlorides) that is used for this purpose comprises the muriate of fumaric acid, toxilic acid, methylfumaric acid, citraconic acid, propene dicarboxylic acid, methylene-succinic acid, vinyl-butane bisgallic acid and 2-propenyl-butane bisgallic acid.
[0089] the diaryl sulfonate of two (a-amino acid) diester can so prepare: mix a-amino acid, to aryl sulfonic acid (for example tosic acid monohydrate) and saturated or unsaturated diol in toluene, be heated to reflux temperature, emit up to water and to reach minimum, cooling then.The unsaturated diol that can be used for this purpose comprises: for example,
2-butylene-1,3-two is pure and mild 1,18-Linolenic Acid-alkene-glycol.
Saturated two tosilate of saturated two p-nitrophenyl esters of dicarboxylic acid and two (a-amino acid) diester can be by United States Patent (USP) the 6th, 503,538 B1 numbers described preparations.
[0090] as the synthesizing of unsaturated polyester (carboxylic acid amide esters) class (UPEAs) of the biodegradable polymers of structure (I) will be described now as mentioned above.Can with United States Patent (USP) the 6th, 503,538 B1 numbers compound (VII) similarly mode prepares the have structure UPEA of (I), the just R of 6,503,538 (III)
4And/or the R of 6,503,538 (V)
1Be aforesaid C
2-C
20Alkenylene.React following carrying out: for example, anhydrous triethylamine is at room temperature added 6,503,538 described (III) and (IV) and 6,503,538 described (V) is at anhydrous N, in the mixture in the N-N,N-DIMETHYLACETAMIDE, be warming up to 80 ℃ and stirred 16 hours then, then reaction soln be cooled to room temperature, use alcohol dilution, pour in the water, isolating polymer washes the polymkeric substance of separating with water, drying under reduced pressure is to about 30 ℃, and subsequent purificn is to the test (negativetest) that is negative for p-NP and tosilate.Preferred reactant (IV) is the tosilate of Methionin benzyl ester, benzyl ester protecting group is preferably removed from (II), to give biological degradability, but it should be as at United States Patent (USP) the 6th, 503, removed by hydrogenolysis like that among 538 the embodiment 22, reason is two keys of the saturated expectation of hydrogenolysis meeting; On the contrary, should carbobenzoxy be converted into acidic group by keeping undersaturated method.Alternatively; the Methionin reactant can be protected with the protecting group that is different from benzyl; this protecting group is in the end removed in the product easily; and maintenance undersaturated condition; for example the Methionin reactant is used tertiary butyl protection (being that reactant can be the tert-butyl ester of Methionin); and by using acid-treated product (II), this tertiary butyl can be converted into H, keeps unsaturated simultaneously.
[0091] passes through with two (L-phenylalanine) 2-butylene-1, the tosilate of 4-diester replaces 6,503, (III) among 538 embodiment 1, perhaps by replacing 6 with FUMARIC ACID TECH GRADE two p-nitrophenyl esters, 503, (V) among 538 embodiment 1, perhaps by using (L-phenylalanine) 2-butylene-1, the tosilate of 3-diester replaces 6,503, III among 538 embodiment 1 and also usefulness FUMARIC ACID TECH GRADE two p-nitrophenyl esters replacement 6, (V) among 503,538 embodiment 1 provides the working example of the compound with structural formula (I).
[0092] in having structural formula (I) or unsaturated compound (III), has following item: utilize phosphinylidyne diimidazole or suitable carbodiimide as condensing agent, can connect amino oxygen base group, for example the amino TEMPO of 4-.Peptide antigen as described herein, adjuvant and peptide antigen/adjuvant conjugate can be connected by two key functionality.Can give wetting ability by being bonded to polyethyleneglycol diacrylate.
[0093] aspect another, considers that the polymkeric substance that is used to form vaccine delivery system of the present invention comprises: at United States Patent (USP) 5,516,881,6,476,204,6,503,538 and at U. S. application 10/096,435,10/101,408,10/143,572 and 10/194, described in 965 those, the full content of they each is incorporated in this as a reference.
[0094] each monomer of biodegradable PEA, PEUR and PEU polymkeric substance and multipolymer can contain up to two amino acid, and each polymer molecule contains a plurality of amino acid, and preferably has the weight-average molecular weight of scope between 10,000 to 125,000; The logarithmic viscosity number that these polymkeric substance and multipolymer generally have under 25 ℃, by standard visicosity measure method mensuration, is in 0.3 to 4.0 scope, for example in 0.5 to 3.5 scope.
[0095] considers that the polymkeric substance that is used in the practice of the present invention can synthesize by several different methods well known in the art.For example, tributyl tin (IV) catalyzer generally is used to form polyester, such as poly-ε-caproic acid lactone, poly-glycollide, polylactide and analogue.Yet, should be appreciated that multiple catalyzer can be used to form the polymkeric substance that is suitable in the present invention's practice.
[0096] considers that the PEA and the PEUR polymkeric substance that are used in the practice of the present invention can synthesize by several different methods well known in the art.For example, tributyl tin (IV) catalyzer generally is used to form polyester, such as poly-ε-caproic acid lactone, poly-glycollide, polylactide and analogue.Yet, should be appreciated that multiple catalyzer can be used to form the polymkeric substance that is suitable in the present invention's practice.
[0097] consider that this polycaprolactone (poly (caprolactones)) that uses has following exemplary configurations formula (XIII):
Formula (XIII)
[0098] consider that the poly-glycollide of using (poly (glycolides)) has following exemplary configurations formula (XIV):
Formula (XIV)
[0099] consider that the polylactide (poly (lactides)) that uses has following exemplary configurations formula (XV):
Formula (XV)
[0100] the suitable rac-Lactide/ε-caproic acid lactone copolymers that comprises amino oxygen (aminoxyl) part is exemplary synthetic as described below.The first step comprise rac-Lactide and ε-caproic acid lactone in the presence of benzylalcohol, do the copolymerization of catalyzer with stannous octoate, to form the polymkeric substance of structural formula (XVI).
Formula (XVI)
[0101] hydroxy-end capped then polymer chain can be used the maleic anhydride end-blocking, has the polymer chain of structural formula (XVII) with formation:
Formula (XVII)
[0102] in this, 4-amino-2,2,6,6-tetramethyl piperidine-1-oxygen can react with carboxylic end group, and is so that pass through the amido linkage that the reaction between 4-amino and the carboxylic end group produces, that the amino oxygen part is covalently bound to multipolymer.Alternatively, the end capped multipolymer of maleic acid can so that other carboxylic moiety to be provided, be used for the more connection subsequently of polyamino oxygen groups with polyacrylic acid grafted.
[0103] is being used for unsaturated compound PEU, that have structural formula (VII), have following: utilize phosphinylidyne diimidazole or suitable carbodiimide as condensing agent, can connect group, for example the amino TEMPO of 4-with amino amino oxygen (N-oxide compound) base that replaces.Randomly, additional bioactive agents and analogue as described herein can be connected by two keys.
[0104] for example, the high molecular hypocrystalline PEU that the present invention has structural formula (VI) can utilize phosgene to be able to interfacial preparation as the parents' electronics monomer in the chloroform/water system, shown in reaction scheme (2) below:
Diagram (2)
[0105] contain L-Methionin ester and have structural formula (VII) the copolyesters urea (copoly (ester ureas), PEUs) can be undertaken by similar diagram (3):
Diagram (3)
[0106] 20% phosgene (ClCOCl) (high toxicity) toluene solution, (Fluka Chemie, GMBH, the Buchs that for example are obtained commercially, Switzerland), can use trichloromethylchloroformate (superpalite) or triphosgene (two (trichloromethyl) carbonic ether) to replace.The phosphinylidyne diimidazole that toxicity is littler also can be used as parents' electronics monomer, replaces phosgene, trichloromethylchloroformate or triphosgene.
PEU synthetic general method
[0107] for obtaining high molecular PEU, it is necessary using the refrigerative monomer solution.For example, to two (the a-amino acid)-α in 150mL water, ω-alkylidene group diester (bis (ce-amino acid)-α, the suspension of two P-TOLUENE SULFO ACID 99's salt ω-alkylene diester) adds anhydrous sodium carbonate, at room temperature stir about is 30 minutes, and be cooled to 2-0 ℃, form first solution.Abreast, second solution of phosgene in chloroform is cooled to about 15-10 ℃.First solution is placed in the reactor that is used for interfacial polycondensation, and second solution is added rapidly at once and about 15 minutes of vigorous stirring.Then, chloroform layer is separated, through anhydrous Na
2SO
4Drying, and filter.Resulting solution is stored, and is standby.
[0108] all prepared exemplary PEU polymkeric substance all are to obtain with chloroformic solution, and these solution are stable between the shelf lives.Yet, some polymkeric substance, for example 1-Phe-4 is being insoluble in chloroform after the separation.For overcoming this problem, by watering slick hydrophobic surface, and make chloroform evaporated to doing, can from chloroformic solution, isolate polymkeric substance.Do not need to be further purified resulting PEU.Summarized in the yield of the PEU that obtains by present method and the characteristic table 1 in this article.
The general method of preparation porous PEU
[0109] the preparation method of PEU polymkeric substance that contains a-amino acid in general formula is described now.For example, for the embodiment of formula (I) or polymkeric substance (II), a-amino acid can be converted to two-(a-amino acid)-α, and omega-diol-diester monomer is for example by a-amino acid and glycol HO-R
1The condensation of-OH.Therefore, ester bond is formed.Then, the acyl chlorides of carbonic acid (phosgene, trichloromethylchloroformate, triphosgene) is added into and two-(a-amino acid)-α, and the polycondensation of two P-TOLUENE SULFO ACID 99's salt of ω-alkylidene group diester is to obtain to have the final polymkeric substance of ester bond and urea key.
[0110] by two-(a-amino acid)-α, the interface solution condensation of two P-TOLUENE SULFO ACID 99's salt of ω-alkylidene group diester can prepare unsaturated PEU, wherein at R
1In contain at least one two key.The unsaturated diol that can be used for this purpose comprises, 2-butylene-1 for example, and 4-two is pure and mild 1,18-Linolenic Acid-alkene-glycol.Before the reaction in alkaline aqueous solution, for example before the reaction in sodium hydroxide solution, unsaturated monomer can be dissolved.Then, the aqueous solution is with the organic solvent layer of the monomer that contains equimolar amount, dimer and tripolymer phosgene chloroform for example, externally cooling down, by violent stirring.Thermopositive reaction is carried out rapidly, and produces polymkeric substance, and its (in most of the cases) remains dissolved in the organic solvent.Organic layer used water washing several times are used anhydrous sodium sulfate drying, filter, and evaporation.Yield is the unsaturated PEU of about 75%-85%, can be by dry in a vacuum, for example under about 45 ℃.
[0111] for obtaining porous, powerful material, can adopt general method described below, preparation L-leucine base PEU, for example 1-L-Leu-4 and 1-L-Leu-6.When being applied to L-Phe base PEU, the success ratio that this type of step forms the powerful material of porous is lower.
[0112] reaction soln or the milk sap (about 100mL) of PEU in chloroform, as obtaining after the interfacial polycondensation, under agitation, dropwise be added in glass beaker 1, in about 80 ℃ of-85 ℃ of water of 000mL; Preferably, beaker dimethyldichlorosilane(DMCS) hydrophobization is to reduce PEU adhering to walls of beaker.Polymers soln is dispersed into droplet in water, and chloroform evaporates quite tempestuously.Little by little, along with chloroform is evaporated, droplet is merged into the piece of similar tar closely, and it changes into heavy-gravity rubbery state product.This rubbery state product is shifted out from beaker, and puts into the cylindrical glass test tube of hydrophobization, and it was descended about 24 hours at about 80 ℃ by thermostatic control.Then, test tube shifts out from this thermostatted, cool to room temperature, and broken, to obtain polymkeric substance.Resulting porous bar is placed in the vacuum drier, under reduced pressure, under about 80 ℃, is dried about 24 hours.Any method of the acquisition porous polymeric materials that is known in the art in addition, also can be used.
[0113] character of the high molecular porous PEU by method for preparing produces the result who summarizes as table 1.
The characteristic of table 1 formula (VI) and PEU polymkeric substance (VII)
| PEU * | Yield (%) | η red a) [dL/g] | M w b) | Mn b) | M w/M n b) | Tg c) [℃] | Tm c) [℃] |
| 1-L-Leu-4 | 80 | 0.49 | 84000 | 45000 | 1.90 | 67 | 103 |
| 1-L-Leu-6 | 82 | 0.59 | 96700 | 50000 | 1.90 | 64 | 126 |
| 1-L-Phe-6 | 77 | 0.43 | 60400 | 34500 | 1.75 | - | 167 |
| [1-L-Leu-6] 0.75- [1-L-Lys(OBn)] 0.25 | 84 | 0.31 | 64400 | 43000 | 1.47 | 34 | 114 |
| 1-L-Leu-DAS | 57 | 0.28 | 55700 d) | 27700 d) | 2.1 d) | 56 | 165 |
*The PEUs of general formula (VI), wherein,
1-L-Leu-4：R
4＝(CH
2)
4，R
3＝i-C
4H
9
1-L-Leu-6：R
4＝(CH
2)
6，R
3＝i-C
4H
9
1-L-Phe-6：R
4＝(CH
2)
6，R
3＝-CH
2-C
6H
5.
1-L-Leu-DAS:R
4=1,4:3,6-two anhydrous sorbyl alcohols, R
3=i-C
4H
A)Under the concentration of 25 ℃ and 0.5g/dL, in DMF, measure reduced viscosity (reduced viscosity)
B)In DMF, carry out gpc measurement, (PMMA)
C)From second heating curve, obtain Tg (10 ℃/minute of heating rate) from dsc measurement
D)In DMAc, carry out gpc measurement, (PS)
[0114] tensile strength of the illustrative synthetic PEU of measurement, the result is summarized in table 2.Use dumb-bell shape PEU film (4 x 1.6cm), obtain the stretching strength measurement value, this PEU film is from the chloroformic solution formation of being cast, mean thickness is 0.125mm, and carrying out Elongation test being combined with on the tensile strength instrument (ChatillonTDC200) of PC (Personal Computer), this PC uses Nexygen FM software (Amtek, Largo, FL), carry out with the pinblock speed of 60mm/min.Described in this article example can expect to have following mechanical property:
Second-order transition temperature be about 30 ℃ to about 90 ℃ scope, for example about 35 ℃ to about 70 ℃ scope;
2. mean thickness will have the tensile stress at yield of about 20Mpa to about 150Mpa for the polymeric film of about 1.6cm, and for example about 25Mpa is to about 60Mpa;
3. mean thickness will have about 10% to about 200% elongation, for example about 50% to about 150% for the polymeric film of about 1.6cm; With
4. mean thickness will have about 500MPa to the interior Young's modulus of about 2000MPa scope for the polymeric film of about 1.6cm.Following table 2 has been summarized the character of the exemplary PEU of the type.
The mechanical property of table 2 PEUs
| The polymkeric substance title | Tg a) [℃] | Tensile stress at yield (MPa) | Elongation (%) | Young's modulus (MPa) |
| 1-L-Leu-6 | 64 | 21 | 114 | 622 |
| [1-L-Leu-6] 0.75- [1-L-Lys(OBn)] 0.25 | 34 | 25 | 159 | 915 |
[0115] the various compositions of vaccine delivery compositions of the present invention can exist with the ratio of the various scopes of broadness.For example, polymer repeat unit: antigen typically is used to the ratio of 50:1 with 1:50, for example 1:10 to 10:1, about 1:3 to 3:1 or about 1:1.Yet other ratio may be more suitably for specific purposes, for example when specific antigen not only had been difficult to mix in the particular polymers, but also when having lower immunogenicity, in this case, higher peptide antigen relative quantity needs.
[0116] in some embodiments, the vaccine delivery compositions of the present invention of Miao Shuing in this article, can provide with particle, it has peptide antigen/adjuvant conjugate, or antigen---contain or do not contain adjuvant, they or mix (dispersion) in particle or be attached on the polymers functionality with physical form, randomly utilize joint, this adopts any the carrying out in well known in the art and some as described herein technology.The particulate size is designed have for example about 10 nanometers to about 1000 microns scope, or about 10 nanometers to be to the interior mean diameter of about 10 microns scope so that be suitable for the APC picked-up.Randomly, described particle further comprises the thin coverture of polymkeric substance, to help their biological degradation of control.Usually, the every polymer molecule of this class particle comprises about 5 to about 150 peptide antigens.
[0117] be used in the polymkeric substance of vaccine delivery compositions of the present invention, for example biological degradation by enzymatic action, takes place on the surface in PEA, PEUR and PEU polymkeric substance.Therefore, polymkeric substance, for example their particle gives antigen to object with controlled release speed, and described speed is specific in the period of an elongated segment and is constant.In addition, because PEA, PEUR and PEU polymkeric substance are by lytic enzyme decomposition in vivo under the situation that does not produce unfavorable by product, so vaccine delivery compositions of the present invention is non-inflammatory basically.As used herein, " biodegradable (biogradable) " is meant that as being used for describing the polymkeric substance of vaccine composition of the present invention polymkeric substance can resolve into product harmless in the normal function of body.In one embodiment, whole vaccine delivery compositions is biodegradable.Preferred biodegradable polymers has provides the hydrolyzable of biodegradable ester bond, and normally mainly uses the end capped chain of amino group.
[0118] as used herein, " dispersive (dispersed) " be meant as disclosed herein peptide antigen or adjuvant in polymkeric substance by dispersion, mixing, dissolving, homogenizing and/or covalent attachment (" dispersive " or load), described polymkeric substance can form or can not form particle.
[0119] need not chemistry although peptide antigen and optional adjuvant can be dispersed in the polymeric matrix is connected on the polymer support, but antigen and/or antigen-adjuvant conjugate can pass through a variety of suitable functional groups, is covalently bound on the biodegradable polymers also to be considered.For example, when biodegradable polymers is polyester, the carboxyl end of the chain can be used to antigen or adjuvant on complementary portion reaction, for example hydroxyl, amino, thio group or analogue.A variety of suitable reagents and reaction conditions are disclosed, for example in March ' sAdvanced Organic Chemistry, Reactions, Mechanisms, and Structure, Fifth Edition, (2001); With Comprehensive Organic Transformations, Second Edition, among the Larock (1999).
[0120] in other embodiments, antigen and/or adjuvant can be connected in structure (I) or the polymkeric substance (III-VII) any one by amido linkage, ester bond, ehter bond, amino, ketonic bond, thioether bond, sulfinyl, alkylsulfonyl, disulfide linkage.Such key can utilize synthetic method as known in the art, is formed from suitable functionalized raw material.
[0121] for example, in one embodiment, polymkeric substance can be connected with peptide antigen or adjuvant by the end or the side chain carboxyl group group (for example COOH) of this polymkeric substance.Particularly, the compound of structure III, V and VII can react with antigenic amido functional group of peptide or hydroxy functional group, is connected with the antigenic biodegradable polymkeric substance of peptide to provide to have respectively by amido linkage or carboxylic acid ester bond.In another embodiment, the carboxyl of polymkeric substance can be converted into carboxylic acid halides, acyl group acid anhydrides/" mixing " acid anhydride or active ester.In other embodiments, the freedom-NH of polymer molecule
2End can be by acidylate, with guarantee peptide antigen will be only by polymkeric substance carboxyl in conjunction with and be not joined to the free end of polymkeric substance.For example, vaccine delivery compositions of the present invention as described in this article, can for example be used acid anhydrides RCOOCOR acidylate, wherein R=(C by the N-terminal free amino group by the PEA of acidylate, PEUR or PEU preparation
1-C
24) alkyl, to guarantee: biologically active agent will only pass through the carboxyl combination of polymkeric substance, and be not joined to the free end of polymkeric substance.
[0122] alternatively, peptide antigen or adjuvant can be connected to polymkeric substance by linkers, for example, are described in the joint in the structural formula (VIII-XI).Really, for improving the surface hydrophobicity of biodegradable polymers, for improving the accessibility of biodegradable polymers to enzyme activation, and for improving the release profiles (release profile) of biodegradable polymers, can utilize joint, indirectly peptide antigen and/or adjuvant are connected on the biodegradable polymers.In some embodiments, linker compounds comprises: polyoxyethylene glycol, the molecular weight (M that it has
W) be about 44 to about 10,000, preferably from 44 to 2000; Amino acid, for example Serine; Polypeptide with 1 to 100 repeating unit; With any other suitable low-molecular weight polymer.Joint is generally separated about 5 dusts with peptide antigen and polymkeric substance, up to about 200 dusts.
[0123] still in further embodiment, joint is the divalent radical of formula W-A-Q, and wherein A is (C
1-C
24) alkyl, (C
2-C
24) thiazolinyl, (C
2-C
24) alkynyl, (C
3-C
8) cycloalkyl or (C
6-C
10) aryl, and W and Q each independently be-N (R) C (=O)-,-C (=O) N (R)-,-OC (=O)-,-C (=O) O ,-O-,-S-,-S (O) ,-S (O)
2-,-S-S-,-N (R)-,-C (=O)-, wherein each R independently is H or (C
1-C
6) alkyl.
[0124] as being used to describe above-mentioned joint, term " alkyl (alkyl) " refers to the straight or branched alkyl, comprises methyl, ethyl, n-propyl, sec.-propyl, normal-butyl, isobutyl-, the tertiary butyl, n-hexyl and analogue.
[0125] as used herein, " thiazolinyl (alkenyl) " that is used to describe above-mentioned joint refers to the straight or branched alkyl with one or more carbon-to-carbon double bonds.
[0126] as used herein, " alkynyl (alkynyl) " that is used to describe above-mentioned joint refers to the straight or branched alkyl with at least one carbon-to-carbon triple bond.
[0127] as used herein, " aryl (aryl) " that is used to describe above-mentioned joint refers to the aromaticity group with 6 to 14 carbon atoms.
[0128] in some embodiments, joint can be have about 2 to about 25 amino acid whose polypeptide.The suitable peptide that consider to use comprises: poly-L-Lysine, poly--L-L-glutamic acid, poly-L-aspartic acid, poly-L-histidine, poly--the L-ornithine, poly--the L-Threonine, poly--L-tyrosine, poly--the L-leucine, poly-L-Lysine-L-phenylalanine, poly--the L-arginine, poly-L-Lysine-L-tyrosine, and analogue.
[0129] in one embodiment, peptide antigen can the covalent cross-linking polymkeric substance, and promptly antigen is attached on the more than one polymer molecule.This covalent cross-linking can carry out under the situation that has or do not have extra polymkeric substance-antigen joint.
What [0130] the peptide antigen molecule also can be by between single macromolecular two parts is covalently bound, forms the intramolecularly bridge.
[0131] see through potential nucleophile on the protection antigen skeleton, and only stay next reactive group to combine with polymkeric substance or polymeric joint structure, can prepare the simple linear polymer peptide conjugate.Go protection (for example Boc and Fmoc chemistry) method according to peptide well known in the art, go protection.
[0132] in an embodiment of the invention, peptide antigen exists with contrapositive (retro-inverso) or the trans peptide of Partial Inverse.
[0133] in other embodiments, peptide antigen mixes with the photic crosslinkable form of polymkeric substance in matrix, and after crosslinked, the size that this material is disperseed (pulverizing) to become can engulf, i.e. 0.1-10 μ m.
[0134] joint can at first be connected to polymkeric substance, perhaps to peptide antigen or adjuvant.Between synthesis phase, joint can be in the form of not protecting, and perhaps utilizes numerous protecting groups that those of ordinary skills know and is in protected form.Under the situation of protection joint, the not protection end of joint can at first be connected to polymkeric substance or peptide antigen.Then, utilize Pd/H
2The acid or the basic hydrolysis of hydrogenolysis, gentleness, any other routine perhaps known in the art is gone guard method, and protecting group can be gone protection.Then, go to protect joint can be connected to peptide antigen, adjuvant, or adjuvant/peptide antigen conjugates.
[0135] exemplary synthetic (molecule wherein to be connected is an amino oxygen) according to biodegradable polymkeric substance of the present invention is described below.Polyester can with the group with amino N-oxide compound free radical (amino oxygen) that replaces, 4-amino-2 for example, 2,6,6-tetramethyl piperidine-1-oxygen, at N, there is reaction down in N '-phosphinylidyne diimidazole, so that replace carboxy moiety on the polyester chain end, make the carbon covalent attachment of amino part and the carbonyl residue of the carboxylic group of polymkeric substance with the amido linkage that contains the amino amino oxygen base radical that replaces.N, N '-phosphinylidyne diimidazole or suitable carbodiimide with the hydroxylic moiety in the carboxylic group on the polyester chain end, are converted into and amino oxygen 4-amino-2,2,6 for example, the intermediate product part of 6-tetramethyl piperidine-1-oxygen reaction.Generally the molar ratio range by reactant and polyester is 1:1 to 100:1, uses the amino oxygen reactant.N, the mol ratio of N '-phosphinylidyne diimidazole and amino oxygen is preferably about 1:1.
[0136] type reaction is as follows.Polyester is dissolved in the reaction solvent, and easily reacts under the dissolved temperature being used for.Reaction solvent can be that polyester will be dissolved in any solvent wherein.When polyester is polyglycolic acid or poly-(glycollide-L-rac-Lactide) (monomer mole ratio of oxyacetic acid and L-lactic acid is greater than 50:50), highly refined (99.9+% purity) methyl-sulphoxide 115 ℃ to 130 ℃ down or methyl-sulphoxide (DMSO) at room temperature, be suitable for dissolving this polyester.When polyester be poly--during L-lactic acid, poly-DL-lactic acid, or during poly-(glycollide-L-rac-Lactide) (have 50:50 or less than the oxyacetic acid of 50:50 and the monomer mole ratio of L-lactic acid), tetrahydrofuran (THF), methylene dichloride and chloroform, under room temperature to 50 ℃, be suitable for dissolving described polyester.
Polymkeric substance/peptide antigen connects
[0137] in one embodiment, the polymkeric substance that is used to make vaccine delivery compositions of the present invention as described herein has at least one and is directly connected to peptide antigen on the polymkeric substance.The residue of polymkeric substance can be connected with the antigenic residue of these one or more peptides.For example, the direct antigenic residue of connection peptides of polymkeric substance residue.Each of polymkeric substance and peptide antigen can have an open valency (open valence).Alternatively, more than one peptide antigen, a plurality of peptide antigen or from the cause a disease antigenic mixture of peptide of primary object of difference can directly be connected with polymkeric substance.Yet because the antigenic residue of each peptide can be connected with the corresponding residue of polymkeric substance, the number of the antigenic residue of described one or more peptides can be corresponding to the valent number of the opening on the polymer residue.
[0138] as used herein, " residue of polymkeric substance (residue of a polymer) " refers to the radical of polymkeric substance, and it has one or more open valencys.Any synthetic feasible atom, a plurality of atom or the functional group (for example on polymer backbone or side group) of polymkeric substance of the present invention can be removed, so that open valency to be provided, condition is that biological activity is retained substantially when this radical is connected to the antigenic residue of peptide.In addition, any synthetic feasible functional group's (for example carboxyl) can be formed at (for example on polymer backbone or side group) on the polymkeric substance, so that open valency to be provided, condition is that biological activity is retained substantially when this radical is connected on the antigenic residue of peptide.Based on required connection, those of ordinary skills can select suitably functionalized raw material, and raw material can be come out by polymkeric substance reasoning of the present invention with methods known in the art.
[0139] as used herein, " structural formula (
*) compound residue (residue of a compound ofstructural formula (
*)) " referring to formula (I) and (III-VII) radical of compound as described herein, it has one or more open valencys.Any synthetic feasible atom, a plurality of atom or the functional group (for example on polymer backbone or side group) of this compound can be removed, so that open valency to be provided, prerequisite is that biological activity is retained substantially when radical is connected on the antigenic residue of peptide.In addition, any synthetic feasible functional group's (for example carboxyl) can be by in formula (I) and (III-VII) (for example on polymer backbone or side group) formation on the compound, so that open valency to be provided, prerequisite is that biological activity is retained substantially when radical is connected to the antigenic residue of peptide.Based on required connection, those of ordinary skills can select suitably functionalized raw material, raw material can with methods known in the art by formula (I) and (III-VII) the compound reasoning come out.
[0140] for example, the antigenic residue of peptide, can pass through acid amides (for example-N (R) C (=O)-or-C (O) N (R)-), ester (for example-OC (=O)-or-C (=O) O-), ether (for example-O-), amino (for example-N (R)-), ketone (for example-C (=O)-), thioether (for example-S-), sulfinyl (for example-S (O)-), sulphonyl (for example-S (O)
2-), (for example-S-S-) or direct (for example C-C key) key, and being connected with the residue of the structural formula (I) and the compound of (III-VII), wherein each R independently is H or (C to two sulphur
1-C
6) alkyl.Utilize synthesis step known in the art, such key can form from suitably functionalized raw material.Key based on expectation, those of ordinary skills can select suitably functionalized raw material, utilize methods known in the art, raw material can derive from structural formula (I) and (III-VII) in the residue of any one compound, and from the given residue of peptide antigen or adjuvant.The residue of peptide antigen or adjuvant, can be connected directly to be arranged in structural formula (I) and (III-VII) any synthetic on the residue of the compound shown in any one feasible position.In addition, the present invention also provides compound, peptide antigen or adjuvant biologically active agent that it has an above residue, be directly connected to structural formula (I) and (III-VII) in any one compound on.
The antigenic number of peptide that [0141] can be connected with polymer molecule generally depends on the molecular weight of this polymkeric substance.For example, for the compound of structural formula (I) or formula (III), wherein n is about 5 to about 150, preferred about 5 to about 70, by making the end group reaction of peptide antigen and polymkeric substance, can directly be connected with polymkeric substance (being its residue) up to about 150 peptide antigen (being its residue).In unsaturated polymer, peptide antigen also can be directly and the reaction of the two keys (or triple bond) in the polymkeric substance.
[0142] PEA described herein, PEUR and PEU polymkeric substance absorb water (on polymeric film, the water of 5 to 25% w/w absorbs) easily, make hydrophilic molecules diffuse through them easily.This characteristic makes PEA, PEUR and PEU polymkeric substance be suitable for use as the covering tegillum on the particle, with sustained release speed.Water absorbs and also to strengthen polymkeric substance and based on the biocompatibility of the vaccine delivery compositions of this base polymer.In addition, because the water-wet behavior of PEA, PEUR and PEU polymkeric substance, when being transferred in vivo, particle becomes and has viscosity and gather together, especially in vivo under the temperature.Therefore, when subcutaneous or intramuscularly during with localized delivery, for example annotate or Needleless injection (needle-less injection) by hypodermic needle, polymer beads forms polymer depot naturally.The mean diameter scope is about 1 micron particle to about 100 microns size, has not allow round-robin size in vivo; Such particle is suitable for forming in vivo this type of polymer depot.Alternatively, for oral administration, gi tract (GI tract) can be stood much bigger particle, for example about 1 micron particulate that arrives big to about 1000 microns mean diameters.
[0143] for example, usually, polymer depot will be degraded in for some time, and this section period is selected from about twenty four hours, about seven days, about 30 days, about 90 days or longer.Longer time span is particularly suitable for providing implantable vaccine delivery compositions, and it has eliminated the duplicate injection vaccine to obtain the needs of suitable immunne response.
[0144] is suitable for the particle of vaccine delivery compositions of the present invention, can utilizes the immiscible solvent technology to be prepared.Generally speaking, these methods require the milk sap of two kinds of immiscible liquids of preparation.Single emulsion method can be used to prepare polymer beads, and it mixes hydrophobic adjuvant and peptide antigen, or their conjugate.In single emulsion method, will be impregnated in the particulate molecule at first in solvent with mixed with polymers, then in the aqueous solution with surface stabilizer---for example tensio-active agent, emulsification together.In this way, the polymer beads with hydrophobic adjuvant, peptide antigen or adjuvant/peptide antigen conjugates is formed, and suspends in the aqueous solution; In this aqueous solution, the hydrophobic conjugate in the described particle---under situation in the aqueous solution is advanced in elution not yet in effect, be stable, but this quasi-molecule will be advanced body tissue by elution, muscle tissue for example.
[0145] most of biotechnological formulations comprise antigenic synthetic peptide, are hydrophilic.Two emulsion methods can be used for preparing and have liquid or the hydrophilic adjuvant/peptide antigenic polymer particle that is dispersed in wherein.In two emulsion methods, be dissolved in liquid or hydrophilic adjuvant/peptide antigen in the water, at first emulsification in polymers soln; And whole milk sap is placed in the water, with emulsification once more, forms the particle with outer polymer coating, and at the liquid adjuvant/peptide antigen of granule interior.Tensio-active agent can be used on and carries out in two kinds of methods of emulsive, to prevent particle aggregation.The solvent that is used as PEA and PEUR polymkeric substance with the chloroform or the methylene dichloride (DCM) of water immiscibility, but afterwards, in preparation, use methods known in the art to remove and desolvate.
[0146] yet, have the peptide antigen or the adjuvant of low water solubility for some, these two kinds of emulsion methods have limitation.In context, " low water solubility (low water solubility) " refers to that such promoting agent---hydrophobicity is than true lipophilic drugs, and for example Taxol is lower; But its wetting ability is than real water soluble drug, and for example many biotechnological formulations are lower.The intermediate compound of these types is for high load and firm ground materialization enter in single emulsion particle, and wetting ability is too high; But for for high load in two emulsions and stability, hydrophobicity is too high.Under these circumstances, by three emulsion process technologies, polymer layer is coated on by polymkeric substance and has on the particle of drugs of low aqueous solubility preparation.This method provides low relatively medicament contg (～10% w/w), but structural stability and controlled drug release rate are provided.
[0147] by promoting agent is mixed in the polymers soln; And in the aqueous solution, use tensio-active agent or lipid, for example two (hexadecanoyl) phosphatidylcholine (di-(hexadecanoyl) phosphatidylcholine, DHPC; The short chain derivative of neutral grease), this mixture of emulsification prepares first milk sap.By this way, the particle that contains promoting agent is formed and is suspended in the water, to form first emulsion.By adding in the polymers soln first emulsion and this mixture of emulsification, second emulsion is formed, and is formed in the polymers soln so that wherein contain polymer particulate water droplet.Water and tensio-active agent or fat will separate particle, and dissolve this particle in polymers soln.Then, by second emulsion is added in the water contain tensio-active agent or fat and this mixture of emulsification being formed on the final particle in the water, thereby the 3rd emulsion is formed.The resulting granules structure as shown in Figure 1, will have one or more particles, it adds that by polymkeric substance the peptide antigen prepd forms, contain or do not contain adjuvant in the center, by water and surface stabilizer---for example tensio-active agent or fat surround, and are coated with the straight polymer shell.Surface stabilizer and water will prevent that the solvent in the polymeric coating from contacting with particle in this coating, and dissolve them.
[0148] for increasing promoting agents by triple emulsion processes---the capacity value of peptide antigen or adjuvant for example, have the promoting agent of low water solubility can be in first emulsion with surface stabilizer dressing in addition, and need not polymer coating and need not the described promoting agent of dissolving in water.In this first emulsion, water, surface stabilizer and promoting agent have similar volume respectively, and perhaps the scope of volume ratio is (1 to 3): (0.2 to about 2): 1.In this case, water is used, and is not for the lytic activity agent, but in order under the help of surface stabilizer, to protect promoting agent.Then, two emulsions and three emulsions are produced (Fig. 1) as mentioned above.
[0149] many emulsifying technologies will work in preparation is used for the emulsion of described particle manufacture.Yet the preferred emulsions preparation method utilizes immiscible solvent in water at present.The emulsification program is made up of following: with described dissolution with solvents polymkeric substance, mix with adjuvant/peptide antigen molecule (one or more), add in the entry, stir with mixing tank and/or ultrasonator then.By the control stirring velocity, and/or the concentration of polymkeric substance, adjuvant/peptide antigen molecule (one or more) and surface stabilizer, granular size can be controlled.By regulating the ratio of second emulsion and the 3rd emulsion, coatings thickness is controlled.In above-mentioned any particle formation method, after particle forms, by being coupled on the polymkeric substance in the described particle, thereby make antigen peptide and optional adjuvant can on particle surface, form coatings.
[0150] suitable emulsion stabilizer can comprise nonionic surface active agent, for example mannide monoleate (mannide monooleate), macrodex, 000, Soxylat A 25-7, polyglycol ether and surfactant-like, all these easily from, Sigma Chemical Co. for example, St.Louis, Mo. is commercially available.Described tensio-active agent will exist with about 0.3% to about 10% concentration, and preferred about 0.5% to about 8%, and more preferably from about 1% to about 5%.
[0151] the antigenic rate of release of the adjuvant/peptide of described composition can be controlled by regulating coating thickness, covering the density of antigen number, granular size, structure and the dressing of described particle outside.The density of described dressing can be controlled by being adjusted in the adjuvant/peptide antigen load in the dressing.When described dressing does not contain adjuvant/peptide antigen, polymer coating is the density maximum, and described adjuvant/peptide antigen elution is the slowest by described dressing.In comparison, when adjuvant/when peptide antigen is loaded into described dressing, in case described adjuvant/peptide antigen is gone out by elution, then this dressing begins to become porous from the outside surface of described dressing, and therefore, the adjuvant/peptide antigen that is in the particle center can be with the speed wash-out that increases progressively.The medicine capacity value is high more, and coatings density is low more, and then elution speed is more high.Adjuvant in described dressing/peptide antigen capacity value can be lower than the adjuvant/peptide antigen capacity value within the particle under the dressing externally.Particulate adjuvant/peptide antigen rate of release also can be mixed by the particle that will prepare as mentioned above, have different rates of release and be controlled.
[0152] detailed description of the method for two emulsion polymers of preparation and three emulsion polymers, can be at PierreAutant et al, Medicinal and/or nutritional microcapsules for oral administration, United States Patent (USP) 6,022,562; Iosif Daniel Rosea et al., Microparticle formation and its mechanism insingle and double emulsion solvent evaporation, Journal of Controlled Release (2004) 99:271-280; L.Mu and S.S.Feng, A novel controlled release formulation for theanticancer drug paclitaxel (Taxol): PLGA nanoparticles containing vitamin E (TPGS, J.Control.Release (2003) 86:33-48; Somatosin containing biodegradable microspheresPrepared by a modified solvent evaporation method based on W/O/W-multipleemulsions, Int.J.Pharm. (1995) 126:129-138 and F.Gabor et al., Ketoprofenpoly (d, 1-lactic-co-glycolic acid) microspheres:influence of manufacturingParameters and type of polymer on the release characteristics, J.Microencapsul. (1999) 16 (1): find among the 1-12, each piece of writing is by the complete this paper that incorporates into.
[0153] in the another embodiment of carrying water soluble peptide antigen and/or adjuvant, described particle can be prepared to mean diameter for the nano particle of about 20nm to about 200nm, is used to be transported to the recycle system.By single emulsion process, utilize to be dispersed in wherein---promptly be mixed in the described emulsion or be coupled to the peptide antigen of polymkeric substance as herein described, can prepare described nano particle.Described nano particle also can be provided with the capsule that contains PEA as herein described or PEUR polymkeric substance.Described capsule shape is formed in the water, and the described proteinic water-soluble antigen of optional adjuvant that has is not loaded in the capsule under having the situation of solvent simultaneously.
[0154] more specifically, biodegradable micelle, it is illustrated elaboration in Fig. 2, by the water soluble ion fluidized polymer chain formation that is coupled on the hydrophobic polymer chain.Although the external portion of described micelle mainly is made up of the water soluble ion sections of described polymkeric substance, the hydrophobic sections of described polymkeric substance mainly is distributed in the inside of described micelle and polymer molecule is kept together.
[0155] is used to prepare the biodegradable hydrophobic sections of the described polymkeric substance of micelle, makes by PEA, PEUR or PEU polymkeric substance, as described herein.For strong hydrophobic PEA, PEUR or PEU polymkeric substance, specific examples of such components, for example 1,4:3,6-two anhydrous-two L-leucine esters of D-mountain plough alcohol, or rigidity aromatic diacid resembles α, ω-two (4-carboxyl phenoxy group)-(C
1-C
8) alkane, can be included in the polymer repeat unit.On the contrary, the water-soluble sections of polymkeric substance comprises: the repetition alternate cells of polyoxyethylene glycol, poly-glycosamino glucan or polysaccharide, and at least a ionizable or polar amino acid, wherein said repetition alternate cells has similar basically molecular weight, and the molecular weight of wherein said polymkeric substance is in about 10kD arrives the scope of about 300kD.The molecular weight of this water-soluble sections is high more, and the porosity of micelle is big more, and wherein long chain can high load water-soluble antigen and optional adjuvant.In addition, polyamino acid (polyamino acids) is stronger than single amino acid immunogenicity.
[0156] repeat alternate cells can have scope about 300D extremely in about 700D, similar molecular weight basically.In the embodiment of polymericular weight therein more than 10kD, at least a in the amino acid unit is ionizable, or is selected from Serine, L-glutamic acid, aspartic acid, Methionin and arginic polare Aminosaeren.In one embodiment, the amino acid whose unit of ionizable comprises the block of at least one ionizable polyamino acid, and for example glutamate or aspartate can be included in the described polymkeric substance.Micelle composition of the present invention may further include: the pharmaceutically acceptable water-bearing media with certain pH value; Under this pH value, at least a portion ionizable amino acid that is in the water-soluble sections of polymkeric substance is ionized.
[0157] biodegradable hydrophobic polymer chain is made by PEA, PEUR or PEU polymkeric substance, as described herein.For strong hydrophobic PEA, PEUR or PEU polymkeric substance, composition---for example 1, two (4-carboxylicesters-phenoxy group)-propane (CPP) and/or 1 of 3-, 4:3, two (L-leucine) diester of the two anhydrous hexitols of 6--D-mountain plough alcohol (DAS) can be comprised in the described hydrophobic polymer chain.In comparison, water-soluble chain is made by following compositions: the repeating unit of many polyoxyethylene glycol (PEG), and ionizable amino acid---for example poly-leucine or polyglutamate, wherein the PEG unit has similar molecular weight with ionizable amino acid unit, for example hundreds of kD (be the PEG unit can have in this scope, the molecular weight of any value basically).Yet, the total molecular weight of the water-soluble sections of polymkeric substance, for example, in about 10kD arrives the scope of about 300kD.The molecular weight of this water-soluble sections is high more, and the porosity of micelle is big more, and characteristics are that long chain makes it possible to high load water-soluble antigen and optional adjuvant.In addition, polyamino acid is stronger than single amino acid immunogenicity.
[0158] alive part in the micelle partly is separated from each other in water, and produce to absorb for example space of peptide antigen and optional protein adjuvant of water-soluble antigen.Ionization chain with same type electric charge is with mutually exclusive, and the generation more space.Ionized polymkeric substance also attracts peptide antigen, for matrix provides stability.In addition, after the ionization site is occupied by therapeutical agent, proteinic bonding in the water-soluble outside prevention micelle of micelle and the body fluid.The micelle of the type has very high voidage, can reach 95% of micelle volume, allows high load water-soluble biological preparation, for example peptide antigen and antigen.The granular size scope of micelle is extremely about 200nm of about 20nm, wherein for the circulation in the blood, is preferably about 20nm to about 100nm.
[0159] the antigenic rate of release of the adjuvant/peptide of described composition can be controlled by the density of regulating coating thickness, granular size, structure and dressing.The density of described dressing can be regulated by the adjuvant/peptide antigen capacity value that changes in dressing.When described dressing does not contain adjuvant or peptide antigen, polymer coating is the density maximum, and described adjuvant/peptide antigen elution is the slowest by described dressing.In comparison, when peptide antigen or adjuvant are loaded into described dressing, in case described peptide antigen or adjuvant are gone out by elution, this dressing then, the outside surface from described dressing becomes porous, and, therefore, can be at the promoting agent (one or more) at particle center with the speed that increases by wash-out.The medicine capacity value is high more, and coatings density is low more, and elution speed is high more.The capacity value of the adjuvant in described dressing/peptide antigen can be lower than the antigenic capacity value of adjuvant/peptide under the dressing externally, granule interior.From particulate adjuvant/peptide antigen rate of release, also can mix by the particle with different rates of release that will prepare as mentioned above and be controlled.
[0160] granular size for example by laser light scattering, is used the spectrometer that for example is integrated with helium-neon laser, can be determined.Generally speaking, granular size is at room temperature determined, and comprises the repeatedly analysis (for example 5-10 time) to the consideration sample, to produce the mean value of particle diameter.(scanning electronmicroscopy SEM), also can easily determine granular size to use scanning electronic microscope.In order to do like this, dried particles to be coated with into the thickness of about 100 dusts, to detect with scanning electronic microscope then with gold/palladium mixture sputter.Alternatively, polymkeric substance---perhaps particle form or non-particulate form---can be by directly covalently bound to peptide antigen, rather than peptide antigen is mixed wherein (" load (loading) " or " matrixization (matrixing) ") not having under the situation that chemistry connects, this use in this area known and as hereinafter any of described several method carry out.The peptide antigenic content exists with certain amount usually, and with respect to polymkeric substance, it is expressed as the about 0.1% peptide antigen to about 40% (w/w), the 1% peptide antigen to about 25% (w/w) more preferably, even the more preferably about 2% peptide antigen to about 20% (w/w).The antigenic percentage composition of peptide will depend on desired amount and institute's situation of being treated, as more specifically discussion below.After having prepared---having or the do not have adjuvant---particle or polymer molecule of load of using peptide antigen, composition can be by freeze-drying, and this exsiccant composition can be suspended in the suitable carriers before immunity.
[0161] is included in suitable and the immunogenicity particle that contains peptide antigen and any adjuvant significant quantity in the vaccine delivery compositions, any or polymer segments and can be in time discharges (comprise the polymer depot that forms in vivo those) from described polymer beads; and will depend on usually: for example; concrete polymkeric substance, peptide antigen, adjuvant or polymkeric substance/peptide antigen connects, if present.Usually, can reach about 100% polymer beads or molecule can discharge from polymer depot.Particularly, can reach that it is about 99%, can reach that it is about 75%, can reach that it is about 50%, can reach maybe that it is about 25%, can from polymer depot, be released.Usually influence from the factor of the speed of polymkeric substance release is: the property quality and quantity of polymkeric substance, the type that polymkeric substance/peptide antigen connects and/or polymkeric substance/biologically active agent connects and other Substance Properties and the amount that exists said preparation.
[0162] in case vaccine delivery compositions of the present invention as above is produced, then said composition is used for subsequently mucous membrane or subcutaneous delivery by preparation.Described composition will comprise that usually one or more are suitable for " the pharmaceutically acceptable vehicle or the carrier " of mucous membrane or subcutaneous delivery, as water, salt solution, glycerol, polyoxyethylene glycol, hyaluronic acid, ethanol etc.In addition, auxiliary substance is wetting agent or emulsifying agent, pH buffer substance and similar substance for example, can exist in examples of such carriers.
[0163] for example, will comprise usually with lung's preparation in the nose: neither cause stimulating nasal mucosa significantly not disturb the carrier of fibre function again.Thinner for example water, aqueous salt or other known substance can be used with the invention of this theme.This nasal preparation also can contain sanitas, for example, but is not limited to butylene-chlorohydrin and Benzalkonii Chloridum.Tensio-active agent can exist, to strengthen the absorption of nasal mucosa.
[0164] for rectum and urethral suppositories, vehicle will comprise traditional tackiness agent and carrier, for example theobroma oil (theobroma oil) or other triglyceride level; The vegetables oil of esterification, hydrogenation and/or fractionation modification; Glycerinated gelatin; Poly-alkaline ethylene glycol (polyalkaline glycols); The mixture of different molecular weight polyethylene glycol and the fatty acid ester of polyoxyethylene glycol.
[0165] carries for vagina, preparation of the present invention can be impregnated in vaginal suppository matrix (pessary bases), for example comprises those vaginal suppository matrix of polyethylene triglyceride level, perhaps is suspended in the oil, for example in Semen Maydis oil or the sesame oil, randomly contain colloidal silica.Referring to, Richardson et al. for example, Inr.J.Pharm. (1995) 115:9-15.
[0166] for the further discussion of the appropriate carrier that is used in the particular delivery pattern, referring to, Remington:The Science and Practice of Pharmacy for example, Mack Publishing Company, Easton, Pa., 19thedition, 1995.Those of ordinary skills can easily be identified for the suitable carrier of specific antigen and delivery site.
[0167] usefulness composition in the methods of the invention can contain the interested peptide antigen of " significant quantity ".That is to say that a certain amount of antigen will be included in the composition, it will cause object to produce enough immunne responses, to prevent, to reduce or eliminate symptom.Except other factors, according to handled object; The age of handled object and overall state; The ability of object-immunity system synthetic antibody; The degree of expectation protection; At the severity of symptom; Selected concrete antigen and mode of administration thereof, required exact amount will change.Suitable effective amount can easily be determined by those of ordinary skills.Therefore, " significant quantity " will fall into wide relatively scope, and it can be determined by routine test.For example, for the purposes of the present invention, effective dose normally every dose to be transferred antigen be about 1 μ g in the scope of about 100mg, for example from about 5 μ g to about 1mg, or about 10 μ g arrive about 500 μ g.
[0168] in case preparation is finished, then adopt standard technique, composition of the present invention is used by mucosal administration or subcutaneous injection.Referring to, Remington:The Science and Practice of Pharmacy for example, MackPublishing Company, Easton, Pa., 19th edition, 1995, about the mucous membrane conveying technology, comprise in the nose, lung, vagina and rectum technology, and No. the 517th, 565, European publication and Illum et al, J.Controlled Rel (1994) 29:133-141 is about the intranasal administration technology.
[0169] dosage treatment can be that the time control of the present invention of single dose discharges vaccine delivery compositions, perhaps multiple doses scheme, as known in the art.Booster dose can adopt and be used to obtain the same preparation that primary immune response gives, and perhaps can adopt and contain antigenic different preparation.Described dosage also will be determined by the demand of object at least in part, and depend on practitioner's judgement.And, if disease prevention expects that vaccine delivery compositions normally was applied before pathogenic agent primary infection interested.If treatment expects that for example reduce symptom or recurrence, vaccine delivery compositions normally is applied after primary infection.
[0170] present composition can carry out the body build-in test at many many animal models that are used to study subcutaneous or mucosa delivery that develop.For example, the sheep model that is in waking state is art-recognized a kind of model that the test substances intranasal is carried.Referring to for example Longenecker et al, J.Pharm.Sci (1987) 76:351-355 and Illum et al., J.Controlled Rel. (1994) 29:133-141.Vaccine delivery compositions, normally pulverous lyophilized form is blown in the nasal cavity.Use standard technique as known in the art, can measure the antibody titers in the blood sample, as mentioned above.Cellullar immunologic response also can be monitored as mentioned above.
[0171] external test method that exists the immunne response of a series of pair cells mediation to test at present, their use the cell from donor.This mensuration comprises the situation of cell from donor, yet a lot of mensuration provides the antigen presenting cell source from other source (for example B clone).These external tests comprise cytotoxic T lymphocyte mensuration; Lymphopoiesis is measured, and for example contains the tritium thymidine and mixes; Protein kinase is measured; Ion transport mensuration and lymphocyte migration inhibit feature are measured (Hickling, J.K.et al. (1987) J.Virol., 61:3463; Hengel, H.et al. (1987) J.Immunol., 139:4196; Thorley-Lawson, D.A.et al. (1987) Proc.Natl.Acad.Sci.USA, 84:5384; Kadival, G.J.et al. (1987) J.Immunol, 139:2447; Samuelson, L.E.et al. (1987) J.Immunol, 139:2708; Cason, J.et al. (1987) J.Immunol Meth., 102:109; And Tsein, R.J.et al. (1982) Nature, 293:68.These mensuration be have insufficient, because they lack at cell-mediated immunocompetent real specificity, they require by the APC of identical MHC type antigen to be processed and presented, they are (sometimes continue several days) slowly, and some are subjective and/or require to use radio isotope.
[0172] whether will activate this T cell and produce immunne response for detecting peptide by the T cell recognition, so-called " function test (functional test) " is used.Enzyme linked immunological spot (enzyme-linked immunospot, ELISpot) measure, be modified, be used for a kind of cytokine or effector molecule being had specific monoclonal antibody, detect the specific cytokine of secretion or the individual cells of other effector molecule by on microwell plate, adhering to.Contacted with immobilized antibody by the cell of antigenic stimulation.After cell and any unconjugated material were removed in washing, the polyclonal antibody of tape label perhaps was more typically monoclonal antibody, and---it is specific to this same cytokine or other effector molecule---is added in the hole.After washing, tinting material that can the combined belt tag antibody is added into, and makes the coloured throw out of indigo plant-Hei (or spot) form at the localized position of cytokine.Can manually or adopt automatization ELISpot reader parts, spot is counted, so that carry out quantitatively replying.Final affirmation to the T cell activation may require to detect in the body for this test peptides, for example in mouse or other animal model.
[0173] as clearly as seen, vaccine delivery compositions of the present invention is for the immunne response of inducing at virus, bacterium, parasite and fungi, disease and infection a variety of for treating and/or preventing, that cause by this class pathogenic agent, and be useful for stimulating at the antigenic immunne response of kinds of tumors.Said composition not only can be by therapeutic or prophylactic application---and aforesaid, said composition also can be used to prepare the purpose of antibody, and promptly polyclone and monoclonal antibody for example are used for diagnostic purpose, and is used for the interested antigen of immune purifying.If polyclonal antibody expects that then selected Mammals (for example mouse, rabbit, goat, horse etc.) is used composition immunity of the present invention.This animal, is used by one or many antigen after week at 2-6, is randomly strengthened.Then, obtain polyclonal antiserum, and handle according to known method from the animal of immunity.Referring to, J ü rgens et al. (1985) J.Chrom.348:363-370 for example.
[0174] use Kohler and Milstein, method or its improvement of Nature (1975) 256:495-96, monoclonal antibody is prepared prevailingly.Usually, mouse or rat are by as above immunity.Then, be not to make this animal bloodletting extracting serum, but that spleen (and randomly, several big lymphoglandula) picked-off and is dissociated into is unicellular.If desired, by cell suspension being administered on the flat board or plate hole with proteantigen bag quilt, spleen cell can screened (after removing non-specific adherent cell).The B cell that expression is specific to described antigenic membrane bound immunoglobulin will be attached on this flat board, and not rinsed by remaining suspension.Then, resulting B cell or all dissociated splenocytes, induced with the myeloma cell and merged, to form hybridoma, and selecting substratum (for example, xanthoglobulin, aminopterin-induced syndrome, Thymine deoxyriboside substratum (hypoxanthine, aminopterin, thymidine medium), " HAT ") the middle cultivation.Resulting hybridoma by limiting dilution by bed board, and the generation of determined antibody (and the irrelevant antigen of its debond) that can the specific combination immunizing antigen.Then, the hybridoma of the selected secrete monoclonal antibody of (as mouse ascites) cultivation in perhaps external (for example in tissue culture flasks or hollow fiber reactor) or the body.Referring to for example M.Schreier et al., Hybridoma Techniques (1980); Hammerling et al., MonoclonalAntibodies and T-cell Hybridomas (1981); Kennett et al., Monoclonal Antibodies (1980); Also referring to United States Patent (USP) the 4th, 341, No. 761; The 4th, 399, No. 121; The 4th, 427, No. 783; The 4th, 444, No. 887; The 4th, 466, No. 917; The 4th, 472, No. 500, the 4th, 491, No. 632; With the 4th, 493, No. 890.Can screen the various character that produced, i.e. isotype, epi-position, affinity etc. at a series of monoclonal antibodies of target polypeptides.
[0175] the following examples intended as illustrative explanation the present invention, and unrestricted the present invention.
Embodiment 1
Synthesizing of PEA-antigen conjugates
[0176] PE4 succinimide ester (PEA-Osu) is synthetic.All examples are all from N-acetylize polymkeric substance (A).PEA 1.392g, 754 μ M calculate molecular weight MW=every repeating unit 1845 (formula I, R
1=(CH
2)
8R
2=H; R
3=(CH
3)
2CHCH
2R
4=(CH
2)
6N=70; M/m+p=0.75 and p/m+p=0.25), it under agitation is dissolved in the 7ml dry DMF.To this a little the PEA solution of viscosity add 0.110g, 955 μ M solid N-maloyl imines (NHS).1-ethyl-3-of 146mg, 759.8 μ M (3 '-dimethylaminopropyl) carbodiimide hydrochloride is transferred with the suspension in DMF.The cumulative volume that is used for the DMF of this reaction is 10ml.This reaction at room temperature, under nitrogen atmosphere, was carried out 24 hours.
Synthesizing of PEA-influenza peptides conjugate:
[0177] B1) be used in 49.5 μ M aliquots containigs of the Acibenzolar (A) among the DMF, and 96mg (49.5 μ M) H-PKYVKQNTLKLAT-OH, be trifluoroacetate, carry out synthetic (formula IV, the R of PEA-peptide conjugate
1=(CH
2)
8R
3=(CH
3)
2CHCH
2R
4=(CH
2)
6R
5=NH; N=70; M/m+p=0.75 and p/m+p=0.25 and R
7=PKYVKQNTLKLAT).Peptide is dissolved and be transferred in the Acibenzolar in 5ml DMSO.Monovalent i.e. ethyl-diisopropylamine of 49.5 μ M is added into, and is reflected under the nitrogen atmosphere and continues 24 hours.Distilled water, 30 μ l in 300 μ l DMSO, are added into; And at room temperature continue to stir other 4 hours.
[0178] precipitin reaction mixture in ether (60ml), and, after centrifugal, the material that is obtained with 15ml ether washing three times.After air-dry, under sound wave, handled the product that obtains one minute with 3 * 5ml distilled water.After centrifugal, resulting material is by freeze-drying.Yield 86mg, 47%.
[0179] B2) be used in Acibenzolar (A) aliquots containig of 37.7 μ M among the DMF (600 μ l), and 74mg (37.7 μ M) H-PKYVKQNTLKLAT-OH, be trifluoroacetate, (formula IX passes through R to carry out PEA-peptide conjugate synthetic
5-R
7-R
5Crosslinked, R wherein
1=(CH
2)
8R
3=(CH
3)
2CHCH
2R
4=(CH
2)
6R
5=NH; N=70; M/m+p=0.75 and p/m+p=0.25 and R
7=PKYVKQNTLKLAT).Peptide is dissolved and transfer in the Acibenzolar in 0.8ml DMSO (dimethyl sulfoxide (DMSO)).Four equivalents i.e. ethyl-diisopropylamine of 198 μ M are added into, and are reflected under the nitrogen atmosphere and continue 48 hours.By decant, this is transparent, gel-like material is separated from organic solvent.After being cut into the bulk of 2-3mm, using 17ml distilled water, handled product 18 hours down at 4 ℃.Behind centrifugal and decant, handle this material twice (each 3 hours) with 17ml distilled water, and after centrifugal the last time, product is frozen drying.Yield: 75mg, 53%.
[0180] B3) with the Acibenzolar of 41.2 μ M, it synthesizes, is among the DMF (600 μ l) in the mode that is similar to (A), and 40mg (20.6 μ M) H-PKYVKQNTLKLAT-OH, and as trifluoroacetate, (formula IX passes through R to carry out PEA-peptide conjugate synthetic
5-R
7-R
5Crosslinked, R wherein
1=(CH
2)
8R
3=(CH
3)
2CHCH
2R
4=(CH
2)
6R
5=NH; N=8; M/m+p=0.75 and p/m+p=0.25 and R
7=PKYVKQNTLKLAT).Peptide is dissolved, and transfers in the Acibenzolar in 5ml DMSO.Four equivalents i.e. ethyl-diisopropylamine of 80 μ M are added into, and are reflected under the nitrogen atmosphere and continue 72 hours.Distilled water, 75 μ l (4.2mM) in 300 μ l DMSO, are added into; And continue to stir other 24 hours.Then, reaction mixture is precipitated in 24ml water/acetone (1:1 v/v).The throw out that under 4 ℃, obtains with the distilled water processing (4 * 12ml), each one hour, centrifugal subsequently.After centrifugal the last time, product is frozen drying.Yield: 50mg, 45%.
The summary of human T-cell's vitro responses flow process
[0181] isolates CD4 from the peripheral blood of people's donor
+T cell and monocyte.In being rich in the substratum of cytokine, cultivated monocyte 48 hours, be divided into dendritic cell (antigen presenting cell) to induce.During in entering this incubation period 24 hours, PEA or PEA-hemagglutinin peptide (307-319) conjugate are added in the substratum.In two hours, free peptide is added in the control wells before beginning common cultivation dendritic cell and T cell.When 48 hours, 72 hours and 96 hours,, measure the activation situation of the T cell of cultivating with dendritic cell by propagation and cytokine secretion.The synoptic diagram of t cell response flow process is described in Fig. 3 herein.
[0182] utilize top flow process, in response to the T cell activation of the dendritic cell that are exposed to polymkeric substance-peptide conjugate, detected.Fig. 4 A shows the T cell proliferation in 96 hours, and wherein with respect to independent peptide or independent PEA, the PEA-peptide conjugate has stimulated significant propagation.The IL-2 that is undertaken by the T cell that Fig. 4 B showed in 96 hours secretes, and wherein than independent peptide or independent PEA, PEA-peptide conjugate (formula III, Embodiment B 1) stimulates significant IL-2 secretion.
[0183] all publication, patent and patent documents are merged in this paper as a reference, as being incorporated herein by reference separately.With reference to various concrete and preferred embodiment and technology, the present invention is described.Yet, should be appreciated that and may make many variations and change, and still keep within the spirit and scope of the present invention.
[0184] although with reference to top embodiment, the present invention is described, should be understood that: modifications and variations still are included within the spirit and scope of the present invention.Therefore, the present invention is only limited by claims.
Sequence table
＜110〉Medivas LLC
W.G. figure Nellie
V.P. Vassilieff
The K.M moral is luxuriant and bdautiful
H. Lee
Z.D. Ge Mulashiweili
R. Ka Chalawa
＜120〉vaccine delivery compositions and using method
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<150>US 60/759,179
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<150>US 60/748,486
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<150>US 60/742,188
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<150>US 60/719,950
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<150>US 60/689,003
<151>2005-06-08
<150>US 60/687,570
<151>2005-06-03
<150>US 60/649,289
<151>2005-02-01
<160>15
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<400>8
<210>9
<211>13
<212>PRT
＜213〉artificial sequence
<220>
＜223〉synthetic construction
<400>9
<210>10
<211>13
<212>PRT
＜213〉artificial sequence
<220>
＜223〉synthetic construction
<400>10
<210>11
<211>9
<212>PRT
＜213〉artificial sequence
<220>
＜223〉synthetic construction
<400>11
<210>12
<211>9
<212>PRT
＜213〉artificial sequence
<220>
＜223〉synthetic construction
<400>12
<210>13
<211>9
<212>PRT
＜213〉artificial sequence
<220>
＜223〉synthetic construction
<400>13
<210>14
<211>9
<212>PRT
＜213〉artificial sequence
<220>
＜223〉synthetic construction
<400>14
<210>15
<211>13
<212>PRT
＜213〉artificial sequence
<220>
＜223〉synthetic construction
<400>15
Claims (68)
1. vaccine delivery compositions, it comprises:
At least a MHC I class of significant quantity or MHC II class peptide antigen, it is coupled on the particle or molecule of following polymkeric substance: have biodegradable polyamidoester (PEA) polymkeric substance of the described chemical structure of structural formula (I),
Formula (I)
Wherein n is between about 5 to about 150; R
1Be independently selected from α, ω-two (4-carboxyl phenoxy group)-(C
1-C
8) alkane, 3,3 '-(alkane two acyl dioxy bases) two styracins or 4,4 '-the residue, (C of (alkane two acyl dioxy bases) two styracins
2-C
20) alkylene hydrocarbon or (C
2-C
20) inferior alkene; R in an independent n monomer
3Be independently selected from hydrogen, (C
1-C
6) alkyl, (C
2-C
6) thiazolinyl, (C
2-C
6) alkynyl, (C
6-C
10) aryl (C
1-C
20) alkyl and-(CH
2)
2S (CH
3); And R
4Be independently selected from (C
2-C
20) alkylidene group, (C
2-C
20) alkenylene, (C
2-C
8) alkoxyl group, (C
2-C
20) alkylidene group, saturated or unsaturated therapeutic glycol residue, structural formula (II) 1,4:3, dicyclo part and their the combination, (C of the two anhydrous hexitols of 6-
2-C
20) alkylidene group and (C
2-C
20) alkenylene;
Formula (II)
The PEA polymkeric substance that perhaps has the described chemical formula of structural formula (III),
Formula (III)
Wherein n is between about 5 to about 150, and m is between about 0.1 to 0.9, and p is between about 0.9 to 0.1; R wherein
1Be independently selected from α, ω-two (4-carboxyl phenoxy group)-(C
1-C
8) alkane, 3,3 '-(alkane two acyl dioxy bases) two styracins or 4,4 '-the residue, (C of (alkane two acyl dioxy bases) two styracins
2-C
20) alkylene hydrocarbon or (C
2-C
20) inferior alkene; Each R
2Be hydrogen, (C independently
1-C
12) alkyl or (C
6-C
10) aryl or protecting group; R in an independent m monomer
3Be independently selected from hydrogen, (C
1-C
6) alkyl, (C
2-C
6) thiazolinyl, (C
2-C
6) alkynyl, (C
6-C
10) aryl (C
1-C
20) alkyl and-(CH
2)
2S (CH
3); And R
4Be independently selected from (C
2-C
20) alkylidene group, (C
2-C
20) alkenylene, (C
2-C
8) alkoxyl group, (C
2-C
20) alkylidene group, saturated or unsaturated therapeutic glycol residue, general formula (II) 1,4:3, the dicyclo part and their combination of the two anhydrous hexitols of 6-;
Polyester urethane (PEUR) polymkeric substance that perhaps has the described chemical formula of structural formula (IV),
Formula (IV)
Wherein n is between about 5 to about 150; R wherein
3Be independently selected from hydrogen, (C
1-C
6) alkyl, (C
2-C
6) thiazolinyl, (C
2-C
6) alkynyl, (C
6-C
10) aryl (C
1-C
20) alkyl and-(CH
2)
2S (CH
3); R
4Be selected from (C
2-C
20) alkylidene group, (C
2-C
20) alkenylene or alkoxyl group, saturated or unsaturated therapeutic glycol residue, structural formula (II) 1,4:3, the dicyclo part and their combination of the two anhydrous hexitols of 6-; And R
6Be independently selected from (C
2-C
20) alkylidene group, (C
2-C
20) alkenylene or alkoxyl group, general formula (II) 1,4:3, the dicyclo part and their combination of the two anhydrous hexitols of 6-;
The PEUR polymkeric substance that perhaps has the described chemical structure of universal architecture formula V
Formula V
Wherein n is between about 5 to about 150, and m is between about 0.1 to about 0.9, and p is between about 0.9 to about 0.1; R
2Be independently selected from hydrogen, (C
6-C
10) aryl (C
1-C
20) alkyl or protecting group; R in an independent m monomer
3Be independently selected from hydrogen, (C
1-C
6) alkyl, (C
2-C
6) thiazolinyl, (C
2-C
6) alkynyl, (C
6-C
10) aryl (C
1-C
20) alkyl and-(CH
2)
2S (CH
3); R
4Be selected from (C
2-C
20) alkylidene group, (C
2-C
20) alkenylene or alkoxyl group, saturated or unsaturated therapeutic glycol residue, structural formula (II) 1,4:3, the dicyclo part and their combination of the two anhydrous hexitols of 6-; And R
6Be independently selected from (C
2-C
20) alkylidene group, (C
2-C
20) alkenylene or alkoxyl group, general formula (II) 1,4:3, the dicyclo part of the two anhydrous hexitols of 6-, the residue of the saturated or unsaturated therapeutic glycol of significant quantity and their combination;
The polyester-urea (PEU) that perhaps has the described chemical formula of universal architecture formula (VI):
Formula (VI),
Wherein n is between about 10 to about 150; R in an independent n monomer
3Be independently selected from hydrogen, (C
1-C
6) alkyl, (C
2-C
6) thiazolinyl, (C
2-C
6) alkynyl, (C
6-C
10) aryl (C
1-C
20) alkyl and-(CH
2)
2S (CH
3); R
4Be independently selected from (C
2-C
20) alkylidene group, (C
2-C
20) alkenylene, (C
2-C
8) alkoxyl group (C
2-C
20) residue of alkylidene group, saturated or unsaturated therapeutic glycol; Or structural formula (II) 1,4:3, the dicyclo part of the two anhydrous hexitols of 6-; The PEU that perhaps has the described chemical formula of structural formula (VII)
Formula (VII),
Wherein m is between about 0.1 to about 1.0, and p is between about 0.9 to about 0.1, and n is between about 10 to about 150; Each R
2Be hydrogen, (C independently
1-C
12) alkyl or (C
6-C
10) aryl; R in an independent m monomer
3Be independently selected from hydrogen, (C
1-C
6) alkyl, (C
2-C
6) thiazolinyl, (C
2-C
6) alkynyl, (C
6-C
10) aryl (C
1-C
20) alkyl and-(CH
2)
2S (CH
3); Each R
4Be independently selected from (C
2-C
20) alkylidene group, (C
2-C
20) alkenylene, (C
2-C
8) alkoxyl group (C
2-C
20) alkylidene group, saturated or unsaturated therapeutic glycol residue, structural formula (II) 1,4:3, the dicyclo part and their combination of the two anhydrous hexitols of 6-.
2. the described composition of claim 1, wherein said peptide antigen contain 5 to about 30 amino acid.
3. the described composition of claim 1, wherein said composition is formulated into the dispersion of described polymer beads or molecule.
4. the described composition of claim 1, wherein said polymkeric substance is structural formula (I) or the described PEA of structural formula (III).
5. the described composition of claim 4, wherein at least one R
1Be α, ω-two (4-carboxyl phenoxy group)-(C
1-C
8) alkane, 3,3 '-(alkane two acyl dioxy bases) two styracins or 4,4 '-residue of (alkane two acyl dioxy bases) two styracins, perhaps at least one R
4Be 1 of structural formula (II), 4:3, the dicyclo part of the two anhydrous hexitols of 6-.
6. the described composition of claim 4, wherein at least one R
1Be α, ω-two (4-carboxyl phenoxy group)-(C
1-C
8) alkane, 3,3 '-(alkane two acyl dioxy bases) two styracins or 4,4 '-residue of (alkane two acyl dioxy bases) two styracins, or their mixture, and at least one R
4Be 1 of structural formula (II), 4:3, the dicyclo part of the two anhydrous hexitols of 6-.
7. the described composition of claim 1, wherein said polymkeric substance is structural formula (IV) or the described PEUR of structure formula V.
8. the described composition of claim 7, wherein at least one R
1Be α, ω-two (4-carboxyl phenoxy group)-(C
1-C
8) alkane, 3,3 '-(alkane two acyl dioxy bases) two styracins or 4,4 '-residue of (alkane two acyl dioxy bases) two styracins, perhaps at least one R
4Be 1 of structural formula (II), 4:3, the dicyclo part of the two anhydrous hexitols of 6-.
9. the described composition of claim 7, wherein at least one R
1Be α, ω-two (4-carboxyl phenoxy group)-(C
1-C
8) alkane, 3,3 '-(alkane two acyl dioxy bases) two styracins or 4,4 '-residue of (alkane two acyl dioxy bases) two styracins, or their mixture, and at least one R
4Be 1 of structural formula (II), 4:3, the dicyclo part of the two anhydrous hexitols of 6-.
10. the described composition of claim 1, wherein said polymkeric substance is structural formula (VI) or the described PEU of structural formula (VII).
11. the described composition of claim 10, at least one R
1Be 1 of structural formula (II), 4:3, the dicyclo part of the two anhydrous hexitols of 6-.
12. the described composition of claim 1, wherein said composition when being applied in vivo, forms time control release polymers bank.
13. the described composition of claim 1, wherein said composition, in twenty four hours, about seven days, about 30 days or about 90 days for some time, biological degradation.
14. the described composition of claim 1, the form of wherein said composition are the particle of mean diameter in about 10 nanometers arrive about 1000 microns scope, and at least a peptide antigen is dispersed in each polymer molecule of described particulate.
15. the described composition of claim 14, wherein said particle further comprises polymer covering.
16. the described composition of claim 1, the mean diameter that wherein said particle has are in about 10 nanometers arrive about 10 microns scope.
17. the described composition of claim 1, wherein said particle comprises: every polymer molecule contains has an appointment 5 to about 150 peptide antigens.
18. the described composition of claim 1, wherein the molecular-weight average that has of polymer molecule is from about 5,000 in about 300,000 scope, and at least one peptide antigen is coupled on the described polymer molecule.
19. the described composition of claim 1, wherein polymer molecule has about 5 to 70 the peptide antigens that are coupled on it.
20. the described composition of claim 1, wherein said polymkeric substance are comprised in the have structural formula polymkeric substance-antigen conjugates of chemical structure of (VIII):
Formula (VIII)
Wherein n, m, p, R
1, R
3And R
4As above, R
5Be selected from-O-,-S-and-NR
8-, R wherein
8Be H or (C
1-C
8) alkyl; And, R
7Be described peptide antigen.
21. the described composition of claim 20, the two or more molecules that are described polymkeric substance are crosslinked to provide-R
5-R
7-R
5-conjugate.
22. the described composition of claim 20 is that described antigen is by described-R
5-R
7-R
5-conjugate is linked on the molecule of described polymkeric substance by covalency, and R
5Be independently selected from-O-,-S-and-NR
8-, R wherein
8Be H or alkyl.
23. the described composition of claim 21, just R
1Be (C independently
2-C
20) alkylidene group or (C
2-C
20) alkenylene, and R wherein
5One of be-X-Y-that wherein X is selected from (C
1-C
18) alkylidene group, substituted alkylene, (C
3-C
8) encircle alkylidene group, substituted ring alkylidene group, contain 1-3 the first heterocyclic system of heteroatomic 5-6, substituted heterocycle, (C that is selected from O, N and S
2-C
18) thiazolinyl, substituted alkenyl, alkynyl, substituted alkynyl, C
6And C
10Aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkylaryl, substituted alkyl aryl, aromatic yl polysulfide yl, substituted aryl alkynyl, aryl alkenyl, substituted aryl thiazolinyl, aromatic yl polysulfide yl, substituted aryl alkynyl, and wherein substituting group is selected from H, F, Cl, Br, I, (C
1-C
6) alkyl ,-CN ,-NO
2,-OH ,-O (C
1-C
4) alkyl ,-S (C
1-C
6) alkyl ,-S[(=O) (C
1-C
6) alkyl)] ,-S[(O
2) (C
1-C
6) alkyl] ,-C[(=O) (C
1-C
6) alkyl], CF
3,-O[(CO)-(C
1-C
6) alkyl)] ,-S (O
2) [N (R
9R
10)] ,-NH[(C=O) (C
1-C
6) alkyl] ,-NH (C=O) N (R
9R
10) and-N (R
9R
10); R wherein
9And R
10Be H or C independently
1-C
6Alkyl; And Y be selected from-O-,-S-,-S-S-,-S (O)-,-S (O
2)-,-NR
8-,-C (=O)-,-OC (=O)-,-C (=O) O-,-OC (=O) NH-,-NR
8C (=O)-,-C (=O) NR
8-,-NR
8C (=O) NR
8-,-NR
8C (=O) NR
8-and-NR
8C (=S) NR
8-.
24. the described composition of claim 23, just each R
5All be-X-Y-.
25. the described composition of claim 23, it comprises two molecules of described polymkeric substance, is two omission R in four repeating units
7, and be crosslinked so that single-R to be provided
5-X-R
5-conjugate, wherein X is selected from (C
1-C
18) alkyl, substituted alkyl, (C
3-C
8) encircle alkylidene group, substituted ring alkylidene group, contain 1-3 the first heterocyclic system of heteroatomic 5-6, substituted heterocycle, (C that is selected from O, N and S
2-C
18) thiazolinyl, substituted alkenyl, alkynyl, substituted alkynyl, C
6And C
10Aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkylaryl, substituted alkyl aryl, aromatic yl polysulfide yl, substituted aryl alkynyl, aryl alkenyl, substituted aryl thiazolinyl, aromatic yl polysulfide yl, substituted aryl alkynyl, and wherein substituting group is selected from H, F, Cl, Br, I, (C
1-C
6) alkyl ,-CN ,-NO
2,-OH ,-O (C
1-C
4) alkyl ,-S (C
1-C
6) alkyl ,-S[(=O) (C
1-C
6) alkyl)] ,-S[(O
2) (C
1-C
6) alkyl] ,-C[(=O) (C
1-C
6) alkyl], CF
3,-O[(CO)-(C
1-C
6) alkyl)] ,-S (O
2) [N (R
9R
10) ,-NH[(C=O) (C
1-C
6) alkyl] ,-NH (C=O) N (R
9R
10) and-N (R
9R
10); R wherein
9And R
10Be H or (C independently
1-C
6) alkyl.
26. the described composition of claim 20, two molecules that are described polymkeric substance are by partial cross-linked, to provide-R
5-X-Y-R
7-R
5-conjugate.
27. the described composition of claim 22, a molecule that is described polymkeric substance passes through-R
5-R
7-Y-X-R
5-bridge is linked to described antigen (formula XI) by covalency:
Formula (XI)
Wherein, X is selected from (C
1-C
18) alkylidene group, substituted alkylene, (C
3-C
8) encircle alkylidene group, substituted ring alkylidene group, contain 1-3 the first heterocyclic system of heteroatomic 5-6, substituted heterocycle, (C that is selected from O, N and S
2-C
18) thiazolinyl, substituted alkenyl, alkynyl, substituted alkynyl, C
6And C
10Aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkylaryl, substituted alkyl aryl, aromatic yl polysulfide yl, substituted aryl alkynyl, aryl alkenyl, substituted aryl thiazolinyl, aromatic yl polysulfide yl, substituted aryl alkynyl, wherein substituting group is selected from H, F, Cl, Br, I, (C
1-C
6) alkyl ,-CN ,-NO
2,-OH ,-O (C
1-C
6) alkyl ,-S (C
1-C
6) alkyl ,-S[(=O) (C
1-C
6) alkyl)] ,-S[(O
2) (C
1-C
6) alkyl] ,-C[(=O) (C
1-C
6) alkyl], CF
3,-O[(CO)-(C
1-C
6) alkyl)] ,-S (O
2) [N (R
9R
10) ,-NH[(C=O) (C
1-C
6) alkyl] ,-NH (C=O) N (R
9R
10) ,-N (R
11R
12); R wherein
9And R
10Be H or (C independently
1-C
6) alkyl, R
11And R
12Be (C independently
2-C
20) alkylidene group or (C
2-C
20) alkenylene.
28. comprising, the described composition of claim 1, wherein said peptide antigen have about 8 to about 12 amino acid whose I class epi-positions.
29. the described composition of claim 28 further comprises adjuvant.
30. the described composition of claim 29, wherein said adjuvant is covalently bound to described polymkeric substance.
31. the described composition of claim 29, wherein said adjuvant and described antigen are coupled on the same polymkeric substance.
32. comprising, the described composition of claim 1, wherein said peptide antigen have about 8 to about 30 amino acid whose II class epi-positions.
33. the described composition of claim 1, wherein said peptide antigen comprises the epi-position of virus, bacterium, fungi or TCSA.
34. the described composition of claim 33, wherein said antigen are the contrapositive peptides.
35. the described composition of claim 34, wherein said antigen are the trans peptides of Partial Inverse.
36. the described composition of claim 1, wherein said peptide antigen comprises virus epitopes.
37. the described composition of claim 36, wherein said virus epitopes are HIV epi-position or influenza virus epi-position.
38. the described composition of claim 37, wherein said HIV epi-position has the aminoacid sequence of SEQ ID NO:8.
39. the described composition of claim 37, wherein said influenza virus epi-position has the aminoacid sequence of SEQ ID NO:9 or 10.
40. the described composition of claim 1, wherein said composition further comprise pharmaceutically acceptable carrier.
41. the described composition of claim 1, wherein said composition is in the form of the dispersed droplets in the mist.
42. the described composition of claim 41, wherein said mist is produced by spraying gun.
43. the described composition of claim 1, wherein said composition is comprised in the spraying gun, and described spraying gun can be driven, and contains the mist of the dispersed droplets of described carrier with generation.
44. the described composition of claim 1, wherein said composition is comprised in the injection device, and described injection device can be driven, to pass through the injection applying said compositions.
45. the method for induce immune response in Mammals, described method comprises:
The described vaccine delivery compositions of claim 1 to described administration immunostimulation amount; described composition is the form of the liquid dispersion of polymer beads or molecule; it is by described mammiferous antigen presenting cell picked-up, with induce immune response in described Mammals.
46. the described method of claim 45, wherein said composition when using in by body, forms time control release polymers bank.
47. the described method of claim 45, the biological degradation in for some time of wherein said composition, described for some time is twenty four hours, about seven days, about 30 days or about 90 days.
48. the described method of claim 45, wherein said composition is in particle form, and described particulate mean diameter is in about 10 nanometers arrive about 1000 microns scope, and at least a peptide antigen is dispersed in the described particle.
49. the described method of claim 45, the mean diameter that wherein said particle has are in about 10 nanometers arrive about 10 microns scope.
50. the described method of claim 45, wherein said particle further comprises polymer covering.
51. the described method of claim 45, wherein particle comprises: about 5 are arrived about 150 peptide antigens/every polymer molecule.
52. the described method of claim 45, wherein polymer molecule has the molecular-weight average that arrives in about 300,000 scopes from about 5,000, and at least a peptide antigen is coupled to described polymer molecule.
53. the described method of claim 45, wherein polymer molecule has about 5 to 70 the peptide antigens that are coupled on it.
54. the described method of claim 45, wherein said peptide antigen comprise about 8 to about 12 amino acid whose I class epi-positions.
55. the described method of claim 45 also comprises adjuvant.
56. the described method of claim 55, wherein said adjuvant is covalently bound to described polymkeric substance.
57. the described method of claim 55, wherein said adjuvant and described antigen are coupled on the same polymkeric substance.
58. the described method of claim 45, wherein said peptide antigen comprise about 8 to about 30 amino acid whose II class epi-positions.
59. the described method of claim 58, wherein said peptide antigen comprises the epi-position of virus, bacterium, fungi or TCSA.
60. carry the method for vaccine, comprise described object is used the described vaccine delivery compositions of claim 1, so that described vaccine is absorbed by the antigen presenting cell of described object to object.
61. vaccine delivery compositions, it comprises at least a MHC I class or the II class peptide antigen of significant quantity, described MHC I class or II class peptide antigen are dispersed in the biodegradable polymkeric substance, each monomer of described biodegradable polymkeric substance comprises the amino acid of at least a type, and described one type amino acid is coupled at least a non-amino acid moiety.
62. the described composition of claim 61, wherein said non-amino acid moiety is between two adjacent amino acids.
63. the described composition of claim 61, wherein said non-amino acid moiety is hydrophobic.
64. the described composition of claim 61, wherein said peptide antigen comprise 5 to about 30 amino acid.
65. the described composition of claim 61, wherein said polymkeric substance comprise at least two kinds of different amino acid.
66. the described composition of claim 61, wherein said polymkeric substance are the segmented copolymers that forms micelle in liquid.
67. the method for induce immune response in Mammals, described method comprises:
To the described vaccine delivery compositions of described administration claim 61; described composition is the form of the liquid dispersion of polymer beads or molecule; described vaccine delivery compositions is by described mammiferous antigen presenting cell picked-up, with induce immune response in described Mammals.
68. the described composition of claim 1, the wherein R at least one monomer
3Further can be-(CH
2)
3-; And, at least one R
3Cyclisation forms the described chemical structure of structural formula (XVIII):
Formula (XVIII).
Applications Claiming Priority (8)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US64928905P | 2005-02-01 | 2005-02-01 | |
| US60/649,289 | 2005-02-01 | ||
| US60/687,570 | 2005-06-03 | ||
| US60/689,003 | 2005-06-08 | ||
| US60/719,950 | 2005-09-22 | ||
| US60/742,188 | 2005-12-02 | ||
| US60/748,486 | 2005-12-07 | ||
| US60/759,179 | 2006-01-13 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101506266A true CN101506266A (en) | 2009-08-12 |
Family
ID=40977673
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2006800035269A Pending CN101506266A (en) | 2005-02-01 | 2006-01-31 | Vaccine delivery compositions and methods of use |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101506266A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102459629A (en) * | 2009-05-19 | 2012-05-16 | 迈阿密大学 | Compositions, kits and methods for in vitro antigen presentation, assessing vaccine efficacy, and assessing immunotoxicity of biologics and drugs |
| CN103980494A (en) * | 2014-04-21 | 2014-08-13 | 国家纳米科学中心 | Polypeptide polymer having antitumor activity and its preparation method and use |
| US9764012B2 (en) | 2009-04-01 | 2017-09-19 | University Of Miami | Vaccine compositions and methods of use thereof |
| CN107596364A (en) * | 2017-09-19 | 2018-01-19 | 洛阳赛威生物科技有限公司 | A kind of w/o/w adjunvant compositions, the vaccine combination prepared and preparation method thereof |
-
2006
- 2006-01-31 CN CNA2006800035269A patent/CN101506266A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9764012B2 (en) | 2009-04-01 | 2017-09-19 | University Of Miami | Vaccine compositions and methods of use thereof |
| CN102459629A (en) * | 2009-05-19 | 2012-05-16 | 迈阿密大学 | Compositions, kits and methods for in vitro antigen presentation, assessing vaccine efficacy, and assessing immunotoxicity of biologics and drugs |
| CN103980494A (en) * | 2014-04-21 | 2014-08-13 | 国家纳米科学中心 | Polypeptide polymer having antitumor activity and its preparation method and use |
| CN103980494B (en) * | 2014-04-21 | 2016-04-13 | 国家纳米科学中心 | A kind of polypeptide polymer with anti-tumor activity and its preparation method and application |
| CN107596364A (en) * | 2017-09-19 | 2018-01-19 | 洛阳赛威生物科技有限公司 | A kind of w/o/w adjunvant compositions, the vaccine combination prepared and preparation method thereof |
| CN107596364B (en) * | 2017-09-19 | 2021-03-12 | 洛阳赛威生物科技有限公司 | W/o/w adjuvant composition, vaccine composition prepared from same and preparation method of vaccine composition |
| CN112843227A (en) * | 2017-09-19 | 2021-05-28 | 洛阳赛威生物科技有限公司 | W/o/w adjuvant composition, vaccine composition prepared from same and preparation method of vaccine composition |
| CN112843227B (en) * | 2017-09-19 | 2024-05-07 | 洛阳赛威生物科技有限公司 | W/o/w adjuvant composition, vaccine composition prepared from w/o/w adjuvant composition and preparation method of vaccine composition |
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