AU2006210863A1 - Vaccine delivery compositions and methods of use - Google Patents
Vaccine delivery compositions and methods of use Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
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- C01G23/00—Compounds of titanium
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/593—Polyesters, e.g. PLGA or polylactide-co-glycolide
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/595—Polyamides, e.g. nylon
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82B—NANOSTRUCTURES FORMED BY MANIPULATION OF INDIVIDUAL ATOMS, MOLECULES, OR LIMITED COLLECTIONS OF ATOMS OR MOLECULES AS DISCRETE UNITS; MANUFACTURE OR TREATMENT THEREOF
- B82B3/00—Manufacture or treatment of nanostructures by manipulation of individual atoms or molecules, or limited collections of atoms or molecules as discrete units
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
Description
WO 2006/083874 PCT/US2006/003412 1 VACCINE DELIVERY COMPOSITIONS AND METHODS OF USE RELATED APPLICATIONS [0001] This application claims priority under §35 U.S.C. 119(e) from provisional application Serial Nos. 60/649,289, filed February 1, 2005; 60/689,003, filed June 8, 2005; 60/742,188, filed December 2, 2005; 60/748,486, filed December 7, 2005; 60/719,950, filed September 22, 2005; 60/687,570, filed June 3, 2005; 60/759,179, filed January 13, 2006. FIELD OF THE INVENTION [0002] This invention relates generally to immunogenic compositions and, in particular to vaccine delivery compositions that bind to MHC alleles. BACKGROUND INFORMATION [0003] Although significant progress in vaccine development and administration has been made, alternative approaches that enhance the efficacy and safety of vaccine preparations remain under investigation. Sub-unit vaccines such as recombinant proteins, synthetic peptides, and polysaccharide-peptide conjugates are emerging as novel vaccine candidates. However, traditional vaccines, consisting of attenuated pathogens and whole inactivated organisms, contain impurities and bacterial components capable of acting as adjuvants, an activity which these subunit vaccines lack. Therefore the efficacy of highly purified sub-unit vaccines delivered as stand-alone formulations will require addition of potent adjuvants. [0004] Currently, aluminum compounds remain the only FDA approved adjuvants for use in human vaccines in the United States. Despite their good safety record, they are relatively weak adjuvants and often require multiple dose regimens to elicit antibody levels associated with protective immunity. Aluminum compounds may therefore not be ideal adjuvants for the induction of protective immune responses to sub-unit vaccines. Although many candidate adjuvants are presently under investigation, they suffer from a WO 2006/083874 PCT/US2006/003412 2 number of disadvantages including toxicity in humans and requirements for sophisticated techniques to incorporate antigens. [00051 Use of peptidic antigens in vaccines is based on knowledge of operation of the immune system in mammals and other animals, especially the major histocompatibility complexes (MHC). MHC molecules are synthesized and displayed by most of the cells of the body. The MHC works coordinately with specialized types of T cell (for example, the cytotoxic T cell) to rid the body of "nonself' or foreign viral proteins. The antigen receptor on T-cells recognizes an epitope that is a mosaic of the bound peptide and portions of the alpha helices that make up the groove flanking it. Following generation of peptide fragments by cleavage of a foreign protein, the presentation of peptide fragments by the MHC molecule allows for antigen-restricted cytotoxic T cells to survey cells for the expression of "nonself or foreign viral proteins. A functional T-cell will exhibit a cytotoxic immune response upon recognition of an MHC molecule containing bound peptidic antigen for which the T-cell is specific. [0006] Exogenous antigens are those from outside cells of the body. Examples include bacteria, free viruses, yeasts, protozoa, and toxins. These exogenous antigens enter antigen-presenting cells or APCs (macrophages, dendritic cells, and B-lymphocytes) through phagocytosis. The microbes are engulfed and protein antigens are degraded by proteases into a series of peptides. These peptides eventually bind to grooves in MHC-II molecules and are transported to the surface of the APC. T4-lymphocytes are then able to recognize peptide/MHC-II complexes by means of their T-cell receptors (TCRs) and CD4 molecules. Peptides that are presented by APCs in class II MHCs are about 10 to about 30 amino acids, for example about 12 to about 24 amino acids in length (Marsh, S. G. E. et al. (2000) The HLA Facts Book, Academic Press, p. 58-59). The effector functions of the activated T4-lymphocytes include production of antibodies by B cells and microbiocidal activities of macrophages, which are the main mechanisms by which extracellular or phagocytosed microbes are destroyed. [00071 One of the body's major defenses against viruses, intracellular bacteria, and cancers is destruction of endogenous infected cells and tumor cells by cytotoxic T lymphocytes or CTLs. These CTLs are effector cells derived from T8-lymphocytes during cell-mediated immunity. However, in order to become CTLs, naive T8- WO 2006/083874 PCT/US2006/003412 3 lymphocytes must become activated by cytokines produced by APCs. This interaction between APCs and naive T8-lymphocytes occurs primarily in the lymph nodes, the lymph nodules, and the spleen. The process involves dendritic cells and macrophages engulfing and degrading infected cells, tumor cells, and the remains of killed infected and tumor cells. It is thought that in this manner, endogenous antigens from diseased cells are able to enter the APC, where proteases and peptidases chop the protein up into a series of peptides, of about 8 to about 10, possibly about 8 to about 11, or about 8 to about 12 amino acids in length. The MHC class I molecules with bound peptide, which appear on the surface of the APCs, can now be recognized by naive T8-lymphocytes possessing TCRs and CD8 molecules with a complementary shape. This recognition of the peptide epitope by the TCR serves as a first signal for activating the naive T8-lymphocyte for cell-mediated immunity function. A single cell may have up to 250,000 molecules of MHC-I with bound epitope on its surface. [00081 Thus, there is still a need in the art for new and better vaccine delivery compositions utilizing peptidic antigens rather than deactivated pathogens and methods for their use to induce an immune response in individuals against pathogenic organisms that are identified by MHC class I and class II alleles. SUMMARY OF THE INVENTION [0009] The present invention is based on the premise that biodegradable polymers that contain amino acids in the polymer chain, such as certain poly (ester amide) (PEA), poly (ester urethane) (PEUR), and poly (ester urea) (PEU) polymers, can be used to formulate completely synthetic and, hence, easy to produce vaccine delivery compositions for stimulating an immune response to a variety of pathogenic organisms in humans and other mammals. [00101 Accordingly, in one embodiment the invention provides a vaccine delivery composition that includes an effective amount of at least one MHC class I or class II peptidic antigen comprising from 5 to about 30 amino acids dispersed in'biodegradable polymer molecules or particles comprising at least one type of amino acid conjugated to at least one non-amino acid moiety per monomer.
WO 2006/083874 PCT/US2006/003412 4 [0011] In another embodiment the invention provides a vaccine delivery composition formulated for administration in the form of a liquid dispersion of polymer particles or molecules conjugated to an effective amount of at least one MHC class I or class II peptidic antigen comprising from 5 to about 30 amino acids and a biodegradable PEA having a structural formula described by structural formula (I), 0 0 HO 0 H 4-Ri-C-N--0C-R4-0- C-N Formula (I) wherein n ranges from about 5 to about 150; R 1 is independently selected from residues of a~w-bis(4-carboxyphenoxy)-(Ci-Cs) alkane, 3,3'-(alkanedioyldioxy)dicinnamic acid or 4,4'-(alkanedioyldioxy)dicinnamic acid, (C 2 - C 20 ) alkylene, or (C 2
-C
20 ) alkenylene; the
R
3 s in individual n monomers are independently selected from the group consisting of hydrogen, (C 1
-C
6 ) alkyl, (C 2
-C
6 ) alkenyl, (C 2
-C
6 ) alkynyl, (C 6
-C
10 ) aryl (C 1
-C
20 ) alkyl, and -(CH 2
)
2
S(CH
3 ); and R 4 is independently selected from the group consisting of (C 2 C 20 ) alkylene, (C 2
-C
20 ) alkenylene, (C 2 -Cs) alkyloxy, (C 2
-C
20 ) alkylene, a residue of a saturated or unsaturated therapeutic diol, bicyclic-fragments of 1,4:3,6-dianhydrohexitols of structural formula (II), and combinations thereof, (C 2 - C 20 ) alkylene, and (C 2
-C
2 0 ) alkenylene; CH O
H
2 C CH 2 0 CH Formula (II) or a PEA polymer having a chemical formula described by structural formula III: O 0 HO O H O O H -- R-C-N-C--0-R 4 ---- N- -R-C-N-C-(CH2)4-N- HR 13 IR3I{ I S la H-O-R2 Formula (III) WO 2006/083874 PCT/US2006/003412 5 wherein n ranges from about 5 to about 150, m ranges about 0.1 to 0.9: p ranges from about 0.9 to 0.1; wherein R1 is independently selected from residues of aCp-bis(4 carboxyphenoxy)-(CI-C 8 ) alkane, 3,3'-(alkanedioyldioxy)dicinnamic acid or 4,4' (alkanedioyldioxy)dicinnamic acid, (C 2 - C 20 ) alkylene, or (C 2
-C
20 ) alkenylene; each R 2 is independently hydrogen, (C 1
-C
12 ) alkyl or (C 6
-C
10 ) aryl or a protecting group; the R3s in individual m monomers are independently selected from the group consisting of hydrogen, (C 1
-C
6 ) alkyl, (C 2
-C
6 ) alkenyl, (C 2
-C
6 ) alkynyl, (C 6 -Cio) aryl (C 1
-C
2 0 ) alkyl, and -(CH 2
)
2
S(CH
3 ); and R 4 is independently selected from the group consisting of (C 2 C 20 ) alkylene, (C 2
-C
20 ) alkenylene, (C 2
-C
8 ) alkyloxy, (C 2
-C
20 ) alkylene, a residue of a saturated or unsaturated therapeutic diol or bicyclic-fragments of 1,4:3,6 dianhydrohexitols of structural formula(II), and combinations thereof. [0012] In another embodiment, the polymer is a PEUR polymer having a chemical formula described by structural formula (IV), 0 0 HO O H C-0-R 6 -O-"-N-C-C-0-R 4 -O0-c--N
R
3 R3 1 Formula IV wherein n ranges from about 5 to about 150; wherein R 3 s in independently selected from the group consisting of hydrogen, (C 1
-C
6 ) alkyl, (C 2
-C
6 ) alkenyl, (C 2
-C
6 ) alkynyl, (C 6 CIO) aryl (C 1
-C
20 ) alkyl, and -(CH 2
)
2
S(CH
3 ); R4 is selected from the group consisting of
(C
2
-C
20 ) alkylene, (C 2
-C
20 ) alkenylene or alkyloxy, a residue of a saturated or unsaturated therapeutic diol, bicyclic-fragments of 1,4:3,6-dianhydrohexitols of structural formula (II); and combinations thereof, and R6 is independently selected from (C 2
-C
20 ) alkylene,
(C
2
-C
20 ) alkenylene or alkyloxy, bicyclic-fragments of 1,4:3,6-dianhydrohexitols of general formula (II), and combinations thereof; or a PEUR polymer having a chemical structure described by general structural formula (V) WO 2006/083874 PCT/US2006/003412 6 O 0 H O OH 0 0 H C-0-R--0C-N----0-R 4 -- 0-C- - - O-R 6 -0-C-N-C-(CH 2
)
4 -N H 3 R3 m H -O-R2 H. O n Formula (V) wherein n ranges from about 5 to about 150, m ranges about 0.1 to about 0.9: p ranges from about 0.9 to about 0.1; R 2 is independently selected from hydrogen, (C 6 -Cio) aryl
(C
1
-C
2 0 ) alkyl, or a protecting group; the R 3 s in an individual m monomer are independently selected from the group consisting of hydrogen, (C 1
-C
6 ) alkyl, (C 2
-C
6 ) alkenyl, (C 2
-C
6 ) alkynyl, (C 6
-C
1 0 ) aryl (C 1
-C
20 ) alkyl and -(CH 2
)
2
S(CH
3 ); R 4 is selected from the group consisting of (C 2
-C
2 0 ) alkylene, (C 2
-C
20 ) alkenylene or alkyloxy, a residue of a saturated or unsaturated therapeutic diol and bicyclic-fragments of 1,4:3,6 dianhydrohexitols of structural formula (II) and combinations thereof; and R6 is independently selected from (C 2
-C
2 0 ) alkylene, (C 2
-C
2 0 ) alkenylene or alkyloxy, bicyclic fragments of 1,4:3,6-dianhydrohexitols of general formula (II), an effective amount of a residue of a saturated or unsaturated therapeutic diol, and combinations thereof. [0013] In still another embodiment, the polymer is a biodegradable PEU polymer having a chemical formula described by general structural formula (VI): O H O 0 H 0-N-60-0-R4-0-0-N R3 Ra n Formula (VI), wherein n is about 10 to about 150; the R 3 s within an individual n monomer are independently selected from hydrogen, (C 1
-C
6 ) alkyl, (C 2
-C
6 ) alkenyl, (C 2
-C
6 ) alkynyl,
(C
6
-C
10 ) aryl (C 1
-C
20 ) alkyl and -(CH 2
)
2
S(CH
3 ); Ri is independently selected from (C 2 C 2 0 ) alkylene, (C 2
-C
2 0 ) alkenylene, (C 2
-C
8 ) alkyloxy (C 2
-C
20 ) alkylene, a residue of a saturated or unsaturated therapeutic diol; or a bicyclic-fragment of a 1,4:3,6 dianhydrohexitol of structural formula (II); or a PEU having a chemical formula described by structural formula (VII) WO 2006/083874 PCT/US2006/003412 7 0 H 0 0 H 0 H -- N--0-0-R 4 -0-C-C-N -- N-C-(CH2)4-N H R R3 H HC-0-R 2 ] Formula (VII), wherein m is about 0.1 to about 1.0; p is about 0.9 to about 0.1; n is about 10 to about 150; each R 2 is independently hydrogen, (C 1
-C
1 2 ) alkyl or (C 6 -Cio) aryl; the R 3 s within an individual m monomer are independently selected from hydrogen, (C 1
-C
6 ) alkyl, (C 2
-C
6 ) alkenyl, (C 2
-C
6 ) alkynyl, (C 6 -Cio) aryl (C 1
-C
20 ) alkyl and -(CH 2
)
2
S(CH
3 ); each R 4 is independently selected from (C 2
-C
20 ) alkylene, (C 2
-C
2 0 ) alkenylene, (C 2 -Cs) alkyloxy
(C
2
-C
2 0 ) alkylene, a residue of a saturated or unsaturated therapeutic diol; a bicyclic fragment of a 1,4:3,6-dianhydrohexitol of structural formula (II), and combinations thereof. [00141 In still another embodiment, the invention provides methods for inducing an immune response in a mammal by administering to the mammal an invention vaccine delivery composition in the form of a liquid dispersion of particles or molecules of a polymer described by structural formulas I and III-VII, which is conjugated'to an effective amount of class I or class II peptidic antigens. The composition is taken up by antigen presenting cells of the mammal so as to induce an immune response in the mammal. [00151 In yet another embodiment, the invention provides methods for delivering a vaccine to a mammal by administering to the mammal an invention vaccine delivery composition in the form of a liquid dispersion of particles or molecules of a polymer described by structural formulas I and III-VII, which is conjugated to class I or class II peptidic antigens. The composition is taken up by antigen presenting cells of the mammal to deliver the class I or class II peptidic antigens to the mammal.
WO 2006/083874 PCT/US2006/003412 8 A BRIEF DESCRIPTION OF THE FIGURES [0016] Fig. 1 is a schematic drawing illustrating the generation of particles of PEA, PEUR or PEU with various types of active agents, such as a peptidic antigen, dispersed therein by double and triple emulsion procedures described herein. [0017] Fig. 2 is a schematic drawing illustrating invention micelles containing dispersed peptidic antigens, as described herein. [0018] Fig. 3 is a flow chart of the process for making an invention vaccine and testing the in vitro human T-Cell response to the invention vaccine. [0019] Figs. 4A-B are graphs showing T Cell activation in response to dendritic cells exposed to polymer-peptide conjugates. Fig. 4A shows T-Cell proliferation over 96 hours in which PEA-peptide conjugates stimulated significant proliferation over peptide or PEA alone. Fig. 4B shows T-Cell IL-2 secretion over 96 hours in which PEA-peptide (Formula III, Example B 1) stimulated significant IL-2 secretion compared to peptide or PEA alone. DETAILED DESCRIPTION OF THE INVENTION [0020] The invention is based on the discovery that biodegradable polymers that contain at least one amino acid per monomer can be used to create a synthetic vaccine delivery composition for subcutaneous or intramuscular injection or mucosal administration that is reproducible in large quantities, safe (containing no attenuated virus), stable, and can be lyophilized for transportation and storage. Due to structural properties of the polymer used, the vaccine delivery composition provides high copy number and local density of antigen. [0021] The polymer can be formulated into vaccine delivery compositions with different properties. In one embodiment, the polymer acts as a time-release polymer depot releasing peptidic antigen and antigen-polymer fragments to be taken up by APCs and presented by MHC class I or class II alleles as the polymer depot biodegrades in vivo. In other embodiments, the polymer acts as a carrier for the peptidic antigen into the APC, WO 2006/083874 PCT/US2006/003412 9 and the peptidic antigen is released for presentation intracellularly. The polymer may actually stimulate the APCs by inducing phagocytosis of polymer-antigen conjugates. [0022] In yet another embodiment, the invention provides methods for inducing an immune response in a mammal by administering to the mammal an effective amount of an invention vaccine delivery composition, which is taken up by antigen presenting cells of the mammal to induce an immune response in the mammal. [00231 In addition to treatment of humans, the invention vaccine delivery compositions are also intended for use in veterinary treatment of a variety of mammalian patients, such as pets (for example, cats, dogs, rabbits, and ferrets), farm animals (for example, swine, horses, mules, dairy and meat cattle) and race horses. [00241 Polymer particles or polymer molecules delivered directly or released from an in vivo polymer depot are sized to be readily taken up by antigen presenting cells (APCs) and contain peptidic antigens, and optionally adjuvants, dispersed within polymer particles or conjugated to functional groups on the polymer molecules. The APCs display the peptidic antigen via MHC complexes and are recognized by T-cells, such as cytotoxic T-cells, to generate and promote endogenous immune responses leading to destruction of pathogenic cells bearing matching or similar antigens. The polymers used in the invention vaccine delivery composition can be designed to tailor the rate of biodegradation of the polymer depots, molecules and particles to result in continuous contact of the peptidic antigen with antigen presenting cells over a selected period of time. For instance, typically, the polymer depot will degrade over a time selected from about twenty-four hours, about seven days, about thirty days, or about ninety days, or longer. Longer time spans are particularly suitable for providing an implantable vaccine delivery composition that eliminates the need to repeatedly inject the vaccine to obtain a suitable immune response. [0025] The present invention utilizes biodegradable polymer-mediated delivery techniques to elicit an immune response against a wide variety of pathogens, including mucosally transmitted pathogens. The composition affords a vigorous immune response, even when the antigen is by itself weakly immunogenic. Although the individual components of the vaccine delivery composition and methods described herein were WO 2006/083874 PCT/US2006/003412 10 known, it was unexpected and surprising that such combinations would enhance the efficiency of antigens beyond levels achieved when the components were used separately and, moreover, that the polymers used in making the vaccine delivery composition would obviate the need for additional adjuvants in some cases. [0026] Although the invention is broadly applicable for providing an immune response against any of the above-mentioned pathogens, the invention is exemplified herein by reference to influenza virus and HIV. [0027] The method of the invention provides for cell-mediated immunity, and/or humoral antibody responses. Accordingly, the methods of the present invention will find use with any antigen for which cellular and/or humoral immune responses are desired, including antigens derived from viral, bacterial, fungal and parasitic pathogens that may induce antibodies, T- helper cell activity and T-cell cytotoxic activity. Thus, "immune response" as used herein means production of antibodies, T-helper cell activity or T-cell cytotoxic activity specific to the peptidic antigen used. Such antigens include, but are not limited to those encoded by human and animal pathogens and can correspond to either structural or non-structural proteins, polysaccharide-peptide conjugates, or DNA. [0028] For example, the present invention will find use for stimulating an immune response against a wide variety of proteins from the herpes virus family, including proteins derived from herpes simplex virus (HSV) types 1 and 2, such as HSV-1 and HSV-2 glycoproteins gB, gD and gH; antigens derived from varicella zoster virus (VZV), Epstein-Barr virus (EBV) and cytomegalovirus (CMV) including CMV gB and gH; and antigens derived from other human herpes viruses such as HHV6 and HHV7. (See, e.g. Chee et al., Cytomegaloviruses (J. K. McDougall, ed., Springer-Verlag 1990) pp. 125 169, for a review of the protein coding content of cytomegalovirus; McGeoch et al., J. Gen. Virol. (1988) 69:1531-1574, for a discussion of the various HSV-1 encoded proteins; U.S. Pat. No. 5,171,568 for a discussion of HSV-1 and HSV-2 gB and gD proteins and the genes encoding therefor; Baer et al., Nature (1984) 310:207-211, for the identification of protein coding sequences in an EBV genome; and Davison and Scott, J. Gen. Virol. (1986) 67:1759-1816, for a review of VZV.) WO 2006/083874 PCT/US2006/003412 11 [0029] Antigens from the hepatitis family of viruses, including hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), the delta hepatitis virus (HDV), hepatitis E virus (HEV) and hepatitis G virus (HGV), can also be conveniently used in the techniques described herein. By way of example, the viral genomic sequence of HCV is known, as are methods for obtaining the sequence. See, e.g., International Publication Nos. WO 89/04669; WO 90/11089; and WO 90/14436. The HCV genome encodes several viral proteins, including El (also known as E) and E2 (also known as E2/NSI) and an N-terminal nucleocapsid protein (termed "core") (see, Houghton et al., Hepatology (1991) 14:381-388, for a discussion of HCV proteins, including El and E2). Each of these proteins, as well as antigenic fragments thereof, will find use in the present methods. Similarly, the sequence for the s-antigen from HDV is known (see, e.g., U.S. Pat. No. 5,378,814) and this antigen can also be conveniently used in the present methods. Additionally, antigens derived from HBV, such as the core antigen, the surface antigen, sAg, as well as the presurface sequences, pre-S 1 and pre-S2 (formerly called pre S), as well as combinations of the above, such as sAg/pre-S1, sAg/pre-S2, sAg/pre Sl/pre-S2, and pre-Sl/pre-S2, will find use herein. See, e.g., "HBV Vaccines--from the laboratory to license: a case study" in Mackett, M. and Williamson, J. D., Human Vaccines and Vaccination, pp. 159-176, for a discussion of HBV structure; and U.S. Pat. Nos. 4,722,840, 5,098,704, 5,324,513, incorporated herein by reference in their entireties; Beames et al., J. Virol. (1995) 69:6833-6838, Bimbaum et al., J. Virol. (1990) 64:3319 3330; and Zhou et al., J. Virol. (1991) 65:5457-5464. [00301 Antigens derived from other viruses will also find use in the claimed methods, such as without limitation, proteins from members of the families Picornaviridae (e.g., polioviruses, etc.); Caliciviridae; Togaviridae (e.g., rubella virus, dengue virus, etc.); Flaviviridae; Coronaviridae; Reoviridae; Birnaviridae; Rhabodoviridae (e.g., rabies virus, etc.); Filoviridae; Paramyxoviridae (e.g., mumps virus, measles virus, respiratory syncytial virus, etc.); Orthomyxoviridae (e.g., influenza virus types A, B and C, etc.); Bunyaviddae; Arenaviridae; Retroviradae (e.g., HTLV-I; HTLV-II; HIV-1 (also known as HTLV-III LAV, ARV, hTLR, etc.)), including but not limited to antigens from the isolates HIVIlb, HIVSF2, HIVLAV, HIVLAI, HIVMN); HIV-1cM235, HIV-lus 4 ; HIV-2; simian immunodeficiency virus (SIV) among others. Additionally, antigens may also be derived from human papillomavirus (HPV) and the tick-borne encephalitis viruses. See, WO 2006/083874 PCT/US2006/003412 12 e.g. Virology, 3rd Edition (W. K. Joklik ed. 1988); Fundamental Virology, 2nd Edition (B. N. Fields and D. M. Knipe, eds. 1991), for a description of these and other viruses. [0031] More particularly, the envelope proteins from any of the above HIV isolates, including members of the various genetic subtypes of HIV, are known and reported (see, e.g., 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, for a comparison of the envelope sequences of a variety of HIV isolates) and antigens derived from any of these isolates will find use in the present methods. Specifically, the synthetic peptide, R15K (Nehete et al. Antiviral Res. (2002) 56:233-251), derived from the V3 loop of gp120 and having the sequence RIQRGPGRAFVTIGK (SEQ ID NO:1), will have use in the invention compositions and methods. Furthermore, the invention is equally applicable to other immunogenic proteins derived from any of the various HIV isolates, including any of the various envelope proteins such as gp 160 and gp41, gag antigens such as p24gag and p55gag, as well as proteins derived from the pol region. Furthermore, multi-epitope cocktails of polymer-peptide conjugates can be envisioned using various epitopes from HIV proteins. For example, 6 conserved peptides from gp120 and gp4l have been shown to reduce viral load and prevent transmission in a rhesus/SHIV model: SVITQACSKVSFE (S13E) (SEQ ID NO:2), GTGPCTNVSTVQC (Gl3C) (SEQ ID NO:3), LWDQSLKPCVKLT (L13T) (SEQ ID NO:4), VYYGVPVWKEA (VI A) (SEQ ID NO:5), YLRDQQLLGIWG (V12G) (SEQ ID NO:6), and FLGFLGAAGSTMGAASLTLTVQARQ (F25Q) (SEQ ID NO:7) (Nehete et al. Vaccine (2001) 20:813-). The amino acid sequence of the antigen tested in by the Applicants in the invention compositions and methods is IFPGKRTIVAGQRGR (SEQ ID NO:8), wherein all amino acids are natural, L-amino acids. [0032] As explained above, influenza virus is another example of a virus for which the present invention will be particularly useful. Specifically, the envelope glycoproteins HA and NA of influenza A are of particular interest for generating an immune response, as are the nuclear proteins . Numerous HA subtypes of influenza A have been identified (Kawaoka et al., Virology (1990) 12:759-767; Webster et al., "Antigenic variation among type A influenza viruses," p. 127-168. In: P. Palese and D. W. Kingsbury (ed.), Genetics WO 2006/083874 PCT/US2006/003412 13 of influenza viruses. Springer-Verlag, New York). Thus, proteins derived from any of -these isolates can also be used in the immunization techniques described herein. In particular, the conserved 13 amino acid sequence of HA can be used in the invention vaccine delivery composition and methods. In H3 strains used in current vaccine formulations, this amino acid sequence is PRYVKQNTLKLAT (SEQ ID NO:9), and in H5 strains it is predominantly PKYVKSNRLVLAT (SEQ ID NO: 10). [00331 The methods described herein will also find use with numerous bacterial antigens, such as those derived from organisms that cause diphtheria, cholera, tuberculosis, tetanus, pertussis, meningitis, and other pathogenic organism, including, without limitation, Meningococcus A, B and C, Hemophilus influenza type B (HIB), and Helicobacter pylori. Examples of parasitic antigens include those derived from organisms causing malaria and Lyme disease. [0034] Furthermore, the methods described herein provide a means for treating a variety of malignant cancers. For example, the composition of the present invention can be used to mount both humoral and cell-mediated immune responses to particular proteins specific to the cancer in question, such as an activated oncogene, a fetal antigen, or an activation marker. Such tumor antigens include any of the various MAGEs (melanoma associated antigen E), including MAGE 1, 2, 3, 4, etc. (Boon, T. Scientific American (March 1993):82-89); any of the various tyrosinases; MART 1 (melanoma antigen recognized by T cells), mutant ras; mutant p53; p 97 melanoma antigen; CEA (carcinoembryonic antigen), among others. Additional melanoma peptidic antigens useful in the invention compositions and compositions include the following: DESIGNATION ANTIGEN PROTEIN SEQUENCE Martl-27 AAGIGILTV MART1 (SEQ ID NO:11) Gp100-209* ITDQVPFSV Melanocyte lineage-specific antigen (SEQ ID NO:12) GP100 Gp100-154 KTWGQYWQV Melanocyte lineage-specific antigen (SEQ ID NO:13) GP100 Gp100-280 YLEPGPVTA Melanocyte lineage-specific antigen (SEQ ID NO:14) GP100 *GP100 is also called melanoma-associated ME20 antigen.
WO 2006/083874 PCT/US2006/003412 14 [0035] It is readily apparent that the subject invention can be used to prevent or treat a wide variety of diseases. [0036] The peptidic antigens dispersed within the polymers in the invention vaccine delivery compositions can have any suitable length, but may incorporate a peptidic antigen segment of 8 to about 30 amino acids that is recognized by a peptide-restricted T lymphocyte. Specifically, the peptidic antigen segment that is recognized by a corresponding class I peptide-restricted cytotoxic T-cell contains 8 to about 12 amino acids, for example 9 to about 11 amino acids and, the peptidic antigen segment that is recognized by a corresponding class II peptide-restricted T-helper cell contains 8 to about 30 amino acids, for example about 12 to about 24 amino acids. [0037] While natural T-cell mediated immunity works via presentation of peptide epitopes by MHC molecules (on the surface of APCs), MHCs can also present peptide adjunct--in particular glycol-peptides and lipo-peptides, in which the peptide portion is held by the MHC so as to display to the T-cell the sugar or lipid moiety. This consideration is particularly relevant in cancer vaccinology because several tumors over express glyco-derivatized proteins or lipo-derivatized proteins, and the glyco- or lipo derivatized peptide fragments of these can, in some cases, be powerful T-cell epitopes. Moreover, the lipid in such T-cell epitopes can be a glyco-lipid. [00381 Unlike the normal peptide-alone presentation, in these cases T-cell recognition is dominated by the sugar or lipid group on the peptide, so much so that short synthetic peptides that bind to MHCs with high affinity, but were not derived from the tumor proteins, yet to which the tumor-associated sugar or lipid molecule is covalently attached synthetically have been successfully used as peptidic antigens. This approach to building an artificial T-cell epitope directed against a natural tumor cell line has recently been adopted by Franco et al., J. Exp. Med (2004) 199(5):707-716. Therefore, synthetic peptide derivatives and even peptidomimetics can be substituted for the peptidic antigen in the invention vaccine delivery compositions to act as high-affinity MHC-binding ligands that form a platform for the presentation to T-cells of peptide branches and non peptidic antigens.
WO 2006/083874 PCT/US2006/003412 15 [00391 Accordingly, the term "peptidic antigen", as used herein, refers to peptides, wholly peptide derivatives (such as branched peptides) and covalent hetero- (such as glyco- and lipo- and glycolipo-) derivatives of peptides. It also is intended to encompass fragments of such materials that are specifically bound by a specific antibody or specific T lymphocyte. [0040] The peptidic antigens can be synthesized using any technique as is known in the art. The peptidic antigens can also include "peptide mimetics." Peptide analogs are commonly used in the pharmaceutical industry as non-peptide bioactive agents with properties analogous to those of the template peptide. These types of non-peptide compound are termed "peptide mimetics" or "peptidomimetics." Fauchere, J. (1986) Adv. Bioactive agent Res., 15:29; Veber and Freidinger (1985) TINS p. 392; and Evans et al. (1987) J. Med. Chem., 30:1229; and are usually developed with the aid of computerized molecular modeling. Generally, peptidomimetics are structurally similar to a paradigm polypeptide (i.e., a polypeptide that has a biochemical property or pharmacological activity), but have one or more peptide linkages optionally replaced by a linkage selected from the group consisting of: - -CH 2 NH--, -- CH 2 S--, CH 2
-CH
2 --, - CH=CH-- (cis and trans), --COCH 2 --, --CH(OH)CH 2 --, and --CH 2 SO--, by methods known in the art and further described in the following 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 review); Morley, J.S., Trends. Pharm. Sci., (1980) pp. 463-468 (general review); Hudson, D. et al., Int. J. Pept. Prot. Res., (1979) 14:177-185 (--CH 2 NH--, CH 2
CH
2 --); Spatola, A.F. et al., Life Sci., (1986) 38:1243-1249 (--CH 2 -S--); Harm, M. M., J. Chem. Soc. Perkin Trans 1(1982) 307-314 (--CH=CH--, cis and trans); Almquist, R.G. et al., J. Med. Chem., (1980) 23:2533 (-
COCH
2 --); 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
(-CH
2 -S-). Such peptide mimetics may have significant advantages over polypeptide embodiments, including, for example: more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, WO 2006/083874 PCT/US2006/003412 16 etc.), altered specificity (e.g., a broad-spectrum of biological activities), reduced antigenicity, and others. [00411 Additionally, substitution of one or more amino acids within a peptide (e.g., with a D-Lysine in place of L-Lysine) may be used to generate more stable peptides and peptides resistant to endogenous proteases. Alternatively, the synthetic peptidic antigens, e.g., covalently bound to the biodegradable polymer, can also be prepared from D-amino acids, referred to as inverso peptides. When a peptide is assembled in the opposite direction of the native peptide sequence, it is referred to as a retro peptide. In general, peptides prepared from D-amino acids are very stable to enzymatic hydrolysis. Many cases have been reported of preserved biological activities for retro-inverso or partial retro-inverso peptides (US patent, 6,261,569 B 1 and references therein; B. Fromme et al., Endocrinology (2003)144:3262-3269. [00421 The selected peptidic antigen is combined with the biodegradable polymer, with or without adjuvant, for subsequent administration to a mammalian subject. The invention vaccine delivery composition can be prepared for intravenous, mucosal, intramuscular, or subcutaneous delivery. For example, useful polymers in the methods described herein include, but are not limited to, the PEA, PEUR and PEU polymers described herein. These polymers can be fabricated in a variety of molecular weights, and the appropriate molecular weight for use with a given antigen is readily determined by one of skill in the art. Thus, e.g., a suitable molecular weight will be on the order of about 5,000 to about 300,000, 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 persistence, protection, and delivery of the peptide into APCs, by the polymer composition itself may be sufficient to provide adjuvant activity. In other embodiments the invention vaccine delivery composition may include an adjuvant that can augment immune responses, especially cellular immune responses, to soluble protein antigen, by increasing delivery of antigen, stimulating cytokine production, and/or stimulating antigen presenting cells. The adjuvants can be administered by dispersing the adjuvant along with the peptidic antigen within the polymer matrix, for example by conjugating the adjuvant to the antigen. Alternatively, the adjuvants can be administered concurrently with the vaccine delivery composition of WO 2006/083874 PCT/US2006/003412 17 the invention, e.g., in the same composition or in separate compositions. For example, an adjuvant can be administered prior or subsequent to the vaccine delivery composition of the invention. Alternatively still, the adjuvant or an adjuvant/peptidic antigen can be chemically bonded to the polymer as described herein for simultaneous delivery. Such adjuvants include, but are not limited to: (1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.; (2) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides or bacterial cell wall components), such as for example (a) MF59 (International Publication No. WO 90/14837), containing 5% Squalene, 0.5% Tween 80TM, and 0.5% Span 85, optionally containing various amounts of MTP-PB, formulated into submicron particles using a microfluidizer such as Model 110Y microfluidizer (Microfluidics, Newton, Mass.), (b) SAF, containing 10% Squalane, 0.4% Tween 8OTM, 5% pluronic blocked polymer L121, and thr-MDP, either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) RibiTM adjuvant composition (RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene, 0.2% Tween 80TM, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (DetoxTM); (3) saponin adjuvants, such as StimulonTM (Cambridge Bioscience, Worcester, Mass.) may be used or particle generated therefrom such as ISCOMs (immunostimulating complexes); (4) Complete Freunds Adjuvant (CFA) and Incomplete Freunds Adjuvant (IFA); (5) cytokines, such as interleukins (IL-1, IL-2 etc.), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.; (6) detoxified mutants of a bacterial ADP-ribosylating toxin such as a cholera toxin (CT), a pertussis toxin (PT), or an E. coli heat-labile toxin (LT), particularly LT-K63 (where lysine is substituted for the wild-type amino acid at position 63) LT-R72 (where arginine is substituted for the wild-type amino acid at position 72), CT-S 109 (where serine is substituted for the wild-type amino acid at position 109), and PT-K9/G129 (where lysine is substituted for the wild-type amino acid at position 9 and glycine substituted at position 129) (see, e.g., International Publication Nos. W093/13202 and W092/19265); and (7) QS21, a purified form of saponin and 3D-monophosphoryl lipid A (MPL), a nontoxic derivative of lipopolysaccharide (LPS), to enhance cellular and humoral immune responses (Moore, et al., Vaccine. 1999 Jun 4;17(20-21):2517-27).
WO 2006/083874 PCT/US2006/003412 18 Other substances that act as immunostimulating agents may also be used to enhance the effectiveness of the composition. [0044] Polymers suitable for use in the practice of the invention bear functionalities that allow facile covalent attachment of the peptidic antigen, adjuvant, or antigen adjuvant conjugate to the polymer. For example, a polymer bearing carboxyl groups can readily react with an amino moiety, thereby covalently bonding the peptide to the polymer via the resulting amide group. As will be described herein, the biodegradable polymer and the peptide or adjuvant may contain numerous complementary functional groups that can be used to covalently attach the peptidic antigen and/or the adjuvant to the biodegradable polymer. [0045] The polymer in the invention vaccine delivery composition plays an active role in the endogenous immune processes at the site of implant by holding the peptidic antigen and optional adjuvant at the site of injection for a period of time sufficient to allow the individual's immune cells to interact with the peptidic antigen and optional adjuvant to affect immune processes, while slowly releasing the particles or polymer molecules containing such agents during biodegradation of the polymer. The fragile biologic peptidic antigen is protected by the more slowly biodegrading polymer to increase half life and persistence of the antigen. [0046] The polymer itself may also have an active role in delivery of the antigen into APCs by stimulating phagocytosis of the polymer-antigen composition. In addition, the polymers disclosed herein (e.g., those having structural formulae (I and III-VIII), upon enzymatic degradation, provide essential amino acids that nurture cells while the other breakdown products can be metabolized in the way that fatty acids and sugars are metabolized. Uptake of the polymer with antigen is safe: studies have shown that the APCs survive, function normally, and can metabolize/clear the polymer degradation products. These polymers and the vaccine delivery composition are, therefore, substantially non-inflammatory to the subject both at the site of injection and systemically, apart from the trauma caused by injection itself. Moreover, in the case of active uptake of polymer by APCs, the polymer may act as an adjuvant for the antigen, so there is no essential requirement to formulate an adjuvant separately.
WO 2006/083874 PCT/US2006/003412 19 [00471 The biodegradable polymers useful in forming the invention biocompatible vaccine delivery compositions include those comprising at least one amino acid conjugated to at least one non-amino acid moiety per monomer. The term "non-amino acid moiety" as used herein includes various chemical moieties, but specifically excludes amino acid derivatives and peptidomimetics as described herein. In addition, the polymers containing at least one amino acid are not contemplated to include polyamino acid segments, including naturally occurring polypeptides, unless specifically described as such. In one embodiment, the non-amino acid is placed between two adjacent amino acids in the monomer. In another embodiment, the non-amino acid moiety is hydrophobic. The polymer may also be a block co-polymer. [0048] Preferred for use in the invention compositions and methods are polyester amides (PEAs) and polyester urethanes (PEURs) that have built-in functional groups on PEA or PEUR backbones, and these built-in functional groups can react with other chemicals and lead to the incorporation of additional functional groups to expand the functionality of PEA or PEUR further. Therefore, such polymers used in the invention methods are ready for reaction with other chemicals having a hydrophilic structure to increase water solubility and with peptidic antigens, adjuvants, and other agents, without the necessity of prior modification. [0049] In addition, the polymers used in the invention vaccine delivery compositions display no hydrolytic degradation when tested in a saline (PBS) medium, but in an enzymatic solution, such as chymotrypsin or CT, a uniform erosive behavior has been observed. In one embodiment the PEAs wherein the polymer is a PEA having a chemical formula described by structural formula (I), 0 O HO 0 H -CRI-C-N--0-0-R4-O-CC-N R R H Formula (I) wherein n ranges from about 5 to about 150; R' is independently selected from residues of cr-bis(4-carboxyphenoxy)-(CI-Cs) alkane, 3,3'-(alkanedioyldioxy)dicinnamic acid or WO 2006/083874 PCT/US2006/003412 20 4,4'-(alkanedioyldioxy)dicinnamic acid, (C 2 - C 20 ) alkylene, or (C 2
-C
20 ) alkenylene; the Rs in individual n monomers are independently selected from the group consisting of hydrogen, (Ci-C 6 ) alkyl, (C 2
-C
6 ) alkenyl, (C 2
-C
6 ) alkynyl, (C 6
-C
10 ) aryl (C 1
-C
20 ) alkyl, and -(CH 2
)
2
S(CH
3 ); and R 4 is independently selected from the group consisting of (C 2 C 20 ) alkylene, (C 2
-C
20 ) alkenylene, (C 2 -Cs) alkyloxy, (C 2
-C
2 0 ) alkylene, a residue of a saturated or unsaturated therapeutic diol, bicyclic-fragments of 1,4:3,6-dianhydrohexitols of structural formula (II), and combinations thereof, (C 2 - C 20 ) alkylene, and (C 2
-C
20 ) alkenylene; CH O
H
2 C O CCH 2 \0 ICH Formula (II) or a PEA polymer having a chemical formula described by structural formula III: O 0 HO O H O H H6H { ~R 1-1[ _ _ -I -R -C-N-C-C-0-R 4 -O-- N -R -C-N-C-(CH 2
)
4
-N-
3 k3 '3 I Z H m H C-O-R2 Formula (III) wherein n ranges from about 5 to about 150, m ranges about 0.1 to 0.9: p ranges from about 0.9 to 0.1; wherein R 1 is independently selected from residues of c-bis(4 carboxyphenoxy)-(CI-Cs) alkane, 3,3'-(alkanedioyldioxy)dicinnamic acid or 4,4' (alkanedioyldioxy)dicinnamic acid, (C 2 - C 2 0 ) alkylene, or (C 2
-C
20 ) alkenylene; each R2 is independently hydrogen, (C 1
-C
1 2 ) alkyl or (C 6 -Cio) aryl or a protecting group; the R 3 s in individual m monomers are independently selected from the group consisting of hydrogen, (Ci-C 6 ) alkyl, (C 2
-C
6 ) alkenyl, (C 2
-C
6 ) alkynyl, (C 6
-C
1 0 ) aryl (C 1
-C
2 0 ) alkyl, and -(CH 2
)
2
S(CH
3 ); and R 4 is independently selected from the group consisting of (C 2 C 20 ) alkylene, (C 2
-C
20 ) alkenylene, (C 2
-C
8 ) alkyloxy, (C 2
-C
2 0 ) alkylene, a residue of a saturated or unsaturated therapeutic diol or bicyclic-fragments of 1,4:3,6 dianhydrohexitols of structural formula (II), and combinations thereof.
WO 2006/083874 PCT/US2006/003412 21 [00501 In another embodiment, the polymer is a PEUR polymer having a chemical formula described by structural formula (IV), 0 0 H O OH C-0-R 6 -0-0-N- --- R 4 -O-0-C-N H R 3 R3 H n Formula IV wherein n ranges from about 5 to about 150; wherein R 3 s in independently selected from the group consisting of hydrogen, (C1-C 6 ) alkyl, (C 2
-C
6 ) alkenyl, (C 2
-C
6 ) alkynyl, (C 6 C 10 ) aryl (C 1
-C
2 0 ) alkyl, and -(CH 2
)
2
S(CH
3 ); R is selected from the group consisting of
(C
2
-C
20 ) alkylene, (C 2
-C
2 0 ) alkenylene or alkyloxy, a residue of a saturated or unsaturated therapeutic diol, bicyclic-fragments of 1,4:3,6-dianhydrohexitols of structural formula (II); and combinations thereof, and R 6 is independently selected from (C 2
-C
20 ) alkylene,
(C
2
-C
20 ) alkenylene or alkyloxy, bicyclic-fragments of 1,4:3,6-dianhydrohexitols of general formula (II), and combinations thereof. [0051] In another embodiment the polymer is a PEUR having a chemical structure described by general structural formula (V) O 0 HO OH 0 0 H -0-0-R -0--N-O-0-0-R4 --- N- -- -0--R-0--N-O-(CH2)4-N HH R.mH.HO-R2 H O "I Formula (V) wherein n ranges from about 5 to about 150, m ranges about 0.1 to about 0.9: p ranges from about 0.9 to about 0.1; R 2 is independently selected from hydrogen, (C 6 -C1o) aryl
(C
1
-C
20 ) alkyl, or a protecting group; the R 3 s in an individual m monomer are independently selected from the group consisting of hydrogen, (C 1
-C
6 ) alkyl, (C 2
-C
6 ) alkenyl, (C 2
-C
6 ) alkynyl, (C 6
-C
10 ) aryl (C 1
-C
2 0 ) alkyl and -(CH 2
)
2
S(CH
3 ); R4 is selected from the group consisting of (C 2
-C
20 ) alkylene, (C 2
-C
20 ) alkenylene or alkyloxy, a residue of a saturated or unsaturated therapeutic diol and bicyclic-fragments of 1,4:3,6 dianhydrohexitols of structural formula (II) and combinations thereof; and R6 is WO 2006/083874 PCT/US2006/003412 22 independently selected from (C 2
-C
20 ) alkylene, (C 2
-C
20 ) alkenylene or alkyloxy, bicyclic fragments of 1,4:3,6-dianhydrohexitols of general formula (II), an effective amount of a residue of a saturated or unsaturated therapeutic diol, and combinations thereof. [00521 In still another embodiment, the polymer is a biodegradable PEU polymer having a chemical formula described by general structural formula (VI): 0 H 0 O H C-N-0-0-0-R4-O-C-0-N 4 R Ra Hn Formula (VI), wherein n is about 10 to about 150; the R 3 s within an individual n monomer are independently selected from hydrogen, (Ci-C 6 ) alkyl, (C 2
-C
6 ) alkenyl, (C 2
-C
6 ) alkynyl,
(C
6 -CIO) aryl (C 1
-C
2 0 ) alkyl and -(CH 2
)
2
S(CH
3 ); R 4 is independently selected from (C 2 C 20 ) alkylene, (C 2
-C
20 ) alkenylene, (C 2
-C
8 ) alkyloxy (C 2
-C
20 ) alkylene, a residue of a saturated or unsaturated therapeutic diol; or a bicyclic-fragment of a 1,4:3,6 dianhydrohexitol of structural formula (II). [0053] In still another embodiment the polymer is a PEU having a chemical formula described by structural formula (VII) H 0 O H 0 H -0-N-0-0-0-R 4 -O-0-0-C - -(CH2)4 R3 R3 Hm H -O-2 Hpn H C ,O-R 2 Formula (VII), wherein m is about 0.1 to about 1.0; p is about 0.9 to about 0.1; n is about 10 to about 150; each R 2 is independently hydrogen, (Ci-C 12 ) alkyl or (C 6 -Cio) aryl; the R 3 s within an individual m monomer are independently selected from hydrogen, (Ci-C 6 ) alkyl, (C 2
-C
6 ) alkenyl, (C 2
-C
6 ) alkynyl, (C 6 -CIO) aryl (C 1
-C
20 ) alkyl and -(CH 2
)
2
S(CH
3 ); each R 4 is independently selected from (C 2
-C
20 ) alkylene, (C 2
-C
2 0 ) alkenylene, (C 2
-C
8 ) alkyloxy
(C
2
-C
20 ) alkylene, a residue of a saturated or unsaturated therapeutic diol; a bicyclic- WO 2006/083874 PCT/US2006/003412 23 fragment of a 1,4:3,6-dianhydrohexitol of structural formula (II), and combinations thereof. [0054] For example, in one alternative in the PEA polymer used in the invention particle delivery composition, at least one R 1 is a residue of Cp-bis(4-carboxyphenoxy) (Ci-Cs) alkane, 3,3'-(alkanedioyldioxy)dicinnamic acid, or 4,4' (alkanedioyldioxy)dicinnamic acid and R 4 is a bicyclic-fragment of a 1,4:3,6 dianhydrohexitol of general formula(II). In another alternative, R 1 in the PEA polymer is either a residue of pc-bis (4-carboxyphenoxy) (C 1
-C
8 ) alkane, 3,3'- (alkanedioyldioxy)dicinnamic acid ,or 4,4'-(alkanedioyldioxy)dicinnamic acid. In yet another alternative, in the PEA polymer R' is a residue pC-bis (4-carboxyphenoxy) (C 1 C 8 ) alkane, such as 1,3-bis(4-carboxyphenoxy)propane (CPP), 3,3' (alkanedioyldioxy)dicinnamic acid or 4,4'-(adipoyldioxy)dicinnamic acid and R 4 is a bicyclic-fragment of a 1,4:3,6-dianhydrohexitol of general formula (II), such as DAS. [00551 In another embodiment, the polymer is a PEUR having a chemical formula described by structural formula (IV), 0 0 H O OH
C-O-R
6 -O-C-N-0-0-0-R 4 -O-C-C-N
HR
3 R3 H Formula (IV) wherein n ranges from about 5 to about 150; wherein Rs in independently selected from the group consisting of hydrogen, (Ci-C 6 ) alkyl, (C 2
-C
6 ) alkenyl, (C 2
-C
6 ) alkynyl, (C 6 C 1 0 ) aryl (C 1
-C
2 0 ) alkyl and -(CH 2
)
2
S(CH
3 ); R 4 is selected from the group consisting of
(C
2
-C
2 0 ) alkylene, (C 2
-C
2 0 ) alkenylene or alkyloxy, a residue of a saturated or unsaturated therapeutic diol and bicyclic-fragments of 1,4:3,6-dianhydrohexitols of structural formula (II); and R 6 is independently selected from (C 2
-C
20 ) alkylene, (C 2
-C
20 ) alkenylene or alkyloxy, bicyclic-fragments of 1,4:3,6-dianhydrohexitols of general formula (II), an effective amount of a residue of a saturated or unsaturated therapeutic diol, and combinations thereof; WO 2006/083874 PCT/US2006/003412 24 or a PEUR polymer having a chemical structure described by general structural formula (V) O O H O OH 0 0 H -0-0-R6-O-0-N---0-0-R4-0-0-0-N-- -0-RI-0-0---(H)4-N ] mH O-R2 H O " Formula (V) wherein n ranges from about 5 to about 150, m ranges about 0.1 to about 0.9: p ranges from about 0.9 to about 0.1; R 2 is independently selected from hydrogen, (C 6
-C
10 ) aryl
(CI-C
2 0 ) alkyl, or a protecting group; the Rs in an individual m monomer are independently selected from the group consisting of hydrogen, (C-C 6 ) alkyl, (C 2
-C
6 ) alkenyl, (C 2
-C
6 ) alkynyl, (C 6
-C
1 0 ) aryl (C-C 20 ) alkyl, and -(CH 2
)
2
S(CH
3 ); R 4 is selected from the group consisting of (C 2
-C
20 ) alkylene, (C 2
-C
20 ) alkenylene or alkyloxy, and bicyclic-fragmenis of 1,4:3,6-dianhydrohexitols of structural formula (II); and R 6 is independently selected from (C 2
-C
20 ) alkylene, (C 2
-C
20 ) alkenylene or alkyloxy, bicyclic fragments of 1,4:3,6-dianhydrohexitols of general formula (II), and combinations thereof. [0056] In one alternative in the PEUR polymer, at least one of R! is a bicyclic fragment of 1,4:3,6-dianhydrohexitol (formula (II)), such as 1,4:3,6-dianhydrosorbitol (DAS); or R6 is a bicyclic fragment of 1,4:3,6-dianhydrohexitol, such as 1,4:3,6 dianhydrosorbitol (DAS). In still alternative in the PEUR polymer, R and/or R6 is a bicyclic fragment of 1,4:3,6-dianhydrohexitol, such as 1,4:3,6-dianhydrosorbitol (DAS). [00571 In yet another embodiment the polymer in the invention particle delivery composition is a PEU polymer having a chemical formula described by general structural formula (VI): O H 0 O H 0-N-C-0--R 4 -0-0--N Formula (VI), WO 2006/083874 PCT/US2006/003412 25 wherein n is about 10 to about 150; each Ras within an individual n monomer are independently selected from hydrogen, (C-C 6 ) alkyl, (C 2
-C
6 ) alkenyl, (C 2
-C
6 ) alkynyl,
(C
6
-C
10 ) aryl (C-C 20 ) alkyl and -(CH 2
)
2
S(CH
3 ); R 4 is independently selected from (C 2 C 20 ) alkylene, (C 2
-C
20 ) alkenylene, (C 2
-C
8 ) alkyloxy (C 2
-C
20 ) alkylene, an effective amount of a residue of a saturated or unsaturated therapeutic diol; or a bicyclic-fragment of a 1,4:3,6-dianhydrohexitol of structural formula (II); or a PEU having a chemical formula described by structural formula (VII) H 0 O H 0 H - N-6-0-0-R 4 -O-C-6-- I-i-(CH2)4 3 R 3 H H CO-R 2 Formula (VII), wherein m is about 0.1 to about 1.0; p is about 0.9 to about 0.1; n is about 10 to about 150; each R2 is independently hydrogen, (C-C 12 ) alkyl or (C 6
-C
1 o) aryl or other protective group; and the R 3 s within an individual m monomer are independently selected from hydrogen, (C-C 6 ) alkyl, (C 2
-C
6 ) alkenyl, (C 2
-C
6 ) alkynyl, (C 6
-C
10 ) aryl (C
C
2 o)alkyl, -(CH 2
)
3 - and -(CH 2
)
2
S(CH
3 ); R4 is independently selected from (C 2
-C
20 ) alkylene, (C 2
-C
2 0 ) alkenylene, (C 2 -Cs) alkyloxy (C 2
-C
2 0 ) alkylene, an effective amount of a residue of a saturated or unsaturated therapeutic diol; or a bicyclic-fragment of a 1,4:3,6-dianhydrohexitol of structural formula (II); [00581 Suitable protecting groups for use in practice of the invention include t-butyl and others as are known in the art. Suitable bicyclic-fragments of 1,4:3,6 dianhydrohexitols can be derived from sugar alcohols, such as D-glucitol, D-mannitol, and L-iditol. For example, 1,4:3,6-dianhydrosorbitol (isosorbide, DAS) is particularly. suited for use as a bicyclic-fragment of 1,4:3,6-dianhydrohexitol. [0059] These PEU polymers can be fabricated as high molecular weight polymers useful for making the invention vaccine delivery compositions for delivery to humans and other mammals of a variety of pharmaceutical and biologically active agents. The invention PEUs incorporate hydrolytically cleavable ester groups and non-toxic, naturally occurring monomers that contain c-amino acids in the polymer chains. The ultimate WO 2006/083874 PCT/US2006/003412 26 biodegradation products of PEUs will be a-amino acids (whether biological or not), diols, and CO 2 . In contrast to the PEAs and PEURs, the invention PEUs are crystalline or semi crystalline and possess advantageous mechanical, chemical and biodegradation properties that allow formulation of completely synthetic, and hence easy to produce, crystalline and semi-crystalline polymer particles, for example nanoparticles. [0060] For example, the PEU polymers used in the invention vaccine delivery compositions have high mechanical strength, and surface erosion of the PEU polymers can be catalyzed by enzymes present in physiological conditions, such as hydrolases. [0061] In one alternative in the PEU polymer, at least one R' is a bicyclic fragment of a 1,4:3,6-dianhydrohexitol, such as 1,4:3,6-dianhydrosorbitol (DAS). [0062] Suitable protecting groups for use in practice of the invention include t-butyl and others as are known in the art. Suitable bicyclic-fragments of 1,4:3,6 dianhydrohexitols can be derived from sugar alcohols, such as D-glucitol, D-mannitol, and L-iditol. For example, dianhydrosorbitol is particularly suited for use as a bicyclic fragment of 1,4:3,6-dianhydrohexitol. [0063] In one alternative, the R 3 s in at least one n monomer are CH 2 Ph and the a amino acid used in synthesis is L-phenylalanine. In alternatives wherein the R 3 s within a monomer are -CH 2
-CH(CH
3
)
2 , the polymer contains the a-amino acid, leucine. By varying the R 3 s, other a-amino acids can also be used, e.g., glycine (when the R 3 s are -H), proline (when the R 3 s are ethylene amide); alanine (when the R 3 s are -CH 3 ), valine (when the R 3 s are -CH(CH 3
)
2 ), isoleucine (when the R 3 s are -CH(CH 3
)-CH
2
-CH
3 ), phenylalanine (when the R 3 s are -CH 2
-C
6
H
5 ); lysine (when the R 3 s are -(CH 2
)
4
-NH
2 ); or methionine (when the R 3 s are -(CH 2
)
2
S(CH
3 ). [0064] In yet a further embodiment wherein the polymer is a PEA, PEUR or PEU of formula I or III-VII, at least one of the R 3 s further can be -(CH 2
)
3 - and the at least one of the R 3 s cyclizes to form the chemical structure described by structural formula Formula H O I Il
-N-C-C-O
I I
H
2
C,C.CH
2 Formula (XVIII)
H
2 WO 2006/083874 PCT/US2006/003412 27 [00651 When the R 3 s are -(CH 2
)
3 , an a-imino acid analogous to pyrrolidine-2 carboxylic acid (proline) is used. [00661 The PEAs, PEURs and PEUs are biodegradable polymers that biodegrade substantially by enzymatic action so as to release the dispersed peptidic antigen and optional adjuvant over time. Due to structural properties of the polymer used, the invention vaccine delivery compositions provide for stable loading of the peptidic antigens and optional adjuvants while preserving the three dimensional structure thereof and, hence, the bioactivity. 100671 As used herein, the terms "amino acid" and "a-amino acid" mean a chemical compound containing an amino group, a carboxyl group and a pendent R group, such as the R3 groups defined herein. As used herein, the term "biological a-amino acid" means the amino acid(s) used in synthesis are selected from phenylalanine, leucine, glycine, alanine, valine, isoleucine, methionine, proline, or a mixture thereof. [0068] In the PEA, PEUR and PEU polymers useful in practicing the invention, multiple different a-amino acids can be employed in a single polymer molecule. These polymers may comprise at least two different amino acids per repeat unit and a single polymer molecule may contain multiple different a-amino acids in the polymer molecule, depending upon the size of the molecule. In one alternative, at least one of the a-amino acids used in fabrication of the invention polymers is a biological a-amino acid. 10069] For example, when the R 3 s are CH 2 Ph, the biological a-amino acid used in synthesis is L-phenylalanine. In alternatives wherein the R 3 s are CH 2
-CH(CH
3 )2, the polymer contains the biological a-amino acid, L-leucine. By varying the R 3 s within co monomers as described herein, other biological a-amino acids can also be used, e.g., glycine (when the R 3 s are H), alanine (when the Rs are CH 3 ), valine (when the R 3 s are
CH(CH
3
)
2 ), isoleucine (when the R3s are CH(CH 3
)-CH
2 -CH3), phenylalanine (when the Rs are CH 2 -- C 6
H
5 ), or methionine (when the R 3 s are -(CH 2
)
2
S(CH
3 ), and mixtures thereof. When the RWs are -(CH 2
)
3 - as in 2-pyrrolidinecarboxylic acid (proline), a biological a-imino acid can be used. In yet another alternative embodiment, all of the WO 2006/083874 PCT/US2006/003412 28 various cx-amino acids contained in the invention vaccine delivery compositions are biological c-amino acids, as described herein. [0070] The polymer molecules may also have the peptidic antigen conjugated thereto via a linker or incorporated into a crosslinker between molecules. For example, in one embodiment, the polymer is contained in a polymer-antigen conjugate having structural formula VIII: 0 0 0 00 0 11H H 4- -1 H 1- 1-H 'H ) C-R-C1-N-C-C-O-R 4 - C- N C R'-C-N-CH- -NH
R
3 In C=O R5 R7 Formula (VIII) wherein n, m, p, R 1 , R 3 , and R 4 are as above, R is selected from the group consisting of 0-, -S-, and -NR-, wherein R 8 is H or (C 1 -Cs)alkyl; and R 7 is the peptidic antigen. [0071] In yet another embodiment, two molecules of the polymer of structural formula (IX) can be crosslinked to provide an -R 5 -R7-R 5 -conjugate. In another embodiment, as shown in structural formula IX below, the peptidic antigen is covalently linked to two parts of a single polymer molecule of structural formula IV through the -R 5
-R
7
-R
5 conjugate and R 5 is independently selected from the group consisting of -0-, -S-, and NR 8 -, wherein R 8 is H or (Ci-C 8 ) alkyl; and R 7 is the peptidic antigen. R 3 R 3 H II H H
-R
1 -C-N-C-C-0-R 4 -0-C-CH-N C-RI-C-N-C-(CH) 4 -NH 11 H If " 11 11 o 0 0 0 0 0 p1n mi p
R
5 R7 R 5 0 O iH -(CH2)4-C-NH-C-R NH-C-C-0-R 4 -O-C-CH-N-C-R-C H H F 0 0 0 0 (Xn Formula (IX) WO 2006/083874 PCT/US2006/003412 29 [0072] Alternatively still, as shown in structural formula (X) below, a linker, -X-Y-, can be inserted between R and peptidic antigen R 7 , in the molecule of structural formula (VIII), wherein X is selected from the group consisting of (C 1
-C
1 8 ) alkylene, substituted alkylene, (C 3 -Cs) cycloalkylene, substituted cycloalkylene, 5-6 membered heterocyclic system containing 1-3 heteroatoms selected from the group 0, N, and S, substituted heterocyclic, (C 2
-C
18 ) alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, C 6 and CI aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkylaryl, substituted alkylaryl, arylalkynyl, substituted arylalkynyl, arylalkenyl, substituted arylalkenyl, arylalkynyl, substituted arylalkynyl and wherein the substituents are selected from the group H, F, Cl, Br, I, (Ci-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[(0 2
)(C
1
-C
6 ) alkyl], -C[(=O)(Ci-C 6 ) alkyl], CF 3 ,-O[(CO)-( C 1 C) alkyl], -S(0 2
)[N(R
9
R'
0 )], -NH[(C=0)(C-C 6 ) alkyl],
-NH(C=O)N(R
9 R"), -N(R 9
R'
0 ); where R 9 and R' 0 are independently H or (C 1
-C
6 ) alkyl; and Y is selected from the group consisting of -O-, -S-, -S-S-, -S(O)-,-S(0 2 )-, -NR C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)NH-, -NR 8 C(=O)-, -C(=O)NR-, -N
R
8 C(=O)NR-, -N R 8
C(=O)NR
8 -, and -NR 8 C(=S)N R'-. 10 0 0 00 0
C-R
1 -- N-C- -O-R 4 -0--C- -Ri-C-N-CH-(CH 2 )4-NH R3 3 m Op n
R
5 Y
R
7 Formula (X) [0073] In another embodiment, two parts of a single peptidic antigen are covalently linked to the bioactive agent through an -R 5
-R
7 -Y-X- R5- bridge (Formula XI): WO 2006/083874 PCT/US2006/003412 30 R 3 R 3 H I H H
-R-C-N-C-C-O-R
4 -O-C-CH-N C-R-C-N-C- (CH2)4-NH 11 I H 11ZI11 I 210KI 2---(H 4 0 0 0 00 R R Y X RG
R
3 Ra I -I H
-(CH
2
)
4 'C-NH-C-R'-C NH-C-C-0--R 4 -O-C-CH-N-C-R-C H H n 0 P00 0 0 n Formula (XI) wherein, X is selected from the group consisting of (C 1
-C
18 ) alkylene, substituted alkylene, (C 3
-C
8 ) cycloalkylene, substituted cycloalkylene, 5-6 membered heterocyclic system containing 1-3 heteroatoms selected from the group 0, N, and S, substituted heterocyclic, (C 2
-C
18 ) alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, (C 6 - C 10 ) aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkylaryl, substituted alkylaryl, arylalkynyl, substituted arylalkynyl, arylalkenyl, substituted arylalkenyl, arylalkynyl, substituted arylalkynyl, wherein the substituents are selected from the group consisting of H, F, Cl, Br, I, (CI-C 6 ) alkyl, -CN,
-NO
2 ,, -011, -O(C 1
-C
6 ) alkyl, -S(C 1
-C
6 ) alkyl, -S[(=O)(C 1
-C
6 ) alkyl], -S[(0 2
)(C
1
-C
6 ) alkyl],
-C[(=O)(C
1
-C
6 ) alkyl], CF 3
,-O[(CO)-(C
1
-C
6 ) alkyl], -S(0 2
)[N(R
9
R"
0 )], -NH[(C=O)(C 1 C 6 ) alkyl], -NH(C=O)N(R 9
R'
0 ), wherein R9 and R 10 are independently H or (C 1
-C
6 ) alkyl, and -N(R"R 12 ), wherein R 1 1 and R1 2 are independently selected from (C 2
-C
2 0 ) alkylene and (C 2
-C
2 0 ) alkenylene. [0074] In yet another embodiment, the vaccine delivery composition contains four molecules of the polymer, except that only two of the four molecules omit RW and are crosslinked to provide a single -R 5
-X-R
5 - conjugate. [00751 The term "aryl" is used with reference to structural formulae herein to denote a phenyl radical or an ortho-fused bicyclic carbocyclic radical having about nine to ten ring atoms in which at least one ring is aromatic. In certain embodiments, one or more of the ring atoms can be substituted with one or more of nitro, cyano, halo, trifluoromethyl, or trifluoromethoxy. Examples of aryl include, but are not limited to, phenyl, naphthyl, and nitrophenyl.
WO 2006/083874 PCT/US2006/003412 31 [00761 The term "alkenylene" is used with reference to structural formulae herein to mean a divalent branched or unbranched hydrocarbon chain containing at least one unsaturated bond in the main chain or in a side chain. [0077] As used herein, a "therapeutic diol" means any diol molecule, whether synthetically produced, or naturally occurring (e.g., endogenously) that affects a biological process in a mammalian individual, such as a human, in a therapeutic or palliative manner when administered to the mammal [0078] As used herein, the term "residue of a therapeutic diol" means a portion of a therapeutic diol, as described herein, which portion excludes the two hydroxyl groups of the diol. The corresponding therapeutic diol containing the "residue" thereof is used in synthesis of the polymer compositions. The residue of the therapeutic diol is reconstituted in vivo (or under similar conditions of pH, aqueous media, and the like) to the corresponding diol upon release from the backbone of the polymer by biodegradation in a controlled manner that depends upon the properties of the PEA, PEUR or PEU polymer selected to fabricate the composition, which properties are as known in the art and as described herein. [0079] Due to the versatility of the PEA, PEUR and PEU polymers used in the invention compositions, the amount of the therapeutic diol incorporated in the polymer backbone can be controlled by varying the proportions of the building blocks of the polymer. For example, depending on the composition of the PEA, loading of up to 40% w/w of 17fl-estradiol can be achieved. Three different regular, linear PEAs with various loading ratios of 17f-estradiol are illustrated in Scheme 1 below: WO 2006/083874 PCT/US2006/003412 32 "homopoly"-bis-Leu-Estradiol-Adipate (40% w/w -estradiol on polymer) CH(CH3)2 CH(CH 3
)
2
OH
2
H
2 C 0 N- CH - O -C-C -NH- -(CH2)4- - n Copolymer: Leu(ED) 3 Lys(OEt)Adip 4 with 38% w/w estradiol loading
CH(CH
3
)
2 9H(CH 3
)
2 H H 2 C - N-C -O O--C-NH-- -(CH2)~ 3n 0 H H -- (CH 2
)
4
-C-N-(CH
2
)
4
-C-NH-
O COOEt In Scheme 1 [00801 Similarly, the loading of the therapeutic diol into PEUR and PEU polymer can be varied by varying the amount of two or more building blocks of the polymer. [0081] In addition, synthetic steroid based diols based on testosterone or cholesterol, such as 4-androstene-3, 17 diol (4-Androstenediol), 5-androstene-3, 17 diol (5 Androstenediol), 19-nor5-androstene-3, 17 diol (19-Norandrostenediol) are suitable for incorporation into the backbone of PEA and PEUR polymers according to this invention. Moreover, therapeutic diol compounds suitable for use in preparation of the invention vaccine delivery compositions include, for example, amikacin; amphotericin B; apicycline; apramycin; arbekacin; azidamfenicol; bambermycin(s); butirosin; carbomycin; cefpiramide; chloramphenicol; chlortetracycline; clindamycin; clomocycline; demeclocycline; diathymosulfone; dibekacin, dihydrostreptomycin; dirithromycin; doxycycline; erythromycin; fortimicin(s); gentamycin(s); glucosulfone solasulfone; guamecycline; isepamicin; josamycin; kanamycin(s); leucomycin(s); lincomycin; lucensomycin; lymecycline; meclocycline; methacycline; micronomycin; midecamycin(s); minocycline; mupirocin; natamycin; neomycin; netilmicin; WO 2006/083874 PCT/US2006/003412 33 oleandomycin; oxytetracycline; paromycin; pipacycline; podophyllinic acid 2 ethylhydrazine; primycin; ribostamycin; rifamide; rifampin; rafamycin SV; rifapentine; rifaximin; ristocetin; rokitamycin; rolitetracycline; rasaramycin; roxithromycin; sancycline; sisomicin; spectinomycin; spiramycin; streptomycin; teicoplanin; tetracycline; thiamphenicol; theiostrepton; tobramycin; trospectomycin; tuberactinomycin; vancomycin; candicidin(s); chlorphenesin; dermostatin(s); filipin; fungichromin; kanamycin(s); leucomycins(s); lincomycin; lvcensomycin; lymecycline; meclocycline; methacycline; micronomycin; midecamycin(s); minocycline; mupirocin; natamycin; neomycin; netilmicin; oleandomycin; oxytetracycline; paramomycin; pipacycline; podophyllinic acid 2-ethylhydrazine; priycin; ribostamydin; rifamide; rifampin; rifamycin SV; rifapentine; rifaximin; ristocetin; rokitamycin; rolitetracycline; rosaramycin; roxithromycin; sancycline; sisomicin; spectinomycin; spiramycin; strepton; otbramycin; trospectomycin; tuberactinomycin; vancomycin; candicidin(s); chlorphenesin; dermostatin(s); filipin; fungichromin; meparticin; mystatin; oligomycin(s); erimycin A; tubercidin; 6-azauridine; aclacinomycin(s); ancitabine; anthramycin; azacitadine; bleomycin(s) carubicin; carzinophillin A; chlorozotocin; chromomcin(s); doxifluridine; enocitabine; epirubicin; gemcitabine; mannomustine; menogaril; atorvasi pravastatin; clarithromycin; leuproline; paclitaxel; mitobronitol; mitolactol; mopidamol; nogalamycin; olivomycin(s); peplomycin; pirarubicin; prednimustine; puromycin; ranimustine; tubercidin; vinesine; zorubicin; coumetarol; dicoumarol; ethyl biscoumacetate; ethylidine dicoumarol; iloprost; taprostene; tioclomarol; amiprilose; romurtide; sirolimus (rapamycin); tacrolimus; salicyl alcohol; bromosaligenin; ditazol; fepradinol; gentisic acid; glucamethacin; olsalazine; S-adenosylmethionine; azithromycin; salmeterol; budesonide; albuteal; indinavir; fluvastatin; streptozocin; doxorubicin; daunorubicin; plicamycin; idarubicin; pentostatin; metoxantrone; cytarabine; fludarabine phosphate; floxuridine; cladriine; capecitabien; docetaxel; etoposide; topotecan; vinblastine; teniposide, and the like. The therapeutic diol can be selected to be either a saturated or an unsaturated diol. [0082] The molecular weights and polydispersities herein are determined by gel permeation chromatography (GPC) using polystyrene standards. More particularly, number and weight average molecular weights (M, and M,) are determined, for example, using a Model 510 gel permeation chromatography (Water Associates, Inc., Milford, WO 2006/083874 PCT/US2006/003412 34 MA) equipped with a high-pressure liquid chromatographic pump, a Waters 486 UV detector and a Waters 2410 differential refractive index detector. Tetrahydrofuran (THF), N,N-dimethylformamide (DMF) or N,N-dimethylacetamide (DMAc) is used as the eluent (1.0 L/mmin). Polystyrene or poly(methyl methacrylate) standards having narrow molecular weight distribution were used for calibration. [0083] As used herein, the terms "amino acid" and "a-amino acid" mean a chemical compound containing an amino group, a carboxyl group and a pendent R group, such as the R3 groups defined herein. As used herein, the term "biological a-amino acid" means the amino acid(s) used in synthesis are selected from phenylalanine, leucine, glycine, alanine, valine, isoleucine, methionine, or a mixture thereof. [0084] Methods for making the polymers of structural formulas (I) and (III-VII), containing an a-amino acid in the general formula are well known in the art. For example, for the embodiment of the polymer of structural formula (I) wherein R 4 is incorporated into an a-amino acid, for polymer synthesis the a-amino acid with pendant R3 can be converted through esterification into a bis-aco-diamine, for example, by condensing the a-amino acid containing pendant R 3 with a diol HO-R 4 -OH. As a result, di-ester monomers with reactive aco-amino groups are formed. Then, the bis-a,O diamine is entered into a polycondensation reaction with a di-acid such as sebacic acid, or its bis-activated esters, or bis-acyl chlorides, to obtain the final polymer having both ester and amide bonds (PEA). Alternatively, for PEUR, instead of the di-acid, a di-carbonate derivative, formula (XII), is used, where R 6 is defined above and R1 3 is independently
(C
6 -Clo)aryl, optionally substituted with one or more of nitro, cyano, halo, trifluoromethyl or trifluoromethoxy. 0 0 R _0C-0-R 6-0-C-0-R 1 Formula (XII) [00851 More particularly, synthesis of the unsaturated poly(ester-amide)s (UPEAs) useful as biodegradable polymers of the structural formula (I) as disclosed above will be described, wherein WO 2006/083874 PCT/US2006/003412 35 () 0 0 0 H (a) R is A - ' H 0 and/or (b) R 4 is -CH 2
-CH=CH-CH
2 -. In cases where (a) is present and (b) is not present,
R
4 in (I) is -C 4
H
8 - or -C 6 Hu 2 -. In cases where (a) is not present and (b) is present, R' in (I) is -C 4
H
8 - or -CHi 6 -. [0086] The UPEAs can be prepared by solution polycondensation of either (1) di-p toluene sulfonic acid salt of bis (alpha-amino acid) diesters, comprising at least 1 double bond in R 4 , and di-p-nitrophenyl esters of saturated dicarboxylic acid or (2) di-p-toluene sulfonic acid salt of bis (alpha-amino acid) diesters, comprising no double bonds in R 4 , and di-nitrophenyl ester of unsaturated dicarboxylic acid or (3) di-p-toluene sulfonic acid salt of bis(alpha-amino acid) diesters, comprising at least one double bond in R 4 , and di nitrophenyl esters of unsaturated dicarboxylic acids. [0087] Salts of p-toluene sulfonic acid are known for use in synthesizing polymers containing amino acid residues. The aryl sulfonic acid salts are used instead of the free base because the aryl sulfonic salts of bis (alpha-amino acid) diesters are easily purified through recrystallization and render the amino groups as unreactive ammonium tosylates throughout workup. In the polycondensation reaction, the nucleophilic amino group is readily revealed through the addition of an organic base, such as triethylamine, so the polymer product is obtained in high yield. [0088] The di-p-nitrophenyl esters of unsaturated dicarboxylic acid can be synthesized from p-nitrophenol and unsaturated dicarboxylic acid chloride, e.g., by dissolving triethylamine and p-nitrophenol in acetone and adding unsaturated dicarboxylic acid chloride drop wise with stirring at -78'C and pouring into water to precipitate product. Suitable acid chlorides useful for this purpose include fumaric, maleic, mesaconic, citraconic, glutaconic, itaconic, ethenyl-butane dioic and 2-propenyl-butanedioic acid chlorides. [0089] The di-aryl sulfonic acid salts of bis(alpha-amino acid) diesters can be prepared by admixing alpha-amino acid, p-aryl sulfonic acid (e.g. p-toluene sulfonic acid monohydrate), and saturated or unsaturated diol in toluene, heating to reflux temperature, WO 2006/083874 PCT/US2006/003412 36 until water evolution is minimal, then cooling. The unsaturated diols useful for this purpose include, for example, 2-butene-1,3-diol and 1,18-octadec-9-en-diol. Saturated di-p-nitrophenyl esters of dicarboxylic acids and saturated di-p-toluene sulfonic acid salts of bis(alpha-amino acid) di-esters can be prepared as described in U. S. Patent No. 6,503,538 B1. [0090] Synthesis of the unsaturated poly(ester-amide)s (UPEAs) useful as biodegradable polymers of the structural formula (I) as disclosed above will now be described. UPEAs having the structural formula (I) can be made in similar fashion to the compound (VII) of U. S. Patent No. 6,503,538 B1, except that R 4 of (III) of 6,503,538 and/or R 1 of (V) of 6,503,538 is (C 2
-C
20 ) alkenylene as described above. The reaction is carried out, for example, by adding dry triethylamine to a mixture of said (III) and (IV) of 6,503,538 and said (V) of 6,503,538 in dry N,N-dimethylacetamide, at room temperature, then increasing the temperature to 80*C and stirring for 16 hours, then cooling the reaction solution to room temperature, diluting with ethanol, pouring into water, separating polymer, washing separated polymer with water, drying to about 30'C under reduced pressure and then purifying up to negative test on p-nitrophenol and p-toluene sulfonate. A preferred reactant (IV) is p-toluene sulfonic acid salt of Lysine benzyl ester, the benzyl ester protecting group is preferably removed from (II) to confer biodegradability, but it should not be removed by hydrogenolysis as in Example 22 of U.S. Patent No. 6,503,538 because hydrogenolysis would saturate the desired double bonds; rather the benzyl ester group should be converted to an acid group by a method that would preserve unsaturation. Alternatively, the lysine reactant (IV) can be protected by a protecting group different from benzyl that can be readily removed in the finished product while preserving unsaturation, e.g., the lysine reactant can be protected with t butyl (i.e., the reactant can be t-butyl ester of lysine) and the t-butyl can be converted to H while preserving unsaturation by treatment of the product (II) with acid. [0091] A working example of the compound having structural formula (I) is provided by substituting p-toluene sulfonic acid salt of bis(L-phenylalanine) 2-butene- 1,4-diester for (III) in Example 1 of 6,503,538 or by substituting di-p-nitrophenyl fumarate for (V) in WO 2006/083874 PCT/US2006/003412 37 Example 1 of 6,503,538 or by substituting p-toluene sulfonic acid salt of L-phenylalanine 2-butene-1,3-diester for III in Example 1 of 6,503,538 and also substituting de-p nitrophenyl fumarate for (V) in Example 1 of 6,503,538. [0092] In unsaturated compounds having either structural formula (I) or (III), the following hold: Aminoxyl radical e.g., 4-amino TEMPO, can be attached using carbonyldiimidazol, or suitable carbodiimide, as a condensing agent. Peptidic antigens, adjuvants and peptidic antigen/adjuvant conjugates, as described herein, can be attached via the double bond functionality. Hydrophilicity can be imparted by bonding to poly(ethylene glycol) diacrylate. [00931 In yet another aspect, polymers contemplated for use in forming the invention vaccine delivery systems include those set forth in U.S. Patent Nos. 5,516, 881; 6,476,204; 6,503,538; and in U.S. Application Nos. 10/096,435; 10/101,408; 10/143,572; and 10/194,965; the entire contents of each of which is incorporated herein by reference. [0094] The biodegradable PEA, PEUR and PEU polymers and copolymers may contain up to two amino acids per monomer, multiple amino acids per polymer molecule, and preferably have weight average molecular weights ranging from 10,000 to 125,000; these polymers and copolymers typically have intrinsic viscosities at 25 C, determined by standard viscosimetric methods, ranging from 0.3 to 4.0, for example, ranging from 0.5 to 3.5. [0095] Polymers contemplated for use in the practice of the invention can be synthesized by a variety of methods well known in the art. For example, tributyltin (IV) catalysts are commonly used to form polyesters such as poly(E-caprolactone), poly(glycolide), poly(lactide), and the like. However, it is understood that a wide variety of catalysts can be used to form polymers suitable for use in the practice of the invention. [00961 PEA and PEUR polymers contemplated for use in the practice of the invention can be synthesized by a variety of methods well known in the art. For example, tributyltin (IV) catalysts are commonly used to form polyesters such as poly(E caprolactone), poly(glycolide), poly(lactide), and the like. However, it is understood that a wide variety of catalysts can be used to form polymers suitable for use in the practice of the invention.
WO 2006/083874 PCT/US2006/003412 38 [00971 Such poly(caprolactones) contemplated for use have an exemplary structural formula (XIII) as follows: 0
O-C-(CH
2
)
5 Sn Formula (XIII) [00981 Poly(glycolides) contemplated for use have an exemplary structural formula (XIV) as follows: O H 0-0-c n Formula (XIV) [0099] Poly(lactides) contemplated for use have an exemplary structural formula (XV) as follows: O Me O-0--C 0-MAn Formula (XV) [01001 An exemplary synthesis of a suitable poly(lactide-co-s-caprolactone) including an aminoxyl moiety is set forth as follows. The first step involves the copolymerization of lactide and s-caprolactone in the presence of benzyl alcohol using stannous octoate as the catalyst to form a polymer of structural formula (XVI). O 0 CH Mel
CH
2 OH +M Me+ r 0 O H O O CH20---- C -(CH2)5--O-H ./Me n -m Formula (XVI) WO 2006/083874 PCT/US2006/003412 39 [01011 The hydroxy terminated polymer chains can then be capped with maleic anhydride to form polymer chains having structural formula (XVII): 0OH 0 O O CH20OC--O- -(CH2)--0-C=C-0-OH ./Men m Formula (XVII) [0102] At this point, 4-amino-2,2,6,6-tetramethylpiperidine- 1 -oxy can be reacted with the carboxylic end group to covalently attach the aminoxyl moiety to the copolymer via the amide bond which results from the reaction between the 4-amino group and the carboxylic acid end group. Alternatively, the maleic acid capped copolymer can be grafted with polyacrylic acid to provide additional carboxylic acid moieties for subsequent attachment of further aminoxyl groups. [0103] In unsaturated compounds having structural formula (VII) for PEU the following hold: An amino substituted aminoxyl (N-oxide) radical bearing group e.g., 4 amino TEMPO, can be attached using carbonyldiimidazole, or suitable carbodiimide, as a condensing agent. Additional bioactive agents, and the like, as described herein, optionally can be attached via the double bond. [0104] For example, the invention high molecular weight semi-crystalline PEUs having structural formula (VI) can be prepared inter-facially by using phosgene as a bis electrophilic monomer in a chloroform/water system, as shown in the reaction scheme (2) below: 1. Na 2
CO
3 / H 2 0 H O O H 2. CICOCI / CHCl 3 HOTos.H 2
N-C-C---R
4
-O-C-C-NH
2 .TosOH 3 k3 Scheme (2) [0105] Synthesis of copoly(ester ureas) (PEUs) containing L-Lysine esters and having structural formula (VII) can be carried out by a similar scheme (3): WO 2006/083874 PCT/US2006/003412 40 H9 9 H H m HOTos.H 2
N-C-C-O-R
4
-O-C-C-NH
2 .TosOH + p HOTos.H 2
N-C-(CH
2
)
4 -- NH 2 .TosOH R3 R3 1O-R 2 1. Na 2
CO
3 / H20 2. CICOCI / CHC 3 V ) Scheme (3) [0106] A 20% solution of phosgene (CICOCI) (highly toxic) in toluene, for example (commercially available (Fluka Chemie, GMBH, Buchs, Switzerland), can be substituted either by diphosgene (trichloromethylchloroformate) or triphosgene (bis(trichloromethyl)carbonate). Less toxic carbonyldiimidazole can be also used as a bis-electrophilic monomer instead of phosgene, di-phosgene, or tri-phosgene. General Procedure for Synthesis of PEUs [0107] It is necessary to use cooled solutions of monomers to obtain PEUs of high molecular weight. For example, to a suspension of di-p-toluenesulfonic acid salt of bis(a amino acid)-apc-alkylene diester in 150 mL of water, anhydrous sodium carbonate is added, stirred at room temperature for about 30 minutes and cooled to about 2 - 0 0 C, forming a first solution. In parallel, a second solution of phosgene in chloroform is cooled to about 15 -10 "C. The first solution is placed into a reactor for interfacial polycondensation and the second solution is quickly added at once and stirred briskly for about 15 min. Then chloroform layer can be separated, dried over anhydrous Na 2 S04, and filtered. The obtained solution can be stored for further use. [01081 All the exemplary PEU polymers fabricated were obtained as solutions in chloroform and these solutions are stable during storage. However, some polymers, for example, 1-Phe-4, become insoluble in chloroform after separation. To overcome this problem, polymers can be separated from chloroform solution by casting onto a smooth hydrophobic surface and allowing chloroform to evaporate to dryness. No further purification of obtained PEUs is needed. The yield and characteristics of exemplary PEUs obtained by this procedure are summarized in Table 1 herein.
WO 2006/083874 PCT/US2006/003412 41 General Procedure for Preparation of porous PEUs. [01091 Methods for making the PEU polymers containing a-amino acids in the general formula will now be described. For example, for the embodiment of the polymer of formula (I) or (II), the a-amino acid can be converted into a bis-(a-amino acid)-Co diol-diester monomer, for example, by condensing the a-amino acid with a diol HO-R 1 OH. As a result, ester bonds are formed. Then, acid chloride of carbonic acid (phosgene, diphosgene, triphosgene) is entered into a polycondensation reaction with a di-p toluenesulfonic acid salt of a bis-(a-amino acid) -alkylene diester to obtain the final polymer having both ester and urea bonds. [0110] The unsaturated PEUs can be prepared by interfacial solution condensation of di-p-toluenesulfonate salts of bis-(a-amino acid)-alkylene diesters, comprising at least one double bond in R'. Unsaturated diols useful for this purpose include, for example, 2 butene- 1,4-diol and 1,1 8-octadec-9-en-diol. Unsaturated monomer can be dissolved prior to the reaction in alkaline water solution, e.g. sodium hydroxide solution. The water solution can then be agitated intensely, under external cooling, with an organic solvent layer, for example chloroform, which contains an equimolar amount of monomeric, dimeric or trimeric phosgene. An exothermic reaction proceeds rapidly, and yields a polymer that (in most cases) remains dissolved in the organic solvent. The organic layer can be washed several times with water, dried with anhydrous sodium sulfate, filtered, and evaporated. Unsaturated PEUs with a yield of about 75%-85% can be dried in vacuum, for example at about 45*C. [0111] To obtain a porous, strong material, L-Leu based PEUs, such as 1-L-Leu-4 and 1 -L-Leu-6, can be fabricated using the general procedure described below. Such procedure is less successful in formation of a porous, strong material when applied to L Phe based PEUs. [01121 The reaction solution or emulsion (about 100 mL) of PEU in chloroform, as obtained just after interfacial polycondensation, is added dropwise with stirring to 1,000 mL of about 80 C -85 *C water in a glass beaker, preferably a beaker made hydrophobic with dimethyldichlorsilane to reduce the adhesion of PEU to the beaker's walls. The polymer solution is broken in water into small drops and chloroform evaporates rather WO 2006/083874 PCT/US2006/003412 42 vigorously. Gradually, as chloroform is evaporated, small drops combine into a compact tar-like mass that is transformed into a sticky rubbery product. This rubbery product is removed from the beaker and put into hydrophobized cylindrical glass-test-tube, which is thermostatically controlled at about 80 C for about 24 hours. Then the test-tube is removed from the thermostat, cooled to room temperature, and broken to obtain the polymer. The obtained porous bar is placed into a vacuum drier and dried under reduced pressure at about 80 C for about 24 hours. In addition, any procedure known in the art for obtaining porous polymeric materials can also be used. [0113] Properties of high-molecular-weight porous PEUs made by the above procedure yielded results as summarized in Table 1. Table 1 Properties of PEU Polymers of Formula (VI) and (VII) PEU* Yield Wered a) , M. M,"/M. b) Tg c) Tm c) [%] [dL/g] [*C] [*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
.
7 5 - [1-L- 84 0.31 64400 43000 1.47 34 114 Lys(OBn)]o.
25 1-L-Leu-DAS 57 0.28 5 5 7 00 d) 27 70 0 d) Ud) 56 165 *PEUs of general formula (VI), where, 1-L-Leu-4: R 4 = (CH 2
)
4 , R 3 = i-C 4
H
9 l-L-Leu-6: R 4 = (CH 2
)
6 , R 3 = i-C 4
H
9 1-L-Phe-6:.R 4 = (CH 2
)
6 , R3 = -CH 2
-C
6
H
5 . 1-L-Leu-DAS: R4 = 1,4:3,6-dianhydrosorbitol, R 3 = i-C 4 H a) Reduced viscosities were measured in DMF at 25'C and a concentration 0.5 g/dL b) GPC Measurements were carried out in DMF, (PMMA) c) Tg taken from second heating curve from DSC Measurements (heating rate 10*C/min). d) GPC Measurements were carried out in DMAc, (PS) [01141 Tensile strength of illustrative synthesized PEUs was measured and results are summarized in Table 2. Tensile strength measurement was obtained using dumbbell shaped PEU films (4 x 1.6 cm), which were cast from chloroform solution with average thickness of 0.125 mm and subjected to tensile testing on tensile strength machine (Chatillon TDC200) integrated with a PC using Nexygen FM software (Amtek, Largo, WO 2006/083874 PCT/US2006/003412 43 FL) at a crosshead speed of 60 mm/min. Examples illustrated herein can be expected to have the following mechanical properties: 1. , A glass transition temperature in the range from about 30 C' to about 90 C*, for example, in the range from about 35 C' to about 70 C 0 ; 2. A film of the polymer with average thickness of about 1.6 cm will have tensile stress at yield of about 20 Mpa to about 150 Mpa, for example, about 25 Mpa to about 60 Mpa; 3. A film of the polymer with average thickness of about 1.6 cm will have a percent elongation of about 10 % to about 200%, for example about 50 % to about 150%; and 4. A film of the polymer with average thickness of about 1.6 cm will have a Young's modulus in the range from about 500 MPa to about 2000 MPa. Table 2 below summarizes the properties of exemplary PEUs of this type. Table 2 Mechanical Properties of PEUs Tga) Tensile Stress Young's Polymer designation Og at Yield Percent Modulus
(
0 C) (MPa) Elongation (%) (MPa) 1-L-Leu-6 64 21 114 622 [1-L-Leu-6] 0
.
7 5 - [1-L-Lys(OBn)1 025 34 25 159 915 [0115] The various components of the invention vaccine delivery composition can be present in a wide range of ratios. For example, the polymer repeating unit:antigen are typically used in a ratio of 1:50 to 50:1, for example 1:10 to 10:1, about 1:3 to 3:1, or about 1:1. However, other ratios may be more appropriate for specific purposes, such as when a particular antigen is both difficult to incorporate into a particular polymer and has a low immunogenicity, in which case a higher relative amount of the peptidic antigen is required. [01161 In certain embodiments, the invention vaccine delivery composition described herein can be provided as particles, with peptidic antigen/adjuvant conjugate, or antigens, with or without adjuvant, either physically incorporated (dispersed) within the particle or attached to polymer functional groups, optionally by use of a linker, using any of several WO 2006/083874 PCT/US2006/003412 44 techniques well known in the art and as described herein. The particles are sized for uptake by APCs, having an average diameter, for example, in the range from about 10 nanometers to about 1000 microns, or in the range from about 10 nanometers to about 10 microns. Optionally, the particles can further comprise a thin covering of the polymer to aid in control of their biodegradation. Typically such particles include from about 5 to about 150 peptidic antigens per polymer molecule. [0117] The polymers used in the invention vaccine delivery compositions, such as PEA, PEUR and PEU polymers, biodegrade by enzymatic action at the surface. Therefore, the polymers, for example particles thereof, administer the antigen to the subject at a controlled release rate, which is specific and constant over a prolonged period. Additionally, since PEA, PEUR and PEU polymers break down in vivo via hydrolytic enzymes without production of adverse side-products, the invention vaccine delivery compositions are substantially non-inflammatory. As used herein, "biodegradable" as used to describe a polymer in the invention vaccine compositions means the polymer is capable of being broken down into innocuous products in the normal functioning of the body. In one embodiment, the entire vaccine delivery composition is biodegradable. The preferred biodegradable polymers have hydrolyzable ester linkages that provide the biodegradability, and are typically chain terminated predominantly with amino groups. [01181 As used herein "dispersed" means a peptidic antigen or adjuvant as disclosed herein is dispersed, mixed, dissolved, homogenized, and/or covalently bound ("dispersed" or loaded) in the polymer, which may or may not be formed into particles. [0119] While the peptidic antigens and optional adjuvants can be dispersed within the polymer matrix without chemical linkage to the polymer carrier, it is also contemplated that the antigen and/or antigen-adjuvant conjugate can be covalently bound to the biodegradable polymers via a wide variety of suitable functional groups. For example, when the biodegradable polymer is a polyester, the carboxyl group chain end can be used to react with a complimentary moiety on the antigen or adjuvant, such as hydroxy, amino, thio, and the like. A wide variety of suitable reagents and reaction conditions are disclosed, e.g., in March's Advanced Organic Chemistry, Reactions, Mechanisms, and WO 2006/083874 PCT/US2006/003412 45 Structure, Fifth Edition, (2001); and Comprehensive Organic Transformations, Second Edition, Larock (1999). [0120] In other embodiments, an antigen and/or adjuvant can be linked to any of the polymers of structures (I) or (III-VII) through an amide, ester, ether, amino, ketone, thioether, sulfinyl, sulfonyl, disulfide linkage. Such a linkage can be formed from suitably functionalized starting materials using synthetic procedures that are known in the art. [01211 For example, in one embodiment a polymer can be linked to the peptidic antigen or adjuvant via an end or pendent carboxyl group (e.g., COOH) of the polymer. Specifically, a compound of structures III, V and VII can react with an amino functional group or a hydroxyl functional group of a peptidic antigen to provide a biodegradable polymer having the peptidic antigen attached via an amide linkage or carboxylic ester linkage, respectively. In another embodiment, the carboxyl group of the polymer can be transformed into an acyl halide, acyl anhydride/"mixed" anhydride, or active ester. In other embodiments, the free -NH 2 ends of the polymer molecule can be acylated to assure that the peptidic antigen will attach only via a carboxyl group of the polymer and not to the free ends of the polymer. For example, the invention vaccine delivery composition described herein can be prepared from PEA, PEUR, or PEU where the N terminal free amino groups are acylated, e.g., with anhydride RCOOCOR, where the R = (Ci-C 2 4 ) alkyl, to assure that the bioactive agent will attach only via a carboxyl group of the polymer and not to the free ends of the polymer. [0122] Alternatively, the peptidic antigen or adjuvant may be attached to the polymer via a linker molecule, for example, as described in structural formulae (VIII - XI). Indeed, to improve surface hydrophobicity of the biodegradable polymer, to improve accessibility of the biodegradable polymer towards enzyme activation, and to improve the release profile of the biodegradable polymer, a linker may be utilized to indirectly attach the peptidic antigen and/or adjuvant to the biodegradable polymer. In certain embodiments, the linker compounds include poly(ethylene glycol) having a molecular weight (Mw) of about 44 to about 10,000, preferably 44 to 2000; amino acids, such as serine; polypeptides with repeat units from 1 to 100; and any other suitable low molecular WO 2006/083874 PCT/US2006/003412 46 weight polymers. The linker typically separates the peptidic antigen from the polymer by about 5 angstroms up to about 200 angstroms. [0123] In still further embodiments, the linker is a divalent radical of formula W-A-Q, wherein A is (Ci-C 24 ) alkyl, (C 2
-C
2 4 ) alkenyl, (C 2
-C
24 ) alkynyl, (C 3
-C
8 ) cycloalkyl, or
(C
6 -Cio) aryl, and W and Q are each independently -N(R)C(=O)-, -C(=O)N(R)-, OC(=0)-, -C(=O)O, -0-, -S-, -S(O), -S(0) 2 -, -S-S-, -N(R)-, -C(=0)-, wherein each R is independently H or (Ci-C 6 )alkyl. [01241 As used to describe the above linkers, the term "alkyl" refers to a straight or branched chain hydrocarbon group including methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, and the like. [0125] As used herein, "alkenyl" as used to describe linkers refers to straight or branched chain hydrocarbon groups having one or more carbon-carbon double bonds. [01261 As used herein, "alkynyl" as used to describe linkers refers to straight or branched chain hydrocarbon groups having at least one carbon-carbon triple bond. [0127] As used herein, "aryl" as used to describe linkers refers to aromatic groups having in the range of 6 up to 14 carbon atoms. [0128] In certain embodiments, the linker may be a polypeptide having from about 2 up to about 25 amino acids. Suitable peptides contemplated for use include poly-L lysine, poly-L-glutamic acid, poly-L-aspartic acid, poly-L-histidine, poly-L-ornithine, poly-L-threonine, poly-L-tyrosine, poly-L-leucine, poly-L-lysine-L-phenylalanine, poly L-arginine, poly-L-lysine-L-tyrosine, and the like. [0129] In one embodiment, the peptidic antigen can covalently crosslink the polymer, i.e. the antigen is bound to more than one polymer molecule. This covalent crosslinking can be done with or without additional polymer-antigen linker. [01301 The peptidic antigen molecule can also form an intramolecular bridge by covalent attachment between two parts of a single macromolecule.
WO 2006/083874 PCT/US2006/003412 47 [0131] A linear polymer peptide conjugate is made by protecting the potential nucleophiles on the antigen backbone and leaving only one reactive group to be bound to the polymer or polymer linker construct. Deprotection is performed according to well known in the art deprotection of peptides (Boc and Fmoc chemistry for example). [0132] In one embodiment of the present invention, the peptidic antigen is presented as retro-inverso or partial retro-inverso peptide. [01331 In other embodiments the peptidic antigen is mixed with a photocrosslinkable version of the polymer in a matrix, and after crosslinking the material is dispersed (ground) to a phagocytosable size, i.e. 0.1-10ptm. [01341 The linker can be attached first to the polymer or to the peptidic antigen or adjuvant. During synthesis, the linker can be either in unprotected form or protected from, using a variety of protecting groups well known to those skilled in the art. In the case of a protected linker, the unprotected end of the linker can first be attached to the polymer or the peptidic antigen. The protecting group can then be de-protected using Pd/H 2 hydrogenolysis, mild acid or base hydrolysis, or any other common de-protection method that is known in the art. The de-protected linker can then be attached to the peptidic antigen, adjuvant, or adjuvant/peptidic antigen conjugate. [0135] An exemplary synthesis of a biodegradable polymer according to the invention (wherein the molecule to be attached is an aminoxyl) is set forth as follows. A polyester can be reacted with an amino substituted N-oxide free radical (aminoxyl) bearing group, e.g., 4-amino-2,2,6,6-tetramethylpiperidine- 1 -oxy, in the presence of N,N' carbonyldiimidazole to replace the carboxylic acid moiety at the chain end of the polyester with an amide bond to the amino substituted aminoxyl-containing radical, so that the amino moiety covalently bonds to the carbon of the carbonyl residue of the carboxyl group of the polymer. The N,N'-carbonyl diimidazole or suitable carbodiimide converts the hydroxyl moiety in the carboxyl group at the chain end of the polyester into an intermediate product moiety that will react with the aminoxyl, e.g., 4-amino-2,2,6,6 tetramethylpiperidine-1-oxy. The aminoxyl reactant is typically used in a mole ratio of reactant to polyester ranging from 1:1 to 100:1. The mole ratio of N,N'-carbonyl diimidazole to aminoxyl is preferably about 1:1.
WO 2006/083874 PCT/US2006/003412 48 [0136] A typical reaction is as follows. A polyester is dissolved in a reaction solvent and reaction is readily carried out at the temperature utilized for the dissolving. The reaction solvent may be any in which the polyester will dissolve. When the polyester is a polyglycolic acid or a poly(glycolide-L-lactide) (having a monomer mole ratio of glycolic acid to L-lactic acid greater than 50:50), highly refined (99.9+% pure) dimethyl sulfoxide at 115 "C to 130 "C or dimethylsulfoxide (DMSO) at room temperature suitably dissolves the polyester. When the polyester is a poly-L-lactic acid, a poly-DL-lactic acid or a poly(glycolide-L-lactide) (having a monomer mole ratio of glycolic acid to L-lactic acid 50:50 or less than 50:50), tetrahydrofuran, methylene chloride and chloroform at room temperature to 50 *C suitably dissolve the polyester. Polymer / Peptidic antigen Linkage [0137] In one embodiment, the polymers used to make the invention vaccine delivery compositions as described herein have at least one peptidic antigen directly linked to the polymer. The residues of the polymer can be linked to the residues of the one or more peptidic antigens. For example, one residue of the polymer can be directly linked to one residue of the peptidic antigen. The polymer and the peptidic antigen can each have one open valence. Alternatively, more than one peptidic antigen, multiple peptidic antigens, or a mixture of peptidic antigens from different pathogenic organisms can be directly linked to the polymer. However, since the residue of each peptidic antigen can be linked to a corresponding residue of the polymer, the number of residues of the one or more peptidic antigens can correspond to the number of open valences on the residue of the polymer. [0138] As used herein, a "residue of a polymer" refers to a radical of a polymer having one or more open valences. Any synthetically feasible atom, atoms, or functional group of the polymer (e.g., on the polymer backbone or pendant group) of the present invention can be removed to provide the open valence, provided bioactivity is substantially retained when the radical is attached to a residue of a peptidic antigen. Additionally, any synthetically feasible functional group (e.g., carboxyl) can be created on the polymer (e.g., on the polymer backbone or pendant group) to provide the open valence, provided bioactivity is substantially retained when the radical is attached to a residue of a peptidic antigen. Based on the linkage that is desired, those skilled in the art can select suitably WO 2006/083874 PCT/US2006/003412 49 functionalized starting materials that can be derived from the polymer of the present invention using procedures that are known in the art. [0139] As used herein, a "residue of a compound of structural formula (*)" refers to a radical of a compound of polymer of formulas (I) and (III-VII) as described herein having one or more open valences. Any synthetically feasible atom, atoms, or functional group of the compound (e.g., on the polymer backbone or pendant group) can be removed to provide the open valence, provided bioactivity is substantially retained when the radical is attached to a residue of a peptidic antigen. Additionally, any synthetically feasible functional group (e.g., carboxyl) can be created on the compound of formulas (I) and (III VII) (e.g., on the polymer backbone or pendant group) to provide the open valance, provided bioactivity is substantially retained when the radical is attached to a residue of a peptidic antigen. Based on the linkage that is desired, those skilled in the art can select suitably functionalized starting materials that can be derived from the compound of formula (I) and (III-VII) using procedures that are known in the art. [0140] For example, the residue of a peptidic antigen can be linked to the residue of a compound of structural formulas (I) and (III-VII) through an amide (e.g., -N(R)C(=0)- or -C(=O)N(R)-), ester (e.g., -OC(=O)- or -C(=O)O-), ether (e.g., -0-), amino (e.g., -N(R) ), ketone (e.g., -C(=0)-), thioether (e.g., -S-), sulfinyl (e.g., -S(O)-), sulfonyl (e.g., -S(0) 2 ), disulfide (e.g., -S-S-), or a direct (e.g., C-C bond) linkage, wherein each R is independently H or (C-C 6 ) alkyl. Such a linkage can be formed from suitably functionalized starting materials using synthetic procedures that are known in the art. Based on the linkage that is desired, those skilled in the art can select suitably functional starting material that can be derived from a residue of a compound of any one of structural formulas (I) and (III-VII) and from a given residue of a peptidic antigen or adjuvant using procedures that are known in the art. The residue of the peptidic antigen or adjuvant can be linked to any synthetically feasible position on the residue of a compound of any one of structural formulas (I) and (III-VII). Additionally, the invention also provides compounds having more than one residue of a peptidic antigen or adjuvant bioactive agent directly linked to a compound of any one of structural formulas (I) and
(III-VII).
WO 2006/083874 PCT/US2006/003412 50 [01411 The number of peptidic antigens that can be linked to the polymer molecule can typically depend upon the molecular weight of the polymer. For example, for a compound of structural formulas (I) or (III), wherein n is about 5 to about 150, preferably about 5 to about 70, up to about 150 peptidic antigens (i.e., residues thereof) can be directly linked to the polymer (i.e., residue thereof) by reacting the peptidic antigen with end groups of the polymer. In unsaturated polymers, the peptidic antigens can also be reacted with double (or triple) bonds in the polymer. [0142] The PEA, PEUR and PEU polymers described herein readily absorb water (5 to 25 % w/w water up-take, on polymer film), allowing hydrophilic molecules to readily diffuse through them. This characteristic makes PEA, PEUR and PEU polymers suitable for use as an over coating on particles to control release rate. Water absorption also enhances biocompatibility of the polymers and the vaccine delivery composition based on such polymers. In addition, due to the hydrophilic properties of the PEA, PEUR and PEU polymers, when delivered in vivo the particles become sticky and agglomerate, particularly at in vivo temperatures. Thus the polymer particles spontaneously form polymer depots when injected subcutaneously or intramuscularly for local delivery, such as by subcutaneous needle or needle-less injection. Particles with average diameter range from about 1 micron to about 100 microns, of a size that will not permit circulation in the body, are suitable for forming such polymer depots in vivo. Alternatively, for oral administration, the GI tract can tolerate much larger particles, for example micro particles of about 1 micron up to about 1000 microns average diameter. [0143] For instance, typically, the polymer depot will degrade over a time selected from about twenty-four hours, about seven days, about thirty days, or about ninety days, or longer. Longer time spans are particularly suitable for providing an implantable vaccine delivery composition that eliminates the need to repeatedly inject the vaccine to obtain a suitable immune response. [01441 Particles suitable for use in the invention vaccine delivery compositions can be made using immiscible solvent techniques. Generally, these methods entail the preparation of an emulsion of two immiscible liquids. A single emulsion method can be used to make polymer particles that incorporate hydrophobic adjuvants and peptidic antigens, or conjugates thereof. In the single emulsion method, molecules to be WO 2006/083874 PCT/US2006/003412 51 incorporated into the particles are mixed with polymer in solvent first, and then emulsified in water solution with a surface stabilizer, such as a surfactant. In this way, polymer particles with hydrophobic adjuvant, peptidic antigen, or adjuvant/peptidic antigen conjugates are formed and suspended in the water solution, in which hydrophobic conjugates in the particles will be stable without significant elution into the aqueous solution, but such molecules will elute into body tissue, such as muscle tissue. 101451 Most biologics, including synthetic peptidic antigens, are hydrophilic. A double emulsion method can be used to make polymer particles with liquid or hydrophilic adjuvant/peptidic antigens dispersed within. In the double emulsion method, liquid or hydrophilic adjuvant/peptidic antigens dissolved in water are emulsified in polymer solution first, and the whole emulsion is put into water to emulsify again to form particles with an external polymer coating and liquid adjuvant/peptidic antigens in the interior of the particles. Surfactant can be used in both methods of emulsification to prevent particle aggregation. Chloroform or dichloromethane (DCM), which are not miscible in water, are used as solvents for PEA and PEUR polymers, but later in the preparation the solvent is removed, using methods known in the art. [0146] For certain peptidic antigens or adjuvants with low water solubility, however, these two emulsion methods have limitations. In this context, "low water solubility" means an active agent that is less hydrophobic than truly lipophilic drugs, such as Taxol, but which is less hydrophilic than truly aqueous-soluble drugs, such as many biologics. These types of intermediate compounds are too hydrophilic for high loading and stable matrixing into single emulsion particles, yet are too hydrophobic for high loading and stability within double emulsions. In such cases, a polymer layer is coated on to particles made of polymer and drugs with low water solubility, by three emulsification process. This method provides relatively low drug loading (~10% w/w), but provides structure stability and controlled drug release rate. [01471 The first emulsion is made by mixing the active agents into a polymer solution and emulsifying the mixture in a water solution with surfactant or lipid, such as di (hexadecanoyl)phosphatidylcholine (DHPC; a short-chain derivative of a natural lipid). In this way, particles containing the active agents are formed and suspended in water to form the first emulsion. The second emulsion is formed by putting the first emulsion into WO 2006/083874 PCT/US2006/003412 52 a polymer solution, and emulsifying the mixture, so that water drops with the polymer/drug particles inside are formed within the polymer solution. Water and surfactant or lipid will separate the particles and dissolve the particles in the polymer solution. The third emulsion is then formed by putting the second emulsion into water with surfactant or lipid, and emulsifying the mixture to form the final particles in water. The resulting particle structure, as illustrated in Fig. 1 will have one or more particles made with polymer plus peptidic antigen, with or without adjuvant, at the center, surrounded by water and surface stabilizer, such as surfactant or lipid, and covered with a pure polymer shell. Surface stabilizer and water will prevent solvent in the polymer coating from contacting the particles inside the coating and dissolving them. [0148] To increase loading of active agents, such as the peptidic antigen or adjuvant, by the triple emulsion method, active agents with low water solubility can be coated with surface stabilizer in the first emulsion, without polymer coating and without dissolving the active agent in water. In this first emulsion, water, surface stabilizer and active agent have similar volume or in the volume ratio range of (1 to 3):(0.2 to about 2):1, respectively. In this case, water is used, not for dissolving the active agent, but rather for protecting the active agent with help of surface stabilizer. Then the double and triple emulsions are prepared as described above (Fig. 1) [0149] Many emulsification techniques will work in making the emulsions used in manufacture of the particles. However, the presently preferred method of making the emulsion is by using a solvent that is not miscible in water. The emulsifying procedure consists of dissolving polymer with the solvent, mixing with adjuvant/peptidic antigen molecule(s), putting into water, and then stirring with a mixer and/or ultra-sonicator. Particle size can be controlled by controlling stir speed and/or the concentration of polymer, adjuvant/peptidic antigen molecule(s), and surface stabilizer. Coating thickness can be controlled by adjusting the ratio of the second to the third emulsion. In any of the methods of particle formation described above, the antigenic peptide and optional adjuvant can form a coating on the surface of the particles by conjugation to the polymers in the particles after particle formation. [0150] Suitable emulsion stabilizers may include nonionic surface active agents, such as mannide monooleate, dextran 70,000, polyoxyethylene ethers, polyglycol ethers, and WO 2006/083874 PCT/US2006/003412 53 the like, all readily commercially available from, e.g., Sigma Chemical Co., St. Louis, Mo. The surface active agent will be present at a concentration of about 0.3% to about 10%, preferably about 0.5% to about 8%, and more preferably about 1% to about 5%. [01511 Rate of release of the adjuvant/peptidic antigen from the compositions can be controlled by adjusting the coating thickness, number of antigens covering the exterior of the particle, particle size, structure, and density of the coating. Density of the coating can be adjusted by adjusting loading of the adjuvant/peptidic antigen in the coating. When the coating contains no adjuvant/peptidic antigen, the polymer coating is densest, and the adjuvant/peptidic antigen elutes through the coating most slowly. By contrast, when adjuvant/peptidic antigen is loaded into the coating, the coating becomes porous once the adjuvant/peptidic antigen has eluted out, starting from the outer surface of the coating and, therefore, the adjuvant/peptidic antigen at the center of the particle can elute at an increased rate. The higher the drug loading, the lower the density of the coating layer and the higher the elution rate. The loading of adjuvant/peptidic antigen in the coating can be lower than that in the interior of the particles beneath the exterior coating. Release rate of adjuvant/peptidic antigen from the particles can also be controlled by mixing particles with different release rates prepared as described above. [0152] A detailed description of methods of making double and triple emulsion polymers may be found in Pierre Autant et al, Medicinal and/or nutritional microcapsules for oral administration, U.S. patent No. 6,022,562; losif Daniel Rosca et al., Microparticle formation and its mechanism in single 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 the anticancer drug paclitaxel (Taxol): PLGA nanoparticles containing vitamin E (TPGS, J. Control. Release (2003) 86:33- 48; Somatosin containing biodegradable microspheres prepared by a modified solvent evaporation method based on W/O/W-multiple emulsions, Int. J. Pharin. (1995) 126:129- 138 and F. Gabor et al., Ketoprofenpoly(d,l-lactic-co-glycolic acid) microspheres: influence of manufacturing parameters and type of polymer on the release characteristics, J. Microencapsul. (1999) 16 (1):1- 12, each of which is incorporated herein in its entirety. [0153] In yet further embodiments for delivery of aqueous-soluble peptidic antigens and/or adjuvant, the particles can be made into nanoparticles having an average diameter WO 2006/083874 PCT/US2006/003412 54 of about 20nm to about 200 nm for delivery to the circulation. The nanoparticles can be made by the single emulsion method with the peptidic antigen dispersed therein, i.e., mixed into the emulsion or conjugated to polymer as described herein. The nanoparticles can also be provided as micelles containing the PEA or PEUR polymers described herein. The micelles are formed in water and the water soluble antigens with optional adjuvant protein are loaded into micelles at the same time without solvent. [0154] More particularly, the biodegradable micelles, which are illustrated in Fig. 2, are formed of a water soluble ionized polymer chain conjugated to a hydrophobic polymer chain. Whereas, the outer portion of the micelle mainly consists of the water soluble ionized section of the polymer, the hydrophobic section of the polymer mainly partitions to the interior of the micelles and holds the polymer molecules together. [01551 The biodegradable hydrophobic section of the polymer used to make micelles is made of PEA, PEUR or PEU polymers, as described herein. For strongly hydrophobic PEA, PEUR or PEU polymers, components such as di- L-leucine ester of 1,4:3,6 dianhydro-D-sorbitol or a rigid aromatic di-acid like O-bis (4-carboxyphenoxy) (C-C 8 ) alkane may be included in the polymer repeat unit. By contrast, the water soluble section of the polymer comprises repeating alternating units of polyethylene glycol, polyglycosaminoglycan or polysaccharide and at least one ionizable or polar amino acid, wherein the repeating alternating units have substantially similar molecular weights and wherein the molecular weight of the polymer is in the range from about 1 OkD to about 300kD. The higher the molecular weight of the water soluble section, the greater the porosity of the micelle, with the longer chains enabling high loading of the water soluble antigens and optional adjuvants. In addition, polyamino acids are more immunogenic than single amino acids. [01561 The repeating alternating units may have substantially similar molecular weights in the range from about 300D to about 700D. In one embodiment wherein the molecular weight of the polymer is over 1 kD, at least one of the amino acid units is an ionizable or polar amino acid selected from serine, glutamic acid, aspartic acid, lysine and arginine. In one embodiment, the units of ionizable amino acids comprise at least one block of ionizable poly(amino acids), such as glutamate or aspartate, can be included in the polymer. The invention micellar composition may further comprise a WO 2006/083874 PCT/US2006/003412 55 pharmaceutically acceptable aqueous media with a pH value at which at least a portion of the ionizable amino acids in the water soluble sections of the polymer are ionized. [0157] The biodegradable hydrophobic polymer chain is made of PEA, PEUR or PEU polymers, as described herein. For a strongly hydrophobic PEA, PEUR or PEU, components such as 1,3-bis(-4-carboxylate- phenoxy)- propane (CPP) and/or bis(-L leucine) diesters of -1,4:3,6-dianhydrohexitoles-D-sorbitol (DAS) may be included in the hydrophobic polymer chain. By contrast, the water soluble chain is made of many repeating units of poly-ethylene glycol (PEG) and an ionizable amino acid, such as (poly)lysine or (poly) glutamate, wherein the PEG unit and the ionizable amino acid unit have similar molecular weights, for example, a few hundred kD (i.e., the PEG unit can have a molecular weight at substantially any value in this range). However, the total molecular weight of the water soluble section of the polymer can be, for example, in the range of about 1OkD to about 300kD. The higher the molecular weight of the water soluble section, the greater the porosity of the micelle, with the longer chains enabling high loading of the water soluble antigens and optional adjuvants. In addition, polyamino acids are more immunogenic than single amino acids. [0158] Charged moieties within the micelles partially separate from each other in water, and create space for absorption of water soluble agents, such as the peptidic antigen and optional protein adjuvant. Ionized chains with the same type of charge will repel each other and create more space. The ionized polymer also attracts the peptidic antigen, providing stability to the matrix. In addition, the water soluble exterior of the micelle prevents adhesion of the micelles to proteins in body fluids after ionized sites are taken by the therapeutic agent. This type of micelle has very high porosity, up to 95% of the micelle volume, allowing for high loading of aqueous-soluble biologics, such as peptidic antigen and antigen. Particle size range of the micelles is about 20 nm to about 200 nm, with about 20 nm to about 100 nm being preferred for circulation in the blood. [0159] Rate of release of the adjuvant/peptidic antigen from the compositions can be controlled by adjusting the coating thickness, particle size, structure, and density of the coating. Density of the coating can be adjusted by varying the loading of the adjuvant/peptidic antigen in the coating. When the coating contains no peptidic antigen or adjuvant, the polymer coating is densest, and the elution of the peptidic antigen and WO 2006/083874 PCT/US2006/003412 56 optional adjuvant through the coating is slowest. By contrast, when peptidic antigen or adjuvant is loaded into the coating, the coating becomes porous once the peptidic antigen or adjuvant has eluted out, starting from the outer surface of the coating and, therefore, the active agent(s) at the center of the particle can elute at an increased rate. The higher the drug loading in the coating layer, the lower the density and the higher the elution rate. The loading of adjuvant/peptidic antigen in the coating can be lower than that in the interior of the particles beneath the exterior coating. Release rate of adjuvant/peptidic antigen from the particles can also be controlled by mixing particles with different release rates prepared as described above. [01601 Particle size can be determined by, e.g., laser light scattering, using for example, a spectrometer incorporating a helium-neon laser. Generally, particle size is determined at room temperature and involves multiple analyses of the sample in question (e.g., 5-10 times) to yield an average value for the particle diameter. Particle size is also readily determined using scanning electron microscopy (SEM). In order to do so, dry particles are sputter-coated with a gold/palladium mixture to a thickness of approximately 100 Angstroms, and then examined using a scanning electron microscope. Alternatively, the polymer, either in the form of particles or not, can be covalently attached directly to the peptidic antigen, rather than incorporating peptidic antigen therein ("loading" or "matrixing") without chemical attachment, using any of several methods well known in the art and as described hereinbelow. The peptidic antigen content is generally in an amount that represents approximately 0.1% to about 40% (w/w) peptidic antigen to polymer, more preferably about 1% to about 25% (w/w) peptidic antigen, and even more preferably about 2% to about 20% (w/w) peptidic antigen. The percentage of peptidic antigen will depend on the desired dose and the condition being treated, as discussed in more detail below. Following preparation of the particles or polymer molecules loaded with peptidic antigen, with or without adjuvant, the composition can be lyophilized and the dried composition suspended in an appropriate vehicle prior to immunization. [01611 Any suitable and effective amount of immunogenic particles or polymer fragments containing the peptidic antigen and any adjuvant included in the vaccine delivery composition can be released with time from the polymer particles (including those in a polymer depot formed in vivo) and will typically depend, e.g., on the specific WO 2006/083874 PCT/US2006/003412 57 polymer, peptidic antigen, adjuvant or polymer/peptidic antigen linkage, if present. Typically, up to about 100% of the polymer particles or molecules can be released from the polymer depot. Specifically, up to about 90%, up to 75%, up to 50%, or up to 25% thereof can be released from the polymer depot. Factors that typically affect the release rate from the polymer are the nature and amount of the polymer, the types of polymer/peptidic antigen linkage and/or polymer/bioactive agent linkage, and the nature and amount of additional substances present in the formulation. [0162] Once the invention vaccine delivery composition is made, as above, the compositions are formulated for subsequent mucosal or subcutaneous delivery. The compositions will generally include one or more "pharmaceutically acceptable excipients or vehicles" appropriate for mucosal or subcutaneous delivery, such as water, saline, glycerol, polyethyleneglycol, hyaluronic acid, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. [0163] For example, intranasal and pulmonary formulations will usually include vehicles that neither cause irritation to the nasal mucosa nor significantly disturb ciliary function. Diluents such as water, aqueous saline or other known substances can be employed with the subject invention. The nasal formulations may also contain preservatives such as, but not limited to, chlorobutanol and benzalkonium chloride. A surfactant may be present to enhance absorption by the nasal mucosa. [0164] For rectal and urethral suppositories, the vehicle will include traditional binders and carriers, such as, cocoa butter (theobroma oil) or other triglycerides, vegetable oils modified by esterification, hydrogenation and/or fractionation, glycerinated gelatin, polyalkaline glycols, mixtures of polyethylene glycols of various molecular weights and fatty acid esters of polyethylene glycol. [0165] For vaginal delivery, the formulations of the present invention can be incorporated in pessary bases, such as those including mixtures of polyethylene triglycerides, or suspended in oils such as corn oil or sesame oil, optionally containing colloidal silica. See, e.g., Richardson et al., Int. J. Pharm. (1995) 115:9-15.
WO 2006/083874 PCT/US2006/003412 58 [0166] For a further discussion of appropriate vehicles to use for particular modes of delivery, see, e.g., Remington: The Science and Practice ofPharmacy, Mack Publishing Company, Easton, Pa., 19th edition, 1995. One of skill in the art can readily determine the proper vehicle to use for the particular antigen and site of delivery. [0167] The compositions used in the invention methods may comprise an "effective amount" of the peptidic antigen of interest. That is, an amount of antigen will be included in the compositions that will cause the subject to produce a sufficient immunological response in order to prevent, reduce or eliminate symptoms. The exact amount necessary will vary, depending on the subject being treated; the age and general condition of the subject to be treated; the capacity of the subject's immune system to synthesize antibodies; the degree of protection desired; the severity of the condition being treated; the particular antigen selected and its mode of administration, among other factors. An appropriate effective amount can be readily determined by one of skill in the art. Thus, an "effective amount" will fall in a relatively broad range that can be determined through routine trials. For example, for purposes of the present invention, an effective dose will typically range from about 1 pg to about 100 mg, for example from about 5 gg to about 1 mg, or about 10 pg to about 500 jg of the antigen delivered per dose. [0168] Once formulated, the compositions of the invention are administered mucosally or subcutaneously by injection, using standard techniques. See, e.g., Remington: The Science and Practice ofPharmacy, Mack Publishing Company, Easton, Pa., 19th edition, 1995, for mucosal delivery techniques, including intranasal, pulmonary, vaginal and rectal techniques, as well as European Publication No. 517,565 and Illum et al., J. Controlled Rel. (1994) 29:133-141, for techniques of intranasal administration. [0169] Dosage treatment may be a single dose of the invention time release vaccine delivery composition, or a multiple dose schedule as is known in the art. A booster may be with the same formulation given for the primary immune response, or may be with a different formulation that contains the antigen. The dosage regimen will also be determined, at least in part, by the needs of the subject and be dependent on the judgment of the practitioner. Furthermore, if prevention of disease is desired, the vaccine delivery composition is generally administered prior to primary infection with the pathogen of interest. If treatment is desired, e.g., the reduction of symptoms or recurrences, the WO 2006/083874 PCT/US2006/003412 59 vaccine delivery compositions are generally administered subsequent to primary infection. [0170] The invention compositions can be tested in vivo in a number of animal models developed for the study of subcutaneous or mucosal delivery. For example, the conscious sheep model is an art-recognized model for testing nasal delivery of substances See, e.g., Longenecker et al., J. Pharm. Sci. (1987) 76:351-355 and Illum et al., J. Controlled Rel. (1994) 29:133-141. The vaccine delivery composition, generally in powdered, lyophilized form, is blown into the nasal cavity. Blood samples can be assayed for antibody titers using standard techniques, known in the art, as described above. Cellular immune responses can also be monitored as described above. [01711 There are currently a series of in vitro assays for cell-mediated immune response that use cells from the donor. The assays include situations where the cells are from the donor, however, many assays provide a source of antigen presenting cells from other sources, e.g., B cell lines. These in vitro assays include the cytotoxic T lymphocyte assay; lymphoproliferative assays, e.g., tritiated thymidine incorporation; the protein kinase assays, the ion transport assay and the lymphocyte migration inhibition function assay (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. Natd. Acad. Sci. USA, 84: 5384; Kadival, G. J. et al. (1987) J. Innunol., 139:2447; Samuelson, L. E. et al. (1987) J. Inmunol., 139:2708; Cason, J. et al. (1987) J. Immunol. Meth., 102:109; and Tsein, R. J. et al. (1982) Nature, 293: 68. These assays are disadvantageous in that they may lack true specificity for cell mediated immunity activity, they require antigen processing and presentation by an APC of the same MHC type, they are slow (sometimes lasting several days), and some are subjective and/or require the use of radioisotopes. [0172] To test whether a peptide recognized by a T-cell will activate the T-cell to generate an immune response, a so-called "functional test" is used. The enzyme-linked immunospot (ELISpot) assay has been adapted for the detection of individual cells secreting specific cytokines or other effector molecules by attachment of a monoclonal antibody specific for a cytokine or effector molecule on a microplate. Cells stimulated by an antigen are contacted with the immobilized antibody. After washing away cells and any unbound substances, a tagged polyclonal antibody or more often, a monoclonal WO 2006/083874 PCT/US2006/003412 60 antibody, specific for the same cytokine or other effector molecule is added to the wells. Following a wash, a colorant that binds to the tagged antibody is added such that a blue black colored precipitate (or spot) forms at the sites of cytokine localization. The spots can be counted manually or with automated ELISpot reader composition to quantitate the response. A final confirmation of T-cell activation by the test peptide may require in vivo testing, for example in a mouse or other animal model. [01731 As is readily apparent, the invention vaccine delivery compositions are useful for eliciting an immune response against viruses, bacteria, parasites and fungi, for treating and/or preventing a wide variety of diseases and infections caused by such pathogens, as well as for stimulating an immune response against a variety of tumor antigens. Not only can the compositions be used therapeutically or prophylactically, as described above, the compositions may also be used in order to prepare antibodies, both polyclonal and monoclonal, for, e.g., diagnostic purposes, as well as for immunopurification of the antigen of interest. If polyclonal antibodies are desired, a selected mammal, (e.g., mouse, rabbit, goat, horse, etc.) is immunized with the compositions of the present invention. The animal is optionally boosted 2-6 weeks later with one or more administrations of the antigen. Polyclonal antisera is then obtained from the immunized animal and treated according to known procedures. See, e.g., Jnrgens et al. (1985) J. Chron. 348:363-370. 101741 Monoclonal antibodies are generally prepared using the method of Kohler and Milstein, Nature (1975) 256:495-96, or a modification thereof. Typically, a mouse or rat is immunized as described above. However, rather than bleeding the animal to extract serum, the spleen (and optionally several large lymph nodes) is removed and dissociated into single cells. If desired, the spleen cells may be screened (after removal of nonspecifically adherent cells) by applying a cell suspension to a plate or well coated with the protein antigen. B cells, expressing membrane-bound immunoglobulin specific for the antigen, will bind to the plate, and are not rinsed away with the rest of the suspension. Resulting B cells, or all dissociated spleen cells, are then induced to fuse with myeloma cells to form hybridomas, and are cultured in a selective medium (e.g., hypoxanthine, aminopterin, thymidine medium, "HAT"). The resulting hybridomas are plated by limiting dilution, and are assayed for the production of antibodies which bind specifically to the immunizing antigen (and which do not bind to unrelated antigens). The selected WO 2006/083874 PCT/US2006/003412 61 monoclonal antibody-secreting hybridomas are then cultured either in vitro (e.g., in tissue culture bottles or hollow fiber reactors), or in vivo (as ascites in mice). See, e.g., M. Schreier et al., Hybridoma Techniques (1980); Hammerling et al., Monoclonal Antibodies and T-cell Hybridomas (1981); Kennett et al., Monoclonal Antibodies (1980); see also U.S. Pat. Nos. 4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,466,917; 4,472,500, 4,491,632; and 4,493,890. Panels of monoclonal antibodies produced against the polypeptide of interest can be screened for various properties; i.e., for isotype, epitope, affinity, etc. 101751 The following example is meant to illustrate, and not to limit, the invention. EXAMPLE 1 Synthesis of PEA-Antigen Conjugate [01761 Synthesis of PEA succinimidyl ester (PEA-OSu). All examples are from N acetylated polymer (A). PEA 1.392g, 754ptM, calculated for MW=1845 per repeating unit (Formula I, R 1 = (CH 2 )s; R2 = H; R3 = (CH 3
)
2
CHCH
2 ; R4 = (CH 2
)
6 ; n = 70; m/m+p=0.75 and p/m+p=0.
2 5 ) was dissolved in 7ml anhydrous DMF while stirring. To the slightly viscous solution of PEA was added N-Hydroxysuccinimide (NHS), 0.11 Og, 955pLM as a solid. 1-Ethyl-3-(3'-dimethylaminopropyl)carbodiimide hydrochloride, 146mg, 759.8pM, was transferred as a suspension in DMF. The total volume of DMF for the reaction was 1 Oml. The reaction was carried out at room temperature under nitrogen atmosphere for 24 hrs. Synthesis of PEA-Influenza Peptide Conjugate: [0177] Bi) The synthesis of PEA-Peptide conjugate (Formula IV , R'= (CH 2
)
8 ;; R3
(CH
3
)
2
CHCH
2 ; 4 = (CH 2
)
6 ; R5 = NH; n = 70; m/m+p=0.75 and p/m+p=0.
2 5 and R7 = PKYVKQNTLKLAT) was performed with 49.5pM aliquot of the activated ester (A) in DMF and 96 mg (49.5pM) H-PKYVKQNTLKLAT-OH, as a trifluoroacetic acid salt. The peptide was dissolved and transferred to the activated ester in 5ml DMSO. One equivalent, i.e. 49.5pM ethyl-diisopropylamine was added and the reaction was continued for 24hrs under nitrogen. Distilled water, 30p1 in 300pl DMSO was added and stirring was continued at room temperature for another 4hrs.
WO 2006/083874 PCT/US2006/003412 62 [0178] The reaction mixture was precipitated in diethyl ether (60 ml) and, after centrifugation, the obtained material was washed three times with 15ml of diethyl ether. After being air- dried, the obtained product was treated with 3x5ml distilled water under sonication for a minute. After centrifugation, the obtained material was lyophilized. Yield 86mg, 47%. [0179] B2) The synthesis of PEA-Peptide conjugate (Formula IX, cross-linked through R 5
-R
7 -R, wherein R' = (CH 2 )8; R3= (CH 3
)
2
CHCH
2 ; R4= (CH 2
)
6 ; W = NH; n 70; m/m+p=0.75 and p/m+p=0.25 and R7 = PKYVKQNTLKLAT) was performed with 37.7pM aliquot of the activated ester (A) in DMF (600ptl) and 74mg (37.7ptM) H PKYVKQNTLKLAT-OH, as trifluoroacetic acid salt. The peptide was dissolved and transferred to the activated ester in 0.8ml DMSO (dimethylsulfoxide). Four equivalents, i.e. 198pM ethyl-diisopropylamine were added and the reaction was continued for 48hrs under nitrogen. The transparent, gel like material was separated from the organic solvents by decantation. After being cut into 2-3mm large pieces, the product was treated with 17 ml distilled water at +4 C for 18 hrs. After centrifugation and decantation, the material was treated two times with 17ml distilled water (3hrs each time) and after the last centrifugation the product was lyophilized. Yield: 75mg, 53% [0180] B3) The synthesis of PEA-Peptide conjugate (Formula IX cross-linked through R 5 -R7-R 5 , wherein R1 = (CH 2 )8; R3 = (CH 3
)
2
CHCH
2 ; R = (CH 2
)
6 ; R = NH; n = 8; m/m+p=0.7 5 and p/m+p=0.25 and R7= PKYVKQNTLKLAT) was performed with 41.2pM of the activated ester, which was synthesized in a way similar to (A) in DMF (600pl) and 40mg (20.6pM) H-PKYVKQNTLKLAT-OH, as trifluoroacetic acid salt. The peptide was dissolved and transferred to the activated ester in 5ml DMSO. Four equivalents, i.e. 80pM ethyl-diisopropylamine were added and the reaction continued for 72hrs under nitrogen. Distilled water, 75il, (4.2mM) in 300d DMSO was added and stirring continued for another 24hrs. Then the reaction mixture was precipitated in 24ml water/acetone (1:1 v/v). The resulting precipitate was treated with distilled water (4x12ml) for about an hour each time at +4 *C followed by centrifugation. After the last centrifugation, the product was lyophilized. Yield 50mg, 45%.
WO 2006/083874 PCT/US2006/003412 63 Summary of In Vitro Human T Cell Response Protocol [01811 ' CD4+ T cells and monocytes are isolated from the peripheral blood of human donors. The monocytes are cultured for 48 hours in a cytokine-rich medium to induce differentiation into dendritic cells (antigen presenting cells). 24 hours into that culture period, PEA or PEA-hemagglutinin peptide (307-319) conjugates are added to the medium. Two hours prior to starting the co-culture of dendritic cells and T cells, free peptide is added to control wells. T cells cultured together with dendritic cells are measured for activation by proliferation and cytokine secretion at 48h, 72h, and 96h. A schematic diagram of the T-cell response protocol is illustrated in Fig. 3 herein. [0182] T-cell activation in response to dendritic cells exposed to polymer-peptide conjugates were tested using the above protocol. Fig. 4A shows T-cell proliferation over 96 hours in which PEA-peptide conjugates stimulated significant proliferation over peptide or PEA alone. Fig. 4B shows secretion of IL-2 by T-cells over 96 hours in which PEA-peptide (Formula III, Example B 1) stimulated significant IL-2 secretion compared to peptide or PEA alone. 10183] All publications, patents, and patent documents are incorporated by reference herein, as though individually incorporated by reference. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications might be made while remaining within the spirit and scope of the invention. [0184] Although the invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.
Claims (66)
1. A vaccine delivery composition comprising: an effective amount of at least one MHC class I or class II peptidic antigen conjugated to particles or molecules of a biodegradable poly(ester amide) (PEA) polymer having a chemical structure described by structural formula (I), 0 0 HO 0 H I~ HI
4-R1-0-NO--R-O-0--O-.N} n Formula (I) wherein n ranges from about 5 to about 150; R 1 is independently selected from residues of cp-bis(4-carboxyphenoxy)-(C-C 8 ) alkane, 3,3'-(alkanedioyldioxy)dicinnamic acid or 4,4'-(alkanedioyldioxy)dicinnamic acid, (C 2 - C 20 ) alkylene, or (C 2 -C 20 ) alkenylene; the R 3 s in individual n monomers are independently selected from the group consisting of hydrogen, (C 1 -C 6 ) alkyl, (C 2 -C 6 ) alkenyl, (C 2 -C 6 ) alkynyl, (C 6 -C 1 0 ) aryl (C 1 -C 2 0 ) alkyl, and -(CH 2 ) 2 S(CH 3 ); and R4 is independently selected from the group consisting of (C 2 C 20 ) alkylene, (C 2 -C 20 ) alkenylene, (C 2 -C 8 ) alkyloxy, (C 2 -C 20 ) alkylene, a residue of a saturated or unsaturated therapeutic diol, bicyclic-fragments of 1,4:3,6-dianhydrohexitols of structural formula (II), and combinations thereof, (C 2 - C 20 ) alkylene, and (C 2 -C 20 ) alkenylene; C H O H 2 C CH 2 0 C H Formula (II) or a PEA polymer having a chemical formula described by structural formula (III): O 0 HO O H O O H -0-R i-0-N--C--0-R 4-0-0-0-N- - -R I-0-N-0-(CH2)4-N 133 I 1 Formula - H- Formula (III) WO 2006/083874 PCT/US2006/003412 65 wherein n ranges from about 5 to about 150, m ranges about 0.1 to 0.9: p ranges from about 0.9 to 0.1; wherein R1 is independently selected from residues of ac-bis(4 carboxyphenoxy)-(CI-Cs) alkane, 3,3'-(alkanedioyldioxy)dicinnamic acid or 4,4' (alkanedioyldioxy)dicinnamic acid, (C 2 - C 20 ) alkylene, or (C 2 -C 20 ) alkenylene; each R 2 is independently hydrogen, (C 1 -C 12 ) alkyl or (C 6 -C 10 ) aryl or a protecting group; the R 3 s in individual m monomers are independently selected from the group consisting of hydrogen, (C 1 -C 6 ) alkyl, (C 2 -C 6 ) alkenyl, (C 2 -C 6 ) alkynyl, (C 6 -Cio) aryl (C 1 -C 20 ) alkyl, and -(CH 2 ) 2 S(CH 3 ); and R 4 is independently selected from the group consisting of (C 2 C 20 ) alkylene, (C 2 -C 2 0 ) alkenylene, (C 2 -C 8 ) alkyloxy, (C 2 -C 20 ) alkylene, a residue of a saturated or unsaturated therapeutic diol or bicyclic-fragments of 1,4:3,6 dianhydrohexitols of structural formula(II), and combinations thereof or a poly(ester urethane) (PEUR) polymer having a chemical formula described by structural formula (IV), 0 0 H O OH C-0-R 6 -O-0-N--C-O--R 4 -0C-0-N HR 3 R 3 H Formula (IV) wherein n ranges from about 5 to about 150; wherein R 3 s in independently selected from the group consisting of hydrogen, (Ci-C 6 ) alkyl, (C 2 -C 6 ) alkenyl, (C 2 -C 6 ) alkynyl, (C 6 CIO) aryl (C 1 -C 20 ) alkyl, and -(CH 2 ) 2 S(CH 3 ); R 4 is selected from the group consisting of (C 2 -C 20 ) alkylene, (C 2 -C 20 ) alkenylene or alkyloxy, a residue of a saturated or unsaturated therapeutic diol, bicyclic-fragments of 1,4:3,6-dianhydrohexitols of structural formula (II); and combinations thereof, and R6 is independently selected from (C 2 -C 20 ) alkylene, (C 2 -C 20 ) alkenylene or alkyloxy, bicyclic-fragments of 1,4:3,6-dianhydrohexitols of general formula (II), and combinations thereof; or a PEUR polymer having a chemical structure described by general structural formula (V) HO OH 0 0 H -0-0-R6--0-N-0-0-0-R4-0-0-0-N--0C-0-R 6 -0-C-N-0-(CH 2 ) 4 ~ R3 R 3 m H C-0-R 2 H O " Formula (V) WO 2006/083874 PCT/US2006/003412 66 wherein n ranges from about 5 to about 150, m ranges about 0.1 to about 0.9: p ranges from about 0.9 to about 0.1; R2 is independently selected from hydrogen, (C 6 -C 1 O) aryl (C 1 -C 20 ) alkyl, or a protecting group; the R 3 s in an individual m monomer are independently selected from the group consisting of hydrogen, (C 1 -C 6 ) alkyl, (C 2 -C 6 ) alkenyl, (C 2 -C 6 ) alkynyl, (C 6 -C 1 0 ) aryl (C 1 -C 20 ) alkyl and -(CH 2 ) 2 S(CH 3 ); R 4 is selected from the group consisting of (C 2 -C 20 ) alkylene, (C 2 -C 20 ) alkenylene or alkyloxy, a residue of a saturated or unsaturated therapeutic diol and bicyclic-fragments of 1,4:3,6 dianhydrohexitols of structural formula (II) and combinations thereof; and R6 is independently selected from (C 2 -C 20 ) alkylene, (C 2 -C 2 o) alkenylene or alkyloxy, bicyclic fragments of 1,4:3,6-dianhydrohexitols of general formula (II), an effective amount of a residue of a saturated or unsaturated therapeutic diol, and combinations thereof; or a poly (ester urea) (PEU) having a chemical formula described by general structural formula (VI): O HO O H 0-N-A--0-R4-0-0-0-N Rj3 R3 H Formula (VI), wherein n is about 10 to about 150; the R 3 s within an individual n monomer are independently selected from hydrogen, (CI-C 6 ) alkyl, (C 2 -C 6 ) alkenyl, (C 2 -C 6 ) alkynyl, (C 6 -C 10 ) aryl (C 1 -C 2 0 ) alkyl and -(CH 2 ) 2 S(CH 3 ); R 4 is independently selected from (C 2 C 20 ) alkylene, (C 2 -C 2 0 ) alkenylene, (C 2 -Cs) alkyloxy (C 2 -C 2 o) alkylene, a residue of a saturated or unsaturated therapeutic diol; or a bicyclic-fragment of a 1,4:3,6 dianhydrohexitol of structural formula (II); or a PEU having a chemical formula described by structural formula (VII) HO OH O H -C -- 0-0-R 4 -0-C-C -- (CH2) 4 H R3 R3 H. H C-0-R2 H3 . p, n Formula (VII), wherein m is about 0.1 to about 1.0; p is about 0.9 to about 0.1; n is about 10 to about 150; each R2 is independently hydrogen, (C 1 -C 12 ) alkyl or (C 6 -C 10 ) aryl; the R 3 s within an WO 2006/083874 PCT/US2006/003412 67 individual m monomer are independently selected from hydrogen, (CI-C 6 ) alkyl, (C 2 -C 6 ) alkenyl, (C 2 -C 6 ) alkynyl, (C 6 -C 10 ) aryl (C 1 -C 2 0 ) alkyl and -(CH 2 ) 2 S(CH 3 ); each RW is independently selected from (C 2 -C 20 ) alkylene, (C 2 -C 20 ) alkenylene, (C 2 -Cs) alkyloxy (C 2 -C 2 0 ) alkylene, a residue of a saturated or unsaturated therapeutic diol; a bicyclic fragment of a 1,4:3,6-dianhydrohexitol of structural formula (II), and combinations thereof. or (III). 2. The composition of claim 1, wherein the peptidic antigen comprises from 5 to about 30 amino acids. 3. The composition of claim 1, wherein the composition is formulated as a dispersion of the polymer particles or molecules. 4. The composition of claim 1, wherein the polymer is a PEA described by structural formula (I) or (III).
5. The composition of claim 4, wherein at least one R 1 is a residue of apyc-bis (4 carboxyphenoxy) (C 1 -Cs) alkane, 3,3'-(alkanedioyldioxy)dicinnamic acid, or 4,4'(alkanedioyldioxy)dicinnamic acid, or at least one R4 is a bicyclic-fragment of a 1,4:3,6-dianhydrohexitol of structural formula(II).
6. The composition of claim 4, wherein at least one R' is a residue of apco-bis (4 carboxyphenoxy) (C 1 -Cs) alkane, 3,3'-(alkanedioyldioxy)dicinnamic acid, or 4,4' (alkanedioyldioxy)dicinnamic acid, or a mixture thereof, and at least one R 4 is a bicyclic fragment of a 1,4:3,6-dianhydrohexitol of structural formula(II).
7. The composition of claim 1, wherein the polymer is a PEUR described by structural formula (IV) or (V).
8. The composition of claim 7, wherein at least one R1 is a residue of ap-bis(4 carboxyphenoxy) (Ci-C 8 ) alkane, 3,3'-(alkanedioyldioxy)dicinnamic acid, or 4,4' (alkanedioyldioxy)dicinnamic acid, or at least one R 4 is a bicyclic-fragment of a 1,4:3,6 dianhydrohexitol of structural formula (II).
9. The composition of claim 7, wherein at least one R' is a residue of aO-bis (4 carboxyphenoxy) (C 1 -Cs) alkane, 3,3'(alkanedioyldioxy)dicinnamic acid or WO 2006/083874 PCT/US2006/003412 68 4,4'(alkanedioyldioxy)dicinnamic acid, or a mixture thereof, and at least one R 4 is a bicyclic-fragment of a 1,4:3,6-dianhydrohexitol of structural formula (II).
10. The composition of claim 1, wherein the polymer is a PEU described by structural formula (VI) or (VII).
11. The composition of claim 10, wherein at least one R1 is a bicyclic-fragment of a 1,4:3,6-dianhydrohexitol of structural formula (II).
12. The composition of claim 1, wherein the composition forms a time release polymer depot when administered in vivo.
13. The composition of claim 1, wherein the composition biodegrades over a period of twenty-four hours, about seven days, about thirty days, or about 90 days.
14. The composition of claim 1, wherein the composition is in the form of particles having an average diameter in the range from about 10 nanometers to about 1000 microns and the at least one peptidic antigen is dispersed in each polymer molecule of the particles.
15. The composition of claim 14, wherein the particles further comprise a covering of a polymer.
16. The composition of claim 1, wherein the particles have an average diameter in the range from about 10 nanometers to about 10 microns.
17. The composition of claim 1, wherein a particle includes from about 5 to about 150 peptidic antigens per polymer molecule.
18. The composition of claim 1, wherein a polymer molecule has an average molecular weight in a range from about 5,000 to about 300,000 and the at least one peptidic antigen is conjugated to the polymer molecule.
19. The composition of claim 1, wherein a polymer molecule has from about 5 to about 70 peptidic antigens conjugated thereto.
20. The composition of claim 1, wherein the polymer is contained in a polymer antigen conjugate having a chemical structure of structural formula (VIII): WO 2006/083874 PCT/US2006/003412 69 0~ 00 H) 101 1H 5-Ri--N-C- -- R 4 -0-C-C-N C-R -C-N-CH-(CH 2 ) 4 -NH C ICNC- - I H) 3 3 m CO P n R 5 R7 Formula (VIII) wherein n, m, p, R', R 3 , and R 4 are as above, R is selected from the group consisting of -0-, -S-, and -N R-, wherein R8 is H or (Ci-Cs)alkyl; and R7 is the peptidic antigen.
21. The composition of claim 20, except that two or more molecules of the polymer are crosslinked to provide an -R 5 -R 7 - R- conjugate.
22. The composition of claim 20, except that the antigen is covalently linked to one molecule of the polymer through the -R 5 -R 7 - R5- conjugate and R is independently selected from the group consisting of -O-, -S-, and NR8-, wherein R is H or alkyl. (Formula (IX).
23. The composition of claim 21, except that R 1 is independently (C 2 -C 2 0 ) alkylene or (C 2 -C 2 0 ) alkenylene, and wherein one of R is -X-Y-, wherein X is selected from the group consisting of (C 1 -Cis) alkylene, substituted alkylene, (C 3 -Cs) cycloalkylene, substituted cycloalkylene, 5-6 membered heterocyclic system containing 1-3 heteroatoms selected from the group 0, N, and S, substituted heterocyclic, (C 2 -C 1 s) alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, C 6 and CIO aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkylaryl, substituted alkylaryl, arylalkynyl, substituted arylalkynyl, arylalkenyl, substituted arylalkenyl, arylalkynyl, substituted arylalkynyl and wherein the substituents are selected from the group consisting of 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[(=0)(C 1 -C 6 ) alkyl)], S[(0 2 )(CI-C 6 ) alkyl], -C[(=0)(C-C 6 ) alkyl], CF 3 ,-O[(CO)-( Ci-C 6 ) alkyl)], S(0 2 )[N(R 9 R' 0 ), -NH[(C=0)(C 1 -C 6 ) alkyl], -NH(C=O)N(R 9 R 10 ), and -N(R 9 R' 0 ); wherein R? and RIO are independently H or Ci-C 6 alkyl) and Y is selected from the group consisting of -O-, -S-, -S-S-, -S(O)-,-S(0 2 )-, -N R 5 -, -C(=O)-, -OC(=O)-, -C(=0)O-, OC(=O)NH-, -N R 8 C(=O)-, -C(=O)N R 8 -,-N R 8 C(=O)N Ri-, -N RC(=0)N R8-, and -N R C(=S)N R 8 -.
24. The composition of claim 23, except that each R5 is -X-Y-. WO 2006/083874 PCT/US2006/003412 70
25. The composition of claim 23, comprising two molecules of the polymer, except that two of the four repeating units omit R 7 and are crosslinked to provide a single -- R-X R- conjugate, wherein X is selected from the group consisting of (CI-C 1 8 ) alkyl, substituted alkyl, (C 3 -Cs) cycloalkyl, substituted cycloalkyl, 5-6 membered heterocyclic system containing 1-3 heteroatoms selected from the group 0, N, and S, substituted heterocyclic, (C 2 -Ci 8 ) alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, C 6 and CIO aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkylaryl, substituted alkylaryl, arylalkynyl, substituted arylalkynyl, arylalkenyl, substituted arylalkenyl, arylalkynyl, substituted arylalkynyl and wherein the substituents are selected from the group consisting of H, F, Cl, Br, I, (C 1 -C 6 ) alkyl, -CN, -NO 2 .-OH, -O(C 1 -C 4 ) alkyl), S(Ci-C6) alkyl), -S[(=O)(Ci-C6) alkyl)], -S[(O2)(C1-C6) alkyl], -C[(=0)(C1-C6) alkyl], CF 3 ,-O[(CO)-( C 1 -C 6 ) alkyl)], -S(0 2 )[N(R 9 R' 0 ), -NH[(C=O)(C 1 -C 6 ) alkyl], NH(C=O)N(R 9 R'"), and -N(R 9 R' 0 ); wherein R 9 and R1 0 are independently H or (Ci-C 6 ) alkyl.
26. The composition of claim 20, except that two molecules of the polymer are partially crosslinked to provide an -R 5 -X-Y-R 7 -R 5 - conjugate.
27. The composition of claim 22, except that one molecule of the polymer is covalently linked to the antigen through an -R-R 7 -Y-X- R- bridge (Formula XI): R3 R3 -RI-C-N-C;- C- 0-R4- 0-C- CH-N C-R1-C-N-C-(CH2)4-NH H R5 - R7 - Y - X R5 O R3 R3 -(CH2)4--C-NH-C-R-C NH---O-R4-O-C-CH-N-C-Ri-C H H 0Q X 0 0 0 0 n Formula (XI) wherein, X is selected from the group consisting of (CI-C 1 ) alkylene, substituted alkylene, (C 3 -Cs) cycloalkylene, substituted cycloalkylene, 5-6 membered heterocyclic system containing 1-3 heteroatoms selected from the group 0, N, and S, substituted heterocyclic, (C 2 -C 18 ) alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, C 6 and CIO aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkylaryl, substituted WO 2006/083874 PCT/US2006/003412 71 alkylaryl, arylalkynyl, substituted arylalkynyl, arylalkenyl, substituted arylalkenyl, arylalkynyl, substituted arylalkynyl, wherein the substituents are selected from the group H, F, Cl, Br, I, (CI-C 6 ) alkyl, -CN, -NO 2 . -OH, -O(C-C 4 ) alkyl, -S(C-C 6 ) alkyl, S[(=O)(C-C 6 ) alkyl], -S[(0 2 )(C1-C 6 ) alkyl], -C[(=0)(C-C 6 ) alkyl], CF 3 ,-O[(CO)-(C-C 6 ) alkyl], -S(0 2 )[N(R 9 R 0 ), -NH[(C=O)(C-C 6 ) alkyl], -NH(C=O)N(R 9 Rl 0 ), -N(R"R 1 2 ), wherein R 9 and R 10 are independently H or (C-C 6 ) alkyl, R" and R 2 are independently (C 2 -C 20 ) alkylene or (C 2 -C 20 ) alkenylene.
28. The composition of claim 1, wherein the peptidic antigen comprises a Class I epitope of about 8 to about 12 amino acids.
29. The composition of claim 28, further comprising an adjuvant.
30. The composition of claim 29, wherein the adjuvant is covalently bound to the polymer.
31. The composition of claim 29, wherein the adjuvant and the antigen are conjugated to the same polymer.
32. The composition of claim 1, wherein the peptidic antigen comprises a Class II epitope of about 8 to about 30 amino acids.
33. The composition of claim 1, wherein the peptidic antigen comprises an epitope of a virus, bacterium, fungus or tumor cell surface antigen.
34. The composition of claim 33, where the antigens are retro-inverso peptides.
35. The composition of claim 34, where the antigens are partially retro-inverso peptides.
36. The composition of claim 1, wherein the peptidic antigen comprises a viral epitope.
37. The composition of claim 36, wherein the viral epitope is an HIV or influenza viral epitope.
38. The composition of claim 37, wherein the HIV epitope has the amino acid sequence of SEQ ID NO: 8. WO 2006/083874 PCT/US2006/003412 72
39. The composition of claim 37, wherein the influenza epitope has the amino acid sequence of SEQ ID NO:9 or 10.
40. The composition of claim 1, wherein the composition further comprises a pharmaceutically acceptable vehicle.
41. The composition of claim 1, wherein the composition is in the form of disperse droplets in a mist.
42. The composition of claim 41, wherein the mist is produced by a nebulizer.
43. The composition of claim 1, wherein the composition is contained within a nebulizer actuatable to produce a mist comprising dispersed droplets of the vehicle.
44. The composition of claim 1, wherein the composition is contained within an injection device that is actuatable to administer the composition by injection.
45. A method for inducing an immune response in a mammal, said method comprising: administering to the mammal an immunostimulating amount of a vaccine delivery composition of claim 1 in the form of a liquid dispersion of polymer particles or molecules, which are taken up by antigen presenting cells of the mammal to induce an immune response in the mammal.
46. The method of claim 45, wherein the composition forms a time release polymer depot when administered in vivo.
47. The method of claim 45, wherein the composition biodegrades over a period of twenty-four hours, about seven days, about thirty days, or about ninety days.
48. The method of claim 45, wherein the composition is in the form of particles having an average diameter in the range from about 10 nanometers to about 1000 microns and the at least one peptidic antigen is dispersed in the particles.
49. The method of claim 45, wherein the particles have an average diameter in the range from about 10 nanometers to about 10 microns. WO 2006/083874 PCT/US2006/003412 73
50. The method of claim 45, wherein the particles further comprise a covering of the polymer.
51. The method of claim 45, wherein a particle includes from about 5 to about 150 peptidic antigens per polymer molecule.
52. The method of claim 45, wherein a polymer molecule has an average molecular weight in range from about 5,000 to about 300,000 and the at least one peptidic antigen is conjugated to the polymer molecule.
53. The method of claim 45, wherein a polymer molecule has from about 5 to about 70 peptidic antigens conjugated thereto.
54. The method of claim 45, wherein the peptidic antigen comprises a Class I epitope of about 8 to about 12 amino acids.
55. The method of claim 45, further comprising an adjuvant.
56. The method of claim 55, wherein the adjuvant is covalently bound to the polymer.
57. The method of claim 55, wherein the adjuvant and the antigen are conjugated to the same polymer.
58. The method of claim 45, wherein the peptidic antigen comprises a Class II epitope of about 8 to about 30 amino acids.
59. The method of claim 58, wherein the peptidic antigen comprises an epitope of a virus, bacterium, fungus or tumor cell surface antigen.
60. A method for delivering a vaccine to a subject comprising administering to the subject a vaccine delivery composition of claim 1 so that the vaccine is taken up by antigen presenting cells of the subject.
61. A vaccine delivery composition comprising an effective amount of at least one MHC class I or class II peptidic antigen dispersed in a biodegradable polymer comprising at least one type of amino acid conjugated to at least one non-amino acid moiety per monomer. WO 2006/083874 PCT/US2006/003412 74
62. The composition of claim 61, wherein the non-amino acid moiety is between two adjacent amino acids.
63. The composition of claim 61, wherein the non-amino acid moiety is hydrophobic.
64. The composition of claim 61, wherein the peptidic antigen comprises from 5 to about 30 amino acids.
65. The composition of claim 61, wherein the polymer comprises at least two different amino acids.
66. The composition of claim 61, wherein the polymer is a block co-polymer that forms micelles in liquid.
67. A method for inducing an immune response in a mammal, said method comprising: administering to the mammal a vaccine delivery composition of claim 61 in the form of a liquid dispersion of polymer particles or molecules, which are taken up by antigen presenting cells of the mammal to induce an immune response in the mammal.
68. The composition of claim 1, wherein the R 3 s in at least one monomer further can be -(CH 2 ) 3 - and the at least one of the R 3 s cyclizes to form the chemical structure described by structural formula (XVIII): H O -N-C--C-O H 2 CC.CH 2 H 2 Formula (XVIII)
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| PCT/US2006/003412 WO2006083874A2 (en) | 2005-02-01 | 2006-01-31 | Vaccine delivery compositions and methods of use |
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Families Citing this family (17)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US20060177416A1 (en) | 2003-10-14 | 2006-08-10 | Medivas, Llc | Polymer particle delivery compositions and methods of use |
| WO2007038246A2 (en) | 2005-09-22 | 2007-04-05 | Medivas, Llc | Solid polymer delivery compositions and methods for use thereof |
| US8445007B2 (en) | 2005-09-22 | 2013-05-21 | Medivas, Llc | Bis-(α-amino)-diol-diester-containing poly (ester amide) and poly (ester urethane) compositions and methods of use |
| WO2008048298A2 (en) * | 2005-11-21 | 2008-04-24 | Medivas, Llc | Polymer particles for delivery of macromolecules and methods of use |
| JP2009525341A (en) * | 2006-01-31 | 2009-07-09 | メディバス エルエルシー | Vaccine delivery compositions and methods of use |
| US20090202620A1 (en) * | 2006-09-05 | 2009-08-13 | Medivas, Llc | Polymer-stabilized liposomal compositions and methods of use |
| EP2017006A1 (en) | 2007-07-20 | 2009-01-21 | Koninklijke Philips Electronics N.V. | Microfluidic methods and systems for use in detecting analytes |
| JP2011525477A (en) * | 2008-03-04 | 2011-09-22 | リクイディア・テクノロジーズ・インコーポレーテッド | Immunomodulator particles and method of treatment |
| EP2274043A4 (en) * | 2008-05-07 | 2013-07-31 | Medivas Llc | Biodegradable metal-chelating polymers and vaccines |
| EP2404618A1 (en) | 2010-07-07 | 2012-01-11 | Stichting Katholieke Universiteit meer in het bijzonder Radboud Universiteit Nijmegen | Immunomodulatory protein constructs with a helical polymeric backbone. |
| KR101327181B1 (en) * | 2011-02-28 | 2013-11-06 | 이연제약주식회사 | Gene Delivery Complex Comprising Renal Cell-Specific Peptide and Polyester Amine and Composition Comprising The Same for Preventing or Treating of Renal Fibrosis. |
| EP3159368A1 (en) | 2011-06-23 | 2017-04-26 | DSM IP Assets B.V. | New biodegradable polyesteramide copolymers for drug delivery |
| US9873765B2 (en) | 2011-06-23 | 2018-01-23 | Dsm Ip Assets, B.V. | Biodegradable polyesteramide copolymers for drug delivery |
| US10434071B2 (en) | 2014-12-18 | 2019-10-08 | Dsm Ip Assets, B.V. | Drug delivery system for delivery of acid sensitivity drugs |
| EP3288538A4 (en) * | 2015-05-01 | 2018-10-24 | ImmunoVaccine Technologies Inc. | Methods for potentiating an immune response using depot-forming and non-depot-forming vaccines |
| IL269830B2 (en) * | 2017-04-04 | 2024-01-01 | Avidea Tech Inc | Peptide-based vaccines, methods of manufacturing, and uses thereof for inducing an immune response |
| AU2019272844A1 (en) * | 2018-05-22 | 2021-01-21 | Barinthus Biotherapeutics North America, Inc. | Improved methods of manufacturing peptide-based vaccines |
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|---|---|---|---|---|
| US6503538B1 (en) * | 2000-08-30 | 2003-01-07 | Cornell Research Foundation, Inc. | Elastomeric functional biodegradable copolyester amides and copolyester urethanes |
-
2006
- 2006-01-31 WO PCT/US2006/003412 patent/WO2006083874A2/en active Application Filing
- 2006-01-31 CA CA002595783A patent/CA2595783A1/en not_active Abandoned
- 2006-01-31 JP JP2007553357A patent/JP2008532929A/en active Pending
- 2006-01-31 AU AU2006210863A patent/AU2006210863A1/en not_active Abandoned
- 2006-01-31 EP EP06734124A patent/EP1865983A2/en not_active Withdrawn
- 2006-01-31 KR KR1020077019121A patent/KR20070101341A/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| WO2006083874A3 (en) | 2009-04-23 |
| EP1865983A2 (en) | 2007-12-19 |
| WO2006083874A2 (en) | 2006-08-10 |
| JP2008532929A (en) | 2008-08-21 |
| CA2595783A1 (en) | 2006-08-10 |
| KR20070101341A (en) | 2007-10-16 |
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