MESOPOROUS SILICA COMPOSITIONS COMPRISING INFLAMMATORY CYTOKINES FOR MODULATING IMMUNE RESPONSES

Abstract

A composition comprising mesoporous silica rods comprising an immune cell recruitment compound and an immune cell activation compound, and optionally comprising an antigen such as a tumor lysate. The composition is used to elicit an immune response to a vaccine antigen.

Claims

1.-18. (canceled)

19. A method of inducing a systemic antigen-specific immune response to a vaccine antigen, comprising administering to a subject a composition comprising: mesoporous silica rods, wherein the mesoporous silica rods have a length of between 5 μm to 500 μm, and wherein the mesoporous silica rods comprise pores of between 2-50 nm in diameter; and an inflammatory cytokine that recruits an immune cell to the mesoporous silica rods, wherein the inflammatory cytokine is loaded into the mesoporous silica rods and is released from the mesoporous silica rods upon administration to a subject, and wherein the mesoporous silica rods are capable of self-assembly in vivo into a three-dimensional scaffold that allows for immune cell infiltration.

20. A method of inducing homing of vaccine antigen-specific immune cells to a lymph node, comprising administering to a subject a composition comprising: mesoporous silica rods, wherein the mesoporous silica rods have a length of between 5 μm to 500 μm, and wherein the mesoporous silica rods comprise pores of between 2-50 nm in diameter; and an inflammatory cytokine that recruits an immune cell to the mesoporous silica rods, wherein the inflammatory cytokine is loaded into the mesoporous silica rods and is released from the mesoporous silica rods upon administration to a subject, and wherein the mesoporous silica rods are capable of self-assembly in vivo into a three-dimensional scaffold that allows for immune cell infiltration.

21. The method of claim 19, wherein the mesoporous silica rods comprise pores of between 5-25 nm in diameter.

22. The method of claim 19, wherein the mesoporous silica rods comprise pores of between 5-10 nm in diameter.

23. The method of claim 19, wherein the mesoporous silica rods comprise pores of approximately 8 nm in diameter.

24. The method of claim 19, wherein the mesoporous silica rods have a length of 5 μm to 25 μm.

25. The method of claim 19, wherein the mesoporous silica rods have a length of 80 μm to 120 μm.

26. The method of claim 19, wherein the inflammatory cytokine comprises granulocyte macrophage-colony stimulating factor (GM-CSF), chemokine (C-C motif) ligand 21 (CCL-21), chemokine (C-C motif) ligand 19 (CCl-19), or a FMS-like tyrosine kinase 3 (Flt-3) ligand.

27. The method of claim 19, wherein the inflammatory cytokine comprises GM-CSF.

28. The method of claim 19, wherein the composition further comprises a vaccine antigen.

29. The method of claim 28, wherein the vaccine antigen comprises a tumor antigen.

30. The method of claim 29, wherein the tumor antigen is selected from the group consisting of MAGE-1, MART-1/melana, tyrosinase, ganglioside, gp100, GD-2, O-acetylated GD-3, GM-2, Mucin 1, Sos1, protein kinase C-binding protein, reverse transcriptase protein, AKAP protein, VRK1, KIAA1735, T7-1, T11-3, T11-9, Homo sapiens telomerase ferment (hTRT), Cytokeratin-19 (CYFRA21-1), squamous cell carcinoma antigen 1 (SCCA-1), Protein T4-A, squamous cell carcinoma antigen 2 (SCCA-2), ovarian carcinoma antigen CA125 (1A1-3B) (KIAA0049), CTCL tumor antigen se1-1, CTCL tumor antigen se14-3, CTCL tumor antigen se20-4, CTCL tumor antigen se20-9, CTCL tumor antigen se33-1, CTCL tumor antigen se37-2, CTCL tumor antigen se57-1, CTCL tumor antigen se89-1, prostate specific membrane antigen, 5T4 oncofetal trophoblast glycoprotein, Orf73 Kaposi's sarcoma-associated herpesvirus, MAGE-C1 (cancer/testis antigen CT7), MAGE-B1 Antigen (MAGE-XP Antigen), DAM10, MAGE-B2 Antigen (DAM6), MAGE-2 Antigen, MAGE-4a antigen, MAGE-4b antigen, colon cancer antigen NY—CO-45, lung cancer antigen NY-LU-12 variant A, cancer associated surface antigen, adenocarcinoma antigen ART1, paraneoplastic associated brain-testis-cancer antigen, onconeuronal antigen MA2, paraneoplastic neuronal antigen, neuro oncological ventral antigen 2 (NOVA2), hepatocellular carcinoma antigen gene 520, tumor-associated antigen CO-029, tumor-associated antigen MAGE-X2, synovial sarcoma, X breakpoint 2, squamous cell carcinoma antigen recognized by T cell, seriologically defined colon cancer antigen 1, seriologically defined breast cancer antigen NY—BR-15, seriologically defined breast cancer antigen NY—BR-16, Chromogranin A, parathyroid secretory protein 1, DUPAN-2, CA 19-9, CA 72-4, CA 195, and carcinoembryonic antigen (CEA).

31. The method of claim 19, wherein the composition further comprises an immune cell activation compound.

32. The method of claim 31, wherein the immune cell activation compound comprises a TLR agonist.

33. The method of claim 32, wherein the TLR agonist comprises CpG ODN.

34. The composition of claim 19, wherein the composition comprises an immune cell activation compound and an antigen.

35. The composition of claim 34, wherein the inflammatory cytokine comprises GM-CSF, the immune cell activation compound comprises CpG-ODN, and the antigen comprises a tumor antigen.

36. The method of claim 19, wherein the mesoporous silica rods are modified with a chemical group or a compound selected from the group consisting of an amine group, thiol group, chloro group, phosphonate group, glycolic acid, and lactic acid.

37. The method of claim 19, wherein the subject is a human.

38. The method of claim 19, wherein the composition is administered by injection.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG. 1 is an SEM image of the MPS rods. The random stacking and self assembly of these rods generate a 3D space that allows for cell infiltration.

[0019] FIG. 2 is a bar graph showing surface marker expression dependent on MPS rods of different lengths. 100 μg of the MPS rods of different lengths were incubated with bone marrow derived dendritic cells for 18 hour. As a marker of inflammation, the percentage of activated cells was determined by staining for cell surface receptors CD11c (DC marker), MHCII (antigen presentation marker) and CD86 (costimulatory receptor). The inflammatory property of the rods increased with increasing length. Since a desired scaffold exhibits inflammatory properties (similar to the PLG scaffold control), rods between 30 and 100 μm or 120 μm were used in subsequent experiments.

[0020] FIGS. 3A-3C is a series of images showing the effect of MPS rods in a mouse. FIG. 3A is a picture depicting a scaffold injection site excised after 7 days after 5 mg of rhodamine labeled MPS rod was injected subcutaneously into a C57b1/6j mouse. The subcutaneous pocket indicates that the rods were localized and have self-assembled to create a 3D microenvironment. FIG. 3B is an SEM image of the excised scaffold site demonstrating cell infiltration into the scaffold site. FIG. 3C depicts live/dead imaging of the cells detached from the MPS scaffold, demonstrating the recruitment of living cells.

[0021] FIG. 4A is a line graph showing GM-CSF release from MPS rods. 1 μg of GM-CSF was loaded into the MPS rods and incubated in 0.1% BSA/PBS. Supernatant was collected periodically and measured for GM-CSF using ELISA (R&D). GM-CSF is being continuously released from the MPS rods. FIG. 4B is a bar graph showing surface marker expression in an in vitro bone marrow derived dendritic cell (BMDC) co-culture with MPS. 100 μg of the unmodified, amine-, thiol-, chloro- and phosphonate—modified MPS rods were incubated with 10.sup.6/ml BMDCs for 18 hours. The cells were then analyzed for the expression of MHC class-II and the costimulatory molecule CD86. An increased expression of MHCII and CD86 of the BMDCs stimulated by MPS indicates that the MPS rods are inflammatory, and this property was modulated by surface-modifying the MPS rods.

[0022] FIG. 5A is a bar graph depicting recruitment of CDs by GM-CSF loaded MPS rods. 5 mg/mouse of MPS rods loaded with or without 1 μg/mouse GM-CSF were injected subcutaneously. Cells from the scaffold site were retrieved and stained for the CD11c receptor (DC marker). The MPS scaffold loaded with GM-CSF was capable of recruiting more DCs than the blank MPS scaffold. FIG. 5B is a bar graph depicting surface marker expression of DCs. The MPS scaffold loaded with GM-CSF and CpG-ODN activated more DCs than without CpG-ODN.

[0023] FIGS. 6A-6B are bar graphs showing (FIG. 6A) percentage of B220+ cells or (FIG. 6B) total number of B220+ cells at the scaffold site. 5 mg/mouse of MPS rods loaded with or without 1 μg/mouse GM-CSF, 100 μg CpG-ODN and 130 μg ovalbumin were injected subcutaneously. Cells from the scaffold site were retrieved periodically and stained for the B220 receptor (B cell marker). The MPS scaffold loaded with GM-CSF, CpG-ODN and Ova was capable of recruiting more B cells by day 3.

[0024] FIGS. 7A-7C are flow cytometry scatterplots (FIG. 7A) and bar graphs (FIGS. 7B-7C) depicting the presence of the MHC—I-SIINFEKL (SEQ ID NO:1) marker on the surface of DCs. The MPS scaffold loaded with GM-CSF, CpG-ODN and the model antigen ovalbumin induced the expansion of antigen specific DCs homed to the draining lymph node (dLN). dLN cells are stained with the dendritic marker CD11c and the marker MHC—I-SIINFEKL, which indicates that a part of the ovalbumin protein, the peptide sequence SIINFEKL, is being presented on the surface of the cell in the MHC-I complex.

[0025] FIGS. 8A-8B are bar graphs showing formation of the germinal center due to the MPS scaffold. The MPS scaffold loaded with GM-CSF, CpG-ODN and the model antigen ovalbumin was able to induce the germinal center, home to activated and antigen primed B cells, in the dLN. The dLN cells are stained with B220 (B cell marker) and GL7 (germinal center marker) to investigate the formation of the germinal center, which hosts only B cells that are activated and in the process of somatic hypermutation and isotype switching.

[0026] FIGS. 9A-9B are bar graphs depicting that the MPS scaffold loaded with GM-CSF, CpG-ODN and the model antigen ovalbumin was able to induce the expansion of CD8+ cytotoxic T lymphocytes (CTLs) that can specifically recognize the model antigen. Splenocytes were stained for CD8 (killer T cell marker) and with a MHCI-tetramer-SIINFEKL antibody, which indicates whether a T cell is able to recognize a specific antigen presented on the MHCI complex.

[0027] FIGS. 10A-10C are histograms showing that the MPS scaffold loaded with GM-CSF, CpG-ODN and the model antigen ovalbumin was able to induce the clonal expansion of antigen specific CD8+ CTLs in the dLN and the spleen. Injected splenocytes from the OT-I mouse were stained the cell tracker dye CFSE, whose fluorescence halves every time a cell divides. The lower fluorescence in the CFSE stained splenocytes in the spleen and the dLN in the fully loaded vaccine group indicates ova-specific CTL proliferation.

[0028] FIGS. 11A-11C are histograms showing that the MPS scaffold loaded with GM-CSF, CpG-ODN and the model antigen ovalbumin was able to induce the clonal expansion of antigen specific CD4+ THs in the dLN and the spleen. Injected splenocytes from the OT-II mouse were stained the cell tracker dye CFSE, whose fluorescence halves every time a cell divides. The lower fluorescence in the CFSE stained splenocytes in the spleen and the dLN in the fully loaded vaccine group and the MPS loaded with only the antigen group indicates ova-specific CD4+TH cell proliferation.

[0029] FIGS. 12A-12B are histograms showing that antibody titer is defined as the degree to which the antibody-serum solution can be diluted and still contain a detectable amount of the antibody. The anti-ovalbumin serum antibodies were titrated. The MPS scaffold loaded with GM-CSF, CpG-ODN and ova elicited a strong and durable TH1 and TH2 antibody response. MPS loaded with OVA alone can elicit a strong TH2 response, indicating the adjuvant potential of the MPS material itself.

[0030] FIGS. 13A-13B are line graphs showing that GM-CSF is released from the MPS microparticles in a controlled and sustained manner FIG. 13A 1 μg of GM-CSF was loaded into 5 mg of MPS rods and incubated in 0.1% BSA/PBS. Supernatant was collected periodically and measured for GM-CSF using ELISA (R&D). GM-CSF was continuously released from the MPS rods, although in small quantities. FIG. 13B 1 μg of GM-CSF was loaded into 5 mg of MPS containing OVA and CpG. It was then injected subcutaneously into C57BL/6j mice. At various time points, the tissue surrounding the injected particle scaffold was harvested and analyzed for GM-CSF level. The level of GM-CSF was maintained throughout a week.

[0031] FIGS. 14A-14B are photographs, and FIG. 14C is a bar graph. Higher aspect ratio MPS microparticles recruit more cells. FIG. 14A shows SEM images of MPS microparticles either between 10 μm and 20 μm or between 80 μm and 120 μm in length. The longer rods resulted in a composition with greater area or space between the rods. FIG. 14B shows MPS compositions that was harvested from mice. 5 μg of MPS microparticles were injected subcutaneously into mice, and the scaffold site was harvested on day 7 post injection. FIG. 14C is a bar graph showing enumeration of cells that were isolated from the scaffold. MPS microparticles of higher aspect ratio recruited more cells.

[0032] FIGS. 15A-15B are bar graphs. Dendritic cells (DCs) are recruited to the MPS microparticle as a response to GM-CSF. MPS microparticles were loaded with GM-CSF (0 ng, 500 ng, 1000 ng, 3000 ng) and injected subcutaneously into mice. On day 7 post injection, the scaffold was harvested and analyzed using flow cytometry. FIG. 15A shows that recruited CD11c+CD11b+ DCs increases with increasing GM-CSF dose. FIG. 15B shows that recruited mature CD11c+ MHCII+ DCs also DCs increases with increasing GM-CSF dose.

[0033] FIG. 16 is a bar graph showing that GM-CSF increases recruited DC trafficking to the draining LN. MPS microparticles were loaded with Alexa 647 labeled ovalbumin (OVA), or Alexa 647 labeled OVA with 1 μg GM-CSF, and injected subcutaneously into mice. On day 7 post injection, the draining lymph node (dLN) was harvested and analyzed using flow cytometry. With the addition of antigen (OVA), there is a modest increase of Alexa 647+CD11c+ DCs that have trafficked to the scaffold and up-taken the OVA. However, the addition of GM-CSF drastically increases DC trafficking from the scaffold to the dLN.

[0034] FIGS. 17A-17C are scatterplots and FIG. 17D is a bar graph. Local delivery of CpG-ODN from the MPS microparticle scaffold increases circulation of activated DCs. MPS microparticles were loaded with 1 μg of GM-CSF, 300 μg of OVA and 100 μg of CpG-ODN. They were then injected subcutaneously into mice. The dLNs were harvested and analyzed after 7 days post injection. LN cells were stained with CD11c and CD86. The addition of CpG-ODN, which is locally released from the MPS scaffold, further increases the percentage and number of activated, mature DCs in the dLN.

[0035] FIGS. 18A-18C are photographs and FIG. 18D is a bar graph. Proteins are released from the MPS microparticle scaffold in a controlled and sustained manner Alexa 647 labeled ovalbumin was injected subcutaneously into the flank of a mouse either in a buffer solution or loaded onto the MPS microparticles. Relative fluorescence was measured using the IVIS at various time points. If injected in a buffer solution, the protein diffuses away from the injection site in less than 1 day. However, if loaded onto the MPS microparticles, the protein is released from the local scaffold site in a sustained manner, e.g., over the course of a week (2, 3, 4, 5, 7 or more days).

[0036] FIGS. 19A-19B are line graphs showing antibody titers. Antibody titer is defined as the degree to which the antibody-serum solution can be diluted and still contain a detectable amount of the antibody. Mice were vaccinated with 300 μg OVA, 100 μg CpG-ODN and 1 μg GM-CSF in the soluble form or loaded in 5 mg of MPS microparticles. The anti-ovalbumin serum antibodies are titrated. The MPS scaffold loaded with GM-CSF, CpG-ODN and ova can elicit a strong and durable TH1 and TH2 antibody response, as indicated by a high titer of the IgG2a and IgG1 antibody, respectively. More impressively, this response is evident beyond 200 days after vaccination with a single injection. This response was compared to OVA delivered using aluminum hydroxide, the only adjuvant approved for human use in the US. While the MPS vaccine is capable of inducing both TH1 and TH2 antibody responses, due to its capability to load small cytokines, proteins and DNAs, the response induced by alum is completely TH2 biased. The MPS vaccine is very versatile and is readily fine tuned and controlled to induce specific immune responses.

[0037] FIGS. 20A-20D are scatterplots showing that vaccination with the MPS vaccine induces the expansion of T follicular helper cells. OT-II splenocytes were stained with CFSE and adoptively transferred into Thy 1.1+ recipient mice. The recipient mice were then vaccinated with MPS and lysozyme, MPS and OVA, and the full form of the vaccine containing GM-CSF and CpG. Three days after vaccination, dLN were harvested and the adoptively transferred cells were analyzed. It is shown here that mice that were vaccinated with MPS and ova, and the full form of the vaccine, generated a strong population of follicular T helper cells, which are the cells directly responsible for “helping” B cells to differentiate and mature into full, functional antigen specific antibody secreting plasma cells.

[0038] FIGS. 21A-21D are line graphs showing that vaccination with the MPS vaccine induces the clonal expansion of CD4+T helper cells. OT-II splenocytes were stained with CFSE and adoptively transferred into Thy 1.1+ recipient mice. The recipient mice were then vaccinated with MPS and lysozyme, MPS and OVA, and the full form of the vaccine containing GM-CSF and CpG. Three days after vaccination, dLN were harvested and the adoptively transferred cells were analyzed. It is shown here that both MPS and OVA, and the full vaccine, induced strong clonal expansion of the CD4+T helper cells, indicating a systemic antigen specific response.

[0039] FIGS. 22A-22B are line graphs showing that glycolic acid and lactic acid modification of the MPS microparticles aids the release of bioactive GM-CSF. 1 μg of GM-CSF was loaded into 5 mg of glycolic acid or lactic acid modified MPS rods and incubated in 0.1% BSA/PBS. Supernatant was collected periodically and measured for GM-CSF using ELISA (R&D). Compared to GM-CSF released from unmodified MPS microparticles, glycolic acid and lactic acid modification increasecumulative release of bioactive GM-CSF by more than 20 fold.

[0040] FIGS. 23A-23D are scatterplots showing that glycolic acid modified MPS increases the percentage of recruited DCs and mature DCs. 5 mg of unmodified or glycolic acid modified MPS microparticles were loaded with 1 μg of GM-CSF for 1 hour at 37C, and injected subcutaneously into mice. The scaffold was harvested at day 7 and analyzed. It is evident here that the modified MPS almost doubles the percentage of recruited CD11c+CD11b+ DCs, and more drastically increases the percentage of CD11c+CD86+ mature DCs. These results indicate that the great surface modification potential of the MPS microparticles permit further manipulation of the phenotype of recruited cells to induce more potent immune responses.

DETAILED DESCRIPTION

[0041] Injectable MPS-based micro-rods randomly self-assemble to form 3D scaffold in vivo. This system is designed such that it releases a cytokine to recruit and transiently house immune cells, present them with an antigen, and activate them with a danger signal. After recruitment and temporary housing or presence of the cells in the structure, these immune cells migrate out of the device structure and homed to a lymph node. Thus, the composition is one in which cells traffic/circulate in and out of, their status of immune activation being altered/modulated as a result of the trafficking through the device.

[0042] A markedly expanded population of antigen specific dendritic cells was found in the lymph node as well as the formation of the germinal center, which is aided by the involvement of follicular T helper cells in the lymph node as a result of the administration of the device. A population of antigen-recognizing CD8+ CTLs in the spleen was also found to be significantly enhanced. The system also induces the clonal expansion of both antigen specific CD4 and CD8 T cells. In addition to the significant amplification of the cellular immune response, a high and durable antibody production and a balanced T.sub.H1/T.sub.H2 response was detected.

[0043] Key elements of the injectable mesoporous silica micro-rod based scaffold system for modulating immune cell trafficking and reprogramming include: [0044] Aspect ratio of the MPS micro rods (10 nm cross sectional area by 100 micron length) prevents their uptake and therefore allows for local formation of a 3D structure. [0045] 8 nm pore size allows for adsorption of small molecules that can be delivered via diffusion in a controlled and continuous manner. [0046] High surface area to volume ratio allows for the control of the loading of various cytokines, danger signal and antigen. [0047] Inflammatory property of the MPS micro rods promotes immune cell recruitment without the need of other inflammatory cytokines. [0048] Versatility in ability of surface chemical modification of the MPS rods that modulate properties of the rods and ways that proteins are bound to the rods. [0049] Controlled release of GM-CSF can modulate the trafficking of immune cells to the site of the scaffold. [0050] Controlled release of CpG-ODN activates the recruited dendritic cells. [0051] Anchored protein antigens are taken up by the recruited immune cells at the site of the scaffold.

[0052] A pore size of approximately 5-10, e.g., 8 nm, was found to be optimal for loading efficiency for small molecules as well as larger proteins, e.g., GM-CSF (which molecule is about 3 nm in diameter).

[0053] Upon subcutaneous injection of the micro rods suspended in a buffer, the rods randomly stack into a 3D structure; the ends of the rods form physical contact with each other, and a micro space is formed between the contacts.

Mps

[0054] Mesoporous silica nanoparticles are synthesized by reacting tetraethyl orthosilicate with a template made of micellar rods. The result is a collection of nano-sized spheres or rods that are filled with a regular arrangement of pores. The template can then be removed by washing with a solvent adjusted to the proper pH. In another technique, the mesoporous particle could be synthesized using a simple sol-gel method or a spray drying method. Tetraethyl orthosilicate is also used with an additional polymer monomer (as a template). Other methods include those described in U.S. Patent Publication 20120264599 and 20120256336, hereby incorporated by reference.

Granulocyte Macrophage Colony Stimulating Factor (GM-CSF)

[0055] Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a protein secreted by macrophages, T cells, mast cells, endothelial cells and fibroblasts. Specifically, GM-CSF is a cytokine that functions as a white blood cell growth factor. GM-CSF stimulates stem cells to produce granulocytes and monocytes. Monocytes exit the blood stream, migrate into tissue, and subsequently mature into macrophages.

[0056] Scaffold devices described herein comprise and release GM-CSF polypeptides to attract host DCs to the device. Contemplated GM-CSF polypeptides are isolated from endogenous sources or synthesized in vivo or in vitro. Endogenous GM-CSF polypeptides are isolated from healthy human tissue. Synthetic GM-CSF polypeptides are synthesized in vivo following transfection or transformation of template DNA into a host organism or cell, e.g. a mammal or cultured human cell line. Alternatively, synthetic GM-CSF polypeptides are synthesized in vitro by polymerase chain reaction (PCR) or other art-recognized methods Sambrook, J., Fritsch, E. F., and Maniatis, T., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY, Vol. 1, 2, 3 (1989), herein incorporated by reference).

[0057] GM-CSF polypeptides are modified to increase protein stability in vivo. Alternatively, GM-CSF polypeptides are engineered to be more or less immunogenic. Endogenous mature human GM-CSF polypeptides are glycosylated, reportedly, at amino acid residues 23 (leucine), 27 (asparagine), and 39 (glutamic acid) (see U.S. Pat. No. 5,073,627). GM-CSF polypeptides of the present invention are modified at one or more of these amino acid residues with respect to glycosylation state.

[0058] GM-CSF polypeptides are recombinant. Alternatively GM-CSF polypeptides are humanized derivatives of mammalian GM-CSF polypeptides. Exemplary mammalian species from which GM-CSF polypeptides are derived include, but are not limited to, mouse, rat, hamster, guinea pig, ferret, cat, dog, monkey, or primate. In a preferred embodiment, GM-CSF is a recombinant human protein (PeproTech, Catalog #300-03). Alternatively, GM-CSF is a recombinant murine (mouse) protein (PeproTech, Catalog #315-03). Finally, GM-CSF is a humanized derivative of a recombinant mouse protein.

[0059] Human Recombinant GM-CSF (PeproTech, Catalog #300-03) is encoded by the following polypeptide sequence (SEQ ID NO:2):

TABLE-US-00001 MAPARSPSPS TQPWEHVNAI QEARRLLNLS RDTAAEMNET VEVISEMFDL QEPTCLQTRL ELYKQGLRGS LTKLKGPLTM MASHYKQHCP PTPETSCATQ IITFESFKEN LKDFLLVIPF DCWEPVQE

[0060] Murine Recombinant GM-CSF (PeproTech, Catalog #315-03) is encoded by the following polypeptide sequence (SEQ ID NO: 3):

TABLE-US-00002 MAPTRSPITV TRPWKHVEAI KEALNLLDDM PVTLNEEVEV VSNEFSFKKL TCVQTRLKIF EQGLRGNFTK LKGALNMTAS YYQTYCPPTP ETDCETQVTT YADFIDSLKT FLTDIPFECK KPVQK

[0061] Human Endogenous GM-CSF is encoded by the following mRNA sequence (NCBI Accession No. NM_000758 and SEQ ID NO: 4):

TABLE-US-00003 1 acacagagag aaaggctaaa gttctctgga ggatgtggct gcagagcctg ctgctcttgg 61 gcactgtggc ctgcagcatc tctgcacccg cccgctcgcc cagccccagc acgcagccct 121 gggagcatgt gaatgccatc caggaggccc ggcgtctcct gaacctgagt agagacactg 181 ctgctgagat gaatgaaaca gtagaagtca tctcagaaat gtttgacctc caggagccga 241 cctgcctaca gacccgcctg gagctgtaca agcagggcct gcggggcagc ctcaccaagc 301 tcaagggccc cttgaccatg atggccagcc actacaagca gcactgccct ccaaccccgg 361 aaacttcctg tgcaacccag attatcacct ttgaaagttt caaagagaac ctgaaggact 421 ttctgcttgt catccccttt gactgctggg agccagtcca ggagtgagac cggccagatg 481 aggctggcca agccggggag ctgctctctc atgaaacaag agctagaaac tcaggatggt 541 catcttggag ggaccaaggg gtgggccaca gccatggtgg gagtggcctg gacctgccct 601 gggccacact gaccctgata caggcatggc agaagaatgg gaatatttta tactgacaga 661 aatcagtaat atttatatat ttatattttt aaaatattta tttatttatt tatttaagtt 721 catattccat atttattcaa gatgttttac cgtaataatt attattaaaa atatgcttct 781 a

[0062] Human Endogenous GM-CSF is encoded by the following amino acid sequence (NCBI Accession No. NP_000749.2 and SEQ ID NO: 5):

TABLE-US-00004 MWLQSLLLLGTVACSISAPARSPSPSTQPWEHVNAIQEARRLLNLSRDTA AEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASH YKQHCPPTPETSCATQIITFESFKENLKDFLLVIPFDCWEPVQE

Cytosine-Guanosine (CpG) Oligonucleotide (CpG-ODN) Sequences

[0063] CpG sites are regions of deoxyribonucleic acid (DNA) where a cysteine nucleotide occurs next to a guanine nucleotide in the linear sequence of bases along its length (the “p” represents the phosphate linkage between them and distinguishes them from a cytosine-guanine complementary base pairing). CpG sites play a pivotal role in DNA methylation, which is one of several endogenous mechanisms cells use to silence gene expression. Methylation of CpG sites within promoter elements can lead to gene silencing. In the case of cancer, it is known that tumor suppressor genes are often silenced while oncogenes, or cancer-inducing genes, are expressed. CpG sites in the promoter regions of tumor suppressor genes (which prevent cancer formation) have been shown to be methylated while CpG sites in the promoter regions of oncogenes are hypomethylated or unmethylated in certain cancers. The TLR-9 receptor binds unmethylated CpG sites in DNA.

[0064] The vaccine composition described herein comprises CpG oligonucleotides. CpG oligonucleotides are isolated from endogenous sources or synthesized in vivo or in vitro. Exemplary sources of endogenous CpG oligonucleotides include, but are not limited to, microorganisms, bacteria, fungi, protozoa, viruses, molds, or parasites. Alternatively, endogenous CpG oligonucleotides are isolated from mammalian benign or malignant neoplastic tumors. Synthetic CpG oligonucleotides are synthesized in vivo following transfection or transformation of template DNA into a host organism. Alternatively, Synthetic CpG oligonucleotides are synthesized in vitro by polymerase chain reaction (PCR) or other art-recognized methods (Sambrook, J., Fritsch, E. F., and Maniatis, T., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY, Vol. 1, 2, 3 (1989), herein incorporated by reference).

[0065] CpG oligonucleotides are presented for cellular uptake by dendritic cells. For example, naked CpG oligonucleotides are used. The term “naked” is used to describe an isolated endogenous or synthetic polynucleotide (or oligonucleotide) that is free of additional substituents. In another embodiment, CpG oligonucleotides are bound to one or more compounds to increase the efficiency of cellular uptake. Alternatively, or in addition, CpG oligonucleotides are bound to one or more compounds to increase the stability of the oligonucleotide within the scaffold and/or dendritic cell. CpG oligonucleotides are optionally condensed prior to cellular uptake. For example, CpG oligonucleotides are condensed using polyethylimine (PEI), a cationic polymer that increases the efficiency of cellular uptake into dendritic cells.

[0066] CpG oligonucleotides can be divided into multiple classes. For example, exemplary CpG-ODNs encompassed by compositions, methods and devices of the present invention are stimulatory, neutral, or suppressive. The term “stimulatory” describes a class of CpG-ODN sequences that activate TLR9. The term “neutral” describes a class of CpG-ODN sequences that do not activate TLR9. The term “suppressive” describes a class of CpG-ODN sequences that inhibit TLR9. The term “activate TLR9” describes a process by which TLR9 initiates intracellular signaling.

[0067] Stimulatory CpG-ODNs can further be divided into three types A, B and C, which differ in their immune-stimulatory activities. Type A stimulatory CpG ODNs are characterized by a phosphodiester central CpG-containing palindromic motif and a phosphorothioate 3′ poly-G string. Following activation of TLR9, these CpG ODNs induce high IFN-α production from plasmacytoid dendritic cells (pDC). Type A CpG ODNs weakly stimulate TLR9-dependent NF-κB signaling.

[0068] Type B stimulatory CpG ODNs contain a full phosphorothioate backbone with one or more CpG dinucleotides. Following TLR9 activation, these CpG-ODNs strongly activate B cells. In contrast to Type A CpG-ODNs, Type B CpG-ODNS weakly stimulate IFN-α secretion.

[0069] Type C stimulatory CpG ODNs comprise features of Types A and B. Type C CpG-ODNs contain a complete phosphorothioate backbone and a CpG containing palindromic motif. Similar to Type A CpG ODNs, Type C CpG ODNs induce strong IFN-α production from pDC. Similar to Type B CpG ODNs, Type C CpG ODNs induce strong B cell stimulation.

[0070] Exemplary stimulatory CpG ODNs comprise, but are not limited to, ODN 1585, ODN 1668, ODN 1826, ODN 2006, ODN 2006-G5, ODN 2216, ODN 2336, ODN 2395, ODN M362 (all InvivoGen). The present invention also encompasses any humanized version of the preceding CpG ODNs. In one preferred embodiment, compositions, methods, and devices of the present invention comprise ODN 1826 (the sequence of which from 5′ to 3′ is tccatgacgttcctgacgtt, wherein CpG elements are bolded, SEQ ID NO: 10).

[0071] Neutral, or control, CpG ODNs that do not stimulate TLR9 are encompassed by the present invention. These ODNs comprise the same sequence as their stimulatory counterparts but contain GpC dinucleotides in place of CpG dinucleotides.

[0072] Exemplary neutral, or control, CpG ODNs encompassed by the present invention comprise, but are not limited to, ODN 1585 control, ODN 1668 control, ODN 1826 control, ODN 2006 control, ODN 2216 control, ODN 2336 control, ODN 2395 control, ODN M362 control (all InvivoGen). The present invention also encompasses any humanized version of the preceding CpG ODNs.

Antigens

[0073] Compositions, methods, and devices described herein comprise tumor antigens or other antigens. Antigens elicit protective immunity or generate a therapeutic immune response in a subject to whom such a device was administered. Preferred tumor antigens are tumor cell lysates (see, e.g., Ali et. Al., 2009, Nature Materials 8, 151-158; hereby incorporated by reference). For example, a whole tumor or tumor biopsy sample is extracted from a human patient (or non-human animal), and digested using collagenase to degrade the extra cellular matrix. Then the tumor cells then undergo three cycles of the freeze thaw process, in which the cells are frozen in liquid N.sub.2 and then thawed in a water bath. This process generates the tumor antigens, which are then loaded onto the MPS particles at the same time with GM-CSF and CpG ODN. For example, a vaccine dose of tumor cell lysate antigen is the amount obtained from 1×10.sup.6 tumor cells. After fabrication of the MPS particles, the particles are suspended in a physiologically accepted buffer, e.g., PBS, and the recruitment compound (e.g., GM-CSF), activating compound (e.g., CpG), and antigen added to the suspension of rods. The mixture is shaken at room temperature overnight and then lyophilized for about 4 hours. Prior to administration, the rods are again suspended in buffer and the suspension loaded into a 1 ml syringe (18 gauge needle). A typical vaccine dose is 150 μl of the mixture per injection.

[0074] Exemplary cancer antigens encompassed by the compositions, methods, and devices of the present invention include, but are not limited to, tumor lysates extracted from biopsies, irradiated tumor cells, MAGE series of antigens (MAGE-1 is an example), MART-1/melana, tyrosinase, ganglioside, gp100, GD-2, O-acetylated GD-3, GM-2, MUC-1, Sos1, Protein kinase C-binding protein, Reverse transcriptase protein, AKAP protein, VRK1, KIAA1735, T7-1, T11-3, T11-9, Homo Sapiens telomerase ferment (hTRT), Cytokeratin-19 (CYFRA21-1), SQUAMOUS CELL CARCINOMA ANTIGEN 1 (SCCA-1), (PROTEIN T4-A), SQUAMOUS CELL CARCINOMA ANTIGEN 2 (SCCA-2), Ovarian carcinoma antigen CA125 (1A1-3B) (KIAA0049), MUCIN 1 (TUMOR-ASSOCIATED MUCIN), (CARCINOMA-ASSOCIATED MUCIN), (POLYMORPHIC EPITHELIAL MUCIN),(PEM),(PEMT),(EPISIALIN), (TUMOR-ASSOCIATED EPITHELIAL MEMBRANE ANTIGEN),(EMA),(H23AG), (PEANUT-REACTIVE URINARY MUCIN), (PUM), (BREAST CARCINOMA—ASSOCIATED ANTIGEN DF3), CTCL tumor antigen se1-1, CTCL tumor antigen se14-3, CTCL tumor antigen se20-4, CTCL tumor antigen se20-9, CTCL tumor antigen se33-1, CTCL tumor antigen se37-2, CTCL tumor antigen se57-1, CTCL tumor antigen se89-1, Prostate-specific membrane antigen, 5T4 oncofetal trophoblast glycoprotein, Orf73 Kaposi's sarcoma-associated herpesvirus, MAGE-C1 (cancer/testis antigen CT7), MAGE-B1 ANTIGEN (MAGE-XP ANTIGEN) (DAM10), MAGE-B2 ANTIGEN (DAM6), MAGE-2 ANTIGEN, MAGE-4a antigen, MAGE-4b antigen, Colon cancer antigen NY—CO-45, Lung cancer antigen NY-LU-12 variant A, Cancer associated surface antigen, Adenocarcinoma antigen ART1, Paraneoplastic associated brain-testis-cancer antigen (onconeuronal antigen MA2; paraneoplastic neuronal antigen), Neuro-oncological ventral antigen 2 (NOVA2), Hepatocellular carcinoma antigen gene 520, TUMOR-ASSOCIATED ANTIGEN CO-029, Tumor-associated antigen MAGE-X2, Synovial sarcoma, X breakpoint 2, Squamous cell carcinoma antigen recognized by T cell, Serologically defined colon cancer antigen 1, Serologically defined breast cancer antigen NY—BR-15, Serologically defined breast cancer antigen NY—BR-16, Chromogranin A; parathyroid secretory protein 1, DUPAN-2, CA 19-9, CA 72-4, CA 195, Carcinoembryonic antigen (CEA). Purified tumor antigens are used alone or in combination with one another.

[0075] The system is also useful to generate an immune response to other antigens such as microbial pathogens (e.g., bacteria, viruses, fungi).

[0076] The following materials and methods were used to generate the data described herein.

MPS Scaffold Fabrication

[0077] The Pluronic P-123 (Sigma-Aldrich) surfactant was dissolved in 1.6M HCl at room temperature, and heated 40 degrees C. 42 mmol of Tetraethyl orthosilicate (TEOS) (Sigma-Aldrich) was added and heated for 20 hours at 40 degrees C. under stirring (600 rpm). The composition was then heated to 100 degrees C. for 24 hours. The rod particles were collected by filtration and air dried at room temperature. The particles were extracted in ethanol/HCl (5 parts HCl to 500 parts EtOH) overnight at 80 degrees C. Alternatively, the particles were calcined at 550 degrees C. for 4 hours in 100% ethanol. The MPS composition may be stored and shipped for use before or after adding recruitment and/or activation compounds and before or after adding antigen. For example, if antigen is a tumor cells lysate made from a biopsy sample taken from a patient, it may be processed and added to MPS particles shortly before administration to the patient.

[0078] For full vaccine composition, 1 μg/mouse GM-CSF, 100 μg/mouse CpG-ODN and 130 μg/mouse of the ovalbumin protein were incubated with 5 mg/mouse MPS in dH.sub.2O for 12 hours, lyophilized for 12 hours, resuspended in 150 μl/mouse PBS and injected subcutaneously using a 18G needle.

[0079] To determine the release kinetics of GM-CSF, and CpG-ODN from the MPS rods, radioactive I-labeled recombinant human GM-CSF was used as a tracer, and standard release studies were carried out. Similarly, the amount of CpG-ODN released into PBS was determined by the absorbance readings using a Nanodrop instrument (ND1000, Nanodrop Technologies).

Modification of MPS Microparticles

[0080] Standard 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) chemistry is used to activate a carboxylic acid on glycolic acid or lactic acid. The MPS particles are amine modified using amine propyl silane. The two components are then mixed together at room temperature overnight to react. The particles are then allowed to air dry. The resulting glycolic acid and/or lactic acid modified particles are then contacted with GM-CSF or other compounds as described above.

In Vitro DC Activation Assay

[0081] Murine Dendritic Cells (DCs) were differentiated from the bone marrow cells using 20 ng/ml GM-CSF. Differentiated DCs, at 10.sup.6/ml, were incubated with 100 μg/ml of the MPS particles for 18 hours, and analyzed for the expression of CD11c (ebiosciences, San Diego, Calif.), CD86 (ebiosciences, San Diego, Calif.) and MHC class-II (ebiosciences, San Diego, Calif.) using flow cytometry.

Analysis of DC Recruitment to MPS Scaffold and Emigration to Lymph Nodes

[0082] The MPS scaffold was retrieved from the animal and digested by mechanically separating the cells from the rods. APC conjugated CD11c (dendritic cell marker), FITC conjugated MHC class-II, and PE conjugated CD86 (B7, costimulatory molecule) stains were used for DC and leukocyte recruitment analysis. Cells were gated according to positive fluorescein isothiocyanate (FITC), Allophycocyanin (APC) and phycoerythrin (PE) using isotype controls, and the percentage of cells staining positive for each surface antigen was recorded.

[0083] To determine the presence of antigen specific DCs in the lymph nodes, the draining lymph node (dLN) was harvested and analyzed using APC conjugated CD11c and PE conjugated SIINFEKL-MHC class-I (ebiosciences, San Diego, Calif.).

Analysis of Antigen Specific CD8+ Spleen T Cells

[0084] The spleen was harvested and digested. After lysing the red blood cells, the splenocytes were analyzed using PE-CY7 conjugated CD3 (ebiosciences, San Diego, Calif.), APC conjugated CD8 (ebiosciences, San Diego, Calif.) and the PE conjugated SIINFEKL (SEQ ID NO:) MHC class-I tetramer (Beckman Coulter). SIINFEKL is an ovalbumin derived peptide [OVA(257-264)].

Analysis of CD4+ or CD8+ T Cell Clonal Expansion

[0085] The spleens from the OT-II (for CD4) or OT-I (for CD8) transgenic C57b1/6 mice (Jackson Laboratories) were harvested, digested, pooled and stained with the cell tracer Carboxyfluorescein succinimidyl ester (CFSE). 20×10.sup.6 stained splenocytes/mouse were IV injected into the C57b1/6 (Jackson Laboratories) mice two days post immunization. The dLNs and spleens were retrieved after four days post IV injection and analyzed using PE conjugated CD8 or CD4 marker.

Characterization of Anti-OVA Humoral Response

[0086] Blood sera were analyzed for IgG1, and IgG2a antibodies by ELISA using ovalbumin-coated plates. Antibody titration was defined as the lowest serum dilution at which the ELISA OD reading is >0.3 (blank).

OTHER EMBODIMENTS

[0087] While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0088] The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. Genbank and NCBI submissions indicated by accession number cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.

[0089] While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.