WO2016057986A1 - Constructions d'épitopes en tandem pour la présentation d'épitopes cd4 et cd8, et leurs utilisations - Google Patents

Constructions d'épitopes en tandem pour la présentation d'épitopes cd4 et cd8, et leurs utilisations Download PDF

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WO2016057986A1
WO2016057986A1 PCT/US2015/055042 US2015055042W WO2016057986A1 WO 2016057986 A1 WO2016057986 A1 WO 2016057986A1 US 2015055042 W US2015055042 W US 2015055042W WO 2016057986 A1 WO2016057986 A1 WO 2016057986A1
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cells
cell
nucleic acid
epitopes
acid construct
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Remi Creusot
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The Trustees Of Columbia University In The City Of New York
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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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0008—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4615—Dendritic cells
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4621—Cellular immunotherapy characterized by the effect or the function of the cells immunosuppressive or immunotolerising
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/46433—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/46443—Growth factors
    • A61K39/464434—Transforming growth factor [TGF]
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • 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
    • A61K2039/577—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 tolerising response
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/605—MHC molecules or ligands thereof
    • 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
    • A61K2039/70—Multivalent vaccine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule

Definitions

  • Self -recognition or immune tolerance is mediated by several mechanisms including presentation of a "self peptide to a CD4+ or CD8+ T cell, eliminating, inhibiting, or converting autoreactive (self-reactive) T cells that would potentially attack a cell producing the autoantigen (self-protein) from which the particular epitope (peptide) is derived.
  • Autoimmune diseases such as Type 1 diabetes (T1D), rheumatoid arthritis (RA), multiple sclerosis (MS), psoriasis, systemic lupus erythematosus (SLE), and inflammatory bowel disease (IBD) can develop when this process is deficient or altered, and the immune system inappropriately attacks the body's own tissues.
  • Immune tolerance which prevents an immunogenic response from developing upon self-recognition, is mediated by several mechanisms, primarily involving the presentation of "self peptides to CD4+ or CD8+ T cells in a manner that results in the elimination, inhibition, or conversion of the autoreactive T cells that otherwise would potentially attack cells and tissues that are the source of autoantigens and/or support the production by B cells of antibodies that react against these autoantigens.
  • the presentation of epitopes peptides or regions of a protein antigen that are recognized by immune cells
  • APCs antigen-presenting cells
  • Antigen- presenting cells that acquire antigens exogenously (from their milieu) more effectively engage CD4+ T cells, whereas those producing antigens endogenously present epitopes mainly to CD8+ T cells.
  • both CD4+ and CD8+ self -reactive T cells should be silenced in order to prevent autoimmunity.
  • a strategy and method for eliciting a broad tolerogenic response in autoreactive (self- reactive) T cells using antigen- specific therapy, which specifically targets the pathogenic T cells to modulate or prevent autoimmune disorders is needed in the art.
  • the invention described herein addresses these needs.
  • the invention relates to tandem epitope constructs (TEC) for optimal presentation of CD4 and CD8 epitopes and uses thereof in which a series of selected epitopes from multiple antigens are expressed, whereby the epitopes can be differentially targeted to the proper intracellular processing machinery for MHC class I (via proteasome) or class II (via endosome) presentation, respectively, and affect CD8+ or CD4+ cells, respectively.
  • constructs can be co-expressed to produce inhibitory ligands or cytokines, which can interfere with the ability of the APC to activate T cells.
  • the invention herein described relates to an isolated nucleic acid construct encoding a polypeptide which comprises (a) a first sequence comprising an MHC class II targeting sequence and one or more different epitope sequences intended for processing in endosomes; (b) a second sequence comprising one or more different epitope sequences intended for processing by cytoplasmic proteasomes; and (c) a cytosolic protease cleavage site located between the first and second sequences.
  • the epitope sequences in the first sequence are processed in endosomes for presentation to CD4+ T cells and the epitope sequences in the second sequence are processed in cytoplasmic proteasomes for presentation to CD8+ T cells.
  • the nucleic acid construct further comprises (a) one or more endosomal proteolytic cleavage sites flanking the one or more epitope sequences of the first sequence; (b) one or more cytoplasmic proteolytic cleavage sites flanking the one or more epitope sequences of the second sequence; or (c) both (a) and (b).
  • the MHC class II targeting sequence is operably linked to the one or more different epitope sequences intended for processing in endosomes.
  • the nucleic acid construct is DNA, or mRNA.
  • the invention also includes an expression vector comprising the nucleic acid construct described above, which can be a viral vector.
  • sequences of the nucleic acid construct are in the order, from 5' to 3' : (a) the first sequence; (b) the cleavage site; (c) the second sequence.
  • the cleavage site as described above is recognized and cleaved by a cytosolic protease.
  • This cleavage site preferably is selected from the group consisting of a T2A cleavage site, a P2A cleavage site, an E2A cleavage site, and an F2A cleavage site.
  • the MHC class II targeting sequence of the nucleic acid construct is selected from the group consisting of an upstream transferrin receptor domain 1-118; an upstream invariant chain domain 1-80; an upstream invariant chain domain 1-214; an upstream CD16 domain 1-23 and a downstream LAMP-1 domain 166-382; a downstream LIMP- 2 domain 459-478; downstream a CDla-d cytoplasmic tail; and a downstream PMEL domain 506-575.
  • the invention includes nucleic acid constructs wherein at least one epitope in the first sequence or the second sequences is from a self antigen, which preferably is a self antigen recognized in an autoimmune disease condition,
  • This self antigen can be selected from the group consisting of type 1 diabetes, rheumatoid arthritis, multiple sclerosis, psoriasis, Crohn's disease and ulcerative colitis.
  • the invention also includes a tolerogenic DNA vaccine comprising the nucleic acid construct as described above.
  • the tolerogenic DNA vaccine has CpG motifs removed from the backbone of the DNA and optionally replaced by GpG motifs.
  • the invention also includes nucleic acid constructs wherein at least one epitope in the first sequence or the second sequences is from a non-self antigen, for example an infectious disease antigen, preferably an antigen from a pathogen selected from the group consisting of a virus, bacteria, fungus, or parasite, or a cancer antigen.
  • a non-self antigen for example an infectious disease antigen, preferably an antigen from a pathogen selected from the group consisting of a virus, bacteria, fungus, or parasite, or a cancer antigen.
  • the invention also includes an immunogenic DNA vaccine comprising the nucleic acid construct as described above.
  • the immunogenic DNA vaccine has CpG motifs are maintained in the backbone of the DNA or optionally added to it.
  • At least one of the epitopes in the first sequence, the second sequence or both is a mimotope.
  • the nucleic acid construct is codon-optimized.
  • the invention also comprises an isolated antigen-presenting cell that expresses the nucleic acid construct of any of the above-mentioned embodiments.
  • the antigen presenting cell preferably is a dendritic cell, a macrophage, a B cell, a mesenchymal stromal cell, an epithelial cell, an endothelial cell and a fibroblastic cell, and most preferably is a dendritic cell.
  • the invention further comprises a peptide encoded by the nucleic acid construct described as any of the above embodiments, and a pharmaceutical composition comprising any of the nucleic acid constructs described above or any of the peptides encoded by these nucleic acid constructs or any of the cells containing the peptides or nucleic acid constructs.
  • a pharmaceutical composition comprising any of the nucleic acid constructs described above or any of the peptides encoded by these nucleic acid constructs or any of the cells containing the peptides or nucleic acid constructs.
  • Such pharmaceutical compositions preferably also contain a pharmaceutically acceptable carrier.
  • the invention also includes a method of inactivating a self-reactive T cell in a subject in need, comprising administering to the subject any of the described nucleic acid constructs or any of the cells that express the described nucleic acid constructs, where the epitope sequences of the first and second sequences comprise an epitope to which the subject is self-reactive, for example a diabetogenic antigen epitope.
  • the invention also includes a method of treating a subject suffering from an autoimmune disease, comprising administering to the subject any of the described nucleic acid constructs or any of the cells that express the described nucleic acid constructs.
  • the autoimmune disease preferably is selected from the group consisting of type 1 diabetes, rheumatoid arthritis, multiple sclerosis, psoriasis, Crohn's disease and ulcerative colitis.
  • the methods of the invention also preferably comprise further administering to the subject an immune inhibiting compound, which can be selected from the group consisting of transforming growth factor beta (TGF- ⁇ ), interleukin 10 (IL-10), interleukin 1 receptor antagonist (IL-1RA), interleukin 4 (IL-4), interleukin 27 (IL-27),interleukin 35 ( IL-35), programmed death-ligand 1 (PD-L1), inducible T-cell co-stimulator ligand (ICOSL), B7-H4, CD39, CD73, FAS, FAS-IL, indoleamine 2,3-dioxygenase 1 (IDOl), indoleamine 2,3- dioxygenase 2 (ID02), acetaldehyde dehydrogenase 1 (ALDHl)/retinaldehyde dehydrogenase (RALDH), arginase 1 (ARG1), arginase 2 (ARG2), nitrous oxide synthase (NOS
  • Preferred methods include those wherein an epitope in the first sequence is an epitope from an antigen selected from the group consisting of proinsulin, insulin, glutamate
  • GAD 65 insulinoma-associated protein 2
  • IA-2 insulinoma-associated protein 2 beta
  • IA-2b heat shock protein 60 islet-specific glucose-6-phosphate catalytic subunit- related protein (IGRP), chromogranin A (ChgA), zinc transporter 8 (ZnT8), islet amyloid polypeptide (IAPP), regenerating islet 3A (REG3A) and islet cell autoantigen 69 (ICA69).
  • the epitope most preferably is insulin B:9-23.
  • the methods of the invention also include a method for treating an autoimmune disease, comprising administering to a subject in need an isolated antigen-presenting cell of claim that expresses any of the nucleic acid constructs described herein or the tolerogenic DNA vaccines described herein.
  • the invention also includes a method for treating an infectious disease, comprising administering to a subject in need an isolated antigen-presenting cell as described herein that expresses any of the nucleic acid constructs described herein or the immunogenic DNA vaccines described herein.
  • FIG. 2 is a more detailed schematic showing an embodiment of the inventive nucleic acid construct, with the indicated epitopes and mimotopes.
  • the figure shows a representative construct (IET4T) according to some embodiments of the invention, comprising an MHC class II targeting sequence, a string of epitopes and mimotopes for processing to CD4+ T cells, a T2A cleavage site, and a string of epitopes for processing to CD8+ T cells.
  • IET4T representative construct according to some embodiments of the invention, comprising an MHC class II targeting sequence, a string of epitopes and mimotopes for processing to CD4+ T cells, a T2A cleavage site, and a string of epitopes for processing to CD8+ T cells.
  • FIG. 3 is a cartoon showing the interactions which are part of the strategy for achieving tolerance using the inventive methods.
  • Tolerogenic interfaces can comprise several of the following: (i) MHC/peptide complexes, (ii) inhibitory ligands displayed on the cell surface, (iii) secreted molecules that regulate T cells, and (iv) enzymes that influence the metabolism of the T cells (e.g. Tryptophan deprivation by IDO or retinoic acid production by ALDH1).
  • Optimal tolerance induction may require integration of all antigenic and tolerogenic signals by the targeted T cells. Thus, these signals must be concomitant.
  • Such interfaces can be created by providing mRNAs coding for all the needed components to antigen-presenting cells serving as the tolerogenic interface.
  • FIG. 4A and FIG. 4B are schematic drawings of exemplary constructs (4A) and the mechanism of peptide cleavage (4B) for those constructs.
  • FIG. 4A shows five exemplary constructs, which can contain natural peptides only, or some or all of the natural epitopes, such as the Ins2 and ChgA peptides, can be replaced with their mimotope counterpart.
  • the MHC class II pathway-targeting sequences are shown in blue.
  • FIG. 4B is a cartoon presenting the mechanism of peptide production intracellularly.
  • FIG. 5 is a table showing a selection of epitopes relevant to T1D, including sequences and characteristics of autoreactive T cells that have been previously isolated as reacting against these epitopes.
  • FIG. 6 is a schematic showing ten constructs, using native sequence epitopes only (NO) or some mimotope substitution (NM), that have been tested and compared upon introduction into dendritic cells by lenti viral transduction using GFP as reporter gene, whereby the expression of GFP is proportional to the expression of the epitopes. .
  • FIG. 7A is a series of graphs showing how transduced DCs were sorted based on intermediate GFP expression levels to correct for variations in transduction efficiencies and normalize expression of epitopes between groups. Data are representative of two experiments. The constructs used are depicted in FIG. 6, except for the superior IET4T construct (containing rearranged epitopes and T2A cleavage site), which is depicted on FIG.2.
  • FIG. 7B is a graph showing GFP MFI for the indicated cells, including All GFP+ (triangle) and sorted GFP+ (circle), from the work shown in FIG. 7A.
  • FIG. 8A is a series of plots showing FACS results of stimulation of ChgA-specific BDC2.5 CD4+ T cells.
  • FIG. 8B is a graph showing the percent divided for the NO series (native epitope) and the NM series (mimotopes) of these experiments. Data are representative of two experiments, and show that these CD4+ T cells only respond to the mimotope and require a targeting signal for maximal response.
  • FIG. 9A is a series of plots showing FACS results of stimulation of Ins-specific BDC12- 4.1 CD4+ T cells.
  • FIG. 9B is a graph showing the percent divided for the NO series (native epitope) and the NM series (mimotopes) of these experiments. Data are representative of two experiments, and show that these CD4+ T cells respond better to the mimotope and require a targeting signal for maximal response.
  • FIG. 10A is a series of FACS plots showing cell sorting results after stimulation of Ins- specific G9C8 CD8+ T cells (TCR alpha-/- background).
  • FIG. 10A is a series of FACS plots showing cell sorting results after stimulation of Ins- specific G9C8 CD8+ T cells (TCR alpha-/- background).
  • 10B is a graph presenting data for the NO series (native epitope only) and NM series (mimo topes included) constructs. Data are combined from two experiments, with the second experiment using frozen T cells. The results show that these CD8+ T cells only respond to the native epitope and respond better if the epitope is not diverted to the endosomal pathway.
  • FIG. 11A is a series of FACS plots showing cell sorting results after stimulation of IGRP- specific NY8.3 CD8+ T cells.
  • FIG. 1 IB and FIG. 11C are graphs presenting data for the NO series (native epitope only) and NM series (mimo topes included) constructs. Data are
  • FIG. 12 is a series of FACS plots showing cell sorting results after stimulation of GAD65-specific G286 CD4+ T cells. Stimulation of cell proliferation dye-labeled T cells after 3 days in culture with transduced DCs is shown. The data show a reduced stimulation for constructs lacking a class II targeting sequence (first column) compared to constructs containing one such sequence (TFR or CD16/LAMP1; second and third column). Because there is no mimotope for this epitope, the sequence is the same between NO and NM series, and the response the same also.
  • FIG. 13 is a series of plots showing FACS results summarizing the response (measured by dilution of a cell proliferation dye) of 5 different autoreactive diabetogenic T cell clones responding to 5 different epitopes, depending on whether native epitopes (N) or mimotopes (M) are used (here again, presented by transduced bone marrow-derived dendritic cells), and whether MHCII- targeting sequences are used (in this case with a domain from the transferrin receptor).
  • Insulin-reactive T cells from BDC12-4.1 and G9C8 are low affinity and weak responders.
  • FIG. 14A shows the percentage of T cells divided after 2.5 days from the indicated mice administered DCs transduced by lentivirus with the indicated constructs.
  • Beads indicate a positive control where maximal stimulation is provided.
  • the DC control contains no construct.
  • TFR indicates the presence of one of the MHC class ⁇ targeting sequence tested.
  • FIG.15 is a schematic drawing of an alternative nucleic acid construct (IET5T) depicting additional epitopes for broader targeting of diabetogenic CD4+ and CD8+ T cells according to the invention.
  • IET5T alternative nucleic acid construct
  • FIG. 16 shows flow cytometry data on stimulation of polyclonal ChgA -reactive T cells in vivo (in non-obese diabetic (NOD) mice).
  • the polyclonal CD4+ T cells recognizing the ChgA mimotope were identified by tetramer staining, and these data confirm the previously shown responses using specific clones obtained from T cell receptor-transgenic mice.
  • the left group of four plots are spleen cells; the right group of four plots are pancreatic lymph node cells.
  • FIG. 17 presents data on the stimulation of ChgA-reactive CD4+ T cells in vivo and in vitro using DCs electrop orated with IET4T mRNA, with or without TGF- ⁇ mRNA.
  • FIG. 17A is a series of plots showing FACS data for CD4+ CD25- T cells isolated from BDC2.5.GFP-Foxp3 mice, labeled with Violet Cell Proliferation Dye (VCPD) and stimulated for 3 days in vitro + exogenous TGF- ⁇ or in vivo in NOD Thyl. l mice.
  • FIG. 17B and FIG. 17C are graphs showing the data for divisions of the cells in vivo (FIG. 17B) and in vitro (FIG. 17C).
  • FIG. 17A is a series of plots showing FACS data for CD4+ CD25- T cells isolated from BDC2.5.GFP-Foxp3 mice, labeled with Violet Cell Proliferation Dye (VCPD) and stimulated for 3 days in
  • FIG. 17D presents data for the % GFP-Foxp3 induced in vitro for the indicated cells/constructs. Arrows indicate exogenous TGF- ⁇ .
  • FIG. 17E shows % GFP-Foxp3 induced in divided CD4+ T cells in vivo for the indicated cells/constructs.
  • the invention relates to constructs, cells and methods for modulating the immune system that optimize presentation of CD4 and CD8 nonself- or self-epitopes to antigen-presenting cells, and in so doing optimize either tolerance or immunogenicity to those respective epitopes.
  • the new constructs referred to as tandem epitope constructs (TEC), encode one or more dominant, disease-driving epitopes targeted for MHC class II processing within the endosomes of a cell and one or more epitopes targeted for MHC class I processing within the cytosol of the cell, to achieve maximum antigen/epitope presentation in the immune system.
  • the two types of epitopes (CD4 and CD8) are separated from one another on the construct and grouped together for optimum processing in the cell.
  • the invention can be used to either increase immune tolerance as is needed in autoimmune disease treatment by including a plurality of relevant epitopes from self antigens in the construct, or to increase an immune response (immune stimulation) for example to treat infectious diseases whereby several non-self epitopes are included in the construct.
  • the TEC optimize presentation of both CD4 and CD8 epitopes to achieve a strong tolerogenic (including tolerance, ignorance or suppression of a response to the epitope(s)) or immunogenic response to both groups of epitopes by optimizing antigen/epitope presentation.
  • optimization is meant increasing epitope presentation and engagement of a T cell, regardless of the eventual outcome of the engagement.
  • T cell help can be direct from the CD4+ T cell to a CD8+ T cell that is brought in close proximity thanks to the bridging APC, or the CD4+ T cell boosts the APC's immunogenicity, and the APC in turn becomes a better stimulator for the CD8+ T cell. If one delivers CD4 and CD8 epitopes on different constructs, there is no way to be sure that the CD4 and CD8 epitopes are co-expressed in the same APC, and thus that linked cooperation occurs.
  • the CD4+ T cells can be regulatory T cells (so-called “Tregs”) that, instead of boosting the immunogenicity of APCs, dampen the APC's function and ensure that both autoreactive CD4+ and autoreactive CD8+ T cells receive tolerogenic signals.
  • Treg cells also can directly suppress the activation of autoreactive T cells upon concomitant interaction with APCs due to the close proximity. This process, known as linked suppression, equally relies on co-expression of both CD4 and CD8 epitopes by the same APC.
  • tissue-specific autoimmune diseases Insufficient induction of tolerance to self antigens from particular tissues is the major cause for tissue-specific autoimmune diseases. Under normal conditions, these tissue-specific antigens are presented by tolerance-inducing (tolerogenic) cells, which program T cells to not respond to these antigens. However, in autoimmune diseases, these antigens are presented improperly, instructing specific T cells to respond instead.
  • tolerance-inducing (tolerogenic) cells which program T cells to not respond to these antigens.
  • autoimmune diseases these antigens are presented improperly, instructing specific T cells to respond instead.
  • Previous studies using antigen- specific immunotherapy have shown that administering certain antigens known to be involved in a particular autoimmune disease, systematically or via the mucosa, was effective in reinstating tolerance in animal models at least in part through the generation of suppressor T cells able to counteract improperly activated pathogenic T cells.
  • autoimmune diseases and particularly the most common types of tissue- specific autoimmune diseases, involve multiple epitopes, a large number of which have been identified (DiLorenzo 2007) and continue being identified.
  • each protein comprising a relevant epitope(s) has to be administered to the subject to induce tolerance, and in contrast to animals, this led to poor efficacy in patients. Without being bound by, this may be because a single antigen was chosen and/or because the native protein cannot produce the modified epitopes produced during the course of disease which can be better simulated by mimotopes.
  • whole native antigens are delivered in these methods, either as protein (which is mostly acquired and processed to MHC class II in vivo) or as a DNA vaccine (which is mostly processed to MHC class I, unless the expressed product is secreted).
  • protein which is mostly acquired and processed to MHC class II in vivo
  • DNA vaccine which is mostly processed to MHC class I, unless the expressed product is secreted.
  • the TEC includes a series of epitopes or mimotopes that are processed in endosomes for presentation on MHC class II to CD4+ T cells, and a series of epitopes or mimotopes processed by the proteasome in the cytoplasm for presentation on MHC class I for CD8+ T cells.
  • the mimotopes in the TEC can optimally include analog peptides (also referred to by some as altered peptide ligands APL), but are not limited to them.
  • Mimotopes that are rationally designed typically differ from the native epitope by a few amino acids, whereas those that are identified via a screening system may have a more substantial dissimilarity.
  • the TEC consists of (a) a first sequence comprising an MHC class II targeting sequence operably linked to one or more different epitope sequences (which epitopes are processed in the endosomes for CD4+ T cells); (b) a second sequence comprising one or more different epitope sequences (these epitopes are processed in the cytoplasm where the construct itself is originally delivered and therefore does not need to have any MHC targeting sequence); and (c) a cleavage site located between the first and second sequences to separate the two polypeptides and allow their differential targeting within the cell.
  • the epitopes are arranged into two groups: a first group of CD4 epitopes (for association with MHC class II) are included in a first sequence of the TEC to be targeted to the endosomes by using an appropriate MHC class II targeting sequence, and a second group of CD8 epitopes (for association with MHC class I) included in a second sequence of the TEC destined for processing in the cytoplasm and presentation on the surface in the context of class I.
  • the position of the MHC class II targeting sequence with respect to the CD4 epitopes on the first sequence is either upstream or downstream of the epitopes, depending on the choice of MHC class II targeting sequence. See FIG. 1. This approach not only permits the expression of multiple disease-driving epitopes in a single construct but also results in each group of epitopes being optimally processed and presented onto their respective MHC molecules (class II for CD4 and class I for CD8).
  • the tolerogenic TEC are delivered to tolerogenic APCs (or alternatively immunogenic TEC are delivered to immunogenic APCs) ex vivo using viral vectors or mRNA electroporation or other vector, or delivered directly in vivo in the form of tolerogenic DNA (or immunogenic) vaccines and the like.
  • agents known to be immunosuppressive such as TGF- ⁇ , IL-10, IL-1RA, IL-4, IL-27, IL-35, PD-L1, ICOSL, B7-H4, CD39, CD73, FAS, FAS-IL, IDOl, ID02, ALDH1/RALDH, ARG1, ARG2, NOS2, galectin-1, galectin-9, semaphorin 4A, and any combination thereof can be used to help these APCs to reprogram autoreactive T cells.
  • agents known to be immunosuppressive such as TGF- ⁇ , IL-10, IL-1RA, IL-4, IL-27, IL-35, PD-L1, ICOSL, B7-H4, CD39, CD73, FAS, FAS-IL, IDOl, ID02, ALDH1/RALDH, ARG1, ARG2, NOS2, galectin-1, galectin-9, semaphorin 4A, and any combination thereof can be used to help these APCs to reprogram autore
  • the tolerogenic or immunogenic TEC-transfected APC such as dendritic cells, mesenchymal cells or other antigen presenting cells, are then inoculated into subjects for therapy of autoimmune or infectious diseases, respectively.
  • the use of mRNA provides the option of co- delivering other mRNAs encoding tolerogenic ligands or immunosuppressive cytokines in the case of tolerogenic cells, and immunogenic ligands in the case of an infectious disease or other disease where an immune response to one or more particular epitopes is needed.
  • this construct will allow reprograming of many self -reactive T cells into programmed cell death, a state of unresponsiveness or suppressor cells.
  • all epitopes would be presented by the same antigen-presenting cells, allowing processes such as linked suppression and infectious tolerance whereby preexisting suppressor CD4+ cells specific to a particular (CD4) epitope can suppress self-reactive CD4+ or CD8+ T cells recognizing other epitopes from the expressed set of antigens, and reprogramed CD4+ T cells can turn into suppressor cells.
  • immunogenic TECs carrying epitopes from non-self antigens such as bacterial epitopes can be delivered into immunogenic (immune- stimulating) APCs ex vivo that are then inoculated into subjects to fight certain infections.
  • exogenous proteins mostly results in presentation of epitopes that are seen by CD4+ T cells, whereas endogenously produced epitopes are mainly recognized by CD8+ T cells, both of which cell types are implicated in autoimmune responses and are important for robust immunological response. Therefore, neither previously known approach is optimal for the treatment for autoimmune disease.
  • tissue-specific diseases such as T1D, MS, RA
  • the autoimmune response targets an increasing number of antigens from the same target tissue (epitope spreading), such that subsequent induction of tolerance to one antigen will not completely block the disease.
  • Tolerance induction to multiple antigens is more difficult to achieve when so many antigens all need to be administered or expressed to achieve immune tolerance (DNA vaccine, lenti virus, mRNA).
  • Exogenous delivery of antigens generally leads to uptake by professional antigen- presenting cells (APCs), the tolerogenic function of which may become compromised under inflammatory conditions.
  • APCs professional antigen-presenting cells
  • these antigens are primarily presented on MHC class II to CD4+ T cells, while CD8+ T cells represent the predominant islet-infiltrating lymphocyte population in human T1D, for example.
  • Endogenous delivery of antigens can achieve low level of expression in a wide range of cells, including non-professional APCs.
  • Non-professional APCs such as fibroblastic and endothelial cells, can mediate CD8+ T cell tolerance and cannot become immunogenic (Lukacs-Kornek & Turley, 2011, Curr.
  • autoreactive T cells can be more responsive to peptides bound to MHC in an uncommon conformation (Stadinski et al., PNAS, 107(24): 10978-83, 2010) or to post-translationally modified peptides (Delong et al., 61(12):3239-46, 2012; van Lummel et al., Curr. Opin. Endocrinol. Diabetes Obes. 20(4):299-306, 2013), it has become clear that mimotopes are beneficial in targeting such T cells.
  • the mimotope that mimics InsB:9-23 bound to I-Ag7 on register #3 was found to be greatly superior to the corresponding native epitope in preventing T1D and inducing Treg cells in NOD mice (Daniel et al., J. Exp. Med. 208(7): 1501-10, 2011).
  • Antigen-specific therapies aimed at increasing tolerance to self antigens currently use methods that target either MHC class I or class II pathways, but not both optimally. This has extremely limited success, especially in autoimmune diseases where epitope spreading occurs (T1D, MS, RA). In such diseases, both the T cell and antibody responses diversify against an increasing number of antigens from the target tissue. In T1D, there is solid evidence that insulin is one of the major driving antigens, if not the initial one, for the disease (Nakayama, Diabetes Metab. Res. Rev. 27(8):773-7, 2011). Antigen- specific therapies in preclinical and clinical studies for diabetes so far have involved administering proinsulin, insulin, glutamate
  • the inventive constructs are called "tandem epitope constructs (TEC)" and contain two groups of epitopes, one group that is intended for processing in cytoplasmic proteasomes for MHC class I presentation to CD8 cells, and one group intended for processing in the endosome for MHC class II presentation to CD8 cells.
  • TEC tandem epitope constructs
  • the inventors here have found that adding an MHC class II targeting sequence to antigens which are intended for processing to both compartments improved stimulation of CD4+ T cells, but diminished that of CD8+ T cells. This is because the MHC class II targeting sequence also caused the CD8 epitopes as well as the CD4 epitopes to bypass cytoplasmic processing and presentation to CD8+ T cells.
  • FIGS. 2 show a TEC in which two groups or strings of epitopes were joined in one sequence, but separated by a proteolytic cleavage site so that one group, having a targeting sequence for endosomal delivery and class II processing was processed and presented to CD4+ T cells in endosomes, while the other group without an added target sequence, was processed and presented to CD8+ cells in the cytoplasm.
  • the new TECs increased presentation of multiple epitopes by both pathways.
  • Autoimmune cells such as diabetogenic T cells can now be targeted by endogenous delivery of a plurality of selected epitopes arranged in such a way that both CD4 and CD8 epitopes can be targeted optimally for proper presentation on MHC class II and class I, respectively.
  • the data presented here show that substitution of certain native epitopes by mimo topes enhances engagement of particular T cell clones.
  • Mimo topes typically allowed for better engagement of target CD4+ T cells than native epitopes, which were poorly or not at all recognized.
  • the native insulin B:9-23 epitope can bind to MHC class II in three different conformations called registers.
  • the register that is recognized by diabetogenic T cells is one that is uncommon because of weak binding.
  • the binding in the desired conformation can be favored so that diabetogenic can be more efficiently engaged by the APC (Stadinski et al., Proc. Natl. Acad. Sci. USA. 107 (24): 10978-10983, 2010).
  • Another example is an epitope from another beta cell antigen, chromagranin A (ChgA).
  • ChgA chromagranin A
  • a well-known diabetogenic CD4+ T cell clone is incapable of reacting to the native peptide, but if the latter is post- translationally modified by the enzyme transglutaminase, it becomes strongly recognized (Delong et al., Diabetes 61(12):3239-3246, 2010).
  • Mimotopes have been identified by screening peptide libraries that are able to efficiently stimulate those T cells, thus in a way simulating those modified antigens (Judkowski et al., J. Immunol. 166 (2):908-917, 2001; You et al., J. Immunol. 170(8):4011-4020, 2003).
  • the insulin B:9-23 CD4 epitope recognized by the BDC12-4.1 CD4+ T cell clone contains the B: 15-23 CD8 epitope recognized by the G9C8 CD8+ T cell clone.
  • the R22E mutation mimotope
  • the CD4+ T clone it abrogated recognition by the CD8+ T clone, indicating a benefit of including multiple variants for each epitope, as done in some embodiments of the TECs (see FIG. 2 and FIG. 15).
  • the technology of this invention is directed primarily to tolerogenic tandem epitope construct (TEC) carrying a fusion peptide sequence encoding a operably linked endosomal MHC class II targeting sequence followed by one or more epitopes for CD4+ T cells (presented on MHC class II), a series of one or more CD8 epitopes (presented on MHC class I), with a cleavable linker separating the two epitope sequences.
  • TEC tandem epitope construct
  • This new construct enables delivery of multiple disease-driving epitopes expressed by a single nucleic acid-based (DNA or RNA) construct that initiates immune tolerance to both autoreactive CD4 and CD8 T cells through optimized antigen presentation and processing.
  • the TEC carries an MHC class II targeting sequence operably linked to the CD4 epitopes intended for processing in endosomes, it is not necessary to include an MHC class I targeting sequence for CD8 epitopes because the construct is delivered to the cytoplasm where these epitopes will be processed via endogenous MHC class I in proteasomes, according to the normal cellular process.
  • the two major groups of epitopes are separated by a cleavage site allowing the CD8 epitope section to be cleaved off and remain cytoplasmic for processing in cytoplasmic proteasomes, while the CD4 epitope section is targeted to the endosomes using an appropriate MHC class II targeting sequence.
  • the invention provides a highly customizable fusion peptide with epitopes for presentation to antigen-presenting cells that can be delivered via a DNA tolerogenic or immunogenic vaccine, lenti virus, mRNA, or any other appropriate vector to carry two series of epitopes, for example from self antigens, to ensure each group of epitopes reaches the appropriate cellular compartment for proper presentation to both CD8+ (cytoplasm) and CD4+ T cells (endosomes).
  • each epitope is flanked on each side by at least two amino acids
  • flank CD4 epitopes contain natural endosomal proteolytic cleavage sites, whereas those that flank CD8 epitopes contain natural proteosomal cleavage sites.
  • Using artificial flanking sequences carries unknown risks that some epitopes may not be processed properly or bind properly on the MHC.
  • flanking sites of the corresponding native peptides can be used and may be compared with artificial flanking residues or spacers.
  • Embodiments of the TEC comprise the natural flanking sequences that flank the epitope in the whole protein, instead of artificial flanking sequences. This is useful because it is known that these epitopes are naturally processed and generated in the cell using these natural sequences. Artificial flanking sequences increase the risk that adjacent amino acids will affect the binding on MHC. For mimo topes, flanking sites of the respective native peptides are used.
  • the construct is codon-optimized for the species in which the construct is used.
  • Codon optimization may include nucleotide changes that reduce or enhance the immunogenicity of the vector without altering the amino acid sequence.
  • vaccines designed to promote immune stimulation with respect to the antigen(s) incorporate adjuvants to induce the necessary signals to promote immunogenicity.
  • adjuvants are taught in the art, for example by Sasaki et al., Methods, 31(3):243-254, 2003. Any of these adjuvants and adjuvant methods are contemplated for use with the invention.
  • the construct contains appropriate targeting signals to allow the cells' natural machinery to deliver and process peptides to endosomes for greater efficiency of MHC class II processing.
  • the TECs further optionally contain appropriate proteolytic cleavage sites flanking each epitope that permits them to be separated easily into individual peptide epitopes once the translated polypeptide has been cleaved between the sections in the cytoplasm and arrived at the appropriate compartment (endosome for class II or proteasome for class I). This arrangement allows each group of epitopes to be optimally processed and presented onto their respective MHC molecules.
  • the TEC allows targeting of both CD4+ and CD8+ diabetogenic T cells for deletion or suppression across multiple beta-cell antigens, using the herein-described tolerogenic DNA vaccination strategy that has a good safety profile in TID patients (Roep et al., 2013).
  • the TEC can be introduced into tolerogenic APCs by transfection or transduction of DNA/RNA material for cell therapy.
  • tolerogenic dendritic cell therapy has been is well-tolerated in TID patients (Giannoukakis et al., Diabetes Care, 34:2026-2032, 2011).
  • the TEC is designed to carry bacterial or viral epitopes or cancer epitopes, for example, to create an immunogenic DNA vaccine to increase immune stimulation of both CD4+ and CD8+ T cells that attack infecting organisms, or tumor cells, respectively.
  • the antigens encoded by the TEC can be customized not only for various diseases requiring either immune tolerance such as autoimmune diseases or immune stimulation such as infectious diseases, but can also be customized for individual patients to elicit the greatest tolerance response or the greatest immune response.
  • Various immunoassays exist to determine whether some immune cells circulating in the blood in a given patient develop an immune response to particular peptides tested.
  • the antigens selected can be based on the most common reactivity seen in a class of patients. Because it is customizable, native peptides may be mutated for better targeting of specific types of self-reactive T cells (those requiring post-translational modifications or an uncommon MHC binding register).
  • the TECs provide a way to ensure endogenous expression of dominant disease epitopes (including modified neoepitopes) that cannot be achieved with simple administration of combined exogenous proteins. Because only the important selected disease epitopes are included in the TEC, a single construct suffices to present a plurality of epitopes from multiple protein antigens to both or either CD4+ and CD8+ T cells, enabling a balanced expression of both CD4 and CD8 epitopes.
  • the DNA/RNA vehicles that carry the TECs can be modified to remove certain motifs that are immunogenic, for example CpG motifs, which can be replaced with GpG motifs.
  • immunogenic motifs for example CpG motifs
  • the TECs can be combined with external standard adjuvants known in the art to promote strong immunogenicity.
  • the term “about” is used herein to mean approximately, roughly, around, or in the region of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20 percent up or down (higher or lower).
  • an "adjuvant” is a compound that enhances or prolongs the antigen- specific immunostimulatory response produced by a vaccine.
  • adjuvants there are three types of adjuvants that can be considered for use: (1) the adjuvant properties of the DNA vector itself, for example the presence and abundance of unmethylated CpG motifs, which act as an adjuvant by stimulation through the TLR-9 receptor, (2) genetic adjuvants, which are proteins expressed alongside antigen by the same or another co-administered DNA vector, including inflammatory cytoVkines and immunomodulatory cytokines that support specific subsets of T cells, and (3) conventional adjuvants, which can be co-injected with the DNA vectors, and include alum-based adjuvants, saponin-based adjuvants, certain bacterial elements known in the art, immunostimulatory complexes, and mineral oil-based adjuvants (Petrovsky and Aguilar, Vaccine adjuvants: current state
  • Immunol. Cell. Biol. 84:488-496, 2004 are considered to be an adjuvant in the context of this invention.
  • Genetic adjuvant plasmids and conventional adjuvants such as aluminum salts, oil-based adjuvants, virosomes or squalene adjuvants, as known in the art also are contemplated for use with the invention.
  • An "antigen,” as the term used herein, is a structural substance, often a protein, that is recognized by the immune system and serves as a target for an immune response.
  • a “self antigen” or “autoantigen” is an antigen derived from an organism which under normal circumstances is not recognized by the immune system of that organism, but which may become a target of immune attack, resulting in an autoimmune disease.
  • a “cancer antigen” is an antigen present on cells of a tumor or other malignancy which is overexpressed or mutated in the tumor or other malignancy and so advantageously can be targeted as a non-self antigen for immune attack.
  • Antigen-presenting cells refer to any cell that can present, or can be induced to present, antigens in the context of MHC class II and/or class I.
  • Professional antigen-presenting cells known in the art express high levels of MHC class I and II and can be highly immunogenic in the context of inflammation and infection. These cells, which typically include dendritic cell subsets, and to some extent macrophages and B-cells, can also be tolerogenic under steady-state conditions and other contexts.
  • Non-professional APCs express MHC class I and some inducible levels of MHC class II, are typically tolerogenic and consist of stromal cells that include certain epithelial cells, fibroblastic cells and endothelial cells.
  • Type 1 diabetes is an autoimmune disease where cells that recognize insulin or other beta-cell antigens have become activated and destroy pancreatic beta cells, leading to diabetes.
  • CD4 epitope refers to an epitope that is processed by or designed to be processed by the MHC class II pathway and presented in the context of MHC class II to CD4+ T cells.
  • a “CD8 epitope,” as used herein, refers to an epitope that is processed by or designed to be processed by the MHC class I pathway and presented in the context of MHC class I to CD8+ T cells.
  • a "cleavage site,” as used herein, is a short sequence of peptides within a sequence which is susceptible to proteolytic cleavage by one or more proteases. The nucleic acid sequence encoding a cleavage site also is referred to as a cleavage site.
  • codon-optimized and “codon optimization” refer to a technique for improving protein expression in a cell, increasing translational efficiency of the gene, by using codons preferentially used in the system expressing the DNA.
  • bacteria use some codons rarely used by humans, therefore if the bacterial codon for a particular amino acid is substituted with the codon preferentially used by human cells, the protein will be translated more efficiently. Therefore, codon optimization refers to a method of using the codons most used in the species of interest for expression of foreign proteins or peptides.
  • construct refers to an artificially synthesized sequence of nucleic acid which encodes a protein or peptide of interest, and generally contains promoters for expression of that protein or peptide in a cell.
  • a "cytosolic proteolytic cleavage site,” as used herein, is a proteolytic cleavage site that is acted upon by a protease located in the cytosol, generally but not necessarily in association with a proteasome such that the site is cleaved in the cytosol.
  • cytosolic proteolytic cleavage is a proteolytic cleavage site that is acted upon by a protease located in the cytosol, generally but not necessarily in association with a proteasome such that the site is cleaved in the cytosol.
  • Diabetogenic T cells are beta-cell antigen-reactive T cells (self-reactive T cells) that have eluded tolerance mechanisms and eventually cause the destruction of beta-cells, leading to Type 1 diabetes.
  • a "DNA vaccine,” in general terms, is DNA in the form of an expression plasmid for administration to a subject, which DNA contains the sequence of one or more antigens to which it is desired to produce an immune reaction in that subject.
  • the desired reaction includes the opposite effects of increasing tolerance to the antigens, such as self antigens, or to stimulate an immune response, such as a response against microbial epitopes.
  • An "endosomal proteolytic cleavage site,” as used herein, is a proteolytic cleavage site that is acted upon, preferably selectively acted upon, by a protease located in the endosome, such that the site is not cleaved appreciably until arrival in the endosome.
  • An “epitope,” (also referred to as an “antigenic determinant”) as the term is used herein, is the part of an antigen which is specifically recognized by the immune system. Epitopes are either in the form of peptides presented by MHC molecules and recognized by T cell through their T cell receptor, or correspond to exposed regions of a complete antigen that are recognized by B cells through their B cell receptor, and later by antibodies that these B cells produce.
  • Epitope spreading occurs when the immune reaction targets epitopes beyond the primary epitope or antigen. Responses can spread from one epitope to another epitope within the same antigen (intramolecular) or from an epitope of one antigen to an epitope of another antigen (intermolecular). Responses to autoantigens tend to become more diverse as the disease progresses. This also is referred to as “determinant spreading” or “antigen spreading.”
  • Immuno response refers to a coordinated response by the immune system to defend an organism against infection or a disease, such as cancer.
  • Immunune tolerance is the mechanism of non-self discrimination which allows the immune system to recognize foreign antigens, but not self antigens. Under normal conditions, tissue-specific self antigens are presented by tolerance-inducing (tolerogenic) cells, which program T cells to not respond to these antigens. Autoimmune disease results when these self antigens are not tolerized.
  • a "immunogenic DNA vaccine” or a “immuno stimulatory DNA vaccine” refers to a DNA vaccine designed to stimulate an immune response to the antigen(s) in the subject, thereby promoting the rejection of the antigen-expressing pathogens, infected cells or tumors.
  • a “mimotope” is a molecule that mimics the three-dimensional structure of an epitope, and therefore has the same or a highly similar binding specificity, but may or may not have a different affinity or avidity.
  • a “mimotope” causes an antibody response similar to that elicited by the epitope which it mimics.
  • An antibody elicited against a particular epitope (antigen) recognizes a mimotope of that particular epitope, and a mimotope of a particular epitope can elicit an antibody response which binds that particular epitope. Therefore, one or more mimotopes can be used as a vaccine.
  • a mimotope may be, as are most epitopes, a portion of a macromolecule, such as a protein, nucleic acid or polysaccharide. Preferably, it is a protein or a portion of a protein, and may be a peptide typically about 9 to about 20 amino acids in length. Mimotopes are either obtained by screening phage-display or peptide libraries, or by directed mutagenesis aimed altering the binding properties of the peptide, according to methods known in the art.
  • naked refers to purified, histone-free DNA or RNA, without associated proteins and free of agents that promote transfection.
  • operably linked or "operable link,” and their cognates, has its usual meaning of an arrangement in which a genetic control sequence, e.g. a promoter, enhancer or terminator, is capable of exerting its function with regard to a polynucleotide being operably linked to it, for example a polynucleotide encoding a polypeptide. It has a further meaning for the purpose of this invention wherein it means that the MHC class II targeting sequence in the first sequence of the TEC is located in a position that is in sufficient proximity to a group of epitopes in the first sequence of embodiments of the invention so as to target the group of epitopes to the endosomes for processing by MHC class II.
  • the targeting sequence can be upstream or downstream, or can be composes of sequences placed both upstream and downstream of the DNA sequence to be targeted depending on the particular targeting sequence selected.
  • a "protease” is an enzyme that cleaves protein sequences.
  • a cytosolic protease is located in the cytosol of a cell; an endosomal protease is located in the endosomes of a cell.
  • proteasomes are protein complexes inside all eukaryot.es and archaea, and in some bacteria. In eukaryotes, they are located in the nucleus and the cytoplasm. The main function of the proteasome is to degrade unneeded or damaged proteins by proteolysis, a chemical reaction that breaks peptide bonds.
  • the proteasomes referred to herein for processing of the TEC are cytoplasmic.
  • Tregs Regulatory T cells secrete regulatory cytokines (such as IL-10, IL-35 or TGF- ⁇ ) which suppress immune responses. Tregs can inactivate T cells in a contact-dependent manner. Finally, Tregs dampen the immunogenic function of antigen-presenting cells, and can potentially render them tolerogenic. Tregs are involved in preventing autoimmune diseases.
  • regulatory T cells such as IL-10, IL-35 or TGF- ⁇
  • sequence refers to the primary structure of a biological
  • a "targeting sequence” refers to a sequence of nucleotides within a sequence that causes targeting to a particular location or compartment of the cell, for example endosomes.
  • T cells are a type of lymphocytes.
  • T helper cells (CD4+ T cells) become activated when they are presented with peptide antigens by MHC class II molecules on the surface of antigen- presenting cells. Once activated, T helper cells divide and secrete cytokines that stimulate an active immune response.
  • Cytotoxic T cells (CD8+ T cells) are activated by binding to antigen associated with MHC class I molecules on the surface of antigen-presenting cells, and destroy virus-infected cells and tumor cells. These cytotoxic T cells also are implicated in transplant rejection.
  • a self-reactive (or autoreactive) T cell is a CD4+ or CD8+ T cell that is or has been activated by a self antigen (or autoantigen).
  • a "tandem epitope construct,” as used herein, refers to a nucleic acid construct that contains two groups of linked epitopes, where the groups are separated from each other by a proteolytic cleavage site, and where one group of epitopes destined for processing in the MHC class II pathway for presentation to CD4+ T cells is operably linked to an endosomal targeting sequence and the other group of epitopes destined for processing in the class I pathway for presentation to CD8+ T cells does not.
  • the two groups of epitopes in the tandem epitope construct are separated so that each group undergoes separate processing within the cell, one onto MHC class II and the other one onto MHC class I.
  • Each epitope in each of the two groups are separated by a proteolytic cleavage site so that once in the appropriate cellular compartment for processing, each epitope is cleaved from the other epitopes in the group.
  • TEC designed to induce immune tolerance to the epitopes carried by the construct, although a TEC also can be designed in such a way as to increase an immune response to certain epitopes, for example those from bacterial antigens.
  • tandem epitope construct TEC achieves separate processing and presentation of epitopes as follows once inside a cell:
  • the translated construct comprising both epitope groups is cleaved to separate the groups of peptide epitopes by action of a cytosolic protease
  • the group of epitopes having the class II targeting sequence is shepherded to the endosome by the targeting sequence as a group, and once in the endosome is further cleaved into their individual constituent epitopes (by endosomal proteases), which are presented on the surface of the cell in the context of MHC class II, and
  • the group of epitopes lacking a targeting sequence remains in the cytosol, where it is shuttled into the proteasome, cleaved into its constituent individual epitopes, and presented on the surface of the cell in the context of MHC class I.
  • terapéuticaally effective amount or "an effective amount” have the standard meanings known in the art and are used interchangeably herein to mean an amount sufficient to treat a subject afflicted with a disease (e.g., diabetes) or to alleviate a symptom or a complication associated with the disease.
  • a disease e.g., diabetes
  • a "tolerogenic DNA vaccine” refers to a DNA vaccine designed to produce tolerance or anergy to the antigen(s), or to achieve reprograming of CD4+ T cells reactive to the antigen(s) to antigen-tolerant and suppressive "Tregs" in the subject, thereby preventing rejection of autoantigen-expressing cells and tissues.
  • treating and “treatment” have the standard meaning known in the art to mean slowing, stopping or reversing the progression of diabetes or other autoimmune diseases, or conversely to mean enhancing or accelerating an immune response meant to eradicate an unwanted pathogen or tumor.
  • Treatment thus, for example, covers any treatment of diabetes in a mammal, particularly in a human.
  • a "vector” generally refers to a DNA molecule used as a vehicle used to carry foreign genetic material into a cell, where it can be expressed.
  • the term refers to any means for inserting a nucleic acid into a cell, and includes mRNA, plasmids, viral vectors, cosmids, artificial chromosomes, expression constructs and the like.
  • MHC major histocompatibility complex
  • MHC/peptide and the T cell receptor plays a key role in the ability of the CD4+ T cell or CD8+ T cell to recognize an infectious organism, or the products of these pathogens, as foreign.
  • Two major types of foreign pathogens include: (i) viruses that take over the replicative machinery of a cell, and (ii) bacteria that replicate in different ways. These two types of pathogens present very different challenges to the immune system.
  • a cytotoxic CD8+ T cell kills a cell containing a virus whereas a bacterium can be eliminated by a phagocyte that has been activated by a CD4+ helper T cell or neutralized by antibodies produced by B cells with the help of CD4+ T cells.
  • the necessity of distinguishing between whether the presence of a pathogen peptide should elicit a killer or helper T cell receptor response is presumed to be the reason for the creation of two specialized forms of MHC molecules: class I and class II. Both class I and class II responses are necessary for a fully functioning immune system.
  • MHC class I molecules are found on almost all tissues of the body, and they play an important role in alerting the immune system to virally infected cells. MHC class I molecules are expressed on the cell surface of all nucleated cells and present peptide fragments derived from intracellular proteins. These peptides are normally derived from the cell's own proteins.
  • peptides derived from viral proteins may also be presented.
  • MHC class I molecules fold and assemble in the endoplasmic reticulum.
  • Antigens for presentation in the context of MHC class I are typically processed into short peptides in the proteasome, peptides which are then translocated into the lumen of the endoplasmic reticulum, where the peptide antigens (epitopes) are loaded onto the MHC class I molecule.
  • CD8+ T cells can recognize these presented peptides that we refer to as CD8 epitopes.
  • MHC class II molecules are found at high levels only on professional antigen -presenting cells. MHC class II molecules present antigens acquired from an exogenous source, which can be a self-tissue or a pathogen. Antigenic molecules enter the cell by endocytosis / phagocytosis and are digested in lysosomes and/or endosomes. The resulting peptide fragments, slightly longer than CD8 epitopes, are loaded onto MHC class II molecules in the endosomal compartment prior to migration to the cell surface for presentation. The MHC class II molecule complexed with the antigenic peptide is then recognized by CD4+ T cells; these peptides are therefore referred to as CD4 epitopes. b. Immune Tolerance
  • Immune tolerance is the mechanism by which potentially harmful self-reactive T cells are eliminated, inhibited or converted into a protective type of cells. Insufficient induction of tolerance to self antigens from particular tissues is the major cause for tissue-specific
  • autoimmune diseases Under normal conditions, these tissue-specific self antigens are presented by tolerance-inducing (tolerogenic) cells, which program any reactive T cells to undergo cell death, unresponsiveness or conversion to a type of Treg. In autoimmune diseases, these same self antigens are either not presented sufficiently, which limit engagement of autoreactive T cells for instruction, or presented improperly, instructing specific T cells to mount an immune response instead of tolerating the antigen as self.
  • Antigen- specific therapies that deliver these self antigens to potent tolerogenic cells, systematically or via the mucosa are partially able to reinstate tolerance in part through the generation of suppressor T cells able to counteract improperly activated pathogenic T cells. c. Autoimmune Diseases
  • Insufficient induction of tolerance to antigens from particular tissues is the major cause for tissue- specific autoimmune diseases.
  • Deficits in central or peripheral tolerance cause cells of the immune system to inappropriately mount an immune response to self antigens, resulting in syndromes such as SLE, RA, T1D, MS, autoimmune polyendocrine syndrome type 1 (APS-1) and immunodysregulation polyendocrinopathy enteropathy X-linked syndrome (IPEX), and potentially contribute to asthma, and IBD.
  • Central tolerance refer to instruction of T cells in the thymus as they develop from precursors to mature T cells
  • peripheral tolerance relates to instruction of mature T cells in secondary lymphoid tissues (spleen, lymph nodes) after their release from the thymus.
  • CD4 T cell epitopes onto MHC class II and CD8 T cell epitopes onto MHC class I) and targeting of certain diabetogenic T cells will be improved when certain mutated epitopes (mimotopes) are included to mimic certain post-translational epitope modifications or favor particular MHC binding registers. d. Other Diseases
  • tandem epitope construct can be designed to hold a plurality of any epitopes
  • the invention can be used to cause any epitope to be presented.
  • other diseases where it would be desirable to tolerize a subject to particular antigens comprising both CD4 and CD8 epitopes can be treated using the invention.
  • the subject Prior to transplanting a tissue or an organ into a subject, the subject can be treated according to the invention using antigens from the prospective transplant so that the subject can be tolerized.
  • pre-exposing transplant patients to epitopes present on donor organs can condition their immune system not to respond to these epitopes, increasing the chance of transplant success.
  • the constructs of the invention can be used to induce immune responses against pathogens or tumors, where multiple epitopes that include both CD4 and CD8 epitopes need to be targeted. If a stimulated immune response is desired, the construct should be administered in a manner that prime antigen-presenting cells to be immunogenic, typically with the help of adjuvants.
  • an immunogenic DNA vaccine or a vector carrying an immunogenic TEC can be engineered to include immuno stimulatory motifs such as CpG, or in the contrary, to remove them (and optionally replace them with GpG motifs) when tolerance is desired.
  • infectious diseases such as influenza and HIV, where antigenic shift (reassortment of antigens) is a problem or multiple epitopes are known.
  • Some viruses such as hepatitis B and C viruses can cause chronic infection by exhausting T cells. Eradication of many viruses rely heavily on optimal cytotoxic T cell responses and B cell-mediated antibody neutralization, both of which depends on robust CD4+ T cell help.
  • Many viral and bacterial pathogens are well characterized in terms of the CD4 and CD8 epitopes recognized in most patients. Better eradication of these infections may depend on a better stimulation of as many epitopes as possible.
  • hyperproliferative diseases such as cancer can be treated using tumor antigens, to increase killing of the tumor cells.
  • Preferred cancers for use with the invention include, but are not limited to melanomas and carcinomas that are characterized by multiple antigens mutated or
  • the tandem epitope construct of the invention is depicted in its most simplistic form in FIG. 1.
  • the square box marked "T” indicates a targeting sequence which enables delivery of the epitopes in that group to the endosome for MHC class II processing for CD4+ T cells.
  • the circle represents a proteolytic cleavage site to separate the two groups of epitopes from each other, allowing one group to be targeted to MHC class I and the other to MHC class II processing.
  • the group of epitopes operably linked to a targeting region can be positioned at either end of the construct.
  • Each of the two sequences, to be targeted to MHC class I or class II pathways and joined to form a tandem construct separated by a protease cleavage site contains at least one epitope.
  • the number of epitopes depends on the number of nucleic acids any given vector can accommodate. The number will depend on the size of the epitopes and the vector being used, and can be determined by one of skill in the art.
  • the epitopes generally are about 7-25 amino acids in length, and preferably 8 or 9 to about 15 or 20 amino acids.
  • a construct of 1,000 to 2,000 bp can easily be expressed by mRNA, DNA plasmid or a viral vector.
  • the construct contains two strings of epitopes, separated by a site for proteolytic cleavage which can separate the two strings of epitopes from each other.
  • Each epitope in the string optionally can be flanked by native sequences that assist in processing (cleaving) of the individual epitopes (to replicate natural processing of the whole antigen), or in some cases, flanked by specific amino acids, for example lysines, which define good proteasomal cleavage sites.
  • Any of the individual native epitopes can be substituted with a mimotope to improve binding and recognition of the epitope by the appropriate T cell.
  • the sequence of a mimotope can be obtained based on "rational design" or from peptide screening studies. The existence of mimotopes or modified antigens is usually known in each field. Modification of beta cell antigens can result from enzymes induced upon beta cell stress in T1D, including
  • the TEC, vectors, and cells containing them can be used to induce either immune tolerance or to increase an immune response to certain epitopes.
  • diseases such as autoimmune diseases can be treated; and where the tandem construct and vector for administration are designed to induce immunity or an immune reaction, diseases such as infectious diseases and malignancies can be treated.
  • the autoimmune diseases that can benefit most from the invention are those autoimmune diseases characterized by multiple protein antigens targeted by the immune system (epitope spreading), in which the antigenic epitopes are known or can be discovered, and in which the epitopes comprise both CD4 and CD8 epitopes. Should there be a disease in which only CD4 or CD8 epitope processing is needed, the construct can be so designed. Persons of skill are aware of diseases of this type and any of these are contemplated as part of the invention. Such
  • autoimmune diseases include, but are not limited to type 1 diabetes (TID), multiple sclerosis (MS), rheumatoid arthritis (RA), psoriasis, inflammatory bowel syndrome (IBS; including Crohn's disease and ulcerative colitis), autoimmune hepatitis, vitiligo, and celiac disease.
  • TID type 1 diabetes
  • MS multiple sclerosis
  • RA rheumatoid arthritis
  • IBS inflammatory bowel syndrome
  • Other autoimmune disorders which involve limited numbers of epitopes can be used with the invention as well, but will not receive the maximum benefits of the invention and hence are not preferred.
  • Proinsulin/insulin (gene: INS): extensive CD4+ T cell responses in NOD mice and TID patients, extensive CD8+ T cell in TID patients, some CD8+ T cell in NOD mice, autoantibodies in NOD mouse and TID patients.
  • GAD65 glutlutamic acid decarboxylase, gene: GAD2
  • GAD65 glutlutamic acid decarboxylase, gene: GAD2
  • IA-2 insulinoma-associated protein 2, or protein tyrosine phosphatase, receptor type, N, gene: PTPRN
  • IA-2 extensive CD4+ T cell responses in NOD mice and TID patients (Peakman et al., J. Clin. Invest. 104(10): 1449-1457, 1999), some CD8+ T cell responses in TID patients, autoantibodies in TID patients (Bonifacio et al., J. Immunol. 155(11):5419-5426, 1995).
  • IGRP islet-specific glucose-6-phosphatase catalytic subunit-related protein
  • G6PC2 some CD4+ T cell responses in NOD mice and TID patients ( Jarchum et al., Clin Immunol, 127(3):359-365, 2008), some CD8+ T cell responses in TID patients, extensive CD8+ T cell responses in NOD mice.
  • Chromogranin A (gene: CHGA): CD4+ T cell responses in NOD mice (Stadinski et al., Proc. Natl. Acad. Sci. USA 107: 10978-10983, 2010; Delong et al., Diabetes 61:3239-3246, 2012
  • IAPP islet amyloid polypeptide; gene: IAPP
  • CD4+ T cell responses in NOD mice see Baker et al., J. Immunol, 191(8):3990-3994, 2013; some CD8+ T cell responses in TID patients.
  • ZnT8 (zinc transporter 8; gene: SLC30A8): autoantibodies in in TID patients, evidence of T cell responses in NOD mice (Dang et al., J. Immunol, 186(10):6056-6063. 2011); (Nayak et al., Diabetes, 63(10):3438-3448, 2014).
  • ICA69 islet cell autoantigen; gene: ICA1: some CD4+ T cell responses in NOD mice and TID patients.
  • ⁇ -2 ⁇ (insulinoma-associated protein 2 beta or phogrin or protein tyrosine phosphatase, receptor type, N polypeptide 2; gene: PTPRN2): some CD4+ T cell responses in NOD mice and TID patients.
  • Regll regenerating islet II, gene: REG3A: T cell responses in NOD mice (Gurr et al., Diabetes, 51(2):339-346, 2002); (Gurr et al., Diabetes, 56(l):34-40, 2007).
  • GPR78 G protein-coupled receptor 78, when citruUinated; gene: GPR78: T cell responses and autoantibodies against citruUinated GPR78 in NOD mice (Rondas et al., Diabetes, 64(2):573-586, 2015).
  • MBP Myelin basic protein
  • PLP Proteolipid protein
  • MOG Myelin oligodendrocyte glycoprotein
  • MAG Myelin-associated antigen
  • MOBP Myelin-associated oligodendrocyte basic protein
  • CNPase 2', 3'-cyclic-nucleotide 3 '-phosphodiesterase
  • Collagen (type II) T cell responses and antibodies
  • Cartilage glycoprotein 39 (Chitinase 3-like 1; gene: CHI3L1) (Verheijden et al., Arthritis Rheum, 40(6): 1115- 1125, 1997)
  • Aggrecan Gl (cartilage-specific proteoglycan core protein, domain Gl; gene ACAN) (Li et al, Cell Res, 10(l):39-49, 2000)
  • Rheumatoid factor autoantibodies to Fc portions of IgG
  • Collagen type XVII (Inokuma et al., Br. J. Dermatol, 160(2):451-454, 2009).
  • Keratin 13, hnRNP-Al and FLJ00294 (Jones et al, J. Invest. Dermatol, 123(1):93-100,
  • SCG, GLCDAC05, alpha-endosulfine, NOL8, GFGR3, dematin, signal recognition particle subunit 14 and EPF as alopecia areata autoantigens (Lueking et al., Mol. Cell.
  • Glycoproteins CUZD1 and GP2 Rost al., Gut, 58(12): 1620-1628, 2009;
  • FAM84A (Vermeulen et al., Inflamm Bowel Dis, 17(6): 1291-1300, 2011).
  • Collagen type VII (Chen et al., J. Invest. Dermatol, 118(6): 1059- 1064, 2002),
  • Ubiquitination factor e4A (UBE4A) (Sakiyama et al., Inflamm Bowel Dis, 14(3):310-7, 2008).
  • Galectin-3 autoantibodies (Jensen-Jarolim et al., J. Clin Immunol, 21(5):348-356, 2001). [0177] Catalase and alpha-enolase (Roozendaal et al., Clin Exp Immunol, 112(1): 10-6, 1998). [0178] Lactoferrin autoantibodies (Roozendaal et al., Adv Exp Med Biol, 443:313-319, 1998).
  • the tandem epitope construct also can be used to treat and produce immune reaction to infectious diseases characterized by multiple protein antigens (conserved antigens) to be targeted by the immune system, for where the immune response benefits from being composed of both CD4+ T cell and CD8+ T cell responses, and for which antigenic epitopes are known or predicted.
  • infectious diseases characterized by multiple protein antigens (conserved antigens) to be targeted by the immune system, for where the immune response benefits from being composed of both CD4+ T cell and CD8+ T cell responses, and for which antigenic epitopes are known or predicted.
  • infectious diseases characterized by multiple protein antigens (conserved antigens) to be targeted by the immune system, for where the immune response benefits from being composed of both CD4+ T cell and CD8+ T cell responses, and for which antigenic epitopes are known or predicted.
  • examples of such diseases are influenza, HIV, HBV, and HCV.
  • Hyperproliferative disorders including malignancies, also can have more than one antigen useful for targeting the tumor cells (i.e. mutated or overexpressed antigens).
  • the inventive methods can be used to target epitopes from a plurality of the known antigens at the same time for optimal results.
  • any known epitope to which one would like to induce tolerance or stimulate an immune reaction in a subject is contemplated for use with the invention.
  • the choice of epitopes is determined based on those most often targeted in the patient population or personalized to individual patients based on diagnostic tests. Because it is customizable, native peptides may be mutated for better targeting of specific types of self-reactive T cells (those requiring post- translational modifications or uncommon MHC binding register).
  • the construct encodes a balance of both CD4 and CD8 epitopes so that tolerance or alternatively a robust immune response is stimulated for both MHC class I and MHC class II antigens at the same time.
  • Preferred antigens for making a tolerogenic TEC include any of those specifically discussed or provided herein, or any epitopes from diabetogenic or autoimmune antigens. Cancer antigens, pathogens and the like also are contemplated for use with in TEC designed to induce a strong immune response to those antigens/epitopes.
  • three antigens have been evaluated individually in T1D clinical trials (proinsulin/insulin, GAD65 and HSP60 p277) using a variety of delivery methods. Overall, these antigen- specific therapies were well-tolerated, but poorly efficacious.
  • Other antigens are targeted in T1D, including but not limited to IA-2, IGRP, ChgA and ZnT8.
  • the data presented are based on epitopes targeted in the NOD mouse model of TID, and include Ins2 B: 15-23 and IGRP 2 06-214 for CD8 epitopes, and Ins2 B:9- 23, Ins2 B:9-23 (R22E), Ins2 B:9-23 (R22E)(E21G), ChgA1040-79, GAD65 286 -3oo, GAD65 52 4-543 for CD4 epitopes/mimo topes.
  • Some of these epitopes have been chosen for proof of principle experiments because tools and reagents exist to assess the T cell responses to these particular epitopes, such as T cell receptor transgenic mice and tetramer reagents.
  • epitopes include those known in the field from insulin, GAD65, and IA-2. A smaller number of epitopes have been identified for other antigens, including IGRP, ChgA, ZnT8, IAPP and ICA69. Epitopes from HSP60/70 proteins are also targeted although those are not beta cell- specific. A widely recognized important epitope for TID is the CD4 epitope insulin B:9-23, which is targeted in both NOD mice and TID patients. In NOD mice, this epitope is involved in initiation of disease (Nakayama et al., Nature. 35(7039):220-3, 2005).
  • any antigen on an invading pathogen such as a viral antigen, bacterial antigen, fungal antigen, parasitic antigen and the like can be used.
  • Preferred pathogen antigens are viral antigens such as influenza antigens and HIV antigens, however any antigen which is known to produce an immune response can take advantage of the compositions and methods of this invention.
  • specific tumor antigens or antigens mutated or overexpressed in tumors can be used with the invention and for treatment of hyperproliferative disorders. Persons of skill in the art are aware of such antigens which may be important in various disease conditions and can choose appropriate epitopes (or design mimotopes) as a matter of routine.
  • any tumor antigens which are overexpressed in tumors or which are mutated in cells to be destroyed can be used.
  • the epitopes used are those which will not affect normal tissues bearing the same epitope but are confined to or mostly confined to the tumor.
  • pathogen epitopes such as viral nucleic acid sequences and the like can be used.
  • the invention may be used with or comprise expression vectors. Such vectors are known per se in the art and are designed as a mode of delivery of a nucleic acid, or to produce endogenous expression of a peptide encoded by a nucleic acid.
  • Vectors for delivering nucleic acids can be viral, non- viral, or physical. See, for example, Rosenberg et al., Science, 242: 1575-1578, 1988, and Wolff et al., Proc. Natl. Acad. Sci. USA 86:9011-9014 (1989).
  • 6,080,728 also provides a discussion of a wide variety of gene delivery methods and compositions.
  • the routes of delivery include, for example, systemic administration and administration in situ.
  • Well-known viral delivery techniques include the use of adenovirus, retrovirus, lentivirus, foamy virus, herpes simplex virus, vaccinia virus and adeno-associated virus vectors.
  • Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which nonessential genes have been replaced with the TEC carrying the nucleic acids encoding the epitopes and targeting sequences of interest.
  • Preferred viruses for certain embodiments of the invention are the adenoviruses and adeno-associated (AAV) viruses, which are double- stranded DNA viruses that have already been approved for human use in gene therapy.
  • preferred vectors for tolerizing do not include immune- stimulating sequences.
  • adenovirus expression vector is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to express a polynucleotide that has been cloned therein in a sense or antisense orientation.
  • expression does not require that the gene product be synthesized.
  • the delivery vector pertains to commercially available ORF of cytochrome b5 reductase 3 (CYB5R3), transcript variant 1 in adenoviral vector pAd, with C terminal Flag and His tag, (Vigene Biosciences Product code AH889428).
  • CYB5R3 cytochrome b5 reductase 3
  • transcript variant 1 in adenoviral vector pAd with C terminal Flag and His tag
  • WIPO Patent Application WO/2015/050364 also teaches vectors with expression constructs including a Cyb5r3 gene.
  • Adenoviral vectors are highly immunogenic and therefore are less preferred for administration to induce tolerance by presenting antigens, or in the case of autoimmune diseases. These vectors can be used, however to induce immunity, for example in treatment of infectious diseases and the like, include, for example, influenza, HBV, HCV and HIV.
  • AAV Adeno- Associated Virus Vectors
  • AAV is a good choice of delivery vehicles due to its safety, i.e., genetically engineered (recombinant) does not integrate into the host genome. Likewise, AAV is not pathogenic and not associated with any disease. The removal of viral coding sequences minimizes immune reactions to viral gene expression, and therefore, rAAV does not evoke an inflammatory response.
  • an AAV vector containing an expression construct that includes a polynucleotide encoding a Cyb5r3 protein is useful for transducing pancreatic beta cells.
  • AAV-Cyb5r3 vectors are commercially available, see, e.g., ABM Cat. No.
  • the construct in the AAV-Cyb5r3 vector may include an insulin promoter to provide for specific gene expression in ⁇ cells, see for example, Wang et al., Diabetes 55(4):875- 884, 2006, which describes methods of developing AAV vectors having ⁇ -cell specific gene expression, as well as routes of administering the same.
  • an insulin promoter for driving expression of the Cyb5r3 can be used in conjunction with other gene delivery vehicles described herein.
  • viral vectors containing TEC are assembled from polynucleotides encoding the desired epitopes, suitable regulatory elements and elements necessary for epitope expression which mediate cell transduction.
  • adeno-associated viral (AAV) vectors are employed.
  • the AAV vector is an AAV1, AAV6, or AAV8.
  • the AAV expression vector which harbors the DNA molecule of interest bounded by AAV ITRs can be constructed by directly inserting the selected sequence(s) into an AAV genome which has had the major AAV open reading frames (“ORFs") excised therefrom.
  • ORFs major AAV open reading frames
  • constitutive promoters which may be included in the AAV of this invention include, without limitation, the exemplified CMV immediate early enhancer/chicken ⁇ -actin (CBA) promoter.
  • CBA CMV immediate early enhancer/chicken ⁇ -actin
  • expression control sequences typically include a promoter, an enhancer, such as one derived from an immunoglobulin gene, SV40, cytomegalovirus, etc., and a polyadenylation sequence which may include splice donor and acceptor sites.
  • polyadenylation sequence generally is inserted following the transgene sequences and before the 3' ITR sequence.
  • the bovine growth hormone polyA may be used.
  • the viral vector may be a retroviral vector.
  • Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse
  • Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are known to those of skill in the art.
  • the retroviral genome contains three genes, gag, pol, and env that code for capsid proteins, polymerase enzyme, and envelope components, respectively.
  • a sequence found upstream from the gag gene contains a signal for packaging of the genome into virions.
  • Retroviral vectors are gene transfer plasmids wherein the heterologous nucleic acid resides between two retroviral LTRs. Retroviral vectors typically contain appropriate packaging signals that enable the retroviral vector, or RNA transcribed using the retroviral vector as a template, to be packaged into a viral virion in an appropriate packaging cell line (see, e.g., U.S. Pat. No. 4,650,764). These two long terminal repeat (LTR) sequences are present at the 5' and 3' ends of the viral genome. These contain strong promoter and enhancer sequences and are also required for integration in the host cell genome (Coffin, 1990).
  • LTR long terminal repeat
  • a nucleic acid encoding one or more oligonucleotide or polynucleotide sequences of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. Also included are episomal or non-integrating forms of retroviral vectors based on lentiviruses (e.g., a type of retrovirus).
  • Lentiviral vectors are useful when stable expression is needed, but lentiviral vectors can be immunogenic, and possibly have other undesirable effects. Therefore, although lentiviral vectors are convenient for research, care should be taken when using them for human administration, particularly where it is desired to induce tolerance rather than immunity.
  • Lentiviruses are more preferred for engineering T cells or dendritic cells or other antigen presenting cells ex vivo for cancer therapy, although mRNA electroporation is preferred for safety reasons.
  • two recent advances have made the use of lentiviruses safer and more clinically translatable.
  • a suicide gene along with the antigens whose products become functional when a drug is administered.
  • a typical example is Herpes simplex virus thymidine kinase (HSV-Tk). Cells that express these genes can metabolize the drug ganciclovir into a cytotoxic product that induces cell death. Thus, in case some transduced cells become malignant, they can be eradicated.
  • Suitable retroviral vectors for use herein are described, for example, in U.S. Patent Nos. 5,399,346 and 5,252,479; and in WIPO publications WO 92/07573, WO 90/06997, WO
  • retroviral vectors include, for example, mouse mammary tumor virus vectors (e.g., Shackleford et al., Proc. Natl. Acad. Sci. U.S.A. 85:9655-9659, 1998), lentiviruses, and the like.
  • An exemplary viral vector is plentilox-IRES-GFP.
  • viral vectors may be employed as expression constructs in the present invention for the delivery of oligonucleotide or polynucleotide sequences to a host cell.
  • Vectors derived from viruses such as vaccinia virus, polioviruses and herpes viruses may be employed. They offer several attractive features for various mammalian cells. Also included are hepatitis B viruses.
  • Constitutive promoters are active in any cell type in which it is introduced, although the activity varies from cell to cell. Some promoters generate higher levels of expression than others, some promoters become silenced over time, and others have a very stable expression.
  • the most common promoters include the CMV promoter (from cytomegalovirus), the composite CAG promoter (CMV early enhancer, chicken beta actin promoter, rabbit beta globin splice acceptor), the ubiquitin C promoter, the PGK promoter (phosphoglycerate kinase 1), the EFl-alpha promoter (elongation factor 1-alpha), the SV40 promoter, the MSCV promoter (from murine stem cell virus), and the composite MND promoter (MPSV LTR, NCR deleted, and d/587 PBS). Any of these are contemplated for use with the invention, but any convenient promoter can be chosen by the person of skill in the art, depending on the system being used.
  • Tissue-specific promoters are promoters that are only active in certain cell types such as the dendritic cell-specific CD 11c promoter, the hepatocyte-specific ET and albumin promoters, the endothelial cell-specific ICAM-2 promoter or the epithelial cell-specific Cytokeratin 18 promoter (Papadakis et al., Current Gene Therapy, 4:89-113, 2004).
  • mRNA does not depends on a promoter as it is already transcribed. It is rapidly translated in any cell, whether it is picked up in vivo or introduced ex vivo by transfection methods such as electroporation.
  • Non- Viral Vectors 1. Plasmid Vectors
  • Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g. Sambrook et al., 1989, cited above. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigen-encoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide epitope encoded by nucleic acid within the plasmid. Other plasmids are well known to those of ordinary skill in the art.
  • Plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by a variety of parenteral, mucosal and topical routes.
  • the DNA plasmid can be injected by intramuscular, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally. It may also be administered into the epidermis or a mucosal surface using a gene-gun.
  • the plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and microencapsulation.
  • a plasmid for expression of the TEC which includes an expression cassette; also referred to as a transcription unit.
  • an expression cassette also referred to as a transcription unit.
  • the transcription unit includes a transcriptional control sequence, which is transcriptionally linked with a cellular immune response element coding sequence.
  • Transcriptional control sequence may include promoter/enhancer sequences such as cytomegalovirus (CMV) promoter/enhancer sequences.
  • CMV cytomegalovirus
  • those skilled in the art will recognize that a variety of other promoter sequences suitable for expression in eukaryotic cells are known and can similarly be used in the constructs disclosed herein.
  • the level of expression of the nucleic acid product will depend on the associated promoter and the presence and activation of an associated enhancer element.
  • a sequence encoding the desired epitopes and targeting sequence can be cloned into an expression plasmid which contains the regulatory elements for
  • transcription, translation, RNA stability and replication i.e., including a transcriptional control sequence.
  • expression plasmids are well known in the art and one of ordinary skill would be capable of designing an appropriate expression construct with a polynucleotide including a sequence encoding a cellular immune response element or fragment thereof in such a manner that the cellular immune response element is expressible.
  • suitable expression plasmids into which a polynucleotide including a sequence could be cloned such as pCI-neo, pUMVC or pcDNA3.
  • the purpose of the plasmid is the efficient delivery of nucleic acid sequences to and expression of therapeutic epitopes in a cell or tissue.
  • the purpose of the plasmid may be to achieve high copy number, avoid potential causes of plasmid instability and provide a means for plasmid selection.
  • the nucleic acid cassette contains the necessary elements for expression of the nucleic acid within the cassette. Expression includes the efficient transcription of an inserted gene, nucleic acid sequence, or nucleic acid cassette with the plasmid. Expression products may be proteins, polypeptides or RNA.
  • the nucleic acid sequence can be contained in a nucleic acid cassette. Expression of the nucleic acid can be continuous or regulated.
  • nucleic acid As an initial step in the process of ultimately obtaining expression of a product encoded by a nucleic acid, is to effect the uptake of the nucleic acid by cells. Uptake of nucleic acid by cells is dependent on a number of factors, one of which is the length of time during which a nucleic acid is in proximity to a cellular surface. For instance, after intramuscular (i.m.) administration of plasmid DNA in buffer, a marked reduction in gene expression was observed if the muscle is massaged, presumably due to DNA leakage out of the muscle either directly or via lymphatic vessels (Human Gene Therapy 4: 151-159; 1993).
  • nucleic acids may be desirable to formulate nucleic acids with compounds which would retard the rate at which nucleic acids diffuse or are carried away from a site at which cellular uptake of the nucleic acid is desired. Further, these compounds could be suitable for administration to an organism by means such as injection while maintaining or regaining the physical characteristics necessary to increase cellular uptake of nucleic acids.
  • an expression construct comprising one or more oligonucleotide or polynucleotide sequences may simply consist of naked recombinant DNA or plasmids.
  • DNA vaccine vectors of any type preferably are engineered to be CpG-rich (to stimulate TLR9 on immune cells) or conversely are engineered to remove CpG, and when possible, replace CpG motifs with GpG motifs (Ho et al., J. Immunol. 71(9):4920-6, 2003; Ho et al., J.
  • DNA vaccines can be engineered to contain the antigen(s)/epitope(s), and also can contain additional genes for co-expression with the antigens to act as adjuvants or immunomodulators (multiple promoter vectors. These DNA vaccines have been found to be safe clinically, for example in T1D patients (Roep et al., Sci. Transl. Med. 5(191): 191ra82, 2013).
  • Additional non-viral delivery methods include but are not limited to mechanical delivery systems that can be used in vitro such as the approach described in Woffendin et al. , Proc. Natl. Acad. Sci. USA 91(24): 11581, 1994; deposition of photopolymerized hydrogel materials or use of ionizing radiation (see, e.g., U.S. Pat. No. 5,206,152 and WO 92/11033); the use of a handheld gene transfer particle gun (see, e.g., U.S. Pat. No. 5,149,655); and the use of ionizing radiation for activating transferred gene (see, e.g., U.S. Pat. No.
  • Delivery devices can also be biocompatible, and may also be biodegradable.
  • the formulation preferably provides a relatively constant level of active component release. On the other hand, a more rapid rate of release immediately upon administration may be desired.
  • the formulation of such compositions is well within the level of ordinary skill in the art using known techniques.
  • DNA also can be encapsulated in liposomes, preferably cationic liposomes, or polymersomes (synthetic liposomes) which can interact with the cell membrane and fuse or undergo endocytosis to effect DNA transfer into the cell.
  • the DNA also can be formed into complexes with polymers (polyplexes) or with dendrimers which can directly release there load into the cytoplasm of a cell.
  • Illustrative carriers useful in this regard include microparticles of poly(lactide-co- glycolide), polyacrylate, latex, starch, cellulose, dextran and the like.
  • Other illustrative delayed- release carriers include supramolecular biovectors, which comprise a non-liquid hydrophilic core (e.g., a cross-linked polysaccharide or oligosaccharide) and, optionally, an external layer comprising an amphiphilic compound, such as a phospholipid (see e.g., U.S. Pat. No. 5,151,254 and PCT applications WO 94/20078, WO/94/23701 and WO 96/06638).
  • the amount of active agent contained within a sustained release formulation depends upon the site of implantation, the rate and expected duration of release and the nature of the condition to be treated or prevented.
  • Biodegradable microspheres may be employed as carriers for compositions. Suitable biodegradable microspheres are disclosed, for example, in U.S. Patent Nos. 4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883; 5,853,763; 5,814,344, 5,407,609 and 5,942,252. Modified hepatitis B core protein carrier systems such as described in WO/99 40934, and references cited therein, will also be useful for many applications. Another illustrative carrier/delivery system employs a carrier comprising particulate-protein complexes, such as those described in U.S. Patent No. 5,928,647, which are capable of inducing an MHC class I-restricted cytotoxic T lymphocyte responses in a host.
  • Biodegradable polymeric nanoparticles facilitate nonviral nucleic acid transfer to human embryonic stem cells (hESCs). Small (approximately 200 nm), positively charged
  • Polynucleotides may also be administered to cells by direct microinjection, temporary cell permeabilizations (e.g., co-administration of repressor and/or activator with a cell permeabilizing agent), fusion to membrane translocating peptides, and the like.
  • mRNA can be used to modify dendritic cells (DC) or other antigen presenting cells for cell therapy of cancer, and are contemplated for use with the invention (Lee J, Boczkowski D, Nair, Methods Mol. Biol. 969: 111-25, 2013). mRNA is less immunogenic than DNA in DCs differentiated in vitro, and possibly is the least immunogenic of all vectors (See Creusot et al., Mol. Ther. 18(12):2112-2120, 2012, and United States Patent No. 8,513,208 B2 for more information on these vectors and their use, particularly for cell therapy for autoimmunity).
  • mRNA is a preferred vector for this invention, although it has a short window of expression.
  • mRNA electrop oration offers many advantages over traditional viral techniques used for DC manipulation, including ability to co-express many genes in a per cell basis, high transfection efficiency, good viability, minimal DC maturation (which would otherwise render them more immunogenic) and absence of genetic disruption.
  • RNA also can be used as a vaccine to administer the treatment of the invention, as well, although it is unstable, particularly outside cells (Challenges and advances towards the rational design of mRNA vaccines. (Pollard et al., J. Trends Mol. Med. 19(12):705-13, 2013).
  • RNA Although less immunogenic than most viral vectors and unmodified plasmid DNA, RNA has nonetheless some immunogenic properties that can be reduced or abrogated with certain modification, for example as described in Kariko et al., "Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA.” Immunity Aug;23(2): 165-75, 2005; Kariko et al., "Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability.” Mol. Ther. 16(11): 1833-1840, 2008; and Anderson et al., "Incorporation of pseudouridine into mRNA enhances translation by diminishing PKR activation.” Nucleic Acids Res.
  • IVT in vitro transcription
  • the IVT mRNA is delivered into a patient's cells ex vivo prior to the cells administered back into the patient, or direct delivered by various routes of inoculation.
  • the mRNA In order to be recognized by the cell as a normal mature mRNA, ready for transcription, the mRNA usually is constructed to have a 5' cap and a poly- A tail, correct start and stop codons, and flanking untranslated regions.
  • Electroporation is a preferred method for introducing mRNA into a cell. This well-known method has been shown to be safe in patients with cancer and is contemplated for use here. Delivery of naked mRNA to the subject also can be used and has been shown to induce immune responses in other contexts.
  • Preferred vectors for use when inducing tolerance are (1) mRNA and plasmid DNA modified for minimal immunogenicity for direct inoculation, (2) mRNA electroporation into tolerogenic APCs ex vivo which are then re-infused, and also (3) non-integrating lentiviral vectors used to infect tolerogenic APCs ex vivo or directly inoculated, Viral vectors with tissue- specific promoters are preferred, such as those described for gene therapy of T1D (Insulin B chain 9-23 gene transfer to hepatocytes protects from type 1 diabetes by inducing Ag-specific FoxP3+ Tregs. Akbarpour et al., Sci. Transl. Med. 7(289):289ra81, 2015). Long-term expression is seen as favorable for maintenance of tolerance, although most vectors do not achieve long- term expression without certain risks (e.g., integrating vectors). Vectors that are non- immunogenic and have limited integration sites like AAV have an advantage at this level.
  • Preferred vectors for use when inducing an active immune response are viral vectors, mRNA or DNA plasmids non-devoid of immunostimulatory motifs and combined with adjuvants that are known in the art.
  • vectors that confer long-term expression may not be favorable or should include a shutdown mechanism for after the pathogens or tumor has been eradicated.
  • Antigen-presenting cells are cells that can, or can be induced to, present antigens on their surface in the context of MHC class I or class II. Dendritic cells, macrophages, and B cells, are considered in the art to be "professional APCs.” These cells, and any other "nonprofessional" APCs that are able to present antigen with MHC class I (and to some extent with class II) are defined here as APCs. Any APC can be used with the invention and are
  • Dendritic cells are most preferred as they have considerable clinical benefits for reprograming immune cells because of their ability to target specific T cells, ability to preferentially migrate to tissues of interest, ease of ex vivo expansion and manipulation, and good safety records. Dendritic cells and their use are well known in the art. These cells are discussed in Creusot et al., Blood 113:6638-6647, 2009 and Creusot et al. Diabetes 63(l):20-30, 2014, as well as methods for handling and culturing the cells and using these cells for presentation of antigen, and a discussion of homing and trafficking of dendritic cells.
  • blood can be harvested from a subject according to methods well-known in the art, from which monocytes can be isolated and differentiated in vitro over one week of culture in presence of granulocyte-macrophage colony-stimulating factor (GM- CSF) and IL-4.
  • GM- CSF granulocyte-macrophage colony-stimulating factor
  • Phenotype can be analyzed and confirmed by FACS according to well-known methods in the art. For suitable methods, see Nava et al., "An optimized method for
  • Tolerogenic (tolerance-inducing) antigen-presenting cells are naturally found throughout lymphoid tissues and function to present self antigens to potential autoreactive T cells and instruct these T cells to undergo apoptosis (deletion), inactivation (anergy) or conversion into regulatory T cells (Tregs) via tolerogenic signals.
  • Different subsets of tolerogenic APCs exist within the dendritic cell and stromal cell populations. Each subset displays its own combination of antigens, acquired exogenously or through endogenous expression, and surface- expressed or secreted molecules (tolerogenic signals).
  • a single tolerogenic cell can simultaneously provide antigenic and multiple tolerogenic signals that can be optimally integrated and interpreted by the target T cells.
  • Immature (non-immunogenic) DCs can be modified to co-express antigens and multiple tolerogenic products, for example by mRNA electroporation.
  • Immunogenic DCs have been tested as therapeutic vaccines in cancer patients for nearly a decade, culminating with the FDA approval of Provenge®.
  • the application of tolerogenic DCs to autoimmune diseases and transplanation has been more recent, leading to the first clinical trials for T1D (Giannoukakis et al, Diabetes Care, 2011) and RA (AutoDECRA trial in UK, Rheumavax trial in Australia: Citrullinated peptide dendritic cell immunotherapy in HLA risk genotype-positive rheumatoid arthritis patients. Benham et al., Sci. Transl. Med. 7(290):290ra87, 2015).
  • compositions for administration, routes, and doses.
  • nucleic acids of the invention are contemplated for administration to a subject in need, and can be administered by any convenient method known to the person of skill in the art.
  • Administration can be by any route, including but not limited to local and systemic methods, for example aerosols for delivery to the lung, oral, rectal, vaginal, buccal, transmucosal, transdermal, intravenous, subcutaneous, intradermal, intratracheal, intramuscular, intraarterial, intraperitoneal, intracranial (e.g., intrathecal or intraventricular) or any known and convenient route.
  • Preferred routes of administration are intravenous, intraperitoneal, oral/nasal and direct injection into the affected organ, tissue, area of infection or tumor.
  • the form of the administration can determine how the active agent is formulated, and this is easily determined by the skilled artisan.
  • Nucleic acid drugs generally are delivered in nanosized drug formulations into the blood stream, and these well-known formulations and methods of administration are preferred.
  • An exemplary nanocarrier is described in Pujol- Autonell et al., "Use of autoantigen-loaded phosphatidylserine- liposomes to arrest autoimmunity in type 1 diabetes.” PloS one 10, e0127057 (2015).
  • compositions of the present invention therefore can include, but are not limited to, solid preparations for oral administration, solid preparations to be dissolved in a liquid carrier for oral or parenteral administration, solutions, suspensions, emulsions, oils, creams, ointments, lotions, gels, powders, granules, cells in suspension, and liposome-containing formulations, and the like, or any convenient form known in the art.
  • These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.
  • Solutions or suspensions used for parenteral, intradermal, subcutaneous or other injection can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylene diamine tetra acetic acid; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where the therapeutic agents are water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor EL® (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that they can pass through a syringe and needle easily enough for administration. It should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms such as bacteria and fungi. All solutions used to solubilize DNA or RNA should also be DNase-free and RNase-free.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active agent in the required amount in an appropriate solvent with one or a combination of the ingredients enumerated above, as required, followed by filter sterilization.
  • dispersions are prepared by incorporating the active agent into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The skilled person is aware of how to use these dried preparations for injection.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. Depending on the specific conditions being treated, pharmaceutical compositions of the present invention for treatment of
  • Atherosclerosis or the other elements of metabolic syndrome can be formulated and administered systemically or locally. Techniques for formulation and administration can be found in
  • the agent can be contained in enteric forms to survive the stomach or further coated or mixed to be released in a particular region of the GI tract by known methods.
  • the active agent can be incorporated with excipients and used in the form of tablets, troches, or capsules.
  • Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.
  • Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as
  • microcrystalline cellulose, gum tragacanth or gelatin an excipient such as starch or lactose, a disintegrating agent such as alginic acid, PRIMOGEL® or corn starch; a lubricant such as magnesium stearate or STEROTES®; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • Systemic administration can also be by transmucosal means to the intestinal or colon, such as by suppository or enema, for example.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active agents are formulated into ointments, salves, gels, or creams as generally known in the art.
  • Preferred administration sites include those that target the lymph nodes draining the site of infection, tumor or autoimmune attack. Persons of skill are aware of those locations and can select an appropriate route of administration for each patient or disease state. For example, pancreatic lymph nodes are targeted efficiently by dendritic cells after intraperitoneal and intravenous administration. See Creusot et al. Blood 113:6638-6647, 2009.
  • the active agents are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release or delayed formulation, including implants and microencapsulated delivery systems.
  • a controlled release or delayed formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • the materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to particular cells with, e.g., monoclonal antibodies) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art.
  • Formulations designed to provide extended or delayed release also are contemplated for use with the invention.
  • the following United States patents contain representative teachings concerning the preparation of uptake, distribution and/or absorption assisting formulations: U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932;
  • the pharmaceutical formulations of the present invention may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • the active agents described herein also can be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption.
  • inventive compounds can be fused to microspheres in suspension for intravenous injection.
  • compositions including the nucleic acid, peptide, composition and cells of the invention can be performed a single time, or repeated at intervals, such as by continuous infusion over a period of time, four times daily, twice daily, daily, every other day, weekly, monthly, or any interval to be determined by the skilled artisan based on the subject involved.
  • Treatment can involve administration over a period of one day only, a week, a month, several months, years, or over a lifetime.
  • Regimens and duration can vary according to any system known in the art, as is known to the skilled person.
  • Cells expressing a DNA or an mRNA, or naked DNA or RNA in a nanocarrier-type pharmaceutical vehicle can be injected into a patient, intravenously or into the tissues and/or organs affected by the disease condition to be treated.
  • Current cell vehicles available for human therapy include tolerogenic or immunogenic dendritic cells, and mesenchymal stromal cells.
  • Dendritic cells are typically differentiated in vitro from blood precursors, but may also be derived from bone marrow precursors.
  • Mesenchymal stromal cells may be derived from bone marrow or adipose tissue.
  • the nanocarrier vehicle can be a liposome, a nanoparticle or microparticle, which can be taken up by APCs in vivo.
  • Doses of the inventive nucleic acid, peptide and cells can be determined by the skilled artisan based on the condition of the subject and the route of administration to be used, but are expected to range from about 100 ⁇ g to about 10 mg, preferably from about 500 ⁇ g to about 10 mg, or about 1 mg to about 10 mg, or about 1 mg to about 5 mg or about 5 mg to about 10 mg and most preferably from about 1 mg to about 5 mg. Optimization/pharmacokinetics can make lower doses effective, therefore even lower doses are contemplated for use with the invention, for example about 10 ⁇ g to about 100 ⁇ g.
  • constructs of the invention can be delivered in situ as plasmid DNA (naked DNA vaccines, nanoparticle-mediated delivery) or mRNA vaccine, or ex vivo into tolerogenic DCs (or other tolerogenic APCs as they become available) as viral vector (transduction) or mRNA (electroporation) .
  • the invention leads to the elimination or inactivation of self-reactive T cells, and the induction of stable regulatory T cells (Tregs), which is most important for long-term tolerance.
  • Tregs stable regulatory T cells
  • Targeting T cells with relevant antigen specificity and focusing the intervention on relevant tissues are advantageous to avert systemic immune suppression and adverse effects.
  • T1D patients for example, vary in the risk alleles that they possess, the antigens that are targeted and the rate of disease progression.
  • therapies preferably impact multiple biological pathways while targeting only specific T cell populations.
  • a construct according to the invention is produced and introduced into an antigen-presenting cell, preferably a dendritic cell, in vivo or ex vivo, using a lentivirus or other viral vector, a naked DNA or RNA method, mRNA electroporation, or any of the methods discussed above. If the introduction of the construct is performed ex vivo, the modified cell is administered to a subject in need by an appropriate route of administration, preferably intravenously.
  • mRNA electroporation is preferred due to its combinatorial versatility which allows combined expression of more products than is possible using other common methods of modification such as viral vectors.
  • Other interesting properties of an mRNA approach include the high transfection efficiency and absence of maturity induction relative to viral methods.
  • rapid and durable effects can be achieved, including induction of protective T cell populations that sustain tolerance long after expression in DCs is lost, resulting in significant delay, reduced incidence, and even reversal of disease in non-obese diabetic (NOD) mice (Creusot et al., Mo. Ther. 18:2112-2190, 2010).
  • NOD non-obese diabetic
  • This approach adds another clinical safety aspect since mRNA cannot disrupt the genome nor lead to prolonged expression of the encoded nucleic acids, as this may not necessarily be desirable where producing immune stimulation. Production of antigen- specific Tregs that are more suppressive and more stable can be achieved.
  • Changing the mode of antigen presentation may enhance immune responses in cases where destruction of a cell is desired, such as, e.g., cancer, viral and bacterial infections.
  • This mode of antigen delivery would favor efficient priming of CD4+ and CD8+ T cells that target multiple epitopes while enabling cooperation between the activated T cells.
  • the following steps preferably are performed: single or multiple treatments with a nucleic acid of choice expressing TEC with or without other tolerogenic products or administration of a tolerogenic APC modified by a nucleic acid of choice to express TEC with or without other tolerogenic products.
  • the following steps preferably are performed: single or multiple treatments with a nucleic acid of choice expressing TEC with or without adjuvants to enhance immunogenicity or administration of an immunogenic APC modified by a nucleic acid of choice to express TEC and activated with immunogenic compounds such as TLR ligands.
  • immune inhibitory compounds can be coadministered with the tandem epitope construct.
  • Such compounds include, but are not limited to immune inhibiting compound selected from the group consisting of TGF- ⁇ , IL-10, IL-4, IL-27, IL-35, PD-L1, ICOSL, B7-H4 or any combination thereof. These can be coadministered, for example, by administration of the compound itself, or by administration of an mRNA encoding the compound, with appropriate sequences necessary for expression of the compound.
  • inventive constructs and approaches of administering epitopes by engaging multiple pathways in antigen- specific T cells as part of a single encounter with an engineered APC can be used in research to design better cell-based therapies, and can be used to develop rational combinations of small drugs and/or biologies that simultaneously impact these pathways.
  • DC manipulation with mRNA allows for co-expression of additional products, homing molecules that would enhance the in vivo targeting of tolerogenic DCs, or inhibitory molecules that dampen the immune system can be used with the invention, mRNA methods are preferred.
  • T cell receptor transgenic mice such as BDC2.5, BDC12-4.1, NY8.3, G9C8 and G286.
  • Spleen and pooled lymph nodes from these mice are produced into single cell suspensions and antigen- specific CD4+ CD25- or CD8+ T cells are purified and injected into recipient NOD or NOD.Thyl. l mice.
  • the recipient mice are treated on the same day or the day after with TEC or DCs modified by lentiviral vector or mRNA to express TEC.
  • Spleen and lymph nodes can be isolated 3 days later to measure stimulation of adoptively transferred T cell clones, and induction of regulatory T cells expressing Foxp3 or IL-10 for example.
  • antigen-specific T cells reactive to specific epitopes expressed by TECs can be identified by tetramer staining for phenotype analysis. Many such reagents are available, for example from the NIH Tetramer Core Facility.
  • Bone marrow from mice or humans is harvested according to methods well-known in the art, and then depleted of T cells, B cells, and granulocytes by magnetic separation using biotinylated anti-CD3, anti-B220 and anti-Gr-1, and antibiotin microbeads.
  • Blood from human subjects is enriched for monocytes using Ficoll and/or magnetic separation methods.
  • the isolated cells are cultured for approximately 1 week in the presence of complete recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 (mouse or human).
  • GM-CSF mouse granulocyte-macrophage colony-stimulating factor
  • IL-4 mouse or human
  • a viral vector is used to transduce dendritic cells, preferably a multiplicity of infection (MOI; estimated number of. viral particles in suspension) of about 10 MOI to about 100 MOI is used, more preferably an MOI of about 15-20. If transduction proves difficult, an MOI of up to about 200 can be used.
  • MOI multiplicity of infection
  • DNA plasmids (vectors) containing TEC are produced in large scale using combined endotoxin-free maxiprep or gigaprep kits and are solubilized in DNase-free saline.
  • 100 ⁇ g of the plasmids/vectors can be injected per mouse per day intramuscularly (50 ⁇ g per quadriceps).
  • intramuscular injection weekly for 12 weeks
  • a preferred method is to use about 30 ⁇ g mRNA per 5 million cells in a 0.4 cuvette using optimized parameters described by Creusot et al., Mol. Ther., 2010. Cells and mRNA are mixed in serum- free medium, and placed back in complete medium after electroporation. The method is essentially the same between mouse and human dendritic cells, although GMP-grade mRNA must be used for human patients. IVT mRNA, including GMP-grade, can be produced custom-made by commercial entities.
  • the cells In vitro, the cells generally are saturated with mRNA. This usually is accomplished by adding about 30-40 micrograms of mRNA per 5 million cells. As the skilled person in the art is aware, the amount used can be from about 1 ⁇ g to about 40 ⁇ g. Although any known and convenient method can be used, it is typical to electroporate about 1 million cells at a time.
  • Epitopes can be delivered to DCs as a single construct or mRNA molecule with the appropriate targeting signal, and are naturally processed inside the DCs and presented onto MHC class I molecules at least (see FIG. 4A, which shows a construct designed to target for class II processing). Variants of these constructs with leader peptides can be used for their ability to target the polypeptides more efficiently to the endosomes for MHC class II presentation (see FIG. 4B).
  • the leader sequences of these exemplary constructs include fragments from CD 16 and lysosome-associated membrane protein-1 (LAMP-1) combined transferrin receptor (TFR) or invariant chain (Ii). These constructs have been codon-optimized.
  • TCR transgenic T cells or T cell clones can be used to determine the degree of presentation of the epitopes on both MHC class I and class II molecules.
  • stimulation can be assessed by a cell proliferation dye dilution or IL-2 or IFN- ⁇ production.
  • Example 3 Alternative Constructs.
  • FIG. 14 shows results for antigens and antigen-reactive T cells used in the methods discussed here.
  • the stimulation of various TCR-tg (TCR-transgenic) T cells was tested by dilution of a cell proliferation dye in response to the different constructs expressed in DCs.
  • Bone marrow-derived dendritic cells from NOD mice were generated over 7 days in the presence of GM-CSF and IL-4. They were transduced on day 5 with the various antigen-expressing lentiviral constructs shown in FIG. 2 and FIG.6, and sorted (FACS) on day 7, prior to culture with purified antigen- specific TCR-transgenic T cells.
  • TS transferrin receptor
  • Ii invariant chain
  • FIG. 8, 9 and 12 show that CD4+ T cells benefited from their epitopes being targeted to endosomes/MHC class II, particularly with TFR1-118 and Iil-80 (compare DC/NO and DC/NM to the results for targeted epitopes).
  • FIG. 10 and 11 show that CD8+ T cells were less efficiently stimulated if their epitopes were preceded by these targeting signals, suggesting that these signals divert epitopes away from the endogenous proteasome (MHC class I) pathway.
  • BDC2.5 CD4+ T cells responded only to the mimotope (the native epitopes appear to require post-translational modifications, such as that mediated by transglutaminase, to be stimulatory).
  • the Ins2 CD4 mimotope is more efficient than the native Ins2 peptide, however the Ins2 CD8 epitope (B: 15- 23) contained within the Ins2 CD4 epitope (B:9-23) is no longer stimulatory if it is mutated in the mimotope (R22E).
  • Mimo topes can allow better targeting of CD4+ T cells than the native epitopes.
  • ChgA- specific BDC2.5 CD4+ T cells (FIG. 8) and Ins-specific BDC12-4.1 T cells (FIG. 9) were stimulated as described above and evaluated by flow cytometry.
  • BDC2.5 CD4+ T cells do not respond at all to the native epitope (see FIG. 8).
  • BDC12-4.1 T cells respond to both native epitope and mimotope, but better with the latter (not shown).
  • GAD65, chromogranin A and IGRP, known to be targeted in T1D was confirmed.
  • Different endosomal targeting signals for enhanced presentation of CD4-processed epitopes
  • several mimo topes were tested and compared using in vitro stimulation assays. All of the epitopes were shown to be processed and presented to antigen-specific T cells when the construct is introduced into antigen presenting cells. See FIG. 13, which shows responsiveness (dilution of CFSE or equivalent) of diabetogenic T cells to native epitopes (N) or mimotopes (M) presented by transduced bone marrow-derived dendritic cells.
  • Epitopes are expressed with (bottom row) or without (top row) MHC class II targeting (transferrin receptor domain in this case).
  • Four endosome targeting signals were compared in total, all of which enhanced presentation of the targeted epitopes to CD4+ T cells but reduced presentation to CD8+ T cells (see data from 6 th and 7 th column of FIG. 13).
  • CD4 epitopes were more poorly presented in absence of targeting signals, while CD8 epitopes were most efficiently expressed in these conditions.
  • the native insulin CD4 epitope (B:9-23) internally contains a CD8 epitope (B: 15-23).
  • CD4+ T cells respond better while the CD8+ T cells are no longer stimulated.
  • Example 5 Constructs for the Presentation of Epitopes, and Sequences Thereof.
  • Constructs for presentation of multiple epitopes contain an endosome (MHC class retargeting sequence, such as transferrin receptor TFRi.ns, invariant chain Ii 1-8 o or Iil_ 21 4, a plurality of native epitopes or corresponding mimotopes known to be recognized by mouse or human CD4+ T cells downstream of this targeting sequence, a plurality of native epitopes or corresponding mimotopes known to be recognized by mouse or human CD8+ T cells, and a cleavage site, such as P2A, T2A, E2A and F2A, located between the group of CD4- and the group of CD8 epitopes. See FIG. 1 and FIG. 2.
  • MHC class retargeting sequence such as transferrin receptor TFRi.ns, invariant chain Ii 1-8 o or Iil_ 21 4
  • a plurality of native epitopes or corresponding mimotopes known to be recognized by mouse or human CD4+ T cells downstream of this targeting sequence,
  • the CD4 epitopes for MHC class II processing and the CD8 epitopes for MHC class I processing can originate from multiple antigens from a given tissue that is targeted by an autoimmune response, or from multiple antigens from a pathogen or a tumor, and the like, as discussed herein.
  • each epitope (native or mimotope) is flanked by additional amino acids, e.g., 2 amino acids, from the corresponding native protein in order to facilitate processing of the peptide antigens in each targeted cellular compartment.
  • the construct IET4T (675 bp) is an exemplary construct according to the invention. A schematic diagram of the construct is given in FIG. 2.
  • This "Islet Epitope Tandem" construct contains a transferrin receptor (TFR) signal (IET4T) and expresses mimo topes for an insulin CD4 epitope and a chromogranin A CD4 epitope, a native GAD65 CD4 epitope, and native insulin and IGRP CD8 epitopes. All these epitopes are recognized by known diabetogenic T cells, and therefore can target them for reprogramming.
  • TFR transferrin receptor
  • IET4T transferrin receptor
  • T2A cleavage site 529-582
  • MDDQRDLIS NHEQLPILGNRPREPERCS RG ALYTG VS VLV ALLLAGQ ATTA YFLYQQQG RLDKLTITS QNLQLES LRMKL (SEQ ID NO: 14)
  • MDDQRDLIS NHEQLPILGNRPREPERCS RG ALYTG VS VLV ALLLAGQ ATTA YFLYQQQG RLDKLTITSQNLQLESLRMKLPKSAKPVSQMRMATPLLMRPMSMDNMLLGPVKNVTK YGNMTQDHVMHLLTRSGPLEYPQLKGTFPENLKHLKNSMDGVNWKIFESWMKQWLLF EMS KNS LEEKKPTE APPKEPLDMEDLS S GLG VTRQELGQ VT (SEQ ID NO: 16)
  • KRWSRMDQLAKELTAEKR (SEQ ID NO: 28; epitope underlined)
  • KRAVRPLWVRMEKR (SEQ ID NO: 30; epitope underlined)
  • ATNFS LLKQ AGD VEENPGP (SEQ ID NO: 36)
  • T2A (from Thosea asigna virus)
  • E2A (from equine rhinitis A virus)
  • F2A (from Foot-and-mouth disease virus)
  • the constructs indicated in FIG 14 were delivered into DCs by lenti viral transduction and co-cultured with several purified diabetogenic clones labeled with a proliferation dye.
  • NO indicates that all the expressed epitopes are native epitopes
  • NM indicates that some of the native epitopes are replaced with mimo topes when available (Ins and ChgA).
  • the construct labeled TFR have the transferrin receptor 1-118 targeting signal.
  • Constructs labeled NO and NM do not have the 2A cleavage site, whereas IET4T (depicted on FIG.2) has both a targeting signal and a cleavage site. Beads coated with anti-CD3 and anti-CD28 were used as positive control ("Beads"); DC control indicates no transduction.
  • TFR1-118 See FIG. 14.
  • CD8+ T cells the response was diminished with targeting signals, indicating the need to segregate CD8 epitopes such that they are not targeted to endosomes with
  • FIG. 15 is a schematic drawing of additional nucleic acid constructs.
  • Construct IET5 contains more epitopes and mimotopes for extended coverage. Although more epitopes can be added, these were chosen because tools and reagents exist to assess the T cell response to these epitopes in vivo, either through adoptively transferred T cell receptor transgenic T cells or tetramer reagents.
  • An example of identification of diabetogenic T cells by tetramer staining and their response to dendritic cells expressing TEC (IET4T in this case) is shown on FIG.16.
  • These TECs will also be cloned into the DNA vaccine vector BHT-568, which has been engineered to promote tolerance rather than immunity by substitution of CpG by GpG motifs.
  • Example 8 Stimulation of BDC2.5 CD4+ T Cells with DCs Electroporated with IET4T mRNA, with or without TGF- ⁇ mRNA.
  • IET4T (with TFR signal) mRNA was combined with codon-optimized TGF- ⁇ mRNA to electroporate DCs (5 ⁇ g IET4T mRNA/5xl0 6 DCs with or without TGF- ⁇ mRNA 20 ug/5xl0 6 DCs). Electroporated DCs were used to stimulate BDC2.5 CD4+ CD25- GFP-Foxp3 T cells in vitro and in vivo (after adoptive transfer of lxlO 6 T cells/mouse i.p.). See FIG. 16.
  • CD4+ CD25- T cells were isolated from BDC2.5.GFP-Foxp3 mice, labeled with Violet Cell Proliferation Dye (VCPD) and stimulated for 3 days in vitro + exogenous TGF- ⁇ (FIG. 17A lower panels and FIG. 17C) or in vivo (in NOD. Thy 1.1 mice) (FIG. 17A upper panels and 17B) with control or electroporated DCs (eDCs).
  • Panels A-C show proliferation
  • panels D-E show GFP-Foxp3 induction (gated on divided cells, as non-stimulated cells do not upregulate Foxp3). Data shown are from gated CD4+ VCPD+ T cells. Arrows in FIG.
  • T cells are injected in congenic NOD.Thyl. l female mice (lxl0 6 /clone/mouse intravenously, 5 mice per group) one day before inoculation of electroporated DCs (lxl0 6 /mouse intraperitoneally, which target DCs to the PLNs). After ⁇ 3 days, PLNs and inguinal lymph nodes (no stimulation control) are collected. Transferred T cells are identified as CD4+ Thy 1.2+ and ⁇ 2+ (BDC12-4.1) or ⁇ 4+ (BDC2.5). DCs electroporated with GFP mRNA (no antigen) or with TEC mRNA only are used for control groups.
  • Induction of GFP-Foxp3, CTLA-4, PD-1, ICOS and Nrp-1 is measured on day 3 and compared to baseline levels on day 0.
  • Expression of IL-10 and LAP/TGF- ⁇ also is measured in T cells on day 3 by intracellular staining. The goal of these assays is to identify combinations that induce the highest frequency of Foxp3+, IL-10+ and/or TFG- ⁇ - ⁇ - T cells.
  • the response of endogenous diabetogenic CD4+ T cells is assessed, using I-A g7 tetramers with Ins or ChgA mimo topes or GAD65 2 86-3oo peptide (FIG.16).
  • Example 10 Assays for T Cell Suppressive Function and Stability.
  • Treg stability in vitro using scaled up cultures for "Treg-Demethylated Region” assays (using EpigenDx or Epiontis platform).
  • Treg-Demethylated Region assays (using EpigenDx or Epiontis platform).
  • CD8+ T cells from NY8.3 mice, which are readily stimulated by endogenous DCs in the PLNs and by TEC constructs. If injected DCs induce functional Tregs, the overall proliferation of CD4+ and CD8+ T cells in the PLNs is expected to be reduced (as seen in FIG. 17A-B).
  • in vitro-activated BDC2.5 or NY8.3 T cells are transferred two weeks after initial DC treatment and then glycemia is measured weekly to determine whether induced Tregs can delay or block the subsequent induction of disease by the in vitro activated diabetogenic T cells (transferred DCs are no longer present by then).
  • Example 11 Functional Tolerance of Diabetogenic T cells by DNA vaccine.
  • T cell receptor-transgenic (TCR-tg) T cells CD4+ or CD8+
  • T cell receptor-transgenic (CD4+ or CD8+) are used as traceable antigen-specific T cells reactive to specific ⁇ -cell antigens after transfer into congenic NOD.Thyl.l mice, or responding antigen- specific T cells can be identified by tetramer staining (FIG.16).
  • mice Two cohorts of mice are used to reflect the most challenging conditions for prevention and treatment of T ID in this model, either advanced peri-insulitis or overt hyperglycemia.
  • a first cohort of female NOD mice (12 weeks of age and euglycemic) are divided into groups according to the DNA vector tested: mice receive either saline (group a), BHT-568 with Prolns only (group b), a mix of BHT-568 expressing Prolns, ChgA, GAD65 and IGRP (25% of total mass each) (group c), or BHT-568 expressing the IET4 construct (FIG.
  • mice In a second cohort of mice (monitored for glycemia starting at 10 weeks of age), each mouse is treated as soon as it reaches blood glucose levels between 250 and 350mg/ml with one of the above treatment weekly for 8 weeks until at least 10 mice in each group have been treated. Glycemia will be measured weekly up to 30 weeks of age and mice with glycemia over 400 mg/dl on two consecutive measurements (3 days apart) will be euthanized. If significant protection is observed (Log Rank survival test applied), additional mice will be treated in order to compare the degree of islet infiltration by histology (H&E staining).
  • Example 13 Incidence of Disease after Tolerogenic DNA Vaccine Treatment.
  • tolerogenic DNA vaccine also called a reverse DNA vaccine
  • the efficacy of various DNA vaccine treatments in preventing the onset of T1D in NOD mice treated during early or late stage of disease (5 versus 10 weeks of age) is assessed. In both cases, mice are treated for 8 weeks and monitored weekly for glycemia.
  • mice are used to reflect either an early or advanced stage of peri-insulitis.
  • Glycemia is monitored every 2 weeks from 6 to 10 weeks, and then weekly thereafter up to 30 weeks of age (Expt 2A).
  • Bone marrow-derived DCs (CD11C+ CD1 lb+) from female NOD mice are generated over 6 days in the presence of GM-CSF and IL-4 and used for electroporation with several mRNA constructs. All mRNA reagents are custom-made by TriLink Biotechnologies and are codon-optimized to improve translation and modified with 5-methylcytidine and pseudo-uridine to reduce immune stimulation.
  • the amount of mRNA used, per 5xl0 6 DCs, will vary between 1 and 5 ⁇ g for antigen mRNA (intracellular IET5, see FIG.
  • Modified DCs then are injected intravenously or intraperitoneally (10 6 /mouse) into recipient NOD.Thyl. l mice that will have received an intravenous transfer of labeled TCR-tg T cells as described in Example 6.
  • n>5/group for each type of tolerogenic DC are compared: (al) saline, (bl) eDC/GFP mRNA control, (cl) eDCs/IET5 mRNA and (dl) non-treated (non- tolerogenic) eDC/IET5.
  • the conditions are: (a2) saline, (b2) eDC/GFP, (c2) eDC/IET5, (d2) eDC/IET5/TGF-p/IL-2, (e2) eDC/IL-10, (f2) eDC/PD-Ll and (g2) a mixture of (d2+e2+f2).
  • the response and phenotype of antigen- specific T cells 3 days or 2 weeks later is analyzed as described in Example E.
  • Treg induction is determined by intracellular staining of Foxp3 and IL-10, and if significant, the suppressive function is confirmed by transfer of splenocytes and lymph node cells from treated mice into NOD.SCID mice. Deletion is assessed by disappearance of antigen- specific T cells after two weeks, following possible expansion on day 3 post-treatment. Anergy is assessed by production of IL-2 upon restimulation ex vivo with specific peptides on both time points.
  • Example 15 Therapeutic Efficacy of Antigen-presenting Tolerogenic DCs.
  • the two cohorts of NOD mice described in Example 8 are used for prevention and treatment (reversal) of diabetes. Because monitoring of treated mice is a lengthy process (from 10 to 30 weeks of age), only groups of DCs that show promising results in their mechanisms of action, along with controls (saline, eDC/GFP, eDC/IET5) are tested.
  • For prevention euglycemic 12-wk old female NOD mice are treated with a single intravenous injection of 10 6 DCs (of one type or a mixture).
  • aliquots of 2xl0 6 DCs for each group are prepared and frozen. Mice are followed weekly for glycemia and treated with thawed DCs upon onset of hyperglycemia (250-350 mg/ml). The animals are monitored weekly for glucose blood levels until 30 weeks of age.
  • Example 16 Combination Strategies for TECs with Tolerogenic Signals.
  • Antigen/cytokine mRNA is compared with expression of cell surface ligands (PD-L1, B7-H4, ICOS-L, Sema4A) that have also been implicated in the induction or boosting of Tregs (PD-L1 and Sema4A for Foxp3 Tregs through PD-1 and Nrp-1, ICOS-L and B7-H4 for Trl through ICOS and an unknown receptor).
  • PD-L1, B7-H4, ICOS-L, Sema4A cell surface ligands
  • Tregs PD-L1 and Sema4A for Foxp3 Tregs through PD-1 and Nrp-1
  • ICOS-L and B7-H4 for Trl through ICOS and an unknown receptor.
  • the TEC expressing the aforementioned epitopes and targeting sequences are synthesized after codon optimization for higher expression levels. These nucleic acids then are in vitro- transcribed into mRNA with antireverse cap analog and polyadenylation for enhanced stability.
  • the DNA constructs for certain genes IL-10, IL-27, IL-35 and Sema4A have already been synthesized.
  • IL-27 and IL-35 are composed of two chains (IL27p28/EBI3 and IL12p35/EBI3 respectively), which are expressed on the same mRNA molecule with a cleavable P2A site between the two chains.
  • the level of expression in DCs is assessed with or without overexpression by mRNA (up to 30 ⁇ g per 5xl0 6 DCs), and the duration of expression over multiple time points (ranging from 4 to 72h) by flow cytometry or ELISA. If "overexpressed" levels are not substantially increased over background, the DCs are considered sufficient for this product.
  • nonobese diabetic mice J. Exp. Med. 198:63-69, 2003.
  • Boks et al., IL-10-generated tolerogenic dendritic cells are optimal for functional regulatory T cell induction— a comparative study of human clinical-applicable DC. Clin. Immunol.
  • CXC chemokine ligand (CXCL) 10 IFN-gamma-inducible protein of 10 kDa
  • CXCL9 IFN-gamma-inducible protein of 10 kDa
  • CXCL9 monokine induced by IFN-gamma
  • Creusot et al. A short pulse of IL-4 delivered by DCs electroporated with modified mRNA can both prevent and treat autoimmune diabetes in NOD mice. Mol. Ther. 18:2112-2120, 2010.
  • Diabetogenic T-cell clones recognize an altered peptide of chromogranin A.
  • PD-L1 regulates the development, maintenance, and function of induced
  • Chromogranin A is a T cell antigen in human type 1 diabetes. J. Autoimmun.
  • Skin-draining lymph nodes contain dermis-derived CD103(-) dendritic cells that constitutively produce retinoic acid and induce Foxp3(+) regulatory T cells.
  • Gurr et al., Regll is a beta-cell protein and autoantigen in diabetes of NOD mice. Diabetes.
  • CCR9 expression defines tolerogenic plasmacytoid dendritic cells able to
  • Plasmacytoid dendritic cells transport peripheral antigens to the thymus to
  • T-cell receptor mice develop autoimmune diabetes dependent upon RAG genotype, H-2g7 homozygosity, and insulin 2 gene knockout. Diabetes 55: 1978-1984, 2006.
  • Kang et al. De novo induction of antigen-specific CD4+CD25+Foxp3+ regulatory T cells in vivo following systemic antigen administration accompanied by blockade of mTOR. J.
  • the antimicrobial peptide LL37 is a T-cell autoantigen in psoriasis. Nat Commun.
  • PD-L2 is a second ligand for PD-1 and inhibits T cell activation. Nat. Immunol.
  • Nayak et al., ZnT8-reactive T cells are weakly pathogenic in NOD mice but can participate in diabetes under inflammatory conditions. Diabetes. 63:3438-3448, 2014.
  • Pen et al. Modulation of regulatory T cell function by monocyte-derived dendritic cells matured through electroporation with mRNA encoding CD40 ligand, constitutively active TLR4, and CD70. J. Immunol. 191: 1976-1983, 2013. Peng et al., Dendritic cells transfected with PD-L1 recombinant adenovirus induces T cell suppression and long-term acceptance of allograft transplantation. Cell. Immunol. 271:73-77, 2011.
  • CD4+CD25+ regulatory T cells during autoimmune diabetes J. Exp. Med. 201: 1333-1346, 2005.
  • Robert et al. Gene therapy to target dendritic cells from blood to lymph nodes. Gene Ther.
  • Citrullinated glucose-regulated protein 78 is an autoantigen in type 1 diabetes.
  • Van Lint et al. mRNA: From a chemical blueprint for protein production to an off-the-shelf therapeutic. Hum. Vaccines Immunother. 9:265-274, 2013. van Lummel et al., Changing faces, unmasking the beta-cell: post-translational modification of antigens in type 1 diabetes. Curr. Opin. Endocrinol. Diabetes Obesity 20:299-306 (2013).
  • Van Meirvenne et al. Efficient genetic modification of murine dendritic cells by electroporation with mRNA. Cancer Gene Ther. 9:787-797, 2002.
  • Verheijden et al. Human cartilage glycoprotein-39 as a candidate autoantigen in rheumatoid arthritis. Arthritis Rheum. 40: 1115-1125, 1997.
  • Neuropilin- 1 distinguishes natural and inducible regulatory T cells among

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Abstract

L'invention concerne des constructions, cellules et méthodes destinées à moduler le système immunitaire qui permettent d'optimiser la présentation des épitopes CD4 et CD8 à des cellules présentatrices d'antigène. Des épitopes soit d'auto-antigènes, soit d'antigènes exogènes peuvent être utilisés pour optimiser soit la tolérance envers ces épitope, soit l'immunogénicité de ces épitopes, respectivement. Les nouvelles constructions, appelées constructions d'épitopes en tandem (TEC, pour tandem epitope constructs), codent pour un ou plusieurs épitopes (CD4) dominants liés à une maladie, ciblés pour une prise en charge par le CMH de classe II dans les endosomes des cellules, et un ou plusieurs épitopes (CD8) ciblés pour une prise en charge par le CMH de classe I dans le cytosol des cellules, afin d'obtenir une présentation antigène/épitope maximale au sein du système immunitaire. Les deux types d'épitopes (CD4 et CD8) sont séparés l'un de l'autre sur la construction et regroupés pour un traitement optimal dans la cellule.
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US10882891B2 (en) 2015-12-23 2021-01-05 Medigene Immunotherapies Gmbh Dendritic cell composition
US11466278B2 (en) 2016-11-01 2022-10-11 Novo Nordisk A/S Temperature based plasmid regulation system
JP2020062057A (ja) * 2016-11-01 2020-04-23 ノヴォ ノルディスク アー／エス 免疫寛容誘導性ｄｎａワクチン
TWI728201B (zh) * 2016-11-01 2021-05-21 丹麥商諾佛 儂迪克股份有限公司 耐受性ｄｎａ疫苗
CN109922827A (zh) * 2016-11-01 2019-06-21 诺和诺德股份有限公司 致耐受性dna疫苗
CN109922827B (zh) * 2016-11-01 2024-03-01 诺和诺德股份有限公司 致耐受性dna疫苗
RU2752608C2 (ru) * 2016-11-01 2021-07-29 Ново Нордиск А/С Толерогенная днк-вакцина
EP3842442A1 (fr) 2016-12-22 2021-06-30 Calithera Biosciences, Inc. Compositions et procédés pour inhiber l'activité d'arginase
WO2018119440A1 (fr) 2016-12-22 2018-06-28 Calithera Biosciences, Inc. Compositions et procédés pour inhiber l'activité de l'arginase
US11279745B2 (en) * 2019-04-26 2022-03-22 Novo Nordisk A/S Tolerogenic DNA vaccine
CN112231987A (zh) * 2020-11-04 2021-01-15 华东交通大学 一种基于VMD与Elman神经网络的电离层预报方法
CN112231987B (zh) * 2020-11-04 2022-05-17 华东交通大学 一种基于VMD与Elman神经网络的电离层预报方法
CN112843255A (zh) * 2021-03-18 2021-05-28 华中科技大学同济医学院附属同济医院 Sema4c在制备抗肿瘤药物中的应用

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