US20190248867A1 - Myelin oligodendrocyte glycoprotein-specific peptide for the treatment or prevention of multiple sclerosis - Google Patents
Myelin oligodendrocyte glycoprotein-specific peptide for the treatment or prevention of multiple sclerosis Download PDFInfo
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- US20190248867A1 US20190248867A1 US16/314,642 US201716314642A US2019248867A1 US 20190248867 A1 US20190248867 A1 US 20190248867A1 US 201716314642 A US201716314642 A US 201716314642A US 2019248867 A1 US2019248867 A1 US 2019248867A1
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Definitions
- MS Multiple sclerosis
- CNS central nervous system
- MS autoimmune animal models experimental autoimmune encephalomyelitis, or EAE
- EAE autoimmune encephalomyelitis
- a composition for treatment of multiple sclerosis comprising: a peptide comprising a partial amino acid sequence of myelin oligodendrocyte glycoprotein, wherein the peptide activates a subset of immune cells.
- a composition is provided wherein the activated immune cells comprise HLA-E-restricted regulatory CD8+ T cells.
- a composition is provided wherein the peptide comprises a MOG-specific MHC 1b epitope.
- a method for treating multiple sclerosis comprising: administering a peptide comprising a partial amino acid sequence of myelin oligodendrocyte glycoprotein; and activating a subset of immune cells, wherein the immune cells comprise HLA-E-restricted regulatory CD8+ T cells.
- a method is provided wherein the peptide comprises a MOG-specific MHC 1b epitope.
- compositions for the treatment or prevention of multiple sclerosis comprise an isolated peptide comprising a partial amino acid sequence of a myelin oligodendrocyte glycoprotein (MOG) protein, wherein the peptide activates regulatory T cells.
- the composition comprises dendritic cells pulsed with a myelin oligodendrocyte glycoprotein (MOG) peptide that activates regulatory T cells.
- the peptide comprises a MOG-specific MHC1b epitope. In some embodiments, the peptide comprises a HLA-E epitope. In some embodiments, the peptide comprises the amino acid sequence IICYNWLHR. In some embodiments, the peptide consists of the amino acid sequence IICYNWLHR.
- the peptide activates HLA-E-restricted regulatory CD8 + T cells.
- the dendritic cells are derived from myelin-specific pathogenic autoimmune cells. In some embodiments, the dendritic cells are derived from monocytes.
- methods of treating or preventing a demyelinating disease comprises administering to a subject a composition as disclosed herein (e.g., an isolated peptide comprising a partial amino acid sequence of MOG (e.g., a peptide comprising or consisting of the amino acid sequence IICYNWLHR), a composition comprising an isolated peptide comprising a partial amino acid sequence of MOG, or a composition comprising dendritic cells pulsed with an isolated peptide comprising a partial amino acid sequence of MOG).
- methods of treating the demyelinating disease are provided.
- methods of preventing or delaying the onset of the demyelinating disease are provided.
- the demyelinating disease is selected from the group consisting of multiple sclerosis, idiopathic inflammatory demyelinating disease, transverse myelitis, Devic's disease, progressive multifocal leukoencephalopathy, optic neuritis, leukoystrophy, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, autoimmune peripheral neuropathy, Charcot-Marie-Tooth disease, acute disseminated encephalomyelitis, adrenoleukodystrophy, adrenomyeloneuropathy, Leber's hereditary optic neuropathy, or HTLV-associated myelopathy.
- the demyelinating disease is multiple sclerosis.
- the composition is administered subcutaneously or intravenously.
- the method comprises administering to a subject having multiple sclerosis a composition comprising dendritic cells pulsed with a myelin oligodendrocyte glycoprotein (MOG) peptide that activates HLA-E-restricted regulatory CD8 + T cells.
- MOG myelin oligodendrocyte glycoprotein
- the peptide comprises or consists of the amino acid sequence IICYNWLHR.
- the dendritic cells are derived from monocytes.
- the dendritic cells are derived from monocytes that are autologous to the subject.
- the dendritic cells are derived from monocytes that are allogeneic to the subject.
- FIG. 1A-1C Portion of CD8+ T cells reactive to the pool of overlapping peptides (OLPs) covering the whole length of mouse MOG was Qa-1 b restricted.
- OLPs overlapping peptides
- FIG. 1A-1C A schematic view of the mouse MOG OLP library. 15mer peptides across the whole length of mouse MOG (247 amino acids) and overlapped by 11 amino acids were synthesized. A pool of the 59 OLPs (“MOG_pool”) at a concentration of 4.2 ⁇ g/ml for each peptide was then generated for stimulating CD8 + T cells purified from K b ⁇ / ⁇ D b ⁇ / ⁇ mice.
- FIG. 2A-2F Recognition of multiple OLPs by the MOG_pool-reactive CD8 + T cell lines depended on Qa-1 b .
- A-D MOG_pool-reactive CD8 + T cell lines were generated as described in FIG. 1 . The 59 individual OLPs were interrogated, using IFN- ⁇ Enzyme-linked ImmunoSpot, for their ability to stimulate the MOG_pool-reactive CD8 + T cell lines in the presence of either C1R or C1R.Qa-1 b cells.
- A 96-well plate layout of the 59 individual OLPs. Numbers in the wells represent identification codes (IDs) for the 59 individual OLPs, i.e., 49-107 from N to C termini.
- IDs identification codes
- FIG. 3 Fine mapping of the minimal and optimal Qa-1 b epitope in OLP105.
- Progressively N- and C-terminally truncated OLP105 peptides were synthesized and interrogated for their ability to stimulate, using IFN- ⁇ Enzyme-linked ImmunoSpot, an OLP105-reactive CD8 + T cell line in the presence of C1R.
- Qa-1 b cells IDs and sequences of the truncated peptides are shown in the left and middle panels, respectively.
- Corresponding bars in the right panel show IFN- ⁇ spot-forming cells (SFCs) per 10 6 CD8 + T cells in response to corresponding peptides.
- SFCs spot-forming cells
- FIG. 4A-4D MOG 196 can bind to Qa-1 b and stimulate MOG 196 -specific Qa-1 b -restricted CD8 + T cells.
- MOG 196 was incubated with recombinant Qa-1 b and ⁇ 2 microglobulin in a protein refolding buffer at 10° C. under gentle agitation (60 rpm) for four days. The solution was separated by a size exclusion column. Peak “A” and “B” represented typical non-specific protein aggregates and correctly refolded MOG 196 /Qa-1 b monomer, respectively.
- Monomers in peak B of panel “A” were further analyzed by an anion exchange chromatography.
- CD8 + T cells were purified from na ⁇ ve C57Bl/6 (B6) mice, individually stimulated weekly with untreated (no peptide) or MOG 196 -pulsed, either B6 (upper panels) or K b ⁇ / ⁇ D b ⁇ / ⁇ (lower panels) macrophages.
- the CD8 + T cells were analyzed for binding to Qa-1 b /MOG 196 tetramer at days 0, 7, and 14. One representative data on day 14 from four individual experiments was shown.
- FIG. 5A-5D Immunization with MOG 196 -pulsed DCs suppressed MOG 35-55 -induced EAE.
- A Experimental design for “B”: C57BL/6 mice were immunized with MOG 35-55 for EAE. One week before and after the EAE induction, the animals received one of the following subcutaneous treatments: 1) no treatment (No Tx); 2) 1 ⁇ 10 6 Qdm-pulsed K b ⁇ / ⁇ D b ⁇ / ⁇ DCs (DC/Qdm); 3) 1 ⁇ 10 6 MOG 196 -pulsed K b ⁇ / ⁇ D b ⁇ / ⁇ DCs (DC/MOG 196 ). The mice were then monitored for paralytic disease daily.
- FIG. 6A-6B Immunization with MOG 196 -pulsed DCs suppressed ongoing MOG 35-55 -induced EAE, which was dependent on CD8 + T cells.
- A Experimental design for “B”: C57BL/6 mice were immunized with MOG 35-55 for EAE on day 0. In one group, the animals were intraperitoneally injected with a monoclonal depleting anti-CD8 antibody (mAb) at days ⁇ 2, ⁇ 1, 7, and 14. At day 10, animals received either no treatment (No Tx) or one intravenous injection of mitomycin C-treated C57BL/6 DCs pulsed with MOG 196 (DC/MOG 196 ). Paralytic disease was monitored daily.
- FIG. 7A-7G Immunization with DCs pulsed with MOG 196 activates Qa-1 b /MOG 196 tetramer + cells that specifically accumulate in cervical lymph nodes.
- C57BL/6 mice (5 mice/group) were immunized with MOG 35-55 for EAE.
- animals received one intravenous immunization with mitomycin C-treated DC2.4 or MOG 196 -pulsed DC2.4 (DC2.4/MOG 196 ).
- mononuclear cells prepared from spleens and cervical lymph nodes were examined for the presence of Qa-1 b /MOG 196 and I-A b /MOG 38-49 tetramer + cells by flow cytometry.
- FIG. 8 MOG 196 sequence is conserved across species.
- the data show an alignment of the sequences surrounding MOG 196 in four different species. “m”: mice; “r”: rats; “C Jacchus”: Callithrix jacchus ; “h”: humans.
- the 9-mer peptide sequence IICYNWLHR is underlined.
- FIG. 9 MOG 196 sequence is located in the intracellular domain of myelin oligodendrocyte glycoprotein (MOG).
- MOG 35-55 The three highlighted extracellular epitopes, MOG 35-55 , MOG 1-22 , and MOG 92-106 , are encephalogenic (or pathogenic) epitopes.
- the intracellular MOG 196 epitope is a regulatory (or protective) Qa-1 b epitope.
- FIG. 10 A model of immune regulation mediated by myelin-specific, Qa-1-restricted CD8 + Treg.
- Myelin-specific, Qa-1-restricted CD8 + Treg cells can recognize and tolerize/eliminate antigen-presenting cells (APCs) that otherwise activate myelin-specific encephalitogenic T cells in the CNS and/or peripheral lymphoid tissues.
- APCs antigen-presenting cells
- Tolerization/elimination of APCs which present myelin epitopes, is mediated by regulatory cytokines (CKs), inhibitory molecules, or direct cytotoxicity. Consequently, activation of myelin-specific encephalitogenic T cells and autoimmune attacks of myelin sheath are thwarted.
- CKs regulatory cytokines
- MS Multiple sclerosis
- CNS central nervous system
- antigen-specific therapy See, Clifford et al., The Lancet Neurology, 2010, 9:438-446; Linda et al., The New England Journal of Medicine, 2009, 361:1081-187.
- antigen-specific therapy See, e.g., Steinman, Mult Scler, 2015, 21:1223-1238; Lutterotti et al., Journal of the Neurological Sciences, 2008, 274:18-22.
- the goal of antigen-specific therapy is to specifically delete, anergize, or deviate myelin-specific pathogenic autoimmune cells that are responsible for MS.
- Treg regulatory T cells
- HLA-E a group of non-classical MHC Ib molecules
- HLA-E-restricted CD8 + T cells another subset of immune cells, are altered or deficient in MS patients. See, e.g., Ben-Nun et al., J. Autoimmun., 2014, 54:33-50. The data therefore indicate that HLA-E-restricted CD8+ T cells contain a subset of Treg (referred to herein as HLA-E-restricted CD8 + Treg).
- HLA-E-restricted CD8 + Treg may be involved in the prevention and/or therapy of MS, and this has been further supported by data gathered from studies of Qa-1-restricted CD8 + Treg (the murine homologue of human HLA-E-restricted CD8 + Treg).
- Qa-1-restricted CD8 + Treg may maintain immune homeostasis under physiological condition.
- the CD8 + Treg recognize specific peptides (also called regulatory Qa-1 epitopes) on and thereby target autoreactive T cells (i.e., the immune cell subset that causes autoimmune diseases).
- the first limitation is that CD8 + Treg activated by current strategies would mainly target autoreactive T cells in the peripheral lymphoid organs and may not have the capacity to specifically accumulate in the diseased CNS. Consequently, efficacy of current strategies may be compromised.
- the second limitation is that the CD8 + Treg's targets in human, i.e., regulatory HLA-E epitopes expressed in autoreactive T cells, can be difficult to determine in patients. Therefore, current strategies are difficult to implement in clinics.
- a regulatory Qa-1 (HLA-E) epitope is disclosed.
- the Qa-1 epitope is a “MOG-specific MHC1b epitope” (or “MSIBE”), that is specifically located in MOG, a protein located in myelin sheath that is attacked by the immune system in EAE.
- MSIBE MOG-specific MHC1b epitope
- immunization with MSIBE but not non-MOG Qa-1 epitopes suppresses EAE.
- data presented herein demonstrate that Qa-1-restricted CD8 + Treg cells activated by MSIBE immunization are involved in efficient suppression of EAE.
- MSIBE Because of MSIBE's unique location in MOG, an autoantigen that is the ultimate target of autoreactive T cells in EAE and in MS, Qa-1-restricted (or HLA-E-restricted) CD8 + Treg activated by MSIBE may specifically accumulate at and arrest the autoimmune attacks on myelin sheath in EAE and in MS. Thus, in some embodiments, MSIBE can be used as a peptide vaccine for specific prevention and therapy of MS.
- a 9mer peptide that is specifically located in myelin oligodendrocyte glycoprotein (MOG), a major autoantigen in multiple sclerosis (MS), is provided.
- the epitope has the amino acid sequence IICYNWLHR.
- the MSIBE epitope can be used for activating a subset of immune cells, specifically HLA-E (Qa-1 in mouse)-restricted regulatory CD8 + T cells (also referred to herein as “HLA-E (Qa-1)-restricted CD8 + Treg”) for the specific prevention and therapy of MS.
- MOG myelin oligodendrocyte glycoprotein
- a MOG protein is a human MOG protein.
- Human MOG is known to have alternatively spliced transcript variants. Sequences for human MOG mRNA are set forth in, e.g., NCBI GenBank Accession Nos.
- Sequences for human MOG protein are set forth in, e.g., NCBI GenBank Accession Nos. NP_001008229.1, NP_001008230.1, NP_001163889.1, NP_002424.3, NP_996532.2, NP_996533.2, NP_996534.2, NP_996535.2, and NP_996537.3.
- a MOG protein or peptide is a homolog or ortholog of a human sequence disclosed herein (e.g., a mouse, rat, cynomolgus monkey, or marmoset ( C. jacchus ) form of MOG or a peptide thereof).
- a MOG protein is a mouse MOG protein.
- the sequence for mouse MOG protein is set forth in, e.g., NCBI GenBank Accession No. NP_034944.2.
- MOG-specific MHC1b epitope refers to an epitope in myelin oligodendrocyte glycoprotein (MOG), e.g., human MOG or mouse MOG, that binds to a non-classical major histocompatibility complex 1b (MHC1b) molecule.
- MOG myelin oligodendrocyte glycoprotein
- MHC1b non-classical major histocompatibility complex 1b
- the MHC1b molecule is the antigen HLA-E (in humans) or Qa-1 (in mice).
- epitope refers to an area or region of a protein that specifically binds to an antigen (e.g., a MHC1b molecule, e.g., HLA-E or Qa-1), and can include a few amino acids, e.g., 5, 6, 7, 8, 9, 10, 11 or more amino acids.
- the epitope is comprised of consecutive amino acids of a protein (e.g., 5, 6, 7, 8, 9, 10, 11 or more consecutive amino acids).
- epitope refers to a peptide (e.g., an isolated peptide) comprising or consisting of the area or region of the protein that specifically binds to the antigen.
- the term “specifically binds” refers to a molecule (e.g., a peptide) that binds to a target (e.g., an antigen) with greater affinity, greater avidity, and/or greater duration to that target in a sample than it binds to another non-target compound (e.g., a structurally different antigen).
- the molecule e.g., peptide
- the target e.g., antigen
- the target e.g., antigen
- the target e.g., antigen
- at least 3-fold greater affinity than other non-target compounds e.g., at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold greater affinity.
- isolated denotes that the protein or peptide is essentially free of other cellular components with which it is associated in the natural state. It is preferably in a homogeneous state. Purity and homogeneity are typically determined using analytical chemistry techniques such as electrophoresis (e.g., polyacrylamide gel electrophoresis) or chromatography (e.g., high performance liquid chromatography). In some embodiments, an isolated protein or peptide is at least 85% pure, at least 90% pure, at least 95% pure, or at least 99% pure.
- demyelinating disease refers to a disease or condition of the nervous system characterized by damage to or loss of the myelin sheath of neurons.
- a demyelinating disease can be a disease affecting the central nervous system or a disease affecting the peripheral nervous system.
- demyelinating diseases include, but are not limited to, multiple sclerosis, idiopathic inflammatory demyelinating disease, transverse myelitis, Devic's disease, progressive multifocal leukoencephalopathy, optic neuritis, leukoystrophy, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, autoimmune peripheral neuropathy, Charcot-Marie-Tooth disease, acute disseminated encephalomyelitis, adrenoleukodystrophy, adrenomyeloneuropathy, Leber's hereditary optic neuropathy, or HTLV-associated myelopathy.
- the demyelinating disease is multiple sclerosis.
- the term “subject” or “patient” refers to a human or a non-human mammal, e.g., a dog, a cat, a mouse, a rat, a cow, a sheep, a pig, a goat, or a non-human primate, or a bird, e.g., a chicken, or any other vertebrate or invertebrate animal.
- treatment refers to administering a compound or pharmaceutical composition to a subject for prophylactic and/or therapeutic purposes.
- prophylactic treatment refers to treating a subject who does not yet exhibit symptoms of a disease or condition, but who is susceptible to, or otherwise at risk of, a particular disease or condition, whereby the treatment reduces the likelihood that the patient will develop the disease or condition.
- therapeutic treatment refers to administering treatment to a subject already having a disease or condition.
- an “effective amount” or a “therapeutically effective amount” refers to an amount of a therapeutic agent that is effective to relieve, to some extent, or to reduce the likelihood of onset of, one or more symptoms of a disease or condition (e.g., multiple sclerosis) and includes curing the disease or condition.
- a prophylactically effective amount refers to an amount that is effective to prevent or delay the onset of one or more symptoms of a disease or condition (e.g., multiple sclerosis), or otherwise reduce the severity of said one or more symptoms, when administered to a subject who does not yet exhibit symptoms of a disease or condition, but who is susceptible to, or otherwise at risk of, a particular disease or condition.
- a therapeutically effective amount will show an increase of therapeutic effect of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%.
- Therapeutic efficacy can also be expressed as “fold” increase or decrease.
- a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a control.
- administer refers to introducing an agent into a subject or patient, such as a human.
- the terms encompass both direct administration, (e.g., self-administration or administration to a patient by a medical professional) and indirect administration (e.g., the act of prescribing a compound or composition to a subject).
- composition refers to a composition suitable for administration to a subject.
- a pharmaceutical composition is sterile, and preferably free of contaminants that are capable of eliciting an undesirable response with the subject.
- Pharmaceutical compositions can be designed for administration to subjects in need thereof via a number of different routes of administration, including oral, intravenous, buccal, rectal, parenteral, intraperitoneal, intradermal, intratracheal, intramuscular, subcutaneous, inhalational, and the like.
- compositions for treating or preventing a demyelinating disease e.g., multiple sclerosis or EAE
- a demyelinating disease e.g., multiple sclerosis or EAE
- the composition comprises an isolated peptide comprising a partial amino acid sequence of a myelin oligodendrocyte glycoprotein (MOG) protein, wherein the peptide activates a subset of immune cells.
- MOG myelin oligodendrocyte glycoprotein
- the peptide comprises a MOG-specific MHC1b epitope. In some embodiments, the peptide comprises a HLA-E epitope (in humans) or a Qa-epitope (in mice). In some embodiments, the peptide comprises a portion of a MOG sequence of set forth in, e.g., NCBI GenBank Accession Nos. NP_001008229.1, NP_001008230.1, NP_001163889.1, NP_002424.3, NP_996532.2, NP_996533.2, NP_996534.2, NP_996535.2, or NP_996537.3, or a homolog thereof.
- the peptide comprises about 7-25, about 7-20, about 7-15, about 8-25, about 8-20, about 8-15, about 9-25, about 9-20, about 9-15, or about 9-13 contiguous amino acids of a MOG sequence as described herein. In some embodiments, the peptide comprises at least about 7, at least about 8, or at least about 9 contiguous amino acids of a MOG sequence as described herein. In some embodiments, the peptide comprises up to about 25, up to about 20, up to about 15, up to about 12, or up to about 10 contiguous amino acids of a MOG sequence as described herein. In some embodiments, the peptide comprises a portion of a human MOG protein as described herein.
- the peptide comprises a portion of a non-human MOG homolog. In some embodiments, the peptide comprises a portion of a mouse MOG protein as described herein. In some embodiments, the peptide comprises the amino acid sequence IICYNWLHR. In some embodiments, the peptide consists of the amino acid sequence IICYNWLHR.
- the amino acid sequence IICYNWLHR is also referred to herein as “MOG 196-204 ” or “MOG 196 ”, in reference to the region of the MOG protein from which the peptide is derived.
- the peptide activates regulatory T cells. In some embodiments, the peptide activates Qa-1-restricted or HLA-E-restricted regulatory CD8 + T cells.
- methods of identifying a peptide that binds to Qa-1 or HLA-E and activates Qa-1-restricted or HLA-E-restricted regulatory CD8 + T cells are provided.
- the method comprises retrieving a myelin oligodendrocyte glycoprotein (MOG)-specific epitope (peptide) that binds to Qa-1 b , a non-classical MHCIb molecule.
- MOG myelin oligodendrocyte glycoprotein
- the method comprises the use of an overlapping peptide (OLP) library of MOG peptide, such as an OLP library of murine MOG that contains 59 OLPs, which is described herein in the Examples section and referred to herein as “MOG_pool.”
- OLP overlapping peptide
- the method comprises the use of the cell line C1R, a human B lymphoblastoid cell line and/or C1R.Qa-1 b , a stable Qa-1 transfectant of C1R cells.
- An OLP library can be used to generate reactive CD8 + T cell lines, which can then be employed to screen individual OLPs in the OLP library and identify peptides that stimulate the reactive CD8 + T cell lines in a Qa-1 b -restricted manner.
- a Qa-1 b -restricted response is determined by an OLP-stimulated increased IFN- ⁇ secretion in the reactive CD8 + T cell lines in the presence of C1R.Qa-1 b but not C1R cells.
- the methods of identifying a peptide that binds to Qa-1 or HLA-E and activates Qa-1-restricted or HLA-E-restricted regulatory CD8 + T cells further comprise confirming whether the peptide binds to HLA-E, the human homolog of murine Qa-1.
- the method comprises the use of a binding assay as described in the Examples section below and/or the use of a HLA-E-expressing cell line as described in the Examples section below (e.g., by incubating the peptide with the cell line 721.21-HLAE and using flow cytometry to determine whether HLA-E expression on the cell surface of 721.221-HLAE is upregulated after co-incubation with the peptide).
- the methods of identifying a peptide that binds to Qa-1 or HLA-E and activates Qa-1-restricted or HLA-E-restricted regulatory CD8 + T cells further comprise confirming that MSIBE can bind to Qa-1 b and activate MSIBE-specific Qa-1 b restricted CD8 + T cells in vitro.
- the method comprises the use of a binding assay as described in the Examples section below (e.g., tetramer formation and detection of tetramer+ cells).
- the methods of identifying a peptide that binds to Qa-1 or HLA-E and activates Qa-1-restricted or HLA-E-restricted regulatory CD8 + T cells further comprise confirming that the peptide can activate specific HLA-E-restricted CD8 + Treg in vitro and thereby potentially suppress MS.
- the method involves utilizing the peptide to activate HLA-E restricted CD8 + Treg in vivo.
- the method includes MSIBE, peripheral blood mononuclear cells (PBMCs), MSIBE/HLA-E tetramer, and human MOG-specific CD4 + T cell lines.
- CD8 + T cells can be purified from peripheral blood mononuclear cells (PBMCs) of healthy control individuals, stimulated with peptide-pulsed autologous monocytes at weekly basis, and monitored for binding to HLA-E (e.g., via formation of a peptide/HLA-E tetramer).
- PBMCs peripheral blood mononuclear cells
- HLA-E peptide/HLA-E tetramer+ cells reach more than 50%
- the CD8 + T cell line is examined for its ability to specifically suppress proliferation of human MOG-but not control protein-specific CD4 + T cells using a CFSE-dilution assay.
- the methods of identifying a peptide that binds to Qa-1 or HLA-E and activates Qa-1-restricted or HLA-E-restricted regulatory CD8 + T cells further comprise confirming that immunization with the peptide can activate Qa-1 b -restricted CD8 + T cells in vivo and suppress EAE.
- Methods of inducing EAE in mice and immunizing mice with peptides or dendritic cells pulsed with peptides are described in the Examples section below.
- suppression of one or more clinical disease symptoms indicates that the peptide is a peptide that binds to Qa-1 or HLA-E and activates Qa-1-restricted or HLA-E-restricted regulatory CD8 + T cells.
- dendritic cells pulsed with a myelin oligodendrocyte glycoprotein (MOG) peptide that activates regulatory T cells are provided.
- the peptide activates HLA-E-restricted or Qa-1-restricted regulatory CD8 + T cells.
- the peptide comprises a MOG-specific MHC1b epitope.
- the peptide comprises a HLA-E epitope.
- the peptide comprises the amino acid sequence IICYNWLHR.
- the peptide consists of the amino acid sequence IICYNWLHR.
- the dendritic cell can be obtained or derived from any suitable source.
- the dendritic cell may be a bone marrow-derived dendritic cell, a cord blood-derived dendritic cell, or a peripheral blood-derived dendritic cell.
- the dendritic cells are derived from monocytes.
- the dendritic cells are derived from myelin-specific pathogenic autoimmune cells. Methods of generating dendritic cells are described herein in the Examples section. Methods of isolating and generating dendritic cells are also known in the art.
- the dendritic cells are immunogenic dendritic cells. In some embodiments, the dendritic cells are tolerogenic dendritic cells.
- the dendritic cell is an activated dendritic cell.
- the dendritic cell is activated by stimulating the cell with a lipopolysaccharide (LPS) or with a cytokine (e.g., TNF- ⁇ ).
- LPS lipopolysaccharide
- TNF- ⁇ cytokine
- the dendritic cell is treated with an anti-proliferative agent.
- the anti-proliferative agent is irradiation (e.g., gamma irradiation).
- the anti-proliferative agent is a chemical compound (e.g., mitomycin C).
- the composition comprises a peptide as described herein or a peptide-pulsed dendritic cell as described herein and further comprises a pharmaceutically acceptable carrier and/or excipient.
- Guidance for preparing formulations for use in the present invention is found in, for example, Remington: The Science and Practice of Pharmacy, 21st Edition, Philadelphia, Pa., Lippincott Williams & Wilkins, 2005.
- a pharmaceutically acceptable carrier includes any solvents, dispersion media, or coatings that are physiologically compatible and that preferably does not interfere with or otherwise inhibit the activity of the therapeutic agent.
- the carrier is suitable for intravenous, intramuscular, oral, intraperitoneal, transdermal, topical, or subcutaneous administration.
- Pharmaceutically acceptable carriers can contain one or more physiologically acceptable compound(s) that act, for example, to stabilize the composition or to increase or decrease the absorption of the active agent(s).
- Physiologically acceptable compounds can include, for example, carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, compositions that reduce the clearance or hydrolysis of the active agents, or excipients or other stabilizers and/or buffers.
- carbohydrates such as glucose, sucrose, or dextrans
- antioxidants such as ascorbic acid or glutathione
- chelating agents such as ascorbic acid or glutathione
- low molecular weight proteins compositions that reduce the clearance or hydrolysis of the active agents, or excipients or other stabilizers and/or buffers.
- Other pharmaceutically acceptable carriers and their formulations are well-known and generally described in, for example, Remington: The Science and Practice of Pharmacy , supra.
- Various pharmaceutically acceptable excipients are well-known in the art and can be found in, for example, Handbook of Pharmaceutical Excipients (7 th edition, Rowe et al. (Eds.
- compositions described herein may be in any of a variety of suitable forms for a variety of routes for administration, for example, for oral, nasal, rectal, topical (including transdermal), ocular, intracerebral, intracranial, intrathecal, intra-arterial, intravenous, intramuscular, or other parental routes of administration.
- routes for administration for example, for oral, nasal, rectal, topical (including transdermal), ocular, intracerebral, intracranial, intrathecal, intra-arterial, intravenous, intramuscular, or other parental routes of administration.
- oral and nasal compositions include compositions that are administered by inhalation, and made using available methodologies.
- a variety of pharmaceutically acceptable carriers well-known in the art may be used.
- Various oral dosage forms can be used, including such solid forms as tablets, capsules, granules and bulk powders. Tablets can be compressed, tablet triturates, enteric-coated, sugar-coated, film-coated, or multiple-compressed, containing suitable binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents.
- Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules, and effervescent preparations reconstituted from effervescent granules, containing suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, melting agents, coloring agents and flavoring agents.
- the compounds and compositions described herein may be dissolved or dispersed in a pharmaceutically acceptable diluent, such as a saline or dextrose solution.
- a pharmaceutically acceptable diluent such as a saline or dextrose solution.
- Suitable excipients may be included to achieve the desired pH, including but not limited to NaOH, sodium carbonate, sodium acetate, HCl, and citric acid.
- the pH of the final composition ranges from 2 to 8, or preferably from 4 to 7.
- Antioxidant excipients may include sodium bisulfite, acetone sodium bisulfite, sodium formaldehyde, sulfoxylate, thiourea, and EDTA.
- excipients found in the final intravenous composition may include sodium or potassium phosphates, citric acid, tartaric acid, gelatin, and carbohydrates such as dextrose, mannitol, and dextran. Further acceptable excipients are described in Powell et al., “Compendium of Excipients for Parenteral Formulations,” FDA J Pharm Sci and Tech, 1998, 52:238-311, and Nema et al., “Excipients and Their Role in Approved Injectable Products: Current Usage and Future Directions,” FDA J Pharm Sci and Tech, 2011, 65:287-332, both of which are incorporated herein by reference in their entirety.
- Antimicrobial agents may also be included to achieve a bacteriostatic or fungistatic solution, including but not limited to phenylmercuric nitrate, thimerosal, benzethonium chloride, benzalkonium chloride, phenol, cresol, and chlorobutanol.
- compositions for intravenous administration may be provided in the form of one more solids that are reconstituted with a suitable diluent such as sterile water, saline or dextrose in water shortly prior to administration.
- a suitable diluent such as sterile water, saline or dextrose in water shortly prior to administration.
- the compositions are provided in solution ready to administer parenterally.
- the compositions are provided in a solution that is further diluted prior to administration.
- a composition as described herein is provided in unit dosage form.
- a “unit dosage form” is a composition containing an amount of a compound that is suitable for administration to an animal, preferably mammal subject, in a single dose, according to good medical practice.
- the preparation of a single or unit dosage form does not imply that the dosage form is administered once per day or once per course of therapy.
- Such dosage forms can be administered once, twice, thrice or more per day and may be administered as infusion over a period of time (e.g., from about 30 minutes to about 2-6 hours), or administered as a continuous infusion, and may be given more than once during a course of therapy, though a single administration is not specifically excluded.
- the skilled artisan will recognize that the formulation does not specifically contemplate the entire course of therapy and such decisions are left for those skilled in the art of treatment.
- kits comprising the peptides and/or dendritic cells disclosed herein are provided.
- a kit further comprises instructional materials containing directions (i.e., protocols) for the practice of the methods of this invention (e.g., instructions for using the kit for treating a demyelinating disease, e.g., multiple sclerosis).
- directions i.e., protocols
- the instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure.
- Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like.
- Such media may include addresses to internet sites that provide such instructional materials.
- methods of treating or preventing a demyelinating disease are provided. In some embodiments, methods of treating a demyelinating disease are provided. In some embodiments, methods of preventing or delaying the onset of a demyelinating disease are provided.
- the method comprises administering to a subject (e.g., a subject having a demyelinating disease or a subject at risk of having or suspected of having a demyelinating disease) a composition as described herein (e.g., a peptide comprising or consisting of a MOG-specific MHC1b epitope, a composition comprising an isolated peptide comprising or consisting of a MOG-specific MHC1b epitope, or a composition comprising dendritic cells pulsed with an isolated peptide comprising or consisting of a MOG-specific MHC1b epitope).
- a composition as described herein e.g., a peptide comprising or consisting of a MOG-specific MHC1b epitope, a composition comprising an isolated peptide comprising or consisting of a MOG-specific MHC1b epitope, or a composition comprising dendritic cells pulsed with an isolated peptide comprising or consisting of a MOG
- the demyelinating disease is selected from the group consisting of multiple sclerosis, idiopathic inflammatory demyelinating disease, transverse myelitis, Devic's disease, progressive multifocal leukoencephalopathy, optic neuritis, leukoystrophy, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, autoimmune peripheral neuropathy, Charcot-Marie-Tooth disease, acute disseminated encephalomyelitis, adrenoleukodystrophy, adrenomyeloneuropathy, Leber's hereditary optic neuropathy, or HTLV-associated myelopathy.
- the demyelinating disease is multiple sclerosis.
- the demyelinating disease is a disease characterized by the expression or overexpression of antibodies against MOG.
- the demyelinating disease that expresses or overexpresses antibodies against MOG is multiple sclerosis, transverse myelitis, optic neuritis, or acute disseminated encephalomyelitis.
- the subject has multiple sclerosis (MS). There are several subtypes of MS, including relapsing-remitting multiple sclerosis (RRMS), secondary progressive multiple sclerosis (SPMS), primary progressive multiple sclerosis (PPMS), and progressive relapsing multiple sclerosis (PRMS).
- RRMS relapsing-remitting multiple sclerosis
- SPMS secondary progressive multiple sclerosis
- PPMS primary progressive multiple sclerosis
- PRMS progressive relapsing multiple sclerosis
- the subject has RRMS.
- SPMS secondary progressive multiple sclerosis
- PPMS primary progressive multiple sclerosis
- PRMS progressive relapsing multiple sclerosis
- the subject has RRMS.
- SPMS secondary progressive multiple sclerosis
- PPMS primary progressive multiple sclerosis
- PRMS progressive relapsing multiple sclerosis
- PRMS progressive relapsing multiple sclerosis
- the subject has RRMS.
- SPMS secondary progressive multiple sclerosis
- PPMS primary progressive multiple
- a MOG peptide or dendritic cells pulsed with a myelin oligodendrocyte glycoprotein (MOG) peptide as described herein are used as a peptide vaccine for the prevention or treatment of MS or EAE.
- the method comprises generating a vaccine comprising MSIBE and human dendritic cells (DCs).
- the method of generating a vaccine comprises generating dendritic cells from monocytes that are autologous to the subject.
- the method comprises generating dendritic cells from monocytes that are allogeneic to the subject.
- the dendritic cells are generated in the presence of one or more cytokines (e.g., GM-CSF or interleukins, e.g., IL-4).
- the dendritic cells are treated with an anti-proliferative agent.
- the dendritic cells are pulsed with a peptide as described herein (e.g., MSIBE) and subsequently administered to a subject having MS.
- dendritic cells e.g., 1 ⁇ 10 6 human DCs
- a peptide as described herein e.g., MSIBE
- the method comprises optimizing immunization for prevention and therapy of MS or EAE.
- immunization with a MOG peptide or dendritic cells pulsed with a MOG peptide can be administered at varying amounts, or multiple immunizations can be performed, to improve the therapeutic response.
- the route of administration of a peptide, dendritic cell, or composition as described herein can be oral, intraperitoneal, transdermal, subcutaneous, intravenous, intramuscular, inhalational, topical, intralesional, rectal, intrabronchial, intralymphatic, intradermal, nasal, transmucosal, intestinal, ocular or otic delivery, or any other methods known in the art.
- a peptide, dendritic cell, or composition as described herein is administered by intravenous injection or by subcutaneous injection. In some embodiments, a peptide, dendritic cell, or composition as described herein is administered systemically. In some embodiments, a peptide, dendritic cell, or composition as described herein is administered locally.
- Dosages and desired concentrations of the peptides or dendritic cells of the disclosure may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration is well within the skill of one in the art. Typically the amount administered to a subject is a therapeutically effective amount. In some embodiments, a therapeutically effective amount of a peptide, dendritic cell, or composition as described herein is an amount that prevents or reverses one or more symptoms of the disease (e.g., MS). In some embodiments, a therapeutically effective amount of a peptide, dendritic cell, or composition as described herein is administered about once per day, once per week, twice per week, once per month, or twice per month.
- the peptides, dendritic cells, and compositions as described herein may be administered to a subject in need thereof for a predetermined time, an indefinite time, or until an endpoint is reached.
- treatment is continued on a continuous daily or weekly basis for at least two to three months, six months, one year, or longer.
- treatment is for at least 30 days, at least 60 days, at least 90 days, at least 120 days, at least 150 days, or at least 180 days.
- treatment is continued for at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least one year.
- treatment is continued for the rest of the patient's life or until administration is no longer effective to provide meaningful therapeutic benefit.
- the peptides, dendritic cells, and compositions as described herein are administered in combination with one or more additional agents (e.g., a therapeutic agent).
- the two or more agents are administered at the same time or substantially the same time.
- the two or more agents are administered sequentially.
- the two or more agents are administered through the same route (e.g., intravenously).
- the two or more agents are administered through different routes (e.g., the peptide, dendritic cell, or composition as described herein is administered intravenously and the additional agent is administered orally).
- Example 1 Targeting Non-Classical Myelin Epitopes to Treat Experimental Autoimmune Encephalomyelitis
- C57BL/6 (B6) and K b ⁇ / ⁇ D b ⁇ / ⁇ mice were obtained from Taconic Farms and housed in a specific pathogen-free animal facility at the University of Texas at El Paso (UTEP) or Loma Linda University (LLU). All experiments were done in compliance with an Institutional Animal Care and Use Protocol approved by UTEP and/or LLU Animal Care and Use Committee.
- C1R is a human B lymphoblastoid cell line.
- C1R.Qa-1 b is a stable transfectant that constitutively expresses Qa-1 b .
- DC2.4 is a DC line generated from bone-marrow-derived DCs.
- ELISPOT plates were coated with an anti-mouse IFN- ⁇ mAb (5 ⁇ g/ml in PBS) at 4° C. overnight, blocked with culture medium for 2 hours at room temperature, and added with desired numbers of CD8+ T cells, peptides, and irradiated antigen-presenting cells (APCs). After cultured at 37° C. and 5% CO 2 for 16 hours, plates were incubated with a biotinylated anti-mouse IFN- ⁇ mAb (2 ⁇ g/ml) for 2 hours followed by streptavidin-conjugated HRP (horseradish peroxidase) for 1 hour. Finally, plates were incubated with a substrate, monitored for spot development, and washed with distilled water when spots were fully developed. After air-drying, plates were analyzed on an ELISPOT plate reader for enumerating spots in each well.
- APCs irradiated antigen-presenting cells
- mice were immunized subcutaneously with 200 ⁇ g MOG 35-55 emulsified in Incomplete Freund Adjuvant (IFA) supplemented with 250 ⁇ g heat-inactivated mycobacterium tuberculosis H37Ra (Difeo Laboratories, Michigan, USA).
- IFA Incomplete Freund Adjuvant
- H37Ra heat-inactivated mycobacterium tuberculosis H37Ra
- DCs Bone Marrow-Derived Dendritic Cells
- Bone marrow single cell suspensions (1 ⁇ 10 6 cells/ml) containing 10 U/ml IL-4 and 100 U/ml GM-CSF were seeded into a 6-well plate (4 ml/well) and cultured at 37° C., 5% CO 2 .
- plates were replenished with fresh media and cytokines.
- non-adherent cells containing fresh media and cytokines were transferred into a new 6-well plate.
- cells were replenished with fresh media and cytokines and stimulated with 0.1 g/ml LPS overnight.
- cells were collected for experiments.
- LPS activated DCs at the concentration of 5 ⁇ 10 7 cells/ml in PBC were treated by mitomycin C (50 ⁇ g/ml) for 20 minutes at 37° C. and 5% CO 2 (DC2.4 cells were treated for 30 minutes).
- the treated DCs were then washed for three times and adjusted to 5 ⁇ 10 6 cells/ml in serum-free medium containing 100 ⁇ g/ml MOG 196 or HSP60 P216 peptide.
- the cells were pulsed with the peptides for three to four hours at room temperature. After pulsing, the cells were washed once, reconstituted in PBS, and intravenously (or subcutaneously) injected into animals at 200 ⁇ l/mouse (5 ⁇ 10 5 cells or 1 ⁇ 10 6 cells/mouse).
- FACS buffer PBS containing 1% FBS and 0.05% sodium azide
- the OLP library contained 59 OLPs in total ( FIG. 1A ).
- a pool of the 59 OLPs (MOG_pool), which contained a final concentration of 4.2 ⁇ g/ml for each individual peptide, was used to stimulate CD8 + T cells purified from K b ⁇ / ⁇ D b ⁇ / ⁇ mice for generating MOG_pool-reactive CD8 + T cell lines in vitro.
- CD8 + T cells that were purified from K b ⁇ / ⁇ D b ⁇ / ⁇ mice were utilized because CD8 + T cells in these mice were restricted mostly by non-classical MHC Ib molecules including Qa-1 b .
- CD8 + T cell lines were monitored weekly for response, using IFN- ⁇ Enzyme-linked ImmunoSpot, to the MOG_pool in the presence of either C1R or C1R.Qa-1 b cells.
- the data showed that most CD8 + T cell lines that were generated specifically responded to the MOG_pool in the presence of both C1R and C1R.Qa-1 b cells ( FIG. 1B and FIG. 1C ).
- response to the MOG_pool was much stronger when C1R.Qa-1 b cells were present.
- the data therefore suggested that portion of the CD8 + T cells in the lines responded to the MOG_pool in a Qa-1 b -dependent (or Qa-1-restricted) manner.
- CD8 + T cells were examined for response, using IFN- ⁇ Enzyme-linked ImmunoSpot, to the corresponding peptides in the presence of either C1R or C1R.Qa-1 b cells.
- the data showed that all three OLPs stimulated CD8 + T cells in a Qa-1 b -restricted manner ( FIGS. 2E and 2F ), suggesting that all three OLPs contained Qa-1 b epitopes.
- MOG 195-204 (“MOG 196 ”) was the Minimal and Optimal Qa-1 b Epitope in OLP105.
- MOG 196 Binds to Qa-1 b and Activates MOG 196 -Specific Qa-1 b -Restricted CD8 + T Cells In Vitro.
- MOG 196 was a Qa-1 b epitope
- MOG 196 could bind to Qa-1 b by addressing whether this 9mer peptide could successfully refold with recombinant Qa-1 b protein in vitro.
- MOG 196 was incubated with recombinant Qa-1 b protein and ⁇ 2m at 10° C. for four days.
- the resulting solution was analyzed in a size exclusion column and displayed a distinct protein peak ( FIG. 4A , peak B), suggesting formation of MOG 196 /Qa-1 b / ⁇ 2m monomer.
- the monomer was further analyzed in an anion exchange column, the monomer displayed two protein peaks ( FIG.
- peaks A, B1, and B2 proteins in peaks A, B1, and B2 were biotinylated. Portions of the biotinylated proteins were incubated with streptavidin that was able to bind four biotinylated proteins to form tetramers. Subsequently, the biotinylated proteins and streptavidin-conjugated tetramers were analyzed in a non-denature protein gel. Data showed that biotinylated proteins in peak A did not show any distinct protein band ( FIG. 4C , lane 1), supporting that this peak contained mainly non-specific protein aggregates.
- biotinylated proteins in peak B1 displayed a single protein band ( FIG. 4C , lane 3), indicating correct formation of MOG 196 /Qa-1 b / ⁇ 2m monomer.
- biotinylated proteins in peak B2 exhibited two protein bands ( FIG. 4C , lane 5), suggesting that some proteins in this peak were not correctly refolded.
- addition of streptavidin successfully conjugated biotinylated proteins in peak B1 and B2 into tetramers FIG. 4C , lanes 4 and 6. The data therefore demonstrated that MOG 196 could bind to Qa-1 b .
- MOG 196 could be presented by antigen-presenting cells to activate MOG 196 -specific Qa-1 b -restricted CD8 + T cells.
- CD8+ T cells purified from C57Bl/6 mice were stimulated in vitro weekly by MOG 196 -pulsed antigen-presenting cells derived from either K b ⁇ / ⁇ D b ⁇ / ⁇ or C57Bl/6 mice.
- Qa-11 b /MOG 196 tetramer+ cells could be detected in the CD8 + T cell lines ( FIG. 4D ), indicating successful activation of MOG 196 -specific Qa-1 b -restricted CD8 + T cells.
- antigen-presenting cells derived from both K b ⁇ / ⁇ D b ⁇ / ⁇ and C57Bl/6 mice were able to present MOG 196 to activate MOG 196 -specific Qa-1 b -restricted CD8 + T cells in vitro ( FIG. 4D ).
- mice were vaccinated with MOG 196 -pulsed B6 DCs on days ⁇ 3, 2, and 7. On day 0, mice were immunized with MOG 35-55 for EAE. The data again showed that vaccination with wild-type DCs pulsed with MOG 196 , but not Qdm or HSP60 p216 , significantly suppressed paralytic disease ( FIGS. 5C-5D and Table 2).
- this epitope may be essential in maintaining immune homeostasis in the central nervous system (CNS).
- CNS central nervous system
- I-A b /MOG 38-49 tetramer+ cells which detected MOG 35-55 -reactive pathogenic autoimmune cells, were significantly reduced in cervical lymph nodes ( FIGS. 7A, 7D, and 7E ).
- the data were consistent with suppression of ongoing paralytic disease following DC2.4/MOG 196 treatment ( FIG. 6 ).
- CD122 and Ly49 were Expressed in a Portion of the Qa-1 b /MOG 196 Tetramer+ Cells.
- MOG 196 is Evolutionarily Conversed and Unique to MOG.
- Qa-1-binding peptides bind to both Qa-1 and HLA-E (Miller et al., J. Immunol., 2003, 171:1369-1375).
- peptide binding motifs of Qa-1 and HLA-E are similar (Miller et al., supra; Kurepa et al., J. Immunol., 1997, 158:3244-3251). It was investigated whether the MOG 196 peptide sequence was evolutionarily conserved. A Blast search of GenBank protein sequence data showed that MOG 196 sequence was evolutionarily conserved ( FIG. 8 ).
- sequence of MOG 196 is specific to MOG and located in the intracellular domain ( FIG. 9 ). Therefore, HLA-E can be utilized to suppress MS in a similar manner as Qa-1 is used to suppress EAE.
- recognition of the regulatory Qa-1/HLA-E myelin epitopes by the CD8 + Treg cells leads to 1) elimination of and/or induction of tolerance in the antigen-presenting cells; 2) elimination and/or induction of tolerance in encephalitogenic T cells that have obtained the epitope complexes from antigen-presenting cells via a process called trogocytosis. Consequently, activation and proliferation of encephalitogenic T cells are thwarted and autoimmune attacks of myelin sheath are stopped.
- MOG 196 i.e., the regulatory Qa-1 epitope
- MOG 35-55 i.e., the pathogenic epitope
- MOG 196 -specific Qa-1-restricted CD8 + Treg functionally enhanced by MOG 196 immunization, can specifically recognize and suppress APCs that have phagocytosed MOG and otherwise initiated or perpetuated EAE.
- the present disclosure provides evidence of an evolutionarily conserved regulatory Qa-1 epitope, MOG 196 , in mouse myelin oligodendrocyte glycoprotein (MOG).
- MOG mouse myelin oligodendrocyte glycoprotein
- the discovery of the regulatory Qa-1 epitope in MOG is significant for several reasons. First, it reveals another potential regulatory pathway wherein Qa-1-restricted CD8 + Treg cells, besides targeting autoreactive T cells, may also target an autoantigen to suppress an autoimmune disease. Second, because of their specificity for an autoantigen, Qa-1-restricted CD8 + Treg cells activated by a regulatory Qa-1 epitope derived from an autoantigen, as compared to autoreactive T cells, may potentially accumulate in the diseased tissues and thereby more efficiently suppress autoimmune attacks. Third, because more autoantigens have been discovered in various autoimmune diseases and because generation of autoreactive T cells is lengthy and tedious, autoantigens are more accessible than autoreactive T cells.
- myelin-specific Qa-1-restricted CD8 + Treg cells described here will have the advantage to cross the BBB and provide in situ suppression of demyelinating inflammation in the CNS.
- the data herein shows that, in the course of EAE, MOG 196 -specific, Qa-1-restricted CD8 + Treg cells specifically accumulated in the cervical lymph nodes (i.e., the draining lymph nodes for CNS).
- the disease suppression involves IFN- ⁇ , perforin, and IL-15 (Beeston et al., J. Neuroimmunol., 2010, 229:91-97; Kim et al., Nature, 2010, 467:516-523). These data support a direct killing of target cells by the Qa-1-restricted CD8 + Treg cells.
- oligodendrocytes normally do not express MHC molecules (Goverman, Nat Rev Immunol, 2009, 9:393-407), it may explain that the MOG 196 -specific Qa-1-restricted CD8 + Treg cells do not damage myelin. It is proposed that the same antigen presenting cell, which phagocytoses myelin, can present both pathogenic and regulatory myelin epitopes to pathogenic CD4 + and regulatory CD8 + T cells respectively because both epitopes are located in the myelin ( FIG. 10 ).
- recognition of the regulatory Qa-1/HLA-E myelin epitopes by the CD8 Treg cells leads to 1) elimination of and/or induction of tolerance in the antigen-presenting cells; 2) elimination and/or induction of tolerance in encephalitogenic T cells that have obtained the epitope complexes from antigen-presenting cells via a process called trogocytosis. Consequently, activation and proliferation of encephalitogenic T cells are thwarted and autoimmune attacks on myelin sheath are stopped.
- neuroantigen-specific CD8 + T cells are regulatory. See, Ortega et al., Neural Neuroimmnunol Neuroinflamm, 2015, 2:e170.
- the neuroantigen-specific CD8 + Treg cells differ from myelin-specific Qa-1-restricted CD8 + Treg cells in requiring classical MHC I molecules for presentation.
- priming of the neuroantigen-specific CD8 + Treg cells requires the presence of regulatory and pathogenic epitopes m the same myelin protein. This second characteristic suggests that active immunization with such a regulatory neuroantigenic epitope is not a suitable therapy for MS.
- the myelin-specific CD8 + Treg cells described here can be primed through active immunization with DCs pulsed with the regulatory epitope alone.
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Abstract
Description
- This application claims priority to U.S. Provisional Patent Application No. 62/357,891, filed Jul. 1, 2016, the entire content of which is incorporated by reference herein.
- Multiple sclerosis (MS) is a chronic and often debilitating disorder of the central nervous system (CNS). Data suggest that MS global prevalence and incidence rate are increasing (Melcon et al., Journal of the Neurological Sciences, 2014, 344:171-181). It is believed that MS is caused by attacks on the myelin sheath by one's own immune system.
- Studies in MS autoimmune animal models (experimental autoimmune encephalomyelitis, or EAE) have led to a series of FDA-approved medications that have significantly slowed disease progression and improved patients' quality of life. However, currently available therapies still fail to halt autoimmune attacks in the CNS. There remains a need for methods of treating MS.
- According to one embodiment, a composition for treatment of multiple sclerosis is provided, the composition comprising: a peptide comprising a partial amino acid sequence of myelin oligodendrocyte glycoprotein, wherein the peptide activates a subset of immune cells. In some embodiments, a composition is provided wherein the activated immune cells comprise HLA-E-restricted regulatory CD8+ T cells. In some embodiments, a composition is provided wherein the peptide comprises a MOG-
specific MHC 1b epitope. - According to another embodiment, a method for treating multiple sclerosis is provided, the method comprising: administering a peptide comprising a partial amino acid sequence of myelin oligodendrocyte glycoprotein; and activating a subset of immune cells, wherein the immune cells comprise HLA-E-restricted regulatory CD8+ T cells. In some embodiments, a method is provided wherein the peptide comprises a MOG-
specific MHC 1b epitope. - In another aspect, compositions for the treatment or prevention of multiple sclerosis are provided. In some embodiments, the composition comprises an isolated peptide comprising a partial amino acid sequence of a myelin oligodendrocyte glycoprotein (MOG) protein, wherein the peptide activates regulatory T cells. In some embodiments, the composition comprises dendritic cells pulsed with a myelin oligodendrocyte glycoprotein (MOG) peptide that activates regulatory T cells.
- In some embodiments, the peptide comprises a MOG-specific MHC1b epitope. In some embodiments, the peptide comprises a HLA-E epitope. In some embodiments, the peptide comprises the amino acid sequence IICYNWLHR. In some embodiments, the peptide consists of the amino acid sequence IICYNWLHR.
- In some embodiments, the peptide activates HLA-E-restricted regulatory CD8+ T cells.
- In some embodiments, the dendritic cells are derived from myelin-specific pathogenic autoimmune cells. In some embodiments, the dendritic cells are derived from monocytes.
- In another aspect, methods of treating or preventing a demyelinating disease are provided. In some embodiments, the method comprises administering to a subject a composition as disclosed herein (e.g., an isolated peptide comprising a partial amino acid sequence of MOG (e.g., a peptide comprising or consisting of the amino acid sequence IICYNWLHR), a composition comprising an isolated peptide comprising a partial amino acid sequence of MOG, or a composition comprising dendritic cells pulsed with an isolated peptide comprising a partial amino acid sequence of MOG). In some embodiments, methods of treating the demyelinating disease are provided. In some embodiments, methods of preventing or delaying the onset of the demyelinating disease are provided.
- In some embodiments, the demyelinating disease is selected from the group consisting of multiple sclerosis, idiopathic inflammatory demyelinating disease, transverse myelitis, Devic's disease, progressive multifocal leukoencephalopathy, optic neuritis, leukoystrophy, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, autoimmune peripheral neuropathy, Charcot-Marie-Tooth disease, acute disseminated encephalomyelitis, adrenoleukodystrophy, adrenomyeloneuropathy, Leber's hereditary optic neuropathy, or HTLV-associated myelopathy. In some embodiments, the demyelinating disease is multiple sclerosis.
- In some embodiments, the composition is administered subcutaneously or intravenously.
- In still another aspect, methods of treating multiple sclerosis are provided. In some embodiments, the method comprises administering to a subject having multiple sclerosis a composition comprising dendritic cells pulsed with a myelin oligodendrocyte glycoprotein (MOG) peptide that activates HLA-E-restricted regulatory CD8+ T cells. In some embodiments, the peptide comprises or consists of the amino acid sequence IICYNWLHR. In some embodiments, the dendritic cells are derived from monocytes. In some embodiments, the dendritic cells are derived from monocytes that are autologous to the subject. In some embodiments, the dendritic cells are derived from monocytes that are allogeneic to the subject.
-
FIG. 1A-1C . Portion of CD8+ T cells reactive to the pool of overlapping peptides (OLPs) covering the whole length of mouse MOG was Qa-1b restricted. (A) A schematic view of the mouse MOG OLP library. 15mer peptides across the whole length of mouse MOG (247 amino acids) and overlapped by 11 amino acids were synthesized. A pool of the 59 OLPs (“MOG_pool”) at a concentration of 4.2 μg/ml for each peptide was then generated for stimulating CD8+ T cells purified from Kb−/−Db−/− mice. (B) Experimental design for Enzyme-linked ImmunoSpot assay in “C”: CD8+ T cells purified from Kb−/−Db−/− mice were stimulated with the MOG_pool in vitro on a weekly basis. Before being stimulated each time, the CD8+ T cells were monitored for response to the MOG_pool, using IFN-γ Enzyme-linked ImmunoSpot, in the presence of either C1R or C1R.Qa-1b cells as antigen presenting cells. (C) Enzyme-linked ImmunoSpot data onday 7 were shown and were representative of three independent experiments. The number at the upper left corner of each well is the absolute number of IFN-γ spot forming cells (SFCs) in the corresponding well (50,000 CD8 T cells/well). -
FIG. 2A-2F . Recognition of multiple OLPs by the MOG_pool-reactive CD8+ T cell lines depended on Qa-1b. (A-D) MOG_pool-reactive CD8+ T cell lines were generated as described inFIG. 1 . The 59 individual OLPs were interrogated, using IFN-γ Enzyme-linked ImmunoSpot, for their ability to stimulate the MOG_pool-reactive CD8+ T cell lines in the presence of either C1R or C1R.Qa-1b cells. (A) 96-well plate layout of the 59 individual OLPs. Numbers in the wells represent identification codes (IDs) for the 59 individual OLPs, i.e., 49-107 from N to C termini. (B)-(C) Responses of one representative MOG_pool-reactive CD8+ T cell line to the 59 individual OLPs in the presence of either C1R (B) or C1R.Qa-1b (C) cells. Numbers represent spot forming cells (SFCs)/106 CD8+ T cells. (D) Fold increases of SFCs/106 CD8+ T cells in the wells that contained C1R.Qa-1b cells (C) as compared to C1R cells (B), i.e., numbers=(SFCs per 106 CD8+ T cells in C)/(SFCs per 106 CD8+ T cells in B). The highlighted boxes are the top three OLPs that stimulated unequivocal Qa-1b-restricted CD8+ T cell response. (E) Experimental design for assay in “F”: Kb−/−Db−/− mice were immunized with OLP68, OLP96, or OLP105. CD8+ T cells purified ten days later were stimulated with the corresponding peptides used for immunization. After one week of in vitro culture, the CD8+ T cells were examined, using IFN-γ Enzyme-linked ImmunoSpot, for specific responses to the corresponding peptides in the presence of either C1R or C1R.Qa-1b cells. (F) Representative data from two independent experiments is shown. -
FIG. 3 . Fine mapping of the minimal and optimal Qa-1b epitope in OLP105. Progressively N- and C-terminally truncated OLP105 peptides were synthesized and interrogated for their ability to stimulate, using IFN-γ Enzyme-linked ImmunoSpot, an OLP105-reactive CD8+ T cell line in the presence of C1R. Qa-1b cells. IDs and sequences of the truncated peptides are shown in the left and middle panels, respectively. Corresponding bars in the right panel show IFN-γ spot-forming cells (SFCs) per 106 CD8+ T cells in response to corresponding peptides. Highlighted bar and sequence displayed the highest response and stimulating peptide sequence respectively, demonstrating that sequence of the optimal and minimal epitope in OLP105 was IICYNWLHR, i.e., MOG196-204 (also referred to herein as MOG196). The data was representative of four independent experiments. -
FIG. 4A-4D . MOG196 can bind to Qa-1b and stimulate MOG196-specific Qa-1b-restricted CD8+ T cells. (A) MOG196 was incubated with recombinant Qa-1b and β2 microglobulin in a protein refolding buffer at 10° C. under gentle agitation (60 rpm) for four days. The solution was separated by a size exclusion column. Peak “A” and “B” represented typical non-specific protein aggregates and correctly refolded MOG196/Qa-1b monomer, respectively. (B) Monomers in peak B of panel “A” were further analyzed by an anion exchange chromatography. (C) Proteins in peaks “A”, “B1”, and “B2” were biotinylated and portions of the biotinylated proteins were incubated with streptavidin (SA) to examine formation of tetramers. The biotinylated proteins and corresponding tetramers were analyzed in a non-denature protein gel. Arrows show protein bands for MOG196/Qa-1b monomers. “−”: without SA; “+”: with SA; “pk A”: peak A; “pk B1”: peak B1; “pk B2”” peak B2; “Std”: a protein standard. (D) CD8+ T cells were purified from naïve C57Bl/6 (B6) mice, individually stimulated weekly with untreated (no peptide) or MOG196-pulsed, either B6 (upper panels) or Kb−/−Db−/− (lower panels) macrophages. The CD8+ T cells were analyzed for binding to Qa-1b/MOG196 tetramer atdays day 14 from four individual experiments was shown. -
FIG. 5A-5D . Immunization with MOG196-pulsed DCs suppressed MOG35-55-induced EAE. (A) Experimental design for “B”: C57BL/6 mice were immunized with MOG35-55 for EAE. One week before and after the EAE induction, the animals received one of the following subcutaneous treatments: 1) no treatment (No Tx); 2) 1×106 Qdm-pulsed Kb−/−Db−/− DCs (DC/Qdm); 3) 1×106 MOG196-pulsed Kb−/−Db−/− DCs (DC/MOG196). The mice were then monitored for paralytic disease daily. (B) Daily mean disease score was shown. N=5. *P<0.05; **P<0.01; ***P<0.001. Two-way ANOVA test. (C) Experimental design for “D”: C57BL/6 mice were immunized with MOG35-55 for EAE onday 0. At days −3, 2, and 7, animals were immunized with 1×106 B6 DCs pulsed with Qdm (DC/Qdm), HSP60p216 (DC/HSP60p216), or MOG196 (DC/MOG196). The animals were then monitored for paralytic disease daily. (D) Daily mean disease score was shown. N=5. *P<0.05. Two-way ANOVA test. Concentration of peptides used for pulsing the DCs was 10 μg/ml. -
FIG. 6A-6B . Immunization with MOG196-pulsed DCs suppressed ongoing MOG35-55-induced EAE, which was dependent on CD8+ T cells. (A) Experimental design for “B”: C57BL/6 mice were immunized with MOG35-55 for EAE onday 0. In one group, the animals were intraperitoneally injected with a monoclonal depleting anti-CD8 antibody (mAb) at days −2, −1, 7, and 14. Atday 10, animals received either no treatment (No Tx) or one intravenous injection of mitomycin C-treated C57BL/6 DCs pulsed with MOG196 (DC/MOG196). Paralytic disease was monitored daily. (B) Daily mean disease score was shown. N=5 (one animal that died from EAE before antibody treatment was excluded from this analysis). *P<0.05; **P<0.01; ***P<0.001. Two-way ANOVA test. Data shown were representative of two independent experiments. -
FIG. 7A-7G . Immunization with DCs pulsed with MOG196 activates Qa-1b/MOG196 tetramer+ cells that specifically accumulate in cervical lymph nodes. (A) C57BL/6 mice (5 mice/group) were immunized with MOG35-55 for EAE. Ten days later, when paralytic symptoms began, animals received one intravenous immunization with mitomycin C-treated DC2.4 or MOG196-pulsed DC2.4 (DC2.4/MOG196). Four days later, mononuclear cells prepared from spleens and cervical lymph nodes were examined for the presence of Qa-1b/MOG196 and I-Ab/MOG38-49 tetramer+ cells by flow cytometry. (B) Representative plots of Qa-1b/MOG196 tetramer+ cells among CD8+ T cells in cervical lymph nodes and spleens. (C) Cumulative data of Qa-1b/MOG196 tetramer+ cells from five mice. *P<0.05. Two-way ANOVA test. (D) Representative plots of I-Ab/MOG38-49 tetramer+ cells among CD8− T cells in cervical lymph nodes and spleens. (E) Cumulative data of I-Ab/MOG38-49 tetramer+ cells from five mice. *P<0.05. Two-way ANOVA test. (F) Experimental design for “G”: C57BL/6 mice were intravenously immunized with MOG196-pulsed DC2.4 cells atdays -
FIG. 8 . MOG196 sequence is conserved across species. The data show an alignment of the sequences surrounding MOG196 in four different species. “m”: mice; “r”: rats; “C Jacchus”: Callithrix jacchus; “h”: humans. The 9-mer peptide sequence IICYNWLHR is underlined. -
FIG. 9 . MOG196 sequence is located in the intracellular domain of myelin oligodendrocyte glycoprotein (MOG). The three highlighted extracellular epitopes, MOG35-55, MOG1-22, and MOG92-106, are encephalogenic (or pathogenic) epitopes. The intracellular MOG196 epitope is a regulatory (or protective) Qa-1b epitope. -
FIG. 10 . A model of immune regulation mediated by myelin-specific, Qa-1-restricted CD8+ Treg. Myelin-specific, Qa-1-restricted CD8+ Treg cells can recognize and tolerize/eliminate antigen-presenting cells (APCs) that otherwise activate myelin-specific encephalitogenic T cells in the CNS and/or peripheral lymphoid tissues. Tolerization/elimination of APCs, which present myelin epitopes, is mediated by regulatory cytokines (CKs), inhibitory molecules, or direct cytotoxicity. Consequently, activation of myelin-specific encephalitogenic T cells and autoimmune attacks of myelin sheath are thwarted. - Multiple sclerosis (MS) is a chronic and often debilitating disorder of the central nervous system (CNS). The ultimate goal of MS therapy is to specifically suppress the immune attacks on myelin sheath (a structure that protects nerve fibers in the CNS), while sparing global immune defense mechanisms (Steinman, Mult Scler, 2015, 21:1223-1238). However, currently available medications have not met this goal. Currently available therapies block one molecule that mediates the inflammatory attacks of myelin sheath in the CNS of MS patients. However, these blocked molecules are essential components in the immune system and participate in immune defense against infections and cancers. Consequently, normal immune defense mechanisms are attenuated and predictable side effects ensue. See, Clifford et al., The Lancet Neurology, 2010, 9:438-446; Linda et al., The New England Journal of Medicine, 2009, 361:1081-187. In principle, the adverse side effects associated with current therapies can be eliminated by antigen-specific therapy. See, e.g., Steinman, Mult Scler, 2015, 21:1223-1238; Lutterotti et al., Journal of the Neurological Sciences, 2008, 274:18-22. The goal of antigen-specific therapy is to specifically delete, anergize, or deviate myelin-specific pathogenic autoimmune cells that are responsible for MS. However, there is currently no FDA-approved antigen-specific therapy for MS.
- Convincing evidence have shown that regulatory T (Treg) cells, a subset of immune cells, are important in preventing and/or arresting autoimmune attacks, e.g., the attacks on myelin sheath in MS patients. Recent studies suggest that HLA-E (a group of non-classical MHC Ib molecules)-restricted CD8+ T cells, another subset of immune cells, are altered or deficient in MS patients. See, e.g., Ben-Nun et al., J. Autoimmun., 2014, 54:33-50. The data therefore indicate that HLA-E-restricted CD8+ T cells contain a subset of Treg (referred to herein as HLA-E-restricted CD8+ Treg). HLA-E-restricted CD8+ Treg may be involved in the prevention and/or therapy of MS, and this has been further supported by data gathered from studies of Qa-1-restricted CD8+ Treg (the murine homologue of human HLA-E-restricted CD8+ Treg). Qa-1-restricted CD8+ Treg may maintain immune homeostasis under physiological condition. In addition, the CD8+ Treg recognize specific peptides (also called regulatory Qa-1 epitopes) on and thereby target autoreactive T cells (i.e., the immune cell subset that causes autoimmune diseases).
- At least two limitations may prevent current preclinical studies from clinical translation. The first limitation is that CD8+ Treg activated by current strategies would mainly target autoreactive T cells in the peripheral lymphoid organs and may not have the capacity to specifically accumulate in the diseased CNS. Consequently, efficacy of current strategies may be compromised. The second limitation is that the CD8+ Treg's targets in human, i.e., regulatory HLA-E epitopes expressed in autoreactive T cells, can be difficult to determine in patients. Therefore, current strategies are difficult to implement in clinics.
- The present disclosure provides compositions and methods that overcome these limitations. As described herein, in one aspect a regulatory Qa-1 (HLA-E) epitope is disclosed. In some embodiments, the Qa-1 epitope is a “MOG-specific MHC1b epitope” (or “MSIBE”), that is specifically located in MOG, a protein located in myelin sheath that is attacked by the immune system in EAE. As described herein in the Examples section below, immunization with MSIBE but not non-MOG Qa-1 epitopes suppresses EAE. Additionally, data presented herein demonstrate that Qa-1-restricted CD8+ Treg cells activated by MSIBE immunization are involved in efficient suppression of EAE. Because of MSIBE's unique location in MOG, an autoantigen that is the ultimate target of autoreactive T cells in EAE and in MS, Qa-1-restricted (or HLA-E-restricted) CD8+ Treg activated by MSIBE may specifically accumulate at and arrest the autoimmune attacks on myelin sheath in EAE and in MS. Thus, in some embodiments, MSIBE can be used as a peptide vaccine for specific prevention and therapy of MS.
- In some embodiments, a 9mer peptide (hereafter epitope) that is specifically located in myelin oligodendrocyte glycoprotein (MOG), a major autoantigen in multiple sclerosis (MS), is provided. In some embodiments, the epitope has the amino acid sequence IICYNWLHR. In some embodiments, the MSIBE epitope can be used for activating a subset of immune cells, specifically HLA-E (Qa-1 in mouse)-restricted regulatory CD8+ T cells (also referred to herein as “HLA-E (Qa-1)-restricted CD8+ Treg”) for the specific prevention and therapy of MS.
- The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, because the scope of the present invention will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not be construed as representing a substantial difference over the definition of the term as generally understood in the art.
- As used in this disclosure, except where the context requires otherwise, the method steps disclosed are not intended to be limiting nor are they intended to indicate that each step is essential to the method or that each step must occur in the order disclosed.
- All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied (+) or (−) by increments of 0.1 or 1.0, as appropriate. It is to be understood, although not always explicitly stated that all numerical designations are preceded by the term “about.”
- The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a compound” includes a plurality of compounds.
- The term “comprising” is intended to mean that the compounds, compositions and methods include the recited elements, but not excluding others. “Consisting essentially of” when used to define compounds, compositions and methods, shall mean excluding other elements that would materially affect the basic and novel characteristics of the claimed invention. “Consisting of” shall mean excluding any element, step, or ingredient not specified in the claim. Embodiments defined by each of these transition terms are within the scope of this invention.
- As used herein, “myelin oligodendrocyte glycoprotein” or “MOG” refers to a glycoprotein that is expressed on the oligodendrocyte cell surface and the outermost surface of myelin sheaths. In some embodiments, a MOG protein is a human MOG protein. Human MOG is known to have alternatively spliced transcript variants. Sequences for human MOG mRNA are set forth in, e.g., NCBI GenBank Accession Nos. NM_001008228.2, NM_001008229.2, NM_001170418.1, NM_002433.4, NM_206809.3, NM_206810.3, NM_206811.3, NM_206812.3, and NM_206814.5. Sequences for human MOG protein are set forth in, e.g., NCBI GenBank Accession Nos. NP_001008229.1, NP_001008230.1, NP_001163889.1, NP_002424.3, NP_996532.2, NP_996533.2, NP_996534.2, NP_996535.2, and NP_996537.3. In some embodiments, a MOG protein or peptide is a homolog or ortholog of a human sequence disclosed herein (e.g., a mouse, rat, cynomolgus monkey, or marmoset (C. jacchus) form of MOG or a peptide thereof). In some embodiments, a MOG protein is a mouse MOG protein. The sequence for mouse MOG protein is set forth in, e.g., NCBI GenBank Accession No. NP_034944.2.
- As used herein, the term “MOG-specific MHC1b epitope” or “MSIBE” refers to an epitope in myelin oligodendrocyte glycoprotein (MOG), e.g., human MOG or mouse MOG, that binds to a non-classical
major histocompatibility complex 1b (MHC1b) molecule. In some embodiments, the MHC1b molecule is the antigen HLA-E (in humans) or Qa-1 (in mice). - As used herein, the term “epitope” refers to an area or region of a protein that specifically binds to an antigen (e.g., a MHC1b molecule, e.g., HLA-E or Qa-1), and can include a few amino acids, e.g., 5, 6, 7, 8, 9, 10, 11 or more amino acids. In some embodiments, the epitope is comprised of consecutive amino acids of a protein (e.g., 5, 6, 7, 8, 9, 10, 11 or more consecutive amino acids). In some embodiments, the term “epitope” as used herein refers to a peptide (e.g., an isolated peptide) comprising or consisting of the area or region of the protein that specifically binds to the antigen.
- As used herein, the term “specifically binds” refers to a molecule (e.g., a peptide) that binds to a target (e.g., an antigen) with greater affinity, greater avidity, and/or greater duration to that target in a sample than it binds to another non-target compound (e.g., a structurally different antigen). In some embodiments, the molecule (e.g., peptide) specifically binds to the target (e.g., antigen) with at least 3-fold greater affinity than other non-target compounds, e.g., at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold greater affinity.
- The term “isolated,” as used herein with reference to a protein or peptide, denotes that the protein or peptide is essentially free of other cellular components with which it is associated in the natural state. It is preferably in a homogeneous state. Purity and homogeneity are typically determined using analytical chemistry techniques such as electrophoresis (e.g., polyacrylamide gel electrophoresis) or chromatography (e.g., high performance liquid chromatography). In some embodiments, an isolated protein or peptide is at least 85% pure, at least 90% pure, at least 95% pure, or at least 99% pure.
- The term “demyelinating disease,” as used herein, refers to a disease or condition of the nervous system characterized by damage to or loss of the myelin sheath of neurons. A demyelinating disease can be a disease affecting the central nervous system or a disease affecting the peripheral nervous system. Examples of demyelinating diseases include, but are not limited to, multiple sclerosis, idiopathic inflammatory demyelinating disease, transverse myelitis, Devic's disease, progressive multifocal leukoencephalopathy, optic neuritis, leukoystrophy, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, autoimmune peripheral neuropathy, Charcot-Marie-Tooth disease, acute disseminated encephalomyelitis, adrenoleukodystrophy, adrenomyeloneuropathy, Leber's hereditary optic neuropathy, or HTLV-associated myelopathy. In some embodiments, the demyelinating disease is multiple sclerosis.
- As used herein, the term “subject” or “patient” refers to a human or a non-human mammal, e.g., a dog, a cat, a mouse, a rat, a cow, a sheep, a pig, a goat, or a non-human primate, or a bird, e.g., a chicken, or any other vertebrate or invertebrate animal.
- As used herein, the terms “treatment,” “treating,” and “treat” refer to administering a compound or pharmaceutical composition to a subject for prophylactic and/or therapeutic purposes. The term “prophylactic treatment” refers to treating a subject who does not yet exhibit symptoms of a disease or condition, but who is susceptible to, or otherwise at risk of, a particular disease or condition, whereby the treatment reduces the likelihood that the patient will develop the disease or condition. The term “therapeutic treatment” refers to administering treatment to a subject already having a disease or condition.
- As used herein, an “effective amount” or a “therapeutically effective amount” refers to an amount of a therapeutic agent that is effective to relieve, to some extent, or to reduce the likelihood of onset of, one or more symptoms of a disease or condition (e.g., multiple sclerosis) and includes curing the disease or condition. A prophylactically effective amount, as used herein, refers to an amount that is effective to prevent or delay the onset of one or more symptoms of a disease or condition (e.g., multiple sclerosis), or otherwise reduce the severity of said one or more symptoms, when administered to a subject who does not yet exhibit symptoms of a disease or condition, but who is susceptible to, or otherwise at risk of, a particular disease or condition. In some embodiments, for a given parameter, a therapeutically effective amount will show an increase of therapeutic effect of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic efficacy can also be expressed as “fold” increase or decrease. For example, a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a control.
- The terms “administer,” “administered,” or “administering,” as used herein, refer to introducing an agent into a subject or patient, such as a human. As used herein, the terms encompass both direct administration, (e.g., self-administration or administration to a patient by a medical professional) and indirect administration (e.g., the act of prescribing a compound or composition to a subject).
- As used herein, the term “pharmaceutical composition” refers to a composition suitable for administration to a subject. In general, a pharmaceutical composition is sterile, and preferably free of contaminants that are capable of eliciting an undesirable response with the subject. Pharmaceutical compositions can be designed for administration to subjects in need thereof via a number of different routes of administration, including oral, intravenous, buccal, rectal, parenteral, intraperitoneal, intradermal, intratracheal, intramuscular, subcutaneous, inhalational, and the like.
- In one aspect, compositions for treating or preventing a demyelinating disease (e.g., multiple sclerosis or EAE) are provided. In some embodiments, the composition comprises an isolated peptide comprising a partial amino acid sequence of a myelin oligodendrocyte glycoprotein (MOG) protein, wherein the peptide activates a subset of immune cells.
- In some embodiments, the peptide comprises a MOG-specific MHC1b epitope. In some embodiments, the peptide comprises a HLA-E epitope (in humans) or a Qa-epitope (in mice). In some embodiments, the peptide comprises a portion of a MOG sequence of set forth in, e.g., NCBI GenBank Accession Nos. NP_001008229.1, NP_001008230.1, NP_001163889.1, NP_002424.3, NP_996532.2, NP_996533.2, NP_996534.2, NP_996535.2, or NP_996537.3, or a homolog thereof. In some embodiments, the peptide comprises about 7-25, about 7-20, about 7-15, about 8-25, about 8-20, about 8-15, about 9-25, about 9-20, about 9-15, or about 9-13 contiguous amino acids of a MOG sequence as described herein. In some embodiments, the peptide comprises at least about 7, at least about 8, or at least about 9 contiguous amino acids of a MOG sequence as described herein. In some embodiments, the peptide comprises up to about 25, up to about 20, up to about 15, up to about 12, or up to about 10 contiguous amino acids of a MOG sequence as described herein. In some embodiments, the peptide comprises a portion of a human MOG protein as described herein. In some embodiments, the peptide comprises a portion of a non-human MOG homolog. In some embodiments, the peptide comprises a portion of a mouse MOG protein as described herein. In some embodiments, the peptide comprises the amino acid sequence IICYNWLHR. In some embodiments, the peptide consists of the amino acid sequence IICYNWLHR. The amino acid sequence IICYNWLHR is also referred to herein as “MOG196-204” or “MOG196”, in reference to the region of the MOG protein from which the peptide is derived.
- In some embodiments, the peptide activates regulatory T cells. In some embodiments, the peptide activates Qa-1-restricted or HLA-E-restricted regulatory CD8+ T cells.
- In some embodiments, methods of identifying a peptide that binds to Qa-1 or HLA-E and activates Qa-1-restricted or HLA-E-restricted regulatory CD8+ T cells are provided. In some embodiments, the method comprises retrieving a myelin oligodendrocyte glycoprotein (MOG)-specific epitope (peptide) that binds to Qa-1b, a non-classical MHCIb molecule. In some embodiments, the method comprises the use of an overlapping peptide (OLP) library of MOG peptide, such as an OLP library of murine MOG that contains 59 OLPs, which is described herein in the Examples section and referred to herein as “MOG_pool.” In some embodiments, the method comprises the use of the cell line C1R, a human B lymphoblastoid cell line and/or C1R.Qa-1b, a stable Qa-1 transfectant of C1R cells. An OLP library can be used to generate reactive CD8+ T cell lines, which can then be employed to screen individual OLPs in the OLP library and identify peptides that stimulate the reactive CD8+ T cell lines in a Qa-1b-restricted manner. In some embodiments, a Qa-1b-restricted response is determined by an OLP-stimulated increased IFN-γ secretion in the reactive CD8+ T cell lines in the presence of C1R.Qa-1b but not C1R cells.
- In some embodiments, the methods of identifying a peptide that binds to Qa-1 or HLA-E and activates Qa-1-restricted or HLA-E-restricted regulatory CD8+ T cells further comprise confirming whether the peptide binds to HLA-E, the human homolog of murine Qa-1. In some embodiments, the method comprises the use of a binding assay as described in the Examples section below and/or the use of a HLA-E-expressing cell line as described in the Examples section below (e.g., by incubating the peptide with the cell line 721.21-HLAE and using flow cytometry to determine whether HLA-E expression on the cell surface of 721.221-HLAE is upregulated after co-incubation with the peptide).
- In some embodiments, the methods of identifying a peptide that binds to Qa-1 or HLA-E and activates Qa-1-restricted or HLA-E-restricted regulatory CD8+ T cells further comprise confirming that MSIBE can bind to Qa-1b and activate MSIBE-specific Qa-1b restricted CD8+ T cells in vitro. In some embodiments, the method comprises the use of a binding assay as described in the Examples section below (e.g., tetramer formation and detection of tetramer+ cells).
- In some embodiments, the methods of identifying a peptide that binds to Qa-1 or HLA-E and activates Qa-1-restricted or HLA-E-restricted regulatory CD8+ T cells further comprise confirming that the peptide can activate specific HLA-E-restricted CD8+ Treg in vitro and thereby potentially suppress MS. In some embodiments, the method involves utilizing the peptide to activate HLA-E restricted CD8+ Treg in vivo. In some embodiments, the method includes MSIBE, peripheral blood mononuclear cells (PBMCs), MSIBE/HLA-E tetramer, and human MOG-specific CD4+ T cell lines. As a non-limiting example, CD8+ T cells can be purified from peripheral blood mononuclear cells (PBMCs) of healthy control individuals, stimulated with peptide-pulsed autologous monocytes at weekly basis, and monitored for binding to HLA-E (e.g., via formation of a peptide/HLA-E tetramer). When peptide/HLA-E tetramer+ cells reach more than 50%, the CD8+ T cell line is examined for its ability to specifically suppress proliferation of human MOG-but not control protein-specific CD4+ T cells using a CFSE-dilution assay.
- In some embodiments, the methods of identifying a peptide that binds to Qa-1 or HLA-E and activates Qa-1-restricted or HLA-E-restricted regulatory CD8+ T cells further comprise confirming that immunization with the peptide can activate Qa-1b-restricted CD8+ T cells in vivo and suppress EAE. Methods of inducing EAE in mice and immunizing mice with peptides or dendritic cells pulsed with peptides are described in the Examples section below. In some embodiments, suppression of one or more clinical disease symptoms indicates that the peptide is a peptide that binds to Qa-1 or HLA-E and activates Qa-1-restricted or HLA-E-restricted regulatory CD8+ T cells.
- In some embodiments, dendritic cells pulsed with a myelin oligodendrocyte glycoprotein (MOG) peptide that activates regulatory T cells are provided. In some embodiments, the peptide activates HLA-E-restricted or Qa-1-restricted regulatory CD8+ T cells. In some embodiments, the peptide comprises a MOG-specific MHC1b epitope. In some embodiments, the peptide comprises a HLA-E epitope. In some embodiments, the peptide comprises the amino acid sequence IICYNWLHR. In some embodiments, the peptide consists of the amino acid sequence IICYNWLHR. Methods for peptide pulsing dendritic cells are described herein in the Examples section. Methods for peptide pulsing dendritic cells are also known in the art. See, e.g., O'Neill et al., Methods Mol Med, 2005, 109:97-112.
- The dendritic cell can be obtained or derived from any suitable source. For example, the dendritic cell may be a bone marrow-derived dendritic cell, a cord blood-derived dendritic cell, or a peripheral blood-derived dendritic cell. In some embodiments, the dendritic cells are derived from monocytes. In some embodiments, the dendritic cells are derived from myelin-specific pathogenic autoimmune cells. Methods of generating dendritic cells are described herein in the Examples section. Methods of isolating and generating dendritic cells are also known in the art. See, e.g., Nair et al., Curr Protoc Immnol, 2012, doi: 10.1002/0471142735.im0732s99; Shen et al., J. Immnol., 1997, 158:2723-2730.
- In some embodiments, the dendritic cells are immunogenic dendritic cells. In some embodiments, the dendritic cells are tolerogenic dendritic cells.
- In some embodiments, the dendritic cell is an activated dendritic cell. In some embodiments, the dendritic cell is activated by stimulating the cell with a lipopolysaccharide (LPS) or with a cytokine (e.g., TNF-α).
- In some embodiments, the dendritic cell is treated with an anti-proliferative agent. In some embodiments, the anti-proliferative agent is irradiation (e.g., gamma irradiation). In some embodiments, the anti-proliferative agent is a chemical compound (e.g., mitomycin C).
- In some embodiments, the composition comprises a peptide as described herein or a peptide-pulsed dendritic cell as described herein and further comprises a pharmaceutically acceptable carrier and/or excipient. Guidance for preparing formulations for use in the present invention is found in, for example, Remington: The Science and Practice of Pharmacy, 21st Edition, Philadelphia, Pa., Lippincott Williams & Wilkins, 2005.
- A pharmaceutically acceptable carrier includes any solvents, dispersion media, or coatings that are physiologically compatible and that preferably does not interfere with or otherwise inhibit the activity of the therapeutic agent. In some embodiments, the carrier is suitable for intravenous, intramuscular, oral, intraperitoneal, transdermal, topical, or subcutaneous administration. Pharmaceutically acceptable carriers can contain one or more physiologically acceptable compound(s) that act, for example, to stabilize the composition or to increase or decrease the absorption of the active agent(s). Physiologically acceptable compounds can include, for example, carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, compositions that reduce the clearance or hydrolysis of the active agents, or excipients or other stabilizers and/or buffers. Other pharmaceutically acceptable carriers and their formulations are well-known and generally described in, for example, Remington: The Science and Practice of Pharmacy, supra. Various pharmaceutically acceptable excipients are well-known in the art and can be found in, for example, Handbook of Pharmaceutical Excipients (7th edition, Rowe et al. (Eds.), Pharmaceutical Press, 2012).
- In addition, various adjuvants such as are commonly used in the art may be included. Considerations for the inclusion of various components in pharmaceutical compositions are described, e.g., in Goodman and Gilman's: The Pharmacological Basis of Therapeutics, 12th edition, Brunton (Ed.), McGraw-Hill Education, 2011, which is incorporated herein by reference in its entirety.
- The compositions described herein may be in any of a variety of suitable forms for a variety of routes for administration, for example, for oral, nasal, rectal, topical (including transdermal), ocular, intracerebral, intracranial, intrathecal, intra-arterial, intravenous, intramuscular, or other parental routes of administration. The skilled artisan will appreciate that oral and nasal compositions include compositions that are administered by inhalation, and made using available methodologies. Depending upon the particular route of administration desired, a variety of pharmaceutically acceptable carriers well-known in the art may be used.
- Various oral dosage forms can be used, including such solid forms as tablets, capsules, granules and bulk powders. Tablets can be compressed, tablet triturates, enteric-coated, sugar-coated, film-coated, or multiple-compressed, containing suitable binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents. Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules, and effervescent preparations reconstituted from effervescent granules, containing suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, melting agents, coloring agents and flavoring agents.
- For intravenous administration, the compounds and compositions described herein may be dissolved or dispersed in a pharmaceutically acceptable diluent, such as a saline or dextrose solution. Suitable excipients may be included to achieve the desired pH, including but not limited to NaOH, sodium carbonate, sodium acetate, HCl, and citric acid. In various embodiments, the pH of the final composition ranges from 2 to 8, or preferably from 4 to 7. Antioxidant excipients may include sodium bisulfite, acetone sodium bisulfite, sodium formaldehyde, sulfoxylate, thiourea, and EDTA. Other non-limiting examples of suitable excipients found in the final intravenous composition may include sodium or potassium phosphates, citric acid, tartaric acid, gelatin, and carbohydrates such as dextrose, mannitol, and dextran. Further acceptable excipients are described in Powell et al., “Compendium of Excipients for Parenteral Formulations,” FDA J Pharm Sci and Tech, 1998, 52:238-311, and Nema et al., “Excipients and Their Role in Approved Injectable Products: Current Usage and Future Directions,” FDA J Pharm Sci and Tech, 2011, 65:287-332, both of which are incorporated herein by reference in their entirety. Antimicrobial agents may also be included to achieve a bacteriostatic or fungistatic solution, including but not limited to phenylmercuric nitrate, thimerosal, benzethonium chloride, benzalkonium chloride, phenol, cresol, and chlorobutanol.
- The compositions for intravenous administration may be provided in the form of one more solids that are reconstituted with a suitable diluent such as sterile water, saline or dextrose in water shortly prior to administration. In other embodiments, the compositions are provided in solution ready to administer parenterally. In still other embodiments, the compositions are provided in a solution that is further diluted prior to administration.
- In some embodiments, a composition as described herein is provided in unit dosage form. As used herein, a “unit dosage form” is a composition containing an amount of a compound that is suitable for administration to an animal, preferably mammal subject, in a single dose, according to good medical practice. The preparation of a single or unit dosage form however, does not imply that the dosage form is administered once per day or once per course of therapy. Such dosage forms can be administered once, twice, thrice or more per day and may be administered as infusion over a period of time (e.g., from about 30 minutes to about 2-6 hours), or administered as a continuous infusion, and may be given more than once during a course of therapy, though a single administration is not specifically excluded. The skilled artisan will recognize that the formulation does not specifically contemplate the entire course of therapy and such decisions are left for those skilled in the art of treatment.
- In another aspect, kits comprising the peptides and/or dendritic cells disclosed herein are provided. In some embodiments, a kit further comprises instructional materials containing directions (i.e., protocols) for the practice of the methods of this invention (e.g., instructions for using the kit for treating a demyelinating disease, e.g., multiple sclerosis). While the instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.
- In another aspect, methods of treating or preventing a demyelinating disease are provided. In some embodiments, methods of treating a demyelinating disease are provided. In some embodiments, methods of preventing or delaying the onset of a demyelinating disease are provided. In some embodiments, the method comprises administering to a subject (e.g., a subject having a demyelinating disease or a subject at risk of having or suspected of having a demyelinating disease) a composition as described herein (e.g., a peptide comprising or consisting of a MOG-specific MHC1b epitope, a composition comprising an isolated peptide comprising or consisting of a MOG-specific MHC1b epitope, or a composition comprising dendritic cells pulsed with an isolated peptide comprising or consisting of a MOG-specific MHC1b epitope).
- In some embodiments, the demyelinating disease is selected from the group consisting of multiple sclerosis, idiopathic inflammatory demyelinating disease, transverse myelitis, Devic's disease, progressive multifocal leukoencephalopathy, optic neuritis, leukoystrophy, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, autoimmune peripheral neuropathy, Charcot-Marie-Tooth disease, acute disseminated encephalomyelitis, adrenoleukodystrophy, adrenomyeloneuropathy, Leber's hereditary optic neuropathy, or HTLV-associated myelopathy. In some embodiments, the demyelinating disease is multiple sclerosis.
- In some embodiments, the demyelinating disease is a disease characterized by the expression or overexpression of antibodies against MOG. In some embodiments, the demyelinating disease that expresses or overexpresses antibodies against MOG is multiple sclerosis, transverse myelitis, optic neuritis, or acute disseminated encephalomyelitis.
- In some embodiments, the subject has multiple sclerosis (MS). There are several subtypes of MS, including relapsing-remitting multiple sclerosis (RRMS), secondary progressive multiple sclerosis (SPMS), primary progressive multiple sclerosis (PPMS), and progressive relapsing multiple sclerosis (PRMS). In some embodiments, the subject has RRMS. In some embodiments, the subject has SPMS. In some embodiments, the subject has PPMS. In some embodiments, the subject has PRMS. A subject may initially be diagnosed as having one subtype of MS (e.g., RRMS), and subsequently the subtype of MS afflicting the subject may convert to another subtype of MS (e.g., from RRMS to SPMS). It is contemplated that the therapeutic methods disclosed herein can be applied to treat a subject whose subtype of MS converts to another subtype of MS.
- In some embodiments, a MOG peptide or dendritic cells pulsed with a myelin oligodendrocyte glycoprotein (MOG) peptide as described herein are used as a peptide vaccine for the prevention or treatment of MS or EAE. In some embodiments, the method comprises generating a vaccine comprising MSIBE and human dendritic cells (DCs). In some embodiments, the method of generating a vaccine comprises generating dendritic cells from monocytes that are autologous to the subject. In some embodiments, the method comprises generating dendritic cells from monocytes that are allogeneic to the subject. In some embodiments, the dendritic cells are generated in the presence of one or more cytokines (e.g., GM-CSF or interleukins, e.g., IL-4). In some embodiments, the dendritic cells are treated with an anti-proliferative agent. In some embodiments, the dendritic cells are pulsed with a peptide as described herein (e.g., MSIBE) and subsequently administered to a subject having MS. As a non-limiting exemplary embodiment, dendritic cells (e.g., 1×106 human DCs) can be generated from autologous monocytes in the presence of GM-CSF and IL-4, pulsed with a peptide as described herein (e.g., MSIBE), and injected subcutaneously or intravenously into MS patients.
- In some embodiments, the method comprises optimizing immunization for prevention and therapy of MS or EAE. For example, in some embodiments, immunization with a MOG peptide or dendritic cells pulsed with a MOG peptide can be administered at varying amounts, or multiple immunizations can be performed, to improve the therapeutic response.
- The route of administration of a peptide, dendritic cell, or composition as described herein (e.g., a peptide comprising or consisting of a MOG-specific MHC1b epitope, a composition comprising an isolated peptide comprising or consisting of a MOG-specific MHC1b epitope, or a composition comprising dendritic cells pulsed with an isolated peptide comprising or consisting of a MOG-specific MHC1b epitope, e.g., as described in Section III above) can be oral, intraperitoneal, transdermal, subcutaneous, intravenous, intramuscular, inhalational, topical, intralesional, rectal, intrabronchial, intralymphatic, intradermal, nasal, transmucosal, intestinal, ocular or otic delivery, or any other methods known in the art. In some embodiments, a peptide, dendritic cell, or composition as described herein is administered by intravenous injection or by subcutaneous injection. In some embodiments, a peptide, dendritic cell, or composition as described herein is administered systemically. In some embodiments, a peptide, dendritic cell, or composition as described herein is administered locally.
- Dosages and desired concentrations of the peptides or dendritic cells of the disclosure may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration is well within the skill of one in the art. Typically the amount administered to a subject is a therapeutically effective amount. In some embodiments, a therapeutically effective amount of a peptide, dendritic cell, or composition as described herein is an amount that prevents or reverses one or more symptoms of the disease (e.g., MS). In some embodiments, a therapeutically effective amount of a peptide, dendritic cell, or composition as described herein is administered about once per day, once per week, twice per week, once per month, or twice per month.
- The peptides, dendritic cells, and compositions as described herein may be administered to a subject in need thereof for a predetermined time, an indefinite time, or until an endpoint is reached. In some embodiments, treatment is continued on a continuous daily or weekly basis for at least two to three months, six months, one year, or longer. In some embodiments, treatment is for at least 30 days, at least 60 days, at least 90 days, at least 120 days, at least 150 days, or at least 180 days. In some embodiments, treatment is continued for at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least one year. In some embodiments, treatment is continued for the rest of the patient's life or until administration is no longer effective to provide meaningful therapeutic benefit.
- In some embodiments, the peptides, dendritic cells, and compositions as described herein are administered in combination with one or more additional agents (e.g., a therapeutic agent). In some embodiments, the two or more agents are administered at the same time or substantially the same time. In some embodiments, the two or more agents are administered sequentially. In some embodiments, the two or more agents are administered through the same route (e.g., intravenously). In some embodiments, the two or more agents are administered through different routes (e.g., the peptide, dendritic cell, or composition as described herein is administered intravenously and the additional agent is administered orally).
- The following example are offered to illustrate, but not to limit, the claimed invention.
- Mice:
- C57BL/6 (B6) and Kb−/−Db−/− mice were obtained from Taconic Farms and housed in a specific pathogen-free animal facility at the University of Texas at El Paso (UTEP) or Loma Linda University (LLU). All experiments were done in compliance with an Institutional Animal Care and Use Protocol approved by UTEP and/or LLU Animal Care and Use Committee.
- Cell Lines:
- C1R is a human B lymphoblastoid cell line. C1R.Qa-1b is a stable transfectant that constitutively expresses Qa-1b. DC2.4 is a DC line generated from bone-marrow-derived DCs.
- Reagents:
- Peptides used m this study were synthesized at Genemed Synthesis, Inc., San Antonio, Tex.
- IFN-γ Enzyme-Linked Immunospot (EL/SPOT) Assay:
- ELISPOT plates were coated with an anti-mouse IFN-γ mAb (5 μg/ml in PBS) at 4° C. overnight, blocked with culture medium for 2 hours at room temperature, and added with desired numbers of CD8+ T cells, peptides, and irradiated antigen-presenting cells (APCs). After cultured at 37° C. and 5% CO2 for 16 hours, plates were incubated with a biotinylated anti-mouse IFN-γ mAb (2 μg/ml) for 2 hours followed by streptavidin-conjugated HRP (horseradish peroxidase) for 1 hour. Finally, plates were incubated with a substrate, monitored for spot development, and washed with distilled water when spots were fully developed. After air-drying, plates were analyzed on an ELISPOT plate reader for enumerating spots in each well.
- In Vitro Refolding of a Peptide with Recombinant Qa-1b Protein:
- Refolding of MOG196 with recombinant Qa-1b protein was performed in the NIH tetramer core facility (Emory University, Atlanta, Ga.) (Altman et al., Science, 1996, 274:94-96).
- Induction of MOG35-55-Induced EAE:
- C57BL/6 mice were immunized subcutaneously with 200 μg MOG35-55 emulsified in Incomplete Freund Adjuvant (IFA) supplemented with 250 μg heat-inactivated mycobacterium tuberculosis H37Ra (Difeo Laboratories, Michigan, USA). On
day - Generation of Bone Marrow-Derived Dendritic Cells (DCs):
- Bone marrow single cell suspensions (1×106 cells/ml) containing 10 U/ml IL-4 and 100 U/ml GM-CSF were seeded into a 6-well plate (4 ml/well) and cultured at 37° C., 5% CO2. On
day 2, after non-adherent cells were carefully removed, plates were replenished with fresh media and cytokines. Onday 4, non-adherent cells containing fresh media and cytokines were transferred into a new 6-well plate. Onday 6, cells were replenished with fresh media and cytokines and stimulated with 0.1 g/ml LPS overnight. Onday 7, cells were collected for experiments. - Mitomycin C Treatment of DCs and Peptide Pulsing:
- LPS activated DCs at the concentration of 5×107 cells/ml in PBC were treated by mitomycin C (50 μg/ml) for 20 minutes at 37° C. and 5% CO2 (DC2.4 cells were treated for 30 minutes). The treated DCs were then washed for three times and adjusted to 5×106 cells/ml in serum-free medium containing 100 μg/ml MOG196 or HSP60P216 peptide. The cells were pulsed with the peptides for three to four hours at room temperature. After pulsing, the cells were washed once, reconstituted in PBS, and intravenously (or subcutaneously) injected into animals at 200 μl/mouse (5×105 cells or 1×106 cells/mouse).
- Multichromatic Flow Cytometry:
- Briefly, about 0.5-1×106 cells in a FACS buffer (PBS containing 1% FBS and 0.05% sodium azide) were stained with various fluorescence-conjugated antibodies specific for the desired cell surface proteins or with tetramers at 4° C. for 30 min. The stained cells were washed twice in the FACS buffer before being analyzed on a BD FACSAria II.
- Statistical Analysis:
- All the statistical analyses were performed using One-Way or Two-Way ANOVA, followed by the SNK-q test or the Dunnett's Multiple Comparison test. A P-value less than 0.05 was considered statistically significant.
- A Portion of CD8+ T Cells in the CD8+ T Cell Lines Reactive to the Pool of Overlapping Peptides (OLPs) Covering the Whole Length of Mouse MOG was Qa-1b Restricted.
- Current data suggest that Qa-1-restricted CD8+ Treg cells can target pathogenic autoimmune cells (Jiang et al., Immunity, 1995, 2:185-194) and suppress EAE, an animal model of human MS. In this case, the CD8+ T cells achieve the targeting by recognizing regulatory Qa-1 epitopes that are expressed in the myelin-specific pathogenic autoimmune cells (Tang et al., J. Immunol., 2006, 177:7645-7655; Wu et al., Proc Natl. Acad Sci USA, 2009, 106:534-549; Panoutsakopoulou et al., J. Clin Invest, 2004, 113:1218-1224). These regulatory Qa-1 epitopes have been difficult to define in humans because pathogenic autoimmune cells by themselves are hard to determine in MS patients. This obstacle had previously prevented further clinical translation of the Qa-1-restricted CD8+ Treg cells.
- Pathogenic autoimmune cells in EAE and MS mainly attack myelin sheath. It was investigated whether Qa-1-restricted CD8+ Treg cells could specifically target myelin sheath as well, and to determine if regulatory Qa-1b epitopes which are the targets of Qa-1-restricted CD8+ Treg cells were present in mouse MOG since MOG is one of the myelin proteins in myelin sheath. In order to map potential Qa-1b epitopes in mouse MOG, a 15mer OLP library that covered the whole length of mouse MOG (247 amino acids) was generated from N- to C-termini. All of the OLPs in this library were 15 amino acids in length and overlapped by 11 amino acids. Hence the OLP library contained 59 OLPs in total (
FIG. 1A ). A pool of the 59 OLPs (MOG_pool), which contained a final concentration of 4.2 μg/ml for each individual peptide, was used to stimulate CD8+ T cells purified from Kb−/−Db−/− mice for generating MOG_pool-reactive CD8+ T cell lines in vitro. Here, CD8+ T cells that were purified from Kb−/−Db−/− mice were utilized because CD8+ T cells in these mice were restricted mostly by non-classical MHC Ib molecules including Qa-1b. During in vitro stimulations, CD8+ T cell lines were monitored weekly for response, using IFN-γ Enzyme-linked ImmunoSpot, to the MOG_pool in the presence of either C1R or C1R.Qa-1b cells. The data showed that most CD8+ T cell lines that were generated specifically responded to the MOG_pool in the presence of both C1R and C1R.Qa-1b cells (FIG. 1B andFIG. 1C ). However, response to the MOG_pool was much stronger when C1R.Qa-1b cells were present. The data therefore suggested that portion of the CD8+ T cells in the lines responded to the MOG_pool in a Qa-1b-dependent (or Qa-1-restricted) manner. - Recognition of Multiple OLPs by the MOG_Pool-Reactive CD8+ T Cell Lines Depended on Qa-1b.
- Next the question of which individual OLPs in the MOG_pool were recognized by the MOG_pool-reactive CD8+ T cell lines in a Qa-1b-restricted manner was investigated. Thus, the 59 individual OLPs were interrogated individually for their ability to stimulate the MOG_pool-reactive CD8+ T cell lines in the presence of either C1R or C1R.Qa-1b cells as antigen-presenting cells (
FIG. 2 ). Data showed that most OLPs provided stronger stimulation of the MOG_pool-reactive CD8+ T cell lines in the presence of C1R.Qa-1b cells as compared to C1R cells (FIGS. 2B-D ). However, only three OLPs, i.e., OLP68, OLP96, and OLP105, consistently stimulated the CD8+ T cell lines in a Qa-1b-restricted manner. A detailed analysis of these three OLPs was performed. Accordingly, Kb−/−Db−/− mice were immunized individually with the OLP68, OLP96, or OLP105. Ten days later, CD8+ T cells were purified from draining lymph nodes and stimulated with corresponding peptides used for immunization. One week later, the CD8+ T cells were examined for response, using IFN-γ Enzyme-linked ImmunoSpot, to the corresponding peptides in the presence of either C1R or C1R.Qa-1b cells. The data showed that all three OLPs stimulated CD8+ T cells in a Qa-1b-restricted manner (FIGS. 2E and 2F ), suggesting that all three OLPs contained Qa-1b epitopes. - MOG195-204 (“MOG196”) was the Minimal and Optimal Qa-1b Epitope in OLP105.
- Since OLP stimulated, in most assays, the highest number of SFCs (spot-forming cells)/106 CD8+ T cells in the MOG_pool-reactive CD8+ T cell lines in the presence of C1R.Qa-1b cells, the minimal and optimal Qa-1b epitope in this OLP was analyzed. Thus, progressively N- and C-terminally truncated peptides of OLP105 down to 6mers were synthesized because MHC I molecules can present epitopes of 7-10mers. These truncated peptides and the original OLP105 were then examined for their ability to stimulate, using IFN-γ Enzyme-linked ImmunoSpot, an OLP105-reactive CD8+ T cell line in the presence of C1R.Qa-1b cells. Our data demonstrated that a 9mer peptide, IICYNWLHR, was the minimal and optimal epitope in OLP105 (
FIG. 3 ). - MOG196 Binds to Qa-1b and Activates MOG196-Specific Qa-1b-Restricted CD8+ T Cells In Vitro.
- To further confirm that MOG196 was a Qa-1b epitope, we next asked whether MOG196 could bind to Qa-1b by addressing whether this 9mer peptide could successfully refold with recombinant Qa-1b protein in vitro. Thus, MOG196 was incubated with recombinant Qa-1b protein and β2m at 10° C. for four days. The resulting solution was analyzed in a size exclusion column and displayed a distinct protein peak (
FIG. 4A , peak B), suggesting formation of MOG196/Qa-1b/β2m monomer. When the monomer was further analyzed in an anion exchange column, the monomer displayed two protein peaks (FIG. 4B , peak B1 and B2). To further address potential reasons for the two protein peaks in the anion exchange column, proteins in peaks A, B1, and B2 were biotinylated. Portions of the biotinylated proteins were incubated with streptavidin that was able to bind four biotinylated proteins to form tetramers. Subsequently, the biotinylated proteins and streptavidin-conjugated tetramers were analyzed in a non-denature protein gel. Data showed that biotinylated proteins in peak A did not show any distinct protein band (FIG. 4C , lane 1), supporting that this peak contained mainly non-specific protein aggregates. In contrast, biotinylated proteins in peak B1 displayed a single protein band (FIG. 4C , lane 3), indicating correct formation of MOG196/Qa-1b/β2m monomer. Interestingly, biotinylated proteins in peak B2 exhibited two protein bands (FIG. 4C , lane 5), suggesting that some proteins in this peak were not correctly refolded. Furthermore, addition of streptavidin successfully conjugated biotinylated proteins in peak B1 and B2 into tetramers (FIG. 4C ,lanes 4 and 6). The data therefore demonstrated that MOG196 could bind to Qa-1b. - To investigate whether MOG196 could be presented by antigen-presenting cells to activate MOG196-specific Qa-1b-restricted CD8+ T cells, CD8+ T cells purified from C57Bl/6 mice were stimulated in vitro weekly by MOG196-pulsed antigen-presenting cells derived from either Kb−/−Db−/− or C57Bl/6 mice. Data demonstrated that, beginning on
day 14 after in vitro re-stimulation, Qa-11b/MOG196 tetramer+ cells could be detected in the CD8+ T cell lines (FIG. 4D ), indicating successful activation of MOG196-specific Qa-1b-restricted CD8+ T cells. In addition, antigen-presenting cells derived from both Kb−/−Db−/− and C57Bl/6 mice were able to present MOG196 to activate MOG196-specific Qa-1b-restricted CD8+ T cells in vitro (FIG. 4D ). - Vaccination with MOG196-Pulsed DCs Suppressed MOG35-55-Induced EAE.
- To address whether MOG196 is a biologically relevant Qa-1b epitope, an investigation was performed to determine if immunization with the epitope-pulsed DCs ameliorated MOG35-55 EAE. Thus, C57Bl/6 (B6) mice were immunized with MOG196-pulsed Kb−/−Db−/− DCs one week before and one week after the EAE induction. The data clearly showed that immunization with MOG196 but not Qdm-pulsed Kb−/−Db−/− DCs significantly ameliorated the paralytic disease (
FIGS. 5A-5B and Table 1). -
TABLE 1 Vaccination with MOG196-pulsed Kb−/−Db−/− dendritic cells suppressed MOG35-55 induced experimental autoimmune encephalomyelitis # of animals with disease/ Mean # of total animals Mean days maximal (peak scores of of disease disease Treatments individual animals) onset score No Tx 1 5/5 (4, 5, 4, 5, 4) 10.8 ± 0.8 4.4 ± 0.5 DCs/ Qdm 24/5 (5, 5, 4, 0, 3) 11.0 ± 0.8 3.4 ± 2.1 DCs/ MOG196 31/5 (0, 0, 0, 0, 3) 11 ± 0.0 0.6 ± 1.3 1No treatment 2Qdm-pulsed Kb−/−Db−/− dendritic cells 3MOG196-pulsed Kb−/−Db−/− dendritic cells - To evaluate the potential for wild-type DCs to ameliorate EAE, B6 mice were vaccinated with MOG196-pulsed B6 DCs on days −3, 2, and 7. On
day 0, mice were immunized with MOG35-55 for EAE. The data again showed that vaccination with wild-type DCs pulsed with MOG196, but not Qdm or HSP60p216, significantly suppressed paralytic disease (FIGS. 5C-5D and Table 2). -
TABLE 2 Vaccination with MOG196-pulsed C57BI/6 dendritic cells suppressed MOG35-55 induced experimental autoimmune encephalomyelitis # of animals with disease/ Mean # of total animals Mean days maximal (peak scores of of disease disease Treatments individual animals) onset score DCS/ HSP60 p216 15/5 (3.5, 3.5, 3.5, 2.5, 2) 14.2 ± 1.0 3.1 ± 0.3 DCs/ Qdm 25/5 (3.5, 3.5, 3.5, 2.5, 2) 15 ± 1.1 3.2 ± 0.3 DCs/ MOG196 33/5 (2.5, 2.5, 1.5, 0, 0) 16.7 ± 0.3 1.3 ± 0.6 1HSP60p216-pulsed C57Bl/6 dendritic cells 2Qdm-pulsed C57Bl/6 dendritic cells 3MOG196-pulsed C57Bl/6 dendritic cells - Therefore, this epitope may be essential in maintaining immune homeostasis in the central nervous system (CNS).
- Vaccination with MOG196-Pulsed DCs Suppressed Ongoing MOG34-55 EAE, which was Dependent on CD8+ T Cells.
- Next, it was investigated whether immunization with the MOG196-pulsed mature B6 DCs could suppress ongoing EAE and whether the disease suppression depended on CD8+ T cells. Hence, C57Bl/6 mice were immunized with MOG35-55 for EAE on
day 0. In one group, the animals received an intra-peritoneal injection of a monoclonal anti-CD8 antibody at days −2, −1, 7 and 14 to deplete CD8+ T cells. Atday 10, animals received no treatment or one intravenous injection of 5×105 MOG196-pulsed B6 DCs. The data showed that, as compared to no treatment, one intravenous injection of 5×105 MOG196-pulsed B6 DCs robustly suppressed the ongoing paralytic disease (FIGS. 6A-6B and Table 3). -
TABLE 3 Suppression of ongoing MOG35-55 induced experimental autoimmune encephalomyelitis by MOG196 immunization was dependent on CD8+ T cells # of animals with disease/ Mean # of total animals Mean days maximal (peak scores of of disease disease Treatments individual animals) onset score No Tx 1 5/5 (3.5, 4, 4, 4, 3) 8.4 ± 0.9 3.7 ± 0.4 DCs/ Qdm 25/5 (3, 1.5, 3, 0.5, 0.5) 8 ± 0 1.7 ± 1.3 DCs/ MOG196 34/5 (3, 5, 3, 3)4 8.3 ± 0.5 3.5 ± 1 1No treatment 2MOG196-pulsed C57Bl/6 dendritic cells 3MOG196-pulsed C57Bl/6 dendritic cells + anti-CD8 mAb 4One animal that died before treatment was excluded from this analysis. - Depletion of CD8+ T cells abrogated the protective effect of the DC/MOG196. Therefore, the data demonstrated that suppression of ongoing EAE by the MOG196-pulsed DC was dependent on CD8+ T cells.
- Immunization with MOG196-pulsed DCs activated Qa-1b/MOG196 tetramer+ cells that accumulated in the cervical lymph nodes in EAE-bearing animals. Next, it was investigated whether immunization with MOG196-pulsed DCs indeed activated Qa-1b/MOG196 tetramer+ cells in vivo in EAE-bearing animals. Hence, C57BL/6 mice were induced for EAE. Ten days later, when the paralytic disease began, animals received mature DC2.4 cells (a bone-marrow-derived DC line) or MOG196-pulsed DC2.4 cells (DC2.4/MOG196). Four days after the treatments, spleens and cervical lymph nodes were analyzed for the presence of Qa-1b/MOG196tetramer+ cells. The data showed that percent of Qa-1b/MOG196tetramer+ cells in spleens between the two treatments was similar, while percent of tetramer+ cells in cervical lymph nodes following the DC2.4/MOG196 treatment, as compared to DC2.4 treatment, was significantly elevated (
FIGS. 7A, 7B and 7C ). The data suggested that the Qa-1b/MOG196tetramer+ cells specifically accumulated in the CNS in EAE-bearing animals. In contrast, I-Ab/MOG38-49 tetramer+ cells, which detected MOG35-55-reactive pathogenic autoimmune cells, were significantly reduced in cervical lymph nodes (FIGS. 7A, 7D, and 7E ). The data were consistent with suppression of ongoing paralytic disease following DC2.4/MOG196 treatment (FIG. 6 ). - CD122 and Ly49 were Expressed in a Portion of the Qa-1b/MOG196 Tetramer+ Cells.
- Recent data suggest that Qa-1-restricted CD8+ Treg cells are among CD122+Ly49+ CD8+ T cells (Tang et al., J. Immunol., 2006, 177:7645-7655; Kim et al., Proc Natl. Acad Sci USA, 2011, 108:2010-2015; Leavenworth et al., J Clin Invest, 2013, 123:1382-1389). Expression of these two markers on the Qa-Ib/MOG196 tetramer+ cells was analyzed. The data showed that about 22.6% of the tetramer+ cells expressed CD122 and about 7.2% of the tetramer+ cells expressed both CD122 and Ly49 (
FIG. 7F-7G ). Therefore, the data suggest that CD122 and Ly49 are two markers for Qa-1-restricted CD8+ Treg cells. - MOG196 is Evolutionarily Conversed and Unique to MOG.
- Some known Qa-1-binding peptides, e.g., Qdm and HSP60p216, bind to both Qa-1 and HLA-E (Miller et al., J. Immunol., 2003, 171:1369-1375). In addition, peptide binding motifs of Qa-1 and HLA-E are similar (Miller et al., supra; Kurepa et al., J. Immunol., 1997, 158:3244-3251). It was investigated whether the MOG196 peptide sequence was evolutionarily conserved. A Blast search of GenBank protein sequence data showed that MOG196 sequence was evolutionarily conserved (
FIG. 8 ). Furthermore, sequence of MOG196 is specific to MOG and located in the intracellular domain (FIG. 9 ). Therefore, HLA-E can be utilized to suppress MS in a similar manner as Qa-1 is used to suppress EAE. - Proposed Mechanism Underlying the Disease Suppression Mediated by the Myelin-Specific, Qa-1 (HLA-E)-Restricted CD8+ Treg Cells.
- Previous data suggest that disease suppression involves IFN-γ, perforin, and IL-15. These data support a direct killing of target cells by the Qa-1-restricted CD8+ Treg cells. Since oligodendrocytes normally do not express MHC molecules (Goverman, Nat Rev Immunol, 2009, 9:393-407), it may explain that the MOG196-specific Qa-1-restricted CD8+ Treg cells do not damage myelin. Thus, the same antigen presenting cell, which phagocytoses myelin, can present both pathogenic and regulatory myelin epitopes to pathogenic CD4+ and regulatory CD8+ T cells respectively because both epitopes are located in the myelin (
FIG. 10 ). In this regard, recognition of the regulatory Qa-1/HLA-E myelin epitopes by the CD8+ Treg cells leads to 1) elimination of and/or induction of tolerance in the antigen-presenting cells; 2) elimination and/or induction of tolerance in encephalitogenic T cells that have obtained the epitope complexes from antigen-presenting cells via a process called trogocytosis. Consequently, activation and proliferation of encephalitogenic T cells are thwarted and autoimmune attacks of myelin sheath are stopped. - Since it has been demonstrated that MOG196 (i.e., the regulatory Qa-1 epitope) and MOG35-55 (i.e., the pathogenic epitope) are both located in MOG that is the autoantigen (
FIG. 10 ), the two types of epitopes should be presented by the same APCs. Consequently, MOG196-specific Qa-1-restricted CD8+ Treg, functionally enhanced by MOG196 immunization, can specifically recognize and suppress APCs that have phagocytosed MOG and otherwise initiated or perpetuated EAE. - The present disclosure provides evidence of an evolutionarily conserved regulatory Qa-1 epitope, MOG196, in mouse myelin oligodendrocyte glycoprotein (MOG). The discovery of the regulatory Qa-1 epitope in MOG is significant for several reasons. First, it reveals another potential regulatory pathway wherein Qa-1-restricted CD8+ Treg cells, besides targeting autoreactive T cells, may also target an autoantigen to suppress an autoimmune disease. Second, because of their specificity for an autoantigen, Qa-1-restricted CD8+ Treg cells activated by a regulatory Qa-1 epitope derived from an autoantigen, as compared to autoreactive T cells, may potentially accumulate in the diseased tissues and thereby more efficiently suppress autoimmune attacks. Third, because more autoantigens have been discovered in various autoimmune diseases and because generation of autoreactive T cells is lengthy and tedious, autoantigens are more accessible than autoreactive T cells.
- Immunization with MOG196-pulsed immunogenic dendritic cells robustly suppressed EAE, which was abrogated by depletion of CD8+ T cells. Therefore, immunization with DCs pulsed with myelin-derived HLA-E epitopes has been shown to enhance function of myelin-specific HLA-E-restricted CD8+ Treg cells and thus represents a novel antigen-specific therapy for MS.
- Studies of Qa-1 (HLA-E)-mediated antigen-specific therapies have been focused on targeting pathogenic autoimmune CD4+ T cells. Because efficient entering of CD8+ T cells into CNS depends on presentation of CNS-antigens at the blood-brain barrier (BBB) (Galea et al., J. Exp. Med, 2007, 204:2023-2030), Qa-1-restricted CD8+ Treg cells which target epitopes in pathogenic autoimmune CD4+ T cells may not efficiently accumulate in the CNS due to a lack of specificity for CNS and therefore may predominantly suppress the pathogenic CD4+ T cells in the peripheral lymphoid organs. In contrast, myelin-specific Qa-1-restricted CD8+ Treg cells described here will have the advantage to cross the BBB and provide in situ suppression of demyelinating inflammation in the CNS. To support this notion, the data herein shows that, in the course of EAE, MOG196-specific, Qa-1-restricted CD8+ Treg cells specifically accumulated in the cervical lymph nodes (i.e., the draining lymph nodes for CNS).
- With respect to potential mechanisms underlying the disease suppression mediated by the myelin-specific, Qa-1 (HLA-E)-restricted CD8+ Treg cells, the disease suppression involves IFN-γ, perforin, and IL-15 (Beeston et al., J. Neuroimmunol., 2010, 229:91-97; Kim et al., Nature, 2010, 467:516-523). These data support a direct killing of target cells by the Qa-1-restricted CD8+ Treg cells. Since oligodendrocytes normally do not express MHC molecules (Goverman, Nat Rev Immunol, 2009, 9:393-407), it may explain that the MOG196-specific Qa-1-restricted CD8+ Treg cells do not damage myelin. It is proposed that the same antigen presenting cell, which phagocytoses myelin, can present both pathogenic and regulatory myelin epitopes to pathogenic CD4+ and regulatory CD8+ T cells respectively because both epitopes are located in the myelin (
FIG. 10 ). In this regard, recognition of the regulatory Qa-1/HLA-E myelin epitopes by the CD8 Treg cells leads to 1) elimination of and/or induction of tolerance in the antigen-presenting cells; 2) elimination and/or induction of tolerance in encephalitogenic T cells that have obtained the epitope complexes from antigen-presenting cells via a process called trogocytosis. Consequently, activation and proliferation of encephalitogenic T cells are thwarted and autoimmune attacks on myelin sheath are stopped. - A recent finding showed that neuroantigen-specific CD8+ T cells are regulatory. See, Ortega et al., Neural Neuroimmnunol Neuroinflamm, 2015, 2:e170. However, the neuroantigen-specific CD8+ Treg cells differ from myelin-specific Qa-1-restricted CD8+ Treg cells in requiring classical MHC I molecules for presentation. In addition, priming of the neuroantigen-specific CD8+ Treg cells requires the presence of regulatory and pathogenic epitopes m the same myelin protein. This second characteristic suggests that active immunization with such a regulatory neuroantigenic epitope is not a suitable therapy for MS. In contrast, the myelin-specific CD8+ Treg cells described here can be primed through active immunization with DCs pulsed with the regulatory epitope alone.
- All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and materials in connection with which the publications are cited.
- The inventions have been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.
- It should be understood that although the present invention has been specifically disclosed by certain aspects, embodiments, and optional features, modification, improvement, and variation of such aspects, embodiments, and optional features can be resorted to by those skilled in the art, and that such modifications, improvements, and variations are considered to be within the scope of this disclosure.
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