Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/0005—Vertebrate antigens
  • A61K39/0008—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
  • A61K40/41—Vertebrate antigens
  • A61K40/416—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55544—Bacterial toxins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59

Definitions

• the present disclosure generally relates to compositions and methods for the treatment, prevention, or reversal of neuroinflammation-related disorders using fragments of MHC -bound proteins.
• MHC major histocompatibility complex
• a pharmaceutical composition which may comprise one or more peptides and a pharmaceutically acceptable excipient.
• Each of the one or more peptides may bind to a Major Histocompatibility Complex (MHC) molecule and may be a fragment of a protein selected from TABLE Cl, or a modified fragment thereof.
• Tire protein or peptide may be expressed at elevated levels in the central nervous system (CNS) relative to a control tissue. In one example, an expression level of the protein or peptide is 5 -fold higher than the control tissue. Alternatively, the protein may not be expressed at elevated levels in the CNS or may be expressed at decreased the levels in the CNS relative to a control tissue.
• the CNS tissue is dural meninges and the control tissue is a lymph node, such as a deep cervical lymph node or a superficial cervical lymph node.
• Each of the one or more peptides may independently comprise or consist of the amino acid sequence set forth in any one of SEQ ID NOS: 1- 14619, or a modified sequence thereof. [005]
• Each of the one or more peptides may independently comprise or consist of the amino acid sequence set forth in any one of SEQ ID NOS:74, 75, 78, 91, 174, 176, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 254, 278, 292, 294.
• Each of the one or more peptides may independently comprise or consist of the amino acid sequence set forth in any one of SEQ ID NOS:597. 770, 1231, 1232, 1709, 1748. 2297.
• Each of the one or more peptides independently comprises or consists of the amino acid sequence set forth in any one of the SEQ ID NOS of TABLE 7, TABLE 8, TABLE 9, or TABLE 10, or a modified sequence thereof.
• Each of the one or more peptides may be a fragment of a myelin basic protein (MBP). tubulin beta 3 class III (TUBB3), or neurofilament medium polypeptide (NEFM).
• MBP myelin basic protein
• TUBB3 tubulin beta 3 class III
• NEFM neurofilament medium polypeptide
• Each fragment of MBP may independently comprise or consist of the amino acid sequence set forth in any one of SEQ ID NOS:5776, 176, 278, 3784, 3785, 3786, 3787, 3788, 5438, 5439, 5773, 5774, 5775, 5777, 5778, 5779, 5780, 5781, 5782, 5783, 5784, 5785, 5786, 5787, 5788, 5789, 5790, 5791, 5792, 5793, 5794, 5795, 5796, 10752, 10753, 10754. 10755,
• At least one of the one or more peptides may comprise or consist of the amino acid sequence of FLPRHRDTGILDSIGR (SEQ ID NO:5776), DTGILDSIGR (SEQ ID NO: 10755), or DTGILDSIGRFFSGDRGAPK (SEQ ID NO: 10759), or a modified sequence thereof.
• the pharmaceutical composition may further comprise one or more extracellular vesicles (EV) or liposomes enclosing the one or more peptides.
• the one or more EV may be an exosome, a microvesicle, or an apoptotic body of a cell, optionally a human cell.
• the one or more EV may oligodendrocyte- derived. optionally from a dendritic cell, which may be of the cell line DC2.4.
• the one or more EV may be synthetic.
• One or more vectors may each comprise at least one of the one or more nucleic acids.
• the vectors may be viral vectors.
• Tire viral vectors may be derived from an adenovirus, adeno-associated virus, or retrovirus, optionally selected from a lentivirus.
• Tire vectors may be non- viral vectors.
• the one or more nucleic acids may be mRNAs.
• the mRNAs may be non-replicating mRNAs, self-amplifying mRNAs (saRNAs), or circular RNAs (circRNA).
• Tire mRNAs may be non-replicating mRNAs; and each rnRNA may comprise a coding region encoding one of the one or more peptides, a first UTR, a second UTR, a 5’ cap. and a poly(A) tail; and each coding region may be flanked by the UTRs.
• Each 5’ cap may comprise a 7-methylguanosine (m 7 G) cap that is connected to a first nucleotide of each mRNA (N) by triphosphates (ppp) to form m 7 GpppNp.
• m 7 G cap may comprise a methylated 2 ’-OH on the first nucleotide connecting the 5’ end of each mRNA to the cap (capl or m 7 GpppNimp).
• Each m 7 G cap may comprise a methylated 2 ’-OH on the first nucleotide and a second nucleotide connecting the 5’ end of each mRNA to the cap (cap2 or m 7 GpppNimpN2mp).
• Each mRNA may comprise one more modified nucleosides, optionally selected from the group consisting of pseudouridine (t
• the nucleic acids may be encapsulated, optionally by lipid nanoparticles (LNPs), polyplexes, polymeric nanoparticles, lipopolyplexes (LPPs), or cationic polypeptides.
• the pharmaceutical composition may comprise the one or more nucleic acids, one or more vectors, or one or more mRNAs.
• the one or more peptides or the pharmaceutical composition for use in treating, preventing, or reversing a neuroinflammation-related disorder, an autoimmune neural disorder, or an acute CNS injury'.
• a method of treating, preventing, or reversing a neuroinflammation-related disorder, an autoimmune neural disorder, or an acute CNS injury in a subject in need thereof which may comprise administering the one or more peptides or the pharmaceutical composition to the subject: and use of the one or more peptides or the pharmaceutical composition in the manufacture of a medicament for ting, preventing, or reversing a neuroinflammation-related disorder, an autoimmune neural disorder, or an acute CNS injury.
• Hie peptides or pharmaceutical composition may be or may be intended to be administered to a subject by infusing or injecting into the cerebrospinal fluid (CSF) of the subject.
• CSF cerebrospinal fluid
• Hie neuroinflammation-related disorder or an autoimmune neural disorder may be selected from the group consisting of acute disseminated encephalomyelitis (ADEM), acute inflammatory demyelinating polyradiculoneuropathy (AIDP; Guillain-Barre syndrome (GBS)), acute motor axonal neuropathy (AMAN), acute motor and sensory axonal neuropathy (AMSAN). acute optic neuritis (AON).
• ADAM acute disseminated encephalomyelitis
• AIDP acute inflammatory demyelinating polyradiculoneuropathy
• GBS Guillain-Barre syndrome
• AMAN acute motor axonal neuropathy
• AMSAN acute motor and sensory axonal neuropathy
• AON acute optic neuritis
• AD Alzheimer’s disease
• ALS amyotrophic lateral sclerosis
• AIE autoimmune encephalitis
• chronic ataxic neuropathy ophthalmoplegia
• immunoglobulin M paraprotein immunoglobulin M paraprotein
• cold agglutinins cold agglutinins
• disialosyl antibodies CANOMAD
• chronic meningitis Behcet's disease
• Central nervous system CNS vasculitis
• CIDP chronic inflammatory demyelinating polyradiculoneuropathy
• CLIPPERS chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids
• GFAP glial fibrillar acidic protein
• Hashimoto’s encephalitis hypertrophic pachymeningitis, lgG4 associated neurological disease, Lambert-Eaton myasthenic syndrome (LEMS), anti-myelin oligodendrocyte glycoprotein antibody disease (MOG), Miller Fisher syndrome (MFS), monoclonal gammopathy of
• FIGS. 1A-1B show that MHC II presentation in the CNS centers on a particular MBP region.
• FIG. 1A demonstrates tire composition of MHC Il-bound peptides derived from proteins labeled as CNS- enriched.
• FIG. IB shows the exact sequences of peptides bound to MHC II molecules deriving from MBP.
• FIGS. 2A-2D show that MBP peptides gamer regulatory T cells to suppress CNS autoimmunity.
• FIG. 2A depicts a schematic for the induction of experimental autoimmune encephalomyelitis (EAE) in C57BL/6J mice.
• EAE experimental autoimmune encephalomyelitis
• FIG. 2B shows clinical EAE score tracked over 21 days for mice immunized with either MOG35-55, MOG35-55 and MBP33-52, or MOG35-55 and MBP (27-42 subscript); two-way ANOVA with Sidak’s multiple comparison test, p-values, where significant, indicated on plots.
• FIG. 2C displays representative flow cytometry plots with associated quantifications as in FIG. 2D for evaluation of regulatory T cells in the deep cervical lymph nodes (dCLNs) when comparing between MOG3555 as well as MOG35-55 and MBP2742 co-immunized groups; unpaired two-tailed Student’s t-test, p-values indicated where relevant.
• dCLNs deep cervical lymph nodes
• FIGS. 3A-3B show that presentation of MBP peptides is generalizable and similarly suppresses neuroinflammation in a different active EAE model.
• FIG. 3A depicts CNS-enriched peptides identified across both the brain and dural meninges in SJL/J male mice as well as in fresh frozen human dura.
• FIG. 3B shows clinical EAE scores tracked over 21 days for SJL/J mice immunized with PLP139-151, PLP139-151 and NEFL160-173, or PLP139-151 and MBP27-42. Statistics done with two-way ANOVA with Sidak’s multiple comparison test, p-values, where significant, indicated on plots..
• FIG. 4 show s the harnessing of MBP peptides as a therapeutic against CNS autoimmunity.
• Visualization of EVs by both electron microscopy (EM) and ZetaView 7 nanoparticle tracker visualization.
• Clinical EAE score tracked over 21 days for mice induced for EAE and with introduction of either empty EVs or MBP27-42 containing EVs via intracistcma magna (i.e.m.) injection prior to onset of disease; two-w ay ANOVA and p-valuc indicated on plot. Peak EAE scores for both conditions also quantified and displayed on the right, unpaired two- tailed Student’s t-test, p-value indicated on plot.
• FIGS. 5A-5B show 7 analysis of the MHC II peptidome.
• FIG. 5A depictsepitope mapping by Gibbs cluster analysis of MHC-II-bound peptides of 8-week-old C57BL/6J.I-Ab mice.
• FIG. 5B depicts distribution for MHC-II-bound peptides enriched for proteins that are expressed at elevated levels in tire CNS. ‘"Other” represents the sum of CNS elevated proteins with only a single MHC-II-bound peptide identified. These include: MAP2, NEFL, DPYSL2, CNP, ISLR2, BCAS1, IRS4, PTGDS, and TBC1D30.
• FIG. 5A depictsepitope mapping by Gibbs cluster analysis of MHC-II-bound peptides of 8-week-old C57BL/6J.I-Ab mice.
• FIG. 5B depicts distribution for MHC-II-bound peptides enriched for proteins that are expressed at elevated levels in tire CNS
• FIG. 6A depicts the experimental scheme for induction of experimental autoimmune encephalomyelitis (EAE).
• FIGS. 7A shows average clinical EAE score in C57BL6/J mice immunized with MOG35-55 (red), MOG35-55 + MBP12-26 (gray). MOG35-55 + MBP27-42 (blue), or MOG35-55 + MBP2742 (citrullinated) (yellow) on day 0; standard EAE scoring to assess mice daily for 20 days post immunization.
• FIG. 9A shows average clinical EAE score in SJL/J mice immunized with PLP139-151 (red), PLP139 151 + NF-L160 173 (gray), or PLP139 151 + MBP 2742 (blue) on day 0* standard EAE scoring to assess mice daily for 21 days post immunization.
• FIG. 9B shows peak EAE score for SJL/J mice as immunized in FIG. 9A.
• N 5 mice per group, p-values are indicated on the graphs.
• FIG. 10A shows an immunoblot performed on both supernatant (SN) and pellet post high-speed ultracentrifugation evidencing enrichment of tetraspanins CD9 and CD63, markers of extracellular vesicles, in the pellet.
• FIG. 10B shows a negative stain by transmission electron microscopy of enriched extracellular vesicles.
• FIG. 10C shows an experimental scheme (top) depicting tire insertion of MBP27-42 peptides into extracellular vesicles prior to intracistema magna (i.e.m.) injection.
• FIG. 11A shows average clinical EAE score in C57BL/6J mice immunized with MOG35-55 (red) or CFA only (black) on day 0; standard EAE scoring to assess mice daily for 16 days post immunization.
• FIG. 11B depicts representative images and
• FIG. 11C depicts quantification of percent coverage of intracistema magna (i.e.m.) injected OVA647 protein in the dCLN of CFA only or CFA + MOG35-55 immunized mice at peak of EAE disease (day 16). Scale bar, 150 pm. P-values indicated on plots.
• FIGS. 12A-12C depicts binding motifs for both MHC I alleles H2-Kb (FIG. 12A)and H2-Db (FIG. 12B)as well as the sole MHC II allele H2-Ab (FIG. 12C) in C57BL/6J mice as predicted by both the netMHCpan and netMHCIIpan algorithms.
• FIGS. 13A-13E show that the CNS MHC II peptidome reveals presentation of endogenous CNS peptides.
• FIG. 13A depicts a schematic illustrating the mass spectrometric identification of MHC II- bound peptides from brain (including leptomeninges), dural meninges (dura), and lymph nodes, including the deep cervical lymph nodes (dCLNs) and superficial cervical lymph nodes (sCLNs), of C57BL/6J male mice.
• FIG. 13B shows assessment of the proportion of total unique identified peptides that could be designated as CNS enriched (teal bar); percentages indicated above each individual bar.
• FIG. 13C depicts a donut plot representation of CNS-enriched, MHC Il-bound peptides identified for each individual tissue. The percent of which is composed by MBP is indicated where relevant.
• FIG. 13D shows a Venn diagram depicting the relationship between CNS-enriched, MHC Il-bound peptides in the brain, dura, dCLNs, and sCLNs.
• FIG. 13E shows a summary of individual peptide sequences contained within the MBP158-195 region as defined by the MHC II peptidome.
• FIGS. 14A-14K show that MBP is non-encephalitogenic and fosters immunosuppression.
• FIG. 14B shows quantification of cell counts (mean ⁇ s.e.m, unpaired two-tailed Student’s t-test).
• FIG. 14C shows an ELISpot assay performed to measure IL-2 production by CD4+ T cells upon recall with peptides as indicated on the x-axis. Data shown as mean ⁇ s.e.m, two-way ANOVA with Sidak’s multiple comparisons test.
• FIG. 14E depicts a UMAP visualization of T cells from the draining (inguinal) lymph nodes of MOG35-55 immunized or MOG35-55 and MBP160-175 co-immunized C57BL/6J mice.
• FIG. 14F shows Log2 fold change determined across the different identified clusters by scRNA-seq when comparing MOG35 55 to MOG35 -55 and MBP160 175 immunizations. Significant differences highlighted on the plot.
• FIG. 14E depicts a UMAP visualization of T cells from the draining (inguinal) lymph nodes of MOG35-55 immunized or MOG35-55 and MBP160-175 co-immunized C57BL/6J mice.
• FIG. 14F shows Log2 fold change determined across the different identified clusters by scRNA-seq when comparing MOG35 55 to MOG35 -55 and MBP160 175 immunizations. Significant differences highlighted on the plot.
• FIG. 14E depicts a
• FIGS. 15A-15F show that therapeutic delivery of guardian MBP peptides guards against CNS autoimmunity.
• FIG. 15A depicts a schematic depiction of isolating peptides bound to MHC II molecules in the brain, dura, and spinal cord at the peak (day 16) of MOG35-55 induced EAE.
• FIG. 15B shows the MHC II peptidomes from EAE and naive mice. Quantified relative abundances for peptides was identified within different antigenic regions to generate a heatmap, illustrating alterations to the repertoire of MHC Il-bound peptides in EAE when compared to naive mice.
• FIG. 15C depicts a bar graph representation of the relative abundances of MBP peptides contained within the MBP158-195 or MBP196-236 regions for the brain (top) and dura (bottom) when comparing EAE to naive mice.
• Relative abundances (normalized peak areas) were measured relative to (Cbln 15772) for the brain and (Mbpi96-23e) for the dura.
• FIG. 15D shows an experimental design depicting i.e.m.
• FIG. 15F depicts an experimental scheme atop illustrating i.e.m.
• FIGS. 16A-16H show characterization of the MHC II peptidome in the CNS of C57BL/6J male mice.
• FIGS. 16A-16D show representative flow cytometry plots depicting the gating strategy used to identify the distribution of MHC II-expressing cells.
• B cells gated on CD19+ CD11c-
• dendritic cells DCs, gated on CD19- CD1 lc+
• macrophages MO, gated on CD19- CD11c-
• FIG. 16B shows the frequency of the aforementioned antigen presenting cells as a percent of CD45+ MHC 11+ cells for the brain (FIG. 16B), dura (FIG. 16C), dCLNs (FIG. 16D), and sCLNs (FIG. 16E).
• FIG. 16F shows a violin plot depicting predicted binding affinities for unique MHC Il-bound peptides. The median is represented by a solid line and the first as well as the third quartiles are represented by dashed lines. P-values indicated on plot (one-way ANOVA with Tukey’s multiple comparison test).
• FIG. 16F shows a violin plot depicting predicted binding affinities for unique MHC Il-bound peptides. The median is represented by a solid line and the first as well as the third quartiles are represented by dashed lines. P-values indicated on plot (one-way ANOVA with Tukey’s multiple comparison test).
• FIG. 16D shows a Venn diagram depicting the relationship between all unique MHC Il-bound peptides in the brain, dura, dCLNs, and sCLNs.
• FIG. 16H shows a summary of individual peptides identified on the MBP sequence as defined by the MHC II peptidome of C57BL/6J male mice.
• FIGS. 17A-17F show the characterization of the MHC II peptidome in the CNS of C57BL/6J female mice.
• FIG. 17A shows a pie chart defining the makeup of CNS-enriched, MHC Il-bound peptides across each individual tissue. Percent of CNS-enriched peptides that are MBP are indicated in the plots, where relevant.
• FIG. 17B shows a summary of all identified individual peptide sequences derived from MBP in the C57BL/6J female MHC 11 peptidome.
• FIG. 17C depicts a Venn diagram representation of individual overlapping CNS-enriched peptide sequences identified between C57BL/6J females and males in the brain (including leptomeninges).
• FIG. 17A shows a pie chart defining the makeup of CNS-enriched, MHC Il-bound peptides across each individual tissue. Percent of CNS-enriched peptides that are MBP are indicated in the plots, where relevant.
• FIG. 17B shows a summary of
• FIG. 17D depict a bar graph showing the relative abundance of MBP peptides in the brain that comprised either the MBPiss-iss or MBP196-236 regions in both C57BL/6J males and females. Relative abundances (normalized peak areas) measured against a common identified peptide sequence, Dagl488-531, from which relativity was ascertained.
• FIG. 17E depicts a Venn diagram representation of individual CNS-enriched overlapping peptide sequences identified between C57BL/6J females and males in the dura.
• FIG. 17F depicts a bar graph showing the relative abundance of MBP peptides in the dura that comprised either the MBP158-195 or MBP196-236 regions in both C57BL/6J males and females.
• FIGS. 18A-18F show the characterization of the MHC II peptidome in the CNS of of SJL/J male mice.
• FIG. 18A depicts a violin plot depicting predicted binding affinities for unique MHC Il-bound peptides in the brain (which also includes the leptomeninges), dura, dCLN, and sCLN of SJL/J mice. The median is represented by a solid line, and the first as well as the third quartiles are represented by dashed lines.
• FIG. 18B shows evaluation of the proportion of total unique identified peptides designated as CNS enriched (teal bar) in the brain, dura, dCLN, and sCLN; percentages indicated above each individual bar.
• FIG. 18C shows evaluation of the proportion of total unique identified peptides designated as CNS enriched (teal bar) in the brain, dura, dCLN, and sCLN; percentages indicated above each individual bar.
• FIG. 18C shows evaluation
• FIG. 18E depicts a donut plot representation of all CNS-enriched peptides identified in the MHC II peptidome of SJL/J male mice for the brain, dura, and sCLN. The part of the whole for which MBP represents is indicated in the plot, where relevant.
• FIG. 18F shows a depiction of all individual peptide sequences deriving from MBP identified in the SJL/J male MHC II peptidome.
• FIGS. 19A-19E show that endogenous guardian MBP peptides protect across different models of neuroinflammation.
• FIG. 19A depicts an experimental design of immunizing either C57BL/6J or SJL/J mice with MOG35-55 or PLP139-151 peptides, respectively, with or without MBP or NEFL peptides to actively induce EAE. Separately, C57BL/6J mice were also immunized individually with myelin associated peptides to perform ELISpot assay.
• Plots display mean ⁇ s.e.m and represent two independent experiments. P-values, where significant, indicated on plot and determined by two-way ANOVA with Sidak’s multiple comparisons test.
• Plots display mean ⁇ s.e.m and represent three independent experiments.
• Plots display mean ⁇ s.e.m and represent two independent experiments. P-values, where significant, indicated on plot and determined by two-way ANOVA with Sidak’s multiple comparisons test.
• FIGS. 20A-20E show that peripheral presentation of MBP peptides induces conventional Tregs.
• FIG. 20A depicts a dot plot of population markers from single cell RNA-sequencing scaled by percentage of cells expressing marker genes for each T cell cluster.
• FIG. 20B shows a representative gating strategy for flow 7 cytometry used to define within CD4+ T cells, CTLA-4+ Foxp3- suppressor T cells as well as conventional Foxp3+ regulatory T cells (Treg). CTLA-4+ Foxp3- CD4+ T cells further gated to assess CD39.
• PD-1 CD279
• IL-10 expression FIGS. 20C-20D show representative flow cytometry plots with relevant quantifications (FIG.
• FIGS. 21A-21G show 7 that neuroinflammation alters the MHC Il-bound repertoire of autoantigens.
• FIG. 21C show s a pie chart depiction of CNS-enriched, MHC Il-bound peptides identified in the brain and spinal cord at the peak of EAE disease. Below is a visual summary of individual MBP sequences identified.
• FIG. 21D shows a population pyramid representation of the distribution of amino acids identified at the C -terminus as a percent of the total MHC Il-bound peptides, indicating cleavage preference between EAE and naive mice.
• FIG. 21E shows UMAP projections of the Jordao ct al. dataset displaying broad cell lineages, color-coded accordingly, when analyzing naive, pre-symptomatic, and EAE mice.
• eDCs conventional dendritic cells.
• migDCs migratory dendritic cells.
• FIG. 21F shows a volcano plot depicting differences in gene expression when comparing microglia in EAE-induced mice to controls. Arrows indicate peptidases that could be identified.
• FIG. 21G shows gene ontologies found to be significantly upregulated in microglia when comparing between EAE and naive mice.
• FIGS. 22A-22F show that delivery of encapsulated MBP peptides directly into the CSF induces unconventional suppressor CD4+ T cells.
• FIG. 22A shows an immunoblot performed on the supernatant (SN) and resuspended pellet after ultracentrifugation evidencing enrichment of tetraspanins, CD9 and CD63, markers of extracellular vesicles (EVs). Below 7 is a negative stain by transmission electron microscopy depicting enriched EVs, scale bar indicated on the panel.
• FIG. 22B shows that gating strategy for flow cytometry analysis to define within CD4+ T cells, CTLA-4+ Foxp3- unconventional suppressor T cells as well as conventional Foxp3+ Treg.
• FIGS. 22C-22E show quantification of unconventional CTLA-4+ Foxp3- suppressor T cells as well as conventional Foxp3+ Treg as a frequency of CD4+ T cells of the deep cervical lymph nodes (FIG. 22C), superficial cervical lymph nodes (FIG. 22D), and spleen (FIG. 22E).
• FIGS. 23A-23G show candidates for regulatory peptides against CNS autoimmunity.
• Clinical EAE scores tracked over 19 days post immunization with MOG35-55 or MOG35-55 and Tubb3 peptide 1 (FIG. 23A); MOG35-55 or MOG35-55 and Tubb3 peptide 2 (FIG. 23B); MOG35-55 or MOG35-55 and Tubb3 peptide 3 (FIG. 23C); MOG35.55 or MOG35-55 and Tubb3 peptide 4 (FIG. 23D); MOG35-55 or MOG35-55 and Map2 (FIG. 23E); MOG35-55 or MOG35-55 and Neftn peptide 1 (FIG.
• autoreactive T cells are well-known for provoking autoimmune disease, their protective roles in limiting tissue damage have been described in heart, skeletal muscle, and central nervous system (CNS) pathologies, including Alzheimer’s disease, stroke, and traumatic injury, among others. With evidence supporting a suppressive and beneficial function for autoreactive T cells, the molecular and cellular mechanisms interconnecting the CNS and the peripheral immune system requires further investigation.
• Tire compositions and methods of the present disclosure are based on the surprising discovery that in an experimental MOG35-55 -induced autoimmune encephalomyelitis (EAE) model, co-immunization of MOG35-55 with MBP33-42 peptides ameliorated EAE disease. Further analyses by flow cytometry prior to the peak of EAE disease unveiled an elevated frequency of regulatory Foxp3-expressing CD4+ T cells in CNS-associated tissues of mice coimmunized with the MBP peptides.
• EAE autoimmune encephalomyelitis
• CSF tire cerebrospinal fluid
• MBP3342 fragment is equivalent to the MBP166-175 fragment, when accounting for Goli numbering differences.
• Tire CNS antigen repertoire used to develop the compositions and methods of the present disclosure overcome the shortcomings of existing methods that have hampered our capacities to efficaciously employ antigen-specific immune -tolerizing therapeutics.
• Inadequate knowledge of the repertoire of antigens presented during homeostasis of the CNS is one barrier overcome by utilizing an optimized technique to unravel native peptides bound to major histocompatibility complex class II (MHC II) molecules in the CNS and its borders.
• MHC II major histocompatibility complex class II
• the present disclosure extensively characterizes the MHC II peptidome in CNS during homeostasis, providing the ability to broadly understand the specificities of autoreactive T cells in the CNS.
• the identified peptides described herein are useful in directing the development of MHC II tetramers, allowing for tracking and the development of phenotype autoreactive CNS-specific T cells.
• the methods described herein provide insights into the function of relevant antigen presentation in the CNS to harbor local populations of autoreactive T cells - an understanding of which illuminates mechanisms the CNS employs to acquire immune privilege.
• Another critical barrier to the development of antigen-specific immunotherapies derives from an insufficient understanding of disease-initiating autoantigens and subsequent epitope spreading often arising as a consequence of chronic autoimmune or inflammatory response.
• the present disclosure overcomes this barrier by leveraging an established platform to define the MHC II peptidome to understand how the repertoire of autoantigens presented on MHC molecules diverges from homeostasis with neuroinflammation.
• existing antigen-specific therapeutic strategies also face a barrier of being constrained to identified pathogenic epitopes and finding ways to manipulate either their sequence or their delivery to drive tolerogenic immune responses.
• the methods disclosed herein circumvent this barrier by taking advantage of the knowledge of CNS-derived endogenous antigen presentation in homeostasis to inform antigen-specific immunotherapies.
• the disclosed methods therapeutically deliver a homeostatic, CNS-specific, MHC Il-bound autoantigen to suppress and protect against CNS autoimmunity.
• compositions and methods described herein provide an expanded comprehension of the repertoire of autoantigens presented and of autoreactive T cells within the CNS, thus furthering understanding of the mechanisms supporting CNS immune privilege and immune tolerance by extension.
• Tire methods disclosed herein foster an understanding that will guide antigen-specific immunotherapies, enabling the induction of immune tolerance in other tissues to protect against allo- or autoimmune attack for clinical benefit.
• the term “about,” as used herein, refers to variation of in the numerical quantity that can occur, for example, through typical measuring techniques and equipment, with respect to any quantifiable variable, including, but not limited to, mass, volume, time, distance, and amount. Further, given solid and liquid handling procedures used in the real world, there is certain inadvertent error and variation that is likely through differences in the manufacture, source, or purity of the ingredients used to make the compositions or carry out the methods and the like. The term “about” also encompasses these variations, which can be up to ⁇ 5%, but can also be ⁇ 4%, 3%. 2%,1%, etc. Whether or not modified by the term “about,” the claims include equivalents to the quantities.
• the term “subject” refers to a mammal, preferably a human.
• the mammals include, but are not limited to, humans, primates, livestock, rodents, and pets.
• a subject may be waiting for medical care or treatment, may be under medical care or treatment, or may have received medical care or treatment.
• control population refers to a subject, or group of subjects, who are clinically determined to not have a disease.
• the medical care may be a diagnostic test, a therapeutic treatment, and/or a prophylactic or preventative measure.
• the object of therapeutic and prophylactic treatments is to prevent or slow down (lessen) an undesired physiological change or disease/disorder.
• Beneficial or desired clinical results of therapeutic or prophylactic treatments include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e. , not worsening) state of disease, a delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.
• Those in need of treatment include those already with the disease, condition, or disorder as well as those prone to have the disease, condition or disorder orthose in which the disease, condition or disorder is to be prevented. Accordingly, a subject in need of treatment may or may not have any symptoms or clinical signs of disease.
• heterologous DNA sequence refers to a sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form.
• a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, tire use of DNA shuffling or cloning.
• the terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence.
• the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous DNA segments are expressed to yield exogenous polypeptides.
• a "homologous" DNA sequence is a DNA sequence that is naturally associated with a host cell into which it is introduced.
• Expression vector, expression construct, plasmid, or recombinant DNA construct is generally understood to refer to a nucleic acid that has been generated via human intervention, including by recombinant means or direct chemical synthesis, with a series of specified nucleic acid elements that permit transcription or translation of a particular nucleic acid in, for example, a host cell.
• the expression vector can be part of a plasmid, virus, or nucleic acid fragment.
• the expression vector can include a nucleic acid to be transcribed operably linked to a promoter.
• a '‘promoter” is generally understood as a nucleic acid control sequence that directs the transcription of a nucleic acid.
• An inducible promoter is generally understood as a promoter that mediates the transcription of an operably linked gene in response to a particular stimulus.
• a promoter can include necessary nucleic acid sequences near tire start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element.
• a promoter can optionally include distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.
• a "transcribable nucleic acid molecule” as used herein refers to any nucleic acid molecule capable of being transcribed into an RNA molecule. Methods are known for introducing constructs into a cell in such a manner that the transcribable nucleic acid molecule is transcribed into a functional mRNA molecule that is translated and therefore expressed as a protein product. Constructs may also be constructed to be capable of expressing antisense RNA molecules, in order to inhibit the translation of a specific RNA molecule of interest.
• compositions and methods for preparing and using constructs and host cells are well known to one skilled in the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754).
• the '‘transcription start site” or "initiation site” is the position surrounding the first nucleotide that is part of the transcribed sequence, which is also defined as position +1. With respect to this site, all other sequences of the gene and its controlling regions can be numbered. Downstream sequences (i.e., further protein-encoding sequences in tire 3' direction) can be denominated positive, while upstream sequences (mostly of the controlling regions in the 5' direction) are denominated negative.
• operably-linked refers preferably to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other.
• a regulatory DNA sequence is said to be “operably linked to” or “associated with” a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
• the two nucleic acid molecules may be part of a single contiguous nucleic acid molecule and may be adjacent.
• a promoter is operably linked to a gene of interest if the promoter regulates or mediates transcription of the gene of interest in a cell.
• a "construct” is generally understood as any recombinant nucleic acid molecule such as a plasmid, cosmid, virus, autonomously replicating nucleic acid molecule, phage, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a nucleic acid molecule where one or more nucleic acid molecule has been operably linked.
• a construct of the present disclosure can contain a promoter operably linked to a transcribable nucleic acid molecule operably linked to a 3' transcription termination nucleic acid molecule.
• constructs can include but are not limited to additional regulatory nucleic acid molecules from, e g., the 3 '-untranslated region (3' UTR).
• Constructs can include but are not limited to the 5' untranslated regions (5' UTR) of an mRNA nucleic acid molecule which can play an important role in translation initiation and can also be a genetic component in an expression construct.
• These additional upstream and downstream regulatory nucleic acid molecules may be derived from a source that is native or heterologous with respect to the other elements present on the promoter construct.
• Tire term "transformation” refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in a genetically stable inheritance.
• Host cells containing the transformed nucleic acid fragments are referred to as “transgenic” cells, and organisms comprising transgenic cells are referred to as “transgenic organisms”.
• Transformed refers to a host cell or organism such as a bacterium, cyanobacterium, animal, or plant into which a heterologous nucleic acid molecule has been introduced.
• Tire nucleic acid molecule can be stably integrated into the genome as generally known in the art and disclosed (Sambrook 1989; Innis 1995; Gelfand 1995; Innis & Gelfand 1999).
• Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially mismatched primers, and the like.
• the term “untransfonned” refers to normal cells that have not been through the transformation process.
• Wild-type refers to a virus or organism found in nature without any known mutation.
• Nucleotide and/or amino acid sequence identity percent is understood as the percentage of nucleotide or amino acid residues that are identical with nucleotide or amino acid residues in a candidate sequence in comparison to a reference sequence when the two sequences are aligned. To determine percent identity, sequences are aligned and if necessary, gaps are introduced to achieve the maximum percent sequence identity. Sequence alignment procedures to determine percent identity are well known to those of skill in the art. Often publicly available computer software such as BLAST, BLAST2, ALIGN2, or Mcgalign (DNASTAR) software is used to align sequences. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
• percent sequence identity X/Y100, where X is tire number of residues scored as identical matches by the sequence alignment program's or algorithm's alignment of A and B and Y is the total number of residues in B. If the length of sequence A is not equal to the length of sequence B. the percent sequence identity of A to B will not equal the percent sequence identity of B to A.
• conservative substitutions can be made at any position so long as the required activity is retained.
• So-called conservative exchanges can be carried out in which the amino acid which is replaced has a similar property as the original amino acid, for example, the exchange of Glu by Asp, Gin by Asn, Vai by He, Leu by He, and Ser by Thr.
• amino acids with similar properties can be Aliphatic amino acids (e.g., Glycine, Alanine, Valine, Leucine, Isoleucine); Hydroxyl or sulfur/selenium- containing amino acids (e.g., Serine, Cysteine, Selenocysteine, Threonine, Methionine); Cyclic amino acids (e.g., Proline); Aromatic amino acids (e.g., Phenylalanine, Tyrosine, Tryptophan); Basic amino acids (e.g., Histidine, Lysine, Arginine); or Acidic and their Amide (e.g., Aspartate, Glutamate, Asparagine, Glutamine).
• Aliphatic amino acids e.g., Glycine, Alanine, Valine, Leucine, Isoleucine
• Hydroxyl or sulfur/selenium- containing amino acids e.g., Serine, Cysteine, Selenocysteine, Threonine, Methionine
• Deletion is the replacement of an amino acid by a direct bond. Positions for deletions include the termini of a polypeptide and linkages between individual protein domains. Insertions are introductions of amino acids into the polypeptide chain, a direct bond formally being replaced by one or more amino acids.
• An amino acid sequence can be modulated with the help of art- known computer simulation programs that can produce a polypeptide with, for example, improved activity or altered regulation. On the basis of these artificially generated polypeptide sequences, a corresponding nucleic acid molecule coding for such a modulated polypeptide can be synthesized in-vitro using the specific codon-usage of the desired host cell.
• Host cells can be transformed using a variety of standard techniques known to the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed.. Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754).
• transfected cells can be selected and propagated to provide recombinant host cells that comprise the expression vector stably integrated in the host cell genome.
• Exemplary nucleic acids which may be introduced to a host cell include, for example. DNA sequences or genes from another species, or even genes or sequences which originate with or are present in the same species but are incorporated into recipient cells by genetic engineering methods.
• exogenous is also intended to refer to genes that are not normally present in the cell being transformed, or perhaps simply not present in the form, structure, etc., as found in tire transforming DNA segment or gene, or genes which are normally present and that one desires to express in a manner that differs from the natural expression pattern, e.g., to over-express.
• tire term “exogenous” gene or DNA is intended to refer to any gene or DNA segment that is introduced into a recipient cell, regardless of whether a similar gene may already be present in such a cell.
• Tire type of DNA included in the exogenous DNA can include DNA that is already present in the cell, DNA from another individual of the same type of organism, DNA from a different organism, or a DNA generated externally, such as a DNA sequence containing an antisense message of a gene, or a DNA sequence encoding a synthetic or modified version of a gene.
• Host strains developed according to the approaches described herein can be evaluated by a number of means known in the art (see e.g., Studier (2005) Protein Expr Purif. 41(1), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).
• RNA interference e.g., small interfering RNAs (siRNA), short hairpin RNA (shRNA), and micro RNAs (miRNA)
• siRNA small interfering RNAs
• shRNA short hairpin RNA
• miRNA micro RNAs
• RNAi molecules are commercially available from a variety of sources (e.g., Ambion, TX; Sigma Aldrich, MO; Invitrogen).
• sources e.g., Ambion, TX; Sigma Aldrich, MO; Invitrogen.
• siRNA molecule design programs using a variety of algorithms are known to the art (see e.g., Ccnix algorithm, Ambion; BLOCK-iTTM RNAi Designer, Invitrogen; siRNA Whitehead Institute Design Tools, Bioinfomiatics & Research Computing).
• Traits influential in defining optimal siRNA sequences include G/C content at the termini of tire siRNAs, Tm of specific internal domains of the siRNA, siRNA length, position of the target sequence within the CDS (coding region), and nucleotide content of the 3' overhangs.
• compositions which comprise a peptide as described herein, or a nucleic acid encoding the peptide (e.g., an rnRNA or a vector encoding the peptide).
• Tire composition may be a phannaceutical composition, which comprises the peptide or nucleic acid, and one or more phannaceutically acceptable excipients.
• peptides which may be used in a composition or a method, or for informing the encoding sequence of a nucleic acid.
• Such peptides are based on those from a discovered repertoire of CNS-derived endogenous regulatory peptides (e.g., guardian peptides), which bind to major histocompatibility complex (MHC) molecules (e.g., MHC class I and MHC class II) where they serve as autoantigens important in autoreactive T cell recognition.
• MHC guardian peptides are presented throughout the path of lymphatic drainage from the brain to its surrounding meninges and its draining cervical lymph nodes, where they function in the protection against autoimmunity.
• guardian peptides are diminished in certain disease states, such as neuroinflammatory disease.
• Administration of a peptide as described herein e.g., a peptide derived from an MHC guardian peptide
• a subject mediates autoimmunity (e.g., CNS autoimmunity) by supplementing the function of MHC guardian peptides in immune modulation.
• the peptide may be used for enhancement or expansion of suppressor CD4+ T cell population (e.g., for reduction of CNS autoimmune disease)
• the peptide may be derived from an MHC guardian protein that is an MHC I guardian peptide, MHC II guardian peptide, or both.
• the peptide may be derived from an MHC guardian protein that is CNS-enriched, or an MHC guardian protein that is not CNS-enriched.
• CNS-enrichment may be elevated expression of the protein in the CNS relative to a control tissue type. In some embodiments, the elevated expression is at least about 2-fold, at least about 3 -fold, at least about 4-fold, or at least about 5 -fold relative to a control.
• the peptide may be derived from an MHC guardian protein that is a human MHC guardian protein, a murine (e.g., mouse) guardian protein, or both.
• the peptide comprises a fragment of a protein, wherein the fragment of the protein binds to an MHC molecule, hr some embodiments, the protein is expressed at elevated levels in the CNS. In some embodiments, the protein is CNS enriched. In some embodiments, the protein is not expressed at elevated levels in the central nervous system (CNS), or is expressed at decreased levels in the CNS. In some embodiments, the protein is not enriched in the CNS. In some embodiments, the peptide is a fragment of a protein selected from TABLE 4.
• a peptide as described herein is an MHC guardian peptide. In some embodiments, the peptide binds to MHC I, MHC II, or to both. [085] In some embodiments, a peptide comprises or consist of the amino acid sequence set forth in any one of SEQ ID NOS: 1-14619, or a modified sequence thereof.
• a modified sequence may comprise (e.g., relative to a wild-type MHC guardian peptide sequence or any one of SEQ ID NOS: 1-14619), one or more of: oxidation of methionine; deamidation of glutamine; deamidation of asparagine; citrullination of arginine to citrulline; oxidation of cysteine to cysteic acid; or pyroglutamic acid formation from glutamine.
• the peptide does not have oxidation of methionine.
• the peptide does not have deamidation of glutamine.
• the peptide does not have deamidation of asparagine.
• the peptide does not have citrullination of arginine to citrulline. In some embodiments, the peptide does not have oxidation of cysteine to cysteic acid. In some embodiments, the peptide does not have pyroglutamic acid formation from glutamine.
• a modified sequence may also be a variant sequence (e.g., relative to a wild-type derived MHC guardian peptide sequence or any one of SEQ ID NOS: 1-14619).
• a variant sequence retains its ability to bind to MHC (e.g., MHC I or MHC II).
• a variant sequence is at least 80%, at least 85%, at least 90%, or at least 95% identical to a wild-type MHC guardian peptide sequence or to any one of SEQ ID NOS: 1-14619.
• a variant sequence may comprise one or more amino acid additions or deletions.
• a variant sequence may comprise one or more conservative amino acid substitutions.
• the conservative amino acid substitution is based on a side chain characteristic at the one or more modified positions. In some embodiments, the conservative amino acid substitution is a substitution set forth in TABLE 1 or TABLE 2. In some embodiments, the conservative amino acid substitution is based on the amino acid identity at the one or more modified positions. In some embodiments, the conservative amino acid substitution is a substitution set forth in TABLE 3.
• a modified sequence may also be a conjugated sequence (e.g., a wild-type derived MHC guardian peptide sequence or any one of SEQ ID NOS: 1-14619), wherein the peptide is conjugated to a moiety.
• the moiety may be for enhancing delivery or enhancing targeting of the peptide to the CNS.
• TABLES 5-10 categorize the peptides of SEQ ID NOS: 1-14619 based on their CNS enrichment and presence in the MHC peptidome of humans, mouse, or both. This information, along with the protein and start/stop positions from which the peptides derive from, are provided in the remarks of each sequence in the sequence listing accompanying this patent.
• a peptide comprises or consists of the amino acid sequence set forth in any one of the SEQ ID NOS of TABLE 5, or a modified sequence thereof.
• a peptide comprises or consists of the amino acid sequence set forth in any one of the SEQ ID NOS of TABLE 6, or a modified sequence thereof.
• a peptide comprises or consists of the amino acid sequence set forth in any one of the SEQ ID NOS of TABLE 7, or a modified sequence thereof. In some embodiments, a peptide comprises or consists of the amino acid sequence set forth in any one of the SEQ ID NOS of TABLE 8, or a modified sequence thereof. In some embodiments, a peptide comprises or consists of the amino acid sequence set forth in any one of the SEQ ID NOS of TABLE 9, or a modified sequence thereof. In some embodiments, a peptide comprises or consists of the amino acid sequence set forth in any one of the SEQ ID NOS of TABLE 10, or a modified sequence thereof.
• the peptide is a fragment of a myelin basic protein (MBP).
• the peptide comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOS:5776, 176, 278, 3784, 3785. 3786, 3787, 3788, 5438, 5439, 5773, 5774, 5775, 5777, 5778, 5779, 5780, 5781, 5782. 5783, 5784, 5785, 5786, 5787, 5788, 5789. 5790, 5791, 5792, 5793, 5794, 5795, 5796, 10752, 10753, 10754.
• the peptide is a fragment of a tubulin beta 3 class III (TUBB3) protein.
• the peptide comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOS: IN 249, 250, 251, 252, 4829, 14149, 14150, 14151, 14152, 14153, 14154, and 14155, or a modified sequence thereof.
• the peptide is a fragment of a neurofilament medium polypeptide (NEFM).
• the peptide comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOS:295, 11267, 11268, 11269. 11270, 11271, 11272, 11273, 11274, 11275, 11276, 11277, 11278. 11279, 11280, 11281, 11282. 11283, 11284, and a modified sequence thereof.
• the peptide comprises or consists of the amino acid sequence of, FLPRHRDTGILDSIGR (SEQ ID NO:5776), DTGILDSIGR (SEQ ID NO: 10755), or DTGILDSIGRFFSGDRGAPK (SEQ ID NO: 10759), or a modified sequence thereof.
• nucleic acids encoding a peptide as described herein, or a modified sequence thereof.
• a nucleic acid encodes a fragment of a protein that binds to MHC (e.g., MHC I or MHC II).
• a nucleic acid encodes an MHC guardian peptide.
• a nucleic acid encodes an MHC I guardian peptide, an MHC II guardian peptide, or an MHC I/II guardian peptide (i.e., both).
• a nucleic acid may encode for a peptide comprising an MHC -binding fragment of a protein set forth in TABLE 4.
• a nucleic acid may encode for a peptide comprising or consisting of an amino acid sequence set forth in any one of the SEQ ID NOS set forth in TABLE 5, 6, 7, 8, 9, or 10.
• a nucleic acid may encode for a peptide comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOS: 1-14619, or a modified sequence thereof.
• a nucleic acid encodes a peptide comprising an MHC-binding fragment of a myelin basic protein (MBP).
• the nucleic acid encodes a peptide comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOS:5776, 176, 278, 3784, 3785, 3786, 3787, 3788, 5438. 5439, 5773, 5774, 5775, 5777, 5778, 5779. 5780, 5781, 5782, 5783, 5784, 5785, 5786, 5787, 5788. 5789.
• a nucleic acid encodes a peptide comprising an MHC -binding fragment of a tubulin beta 3 class III (TUBB3) protein.
• the nucleic acid encodes a peptide comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOS: IN 249, 250, 251, 252, 4829, 14149, 14150, 14151, 14152, 14153, 14154, or 14155, or a modified sequence thereof
• a nucleic acid encodes a peptide comprising an MHC-binding fragment of a neurofilament medium polypeptide (NEFM).
• the nucleic acid encodes a peptide comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOS:295, 11267, 11268, 11269, 11270, 11271, 11272, 11273. 11274, 11275, 11276, 11277, 11278, 11279, 11280, 11281, 11282, 11283. 11284, or a modified sequence thereof.
• the nucleic acid encodes a peptide comprising or consisting of the amino acid sequence of: FLPRHRDTGILDSIGR (SEQ ID NO:5776), DTGILDSIGR (SEQ ID NO: 10755), or DTGILDSIGRFFSGDRGAPK (SEQ ID NO: 10759), or a modified sequence thereof.
• vectors comprising a nucleic acid encoding a peptide as described herein.
• There may be one or more vectors, wherein each of the one or more vectors comprises at least one or more nucleic acids, as described herein.
• a vector or multiple vectors may encode multiple different peptides. Such vectors enable production of a transcribable nucleic acid molecule that encodes a peptide as described herein.
• a vector may further comprise a transcription initiation site, and/or a promoter operably-linked to a transcribable nucleic acid molecule encoding a peptide described herein.
• a vector may be a non-viral vector.
• the non-viral vector is a physical vector (e.g., electroporation, sonoporation, magnetofection: or hy droporation).
• the non-viral vector is a chemical vector.
• the chemical vector is an inorganic particle based, lipid based (e.g., solid or liquid), polymer based, or polypeptide based.
• the inorganic particle based vector is selected from calcium phosphate, silica, gold, or magnetic nanoparticle.
• the nucleic vector is a nucleic acid/cationic lipid (lipoplex), nucleic acid/cationic polymer (polyplex), or nucleic acid/cationic polymer/cationic lipid (lipopolyplex).
• the vector is selected from a cationic lipid, lipid nano emulsion, or solid lipid nanoparticle.
• the vector is a polypeptide based vector, a polymer based vector, or a synthetic vector.
• the synthetic vector is selected from a polyethylene imine (PEI), chitosal, poly (DL- Lactide) (PLA) and Poly ( DL-Lactide- co- glycoside) (PLGA), dendrimer, polyphosphoester, or polymethacrylate .
• PEI polyethylene imine
• PLA poly (DL- Lactide)
• PLGA Poly ( DL-Lactide- co- glycoside)
• dendrimer polyphosphoester
• polymethacrylate polymethacrylate
• a vector may be a non-viral vector.
• the non-viral vector is a physical vector (e.g., electroporation, sonoporation, magnetofection; or hy droporation).
• the non-viral vector is a chemical vector.
• the chemical vector is an inorganic particle based, lipid based (e.g., solid or liquid), polymer based, or polypeptide based.
• the inorganic particle based vector is selected from calcium phosphate, silica, gold, or magnetic nanoparticle.
• the nucleic vector is a nucleic acid/cationic lipid (lipoplex), nucleic acid/cationic polymer (polyplex), or nucleic acid/cationic polymer/cationic lipid (lipopolyplex).
• the vector is selected from a cationic lipid, lipid nano emulsion, or solid lipid nanoparticle.
• the vector is a polypeptide based vector, a polymer based vector, or a synthetic vector.
• the synthetic vector is selected from a polyethylene imine (PEI), chitosal. poly (DL- Lactide) (PLA) and Poly ( DL-Lactide- co- glycoside) (PLGA). dendrimer, polyphosphoester, or poly ethacrylate.
• the nucleic acid may comprise a mRNA encoding a peptide disclosed herein.
• a nucleic acid as described herein may also be encapsulated.
• a nucleic is encapsulated by a lipid nanoparticle (LNP), polyplex, polymeric nanoparticle, lipopolyplex (LPP), or cationic polypeptide.
• LNP lipid nanoparticle
• a nucleic acid may be encapsulated by any particle that is phagocytosed (e.g., for delivery ).
• a nucleic acid may be encapsulated by any particle that can be up taken by antigen presenting cells.
• a vector may a viral vector.
• the viral vectors may? be derived from a virus having CNS or brain tissue specificity (e.g. tropism).
• the viral vector is derived from a virus having CNS or brain tissue specificity and not having liver, heart, and/or muscle tropism.
• the viral vector may be derived from an adenovirus, adeno-associated virus, or retrovirus.
• the retrovirus is a lentivirus.
• host cells comprising a nucleic (e.g., a vector or construct) encoding a peptide as described herein.
• Tire host cells may be used for expression (e.g., production) of a peptide as described herein, or for maintaining a nucleic (e.g., vector or construct) as described herein.
• the host cell may or may not be transformed (e.g., transgenic
• compositions described herein can be formulated by any conventional manner using one or more pharmaceutically acceptable carriers or excipients as described in, for example. Remington’s Pharmaceutical Sciences (A.R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005), incorporated herein by reference in its entirety. Such fonnulations will contain a therapeutically effective amount of a peptide as described herein, or a nucleic acid encoding the peptide, which can be in purified form, together with a suitable amount of one or more pharmaceutically acceptable carriers or excipients so as to provide the form for proper administration to the subject.
• formulation refers to preparing a drug in a form suitable for administration to a subject, such as a human.
• a “formulation” can include pharmaceutically acceptable excipients, including diluents or carriers.
• Tire term "pharmaceutically acceptable” as used herein can describe substances or components that do not cause unacceptable losses of pharmacological activity or unacceptable adverse side effects. Generally, they include substances that are compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. Examples of pharmaceutically acceptable ingredients can be those having monographs in United States Pharmacopeia (USP 29) and National Formulary (NF 24), United States Pharmacopeial Convention, Inc, Rockville, Maryland, 2005 (“USP/NF”), or a more recent edition, and the components listed in the continuously updated Inactive Ingredient Search online database of the FDA. Other useful components that are not described in the USP/NF. etc. may also be used.
• compositions can include a solvent, dispersion media, coating, antibacterial and antifungal agent, isotonic, or absorption-delaying agent.
• a solvent dispersion media
• coating antibacterial and antifungal agent
• isotonic or absorption-delaying agent.
• absorption-delaying agent a solvent, dispersion media, coating, antibacterial and antifungal agent, isotonic, or absorption-delaying agent.
• the use of such media and agents for pharmaceutically active substances is well known in the art (see generally Remington’s Pharmaceutical Sciences (A.R. Gennaro, Ed ), 21st edition, ISBN: 0781746736 (2005)). Except insofar as any conventional media or agent is incompatible with an active ingredient, its use in therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
• a “stable" formulation or composition can refer to a composition having sufficient stability to allow storage at a convenient temperature, such as between about 0 °C and about 60 °C, for a commercially reasonable period of time, such as at least about one day, at least about one week, at least about one month, at least about three months, at least about six months, at least about one year, or at least about two years.
• Tire formulation should suit the mode of administration.
• the agents of use with the current disclosure can be formulated by known methods for administration to a subject using several routes which include, but are not limited to. parenteral, pulmonary, oral, topical, intradermal, intratumoral, intranasal, inhalation (e.g., in an aerosol), implanted, intramuscular, intraperitoneal, intravenous, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, intrathecal, ophthalmic, transdermal, buccal, and rectal.
• the individual agents may also be administered in combination with one or more additional agents or together with other biologically active or biologically inert agents.
• Such biologically active or inert agents may be in fluid or mechanical communication w itli the agent(s) or attached to tire agent(s) by ionic, covalent. Van der Waals, hydrophobic, hydrophilic, or other physical forces.
• Controlled-release (or sustained-release) preparations may be formulated to extend the activity of the agent(s) and reduce dosage frequency. Controlled-release preparations can also be used to affect the time of onset of action or other characteristics, such as blood levels of the agent, and consequently affect the occurrence of side effects. Controlled-release preparations may be designed to initially release an amount of an agent(s) that produces the desired therapeutic effect, and gradually and continually release other amounts of the agent to maintain the level of therapeutic effect over an extended period of time. In order to maintain a near-constant level of an agent in the body, the agent can be released from the dosage form at a rate that will replace the amount of the agent being metabolized or excreted from the body. The controlled release of an agent may be stimulated by various inducers, e.g.. change in pH, change in temperature, enzymes, water, or other physiological conditions or molecules.
• inducers e.g. change in pH, change in temperature, enzymes, water, or other physiological conditions or molecules.
• Agents or compositions described herein can also be used in combination with other therapeutic modalities, as described further below.
• therapies described herein one may also provide to the subject other therapies known to be efficacious for the treatment of the disease, disorder, or condition.
• the peptide may in a composition (e.g., a pharmaceutical composition) as described herein, which is administered to the subject.
• the neuroinflammation-related disorder or autoimmune neural disorder is selected from the group consisting of acute disseminated encephalomyelitis (ADEM), acute inflammatory demyelinating polyradiculoneuropathy (AIDP; Guillain-Barre syndrome (GBS)), acute motor axonal neuropathy (AMAN), acute motor and sensory axonal neuropathy (AMSAN), acute optic neuritis (AON).
• ADAM acute disseminated encephalomyelitis
• AIDP acute inflammatory demyelinating polyradiculoneuropathy
• GBS Guillain-Barre syndrome
• AMAN acute motor axonal neuropathy
• AMSAN acute motor and sensory axonal neuropathy
• AON acute optic neuritis
• AD Alzheimer's disease
• ALS amyotrophic lateral sclerosis
• AIE autoimmune encephalitis
• chronic ataxic neuropathy ophthalmoplegia
• immunoglobulin M paraprotein immunoglobulin M paraprotein
• cold agglutinins cold agglutinins
• disialosyl antibodies CANOMAD
• chronic meningitis Behcet's disease
• Central nervous system CNS vasculitis
• CIDP chronic inflammatory demyelinating polyradiculoneuropathy
• CLIPPERS chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids
• GFAP glial fibrillar acidic protein
• Hashimoto’s encephalitis hypertrophic pachymeningitis, IgG4 associated neurological disease, Lambert-Eaton myasthenic syndrome (LEMS), anti-myelin oligodendrocyte glycoprotein antibody disease (MOG), Miller Fisher syndrome (MFS), monoclonal gammopathy of un
• the acute CNS injury' is selected from a spinal cory injury, traumatic brain injury’, spinal cord injury’, optic nerve injury, and stroke.
• Methods described herein are generally performed on a subject in need thereof.
• a subject in need of the therapeutic methods described herein can be a subject having, diagnosed with, suspected of having, or at risk for developing a neuroinflammation-related disorder, autoimmune neural disorder, or an acute CNS injury.
• a determination of the need for treatment will typically be assessed by a history, physical exam, or diagnostic tests consistent with the disease or condition at issue. Diagnosis of the various conditions treatable by the methods described herein is within the skill of the art.
• the subject can be an animal subject, including a mammal, such as horses, cows, dogs, cats, sheep, pigs, mice, rats, monkeys, hamsters, guinea pigs, and humans or chickens.
• the subject can be a human subject.
• a safe and effective amount of the peptide is, for example, an amount that would cause the desired therapeutic effect in a subject while minimizing undesired side effects.
• an effective amount of the peptide (e.g., MBP peptide) described herein can substantially inhibit a neuroinflammation-related disorder or autoimmune neural disorder, slow the progress of a neuroinflammation-related disorder or autoimmune neural disorder, or limit the development of a neuroinflammation-related disorder or autoimmune neural disorder.
• administration can be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, intratumoral, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectal administration.
• the disclosed composition is infused, injected, or otherwise introduced into the cerebrospinal fluid (CSF) of the subject.
• CSF cerebrospinal fluid
• the peptide e.g., MBP peptide
• the extracellular vesicles may be derived from dendritic cells.
• a therapeutically effective amount of the peptide e.g., MBP peptide
• the compounds of the present disclosure can be administered, at a reasonable benefit/risk ratio applicable to any medical treatment, in a sufficient amount to prevent, reduce, or reverse a neuroinflammation-related disorder or autoimmune neural disorder.
• compositions described herein that can be combined with a pharmaceutically acceptable carrier to produce a single dosage form will vary depending upon the subject or host treated and the particular mode of administration. It will be appreciated by those skilled in the art that the unit content of agent contained in an individual dose of each dosage form need not in itself constitute a therapeutically effective amount, as the necessary therapeutically effective amount could be reached by administration of a number of individual doses.
• Toxicity and therapeutic efficacy of compositions described herein can be detennined by standard pharmaceutical procedures in cell cultures or experimental animals for determining the LD50 (the dose lethal to 50% of the population) and the ED50, (the dose therapeutically effective in 50% of the population).
• the dose ratio between toxic and therapeutic effects is the therapeutic index that can be expressed as the ratio LD50/ED50, where larger therapeutic indices are generally understood in the art to be optimal.
• the specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration; the route of administration; the rate of excretion of the composition employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts (see e.g., Koda-Kimble et al.
• treating a state, disease, disorder, or condition includes preventing, reversing, or delaying the appearance of clinical symptoms in a mammal that may be afflicted with or predisposed to the state, disease, disorder, or condition but does not yet experience or display clinical or subclinical symptoms thereof. Treating can also include inhibiting the state, disease, disorder, or condition, e.g., arresting or reducing the development of the disease or at least one clinical or subclinical symptom thereof.
• treating can include relieving the disease, e.g., causing regression of the state, disease, disorder, or condition or at least one of its clinical or subclinical symptoms.
• a benefit to a subject to be treated can be either statistically significant or at least perceptible to the subject or to a physician.
• Administration of the peptide can occur as a single event or over a time course of treatment.
• meningeal immune cells can be administered daily, weekly, bi-weekly, or monthly.
• the time course of treatment will usually be at least several days. Certain conditions could extend treatment from several days to several weeks. For example, treatment could extend over one week, two weeks, or three weeks. For more chronic conditions, treatment could extend from several weeks to several months or even a year or more.
• Treatment in accordance with the methods described herein can be performed prior to, concurrent with, or after conventional treatment modalities for prevention, reduction, or reversal of the neuroinflammation-related disorder or autoimmune neural disorder.
• the peptide can be administered simultaneously or sequentially with another agent, such as an antibiotic, an anti-inflammatory, or another agent.
• the peptide can be administered simultaneously with another agent, such as an antibiotic or an anti-inflammatory.
• Simultaneous administration can occur through the administration of separate compositions, each containing one or more of peptide, an antibiotic, an anti-inflammatory, or another agent.
• Simultaneous administration can occur through the administration of one composition containing two or more peptides, an antibiotic, an anti-inflammatory, or another agent.
• the peptides e g., MBP peptides
• the peptide can be administered before or after tire administration of an antibiotic, an anti-inflammatory, or another agent.
• Agents and compositions described herein can be administered according to methods described herein in a variety of means known to the art.
• the agents and composition can be used therapeutically either as exogenous materials or as endogenous materials.
• Exogenous agents are those produced or manufactured outside of the body and administered to the body.
• Endogenous agents are those produced or manufactured inside the body by some type of device (biologic or other) for delivery within or to other organs in the body.
• administration can be parenteral, pulmonary, oral, topical, intradermal, intratumoral, intranasal, inhalation (e.g., in an aerosol), implanted, intramuscular, intraperitoneal, intravenous, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, intrathecal, ophthalmic, transdermal, buccal, and rectal.
• Agents and compositions described herein can be administered in a variety of methods well- known in the arts. Administration can include, for example, methods involving oral ingestion, direct injection (e.g., systemic or stereotactic), implantation of cells engineered to secrete the factor of interest, drug-releasing biomaterials, polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, implantable matrix devices, mini-osmotic pumps, implantable pumps, injectable gels and hydrogels, liposomes, micelles (e.g., up to 30 pm), nanospheres (e.g., less than 1 pm), microspheres (e.g., 1-100 pm), reservoir devices, a combination of any of the above, or other suitable delivery vehicles to provide the desired release profile in varying proportions. Other methods of controlled-release delivery' of agents or compositions will be known to the skilled artisan and are within the scope of the present disclosure.
• Delivery systems may include, for example, an infusion pump which may be used to administer the agent or composition in a manner similar to that used for delivering insulin or chemotherapy to specific organs or tumors.
• an agent or composition can be administered in combination with a biodegradable, biocompatible polymeric implant that releases the agent over a controlled period of time at a selected site.
• polymeric materials include polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, and copolymers and combinations thereof.
• a controlled release system can be placed in proximity of a therapeutic target, thus requiring only a fraction of a systemic dosage.
• Agents can be encapsulated and administered in a variety of carrier delivery systems.
• carrier delivery systems include microspheres, hydrogels, polymeric implants, smart polymeric carriers, and liposomes (see generally, Uchegbu and Schatzlein, eds. (2006) Polymers in Drug Delivery, CRC, ISBN-10: 0849325331).
• Carrier-based systems for molecular or biomolecular agent delivery can: provide for intracellular delivery; tailor biomolecule/agent release rates; increase the proportion of biomolecule that reaches its site of action; improve tire transport of tire drug to its site of action; allow colocalized deposition with other agents or excipients; improve the stability of the agent in vivo; prolong the residence time of the agent at its site of action by reducing clearance; decrease the nonspecific delivery of the agent to nontarget tissues; decrease irritation caused by the agent; decrease toxicity due to high initial doses of the agent; alter the immunogenicity of the agent; decrease dosage frequency, improve the taste of the product; or improve the shelf life of the product.
• Candidate substances for screening according to the methods described herein include, but are not limited to, fractions of tissues or cells, nucleic acids, polypeptides, siRNAs, antisense molecules, aptamers, ribozymes, triple helix compounds, antibodies, and small (e.g., less than about 2000 mw, or less than about 1000 mw, or less than about 800 mw) organic molecules or inorganic molecules including but not limited to salts or metals.
• Candidate molecules encompass numerous chemical classes, for example, organic molecules, such as small organic compounds having a molecular weight of more than 50 and less than about 2,500 Daltons.
• Candidate molecules can comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, and usually at least two of the functional chemical groups.
• the candidate molecules can comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
• a candidate molecule can be a compound in a library database of compounds.
• One of skill in the art will be generally familiar with, for example, numerous databases for commercially available compounds for screening (see e.g., ZINC database, UCSF, with 2.7 million compounds over 12 distinct subsets of molecules; Irwin and Shoichet (2005) J Chern Inf Model 45, 177-182).
• Candidate molecules for screening according to the methods described herein include both lead- like compounds and drug-like compounds.
• a lead-like compound is generally understood to have a relatively smaller scaffold-like structure (e.g., molecular weight of about 150 to about 350 kD) with relatively fewer features (e.g., less than about 3 hydrogen donors and/or less than about 6 hydrogen acceptors; hydrophobicity character xlogP of about -2 to about 4) (see e.g., Angewante (1999) Chemie Int. ed. Engl. 24, 3943-3948).
• a drug -like compound is generally understood to have a relatively larger scaffold (e.g., molecular weight of about 150 to about 500 kD) with relatively more numerous features (e.g.. less than about 10 hydrogen acceptors and/or less than about 8 rotatable bonds; hydrophobicity character xlogP of less than about 5) (see e.g., Lipinski (2000) J. Phann. Tox. Methods 44, 235-249).
• Initial screening can be performed with lead-like compounds.
• kits can include an agent or composition described herein and, in certain embodiments, instructions for administration. Such kits can facilitate the performance of the methods described herein.
• the different components of the composition can be packaged in separate containers and admixed immediately before use.
• Components include, but are not limited to compositions containing a peptide as described herein (e.g., MBP peptide) optionally enclosed in extracellular vesicles as described herein.
• Such packaging of the components separately can, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the composition.
• the pack may, for example, comprise metal or plastic foil such as a blister pack.
• Such packaging of the components separately can also, in certain instances, permit long-term storage without losing activity of the components.
• Kits may also include reagents in separate containers such as, for example, sterile water or saline to be added to a lyophilized active component packaged separately.
• sealed glass ampules may contain a lyophilized component and in a separate ampule, sterile water, and sterile saline each of which has been packaged under a neutral non-reacting gas, such as nitrogen.
• Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, ceramic, metal, or any other material typically employed to hold reagents.
• suitable containers include bottles that may be fabricated from similar substances as ampules, and envelopes that may consist of foil- lined interiors, such as aluminum or an alloy.
• Other containers include test tubes, vials, flasks, bottles, syringes, and the like.
• Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle.
• Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix.
• Removable membranes may be glass, plastic, rubber, and the like.
• kits can be supplied with instructional materials. Instructions may be printed on paper or other substrate, and/or may be supplied as an electronic -readable medium or video. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an Internet website specified by the manufacturer or distributor of the kit.
• compositions and methods described herein utilizing molecular biology protocols can be according to a variety of standard techniques known to the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed.. Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988.
• numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term “about.”
• the term “about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value.
• the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment.
• the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
• Example 1 Investigating CNS immune privilege - autoantigens fend against CNS autoimmunity
• MHC major histocompatibility complex
• autoreactive T cells are well-known for provoking autoimmune disease, their protective roles in limiting tissue damage have been described in heart, skeletal muscle, and central nervous system (CNS) pathologies, including Alzheimer’s disease, stroke, traumatic injury, among others.
• CNS central nervous system
• the molecular and cellular mechanisms interconnecting the CNS and the peripheral immune system requires further investigation. This search has benefited from the recent rediscovery of a bona fide lymphatic network in the meninges, the membranous coverings that envelop the CNS.
• the brain meninges provide a landscape of opportunities to examine the local presentation of autoantigens to autoreactive T cells.
• meningeal lymphatic vessels serve as conduits for the drainage of antigens and cells to secondary lymphoid organs, specifically the deep cervical lymph nodes (dCLNs).
• dCLNs deep cervical lymph nodes
• the preliminary' data utilized a specialized platform for immunopeptidomics, spearheading an understanding of the CNS MHC II peptidome.
• MBP myelin basic protein
• an abundance of peptides derived from myelin basic protein (MBP) were identified that centered around a common sequence, MBP33-42, was identified.
• EAE experimental autoimmune encephalomyelitis
• co-immunization of MOG35-55 with our identified MBP peptides ameliorated EAE disease.
• Further analyses by flow cytometry prior to the peak of EAE disease unveiled an elevated frequency of regulatory Foxp3 -expressing CD4+ T cells in CNS-associated tissues of mice co-immunized with MBP peptides.
• Aim 1 is to address a hypothesis that CNS derived endogenous peptides promote differentiation of regulatory T cells, and thus, modulate CNS autoimmunity. Inducing EAE by co-immunizing MOG with MBP peptides resulted in increased frequencies of regulatory T cells. It was hypothesized that MBP specific T cells within the CNS possess regulatory' features that contribute to CNS immune tolerance. To bolster the idea that MBP peptides cultivate Foxp3+ regulatory T cells to ameliorate disease, EAE is induced with MOG and MBP peptides in Foxp3DTR mice with or without diphtheria toxin. Furthermore, the specificity of Foxp3+ regulatory T cells for MBP peptides in the meninges and dCLN is examined utilizing MBP-specific MHC II tetramers.
• Aim 2 is to evaluate a hypothesis that neuropathology limits the presentation of homeostatic epitopes on MHC II molecules. Tissue damage and inflammation lead to epitope spreading, which is thought to further contribute to autoimmune pathology. However, it remains elusive whether the presentation of homeostatic epitopes, such as MBP, are also affected by inflammation. It was expected that neuropathology limits the presentation of MBP peptides, especially those containing the MBP33-42 sequence. MHC II peptidomics of the brain meninges and draining dCLNs was performed at the peak of EAE disease.
• Aim 3 is to test a hypothesis that delivery of CNS derived endogenous peptides broadly safeguards against neuroinflammation -induced pathology. Introducing MBP-containing vesicles into the CSF could suppress EAE disease. Moreover, co-immunization of our MBP peptides with my'elin proteolipid protein (PLP)i39-i5i mitigated EAE disease in SJL/I mice. Thus, it was hypothesized that MBP peptides can be implemented therapeutically to broadly limit neuroinflammation and restore immune tolerance. To demonstrate this, MBP containing EVs were delivered at different time points after the onset of EAE disease.
• EAE was actively induced in different strains, including C57BL/6J, SJL/J, and BIO.
• PL mice while delivering MBP-containing vesicles into the CSF to demonstrate broad treatment efficacy of our identified MBP peptides.
• our identified peptides would help direct the development of MHC II tetramers, allowing the tracking and phenotyping of autoreactive CNS-specific T cells. This provides insight into the function of relevant antigen presentation in the CNS to harbor local populations of autoreactive T cells - an understanding of which elucidates mechanisms the CNS employs to acquire immune privilege. Aim Isought to address these two major barriers.
• Another critical barrier to the development of antigen-specific immunotherapies derives from an insufficient understanding of disease-initiating autoantigens and subsequent epitope spreading often arising as a consequence of chronic autoimmune or inflammatory response 1 ’ 5 .
• These studies overcome this barrier by leveraging our platform technology to define the MHC II peptidome to understand how the repertoire of autoantigens presented on MHC molecules diverges from homeostasis with neuroinflammation.
• Aim 2 provides a strategy to address this.
• antigen-specific therapeutic strategies also face a critical barrier of being constrained to identified pathogenic epitopes and finding ways to manipulate either their sequence or their delivery to drive tolerogenic immune responses 6 ’ 10 .
• autoimmune diseases remain a major public health concern, incapacitating many individuals with notably increasing rates, ranging from 3-6% annually 11 12 . In fact, 4.5% of individuals w orldw ide and more than 24 million people in the United States alone are afflicted by a variety of autoimmune disorders 13 14 . Moreover, with enhanced risks for developing another autoimmune condition 15 16 , cancers 17 , and psychiatric disorders 18-21 , autoimmune diseases negatively impact an individual's quality of life, often leading to lifelong disabilities with undeniable socioeconomic burden 22,23 .
• MHC major histocompatibility complex
• HLA human leukocyte antigen
• antigen-specific immunotherapies offer an appealing alternative to precisely target organ-specific autoimmunity- and has shown promise in different autoimmune conditions 37-41 .
• antigen-specific approaches have not always been effective and in some cases have been detrimental 42-45 , exemplifying an incomplete mechanistic understanding of autoantigen presentation in maintaining immune tolerance.
• MHC molecules readout the state of the cell and the physiology of the tissue at large through shaping the repertoire of antigens presented.
• antigens were thought to predominantly derive from endogenous sources for MHC I and exogenous sources for MHC II molecules, in recent years, this distinct separation for peptide sources has been blurred.
• Exogenously derived antigens have been described to funnel through different intracellular pathways to present on MHC I molecules - a process termed cross-presentation 52-55 .
• cross-presentation 52-55 On the other hand, it is also becoming more and more appreciated the abundance of endogenously derived peptides presented on MHC II molecules through which autophagy is thought to play a contributing role 56-58 .
• Aim 1 builds upon preliminary findings to elucidate regulatory mechanisms of autoantigen dependent protection against autoimmunity.
• Aim 2 contextualizes the repertoire of autoantigens presented on MHC II molecules in physiology and pathology.
• Aim 3 advances the therapeutic delivery of autoantigens to reinforce immune tolerance, effectively defending against autoimmune pathology.
• MBP-derived peptides were prominently presented in tire brain meninges, comprising 77% of peptides that could be defined as CNS elevated (FIG. 5B).
• the abundance of MBP presentation in the brain meninges was surprising and elucidation of its role in CNS immune tolerance was sought.
• MBP33-42 a vast majority of MBP-derived peptides centered around a common amino acid sequence, MBP33-42.
• MBP peptides that were identified in our MHC II peptidome were synthesized, which either included or excluded the common MBP33-42 sequence.
• Endogenous peptide suppresses myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE)
• MBP2742 regulated the pathogenicity of MOG ⁇ ⁇ -induccd EAE by modulating the expression or functionality of RORyt.
• 8-week-old C57BL/6J mice were immunized with either MOG35-55 or MOG35-55 + MBP27-42 to induce EAE.
• dCLNs were harvested at day 13 and the brain meninges at day 16 post EAE induction and flow cytometry was perfonned to assess for any changes to T cells.
• MHC II peptidome analysis of the brain meninges of SJL/J mice revealed an abundant presentation of MBP peptides that similarly contained the MBP33-42 sequence.
• MBP27-42-mediated protection against neuroinflammation could be generalizable, we utilized another active EAE model with SJL/J mice.
• PLPjug 151 myelin proteolipid protein
• NF-L neurofilament light polypeptide
• NF-L160-173 an endogenous peptide of similar length to MBP27-42, was selected as an appropriate control peptide with evidence supporting its presentation on MHC II molecules in EAE with limited T cell reactivity in SJL/J micegs.ge. Moreover, NF-L160-173 was identified as an MHC II -bound peptide in the brain meninges of SJL/J mice. Consistent with literature, immunization of PLP139-151 in young male SJL/J mice exhibited monophasic EAE disease 97 . To our surprise, co-immunization with MBP2742 but not NF-Li 60 -i73 demonstrated significant suppression of EAE with a notable downward trend to peak EAE scores (FIGS.
• mice in the experiments were used in the experiments. If mice in the experimental group were obtained from an outside provider, so were the respective controls. Transgenic mice and their wild-type littermate controls were bred in the same facility. Assessment was made using both males and females.
• Aim 1 CNS derived endogenous peptides promotes differentiation of regulatory T cells, and modulate CNS autoimmunity
• EAE enhanced green fluorescent protein
• mice 8- yveek-old Foxp3EGFP mice are utilized, yvhich only express enhanced green fluorescent protein (EGFP) under the control of the Foxp3 promoter.
• EAE is induced in both transgenic mice with either MOG35-55 alone or MOG35 55 + MBP27-42. Diphtheria toxin is injected intraperitoneally and intravenously (200 ng) on day 3 post EAE onset. All experimental groups are subjected to standard EAE scoring for up to 21 days. Tissues are harvested, including brain meninges, at day 16, the peak of EAE disease, for analysis by both floyv cytometry and immunohistochemistry.
• Foxp3EGFP control mice recapitulate of preliminary findings is expected, demonstrating an upward trend to Foxp3 expression in the brain meninges following co-immunization of MOG35-55 + MBP27-42 when compared to MOG35-55.
• Specific ablation of Foxp3+ regulatory T cells in Foxp3DTR mice is expected to abrogate MBP2742 dependent amelioration ofEAE, exhibiting a mechanism that relies on Foxp3 -mediated suppression.
• MBP2742 is presented on MHC class II molecules in the CNS during homeostasis and that MBP27-42 affords protection against MOG3555 -induced EAE.
• MHC II tetramers were recently developed using the shared amino acid sequence, MBP3342. These tetramers are tested and calibrated using lymphocytes isolated from the C57BL/6J mice immunized with MBP27-42 peptides boosted with complete Freund’s adjuvant (CFA) bilaterally in flank regions. One week is allowed for generation of a sufficient immune response prior to isolation of the draining inguinal lymph nodes.
• CFA complete Freund’s adjuvant
• T cell hybridoma clones In tandem, the isolated T cells are fused with BW5147, a thymic lymphoma cell line, to generate T cell hybridoma clones.
• In vitro antigen presentation assays are performed by co-culturing individual T cell clones with M12.C3, a B lymphoma cell line serving as the antigen presenting cell, in the presence of MBP2742 Using IL-2 enzyme linked immunosorbent assay (ELISA) as our readout for T cell activation, T cell clones specific to MBP2742 are selected. With guidance by MHC II binding affinity algorithms, key amino acids of MBP2742 are mutated to identify residues that abrogate activation of MBP2742 specific T cell clones.
• ELISA enzyme linked immunosorbent assay
• MBP27-42 specific MHC II tetramers By discerning critical recognition events for peptide-MHC II and T cell receptor interaction, the development of MBP27-42 specific MHC II tetramers can be better directed.
• MBP2742 specific T cells are phenotyped in the brain meninges and dCLN by flow cytometry of wild-type mice, mice immunized with MBP2742 alone, and mice co-immunized with MOG35-55 + MBP2742. Cell surface markers and transcription factors, including Foxp3, are stained. In doing so, whether the CNS harbors a population of MBP27-42 specific CD4+ T cells that express markers of regulation, such as Foxp3, contributing to CNS immune tolerance, can be detennined.
• MBP27-42 recruits a reservoir of Foxp3+ rcgulatory T cells for the upkeep of CNS immune tolerance.
• MBP27-42 specific T cells may enforce immune regulation through other regulatory mechanisms.
• prior studies have demonstrated a role for Foxp3- T regulatory type 1 (TRI) cells in regulating autoimmunity, including EAE, through production of regulatory cytokines, including IL-10 and TGFP103.104.
• TRI Foxp3- T regulatory type 1
• intracellular cytokine staining for IL- 10 and TGFp is included when phenotyping MBP2742 specific T cells by flow cytometry.
• IL- 1 Ofl/fl and CD4-CreERT2 are both commercially available and permit conditional deletion of IL-10 production in CD4+ T cells.
• Aim 2 Neuropathology limits the presentation of homeostatic epitopes on MHC II molecules.
• Hie lysate pre-cleared of debris by centrifugation will then be incubated with sepharose conjugated to an antibody specific to the MHC class II molecule.
• peptides are eluted from MHC class II molecules with 10% acetic acid. Eluted peptides are then cleaned further with C 18 spin columns prior to being subjected to mass spectrometry and immunological analyses.
• Aim 3 Therapeutic delivery of CNS derived endogenous peptides broadly safeguards against neuroinflammation-induced pathology
• Exo-spinTM Using the Exo-spinTM system, EVs are precipitated and subjected through size exclusion chromatography to purify for EVs specifically. Confirmation is by immunoblot and ZetaView nanoparticle tracking analyzer prior to further downstream application. Following confinnation, exosomes undergo at least five freeze-thaw cycles to release intracellular contents; which is purified by C18 columns enriching for peptides. The collected material is subjected to mass spectrometry and immunological analyses.
• Study #2 Assessing the therapeutic capacity of EVs containing MBP27-42 peptides to treat neuroinflammation
• EVs arc enriched from DC2.4 cells, an immortalized dendritic cell line, with 100,000xg ultracentrifugation and confinn purity by immunoblot, TEM, and ZetaView nanoparticle tracking analyzer. Enriched vesicles are sonicated equally in the absence or presence of endogenous MBP27 42 and encephalitogenic MOG35-55 peptides.
• Vesicles are further purified by both size exclusion chromatography and high-speed ultracentrifugation to remove any unincorporated peptides, effectively isolating intact control vesicles or vesicles containing our peptides of interest. Quality of enriched EVs is assessed similarly as mentioned above and further quantified by ZetaView nanoparticle tracking analyzer to control for the total number of vesicles injected into the CSF. Moreover, loaded peptides are fluorochrome conjugated enabling control for loading efficiency by nanoparticle flow cytometry. EAE is induced with MOG35-55 immunization in 8-week-old C57BL/6J mice.
• control vesicles or vesicles containing either MBP27-42 or MOG35-55 are introduced into the CSF via intracistema magna (i.e.m.) injection on day 5 (prior to disease onset), day 10 (onset of paralytic symptoms), or day 16 (peak disease). All experimental groups with their respective littermate controls are subjected to standard EAE scoring for up to 25 days. In doing so, only the delivery of MBP27-42-containing vesicles across different time points is expected to exhibit therapeutic benefit, promoting the restoration of CNS immune tolerance. Furthermore, intravenous injection of EVs are also examined as it presents with a minimally invasive and convenient mode of delivery.
• MBP27-42 an epitope abundantly presented on MHC II molecules, in regulating CNS immune homeostasis.
• MBP2742 sufficiently suppressed against PLPw-isi-induced EAE in SJL/J mice.
• the MBP27-42 sequence itself is highly conserved across different vertebrate species, including humans 106 107 . For these reasons, interrogation of the capability of MBP 2 7-42-containing vesicles to dampen neuroinflammation-induced damage in different active EAE models is sought.
• mice receive i.c.m. injections of control vesicles or vesicles containing peptides on day 5 after which they are subjected to standard EAE scoring for up to 25 days.
• MBP peptides are predicted to exhibit therapeutic efficacy at different stages of disease and in different strains of mice.
• the observed suppression of EAE disease may remain restricted to i.c.m. delivery of MBP2742 at day 5, prior to the onset of paralysis.
• EVs reflect the cell type and environment from which they are derived 108 109 , modulating the character of vesicles may bolster our antigen-specific immunotherapeutic approach.
• a recent study demonstrated that vesicles derived from dendritic cell cultures treated with low dose IFNy reduced oxidative stress and enhanced CNS myelination processes in vivo 110 .
• EVs derived from mesenchymal stem cells mildly but significantly reduced clinical EAE scores when administered intravenously at the peak of disease 11 this strengthens confidence in treating CNS autoimmunity according to the invention at a more advanced stage of disease.
• MBP27-42 and control peptides are also loaded into vesicles derived from IFNy-stimulated dendritic cells and mesenchymal stem cells.
• these studies further advance MBP27-42-containing vesicles as an antigen-specific immunotherapeutic for autoimmune disease.
• CD4+ T cells support glial neuroprotection, slow disease progression, and modify glial morphology in an animal model of inherited ALS.
• Interleukin-23 rather than interleukin- 12 is the critical cytokine for autoimmune inflammation of the brain. Nature 421, 744-748 (2003). https://doi.org: 10.1038/nature01355 Zhou, L. et al. TGF-beta-induced Foxp3 inhibits T(H)17 cell differentiation by antagonizing ROR- gammat function. Nature 453, 236-240 (2008). https://doi.org: 10.1038/nature06878 Ichiyama, K. et al. Foxp3 inhibits RORgammat-mediated IL-17A mRNA transcription through di-rect interaction with RORgammat. J Biol Chem 283, 17003-17008 (2008). https://doi.org: 10.
• MHC I molecules place tyrosine residues at the end of the peptide binding pocket, often restricting the peptide length to 8-10 amino acid residues, although longer peptides bound to MHC I molecules have been reported.
• the MHC II molecules are more open at the ends allowing for peptides to extrude beyond the confines of the binding cleft.
• the MHC II molecules are more amenable and accommodate peptide lengths of around 15-25 amino acid residues.
• the portions of the peptide not directly involved with the peptide binding groove of the MHC II molecule have been posited to play a role in influencing the stability of peptide binding. This adds an extra element of peptide association for MHC II molecules that of which is currently thought to be absent for MHC I molecules.
• the cell pellet Prior to lysing, optionally wash the cell pellet (e.g., once with PBS buffer).
• lysis buffer e.g., MEGA8/9 lysis buffer
• protease inhibitors e.g., Roche protease inhibitor cocktail, PMSF, lodoacetamide, Leupeptin. Pipet up and down to thoroughly suspend cell pellet.
• Lyse cells (e.g., for 1 hour in cold room while rocking).
• wash samples e.g., as follows: a. 5-10 mL 150 mM NaCl 20 mM Tris pH 7.4; b. 5-10 mL 400 mM NaCl 20 mM Tris pH 7.4; c. 5-10 mL 150 mM NaCl 20 mM Tris pH 7.4: d. 5-10 mL 20 mM Tris pH 8.0)
• Steps include:
• Samples can analyzed using LC-MS/MS for identification of peptidome.
• Example 3 Analyzing MHC I and II peptidomes in both humans and mice
• peptidomes of antigens presented by MHC molecules in brain and lymphatic tissues of humans and mice were investigated by purification and identification using LC/MS. 14,609 peptides were identified in a protcomic assay of autoantigens presented on major histocompatibility complex classes I and II (MHC I and MHC II) in human and mouse and peripheral and CNS immune cells (i.e., SEQ ID NOS: 1-14,609). Peptides were analyzed for the following properties: a. whether shared by both humans and mice, or in humans only, or in mice only; b. binding to MHC I, to MHC II, or to both; c. enrichment in the CNS; and d. identification of the protein from which the peptide derives from, including beginning and end amino acids for the peptide (fragment) of the protein.
• the central nervous system despite the presence of strategically positioned anatomical barriers designed to protect it, is not entirely isolated from the immune system. In fact, it remains physically connected to and can be influenced by the peripheral immune system. How the CNS retains such responsiveness while maintaining an immunologically unique status remains an outstanding conundrum.
• CNS-derived endogenous regulatory (guardian) peptides presented on major histocompatibility complex (MHC) II molecules at the CNS borders.
• guardian peptides During homeostasis, a preponderance of these guardian peptides were found to be bound to MHC II molecules throughout the path of lymphatic drainage from the brain to its surrounding meninges and its draining cervical lymph nodes. With neuroinflammatory disease, however, the presentation of guardian peptides was diminished. By then boosting the presence of these guardian peptides, a population of suppressor CD4+ T cells could be expanded and CNS autoimmune disease significantly reduced.
• This unexpected discovery of CNS-derived autoimmune guardian peptides may be the molecular key adapting the CNS to receive information and to maintain continuous dialogue with the immune system while balancing overt autoreactivity. This sheds new light on how we conceptually think about and therapeutically target neuroinflammatory and neurodegenerative diseases.
• the CNS seeks immunosurveillance yvhile actively providing immune cells with molecular cues to foster its tolerance to autoreactivity.
• a thorough interrogation was performed of the CNS, its borders, and its draining lymph nodes for peptides bound to MHC II molecules.
• CNS-de rived endogenous, regulatory self-peptides were identified, which were termed “guardian peptides”.
• CD1 lb+ CD11c- macrophages were the predominant MHC Il-expressing cells (antigen-presenting cells, APCs) in the brain, which also includes the leptomeninges, and the dura
• CD 19+ B cells were the main APCs in the deep cervical lymph nodes (dCLNs) and in tire superficial cervica lymph nodes (sCLNs) (FIGS. 16A-16E).
• algorithmic predictions of MHC II- binding affinities utilizing three independent models 14 16 identified a propensity for peptides with relatively weaker affinities to be bound on MHC II molecules in the brain. Whereas, further downstream along the path of lymphatic drainage their binding affinities increased, suggesting heterogeneity to the repertoire of peptides bound to MHC II molecules in different compartments (FIG. 16F).
• MBP peptides The abundant presentation of MBP peptides captivated our interest in understanding their potential function in the CNS during homeostasis. Identified MBP peptides were screened by immunizing with them to assess their encephalitogenicity. Whereas immunization of rodents with myelin oligodendrocyte glycoprotein (MOG)s5 55 induced paralysis ty pical of experimental autoimmune encephalomyelitis (EAE) 20 — a rodent model of multiple sclerosis (MS) 21 22 — immunization with endogenous MBP peptides, such as MBP160-175, did not induce paralysis (FIG. 14A).
• MOG myelin oligodendrocyte glycoprotein
• EAE experimental autoimmune encephalomyelitis
• mice that had received MBPiso 175 peptides exhibited decreased cellularity relative to those immunized with MOG35 55 peptides (FIG. 14B).
• MBP160-175 unlike MOG35-55, lacked an antigen-specific effector T cell response when assaying for IL-2 production by enzyme-linked immune absorbent sport (ELISpot) (FIG. 14C).
• ELISpot enzyme-linked immune absorbent sport
• mice were co-immunized with encephalitogenic MOG35-55 peptide and our endogenous MBP peptides.
• the MBP160-175 sequence was directly modified by converting its arginine residues to citrulline.
• T cells were individually sorted from draining lymph nodes of MOG35 55 only and of MOG35 55 + MBPiso 175 immunization groups, and then performed single-cell RNA sequencing (scRNA-seq) using the 10X Genomics Chromium -gene expression platform. Unsupervised clustering and dimensionality reduction was perfonned to project the cells into two dimensions using the uniform manifold approximation and projection (UMAP) algorithm.
• UMAP uniform manifold approximation and projection
• guardian MBP peptides may broadly apply to the treatment of neuroinflammatory as well as of neurodegenerative disorders.
• MHC e.g.. MHC II
• MHC II MHC Il-bound MBP peptides pervading the meningeal lymphatic network, extending from the brain parenchyma to the draining cervical ly ph nodes.
• tissue-specific antigens are presented on MHC II molecules during homeostasis in their respective draining lymph nodes for the skin and intestines 36 as well as the pancreas 37 .
• C57BL/6J (WT: JAX000664) and SJL/J (WT; JAX000686) were purchased from the Jackson Laboratory; mice were maintained in standard housing conditions (12 hour light/dark cycle and provided with sterilized water and regular rodent chow ad libitum unless stated otherwise). Mice were allowed to acclimate for at least one week in the animal facility prior to the beginning of any experiment. Adult males and females between 8-12 weeks of age were primarily used for our studies unless stated otherwise. Sample sizes were determined on the basis of a power analysis in accordance with previously published experiments. Experimenters, where necessary, were blinded to experimental groups during both scoring and quantification. All experiments were approved by the Institutional Animal Care and Use Committee at the Washington University in St. Louis. ii) Single Cell Isolations
• mice were perfused with transcardial perfusion with PBS containing heparin (0.025%); samples were collected into ice-cold RPMI and maintained on ice for the entirety of tissue collection. Spleens were removed from the surrounding tissues and placed on ice-cold RPMI (Gibco) until further use.
• lymph nodes i.e. deep cervical lymph nodes, superficial cervical lymph nodes, and inguinal lymph nodes
• spleen isolations were performed following lethal i.p. injection of Euthasol. Subsequently, mice were perfused with transcardial perfusion with PBS containing heparin (0.025%); samples were collected into ice-cold RPMI and maintained on ice for the entirety of tissue collection. Spleens were removed from the surrounding tissues and placed on ice-cold RPMI (Gibco) until further use.
• Tire whole spleen was digested and mashed through a 70 pm cell strainer with a glass pestle and washed with 5 mL of RPMI. Cells were then centrifuged at 450g for 5 minutes. RBC lysis was performed with 1 mL of ACK lysis buffer (Quality’ Biological); cells were incubated for 2 minutes and then 2 mL of ice-cold PBS was supplemented to the sample. Samples were subsequently centrifuged at 450g for 5 minutes and lysed red blood cells were aspirated. The cell pellets were then resuspended in FACS buffer (2% BSA.
• EDTA ethylenediamine acetic acid
• Lymph nodes were similarly digested and mashed through a 70 pm cell strainer, washed with 2 mL RPMI, centrifuged, resuspended in FACS buffer, and kept on ice until further use. Choroid plexi were removed from the brain; similarly, the dural meninges were peeled from the skull cap using Dumont #5 forceps (Fine Science Tools). Tire dural meninges were kept in ice-cold RPMI for the entirety of the tissue harvest.
• Dural meninges were then digested for 15 minutes at 37°C with constant agitation using 1 mL of prewarmed digestion buffer (RPMI-1640 medium with 2% FBS, 1 mg/mL Collagenase VIII, and 0.5 mg/mL DNase I). They were then filtered similarly through a 70 pm cell strainer and provided with 1 mL of complete medium (RPMI with 10% FBS) to neutralize the enzymes. Samples were then centrifuged at 450g for 5 minutes, resuspended in FACS buffer and kept on ice. Lastly, brains and spinal cords were harvested and placed in ice-cold RPMI for the entirety of the collection.
• prewarmed digestion buffer RPMI-1640 medium with 2% FBS, 1 mg/mL Collagenase VIII, and 0.5 mg/mL DNase I.
• Brains were mechanically dissociated using sterile surgical scalpels into ⁇ 1 mm 3 cubes and digested with constant agitation in 2 mL of pre-warmed digestion buffer for 20 minutes at 37°C, triturated with a 10 mL serological pipette, digested for another 20 minutes, triturated with a 5 mL serological pipette, and digested for an additional 20 minutes.
• a 1: 1 ratio of 22% bovine serum albumin (BSA) in PBS was added and centrifuged at 1000g for 10 minutes. Following centrifugation, the floating myelin layer was aspirated. Cell pellets were then neutralized with RPMI with 10% FBS to halt the enzymatic digestion.
• BSA bovine serum albumin
• Cells were isolated as described in '‘single cell isolations,” suspended in lysis buffer (40 mM MEGA 8, 40 mM MEGA 9, 1 mM phenylmethylsulfonyl fluoride, 0.2 mM iodoacetamide, 20 pg ml— 1 leupeptin and Roche cOmplete Protease Inhibitor cocktail in phosphate-buffered saline), and rocked for 1 h at 4 °C. The cell lysate was spun in a centrifuge at 20,000g for 25 min at 4°C.
• the supernatant was first incubated with polyclonal mouse immunoglobulin G (Bio X Cell; 1.5 mg antibody per sample) bound to Sepharose 4B at 4 °C for 30 min. The flow through containing peptide-MHC II complexes was collected, added to a tube containing phosphate-buffered saline-washed sepharose conjugated to the anti-I-A antibody (Y-3P;
• Mass spectrometry scans were acquired in profile mode and tandem mass spectrometry scans were acquired in centroid mode, for ions with charge states 2-7, with a cycle time of 1.5 s.
• Mass spcctromct ’ spectra were recorded from 375-1,500 Da at 120-K resolution (at m/z 200), and higher- energy collisional dissociation tandem mass spectrometry was triggered above a threshold of 2.0 x 104 . with quadrupole isolation (1.4 Da) at 15-K resolution and a collision energy of 30%. Dynamic exclusion was used (60 s), and monoisotopic precursor selection was on. vi) Mass spectrometry data analysis
• EAE was induced in C57BL/6J mice by subcutaneous injection of MOG35-55 peptide (100 pg, CSBio) and/or different synthesized peptides including, MBP160-175, citrullinated MBPieo-ns. MBPiee-m. and MBPi 92-216 (GenScript) emulsified in Freund s adjuvant (Sigma .Aldnch) supplemented with 2 mg/mL of Mycobacterium tuberculosis (BD). Pertussis toxin (200 ng. List Biologicals) was injected i.p. on day 0 and day 2 following immunization with MOG and/or MBP.
• EAE was induced in SJL/J mice by subcutaneous injection of PLP139-151 (100 pg, Vivitide) and/or different synthesized peptides including, MBP160 175, neurofilament light polypeptide (NEFL)i6o-i73 (GenScript) emulsified in Freund's adjuvant (Sigma Aldrich) supplemented with 2 mg/mL of Mycobacterium tuberculosis (BD). Pertussis toxin (400 ng, List Biologicals) was injected i.p. on day 0 and day 2 following immunization with PLP and/or MBP and NEFM.
• PLP139-151 100 pg, Vivitide
• NEFL neurofilament light polypeptide
• GenScript neurofilament light polypeptide
• BD Mycobacterium tuberculosis
• 0 no clinical disease
• 1 limp tail
• 2 hindlimb weakness
• 3 hindlimb paralysis
• 4 partial front limb paralysis
• 5 moribund.
• mice were subcutaneously immunized with different peptides, including MOG35 55, MBP160-175, MBP166-185, and MBP192-216, among others, emulsified in Freund’s adjuvant.
• 96- well polyvinylidene fluoride membrane plates (Millipore) were coated with IL-2 capture antibodies (BD Biosciences).
• IL-2 capture antibodies (BD Biosciences). 7 days after immunization, cells were isolated from the draining lymph nodes and counted such that 5 x 105 cells would be loaded per well (each condition performed in triplicates). Cells were stimulated with their appropriate antigens and controls and incubated at 37°C in 5% CO2 for 24 hours.
• IL-2 secreting cells were identified with IL-2 detection antibodies (BD Biosciences) and plates were developed following manufacturer’s directions (BD Biosciences). Spots quantified with CTL ImmunoSpot S6 Universal machines and Professional 6.0.0 software. ix) Single-cell RNA Sequencing.
• the filtered and normalized matrix was used as input to the Seurat pipeline and cells were scaled across each gene, regressing out sequencing depth per cell, number of unique features, and percent mitochondrial reads, before the selection of the top 2,000 most highly variable genes using variance stabilizing transformation.
• Principal Components Analysis was conducted and an elbow plot was used to select components for UMAP analysis and clustering.
• Shared Nearest Neighbor (SNN) clustering optimized with the Louvain algorithm, as implemented by the Seurat FindClusters function was performed before manual annotation of clusters based on expression of canonical gene markers.
• CD4 and CD8 cells were subset based on sub-cluster identity and gene expression and each subset was filtered to include genes that had at least 4 transcripts in at least 4 cells. Then the top 2000 highly variable genes were determined and included for further analysis using the SingleCellExperiment modelGeneVar and getTopHVGs functions. After filtering, limma and edgeR were used to build a model and conduct differential expression testing with the ImFit, contrasts.fit, and eBayes functions. Results were then filtered using a Benjamini- Hochberg adjusted p-value threshold of less than 0.05 as statistically significant.
• CSV files containing single-cell transcript counts were downloaded from the Gene Expression Omnibus accession GSE118948, read into R and converted to matrices. Blood and choroid plexus tissue samples were discarded and the remaining leptomeningeal and parenchymal perivascular space samples were manually filtered for quality control. Filtering was performed to remove cells with less than 200 unique genes, less than 1,000 or greater than 25,000 transcripts, and mitochondrial transcript percentage greater than 30 percent. Expression values were then normalized using the scran and scater packages. The resulting log2 values were transformed to the natural log scale for compatibility with the Seurat (v3) pipeline42-44. Initial contamination in the fonn of lowly sequenced clusters were manually removed and the finalized subset of cells was scaled, underwent PCA, clustering, and annotation as described above.
• the mean (or median) value of percentiles across all models was then used as the main source for the given antigen. We further represented these values as inverted percentile ranks such that larger values indicate peptides with increased likelihood to be a binder.
• the decoy set was generated from the mouse proteome and consists of about 1 x 10 6 peptide fragments.
• the DC2.4 cell line was purchased from the American Type Culture Collection (ATCC). Cells were maintained by culturing in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco), 1% penicillin/streptomycin (Gibco) and 2 mM L-glutamine (Gibco). All cells were grown in an incubator set at 37°C in 5% CO2. xiii) Extracellular vesicles isolation and preparation
• the DC2.4 cells were cultured as stated above in complete DMEM medium in T-75 flasks to 80- 90% confluency. Cells were thoroughly washed with dPBS prior to replacing the medium with serum depleted DMEM. They were further cultured overnight. Supernatants were collected next day and spun at varying speeds, including 1.000g for 10 mins to rid of any cell contaminants as well as 20,000g for 30 mins to rid of any smaller debris. The supernatant, at this point, was further filtered with a 0.22 pm filter and ultracentrifuged at 100,000g for 2 hours to pellet down extracellular vesicles (EVs). All spins were performed at 4°C.
• the pellet was resuspended in 500 pl PBS and visualized by either transmission electron microscope (JEOL JEM-1400, Phillips) or nanoparticle tracking analysis (ZetaView) to confirm their isolation, to measure their size, and to approximate the number of particles per milliliter
• mice were anesthetized using ketamine/xylazine (100 mg/kg ketamine and 10 mg/kg xylazine). Their necks were shaved and cleaned appropriately with 70% iodine prior to placing their heads securely into the stereotactic frame. Ophthalmic solution was applied to prevent drying of their eyes prior to the procedure. Following this, a longitudinal incision of the skin was made at the back of their necks: the underlying muscles were retracted with hooks to expose the cistema magna. A 5pL Hamilton syringe with a 33-gauge needle was utilized to inject 5pL of PBS or extracellular vesicles (i.e.
• Tire MHC-II peptidome of pancreatic islets identifies key features of autoimmune peptides. Nat Immunol 21, 455-463 (2020). Cohen, I. R. The cognitive paradigm and the immunological homunculus. Immunol Today 13, 490-494 (1992). Wucherpfennig, K. W. & Strominger, J. L. Molecular mimicry in T cell-mediated autoimmunity: viral peptides activate human T cell clones specific for myelin basic protein. Cell 80, 695-705 (1995). Lunemann, J. D. et al. EBNA1 -specific T cells from patients with multiple sclerosis cross react with myelin antigens and co-produce IFN-gamma and IL-2.
• Example 5 Assessment of Tubb 3, Map2, and Nefm as MHC guardian peptides in an EAE animal

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