Compositions and methods of treatment for neuroinflammation-related disorders

Abstract

Compositions and methods for treating, preventing, or reversing neuroinflammation-related disorders or autoimmune neurological disorders in a subject in need thereof are disclosed. The methods comprise administering a therapeutically effective amount of a composition comprising a myelin basic protein (MBP) peptide.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202380094372.2 (22) Application Date 2023.12.18 (30) Priority Data 63 / 387905 2022.12.16 US 63 / 518333 2023.08.09 US (85) PCT International Application Entering National Phase Date 2025.08.18 (86) PCT International Application Application Data PCT / US2023 / 084623 2023.12.18 (87) PCT International Application Publication Data WO2024 / 130249 EN 2024.06.20 (71) Applicant: University of Washington, Missouri, USA (72) Inventor: J. Kipnis, M.W. King (74) Patent Agency: China Patent Agency (Hong Kong) Limited, 72001 Patent Attorney: Luo Wenfeng, Zhang Hua (51) Int.Cl. A61K 38 / 17 (2006.01) A61K 47 / 69 (2017.01) A61K 9 / 127 (2025.01) C07K 14 / 705 (2006.01) A61P 25 / 28 (2006.01) A61P 37 / 04 (2006.01) (54) Invention Title: Compositions and Methods for Treating Neuroinflammatory-Related Disorders (57) Abstract: This invention discloses compositions and methods for treating, preventing, or reversing neuroinflammatory-related disorders or autoimmune neurological disorders in patients in need. The method comprises administering a therapeutically effective amount of a composition containing a myelin basic protein (MBP) peptide. Claims 7 pages, Description 91 pages, Sequence Listing (electronic publication), Figures 53 pages. CN 121240870 A 2025.12.30 CN 1 21 24 08 70 A 1. A pharmaceutical composition comprising one or more peptides and a pharmaceutically acceptable excipient, wherein each of the one or more peptides binds to a major histocompatibility complex (MHC) molecule and is a fragment of a protein selected from Table C1 or a modified fragment thereof. 2. The pharmaceutical composition of claim 1, wherein the protein is expressed at an elevated level in the central nervous system (CNS) relative to a control tissue. 3. The pharmaceutical composition of claim 1, wherein the protein is not expressed at an elevated level in the central nervous system (CNS) or is expressed at a decreased level in the CNS relative to a control tissue. 4. The pharmaceutical composition of claim 1, wherein each of the one or more peptides binds to an MHC class I molecule, an MHC class II molecule, or both.5. The pharmaceutical composition of claim 1, wherein each of the one or more peptides independently comprises, or is composed of, the amino acid sequence shown in any one of SEQ ID NO: 1-14619 or a modified sequence thereof. 6. The pharmaceutical composition of claim 1, wherein each of the one or more peptides independently comprises, or is composed of, the amino acid sequence shown in any one of SEQ ID NO: 74, 75, 78, 91, 174, 176, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 254, 278, 292, 294, 295, 296, 297, 298, 299, 300, 301, and 5438 or a modified sequence thereof. 7. The pharmaceutical composition according to claim 1, wherein each of the one or more peptides independently comprises SEQ ID NO: 597, 770, 1231, 1232, 1709, 1748, 2297, 2324, 3765, 3766, 3767, 3768, 3769, 3770, 3771, 3772, 3773, 3774, 3775, 3784, 3785, 3786, 3787, 3788, 4514, 4534, 4544, 4545, 4546, 4547, 4548, 4549, 4550, 4551, 4552, 4553, 4554, 4555, 4556, 4557, 4558, 4642, 4643, 4644, 4645, 4646, 4657, 4683, 4762, 4763, 4766, 4767, 4768, 4769, 4770, 4771, 4772, 4774, 4775, 4776, 4777, 4778, 4779, 4780, 4781, 4782, 4783, 4784, 4785, 4786, 4787, 4788, 4789, 4790, 4791, 4792, 4793, 4794, 4795, 4796, 4797, 4798, 4799, 4800, 4801, 4802, 4803, 4804, The amino acid sequence or its modified sequence, or a combination thereof, shown in any one of 4805, 4806, 4807, 4809, 4810, 4811, 4812, 4813, 4819, 4820, 4822, 4823, 4825, 4826, 4828, 4829, 5067, 5439, 5574, 5575, 14519, 14520, 14521, 14522, 14523, 14524, 14525, and 14526. 8. The pharmaceutical composition according to claim 1, wherein each of the one or more peptides independently comprises the SEQ IDs in Table 7.9. The pharmaceutical composition of claim 1, wherein each of the one or more peptides independently comprises, or consists of, any one of the amino acid sequences shown in SEQ ID NO of Table 8, or a modified sequence thereof. 10. The pharmaceutical composition of claim 1, wherein each of the one or more peptides independently comprises, or consists of, any one of the amino acid sequences shown in SEQ ID NO of Table 9, or a modified sequence thereof. 11. The pharmaceutical composition of claim 1, wherein each of the one or more peptides independently comprises, or consists of, any one of the amino acid sequences shown in SEQ ID NO of Table 10, or a modified sequence thereof. 12. The pharmaceutical composition of claim 1, wherein each of the one or more peptides is a fragment of myelin basic protein (MBP), tubulin β3III (TUBB3), or neurofilament medium polypeptide (NEFM). 13. The pharmaceutical composition according to claim 12, wherein each fragment of MBP independently comprises SEQ ID NO: 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, 10756, 10757, 10758, 10759, 10760, 10761, Claims 1 / 7 page 2 CN 121240870 A 10762, 10763, 10764, 10765, 10766, 10767, 10768, 10769, 10770, 10771, 10772, 10773, 10774, 10775, 10776, 10777, 10778, 10779, 10780, 10781, 10782, 10783, 10784, 10785, 10786, 10787, 10788, 10789, 10790, 10791, 10792, 10793, 10794, 10795, 10796, 10797, 10798, 10799, 10800, 10801, 10802, 10803, 10804, 10805, 10806, 10807, 10808, 1080910810, 10811, 10812, 10813, 10814, 10815, 10816, 10817, 10818, 10819, 10820, 10821, 10822, 10823, 10824, 10825, 10826, 10827, 10828, 10829, 10830, 10831, 10832, 10833, 10834, 10835, 10836, 10837, 10838, 10839, 10840, 10841, 10842, 10843, 10844, 10845, 12162, 14537, 14538, 14539, 14540, 14541, 14542, 14543, 14544, 14545, 14546, 14547, 14548, 14549, 14550, 14551, 14552, 14553, 14554, 14555, 14556, 14557, 14558, 14559, 14560, 14561, 14562, 14563, 14564, 14565, 14566, 14567, 14568, 14569, 14570, 14571, The amino acid sequence shown in any one of 14572, 14573, 14574, 14575, 14576, 14577, 14578, 14579, 14580, 14581, 14582, 14583, 14584, 14585, 14586, 14587, 14588, 14589, 14590, 14591, 14592, 14593, 14594, 14595, 14596, 14610, 14611, 14612 and 14613, or a sequence thereof. 14. The pharmaceutical composition of claim 1, wherein at least one of the one or more peptides comprises, or is composed of, an 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. 15. The pharmaceutical composition of claim 1, further comprising one or more extracellular vesicles (EVs) or liposomes encapsulating the one or more peptides. 16. The method of claim 15, wherein the pharmaceutical composition comprises one or more EVs, and wherein the one or more EVs are exosomes, microvesicles, or apoptotic bodies of cells, optionally human cells. 17. The method of claim 15, wherein the pharmaceutical composition comprises one or more EVs, and wherein the...The one or more EVs are of oligodendrocyte origin, optionally derived from cell line DC2.4. 18. The method of claim 15, wherein the pharmaceutical composition comprises one or more EVs, and wherein the one or more EVs are synthetic. 19. The pharmaceutical composition of any one of claims 1-18, for treating, preventing, or reversing neuroinflammatory conditions, autoimmune neurological conditions, or acute CNS injury. 20. The pharmaceutical composition of claim 19, wherein the neuroinflammatory-related condition or autoimmune neuropathy is selected from acute disseminated encephalomyelitis (ADEM), acute inflammatory demyelinating polyradiculoneuropathy (AIDP; Guillain-Barré syndrome (GBS)), acute motor axonopathy (AMAN), acute motor-sensory axonopathy (AMSAN), acute optic neuritis (AON), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), autoimmune encephalitis (AIE), chronic ataxia neuropathy, oculomotor palsy, immunoglobulin M paraprotein, cold agglutinin and disialic acid antibody (CANOMAD), chronic meningitis, Bechtel's disease, central nervous system (CNS) vasculitis, and chronic inflammatory demyelinating polyradiculoneuropathy. (CIDP), Steroid-Reactive Chronic Lymphocytic Inflammation with Perivascular Enhancement of the Pontine Clippers (CLIPPERS), Glial Fibrillary Acidic Protein (GFAP), Hashimoto's Encephalitis, Hypertrophic Pain Meningitis, IgG4-Related Neurological Disorders, Lambert-Eton Myasthenic Syndrome (LEMS), Antimyelin Oligodendrocyte Glycoprotein Antibody Disease (MOG), Miller Fisher Syndrome (MFS), Monoclonal Gammaglobulinosis of Undetermined Significance (MGUS), Multifocal Motor Neuropathy (MMN), Myasthenia Gravis (MG), Multiple Sclerosis (MS), Neuromyelitis Optic (NMO), Neurosarcoidosis, Paraneoplastic Neurological Syndrome (PNS), Parkinson's Disease (PD), Steroid-Reactive Encephalopathy Associated with Autoimmune Thyroiditis (SREAT), Stiff-Person Syndrome, Sussac Syndrome, and Transverse Myelitis. 21. The pharmaceutical composition of claim 19, wherein the acute CNS injury is selected from spinal cord injury, traumatic brain injury, spinal cord injury, optic nerve injury, and stroke. 22. A method of treating, preventing, or reversing neuroinflammatory conditions, autoimmune neurological conditions, or acute CNS injury in a subject of need, comprising administering to the subject one or more peptides, each peptide independently comprising, or consisting of, an amino acid sequence shown in any one of SEQ ID NO: 1-14619 or a modified sequence thereof.23. The method of claim 22, wherein the neuroinflammatory-related condition or autoimmune neuropathy is selected from acute disseminated encephalomyelitis (ADEM), acute inflammatory demyelinating polyradiculoneuropathy (AIDP; Guillain-Barré syndrome (GBS)), acute motor axononeuropathy (AMAN), acute motor-sensory axononeuropathy (AMSAN), acute optic neuritis (AON), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), autoimmune encephalitis (AIE), chronic ataxia neuropathy, oculomotor palsy, immunoglobulin M paraprotein, cold agglutinin and disialic acid antibody (CANOMAD), chronic meningitis, Bechtel's disease, central nervous system (CNS) vasculitis, and chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). ), steroid-responsive chronic lymphocytic inflammation with perivascular enhancement in the pons (CLIPPERS), glial fibrillary acidic protein (GFAP), Hashimoto's encephalitis, hypertrophic pachymeningitis, IgG4-related neurological disorders, Lambert-Eton myasthenic syndrome (LEMS), anti-myelin oligodendrocyte glycoprotein antibody disease (MOG), Miller Fisher syndrome (MFS), monoclonal gammopathy of undetermined significance (MGUS), multifocal motor neuropathy (MMN), myasthenia gravis (MG), multiple sclerosis (MS), neuromyelitis optica (NMO), neurosarcoidosis, paraneoplastic neurological syndromes (PNS), Parkinson's disease (PD), steroid-responsive encephalopathy associated with autoimmune thyroiditis (SREAT), stiff-person syndrome, Sussac syndrome, and transverse myelitis. 24. The method of claim 22, wherein the acute CNS injury is selected from spinal cord injury, traumatic brain injury, spinal cord injury, optic nerve injury, and stroke. 25. The method of claim 22, wherein each of the one or more peptides independently comprises, or is composed of, the amino acid sequence shown in any one of SEQ ID NO: 74, 75, 78, 91, 174, 176, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 254, 278, 292, 294, 295, 296, 297, 298, 299, 300, 301, and 5438, or a modified sequence thereof. 26. The method of claim 22, wherein each of the one or more peptides independently comprises, SEQ ID NO: 597, 770, 1231, 1232, 1709, 1748, 2297, 2324, 3765, 3766, 3767, 3768, 3769, 3770,3771, 3772, 3773, 3774, 3775, 3784, 3785, 3786, 3787, 3788, 4514, 4534, 4544, 4545, 4546, 4547, 4548, 4549, 4550, 4551, 4552, 4553, 4554, 4555, 4556, 4557, 4558, 4642, 4643, 4644, 4645, 4646, 4657, 4683, 4762, 4763, 4766, 4767, 4768, 4769, 4770, 4771, 4772, 4774, 4775, 4776, 4777, 4778, 4779, 4780, 4781, 4782, 4783, 4784, 4785, 4786, 4787, 4788, 4789, 4790, 4791, 4792, 4793, 4794, 4795, 4796, 4797, 4798, 4799, 4800, 4801, 4802, 4803, 4804, 4805, 4806, 4807, 4809, 4810, 4811, 4812, 4813, 4819, 4820, 4822, 4823, 4825, 4826, 4828, 4829, 27. The method of claim 22, wherein each of the one or more peptides independently comprises, or consists of, any one of the amino acid sequences shown in SEQ ID NO of Table 7, or a modified sequence thereof. 28. The method of claim 22, wherein each of the one or more peptides independently comprises, or consists of, any one of the amino acid sequences shown in SEQ ID NO of Table 8, or a modified sequence thereof. Claims 3 / 7 Page 4 CN 121240870 A 29. The method of claim 22, wherein each of the one or more peptides independently comprises, or consists of, any one of the amino acid sequences shown in SEQ ID NO of Table 9, or a modified sequence thereof. 30. The method of claim 22, wherein each of the one or more peptides independently comprises, or is composed of, the amino acid sequence shown in any one of SEQ ID NOs of Table 10 or a modified sequence thereof. 31. The method of claim 22, wherein each of the one or more peptides is a fragment of myelin basic protein (MBP), tubulin β3III (TUBB3), or neurofilament medium polypeptide (NEFM).32. The method of claim 22, wherein each fragment of the MBP independently comprises SEQ ID NO: 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, 10756, 10757, 10758, 10759, 10760, 10761, 10762, 10763, 10764, 10765, 10766, 10767, 10768, 10769, 10770, 10771, 10772, 10773, 10774, 10775, 10776, 10777, 10778, 10779, 10780, 10781, 10782, 10783, 10784, 10785, 10786 10787, 10788, 10789, 10790, 10791, 10792, 10793, 10794, 10795, 10796, 10797, 10798, 10799, 10800, 10801, 10802, 10803, 10804, 10805, 10806, 10807, 10808, 10809, 10810, 10811, 10812, 10813, 10814, 10815, 10816, 10817, 10818, 10819, 10820, 10821, 10822, 10823, 10824, 10825, 10826, 10827, 10828, 10829, 10830, 10831, 10832, 10833, 10834, 10835, 10836, 10837, 10838, 10839, 10840, 10841, 10842, 10843, 10844, 10845, 12162, 14537, 14538, 14539, 14540, 14541, 14542, 14543, 14544, 14545, 14546, 14547, 14548, 14549, 14550, 14551, 14552, 14553, 14554, 14555, 14556, 14557, 14558, 14559, 1456014561, 14562, 14563, 14564, 14565, 14566, 14567, 14568, 14569, 14570, 14571, 14572, 14573, 14574, 14575, 14576, 14577, 14578, 14579, 14580, 14581, 14582, 14583, 14584, 14585, 14586, 14587, 14588, 14589, 14590, 14591, 14592, 14593, 14594, 14595, 14596 33. The method of claim 22, wherein at least one of the one or more peptides comprises, or is composed of, the amino acid sequence shown in any one of 14610, 14611, 14612, and 14613, or a modified sequence thereof. 34. The method of claim 22, wherein the one or more peptides are encapsulated in one or more extracellular vesicles (EVs) or liposomes. 35. The method of claim 34, wherein the pharmaceutical composition comprises one or more EVs, and wherein the one or more EVs are exosomes, microvesicles, or apoptotic bodies of cells, optionally human cells. 36. The method of claim 34, wherein the pharmaceutical composition comprises one or more EVs, and wherein the one or more EVs are of oligodendrocyte origin. 37. The method of claim 34, wherein the pharmaceutical composition comprises one or more EVs, and wherein the one or more EVs are synthetic. 38. The method of claim 21, wherein the one or more peptides are administered to the subject by infusion or injection into the subject's cerebrospinal fluid (CSF). 39. One or more nucleic acids encoding one or more peptides, said peptides independently comprising, or consisting of, the amino acid sequence shown in any one of SEQ ID NO: 1-14619 or a modified sequence thereof. 40. The one or more nucleic acids of claim 39, wherein each of said one or more peptides independently comprises SEQ ID NO: 74, 75, 78, 91, 174, 176, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, ...The amino acid sequence shown in any one of 251, 252, 254, 278, 292, 294, 295, 296, 297, 298, 299, 300, 301 and 5438 or a modified sequence thereof, or composed of thereof. 41. The one or more nucleic acids according to claim 39, wherein each of the one or more peptides independently comprises SEQ ID NO: 597, 770, 1231, 1232, 1709, 1748, 2297, 2324, 3765, 3766, 3767, 3768, 3769, 3770, 3771, 3772, 3773, 3774, 3775, 3784, 3785, 3786, 3787, 3788, 4514, 4534, 4544, 4545, 4546, 4547, 4548, 4549, 4550, 4551, 4552, 4553, 4554, 4555, 4556, 4557, 4558, 4642, 4643, 4644, 4645, 4646, 4657, 4683, 4762, 4763, 4766, 4767, 4768, 4769, 4770, 4771, 4772, 4774, 4775, 4776, 4777, 4778, 4779, 4780, 4781, 4782, 4783, 4784, 4785, 4786, 4787, 4788, 4789, 4790, 4791, 4792, 4793, 4794, 4795, 4796, 4797, 4798, 4799, 4800, 4801, 4802, 4803 4804, 4805, 4806, 4807, 4809, 4810, 4811, 4812, 4813, 4819, 4820, 4822, 4823, 4825, 4826, 4828, 4829, 5067, 5439, 5574, 5575, 14519, 14520, 14521, 14522, 14523, 14524, 14525, and 14526, or a modified sequence thereof, or composed thereof. 42. The one or more nucleic acids according to claim 39, wherein each of the one or more peptides independently comprises, or is composed of, any one of the amino acid sequences shown in SEQ ID NO of Table 7, or a modified sequence thereof, or a modified sequence thereof. 43. The one or more nucleic acids of claim 39, wherein each of the one or more peptides independently comprises, or is composed of, any one of the amino acid sequences shown in SEQ ID NO of Table 8, or a modified sequence thereof or a modified sequence thereof. 44. The one or more nucleic acids of claim 39, wherein each of the one or more peptides independently comprises, the SEQ ID NO of Table 9,45. The one or more nucleic acids of claim 39, wherein each of the one or more peptides independently comprises, or consists of, any one of the amino acid sequences or modified sequences or modified sequences shown in SEQ ID NO of Table 10. 46. The one or more nucleic acids of claim 39, wherein each of the one or more peptides is a fragment of myelin basic protein (MBP), tubulin β3III (TUBB3), or neurofilament medium polypeptide (NEFM). 47. One or more nucleic acids according to claim 46, wherein each fragment of MBP independently comprises SEQ ID NO: 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, 10756, 10757, 10758, 10759, 10760, 10761, 10762, 10763, 10764, 10765, 10766, 10767, 10768, 10769, 10770, 10771, 10772, 10773, 10774, 10775, 10776, 10777, 10778, 10779, 10780, 10781, 10782, 10783, 10784, 10785 10786, 10787, 10788, 10789, 10790, 10791, 10792, 10793, 10794, 10795, 10796, 10797, 10798, 10799, 10800, 10801, 10802, 10803, 10804, 10805, 10806, 10807, 10808, 10809, 10810, 10811, 10812, 10813, 10814, 10815, 10816, 10817, 10818, 10819, 10820, 10821, 10822, 10823, 10824, 10825, 10826, 10827, 10828, 10829, 10830, 10831, 10832, 10833, 10834, 10835, 10836, 10837, 10838, 10839, 10840, 10841, 10842, 10843, 10844, 10845,12162, 14537, 14538, 14539, 14540, 14541, 14542, 14543, 14544, 14545, 14546, 14547, Claims 5 / 7, Page 6, CN 121240870 A, 14548, 14549, 14550, 14551, 14552, 14553, 14554, 14555, 14556, 14557, 14558, 14559, 14560, 14561, 14562, 14563, 14564, 14565, 14566, 14567, 14568, 14569, 14570, 14571, The amino acid sequence shown in any one of 14572, 14573, 14574, 14575, 14576, 14577, 14578, 14579, 14580, 14581, 14582, 14583, 14584, 14585, 14586, 14587, 14588, 14589, 14590, 14591, 14592, 14593, 14594, 14595, 14596, 14610, 14611, 14612 and 14613, or a modified sequence thereof, or composed of thereof. 48. One or more nucleic acids according to claim 39, wherein at least one of the one or more peptides comprises, or is composed of, the amino acid sequence of FLPRHRDTGILDSIGR (SEQ ID NO: 5776), DTGILDSIGR (SEQ ID NO: 10755), or DTGILDSIGRFFSGDRGAPK (SEQ ID NO: 10759). 49. One or more vectors, each comprising at least one of the one or more nucleic acids according to any one of claims 39-48. 50. One or more vectors according to claim 49, wherein the one or more vectors are viral vectors. 51. The viral vector according to claim 50, derived from adenovirus, adeno-associated virus, or retrovirus, optionally selected from lentiviruses. 52. One or more vectors according to claim 49, wherein the vector is a non-viral vector. 53. One or more nucleic acids according to any one of claims 39-48, wherein the nucleic acid is mRNA. 54. One or more nucleic acids according to claim 53, wherein the mRNA is non-replicating mRNA, self-amplifying mRNA (saRNA), or circular RNA (circRNA). 55. One or more nucleic acids according to claim 54, wherein the mRNA is a non-replicating mRNA, wherein each mRNA comprises 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, andEach coding region is flanked by UTRs. 56. One or more nucleic acids according to claim 55, wherein each 5' cap comprises a 7-methylguanosine (m7G) cap, which is linked to the first nucleotide (N) of each mRNA via a triphosphate (ppp) to form m7GpppNp. 57. One or more nucleic acids according to claim 56, wherein each m7G cap comprises a methylated 2'-OH on the first nucleotide, which links the 5' end of each mRNA to the cap (cap1 or m7GpppN1mp). 58. One or more nucleic acids according to claim 56, wherein each m7G cap comprises a methylated 2'-OH on the first nucleotide and a second nucleotide, which links the 5' end of each mRNA to the cap (cap2 or m7GpppN1mpN2mp). 59. One or more nucleic acids according to any one of claims 53-58, wherein each mRNA comprises one or more modified nucleosides, optionally selected from pseudouridine (ψ), N1-methylpseudouridine (m1ψ), 5-methoxyuridine (mo5U), 2-thiouridine (s2U), 5-methylcytidine (m5C), and N6-methyladenosine (m6A). 60. One or more nucleic acids according to any one of claims 53-59, wherein the nucleic acid is encapsulated, optionally encapsulated with lipid nanoparticles (LNP), polyplex, polymer nanoparticles, lipopolyplex (LPP), or cationic polypeptide. 61. One or more nucleic acids according to claim 60, wherein they are encapsulated with LNP. 62. A pharmaceutical composition comprising a nucleic acid according to any one of claims 39-48, a carrier according to any one of claims 49-52, or an mRNA according to any one of claims 53-61. 63. The pharmaceutical composition according to claim 62, formulated for infusion or injection into cerebrospinal fluid (CSF). 64. The pharmaceutical composition according to any one of claims 62-63, used to treat, prevent, or reverse neuroinflammatory conditions, autoimmune neurological conditions, or acute CNS injury in a subject requiring claims 6 / 7 pages 7 CN 121240870 A. 65. The pharmaceutical composition according to claim 64, wherein the neuroinflammatory conditions or autoimmune neurological conditions are selected from acute disseminated encephalomyelitis (ADEM), acute inflammatory demyelinating polyradiculoneuropathy (AIDP; Guillain-Barré syndrome (GBS)), acute motor axonal neuropathy (AMAN), acute motor sensory axonal neuropathy (AMSAN), acute optic neuritis (AON), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and autoimmune diseases.Encephalitis (AIE), chronic ataxic neuropathy, ophthalmoplegia, immunoglobulin M paraprotein, cold agglutinin and disialic acid antibody (CANOMAD), chronic meningitis, Bechtel's disease, central nervous system (CNS) vasculitis, chronic inflammatory demyelinating polyradiculopathy (CIDP), steroid-responsive chronic lymphocytic inflammatory disease with perivascular enhancement in the pons (CLIPPERS), glial fibrillary acidic protein (GFAP), Hashimoto's encephalitis, hypertrophic pachymeningitis, IgG4-related neurological disorders, Lambert-Eaton myasthenia gravis Neuromyelopathy syndrome (LEMS), myasthenia gravis (MG), Miller Fisher syndrome (MFS), monoclonal gammopathy of undetermined significance (MGUS), multifocal motor neuropathy (MMN), myasthenia gravis (MG), multiple sclerosis (MS), neuromyelitis optica (NMO), neurosarcoma, paraneoplastic neurological syndrome (PNS), Parkinson's disease (PD), steroid-reactive encephalopathy associated with autoimmune thyroiditis (SREAT), stiff-person syndrome, Sussac syndrome, and transverse myelitis. 66. The pharmaceutical composition according to claim 64, wherein the acute CNS injury is selected from spinal cord injury, traumatic brain injury, spinal cord injury, optic nerve injury, or stroke. Claims 7 / 7 Page 8 CN 121240870 A Compositions and methods for treating neuroinflammatory-related conditions

[0001] Statement regarding federally funded research or development

[0002] This invention was made with government support under grant granted by the National Institutes of Health (DPI AT010416). The United States government has certain rights to this invention. Technical Field

[0003] This disclosure generally relates to compositions and methods for treating, preventing, or reversing neuroinflammatory-related conditions using fragments of MHC-binding proteins. Background Art

[0004] With increasing morbidity and prevalence, autoimmune diseases continue to debilitate countless people, increase the burden of healthcare, and reduce quality of life. To date, current interventions have focused on nonspecific immunosuppressive therapies with significant side effects. Therefore, antigen-specific immunotherapy offers an attractive alternative to accurately target organ-specific autoimmunity. For personalized medicine, a deeper understanding of the repertoire of autoantigens presented on major histocompatibility complexes (MHCs) (e.g., MHC class II) will guide the development of antigen-specific immunotherapy.

[0005] This invention provides a pharmaceutical composition that 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 selectively...A fragment of the protein or a modified fragment thereof from Table C1. The protein or peptide may be expressed at an elevated level in the central nervous system (CNS) relative to a control tissue. In one example, the expression level of the protein or peptide is 5 times that of the control tissue. Alternatively, the protein may not be expressed at an elevated level or may be expressed at a reduced level in the CNS relative to a control tissue. In one example, the CNS tissue is the dura mater, 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 shown in any one of SEQ ID NO:1-14619 or a modified sequence thereof.

[0006] Each of the one or more peptides may independently comprise, or consist of, the amino acid sequence shown in any one of SEQ ID NO: 74, 75, 78, 91, 174, 176, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 254, 278, 292, 294, 295, 296, 297, 298, 299, 300, 301 and 5438 or a modified sequence thereof. Each of the one or more peptides may independently comprise SEQ ID NO: 597, 770, 1231, 1232, 1709, 1748, 2297, 2324, 3765, 3766, 3767, 3768, 3769, 3770, 3771, 3772, 3773, 3774, 3775, 3784, 3785, 3786, 3787, 3788, 4514, 4534, 4544, 4545, 4546, 4547, 4548, 4549, 4550, 4551, 4552, 4553, 4554, 4555, 4556, 4557, 4558, 4642, 4643, 4644, 4645, 4646, 4657, 4683, 4762, 4763, 4766, 4767, 4768, 4769, 4770, 4771, 4772, 4774, 4775, 4776, 4777, 4778, 4779, 4780, 4781, 4782, 4783, 4784, 4785, 4786, 4787, 4788, 4789, 4790, 4791, 4792, 4793, 4794, 4795, 4796, 4797, 4798 4799, 4800, 4801, 4802, 4803, 4804, 4805, 4806, 4807, 4809, 4810, 4811, 4812, 4813, 4819The amino acid sequence or its modified sequence shown in any one of SEQ ID NOs in Tables 7, 8, 9 or 10, or composed of the amino acid sequence shown in Tables 7, 8, 9 or 10, or composed of the amino acid sequence shown in Tables 1 / 91 or 9 CN 121240870 A. Each of the one or more peptides independently comprises, or is composed of, the amino acid sequence or its modified sequence shown in Tables 7, 8, 9 or 10, or composed of the amino acid sequence shown in Tables 1 / 91 or 9 CN 121240870 A.

[0007] Each of the one or more peptides may be a fragment of myelin basic protein (MBP), tubulin β3III (TUBB3) or neurofilament medium polypeptide (NEFM). Each fragment of the MBP can independently contain SEQ ID NO: 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, 10756, 10757. 10758, 10759, 10760, 10761, 10762, 10763, 10764, 10765, 10766, 10767, 10768, 10769, 10770, 10771, 10772, 10773, 10774, 10775, 10776, 10777, 10778, 10779, 10780, 10781, 10782, 10783, 10784, 10785, 10786, 10787, 10788, 10789, 10790, 10791, 10792, 10793 10794, 10795, 10796, 10797, 10798, 10799, 10800, 10801, 10802, 10803, 10804, 10805, 10806, 10807, 10808, 10809, 10810, 10811, 10812, 10813, 10814, 10815, 10816, 10817, 10818, 10819, 10820, 10821, 10822, 10823, 10824, 10825, 10826, 10827, 10828, 1082910830, 10831, 10832, 10833, 10834, 10835, 10836, 10837, 10838, 10839, 10840, 10841, 10842, 10843, 10844, 10845, 12162, 14537, 14538, 14539, 14540, 14541, 14542, 14543, 14544, 14545, 14546, 14547, 14548, 14549, 14550, 14551, 14552, 14553, 14554, 14555, 14556, 14557, 14558, 14559, 14560, 14561, 14562, 14563, 14564, 14565, 14566, 14567, 14568, 14569, 14570, 14571, 14572, 14573, 14574, 14575, 14576, 14577, 14578, 14579, 14580, 14581, 14582, 14583, 14584, 14585, 14586, 14587, 14588, 14589, 14590, 14591, The amino acid sequence shown in any one of 14592, 14593, 14594, 14595, 14596, 14610, 14611, 14612, and 14613, or composed thereof. At least one of the one or more peptides may contain, or be composed 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.

[0008] The pharmaceutical composition may further comprise one or more extracellular vesicles (EVs) or liposomes encapsulating the one or more peptides. The one or more EVs may be exosomes, microvesicles, or apoptotic bodies of cells, optionally human cells. The one or more EVs may be of oligodendrocyte origin, optionally derived from dendritic cells, which may belong to the cell line DC2.4. The one or more EVs may be synthetic.

[0009] This document provides one or more nucleic acids encoding the one or more peptides. One or more vectors may each contain at least one of the one or more nucleic acids. The vector may be a viral vector. The viral vector may be derived from adenovirus, adeno-associated virus, or retrovirus, optionally selected from lentiviruses. The vector may be a non-viral vector.

[0010] The one or more nucleic acids may be mRNA. The mRNA may be non-replicating mRNA or self-amplifying mRNA.(saRNA) or circular RNA (circRNA). The mRNA may be a non-replicating mRNA; and each mRNA may contain 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 UTRs. Each 5' cap may contain a 7-methylguanosine (m7G) cap, which is linked to the first nucleotide (N) of each mRNA via a triphosphate (ppp) to form m7GpppNp. Each m7G cap may contain a methylated 2'-OH on the first nucleotide, which links the 5' end of each mRNA to the cap (cap1 or m7GpppN1mp). Each m7G cap may contain a methylated 2'-OH on the first nucleotide and the second nucleotide, which links the 5' end of each mRNA to the cap (cap2 or m7GpppN1mpN2mp). Each mRNA may contain one or more modified nucleosides, optionally selected from pseudouridine (ψ), N1-methylpseudouridine (m1ψ), 5-methoxyuridine (mo5U), 2-thiouridine (s2U), 5-methylcytidine (m5C), and N6-methyladenosine (m6A). The nucleic acid may be encapsulated, optionally with lipid nanoparticles (LNP), polyplex, polymer nanoparticles, lipopolyplex (LPP), or cationic peptides. The pharmaceutical composition may contain one or more of the aforementioned nucleic acids, one or more carriers, or one or more mRNAs.

[0011] This document provides one or more of the aforementioned peptides or the aforementioned pharmaceutical compositions for the treatment, prevention, or reversal of neuroinflammatory conditions, autoimmune neuropathies, or acute CNS injury. This article also provides a method for treating, preventing, or reversing neuroinflammatory-related conditions, autoimmune neuropathies, or acute CNS injury in a subject of need, which may include administering the one or more peptides or the pharmaceutical composition to the subject; and the use of the one or more peptides or the pharmaceutical composition in the preparation of a medicament for treating, preventing, or reversing neuroinflammatory-related conditions, autoimmune neuropathies, or acute CNS injury. The peptides or pharmaceutical compositions may be administered to the subject by infusion or injection into the subject's cerebrospinal fluid (CSF), or may be intended to be administered to the subject by infusion or injection into the subject's CSF.

[0012] Neuroinflammatory-related conditions or autoimmune neuropathies may be selected from acute disseminated encephalomyelitis (ADEM), acute inflammatory demyelinating polyradiculoneuropathy (AIDP; Guillain-Barré syndrome (GBS)), acute motor axonal neuropathy (AMAN), acute motor sensory axonal neuropathy (AMSAN), acute optic neuritis (AON), and Alzheimer's disease.(AD), Amyotrophic Lateral Sclerosis (ALS), Autoimmune Encephalitis (AIE), Chronic Ataxic Neuropathy, Ophthalmoplegia, Immunoglobulin M Paraprotein, Cold Aggregate and Disialic Acid Antibody (CANOMAD), Chronic Meningitis, Bechtel Disease, Central Nervous System (CNS) Vasculitis, Chronic Inflammatory Demyelinating Polyradiculoneuropathy (CIDP), Steroid-Reactive Chronic Lymphocytic Inflammation with Pontine Perivascular Enhancement (CLIPPERS), Glial Fibrillary Acidic Protein (GFAP), Hashimoto's Encephalitis, Hypertrophic Pain Meningitis, IgG4-Related Neurological Disorders, Lambert-Eton Myasthenic Syndrome (LEMS), Anti-Myelin Oligodendrocyte Glycoprotein Antibody Disease (MOG), Miller Fisher Syndrome (MFS), Monoclonal Gamma Globulinosis of Undetermined Significance (MGUS), Multifocal Motor Neuropathy (MMN), Myasthenia Gravis (MG), Multiple Sclerosis (MS), Neuromyelitis Optic (NMO) Neurosarcoidosis, paraneoplastic neurological syndrome (PNS), Parkinson's disease (PD), steroid-reactive encephalopathy associated with autoimmune thyroiditis (SREAT), stiff-person syndrome, Susac's syndrome, and transverse myelitis. Acute CNS injury can be caused by spinal cord injury, traumatic brain injury, spinal cord injury, optic nerve injury, and stroke. Brief Description of the Drawings

[0013] Those skilled in the art will understand that the drawings described below are for illustrative purposes only. The drawings are not intended to limit the scope of this teaching in any way.

[0014] Figures 1A-1B show the presentation of MHC II in specific MBP regions in the CNS central nervous system. Figure 1A confirms the composition of MHC II-binding peptides derived from proteins labeled as CNS-enriched. Figure 1B shows the exact sequence of MHC II-binding peptides derived from MBP.

[0015] Figures 2A-2D show the acquisition of regulatory T cells by MBP peptides to suppress CNS autoimmunity. Figure 2A depicts a schematic diagram of the induction of experimental autoimmune encephalomyelitis (EAE) in C57BL / 6J mice. Figure 2B shows the clinical EAE scores tracked for 21 days in mice immunized with MOG35-55, MOG35-55 and MBP33-52, or MOG35-55 and MBP (27-42 subscript); two-way ANOVA and multiple comparison tests, with p-values ​​indicated on the plot in cases of significance. Figure 2C shows a representative flow cytometry plot of regulatory T cells in the deep cervical lymph nodes (dCLN) when comparing the MOG35-55 group and the MOG35-55 and MBP27-42 co-immunization group, with relevant quantifications in Figure 2D; unpaired two-tailed Student's t-test, with p-values ​​indicated in cases of relevance.

[0016] Figures 3A-3B show that the presentation of MBP peptides is generalizable and similarly inhibits neuroinflammation in different active EAE models. Figure 3A depicts CNS-enriched peptides identified in the brain and dura mater of male SJL / J mice and freshly frozen human dura mater. Figure 3B shows the 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. Statistical analysis was performed using two-way ANOVA and multiple comparison tests, with p-values ​​marked on the plot in cases of significance.

[0017] Figure 4 illustrates the use of MBP peptides as a therapeutic agent against CNS autoimmunity. The schematic diagram shows the purification of extracellular vesicles (EVs) and their subsequent packaging with peptides via sonication. Fluorescently labeled (Alexa Fluor 488) ovalbumin (OVA) was incorporated into the vesicles, and cellular uptake was confirmed by flow cytometry. EVs were visualized by electron microscopy (EM) and a ZetaView nanoparticle tracker. Clinical EAE scores were tracked for 21 days in mice that were induced for EAE and introduced with empty EVs or EVs containing MBP27-42 via intracranial injection before disease onset; two-way ANOVA was used, and p-values ​​were plotted on the graph. For both conditions, peak EAE scores were also quantified and shown on the right, with unpaired two-tailed Student's t-tests and p-values ​​plotted on the graph.

[0018] Figures 5A-5B show the analysis of the MHC II peptide genome. Figure 5A depicts epitope mapping of MHC-II binding peptides by Gibbs clustering analysis of 8-week-old C57BL / 6J.I-Ab mice. Figure 5B depicts the distribution of MHC-II binding peptides enriched for proteins expressed at elevated levels in the CNS. "Other" indicates the sum of CNS-elevating proteins, and only a single MHC-II binding peptide was identified. These include: MAP2, NEFL, DPYSL2, CNP, ISLR2, BCAS1, IRS4, PTGDS, and TBC1D30.

[0019] Figure 6A depicts the experimental protocol used to induce experimental autoimmune encephalomyelitis (EAE). Figure 6B depicts the mean clinical EAE score in C57BL / 6J mice immunized on day 0 with MOG33-55 (red), MBP12-26 (grey), or MBP27-42 (blue); mice were intraperitoneally injected with pertussis toxin (PTx) on days 0 and 2. Standardized EAE scores were evaluated daily for 20 days after immunization. N = 5 mice / group, and p-values ​​are marked on the figure.

[0020] Figure 7A shows the mean clinical EAE score in C57BL6 / J mice immunized on day 0 with MOG35-55 (red), MOG35-55+MBP12-26 (grey), MOG35-55+MBP27-42 (blue), or MOG35-55+MBP27-42 (citrullinated) (yellow); standard EAE scores were evaluated daily for 20 days post-immunization. Figure 7B shows the peak EAE score of C57BL6 / J mice immunized according to Figure 7A. N = 5 mice / group, p-values ​​are marked on the figures.

[0021] Figures 8A-8D show mice immunized with MOG35-55 or MOG35-55+MBP27-42 to induce EAE (N = 4 mice / group). dCLN and meninges were evaluated by flow cytometry on days 13 and 16 post-EAE induction. Representative flow cytometry plots for live CD45+, TCRβ+, and CD4+ gated cells show Foxp3 and RORγt, with frequencies indicated for dCLN (Fig. 8A) and meninges (Fig. 8C). For dCLN (Fig. 8B) and meninges (Fig. 8D), pooled data show the frequencies of RORγt and Foxp3 expression as CD4+ T cells, with p-values ​​indicated on each plot.

[0022] Fig. 9A shows the mean clinical EAE score in SJL / J mice immunized on day 0 with PLP139-151 (red), PLP139-151+NF-L160-173 (grey), or PLP139-151+ MBP27-42 (blue); standard EAE scores were used to evaluate mice daily for 21 days post-immunization. Fig. 9B shows the peak EAE score of SJL / J mice immunized according to Fig. 9A. N = 5 mice / group, p-values ​​are marked on the figure.

[0023] Figure 10A shows the immunoblotting of the supernatant (SN) and precipitate after high-speed ultracentrifugation, demonstrating the enrichment of extracellular vesicle markers tetraspan membrane proteins CD9 and CD63 in the precipitate. Figure 10B shows negative staining by transmission electron microscopy of the enriched extracellular vesicles. Figure 10C shows the experimental protocol depicting the insertion of the MBP27-42 peptide into extracellular vesicles prior to intracephalic magnum (ICM) injection (top). Mean clinical EAE score in C57BL / 6J mice immunized with MOG35-55 by ICM injection of empty vesicles (red) or vesicles containing MBP27-42 (blue) on day 5; standard EAE scores were used to evaluate mice daily for 21 days post-immunization. N = 5 mice / group, p-values ​​are marked on the figure.

[0024] Figure 11A shows the results of immunization with MOG35-55 (red) or CFA only (black) on day 0 in C57BL / 6J mice. (Instructions for Use, page 4 / 91, CN 121240870 A)All clinical EAE scores were used; standard EAE scores were used to evaluate mice daily for 16 days post-immunization. Figure 11B depicts representative images and Figure 11C depicts the quantification of the percentage coverage of OVA647 protein injected into the cisterna magna (icm) of dCLN in mice immunized with CFA only or CFA+MOG35-55 at the peak of EAE disease (day 16). Scale bar, 150 μm. P-values ​​are marked on the plot.

[0025] Figures 12A-12C depict the binding motifs of the MHCI alleles H2-Kb (Figure 12A) and H2-Db (Figure 12B) and the unique MHC II allele H2-Ab (Figure 12C) in C57BL / 6J mice, as predicted by the netMHCpan and netMHCIIpan algorithms.

[0026] Figures 13A-13E show the presentation of endogenous CNS peptides revealed by the CNS MHC II peptidomome. Figure 13A depicts a schematic diagram illustrating the mass spectrometry identification of MHC II-binding peptides from the brain (including the pia mater), dura mater (dura), and lymph nodes (including the deep cervical lymph nodes (dCLN) and superficial cervical lymph nodes (sCLN)) of male C57BL / 6J mice. Figure 13B shows an evaluation of the proportion of all uniquely identified peptides that can be designated as CNS enriched (teal columns); the percentage is shown above each individual column. Figure 13C depicts a circular diagram of CNS-enriched MHC II-binding peptides identified for each individual tissue. The percentage of MBP is indicated where applicable. Figure 13D shows a Venn diagram depicting the relationship between CNS-enriched MHC II-binding peptides in the brain, dura mater, dCLN, and sCLN. Figure 13E shows a summary of the individual peptide sequences contained within the MBP 158–195 region as defined by the MHC II peptidome.

[0027] Figures 14A-14K show that MBP is non-encephalitis-derived and promotes immunosuppression. Figure 14A shows the mean clinical EAE score evaluated by immunization with MOG35-55 or MBP160-175 in C57BL / 6J mice (n = 5 mice / group). The plot shows mean ± sem and represents two independent experiments. p-values ​​are marked on the plot and determined by two-way ANOVA. Mice were immunized with MOG35-55 or MBP160-175, and draining (groin) lymph nodes were removed on day 7 post-immunization (n = 3 mice / group). Figure 14B shows the quantification of cell counts (mean ± sem, unpaired two-tailed Student's t-test). Figure 14C shows the ELISpot assay performed after peptide recall indicated on the x-axis to measure IL-2 production of CD4+ T cells. Data are shown as mean ± sem, two-way ANOVA, and multiple comparison tests. Figure 14D shows the results obtained through C57BL / 6J males.Mean clinical EAE scores were evaluated in mice (n = 5 mice / group) immunized with MOG35-55 alone, or with MOG35-55 and citrullinated MBP160-175 (MBP160-175 (cit.)), or co-immunized with MOG35-55 and MBP160-175. The plot shows mean ± sem and represents three independent experiments. In cases of significance, p-values ​​are indicated on the plot and determined by two-way ANOVA and multiple comparison tests. Figure 14E depicts UMAP visualization of T cells from draining (groin) lymph nodes of C57BL / 6J mice immunized with MOG35-55 or co-immunized with MOG35-55 and MBP160-175. Figure 14F shows the Log2 fold change measured by scRNA-seq in different identification clusters when comparing immunization with MOG35-55 versus MOG35-55 and MBP160-175. Significant differences are highlighted in the plots. Figure 14G shows the genetic ontology for identifying CD4+ (left) or CD8+ (right) T cells that were significantly upregulated in the MOG35-55 and MBP160-175 groups compared to the MOG35-55 group. Figures 14H-14I depict representative flow cytometry plots with relevant quantification (Figure 14H) and depict the proportion of CTLA-4+Foxp3– unconventional suppressor T cells or Foxp3+ regulatory T cells (Tregs) in the CD4+ T cell population in the dCLN at 13 days post-immunization (Figure 14I) (n = 3 mice / group, mean ± sem, unpaired two-tailed Student's t-test). Figures 14J-14K show representative flow cytometry plots (Figure 14J) and associated quantifications (Figure 14K), confirming the proportion of CTLA-4+Foxp3–CD39+ in the CD4+ T cell population in the spinal cord 13 days post-immunization (n = 5 mice / group, mean ± sem, unpaired two-tailed Student's t-test).

[0028] Figures 15A-15F show the therapeutic delivery of protective MBP peptides to avoid CNS autoimmunity. Figure 15A depicts a schematic diagram of the isolation of MHC II-binding peptides in the brain, dura mater, and spinal cord at the peak of MOG35-55-induced EAE (day 16). Figure 15B shows the MHC II peptide group from EAE and juvenile mice. The quantitative relative abundance of peptides was identified in different antigenic regions to generate heatmaps illustrating changes in the MHC II-binding peptide library in EAE compared to juvenile mice. For brain relative to (Cbln157-72) and for dura mater relative to (Mbp196-236), the relative abundance of the peptides specified in the heatmap was measured. (Instructions 5 / 91, page 13, CN 121240870 A)(Normalized peak area). Figure 15C depicts a bar chart of the relative abundance of MBP peptides in the MBP158–195 or MBP196–236 regions for the brain (top) and dura mater (bottom) when comparing EAEs with juvenile mice. Relative abundance (normalized peak area) was measured for the brain relative to (Cbln157–72) and for the dura mater relative to (Mbp196–236). Figure 15D shows the experimental design depicting ICM injection of extracellular vesicles (empty, MBP160–175, or MBP160–175 (cit.)) followed by flow cytometry evaluation of the dura mater two days later. e. Representative flow cytometry plots (top) and related quantifications (bottom) reveal the proportion of cells expressing CTLA-4+Foxp3– unconventional suppressor T cells (left) or Foxp3+Treg (right) within a CD4+ T cell population (n = 5 mice / group, mean ± s.e.m., one-way ANOVA and Tukey multiple comparison test). Figure 15F depicts the experimental protocol illustrating the induction of EAE by immunization with MOG35-55 in C57BL / 6J mice 7 days post-immunization with i.c.m. of PBS, MBP160-175 extracellular vesicles, or MBP160-175 (cit.) extracellular vesicles (top). Mean clinical EAE scores were evaluated up to day 20 post-immunization (n = 5 mice / group). The plots show mean ± sem and represent three independent experiments. p-values ​​are marked on the plots and determined by two-way ANOVA and multiple comparison tests.

[0029] Figures 16A-16H show the characterization of the MHC II peptidome in the CNS of male C57BL / 6J mice. Figure 16A shows the peptide length distribution as a percentage of the total number of unique peptides identified by the MHC II peptidome for the brain (including the pia mater), dura mater, dCLN, and sCLN. Figures 16B-16D show representative flow cytometry plots depicting the gating strategies used to identify the distribution of MHC II-expressing cells. B cells (gated against CD19+CD11c–), dendritic cells (DCs, gated against CD19–CD11c+), and macrophages (MΦ, gated against CD19–CD11c–) were identified within the MHC II+ gate. Macrophages were further confirmed by F4 / 80+ and CD11b+ staining (not shown). To the right, the bar chart shows the frequency of the aforementioned antigen-presenting cells as a percentage of CD45+MHC II+ cells for the brain (Fig. 16B), dura mater (Fig. 16C), dCLN (Fig. 16D), and sCLN (Fig. 16E). Fig. 16F shows a violin plot depicting the predicted binding affinity for unique MHC II-binding peptides. Median passSolid lines represent the quartiles, and dashed lines represent the first and third quartiles. p-values ​​(one-way ANOVA and Tukey multiple comparison test) are plotted on the graph. Figure 16D shows a Venn diagram depicting the relationships between all unique MHCII-binding peptides in the brain, dura mater, dCLN, and sCLN. Figure 16H shows a summary of the individual peptides identified on MBP sequences as defined by the MHC II peptidomome in male C57BL / 6J mice.

[0030] Figures 17A-17F show the characterization of the MHC II peptidomome in the CNS of female C57BL / 6J mice. Figure 17A shows a pie chart of the composition of MHC II-binding peptides enriched in the CNS in each individual tissue. Where applicable, the percentage of CNS-enriched peptides as MBP is plotted. Figure 17B shows a summary of all identified individual peptide sequences derived from MBP in the MHC II peptidomome of female C57BL / 6J mice. Figure 17C depicts a Venn diagram of the various overlapping CNS-enriched peptide sequences identified in the brain (including the pia mater) between female and male C57BL / 6J individuals. Figure 17D depicts a bar chart showing the relative abundance of MBP peptides in the brain of both male and female C57BL / 6J individuals containing the MBP158-195 or MBP196-236 regions. The relative abundance (normalized peak area) was determined from the peptide sequence Dag1488-531, which was identified as a common peptide. Figure 17E depicts a Venn diagram of the various CNS-enriched overlapping peptide sequences identified in the dura mater between female and male C57BL / 6J individuals. Figure 17F depicts a bar chart showing the relative abundance of MBP peptides in the dura mater of both male and female C57BL / 6J individuals containing the MBP158-195 or MBP196-236 regions. Relative abundance (normalized peak area) was measured for the identified common peptide sequence Sptn1381-389 to determine relativity from the peptide sequence.

[0031] Figures 18A-18F show the characterization of the MHCII peptidome in the CNS of male SJL / J mice. Figure 18A depicts a violin plot illustrating the predicted binding affinity for unique MHC II binding peptides in the brain (including the pia mater), dura mater, dCLN, and sCLN of SJL / J mice. The median is represented by a solid line, and the first and third quartiles are represented by dashed lines. p-values ​​(one-way ANOVA and Tukey multiple comparison test) are marked on the plot. Figure 18B shows the evaluation of the proportions of all uniquely identified peptides designated as CNS enriched (teal-colored columns) in the brain, dura mater, dCLN, and sCLN; the percentages are marked above each individual column on page 6 / 91 of the specification, CN 121240870 A. Figures 18C-18D depict the relationship between MHC and different tissues in male SJL / J mice.A Venn diagram showing the relationship between all peptides bound to MHC II molecules (Fig. 18C) or CNS-enriched peptides (Fig. 18D). Fig. 18E depicts a circular diagram of all CNS-enriched peptides identified in the MHC II peptidomite of male SJL / J mice for the brain, dura mater, and sCLN. Where applicable, the whole portion represented by MBP is indicated in the drawing. Fig. 18F shows a depiction of all individual peptide sequences derived from MBP identified in the MHC II peptidomite of male SJL / J mice.

[0032] Figs. 19A-19E show the protection of endogenous protective MBP peptides in different neuroinflammatory models. Fig. 19A depicts experimental designs for actively inducing EAE by immunizing C57BL / 6J or SJL / J mice with MOG35-55 or PLP139-151 peptides, with or without MBP or NEFL peptides, respectively. Individually, C57BL / 6J mice were also immunized with myelin-related peptides for ELISpot assays. Figure 19B shows mice immunized with either MBP166-185 or MBP192-216, with draining (groin) lymph nodes removed on day 7 post-immunization (n = 3 mice / group). ELISpot assays were performed to measure IL-2 production by CD4+ T cells after recall using peptides specified on the x-axis. Data are presented as mean ± sem, two-way ANOVA, and multiple comparison tests. Figure 19C shows the mean clinical EAE score in C57BL / 6J male mice (n = 5 mice / group) evaluated by immunization with MOG35-55 alone or by co-immunization with MOG35-55 and MBP166-185 or MOG35-55 and MBP192-216. The plot shows mean ± sem and indicates two independent experiments. In cases of significance, p-values ​​are indicated on the plot and determined by two-way ANOVA and multiple comparison tests. Figure 19D shows the mean clinical EAE scores in C57BL / 6J female mice (n = 5 mice / group) evaluated by immunization with MOG35-55 alone, or by co-immunization with MOG35-55 and MBP160-175 (cit.), or by co-immunization with MOG35-55 and MBP160-175. The plot shows mean ± sem and represents three independent experiments. In cases of significance, p-values ​​are marked on the plot and determined by two-way ANOVA and multiple comparison tests. Figure 19E shows the mean clinical EAE scores in SJL / J male mice (n = 5 mice / group) evaluated by immunization with PLP139-151 alone, or by co-immunization with PLP139-151 and MBP160-175, or by co-immunization with PLP139-151 and NEFL160-173. The plot shows mean ± sem and represents two independent experiments. In cases of significance, the p-value is plotted on the graph and determined using two-way ANOVA and multiple comparison tests.

[0033] Figures 20A-20E show peripherally presented MBP peptide-induced conventional Tregs. Figure 20A depicts a dot plot of population markers from single-cell RNA sequencing, measured by the percentage of cells expressing the marker gene for each T cell cluster. Figure 20B shows representative gating strategies used in flow cytometry to define CTLA-4+Foxp3–repressive T cells and conventional Foxp3+ regulatory T cells (Tregs) within CD4+ T cells. CTLA-4+Foxp3–CD4+ T cells were further gated to evaluate CD39, PD-1 (CD279), and IL-10 expression. Figures 20C-20D show representative flow cytometry plots and related quantifications (Figure 20C) confirming the proportions of CTLA-4+ Foxp3– suppressor T cells and Foxp3+ Tregs in the CD4+ T cell population in the inguinal (draining) lymph nodes 13 days post-immunization (Figure 20D) (n = 3 mice / group, mean ± sem, unpaired two-tailed Student's t-test). Figure 20E shows the quantification of the frequency of conventional Foxp3+ Tregs as CD4+ T cells in the spinal cord 13 days post-immunization (n = 5 mice / group, mean ± sem, unpaired two-tailed Student's t-test).

[0034] Figures 21A-21G show the MHCII binding library of autoantigens altered by neuroinflammation. Figures 21A-21B show the changes in MHCII expression in mice immunized with CFA alone or CFA+MOG35-55, as shown by representative flow cytometry plots (Figure 21A) and related quantifications (Figure 21B), n = 5 mice / group, mean ± sem, unpaired two-tailed Student's t-test. Figure 21C shows a pie chart depicting the CNS-enriched MHCII-binding peptides identified in the brain and spinal cord at the peak of EAE disease. Below is a visual summary of the individual MBP sequences identified. Figure 21D shows a population pyramid diagram of the distribution of amino acids identified at the C-terminus as a percentage of all MHCII-binding peptides, indicating cleavage preference between EAE and naive mice. Figure 21E shows the UMAP projection of human datasets when analyzing naive, pre-symptomatic, and EAE mice, showing the corresponding color-coded extensive cell lineages. cDCs = conventional dendritic cells. migDCs = migratory dendritic cells. Figure 21F shows a volcano plot depicting the differences in gene expression when comparing EAE-induced mice with control microglia on page 7 / 91 of the specification, CN 121240870 A. Arrows indicate identifiable peptidases. Figure 21G shows the genes that were significantly upregulated in microglia when comparing EAE and juvenile mice.

[0035] Figures 22A-22F show the induction of unconventional repressive CD4+ T cells by direct delivery of encapsulated MBP peptides into CSF.Figure 22A shows immunoblotting of the supernatant (SN) and resuspended precipitate after ultracentrifugation, demonstrating enrichment of the tetraspan membrane proteins CD9 and CD63, markers of extracellular vesicles (EVs). Below, negative staining by transmission electron microscopy depicts the enriched EVs, with scale bars marked on the subplot. Figure 22B shows the gating strategy for flow cytometry analysis defining CTLA-4+ Foxp3– unconventional suppressor T cells and conventional Foxp3+ Tregs within CD4+ T cells. At the bottom, representative flow cytometry plots of dCLN depict the gating of these populations injected with ICM: empty EVs, MBP160–175 EVs, and MBP160–175(cit.) EVs. Figures 22C-22E show the quantification of unconventional CTLA-4+Foxp3– suppressor T cells and conventional Foxp3+ Tregs as CD4+ T cell frequencies in deep cervical lymph nodes (Figure 22C), superficial cervical lymph nodes (Figure 22D), and spleen (Figure 22E). Figure 22F shows mice immunized with MOG35-55 to induce EAE, followed by administration of PBS or free MBP160-175 peptide via ICM injection on day 7. Mean clinical EAE scores were evaluated until day 20 post-immunization (n = 5 mice / group). Plots show mean ± sem and represent two independent experiments. p-values ​​are marked on the plots and determined by two-way ANOVA.

[0036] Figures 23A-23G show candidates for regulatory peptides against CNS autoimmunity. Following immunization with MOG35-55 or MOG35-55 and Tubb3 peptide 1 (Figure 23A); MOG35-55 or MOG35-55 and Tubb3 peptide 2 (Figure 23B); MOG35-55 or MOG35-55 and Tubb3 peptide 3 (Figure 23C); MOG35-55 or MOG35-55 and Tubb3 peptide 4 (Figure 23D); MOG35-55 or MOG35-55 and Map2 (Figure 23E); MOG35-55 or MOG35-55 and Nefm peptide 1 (Figure 23F); and MOG35-55 or MOG35-55 and Nefm peptide 2 (Figure 23G), clinical EAE scores were tracked for 19 days. Statistical analysis was performed using two-way ANOVA. The asterisks shown on the graph represent p-values ​​(* indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001). Detailed Implementation

[0037] Although autoreactive T cells are well known to cause autoimmune diseases, their protective role in limiting tissue damage has been described in cardiac, skeletal muscle, and central nervous system (CNS) pathologies, including Alzheimer's disease, stroke, and traumatic injury, etc. Given the evidence supporting the suppression and beneficial functions of autoreactive T cells, therefore, CNS...The molecular and cellular mechanisms by which the brain interacts with the peripheral immune system require further investigation.

[0038] This research has benefited from the recent rediscovery of the true lymphatic network in the meninges (the membranous covering that encloses the CNS). Emphasizing the role of the brain boundary as an intimate space for interactions between CNS-resident and peripheral immune cells, the meninges offer a compelling picture for examining the local presentation of self-antigens to self-reactive T cells.

[0039] Furthermore, the lymphatic vessels of the meninges act as conduits for the drainage of antigens and cells to secondary lymphoid organs, particularly the deep cervical lymph nodes (dCLN). For these reasons, the inventors have the insight to examine CNS-specific self-antigen presentation in CNS-related tissues and how it facilitates immune surveillance, which will inform new mechanisms for maintaining tissue homeostasis and preventing overt autoimmunity.

[0040] The compositions and methods of this disclosure are based on the surprising finding that co-immunization with MOG35-55 and the MBP33-42 peptide improved EAE disease in an experimental MOG35-55-induced autoimmune encephalomyelitis (EAE) model. Further analysis by flow cytometry prior to the peak of EAE disease revealed an increased frequency of regulatory Foxp3-expressing CD4+ T cells in CNS-related tissues of mice co-immunized with the MBP peptide. Furthermore, therapeutic delivery of extracellular vesicles packaged with the MBP peptide into cerebrospinal fluid (CSF) similarly suppressed MOG35-55-induced EAE disease when compared with control vesicles without the MBP peptide.

[0041] Throughout the specification, fragments of MBP are sometimes described according to conventional amino acid position numbering, while in other specifications, such as page 8 / 91 of CN 121240870 A, they are described according to Gölly numbering. As those skilled in the art will recognize, when taking into account Golly numbering differences, the MBP33-42 fragment is equivalent to the MBP166-175 fragment.

[0042] The data forming the basis of this disclosure utilizes a specialized platform of immunopeptidomics, leading to our understanding of the CNSMHC peptidomome (e.g., MHC1 and MHCII). In doing so, we identified a large number of peptides derived from myelin basic protein (MBP) centered around the common sequence MBP33-42. Without being bound by any particular theory, the presentation of endogenous CNS autoantigens is considered to be crucial for maintaining and restoring the regulatory T cell reserve, both physiologically and pathologically, for maintaining and restoring CNS immune tolerance.

[0043] The CNS antigen library used to develop the compositions and methods of this disclosure overcomes the deficiencies of existing methods that have limited our ability to effectively utilize antigen-specific immune tolerance therapies. Insufficient understanding of the antigen library presented during CNS homeostasis is an obstacle that can be overcome by utilizing an optimized technique to resolve the major histocompatibility complex class II (MHC) in the CNS and its boundaries.II) The limitations of molecularly bound native peptides are overcome. Furthermore, instead of focusing on a limited number of peptides to study self-reactive T cells as previous approaches have done, this disclosure broadly characterizes the MHC II peptidomome in the CNS during homeostasis, providing a broad understanding of the specificity of self-reactive T cells in the CNS. Additionally, the identified peptides described herein can be used to guide the development of MHC II tetramers, allowing for the tracking and development of phenotypically self-reactive CNS-specific T cells. The methods described herein provide insights into the function of local populations of relevant antigen-presenting T cells in the CNS—a realization that elucidates the mechanisms employed by the CNS to acquire immune immunity.

[0044] Another key obstacle to developing antigen-specific immunotherapies stems from a lack of understanding of disease-causing autoantigens and subsequent epitope expansion, often due to chronic autoimmune or inflammatory responses. This disclosure overcomes this obstacle by utilizing an established platform to define the MHC II peptidomome to understand how a library of autoantigens presented on MHC molecules deviates from homeostasis in cases of neuroinflammation. Furthermore, existing antigen-specific therapeutic strategies also face obstacles limited to identified pathogenic epitopes and the search for methods to manipulate their sequences or their delivery to drive tolerance-inducing immune responses. The methods disclosed herein circumvent this obstacle by utilizing knowledge of CNS-derived endogenous antigen presentation at steady state to provide information for antigen-specific immunotherapy. Thus, instead of targeting pathogenic autoantigens to directly induce tolerance, the disclosed methods therapeutically deliver steady-state, CNS-specific, MHC II-binding autoantigens to suppress and prevent CNS autoimmunity.

[0045] The compositions and methods disclosed herein provide an expanded understanding of the CNS-presented library of autoantigens and autoreactive T cells, thereby facilitating the understanding of mechanisms supporting CNS immune exemption and immune tolerance. The methods disclosed herein facilitate the understanding of guiding antigen-specific immunotherapy, enabling the induction of immune tolerance in other tissues to prevent allogeneic or autoimmune attacks for clinical benefit.

[0046] 1. Definitions

[0047] To make the invention more readily understood, certain terms are defined first. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the invention pertain. Many methods and materials similar to, modified, or equivalent to those described herein can be used in the practice of embodiments of the invention without excessive experimentation; preferred materials and methods are described herein. In describing and claiming embodiments of the invention, the following terms will be used as defined below.

[0048] The terms “a”, “an”, “the”, and similar references used in the context of describing specific embodiments (particularly in the context of certain subsequent claims) are to be interpreted as including both singular and plural, unless otherwise expressly stated.In some embodiments, the term “or” as used herein (including the claims) is used to mean “and / or” unless explicitly indicated to refer only to alternatives or the alternatives are mutually exclusive. Therefore, the term “and / or” as used herein, such as in phrases like “A and / or B”, is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Specification 9 / 91 page 17 CN 121240870 A

[0049] The terms “comprises,” “have,” and “include” are open-ended copulas. Any form or tense of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes,” and “including,” is also open-ended. For example, any method that “comprises,” “have,” or “includes” one or more steps is not limited to having only said one or more steps and may also cover other unlisted steps. Similarly, any composition or device that “comprises,” “have,” or “includes” one or more features is not limited to having only those one or more features and may cover other unlisted features.

[0050] As used herein, the term “about” refers to any quantifiable variable, including but not limited to mass, volume, time, distance, and quantity, such as quantitative variations that may occur through typical measuring techniques and equipment. Furthermore, given the solid and liquid handling procedures used in the real world, some accidental errors and variations may exist due to differences in the manufacture, source, or purity of the ingredients used to prepare the composition or to carry out the method, etc. The term “about” also includes these variations, which may be at most ±5%, but may also be ±4%, 3%, 2%, 1%, etc. Whether modified by the term “about” or not, the claims include the equivalent of the stated quantity.

[0051] As used herein, the term “subject” refers to a mammal, preferably a human. Mammals include, but are not limited to, humans, primates, livestock, rodents, and pets. A subject may be awaiting medical care or treatment, may be undergoing medical care or treatment, or may have already received medical care or treatment.

[0052] As used herein, the terms “control group,” “normal group,” or “sample from a “healthy” subject” refer to a subject or group of subjects who are clinically determined to be free from disease.

[0053] As used herein, the terms “treat,” “treating,” or “treatment” refer to the provision of medical care to a subject in need by a trained and licensed professional. Medical care may include diagnostic testing, therapeutic treatment, and / or preventative or preventative measures. The goal of therapeutic and preventative treatments is to prevent or slow down (reduc) [the rate of improvement / deterioration].(Less) Unnecessary physiological changes or diseases / conditions. Beneficial or desired clinical outcomes of therapeutic or preventive treatment include, but are not limited to, symptom relief, disease severity reduction, disease status stabilization (i.e., no worsening), disease progression delay or slowing, disease status improvement or relief and remission (whether partial or complete), whether detectable or undetectable. “Treatment” may also mean a longer survival time compared to the expected survival time without treatment. Those who require treatment include those who already have a disease, condition or symptom, and those who are predisposed to having a disease, condition or symptom, or those who will take preventive measures against a disease, condition or symptom. Thus, subjects who require treatment may or may not have any symptoms or clinical signs of disease.

[0054] The components used to prepare the disclosed compositions and the compositions themselves used in the methods disclosed herein are discussed below. These and other materials are disclosed herein, and it should be understood that when combinations, subsets, interactions, groups, etc., of these materials are disclosed, although specific references to every various individual and collective combination and arrangement of these compounds may not be explicitly disclosed herein, each is particularly considered and described herein. For example, if a particular compound is disclosed and discussed, and numerous modifications that can be made to many molecules of that compound are discussed, then every and all possible combinations and permutations of the compound and modifications are explicitly considered, unless explicitly indicated to the contrary. Thus, if a class of molecules A, B, and C and a class of molecules D, E, and F are disclosed, and examples of combinations of molecules A-D are disclosed, then each is considered individually and collectively, even if not individually described. This means that combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered to be disclosed. Similarly, any subsets or combinations of these are also disclosed. Thus, for example, subgroups of A-E, B-F, and C-E are considered to be disclosed. This concept applies to all aspects of this application, including but not limited to the steps of methods for preparing and using the disclosed compositions. Therefore, where various additional steps are possible, it should be understood that each of these additional steps can be performed using any particular embodiment or combination of embodiments of the disclosed methods. Specification 10 / 91 pages 18 CN 121240870 A

[0055] The grouping of alternative elements or embodiments of the disclosure herein shall not be construed as limiting. Each member of a group may be mentioned and claimed individually or in any combination with other members of that group or other elements found herein. For convenience or patentability reasons, one or more members of a group may be included in or removed from the group. When any such inclusion or removal occurs, this specification is hereby deemed to contain the modified group, thereby satisfying the written description requirements of all Markush groups used in the appended claims.

[0056] All publications, patents, patent applications and other references cited in this application are provided for all purposes by way of reference.The references herein are incorporated herein in their entirety to the extent that each individual publication, patent, patent application or other reference is expressly and individually indicated for all purposes by reference in its entirety. References herein should not be construed as an admission that they are prior art to this disclosure.

[0057] The language in this specification should not be construed as indicating that any unclaimed element is necessary for the practice of this disclosure.

[0058] 2. Molecular Engineering Modification

[0059] The following definitions and methods are provided to better define the invention and guide those skilled in the art in practicing it. Unless otherwise specified, the terms should be understood in accordance with their conventional use by those skilled in the art.

[0060] As used herein, the terms “heterologous DNA sequence,” “exogenous DNA segment,” or “heterologous nucleic acid” each refer to a sequence that is of a foreign source for a particular host cell, or, if from the same source, a sequence modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous for that particular host cell but has been modified, for example, by using DNA shuffling or cloning. The term also includes multiple non-natural copies of a naturally occurring DNA sequence. Therefore, the term refers to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but located in a position within the host cell's nucleic acid where the element is not normally present. The foreign DNA segment is expressed to obtain a foreign polypeptide. A "homologous" DNA sequence is a DNA sequence naturally associated with the host cell to which it is introduced.

[0061] Expression vectors, expression constructs, plasmids, or recombinant DNA constructs are generally understood to refer to nucleic acids produced through human intervention, including by recombination or direct chemical synthesis, having a specified set of nucleic acid elements that allow transcription or translation of a particular nucleic acid in, for example, a host cell. An expression vector may be part of a plasmid, virus, or nucleic acid fragment. Typically, an expression vector may include a nucleic acid to be transcribed, operatively linked to a promoter.

[0062] A "promoter" is generally understood to be a nucleic acid control sequence that directs the transcription of a nucleic acid. An inducible promoter is generally understood to be a promoter that mediates the transcription of an operatively linked gene in response to a specific stimulus. A promoter may include a necessary nucleic acid sequence near the transcription start site, such as a TATA element in the case of a polymerase II promoter. The promoter may optionally include distal enhancer or repressor elements, which may be located up to several thousand base pairs away from the transcription start site.

[0063] As used herein, “transcribed nucleic acid molecule” means any nucleic acid molecule capable of being transcribed into an RNA molecule. Methods for introducing constructs into cells in such a way that transcribed nucleic acid molecules are transcribed into functional mRNA molecules that are translated and thus expressed as protein products are known. Constructs may also be constructed to express antisense RNA molecules,To inhibit the translation of specific target RNA molecules. For the practice of this disclosure, conventional compositions and methods for preparing and using constructs and host cells are well known to those skilled in the art (see, for example, Sambrook and Russell (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 Russell (2001) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, ISBN-10:0879695773; Elhai, J. and Wolk, Specification 11 / 91 pages 19 CN 121240870 A). CP1988. Methods in Enzymology 167, 747-754).

[0064] A “transcription start site” or “start site” is the position surrounding the first nucleotide that is part of the transcribed sequence, and it is also defined as position +1. All other sequences of the gene and its control region can be numbered relative to this site. Downstream sequences (i.e., further protein-coding sequences in the 3' direction) can be named with a positive sign, while upstream sequences (the majority of the control region in the 5' direction) are named with a negative sign.

[0065] “Operationally linked” or “functionally linked” preferably refers to the association of nucleic acid sequences on a single nucleic acid fragment such that the function of one sequence is affected by the other. For example, if the positions of the two sequences allow the regulatory DNA sequence to affect the expression of the coding DNA sequence (i.e., the coding sequence or functional RNA under the transcriptional control of the promoter), the regulatory DNA sequence is considered “operationally linked” or “associated” with the DNA sequence encoding RNA or polypeptide. The coding sequence can be operationally linked to the regulatory sequence in either the sense or antisense direction. The two nucleic acid molecules can be part of a single, continuous nucleic acid molecule and can be adjacent. For example, if the promoter regulates or mediates the transcription of the target gene in a cell, the promoter is operatively linked to the target gene.

[0066] "Construct" is generally understood to be any recombinant nucleic acid molecule, such as plasmids, granules, viruses, autonomously replicating nucleoids, etc.Acid molecules, bacteriophages, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecules of any origin capable of genome integration or autonomous replication, comprising nucleic acid molecules in which one or more nucleic acid molecules have been operatively linked.

[0067] Constructs of this disclosure may contain a promoter operatively linked to a transcribed nucleic acid molecule operatively linked to a 3' transcription termination nucleic acid molecule. Furthermore, constructs may include, but are not limited to, additional regulatory nucleic acid molecules from, for example, the 3' untranslated region (3'UTR). Constructs may include, but are not limited to, the 5' untranslated region (5'UTR) of an mRNA nucleic acid molecule, which may play an important role in translation initiation and may also serve as genetic components in the expression construct. These additional upstream and downstream regulatory nucleic acid molecules may be derived from sources that are natural or heterologous relative to other elements present on the promoter construct.

[0068] The term "transformation" refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance. A host cell containing the transformed nucleic acid fragment is called a "transgenic" cell, and an organism containing a transgenic cell is called a "transgenic organism."

[0069] “Transformation,” “transgenic,” and “recombinant” refer to the introduction of a heterologous nucleic acid molecule into a host cell or organism, such as bacteria, cyanobacteria, animals, or plants. The nucleic acid molecule can be stably integrated into the genome, as is generally known and disclosed in the art (Sambrook 1989; Innis 1995; Gelfand 1995; Innis & Gelfand 1999). Known PCR methods 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, etc. The term “untransformed” refers to normal cells that have not undergone the transformation process.

[0070] “Wild-type” refers to a naturally occurring virus or organism without any known mutations.

[0071] The design, production, and testing of variant nucleotides and their encoded polypeptides that have the percentage identity required above and retain the desired expression protein activity are within the skill of the art. For example, directed evolution and rapid segregation of mutants can be carried out according to methods described in the literature, including but not limited to, Link et al. (2007) Nature Reviews 5(9), 680-688; Sanger et al. (1991) Gene 97(1), 119-123; Ghadessy et al. (2001) Proc Natl Acad Sci USA 98(8), 4552-4557. Therefore, those skilled in the art can generate a large number of nucleotide and / or polypeptide variants having, for example, at least 95-99% identity with the reference sequence described herein, and screen them for the desired phenotype according to methods conventional in the art.

[0072] The percentage of nucleotide and / or amino acid sequence identity (%) is understood as the percentage of nucleotide or amino acid residues identical to those in the candidate sequence compared to a reference sequence when two sequences are aligned. [Specification 12 / 91, page 20, CN 121240870 A] To determine percentage identity, sequences are aligned, and gaps are introduced if necessary to achieve maximum percentage sequence identity. Sequence alignment procedures for determining percentage identity are well known to those skilled in the art. Sequences are aligned using generally publicly available computer software, such as BLAST, BLAST2, ALIGN2, or Megalign (DNASTAR) software. Those skilled in the art can determine suitable parameters for measuring alignment, including any algorithms required to achieve maximum alignment across the entire length of the sequences being compared. When aligning sequences, the percentage sequence identity of a given sequence A with respect to, or against, a given sequence B (or, it can be expressed as a given sequence A having or containing a certain percentage sequence identity with, or against, a given sequence B) can be calculated as: Percentage sequence identity = X / Y × 100, where X is the number of residues that are identified as identical matches by a sequence alignment program or algorithm for 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 percentage sequence identity of A with B will not be equal to the percentage sequence identity of B with A.

[0073] In general, conserved substitutions can be made at any position, as long as the desired activity is preserved. So-called conserved exchanges can be made, where the substituted amino acid has properties similar to the original amino acid, for example, Glu is exchanged for Asp, Gln for Asn, Val for Ile, Leu for Ile, and Ser for Thr. For example, amino acids with similar properties can be aliphatic amino acids (e.g., glycine, alanine, valine, leucine, isoleucine); hydroxyl-containing 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 amino acids and their amides (e.g., aspartic acid, glutamic acid, asparagine, glutamine). Deletion is the substitution of an amino acid with a direct bond. Deletion sites include polypeptide ends and connections between various protein domains. Insertion is the introduction of an amino acid into a polypeptide chain, where a direct bond is formally substituted by one or more amino acids. The amino acid sequence can be modulated with the aid of computer simulation programs known in the art, which can produce polypeptides with, for example, improved activity or altered regulatory effects. Based on these artificially generated polypeptide sequences, the corresponding nucleic acid molecules encoding such modulated polypeptides can be synthesized in vitro using specific codon usages of the desired host cell.

[0074] The host cells can be transformed using a variety of standard techniques known in the art (see, for example, Sambrook and Russell (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 Russell (2001) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, ISBN-10:0879695773; Elhai, J. and Wolf, CP1988. Methods in Enzymology 167, 747-754). Such technologies include, but are not limited to, viral infection, calcium phosphate transfection, liposome-mediated transfection, microelastic-mediated delivery, receptor-mediated uptake, cell fusion, and electroporation. Transfected cells can be selected and proliferated to provide recombinant host cells containing expression vectors stably integrated into the host cell genome.

[0075] Table 1: Conservative Substitution I

[0076] Side Chain Characteristic Amino Aliphatic Nonpolar GAPILV Polar - Uncharged CSTMNQ Polar - Charged DEKR Aromatic HFWY Other NQDE

[0077] Table 2: Conservative Substitution II Specification 13 / 91 pages 21 CN 121240870 A

[0078] Side Chain Characteristic Amino Aliphatic Nonpolar (Hydrophobic) A. Aliphatic: ALIVP B. Aromatic: FW C. Sulfur-containing: M D. Critical: G Uncharged - Polar A. Hydroxyl: STY B. Amide: NQ C. Thiol group: C D. Critical: G Positively charged (basic): KRH Negatively charged (acidic): DE

[0079] Table 3: Conservative Substitution III

[0080] Exemplary Substitution of Original Residue Ala(A) Val, Leu, Ile Arg(R) Lys, Gln, Asn Asn(N) Gln, His, Lys, Arg Asp(D) Glu Cys(C) Ser Gln(Q) AsnGlu(E) Asp His(H) Asn,Gln,Lys,Arg Ile(I) Leu,Val,Met,Ala,Phe, Leu(L) Ile,Val,Met,Ala,Phe Lys(K) Arg,Gln,Asn Met(M) Leu,Phe,Ile Phe(F) Leu,Val,Ile,Ala Pro(P) Gly Ser(S) Thr Thr(T) Ser Trp(W) Tyr,Phe Tyr(Y) Trp,Phe,Tur,Ser Val(V) Ile,Leu,Met,Phe,Ala

[0081] Exemplary nucleic acids that can be introduced into host cells include, for example, DNA sequences or genes from another species, or even genes or sequences that are derived from or exist in the same species but incorporated into recipient cells by genetic engineering methods. The term "exogenous" also refers to genes that are not normally present in the transformed cells or may simply not exist in the form, structure, etc., found in the transformed DNA segment or gene, or genes that are normally present and require expression (e.g., overexpression) in a manner different from their natural expression pattern. Therefore, the term "exogenous" gene or DNA refers to any gene or DNA segment introduced into the recipient cell, regardless of whether similar genes may already exist in such cells. The types of DNA included in exogenous DNA may include DNA already present in the cell, DNA from another individual of the same type of organism, DNA from a different organism, or externally generated DNA, such as DNA sequences containing antisense messengers of genes or DNA sequences encoding synthetic or modified forms of genes.

[0082] Host strains developed according to the methods described herein can be evaluated in many ways known in the art (see, for example, 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).

[0083] Methods for downregulating or silencing genes are known in the art. For example, protein expression activity can be assessed using antisense oligonucleotides.Acids (ASO), protein aptamers, nucleotide aptamers, and RNA interference (RNAi) (e.g., downregulation or elimination of small interfering RNA (siRNA), short hairpin RNA (shRNA), and microRNA (miRNA)) (see, for example, Rinaldi and Wood (2017) Nature Reviews Neurology 14, describing ASO therapy; Fanning and Symonds (2006) Handb Exp Pharmacol. 173, 289–303G, describing hammerhead ribozymes and small hairpin RNA; Helene et al. (1992) Ann. NYAcad. Sci. 660, 27–36; Maher (1992) Bioassays 14(12): 807–15, describing targeting deoxyribonucleotide sequences; Lee et al. (2006) Curr Opin Chem Biol. 10, 1–8, describing aptamers; Reynolds et al. (2004) Nature Biotechnology 22(3)). RNAi is described in references to: 326–330; Pushparaj and Melendez (2006) Clinical and Experimental Pharmacology and Physiology 33(5–6), 504–510; Dillon et al. (2005) Annual Review of Physiology 67, 147–173; and Dykxhoorn and Lieberman (2005) Annual Review of Medicine 56, 401–423. RNAi molecules are commercially available from many sources (e.g., Ambion, TX; Sigma Aldrich, MO; Invitrogen). Several siRNA molecule design programs using various algorithms are known in the art (see, for example, Cenix algorithm, Ambion; BLOCK-iT™ RNAi Designer, Invitrogen; siRNA Whitehead Institute Design Tools, Bioinformatics & Research). Computing). Influential properties in defining optimal siRNA sequences include the G / C content at the siRNA ends, the Tm of specific internal domains of the siRNA, the siRNA length, the target sequence location within the CDS (coding region), and the nucleotide content of the 3' overhang.

[0084] 3. Composition

[0085] This invention provides compositions comprising the peptides described herein or nucleic acids encoding said peptides (e.g., mRNA or vectors encoding said peptides). The compositions may be pharmaceutical compositions comprising said peptides or nucleic acids and one or more pharmaceutically acceptable excipients.

[0086] A. Peptides

[0087] This invention provides peptides that can be used in compositions or methods or for providing information for the coding sequences of nucleic acids. Such peptides are based on those from discovered libraries of CNS-derived endogenous regulatory peptides (e.g., protective peptides) that bind to major histocompatibility complex (MHC) molecules (e.g., MHC class I and MHC class II), where they function as self-antigens important in the recognition of self-reactive T cells. During homeostasis, MHC protective peptides are presented along the entire lymphatic drainage pathway from the brain to the meninges surrounding it and its draining cervical lymph nodes, where they play a role in protection against autoimmunity.

[0088] The presentation of protective peptides is attenuated in certain disease states, such as neuroinflammatory diseases. Administering the peptides described herein (e.g., peptides derived from MHC protective peptides) to subjects mediates autoimmunity (e.g., CNS autoimmunity) by supplementing the function of MHC protective peptides in immune regulation. The peptides can be used to enhance or proliferate a population of repressive CD4+ T cells (e.g., to reduce CNS autoimmune diseases).

[0089] The peptides may be derived from MHC protective proteins, such as MHC I protective peptides, MHC II protective peptides, or both. The peptides may be derived from MHC protective proteins that are CNS-enriched; or MHC protective proteins that are not CNS-enriched. CNS enrichment can be an increase in protein expression in the CNS relative to a control tissue type. In some embodiments, the increased 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 peptides may be derived from MHC protective proteins, such as human MHC protective proteins, mouse (e.g., mouse) protective proteins, or both.

[0090] In some embodiments, the peptide comprises a fragment of a protein, wherein the fragment of the protein binds to MHC molecules. In some embodiments, the protein is expressed at elevated levels in the CNS. In some embodiments, the protein is enriched in the CNS. In some embodiments, the protein is not expressed at elevated levels in the central nervous system (CNS), or is expressed at reduced 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.

[0091] Table 4: Proteins for binding fragments of MHC class I and II molecules

[0092] Specification 16 / 91 pages 24 CN 121240870 A

[0093] DescriptionBook 17 / 91 pages 25 CN 121240870 A

[0094] Specification 18 / 91 pages 26 CN 121240870 A

[0095] Specification 19 / 91 pages 27 CN 121240870 A

[0096] Specification 20 / 91 pages 28 CN 121240870 A

[0097]

[0098] In some embodiments, the peptide described herein is an MHC protective peptide. In some embodiments, the peptide binds to MHC I, MHC II, or both.

[0099] In some embodiments, the peptide comprises, or is composed of, the amino acid sequence shown in any one of SEQ ID NO:1-14619 or a modified sequence thereof. The modified sequence may include (e.g., relative to any one of the wild-type MHC protective peptide sequences or SEQ ID NO: 1-14619) one or more of the following: oxidation of methionine; deamidation of glutamine; deamidation of asparagine; citrullination of arginine to citrulline; oxidation of cysteine ​​to sulfoalanine; or formation of pyroglutamic acid from glutamine. In some embodiments, the peptide does not have methionine oxidation. In some embodiments, the peptide does not have glutamine deamidation. In some embodiments, the peptide does not have asparagine deamidation. In some embodiments, the peptide does not have arginine to citrulline citrullination. In some embodiments, the peptide does not have cysteine ​​to sulfoalanine oxidation. In some embodiments, the peptide does not have the formation of pyroglutamic acid from glutamine.

[0100] The modified sequence may also be a variant sequence (e.g., relative to any one of the wild-type MHC protective peptide sequences or SEQ ID NO: 1-14619). The variant sequence retains its ability to bind MHC (e.g., MHC I or MHC II). In some embodiments, the variant sequence has at least 80%, at least 85%, at least 90%, or at least 95% identity with any of the wild-type MHC protective peptide sequences or SEQ ID NO: 1-14619. The variant sequence may contain one or more amino acid additions or deletions. The variant sequence may contain one or more conserved amino acid substitutions. In some embodiments, the conserved amino acid substitutions are based on side chain characteristics at one or more modification sites. In some embodiments, the conserved amino acid substitutions are the substitutions shown in Table 1 or Table 2. In some embodiments, the conserved amino acid substitutions are based on amino acid identity at one or more modification sites. In some embodiments, the conserved amino acid substitutions are the substitutions shown in Table 3.

[0101] The modified sequence may also be a conjugated sequence (e.g., a wild-type source MHC protective peptide sequence or SEQ ID NO: 1-14619).(Any one of SEQ ID NO: 1-14619), wherein the peptide is conjugated with a portion. The portion can be used to enhance peptide delivery to the CNS or enhance peptide targeting to the CNS.

[0102] Tables 5-10 classify the peptides of SEQ ID NO: 1-14619 based on their CNS enrichment and presence in the MHC peptidases of humans, mice, or both. This information, along with the protein from which the peptide is derived and the start / stop position, is provided in the notes of each sequence in the sequence listing accompanying this patent. In some embodiments, the peptide comprises, or is composed of, the amino acid sequence shown in any one of SEQ ID NO in Table 5 or a modified sequence thereof. In some embodiments, the peptide comprises, or is composed of, the amino acid sequence shown in any one of SEQ ID NO in Table 6 or a modified sequence thereof. In some embodiments, the peptide comprises, or is composed of, the amino acid sequence shown in any one of SEQ ID NO in Table 7 or a modified sequence thereof. In some embodiments, the peptide comprises, or is composed of, the amino acid sequence shown in any one of SEQ ID NO in Table 8 or a modified sequence thereof. In some embodiments, the peptide comprises, or is composed of, the amino acid sequence shown in any of the SEQ ID NOs in Table 9 or a modified sequence thereof. In some embodiments, the peptide comprises, or is composed of, the amino acid sequence shown in any of the SEQ ID NOs in Table 10 or a modified sequence thereof.

[0103] Table 5: CNS-enriched amino acid sequences identified in both human and mouse peptidommes

[0104]

[0105] Table 6: CNS-enriched amino acid sequences identified in the human peptidomme

[0106]

[0107] Table 7: Non-CNS-enriched amino acid sequences identified in both human and mouse peptidommes Specification 22 / 91 page 30 CN 121240870 A

[0108]

[0109]

[0110] Table 8: Non-enriched amino acid sequences identified in the human peptidomme Specification 23 / 91 page 31 CN 121240870 A

[0111] Specification 24 / 91 page 32 CN 121240870 A

[0112] Specification 25 / 91 page 33 CN 121240870 A

[0113] Specification 26 / 91 page 34 CN 121240870 A

[0114] Specification 27 / 91 pages 35 CN 121240870 A

[0115] Specification 28 / 91 pages 36 CN 121240870 A

[0116] Specification 29 / 91 pages 37 CN 121240870 A

[0117]

[0118] Table 9: CNS-enriched amino acid sequences identified in mouse peptidomimetics

[0119] Specification 30 / 91 pages 38 CN 121240870A

[0120]

[0121] Table 10: Non-enriched amino acid sequences identified in mouse peptide sequences

[0122] Specification 31 / 91 pages 39 CN 121240870 A

[0123] Specification 32 / 91 pages 40 CN 121240870 A

[0124] Specification 33 / 91 pages 41 CN 121240870 A

[0125] Specification 34 / 91 pages 42 CN 121240870 A

[0126] Specification 35 / 91 pages 43 CN 121240870 A

[0127] Specification 36 / 91 pages 44 CN 121240870 A

[0128] Specification 37 / 91 pages 45 CN 121240870 A

[0129] Specification 38 / 91 pages 46 CN 121240870 A

[0130] Specification 39 / 91 pages 47 CN 121240870 A

[0131] Specification 40 / 91 pages 48 CN 121240870 A

[0132] Specification 41 / 91 pages 49 CN 121240870 A

[0133] Specification 42 / 91 pages 50 CN 121240870 A

[0134] Specification 43 / 91 pages 51 CN 121240870 A

[0135] Specification 44 / 91 pages 52 CN 121240870 A

[0136] Specification 45 / 91 pages 53 CN 121240870 A

[0137]

[0138] In some embodiments, the peptide is a fragment of myelin basic protein (MBP). In some embodiments, the peptide comprises SEQ ID NO: 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, 10756, 10757, 10758, 10759, 10760, 10761, 10762, 10763, 10764, 10765, 10766, 10767, 10768, 10769, 1077010771, 10772, 10773, 10774, 10775, 10776, 10777, 10778, 10779, 10780, 10781, 10782, 10783, 10784, 10785, 10786, 10787, 10788, 10789, 10790, 10791, 10792, 10793, 10794, Instruction Manual 46 / 91 pages 54 CN 121240870 A 10795, 10796, 10797, 10798, 10799, 10800, 10801, 10802, 10803, 10804, 10805, 10806, 10807, 10808, 10809, 10810, 10811, 10812, 10813, 10814, 10815, 10816, 10817, 10818, 10819, 10820, 10821, 10822, 10823, 10824, 10825, 10826, 10827, 10828, 10829, 10830, 10831, 10832, 10833, 10834, 10835, 10836, 10837, 10838, 10839, 10840, 10841, 10842, 10843, 10844, 10845, 12162, 14537, 14538, 14539, 14540, 14541, 14542, 14543, 14544, 14545, 14546, 14547, 14548, 14549, 14550, 14551, 14552, 14553, 14554, 14555, 14556, 14557, 14558, 14559, 14560, 14561, 14562, 14563, 14564, 14565, 14566, 14567, 14568 The amino acid sequence shown in any one of 14569, 14570, 14571, 14572, 14573, 14574, 14575, 14576, 14577, 14578, 14579, 14580, 14581, 14582, 14583, 14584, 14585, 14586, 14587, 14588, 14589, 14590, 14591, 14592, 14593, 14594, 14595, 14596, 14610, 14611, 14612, and 14613, or a modified sequence thereof, or composed thereof.

[0139] In some embodiments, the peptide is a fragment of tubulin β3III (TUBB3) protein. In some embodiments, the peptide comprises SEQ ID.NO:IN249, 250, 251, 252, 4829, 14149, 14150, 14151, 14152, 14153, 14154, and 14155, or their modified sequences, or constitute thereof.

[0140] In some embodiments, the peptide is a fragment of a neurofilament medium polypeptide (NEFM). In some embodiments, the peptide comprises, or constitutes, an amino acid sequence and its modified sequences, or any one of SEQ ID NO:295, 11267, 11268, 11269, 11270, 11271, 11272, 11273, 11274, 11275, 11276, 11277, 11278, 11279, 11280, 11281, 11282, 11283, and 11284.

[0141] In some embodiments, the peptide comprises, or is composed of, the amino acid sequence of FLPRHRDTGILDSIGR (SEQ ID NO: 5776), DTGILDSIGR (SEQ ID NO: 10755), or DTGILDSIGRFFSGDRGAPK (SEQ ID NO: 10759).

[0142] B. Nucleic Acids

[0143] This document provides nucleic acids encoding the peptides described herein or their modified sequences. In some embodiments, the nucleic acid encodes a fragment of a protein that binds to MHC (e.g., MHC I or MHC II). In some embodiments, the nucleic acid encodes an MHC protective peptide. In some embodiments, the nucleic acid encodes an MHC I protective peptide, an MHC II protective peptide, or an MHC I / II protective peptide (i.e., both).

[0144] The nucleic acid may encode a peptide comprising an MHC-binding fragment of a protein shown in Table 4. The nucleic acid may encode a peptide comprising, or is composed of, any of the amino acid sequences shown in SEQ ID NOs shown in Tables 5, 6, 7, 8, 9, or 10. Nucleic acid may encode an amino acid sequence comprising any one of the amino acid sequences shown in SEQ ID NO:1-14619 or a modified sequence thereof or a peptide composed thereof.

[0145] In some embodiments, the nucleic acid encodes a peptide comprising an MHC-binding fragment of myelin basic protein (MBP). In some implementations, the nucleic acid codes include SEQ ID NO: 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, 10755,10756, 10757, 10758, 10759, 10760, 10761, 10762, 10763, 10764, 10765, 10766, 10767, 10768, 10769, 10770, 10771, 10772, 10773, 10774, 10775, 10776, 10777, 10778, 10779, 10780, 10781, 10782, 10783, 10784, 10785, 10786, 10787, 10788, 10789, 10790, 10791 10792, 10793, 10794, 10795, 10796, 10797, 10798, 10799, 10800, 10801, 10802, 10803, 10804, 10805, 10806, 10807, 10808, 10809, 10810, 10811, 10812, 10813, 10814, 10815, 10816, 10817, 10818, 10819, 10820, 10821, 10822, 10823, 10824, 10825, 10826, 10827, Instruction Manual, Page 47 / 91, 55, CN 121240870 A 10828, 10829, 10830, 10831, 10832, 10833, 10834, 10835, 10836, 10837, 10838, 10839, 10840, 10841, 10842, 10843, 10844, 10845, 12162, 14537, 14538, 14539, 14540, 14541, 14542, 14543, 14544, 14545, 14546, 14547, 14548, 14549, 14550, 14551, 14552, 14553, 14554, 14555, 14556, 14557, 14558, 14559, 14560, 14561, 14562, 14563, 14564, 14565, 14566, 14567, 14568, 14569, 14570, 14571, 14572, 14573, 14574, 14575, 14576, 14577, 14578, 14579, 14580, 14581, 14582, 14583, 14584, 14585, 14586, 14587, 14588, 14589 The amino acid sequence shown in any one of 14590, 14591, 14592, 14593, 14594, 14595, 14596, 14610, 14611, 14612 or 14613, or a modified sequence thereof, or a peptide thereof.

[0146] In some embodiments, the nucleic acid encodes a peptide comprising an MHC-binding fragment of tubulin β3III (TUBB3) protein. In some embodiments, the nucleic acid encodes an amino acid sequence comprising any one of the amino acid sequences shown in SEQ ID NO: 1N 249, 250, 251, 252, 4829, 14149, 14150, 14151, 14152, 14153, 14154, or 14155, or a modified sequence thereof, or a peptide composed thereof.

[0147] In some embodiments, the nucleic acid encodes a peptide comprising an MHC-binding fragment of neurofilament intermediate polypeptide (NEFM). In some embodiments, the nucleic acid encodes an amino acid sequence or a modified sequence thereof, or a peptide thereof, comprising any one of SEQ ID NO:295, 11267, 11268, 11269, 11270, 11271, 11272, 11273, 11274, 11275, 11276, 11277, 11278, 11279, 11280, 11281, 11282, 11283, 11284.

[0148] In some embodiments, the nucleic acid encodes an amino acid sequence or a modified sequence thereof, or a peptide thereof, comprising FLPRHRDTGILDSIGR (SEQ ID NO:5776), DTGILDSIGR (SEQ ID NO:10755), or DTGILDSIGRFFSGDRGAPK (SEQ ID NO:10759).

[0149] Vectors (e.g., expression vectors) comprising nucleic acids encoding peptides described herein are also provided. One or more vectors may be present, each of which contains at least one or more nucleic acids described herein. One or more vectors may encode a variety of different peptides. Such vectors are capable of producing transcribed nucleic acid molecules encoding peptides described herein. The vector may further contain a transcription start site and / or promoter operatively linked to the transcribed nucleic acid molecule encoding the peptides described herein.

[0150] The vector may be a non-viral vector. In some embodiments, the non-viral vector is a physical vector (e.g., electroporation, acoustic perforation, magnetic transfection, or hydroporation). In some embodiments, the non-viral vector is a chemical vector. In some embodiments, the chemical vector is inorganic particle-based, lipid-based (e.g., solid or liquid), polymer-based, or peptide-based. In some embodiments, inorganic particle-based vectors are selected from calcium phosphate, silica, gold, or magnetic nanoparticles. In some embodiments, the nucleic acid vector is a nucleic acid / cationic lipid (lipoplex), a nucleic acid / cationic polymer (polyplex), or a nucleic acid / cationic polymer / cationic lipid (lipopolyplex). In some embodiments, the vector...The carrier is selected from cationic lipids, lipid nanoemulsions, or solid lipid nanoparticles. In some embodiments, the carrier is a peptide-based carrier, a polymer-based carrier, or a synthetic carrier. In some embodiments, the synthetic carrier is selected from polyethyleneimine (PEI), chitosan, poly(DL-lactide) (PLA) and poly(DL-lactide-glycolic acid) copolymer (PLGA), dendritic polymers, polyphosphates, or polymethacrylates.

[0151] The carrier may be a non-viral carrier. In some embodiments, the non-viral carrier is a physical carrier (e.g., electroporation, acoustic perforation, magnetic transfection, or hydroporation). In some embodiments, the non-viral carrier is a chemical carrier. In some embodiments, the chemical carrier is based on inorganic particles, lipids (e.g., solid or liquid), polymers, or peptides. In some embodiments, the inorganic particle-based carrier is selected from calcium phosphate, silica, gold, or magnetic nanoparticles. In some embodiments, the nucleic acid carrier is a nucleic acid / cationic lipid (lipoplex), a nucleic acid / cationic polymer (polyplex), or a nucleic acid / cationic polymer / cationic lipid (lipopolyplex). In some embodiments, the carrier is selected from cationic lipids, lipid nanoemulsions, or solid lipid nanoparticles. In some embodiments, the carrier is a peptide-based carrier, a polymer-based carrier, or a synthetic carrier. In some embodiments, the synthetic carrier is selected from polyethyleneimine (PEI), chitosan, poly(DL-lactide) (PLA) and poly(DL-lactide-glycolic acid) copolymer (PLGA), dendritic polymers, polyphosphates, or polymethacrylates.

[0152] In one example, the nucleic acid may comprise mRNA encoding the peptides disclosed herein. The nucleic acids described herein may also be encapsulated. For example, in some embodiments, the nucleic acid is encapsulated by lipid nanoparticles (LNP), polyplex, polymer nanoparticles, lipopolyplex (LPP), or cationic peptides. The nucleic acid may be encapsulated by any particle that is phagocytosed (e.g., for delivery). Nucleic acids can be encapsulated by any particle that can be taken up by antigen-presenting cells.

[0153] The vector can be a viral vector. The nucleic acids described herein can be encapsulated by a viral vector. The viral vector can be derived from a virus having CNS or brain tissue specificity (e.g., tropism). In some embodiments, 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 can be derived from adenovirus, adeno-associated virus, or retrovirus. In some embodiments, the retrovirus is a lentivirus.

[0154] C. Host Cell

[0155] A host cell comprising a nucleic acid (e.g., a vector or construct) encoding the peptide described herein is also provided. HostCells may be used to express (e.g., produce) the peptides described herein, or to maintain the nucleic acids described herein (e.g., vectors or constructs). The host cell may or may not be transformed (e.g., transgenic).

[0156] 4. Formulations

[0157] The reagents and compositions described herein may be formulated in any conventional manner using one or more pharmaceutically acceptable carriers or excipients as described, for example, Remington's Pharmaceutical Sciences (ARGennaro, Ed.), 21st edition, ISBN: 0781746736 (2005), incorporated herein by reference in its entirety. Such formulations will contain a therapeutically effective amount of the peptides described herein or the nucleic acids encoding said peptides (which may be in purified form), and a suitable amount of one or more pharmaceutically acceptable carriers or excipients to provide a form suitable for administration to a subject.

[0158] The term “formulation” means a pharmaceutical preparation prepared in a form suitable for administration to a subject, such as a human. Thus, a “formulation” may include pharmaceutically acceptable excipients, including diluents or carriers.

[0159] The term “pharmaceuticalally acceptable” as used herein describes a substance or component that does not cause an unacceptable loss of pharmacological activity or unacceptable adverse side effects. Generally, they include substances that are compatible with other components of the formulation and are not harmful to the recipient. Examples of pharmaceutically acceptable components may be those monographs in the United States Pharmacopeia (USP 29) and National Formulary (NF 24), United States Pharmacopeial Convection, Inc., Rockville, Maryland, 2005 ("USP / NF") or later, as well as components listed in the FDA's continuously updated online database of inactive ingredients. Other useful components not described in USP / NF, etc., may also be used.

[0160] As used herein, the term “pharmaceuticalally acceptable excipient” may include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, or absorption delay agents. Such media and reagents are well known in the art for the use of pharmaceutically active substances (for general information see Remington's Pharmaceutical Sciences (A.R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005)). Their use in therapeutic compositions is considered except for any conventional media or reagents that are incompatible with the active ingredient. Additional active ingredients may also be incorporated into the composition.

[0161] A “stable” formulation or composition may refer to a composition having sufficient stability to allow storage at convenient temperature settings (e.g., from about 0°C to about 60°C) for a commercially reasonable period of time, such as at least about 1 day, at least about 1 week, at least about 1 month, at least about 3 months, at least about 6 months, at least about 1 year, or at least about 2 years.

[0162] The formulation should be suitable for the manner of administration. The reagents used in this disclosure can be formulated by known methods for administration to a subject via several routes, including but not limited to, parenteral, pulmonary, oral, topical, intradermal, intratumoral, intranasal, inhalation (e.g., in an aerosol), implantation, intramuscular, intraperitoneal, intravenous, intrathecal, intracranial, intraventricular, subcutaneous, intranasal, epidural, intrathecal, ocular, transdermal, sublingual, and rectal. Each reagent may also be administered in combination with one or more other reagents or with other biologically active or biologically inert reagents. Such biologically active or inert reagents may be fluidly or mechanically connected to the reagent, or connected to the reagent by ionic, covalent, van der Waals, hydrophobic, hydrophilic, or other physical forces.

[0163] Controlled-release (or sustained-release) formulations may be formulated to prolong the activity of the reagent and reduce the frequency of administration. Controlled-release formulations may also be used to influence the onset time of action or other characteristics (e.g., blood levels of the reagent) and thus the occurrence of side effects. Controlled-release formulations may be designed to initially release an amount of reagent that produces the desired therapeutic effect and to gradually and continuously release additional amounts of reagent to maintain the level of therapeutic effect over a prolonged period of time. In order to maintain a nearly constant level of reagent in vivo, the reagent may be released from the dosage form at a rate that replaces the amount of reagent that is metabolized or excreted from the body. Controlled release of the reagent may be stimulated by various inducers, such as pH changes, temperature changes, enzymes, water, or other physiological conditions or molecules.

[0164] The reagents or compositions described herein may also be used in combination with other forms of treatment further described below. Thus, in addition to the therapies described herein, other therapies known to be effective in treating diseases, symptoms, or conditions may be provided to subjects.

[0165] 5. Treatment Methods

[0166] A method is also provided for treating, preventing, or reversing neuroinflammatory-related conditions, autoimmune neurological conditions, or acute CNS injury in a subject of need by administering a therapeutically effective amount of the peptide described herein. The peptide may be in a composition described herein (e.g., a pharmaceutical composition) administered to a subject. The use of the peptide in treating, preventing, or reversing neuroinflammatory-related conditions, autoimmune neurological conditions, or acute CNS injury is also provided; as well as the use of the peptide in the preparation of a medicament for treating, preventing, or reversing neuroinflammatory-related conditions, autoimmune neurological conditions, or acute CNS injury.

[0167] In some implementations, neuroinflammatory-related conditions or autoimmune neuropathies are selected from acute disseminated encephalomyelitis (ADEM), acute inflammatory demyelinating polyradiculoneuropathy (AIDP; Guillain-Barré syndrome (GBS)), acute motor axonal neuropathy (AMAN), acute motor-sensory axonal neuropathy (AMSAN), acute optic neuritis (AON), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), autoimmune encephalitis (AIE), chronic ataxia neuropathy, oculomotor palsy, immunoglobulin M paraprotein, cold agglutinin and disialic acid antibody (CANOMAD), chronic meningitis, Bechtel's disease, central nervous system (CNS) vasculitis, chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), steroid-induced encephalopathy, and other related conditions. Reactive chronic lymphocytic inflammation with perivascular enhancement of the pons (CLIPPERS), glial fibrillary acidic protein (GFAP), Hashimoto's encephalitis, hypertrophic pachymeningitis, IgG4-related neurological disorders, Lambert-Eton myasthenic syndrome (LEMS), anti-myelin oligodendrocyte glycoprotein antibody disease (MOG), Miller Fisher syndrome (MFS), monoclonal gammaglobulinosis of undetermined significance (MGUS), multifocal motor neuropathy (MMN), myasthenia gravis (MG), multiple sclerosis (MS), neuromyelitis optica (NMO), neurosarcoidosis, paraneoplastic neurological syndrome (PNS), Parkinson's disease (PD), steroid-reactive encephalopathy associated with autoimmune thyroiditis (SREAT), stiff-person syndrome, Sussac syndrome, and transverse myelitis.

[0168] In some embodiments, acute CNS injury is selected from spinal cord injury, traumatic brain injury, spinal cord injury, optic nerve injury, and stroke.

[0169] The methods described herein are generally performed on subjects in need. Subjects in need of the treatment methods described herein may be subjects who have, are diagnosed with, are suspected of having, or are at risk of developing neuroinflammatory conditions, autoimmune neurological conditions, or acute CNS injury. The determination of the need for treatment is generally evaluated by history, physical examination, or diagnostic tests consistent with the disease or condition in question. The diagnosis of the various conditions treatable by the methods described herein is within the skill of the art. Subjects may be animal subjects, including mammals such as horses, cattle, dogs, cats, sheep, pigs, mice, rats, monkeys, hamsters, guinea pigs, and humans, or chickens. For example, subjects may be human subjects.

[0170] Typically, a safe and effective amount of the peptide (e.g., myelin basic protein (MBP) peptide) is, for example, in the subjectThe amount of peptides described herein to induce the desired therapeutic effect while minimizing unwanted side effects is determined. In various embodiments, an effective amount of the peptides described herein (e.g., MBP peptides) can significantly inhibit neuroinflammatory-associated conditions or autoimmune neuropathies, slow the progression of neuroinflammatory-associated conditions or autoimmune neuropathies, or limit the development of neuroinflammatory-associated conditions or autoimmune neuropathies.

[0171] According to the methods described herein, administration can be parenteral, pulmonary, oral, local, intradermal, intramuscular, intraperitoneal, intravenous, intratumoral, intrathecal, intracranial, intraventricular, subcutaneous, intranasal, epidural, ocular, sublingual, or rectal administration. In one aspect, the disclosed composition is infused, injected, or otherwise introduced into the cerebrospinal fluid (CSF) of a subject. In some aspects, the peptides (e.g., MBP peptides) are encapsulated in extracellular vesicles or liposomes. Extracellular vesicles may be derived from dendritic cells.

[0172] When used in the treatments described herein, therapeutically effective amounts of the peptide (e.g., MBP peptide) may be used in its pure form, or, where such a form is available, in a pharmaceutically acceptable salt form, and with or without a pharmaceutically acceptable excipient. For example, the compounds of this disclosure may be applied in amounts sufficient to prevent, alleviate, or reverse neuroinflammatory-related conditions or autoimmune neurological conditions, based on a reasonable benefit / risk ratio applicable to any medical treatment.

[0173] The amount of the compositions described herein that may be combined with a pharmaceutically acceptable carrier to produce a single dosage form will vary depending on the subject or host being treated and the specific manner of administration. Those skilled in the art will understand that the amount of reagent units contained in a single dose of each dosage form need not, on its own, constitute a therapeutically effective amount, as the required therapeutically effective amount can be achieved by administering many single doses.

[0174] The toxicity and therapeutic efficacy of the compositions described herein can be determined by standard pharmaceutical procedures used in cell cultures or laboratory animals to determine the LD50 (50% population lethal dose) and ED50 (50% population therapeutically effective dose). The dose ratio between toxicity and therapeutic effect is the therapeutic index, which can be expressed as the ratio LD50 / ED50, where a larger therapeutic index is generally considered optimal in the art.

[0175] The specific therapeutically effective dose level for any particular subject will depend on a variety of factors, including the condition being treated and its severity; the activity of the specific compound used; the specific composition used; the subject's age, weight, general health, sex, and diet; the time of administration; the route of administration; the excretion rate of the composition used; the duration of treatment; the drugs used in combination with or concurrently with the specific compound used; and similar factors well known in the medical field (see, for example, Koda-Kimble et al. (2004) Applied Therapeutics: The Clinical Use of Drugs,Lippincott Williams & Wilkins, ISBN 0781748453; Winter (2003) Basic Clinical Pharmacokinetics, 4th ed., Lippincott Williams & Wilkins, ISBN 0781741475; Sharqel (2004) Applied Biopharmaceutics & Pharmacokinetics, McGraw-Hill / Appleton & Lange, ISBN 0071375503). For example, it is perfectly within the skill of the art to start the dose of the composition at a level below that required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. If necessary, for the purpose of administration as per the instructions on page 51 / 91 of CN 121240870 A, the effective daily dose may be divided into multiple doses. Thus, a single-dose composition may contain such an amount or approximation thereof to constitute the daily dose. However, it will be understood that the total daily use of the compounds and compositions of this disclosure will be determined by the attending physician within the bounds of reasonable medical judgment.

[0176] Similarly, each of the conditions, diseases, symptoms, and ailments described herein may benefit from the compositions and methods described herein. Generally, treating a condition, disease, symptom, or ailment includes preventing, reversing, or delaying the onset of clinical symptoms in mammals that are susceptible to or prone to the condition, disease, symptom, or ailment but have not yet experienced or displayed its clinical or subclinical symptoms. Treatment may also include suppressing a condition, disease, symptom, or ailment, such as preventing or reducing the development of the disease or at least one of its clinical or subclinical symptoms. Furthermore, treatment may include alleviating a disease, such as causing the remission of a condition, disease, symptom, or ailment or at least one of its clinical or subclinical symptoms. The benefit to the subject being treated may be statistically significant or at least perceptible to the subject or physician.

[0177] Administration of the peptide (e.g., MBP peptide) may occur as a single event or over a course of treatment. For example, meningeal immune cells may be administered daily, weekly, bi-weekly, or monthly. For the treatment of acute conditions, the treatment course is typically at least several days. For some conditions, treatment may be extended from several days to several weeks. For example, treatment may be extended to one, two, or three weeks. For more chronic conditions, treatment may be extended from several weeks to several months or even a year or longer.

[0178] Treatment according to the methods described herein may be performed before, simultaneously with, or after conventional treatments for the prevention, relief, or reversal of neuroinflammatory conditions or autoimmune neurological disorders.

[0179] The peptide may be administered simultaneously or sequentially with another agent, such as an antibiotic, an anti-inflammatory agent, or another agent. ExampleFor example, the peptide may be administered simultaneously with another agent, such as an antibiotic or an anti-inflammatory agent. Simultaneous administration may occur by administering a single composition, each containing one or more of the peptide, antibiotic, anti-inflammatory agent, or another agent. Simultaneous administration may occur by administering a composition containing two or more peptides, antibiotics, anti-inflammatory agents, or another agent. The peptide (e.g., MBP peptide) may be administered sequentially with an antibiotic, anti-inflammatory agent, or another agent. For example, the peptide may be administered before or after the administration of an antibiotic, anti-inflammatory agent, or another agent.

[0180] All methods described herein may be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by the context. The use of any and all examples or exemplary language (e.g., “for example”) provided herein with respect to certain embodiments is intended only to better illustrate the disclosure and does not constitute a limitation on the scope of the disclosure otherwise claimed.

[0181] 6. Administration

[0182] The agents and compositions described herein may be administered in various ways known in the art according to the methods described herein. The agents and compositions may be used therapeutically as exogenous materials or as endogenous materials. Exogenous agents are those produced or manufactured in vitro and administered in vivo. Endogenous reagents are those produced or manufactured in vivo by a certain type of device (biological or other) for delivery within or to other organs of the body.

[0183] As described above, administration can be parenteral, pulmonary, oral, topical, intradermal, intratumoral, intranasal, inhalation (e.g., in an aerosol), implantation, intramuscular, intraperitoneal, intravenous, intrathecal, intracranial, intraventricular, subcutaneous, intranasal, epidural, intrathecal, ocular, percutaneous, sublingual, and rectal.

[0184] The reagents and compositions described herein can be administered by a variety of methods well known in the art. Administration may include, for example, oral ingestion, direct injection (e.g., systemic or stereotactic), implantation into cells modified to secrete factors of interest, release of drugs by biomaterials, polymer matrices, gels, permeable membranes, permeation systems, multilayer coatings, microparticles, implantable matrix devices, mini-osmotic pumps, implantable pumps, injectable gels and hydrogels, liposomes, micelles (e.g., up to 30 μm), nanospheres (e.g., less than 1 μm), microspheres (e.g., 1–100 μm), storage devices, any combination of the above, or other suitable delivery media to provide the desired release profile in varying proportions. Other methods of controlled-release delivery of reagents or compositions are known to those skilled in the art and are within the scope of this disclosure.

[0185] Delivery systems may include, for example, infusion pumps that can be used to administer reagents or compositions in a manner similar to that used to deliver insulin or chemotherapy to a specific organ or tumor. Typically, using such systems, reagents or compositions can be...The agent is administered in combination with a biodegradable, biocompatible polymer implant that releases the agent at a selected site over a controlled period of time. Examples of polymeric materials include polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyvinyl acetate, and copolymers and combinations thereof. Furthermore, the controlled-release system can be placed near the therapeutic target, thus requiring only a portion of the systemic dose.

[0186] The agent can be encapsulated and administered in a variety of carrier delivery systems. Examples of carrier delivery systems include microspheres, hydrogels, polymer implants, smart polymer carriers, and liposomes (for general information see, Uchegbu and Schatzlein, eds. (2006) Polymers in Drug Delivery, CRC, ISBN-10:0849325331). Carrier-based systems for the delivery of molecular or biomolecular reagents can: provide intracellular delivery; customize the release rate of biomolecules / reagents; increase the proportion of biomolecules reaching their sites of action; improve drug delivery to their sites of action; allow co-localization deposition with other reagents or excipients; improve reagent stability in vivo; prolong the residence time of reagents at their sites of action by reducing clearance; reduce nonspecific delivery of reagents to non-target tissues; reduce reagent-induced irritation; reduce toxicity due to high initial doses of reagents; alter the immunogenicity of reagents; reduce dosing frequency; improve product taste; or improve product shelf life.

[0187] 7. Screening

[0188] Screening methods are also provided. The methods of this subject can be used to screen a variety of different candidate molecules (e.g., potential therapeutic candidate molecules). Candidate substances screened according to the methods described herein include, but are not limited to, fractions of tissues or cells, nucleic acids, peptides, siRNAs, antisense molecules, aptamers, ribozymes, triple-helix compounds, antibodies, and small (e.g., less than about 2000 mw, less than about 1000 mw, or less than about 800 mw) organic or inorganic molecules, including but not limited to salts or metals.

[0189] Candidate molecules include many chemical classes, such as organic molecules, for example small organic molecules having a molecular weight greater than 50 and less than about 2,500 Daltons. Candidate molecules may contain functional groups required for structural interactions with proteins, particularly hydrogen bonding, and generally include at least amine, carbonyl, hydroxyl, or carboxyl groups, and generally at least two of the aforementioned chemical functional groups. Candidate molecules may contain cyclic carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the aforementioned functional groups.

[0190] Candidate molecules may be compounds from a compound library database. Those skilled in the art are generally familiar with, for example, numerous commercially available compound databases for screening (see, for example, the ZINC database, UCSF, containing 2.7 million compounds in molecules with 12 distinct subsets; Irwin and Shoichet (2005) J ChemInfModel45, 177-182). Those skilled in the art are also familiar with various search engines to identify compounds and compound classes of commercial origin or desired for further testing (see, for example, the ZINC database; eMolecules.com; and electronic libraries of commercial compounds provided by vendors (e.g., ChemBridge, Princeton BioMolecular, Ambinter SARL, Enamine, ASDI, LifeChemicals, etc.).

[0191] Candidate molecules used for screening according to the methods described herein include both lead-like compounds and drug-like compounds. Lead-like compounds are generally understood to have a relatively small scaffold-like structure (e.g., molecular weight of about 150 to about 350 kD) and relatively few features (e.g., fewer than about 3 hydrogen donors and / or fewer than about 6 hydrogen acceptors; hydrophobic feature xlogP of about -2 to about 4) (see, for example, Angewante (1999) Chemie Int. ed. Engl. 24, 3943-3948). In contrast, drug-like compounds are generally understood to have a relatively large scaffold (e.g., molecular weight of about 150 to about 500 kD) and relatively more features (e.g., fewer than about 10 hydrogen acceptors and / or fewer than about 8 rotatable bonds; hydrophobicity feature specification 53 / 91 pages 61 CN 121240870 A xlogP less than about 5) (see, for example, Lipinski (2000) J. Pharm. Tox. Methods 44, 235-249). Initial screening can be performed with lead-like compounds.

[0192] When designing leads from spatially located data, it may be useful to understand that certain molecular structures are characterized as “drug-like”. Such characterization can be based on a set of empirically recognized qualities obtained by comparing similarities across a width of known drugs within the pharmacopoeia. Although a drug does not need to meet all or even any of these characteristics, if a drug candidate is drug-like, the likelihood of the drug candidate achieving clinical success is much greater.

[0193] Several of these “drug-like” characteristics have been summarized into four rules of Lipinski (often referred to as the “rules of five” because of the prevalence of the number 5). While these rules generally concern oral absorption and are used to predict the bioavailability of compounds during lead optimization, they can serve as effective guidelines for constructing lead molecules during rational drug design attempts (e.g., by using the methods of this disclosure).

[0194] The four “rules of five” state that candidate drug-like compounds should have at least three of the following characteristics: (i) weight less than 500 Daltons; (ii) logP less than 5; (iii) no more than 5 hydrogen bond donors (expressed as the sum of OH and NH groups);(iv) No more than 10 hydrogen bond acceptors (the sum of N and O atoms). Furthermore, drug-like molecules typically have a span (width) of about to approximately [missing information].

[0195] 8. Kits

[0196] Kits are also provided. Such kits may include the reagents or compositions described herein, and in some embodiments, instructions for administration. Such kits facilitate the performance of the methods described herein. When provided as a kit, the different components of the composition may be packaged in separate containers and mixed immediately before use. Components include, but are not limited to, compositions containing peptides (e.g., MBP peptides) described herein, optionally encapsulated in extracellular vesicles described herein. Such individual packaging of components may be provided in a packaging or dispensing device, if desired, which may contain one or more unit dosage forms containing the composition. The packaging may, for example, contain metal or plastic foil, such as blister packs. In some cases, such individual packaging of components may also allow for long-term storage without loss of component activity.

[0197] The kit may also include reagents in separate containers, such as sterile water or saline to be added to the individually packaged lyophilized active component. For example, sealed glass ampoules may contain lyophilized components, and in separate ampoules, sterile water and sterile saline solution, each packaged under a neutral, non-reactive gas, such as nitrogen. Ampoules may be composed of any suitable material, such as glass, organic polymers like polycarbonate, polystyrene, ceramics, metals, or any other material commonly used to contain reagents. Other examples of suitable containers include bottles that may be made from substances similar to ampoules, and sleeves that may be made of foil linings, such as aluminum or alloys. Other containers include test tubes, vials, flasks, bottles, syringes, etc. Containers may have sterile access ports, such as bottles with stoppers that can be punctured by a hypodermic needle. Other containers may have two compartments separated by an easily removable membrane that allows the components to mix upon removal. The removable membrane may be glass, plastic, rubber, etc.

[0198] In some embodiments, the kit may be provided with instruction manual materials. The instructions may be printed on paper or other substrates and / or may be provided as an electronically readable medium or video. The detailed instruction manual may not be physically associated with the kit; instead, the user may be directed to an internet website designated by the kit manufacturer or distributor.

[0199] The compositions and methods utilizing molecular biology protocols described herein may be performed according to various standard techniques known in the art (see, for example, Sambrook and Russell (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 Russell (2001) Molecular Cloning: Manual 54 / 91 pages 62 CN 121240870 A A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, CP1988. Methods in Enzymology 167, 747-754; 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).

[0200] The definitions and methods described herein are provided to better define this disclosure and to guide those skilled in the art in practicing it. Unless otherwise indicated, those skilled in the art should understand the terms as commonly used.

[0201] In some embodiments, the numbers representing the amount, properties, such as molecular weight, reaction conditions, etc., of components used to describe and claim certain embodiments of this disclosure should be understood to be modified by the term "about" in some cases. In some embodiments, the term "about" is used to indicate that the value includes the standard deviation of the mean for the device or method used to determine the value. In some embodiments, the numerical parameters set forth in the specification and the appended claims are approximate values ​​that may vary depending on the desired properties sought to be obtained by a particular embodiment. In some embodiments, numerical parameters should be interpreted based on the number of significant figures reported and by applying conventional rounding. While the wide range of numerical ranges and parameters described in some embodiments of this disclosure are approximate, the numerical values ​​described in specific embodiments are reported as precisely as possible. The numerical values ​​provided in some embodiments of this disclosure may contain inaccuracies due to variations in their respective test measurements.The standard deviation of the standard deviation inevitably results in certain errors. The description of the range of values ​​in this document is intended only as a shorthand method for referring to each individual value falling within that range. Unless otherwise specified herein, each individual value is incorporated into the specification as if it were described separately herein. The description of discrete values ​​is understood to include the range between each value.

[0202] Although the present disclosure has been described in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing from the scope of the present disclosure as defined by the appended claims. Furthermore, it should be understood that all embodiments in the present disclosure are provided as non-limiting embodiments.

[0203] Embodiments

[0204] The following non-limiting embodiments are provided to further illustrate the present disclosure. Those skilled in the art will understand that the techniques disclosed in the following embodiments represent methods that the inventors have found to work adequately in the practice of the present disclosure and can therefore be considered as embodiments constituting their mode of practice. Those skilled in the art will understand from the present disclosure that variations can be made to the specific embodiments described herein without departing from the spirit and scope of the invention and still obtaining the same or similar results. All content set forth or shown in the drawings should be interpreted as illustrative and not restrictive.

[0205] 1. Example 1: Study of CNS Immune Exemption - Self-antigens Resist CNS Autoimmunity

[0206] Experiments were conducted to demonstrate that CNS-derived endogenous peptides promote the differentiation of regulatory T cells and the regulation of CNS autoimmunity; and that the delivery of CNS-derived endogenous peptides broadly resists neuroinflammation-induced pathology.

[0207] A. Specific Objectives:

[0208] With rising incidence and prevalence, autoimmune diseases continue to debilitate countless individuals, increase the burden of healthcare, and reduce quality of life. To date, current interventions have focused on nonspecific immunosuppressive therapies with significant side effects. Therefore, antigen-specific immunotherapy offers an attractive alternative to precisely target organ-specific autoimmunity. In the pursuit of personalized medicine, a deeper understanding of the repertoire of self-antigens presented on major histocompatibility complexes (MHCs) (e.g., MHC class I and MHC class II) will guide the development of antigen-specific immunotherapy.

[0209] Although autoreactive T cells are well known to cause autoimmune diseases, their protective role in limiting tissue damage has been described in cardiac, skeletal muscle, and central nervous system (CNS) pathologies, including Alzheimer's disease, stroke, and traumatic injury, etc. Given the evidence supporting both the suppression and beneficial functions of autoreactive T cells, further research is needed into the molecular and cellular mechanisms that connect the CNS and the peripheral immune system. This research has benefited from recent re-exploration...A true lymphatic network was discovered in the meninges (the membranous covering that encloses the CNS). Emphasizing the role of the brain boundary as an intimate space for interactions between CNS-resident and peripheral immune cells, the meninges provide a rich panorama for examining the local presentation of self-antigens to self-reactive T cells. Furthermore, the lymphatic vessels of the meninges act as conduits for the drainage of antigens and cells to secondary lymphoid organs, particularly the deep cervical lymph nodes (dCLN). For these reasons, examining CNS-specific self-antigen presentation in CNS-related tissues and how it promotes immune surveillance will inform new mechanisms for self-antigens to maintain tissue homeostasis and prevent overt autoimmunity.

[0210] Preliminary data, utilizing a specialized platform of immunopeptidomics, are leading the understanding of the CNS MHC II peptidomome. In doing so, a large number of peptides derived from myelin basic proteins (MBP) centered around the common sequence MBP33-42 were identified. In investigating the effects of these peptides, it was found that co-immunization with MOG35-55 and the identified MBP peptide improved EAE disease in experimental autoimmune encephalomyelitis (EAE). Further analysis by flow cytometry prior to the peak of EAE disease revealed an increased frequency of regulatory Foxp3-expressing CD4+ T cells in CNS-related tissues of mice co-immunized with the MBP peptide. Furthermore, therapeutic delivery of extracellular vesicles (EVs) packaged with the MBP peptide into cerebrospinal fluid (CSF) similarly suppressed MOG35-55-induced EAE disease when compared with control EVs without the MBP peptide. In light of these findings, it is hypothesized that the presentation of endogenous CNS autoantigens leads to a reserve of regulatory T cells that is critical for maintaining and restoring CNS immune tolerance, both physiologically and pathologically.

[0211] Objective 1 is to address the hypothesis that CNS-derived endogenous peptides promote the differentiation of regulatory T cells and thus modulate CNS autoimmunity. Co-immunization with MOG and MBP peptides induces EAE leading to an increase in the frequency of regulatory T cells. It is hypothesized that MBP-specific T cells within the CNS possess regulatory features that promote CNS immune tolerance. To support the idea of ​​culturing Foxp3+ regulatory T cells with MBP peptides to improve disease, EAE was induced in Foxp3DTR mice with or without diphtheria toxin using MOG and MBP peptides. Furthermore, the specificity of Foxp3+ regulatory T cells for MBP peptides in the meninges and dCLN was examined using MBP-specific MHC II tetramers.

[0212] Objective 2 is to evaluate the hypothesis that neuropathology restricts the presentation of homeostatic epitopes on MHC II molecules. Tissue damage and inflammation lead to epitope expansion, which is thought to further promote autoimmune pathology. However, it remains elusive whether the presentation of homeostatic epitopes, such as MBP, is also affected by inflammation. Neuropathology is expected to restrict MBP peptides, particularly those containing the MBP33-42 sequence.The presentation of those. MHC II peptidomics of the meninges and draining dCLN were performed at the peak of EAE disease.

[0213] Objective 3 was to test the hypothesis that delivery of CNS-derived endogenous peptides broadly prevents neuroinflammation-induced pathology. Introducing MBP-containing vesicles into CSF ​​inhibited EAE disease. Furthermore, co-immunization of our MBP peptide with myelin lipoprotein (PLP) 139-151 alleviated EAE disease in SJL / J mice. Therefore, it was hypothesized that the MBP peptide could be therapeutically implemented to broadly limit neuroinflammation and restore immune tolerance. To confirm this, MBP-containing EVs were delivered at different time points after the onset of EAE disease. In addition, EAE was actively induced in different strains (including C57BL / 6J, SJL / J, and B10.PL mice) while delivering MBP-containing vesicles into CSF ​​to confirm the broad therapeutic efficacy of the MBP peptide we identified.

[0214] The overall significance of this study is to dissect the functional importance of CNS autoantigen presentation in CNS immune surveillance. Understanding this holds promise for the artificial induction of immune tolerance in other organ systems to prevent allogeneic and autoimmune attacks for clinical benefit.

[0215] B. Significance:

[0216] By studying the CNS antigen library, these studies have addressed obstacles that have limited our ability to effectively utilize antigen-specific immune tolerance therapy. The first major obstacle stems from insufficient understanding of the antigen library presented during homeostasis. These studies have overcome this obstacle by utilizing optimized techniques to resolve native peptides that bind to MHC class II (MHC II) molecules in and around the CNS. The second major obstacle arises from the limited number of peptides available for studying autoreactive T cells. Given that the MHC II peptide group in the CNS during homeostasis has been extensively characterized, we now have the ability to broadly understand the specificity of autoreactive T cells in the CNS. Furthermore, the peptides we have identified help guide the development of MHC II tetramers, allowing for the tracking and phenotyping of autoreactive CNS-specific T cells. This provides insight into the function of local populations of self-reactive T cells in the CNS by relevant antigen presentation – an understanding that elucidates the mechanisms employed by the CNS to gain immune immunity. Goal 1 aims to address these two major obstacles.

[0217] Another significant obstacle to developing antigen-specific immunotherapies stems from insufficient understanding of the disease-causing autoantigens and subsequent epitope expansion (often resulting from chronic autoimmunity or inflammatory responses)1-5 These studies have overcome this obstacle by leveraging our platform technology to define the MHC II peptide matrix to understand how the library of self-antigens presented on MHC molecules deviates from homeostasis under neuroinflammation. Goal 2 provides strategies to address this. Along these lines, antigen-specific therapiesThe strategy also faces a key hurdle: the identification of pathogenic epitopes and the discovery of methods to manipulate their sequences or their delivery to drive toxic immune responses.6-10 These studies circumvent this hurdle by leveraging our knowledge of CNS-derived endogenous antigen presentation in homeostasis to provide information on antigen-specific immunotherapy. Thus, instead of targeting pathogenic autoantigens to directly induce tolerance, these studies provide the delivery of therapeutic homeostasis-specific, CNS-specific, MHCII-binding autoantigens to suppress and prevent CNS autoimmunity. Therefore, goal 3 provides a strategy to overcome this hurdle.

[0218] These studies provide an expanded understanding of the CNS-presented pool of autoantigens and autoreactive T cells. This, in turn, advances our understanding of the mechanisms supporting CNS immune exemption and immune tolerance. This advances our understanding of antigen-specific immunotherapy that can induce immune tolerance in other tissues to prevent allogeneic or autoimmune attacks for clinical benefit.

[0219] C. Background:

[0220] i) Autoimmune Diseases:

[0221] Autoimmune diseases remain a major public health concern, causing significant disability in many individuals, ranging from 3 to 6% per year.11,12 In fact, 4.5% of individuals worldwide and more than 24 million in the United States alone suffer from various autoimmune conditions.13,14 Furthermore, given the increased risk of developing another autoimmune condition,15,16, cancer,17 and mental illness,18-21 autoimmune diseases negatively impact an individual's quality of life, often resulting in lifelong disability and carrying an undeniable socioeconomic burden.22,23 Major histocompatibility complex (MHC) molecules, also known as human leukocyte antigens (HLA) in humans, have long been considered to play a significant role in the genetic susceptibility to autoimmune diseases.24-27 These molecules are widely believed to promote autoimmune reactivity through the unfavorable presentation of self-antigens or foreign antigens (also known as molecular mimics) that cross-react with self-peptides.28-31 Because identifying target autoantigens for the pathogenesis of autoimmune diseases is challenging, current interventions have focused to date on nonspecific immunosuppressive therapies with significant side effects.32-36 This indicates a need for strategies demonstrating robust efficacy and tolerability. To this end, antigen-specific immunotherapy offers an attractive alternative for precisely targeting organ-specific autoimmunity and has shown promise in various autoimmune conditions.37-41 Nevertheless, antigen-specific approaches are not always effective and in some cases are detrimental,42-45 exemplified by the incomplete understanding of the mechanisms by which autoantigen presentation maintains immune tolerance.

[0222] ii) Antigen presentation and T cell recognition:

[0223] A series of pioneering studies conducted during the 1970s and 1980s dramatically changed immunology’s understanding of how T cells recognize antigens. Several lines of evidence began to suggest that T cells, as entities, possess the ability to respond to foreign materials, but this response depends on the simultaneous recognition of their own MHC molecules 46-48. Subsequent studies then elucidated the functional role of MHC molecules in presenting peptide antigens to T cells, and structural studies of MHC molecules allowed the observation of peptides 49-51 positioned within the binding grooves of both MHC I and II molecules, as described on pages 57 / 91 of the specification 65 CN 121240870 A. Overall, these observations improved our understanding of the key interactions between T cell receptors and peptide-MHC complexes in defining adaptive immune responses, inducing self-tolerance, and initiating effective immune responses to foreign materials.

[0224] Furthermore, MHC molecules read out cellular states and overall tissue physiology by shaping the presented antigen library. However, in the past, antigens were thought to be primarily of endogenous origin for MHC II molecules and primarily of exogenous origin for MHC III molecules. In recent years, this clear distinction regarding peptide origin has become blurred. Exogenous antigens have been described as converging on MHC II molecules via different intracellular pathways, a process known as cross-presentation.52-55 On the other hand, the abundance of endogenous peptides presented on MHC II molecules is increasingly recognized, with autophagy thought to play a promoting role through MHC II molecules.56-58 Under inflammation, these processes change to adapt to and provide contextual information for the immune response, including enhanced and stable expression of MHC molecules on the surface,59-61 improved antigen processing capacity,62,63 and so on. This helps to focus antigen presentation on key molecules requiring an immune response. Nevertheless, even in inflammation, endogenous peptides still constitute a significant proportion of the immune peptidestome.64,65 Whether this presentation promotes the maintenance or destruction of self-tolerance remains elusive.

[0225] iii) CNS Immune Exemption

[0226] Interestingly, early observations noted that certain tissues, such as the CNS, possess enhanced tolerance to skin grafts and xenografts, suggesting they are immune-exempt.66-68 However, significant gaps remain in the understanding of the meaning of immune exemption in the context of systemic immune tolerance, which is primarily explained by the removal or functional inactivation of autoreactive T cells through central (thymus) and peripheral (non-thymus) mechanisms.69 In fact, this biological need to suppress autoreactive T cells has been challenged over the past two decades, with studies demonstrating the beneficial effects of autoreactive T cells in the heart, skeletal muscle, and CNS (including injury, chronic neurodegenerative conditions, etc.).70-76 Furthermore, in the past, brain drainage lymphatic...In the absence of an explanation for CNS immune exemption, the rediscovery of the lymphatic network in the meninges (the membranous covering of the brain) establishes it as a unique neuroimmune interface.77-79 Given its direct and functional connection with the peripheral immune system, the mechanisms that allow the CNS to achieve immune exemption provide an attractive framework for reconceptualizing immune tolerance. In summary, whether the antigen pool presented in the CNS promotes its immune-exempt properties remains an important unanswered question. Therefore, the goal of these studies is to elucidate the mechanisms by which CNS autoantigens entrain autoreactive T cells to maintain and restore immune homeostasis for clinical benefit.

[0227] D. Methods

[0228] These studies enhance our understanding of immune tolerance and have the potential to guide antigen-specific therapies. Goal 1 is based on preliminary findings to elucidate the regulatory mechanisms of autoantigen-dependent protection against autoimmunity. Goal 2 provides background on the autoantigen pool presented on MHC II molecules in physiology and pathology. Goal 3 advances the therapeutic delivery of autoantigens to enhance immune tolerance and effectively combat autoimmune pathologies.

[0229] E. Preliminary Results

[0230] i) Massive Presentation of Myelin Basic Protein (MBP) on MHC Class II Molecules in the CNS During Homeostasis

[0231] Knowledge of antigens locally presented on MHC Class II molecules provides valuable information to expand our understanding of immune tolerance, which remains largely unexplored. The CNS is a fascinating system for exploring this issue due to its immune-immune properties. In particular, recent studies have described a true brain lymphatic network draining to the deep cervical lymph nodes (dCLN), connecting the CNS to the peripheral immune system.77,78 Furthermore, the meninges at the brain boundary have been confirmed as key sites for the functional presentation of CNS-derived antigens to patrolling T cells.79 Thus, the library of antigens presented in the CNS during homeostasis is unknown. MHC Class II peptidomimetics analysis of the meninges and dCLN of 8-week-old C57BL / 6J mice was performed. Gibbs clustering analysis revealed I-Ab binding motifs and was consistent with previous studies (Fig. 5A).80,81 We then analyzed a complete list of MHC-II binding peptides in the dCLN and meninges when compared with other tissues (see page 66 of the manual, page 58 / 91, CN 121240870 A). 6.28% and 17.44% of the MHC-II binding peptides in the dCLN and meninges, respectively, were derived from CNS-elevated proteins (Figure 5B). Interestingly, MBP-derived peptides were primarily presented in the meninges, accounting for 77% of peptides that could be defined as CNS-elevated (Figure 5B). The abundance of MBP presentation in the meninges is surprising, and we sought to elucidate its role in CNS immune tolerance. Furthermore, when the identified MHC-II binding peptides were compared with MBP sequences...During the alignment, it was found that the vast majority of MBP-derived peptides revolved around the common amino acid sequence MBP33-42. To investigate this, MBP peptides identified in our MHC II peptidomimetry, including or excluding the common MBP33-42 sequence, were synthesized.

[0232] ii) Endogenous peptides inhibit experimental autoimmune encephalomyelitis (EAE) induced by oligodendrocyte glycoprotein (MOG)

[0233] Given the advantage of MBP presentation in the meninges, we wanted to know whether the MBP peptides we identified from the CNS MHC II peptidomimetry could induce or regulate the progression of CNS autoimmune diseases. To answer this question, EAE, a multiple sclerosis (MS) animal model, was used. Eight-week-old C57BL / 6J mice were immunized with MOG35-55 alone (as our positive control), MBP12-26 (i.e., KYLATASTMDHARHG (SEQ ID NO:14612)) or MBP27-42. Mice were tracked 20 days (dpi) post-immunization using standard EAE scoring (Figure 6A). The control group immunized with MOG35-55 exhibited hindlimb motor symptoms between 9 and 12 dpi, peaking severity between 14 and 18 dpi, consistent with previous studies.83 This contrasts sharply with the groups immunized with MBP12-26 or MBP27-42, in which none of the five mice showed clinical or pathological signs of EAE (Figure 6B). These findings are consistent with previous observations that the C57BL / 6J strain is particularly resistant to MBP-induced EAE.84,85 Although immunization with MBP peptides alone does not induce pathology, it was considered whether MBP peptides could modulate the disease progression of MOG35-55-induced EAE. To address this question, 8-week-old C57BL / 6J mice were co-immunized with MOG35-55 and MBP12-26, MBP27-42, or MBP27-42 (citrullinated). Interestingly, while co-immunization with MBP27-42 and MOG35-55 showed a statistically significant inhibitory effect on the EAE phenotype, co-immunization with either MBP12-26 or MBP27-42 (citrullinated) did not attenuate the disease, showing results similar to the positive control (Figure 7A). Furthermore, the peak EAE score was significantly lower in the MOG35-55+MBP27-42 group compared to all other treatment modalities (Figure 7B). Therefore, this finding highlights that the observed inhibitory effect of MBP27-42 on MOG35-55-induced EAE is not solely due to co-immunization with another peptide – co-immunization with MBP12-26 (a similar-length autoantigen) failed to inhibit the disease. Moreover, a control experiment was conducted by directly modifying the MBP27-42 sequence with citrullinated (a disease-associated enzyme modification). In fact, peptide-based arginine…Acid deiminases (PADs), a family of enzymes that catalyze the conversion of arginine to citrulline residues, become dysfunctional in many autoimmune diseases, including MS.86,87 Previous studies have also confirmed that MBP itself becomes excessively citrullinated in MS and EAE, and that this is associated with increased disease severity.87,88 Notably, co-immunization of MOG35-55 with citrullinated MBP27-42 abolishes the ability of MBP27-42 to alleviate EAE disease (Figures 7A-7B). Although citrullination induces new epitopes, triggering the hypothesis of autoreactivity, recent studies have instead described a lack of T cell responsiveness to citrullinated myelin-associated peptides.89 Combining this with our findings, it is foreseeable that citrullination of self-antigens may instead impede regulatory immune responses, releasing the brakes set up to fight autoimmunity. These preliminary findings support the specific immunomodulatory function of MBP27-42 and prompt targeted evaluation of its binding epitopes to better understand the T cell responses it generates. The experiments to achieve this are described in Objective 1.

[0234] iii) Co-immunization with MBP peptides in MOG-induced EAE biases the quality of T cell responses toward a regulatory phenotype

[0235] Co-immunization with MBP27-42 and MOG35-55 significantly reduced the severity of clinical EAE, raising the question: Does the MBP27-42 peptide regulate T cell responses in EAE? This question was initially explored by evaluating changes in T cell differentiation, focusing on transcription factor expression. This evaluation was based on the established key role of RORγt+Th17 cells in the pathogenesis of EAE.90 Furthermore, numerous studies have confirmed that Foxp3 can inhibit T cell effector function and, more specifically, antagonize the function of RORγt.91-94 It is hypothesized that MBP27-42 regulates the pathogenicity of MOG35-55-induced EAE by modulating the expression or function of RORγt. To test this hypothesis, 8-week-old C57BL / 6J mice were immunized with MOG35-55 or MOG35-55+MBP27-42 to induce EAE. dCLNs were harvested on day 13 post-EAE induction and meninges on day 16 for flow cytometry evaluation of any changes in T cells. Prior to the peak of EAE, dCLNs from co-immunized MOG35-55+MBP27-42 mice, compared to MOG35-55, reflected a statistically significant increase in Foxp3+CD4+ T cell frequency, with a simultaneous decreasing trend in RORγt+ expression frequency (Figures 8A-8B). This is similar to observations in the meninges at the peak of EAE disease. In the meninges, RORγt+ expression was significantly higher in the MOG35-55+MBP27-42 group.The proportion of CD4+ T cells was significantly reduced, with a corresponding increase in the frequency of Foxp3+CD4+ T cells (Figs. 8C-8D). In summary, our preliminary results noted a significant increase in regulatory Foxp3+CD4+ T cells in the dCLN at the peak of the disease, followed by a decrease in the presence of pathogenic RORγt+CD4+ T cells in the meninges. This provides evidence for the regulation of pathogenic T cell responses in EAE by MBP27-42 (possibly mediated by Foxp3, as described in Target 1).

[0236] iv) Suppression of EAE in different mouse strains by co-immunization with MBP peptides

[0237] Interestingly, MHCII peptidomome analysis of the meninges of SJL / J mice revealed a large presentation of MBP peptides containing the MBP33-42 sequence. Curious about whether the protective effect against neuroinflammation mediated by MBP27-42 could be universal, we used another active EAE model of SJL / J mice. Here, we immunized 8-week-old male SJL / J mice with myelin lipoprotein (PLP) 139-151 or co-immunized them with MBP27-42 or neurofilament light chain polypeptide (NF-L) 160-173 to induce EAE disease. NF-L160-173, an endogenous peptide of similar length to MBP27-42, was chosen as a suitable control peptide, supported by evidence of its presentation on MHC II molecules in EAE and restricted T-cell responsiveness in SJL / J mice.95,96 Furthermore, NF-L160-173 was identified as an MHC II-binding peptide in the meninges of SJL / J mice. Consistent with the literature, immunization with PLP139-151 in young male SJL / J mice resulted in monophasic EAE disease.97 Surprisingly, co-immunization with MBP27-42 instead of NF-L160-173 confirmed a significant EAE inhibition and a significant downregulation trend in peak EAE scores (Figs. 9A-9B). Similar to what was observed in C57BL / 6J mice, co-immunization with another peptide, NF-L160-173, and PLP139-151 could not explain the inhibitory effect observed when co-immunized with MBP27-42. Overall, this observation not only further supports the specific immunomodulatory function of the MBP27-42 peptide, but also hypothesizes the potential universal role of MBP peptides in broad defense against CNS autoimmunity.

[0238] v) Therapeutic delivery of MBP peptides in extracellular vesicles (EVs) alleviates CNS autoimmune damage

[0239] The role of EVs in regulating immune responses has been increasingly recognized over the past two decades.98 From loading nucleic acids to small molecule drugs, EVs have enormous immunotherapeutic potential.99,100 Recent studies have shown that EVs derived from oligodendrocytes can suppress autoimmune neuroinflammation.101. Following this therapeutic approach, EVs were isolated from dendritic cells using ultracentrifugation, and enrichment was confirmed by both immunoblotting and transmission electron microscopy (Figs. 10A-10B). Following established protocols 100, 102, the MBP27-42 peptide was introduced into the EVs via sonication. Interestingly, when compared to injection of EVs lacking MBP27-42, injection of EVs containing MBP27-42 into the CSF on day 5 after induced EAE via MOG35-55 significantly reduced the clinical EAE score (Figs. 10C-10D). This finding confirms the exciting immunotherapeutic potential of the MBP peptide, as explored in target 3.

[0240] F. Experimental Design:

[0241] Age-matched mice were used for the experiments. If mice in the experimental group were obtained from an external supplier, then the corresponding controls were also obtained. Transgenic mice and their wild-type littermates were housed in the same facility. Both males and females were used for evaluation. For in vivo studies, 10 animals / group were used based on the following statistical criteria: expected difference of approximately 50%, SD 0.33, two-sided, α-efficacy 0.9, p-value 0.05: N = 10. For imaging and flow cytometry analyses, 5 animals / group were used based on the following statistical criteria: expected difference of approximately 37%, SD 0.20 for each, two-sided, α-efficacy 0.8, p-value 0.05: N = 5. To enhance rigor, all measurements were performed without the researchers' knowledge of the group identities; group identities were only revealed after data recording was completed.

[0242] i) Objective 1: CNS-derived endogenous peptides promote the differentiation of regulatory T cells and regulate CNS autoimmunity.

[0243] Preliminary studies have shown that the presentation of endogenous peptides regulates T cell responses to reduce neuroinflammation in EAEs. However, it remains unclear whether MBP peptides contribute to the regulatory T cell population. By specifically removing Foxp3+ regulatory T cells and using tetramers to phenotypically evaluate MBP-specific T cells, the regulatory mechanism by which endogenous peptide presentation confers protection of the CNS from significant autoimmunity can be determined.

[0244] Study #1: MBP27-42-dependent regulation of T cell response by removing Foxp3

[0245] Preliminary results revealed that MOG35-55+ MBP27-42 co-immunosuppressed EAE, and the ratio between RORγt and Foxp3 expression on CD4+ T cells changed significantly. To confirm the role of MBP27-42 in cultivating a population of Foxp3+ regulatory T cells, 8-week-old Foxp3DTR transgenic mice expressing diphtheria toxin receptor (DTR) under the Foxp3 promoter were used.Foxp3+ T cells were selectively eliminated after treatment with Foxp3DTR mice. Eight-week-old Foxp3EGFP mice, expressing only enhanced green fluorescent protein (EGFP) under the control of the Foxp3 promoter, were used as our control. EAE was induced in transgenic mice with either MOG35-55 alone or MOG35-55+MBP27-42. Diphtheria toxin (200 ng) was administered intraperitoneally and intravenously on day 3 post-EAE onset. All experimental groups were assessed using standard EAE scoring for up to 21 days. Tissues, including the meninges, were harvested on day 16 (peak EAE disease) for analysis by flow cytometry and immunohistochemistry. The initial findings were expected to be reproduced using Foxp3EGFP control mice, confirming the increased trend of Foxp3 expression in the meninges after co-immunization with MOG35-55+MBP27-42 compared to MOG35-55. The expected depletion of Foxp3+ regulatory T cells in Foxp3DTR mice abolished MBP27-42-dependent EAE improvement, demonstrating a mechanism of Foxp3-mediated inhibition.

[0246] Study #2: Determining the role of MBP27-42-specific CD4+ T cells in regulating CNS immune tolerance

[0247] Preliminary data confirmed the presentation of MHC class II molecules in the CNS during homeostasis and that MBP27-42 provides protection against MOG35-55-induced EAE. To characterize the function of MBP27-42-specific CD4+ T cells, an MHC II tetramer was recently developed using the common amino acid sequence MBP33-42. These tetramers were tested and calibrated using lymphocytes isolated from C57BL / 6J mice immunized with a peptide of MBP27-42 enhanced with complete Freund's adjuvant (CFA) in the bilateral flank region. A week was allowed to generate a sufficient immune response before inguinal lymph nodes were isolated and drained. Subsequently, the isolated T cells were fused with BW5147 (a thymic lymphoma cell line) to generate T cell hybridoma clones. In vitro antigen presentation assays were performed by co-culturing individual T cell clones with M12.C3 (a B-lymphoma cell line used as antigen-presenting cells) in the presence of MBP27-42. T cell clones specific to MBP27-42 were selected using IL-2 enzyme-linked immunosorbent assay (ELISA) as our T cell activation readout. Key amino acid mutations in MBP27-42 were performed to identify residues that deactivate MBP27-42-specific T cell clones, guided by an MHC II binding affinity algorithm. The development of MBP27-42-specific MHC II tetramers can be better guided by identifying key recognition events for peptide-MHC II and T cell receptor interactions. Using our developed tetramer…MBP27-42-specific T cells were phenotypically classified in the meninges and dCLN using flow cytometry in wild-type mice, mice immunized with MBP27-42 alone, and mice co-immunized with MOG35-55+ MBP27-42. Cell surface markers and transcription factors (including Foxp3) were stained. In doing so, it was possible to determine whether the CNS possessed a population of MBP27-42-specific CD4+ T cells expressing regulatory markers that promote CNS immune tolerance, such as Foxp3.

[0248] In summary, the experiments described in Objective 1 identified the role of endogenous peptide presentation in regulating T cell responses and explored Foxp3-mediated regulation as the mechanistic basis for this phenotype.

[0249] Based on the preliminary results, it is hypothesized that MBP27-42 recruits a reservoir of Foxp3+ regulatory T cells to maintain CNS immune tolerance. However, it is possible that MBP27-42-specific T cells may enhance immunomodulation through other regulatory mechanisms. For example, previous studies have confirmed the role of Foxp3-T regulatory type 1 (TR1) cells in regulating autoimmunity (including EAE) by producing regulatory cytokines (including IL-10 and TGFβ)103,104. For this purpose, intracellular cytokine staining for IL-10 and TGFβ was included when MBP27-42-specific T cell phenotypes were classified by flow cytometry. In addition, IL-10 fl / fl and CD4- CreERT2 are commercially available and allow for conditional loss of IL-10 production in CD4+ T cells.

[0250] ii) Objective 2: Neuropathological restriction of homeostatic epitope presentation on MHC II molecules

[0251] The aids to inflammation-induced tissue damage, epitope expansion and their contribution to autoimmune pathology have been described, including in EAE1-5. However, the effects of epitope expansion and inflammation in the CNS on the autoantigen pool are unknown. Furthermore, their impact on the homeostatic presentation of MHC-II binding peptides remains elusive. To evaluate any changes in meningeal lymphatic drainage, fluorescently conjugated ovalbumin protein was introduced into the CSF via intracephalic magnum (icm) injection at the peak of EAE disease, and the drainage dCLN was evaluated. In doing so, a statistically significant increase in ovalbumin coverage was found in the dCLN of EAE mice compared to the CFA control (Figures 11A-11C). Furthermore, previous studies have confirmed that linking afferent lymph nodes to the dCLN alleviates EAE disease, demonstrating its relevance to EAE disease progression.78 Therefore, in this objective, an extensive survey of the immune peptidomome was attempted to evaluate the divergence of the CNSMHCII peptidomome between inflammation and homeostasis, anticipating a reduction in the presentation of the MBP peptides we identified.

[0252] Study #1: Establishment of the CNS MHC II Peptideome During Neuroinflammation

[0253] Preliminary data revealed a significant increase in meningeal lymphatic drainage at the peak of EAE disease, suggesting the need to investigate the autoantigen library presented in the meninges and their draining peripheral lymph nodes (dCLNs). To address this, C57BL / 6J mice were immunized with either CFA alone or CFA+MOG35-55; pertussis toxin was administered on the day of immunization and two days thereafter. All experimental groups underwent standard EAE scoring for 16 days, after which tissues (including the meninges and dCLNs) were harvested and processed. Individual tissues were dissolved in a mild detergent in the presence of protease inhibitors to preserve the peptide-MHC complex. The dissolved products of fragments were pre-clarified by centrifugation and then incubated with agarose conjugated with MHC class II molecule-specific antibodies. After a series of washing steps, peptides were eluted from MHC class II molecules with 10% acetic acid. The eluted peptides were then further clarified with a C18 spinning column and then subjected to mass spectrometry and immunological analysis. By establishing an MHC II peptidomome during active EAE disease, the presentation of MBP peptides, particularly those containing the common sequence MBP33-42, was expected to be reduced due to epitope expansion and diversification.

[0254] The experiments of Objective 2 aimed to reveal changes in the autoantigen pool, particularly in the CNS, when comparing homeostatic and inflammatory diseases. In doing so, the study confirmed a reduction in the presentation of homeostatic autoantigens, which play a key role in maintaining CNS immune tolerance.

[0255] It is possible that CFA immunization in the absence of MOG35-55 and administration of two doses of pertussis toxin can induce changes in CNS boundary tissues, which confounds EAE disease-specific differences. With this in mind, naive C57BL / 6J mice were included as another control when harvesting tissues, and they were subjected to the same pipeline for immunopeptidomics. In addition, given that the EAE disease progression in C57BL / 6J mice is monophasic, it is possible that the autoantigen pool has stabilized at the peak of EAE disease as it enters the chronic phase of the disease. To address this issue, harvests were staggered to target collection of the meninges and dCLN before disease peak (day 10) and after disease peak (day 21). This further utilized an immunopeptidomics platform in the CNS to provide a more comprehensive picture of the dynamics of the MHC II peptidome in CNS-related tissues in EAE disease over time.

[0256] iii) Objective 3: Therapeutic delivery of CNS-derived endogenous peptides to broadly combat neuroinflammation-induced pathology

[0257] Preliminary results suggest that the potential universal role of MBP-derived peptides in regulating neuroinflammation makes them an attractive therapeutic target. In SJL / J mice (a different active EAE model), MBP27-42 peptides were co-immunized with PLP139-151.Significantly reduced disease progression. In addition, injection of EVs containing MBP27-42 into CSF ​​prior to the onset of paralysis symptoms attenuated the severity of EAE disease. Both observations highlight the promising immunotherapeutic potential of MBP peptides. Furthermore, the targeting nature and high biocompatibility of EVs provide a modifiable carrier for further fine-tuning of antigen-specific immunotherapy. The efficacy of vesicles containing MBP27-42 delivered into CSF ​​was examined to improve pathology, even when administered after the onset of paralysis symptoms, and whether it broadly resisted CNS autoimmunity in different mouse strains.

[0258] Study #1: Evaluation of the peptide contents of EVs floating freely in CSF during steady state

[0259] Recent studies in humans have confirmed that myelin-associated proteins, including MOG, PLP, and MBP, can be found in EVs in serum and CSF derived from healthy individuals and patients with MS. Furthermore, given the preliminary data indicating the beneficial function of the MBP peptide encapsulated in EVs, it is necessary to investigate the contents of EVs present in CSF during steady state. To evaluate this, CSF was collected from naive C57BL / 6J mice using a glass microcapillary pipette. Specifically, EVs were precipitated and purified by size exclusion chromatography using the Exo-spin™ system. Confirmation was performed by immunoblotting and a ZetaView nanoparticle tracking analyzer prior to further downstream applications. After confirmation, exosomes underwent at least 5 freeze-thaw cycles to release the intracellular contents; these were purified by a C18 column, enriching the peptides. The collected material was subjected to mass spectrometry and immunological analysis.

[0260] Study #2: Evaluation of the therapeutic capacity of EVs containing MBP27-42 peptides for neuroinflammation

[0261] Preliminary data support the beneficial function of vesicle-mediated delivery of MBP27-42 peptides when injected into CSF ​​before the onset of any EAE symptoms. However, its ability to suppress neuroinflammation after the onset of paralysis remains uninvestigated. To investigate this, EVs were enriched from DC2.4 cells (an immortalized dendritic cell line) by 100,000 xg ultracentrifugation, and purity was confirmed by Western blotting, TEM, and a ZetaView nanoparticle tracking analyzer. The enriched vesicles were subjected to parallel sonication in the absence or presence of endogenous MBP27-42 and encephalitis-derived MOG35-55 peptides. The vesicles were further purified by size exclusion chromatography and high-speed ultracentrifugation to remove any unincorporated peptides, effectively separating intact control vesicles or vesicles containing our target peptide. The quality of the enriched EVs was evaluated similarly to that described above, and further quantified using a ZetaView nanoparticle tracking analyzer to control the total number of vesicles injected into the CSF. Furthermore, the loaded peptides were conjugated with fluorescent dyes, enabling flow cytometry analysis using nanoparticles.Cytological control of loading efficiency was employed. EAE was induced in 8-week-old C57BL / 6J mice with MOG35-55. Subsequently, control vesicles or vesicles containing MBP27-42 or MOG35-55 were introduced into the CSF via intracranial injection at day 5 (before disease onset), day 10 (onset of paralytic symptoms), or day 16 (peak disease). All experimental groups with their corresponding littermate controls were assessed using standard EAE scoring for up to 25 days. In doing so, it was anticipated that delivery of vesicles containing only MBP27-42 would demonstrate a therapeutic benefit and promote the recovery of CNS immune tolerance at different time points. In addition, intravenous injection of EVs was also examined because it provided a minimally invasive and convenient delivery method.

[0262] Study #3: Elucidating the function of endogenous MBP27-42 peptide in broadly suppressing CNS autoimmunity

[0263] Preliminary findings confirmed the more general immunomodulatory role of MBP27-42 (an epitope presented extensively on MHCII molecules) in regulating CNS immune homeostasis. In completely different mouse strains with active EAE induced by different peptides, MBP27-42 adequately suppressed PLP139-151-induced EAE in SJL / J mice. Furthermore, the MBP27-42 sequence itself is highly conserved in different vertebrate species, including humans [106, 107]. For these reasons, an inquiry was made into the ability of vesicles containing MBP27-42 to reduce neuroinflammation-induced damage in different active EAE models. The pipeline established as described above in Study #1 was followed to enrich control vesicles and vesicles containing MBP27-42 or suitable control peptides. Active EAE was induced in C57BL / 6J, SJL / J, or B10.PL mice by immunization with MOG35-55, PLP139-151, or N-terminal acetylated MBP1-11, respectively. Shortly thereafter, on day 5, the mice received an ICM injection of either control vesicles or peptide-containing vesicles, which were then evaluated for standard EAE for up to 25 days. Given that co-immunization with MBP27-42 and PLP139-151 in SJL / J mice and delivery of MBP27-42-containing vesicles to the CSF of MOG35-55-immunized C57BL / 6J mice effectively suppressed EAE disease, therapeutic delivery of MBP27-42-containing vesicles is expected to similarly resist different active EAE models.

[0264] In summary, the experiments described in Objective 3 revealed the broad immunomodulatory function of endogenous MBP27-42 peptide in resisting CNS autoimmunity, confirming its immunotherapeutic potential in controlling neuroinflammation-induced damage and restoring CNS immune tolerance.

[0265] Based on the preliminary results, it is predicted that MBP peptide will exhibit therapeutic efficacy in different disease stages and different mouse strains. (Specification 63 / 91 pages, 71 CN)121240870 A However, the observed disease-suppressive effect of EAE may still be limited to ICM delivery of MBP27-42 on day 5 before the onset of paralysis. Since vesicles reflect cell type and their environment of origin [108, 109], modulating vesicle characteristics could support our antigen-specific immunotherapy approach. Recent studies have confirmed that vesicles derived from dendritic cell cultures treated with low-dose IFNγ reduce oxidative stress and enhance CNS myelination processes in vivo

[110] . Furthermore, vesicles derived from mesenchymal stem cells, when administered intravenously at disease peak, mildly but significantly reduce clinical EAE scores

[111] ; this enhances confidence in treating CNS autoimmune diseases at later stages according to the present invention. Therefore, by utilizing the modulatory capacity of vesicles and the antigen specificity of the methods described herein, MBP27-42 and control peptides were also loaded into vesicles derived from IFNγ-stimulated dendritic cells and mesenchymal stem cells. In summary, these studies further advance the use of MBP27-42-containing vesicles as antigen-specific immunotherapy for autoimmune diseases.

[0266] G. References for Example 1:

[0267] 1. Vanderlugt, C.J. & Miller, S.D. Epitope spreading. Curr Opin Immunol 8, 831-836 (1996). https: / / doi.org:10.1016 / s0952-7915(96)80012-4

[0268] 2. Vanderlugt, C.L. & Miller, S.D. Epitope spreading in immune-mediated diseases: implications for immunotherapy. Nat Rev Immunol 2, 85-95 (2002). https: / / doi.org:10.1038 / nri724

[0269] 3. Genain, CP et al. Late complications of immunoepileptic therapy in nonhuma n prima te. Science 274 ,2054‑ 2057 (1 996) .h tt ps : / / d oi .o rg :1 0 .11 26 / science.274.5295.2054

[0270] 4.Lehmann,PV,Forsthuber,T.,Miller,A.&Sercarz,E.E.Spreading of T‑ cell autoimmunity to crypticdeterminants of an autoantigen. Nature 358, 155‑157 (1992). https: / / doi.org:10.1038 / 358155a0

[0271] 5. McRae, B. L., Vanderlugt, C. L., DalCanto, M. C. & Miller, S. D. Functional evidence for epitope spreading in the relapsing pathology of experimental autoimmune encephalomyelitis. J Exp Med 182, 75‑85 (1995). https: / / doi.org:10.1084 / jem.182.1.75

[0272] 6. Anderton, S.M. Peptide‑based immunotherapy of autoimmunity: a path of puzzles, paradoxes and possibilities. Immunology 104, 367‑376 (2001). https: / / doi.org:10.1046 / j.1365‑2567.2001.01324.x

[0273] 7. Shepard, E. R. et al. The Mechanism of Action of Antigen Processing Independent T Cell Epitopes Designed for Immunotherapy of Autoimmune Diseases. Front Immunol 12, 654201 (2021). https: / / doi .org:10 .3389 / fimmu.2021.654201

[0274] 8. Herzog, R. W. et al. Oral Tolerance Induction in Hemophilia B Dogs Fed with Transplastomic Let‑tuce. Mol Ther 25, 512‑522 (2017). https: / / doi .org: 10.1016 / j.ymthe.2016.11.009

[0275] 9. Hawiger, D. et al. Dendritic cells induce peripheral Tcell unresponsiveness under steady state conditions in vivo. J Exp Med 194, 769-779 (2001). https: / / doi.org:10.1084 / jem.194.6.769

[0276] 10. Zhang, A. H., Rossi, R. J., Yoon, J., Wang, H. & Scott, D. W. Tolerogenic nanoparticles to induce immunologic tolerance: Prevention and reversal of FVIII Description Page 64 / 91 72 CN 121240870 A inhibitor formation. Cell Immunol 301, 74-81 (2016). https: / / doi.org:10.1016 / j.cellimm.2015.11.004

[0277] 11. Lerner, A., Jeremias, P. & Matthias, T. The World Incidence and Prevalence of Autoimmune Diseases is Increasing. International Journal of Celiac Disease 3, 151-155 (2016). https: / / doi.org:10.12691 / ijcd-3-4-8

[0278] 12. Cooper, G. S., Bynum, M. L. & Somers, E. C. Recent insights in the epidemiology of autoimmune diseases: improved prevalence estimates and understanding of clustering of diseases. J Autoimmun 33, 197-207 (2009). https: / / doi.org:10.1016 / j.jaut.2009.09.008

[0279] 13. Hayter, S. M. & Cook, M. C. Updated assessment of the prevalence, spectrum and case definition of autoimmunedisease . Autoimmun Rev 11 , 754‑765 (2012) . https: / / doi.org:10.1016 / j.autrev.2012.02.001

[0280] 14 . U.S. Department of Health and Human Services , N .I .o .A.a .I .D. Progress in Autoimmune Diseases Research.(2005) .

[0281] 15 . Cho ,J .H .&Feldman ,M . Heterogeneity of autoimmune diseases: pathophysiologic insights from genetics and implications for new therapies. Nat Med 21, 730‑738 (2015) . https: / / doi.org:10.1038 / nm.3897

[0282] 16. Somers, E.C., Thomas, S.L., Smeeth, L.&Hall, A.J. Are individuals with an autoimmune disease at higher risk of a second autoimmune disorder?Am J Epidemiol 169, 749‑755 (2009) . https: / / doi.org:10.1093 / aje / kwn408

[0283] 17 . Giat ,E .,Ehrenfeld ,M .&Shoenfeld ,Y . Cancer and autoimmune diseases . Autoimmun Rev 16 , 1049‑1057 (2017) . https: / / doi .org:10 .1016 / j.autrev.2017.07.022

[0284] 18. Cullen ,A.E . et al. Associations Between Non‑neurological Autoimmune Disorders and Psychosis: A Meta‑analysis . Biol Psychiatry 85 , 35‑48 (2019) . https: / / doi.org:10.1016 / j.biopsych.2018.06.016

[0285] 1919. Marrie, R. A. et al. Mental comorbidity and multiple sclerosis: validating administrative data to support population‑based surveillance. BMC Neurol 13, 16 (2013). https: / / doi.org:10.1186 / 1471‑2377‑13‑16

[0286] 20. Hauser, W., Janke, K. H., Klump, B. & Hinz, A. Anxiety and depression in patients with inflammatory bowel disease: comparisons with chronic liver disease patients and the general population. Inflamm Bowel Dis 17, 621‑632 (2011). https: / / doi.org:10.1002 / ibd.21346

[0287] 21. Matcham, F., Rayner, L., Steer, S. & Hotopf, M. The prevalence of depression in rheumatoid arthritis: a systematic review and meta‑analysis. Rheumatology (Oxford) 52, 2136‑2148 (2013). https: / / doi.org:10.1093 / rheumatology / ket169

[0288] 22. Shoenfeld, Y., Selmi, C., Zimlichman, E. & Gershwin, M. E. The autoimmunologist: geoepidemiology, a new center of gravity, and prime time for autoimmunity. J Autoimmun 31, 325‑330 (2008). https: / / doi.org:10.1016 / Description Page 65 / 91 73 CN 121240870 A j.jaut.2008.08.004

[0289] 23. Jacobs, P., Bissonnette, R. & Guenther, L. C. Socioeconomic burden of immune‑mediated inflammatory diseases‑‑focusing on work productivity and disability. J Rheumatol Suppl 88, 55‑61(2011). https: / / doi.org / 10.3899 / jrheum.110901

[0290] 24. Ryder, L. P., Svejgaard, A. & Dausset, J. Genetics of HLA disease association. Annu Rev Genet 15, 169‑187(1981). https: / / doi.org / 10.1146 / annurev.ge.15.120181.001125

[0291] 25. Todd, J. A. & Wicker, L. S. Genetic protection from the inflammatory disease type 1 diabetes in humans and animal models. Immunity 15, 387‑395(2001). https: / / doi.org / 10.1016 / s1074‑7613(01)00202‑3

[0292] 26. MacKay, K. et al. Whole‑genome linkage analysis of rheumatoid arthritis susceptibility loci in 252 affected sibling pairs in the United Kingdom. Arthritis Rheum 46, 632‑639(2002). https: / / doi.org / 10.1002 / art.10147

[0293] 27. International Multiple Sclerosis Genetics, C. et al. Risk alleles for multiple sclerosis identified by a genomewide study. N Engl J Med 357, 851‑862 (2007). https: / / doi.org / 10.1056 / NEJMoa073493

[0294] 28. Nepom, G.T. & Kwok, W.W. Molecular basis for HLA‑DQ associations with IDDM. Diabetes 47, 1177‑1184(1998). https: / / doi.org:10.2337 / diab.47.8.1177

[0295] 29. Oldstone, M. B. Molecular mimicry: its evolution from concept to mechanism as a cause of auto‑immune diseases. Monoclon Antib Immunodiagn Immunother 33, 158‑165(2014). https: / / doi.org:10.1089 / mab.2013.0090

[0296] 30. Ridgway, W. M. & Fathman, C. G. The association of MHC with autoimmune diseases: under‑standing the pathogenesis of autoimmune diabetes. Clin Immunol Immunopathol 86, 3‑10(1998). https: / / doi.org:10.1006 / clin.1997.4449

[0297] 31. Yin, L. et al. Recognition of self and altered self by T cells in autoimmunity and allergy. Protein Cell 4, 8‑16(2013). https: / / doi.org:10.1007 / s13238‑012‑2077‑7

[0298] 32. Sepriano, A. et al. Safety of synthetic and biological DMARDs: a systematic literature review informing the 2019 update of the EULAR recommendations for the management of rheumatoid arthritis. AnnRheum Dis 79, 760‑770 (2020) .https: / / doi.org:10.1136 / annrheumdis‑2019‑216653

[0299] 33.Lallana, E.C. & Fadul, C.E. Toxicities of immunosuppressive treatment of autoimmune neurologic diseases .Curr Neuropharmacol 9, 468‑477 (2011) .https: / / doi.org:10.2174 / 157015911796557939

[0300] 34 .Meyer, K .C .,Decker, C . & Baughman, R . Toxicity and monitoring ofimmunosuppressive therapy used in systemic autoimmune diseases.Clin Chest Med 31, 565‑588 (2010) .https: / / doi.org:10.1016 / j.ccm.2010.05.006

[0301] 35 .Quistrebert, J. et al. Incidence and risk factors for adalimumab and DESCRIPTION Page 66 / 91 74 CN 121240870 A infliximab anti‑drug antibodies in rheumatoid arthritis: A European retrospective multicohort analysis .Semin Arthritis Rheum 48, 967‑975 (2019) .https: / / doi.org:10.1016 / j.semarthrit.2018.10.006

[0302] 36.Nash, P. et al. Points to consider for the treatment of immune‑mediated inflammatory diseases with Janus kinase inhibitors: a consensus statement.Ann Rheum Dis 80, 71‑87 (2021).https: / / doi.org:10.1136 / annrheumdis‑2020‑218398

[0303] 37.Goel,G. et al. Epitope‑specific immunotherapy targeting CD4‑positive T cells in coeliac disease: two randomised, double‑blind, placebo‑controlled phase 1 studies .Lancet Gastroenterol Hepatol 2 ,479‑493(2017) .https: / / doi .org: 10.1016 / S2468‑1253(17)30110‑3

[0304] 38 .Alhadj Ali ,M . et al. Metabolic and immune effects of immunotherapy with proinsulin peptide in human new‑onset type 1 diabetes .Sci Transl Med 9 (2017) .https: / / doi.org:10.1126 / scitranslmed.aaf7779

[0305] 3 9 .Benham ,H . et al. Citrullinated peptide dendritic cell immunotherapy in HLA risk genotype‑positive rheumatoid arthritis patients .Sci Transl Med 7,290ra287(2015) .https: / / doi.org:10.1126 / scitranslmed.aaa9301

[0306] 40.Chataway,J. et al. Effects of ATX‑MS‑1467 immunotherapy over 16 weeks in relapsing multiple sclerosis .Neurology 90,e955‑e962(2018) .https: / / doi.org: 10.1212 / WNL.0000000000005118

[0307] 41.Pearce,S.H.S. et al. Antigen‑SpecificImmunotherapy with Thyrotropin Receptor Peptides in Graves' Hyperthyroidism: A Phase I Study. Thyroid 29, 1003‑1011 (2019). https: / / doi.org:10.1089 / thy.2019.0036

[0308] 42. Campbell, B., Vogel, P. J., Fisher, E. & Lorenz, R. Myelin basic protein administration in multiple sclerosis. Arch Neurol 29, 10‑15 (1973). https: / / doi.org:10.1001 / archneur.1973.00490250028003

[0309] 43. Fontoura, P. & Garren, H. Multiple sclerosis therapies: molecular mechanisms and future. Results Probl Cell Differ 51, 259‑285 (2010). https: / / doi.org:10.1007 / 400_2010_36

[0310] 44. Ludvigsson, J. et al. GAD65 antigen therapy in recently diagnosed type 1 diabetes mellitus. N Engl J Med 366, 433‑442 (2012). https: / / doi.org:10.1056 / NEJMoa1107096

[0311] 45. Bielekova, B. et al. Encephalitogenic potential of the myelin basic protein peptide (amino acids 83‑99) in multiple sclerosis: results of a phase II clinical trial with an altered peptide ligand. Nat Med 6, 1167‑1175 (2000). https: / / doi.org:10.1038 / 80516

[0312] 46. Leclerc, J. C., Gomard, E.&Levy, J. P. Cell‑mediated reaction against tumors induced by oncorna‑viruses. I. Kinetics and specificity of the immune response in murine sarcoma virus (MSV)‑induced tu‑mors and transplanted lymphomas. Int J Cancer 10, 589‑601 (1972). https: / / doi.org:10.1002 / Description Page 67 / 91 Page 75 CN 121240870 A ijc.2910100318

[0313] 47. Katz, D. H., Hamaoka, T., Dorf, M. E., Maurer, P. H. & Benacerraf, B. Cell interactions between histoincompatible T and B lymphocytes. IV. Involvement of the immune response (Ir) gene in the con‑trol of lymphocyte interactions in responses controlled by the gene. J Exp Med 138, 734‑739 (1973). https: / / doi.org: 10.1084 / jem.138.3.734

[0314] 48. Zinkernagel, R. M. & Doherty, P. C. Restriction of in vitro T cell‑ mediated cytotoxicity in lympho‑cytic choriomeningitis within a syngeneic or semiallogeneic system. Nature 248, 701‑702 (1974). https: / / doi.org:10.1038 / 248701a0

[0315] 49. Babbitt, B.P., Allen, P.M., Matsueda, G., Haber, E. & Unanue, E.R. Binding of immunogenic peptides to Iahistocompatibility molecules. Nature 317, 359‑361 (1985). https: / / doi.org:10.1038 / 317359a0

[0316] 50. 50. Bjorkman, P. J. et al. Structure of the human class I histocompatibility antigen, HLA‑A2. Nature 329, 506‑512 (1987). https: / / doi.org: 10.1038 / 329506a0

[0317] 51. Brown, J. H. et al. Three‑dimensional structure of the human class II histocompatibility antigen HLA‑DR1. Nature 364, 33‑39 (1993). https: / / doi.org: 10.1038 / 364033a0

[0318] 52. Carbone, F. R. & Bevan, M. J. Class I‑ restricted processing and presentation of exogenous cell‑associated antigen in vivo. J Exp Med 171, 377‑387 (1990). https: / / doi.org:10.1084 / jem.171.2.377

[0319] 53. Huang, A. Y. et al. Role of bone marrow‑derived cells in presenting MHC class I‑restricted tumor antigens. Science 264, 961‑965 (1994). https: / / doi.org: 10.1126 / science.7513904

[0320] 54. Sigal, L. J., Crotty, S., Andino, R. & Rock, K. L. Cytotoxic T‑cell immunity to virus‑infected non‑haematopoietic cells requires presentation of exogenousantigen. Nature 398, 77‑80 (1999). https: / / doi.org:10.1038 / 18038

[0321] 55. Kurts, C., Kosaka, H., Carbone, F. R., Miller, J. F. & Heath, W. R. Class I‑ restricted cross‑presentation of exogenous self‑antigens leads to deletion of autoreactive CD8(+) T cells. J Exp Med 186, 239‑245 (1997). https: / / doi.org: 10.1084 / jem.186.2.239

[0322] 56. Mommen, G.P. et al. Sampling From the Proteome to the Human Leukocyte Antigen‑DR (HLA‑DR) Ligandome Proceeds Via High Specificity. Mol Cell Proteomics 15, 1412‑1423 (2016). https: / / doi.org:10.1074 / mcp.M115.055780

[0323] 57. Adamopoulou, E. et al. Exploring the MHC‑peptide matrix of central tolerance in the human thy‑mus. Nat Commun 4, 2039 (2013). https: / / doi.org: 10.1038 / ncomms3039

[0324] 58. Sofron, A., Ritz, D., Neri, D. & Fugmann, T. High‑resolution analysis of the Description Page 68 / 91 76 CN 121240870 A murine MHC class II immunopeptidome. Eur J Immunol 46, 319‑328 (2016). https: / / doi.org:10.1002 / eji.201545930

[0325] 59. Cella, M., Engering, A., Pinet, V., Pieters, J. & Lanzavecchia, A.Inflammatory stimuli induce accumulation of MHC class II complexes on dendritic cells.Nature 388,782‑787(1997) .https: / / doi.org:10.1038 / 42030

[0326] 60.Pierre, P. et al. Developmental regulation of MHC class II transport in mouse dendritic cells.Nature 388,787‑792(1997) .https: / / doi.org: 10.1038 / 42039

[0327] 61.Shin,J.S. et al. Surface expression of MHC class II in dendritic cells is controlled by regulated ubiquitination.Nature 444 ,115‑118(2006) .https: / / doi.org:10.1038 / nature05261

[0328] 62.Lautwein,A. et al. Inflammatory stimuli recruit cathepsin activity to late endosomal compartments in human dendritic cells.Eur J Immunol 32,3348‑ 3357(2002) .https: / / doi.org:10.1002 / 1521‑4141(200212)32:12<3348::AID‑IMMU3348> 3.0.CO;2‑S

[0329] 63.Blander ,J. M. & Medzhitov ,R. Toll‑dependent selection of microbial antigens for presentation by dendritic cells .Nature 440 ,808‑812(2006) .https: / / doi.org:10.1038 / nature04596

[0330] 64.Fugmann ,T .,Sofron ,A .,Ritz ,D .,Bootz ,F.&Neri ,D .The MHC Class II Immunopeptidome of Lymph Nodes in Health and in Chemically Induced Colitis.J Immunol 198,1357‑1364(2017) .https: / / doi.org:10.4049 / jimmunol.1601157

[0331] 65 .Draheim ,M .et al.Profiling MHC II immunopeptidome of blood‑stage malaria reveals that cDC1 control the functionality of parasite‑specific CD4 T cells.EMBO Mol Med 9,1605‑1621(2017) .https: / / doi.org:10.15252 / emmm.201708123

[0332] 6 6 .S h i r a i ,Y .On the transplantation of the rat sarcoma in adult heterogeneous animals.Jap Med World 1,14‑15(1921) .

[0333] 67.Murphy ,J .B.&Sturm ,E.Conditions Determining the Transplantability of Tissues in the Brain.J Exp Med 38,183‑197(1923) .https: / / doi.org:10.1084 / jem.38.2.183

[0334] 68.Medawar,P.B.Immunity to homologous grafted skin;the fate of skin homografts transplanted to the brain ,to subcutaneous tissue ,and to the anterior chamber of the eye.Br J Exp Pathol 29,58‑69(1948) .

[0335] 69.Xing ,Y.&Hogquist,K.A.T‑cell tolerance:central andperipheral. Cold Spring Harb Perspect Biol 4 (2012) .https: / / doi.org:10.1101 / cshperspect.a006957

[0336] 70 .Rieckmann ,M .et al. Myocardial infarction triggers cardioprotective antigen‑specific T helper cell responses .J Clin Invest 129,4922‑4936(2019) .https: / / doi.org:10.1172 / JCI123859

[0337] 71 .Mendell ,J .R .et al. Dystrophin immunity in Duchenne 's muscular dystrophy. N Engl J Med 363,1429‑1437(2010) .https: / / doi.org:10.1056 / NEJMoa1000228 DESCRIPTION Page 69 / 91 77 CN 121240870 A

[0338] 72 .Eghtesad ,S .,Morel ,P .A .&Clemens ,P .R .The companions:regulatory T cells and gene therapy .Immunology 127 ,1‑7(2009) .https: / / doi .org:10 .1111 / j.1365‑2567.2009.03069.x

[0339] 73 .Moalem ,G .et al. Autoimmune T cells protect neurons from secondary degeneration after central nervous system axotomy .Nat Med 5 ,49‑55(1999) .https: / / doi.org:10.1038 / 4734

[0340] 74 .Hauben ,E .et al. Autoimmune T cells as potential neuroprotective therapy for spinal cord injury .Lancet 355 ,286‑287(2000) .https: / / doi .org:10.1016 / s0140‑6736(99)05140‑5

[0341] 75 .Kipnis, J., Mizrahi, T., Yoles, E., Ben‑Nun, A. & Schwartz, M. Myelin specific Th1 cells are necessary for post‑traumatic protective autoimmunity. J Neuroimmunol 130, 78‑85(2002) .https: / / doi.org:10.1016 / s0165‑5728(02)00219‑9

[0342] 76 .Beers, D. R., Henkel, J. S., Zhao, W., Wang, J. & Appel, S. H. CD4+T cells support glial neuro‑protection, slow disease progression, and modify glial morphology in an animal model of inherited ALS. Proc Natl Acad Sci U S A 105, 15558‑15563(2008) .https: / / doi.org:10.1073 / pnas.0807419105

[0343] 77 .Louveau, A. et al. Structural and functional features of central nervous system lymphatic vessels .Nature 523, 337‑341(2015) .https: / / doi.org: 10.1038 / nature14432

[0344] 78 .Louveau, A. et al. CNS lymphatic drainage and neuroinflammation are regulated by meningeal lym‑phatic vasculature .Nat Neurosci 21 , 1380‑1391 (2018) .https: / / doi.org:10.1038 / s41593‑018‑0227‑9

[0345] 79.Rustenhoven, J. et al. Functional characterization of the dural sinusesas a neuroimmune interface .Cell 184 ,1000‑1016e1027(2021) .https: / / doi .org: 10.1016 / j.cell.2020.12.040

[0346] 80 .Zhu ,Y .,Rudensky ,A .Y .,Corper ,A .L .,Teyton ,L .&Wilson ,I .A .Crystal structure of MHC class II I‑Ab in complex with a human CLIP peptide: prediction of an I‑Ab peptide‑binding motif .J Mol Biol 326 ,1157‑1174(2003) .https: / / doi.org:10.1016 / s0022‑2836(02)01437‑7

[0347] 81 .Nanaware, P. P. et al. Distinguishing Signal From Noise in Immunopeptidome Studies of Limiting‑Abundance Biological Samples: Peptides Presented by I‑A(b) in C57BL / 6 Mouse Thymus .Front Immu‑nol 12 ,658601(2021) .https: / / doi.org:10.3389 / fimmu.2021.658601

[0348] 82.Sjostedt,E. et al. An atlas of the protein‑coding genes in the human, pig, and mouse brain.Science 367(2020) .https: / / doi.org:10.1126 / science.aay5947

[0349] 83 .Stromnes ,I .M .&Goverman ,J .M .Active induction of experimental allergic encephalomyelitis .Nat Protoc 1 ,1810‑1819(2006) .https: / / doi .org: 10.1038 / nprot.2006.285

[0350] 84 .Skundric,D.S.,Huston ,K .,Shaw ,M .,Tse ,H.Y .&Raine ,C.S.Experimental Description Page 70 / 91 78 CN 121240870 A allergic encepha‑lomyelitis . T cell trafficking to the central nervous systemin a resistant Thy‑1congenic mouse strain.Lab Invest 71,671‑679(1994) .

[0351] 85 .Fritz ,R .B .&Zhao ,M .L .Active andpassive experimental autoimmune encephalomyelitis in strain 129 / J(H‑2b)mice.J Neurosci Res 45,471‑474(1996) .https: / / doi.org:10.1002 / (SICI)1097‑4547(19960815)45:4<471::AID‑JNR17>3.0.CO; 2‑3

[0352] 86.Valesini,G. et al. Citrullination and autoimmunity.Autoimmun Rev 14, 490‑497(2015) .https: / / doi.org:10.1016 / j.autrev.2015.01.013

[0353] 87 .Moscarello ,M .A .,Mastronardi ,F .G .&Wood ,D .D .The role of citrullinated proteins suggests a novel mechanismin the pathogenesis ofmultiple sclerosis.Neurochem Res 32,251‑256(2007) .https: / / doi.org / 10.1007 / s11064‑006‑9144‑5

[0354] 88.Raijmakers,R. et al. Citrullination of central nervous system proteins during the development ofexperimental autoimmune encephalomyelitis .JComp Neurol 486, 243‑253 (2005). https: / / doi.org:10.1002 / cne.20529

[0355] 89. Faigle, W. et al. Brain Citrullination Patterns and T Cell Reactivity of Cerebrospinal Fluid‑Derived CD4(+) T Cells in Multiple Sclerosis. Front Immunol 10, 540 (2019). https: / / doi.org:10.3389 / fimmu.2019.00540

[0356] 90. Cua, D. J. et al. 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

[0357] 91. 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

[0358] 92. Ichiyama, K. et al. Foxp3 inhibits RORgammat‑mediated IL‑17A mRNA transcription through direct interaction with RORgammat. J Biol Chem 283, 17003‑17008 (2008). https: / / doi.org:10.1074 / jbc.M801286200

[0359] 93. Fontenot, J. D., Gavin, M. A. & Rudensky, A. Y. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol4,330‑336 (2003) .https: / / doi.org / 10.1038 / ni904

[0360] 94 .Hori ,S .,Nomura ,T .&Sakaguchi ,S .Control of regulatory T cell development by the transcription factor Foxp3 .Science 299 ,1057‑1061(2003) .https: / / doi.org / 10.1126 / science.1079490

[0361] 95 .Huizinga ,R .et al.Phagocytosis of neuronal debris by microglia is associated with neuronal damage in multiple sclerosis .Glia 60 ,422‑431(2012) .https: / / doi.org / 10.1002 / glia.22276

[0362] 96.Puentes,F. et al.Characterization of immune response to neurofilament light in experimental auto‑immune encephalomyelitis.J Neuroinflammation 10, 118(2013) .https: / / doi.org / 10.1186 / 1742‑2094‑10‑118 Description Page 71 / 91 79 CN 121240870 A

[0363] 97 .Bebo ,B .F .,Jr .,Vandenbark ,A .A .&Offner ,H .Male SJL mice do not relapse after induction of EAE with PLP 139‑151 .J Neurosci Res 45 ,680‑689 (1996) .https: / / doi.org / 10.1002 / (SICI)1097‑4547(19960915)45:6<680::AID‑JNR4> 3.0.CO;2‑4

[0364] 98 .Buzas ,E .I .The roles of extracellular vesicles in the immune system.Nat RevImmunol,1‑15(2022) .https: / / doi.org:10.1038 / s41577‑022‑00763‑8

[0365] 99.Zhang ,B.,Yin ,Y .,Lai ,R .C .&Lim ,S.K .Immunotherapeutic potential of extracellular vesicles .Front Immunol 5 ,518(2014) .https: / / doi .org:10 .3389 / fimmu.2014.00518

[0366] 100 .Xu ,M .,Yang ,Q .,Sun ,X .&Wang ,Y .Recent Advancements in the Loading and Modification of Therapeutic Exosomes .Front Bioeng Biotechnol 8,586130 (2020) .https: / / doi.org:10.3389 / fbioe.2020.586130

[0367] 101 .Casella ,G .et al.Oligodendrocyte‑derived extracellular vesicles as antigen‑specific therapy for autoimmune neuroinflammation in mice.Sci Transl Med 12(2020) .https: / / doi.org:10.1126 / scitranslmed.aba0599

[0368] 102 .Chen ,C .et al.Single‑particle assessment of six different drug‑ loading strategies for incorporating doxorubicin into small extracellular vesicles.Anal Bioanal Chem(2022) .https: / / doi.org:10.1007 / s00216‑022‑04248‑4

[0369] 103.Wildbaum,G.,Netzer,N.&Karin,N.Tr1 cell‑dependent active tolerance blunts the pathogenic effects ofdeterminant spreading. J Clin Invest 110, 701‑ 710 (2002) . https: / / doi.org:10.1172 / JCI15176

[0370] 104 .Clemente‑Casares ,X . et al. Expanding antigen‑specific regulatory networks to treat autoimmunity .Nature 530 , 434‑440 (2016) . https: / / doi .org: 10.1038 / nature16962

[0371] 105 .Galazka ,G ., Mycko ,M.P., Selmaj,I ., Raine ,C . S. & Selmaj,K . W . Multiple sclerosis: Serum‑derived exosomes express myelin proteins. Mult Scler 24, 449‑ 458 (2018) . https: / / doi.org:10.1177 / 1352458517696597

[0372] 106. Streicher,R. & Stoffel,W. The organization of the human myelin basic protein gene. Comparison with the mouse gene. Biol Chem Hoppe Seyler 370, 503‑ 510 (1989) . https: / / doi.org:10.1515 / bchm3.1989.370.1.503

[0373] 107 .Stoner ,G .L . Conservation throughout vertebrate evolution of the predicted beta‑strands in myelin basic protein. J Neurochem 55, 1404‑1411 (1990) . https: / / doi.org:10.1111 / j.1471‑4159.1990.tb03153.x

[0374] 108. Harting,M.T. et al. Inflammation‑Stimulated Mesenchymal Stromal Cell‑ Derived Extracellular Vesicles AttenuateInflammation. Stem Cells 36, 79-90 (2018). https: / / doi.org:10.1002 / stem.2730

[0375] 109. Ojeda-Hernandez, D.D. et al. Exosomes and Biomaterials: In Searchofa New Therapeutic Strategy for Multiple Sclerosis. Life (Basel) 12 (2022). https: / / doi.org:10.3390 / life12091417 Specification 72 / 91 pages 80 CN 121240870 A

[0376] 110. Pusic, A.D., Pusic, K.M., Clayton, B.L. & Kraig, R.P. IFNgamma- Stimulate dendritic cell exosomes as a potential therapeutic for remyelination. J Neuroimmunol 266, 12-23 (2014). https: / / doi.org:10.1016 / j.jneuroim.2013.10.014

[0377] 111. Riazifar, M. et al. Stem Cell-Derived Exosomes as Nanotherapeutics for Autoimmune and Neuro-degenerative Disorders. ACS Nano 13, 6670-6688 (2019). https: / / doi.org:10.1021 / acsnano.9b01004

[0378] 2. Example 2: Identification and purification of MHC-binding peptides that regulate neuroinflammation

[0379] Experimental methods and results for identifying and purifying peptides that regulate neuroinflammation are provided.

[0380] A. Background:

[0381] A series of groundbreaking studies conducted during the 1970s and 1980s transformed the immunological understanding of T cell antigen specificity. Indeed, several pieces of evidence confirmed T cells as entities capable of responding to foreign materials, but this response relied on the simultaneous recognition of major histocompatibility complex (MHC) molecules. Subsequent research...The functional role of MHC molecules in presenting peptide antigens to T cells has been clarified. Structural studies of MHC molecules allow observation of peptides housed in the binding grooves of MHC I and II molecules. Despite sharing similar structures, the requirements for peptide binding to MHC I or II molecules result in completely different binding motifs; this avoids amino acid preference at certain positions along the peptide binding groove (Figures 12A-12C).

[0382] MHC I molecules have tyrosine residues at the ends of the peptide binding pocket, which typically limits peptide length to 8-10 amino acid residues, although longer peptides bound to MHC I molecules have been reported. In contrast, MHC II molecules are more open at the ends, allowing peptides to protrude beyond the boundaries of the binding groove. Therefore, MHC II molecules are more adaptable and accommodate peptide lengths of approximately 15-25 amino acid residues. Furthermore, it has been assumed that peptide portions not directly involved in the peptide binding groove of MHC II molecules play a role in influencing peptide binding stability. This adds an additional factor to peptide association for MHC II molecules, which is currently considered absent for MHC I molecules.

[0383] In addition to the differences in peptide binding requirements between MHC I and II molecules, the source and processing mechanism of antigens are also thought to shape the characteristics of antigens presented by MHC I or II molecules. Indeed, it was previously thought that for MHC I molecules, antigens were primarily of endogenous origin, and for MHC II molecules, antigens were primarily of exogenous origin. However, in recent years, this clear distinction regarding peptide origin has become blurred. In fact, exogenous antigens have been described as converging on MHC I molecules via different intracellular pathways, a process known as cross-presentation. On the other hand, the abundance of endogenous peptides presented on MHC II molecules is increasingly recognized, and autophagy is thought to play a facilitating role through MHC II molecules. Under inflammation, these processes naturally change to adapt to and provide contextual information for the immune response, including enhanced and stable MHC molecule surface expression, improved antigen processing capacity, and so on. This helps to focus antigen presentation on key molecules that require an immune response. Nevertheless, even in inflammation, endogenous peptides still constitute a significant proportion of the immune peptidest group. The function of this presentation and whether it promotes the maintenance or disruption of self-tolerance remains elusive. To address this issue, we established MHC I and II peptide groups in both mice and humans and evaluated changes in the autoantigen library under different conditions.

[0384] B. Protocol:

[0385] Tissues were harvested and processed according to the established protocol to obtain cell pellets per sample. Following this step, downstream processing of the samples can be performed as follows.

[0386] i) Day 1 (Immunopurification of MHC I or MHC II)

[0387] The cell pellet was optionally washed (e.g., once with PBS buffer) before dissolution.

[0388] 1. Rotate the cells (e.g., 1,000 g, 5 min). Instructions 73 / 91 pages 81 CN 121240870 A

[0389] 2. Aspirate and add 1 mL / sample of lysis buffer (e.g., MEGA8 / 9 lysis buffer) and one or more protease inhibitors (e.g., Roche protease inhibitor mixture, PMSF, iodoacetamide, leucopeptide). Aspirate up and down to fully suspend the cell pellet.

[0390] 3. Lyse the cells (e.g., in a cold room for 1 hour while shaking).

[0391] 4. Centrifuge the dissolved product (e.g., 20,000 g, 25 min, at 4°C).

[0392] a. Fragments / nucleic acids / etc. will precipitate, leaving the target protein in the supernatant.

[0393] 5. Select the supernatant (avoiding contact with the pellet) and apply it to a purification medium (e.g., IgG agarose).

[0394] 6. Shake at 4°C for 30 min.

[0395] 7. Unscrew the sample (e.g., 500g, 5 minutes).

[0396] 8. Select the supernatant and apply it to the specific MHC antibody conjugated with agarose resin. Specific information about the MHC antibody is provided below. Shake overnight in a cold room.

[0397] a. Human MHC I (pan-HLA-A / B / C) = clone W6 / 32

[0398] b. Human MHC II (HLA-DR) = clone L243

[0399] c. Human MHC II (HLA-DQ) = clone 1a3

[0400] d. Mouse MHC I (H2-Kb) = clone Y3

[0401] e. Mouse MHC I (H2-Db) = clone B22 / 249

[0402] f. Mouse MHC II (H2-Ab) = clone Y3-P

[0403] ii) Day 2 (column purification)

[0404] 1. Apply the sample from Day 1 to the chromatographic column (e.g., BioRad column).

[0405] 2. Wash the sample (e.g., as follows:

[0406] a. 5-10 mL 150 mM NaCl 20 mM Tris pH 7.4;

[0407] b. 5-10 mL 400 mM NaCl 20 mM Tris pH 7.4;

[0408] c. 5-10 mL 150 mM NaCl 20 mM Tris pH 7.4;

[0409] d. 5-10 mL 20 mM Tris pH 8.0)

[0410] 3. Elute the peptide from MHC I- or MHC II- conjugated agarose (e.g., with 10% acetic acid).

[0411] 4. Freeze the sample (e.g., -20°C).

[0412] 5. Apply vacuum to the sample (e.g., SpeedVac) to dry the peptide (approximately 6 hours).

[0413] a. After completion, a precipitate is present (e.g., chalky white or sometimes gel-like).

[0414] iii) Day 3 (optional peptide purification):

[0415] At this point (up to Day 2), the peptides bound to MHC I or MHC II molecules have been purified, but residual detergent or smaller interfering molecules may be present, which can be removed. The steps include:

[0416] Detergent removal from the column (Pierce) (according to the manufacturer's protocol, simply put):

[0417] 1. Unscrew the initial storage buffer of the column (e.g., 1,500 g / 1 min).

[0418] 2. Wash the column 2-3 times with 20 mM Tris pH 8 to prepare the column for use.

[0419] 3. Resuspend each sample in buffer (e.g., ~20 mM Tris pH 8) and apply to the resin.

[0420] 4. Unscrew (e.g., 1,500 g / 2 min).

[0421] a. The resin will bind the detergent, allowing the peptide to flow through.

[0422] C18 spinning column (according to the manufacturer's protocol, simply put):

[0423] 1. Replenish the eluted sample from step 4 (above) with a 20% acetonitrile / 2% TFA solution. Hold the sample on ice. (Instructions 74 / 91, page 82, CN 121240870 A)

[0424] 2. Activate the C18 spinning column with a 50% acetonitrile solution.

[0425] 3. Equilibrate the C18 spinning column with a solution containing 5% acetonitrile / 0.5% TFA.

[0426] 4. Load the sample from ice onto the C18 spinning column and spin it down (e.g., 1,500 g / 1 min).

[0427] 5. Optionally, apply the flow-through to the C18 spinning column again to maximize peptide resin binding.

[0428] 6. Wash the column with a solution containing 5% acetonitrile / 0.5% TFA.

[0429] 7. Elute the sample with 95% acetonitrile solution. Vortex (e.g., 1,500 g / min).

[0430] 8. Freeze the sample on dry ice (5–10 min).

[0431] 9. Apply vacuum to the sample (e.g., SpeedVac) to dry the peptides (approximately 4 hours).

[0432] The sample can be analyzed using LC-MS / MS to identify the peptide group.

[0433] 3. Example 3: Analysis of MHC I and II Peptides in Both Humans and Mice

[0434] The peptide groups of antigens presented by MHC molecules in the brain and lymphoid tissues of humans and mice were studied using LC / MS purification and identification. 14,609 peptides (i.e., SEQ ID NO: 1–14,609) were identified in proteomic assays of self-antigens presented on major histocompatibility complexes I and II (MHC1 and MHCII) in humans, mice, and peripheral and CNS immune cells. The peptides were analyzed for the following properties:

[0435] a. Whether it is shared by both humans and mice, or only in humans, or only in mice;

[0436] b. Whether it binds to MHC I, MHC II, or both;

[0437] c. Whether it is enriched in the CNS; and

[0438] d. Whether it is derived from the protein, including the start and end amino acids of the peptide (fragment) for the protein.

[0439] The resulting sequences of peptides identified in the MHC I and MHC II peptidases are provided in the sequence listing in ST.26 format accompanying this patent (see in particular SEQ ID NO: 1-14619). The properties of the analysis of each peptide are provided in the sequence listing (i.e., in the respective notes of SEQ ID NO: 1-14619), including information about the protein from which the identified peptide is derived, the start and end positions of each peptide (fragment), their human / mouse specificity, and their specificity for MHC I, II, or both.

[0440] 4. Example 4: MHC-II Peptideome of CNS Reveals Endogenous Regulatory (Protective) Peptides

[0441] A. Abstract:

[0442] The central nervous system (CNS), despite the presence of strategically located anatomical barriers designed to protect it, is not entirely isolated from the immune system. In fact, it remains physically connected to and influenced by the peripheral immune system. How the CNS maintains such reactivity while preserving its immunologically unique state remains a prominent challenge. In searching for molecular clues originating from the CNS that allow it to communicate directly with the immune system, we discovered a library of CNS-derived endogenous regulatory (protective) peptides presented on major histocompatibility complex (MHC) II molecules at the CNS boundary. During steady state, these protective peptides were found to bind to MHC II molecules throughout the lymphatic drainage pathway from the brain to its surrounding meninges and its draining cervical lymph nodes. However, the presentation of protective peptides is reduced in the case of neuroinflammatory diseases. Then, by increasing the presence of these protective peptides, the population of repressive CD4+ T cells can be expanded, and CNS autoimmune diseases are significantly reduced. This unexpected discovery of CNS-derived autoimmune protective peptides may be the molecular key to enabling the CNS to adapt to receiving information and maintaining a continuous dialogue with the immune system while balancing pronounced autoimmune responses. This provides new clues for how we can conceptually think about and therapeutically target neuroinflammatory and neurodegenerative diseases.

[0443] B. Introduction:

[0444] The concept of immune immunity has long been the framework by which to understand the interaction between the central nervous system (CNS) and the periphery. Given that the CNS can tolerate the transplantation of autologous grafts or sarcomas1-3, it is widely believed that the brain isolates itself from the peripheral immune system4. Although the physical barriers in the brain and its lack of lymphatic drainage are thought to help the CNS maintain its immune immunity.The rediscovery of a true lymphatic network at the brain boundary, however, begins to break this assumed isolation from the periphery.5,6 Indeed, alongside the immunologically free brain parenchyma, there are immunologically rich niches for immune cells in the meninges, particularly in the dura mater, the outermost membrane covering and encapsulating the CNS.7-9

[0445] Furthermore, recent work has confirmed the availability of CNS-derived antigens within these neuroimmune hubs, making it possible for them to be presented to circulating autoreactive T cells on major histocompatibility complex (MHC) II molecules.7 In fact, the presence of circulating autoreactive T cells was reported more than 30 years ago, even in healthy individuals.10 Therefore, although autoreactive CD4+ T cells are now fully recognized for causing autoimmune diseases, their potential beneficial role in many diseases and in the normal functioning of the CNS has only recently been recognized.11-13 This raises the puzzle of how the CNS can maintain immune tolerance while enhancing its visibility to the immune system. It is anticipated that the CNS seeks immune surveillance while actively providing molecular cues to immune cells to promote their tolerance to self-reaction.

[0446] To begin examining rational molecular cues from the CNS to self-reactive CD4+ T cells, a comprehensive search was conducted on peptides that bind to MHC II molecules within the CNS, its boundaries, and its draining lymph nodes. In the MHC II peptidomome that establishes the CNS, endogenous regulatory autopeptides derived from the CNS were identified, termed "protective peptides." These autoantigens were found to be presented on MHC II molecules under steady-state conditions and to generate a population of repressive CD4+ T cells that protect the CNS from significant self-reaction. Neuroinflammatory diseases are characterized by a deficiency of protective peptides, and their restoration improves the disease. In summary, this demonstrates that the CNS intentionally presents itself on MHC II molecules via endogenous protective peptides to communicate with and reduce the response of self-reactive T cells, thereby ensuring CNS immune surveillance.

[0447] C. Results:

[0448] i) MHC II peptidome reveals presentation of endogenous peptides from the CNS

[0449] Driven by the hypothesis that the CNS can manipulate antigen presentation during homeostasis to communicate with the immune system, the study began by inquiring about the self-antigen library presented on MHC II molecules. To address this, mass spectrometry was performed to comprehensively evaluate peptides bound to MHC II molecules in the CNS and related tissues of C57BL / 6J male mice (Fig. 13A). We found that CD11b+CD11c– macrophages were the main MHC II expressing cells (antigen-presenting cells, APCs) in the brain (including the pia mater) and dura mater, while CD19+ B cells were the main APCs in the deep cervical lymph nodes (dCLN) and superficial cervical lymph nodes (sCLN) (Fig. 16A-16E). In addition, algorithms predicting MHC II binding affinity using three independent models were used 14-16Peptides with relatively weak affinity for MHC II molecules in the brain were identified as having a tendency to bind. However, their binding affinity increases further downstream along the lymphatic drainage pathway, indicating heterogeneity of the peptide library that binds to MHC II molecules in different compartments (Fig. 16F).

[0450] We then used publicly available transcriptome data from the mammalian brain 17 to distinguish those peptides derived from proteins whose expression is enriched in the CNS. In doing so, we found that 18.0%, 12.7%, 2.5%, and 0.5% of the total MHC II-binding peptides in the brain, dura mater, dCLN, and sCLN, respectively, could be annotated as CNS-enriched (Fig. 13B). Analysis of the CNS-enriched autoantigen library in different tissues revealed a large number of peptides derived from myelin basic protein (MBP) in the brain, dura mater, and dCLN, while they were significantly lacking in the sCLN (Fig. 13C). By comparing overlapping peptide sequences across tissues, we further validated the dCLN as the primary pathway for lymphatic drainage from the brain during homeostasis (Fig. 13D, Fig. 16G)18,19. Furthermore, comparison of the amino acid sequences of different MBP peptides identified in the brain, dura mater, and dCLN highlighted two distinct regions: MBP158–195 and MBP196–236 (Fig. 13E, Fig. 16H). The vast majority of MHC II-binding MBP peptides were contained within the upstream MBP158–195 sequence, representing the only region draining to the dCLN (Fig. 16H).

[0451] The experiment was repeated in female C57BL / 6J mice and male SJL / J mice. Compared to male C57BL / 6J mice, female C57BL / 6J mice exhibited a large number of MHC II-binding MBP peptides in the brain and dura mater (Fig. 17A). Furthermore, in the female C57BL / 6J mice (pages 76 / 91, CN 121240870 A), a similar overpresentation of MHCII-binding peptides containing the MBP158-195 region was observed, compared to the MBP196-236 region (Fig. 17B). Indeed, a considerable amount of the same CNS-enriched peptide sequences were found in both the brain and dura mater between female and male C57BL / 6J mice (Figs. 17C-17F). In evaluating the immunopeptidome of male SJL / J mice, we similarly observed an increase in MHC II-binding peptides with relatively weak binding affinity in the brain, which shifted towards draining lymph nodes, further reaffirming the distinct characteristics of endogenous antigens binding to MHC II molecules in the brain and dura mater compared to draining lymph nodes (Fig. 18A). Additionally, similar to C57BL / 6J mice, we observed increased presentation of CNS-enriched autoantigens in the dura mater (Figs. 18B-18D). However, unlike C57BL / 6J mice, SJL / J mice exhibited increased diversity of CNS-enriched proteins that bind to MHC II molecules.SJL / J mice also exhibited peptides derived from neurofilaments, microtubule-associated proteins, and β-synuclein, among others (Fig. 18E). Despite this increased diversity, peptides containing the same MBP158–195 and MBP196–236 regions found in C57BL / 6J mice were still identified in the brain and dura mater of SJL / J mice (Figs. 18E–18F). In summary, these data confirm the surprising conservation of MBP-derived peptides presented on MHC II molecules in the CNS (Table 11).

[0452] Table 11: Overlapping CNS-enriched peptide families identified in the MHC II peptidome

[0453] Specification 77 / 91 pages 85 CN 121240870 A

[0454]

[0455] ii) MBP peptides are non-encephalitis-derived and suppress immune responses

[0456] The abundant presentation of MBP peptides has prompted our interest in understanding their potential functions in the CNS during homeostasis. They were screened for encephalitis-derived by immunization with identified MBP peptides. Although immunization with myelin oligodendrocyte glycoprotein (MOG) 35-55 induced typical paralysis in rodents of experimental autoimmune encephalomyelitis (EAE) (a rodent model of multiple sclerosis (MS) 21,22), immunization with endogenous MBP peptides (e.g., MBP160-175) did not induce paralysis (Fig. 14A). As expected, these findings were consistent with previous observations that the C57BL / 6J strain was particularly resistant to MBP-induced EAE23. When we compared their draining lymph nodes after immunization, mice that had received the MBP160-175 peptide showed reduced cellularity compared to those immunized with the MOG35-55 peptide (Fig. 14B). Furthermore, unlike MOG35-55, MBP160-175 lacked an antigen-specific effector T cell response when IL-2 production was measured by enzyme-linked immunosorbent assay (ELISpot) (Fig. 14C). To determine whether the low immunogenicity of MBP160-175 was specific to this particular peptide, we further tested different MBP peptides identified by our immunopeptidome. Similar to what was seen with MBP160-175, immunization with MBP166-185 (a peptide contained within the MBP158-195 region) also showed a lack of IL-2 production. In contrast, immunization with MBP192-216 elicited an activated T cell response (Figures 19A-19B).

[0457] Based on the above findings, we wanted to know whether the MBP peptides in the MBP158-195 region could be used to generate more inhibitory T cells and thus suppress the immune response. To test this possibility, mice were immunized with encephalitis-derived MOG35-55 peptide and ICo-immunization with endogenous MBP peptides was performed. Co-immunization with MBP160-175 or MBP166-185, but not with MBP192-216, significantly reduced neuroinflammatory diseases (Fig. 14D, Fig. 19C). To further confirm the need for peptide specificity in the observed protection, the MBP160-175 sequence was directly modified by changing its arginine residues to citrulline. Citrullination is involved in a variety of autoimmune diseases, and studies have confirmed that MBP itself becomes excessively citrullinated and this is associated with increased disease severity in MS and EAE.24,25 We found that citrullinated MBP160-175 did not inhibit EAE when co-immunized with MOG35-55, suggesting a specific immunomodulatory function of naked MBP160-175 peptide in regulating neuroinflammation (Fig. 14D). Interestingly, the specific immunosuppression of neuroinflammatory diseases by the MBP160-175 peptide was observed in both C57BL / 6J females and completely different active EAE models using SJL / J male mice (Figs. 19D-19E).

[0458] Having observed a more significant protective effect of MBP160-175, the study subsequently focused on this specific peptide to determine the potential mechanism of its immunosuppression. To begin investigating the T cell phenotype induced in the presence of MBP160-175 in our co-immunization paradigm, T cells were sorted from draining lymph nodes in both the MOG35-55-only and MOG35-55+MBP160-175 immunization groups, and then single-cell RNA sequencing (scRNA-seq) was performed using the 10X Genomics Chromium gene expression platform. Unsupervised clustering and dimensionality reduction analysis were performed using the Uniform Manifold Approximation and Projection (UMAP) algorithm to project the cells to two dimensions. Differential gene expression analysis revealed 15 distinct clusters that differentiated CD4+ T cells from CD8+ T cells (Fig. 14E, Fig. 20A). By comparing the distribution of identified clusters between the MOG35-55 and MOG35-55+MBP160-175 groups, we observed differences between regulatory Foxp3+CD4+ T cells and activated Cxcr3+Ccl5+CD8+ T cells, both of which are described as being involved in autoimmune diseases, including MS26-28. Interestingly, co-immunization with MOG35-55+MBP160-175 significantly increased the frequency of regulatory CD4+ T cells and markedly reduced the Cxcr3+Ccl5+CD8+ T cell clusters (Fig. 14F). These observations are consistent with the gene ontology analysis, revealing pathways upregulated in the MOG35-55+MBP160-175 group, such as negative regulation of T cell activation and proliferation and enhanced repressive activity.Cytokine (IL-10 and TGF-β) production (Fig. 14G). In summary, scRNA-seq of both CD4+ and CD8+ T cells showed that changes induced by MBP160-175 occurred in the draining lymph nodes prior to the onset of EAE, shaping the immune response toward a suppressive one.

[0459] The MBP peptide on the MHC II molecule has been identified, and subsequent studies focused on understanding its regulation of CD4+ T cell activity. Notably, consistent with the scRNA-seq data, co-immunization with MBP160-175 with the MOG35-55 group maintained an increased Foxp3+CD4+ regulatory T cell population in both the draining lymph nodes and dCLN at the onset of EAE (Fig. 14H-14I, Fig. 20B-20E). In the MBP160-175 co-immunization group, a significant increase in the CTLA-4+Foxp3–CD4+ T cell population was observed, which expanded in both the dCLN and the spinal cord (Fig. 14H-14K). Indeed, this same T cell population in the MBP160-175 co-immunization group also confirmed increased CD39 expression in the spinal cord, indicating enhanced anti-inflammatory activity through extracellular ATP consumption (Fig. 14J-14K)29. However, these cells do not express PD-1 or IL-10, meaning they differ from previously reported Foxp3–unconventional suppressive T cells, such as regulatory T type 1 (Tr1)30 and PD-1hi CD4+ T cells31 (Fig. 20B). In summary, this demonstrates that MBP protective peptides enhance both conventional and unconventional immunosuppressive CD4+ T cells to adequately defend against CNS autoimmune diseases.

[0460] iii) Therapeutic Delivery of Protective MBP Peptides Modulates CNS Autoimmunity

[0461] The above-mentioned interesting observations suggest that endogenous protective MBP peptides shape immune responses toward suppression, which led us to wonder whether their presentation was altered in any way during neuroinflammation. To address this question, brain, dura mater, and spinal cord were harvested from C57BL / 6J male mice at EAE peak (day 16) to establish a pathological MHC II peptide profile (Fig. 15A). A significant increase in MHCII expression was found under neuroinflammation, allowing for full recovery of MHC II-binding peptides using fewer mice (Figs. 21A-21B). When comparing the immune peptidome of EAE-induced and naive C57BL / 6J mice, significant changes were observed in the autoantigen library presented on MHCII molecules (Fig. 15B): MHC II-binding peptides derived from apolipoprotein E204 (Apoe) and prostaglandin D2 synthase (Ptgds), among others, were consistently found to be elevated in the brain and dura mater of EAE mice compared to naive controls. Also of interest were the presentation of peptides in the MBP158–195 region in the brain and dura mater under neuroinflammatory conditions.The presentation of MBP158-195 on the MHC II molecule was drastically reduced, but not in the downstream MBP196-236 antigen region (Fig. 15B-15C). In fact, the only MBP-derived peptides identifiable in the brain or spinal cord of EAE mice were those containing the MBP196-236 region (Fig. 21C).

[0462] To understand the potential cause of this unexpected loss of MBP158-195 presentation on the MHC II molecule, the possibility that changes in antigen processing might be responsible was investigated. By comparing the C-terminus of peptides in EAE and juvenile mice, a significant change in the cleavage signature of MHC II-binding peptides was observed. Specifically, EAE-induced mice showed a significantly reduced preference for basic residues at the C-terminus compared to juvenile mice (Fig. 21D). Myeloid cells in the CNS are known to be major promoting APCs for the initiation and exacerbation of neuroinflammatory diseases, and publicly available scRNA-seq datasets were used to understand changes in their antigen processing mechanisms in the context of EAE. Under homeostasis, most microglia lack MHC II expression, but neuroinflammation induces a disease-associated microglial phenotype with significantly enhanced MHC II expression and genes involved in antigen processing and presentation (Fig. 21E and 21G)33. Furthermore, we found increased expression of peptidases (including cathepsins and caspases) at the peak of EAE disease (Fig. 21F). This change in the peptidases' environment during inflammation not only explains the altered pool of self-antigens presented on MHC II molecules but also the reduced ability to present the MBP158-195 region.

[0463] The abundance of MBP158-195 presentation on MHC II molecules during homeostasis and its significant reduction during acute neuroinflammation prompted us to wonder if we could functionally utilize these endogenous protective MBP peptides to improve CNS autoimmunity. Leveraging advancements in the therapeutic use of extracellular vesicles (EVs) and their recently demonstrated ability to modulate EAE disease,34,35 we encapsulated the MBP160-175 peptide within EVs to test their efficacy as potential antigen-specific immunotherapies (Fig. 22A). Surprisingly, the introduction of EVs containing MBP160-175 into the CSF resulted in a significant expansion of CTLA-4+Foxp3–CD4+ suppressor T cells, which we had previously observed in the dura mater (Fig. 15D–15E). Injection of empty EVs or EVs containing citrullinated MBP160-175 did not upregulate this CTLA-4+Foxp3–CD4+ suppressor T cell population (Fig. 15E), and in fact, the frequency of regulatory Foxp3+CD4+ T cells was not different under all tested conditions (Fig. 15E). Furthermore, in multiple lymph nodes…Similar changes were consistently observed in both the dCLN and sCLN, but without effect on the spleen (Figs. 22B-22E). Therefore, intracisional injection of EVs containing MBP160-175 affected both local and peripheral draining lymph nodes, enhancing a specific population of suppressive CD4+ T cells. It was considered whether this was sufficient to protect against CNS autoimmune diseases. Consistent with our observations, CNS autoimmunity was significantly suppressed in EAE-induced mice that had received EVs containing the MBP160-175 peptide prior to the peak of EAE disease, compared to controls receiving EVs with citrullinated MBP160-175 (Fig. 15F). Furthermore, extracellular vesicle-mediated delivery of MBP160-175 is essential for its protective effect, as intracisional injection of unencapsulated MBP160-175 peptide did not significantly alter disease progression (Fig. 22F).

[0464] In summary, it was demonstrated that by providing endogenous protective MBP peptides to the CSF, the presence of repressive CD4+ T cells can be enhanced, which provides sufficient protection for the CNS against autoimmune reactions. These results lead us to further propose that supplementing the deficiency of protective MBP peptides could be widely used to treat neuroinflammatory and neurodegenerative diseases.

[0465] D. Discussion

[0466] In this study, the MHCII peptidomome of the CNS during homeostasis was established, and endogenous protective peptides were identified, which were presented primarily along the brain boundary and within its draining lymph nodes. These protective peptides directed the immune response toward suppression and, in particular, promoted the unconventional repressive Foxp3–CD4+ T cell population. Surprisingly, neuroinflammation led to a loss of presentation of protective peptides, and supplementing the CSF with these protective peptides significantly improved CNS autoimmune diseases. This raises the possibility that the CNS seeks to engage with the adaptive immune system to obtain “immune exemption.”

[0467] This paper is the first to demonstrate that endogenous peptides derived from the CNS bind directly to MHC (e.g., MHC II) molecules during homeostasis. We further focus particularly on the increased prevalence of MHC II-binding MBP peptides throughout the meningeal lymphatic network, extending from the brain parenchyma to the draining cervical lymph nodes. Studies in other systems have indeed confirmed the presentation of tissue-specific antigens on MHC II molecules during homeostasis in the skin and intestines,36 and the pancreas,37 in their respective draining lymph nodes. Therefore, we consider that organ systems have an intentional need to provide their own “image” to calibrate the immune system toward self-tolerance.38 Specification 80 / 91 pages 88 CN 121240870 A

[0468] It is possible that these self-snapshots are delivered to self-reactive CD4+ T cells through their presentation on MHC II molecules. Strong support for this hypothesis comes from the current finding that the endogenous protective peptides identified herein can enhance the activity of MHC II molecules in the CNS.A specific, unconventional, suppressive population of CD4+ T cells in the CNS can induce an immune response that is suppressive. Therefore, the CNS may have evolved a parallel mechanism that manipulates the adaptive immune system to ensure CNS immune immunity, thus adding an extra layer of protection to the physical barriers that strengthen the CNS.

[0469] This exchange with the peripheral immune system carries risks. In fact, to effectively initiate an immune response against peripheral damage, tissues focus on providing an “image” of the damage on MHC II molecules to appropriately tailor the adaptive immune response. Indeed, the immune response has evolved to sometimes require the replacement of self-antigens presented during homeostasis to address a more pressing issue, namely inflammation. This introduces vulnerability into the system, as there is a potential for pathological self-reactions through molecular mimicry when the immune response targets damage.39-41 Here, we demonstrate that neuroinflammation induced by peripheral immunity alters antigen processing in the CNS. This significantly alters the MHC II peptide group of the CNS and its boundaries, leading to a loss of presentation of these endogenous protective peptides. These findings may provide new clues to the etiology of multiple sclerosis and other autoimmune diseases, in which peripheral infections alter the presentation of protective peptides, thereby exposing tissues to self-reactive attacks.

[0470] Interestingly, in the context of neuroinflammation, the direct reintroduction of unmodified protective peptides into the CSF attenuated CNS autoimmune diseases. Therefore, the findings of this study highlight the feasibility of utilizing endogenous protective peptides to rebalance and restore immune tolerance in tissues. Furthermore, this underscores an attractive pathway along which these protective peptides could be therapeutically administered as antigen-specific immunotherapy for autoimmune diseases. In summary, we demonstrate that the CNS intentionally presents itself on MHC II molecules at the CNS boundary (meninges), and that such presentation is crucial for maintaining immune tolerance.

[0471] E. Methods:

[0472] i) Mice

[0473] C57BL / 6J (WT; JAX000664) and SJL / J (WT; JAX000686) were purchased from Jackson Laboratory; mice were maintained under standard feeding conditions (12-hour light / dark cycle with sterile water and standard rodent feed provided as needed, unless otherwise specified). Mice were allowed to acclimatize in the animal facility for at least one week before starting any experiments. Adult males and females between 8 and 12 weeks of age were initially used in our studies, unless otherwise specified. Sample size was determined based on power analysis according to previously published experiments. Experimenters were unaware of the experimental groups when necessary during scoring and quantification. All experiments were approved by the Institutional Animal Care and Use Committee of Washington University in St. Louis.

[0474] ii) Single-cell isolation

[0475] Following a lethal intraperitoneal injection of Euthasol, the brain (including the pia mater), dura mater, choroid plexus, spinal cord, lymph nodes (i.e., deep cervical lymph nodes, superficial cervical lymph nodes, and inguinal lymph nodes), and spleen were isolated. Mice were then perfused with PBS containing heparin (0.025%) via cardiac perfusion; samples were collected into ice-cold RPMI and kept on ice for complete tissue collection. The spleen was removed from the surrounding tissue and placed on ice-cold RPMI (Gibco) until further use. The entire spleen was digested and homogenized using a glass pestle through a 70 μm cell filter and washed with 5 mL of RPMI. Cells were then centrifuged at 450 g for 5 min. RBCs were lysed with 1 mL of ACK lysis buffer (Quality Biological); cells were incubated for 2 min, then 2 mL of ice-cold PBS was added to the sample. The sample was then centrifuged at 450 g for 5 min, and the lysed red blood cells were aspirated. The cell pellet was then resuspended in FACS buffer (2% BSA, 1 mM EDTA, 20 mM HEPES) and kept on ice until further use. Similarly, lymph nodes were digested and homogenized through a 70 μm cell filter, washed with 2 mL RPMI, centrifuged, resuspended in FACS buffer, and kept on ice until further use. The choroid plexus was removed from the brain; similarly, the dura mater was peeled off from the cranial tectum using Dumont #5 surgical forceps (Fine Science Tools). For whole tissue harvests, the dura mater was kept in ice-cold RPMI. (Instructions for use, pages 81 / 91, 89, CN 121240870 A) The dura mater was then digested for 15 minutes at 37°C with constant stirring using 1 mL of pre-warmed digestion buffer (RPMI-1640 medium containing 2% FBS, 1 mg / mL collagenase VIII, and 0.5 mg / mL DNase I). They were then filtered through a 70 μm cell filter and provided with 1 mL of complete culture medium (RPMI containing 10% FBS) to neutralize the enzymes. The samples were then centrifuged at 450 g for 5 min, resuspended in FACS buffer, and kept on ice. Finally, the brain and spinal cord were harvested and, for the whole collection, placed in ice-cold RPMI. The brain was mechanically dissociated into ~1 mm³ cubes using a sterile scalpel and digested at 37 °C with constant stirring in 2 mL of pre-warmed digestion buffer for 20 min, homogenized with a 10 mL serum pipette, digested for another 20 min, homogenized with a 5 mL serum pipette, and digested for another 20 min. To remove myelin, PBS containing 22% bovine serum albumin (BSA) was added at a 1:1 ratio and centrifuged at 1000 g for 10 min. After centrifugation, the floating myelin layer was aspirated. The cell pellet was then neutralized with RPMI containing 10% FBS to stop enzymatic digestion. An additional 5 mL of RPMI was added, and the cells were washed with a glass pestle and 5 mL of RPMI. Then at 450gCells were centrifuged for 5 minutes, resuspended in FACS buffer, and kept on ice until use.

[0476] iii) Flow cytometry

[0477] As described above in “Single Cell Isolation,” a single-cell suspension was obtained and initially incubated at 4°C for 15 minutes with Zombie NIR (Biolegend) diluted 1:800 in ice-cold PBS for viability staining. The sample was then centrifuged and resuspended in FACS buffer for 5 minutes, the buffer containing anti-CD16 / 32 (Fc blocking agent; Biolegend) diluted 1:100 in FACS buffer. Then, for surface markers, cells were stained at 4°C for 30 minutes with an antibody appropriately diluted in FACS buffer. For surface staining only, the sample was washed in FACS buffer and run on an Aurora Spectral flow cytometer (Cytek), and then analyzed using FlowJo software (Tree Star). Data processing and statistical analysis were performed using GraphPad Prism. For samples requiring intracellular staining, surface staining was first performed as described above. Cells were then fixed and permeabilized using a Foxp3 / transcription factor staining buffer kit (eBioscience); staining was then performed at 4°C for 30 minutes with fluorescently conjugated antibodies targeting intracellular molecules. Similar to the above, samples were washed with FACS buffer after staining and run on an Aurora Spectral flow cytometer. Data were analyzed using FlowJo, followed by further processing and statistical analysis using GraphPadPrism. A complete list of antibodies used is shown in Table 12 below.

[0478] Table 12: Antibody instructions used in flow cytometry experiments 82 / 91 pages 90 CN 121240870 A

[0479]

[0480] iv) Isolation of MHC-II peptide group

[0481] Tissues were harvested from 110 male C57BL / 6J mice, female C57BL / 6J mice, male SJL / J mice, and 20 male C57BL / 6J mice induced with EAE. Cells were isolated as described in “Single cell isolation”, suspended in lysis buffer (40 mM MEGA 8, 40 mM MEGA 9, 1 mM phenylmethylsulfonyl chloride, 0.2 mM iodoacetamide, 20 μg ml-1 mixture of leucopeptide and RochecOmplete protease inhibitor in phosphate-buffered saline) and shaken at 4°C for 1 h. Cell lysates were centrifuged at 20,000 g for 25 minutes at 4°C. To remove peptides that are non-specifically bound to agarose and / or immunoglobulins, the supernatant was first treated with polyclonal mouse immunoglobulin G (BioX) bound to agarose 4B.Cell (1.5 mg antibody / sample) was incubated at 4°C for 30 minutes. The flow-through containing the peptide-MHCII complex was collected and added to a tube containing phosphate-buffered saline-washed agarose conjugated with anti-I-A antibody (Y-3P; 1.5 mg / sample), and incubated overnight at 4°C. The I-A conjugated agarose was applied to the column and washed four times as follows: 10 mL 150 mM NaCl and 20 mM Tris (pH 7.4); 10 mL 400 mM NaCl and 20 mM Tris (pH 7.4); 10 mL 150 mM NaCl and 20 mM Tris (pH 7.4); and 10 mL 20 mM Tris (pH 8.0). The peptide was then eluted with 10% acetic acid and dried using a SpeedVac. The eluted peptides were removed by a detergent removal spinning column (Pierce) to remove trace amounts of residual detergent and further purified using a C18 spinning column from Thermo Fisher Scientific (Pierce).

[0482] v) Mass Spectrometry Manual 83 / 91 pages 91 CN 121240870 A

[0483] The Dionex UltiMate 1000 system (Thermo Fisher Scientific) was coupled to an Orbitrap Fusion Lumos (Thermo Fisher Scientific) using an EASY-Spray ion source (Thermo Fisher Scientific). Prior to loading, the peptide sample was reconstructed in 2% acetonitrile (ACN) / 0.1% formic acid (17 μl). They were then loaded (15 μl, 15 μl min⁻¹; 3 min) onto a capture column (100 μm × 2 cm; 5 μm Acclaim PepMap 100C18; 50 °C), eluted (0.2 μl min⁻¹) onto an EASY-Spray PepMap RSLC C18 column (2 μm; 50 cm × 75 μm ID; 50 °C; Thermo Fisher Scientific) and separated using the following gradient (all % buffer B (i.e., 0.1% formic acid / acetonitrile)): 0–110 min: 2–22%; 110–120 min: 22–35%; 120–130 min: 35–95%; 130–150 min: at 95% isocratic; 151–153 min: 95–2%; 153–171 min: at 2% isocratic. The spray voltage was 1,700 V, the ion transfer tube temperature was 275 °C, and the RF lens was 30%. For ions with charge states 2–7, mass spectrometry scans were obtained in profile mode and tandem mass spectrometry scans were obtained in center mode, with a cycle time of 1.0.5s. Mass spectra from 375–1,500 Da were recorded at 120-K resolution (m / z 200), and high-energy collisional dissociation tandem mass spectrometry was triggered at 15-K resolution and 30% collision energy with quadrupole isolation (1.4 Da) above a threshold of 2.0 × 10⁴. Dynamic exclusion (60s) was used, and single isotope precursor ion selection was enabled.

[0484] vi) Mass Spectrometry Data Analysis

[0485] Data files were uploaded to PEAKS Studio 10.6 (Bioinformatics Solutions) for processing, updating sequencing, and database retrieval. Sequences were retrieved from the UniProt mouse database (downloaded January 4, 2022; 22,102 entries) with mass error tolerances of 10 ppm and 0.02 Da for the parent and fragment, respectively, no enzyme specificity, and oxidation (M), deamidation (NQ), and cysteine ​​oxidation to sulfoalanine as variable modifications. Any contaminating proteins were identified using the incidental protein database public library (www.thegpm.org / crap / ). FDR estimation was enabled. Peptides were screened with either a 5% FDR or a -10lgP score of 15 (whichever is higher), and protein screening was disabled by setting a protein's -10lgP score to 0, accompanied by a unique peptide and a required effective peptide. For the relative quantification of different antigenic regions found in our peptide group, peak areas were normalized to total ion current (TIC), overlapping peptide sequences were grouped into families, and their peak areas were summed where relevant. We then selected common peptide family sequences that could identify similar peak areas to allow comparisons between peptide groups of interest. "Relative abundance" was determined relative to the aforementioned common peptide family sequences.

[0486] vii) Experimental Autoimmune Encephalomyelitis (EAE)

[0487] EAE was induced in C57BL / 6J mice by subcutaneous injection of MOG35-55 peptide (100 μg, CSBio) emulsified in Freund's adjuvant (Sigma-Aldrich) supplemented with 2 mg / mL Mycobacterium tuberculosis (BD) and / or various synthetic peptides (including MBP160-175, citrullinated MBP160-175, MBP166-185, and MBP192-216 (GenScript)). Pertussis toxin (200 ng, ListBiologicals) was injected i.p. on day 0 and day 2 after immunization with MOG and / or MBP. PLP139-151 (100 μg, Vivitide) and / or various synthetic peptides (including MBP160-175, neurofilament light chain polypeptide (NEFL) 160-173 (GenScript)) emulsified in Freund's adjuvant (Sigma Aldrich) supplemented with 2 mg / mL Mycobacterium tuberculosis (BD) were administered subcutaneously.EAE was induced in SJL / J mice. Pertussis toxin (400 ng, ListBiologicals) was injected intraperitoneally on day 0 and day 2 after immunization with PLP and / or MBP and NEFM. For clinical evaluation, mice were scored daily as follows: 0 = no clinical disease, 1 = tail weakness, 2 = hind limb weakness, 3 = hind limb paralysis, 4 = partial forelimb paralysis, 5 = dying. For a complete list of peptide sequences used, see Table 13 below.

[0488] Table 13: Peptide sequences used for immunization Specification 84 / 91 pages 92 CN 121240870 A

[0489]

[0490] viii) Enzyme-linked immunosorbent assay (ELISpot)

[0491] Mice were subcutaneously immunized with different peptides (including MOG35-55, MBP160-175, MBP166-185 and MBP192-216, etc.) emulsified in Freund's adjuvant. One day before harvest, 96-well polyvinylidene fluoride (PVDF) plates (Millipore) were coated with IL-2 capture antibody (BDBiosciences). Seven days after immunization, cells were isolated from draining lymph nodes and counted so that 5 x 10⁵ cells were loaded per well (triple for each condition). Cells were stimulated with their appropriate antigens and controls and incubated at 37°C in 5% CO₂ for 24 hours. Cells secreting IL-2 were identified with IL-2 detection antibody (BDBiosciences) and the plates were developed according to the manufacturer’s instructions (BDBiosciences). Spots were quantified using a CTL ImmunoSpot S6 general-purpose instrument and Professional 6.0.0 software.

[0492] ix) Single-cell RNA sequencing

[0493] Inguinal lymph node T cells. C57BL / 6J male mice were immunized with MOG35-55 or MOG35-55 and MBP160-175 (n = 3 mice / group) and cells were harvested from draining (groin) lymph nodes 10 days later. Cells were isolated as described in “Single Cell Isolation” and sorted to capture those negative for DAPI (live cells), Thy1.2, and TCRβ to capture CD4+ and CD8+ T cells for single-cell sequencing. Sample loading and library construction were performed using the 10X Genomics Chromium platform and Chromium Single Cell 3' Library & Gel Bead Kit version 3, and the libraries were sequenced on Illumina NextSeq 500.

[0494] Reads were aligned to the mm10 genome using the Cellranger software pipeline provided by 10xgenomics. The reads obtained by counting the cell matrix through UMI were aligned using the read10xCounts function from the Droplet Utils package.The data was read into R. Cells were first screened manually by removing cells that deviated from the mean by more than 2.5 standard deviations in total RNA count or unique features in both directions, or by more than 3 standard deviations from the mean in mitochondrial transcript percentage. The expression values ​​of the final subset were then normalized using the scran and scater packages. The resulting log2 values ​​were converted to a natural logarithmic scale to fit the Seurat (v3) pipeline 42–44. The screening and normalized matrices were used as input to the Seurat pipeline, and cell scaling was performed on each gene, with regressions for sequencing depth, number of unique features, and mitochondrial readout percentage for each cell. Variance stabilization transformation was then used to select the top 2,000 most highly variable genes. Principal component analysis was performed, and elbow plots were used to select components for UMAP analysis and clustering. SharedNearestNeighbor (SNN) clustering, optimized with the Louvain algorithm, was performed as implemented using the Seurat FindClusters function, and the clusters were manually annotated based on the expression of typical gene markers. Initial contamination with B-cell or myeloid lineage markers and duplex clusters was manually removed, and the final cell subsets were scaled and subjected to PCA, clustering, and annotation as described above. Statistical analysis of cluster proportions was performed using a 1,000-permutation test, followed by bootstrapping specification page 85 / 91 93 CN 121240870 A method to generate p-values ​​and confidence intervals.

[0495] For differentially expressed genes between analysis conditions, CD4 and CD8 cells were subsetted based on sub-cluster identity and gene expression, and each subset was screened to include genes with at least 4 transcripts in at least 4 cells. The top 2000 highly variable genes were then identified and included for further analysis using the SingleCellExperimentmodelGeneVar and getTopHVGs functions. After screening, models were built and differential expression tests were performed using limma and edgeR with the lmFit, contrasts.fit, and eBayes functions. Then, a Benjamini-Hochberg adjusted p-value threshold of less than 0.05 was used to screen the results for statistical significance. For significantly differentially expressed gene sets, overpresentation enrichment analysis with Fisher's exact test was used to determine significantly enriched gene ontology (GO) terms (adjusted p < 0.05). For each gene set, genes were divided into upregulated and downregulated groups, and enriched using the enrichGO function from the clusterProfiler package, with gene set sizes set between 10 and 500 genes, and p-values ​​adjusted using Benjamini-Hochberg correction.

[0496] x) JordaoEAE Microglia Analysis

[0497] A CSV file containing single-cell transcript counts was downloaded from the Gene Expression Omnibus search number GSE118948, read into R, and converted into a matrix. Blood and choroid plexus tissue samples were removed, and the remaining pia mater and parenchymal perivascular space samples were manually screened for quality control. Screening was performed to remove cells with fewer than 200 unique genes, fewer than 1,000 or more than 25,000 transcripts, and a mitochondrial transcript percentage greater than 30%. Expression values ​​were then normalized using the scran and scater packages. The resulting log2 values ​​were converted to a natural logarithmic scale to fit the Seurat (v3) pipeline 42–44. Initial contamination in the form of low-sequencing clusters was manually removed, and the final cell subset was scaled as described above, undergoing PCA, clustering, and annotation.

[0498] The filtered and normalized matrix was used as input to the Seurat pipeline, and cells were scaled for each gene, with regressions performed for sequencing depth, number of unique features, and percentage of mitochondrial readouts per cell. The top 2,000 most highly variable genes were then selected using variance-stabilized transformation. Principal component analysis was performed, and elbow plots were used to select components for UMAP analysis and clustering. SharedNearestNeighbor (SNN) clustering optimized with the Louvain algorithm was performed, as implemented via the SeuratFindClusters function, and the clusters were then manually annotated based on the expression of typical gene markers. For differential gene expression analysis, samples were grouped into naive, pre-symptom (including preclinical 4d and preclinical 8d from the raw data analysis), and EAE (including flare-up and acute phases from the raw data analysis). Microglia were subsetted and screened to include genes with at least 4 transcripts in at least 4 cells. Then, using the SingleCellExperiment modelGeneVar and getTopHVGs functions, the top 2000 highly variable genes were identified and included for further analysis. After screening, models were built using limma and edgeR with the lmFit, contrasts.fit, and eBayes functions, and differential expression tests were performed. A Benjamini-Hochberg p-value threshold of less than 0.05 was then used to screen the results for statistical significance. For significantly differentially expressed gene sets, overpresentation enrichment analysis with Fisher's exact test was used to identify significantly enriched gene ontology (GO) terms (adjusted p < 0.05). For each gene set, genes were divided into upregulated and downregulated groups, and 45 genes were further subdivided into groups.The enrichGO function from the clusterProfiler package was used, the gene set size was set between 10 and 500 genes, and the p-value was adjusted using Benjamini-Hochberg correction.46 Upregulated or downregulated peptidases were identified using the publicly available MEROPS database.47

[0499] xi) Epitope Prediction

[0500] To predict the binding affinity of MHCII-binding peptides in C57BL / 6J mice (I-Ab haplotype) in the CNS and related tissues, we applied three independent models. These included: an algorithm we developed using a hidden Markov model (HMM) and trained on the latest immune epitope database (IEDB)14, and publicly available platforms such as netMHCIIpan-4.015 and MHCnuggets16. Since scores from different models could not be directly merged, we calculated percentile rankings for the outcome scores of each peptide and model for a large set of baits, 86 / 91 pages 94 CN 121240870 A. The mean (or median) value across all models was then used as the primary source for a given antigen. We further represented these values ​​as inverted percentile rankings, such that larger values ​​indicated a higher likelihood that the peptide was a conjugate. The bait set was generated from the mouse proteome and consisted of approximately 1 x 10⁶ peptide fragments.

[0501] xii) Cell Culture

[0502] 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.

[0503] xiii) Isolation and preparation of extracellular vesicles

[0504] As described above, DC2.4 cells were cultured in T-75 flasks in complete DMEM medium to 80-90% confluence. The cells were thoroughly washed with dPBS and then the medium was replaced with serum-free DMEM. They were cultured overnight. The next day, the supernatant was collected and rotated at varying speeds, including 1,000g for 10 minutes to remove any cellular contaminants and 20,000g for 30 minutes to remove any smaller debris. At this point, the supernatant was further filtered through a 0.22 μm filter and centrifuged at 100,000g for 2 hours to precipitate extracellular vesicles (EVs). All rotations were performed at 4°C. The precipitate was resuspended in 500 μl of PBS and examined by transmission electron microscopy (JEOL).Visualization was performed using JEM-1400 (Phillips) or nanoparticle tracking analysis (ZetaView) to confirm their separation, measure their size, and estimate the number of particles per milliliter (mean ± SD). The separated EVs were then sonicated at 20% amplitude for 6 cycles (30 s on, 2 min off) without antigen (empty EVs), MBP160-175, or citrullinated MBP160-175 peptides. Afterward, they were allowed to recover for 1 hour in an incubator set at 37°C with 5% CO2. To remove any unincorporated peptides and debris, we used an Exo-spin™ column and precipitated the EVs of interest according to the manufacturer's protocol (Cell Guidance Systems). Similar to the above, successful purification of the EVs was evaluated by TEM and nanoparticle tracking analysis. The EVs were held at -80°C prior to intracerebrospinal injection.

[0505] xiv) Western Blotting

[0506] The supernatant or precipitate of the EVs after ultracentrifugation was resuspended in RIPA buffer. The samples were then appropriately diluted with 4x NuPAGET MLDS sample buffer (NP0007, Thermo Fisher Scientific) and boiled at 70 °C for 10 min. The samples were then loaded onto 15-well pre-prepared SDS-PAGE gels (4–15% acrylamide gradient, #4561086, Bio-Rad) and electrophoresed in Bio-Rad tanks. Gel electrophoresis was run at 100 V for 1 h, followed by transfer on ice to methanol-activated PVDF membranes and continued at 30 V for 2 h. The membranes were then blocked for 1 h at room temperature with 5% (w / v) milk in 0.1% Tween-20 (TBS-T) buffered saline. The first antibody, comprising anti-mouse CD9 (1:1000, 124802, BioLegend) and anti-mouse CD63 (1:1000, 143901, BioLegend), was diluted in 5% milk / TBS-T and used to stain the membrane overnight at 4°C. The membrane was then washed three times with TBS-T. A suitable second antibody was diluted 1:10000 in 5% milk / TBS-T. This was used to stain the blot for 2 hours at 4°C. The membrane was washed three more times with TBS-T and then developed using a Pierce™ ECL Western Blotting Substrate Kit (32106, ThermoFisherScientific). Finally, the blot was imaged using a ChemiDoc™ MP gel imaging system (Bio-Rad).

[0507] xv) Intracisional injection

[0508] Mice were anesthetized with ketamine / xylazine (100 mg / kg ketamine and 10 mg / kg xylazine). Before fixing the mice's heads to a stereotactic frame, their neck hair was shaved and properly cleaned with 70% iodine. An eye solution was applied before surgery to prevent their eyes from drying out. Subsequently, a longitudinal skin incision was made at the back of their necks, and the underlying muscles were retracted with retractors to expose the cisterna magna. Using a 5 μL Hamilton syringe with a 33-gauge needle, 5 μL of PBS or extracellular vesicles (i.e., empty, containing MBP160-175, or containing citrullinated MBP160-175) were injected at a rate of approximately 2.5 μL / min, 105 vesicles / μL. To prevent backflow, the needle was not immediately withdrawn after injection but left in the cisterna magna for an additional 1 minute. The skin was sutured, and the mice were held on a heating pad until fully awake. Following surgery, mice were subcutaneously injected with ketoprofen (2.5 mg / kg).

[0509] xvi) Statistical Methods

[0510] Sample size was selected as necessary based on standard power calculations (α = 0.05, power 0.8). Generally, statistical methods were not used to recalculate or predetermine sample size. Variances were similar within comparable experimental groups. To ensure randomness, animals were selected from different cages but under the same experimental conditions. Experimenters remained unaware of group identities until data collection and analysis. Statistical tests applied to each graph were considered appropriate as necessary. Unpaired t-tests were performed when comparing two independent groups. One-way ANOVA and appropriate multiple comparison tests were performed when comparing two independent groups. Two-way ANOVA with repeated measures and appropriate multiple comparison tests were performed when comparing three or more independent groups requiring clinical symptom assessment and consistent observations. Statistical analysis (data are expressed as mean ± s.e.m.) was performed using GraphPadPrism.

[0511] xvii) Brain Data Availability

[0512] The raw mass spectrometry data and peptide matching have been stored in the MassIVE public proteomics database (http: / / massive.ucsd.edu), with the identifier MSV000092643, and can be obtained at ftp: / / massive.ucsd.edu / MSV000092643. Single-cell RNA sequencing can be obtained in Gene Expression Omnibus with the search number GSE240691.

[0513] F. References for Example 4:

[0514] 1. Shirai, Y. On the transplantation of the rat sarcoma in adult heterogenous animals. Jap MedWorld1, 14‑15 (1921).

[0515] 2. Murphy, J. B. & Sturm, E. Conditions Determining the Transplantability of Tissues in the Brain. J Exp Med 38, 183‑197 (1923).

[0516] 3. Medawar, P. B. Immunity to homologous grafted skin; the fate of skin homografts transplanted to the brain, to subcutaneous tissue, and to the anterior chamber of the eye. Br J Exp Pathol 29, 58‑69 (1948).

[0517] 4. Rustenhoven, J. & Kipnis, J. Bypassing the blood‑brain barrier. Science 366, 1448‑1449 (2019).

[0518] 5. Louveau, A. et al. Structural and functional features of central nervous system lymphatic vessels. Nature 523, 337‑341 (2015).

[0519] 6. Absinta, M. et al. Human and nonhuman primate meninges harbor lymphatic vessels that can be visualized noninvasively by MRI. Elife 6 (2017).

[0520] 7. Rustenhoven, J. et al. Functional characterization of the dural sinuses as a neuroimmune interface. Cell 184, 1000‑1016e1027 (2021).

[0521] 8. Rustenhoven, J. & Kipnis, J. Brain borders at the central stage of neuroimmunology. Nature612,417‑429(2022) .

[0522] 9.Castellani,G.,Croese,T.,Peralta Ramos,J.M.&Schwartz,M.Transforming the understanding of brain immunity.Science 380,eabo7649(2023) .

[0523] 10 .Ota ,K . et al.T‑cell recognition of an immunodominant myelin basic protein epitope in multiple sclerosis.Nature 346,183‑187(1990) . Description Page 88 / 91 96 CN 121240870 A

[0524] 11 .Kipnis ,J .,Gadani ,S .&Derecki ,N .C .Pro‑cognitive properties of T cells.Nat Rev Immunol 12,663‑669(2012) .

[0525] 12.Schwartz,M.&Raposo,C.Protective Autoimmunity:A Unifying Model for the Immune Network Involved in CNS Repair.Neuroscientist 20,343‑358(2014) .

[0526] 13 .Richards ,D .M .,Kyewski ,B .&Feuerer ,M .Re‑examining the Nature and Function of Self‑Reactive T cells.Trends Immunol 37,114‑125(2016) .14.Alspach, E . et al.MHC‑II neoantigens shape tumour immunity and response to immunotherapy.Nature 574,696‑701(2019) .

[0527] 15.Reynisson,B. et al.Improved Prediction of MHC II Antigen Presentation through Integration and Motif Deconvolution of Mass SpectrometryMHC Eluted Ligand Data. *J Proteome Res* 19, 2304‑2315 (2020) .

[0528] 16. Shao, X.M. *et al* High‑Throughput Prediction of MHC Class I and II

[0529] Neoantigens with MHCnuggets. *Cancer Immunol Res* 8, 396‑408 (2020) . 17. Sjostedt, E. *et al* An atlas of the protein‑coding genes in the human, pig, and mouse brain. *Science* 367 (2020) .

[0530] 18. Cserr, H.F., Harling‑Berg, C.J. & Knopf, P.M. Drainage of brain

[0531] extracellular fluid into blood and deep cervical lymph and its immunological significance. *Brain Pathol* 2, 269‑276 (1992) .

[0532] 19. Louveau, A. *et al* CNS lymphatic drainage and neuroinflammation are

[0533] regulated by meningeal lymphatic vasculature. *Nat Neurosci* 21, 1380‑1391 (2018) .

[0534] 20. Mendel, I., Kerlero de Rosbo, N. & Ben‑Nun, A. A myelin

[0535] oligodendrocyte glycoprotein peptide induces typical chronic

[0536] experimental autoimmune encephalomyelitis in H‑2b mice: fine

[0537] specificity and T cell receptor V beta expression of encephalitogenic T cells. *Eur J Immunol* 25, 1951‑1959 (1995) .

[0538] 21. Weiner, H.L. Thechallenge of multiple sclerosis: how do we cure a chronic heterogeneous disease?Ann Neurol 65, 239‑248(2009) .

[0539] 22 .Steinman , L .Immunology of relapse and remission in multiple sclerosis.Annu Rev Immunol 32, 257‑281(2014) .

[0540] 23 .Bernard , C . C .Experimental autoimmune encephalomyelitis in mice: genetic control of susceptibility.J Immunogenet 3, 263‑274(1976) .

[0541] 24. Valesini, G. et al. Citrullination and autoimmunity.Autoimmun Rev 14, 490‑497(2015) .

[0542] 25. Sospedra, M.&Martin, R.Immunology of multiple sclerosis.Annu Rev

[0543] Immunol 23, 683‑747(2005) .

[0544] 26. Balashov, K.E., Rottman, J.B., Weiner, H.L.&Hancock, W.W.

[0545] CCR5(+)and CXCR3(+)T cells are increased in multiple sclerosis and

[0546] their ligands MIP‑1alpha and IP‑10 are expressed in demyelinating DESCRIPTION Page 89 of 91 97 CN 121240870 A brain lesions.Proc Natl Acad Sci U S A 96, 6873‑6878(1999) .

[0547] 27. Mackay , C.R . CXCR3(+)CCR5(+)T cells and autoimmune diseases: guilty as charged?J Clin Invest 124, 3682‑3684(2014) .

[0548] 28. Dominguez‑Villar, M. & Hafler, D. A. Regulatory T cells in autoimmune disease. Nat Immunol 19, 665‑673(2018).

[0549] 29. Antonioli, L., Pacher, P., Vizi, E. S. & Hasko, G. CD39 and CD73 in immunity and inflammation. Trends Mol Med 19, 355‑367(2013).

[0550] 30. Roncarolo, M. G., Gregori, S., Bacchetta, R., Battaglia, M. & Gagliani, N.

[0551] The Biology of T Regulatory Type 1 Cells and Their Therapeutic

[0552] Application in Immune‑Mediated Diseases. Immunity 49, 1004‑1019(2018).

[0553] 31. Zappasodi, R. et al. Non‑conventional Inhibitory CD4(+)Foxp3(‑)PD‑

[0554] 1 (hi) T Cells as a Biomarker of Immune Checkpoint Blockade Activity. Cancer Cell 33, 1017‑1032e1017(2018).

[0555] 32. Jordao, M. J. C. et al. Single‑cell profiling identifies myeloid cell subsets with distinct fates during neuroinflammation. Science 363(2019).

[0556] 33. Butovsky, O. & Weiner, H. L. Microglial signatures and their role in health and disease. Nat Rev Neurosci 19, 622‑635(2018).

[0557] 34. Wiklander, O. P. B., Brennan, M. A., Lotvall, J., Breakefield, X. O. & El Andaloussi, S. Advances in therapeutic applications of extracellular vesicles. Sci Transl Med 11(2019).

[0558] 35. Casella, G. et al. Oligodendrocyte‑derived extracellular vesicles as

[0559] antigen‑specific therapy for autoimmune neuroinflammation in mice. Sci Transl Med 12(2020).

[0560] 36. Fugmann, T., Sofron, A., Ritz, D., Bootz, F. & Neri, D. The MHC Class II Immunopeptidome of Lymph Nodes in Health and in Chemically Induced Colitis. J Immunol 198, 1357‑1364(2017).

[0561] 37. Wan, X. et al. The MHC‑II peptidome of pancreatic islets identifies key features of autoimmune peptides. Nat Immunol 21, 455‑463(2020).

[0562] 38. Cohen, I. R. The cognitive paradigm and the immunological homunculus. Immunol Today 13, 490‑494(1992).

[0563] 39. 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).

[0564] 40. Lunemann, J.D. et al. EBNA1‑specific T cells from patients with multiplesclerosis cross react with myelin antigens and co‑produce IFN‑gamma and IL‑ 2.J Exp Med 205,1763‑1773(2008) .

[0565] 41.Lanz,T.V. et al. Clonally expanded B cells in multiple sclerosis bind EBV EBNA1 and GlialCAM.Nature 603,321‑327(2022) .

[0566] 42.Butler,A.,Hoffman,P.,Smibert,P.,Papalexi,E.&Satija ,R .Integrating Description 90 / 91 Page 98 CN 121240870 A single‑cell transcriptomic data across different conditions,technologies,and species.Nat Biotechnol 36,411‑420(2018) .

[0567] 43 .Lun ,A .T .,McCarthy ,D .J .&Marioni ,J .C .A step‑by‑step workflow for low‑level analysis of single‑cell RNA‑seq data with Bioconductor.F1000Res 5, 2122(2016) .

[0568] 44 .McCarthy ,D .J .,Campbell ,K .R .,Lun ,A .T .&Wills ,Q .F .Scater:pre‑ processing,quality control,normalization and visualization of single‑cell RNA‑ seq data in R.Bioinformatics 33,1179‑1186(2017) .

[0569] 45.Hong ,G.,Zhang ,W.,Li,H.,Shen,X.&Guo,Z.Separate enrichment analysis of pathways for up‑and downregulated genes.J R Soc Interface11,20130950(2014) .

[0570] 46. Yu, G., Wang, L.G., Han, Y. & He, Q.Y. clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS 16, 284-287 (2012).

[0571] 47. Rawlings, ND et al. The MEROPS database of proteolytic enzymes, their substrates and inhibitors in 2017 and a comparison with peptidases in the PANTHER database. Nucleic Acids Res 46, D624-D632 (2018).

[0572] 5. Example 5: Evaluation of Tubb 3, Map2 and Nefm as MHC protective peptides in EAE animals

[0573] As previously described, in the MOG33-55-induced EAE animal model, fragments of Map2, Nefm, and Tubb3 were evaluated as candidates for regulatory peptides (e.g., MHC protective peptides) against CNS autoimmunity. Clinical EAE scores were tracked for 19 days following immunization with MOG33-55 alone or with one of the following: one of the four Tubb3 peptides; a Map2 peptide; or one of the two Nefm peptides. Mean clinical scores and statistical (two-way ANOVA) are shown in Figures 23A-23G. The sequences of the peptides tested are shown in Table 14 below.

[0574] Table 14: Tubb3, Map2 and Nefm peptides tested in MOG33-55 induced EAE animal model

[0575] Specification 91 / 91 pages 99 CN 121240870 A Figure 1A Specification Figure 1 / 53 pages 100 CN 121240870 A Figure 1B Specification Figure 2 / 53 pages 101 CN 121240870 A Figure 2A Figure 2B Figure 2C Specification Figure 3 / 53 pages 102 CN 121240870 A Figure 2D Figure 3A Specification Figure 4 / 53 pages 103 CN 121240870 A Figure 3B Figure 4A Figure 4B Specification Figure 5 / 53 pages 104 CN 121240870 A Figure 4C Specification Figure 6 / 53 pages 105 CN 121240870 A Figure 4D Figure 4E Instruction manual, page 7 / 53 (with illustrations)106 CN 121240870 A Figure 5A Figure 5B Instruction Manual Drawings 8 / 53 pages 107 CN 121240870 A Figure 6 Figure 7A Instruction Manual Drawings 9 / 53 pages 108 CN 121240870 A Figure 7B Figure 8A Instruction Manual Drawings 10 / 53 pages 109 CN 121240870 A Figure 8B Figure 8C Instruction Manual Drawings 11 / 53 pages 110 CN 121240870 A Figure 8D Figure 9A Instruction Manual Drawings 12 / 53 pages 111 CN 121240870 A Figure 9B Figure 10A Instruction Manual Drawings 13 / 53 pages 112 CN 121240870 A Figure 10B Figure 10C Instruction Manual Drawings 14 / 53 pages 113 CN 121240870 A Figure 11A Figure 11B Instruction Manual Drawings 15 / 53 pages 114 CN 121240870 A Figure 11C Instruction Manual Drawings 16 / 53 Page 115 CN 121240870 A Figure 12A Figure 12B Instruction Manual Drawings 17 / 53 Page 116 CN 121240870 A Figure 12C Figure 13A Instruction Manual Drawings 18 / 53 Page 117 CN 121240870 A Figure 13B Instruction Manual Drawings 19 / 53 Page 118 CN 121240870 A Figure 13C Instruction Manual Drawings 20 / 53 Page 119 CN 121240870 A Figure 13D Figure 13E Instruction Manual Drawings 21 / 53 Page 120 CN 121240870 A Figure 14A Figure 14B Instruction Manual Drawings 22 / 53 Page 121 CN 121240870 A Figure 14C Figure 14D Instruction Manual Drawings 23 / 53 Page 122 121240870 A Figure 14E Figure 14F Instruction Manual Drawings 24 / 53 Page 123 CN 121240870 A Figure 14G Figure 14H Instruction Manual Drawings 25 / 53 Page 124 CN 121240870 A Figure 14I Figure 14J Instruction Manual Drawings 26 / 53 Page 125 CN 121240870 A Figure 14K Figure 15A Instruction Manual Drawings 27 / 53 Page 126 CN 121240870 A Figure 15B Figure 15C Instruction Manual Drawings 28 / 53 Page 127 CN 121240870 A Figure 15D Instruction Manual Drawings 29 / 53 Page128 CN 121240870 A Figure 15E Instruction Manual Drawings, Page 30 / 53 129 CN 121240870 A Figure 15F Instruction Manual Drawings, Page 31 / 53 130 CN 121240870 A Figure 16A Instruction Manual Drawings, Page 32 / 53 131 CN 121240870 A Figure 16B Figure 16C Figure 16D Figure 16E Instruction Manual Drawings, Page 33 / 53 132 CN 121240870 A Figure 16F Figure 16G Figure 16H Instruction Manual Drawings, Page 34 / 53 133 CN 121240870 A Figure 17A Figure 17B Figure 17C Instruction Manual Drawings, Page 35 / 53 134 CN 121240870 A Figure 17D Figure 17E Figure 17F Instruction Manual Drawings, Page 36 / 53 135 CN 121240870 A Figure 18A Figure 18B Instruction Manual Drawings 37 / 53 Page 136 CN 121240870 A Figure 18C Figure 18D Instruction Manual Drawings 38 / 53 Page 137 CN 121240870 A Figure 18E Figure 18F Figure 19A Instruction Manual Drawings 39 / 53 Page 138 CN 121240870 A Figure 19B Figure 19C Instruction Manual Drawings 40 / 53 Page 139 CN 121240870 A Figure 19D Figure 19E Instruction Manual Drawings 41 / 53 Page 140 CN 121240870 A Figure 20A Instruction Manual Drawings 42 / 53 Page 141 CN 121240870 A Figure 20B Figure 20C Instruction Manual Drawings 43 / 53 Page 142 CN 121240870 A Figure 20D Figure 20E Instruction manual figures 44 / 53, page 143, CN 121240870 A, Figure 21A, Figure 21B; Instruction manual figures 45 / 53, page 144, CN 121240870 A, Figure 21C, Figure 21D; Instruction manual figures 46 / 53, page 145, CN 121240870 A, Figure 21E, Figure 21F; Instruction manual figures 47 / 53, page 146, CN 121240870 A, Figure 21G, Figure 22A; Instruction manual figures 48 / 53, page 147, CN 121240870 A, Figure 22B, Figure 22C; Instruction manual figures 49 / 53, page 148, CN 121240870 A, Figure 22D, Figure 22E; Instruction manual figures 50 / 53, page 149.CN 121240870 A Figure 22F Figure 23A Figure 23B Instruction Manual Drawings, Page 51 / 53, 150 CN 121240870 A Figure 23C Figure 23D Figure 23E Instruction Manual Drawings, Page 52 / 53, 151 CN 121240870 A Figure 23F Figure 23G Instruction Manual Drawings, Page 53 / 53, 152 CN 121240870 A

Claims

1. A pharmaceutical composition comprising one or more peptides and a pharmaceutically acceptable excipient, wherein each of the one or more peptides binds to a major histocompatibility complex (MHC) molecule and is a fragment of a protein selected from Table CI or a modified fragment thereof.

2. The pharmaceutical composition of claim 1, wherein the protein is expressed at an elevated level in the central nervous system (CNS) relative to control tissue.

3. The pharmaceutical composition of claim 1, wherein the protein is not expressed at an elevated level in the central nervous system (CNS) or is expressed at a reduced level in the CNS relative to control tissue.

4. The pharmaceutical composition of claim 1, wherein each of the one or more peptides binds to a MHC class I molecule, a MHC class II molecule, or both.

5. The pharmaceutical composition of claim 1, wherein each of the one or more peptides independently comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 1-14619, or a modified sequence thereof.

6. The pharmaceutical composition of claim 1, wherein each of the one or more peptides independently comprises or consists 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, 295, 296, 297, 298, 299, 300, 301, and 5438, or a modified sequence thereof.

7. The pharmaceutical composition of claim 1, wherein each of the one or more peptides independently comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 597, 770, 1231, 1232, 1709, 1748, 2297, 2324, 3765, 3766, 3767, 3768, 3769, 3770, 3771, 3772, 3773, 3774, 3775, 3784, 3785, 3786, 3787, 3788, 4514, 4534, 4544, 4545, 4546, 4547, 4548, 4549, 4550, 4551, 4552, 4553, 4554, 4555, 4556, 4557, 4558, 4642, 4643, 4644, 4645, 4646, 4657, 4683, 4762, 4763, 4766, 4767, 4768, 4769, 4770, 4771, 4772, 4774, 4775, 4776, 4777, 4778, 4779, 4780, 4781, 4782, 4783, 4784, 4785, 4786, 4787, 4788, 4789, 4790, 4791, 4792, 4793, 4794, 4795, 4796, 4797, 4798, 4799, 4800, 4801, 4802, 4803, 4804, 4805, 4806, 4807, 4809, 4810, 4811, 4812, 4813, 4819, 4820, 4822, 4823, 4825, 4826, 4828, 4829, 5067, 5439, 5574, 5575, 14519, 14520, 14521, 14522, 14523, 14524, 14525, and 14526, or a modified sequence thereof.

8. The pharmaceutical composition of claim 1, wherein 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, or a modified sequence thereof.

9. The pharmaceutical composition of claim 1, wherein 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 8, or a modified sequence thereof.

10. The pharmaceutical composition of claim 1, wherein 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 9, or a modified sequence thereof.

11. The pharmaceutical composition of claim 1, wherein 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 10, or a modified sequence thereof.

12. The pharmaceutical composition of claim 1, wherein each of the one or more peptides is a fragment of myelin basic protein (MBP), tubulin beta 3 class III (TUBB3), or neurofilament medium polypeptide (NEFM).

13. The pharmaceutical composition of claim 12, wherein each fragment of MBP independently comprises SEQ ID NO: 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, 10756, 10757, 10758, 10759, 10760, 10761, 10762, 10763, 10764, 10765, 10766, 10767, 10768, 10769, 10770, 10771, 10772, 10773, 10774, 10775, 10776, 10777, 10778, 10779, 10780, 10781, 10782, 10783, 10784, 10785, 10786, 10787, 10788, 10789, 10790, 10791, 10792, 10793, 10794, 10795, 10796, 10797, 10798, 10799, 10800, 10801, 10802, 10803, 10804, 10805, 10806, 10807, 10808, 10809, 10810, 10811, 10812, 10813, 10814, 10815, 10816, 10817, 10818, 10819, 10820, 10821, 10822, 10823, 10824, 10825, 10826, 10827, 10828, 10829, 10830, 10831, 10832, 10833, 10834, 10835, 10836, 10837, 10838, 10839, 10840, 10841, 10842, 10843, 10844, 10845, 12162, 14537, 14538, 14539, 14540, 14541, 14542, 14543, 14544, 14545, 14546, 14547, 14548, 14549, 14550, 14551, 14552, 14553, 14554, 14555, 14556, 14557, 14558, 14559, 14560, 14561, 14562, 14563, 14564, 14565, 14566, 14567, 14568, 14569, 14570, 14571, 14572, 14573.14574, 14575, 14576, 14577, 14578, 14579, 14580, 14581, 14582, 14583, 14584, 14585, 14586, 14587, 14588, 14589, 14590, 14591, 14592, 14593, 14594, 14595, 14596, 14610, 14611, 14612, and 14613, or consists of the amino acid sequence set forth in any of 14. The pharmaceutical composition of claim 1, wherein at least one of the one or more peptides comprises or consists of an 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.

15. The pharmaceutical composition of claim 1, further comprising one or more extracellular vesicles (EVs) or liposomes encapsulating the one or more peptides.

16. The method of claim 15, wherein the pharmaceutical composition comprises one or more EVs, and wherein the one or more EVs are exosomes, microvesicles, or apoptotic bodies of cells, optionally human cells.

17. The method of claim 15, wherein the pharmaceutical composition comprises one or more EVs, and wherein the one or more EVs are oligodendrocyte-derived, optionally from cell line DC2.

4.

18. The method of claim 15, wherein the pharmaceutical composition comprises one or more EVs, and wherein the one or more EVs are synthetic.

19. The pharmaceutical composition of any one of claims 1-18, for use in treating, preventing, or reversing a neuroinflammation-related disorder, an autoimmune neurological disorder, or an acute CNS injury.

20. The pharmaceutical composition of claim 19, wherein the neuroinflammation-related disorder or autoimmune neurological disorder is selected from acute disseminated encephalomyelitis (ADEM), acute inflammatory demyelinating polyradiculoneuropathy (AIDP; Guillain-Barre syndrome (GBS)), acute motor axonal neuropathy (AMAN), acute motor-sensory axonal neuropathy (AMSAN), acute optic neuritis (AON), Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), autoimmune encephalitis (AIE), chronic ataxic neuropathy, ophthalmoplegia, immunoglobulin M paraprotein, cryoglobulin, and bisialyl antibodies (CANOMAD), chronic meningoencephalitis, Behcet’s disease, central nervous system (CNS) vasculitis, chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids (CLIPPERS), glial fibrillary acidic protein (GFAP), Hashimoto’s encephalitis, pachymeningitis, IgG4-related nervous system disease, Lambert-Eaton myasthenic syndrome (LEMS), anti-myelin oligodendrocyte glycoprotein antibody disease (MOG), Miller Fisher syndrome (MFS), monoclonal gammopathy of undetermined significance (MGUS), multifocal motor neuropathy (MMN), myasthenia gravis (MG), multiple sclerosis (MS), neuromyelitis optica (NMO), neurosarcoidosis, paraneoplastic neurological syndrome (PNS), Parkinson’s disease (PD), steroid-responsive encephalopathy associated with autoimmune thyroiditis (SREAT), stiff person syndrome, Susac’s syndrome, and transverse myelitis.

21. The pharmaceutical composition of claim 19, wherein the acute CNS injury is selected from spinal cord injury, traumatic brain injury, spinal cord injury, optic nerve injury, and stroke.

22. A method of treating, preventing, or reversing a neuroinflammation-related disorder, autoimmune neurological disorder, or acute CNS injury in a subject in need thereof, comprising administering to the subject one or more peptides each independently comprising or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 1-14619, or a modified sequence thereof.

23. The method of claim 22, wherein the neuroinflammation-related disorder or autoimmune neurological disorder is selected from acute disseminated encephalomyelitis (ADEM), acute inflammatory demyelinating polyradiculoneuropathy (AIDP; Guillain-Barre syndrome (GBS)), acute motor axonal neuropathy (AMAN), acute motor-sensory axonal neuropathy (AMSAN), acute optic neuritis (AON), Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), autoimmune encephalitis (AIE), chronic ataxic neuropathy, ophthalmoplegia, immunoglobulin M paraprotein, cryoglobulin, and bisialyl antibodies (CANOMAD), chronic meningoencephalitis, Behcet’s disease, central nervous system (CNS) vasculitis, chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids (CLIPPERS), glial fibrillary acidic protein (GFAP), Hashimoto’s encephalitis, pachymeningitis, IgG4-related nervous system disease, Lambert-Eaton myasthenic syndrome (LEMS), anti-myelin oligodendrocyte glycoprotein antibody disease (MOG), Miller Fisher syndrome (MFS), monoclonal gammopathy of undetermined significance (MGUS), multifocal motor neuropathy (MMN), myasthenia gravis (MG), multiple sclerosis (MS), neuromyelitis optica (NMO), neurosarcoidosis, paraneoplastic neurological syndrome (PNS), Parkinson’s disease (PD), steroid-responsive encephalopathy associated with autoimmune thyroiditis (SREAT), stiff person syndrome, Susac’s syndrome, and transverse myelitis.

24. The method of claim 22, wherein the acute CNS injury is selected from spinal cord injury, traumatic brain injury, spinal cord injury, optic nerve injury, and stroke.

25. The method of claim 22, wherein each of the one or more peptides independently comprises or consists 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, 295, 296, 297, 298, 299, 300, 301, and 5438, or a modified sequence thereof.

26. The method of claim 22, wherein each of the one or more peptides independently comprises or consists of the amino acid sequence set forth in any of SEQ ID NOs: 597, 770, 1231, 1232, 1709, 1748, 2297, 2324, 3765, 3766, 3767, 3768, 3769, 3770, 3771, 3772, 3773, 3774, 3775, 3784, 3785, 3786, 3787, 3788, 4514, 4534, 4544, 4545, 4546, 4547, 4548, 4549, 4550, 4551, 4552, 4553, 4554, 4555, 4556, 4557, 4558, 4642, 4643, 4644, 4645, 4646, 4657, 4683, 4762, 4763, 4766, 4767, 4768, 4769, 4770, 4771, 4772, 4774, 4775, 4776, 4777, 4778, 4779, 4780, 4781, 4782, 4783, 4784, 4785, 4786, 4787, 4788, 4789, 4790, 4791, 4792, 4793, 4794, 4795, 4796, 4797, 4798, 4799, 4800, 4801, 4802, 4803, 4804, 4805, 4806, 4807, 4809, 4810, 4811, 4812, 4813, 4819, 4820, 4822, 4823, 4825, 4826, 4828, 4829, 5067, 5439, 5574, 5575, 14519, 14520, 14521, 14522, 14523, 14524, 14525, and 14526, or a modified sequence thereof.

27. The method of claim 22, wherein each of the one or more peptides independently comprises or consists of the amino acid sequence set forth in any of the SEQ ID NOs of Table 7, or a modified sequence thereof.

28. The method of claim 22, wherein each of the one or more peptides independently comprises or consists of the amino acid sequence set forth in any of the SEQ ID NOs of Table 8, or a modified sequence thereof.

29. The method of claim 22, wherein each of the one or more peptides independently comprises or consists of the amino acid sequence set forth in any of the SEQ ID NOs of Table 9, or a modified sequence thereof.

30. The method of claim 22, wherein each of the one or more peptides independently comprises or consists of the amino acid sequence set forth in any of the SEQ ID NOs of Table 10, or a modified sequence thereof.

31. The method of claim 22, wherein each of the one or more peptides is a fragment of myelin basic protein (MBP), tubulin beta 3 class III (TUBB3), or neurofilament medium polypeptide (NEFM).

32. The method of claim 22, wherein each fragment of MBP independently comprises SEQ ID NO: 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, 10756, 10757, 10758, 10759, 10760, 10761, 10762, 10763, 10764, 10765, 10766, 10767, 10768, 10769, 10770, 10771, 10772, 10773, 10774, 10775, 10776, 10777, 10778, 10779, 10780, 10781, 10782, 10783, 10784, 10785, 10786, 10787, 10788, 10789, 10790, 10791, 10792, 10793, 10794, 10795, 10796, 10797, 10798, 10799, 10800, 10801, 10802, 10803, 10804, 10805, 10806, 10807, 10808, 10809, 10810, 10811, 10812, 10813, 10814, 10815, 10816, 10817, 10818, 10819, 10820, 10821, 10822, 10823, 10824, 10825, 10826, 10827, 10828, 10829, 10830, 10831, 10832, 10833, 10834, 10835, 10836, 10837, 10838, 10839, 10840, 10841, 10842, 10843, 10844, 10845, 12162, 14537, 14538, 14539, 14540, 14541, 14542, 14543, 14544, 14545, 14546, 14547, 14548, 14549, 14550, 14551, 14552, 14553, 14554, 14555, 14556, 14557, 14558, 14559, 14560, 14561, 14562, 14563, 14564, 14565, 14566, 14567, 14568, 14569, 14570, 14571, 14572, 14573.14574, 14575, 14576, 14577, 14578, 14579, 14580, 14581, 14582, 14583, 14584, 14585, 14586, 14587, 14588, 14589, 14590, 14591, 14592, 14593, 14594, 14595, 14596, 14610, 14611, 14612, and 14613, or a modified sequence thereof.

33. The method of claim 22, wherein at least one of the one or more peptides comprises or consists of an 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.

34. The method of claim 22, wherein the one or more peptides are encapsulated in one or more extracellular vesicles (EVs) or liposomes.

35. The method of claim 34, wherein the pharmaceutical composition comprises one or more EVs, and wherein the one or more EVs are exosomes, microvesicles, or apoptotic bodies of cells, optionally human cells.

36. The method of claim 34, wherein the pharmaceutical composition comprises one or more EVs, and wherein the one or more EVs are oligodendrocyte-derived.

37. The method of claim 34, wherein the pharmaceutical composition comprises one or more EVs, and wherein the one or more EVs are synthetic.

38. The method of claim 21, wherein the one or more peptides are administered to the subject by infusion or injection into the cerebrospinal fluid (CSF) of the subject.

39. One or more nucleic acids encoding one or more peptides comprising or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 1-14619, or a modified sequence thereof.

40. The one or more nucleic acids of claim 39, wherein each of the one or more peptides independently comprises or consists of an 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, 295, 296, 297, 298, 299, 300, 301, and 5438, or a modified sequence thereof.

41. The one or more nucleic acids of claim 39, wherein each of the one or more peptides independently comprises or consists of the amino acid sequence set forth in any of SEQ ID NOs: 597, 770, 1231, 1232, 1709, 1748, 2297, 2324, 3765, 3766, 3767, 3768, 3769, 3770, 3771, 3772, 3773, 3774, 3775, 3784, 3785, 3786, 3787, 3788, 4514, 4534, 4544, 4545, 4546, 4547, 4548, 4549, 4550, 4551, 4552, 4553, 4554, 4555, 4556, 4557, 4558, 4642, 4643, 4644, 4645, 4646, 4657, 4683, 4762, 4763, 4766, 4767, 4768, 4769, 4770, 4771, 4772, 4774, 4775, 4776, 4777, 4778, 4779, 4780, 4781, 4782, 4783, 4784, 4785, 4786, 4787, 4788, 4789, 4790, 4791, 4792, 4793, 4794, 4795, 4796, 4797, 4798, 4799, 4800, 4801, 4802, 4803, 4804, 4805, 4806, 4807, 4809, 4810, 4811, 4812, 4813, 4819, 4820, 4822, 4823, 4825, 4826, 4828, 4829, 5067, 5439, 5574, 5575, 14519, 14520, 14521, 14522, 14523, 14524, 14525, and 14526, or a modified sequence thereof.

42. The one or more nucleic acids of claim 39, wherein each of the one or more peptides independently comprises or consists of the amino acid sequence set forth in any of the SEQ ID NOs of Table 7, or a modified sequence thereof.

43. The one or more nucleic acids of claim 39, wherein each of the one or more peptides independently comprises or consists of the amino acid sequence set forth in any of the SEQ ID NOs of Table 8, or a modified sequence thereof.

44. The one or more nucleic acids of claim 39, wherein each of the one or more peptides independently comprises or consists of the amino acid sequence set forth in any of the SEQ ID NOs of Table 9, or a modified sequence thereof.

45. The one or more nucleic acids of claim 39, wherein each of the one or more peptides independently comprises, or consists of, the amino acid sequence set forth in any one of SEQ ID NOs of Table 10, or a modified sequence thereof, or a modified sequence thereof.

46. The one or more nucleic acids of claim 39, wherein each of the one or more peptides is a fragment of myelin basic protein (MBP), tubulin beta 3 class III (TUBB3), or neurofilament medium polypeptide (NEFM).

47. The one or more nucleic acids of claim 46, wherein each fragment of MBP independently comprises SEQ ID NO: 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, 10756, 10757, 10758, 10759, 10760, 10761, 10762, 10763, 10764, 10765, 10766, 10767, 10768, 10769, 10770, 10771, 10772, 10773, 10774, 10775, 10776, 10777, 10778, 10779, 10780, 10781, 10782, 10783, 10784, 10785, 10786, 10787, 10788, 10789, 10790, 10791, 10792, 10793, 10794, 10795, 10796, 10797, 10798, 10799, 10800, 10801, 10802, 10803, 10804, 10805, 10806, 10807, 10808, 10809, 10810, 10811, 10812, 10813, 10814, 10815, 10816, 10817, 10818, 10819, 10820, 10821, 10822, 10823, 10824, 10825, 10826, 10827, 10828, 10829, 10830, 10831, 10832, 10833, 10834, 10835, 10836, 10837, 10838, 10839, 10840, 10841, 10842, 10843, 10844, 10845, 12162, 14537, 14538, 14539, 14540, 14541, 14542, 14543, 14544, 14545, 14546, 14547, 14548, 14549, 14550, 14551, 14552, 14553, 14554, 14555, 14556, 14557, 14558, 14559, 14560, 14561, 14562, 14563, 14564, 14565, 14566, 14567, 14568, 14569, 14570, 14571, 14572, 14573.14574, 14575, 14576, 14577, 14578, 14579, 14580, 14581, 14582, 14583, 14584, 14585, 14586, 14587, 14588, 14589, 14590, 14591, 14592, 14593, 14594, 14595, 14596, 14610, 14611, 14612, and 14613, or a modified sequence thereof.

48. The one or more nucleic acids of claim 39, wherein at least one of the one or more peptides 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.

49. One or more vectors each comprising at least one of the one or more nucleic acids of any one of claims 39-48.

50. The one or more vectors of claim 49, wherein the one or more vectors are viral vectors.

51. The viral vector of claim 50, derived from an adenovirus, an adeno-associated virus, or a retrovirus, optionally selected from a lentivirus.

52. The one or more vectors of claim 49, wherein the vector is a non-viral vector.

53. The one or more nucleic acids of any one of claims 39-48, wherein the nucleic acid is an mRNA.

54. The one or more nucleic acids of claim 53, wherein the mRNA is a non-replicating mRNA, a self-amplifying mRNA (saRNA), or a circular RNA (circRNA).

55. The one or more nucleic acids of claim 54, wherein the mRNA is a non-replicating mRNA, wherein each mRNA comprises 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 wherein each coding region is flanked by a UTR.

56. The one or more nucleic acids of claim 55, wherein each 5’ cap comprises a 7- methylguanosine (m 7 G) cap linked by triphosphate (ppp) to a first nucleotide (N) of each mRNA to form a m 7 GpppNp.

57. The one or more nucleic acids of claim 56, wherein each m 7 The G cap comprises a methylated 2’-OH on the first nucleotide that links the 5’ end of each mRNA to a cap (cap1or m 7 GpppN1mp).

58. The one or more nucleic acids of claim 56, wherein each m 7 The G cap comprises a methylated 2’-OH on the first and second nucleotides that links the 5’ end of each mRNA to a cap (cap2 or m 7 GpppN1mpN2mp).

59. The one or more nucleic acids of any one of claims 53-58, wherein each mRNA comprises one or more modified nucleosides, which are optionally selected from pseudouridine (ψ), N 1 -methylpseudouridine (m 1 ψ), 5-methoxyuridine (mo 5 U), 2-thiouridine (s 2 U), 5-methylcytidine (m 5 C), and N 6 -methyladenosine (m 6 A).

60. The one or more nucleic acids of any one of claims 53-59, wherein the nucleic acid is encapsulated, optionally encapsulated by a lipid nanoparticle (LNP), a polyplex, a polymeric nanoparticle, a lipopolyplex (LPP), or a cationic polypeptide.

61. The one or more nucleic acids of claim 60, encapsulated by an LNP.

62. A pharmaceutical composition comprising the nucleic acid of any one of claims 39-48, the vector of any one of claims 49-52, or the mRNA of any one of claims 53-61.

63. The pharmaceutical composition of claim 62, formulated for infusion or injection into the cerebrospinal fluid (CSF).

64. The pharmaceutical composition of any one of claims 62-63 for use in treating, preventing, or reversing a neuroinflammation-related disorder, an autoimmune neurological disorder, or an acute CNS injury in a subject in need thereof.

65. The pharmaceutical composition for use of claim 64, wherein the neuroinflammation-related disorder or autoimmune neurological disorder is selected from acute disseminated encephalomyelitis (ADEM), acute inflammatory demyelinating polyradiculoneuropathy (AIDP; Guillain-Barre syndrome (GBS)), acute motor axonal neuropathy (AMAN), acute motor-sensory axonal neuropathy (AMSAN), acute optic neuritis (AON), Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), autoimmune encephalitis (AIE), chronic ataxic neuropathy, ophthalmoplegia, immunoglobulin M paraprotein, cryoglobulin, and bisialylated antibodies (CANOMAD), chronic meningoencephalitis, Behcet’s disease, central nervous system (CNS) vasculitis, chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids (CLIPPERS), glial fibrillary acidic protein (GFAP), Hashimoto’s encephalitis, pachymeningitis, IgG4-related nervous system disease, Lambert-Eaton muscle weakness syndrome (LEMS), anti-myelin oligodendrocyte glycoprotein antibody disease (MOG), Miller Fisher syndrome (MFS), monoclonal gammopathy of undetermined significance (MGUS), multifocal motor neuropathy (MMN), myasthenia gravis (MG), multiple sclerosis (MS), neuromyelitis optica (NMO), neurosarcoidosis, paraneoplastic neurological syndrome (PNS), Parkinson’s disease (PD), steroid-responsive encephalopathy associated with autoimmune thyroiditis (SREAT), stiff person syndrome, Susac’s syndrome, and transverse myelitis.

66. The pharmaceutical composition for use of claim 64, wherein the acute CNS injury is selected from spinal cord injury, traumatic brain injury, spinal cord injury, optic nerve injury, or stroke.

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