Chimeric autoantibody receptors (CAARS) targeting EBNA-1 molecular mimics - glialcam, and linked biomarkers

Abstract

The present disclosure relates to chimeric autoantibody receptors (CAARs) specific for an autoantibody that targets Epstein-Barr Virus nuclear antigen 1 (EBNA1) molecular mimics - GlialCAM, aB Crystallin (CRYAB), and Anoctamin-2 (ANO-2), vectors comprising the CAARs, and genetically modified cells comprising the CAARs. Pharmaceutical compositions and kits comprising the same are provided, as well as methods of diagnosing and methods of treating, preventing, and / or managing multiple sclerosis are provided.

Description

LST-IOOIWO PATENT APPLICATION CHIMERIC AUTOANTIBODY RECEPTORS (CAARs) TARGETING EBNA-1 MOLECULAR MIMICS - GLIALCAM, AB CRYSTALLIN (CRYAB), AND ANOCTAMIN-2 (ANO-2), AND LINKED BIOMARKERSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U. S. provisional application no. 63 / 744,458, filed on January 13, 2025. the disclosure of which is hereby incorporated by reference in its entirety herein.INCORPORATION OF MATERIAL OF XML SEQUENCE LISTING BY REFERENCE

[0002] The sequence listing submitted herewith as an XML file named ■‘LSTlOOlUSPSequenceListing’’ created on January 9, 2026, which is 47,000 bytes in size, is hereby incorporated by reference in its entirety.FIELD OF THE DISCLOSURE

[0003] This disclosure relates to chimeric autoantibody receptors (CAARs) specific for an autoantibody that targets Epstein-Barr Virus nuclear antigen 1 (EBNA1) molecular mimics -GlialCAM, aB Crystallin (CRY AB), and Anoctamin-2 (ANO-2), as well as compositions comprising the CAARs, vectors comprising the CAARs, and recombinant T cells comprising the CAARs. The present disclosure also relates to uses of these CAARs in diagnostic and therapeutic methods. The CAARs block or inhibit the binding of autoantibodies induced by an Epstein-Barr Virus (EBV) infection against CNS self-antigens GlialCAM, aB Cry stallin (CRY AB), and Anoctamin-2 (ANO-2) that cause development of MS. The cytotoxic CAARs kill the plasmablasts and plasma cells that are producing the autoantibodies targeting GlialCAM, aB crystallin (CRY AB), and anoctamin-2.BACKGROUND OF THE DISCLOSURE

[0004] Multiple sclerosis (MS) is the most common cause of nontraumatic disability in young adults. It is an autoimmune demyelinating disorder, where aberrantly activated B and T cells from the adaptive arm of the immune system, and macrophages from the innate arm of the immune system, attack the myelin sheaths in the central nervous system (CNS). Demyelination is followed by neuronal loss and widespread activation of the brain’s glial cells. While tremendous progress has been made in controlling disease activity and delaying progression of disability,, much of the pathophysiology of MS remains to be elucidated ( S. L. Hauser, B. A. C. Cree, Treatment of multiple sclerosis: A review. Am. J. Med. 133, 1380-1390.e2 (2020)). The ubiquitous herpesvirus Epstein-Barr Virus (EBV) is strongly associated with MS and isLST-IOOIWO PATENT APPLICATION considered a prerequisite for developing the disease (Bjomevik K. et al., Longitudinal analysis reveals high prevalence of Epstein-Barr virus associated with multiple sclerosis. Science 375, 296-301 (2022)). However, a conundrum arises because only a small percentage of individuals infected with EBV develop MS ( S. L. Atkins, et al., Small molecule screening identifies inhibitors of the Epstein-Barr vims deubiquitinating enzyme, BPLF1. Antiviral Res. 173, 104649 (2020)). Thus, additional genetic and environmental factors are involved before clinical disease appears. In addition to EBV infection, symptomatic infectious mononucleosis (IM) and highly elevated titers of serum antibodies against EBV nuclear antigen 1 (EBNA1) are additional independent risk factors for MS ( A. K. Hedstrom, et al., High Levels of Epstein-Barr Virus Nuclear Antigen- 1 -Specific Antibodies and Infectious Mononucleosis Act Both Independently and Synergistically to Increase Multiple Sclerosis Risk. Frontiers in Neurology [Preprint] (2020). Available at: http: / / dx.doi.org / 10.3389 / fneur.2019.01368; M. Cortese, et al., Serologic response to the Epstein-Barr vims peptidome and the risk for multiple sclerosis. JAMA Neurol.81, 515-524 (2024)). Other risk factors include low vitamin D levels, smoking, and carrying MS risk genes, including the human leukocyte antigen (HLA) class II allele HLA-DRB1 *15:01. the most significant risk gene for MS. Most of these environmental / lifestyle factors interact with MS HLA risk genes, leading to substantially increased Odds ratios (ORs). Since HLA class II genes regulate CD4+ T cells, the interactions with such genes argues for mechanisms of the environmental factors acting through adaptive immunity, just as MS risk genes ( A. K.Hedstrom, L. Alfredsson, T. Olsson, Environmental factors and their interactions with risk genotypes in MS susceptibility. Curr. Opin. Neurol. 29, 293-298 (2016); L. Moutsianas, et al., Class II HLA interactions modulate genetic risk for multiple sclerosis. Nat. Genet. 47, 1107— 1113 (2015); T. Olsson, L. F. Barcellos, L. Alfredsson, Interactions between genetic, lifestyle and environmental risk factors for multiple sclerosis. Nat. Rev. Neurol. 13, 25-36 (2017)).

[0005] On a molecular level, the link between EBV and MS is incompletely understood.Studies found evidence for several mechanisms, including altered anti-EBV T cell responses in MS patients ( Pender M.P. et al., Defective T-cell control of Epstein-Barr vims infection in multiple sclerosis. Clin. Transl. Immunology 6, e126 (2017); Thomas O. G. et al., Epstein-Barr virus and multiple sclerosis: moving from questions of association to questions of mechanism. Clin. Transl. Immunology 12, e1451 (2023); Gottlieb A. et al., Expanded T lymphocytes in the cerebrospinal fluid of multiple sclerosis patients are specific for Epstein-Barr-vims-infected B cells. Proc. Natl. Acad. Sci. U. S. A. 121, e2315857121 (2024)-12); and Schneider-Hohendorf T. et al., Broader Epstein-Barr virus-specific T cell receptor repertoire in patients with multiple sclerosis. J. Exp. Med. 219 (2022)), unstable EBV latency in infected B cells ( Soldan S.S. et al., Multiple sclerosis patient-derived spontaneous B cells have distinct EBV and host geneLST-IOOIWO PATENT APPLICATION expression profiles in active disease. Nat. Microbiol. 9, 1540-1554 (2024)), and dysregulation of genes associated with autoimmunity, largely mediated by the EBV transcription factor EBNA2 ( Harley J. B., et al., Transcription factors operate across disease loci, with EBNA2 implicated in autoimmunity. Nat. Genet. 50, 699-707 (2018)).

[0006] One mechanistic explanation is molecular mimicry where EBV-induced immune responses cross-target CNS proteins that contain epitopes with similar amino acid proteins.

[0007] The present inventors and others described molecular mimicry between the EBV transcription factor EBNA1 and CNS antigens, including anoctamin 2 (ANO2), glial cellular adhesion molecule (GlialCAM), Alpha-B crystallin (CRY AB), and myelin basic protein (MBP) (Lanz T. V. et al.. Clonally expanded B cells in multiple sclerosis bind EBV EBNA1 and GlialCAM. Nature 603, 321-327 (2022); Jog N. R. et al., Epstein Barr virus nuclear antigen 1 (EBNA-1) peptides recognized by adult multiple sclerosis patient sera induced neurologic symptoms in a murine model. J. Autoimmun. 106, 102332 (2020); Thomas O. G. et al., Cross-reactive EBNA1 immunity targets alpha-crystallin B and is associated with multiple sclerosis. Sci. Adv. 9, eadg3032 (2023); and Tengvall K. et al.. Molecular mimicry between Anoctamin 2 and Epstein-Barr virus nuclear antigen 1 associates with multiple sclerosis risk. Proc. Natl. Acad. Sci. U. S. A. 116, 16955-16960 (2019)).

[0008] The pathogenic relevance of several of these mimics was supported by animal models of MS ((Lanz T. V. et al., Nature 603, 321-327 (2022); Jog N. R. et al., J. Autoimmun. 106, 102332 (2020)). In most instances, striking sequence homology between EBNA1 and the respective CNS mimic was identified. Interestingly, most mimics are concentrated in a narrow region on EBNA1 between the second glycine-arginine repeat region and the C-terminal DNA binding domain (amino acid residues ~AA380–450). Elevated antibody reactivities against this region are strongly associated with MS ((Bjomevik K. et al., Science 375, 296-301 (2022); Ruprecht K. et al., Multiple sclerosis: the elevated antibody response to Epstein-Barr virus primarily targets, but is not confined to, the glycine-alanine repeat of Epstein-Barr nuclear antigen-1. J. Neuroimmunol. 272, 56–61 (2014); Jafari N. et al., No evidence for intrathecal IgG synthesis to Epstein Barr virus nuclear antigen- 1 in multiple sclerosis. J. Clin. Virol. 49, 26-31 (2010); Salzer J. et al., Epstein-Barr virus antibodies and vitamin D in prospective multiple sclerosis biobank samples. Mult. Scler. 19, 1587-1591 (2013); and Sundqvist E. et al., Epstein-Barr virus and multiple sclerosis: interaction with HL A. Genes Immun. 13, 14-20 (2012)).

[0009] MS treatments with natalizumab (NAT) and autologous hematopoietic stem cell transplantation (aHSCT) were found to be associated with changes in EBNA1 -directed B cell response, and in vitro studies confirmed that levels of autoantibodies against MS autoantigens ANO2, CRY AB. and GlialCAM were elevated in NAT-treated MS patients and a cross-LST-IOOIWO PATENT APPLICATION reactivity between EBNA1380-641and two MS autoantigens ANO21-275and GlialCAM262-428, revealing treatment-associated changes in the immunoglobulin repertoire in MS. Marti Z. et al., Enhanced and cross-reactive in vitro memory B cell response against Epstein-Barr virus nuclear antigen 1 in multiple sclerosis, Front. Immunol., 15: 1334720 (August 27, 2024).SUMMARY OF THE INVENTION

[0010] In one aspect, the present disclosure provides a chimeric autoantibody receptor (CAAR) comprising an extracellular domain having at least one autoantigen or fragment thereof selected from the group consisting of GlialCAM. CRY AB, and ANO2, a transmembrane domain, and an intracellular signaling domain.

[0011] In a second aspect, the present disclosure provides a vector comprising a nucleic acid sequence encoding a chimeric autoantibody receptor (CAAR) comprising an extracellular domain having at least one autoantigen or a fragment thereof selected from the group consisting of GlialCAM, CRY AB, and ANO2, a transmembrane domain, and an intracellular signaling domain.

[0012] In a third aspect, the present disclosure provides a genetically modified cell comprising a chimeric autoantibody receptor (CAAR) comprising an extracellular domain having at least one autoantigen or fragment thereof selected from the group consisting of GlialCAM, CRY AB, and ANO2, a transmembrane domain, and an intracellular signaling domain, wherein the cell expresses the CAAR and binds an autoantibody-expressing cell.

[0013] In a fourth aspect, the present disclosure provides a method of treating, preventing, and / or managing multiple sclerosis in a subject in need thereof, comprising administering to the subject a genetically modified cell comprising a chimeric autoantibody receptor (CAAR) comprising an extracellular domain having at least one autoantigen or fragment thereof selected from the group consisting of GlialCAM, CRY AB, and ANO2, a transmembrane domain, and an intracellular signaling domain, wherein the cell expresses the CAAR and binds to an autoantibody-expressing cell.

[0014] In any one of the above aspects, the autoantigen or fragment thereof is GlialCAM comprising an amino acid sequence of X₁SPPRAP (SEQ ID NO: 3) wherein X₁ is S that is phosphorylated or non-phosphorylated.

[0015] In the embodiment of ^

[0013] , the autoantigen or fragment thereof is GlialCAM comprising an amino acid sequence selected from the group consisting of GlialCAM AA365-394 (SEQ ID NO: 32), GlialCAM AA365-394-pSer376 (SEQ ID NO: 33), GlialCAM AA376-389 (SEQ ID NO: 34), GlialCAM AA376-389-pSer376 (SEQ ID NO: 35), and GlialCAM AA385-416 (SEQ ID NO: 36).LST-IOOIWO PATENT APPLICATION

[0016] In any one of the above aspects, the autoantigen or fragment thereof is ANO2 comprising an amino acid sequence of PGDIELGPLD (SEQ ID NO: 12).

[0017] In the embodiment of ^

[0015] , the autoantigen or fragment thereof is ANO2 comprising an amino acid sequence selected from the group consisting of ANO2 AA1-275 (SEQ ID NO: 37) and ANO2 AA134-153 (SEQ ID NO: 38).

[0018] In any one of the above aspects, the autoantigen or fragment thereof is CRY AB comprising an amino acid sequence of PPGRRPFF (SEQ ID NO: 8).

[0019] In the embodiment of ^

[0017] , the autoantigen or fragment thereof is CRY AB comprising an amino acid sequence selected from the group consisting of CRY AB AA1-275 (SEQ ID NO: 39) and CRY AB AA2-33 (SEQ ID NO: 40).

[0020] In any one of the above aspects, the autoantigen or fragment thereof comprises GlialCAM AA376-389-pSer376 (SEQ ID NO: 35), CRY AB AA8-15 (SEQ ID NO: 8), and ANO2 AA140-149 (SEQ ID NO: 12).

[0021] In the embodiment of ^

[0019] , the extracellular domain further comprises a linker that forms a linkage between the GlialCAM AA376-389-pSer376 and CRY AB AA8-15, and a linker that forms a linkage between CRY AB AA8-15 and ANO2 AA140-149.

[0022] In the embodiment of • 10020|, the linker is a glycine rich linker selected from the group consisting of GGGGG. GGSSG. GGGGSLVPRGSGGGGS, (GS)nwhere n is 2-5, GGSGGHMGSGG, GGSGGSGGSGG, GGSGpwhere p is 1-5, GSGGGTGGGSG, and GSGGSGGSGGSGGS.

[0023] In any one of the above aspects, the invention may further include a co-stimulatory domain selected from the group consisting of CD28 and tumor necrosis factor ligand superfamily member 9 (4-1BB).

[0024] In any one of the above aspects, the intracellular signaling domain may include a CD3 zeta signaling domain.

[0025] In any one of the above aspects, the transmembrane domain may include a CD8 alpha chain hinge and transmembrane domain.

[0026] In one embodiment of any one of the third and fourth aspects, the autoantibody expressing cell is a plasmablast, a plasma cell, and a B cell.

[0027] In one embodiment of the third and fourth aspect, the CAAR further induces killing of the plasmablast cell.

[0028] In one embodiment of the third and fourth aspect, the CAAR further induces killing of the plasma cell.

[0029] In one embodiment of the third and fourth aspect, the CAAR further induces killing of the B cell expressing autoantibody.LST-IOOIWO PATENT APPLICATION

[0030] In one embodiment of any one of the third and fourth aspects, the cell is selected from the group consisting of a helper T cell, a cytotoxic T cell, a memory T cell, a regulatory T cell, a gamma delta T cell, a natural killer cell, a cytokine induced killer cell, and a cell line thereof.

[0031] In the embodiment of 1

[0029] , the genetically modified cell is a helper T cell, a cytotoxic T cell, a memory T cell, a regulatory T cell, or a gamma delta T cell.

[0032] In one embodiment of the fourth aspect, the method further includes measuring at least one anti-EBNAl antibody, anti-GlialCAM antibody, anti-CRYAB antibody, or anti-ANO2 antibody level in a tissue of a mammal.

[0033] In one embodiment of the fourth aspect, the mammal includes a human.

[0034] In the embodiment of 1

[0032] , the human tissue includes plasma, serum, blood, or cerebrospinal fluid (CSF).

[0035] In one embodiment of the fourth aspect, the method further includes determining an antibody level of at least one antibody selected from the group consisting of anti-EBNAl antibody, anti-GlialCAM antibody, anti-CRYAB antibody, and anti-ANO2 antibody.

[0036] In the embodiment of 1

[0034] , the method includes after the determining step, administering the genetically modified cell comprising the CAAR to the subject based on a criteria wherein:(a) when anti-EBNAl antibody and / or anti-GlialCAM antibody levels are elevated in the subject, the subject is administered the CAAR comprising the extracellular domain comprising GlialCAM having an amino acid sequence of X1SPPRAP (SEQ ID NO: 3) wherein X1is S that is phosphory lated or non-phosphorylated; or(b) when anti-CRYAB antibody and anti-ANO2 antibody levels are elevated in the subject, the subject is administered the CAAR comprising the extracellular domain comprising CRY AB having an amino acid sequence of PPGRRPFF (SEQ ID NO: 8); or(c) when anti-ANO2 antibody levels are elevated in the subject, the subject is administered the CAAR comprising the extracellular domain comprising ANO2 having an amino acid sequence of PGDIELGPLD (SEQ ID NO: 12); or(d) when anti-GlialCAM antibody, anti-CRYAB antibody, and anti-ANO2 antibody levels are elevated in the subject, the subject is administered the CAAR comprising the extracellular domain having GlialCAM AA376-389-pSer376 (SEQ ID NO: 35), CRY AB AA8-15 (SEQ ID NO: 8), and ANO2 AA140-149 (SEQ ID NO: 12), and a linker that forms a linkage between GlialCAM AA376-389-pSer376 and CRY AB AA8-15, and a linkage between CRY AB AA8-15 and ANO2 AA140-149.

[0037] In one embodiment of the fourth aspect, the genetically modified cell is administered once a month for the first two months, and then once every six months.LST-IOOIWO PATENT APPLICATION

[0038] In one embodiment of the fourth aspect, the subject is administered a first dose of 1 x 108cell / kg body weight with lymphodepletion or a dose of 1 x 108of the genetically modified cells without lymphodepletion.

[0039] In another embodiment of the fourth aspect, the subject is administered a dosage of 104to 109cells / kg body weight.

[0040] In a fifth aspect, the present disclosure provides a pharmaceutical composition including the chimeric autoantibody receptor (CAAR) as described in any one of the above aspects and embodiments, the vector as described in any one of the above aspects and embodiments, or the genetically modified cell as described in any one of the above aspects and embodiments, and at least one pharmaceutically acceptable carrier or diluent.

[0041] In a sixth aspect, the present disclosure further provides a kit for treatment, said kit comprising any one of the aspects and embodiments described above, and instructions for using the chimeric autoantibody receptor (CAAR) as described in any one of the above aspects and embodiments, the vector as described in any one of the above aspects and embodiments, or the genetically modified cell as described in any one of the above aspects and embodiments, for treatment of an autoimmune disease or disorder such as multiple sclerosis.

[0042] In a seventh aspect, the present disclosure further provides a kit for diagnosis, said kit comprising one or more of the peptides set forth in Supplementary Table SI, and instructions for using one or more of the peptides for diagnosis of an autoimmune disease or disorder such as multiple sclerosis.

[0043] In an eighth aspect, the present disclosure further provides a method of diagnosing an autoimmune disorder such as multiple sclerosis, the method includes applying one or more of the peptides set forth in Supplementary Table S 1 to a tissue of a mammal.

[0044] In one embodiment of the eighth aspect, the mammal includes a human.

[0045] In one embodiment of

[0043] , the human tissue includes plasma, serum, blood, or cerebrospinal fluid (CSF).BRIEF DESCRIPTION OF THE DRAWINGS

[0046] FIG. 1 shows individual antibody reactivities associated with MS risk. Odds ratios of MS vs. controls for the indicated antibody reactivities. All data were derived from bead-based assay measuring mean fluorescence intensities. Data were adjusted for age, gender, batch effects and PCA1-5. ORs were calculated using logistic regression analysis. ORs ±95% CI are shown.

[0047] FIG. 2 shows correlations of antibody reactivities. Lower Left: Correlation matrix of all tested antigens in all subjects, correlation coefficients of plate-corrected mean MFIs are shown,LST-IOOIWO PATENT APPLICATION calculated using Pearson correlation analysis. Top right: Matrix showing the ratio of correlation coefficients of the MS vs. control group. Yellow and purple squares represent ratios >4 and <-4, respectively.

[0048] FIG. 3A shows alignment of the epitope regions for the molecular mimics GlialCAM, CRY AB. and AN02 on the EBNA1 sequence. Identical residues span the sequences. Central epitopes are highlighted in grey. Amino acid colors based on side chain chemistry.

[0049] FIGS. 3B-3E show bead-based competitive blocking assay, measuring reactivity to (FIG.3B) GlialCAM AA370-389, (FIG. 3C) GlialCAM AA370-389 pSer376, (FIG. 3D) EBNA1 AA386-405, and (FIG. 3E) EBNA1 AA1-120 (negative control peptide) after pre-incubation of selected MS plasma samples (n=10) with the peptides indicated on the x-axis. Negative fold change of MFI over samples incubated with BSA indicating increased inhibition. *p < 0.05, **p < 0.005 according to Wilcoxon signed-rank test.

[0050] FIG. 3B shows EBNA1 AA386-405 efficiently blocked antibody binding to GlialCAM AA370-389, confirming the presence of cross-reactive antibodies.

[0051] FIG. 3C shows phosphorylated GlialCAM AA370-386 was more robustly blocked by EBNA1 AA386-405, suggesting stronger molecular mimicry.

[0052] FIG. 3D shows binding to EBNA1 AA386-405 was partially blocked by GlialCAM AA370-389.

[0053] FIG. 3E shows EBNA1 AA1-120 was used as anegative control peptide.

[0054] FIG. 3F shows heatmap summarizing FIGS. 3B-3E, and demonstrate mean negative fold changes over samples blocked with BSA. x-axis: blocking antigen, y-axis: bead-bound antigen measurement.

[0055] FIG. 4A shows association of HLA-DRB1 *15:01 with anti-EBNAl AA381-410 reactivity in all individuals.

[0056] FIG. 4B shows association of HLA-DRB1*15: O1 with anti-EBNAl AA381-410 reactivity in MS individuals.

[0057] FIG. 4C shows association of HLA-DRB1*15:01 with anti-EBNAl AA381-410 reactivity in healthy controls.

[0058] FIG. 4D shows association of HLA-DRB1*15:01 with GlialCAM AA70-389 in all individuals.

[0059] FIG. 4E shows association of HLA-DRB1*15:01 with GlialCAM AA70-389 in MS individuals.

[0060] FIG. 4F shows association of HLA-DRB1*15:01 with GlialCAM AA70-389 in healthy controls.LST-IOOIWO PATENT APPLICATION

[0061] FIGS. 4G-I show plots representing odds ratios for MS risk for combinations of multiple parameters, including in column 1: positive HLA-DRB1 *15:01 status, in column 2: elevated (FIGS. 4G and 4H) anti-EBNAl AA381-410 and (FIG. 41) anti-EBNAl AA421-450 reactivity’, and in column 3: (FIG. 4G) anti-GlialCAM AA365-394, (FIG. 4H) anti-CRYAB AA2-33, and (FIG. 4G) anti-ANO2 AA134-153. Data were adjusted for age, gender, plate-based batch effects, and PCA 1-5. ORs were calculated using logistic regression analysis with the group not having any increased antibody level or being HLA*DRB15:01 positive (lowest group in each panel) as a reference. ORs ±95% CI are shown.

[0062] FIG. 4G shows positive HLA-DRB1 *15:01 status and elevated antibody levels against EBNA1 AA381-410 and GlialCAM AA365-394 are three independent risk factors that together increase the MS risk to an OR of 9.40 (CI: 6.04 - 14.86).

[0063] FIGs. 4H-4I show a cumulative increase in MS risk for HLA-DRB1*15: O1 and anti-EBNAl AA381-410 in combination with anti-CRYAB AA2-33 (OR: 10.01, CI: 6.38 - 15.97) and HLA-DRB1*15:01 and anti-EBNAl AA421-450 anti-ANO2 AA134-153 (OR: 5.42. CI: 3.65 - 8.13).

[0064] FIGS. 5A and 5B show distribution of individuals with 0-3 elevated antibody reactivities to three different EBNA1 peptides (EBNA1 AA386-405, EBNA1 AA401-430, and EBNA1 AA421-450) (left), and three different CNS-specific antigens (GlialCAM AA386-405. CRY AB AA2-33, and ANO2 AA134-153) (right). Comparisons between healthy controls and MS are shown. P values according to Chi-square test.

[0065] FIG. 5C provide Odds Ratio showing cumulative effects of 1-3 antibody reactivities. Data were adjusted for age, gender, batch effects, and PCA1-5. ORs were calculated using logistic regression analysis. ORs ±95% CI are shown.

[0066] FIG. 6 shows multiple molecular mimics in EBNA1.

[0067] FIG. 7 shows GlialCAM p376-382 peptide mimic (EBNA1 sequence 386-399).

[0068] FIG. 8 shows CRYAB p8-15 peptide mimic (EBNA1 sequence 399-406).

[0069] FIG. 9 shows ANO-2 p140-149 peptide mimic (EBNA1 sequence 431-440).

[0070] FIGS. 10A-10B show sensitivity analysis for regression models. Sensitivity analyses reported as OR derived from logistic regression models, along with their 95% Cis. Adjustments were made for key covariates, including gender, age, plates, and PCA 1-5. Sensitivity' analyses include the use of the cutoff value from the (FIG. 10A) continuous analysis and (FIG. 10B) the AUC cutoff value.

[0071] FIGS. 11A-1 IB show gender-stratified odds ratios for MS risk associated with individual antibody reactivities. Odds ratios of MS vs. controls in (FIG. 11 A) females and (FIG. 1 IB) males for the indicated antibody reactivities. All data derived from bead-based assay measuringLST-IOOIWO PATENT APPLICATION mean fluorescence intensities (MFI). Data were adjusted for age, sex, plate-based batch effects, and PCA1-5. ORs were calculated using logistic regression analysis. Mean ORs ±95% CI are shown.

[0072] FIG. 12 shows the sequence for a Compound CAAR construct.

[0073] FIG. 13 shows the full length amino acid sequence for EBNA1 (UniProt ID P03211) (SEQ ID NO: 16).

[0074] FIG. 14 shows the full length amino acid sequence for GlialCAM (UniProt ID Q14CZ8) (SEQ ID NO: 26).

[0075] FIG. 15 shows the amino acid sequence of ANO2 (UniProt ID Q9NQ90) from amino acid residues 1-175 (SEQ ID NO: 37).

[0076] FIG. 16 shows the amino acid sequence of CRY AB (UniProt ID P02511) (SEQ ID NO: 39).

[0077] FIG. 17 shows the amino acid sequence of EBNA3C (UniProt ID Q3KST0) (SEQ ID NO: 41).

[0078] FIGS. 18A-18C showthe association of HLA-DR2, and HLA-DRB1*15:01 status with anti-EBNAl and anti-GlialCAM antibody reactivity. Plots representing odds ratios for MS risk for combinations of multiple parameters, including in column 1: absence of HLA-A *02: 01 (MS protection allele, absence indicated in red), column 2: presence oiHLA-DRBl*15:01 (MS risk allele, presence indicated in red), in column 3: elevated (FIG. 18A, 18B) anti-EBNAl AA381-410 and (FIG. 18C) EBNA1 AA421-450, and in column 4: (FIG. 18A) GlialCAM AA365-394, (FIG. 18B) CRY AB AA2-33, and (FIG. 18C) ANO2 AA134-153. Data were adjusted for age, gender, batch effects, and PCA1-5. ORs were calculated using logistic regression. Mean ORs ±95% CI are shown.

[0079] FIGS. 19A-19B are scatter plots showing the association between plasma neurofilament light chain (Nfl) levels and antibody reactivity to EBNA1 AA381-410 and GlialCAM AA365-394. (FIG. 19A) Correlation analysis between Nfl and antibody reactivity to EBNA1 AA381-410 (n=519, r=0.048, p=0.958) and to (FIG. 19B) GlialCAM AA365-394 (n=513, r=0.036, p=0.547) was conducted using Spearman correlation analysis to investigate the strength and direction of any associations. Each data point represents an individual sample, with antibody reactivity as mean fluorescence intensity (MFI) plotted on the x-axis and Nfl levels plotted as log values on the y-axis. No statistically significant correlation was observed between Nfl and either antigen.

[0080] FIGS. 20A-20C show cumulative MS risk with multiple combined antibody reactivities against EBNA1 and its three mimics using different analytical approaches. Odds ratios (ORs) demonstrate the cumulative effects of 1-3 antibody reactivities, including three differentLST-IOOIWO PATENT APPLICATION EBNA1 peptides (EBNA1 AA386-405, EBNA1 AA401-430, and EBNA1 AA421-450) and three CNS-specific antigens (GlialCAM AA386-405, CRY AB AA2-33, and ANO2 AA134-153). Different methods were applied to determine the cut-off values for high reactivity: (FIG.20A) median and (FIGS. 20B and 20C) area under the curve (AUC). These approaches identified individuals with 0-3 elevated antibody reactivities, with comparisons made between healthy controls and MS patients. Initially, data were adjusted for age, gender, plate-based batch effects, and PCA1-5 (FIGS. 20A and 20B). In a subsequent analysis, adjustments were limited to age and gender (FIG. 20C). Odds ratios (ORs) and their 95% confidence intervals (Cis) were calculated using logistic regression, and the results are presented as ORs ±95% CI.

[0081] FIG. 21 shows EBNA1 AA361-470 (SEQ ID NO: 1).DEFINITIONS

[0082] In order to facilitate understanding of the examples provided herein, certain frequently occurring terms are defined herein.

[0083] The articles “a” and ‘'an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

[0084] The term “about” as used herein refers a normal variation in that measured quantity that would be expected by a skilled person making the measurement and exercising a level of care commensurate with the objective of the measurement and the precision of the measuring equipment used. Unless otherwise indicated, “about” refers to a variation of ±20% or ±10% of the value provided, in some instances ±5%, in some instances ±1%, and in some instance ±0.1% from the specified value.

[0085] The term “multiple sclerosis” or “MS” as used herein refers to any one or more of the types of recognized MS: relapsing-remitting MS (RRMS), benign MS (a version of relapsing remitting MS with very mild attacks separated by long periods with no symptoms), primary progressive (PPMS), secondary progressive MS (SPMS), progressive relapsing MS (PRMS), clinically isolated syndrome (CIS; refers to the first episode of a condition that affects the myelin but after further testing the syndrome may be diagnosed as MS or a different condition), or radiologically isolated syndrome (refers to findings on MRIs of the brain and spinal cord that look like MS in someone without classic symptoms of MS).

[0086] The term “antibody,” as used herein, refers to intact immunoglobulin molecules, as well as fragments of immunoglobulin molecules, such as Fab, Fab1, (Fab’)2, Fv, and SCA fragments, that are capable of binding to an epitope of an antigen. These antibody fragments, which retain some ability to selectively bind to an antigen (e.g., a polypeptide antigen) of the antibody from which they are derived, can be made using well known methods in the art (see, e.g., Harlow andLST-IOOIWO PATENT APPLICATION Lane, supra), and are described further, as follows. Antibodies can be used to isolate preparative quantities of the antigen by immunoaffinity chromatography.

[0087] The term “high affinity ” as used herein refers to high specificity in binding or interacting or attraction of one molecule to a target molecule.

[0088] The term “antigen” or “Ag” as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial amino acid sequences of one or more than one autoantigen, and that these amino acid sequences are arranged in various combinations to encode polypeptides that elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.

[0089] The term “autoantigen” means an endogenous antigen that stimulates production of an autoimmune response, such as production of autoantibodies. Autoantigen also includes a selfantigen or antigen from a normal tissue that is the target of a cell-mediated or an antibody-mediated immune response that may result in the development of an autoimmune disease. Examples of autoantigens include, but are not limited to, EBV nuclear antigen 1 (EBNA1) and fragments thereof, and EBNA-1 molecular mimics including bur not limited to, GlialCAM, aB Crystallin (CRY AB), and Anoctamin-2 (ANO-2) and fragments thereof.

[0090] “Autoantibody” refers to an antibody that is produced by an immunoreactive cell specific for an autoantigen, including but not limited to, an autoantibody-expressing cell such as a plasmablast, a plasma cell, or a B cell.

[0091] “Chimeric autoantibody receptor” or “CAAR” refers to an engineered receptor that is expressed on a T cell or any other effector cell type capable of cell-mediated cytotoxicity. In the present disclosure, the CAAR includes an autoantigen or fragment thereof that is specific for an autoantibody induced by an Epstein-Barr vims infection. The CAAR also includes aLST-IOOIWO PATENT APPLICATION transmembrane domain, an intracellular domain (such as a co-stimulatory domain), and a signaling domain.

[0092] As used herein, the term “conservative sequence modifications” is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan). nonpolar side chains (e.g.. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, for example, one or more amino acid residues within the extracellular regions of the CAAR of the invention can be replaced with other amino acid residues having a similar side chain or charge and the altered CAAR can be tested for the ability to bind autoantibodies using the functional assays described herein.

[0093] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.

[0094] The term “therapeutic” as used herein means a treatment and / or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state.

[0095] “Effective amount” or “therapeutically effective amount” are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result. Such results may include, but are not limited to, the inhibition of virus infection as determined by any means suitable in the art.

[0096] When “an immunologically effective amount,” “an anti-autoantibody effective amount,” “an autoimmune disease-inhibiting effective amount,” or "therapeutic amount” is indicated, the precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). It can generally beLST-IOOIWO PATENT APPLICATION stated that a pharmaceutical composition comprising the T cells described herein may be administered at a dosage of 104to 109cells / kg body weight, in some instances 105to 106cells / kg body weight, including all integer values within those ranges. T cell compositions may also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see. e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988, herein incorporated by reference in its entirety). The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly. For example, the first dose of T cells may be 1 x 108cells / kg body weight following lymphodepletion with fludarabine (30 mg / m2 on days -5, -4, -3, etc.) and cyclosphosamide (300 mg / m2 on days -5, -4, -3, etc.). Further doses of 1 x 108cells / kg body weight may be given without lymphodepletion. Another example is therapy may be given monthly for the first two months, and then every six months thereafter.

[0097] As used herein "endogenous’’ refers to any material from or produced inside an organism, cell, tissue or system.

[0098] As used herein, the term “exogenous” refers to any material introduced from or produced outside an organism, cell, tissue or system.

[0099] “Expression vector” or “vector” refers to a vector comprising a recombinant or isolated polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector or vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. The term “expression vector” or “vector” should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Thus, expression vectors or vectors may include but are not limited to all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant or isolated polynucleotide. The expression vector or vector can be used to deliver the recombinant or isolated nucleic acid to the interior of a cell.

[0100] “Identity” as used herein refers to the subunit sequence identity between two polymeric molecules particularly between two amino acid molecules, such as, between two polypeptide molecules. The identity or extent to which two amino acid sequences have the same residues at the same positions in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of matching or identical positions; e.g.,LST-IOOIWO PATENT APPLICATION if half (e.g., five positions in a polymer ten amino acids in length) of the positions in two sequences are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 of 10), are matched or identical, the two amino acids sequences are 90% identical.

[0101] “Homologous’' refers to the subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules, such as. two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit; e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two sequences are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 of 10), are matched or homologous, the two sequences are 90% homologous. For example, as described herein, there is high sequence homology between certain amino acid sequences in EBNA1 and certain amino acid sequences of CNS autoantigens such as GlialCAM, aB Crystallin (CRY AB), and Anoctamin-2 (ANO-2). In some instances, the core homologous region is within the binding epitope of the CNS autoantigen. For example, as shown in FIG. 3A, in the case of EBNA1 and CRY AB - the core homologous region is within the binding epitope of CRY AB amino acids 7 and 16 defined by epitope mapping. Likewise, in the case of EBNA1 and GlialCAM, there is significant overlap between the EBNA1 epitope AA393-398 “SPPRRP” and the GlialCAM epitope AA377-382 “SPPRAP” (FIG. 3 A). In the case of EBNA1 and ANO-2, there is significant overlap within EBNA1 AA431-440 and ANO-2 AA140-149 (FIG. 3A).

[0102] As used herein, an “instructional material" includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the compositions and methods of the invention. The instructional material of the kit of the invention may, for example, be affixed to a container which contains the nucleic acid, peptide, and / or composition of the invention or be shipped together with a container which contains the nucleic acid, peptide, and / or composition. Alternatively, the instructional material may be shipped separately from the container with the intention that the instructional material and the compound be used cooperatively by the recipient.

[0103] “Intracellular domain” refers to a portion or region of a molecule that resides inside a cell.

[0104] “Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state isLST-IOOIWO PATENT APPLICATION ■‘isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0105] The term “polynucleotide” as used herein is defined as a chain of nucleotides.Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art. including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR™, and the like, and by synthetic means.

[0106] As used herein, the terms “peptide,” “poly peptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0107] “Signaling domain” refers to the portion or region of a molecule that recruits and interacts with specific proteins in response to an activating signal.

[0108] “Transmembrane domain” refers to a portion or a region of a molecule that spans a lipid bilayer membrane.

[0109] The term “subject” is intended to include living organisms in which an immune response can be elicited (e.g., mammals).

[0110] As used herein, a “substantially purified” cell is a cell that is essentially free of other cell types. A substantially purified cell also refers to a cell which has been separated from other cell types with which it is normally associated in its naturally occurring state. In some instances, a population of substantially purified cells refers to a homogenous population of cells. In other instances, this term refers simply to cells that have been separated from the cells with which theyLST-IOOIWO PATENT APPLICATION are naturally associated in their natural state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.[OHl] The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0112] By the term “specifically binds,” as used herein, is meant an antibody, or a ligand, which recognizes and binds with a cognate binding partner (e.g.. a stimulatory and / or costimulatory molecule present on a T cell) protein present in a sample, but which antibody or ligand does not substantially recognize or bind other molecules in the sample.DETAILED DESCRIPTION

[0113] Multiple sclerosis (MS) is an autoimmune demyelinating disorder of the central nervous system (CNS), which is linked to Epstein-Barr virus (EBV) infection, preceding the disease. The molecular mechanisms that underly this link are incompletely understood. The present inventors have found molecular mimicry between the EBV transcription factor Epstein-Barr nuclear antigen 1 (EBNA1) and three human CNS autoantigens: anoctamin-2 (AN02). alpha-B crystallin (CRY AB), and glial cellular adhesion molecule (GlialCAM). Elevated antibody reactivity against EBNA1 and either GlialCAM, CRY AB, or ANO2 increases the risk for MS.

[0114] Chimeric Autoantibody Receptor (CAAR)

[0115] The present disclosure provides chimeric autoantibody receptor (CAAR) specific to block binding of an autoantibody that binds to at least one of the three CNS autoantigens (EBNA1 mimics) that cause an autoimmune disorder / disease such as multiple sclerosis, recombinant DNA constructs comprising the CAAR, vectors comprising the CAAR, compositions comprising CAAR vectors, and recombinant T cells comprising the CAAR. The present disclosure also includes methods of making a genetically modified T cell expressing the CAAR (CAART).

[0116] The CAAR of the present disclosure comprises an autoantibody binding domain referred to as an autoantigen or a fragment thereof. The autoantibody binding domain portion may comprise an epitope of the autoantigen that binds the autoantibody, as the epitope is the part of the autoantigen that is specifically recognized by the autoantibody. For example, the autoantigen may recognize an autoantibody on autoantibody-expressing cell including, but not limited to, an immunoreactive cell (such as a B cell), a plasmablast, and a plasma cell, associated with an autoimmune disease such as multiple sclerosis. The autoantibody bindingLST-IOOIWO PATENT APPLICATION domain portion or the domain having the autoantigen or a fragment thereof of the CAAR, acts as a “bait” to bind and capture the autoantibody expressing cell such as plasmablasts, plasma cells, and / or B cells by binding to the specific autoantibody through the extracellular domain. This binding may further induce the immune cell to activate and destroy the autoantibody expressing cell.

[0117] In one aspect, the present disclosure provides a genetically engineered chimeric autoantibody receptor (CAAR) comprising an extracellular domain having at least one autoantigen or a fragment thereof selected from the group consisting of GlialCAM, CRY AB, and ANO2, a transmembrane domain, and an intracellular signaling domain.

[0118] In one embodiment, the autoantigen or fragment thereof may be GlialCAM comprising an amino acid sequence selected from GlialCAM AA34-416 (SEQ ID NO: 26), GlialCAM AA34-240 (SEQ ID NO: 27), GlialCAM AA262-416 (SEQ ID NO: 28), GlialCAM AA305-334 (SEQ ID NO: 29), GlialCAM AA325-354 (SEQ ID NO: 30), GlialCAM AA345-374 (SEQ ID NO: 31). GlialCAM AA365-394 (SEQ ID NO: 32). GlialCAM AA365-394-pSer376 (SEQ ID NO: 33), GlialCAM AA376-389 (SEQ ID NO: 34), GlialCAM AA376-389-pSer376 (SEQ ID NO: 35), and GlialCAM AA385-416 (SEQ ID NO: 36).

[0119] In one embodiment, the autoantigen or fragment thereof may be GlialCAM comprising an amino acid sequence selected from GlialCAM AA305-334 (SEQ ID NO: 29), GlialCAM AA325-354 (SEQ ID NO: 30), GlialCAM AA345-374 (SEQ ID NO: 31), GlialCAM AA365-394 (SEQ ID NO: 32), GlialCAM AA365-394-pSer376 (SEQ ID NO: 33), GlialCAM AA376-389 (SEQ ID NO: 34), GlialCAM AA376-389-pSer376 (SEQ ID NO: 35), and GlialCAM AA385-416 (SEQ ID NO: 36).

[0120] In one embodiment, the autoantigen or fragment thereof may be GlialCAM comprising an amino acid sequence of X₁SPPRAP (SEQ ID NO: 3) wherein X₁ is S that is phosphorylated or non-phosphorylated.

[0121] In one embodiment, the autoantigen or fragment thereof may be GlialCAM comprising an amino acid sequence selected from GlialCAM AA365-394 (SEQ ID NO: 32). GlialCAM AA365-394-pSer376 (SEQ ID NO: 33), GlialCAM AA376-389 (SEQ ID NO: 34), GlialCAM AA376-389-pSer376 (SEQ ID NO: 35), and GlialCAM AA385-416 (SEQ ID NO: 36).

[0122] In one embodiment, the autoantigen or fragment thereof may be ANO2 comprising an amino acid sequence of PGDIELGPLD (SEQ ID NO: 12).

[0123] In one embodiment, the autoantigen or fragment thereof may be ANO2 comprising an amino acid sequence selected from ANO2 AA1-275 (SEQ ID NO: 37) and ANO2 AA134-153 (SEQ ID NO: 38).LST-IOOIWO PATENT APPLICATION

[0124] In one embodiment, the autoantigen or fragment thereof is CRY AB comprising an amino acid sequence of PPGRRPFF (SEQ ID NO: 8).

[0125] In one embodiment, the autoantigen or fragment thereof is CRY AB comprising an amino acid sequence selected from CRY AB AA1-275 (SEQ ID NO: 39) and CRY AB AA2-33 (SEQ ID NO: 40).

[0126] In another embodiment of the above aspect, the autoantigen or a fragment thereof comprises all three CNS autoantigens GlialCAM, CRY AB, and ANO2. Preferably, the autoantigen or a fragment thereof comprises GlialCAM AA376-389-pSer376 (SEQ ID NO: 35), CRY AB AA8-15 (SEQ ID NO: 8), and ANO2 AA140-149 (SEQ ID NO: 12).

[0127] In the above embodiment, the CAAR may further include a short oligo- or polypeptide linker that forms a linkage between the GlialCAM AA376-389-pSer376 (SEQ ID NO: 35) and CRY AB AA8-15 (SEQ ID NO: 8), and a linkage between the CRY AB AA8-15 (SEQ ID NO: 8), and ANO2 AA140-149 (SEQ ID NO: 12) in the extracellular domain of the CAAR.

[0128] Examples of a suitable linker includes, but is not limited to glycine rich linkers such as GGGGG, GGSSG, GGGGSLVPRGSGGGGS, (GS)nwhere n is 2-5, GGSGGHMGSGG, GGSGGSGGSGG, GGSGpwhere p is 1-5, GSGGGTGGGSG, and GSGGSGGSGGSGGS.

[0129] In any one of the above embodiments, the CAAR comprises a transmembrane domain, that is fused to the extracellular domain of the CAAR. In one embodiment, the CAAR comprises a transmembrane domain that naturally is associated with one of the domains in the CAAR. In some instances, the transmembrane domain is be selected or modified by amino acid substitution to avoid binding to the transmembrane domains of the same or different surface membrane proteins in order to minimize interactions with other members of the receptor complex.

[0130] The transmembrane domain may be derived either from a natural or from a synthetic source. When the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. In one embodiment, the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. In one aspect a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.

[0131] A variety of human hinges may also be included such as the human Ig (immunoglobulin) hinge. Other examples of a hinge and / or transmembrane domain include, but are not limited to, a hinge and / or transmembrane domain of an alpha, beta or zeta chain of a T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIR, 0X40, CD2, CD27, LFA-1 (CDlla, CD18). ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7,LST-IOOIWO PATENT APPLICATION NKp80 (KLRF1), CD 160, CD 19, IL2R beta, IL2R gamma, IL7R α, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD103, ITGAL, CDlla, LFA-1, ITGAM, CDllb, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM. Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, LylO8), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C.

[0132] In one embodiment, the transmembrane domain comprises a CD8 alpha chain hinge and / or transmembrane domain.

[0133] The cytoplasmic domain or the intracellular signaling domain of the CAAR described herein in any one of the above embodiments, is responsible for activation of at least one of the normal effector functions of the immune cell in which the CAAR has been placed in. Examples of intracellular signaling domains for use in the CAAR described herein include, but are not limited to, a fragment or domain from one or more molecules or receptors including, but are not limited to, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcR gamma, FcR beta (Fc Epsilon Rib), CD79a, CD79b, Fcgamma Rlla, DAP10, DAP12, T cell receptor (TCR), CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CD5, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CDlla, LFA-1, ITGAM, CDllb. ITGAX, CDllc, ITGB1, CD29, ITGB2, CD 18. LFA-1. ITGB7, TNFR2. TRANCE / RANKL, DNAM1 (CD226). SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyi08), SLAM (SLAMF1, CD150, IPO-3). BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and any combination thereof.

[0134] In any one of the above embodiments, the intracellular signaling domain comprises a CD3-zeta signaling domain by itself or in combination with any other desired cytoplasmic domain(s) useful in the context of the CAAR described herein. For example, the intracellular signaling domain of the CAAR can further comprise a CD3 zeta chain portion and a costimulatory signaling region.

[0135] The any one of the above embodiments, the costimulatory signaling region refers to a portion of the CAAR comprising the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligandsLST-IOOIWO PATENT APPLICATION that is required for an efficient response of lymphocytes to an antigen. In one embodiment, the CAAR may further include a co-stimulatory domain selected from the group consisting of CD28 and tumor necrosis factor ligand superfamily member 9 (4-1BB).

[0136] Vectors and Recombinant DNA Constructs

[0137] In a second aspect, the present disclosure provides vectors comprising the CAAR described herein, and compositions comprising such CAAR vectors.

[0138] In one embodiment, the present disclosure includes an isolated nucleic acid sequence encoding a chimeric autoantibody receptor (CAAR), wherein the isolated nucleic acid sequence comprises an extracellular domain comprising at least one autoantigen or a fragment thereof selected from the group consisting of GlialCAM, CRY AB, and ANO2, a nucleic acid sequence of a transmembrane domain, and a nucleic acid sequence of an intracellular signaling domain.

[0139] In any one of the above embodiments, the present disclosure encompasses a recombinant DNA construct comprising nucleic acid sequences that encode an extracellular domain comprising at least one autoantigen or a fragment thereof selected from the group consisting of GlialCAM, CRY AB, and ANO2, wherein the nucleic acid sequence encoding the at least one autoantigen or a fragment thereof is operably linked to a nucleic acid sequence of an intracellular signaling domain.

[0140] In any one of the above embodiments, the intracellular signaling domain (or cytoplasmic domain) may comprise a costimulatory signaling region. The costimulatory signaling region refers to a portion of the CAAR comprising the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules that are required for an efficient T cell activation.

[0141] In any one of the above embodiments, the nucleic acid sequences may encode an extracellular domain having components of GlialCAM, aB Crystallin (CRY AB), and / or Anoctamin-2 (ANO-2).

[0142] In any one of the above embodiments, the nucleic acid sequences may encode an extracellular domain having GlialCAM comprising an amino acid sequence selected from GlialCAM AA34-416 (SEQ ID NO: 26), GlialCAM AA34-240 (SEQ ID NO: 27), GlialCAM AA262-416 (SEQ ID NO: 28), GlialCAM AA305-334 (SEQ ID NO: 29), GlialCAM AA325-354 (SEQ ID NO: 30), GlialCAM AA345-374 (SEQ ID NO: 31), GlialCAM AA365-394 (SEQ ID NO: 32), GlialCAM AA365-394-pSer376 (SEQ ID NO: 33), GlialCAM AA376-389 (SEQ ID NO: 34), GlialCAM AA376-389-pSer376 (SEQ ID NO: 35), and GlialCAM AA385-416 (SEQ ID NO: 36).

[0143] In any one of the above embodiments, the nucleic acid sequences may encode an extracellular domain having GlialCAM comprising an amino acid sequence selected fromLST-IOOIWO PATENT APPLICATION GlialCAM AA305-334 (SEQ ID NO: 29), GlialCAM AA325-354 (SEQ ID NO: 30), GlialCAM AA345-374 (SEQ ID NO: 31), GlialCAM AA365-394 (SEQ ID NO: 32), GlialCAM AA365-394-pSer376 (SEQ ID NO: 33), GlialCAM AA376-389 (SEQ ID NO: 34), GlialCAM AA376-389-pSer376 (SEQ ID NO: 35), and GlialCAM AA385-416 (SEQ ID NO: 36).

[0144] In any one of the above embodiments, the nucleic acid sequences may encode an extracellular domain having GlialCAM comprising an amino acid sequence of X₁SPPRAP (SEQ ID NO: 3) wherein X₁ is S that is phosphorylated or non-phosphorylated.

[0145] In any one of the above embodiments, the nucleic acid sequences may encode an extracellular domain having GlialCAM comprising an amino acid sequence selected from GlialCAM AA365-394 (SEQ ID NO: 32), GlialCAM AA365-394-pSer376 (SEQ ID NO: 33), GlialCAM AA376-389 (SEQ ID NO: 34), GlialCAM AA376-389-pSer376 (SEQ ID NO: 35), and GlialCAM AA385-416 (SEQ ID NO: 36).

[0146] In any one of the above embodiments, the nucleic acid sequences may encode an extracellular domain having ANO2 comprising an amino acid sequence of PGDIELGPLD (SEQ ID NO: 12).

[0147] In any one of the above embodiments, the nucleic acid sequences may encode an extracellular domain having ANO2 comprising an amino acid sequence selected from ANO2 AA1-275 (SEQ ID NO: 37) and ANO2 AA134-153 (SEQ ID NO: 38).

[0148] In any one of the above embodiments, the nucleic acid sequences may encode an extracellular domain having CRY AB comprising an amino acid sequence of PPGRRPFF (SEQ ID NO: 8).

[0149] In any one of the above embodiments, the nucleic acid sequences may encode an extracellular domain having CRY AB comprising an amino acid sequence selected from CRY AB AA1-275 (SEQ ID NO: 39) and CRY AB AA2-33 (SEQ ID NO: 40).

[0150] In any one of the above embodiments, the nucleic acid sequences may encode an extracellular domain having GlialCAM AA376-389-pSer376 (SEQ ID NO: 35), CRY AB AA8-15 (SEQ ID NO: 8), and ANO2 AA140-149 (SEQ ID NO: 12). Preferably, the autoantigen or a fragment thereof comprises GlialCAM AA376-389-pSer376 (SEQ ID NO: 35), CRY AB AA8-15 (SEQ ID NO: 8), and ANO2 AA140-149 (SEQ ID NO: 12).

[0151] In the above embodiment, the nucleic acid sequences may further include a short oligo-or polypeptide linker as described herein, that forms a linkage between the GlialCAM AA376-389-pSer376 (SEQ ID NO: 35) and CRY AB AA8-15 (SEQ ID NO: 8), and a linkage between the CRY AB AA8-15 (SEQ ID NO: 8), and ANO2 AA140-149 (SEQ ID NO: 12) in the extracellular domain of the CAAR.LST-IOOIWO PATENT APPLICATION

[0152] In any one of the above embodiments, recombinant DNA construct may comprise a hinge and / or transmembrane domain as described herein, that is attached to the extracellular domain of the CAAR, or that is associated with one of the domains in the CAAR.

[0153] A variety of human hinges may also be included such as the human Ig (immunoglobulin) hinge. Other examples of a hinge and / or transmembrane domain include, but are not limited to, a hinge and / or transmembrane domain of an alpha, beta or zeta chain of a T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIR, 0X40, CD2, CD27, LFA-1 (CDlla, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR. HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7R α, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD103, ITGAL, CDlla, LFA-1, ITGAM, CD1 lb, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAMl, CRTAM, Ly9 (CD229). CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C.

[0154] In any one of the above embodiments, the transmembrane domain comprises a CD8 alpha chain hinge and / or transmembrane domain.

[0155] The any one of the above embodiments, cytoplasmic domain or the intracellular signaling domain of the CAAR described herein, is responsible for activation of at least one of the normal effector functions of the immune cell in which the CAAR has been placed in.

[0156] Examples of intracellular signaling domains for use in the CAAR described herein include, but are not limited to, a fragment or domain from one or more molecules or receptors including, but are not limited to, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcR gamma, FcR beta (Fc Epsilon Rib), CD79a, CD79b, Fcgamma Rlla, DAP10, DAP 12, T cell receptor (TCR), CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT. NKG2C. B7-H3, a ligand that specifically binds with CD83, CD5, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f. ITGAD, CDlld. ITGAE, CD103, ITGAL, CDlla, LFA-1, ITGAM, CD1 lb, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyi08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT.LST-IOOIWO PATENT APPLICATION GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, other co-stimulatory molecules described herein, any derivative, variant, or fragment thereof, any synthetic sequence of a costimulatory molecule that has the same functional capability, and any combination thereof.

[0157] In any one of the above embodiments, the intracellular signaling domain comprises a CD3-zeta signaling domain by itself or in combination with any other desired cytoplasmic domain(s) useful in the context of the CAAR described herein. For example, the intracellular signaling domain of the CAAR can comprise a CD3 zeta chain portion and a costimulatory signaling region.

[0158] In any one of the above embodiments, the costimulatory signaling region refers to a portion of the CAAR comprising the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligands that is required for an efficient response of lymphocytes to an antigen. In one embodiment, the CAAR may further include a co-stimulatory domain selected from the group consisting of CD28 and tumor necrosis factor ligand superfamily member 9 (4-1BB).

[0159] The present invention also provides a vector in which DNA encoding the CAAR of the present invention is inserted. Vectors, including those derived from retroviruses such as lentivirus, are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses, such as murine leukemia viruses, in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of resulting in low immunogenicity in the subj ect into which they are introduced.

[0160] In any one of the above embodiments, the vector comprises a plasmid vector, viral vector, retrotransposon, site directed insertion vector (e.g. CRISPR. zn finger nucleases, TALEN), or suicide expression vector, or other known vector in the art.

[0161] In any one of the above embodiments, the nucleic acid can be cloned into any number of different types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0162] In any one of the above embodiments, the expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, N. Y.), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitableLST-IOOIWO PATENT APPLICATION vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01 / 96584; WO 01 / 29058; and U. S. Pat. No. 6,326,193; the disclosure of each is herein incorporated by reference in its entirety).

[0163] In any one of the above embodiments, the expression of nucleic acids encoding CAARs is achieved by operably linking a nucleic acid encoding the CAAR polypeptide or portions thereof to a promoter, and incorporating the construct into an expression vector. The vector is one generally capable of replication in a mammalian cell, and / or also capable of integration into the cellular genome of the mammal. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.

[0164] Examples of viral promoters that may be used include, but are not limited to, cytomegalovirus (CMV) promoter sequence, simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, and the elongation factor- la promoter. Examples of human gene promoters that may be used, include but are not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, in addition to constitutive promoters, inducible promoters may also be used. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0165] In any one of the above embodiments, the vector may also include a selectable marker gene or a reporter gene or both, to assess the expression of a CAAR polypeptide or portions thereof and / or to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. Useful selectable markers include, for example, antibiotic-resistance genes, such as neo and the like. Suitable reporter genes may include, but are not limited to, genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, and the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and may be prepared using known techniques or obtained commercially.

[0166] In any one of the above embodiments, the present disclosure includes a vector comprising an isolated nucleic acid sequence encoding a chimeric autoantibody receptor (CAAR) comprising an extracellular domain having at least one autoantigen or a fragment thereof selected from the group consisting of GlialCAM, CRY AB, and ANO2, and a transmembrane domain.LST-IOOIWO PATENT APPLICATION

[0167] In any one of the above embodiments, the vector may further comprise a nucleic acid sequence of an intracellular signaling domain.

[0168] All constructs mentioned herein comprising nucleic acid sequences that encode an extracellular domain comprising at least one autoantigen or a fragment thereof selected from the group consisting of GlialCAM, CRY AB, and ANO2, are capable of use with vectors discussed above.

[0169] Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.

[0170] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well-known in the art. See. for example, Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, N. Y.; the disclosure of which is herein incorporated in its entirety).

[0171] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. RNA vectors include vectors having a RNA promoter and / other relevant domains for production of a RNA transcript. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors may be derived from lentivirus, poxviruses, herpes simplex virus, adenoviruses and adeno-associated viruses, and the like. See. for example, U. S. Pat. Nos. 5,350,674 and 5,585,362; the disclosure of each is herein incorporated by reference in its entirety.

[0172] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0173] A non-viral delivery system including, but not limited to, a liposome and lipids, may also be used. Lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes. Examples of lipids suitable for use include, but are not limited to, dimyristyl phosphatidylcholine (“DMPC”), dicetyl phosphate (“DCP'’), cholesterol (“Choi”), and dimyristyl phosphatidylglycerol (“DMPG”).LST-IOOIWO PATENT APPLICATION

[0174] Modified Cells Comprising CAARs

[0175] In a third aspect, the present disclosure encompasses a genetically modified T cell expressing a CAAR (CAART) wherein the expressed CAAR comprises a construct as described above, and methods of making the same.

[0176] The present disclosure includes a genetically modified cell, such as a helper T cell, a cytotoxic T cell, a memory T cell, regulatory T cell, gamma delta. T cell, a natural killer cell, cytokine induced killer cell, a cell line thereof, and other effector cell, comprising a chimeric autoantibody receptor (CAAR) comprising an extracellular domain having an autoantigen or a fragment thereof, a transmembrane domain, and an intracellular signaling domain, wherein the cell expresses the CAAR and binds an autoantibody-expressing cell.

[0177] In one embodiment the autoantibody-expressing cell is at least one selected from the group consisting of a plasmablast, a plasma cell, and a B cell.

[0178] In any one of the above embodiments, the genetically modified cell comprises a chimeric autoantibody receptor (CAAR) comprising an extracellular domain having at least one autoantigen or fragment thereof selected from the group consisting of GlialCAM, CRY AB, and ANO2, a transmembrane domain, and an intracellular signaling domain, wherein the cell expresses the CAAR and binds an autoantibody-expressing cell.

[0179] In any one of the above embodiments, the autoantibody-expressing cell is at least one selected from the group consisting of plasmablast, a plasma cell, and a B cell.

[0180] In any one of the above embodiments, the genetically modified cell expresses the CAAR. In this embodiment, the cell has high affinity binding for autoantibodies expressed on cells, such as plasmablasts, plasma cells, and B cells. In another embodiment, the cell has no or limited toxicity toward a healthy cell.

[0181] In any one of the above embodiments, the CAAR induces killing of the autoantibodyexpressing cell, including but not limited to, a plasmablast, a plasma cell, and a B cell.

[0182] In any one of the above embodiments, the genetically modified cell is a T cell. In this embodiment, the T cell expresses a CAAR comprising at least one autoantigen or a fragment thereof selected from the group consisting of GlialCAM, CRY AB, and ANO2.

[0183] In any one of the above embodiments, the extracellular domain comprises GlialCAM having an amino acid sequence of X₁SPPRAP (SEQ ID NO: 3) wherein X₁ is S that is phosphorylated or non-phosphorylated.

[0184] In any one of the above embodiments, the extracellular domain comprises GlialCAM or a fragment thereof having an amino acid selected from the group consisting of GlialCAM AA365-394 (SEQ ID NO: 32), GlialCAM AA365-394-pSer376 (SEQ ID NO: 33), GlialCAMLST-IOOIWO PATENT APPLICATION AA376-389 (SEQ ID NO: 34), GlialCAM AA376-389-pSer376 (SEQ ID NO: 35), and GlialCAM AA385-416 (SEQ ID NO: 36).

[0185] In any one of the above embodiments, the extracellular domain comprises ANO2 having an amino acid sequence of PGDIELGPLD (SEQ ID NO: 12).

[0186] In any one of the above embodiments, the extracellular domain comprises ANO2 or a fragment thereof having an amino acid sequence selected from the group consisting of ANO2 AA1-275 (SEQ ID NO: 37) and ANO2 AA134-153 (SEQ ID NO: 38).

[0187] In any one of the above embodiments, the extracellular domain comprises CRY AB having an amino acid sequence of PPGRRPFF (SEQ ID NO: 8).

[0188] In any one of the above embodiments, the extracellular domain comprises CRY AB or a fragment thereof having an amino acid sequence selected from the group consisting of CRY AB AA1-275 (SEQ ID NO: 39) and CRY AB AA2-33 (SEQ ID NO: 40).

[0189] In any one of the above embodiments, the extracellular domain comprises GlialCAM AA376-389-pSer376 (SEQ ID NO: 35). CRY AB AA8-15 (SEQ ID NO: 8), and ANO2 AA140-149 (SEQ ID NO: 12). The extracellular domain may further comprise a linker as described herein that forms a linkage between the GlialCAM AA376-389-pSer376 and CRY AB AA8-15, and a linkage between CRY AB AA8-15 and ANO2 AA140-149 in the extracellular domain.

[0190] In the above embodiment, the extracellular domain may further include a short oligo- or polypeptide linker as described herein, that forms a linkage between the GlialCAM AA376-389-pSer376 (SEQ ID NO: 35) and CRY AB AA8-15 (SEQ ID NO: 8), and a linkage between the CRY AB AA8-15 (SEQ ID NO: 8), and ANO2 AA140-149 (SEQ ID NO: 12) in the extracellular domain of the CAAR. The linker may include, but is not limited to, a glycine rich linker selected from the group consisting of GGGGG. GGSSG, GGGGSLVPRGSGGGGS. (GS)nwhere n is 2-5, GGSGGHMGSGG, GGSGGSGGSGG, GGSGpwhere p is 1-5, GSGGGTGGGSG, and GSGGSGGSGGSGGS.

[0191] In any one of the above embodiments, genetically modified cell may comprise a hinge and / or transmembrane domain as described herein, that is attached to the extracellular domain of the CAAR, or that is associated with one of the domains in the CAAR. A hinge and / or transmembrane domain may include, but not be limited to, human hinges such as the human Ig (immunoglobulin) hinge, and other hinges and / or transmembrane domains including, but not limited to, a hinge and / or transmembrane domain of an alpha, beta or zeta chain of a T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIR, 0X40, CD2, CD27, LFA-1 (CDlla, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7R α, ITGA1, VLA1, CD49a,LST-IOOIWO PATENT APPLICATION ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD 103, ITGAL, CDlla, LFA-1, ITGAM, CDllb, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A. LylO8), SLAM (SLAMF1, CD 150, IPO-3). BLAME (SLAMF8), SELPLG (CD 162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C.

[0192] In any one of the above embodiments, the transmembrane domain comprises a CD8 alpha chain hinge and / or transmembrane domain.

[0193] In any one of the above embodiments, the intracellular signaling domains may include, but are not limited to, a fragment or domain from one or more molecules or receptors including, but are not limited to, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcR gamma, FcR beta (Fc Epsilon Rib), CD79a, CD79b, Fcgamma Rlla, DAP10, DAP12, T cell receptor (TCR), CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS. lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CD5, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD 103, ITGAL, CD1 la. LFA-1. ITGAM. CD1 lb, ITGAX. CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyi08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162). LTBR. LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and any combination thereof.

[0194] In any one of the above embodiments, the intracellular signaling domain may comprise a CD3-zeta signaling domain by itself or in combination with any other desired cytoplasmic domain(s) useful in the context of the CAAR described herein. For example, the intracellular signaling domain of the CAAR can comprise a CD3 zeta chain portion and a costimulatory signaling region.

[0195] In one of the above embodiments, the genetically modified cell may further include a costimulatory domain selected from the group consisting of CD28 and tumor necrosis factor ligand superfamily member 9 (4- IBB).

[0196] Treatment of Autoimmune Diseases

[0197] In a fourth aspect, the present disclosure also provides methods for the prevention, treatment, and / or management an autoimmune disorder or disease which includes, but is not limited to, multiple sclerosis.LST-IOOIWO PATENT APPLICATION

[0198] The methods comprise administering to a subject in need thereof a genetically modified cell described herein in the present disclosure that binds to an autoantibody-expressing cell.

[0199] In one embodiment of the method, the present disclosure includes a method of treating, preventing, or managing multiple sclerosis in a subject in need thereof, comprising administering to the subject a genetically modified cell comprising a chimeric autoantibody receptor (CAAR) comprising an extracellular domain having at least one autoantigen or fragment thereof selected from the group consisting of GlialCAM, CRY AB, and ANO2, a transmembrane domain, and an intracellular signaling domain, wherein the cell expresses the CAAR and binds to an autoantibody-expressing cell.

[0200] In any one of the above embodiments of the method, the genetically modified cell is selected from the group consisting of a helper T cell, a cytotoxic T cell, a memory T cell, a regulatory T cell, a gamma delta cell, a natural killer cell, a cytokine induced killer cell, and a cell line thereof.

[0201] In any one of the above embodiments of the method, T cells isolated from a subject can be modified to express the appropriate CAAR, expanded ex vivo and then reinfused into the subject. The modified T cells recognize target cells, and become activated, resulting in killing of the autoimmune target cells.

[0202] In any one of the above embodiments, the autoantibody-expressing cell is at least one selected from the group consisting of a plasmablast, a plasma cell, and a B cell.

[0203] In any one of the above embodiments, the subject is a human.

[0204] In any one of the above embodiments of the method, non-limiting examples of disorders associated with autoantibody-expressing cells include MS which includes any one or more of the types of recognized MS: relapsing-remitting MS (RRMS), benign MS (a version of relapsing remitting MS with very mild attacks separated by long periods with no symptoms), primary progressive (PPMS), secondary progressive MS (SPMS), progressive relapsing MS (PRMS), clinically isolated syndrome (CIS; refers to the first episode of a condition that affects the myelin but after further testing the syndrome may be diagnosed as MS or a different condition), or radiologically isolated syndrome (refers to findings on MRIs of the brain and spinal cord that look like MS in someone without classic symptoms of MS).

[0205] In any one of the above embodiments of the method, the extracellular domain comprises GlialCAM having an amino acid sequence of X₁SPPRAP (SEQ ID NO: 3) wherein X₁ is S that is phosphorylated or non-phosphorylated.

[0206] In any one of the above embodiments of the method, the extracellular domain comprises GlialCAM or a fragment thereof having an amino acid sequence selected from the group consisting of GlialCAM AA365-394 (SEQ ID NO: 32), GlialCAM AA365-394-pSer376 (SEQLST-IOOIWO PATENT APPLICATION ID NO: 33), GlialCAM AA376-389 (SEQ ID NO: 34), GlialCAM AA376-389-pSer376 (SEQ ID NO: 35), and GlialCAM AA385-416 (SEQ ID NO: 36).

[0207] In any one of the above embodiments of the method, the extracellular domain comprises ANO2 having an amino acid sequence of PGDIELGPLD (SEQ ID NO: 12).

[0208] In any one of the above embodiments of the method, the extracellular domain comprises ANO2 or a fragment thereof having an amino acid sequence selected from the group consisting of ANO2 AA1-275 (SEQ ID NO: 37) and ANO2 AA134-153 (SEQ ID NO: 38).

[0209] In any one of the above embodiments of the method, the extracellular domain comprises CRY AB having an amino acid sequence of PPGRRPFF (SEQ ID NO: 8).

[0210] In any one of the above embodiments of the method, the extracellular domain comprises CRY AB or a fragment thereof having an amino acid sequence selected from the group consisting of CRYAB AA1-275 (SEQ ID NO: 39) and CRY AB AA2-33 (SEQ ID NO: 40).

[0211] In any one of the above embodiments of the method, the extracellular domain comprises GlialCAM AA376-389-pSer376 (SEQ ID NO: 35), CRY AB AA8-15 (SEQ ID NO: 8), and ANO2 AA140-149 (SEQ ID NO: 12).

[0212] In the above embodiment of the method, the extracellular domain further comprises a linker that forms a linkage between the GlialCAM AA376-389-pSer376 and CRY AB AA8-15, and a linkage between CRY AB AA8-15 and ANO2 AA140-149.

[0213] In this embodiment of the method, the linker is a glycine rich linker selected from the group consisting of GGGGG, GGSSG, GGGGSLVPRGSGGGGS, (GS)nwhere n is 2-5, GGSGGHMGSGG, GGSGGSGGSGG, GGSGpwhere p is 1-5, GSGGGTGGGSG, and GSGGSGGSGGSGGS.

[0214] In any one of the above embodiments of the method, the genetically modified cell further comprises a co-stimulatory domain selected from the group consisting of CD28 and tumor necrosis factor ligand superfamily member 9 (4-1BB).

[0215] In any one of the above embodiments of the method, the intracellular signaling domain comprises a CD3 zeta signaling domain.

[0216] In any one embodiment of the method, the transmembrane domain comprises a CD8 alpha chain hinge and transmembrane domain.

[0217] In any one of the above embodiments of the method, the method further comprises measuring at least one anti-EBNAl antibody, anti-GlialCAM antibody. anti-CRYAB antibody, or anti-ANO2 antibody level in a human tissue. In this embodiment, the human tissue comprises plasma, serum, blood, or cerebrospinal fluid (CSF).LST-IOOIWO PATENT APPLICATION

[0218] In any one of the above embodiments of the method, the method further comprises determining an antibody level of at least one antibody selected from the group consisting of anti-EBNA1 antibody, anti-GlialCAM antibody, anti-CRYAB antibody, and anti-ANO2 antibody.

[0219] In the above embodiment, the method further comprises following the determining step, administering the genetically modified cell comprising the CAAR to the subject based on a criteria wherein:(e) when anti-EBNAl antibody and / or anti-GlialCAM antibody levels are elevated in the subject, the subject is administered the CAAR comprising the extracellular domain comprising GlialCAM having an amino acid sequence of X1SPPRAP (SEQ ID NO: 3) wherein X1is S that is phosphorylated or non-phosphorylated; or(f) when anti-CRYAB antibody and anti-ANO2 antibody levels are elevated in the subject, the subject is administered the CAAR comprising the extracellular domain comprising CRY AB having an amino acid sequence of PPGRRPFF (SEQ ID NO: 8); or(g) when anti-ANO2 antibody levels are elevated in the subject, the subject is administered the CAAR comprising the extracellular domain comprising ANO2 having an amino acid sequence of PGDIELGPLD (SEQ ID NO: 12); or(h) when anti-GlialCAM antibody, anti-CRYAB antibody, and anti-ANO2 antibody levels are elevated in the subject, the subject is administered the CAAR comprising the extracellular domain having GlialCAM AA376-389-pSer376 (SEQ ID NO: 35), CRY AB AA8-15 (SEQ ID NO: 8), and ANO2 AA140-149 (SEQ ID NO: 12), and a linker that forms a linkage between GlialCAM AA376-389-pSer376 and CRYAB AA8-15, and a linkage between CRYAB AA8-15 and ANO2 AA140-149.

[0220] In any one of the above embodiments of the method, the genetically modified cell is administered once a month for the first two months, and then once every six months.

[0221] In any one of the above embodiments, the genetically modified cells of the present disclosure may be a type of vaccine for ex vivo immunization and / or in vivo therapy in a mammal. In this embodiment, the genetically modified cells are T cells. In this embodiment, the mammal is a human.

[0222] In any one of the embodiments, the modified T cells may be administered either alone, or as a pharmaceutical composition in combination with diluents and / or with other components such as IL-2 or other cytokines or cell populations.

[0223] Compositions / Pharmaceutical Compositions

[0224]

[0225] Pharmaceutical compositions of the present disclosure may comprise a CAAR as described herein in any one of the above embodiments, a vector comprising a CAAR asLST-IOOIWO PATENT APPLICATION described herein in any one of the above embodiments, or a population of genetically modified cells as described herein in any one of the above embodiments, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions of the present disclosure are in one aspect formulated for intravenous administration.

[0226] “An immunologically effective amount,’" “an anti-autoantibody effective amount,” “an autoimmune disease-inhibiting effective amount,” or “therapeutically effective amount” may be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject).

[0227] In one embodiment, a pharmaceutical composition may comprise a genetically modified cell such as T cells, as described herein, administered at a dosage of 104to 109cells / kg body weight, in some instances 105to 106cells / kg body weight, including all integer values within those ranges. T cell compositions may also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see. e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.

[0228] In one embodiment, a first dose 1 x 108cells / kg body weight with lymphodepletion or a dose of 1 x 108of the genetically modified cells without lymphodepletion.

[0229] Administration of the cells of the invention may be carried out using any convenient means, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient transarterially, subcutaneously, intradermally, intratumorally. intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In one embodiment, the T cell compositions of the present invention are administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell compositions of the present invention are administered by i.v. injection. The compositions of T cells may be injected directly into a tumor, lymph node, or site of infection.LST-IOOIWO PATENT APPLICATION

[0230] Methods and Compositions for Diagnostics and Detection

[0231] As demonstrated in the EXAMPLES below, positive HLA-DRB1 *15:01 status together with elevated antibody reactivity' against EBNA1 and either one or more of GlialCAM, CRY AB, or ANO2 peptides increases the risk for MS. EBNA1 and either one or more of GlialCAM, CRY AB. or ANO2 peptides may be used for detecting the presence of antibody reactivity with allele dose of HLA-DRB1 *15:01, the strongest MS risk allele in a biological sample, either quantitatively or qualitatively.

[0232] In one embodiment, a biological sample comprises a cell or tissue, such as plasma, serum, blood, or cerebrospinal fluid (CSF).

[0233] In one embodiment, any of the EBNA1, GlialCAM, CRY AB, and / or ANO2 peptides described herein may be labelled with a detectable molecule or substance, such as a fluorescent molecule, a radioactive molecule or any other label known in the art.

[0234] For example, any of the EBNA1, GlialCAM, CRY AB, and / or AN02 peptides described herein may be labelled with a radioactive molecule. Examples of suitable radioactive molecules include but are not limited to radioactive atoms used for scintigraphic studies such as123I,124I,111In,186Re, and188Re.

[0235] The present disclosure includes a method of diagnosing multiple sclerosis including steps of:1) contacting a biological sample of a subject with one or more of the EBNA1, GlialCAM, CRYAB, and / or ANO2 peptides described herein; and2) detecting and / or quantifying antibody reactivity' to one or more of the EBNA1, GlialCAM, CRY AB. and / or ANO2 peptides, whereby detection of elevated levels of anti- EBNA1 antibody, anti-GlialCAM antibody, anti-CRYAB antibody, and / or anti-ANO2 antibody is indicative of an increased risk of multiple sclerosis (MS) or a diagnosis of MS.

[0236] In one embodiment of the method, the peptides useful for detecting risk of MS or for diagnosing MS are exemplified in Supplementary Table SI.

[0237] In any one of the above embodiments, the peptides useful for detecting risk of MS or for diagnosing MS include GlialCAM AA365-394 (SEQ ID NO: 32), GlialCAM AA365-394-pSer376 (SEQ ID NO: 33), GlialCAM AA376-389 (SEQ ID NO: 34), GlialCAM AA376-389-pSer376 (SEQ ID NO: 35), and GlialCAM AA385-416 (SEQ ID NO: 36), ANO2 AA140-149 (SEQ ID NO: 12), ANO2 AA1-275 (SEQ ID NO: 37), ANO2 AA134-153 (SEQ ID NO: 38), CRY AB AA8-15 (SEQ ID NO: 8), CRY AB AA1-275 (SEQ ID NO: 39) and CRY AB AA2-33 (SEQ ID NO: 40).

[0238] Articles of Manufacture and KitsLST-IOOIWO PATENT APPLICATION

[0239] In another aspect of the disclosure, an article of manufacture containing an isolated peptide as described herein and exemplified in Supplementary Table SI, and other materials useful for the diagnosis, treatment / prophylaxis, and / or management of an autoimmune disorder such as multiple sclerosis is provided.

[0240] The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or may be combined w ith another composition effective for treating, preventing and / or diagnosing the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an isolated peptide as described herein (and exemplified in Supplementary Table SI) of the disclosure.

[0241] The label or package insert may indicate that the composition is used for diagnosing the autoimmune disorder or disease such as multiple sclerosis. Moreover, the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises an isolated peptide as described herein and exemplified in Supplementary Table SI; and (b) a second container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0242] In another aspect of the disclosure, an article of manufacture containing a CAAR, a vector, or a genetically modified cell as described herein, and other materials useful for the treatment, prevention, and / or management of an autoimmune disorders such as multiple sclerosis is provided.

[0243] In this embodiment, the article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or may be combined with another composition effective for treating, preventing and / or managing an autoimmune disorder or disease such as multiple sclerosis, and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is a CAAR, a vector, or a genetically modified cell as described herein of the disclosure.LST-IOOIWO PATENT APPLICATION

[0244] In this embodiment, the label or package insert indicates that the composition is used for treating an autoimmune disorder or disease such as multiple sclerosis. Moreover, the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises a CAAR, a vector, or a genetically modified cell as described herein of the disclosure; and (b) a second container with a composition contained therein, wherein the composition comprises a further therapeutic agent. Alternatively, or additionally, the article of manufacture may further comprise a third container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0245] Finally, the disclosure also provides kits comprising at least one isolated peptide as described herein and exemplified in Supplementary Table SI. Kits containing an isolated peptide as described herein and exemplified in Supplementary Table SI of the disclosure find use in detecting increased or decreased reactivity of antibodies associated with an autoimmune disease or disorder such as MS for diagnostic assays. Kits of the disclosure can contain an isolated peptide as described herein and exemplified in Supplementary Table SI, coupled to a solid support, e.g., a tissue culture plate or beads (e.g., sepharose beads). Kits can be provided which contain such peptides for detection and quantification in vitro. Such peptides useful for detection may be provided with a label such as a fluorescent or radiolabel.

[0246] EXAMPLES

[0247] As demonstrated herein, antibody responses against EBNA1 and GlialCAM in a large cohort of 650 MS patients and 661 matched population controls were measured and compared to responses against CRY AB and ANO2. It is confirmed that elevated IgG responses against EBNA1 and all three CNS-mimic antigens associate with increased MS risk. Blocking experiments confirmed the presence of cross-reactive antibodies and molecular mimicry between EBNA1 and GlialCAM, and accompanying antibody responses against adjacent peptide regions of GlialCAM suggest epitope spreading. Antibody responses against EBNA1, GlialCAM, CRY AB, and ANO2 are elevated in MS patients carrying the main risk allele HLA-DRB1 *15:01. and combinations of HLA-DRB1 *15:01 with anti-EBNAl and anti-GlialCAM antibodies increase MS risk significantly and in an additive fashion. In addition, antibody reactivities against more than one EBNA1 peptide and more than one CNS-mimic increased the MS risk significantly but modestly. Overall, it was shown that molecular mimicry between EBNA1 and GlialCAM is likely an important molecular mechanism contributing to MS pathology.LST-IOOIWO PATENT APPLICATION

[0248] Multiple sclerosis is an autoimmune disease of the brain and spinal cord, leading to disability in young adults. Infection with Epstein Barr virus (EBV) is a pre-requisite for developing the disease. The present inventors have demonstrated that antibody responses against the virus protein EBNA1 cross-react with brain proteins of MS patients and contribute to the disease. In addition, the present inventors have confirmed in a large cohort of MS patients and controls that the presence of these antibodies increases the risk for MS, and correlate them with the major genetic risk factor for MS. A combination of multiple antibodies and the genetic risk factor increases the risk for MS in an additive fashion.

[0249] Increased serum anti-EBNAl IgG titers are delayed for several weeks to months after an infection with EBV and their development indicate the end of the acute lytic phase of infection and establishment of viral latency in B cells (De Paschale M. and P. Clerici, Serological diagnosis of Epstein-Barr virus infection: Problems and solutions. World J. Virol. 1, 31–43 (2012); and Henle W. et al., Antibody responses to Epstein-Barr virus-determined nuclear antigen (EBNA)-1 and EBNA-2 in acute and chronic Epstein-Barr virus infection. Proc. Natl. Acad. Sci. U. S. A. 84, 570-574 (1987)). In most healthy EBV-infected individuals, titers stay elevated for life and MS patients generally have higher anti-EBNAl IgG titers than healthy individuals ( Bjomevik, et al., Science 375, 296-301 (2022); Hedstrom, et al., Frontiers in Neurology [Preprint] (2020). Available at: http: / / dx.doi.org / 10.3389 / fneur.2019.01368; and Cortese, et al., JAMA Neurol. 81, 515-524 (2024)).

[0250] EBNA1 is a key EBV transcription factor, the only EBV protein that is expressed during all forms of latency (except latency 0), and the only nuclear antigen expressed during the lytic phase of infection (34). In addition to regulating gene expression, it tethers the viral episome to the human chromosome during mitosis and is essential for viral replication and survival during the latent phase. As an immunogen, EBNA1 is unique for several reasons:(i) EBNA1 is chronically expressed in B cells at low levels, resulting in repeated antigen exposures over years and decades. This provides the B cell response with ample time to develop from an anti-viral response into an autoimmune response through molecular mimicry ( Bjomevik, et al., Science 375, 296-301 (2022); and Jons D. et al., Seroreactivity against lytic, latent and possible cross-reactive EBV antigens appears on average 10 years before MS induced preclinical neuroaxonal damage. J. Neurol. Neurosurg. Psychiatry 95, 325-332 (2024)).(ii) EBNA1 tightly binds human and viral nucleic acids ( Dheekollu J. et al., Cell-cycle-dependent EBNA1-DNA crosslinking promotes replication termination at oriP and viral episome maintenance. Cell 184, 643-654.e13 (2021)), which likely increases its immunogenicity, akin to several autoantigens that are elevated in rheumatic diseases including systemic lupus erythematosus (SLE) (37).LST-IOOIWO PATENT APPLICATION (iii) Lastly, EBNA1 harbors an immunogenic region between the second glycine-arginine repeat region and the C-terminal DNA binding domain (-AA380-450), with antibodies against this region eliciting molecular mimicry with several CNS antigens in MS as well as lupus antigens in SLE (15-18, 31, 38). Antibody reactivity against this region is higher in MS patients than in healthy EBV -infected individuals ( Bjomevik. et al., Science 375, 296-301 (2022): Ruprecht K. et al., J. Neuroimmunol. 272, 56-61 (2014)-22, 31); Jafari N., et al., J. Clin. Virol. 49, 26-31 (2010); Salzer J. et al., Mult. Scler. 19, 1587-1591 (2013); and Sundqvist E. et al., Genes Immun. 13, 14-20 (2012).

[0251] The present inventors found elevated antibody reactivities against full-length protein as well as the truncated domains EBNA1 AA1-120 and EBNA1 Mimic-region+DBD (AA380-641), but not the N-terminal GR-repeat region (AA2-89). As expected, the EBNA1 Mimic-region+DBD yielded the highest relative MS risk of all tested proteins (OR: 2.67, CI: 2.06 -3.49), superseding EBNA1 Full-Length. An even higher MS risk was observed for the 30mer EBNA1 peptide AA381-410 (OR: 3.27, CI: 2.50 - 4.28), which encompasses the regions that mimic GlialCAM (central epitope: AA393-398) (15) and CRY AB (central epitope: AA399-406, wider epitope: AA399-415) (17) (Fig. 3A).

[0252] It was previously shown that, despite both autoantigens mimicking a common viral protein, MS patients usually have autoantibodies against either CRY AB or ANO2, but less frequently against both antigens (Thomas O. G. et al., Sci. Adv. 9, eadg3032 (2023)). This partial exclusion was confirmed here for the regions CRY AB Full-Length and ANO2 AA1-275.

[0253] It was also confirmed that there are relatively low autoantibody levels to CRY AB Full-Length, but higher antibody reactivities to linear epitopes of CRY AB and these are associated with MS (Thomas O. G. et al., Sci. Adv. 9, eadg3032 (2023)). CRYAB Full-Length tertiary structures seemingly obscure linear epitopes, and a breakdown of CRY AB in inflamed tissues might be necessary' for the autoantibody response against CRY AB peptides. Reactivity' against all GlialCAM peptides correlated more strongly with ANO2 AA134-153 than with CRY AB AA2-33. In MS patients. GlialCAM ICD and the main GlialCAM epitope AA370-389 correlated strongly with ANO2 AA134-153, but less with CRY AB AA2-33. This might seem counterintuitive, as the mimic regions for GlialCAM and CRY AB on EBNA1 are directly adjacent, whereas ANO2 is located further C-terminal at a distance of approximately 25 amino acids from CRY AB (Fig. 3A). A possible explanation for this observation could be steric epitope masking of one EBNA1 epitope by antibodies against the adjacent epitopes during B cell maturation in the germinal center (40). In addition, it is interesting that CRY AB has the properties of an immune checkpoint inhibitor, and an anti-CRYAB response might therefore initiate additional immunomodulatory mechanisms that skew the B cell response, unlike ANO2LST-IOOIWO PATENT APPLICATION and GlialCAM, which share functions as an ion channel and an auxiliary subunit of a of an ion channel (CLCN2), respectively ( Lanz T. V. et al., Nature 603, 321-327 (2022); Thomas O. G. et al., Sci. Adv. 9, eadg3032 (2023); Tengvall K. et al.. Proc. Natl. Acad. Sci. U. S. A. 116, 16955-16960 (2019); Ousman S. S. et al., Protective and therapeutic role for alphaB-crystallin in autoimmune demyelination. Nature 448, 474-479 (2007) and Jeworutzki E. et al., GlialCAM, a protein defective in a leukodystrophy, serves as a C1C-2 Cl(-) channel auxiliary subunit. Neuron 73, 951-961 (2012)).

[0254] Anti-GlialCAM antibody reactivity is not restricted to the peptide region AA370-389 but was observed for all adjacent peptides ranging from AA305 to AA416, and antibody reactivities against these regions were highly correlated. The data in the EXAMPLES below demonstrate that reactivity against GlialCAM AA370-389 can be blocked effectively by EBNA1 AA386-405 (SEQ ID NO: 2 or SEQ ID NO: 18), indicating that the anti-GlialCAM response is spawned from an ant-EBNAl response. Antibodies against the adjacent regions are likely a result of intramolecular epitope spreading.

[0255] As shown in the EXAMPLES, while plasma incubation with EBNA1 AA386-405 (SEQ ID NO: 2 or SEQ ID NO: 18) robustly blocked antibody binding to GlialCAM AA370-389, the inverse experiment showed only partial inhibition, aligning with the understanding of directionality in the development of the anti-GlialCAM antibody response, i.e.. stemming from an initial anti-EBNAl response. This result also indicates an abundance of anti-EBNAl AA386-405 antibodies, of which only a fraction cross-bind to GlialCAM AA370-389 (SEQ ID NO: 3, SEQ ID NO: 30 or 31). In contrast, the majority of anti-GlialCAM AA370-389 seemingly develops from the response against EBNA1 AA386-405 and can therefore be crossblocked. A similar level of directionality was previously observed for ANO2 ( Tengvall K. et al., Proc. Natl. Acad. Sci. U. S. A. 116, 16955-16960 (2019)).

[0256] Interestingly, the data in the EXAMPLES showed that antibody responses against GlialCAM ECD did not correlate with responses against the peptides mentioned above spanning the intracellular domain, but rather against the N-terminal GR-repeat region of EBNA1 and EBNA3C. EBNA3C is recognized as an important CD8 T cell antigen (43), but to our knowledge not as a relevant molecular mimic. Anti-ANO2 AA1-275 correlates with EBNA3C and this particular correlation seems to be specific for the MS group. However, the antigens GlialCAM ECD. EBNA1 GR-repeat, and EBNA3C individually are not associated with increased MS risk, diminishing the likelihood for their pathological relevance in MS.

[0257] An initial study on molecular mimicry between EBNA1 and GlialCAM in MS demonstrated that phosphorylation of serine at residue 376 in the GlialCAM protein facilitates cross-binding between the two epitopes. The current data in the EXAMPLES, derived from aLST-IOOIWO PATENT APPLICATION significantly larger patient cohort, shows similarly elevated antibody responses against the phosphorylated and unphosphorylated versions of the 30mer GlialCAM AA365-394 (SEQ ID NOS: 29 and 28, respectively) and the 20mer GlialCAM AA370-389 (SEQ ID NO: 3, 30, or 31), and reactivities against the phosphorylated and unphosphorylated versions strongly correlate with each other. The magnitude of blocking by EBNA1 AA386-405 however was significantly larger for the phosphorylated version, indicating that this posttranslational modification indeed facilitates molecular mimicry - albeit this might not be essential for the mechanism, or might quickly be overcome by epitope spreading to the non-phosphorylated peptide.

[0258] Neuroaxonal damage, indicated by elevated Nfl levels, was reported in pre-clinical MS approximately a decade after primary EBV infection with or without clinical IM ( Bjomevik K. et al.. Science 375, 296-301 (2022); and Jons D. et al., J. Neurol. Neurosurg. Psychiatry 95, 325-332 (2024)). Elevated Nfl levels coincide with the emergence of antibodies against latent and lytic EBV antigens and in some patients with reactivity to ANO2 (Jons D. et al., J. Neurol. Neurosurg. Psychiatry 95, 325-332 (2024))). Association of elevated Nfl levels with antibody reactivity to GlialCAM would suggest a direct impact of anti-GlialCAM antibodies on demyelination and neurodegeneration. Animal studies by the present inventors and others indicated that immunization with EBNA1 peptides (AA386-405 and AA411-426) aggravated mouse models of MS and these effects were attributed to increased anti-GlialCAM and anti-MBP B cell responses ( Lanz T. V. et al., Nature 603, 321-327 (2022); and Jog N. R. et al., J. Autoimmun. 106, 102332 (2020)). However, the present data in the EXAMPLES found no correlation with Nfl in the dataset, indicating that anti-GlialCAM antibodies do not drive neuronal damage at the time of the blood draws of our cohort.

[0259] HLA-DRB1 *15:01 is the major genetic risk factor for MS (Moutsianas L. et al., Nat. Genet. 47, 1107–1113 (2015)). Preferred presentation of myelin peptides by antigen presenting cells and activation of CD4 T cells is a likely mechanistic explanation for its pathological relevance in the disease. Multiple studies found the epitope AA83-99 of myelin basic protein (MBP) to be a relevant epitope presented on HLA-DRB1 *15:01 (Ousman S. S. et al., Nature 448, 474-479 (2007); and Jeworutzki E. et al., Neuron 73, 951-961 (2012)). Additionally, there is evidence that humanized HLA-DRB1 *15: 01 -positive mice have diminished immune control of EBV despite increased numbers of CD8+ T cells, indicating insufficient CD4+ T cell help to CD8+ T cells ( Martin R. et al., Trends Genet. 37, 784-797 (2021)). In the same mouse model, CD4+ T cell responses against several myelin proteins were induced by EBV infection. Prior studies have found that antibody responses against EBNA1 were elevated in individuals cartying HLA-DRB1 *15:01, and this included EBNA1 Full-Length protein and fragments spanning theLST-IOOIWO PATENT APPLICATION mimicking region AA380-450 (e.g. AA325-641, AA385-420, and AA393-412) ( Jog N. R. et al., J. Autoimmun. 106, 102332 (2020); Dheekollu J. et al., Cell-cycle-dependent EBNA1-DNA crosslinking promotes replication termination at oriP and viral episome maintenance. Cell 184, 643-654.e13 (2021); Tamaki H. et al., Major histocompatibility complex class I-restricted cytotoxic T lymphocyte responses to Epstein-Barr virus in children. J. Infect. Dis. 172. 739-746 (1995); and Patterson N. et al. Population structure and eigenanalysis. PLoS Genet. 2, e190 (2006)). Similarly, antibody reactivity against CRY AB AA3-17 was associated with HLA-DRB1*15:01 (Jog N. R. et al., J. Autoimmun. 106, 102332 (2020)).

[0260] The data in the present EXAMPLES confirm elevated anti-EBNAl AA381-410 reactivity in MS patients heterozygous and homozygous for HLA -DRB1 *15:01, and in addition, it was shown that anti-GlialCAM AA370-389 reactivity is similarly correlated with HLA-DRB1 *15:01 status. Both relationships are more significant in MS patients than in controls, suggesting that HLA-DRB1 *15:01 promotes the development of molecular mimicry between EBNA1 and GlialCAM, and that this mechanism is relevant in MS. We also demonstrate an additive increase of MS risk with (i) positive HLA-DRB1 *15:01 status, (ii) anti-EBNAl peptide reactivity, and (iii) the corresponding reactivity against the CNS-mimicking peptide, and this is the case for each of the three EBNA1 mimics (HLA-DRB1 *15:01 + EBNA1 AA381-410 + GlialCAM AA365-394, OR: 9.40. CI 6.04 - 14.86; HLA-DRBl*15:01 + EBNA1 AA381-410 + CRY AB AA2-33, OR: 10.01, CI 6.38 - 15.97; HLA-DRB1 *15:01 + EBNA1 AA421-450 + ANO2 AA134-153, OR: 5.42, CI 3.65 - 8.13). Notably, the absence of the protective HLA class I allele HLA-A *02:01 is another additive factor that increases the MS risk by a factor of approximately two.

[0261] Notably, a prior study had described highly significant differences in anti-GlialCAM AA370-389 IgG levels between MS patients and healthy controls in a cohort of 540 individuals, with p-values below 0.0001 (30). 100% of MS patients in this study had relatively high anti-GlialCAM antibody levels, and these were distinct from 60% of healthy controls whose antibody reactivities were approximately 2-fold lower. Interestingly, anti-GlialCAM AA370-389 antibody reactivity in this study was not associated with HLA-DRB1 *15:01 (30). This clear distinction of all MS patients from the majority of healthy individuals is different from the data presented here. While significantly elevated antibody responses against GlialCAM are also described, the significance level is lower, and there is a sizeable number of MS patients in the studies conducted in the EXAMPLES who do not have elevated anti-GlialCAM antibodies. However, as pointed out above, anti -GlialCAM reactivity is significantly correlated with HLA-DRB1 *15:01. A follow-up study by the same authors suggested that the combination of multiple highly elevated antibody reactivities against four EBNA1 peptides spanning the region AA386-LST-IOOIWO PATENT APPLICATION 445 is highly associated with increased MS risk. Similarly, combined elevated antibody reactivities against the three mimicking CNS peptides GlialCAM AA370-389, CRY AB AA2-21, and AN02 AA135-154, as well as the MBP region AA205-224, are highly associated with MS (Vietzen H. et al., Accumulation of Epstein-Barr virus-induced cross-reactive immune responses is associated with multiple sclerosis. J. Clin. Invest. 134 (2024)).

[0262] In this study, over 98% of MS patients had three or more antibody reactivities against either set of peptides, while in healthy individuals either combination was only found in 11.5% and 21.5% of subjects, respectively. The difference between groups that were derived from the data in the EXAMPLES - analyzed in a similar way - reaches significance as well, but it is distinctly smaller: 53.8% of MS patients vs. 31.9% of controls have >3 antibodies against EBNA1 peptides, and 56.5% of MS patients vs. 39.3% of controls have >3 mimic peptides. Based on their data, the prior study calculated an OR of 655.8 for MS vs. healthy with >2 elevated anti-EBNA antibodies, and an OR of 1,366 with >3 antibodies; with >2 anti -mimic antibodies the OR was 459.8 and with >3 antibodies it was 243.1. These are impressive numbers that would make tests for these combinations of antibodies valuable supportive biomarkers for MS diagnosis, and could even be considered as early detection parameter for at-risk family members of MS patients.

[0263] The present study, however, does not replicate the increased MS risk associated with these antibodies at the same magnitude. In the cohort of the present study, the odds ratio (MS vs. healthy) with >1 elevated anti-EBNA antibodies is elevated at 3.16 (CI: 2.23-4.51), with >2 antibodies at 2.41 (CI: 1.82-3.16), and with >3 at 2.49 (CI: 1.9-3.27). With >1 anti-CNS-mimic antibody the odds ratio is 2.36 (CI: 1.74-3.22), with >2 antibodies it is 2.1 (CI: 1.6-2.76), and with >3 it is 2.13 (CI: 1.62-2.8). While significant, the numbers are more than two orders of magnitude lower than those previously described (Vietzen H. et al., J. Clin. Invest. 134 (2024)). Potential reasons for the observed discrepancy include differences in patient and control cohorts, e.g. the rates of IM were different. However, even when the present data was adjusted for IM history, it did not significantly alter the results. In addition, differences could stem from the peptides antigens used: EBNA1 AA386-405 and GlialCAM AA370-389 were used in both studies; EBNA1 AA393-412, EBNA1 AA409-428, CRY AB AA2-21, and AN02 AA135-154 were used in the prior study (Vietzen H. et al., J. Clin. Invest. 134 (2024)).

[0264] In contrast, EBNA1 AA401-430 (SEQ ID NO: 19), EBNA1 AA421-450 (SEQ ID NO: 20), CRY AB AA2-33 (SEQ ID NO: 36), and ANO2 AA134-153 (SEQ ID NO: 34) were used in by the present inventors; EBNA1 AA426-445 and MBP AA205-224 were not included in the present studies. Different IgG detection methods were used (ELISA vs. bead-based method), and differences in statistical analyses could have influenced the results to some degree. The analysisLST-IOOIWO PATENT APPLICATION conducted herein was adjusted for population stratification, sex, age, and plate-based batch effects. However, repeating the analysis adjusting only for sex and age as done previously (Vietzen H. et al., J. Clin. Invest. 134 (2024)), generated similar results to those in FIG. 5A-5C (see also FIGS. 20A-20C). Additional validation studies in large independent cohorts will be necessary to determine the magnitude of increased MS risk levels associated with multiple elevated anti-EBNAl and anti-mimic antibodies, and their potential usefulness as biomarkers.

[0265] In summary. our study demonstrates in a large cohort of MS patients and matched population-based controls that elevated anti-EBNAl and anti-GlialCAM antibody levels are associated with increased risk for MS. Both elevated reactivities are associated with HLA-DRB1*15: O1. and a combination of positive HLA-DRB1 *15:01 status, anti-EBNAl peptide reactivity and the matching anti-GlialCAM reactivity further increases MS risk in an additive fashion. The same combination with anti-ANO2 and anti -CRY AB reactivities also increases MS risk. While our data is significant, the overall odds ratios are modest and range from an OR of 3.27 (EBNA1 AA381-410) and an OR of 2.63 (GlialCAM AA385-416) to an OR of 9.40 (combination of HLA-DRB1*15: O1, anti-EBNAl AA381-410, anti-GlialCAM AA365-394). Significantly more MS patients harbor between 1 and 3 anti-EBNAl peptide antibodies and antimimic antibodies than controls, but with odds ratios between 2.1 and 3.16, the increase in MS risk conveyed by multiple reactivities is also modest. Blocking experiments confirmed cross-reactive antibodies and molecular mimicry between EBNA1 and GlialCAM, and antibody reactivities against multiple intracellular peptides suggests that intramolecular epitope spreading broadens the immune response. Molecular mimicry between EBNA1 and AN02, CRY AB, and GlialCAM is likely an important molecular mechanism contributing to MS pathology.

[0266] EXAMPLE 1 - Antibody reactivity to EBNA1 and GlialCAM is associated with increased MS risk

[0267] The present study followed up on the initial data that described cross -reactivity between EBNA1 and GlialCAM. A bead-based assay was utilized to screen for IgG reactivity against EBNA1 and GlialCAM in a large cohort that included plasma samples of 650 MS patients and 661 sex and age matched population-based controls (Table 1).

[0268] TABLE 1LST-IOOIWO PATENT APPLICATION Table 1: Demographies and clinical characteristics of the study cohort.Characteristic Case (n=650) Control (n=661)Age (years), mean (SD) 40.21 (10.32) 42.93 (10.43) BMI (kg / m²), mean (SD) 25.06 (4.67) 24.83 (4.32) Disease Duration (years), mean (SD) 4.96 (5.95) NAMSSS, median (25th, 75thquartile) 3.65 (1.67, 5.58) NAGender, n (%)Male 187 (28.77) 163 (24.66) Female 463 (71.23) 498 (75.34) MS course, n (%)Control NA 661 (100)PPMS 18 (2.77) NAPRMS 9 (1.38) NARRMS 536 (82.46) NASPMS 68 (10.46) NANA 19 (2.92) NAinfectious mononucleosis. n (%)Yes 88 (13.54) 61 (9.23)No 468 (72.00) 573 (86.69)Do not know 47 (7.23) 26 (3.93)NA 47 (7.23) 1 (0.15) Treatment, n (%)None 406 (62.46) NA1st Line 182 (28.00) NA2nd Line 30 (4.62) NANA 32 (4.92) NASD, Standard Deviation; BMI, Body Mass Index; MS, Multiple Sclerosis; PPMS, Primary Progressive MS; PRMS, Progressive Relapsing MS; RRMS, Relapsing-Remitting MS; SPMS, Secondary Progressive MS; MSSS, Multiple Sclerosis Severity Score.

[0269] EBNA1 and GlialCAM proteins were included, as well as individual peptides spanning the regions of interest, and differential levels of reactivities and the individual relationships between reactivities against each region were evaluated. The data corroborate prior studies that MS risk is associated with elevated antibody levels against EBNA1 AA386-405 and GlialCAM AA370-389 and the adjacent regions of both proteins. Antibody reactivities against the broader regions of both proteins are strongly associated with each other. Anti-GlialCAM reactivity against the initially described peptide epitope GlialCAM AA370-389 can be blocked with the EBNA1 peptide AA386-405 (SEQ ID NO: 22), confirming the presence of cross-reactive antibodies. However, the antibody response against GlialCAM extends beyond this narrow region and encompasses adjacent peptideLST-IOOIWO PATENT APPLICATION regions, likely generated by epitope spreading (Lehmann P. V. et al., Spreading of T-cell autoimmunity to cryptic determinants of an autoantigen. Nature 358, 155-157 (1992); and Dai Y. D. et al., Antigen processing by autoreactive B cells promotes determinant spreading. Cell. Mol. Immunol. 2, 169-175 (2005)).

[0270] In addition, the antibody reactivity was correlated with allele dose of HLA-DRB1*15:01, the strongest MS risk allele. HLA-DRB1*15:01 conveys a four-fold (heterozy gous) and eight-fold (homozygous) increased risk for developing MS (7). The allele shapes the adaptive immune response by enabling preferential presentation of certain peptide antigens on antigen-presenting cells (APC). B cells are effective APCs for their cognate antigens and can promote autoreactive CD4 T cell responses. CD4 T cell help, in turn, promotes maturation of B cells and secretion of antibodies. Enhanced binding of myelin antigens and CD4 T cell responses against CNS antigens have been shown (Martin R. et al., Multiple sclerosis: doubling down on MHC. Trends Genet. 37, 784-797 (2021); and Zdimerova H. et al., Attenuated immune control of Epstein-Barr virus in humanized mice is associated with the multiple sclerosis risk factor HLA-DR15. Eur. J. Immunol. 51, 64-75 (2021)), and recent data suggest that the HLA-DRB1*15:01 allele facilitates cross-presentation of intracellular EBV antigens in EBV -infected B cells to be presented on HLA class II (DrosuN. et al., CD4 T cells restricted to DRB 1*15:01 recognize two Epstein-Barr virus glycoproteins capable of intracellular antigen presentation. Proc. Natl. Acad. Sci. U. S. A. 121 (2024)). HLA-DRB1 *15:01 could be an important factor that facilitates the development of molecular mimicry from an anti-EBNAl antibody response towards GlialCAM.

[0271] The present inventors showed that positive HLA-DRB1 *15:01 status together with elevated antibody reactivity against EBNA1 and either GlialCAM, CRY AB, or AN02 synergistically increases the risk for MS.

[0272] The present case-control study included plasma samples from 1,311 individuals, comprising 650 MS cases and 661 age- and sex-matched population-based controls from the Swedish Nationwide Epidemiological Investigation of MS (EIMS) cohort (Table 1) ( A. K. Hedstrom, M. Baamhielm, T. Olsson, L. Alfredsson, Tobacco smoking, but not Swedish snuff use, increases the risk of multiple sclerosis. Neurology 73, 696–701 (2009)).

[0273] The average age was 40.2 ± 10.32 and 42.9 ± 10.43 years for the MS and control groups, respectively. The female-to-male ratio was 2.47 among cases and 3.05 in the control group, consistent with the gender distribution observed in typical MS cohorts. The majority of MS patients had a relapsing-remitting disease course (RRMS, 82.5%), followed byLST-IOOIWO PATENT APPLICATION secondary progressive MS (SPMS, 10.5%), primary progressive MS (PPMS, 2.8%), and progressive relapsing MS (PRMS, 1.4%). IM rates, body mass index (BMI), and Multiple Sclerosis Severity Score (MSSS) are summarized in Table 1.

[0274] All antibody reactivities in plasma samples were measured using a fluorescence-activated bead-based assay (Supplementary Table SI).LST-IOOIWO PATENT APPLICATIONSupplementary Table SI. List of EBV antigens and CNS self-antigens included in the bead-based array.SEQ ID NO: 16 SEQ ID NO: 17SEQ IDNO: 18SEQ IDNO: 19 SEQ ID NO: 20SEQ ID NO: 21SEQ ID NO: 22SEQ ID NO: 23SEQ ID NO: 24SEQ ID NO: 25SEQ ID NO: 26 SEQ ID NO: 27SEQ ID NO: 28 SEQ ID NO: 29 SEQ ID NO: 30 SEQ ID NO: 31SEQIDNO: 32 SEQIDNO: 33 SEQIDNO: 34 SEQIDNO: 35.. SEQ ID NO: 36SEQ ID NO: 37SEQ ID NO: 38SEQ ID NO: 39 SEQ ID NO: 401 LLJL „..x....>_ SEQ ID NO: 41 EBV and CNS antigens, including EBNA1, EBNA3C, GlialCAM, CRYAB, and ANO2, were included in tire bead-based array as proteins, protein fragments, and peptides. Information for each antigen is provided as UniProt ID, vendor, catalog ID, peptide sequences, and the type of carrier magnetic bead.LST-IOOIWO PATENT APPLICATION

[0275] In agreement with prior findings, reactivity to full-length EBNA1 protein was increased in MS patients over healthy (odds ratio (OR): 1.97, confidence interval (CI): 1.53 – 2.54) (Fig. 1, Supplementary Table S2), and a similar difference was observed for all included truncated EBNA1 protein versions except for the N-terminal glycine / arginine repeat region (EBNA1 GR-repeat, residues AA2-89).

[0276] The difference was most pronounced for the C-terminal EBNA1 Mimic-Region+DBD (DBD, DNA-binding domain) (OR: 2.67, CI: 2.06 - 3.49, residues AA380-641), which includes the region that resembles GlialCAM, ANO2, CRY AB, and MBP (Lanz T. V. et al., Nature 603, 321-327 (2022); Jog N. R. et al., J. Autoimmun. 106, 102332 (2020): Thomas O. G. et al., Sei. Adv. 9, eadg3032 (2023); and Tengvall K. et al.. Proc. Natl. Acad. Sci. U. S. A. 116, 16955-16960 (2019)), and this result was corroborated on a peptide level, as all EBNA1 30mer peptides covering this region (AA361-470) (SEQ ID NOS: 20-25) showed differentially elevated antibody reactivities in the MS group, with the most pronounced difference for EBNA1 peptide AA381-410 (SEQ ID NO: 21) (OR: 3.27, CI: 2.50 - 4.28).LST-IOOIWO PATENT APPLICATION

[0277] Supplementary Table S2. Association of antibody reactivities against the EBV antigens EBNA1 and EBNA3C, and the self-antigens GlialCAM, CRY AB, and ANO2 with MS risk using logistic regression.Antigen Cases(n) Controls(n) OR SE CI95_Low CI95-High P_value GlialCAM_ECD 579 606 1.22 0.13 0.96 1.56 nsGlialCAM_FL 536 572 1.08 0.13 0.84 1.39 ns GlialCAM_ICD 527 562 1.81 0.13 1.40 2.34 0.00003749 GlialCAMpp305to334 541 559 2.30 0.13 1.78 2.99 3.55E-10 GlialCAMpp325to354 546 567 2.37 0.13 1.83 3.08 6.016E-11 GlialCAMpp345to374 544 565 1.96 0.13 1.52 2.54 2.215E-07 GlialCAMpp365to394 539 557 2.30 0.13 1.78 2.99 3.597E-10 GlialCAMpp365to394_Phos376 543 566 2.35 0.13 1.82 3.06 1.057E-10 GlialCAMpp370to389 541 562 1.91 0.13 1.48 2.46 6.287E-07 GlialCAMpp370to389_Phos376 536 570 2.14 0.13 1.65 2.77 8.146E-09 GlialCAMpp385to416 536 566 2.63 0.13 2.02 3.43 7.473E-13 EBNA1_1_aa1to120 538 562 1.58 0.13 1.23 2.03 0.0003432 EBNA1_2_aa380to641 541 566 2.67 0.14 2.06 3.49 3.448E-13 EBNA1_FL 550 570 1.97 0.13 1.53 2.54 1.362E-07 EBNANtermGRegion 543 581 0.97 0.13 0.75 1.25 ns542 Ml 8.87 8.13 87§ 1.25 M 52mp838318W M3 383 2-01 8.13 i. W 25® 74518-88 SSHAlp^MlWl® 54g 558 3.2? 8,14 258 4.25 S. MM-13 £SmippS«MS5 532 888 3M 5.13 285 3.45 3,775543 s5SAlp^811s430 834 555 3,65 9,13 2, M 3,4® 2.3318-13 £OW8421’»433 522 556 3.13 0.13 3.88 7JSK49 5mipM4W478 523 553 2.19 5.13 4.55 3-72 2.8145-58 e& NASO 554 574 1,83 8,13 5.38 1,32 4S MO2jm«8275 §53 577 1.4$ 8.13 2 18 l. M 6. M1M8 A5SG2p$mnlS2 MS 555 2.32 8.13 1.73 331 2 118L18 tWB..a 557 §33 X83 8.13 8.73 L27 as 08Wj!p2um 537 §85 1,34 8.13 4,48 253 223«7 AW2. A81M«1M 525 M3 8.33 8.13 &. T2 1.28 m AHG2aa238te321 5S8 §37 7.82 5.13 0.38 1,44 as CWAfcjspll&elSO 833 §55 2.22 8.12 1.72 238 L8MMS CftTM^MlteiTS M2 §81 2,87 8.13 1.58 2.88 4.023L83CRYABpp72to127 541 568 2.26 0.13 1.75 2.94 7.322E-10 Logistic regression analysis was conducted to evaluate the association between reactivity to EBV antigens (EBNA1 and EBNA3C) and CNS antigens (GlialCAM, CRY AB, and AN02) with MS risk. The model was adjusted for gender, age, plate-based batch effects, and principal components (PCA-1-5). The results are reported as odds ratio (OR) with standard error (SE) and 95% confidence interval (CI95) upper and lower limits.

[0278] The data confirmed that elevated antibody levels against the intracellular domain of GlialCAM (GlialCAM ICD, residues AA262-415) were associated with an increased risk for MS (OR: 1.81, CI: 1.40 – 2.34), and the 30mer GlialCAM peptides covering the larger mimicLST-IOOIWO PATENT APPLICATION region (AA305-416) (SEQ ID NOs: 29-36) were even more strongly associated with MS risk (most strongly associated peptide: GlialCAM AA385-416 (SEQ ID NO: 36), OR: 2.63, CI: 2.02 - 3.43). Interestingly, reactivities against the two GlialCAM peptides AA365-394 (SEQ ID NO: 32) and AA370-389 (SEQ ID NO: 34) were elevated independently of phosphorylation at serine 376. The initial publication had described this posttranslational modification to facilitate molecular mimicry with EBNA1 AA393-398 ( Lanz T V. et al., Nature 603, 321-327 (2022)). Consistent with prior publications, MS risk associated with elevated serum antibody levels against CRYAB AA2-23 (OR: 1.94, CI: 1.49 – 2.53) and ANO2 AA134-153 (OR: 2.32, CI: 1.79 – 3.01) were confirmed ( Thomas O.G. et al., Sci. Adv. 9, eadg3032 (2023); and Tengvall K. et al., Proc. Natl. Acad. Sci. U. S. A. 116, 16955–16960 (2019)).

[0279] The data were adjusted for age, gender, batch effects, and dimensions 1-5 of a principal component analysis (PCA) of genetic datasets available for the cohort ( International Multiple Sclerosis Genetics Consortium. Multiple sclerosis genomic map implicates peripheral immune cells and microglia in susceptibility. Science 365, eaav7188 (2019)). The threshold for significance in Fig. 1 was calculated using the median value among controls. For better comparison with prior publications (Vietzen H. et al., Ineffective control of Epstein-Barr-virus-induced autoimmunity increases the risk for multiple sclerosis. Cell 186, 5705-5718. el3 (2023); and Vietzen H. et al.. Accumulation of Epstein-Barr virus-induced cross-reactive immune responses is associated with multiple sclerosis. J. Clin. Invest.134 (2024)), reactivity thresholds were recalculated using the level at which the largest change in MS risk was observed in a sliding window analysis, as well as the largest area under the curve (AUC) metrics, without a significant change in results (FIGs. 10A-10B).

[0280] Stratifying MS risk by gender reduces group sizes and subsequently decreases significance levels, but gender did not have a significant influence on our results overall (FIGs. 11A-11B).LST-IOOIWO PATENT APPLICATION

[0281] EXAMPLE 2 - Overlap between anti-EBNAl and anti-GlialCAM reactivity

[0282] Antibody reactivities against EBNA1 and GlialCAM antigens were compared. Strong correlations of multiple peptides from one antigen supported the mechanism of intramolecular epitope spreading, and correlations between EBNA1 and GlialCAM antigens supported the mechanism of molecular mimicry between the viral and myelin antigens.

[0283] Strong correlations were observed amongst most GlialCAM peptides and EBNA1 peptides, respectively (FIG. 2, lower left panel; and FIGs. 10A-10B), indicating that isolated reactivity against a single epitope is rare and that intramolecular epitope spreading is a common phenomenon in individuals with antibody responses to GlialCAM. There was a significant overlap between anti-EBNAl and anti-GlialCAM responses, which included peptides representing the central EBNA1 epitope AA393-398 “SPPRRP’’ (SEQ ID NO: 4), which mimics the central GlialCAM epitope AA377-382 “SPPRAP” (SEQ ID NO: 5) (Fig.3A) (Lanz T. V. et al., Nature 603, 321-327 (2022)), but also peptides representing the broader EBNA1 region (AA361-470) (SEQ ID NOS: 20-25) and the broader GlialCAM region (AA305-416) (SEQ ID NOS: 29-36) (Fig. 2).

[0284] It supported a close relationship between B cell responses to EBNA1 and GlialCAM, and it was hypothesized that once initiated by molecular mimicry, intramolecular epitope spreading increases the breadth of the antibody response. Interestingly, the breadth of the antibody response did not extend to regions far away from the central epitopes, as correlations with anti-EBNAl AA1-120, anti-EBNAl GR-repeat (AA2-89), and anti-GlialCAM extracellular domain (ECD) were significantly weaker. Antibody responses against ANO2 AA134-153 (SEQ ID NO: 38) overlapped with anti-GlialCAM responses more strongly than anti-CRYAB AA2-33 responses (FIG. 2). This is in line with a previously observed divergence of anti-CRYAB and anti-ANO2 responses (Thomas O. G. et al., Cross-reactive EBNA1 immunity targets alpha-crystallin B and is associated with multiple sclerosis. Sci. Adv. 9, eadg3032 (2023)).

[0285] A notable association was detected between the EBNA1 GR-repeat region, GlialCAM ECD, and the EBV antigen EBNA3C (FIG. 2), three antigens that were not significantly associated with MS risk. Most of the described correlations were found across the whole cohort and are not unique to MS patients. However, the vast majority of correlations are more enhanced in MS patients than controls (FIG. 2, top right panel, showing values >0 for ratio of MS patients to controls for almost all antibody correlations). A few correlations of reactivities are worth pointing out, including GlialCAM Full-Length and GlialCAM AA370-389 (FIGS.3A and 7), which associate with several other GlialCAM and EBNA1 antigens moreLST-IOOIWO PATENT APPLICATION prominently in MS patients than in controls. In particular, GlialCAM Full-Length was much more strongly correlated with EBNA1 AA381-410 and EBNA1 AA401-430 in MS patients compared to controls (ratios of correlation coefficients MS vs. controls: 8.07 and 143.9, respectively). GlialCAM AA370-389 is prominently correlated with almost all EBNA1 peptides, and also with ANO2 AA1-275, but less so with CRY AB Full-Length (FIG. 2). Interestingly, there are also a few antigens that correlate negatively in the MS vs. control groups, in particular ANO2 AA1-275 (SEQ ID NO: 33; FIG. 15), which is negatively correlated with EBNA1 AA1-120, GlialCAM Full-Length (SEQ ID NO: 22), and CRY AB Full-Length (SEQ ID NO: 35). In addition, there is a strong positive correlation between ANO2 AA1-275 (SEQ ID NO: 33) and EBNA3C (SEQ ID NO: 37) in MS patients (ratio of correlation coefficients: 11.96), although EBNA3C did not reach statistical significance in association to MS (FIG. 1). An associated antibody response between ANO2 and EBNA3C had not been described previously.

[0286] EXAMPLE 3 - Cross-reactivity between antibodies against EBNA1 and GlialCAM

[0287] The original discovery of molecular mimicry between EBNA1 and GlialCAM described the core epitope regions EBNA1 AA393-398 and GlialCAM AA376-382 (Fig. 3A) ( Lanz T. V. et al.. Nature 603, 321-327 (2022)). To determine if cross-reactive antibodies bind EBNA1 and GlialCAM in this new cohort, plasma from 10 MS patients with high reactivity to both EBNA1 and GlialCAM was subsampled, and incubated with the mimicking or control peptides before measuring binding. EBNA1 AA386-405 (SEQ ID NO: 22) was shown to efficiently block antibody binding to GlialCAM AA370-389 (FIGs. 3B, F), confirming the presence of cross-reactive antibodies. In line with our finding that phosphorylation of serine 376 facilitates this molecular mimicry, phosphorylated GlialCAM AA386-405 was more robustly blocked by EBNA1 AA386-405 (SEQ ID NO: 22), suggesting stronger molecular mimicry (Fig. 3C, F). Interestingly, GlialCAM AA370-389 (SEQ ID NO: 34) only partially blocks antibody binding to EBNA1 AA386-405 (Fig. 3C, F), which reflects the direction of the development of the B cell response from an initial anti-EBNA1 response towards an anti-GlialCAM response. EBNA1 AA1-120 (SEQ ID NO: 18) was used as a negative control peptide (Fig. 3E, F).LST-IOOIWO PATENT APPLICATION

[0288] EXAMPLE 4 - Impact of *HLA-DRB1*15:01* on anti-EBNAl and anti-GlialCAM reactivity

[0289] HLA-DRB1 *15:01 is the main genetic risk factor for MS and has a major impact on the autoimmune B and T cell response (7, 24-26). To determine if HLA-DRB1 *15:01 promotes antibody reactivity against GlialCAM in MS patients, antibody reactivities against EBNA1 AA381-410 (SEQ ID NO: 21) and GlialCAM AA370-389 (SEQ ID NO: 34) were analyzed in groups matched for HLA-DRB1 *15: 01 status. Significantly higher IgG levels against both antigens were detected in MS patients hetero- and homozygous for HL A-DRB1 *15:01, but the difference was less pronounced in the control group (Fig. 4A-F).

[0290] Positive HLA-DRB1 *15:01 status and elevated antibody levels against EBNA1 AA381-410 and GlialCAM AA365-394 are three independent risk factors that together increase the MS risk to an OR of 9.40 (CI: 6.04 - 14.86) (Fig. 4G). Likewise, there is a cumulative increase in MS risk for HLA-DRB1 *15:01 and anti-EBNAl AA381-410 in combination with anti-CRYAB AA2-33 (OR: 10.01, CI: 6.38 - 15.97) andHLA-DRB1 *15:01 and anti-EBNAl AA421-450 anti-ANO2 AA134-153 (OR: 5.42, CI: 3.65 -8.13) (Fig. 4H, I). The protective HLA class I allele HLA-A *02: 01 has the reverse effect, and in combination with elevated antibody levels against EBNA1 AA486-405 and either of the other three antigens, its absence adds additional risk (GlialCAM AA365-394, OR: 19.32 CI: 10.23 - 38.07; CRYAB AA2-33, OR: 17.53, CI: 9.26 - 34.60; ANO2 AA134-153 OR: 11.98, CI: 6.62 - 22.42) (FIGS. 18A-18C). No significant effect was detected for the additional alleles HLA-DRB1 *03:01, HLA-DRB1 *08:01, HLA-B*38:01, and HLA-B*44:02.

[0291] Neurofilament light chain (Nfl) is a marker of neuronal damage that is elevated in EBV-infected MS patients as much as 10 years prior to the onset of MS (2, 30). A correlation between elevated anti-EBNAl and anti-GlialCAM antibodies might indicate a direct impact of these antibodies on neuronal damage. No significant correlations were observed between antibody reactivity to GlialCAM AA365-394 (r = 0.036, p = 0.55) or EBNA1 AA381-410 (r = 0.048, p = 0.96) with Nfl levels (FIGS. 19A-19B).

[0292] EXAMPLE 5 - Cumulative effects of multiple cross-reactive antibody reactivities

[0293] Prior studies suggested that combinations of multiple antibodies against peptides in the EBNA1 region AA381-452 or antibodies against several of the molecular mimics GlialCAM AA370-389, CRY AB AA2-21, MBP AA205-224, and ANO2 AA135-154 increased the MS risk by 240- to over 1,300-fold (Vietzen H. et al., Accumulation of Epstein-LST-IOOIWO PATENT APPLICATION Barr virus-induced cross-reactive immune responses is associated with multiple sclerosis. J. Clin. Invest. 134 (2024)). The present study calculated the cumulative risk when combining one or more elevated reactivities against the EBNA1 peptides AA386-405 (SEQ ID NO: 22), AA401-430 (SEQ ID NO: 23), and AA421-450 (SEQ ID NO: 24) and the mimicking peptides GlialCAM AA370-389 (SEQ ID NO: 34 or 35, or both?), CRY AB AA2-33 (SEQ ID NO: 40), and ANO2 AA 134-153 (SEQ ID NO: 38).

[0294] A significant but moderately elevated cumulative risk over the individual reactivities shown in Fig. 1 was determined with ORs ranging from 2.1 to 3.16 (CI: 1.6-2.76 and 2.23-4.51, respectively) (Figs. 5A-5C).

[0295] EXAMPLE 6Prophetic Clinical Trial with CAAR to Molecular Mimics in EBNA-1 in the Treatment of Multiple Sclerosis

[0296] A study of 50 patients with clinically definite forms of MS is undertaken, following screening procedures listed in Steinman et al NEJM 2022 attached.

[0297] Key eligibility criteria were an age of 18 to 55 years; a diagnosis of MS (meeting 2010 revised McDonald criteria, reference 12 Steinman et al NEJM); a score on the Expanded Disability Status Scale (EDSS) of 3 to 5.5 at screening (scores range from 0 to 10.0, with higher scores indicating greater disability ref 13 from Steinman et al NEJM); and neurologic stability’ for at least 30 days before screening and the baseline assessment. Patients must have oligocl onal immunoglobulin to EBNA-1. Key exclusion criteria, including the use of previous disease-modifying treatments and the durations of washout periods, are provided in the Additional Methodology Details section in the Supplementary Appendix for Steinman et al NEJM 2022.

[0298] We refer here to five different constructs: Design of five individual CAAR Targeting Molecular Mimics on EBNA-1 from amino acids 370 to 450. Construct one contains components of GlialCAM p376-382 where ser376 is phosphorylated. Construct 2 contains GlialCAM p376-382, where Ser376 is not phosphorylated. Construct 3 contains CRY AB p8-15 and Construct 4 contains ANO-2pl40-149 are described. CAAR as bait for the autoantibody producing cells that target plasma cells and plasmablasts producing these clonal antibodies in the cerebrospinal fluid are constructed. These CAAR cell lines are transduced to encode various peptide constructs that bind to autoantibody produced on plasmablasts and plasma cells. These clonal antibodies recognize- GlialCAM 377-382, CRYABp8-15 and ANO-2 pl40-149, respectively and recipients for each construct can be detected with aLST-IOOIWO PATENT APPLICATION diagnostic kit. Each cell CAAR cell line has a hinge / transmembrane domain, a co-stimulatory domain-either CD28 or 4-1BB, and a CD3 zeta domain. The CD8 4-1BB costimulatory domain is optimal. The peptide domains for the four constructs GlialCAM, CRY AB and ANO-2 are shown in figures 1-4 from the PNAS paper in press below. {Note figure 3 is particularly informative. Figure 3 has a typo in 3F, where 470 should be written instead as 370], A fifth construct contains a CAAR T cell that contains all three regions of mimicry to GlialCAM 377-382 with Serine 377 phosphorylated, CRYABp8-15 and ANO-2pl40-149. In this “compound CAAR construct the GlialCAM mimicry peptide is separated from the CRY AB peptide with a poly glycine spacer consisting of 5 amino acids, the CRY AB construct and the ANO-2 construct are separated with a polyglycine spacer consisting of 5 amino acids.

[0299] Cohort 1

[0300] In the first cohort 20 patients with MS with Clonal antibody to GlialCAM in their spinal fluid and blood are tested, with Constructs 1 or 2.

[0301] Therapy is delivered monthly for the first two months, and then every' six months thereafter.

[0302] The first dose is 1x108CAAR T cells following lymphodepletion with fludarabine (30 mg / m2on days -5, -4, -3) and cyclophosphamide (300 mg / m2on days -5, -4, -3).

[0303] Further doses of 1x108CAAR T are given without lymphodepletion.

[0304] Measurement of clinical status included monitoring of EDSS status at the onset and then after six and 12 months, measurement of relapse rate, and measurement of MRI activity including change in number and volume of T1 and T2 lesions, change in number of Gadolinium enhancing lesions.

[0305] Patients are monitored every 3 months w ith assessment of relapse rate, progression via EDSS and measurement of No Evidence of Diseae Activity and MRI. as described in Alvarez E, Steinman L, Fox EJ, Hartung HP, Qian P, Wray S. Robertson D, Selmaj K. Wynn D, Mok K, Xu Y, Bodhinathan K, Miskin HP, Cree BAG Improvements in no evidence of disease activity with ublituximab vs. teriflunomide in the ULTIMATE phase 3 studies in relapsing multiple sclerosis. Front Neurol. 2024 Oct 24; 15: 1473284. doi:10.3389 / fneur.2024.1473284, attached

[0306] Levels of anti-GlialCAM antibody in blood and CSF are measured as in PNAS in press.LST-IOOIWO PATENT APPLICATION

[0307] Cohort 2

[0308] The next cohort [Cohort 2] of twenty patients is then tested based on the first cohort. For Cohort 2, if an individual with MS has clonal antibodies to GlialCAM and to CRY AB in their CSF, they are treated with CAAR T from either construct 1 or 2, depending on the best performance in the initial 20 patients. In addition to construct 1 or 2. they are also treated with Construct 3 containing Cryab 8-15. Each dose is split with 0.5x108CAAR T cells from Construct 1 or 2 PLUS 0.5x108CAAR T cells from Construct 3. The split does is given following lymphodepletion with fludarabine (30 mg / m2on days 5, 4, 3)and cyclophosphamide (300 mg / m2on days ~5, ~4, ~3).

[0309] Further doses of 1x108CAAR T split half:half between Construct 1 or 2 and Construct 3 are given without lymphodepletion, monthly for the first two months, and six months thereafter.

[0310] Measurement of clinical status included monitoring of EDSS status at the onset and then after six and 12 months, measurement of relapse rate, and measurement of MRI activity including change in number and volume of T1 and T2 lesions, change in number of Gadolinium enhancing lesions.

[0311] Patients are monitored every 3 months with assessment of relapse rate, progression via EDSS and measurement of No Evidence of Diseae Activity and MRI. as described in Alvarez E, Steinman L, Fox EJ, Hartung HP, Qian P, Wray S, Robertson D, Selmaj K, Wynn D, Mok K, Xu Y. Bodhinathan K, Miskin HP, Cree BAG'. Improvements in no evidence of disease activity with ublituximab vs. teriflunomide in the ULTIMATE phase 3 studies in relapsing multiple sclerosis. Front Neurol. 2024 Oct 24:15: 1473284. doi:10.3389 / fneur, 2024, 1473284, attached

[0312] Levels of anti-GlialCAM and CRY AB antibody in blood and CSF are measured as in PNAS in press.

[0313] Cohort 3

[0314] The third and final cohort would test patients with antibodies to GlialCAM, CRY AB and ANO-2 and w ould be treated with Construct 5.

[0315] Therapy is delivered monthly for the first two months, and then every six months thereafter. The first dose is 1x108CAAR T cells following lymphodepletion with fludarabine (30 mg / m2on days -5, -4, -3) and cyclophosphamide (300 mg / m2on days -5, -4, -3).

[0316] Further doses of 1 xl 08CAAR T are given without lymphodepletion.LST-IOOIWO PATENT APPLICATION

[0317] Measurement of clinical status included monitoring of EDSS status at the onset and then after six and 12 months, measurement of relapse rate, and measurement of MRI activity including change in number and volume of T1 and T2 lesions, change in number of Gadolinium enhancing lesions.

[0318] Patients are monitored every 3 months with assessment of relapse rate, progression via EDSS and measurement of No Evidence of Diseae Activity and MRI, as described in Alvarez E, Steinman L, Fox EJ, Hartung HP, Qian P, Wray S, Robertson D, Selmaj K, Wynn D, Mok K, Xu Y, Bodhinathan K, Miskin HP, Cree BAC. Improvements in no evidence of disease activity with ublituximab vs. teriflunomide in the ULTIMATE phase 3 studies in relapsing multiple sclerosis. FrontNeurol. 2024 Oct 24:15:1473284. doi:10.3389 / fneur.2024.1473284, attached

[0319] Levels of anti-GlialCAM and CRY AB as well as antibody to ANO-2 in blood and CSF are measured as in PNAS in press.

[0320] MATERIALS AND METHODS

[0321] Patient cohort. Plasma samples were collected from participants in the Swedish Nationwide Epidemiological Investigation of Multiple Sclerosis cohort (DrosuN. et al., CD4 T cells restricted to DRBl*15:01 recognize two Epstein-Barr virus glycoproteins capable of intracellular antigen presentation. Proc. Natl. Acad. Sci. U. S. A. 121 (2024)). The study included 650 MS patients and 661 population-based controls, matched to MS cases by sex, age, and geographic region. All participants provided written informed consent for sample collection and data analysis. Cohort characteristics are detailed in Table 1. HLA data were previously obtained from studies conducted at the Karolinska Institute (Thomas O. G. et al., Sci. Adv. 9, eadg3032 (2023)). Information on IM history and BMI was self-reported via a questionnaire administered at the time of consent and sampling (Salzer J. et al., Mult. Scler.19, 1587-1591 (2013); DrosuN. et al., Proc. Natl. Acad. Sci. U. S. A. 121 (2024); and Porszasz J. et al.. Pharmacological investigation on the neurohumoral transmission of the vasomotor regulation. Acta Physiol. Acad. Sci. Hung. 49, 139-165 (1977)). Plasma Nfl levels were quantified using the Single Molecule Array (Simoa™) NF-Light Advantage Kit and normalized for age, sex, and BMI, employing reference data from 1,026 Sw edish controls (46).

[0322] Antigen Selection. Protein and peptide antigens were selected based on prior studies by the present inventors and others (Pender M. P. et al., Defective T-cell control of Epstein-Barr virus infection in multiple sclerosis. Clin. Transl. Immunology 6, el 26 (2017); HarleyLST-IOOIWO PATENT APPLICATION J. B. et al., Transcription factors operate across disease loci, with EBNA2 implicated in autoimmunity. Nat. Genet. 50, 699-707 (2018); 47, 48). The present inventors focused on the EBV transcription factor EBNA1 and its molecular mimics, which include GlialCAM, CRY AB and ANO2. Other EBV antigens included in the array are listed in Supplementary Table SI. Several antigens were represented in multiple versions, including full-length proteins, protein fragments, and peptides. The final array consisted of 26 antigens including 11 GlialCAM, 10 EBNA1, one EBNA3C, two CRY AB full length and CRY AB AA2-33, and two ANO2 AA1-275 and ANO2 AA134-153.

[0323] Protein Expression. EBNA1 was included as full-length protein (AA1-641), two N-terminal regions (AA2-89 and AA1-120), and the C-terminal DNA-binding domain (AA380 - 641). GlialCAM was represented by its full-length form (AA34-416), as well as intracellular (AA262-416) and extracellular domain fragments (AA34-240). EBNA1 AA1-641 and AA2-89, and the extracellular domain of GlialCAM (ECD, AA34-240), were expressed in Freestyle 293-F cells (Thermo Fisher Scientific R79007) using second-generation lentiviral transduction. Briefly, constructs were cloned into a custom lentiviral vector (49) and co-transfected into HEK293T cells with second-generation lentiviral packaging plasmids pMD2. G and psPAX2 (Addgene 12259, 12260), and FuGENE (Promega E2312). Lentivirus in supernatants was harvested 72h post transfection and used to transduce Freestyle 293-F cells with Polybrene (EMD Millipore TR-1003-G). Cells were cultured to a density of 3 x 106cells / mL, lysed via sonication, and his-tagged proteins were purified with Ni-INDIGO resin (Cube Biotech 75103), followed by size-exclusion chromatography on a Superdex 75 Increase 10 / 300 GL column (Cytiva 29148721). The N-terminally His-tagged intracellular domain of GlialCAM (AA262-416) was expressed in E. coli as previously described (14). EBNA1 (AA1-120), EBNA1 (AA380-641) ANO2 (AA1-275) and CRY AB (full length) proteins were produced as previously described (16, 50). Briefly, genes were ordered from Eurofins (Luxembourg) and subcloned into a modified vector containing an 8XHis tag. The plasmid was then transformed into BL21-A1 E. coli (Thermo Fisher Scientific, cat. no.: C607003) and grown in vegetone SB medium supplemented with carbenicillin lOOmg / L, ImM MgSO4, 0.6% glycerol for 3h, before transferring to autoinduction medium (vegetone SB medium), supplemented with 1 mM MgSO4, 0.6% glycerol, carbenicillin (100 mg / L). 0.015% glucose. 0.2% arabinose and 0.2% lactose and incubated overnight at 25°C. The following day, bacterial pellets were centnfuged at 7000xg for 30 minutes and lysed using 6M Gua-Cl buffer and a freeze-thaw cycle. Lysates were then centrifuged at 20,000xg for Ih and protein w as purified from supernatants using HisMagLST-IOOIWO PATENT APPLICATION Sepharose beads (Cytiva, cat. no. 29104065). SDS-PAGE was used to check protein purity and concentrations were determined by NanoDrop (Thermofisher Scientific).

[0324] Peptide Synthesis. For more granular epitope mapping of selected regions, 30-mer peptides with 10-amino-acid overlaps were designed (Supplementary Table SI) and biotinylated with an amino hexanoic acid spacer (Biomatik. Kitchener, Ontario, Canada). For EBNA1, 30-mers covered the region AA361-470 (SEQ ID NOS: 20-21 and 23-25) with five peptides and in addition the 20-mer AA386-405 (SEQ ID NO: 22) was included from our previous work (Harley J. B et al.. Transcription factors operate across disease loci, with EBNA2 implicated in autoimmunity. Nat. Genet. 50, 699-707 (2018)). For GlialCAM, 30-mer peptides spanned the region AA305-389 (SEQ ID NOS: 29-31), with AA365-394 (SEQ ID NOS: 32-33) and AA370-389 (SEQ ID NOS: 34 AND 35) included in both nonphosphorylated and phosphory lated versions (at serine 376). The CRY AB AA2-33 (SEQ ID NO: 40) and ANO2 AA134-153 (SEQ ID NO: 38) peptides were included, based on their immunogenic potential in previous studies (Thomas O. G. et al.. Cross-reactive EBNA1 immunity' targets alpha-crystallin B and is associated with multiple sclerosis. Sci. Adv. 9, eadg3032 (2023); Tengvall K. et al., Molecular mimicry' between Anoctamin 2 and Epstein-Barr virus nuclear antigen 1 associates with multiple sclerosis risk. Proc. Natl. Acad. Sci. U. S. A. 116, 16955-16960 (2019); and Huang J. et al.. Genetics of immune response to Epstein-Barr virus: prospects for multiple sclerosis pathogenesis. Brain 147, 3573-3582 (2024)).

[0325] Array Preparation. Recombinant proteins were coupled to barcoded carboxylated magnetic beads (MagPlex-C, Luminex Corp.), while biotinylated peptides were coupled to streptavidin-coated magnetic beads (MagPlex-Avidin, Luminex Corp.). For both carboxylated and streptavidin bead coupling, 8 pg of each antigen was coupled to 1 x 106magnetic beads per bead ID. Beads were washed with an automated Biotek plate washer in 96-well plates (Greiner Bio-One, 650201).

[0326] Carboxylated beads were washed, resuspended in activation phosphate buffer (0.1 M NaH2PO4, pH 6.2; Sigma-Aldrich; S3139-250G). and then incubated with 0.5 mg / ml 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (Thermo Scientific; 77149) and 0.5 mg / ml N-hydroxy succinimide (Thermo Scientific Pierce; 24510) in phosphate buffer for 20 minutes at room temperature (RT) while shaking. After washing, beads were resuspended with antigens diluted in coupling buffer (50 mM MES, pH 5.0; Sigma- Aldrich; M2933-100G) and incubated for 120 minutes at RT while shaking. Coupled beads were washed twice and then stored in PBS-TBN buffer (PBS, 0.1% BSA, 0.02% Tween 20) at 4°C in the dark until use.LST-IOOIWO PATENT APPLICATION

[0327] Streptavidin beads were washed, resuspended in antigen solution diluted in PBS-1%BSA (Sigma-Aldrich, A8412) and incubated at RT for 30 minutes while shaking. Beads were then washed twice with PBS-TBN, resuspended, and stored at 4°C in the dark until use.

[0328] Coupling efficacy was validated using human plasma with known reactivity to EBNA1, GlialCAM. and CRY AB, along with monoclonal antibodies against EBNA1 and GlialCAM (14). Bead counts for each bead region were adjusted to ensure uniform counts in the multiplex array.

[0329] Array Probing. Plasma samples from MS patients and healthy controls were diluted 1: 100 in PBS-1% BSA (Sigma-Aldrich) and 45 pL / well was used in 384-well plates, mixed w ith 5 pL of bead suspension, and incubated at RT for 60 minutes in the dark while shaking. After w ashing, beads were resuspended with secondary antibodies (1: 1000 diluted goat antihuman IgG, (Jackson ImmunoResearch; 109-117-008) and incubated for 30 minutes in the dark while shaking. Beads were then washed, resuspended in 50 pL of PBST and read on the FlexMap 3D system (Luminex Corp.). Results were reported in Median Fluorescent Intensity (MFI), with background MFI values subtracted. Samples with coefficients of variation (CV%) over 40% w ere re-assayed. Researchers were blinded with regard to control vs. patient samples.

[0330] Batch control. MFI of batch control samples (n=12 / plate) were used to normalize average MFI between plates. Samples that after re-assay still had high CV (over 30%) were removed. A reference plate was selected for each antigen based on the degree of variability of MFI values on each plate. MFI values of batch control samples in the reference plate with the lowest variability were log-transformed, considering the uncertainty’ of linear and non-linear relationships between MFI values in different plates for each antigen. Pearson correlation tests were performed to estimate the possible linear association between MFI values of batch control samples in the reference plate and other plates. Coefficients between the reference plate and other plates were generated for each antigen by linear regression with an intercept as 0. Batch correction was achieved by using a log-transformed average MFI value multiplied with the coefficient of batch control samples in various plates, which w as then exponentiated.

[0331] EBNA1 / GlialCAM blocking. To confirm cross-reactivity between EBNA1 and GlialCAM, blocking competition assays w ere conducted. Plasma from 10 selected MS patients (n=10) were pre-incubated with EBNA1 AA386-405 (SEQ ID NO: 22), GlialCAM AA370-389 (SEQ ID NO: 34) (with highest homology to EBNA1 AA386-405), GlialCAM AA370-389-PhosSerine376 (SEQ ID NO: 35) (increased antibody binding of GlialCAM with Serine 376 phosphorylation) (Harley J. B. et al., Transcription factors operate across diseaseLST-IOOIWO PATENT APPLICATION loci, with EBNA2 implicated in autoimmunity. Nat. Genet. 50, 699-707 (2018)), EBNA1 AA1-120 (a non-homologous EBNA1 region), or BSA (control) in O.lmg / ml of the respective antigen in PBS + 1% BSA, for 1 hour. Following the pre-incubation period signal intensity was probed and analyzed as described in the previous section. Results were evaluated as the fold change in MFI compared to control MFIs (FIGS. 3A-3F).

[0332] Statistical Analysis. Correlations were tested with Pearson correlation tests for continuous variables and Spearman correlation tests for testing monotonic relationships. For continuous variables, T-tests were used for two group comparisons and ANOVA were used for multiple comparisons. Kruskal-Wallis and Dunn's multiple comparison tests were used when normal distribution and homogeneity of variation were violated. Median among controls was used as a cut-off to define individuals with high antibody levels, but as a sensitivity analysis we used two other cutoffs: the level with the highest change in risk and the level with the highest area under the curve. Chi-square tests were performed for categorical variables. Linear and logistic regressions were conducted for adjusted comparisons, coefficients, and OR estimation. All statistical analyses were performed in R. Additional details in statistical analysis are available in supplementary materials.

[0333] SUPPLEMENTAL METHODS IN EXAMPLES

[0334] 1) Supplementary Methods Correlation test. Pearson correlation tests were performed for MFI for all the 26 antigens in all individuals as well as in MS cases and controls separately (Fig. 2 and FIGs. 10A-10B). Gender stratification was also conducted to compare the correlation difference between females and males (FIG. 11A-11B and Supplementary Table S5). Ratios of the correlation coefficients in the MS population and correlation coefficients in the control population were calculated and shown in Fig. 2.Extreme values in ratios of MS cases and controls w ere presented in different colors from the heatmap scale to avoid masking association of other antibodies levels (Fig. 2).LST-IOOIWO PATENT APPLICATION

[0335] Principal component analysis (PCA). PCA was conducted using SmartPCA with default settings (International Multiple Sclerosis Genetics Consortium, Multiple sclerosis genomic map implicates peripheral immune cells and microglia in susceptibility. Science365, eaav7188 (2019); and Porszasz J. et al., Acta Physiol. Acad. Sci. Hung. 49, 139-165LST-IOOIWO PATENT APPLICATION (1977)) using ancestry informative single nucleotide polymorphism markers (n= 3529), excluding the HLA region, genotyped in the custom-made MS replication chip (45). Since sample sizes of antibody association analysis (n=1214) and HLA association analysis (n=1189) were different, PCA were conducted twice for better accuracy using respective sample groups. By generating scree plot and eigenvalues. PCA 1-5 were used as adjustment in the following analysis.

[0336] Defining cutoffs for risk estimation. It is not obvious what cutoff should be used for MFI measures of antibody levels to estimate risk of MS. We therefore decided to compare three different methods. The main analysis is carried out using the median among controls as cutoff and the other methods are used for sensitivity analysis (Supplementary Table S4 and FIG. 10A-10B). The proportion of seropositives in MS cases and controls were also compared between the different methods of defining cutoff values.LST-IOOIWO PATENT APPLICATIONSupplementary Table S3< Association of antibody reactivity to EBV-antigens EBHA1 and EBNA3C, and self«anttga GlialCAM, CRYAB, and AN02 with MS risk using linear regression.Antigen legn£^*»«i«-HS-3^SE? tsate 8?esa« MS $8 MS 5teas.s«M SB cect r®SiC3M.£C8 -3,?7 ilggyiSASS 1.53 -2.51, 4 ';; 1881? 285.74 7'5,32 SSsKAMJi. -S. SS -4,38833454 VVi V.83 125,25 338.33 388,38 m,8S SgsCVUA'B 3.75 ■4,431334883 4.83 <5.47 883,*4 47SM 488,33 31? M GUtKWWWfcSS* -4,88 -5,548478555?,?3 18.53 888838 811154 3855,28 4882,88 2&2iCAM34S325ic3£4 -4.82 5,853322535?.3« 13.33 8882,88 313133 77X 8323,37 883ijVMgg345i83?4 4.24 ■433333335 5.52 -781 1783.?5 1517,25 13*4.3? 1333.33 8231CAM583SSic384 4,5? -5.4545:1525? S? 13.43 522277 478854 3382.22 3545.85 68slC6M55382ic384.?8ss3? S -4.3S 5.855725267 3.3? -1135 8882,81 883846 4353,32 8335,87 8S3®A5U33?i4s343 4, 31 -4, 344333355 4.32 477 1388.88 2382,2:1 1331.3? 1333.33-4,23 -AWSIMSS? 5,33 -8.38 178375 1888 11: J223,3S 1333.32 SSUCAM53SSV«438 -4.38 -3, 433834573 8,?3 13.53 4333,33 4488,58 2385,35 3552,24 £S? U1,1, S52?«325 5,18 ■5,4?5?854i?3 2.18 -3,44 -WO1 4858,42 3585,53 4355.54 £8U4i„2.aa358i5841 -4,78 -S llSSASSaS 18,35 -3138 18332.73 8??83? 33232,35 3235,44 £8m. A V.33 ■8,834332355 5,83 -8.88 37848, 88 18358,38 33743,85 38387,238.,’S S,81?8S3213 388. -8.34 138,51 382,78 183,33 88,38 E8.5Ai8»3S3U335 -4,82 ■ 5,555222274 4.85 -8.43 4858,28 588184?<432,33 4455,52 EB24SS3383i:5MS -4.88 ■ 5,835788384 1-335 -38,83 2;3S8,53 11818,88 38583,83 8724,48 £&3Ul*8-.21U3->825 '4,83 ■5,?3?488618 5,32 8,82 *484. *3 5237,58 3348,83 5831,88 E5NA38845381435 -5,iS -8, 325534823 8,83 -31,38 22888,24 33338,41 32232,56 388808 WWZIW -3.25 S-SSSSSSIS? 8.53 V88 28823,23 18338,38 3338838 33787,38 £8? Wl«S>m«>3?2 4,?» -5,3W?2m VU- -827 3571,88 4287,88 3335,43 3353,43 E8N438 -4,31 -5,75888533 3,28 8.11 358,18 31,45 348,32 52,52 4?®i,34V»2"5 -4,84 -5,545582422 4,51 -4.28 24-.2, 54 5888,?3 3222,32 4473,834.55 5.74 -38,52 4VU,?3 3752,65 3344,83 335?.83 C5W.. F1 -4,82 ■4,325334232 1,?3 1.13 252,35 38535 332,32 232,23 C5Wp8-2to33 -4.82 -4,738834534 4,33 -8.18 882,42 818,34 438,35 555,52 M4C2,;5418832188 8.84 -IVaSSSTOU 5,27 -8,38 1088.*? 4788,25 3183.4? 41.-V.88 V®844*88te-331 -5,?? -5, 825855585 V.31 V.88 3281,35 183588 3382,34 3772,42 CSWppi 1817155 -4.7J.5,388852434?,15 11.73 282788 3438.58 3883,88 3848,38 88? W?il42ss2?2 4.15 -IVlSSSrSSS 5,22 8.58 143*38 3282,57 881,8? 1183,18 CSWppTjxji**,4,4g.5,343225253?,38 -12.18 4668>34 38*? 83 2575,64 2574,53Linear regression analysis was conducted to evaluate the association between reactivity to EBV antigens (EBNA1 and EBNA3C) and CMS antigens (GlialCAM, CRYAB. and AN02) with MS risk. The model was adjusted for gender, age, plate effects, and principal components (PCA 1-5). Since the estimate. SE, and p value were small, log(|estimate|), log(SE) and log(p) were used here. Mean. MS, mean of MS group: SD. MS, standard deviation of MS group; Mean cont mean of control group; SD_cont standard deviation of control group.LST-IOOIWO PATENT APPLICATION

[0337] Continuous association (CAC). The continuous association was one of the methodsused to define the threshold of seropositivity as described previously (Porszasz J. et al., ActaPhysiol. Acad. Sci. Hung. 49, 139-165 (1977)). It uses a sliding window approach where afixed window with the lowest antibody levels is used as a reference when calculating the riskof MS with windows of increasing level of antibody levels. ORs for each window weregenerated in a continuous plot showing the increasing or decreasing trend with the increase of antibody levels. Thresholds of each antibody level are defined manually based on the greatestLST-IOOIWO PATENT APPLICATION increase of OR in an increasing trend and the greatest decrease of OR in a decreasing trend. If there were multiple greater changes close to each other, the smallest value was chosen as the threshold value since that enlarged the sample size of cases and saved more statistical power.

[0338] Area under the curve (AUC). An alternative way to define the cutoff value is to use receiver operating characteristic (ROC) curve for antibody levels as was done in a recent publication investigating role of autoantigen and EBV antibodies in MS risk (Vietzen H. et al., J. Clin. Invest. 134 (2024)). Specificity and sensitivity were calculated based on MS risk varying the antibody level. ROC curves were generated and the greatest AUC were defined using the Youden Index which combined the maximum specificity and sensitivity.

[0339] Linear and logistic regression and sensitivity analysis. For each antigen, coefficients of variation (CV) greater than 30% were removed during the analysis. Using MS as outcome, corrected MFI values of each antigen and other covariates as independent variables, including age, gender, plate-based batch effects, and PCA1-5, linear regressions were conducted. For logistic regression in the main analysis, medians of each antigen MFI values in the control group were used as the cutoff value for defining antibody levels as seropositive and seronegative. Age, gender, plate, and PCA1-5 were used as covariates. Cut off values from CAC and AUC were used in sensitivity analysis while other conditions were the same.

[0340] HLA association with antibody levels. Control of antibody levels by MS associated HLA alleles was estimated (Moutsianas L. et al., Nat. Genet. 47, 1107-1113 (2015)). MS associated HLA alleles were included with reasonable allele frequency in the Swedish population: DRB1 *15:01, DRB1 *03:01, DRB1 *08:01, A *02:01, B*38:01 and B*44:02. For HLA-A *02: 01. HLA-DRB1 *15: 01 and HLA-DRB1 *03: 01. The unadjusted difference between non-carrier, heterozygotes, and homozygotes in antibody levels were estimated, as for these alleles it has been shown that the number of alleles affect MS risk (Moutsianas L. et al., Nat. Genet. 47, 1107-1113 (2015)). Non-parametric statistical tools were used to compare group differences and multiple comparisons, such as Kruskal-Wallis and Dunn’s multiple comparison tests. Frequency associations were also analyzed by linear regressions, adjusted by age, gender, plate, and PCA1-5. The association of antibody levels with carrier and non-carrier of HLA-B* 38:01, HLA-B*44:02, and HLA-DRB1 *08:01 were analyzed separately using the t test, which did not show a correlation between any of these HLA alleles with antibody reactivity (data not shown). Linear regressions were conducted to estimate the adjusted difference in antibody level between carrier and non-carrier groups, adjusted by age,LST-IOOIWO PATENT APPLICATION gender, plate, and PCA1-5. Subgroup analysis of the control group and MS cases group were performed in each HLA allele.

[0341] Association of Nil with antibody levels. Pearson correlation coefficient tests and Spearman correlation coefficient tests were performed to check the association of Nil and antibody levels against GlialCAM AA365-394 and EBNA1 AA381-410. (FIG. 19A-19B).

[0342] Combination of HLA and antibody seropositivity and MS risk. The association of HLA-DRB1 *15:01 with seropositive antibody levels against EBNA1 and GlialCAM, CRY AB, and AN02 were estimated. Medians were used here as the cutoff value definition. Antibodies against EBNA1 AA381-410 and GlialCAM AA365-394, EBNA1 AA381-410 and CRY AB AA2-33, and EBNA1 AA421-450 and AN02 AA134-153 were selected to represent antibodies against EBNA1 peptide regions and its structural homologous CNS epitopes in GlialCAM, CRY AB, and ANO2 (FIGS. 4A-4I). Logistic regression was used adjusting age, gender, plate, and PCA1-5 to estimate OR, with the group carrying no risk factors as reference. ORs under different condition combinations were shown in HLA association plots (FIGS. 4A-4I). Furthermore, the associations of GlialCAM AA365-394 with EBNA1 AA381-410, CRY AB AA2-33 with EBNA1 AA381-410, and AN02 AA134-153 with EBNA1 AA421-450 were also estimated with the combination of HLA-A* 02:01 with HLA-DRB1 *15: O1 (FIG. 18A-18C).

[0343] Accumulation of EBNA1 and CNS specific antibody association with MS incidence. The association of MS with accumulation of high EBNA1 -specific and CNS-specific antibody levels were estimated. For EBNA1 -specific IgG, we selected EBNA1 AA386- 405. EBNA1 AA381-410, and EBNA1 AA421-450, since they overlapped with sequence homology regions for GlialCAM AA370-389, CRY AB AA2-33, and ANO2 AA134-153 respectively. The distribution of accumulation of anti-EBNAl and anti-CNS-mimic antibody status amongst the control group and MS case group were calculated and compared using chi square test. Median values were used as the cutoff value and logistic regression was performed to calculate ORs, adjusted by age, gender, plate, and PCA1-5.

[0344] REFERENCESEach of the following references are herein incorporated by reference in their entirety.1. S. L. Hauser, B. A. C. Cree, Treatment of multiple sclerosis: A review. Am. J. Med. 133,1380-1390.e2 (2020).2. K. Bjomevik, et al., Longitudinal analysis reveals high prevalence of Epstein-Barr virus associated with multiple sclerosis. Science 375, 296-301 (2022).LST-IOOIWO PATENT APPLICATION 3. S. L. Atkins, et al., Small molecule screening identifies inhibitors of the Epstein-Barr virus deubiquitinating enzyme, BPLF1. Antiviral Res. 173, 104649 (2020).4. A. K. Hedstrom, et al., High Levels of Epstein-Barr Virus Nuclear Antigen- 1 -Specific Antibodies and Infectious Mononucleosis Act Both Independently and Synergistically to Increase Multiple Sclerosis Risk. Frontiers in Neurology) [Preprint] (2020). Available at: http: / / dx.doi.org / 10.3389 / fneur.2019.01368.5. M. Cortese, et al.. Serologic response to the Epstein-Barr virus peptidome and the risk for multiple sclerosis. JAMA Neurol. 81, 515-524 (2024).6. A. K. Hedstrom, L. Alfredsson. T. Olsson, Environmental factors and their interactions with risk genotypes in MS susceptibility. Curr. Opin. Neurol. 29, 293-298 (2016). 7. L. Moutsianas, et al., Class II HLA interactions modulate genetic risk for multiple sclerosis. Nat. Genet. 47, 1107-1113 (2015).8. T. Olsson, L. F. Barcellos, L. Alfredsson, Interactions between genetic, lifestyle and environmental risk factors for multiple sclerosis. Nat. Rev. Neurol. 13, 25-36 (2017). 9. M. P. Pender. P. A. Csurhes, J. M. Burrows, S. R. Burrows, Defective T-cell control of Epstein-Barr virus infection in multiple sclerosis. Clin. Transl. Immunology 6, el 26 (2017).10. O. G. Thomas, A. Rickinson. U. Palendira. Epstein-Barr virus and multiple sclerosis: moving from questions of association to questions of mechanism. Clin. Transl.Immunology 12, el 451 (2023).11. A. Gottlieb. H. P. T. Pham, J. G. Saltarrelli, J. W. Lindsey, Expanded T lymphocytes in the cerebrospinal fluid of multiple sclerosis patients are specific for Epstein-Barr-virus- infected B cells. Proc. Natl. Acad. Sci. U. S. A. 121, e2315857121 (2024).12. T. Schneider-Hohendorf, et al.. Broader Epstein-Barr virus-specific T cell receptor repertoire in patients with multiple sclerosis. J. Exp. Med. 219 (2022).13. S. S. Soldan, et al., Multiple sclerosis patient-derived spontaneous B cells have distinct EBV and host gene expression profiles in active disease. Nat. Microbiol. 9, 1540-1554 (2024).LST-IOOIWO PATENT APPLICATION 14. J. B. Harley, et al., Transcription factors operate across disease loci, with EBNA2 implicated in autoimmunity. Nat. Genet. 50, 699-707 (2018).15. T. V. Lanz, et al., Clonally expanded B cells in multiple sclerosis bind EBV EBNA1 and GlialCAM. Nature 603, 321-327 (2022).16. N. R. Jog, et al., Epstein Barr virus nuclear antigen 1 (EBNA-1) peptides recognized by¬ adult multiple sclerosis patient sera induce neurologic symptoms in a murine model. J. Autoimmun. 106, 102332 (2020).17. O. G. Thomas, et al., Cross-reactive EBNA1 immunity targets alpha-crystallin B and is associated with multiple sclerosis. Sci. Adv. 9, eadg3032 (2023).18. K. Tengvall, et al., Molecular mimicry between Anoctamin 2 and Epstein-Barr virus nuclear antigen 1 associates with multiple sclerosis risk. Proc. Natl. Acad. Sci. U. S. A.116. 16955-16960 (2019).19. K. Ruprecht, et al., Multiple sclerosis: the elevated antibody response to Epstein-Barr virus primarily targets, but is not confined to, the glycine-alanine repeat of Epstein-Barr nuclear antigen- 1. J. Neuroimmunol. 272, 56-61 (2014).20. N. Jafari, et al., No evidence for intrathecal IgG synthesis to Epstein Barr virus nuclear antigen-1 in multiple sclerosis. J. Clin. Virol. 49, 26-31 (2010).21. J. Salzer, et al., Epstein-Barr virus antibodies and vitamin D in prospective multiple sclerosis biobank samples. Mult. Scler. 19, 1587-1591 (2013).22. E. Sundqvist, et al., Epstein-Barr virus and multiple sclerosis: interaction with HLA.Genes Immun. 13, 14-20 (2012).23. P. V. Lehmann, T. Forsthuber, A. Miller, E. E. Sercarz, Spreading of T-cell autoimmunity to cryptic determinants of an autoantigen. Nature 358, 155-157 (1992).24. Y. D. Dai, G. Carayanniotis, E. Sercarz, Antigen processing by autoreactive B cells promotes determinant spreading. Cell. Mol. Immunol. 2, 169-175 (2005).25. R. Martin, M. Sospedra, T. Eiermann, T. Olsson. Multiple sclerosis: doubling down on MHC. Trends Genet. 37, 784-797 (2021).LST-IOOIWO PATENT APPLICATION 26. H. Zdimerova, et al., Atenuated immune control of Epstein-Barr virus in humanized mice is associated with the multiple sclerosis risk factor HLA-DR15. Eur. J. Immunol.51, 64-75 (2021).27. N. Drosu, et al., CD4 T cells restricted to DRBl*15:01 recognize two Epstein-Barr virus glycoproteins capable of intracellular antigen presentation. Proc. Natl. Acad. Sci. U. S. A. 121 (2024).28. A. K. Hedstrom, M. Baamhielm, T. Olsson, L. Alfredsson, Tobacco smoking, but not Swedish snuff use, increases the risk of multiple sclerosis. Neurology 73, 696-701 (2009).29. International Multiple Sclerosis Genetics Consortium, Multiple sclerosis genomic map implicates peripheral immune cells and microglia in susceptibility. Science 365, eaav7188 (2019).30. H. Vietzen, et al.. Ineffective control of Epstein-Barr- virus -induced autoimmunity’ increases the risk for multiple sclerosis. Cell 186, 5705-5718.e13 (2023).31. H. Vietzen, et al., Accumulation of Epstein-Barr virus-induced cross-reactive immune responses is associated with multiple sclerosis. J. Clin. Invest. 134 (2024).32. M. De Paschale, P. Clerici, Serological diagnosis of Epstein-Barr virus infection:Problems and solutions. World J. Virol. 1, 31-43 (2012).33. W. Henle, et al.. Antibody responses to Epstein-Barr virus-determined nuclear antigen (EBNA)-l and EBNA-2 in acute and chronic Epstein-Barr virus infection. Proc. Natl. Acad. Sci. U. S. A. 84, 570-574 (1987).34. M.-S. Kang, E. Kieff, Epstein-Barr virus latent genes. Exp. Mol. Med. 47, e131 (2015).35. D. Jons, et al., Seroreactivity against lytic, latent and possible cross-reactive EBV antigens appears on average 10 years before MS induced preclinical neuroaxonal damage. J. Neurol. Neurosurg. Psychiatry 95, 325-332 (2024).36. J. Dheekollu, et al., Cell-cycle-dependent EBNA1-DNA crosslinking promotes replication termination at oriP and viral episome maintenance. Cell 184. 643-654. el 3 (2021).LST-IOOIWO PATENT APPLICATION 37. E. A. Leadbetter, et al., Chromatin-IgG complexes activate B cells by dual engagement of IgM and Toll-like receptors. Nature 416, 603-607 (2002).38. V. Laurynenka, L. Ding, K. M. Kaufman, J. A. James, J. B. Harley, A high prevalence of anti-EBNAl heteroantibodies in systemic lupus erythematosus (SLE) supports anti- EBNA1 as an origin for SLE autoantibodies. Front. Immunol. 13, 830993 (2022).39. J. Huang, et al., Genetics of immune response to Epstein-Barr virus: prospects for multiple sclerosis pathogenesis. Brain 147, 3573-3582 (2024).40. V. I. Zarnitsyna, et al., Masking of antigenic epitopes by antibodies shapes the humoral immune response to influenza. Philos. Trans. R. Soc. Lond. B Biol. Sci. 370, 20140248 (2015).41. S. S. Ousman, et al., Protective and therapeutic role for alphaB-crystallin in autoimmune demyelination. Nature 448, 474-479 (2007).42. E. Jeworutzki, et al., GlialCAM, a protein defective in a leukodystrophy, serves as a C1C-2 Cl(-) channel auxiliary subunit. Neuron 73, 951-961 (2012).43. H. Tamaki, B. L. Beaulieu, M. Somasundaran. J. L. Sullivan, Major histocompatibility complex class I-restricted cytotoxic T lymphocyte responses to Epstein-Barr virus in children. J. Infect. Dis. 172, 739-746 (1995).44. N. Patterson, A. L. Price, D. Reich. Population structure and eigenanalysis. PLoS Genet.2, el90 (2006).45. J. Porszasz, K. P. Gibiszer, J. Porszasz Jr, Pharmacological investigation on the Neurohumoral transmission of the vasomotor regulation. Acta Physiol. Acad. Sci. Hung. 49, 139-165 (1977).

Claims

LST-IOOIWO PATENT APPLICATION CLAIMS:

1. A chimeric autoantibody receptor (C AAR) comprising an extracellular domain having at least one autoantigen or fragment thereof selected from the group consisting of GlialCAM, CRY AB. and AN02, a transmembrane domain, and an intracellular signaling domain.

2. The CAAR of claim 1, wherein the autoantigen or fragment thereof is GlialCAM comprising an amino acid sequence of X₁SPPRAP (SEQ ID NO: 3) wherein X₁ is S that is phosphorylated or non-phosphorylated.

3. The CAAR of claim 2. wherein the autoantigen or fragment thereof is GlialCAM comprising an amino acid sequence selected from the group consisting of GlialCAM AA365-394 (SEQ ID NO: 32), GlialCAM AA365-394-pSer376 (SEQ ID NO: 33), GlialCAM AA376-389 (SEQ ID NO: 34), GlialCAM AA376-389-pSer376 (SEQ ID NO: 35), and GlialCAM AA385-416 (SEQ ID NO: 36).

4. The CAAR of claim 1, wherein the autoantigen or fragment thereof is ANO2 comprising an amino acid sequence of PGDIELGPLD (SEQ ID NO: 12).

5. The CAAR of claim 4, wherein the autoantigen or fragment thereof is ANO2 comprising an amino acid sequence selected from the group consisting of ANO2 AA1-275 (SEQ ID NO: 37) and ANO2 AA134-153 (SEQ ID NO: 38).

6. The CAAR of claim 1, wherein the autoantigen or fragment thereof is CRYAB comprising an amino acid sequence of PPGRRPFF (SEQ ID NO: 8).

7. The CAAR of claim 6, wherein the autoantigen or fragment thereof is CRYAB comprising an amino acid sequence selected from the group consisting of CRYAB AA1- 275 (SEQ ID NO: 39) and CRYAB AA2-33 (SEQ ID NO: 40).

8. The CAAR of claim 1, wherein the autoantigen or fragment thereof comprises GlialCAM AA376-389-pSer376 (SEQ ID NO: 35), CRY AB AA8-15 (SEQ ID NO: 8), and ANO2 AA140-149 (SEQ ID NO: 12).

9. The CAAR of claim 8, wherein the extracellular domain further comprises a linker that forms a linkage between the GlialCAM AA376-389-pSer376 and CRY AB AA8-15, and a linkage between CRY AB AA8-15 and ANO2 AA140-149.

10. The CAAR of claim 9, wherein the linker is a glycine rich linker selected from the group consisting of GGGGG. GGSSG. GGGGSLVPRGSGGGGS, (GS)nwhere n is 2-5, GGSGGHMGSGG, GGSGGSGGSGG, GGSGpwhere p is 1-5, GSGGGTGGGSG, and GSGGSGGSGGSGGS.LST-IOOIWO PATENT APPLICATION 11. The CAAR of claim 1, further comprising a co-stimulatory domain selected from the group consisting of CD28 and tumor necrosis factor ligand superfamily member 9 (4- 1BB)12. The CAAR of claim 1, wherein the intracellular signaling domain comprises a CD3 zeta signaling domain.

13. The CAAR of claim 1, wherein the transmembrane domain comprises a CD8 alpha chain hinge and transmembrane domain.

14. A vector comprising a nucleic acid sequence encoding a chimeric autoantibody receptor (CAAR) comprising an extracellular domain having at least one autoantigen or a fragment thereof selected from the group consisting of GlialCAM, CRY AB, and AN02, a transmembrane domain, and an intracellular signaling domain.

15. The vector of claim 14, wherein the extracellular domain has GlialCAM comprising an amino acid sequence of X₁SPPRAP (SEQ ID NO: 3) wherein X₁ is S that is phosphorylated or non-phosphorylated.

16. The vector of claim 15, wherein GlialCAM comprises an amino acid selected from the group consisting of GlialCAM AA365-394 (SEQ ID NO: 32), GlialCAM AA365-394- pSer376 (SEQ ID NO: 33), GlialCAM AA376-389 (SEQ ID NO: 34), GlialCAM AA376-389-pSer376 (SEQ ID NO: 35). and GlialCAM AA385-416 (SEQ ID NO: 36).

17. The vector of claim 14, wherein the extracellular domain has AN02 comprising an amino acid sequence of PGDIELGPLD (SEQ ID NO: 12).

18. The vector of claim 17, wherein AN02 comprises an amino acid selected from the group consisting of AN02 AA1-275 (SEQ ID NO: 37) and AN02 AA134-153 (SEQ ID NO: 38).

19. The vector of claim 14, wherein the extracellular domain has CRY AB comprising an amino acid sequence of PPGRRPFF (SEQ ID NO: 8).

20. The vector of claim 19, wherein CRY AB comprises an amino acid selected from the group consisting of CRY AB AA1-275 (SEQ ID NO: 39) and CRY AB AA2-33 (SEQ ID NO: 40).

21. The vector of claim 14, wherein the extracellular domain comprises GlialCAM AA376- 389-pSer376 (SEQ ID NO: 35), CRY AB AA8-15 (SEQ ID NO: 8). and ANO2 AA140- 149 (SEQ ID NO: 12).

22. The vector of claim 21, wherein the extracellular domain further comprises a linker that forms a linkage between the GlialCAM AA376-389-pSer376 and CRY AB AA8-15, and a linkage between CRY AB AA8-15 and ANO2 AA140-149.LST-IOOIWO PATENT APPLICATION 23. The vector of claim 22, wherein the linker is a glycine rich linker selected from the group consisting of GGGGG, GGSSG, GGGGSLVPRGSGGGGS, (GS)nwhere n is 2-5, GGSGGHMGSGG, GGSGGSGGSGG, GGSGpwhere p is 1-5, GSGGGTGGGSG, and GSGGSGGSGGSGGS.

24. The vector of claim 14, further comprising a co-stimulatory domain selected from the group consisting of CD28 and tumor necrosis factor ligand superfamily member 9 (4- 1BB)25. The vector of claim 14, wherein the intracellular signaling domain comprises a CD3 zeta signaling domain.

26. The vector of claim 14, wherein the transmembrane domain comprises a CD8 alpha chain hinge and transmembrane domain.

27. A genetically modified cell comprising a chimeric autoantibody receptor (CAAR) comprising an extracellular domain having at least one autoantigen or fragment thereof selected from the group consisting of GlialCAM, CRY AB, and AN02, a transmembrane domain, and an intracellular signaling domain, wherein the cell expresses the CAAR and binds an autoantibody -expressing cell.

28. The cell of claim 27, wherein the autoantibody-expressing cell is at least one selected from the group consisting of a plasmablast, a plasma cell, and a B cell.

29. The cell of claim 28, wherein the CAAR further induces killing of the autoantibodyexpressing cell.

30. The cell of claim 27, wherein the cell is selected from the group consisting of a helper T cell, a cytotoxic T cell, a memory T cell, a regulatory T cell, a gamma delta T cell, a natural killer cell, a cytokine induced killer cell, and a cell line thereof.

31. The cell of claim 29, wherein the genetically modified cell is a T cell.

32. The cell of claim 27, wherein the extracellular domain comprises GlialCAM or a fragment thereof having an amino acid selected from the group consisting of GlialCAM AA365-394 (SEQ ID NO: 32). GlialCAM AA365-394-pSer376 (SEQ ID NO: 33).GlialCAM AA376-389 (SEQ ID NO: 34), GlialCAM AA376-389-pSer376 (SEQ ID NO: 35), and GlialCAM AA385-416 (SEQ ID NO: 36).

33. The cell of claim 27, wherein the extracellular domain comprises GlialCAM having an amino acid sequence of X₁SPPRAP (SEQ ID NO: 3) wherein X₁ is S that is phosphorylated or non-phosphorylated.

34. The cell of claim 32, wherein the extracellular domain comprises GlialCAM or a fragment thereof having an amino acid sequence selected from the group consisting ofLST-IOOIWO PATENT APPLICATION GlialCAM AA365-394 (SEQ ID NO: 32), GlialCAM AA365-394-pSer376 (SEQ ID NO: 33), GlialCAM AA376-389 (SEQ ID NO: 34), GlialCAM AA376-389-pSer376 (SEQ ID NO: 35), and GlialCAM AA385-416 (SEQ ID NO: 36).

35. The cell of claim 27, wherein the extracellular domain comprises ANO2 having an amino acid sequence of PGDIELGPLD (SEQ ID NO: 12).

36. The cell of claim 34, wherein the extracellular domain comprises ANO2 or a fragment thereof having an amino acid sequence selected from the group consisting of AN02 AA1-275 (SEQ ID NO: 37) and ANO2 AA134-153 (SEQ ID NO: 38).

37. The cell of claim 27, wherein the extracellular domain comprises CRY AB having an amino acid sequence of PPGRRPFF (SEQ ID NO: 8).

38. The cell of claim 36, wherein the extracellular domain comprises CRY AB or a fragment thereof having an amino acid sequence selected from the group consisting of CRY AB AA1-275 (SEQ ID NO: 39) and CRYAB AA2-33 (SEQ ID NO: 40).

39. The cell of claim 27, wherein the extracellular domain comprises GlialCAM AA376-389- pSer376 (SEQ ID NO: 35), CRY AB AA8-15 (SEQ ID NO: 8), and ANO2 AA140-149 (SEQ ID NO: 12).

40. The cell of claim 38, wherein the extracellular domain further comprises a linker that forms a linkage between the GlialCAM AA376-389-pSer376 and CRY AB AA8-15, and a linkage between CRY AB AA8-15 and ANO2 AA140-149.

41. The cell of claim 27, wherein the linker is a glycine rich linker selected from the group consisting of GGGGG, GGSSG, GGGGSLVPRGSGGGGS, (GS)nwhere n is 2-5, GGSGGHMGSGG, GGSGGSGGSGG, GGSGpwhere p is 1-5. GSGGGTGGGSG, and GSGGSGGSGGSGGS.

42. The cell of claim 27, further comprising a co-stimulatory domain selected from the group consisting of CD28 and tumor necrosis factor ligand superfamily member 9 (4-1BB).

43. The cell of claim 27, wherein the intracellular signaling domain comprises a CD3 zeta signaling domain.

44. The cell of claim 27, wherein the transmembrane domain comprises a CD8 alpha chain hinge and transmembrane domain.

45. A method of treating, preventing, or managing multiple sclerosis in a subject in need thereof, comprising administering to the subject a genetically modified cell comprising a chimeric autoantibody receptor (CAAR) comprising an extracellular domain having at least one autoantigen or fragment thereof selected from the group consisting of GlialCAM, CRY AB, and ANO2, a transmembrane domain, and an intracellular signalingLST-IOOIWO PATENT APPLICATION domain, wherein the cell expresses the CAAR and binds to an autoantibody-expressing cell.

46. The method of claim 45, wherein the autoantibody-expressing cell is at least one selected from the group consisting of a plasmablast, a plasma cell, and a B cell.

47. The method of claim 45. wherein the genetically modified cell is selected from the group consisting of a helper T cell, a cytotoxic T cell, a memory T cell, a regulator}' T cell, a gamma delta cell, a natural killer cell, a cytokine induced killer cell, and a cell line thereof.

48. The method of claim 45, wherein the extracellular domain comprises GlialCAM having an amino acid sequence of X₁SPPRAP (SEQ ID NO: 3) wherein X₁ is S that is phosphorylated or non-phosphorylated.

49. The method of claim 45, wherein the extracellular domain comprises GlialCAM or a fragment thereof having an amino acid sequence selected from the group consisting of GlialCAM AA365-394 (SEQ ID NO: 32). GlialCAM AA365-394-pSer376 (SEQ ID NO: 33), GlialCAM AA376-389 (SEQ ID NO: 34), GlialCAM AA376-389-pSer376 (SEQ ID NO: 35), and GlialCAM AA385-416 (SEQ ID NO: 36).

50. The method of claim 45, wherein the extracellular domain comprises ANO2 having an amino acid sequence of PGDIELGPLD (SEQ ID NO: 12).

51. The method of claim 50, wherein the extracellular domain comprises ANO2 or a fragment thereof having an amino acid sequence selected from the group consisting of ANO2 AA1-275 (SEQ ID NO: 37) and ANO2 AA134-153 (SEQ ID NO: 38).

52. The method of claim 45, wherein the extracellular domain comprises CRY AB having an amino acid sequence of PPGRRPFF (SEQ ID NO: 8).

53. The method of claim 52, wherein the extracellular domain comprises CRY AB or a fragment thereof having an amino acid sequence selected from the group consisting of CRY AB AA1-275 (SEQ ID NO: 39) and CRY AB AA2-33 (SEQ ID NO: 40).

54. The method of claim 45. wherein the extracellular domain comprises GlialCAM AA376- 389-pSer376 (SEQ ID NO: 35), CRY AB AA8-15 (SEQ ID NO: 8), and ANO2 AA140- 149 (SEQ ID NO: 12).

55. The method of claim 54, wherein the extracellular domain further comprises a linker that forms a linkage between the GlialCAM AA376-389-pSer376 and CRY AB AA8-15, and a linkage between CRY AB AA8-15 and ANO2 AA140-149.

56. The method of claim 55, wherein the linker is a glycine rich linker selected from the group consisting of GGGGG, GGSSG, GGGGSLVPRGSGGGGS, (GS)nwhere n is 2-5,LST-IOOIWO PATENT APPLICATION GGSGGHMGSGG, GGSGGSGGSGG, GGSGpwhere p is 1-5, GSGGGTGGGSG, and GSGGSGGSGGSGGS.

57. The method of claim 45, further comprising a co-stimulatory domain selected from the group consisting of CD28 and tumor necrosis factor ligand superfamily member 9 (4- 1BB).

58. The method of claim 45, wherein the intracellular signaling domain comprises a CD3 zeta signaling domain.

59. The method of claim 45, wherein the transmembrane domain comprises a CD8 alpha chain hinge and transmembrane domain.

60. The method of claim 45, further comprising measuring at least one anti-EBNAl antibody, anti-GlialCAM antibody, anti-CRYAB antibody, or anti-ANO2 antibody level in a human tissue.

61. The method of claim 60, wherein the human tissue comprises plasma, serum, blood, or cerebrospinal fluid (CSF).

62. The method of claim 45, further comprising determining an antibody level of at least one antibody selected from the group consisting of anti-EBNAl antibody, anti-GlialCAM antibody, anti-CRYAB antibody, and anti-ANO2 antibody.

63. The method of claim 62, wherein following the determining step, the genetically modified cell comprising the CAAR is administered to the subject based on a criteria wherein: (i) when anti-EBNAl antibody and / or anti-GlialCAM antibody levels are elevated in the subject, the subject is administered the CAAR comprising the extracellular domain comprising GlialCAM having an amino acid sequence of X1SPPRAP (SEQ ID NO: 3) wherein X1is S that is phosphorylated or non-phosphorylated; or(j) when anti-CRYAB antibody and anti-ANO2 antibody levels are elevated in the subject, the subject is administered the CAAR comprising the extracellular domain comprising CRY AB having an amino acid sequence of PPGRRPFF (SEQ ID NO: 8); or(k) when anti-ANO2 antibody levels are elevated in the subject, the subject is administered the CAAR comprising the extracellular domain comprising ANO2 having an amino acid sequence of PGDIELGPLD (SEQ ID NO: 12); or(l) when anti-GlialCAM antibody, anti-CRYAB antibody, and anti-ANO2 antibody levels are elevated in the subject, the subject is administered the CAAR comprising the extracellular domain having GlialCAM AA376-389-pSer376 (SEQ ID NO: 35), CRY AB AA8-15 (SEQ ID NO: 8), and ANO2 AA140-149 (SEQ ID NO: 12), and aLST-IOOIWO PATENT APPLICATION linker that forms a linkage between GlialCAM AA376-389-pSer376 and CRY AB AA8-15, and a linkage between CRY AB AA8-15 and ANO2 AA140-149.

64. The method of claim 45, wherein the genetically modified cell is administered once a month for the first two months, and then once every six months.

65. The method of claim 45. wherein the subject is administered a first dose 1 x 108cells / kg body weight with lymphodepletion or a dose of 1 x 108of the genetically modified cells without lymphodepletion.

66. The method of claim 45, wherein the subject is administered a dosage of 104to 109cells / kg body weight.

67. A pharmaceutical composition comprising the chimeric autoantibody receptor (CAAR) of any one of claims 1-13, the vector of any one of claims 14-26, or the genetically modified cell of any one of claims 27-44, and at least one pharmaceutically acceptable carrier or diluent.

Images