Cross-reactive epitopes for multiple sclerosis

Abstract

Provided herein are methods and compositions for the diagnosis and treatment of multiple sclerosis. Provided are compositions and methods relating to novel peptides involved in autoimmune diseases. In the experiments described herein, an unbiased approach was used to isolate pathogenic CD4 peptides from patients with multiple sclerosis (MS). + T cells are screened to determine the antigen specificity of the T cell receptors (TCRs) expressed by these pathogenic cells.

Description

[Background technology]

[0001] There has been a long-standing interest in manipulating cells of the immune system to achieve control of autoimmune diseases and other inflammatory diseases. Traditional treatment methods have generally been non-antigen specific. For example, general immunosuppression utilizes drugs such as methylprednisolone, other steroids, methotrexate, cladribine, and cyclophosphamide. However, the global immunosuppression provided by these therapies has significant undesirable side effects.

[0002] More selective modulation of the immune system utilizes cytokine blockers, such as anti-TNFα antibodies, soluble TNFα receptors, soluble IL-1 receptors (anakinra), and anti-IL-6R antibodies (tocilizumab), T cell-targeted therapies (CTLA4-Ig [abatacept]), and B cell-targeted therapies (anti-CD20 [rituximab]), as well as anti-inflammatory cytokines such as interferon beta (IFNβ)-1b (Betaferon / Betaseron).

[0003] However, although these therapies are more targeted, there is still immunosuppression of the overall class of responses, rather than specifically the undesired responses.

[0004] Thus, the promise of highly selective antigen-specific therapy remains attractive, but difficult. Such specificity could provide effective treatment of unwanted immune responses without involving the entire immune system population or response. Identification of the initiating antigen is of clinical interest for this and for diagnostic purposes, and is addressed herein. Summary of the Invention

[0005] Compositions and methods are provided relating to novel peptides involved in autoimmune diseases. In the experiments described herein, an unbiased approach was used to isolate pathogenic CD4 peptides from patients with multiple sclerosis (MS). +T cells were screened to determine the antigen specificity of the T cell receptors (TCRs) expressed by these pathogenic cells. Surprisingly, this method found that peptide epitopes present in human adenoviruses can activate MS pathogenic T cells in an MHC-associated context, and that the TCRs activated by these peptides also react with myelin basic protein (MBP) epitopes, which are thought to be targets for MS autoimmune responses. Because the adenovirus and MBP amino acid sequences are dissimilar, cross-reactivity was unlikely to be found using conventional screening methods. Adenoviruses and related peptides may be referred to herein as "cross-reactive" peptides.

[0006] Without being bound by theory, it can be hypothesized that in certain MHC contexts, including but not limited to the human DR15 protein, the initial T cell response to viral proteins can initiate a spillover response to self-antigens that lack overt sequence similarity yet share the same TCR activation.

[0007] Knowledge of the initiation of autoantigens can be used to develop specific therapies and diagnostics for MS, instead of the non-specific immunomodulation that has traditionally been used. The present invention provides important candidate antigens for involvement in the initiation of MS development, providing targets for diagnostic and therapeutic intervention.

[0008] Compositions of cross-reactive peptides for multiple sclerosis are provided. In some embodiments, the cross-reactive peptides comprise or consist of the amino acid sequence (SEQ ID NO: 1) ATFTSYRSWYLA, or variants thereof, e.g., peptides modified by deletion or substitution of one, two, three, or more amino acids. In other embodiments, the peptides comprise or consist of the amino acid sequence (SEQ ID NO: 2) ANYGKARSWYLK, (SEQ ID NO: 3) IDRHMYHSYLK, or (SEQ ID NO: 4) DKGQQYRNWFLK, or variants thereof, e.g., peptides modified by deletion or substitution of one, two, three, or more amino acids. In some embodiments, the cross-reactive peptides comprise or consist of the peptide sequences set forth in Figure 2 or Figure 4, SEQ ID NOs: 23-75, or variants thereof, e.g., peptides modified by deletion or substitution of one, two, three, or more amino acids. In certain embodiments, the variant peptide retains the same binding specificity and / or affinity for the TCR as the amino acid sequence of the peptide from which it is derived, i.e., SEQ ID NO: 1, 2, 3, 4, etc. In other embodiments, the variant peptide retains the T cell receptor specificity but with altered affinity as the peptide from which it is derived, i.e., SEQ ID NO: 1, 2, 3, 4, etc.

[0009] The T cell response to cross-reactive peptides can serve as a diagnostic marker for MS.A variety of methods can be used to detect enhanced T cell response, such as screening for activation markers on cross-reactive peptide-specific T cells in body fluids, including cerebrospinal fluid, examining the frequency of specific T cells, or enhancing the production of cytokines that respond to cross-reactive peptides.The increase in immune response to either humoral or cellular cross-reactive peptides can be specifically related to MS and can be used to aid diagnosis.

[0010] Antibodies can be made against cross-reactive peptides, complexes of cross-reactive peptides with human MHC proteins, including but not limited to DR15 protein, or TCRs that respond to the cross-reactive peptides described herein, e.g., the TCR sequences shown in Figure 1G, SEQ ID NOS: 5-22, or SEQ ID NOS: 76-83 of Figure 9. Such antibodies can be therapeutic in blocking the activation of pathogenic T cells.

[0011] Cross-reactive peptides are also used in tolerization strategies to reduce pathogenic responses, for example, through engineered peptide ligands (APLs), manipulation of dendritic cell responses, biasing T cell responses towards non-pathogenic responses, etc. [Brief explanation of the drawings]

[0012] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication and color drawings will be provided by the Patent Office upon request and payment of the necessary fee. It is emphasized that, according to common practice, the various features of the drawings are not to scale. Conversely, the dimensions of the various features are arbitrarily enlarged or reduced for clarity. The drawings include the following figures:

[0013] [Figure 1]We analyzed PBMCs from 18 newly diagnosed MS patients and four healthy controls (HCs). We first performed CytoF to determine whether differences existed in T cell populations between HCs and MS patients. MS patients were found to have a higher frequency of brain-homing activated T cells, which we analyzed by single-cell sorting and paired TCR sequencing. We found the following: 1. MS CD8 T cells proliferated massively; 2. MS CD4 T cells showed minimal proliferation; and 3. γδ T cells proliferated at levels similar to HCs. Because MS is highly associated with DR150101 (20-30% susceptibility), we focused on CD4 TCR specificity. To identify antigen specificity, we clustered CD4 TCRs with GLIPH. Using GLIPH, we were able to identify CD4 TCR clusters enriched in DR150101 MS individuals. As shown above, we selected nine CD4 TCRs clustered with DR15 for antigen discovery. These nine CD4 TCRs were produced as soluble recombinant proteins and screened against 12-MER and 15-MER DR15 yeast libraries. As shown above in B–E, the screen reveals enrichment (A647-Myc) and tetramer (PE-TCR Tet) staining for four of the nine CD4 TCRs (MS-1 through MS-4 TCRs). In A and F, no peptide enrichment was found for some of the TCRs (DR15-TCR1 and DR15-TCR9). G shows a table listing the CD4 TCR sequences, SEQ ID NOs: 5–22, screened against the DR15 yeast library. [Figure 2]We generated an MS1-TCR cell line and co-cultured it with a T2-DR15 antigen-presenting cell line loaded with peptides generated in the yeast library to examine activation. CD69 was used as a marker of T cell activation. In A, the majority of the enriched peptides in the library, as shown in SEQ ID NOs: 23-52, stimulated the MS-1 TCR cell line. Using the yeast library enrichment data, we predicted the actual peptides / antigens (self or non-self) for the MS-1 CD4 TCR. Surprisingly, we found that an adenovirus peptide (adeno-ATFTSYRSWYLA, SEQ ID NO: 1) was nearly identical to the enriched peptide, and it also stimulated the MS-1 TCR cell line. [Figure 3] In addition to enriching the yeast library peptides, we added an additional layer to help the algorithm determine wild-type peptides. Essentially, we created single-point mutations in the top, most enriched library peptides and used the T cell stimulation data of these positionally mutated peptides to generate a substitution matrix, which was then used by the algorithm to search for actual peptides from the human proteome. We used T cell receptor 2, TCR2; DR15-expressing T2 lymphoblastoid cell line, DR15T2; myelin basic protein, MBP; adeno-peptide, adeno; library peptide, Lib-PP; and modified library peptide, ALIBPP. Using this positional mutation / refined substitution matrix data, we were able to identify three self-peptides for the TCR2 / MS-1 TCR. [Figure 4] Single-point mutations on yeast library peptides (A) were used to generate an improved substitution matrix. Using the new substitution matrix (B), candidate self-peptides were predicted, and these newly predicted peptides, SEQ ID NOs: 53-75, were used for MS-1 TCR stimulation. [Figure 5] Positional mutation / refinement substitution matrix data was used to identify three self-peptides, SEQ ID NOs: 2, 3, and 4, for the TCR2 / MS-1 TCR shown in A and B. [Figure 6]To detect and determine adeno-specific T cells directly from MS patients (n=9) and healthy controls (n=9) (A), pMHC tetramers were generated (B), enriched, and CD4 T cells specific for adeno-peptides were enumerated. (C) We were able to detect higher numbers of adeno-specific CD4 T cells in MS patients compared to healthy controls (D). We also single-cell sorted these adeno-specific CD4 T cells, sequenced their TCRs, and generated TCR cell lines (adeno-specific TCR). [Figure 7] TCR cell lines derived from adenotetramer-selected CD4 TCRs were tested for cross-reactivity with the MBP85-99 peptide. The MS-1 TCR was also tested for cross-reactivity with MBP. Adenospecific TCRs were stimulated with adeno-peptide and MBP. (A) TCR2 / ms-1 TCR and (B) some of the adeno-TCRs (adeno-TCR1 and 9) cross-react with the MBP peptide. [Figure 8] Sera from MS (n=28) and HC (n=10) were tested for adenovirus titers in the serum. A subset of MS sera had higher adenovirus compared to HC sera. [Figure 9] The TCRαβ sequences for DR15 TCR2 / MS-1 TCR, DR15-adeno TCR2, SEQ ID NOs: 76-83, and adeno TCR9 are shown, as well as the peptide sequences for MBP, adeno, and three self-peptides, SEQ ID NOs: 84, 1-4. [Figure 10] We sorted CD3+ T cells from eight MS patients and five HCs and performed TCR and RNA-seq using a 10x platform. Additionally, we sequenced T cells from cerebrospinal fluid (CSF) of four of the eight MS patients. [Figure 11] Cerebrospinal fluid-derived T cells were sequenced in four of eight MS patients. Clonal expansion was found in (A) CD4+ and (B) CD8+ T cells within the CSF of each MS patient. DETAILED DESCRIPTION OF THE INVENTION

[0014] Before the present methods are described, it is to be understood that this invention is not limited to particular methods described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

[0015] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range, and any other stated or intervening value within this stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, subject to any specifically excluded limit in the stated range. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Although any method and material similar or equivalent to those described herein can also be used to carry out or test the present invention, preferred methods and materials are described below.All publications mentioned herein are incorporated by reference to disclose and describe the method and / or material in connection with which the publication is cited.

[0017] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0018] General methods in molecular and cellular biochemistry are covered in Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001), Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999), Protein Methods (Bollag et al., John Wiley & Sons 1996), Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999), Viral Vectors (Kaplift & Loewy eds., Academic Press 1995), Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997), and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons Genetic engineering reagents, cloning vectors, and kits referred to in this disclosure are available from commercial vendors such as BioRad, Stratagene, Invitrogen, Sigma-Aldrich, and ClonTech.

[0019] The present invention has been described in terms of particular embodiments discovered or contemplated by the inventors to comprise preferred modes for carrying out the invention. Those skilled in the art will understand, in light of this disclosure, that numerous modifications and variations can be made in the particular embodiments exemplified without departing from the intended scope of the invention. All such modifications are intended to be within the scope of the appended claims.

[0020] Compositions and methods are provided for the characterization, use, and manipulation of immunogenic peptides associated with autoimmune diseases.

[0021] The subject method can be used for diagnostic, preventive, or therapeutic purposes. As used herein, the term "treating" refers to both the prevention of recurrence and the treatment of an existing condition. For example, the prevention of autoimmune disease can be achieved by administering a drug before the onset of a recurrence. As used herein, "treatment" covers any treatment of disease in mammals, particularly humans, and includes (a) preventing a disease or condition from occurring in a subject who may be susceptible to the disease or condition but has not yet been diagnosed as having the disease or condition; (b) inhibiting disease symptoms, i.e., inhibiting their progression; or (c) reducing disease symptoms, i.e., causing regression of the disease or condition. Treatment of ongoing disease, in which treatment stabilizes or improves the patient's clinical symptoms, is of particular interest.

[0022] "Inhibiting" the onset of a disorder means either reducing the likelihood of the onset of the disorder or completely preventing the onset of the disorder. Reducing the severity of relapse means that the clinical signs associated with relapse are less severe in the presence of therapy than in untreated disease. As used herein, onset may refer to a relapse in a patient with ongoing relapsing-remitting disease. The method of the present invention is particularly applicable to patients diagnosed with inflammatory diseases, including, for example, autoimmune diseases. Treatment may be aimed at treating or reducing the severity of relapse, which is an exacerbation of an existing condition.

[0023] As used herein, "diagnosis" generally includes determining a subject's susceptibility to a disease or disorder, determining whether a subject is currently affected by a disease or disorder, prognosis of a subject affected by a disease or disorder (e.g., identifying the disease state, stage of the disease, or responsiveness of the disease to a therapy), and use of therapeutic criteria (e.g., monitoring a subject's condition to provide information regarding the efficacy or effectiveness of a therapy).

[0024] The term "biological sample" encompasses a variety of sample types obtained from an organism and can be used in diagnostic or monitoring assays. The term includes blood, cerebrospinal fluid, and other liquid samples of biological origin, solid tissue samples such as biopsy specimens or tissue cultures or cells derived therefrom, and their progeny. The term also encompasses samples that have been manipulated in any way after procurement, such as by treatment with reagents, solubilization, or enrichment for particular components. The term encompasses clinical samples, and also includes cells in cell culture, cell supernatants, cell lysates, serum, plasma, body fluids, and tissue samples.

[0025] The terms "individual," "subject," "host," and "patient" are used interchangeably herein and refer to any mammalian subject, e.g., humans, non-human primates, mice, rats, guinea pigs, rabbits, etc., for which diagnosis, treatment, or therapy is desired.

[0026] As used herein, the term "agent" includes any substance, molecule, element, compound, entity, or combination thereof. Examples include, but are not limited to, proteins, oligopeptides, small organic molecules, polysaccharides, polynucleotides, etc. It may be a natural product, a synthetic compound, or a chemical compound, or a combination of two or more substances. Unless otherwise specified, the terms "agent," "substance," and "compound" may be used interchangeably.

[0027] "Favorable conditions" shall have a meaning that depends on the context in which the term is used. That is, when used in connection with an antibody, the term shall mean a condition that allows the antibody to bind to its corresponding antigen. When used in connection with contacting an agent with a cell, the term shall mean a condition that allows the agent, capable of doing so, to enter the cell and perform its intended function. In one embodiment, as used herein, the term "favorable conditions" refers to a physiological condition.

[0028] A "subject" or "patient" in the context of the present teachings is generally a mammal. Mammals other than humans can be advantageously used as subjects that represent animal models of inflammation. The subject can be male or female.

[0029] "Analyzing" includes determining a set of values ​​associated with a sample by measuring a marker in the sample (e.g., the presence or absence of the marker or constitutive expression level) and comparing the measurements to measurements in a sample or set of samples from the same subject or other control subjects. In particular, the cell surface markers of the present teachings can be analyzed by any of a variety of conventional methods known in the art. "Analyzing" can include, for example, performing a statistical analysis to determine whether a subject is a responder or non-responder to a therapy (e.g., administration of a peptide therapy described herein).

[0030] "Pharmaceutically acceptable excipient," "pharmaceutically acceptable diluent," "pharmaceutically acceptable carrier," and "pharmaceutically acceptable adjuvant" generally refer to excipients, diluents, carriers, and adjuvants that are safe, non-toxic, and not biologically or otherwise undesirable and are useful in preparing pharmaceutical compositions, and include excipients, diluents, carriers, and adjuvants that are acceptable for veterinary and human pharmaceutical use. As used herein and in the claims, "pharmaceutically acceptable excipients, diluents, carriers, and adjuvants" includes both one and more than one such excipient, diluent, carrier, and adjuvant.

[0031] As used herein, "pharmaceutical composition" is meant to encompass compositions suitable for administration to a subject, such as a mammal, particularly a human. Generally, a "pharmaceutical composition" is sterile and preferably free of contaminants capable of eliciting an undesirable response in a subject (e.g., the compounds in the pharmaceutical composition are pharmaceutical grade). Pharmaceutical compositions can be designed to be administered to a subject or patient in need thereof via many different routes of administration, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, intratracheal, intramuscular, subcutaneous, etc.

[0032] "Dosage unit" refers to a physically discrete unit suitable for unitary dosage for a particular individual to be treated. Each unit can contain a predetermined quantity of active compound calculated to produce a desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the dosage unit form can be determined by (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such active compounds.

[0033] "Pharmaceutically acceptable excipient" means an excipient that is generally safe, non-toxic, and useful in preparing the desired pharmaceutical composition, and includes excipients acceptable for veterinary and human pharmaceutical use. Such excipients can be solid, liquid, semi-solid, or, in the case of an aerosol composition, gaseous.

[0034] "Pharmaceutically acceptable salts and esters" refers to salts and esters that are pharmaceutically acceptable and have the desired pharmacological properties. Such salts include salts that can be formed when acidic protons present in the compound can react with inorganic or organic bases. Suitable inorganic salts include those formed with alkali metals, e.g., sodium and potassium, magnesium, calcium, and aluminum. Suitable organic salts include those formed with organic bases such as amine bases, e.g., ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. Such salts also include acid addition salts formed with inorganic acids (e.g., hydrochloric acid and hydrobromic acid) and organic acids (e.g., acetic acid, citric acid, maleic acid, and alkane and arenesulfonic acids, such as methanesulfonic acid and benzenesulfonic acid). Pharmaceutically acceptable esters include esters formed from carboxy, sulfonyloxy, and phosphonoxy groups present in the compound, e.g., C 1-6Alkyl esters are included. When two acidic groups are present, the pharmaceutically acceptable salt or ester may be a mono-acid-mono-salt or ester, or a di-salt or ester; similarly, when more than two acidic groups are present, some or all of such groups may be salified or esterified. The compounds named in this invention may exist in unsalted or unesterified form, or salified and / or esterified form, and the naming of such compounds is intended to include both the parent (unsalted and unesterified) compound and its pharmaceutically acceptable salts and esters. Also, certain compounds named in this invention may exist in two or more stereoisomeric forms, and the naming of such compounds is intended to include all single stereoisomers and all mixtures (whether racemic or not) of such stereoisomers.

[0035] The terms "pharmaceutically acceptable" and "physiologically acceptable," and grammatical variations thereof, are used interchangeably when referring to compositions, carriers, diluents, and reagents, and indicate that the material can be administered to or on a human without producing undesirable physiological effects to an extent that would prohibit administration of the composition.

[0036] A "therapeutically effective amount" means the amount that, when administered to a subject for treating a disease, is sufficient to effect treatment for that disease.

[0037] As used herein, the term "in combination" refers to the use of more than one prophylactic and / or therapeutic agent. The use of the term "combination" does not limit the order in which prophylactic and / or therapeutic agents are administered to a subject with a disorder. A first prophylactic or therapeutic agent can be administered before (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before) or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second prophylactic or therapeutic agent to a subject with a disorder.

[0038] Immune tolerance, or immunological tolerance, or immunotolerance, is a state of immune system unresponsiveness to substances or tissues capable of eliciting an immune response in a given organism. A tolerogenic regimen or formulation is one that induces tolerance to a target antigen, for example, tolerance to a self-antigen such as myelin basic protein. A tolerogenic dose is a dose of a drug, such as a peptide, modified peptide ligand, DNA vector, etc., sufficient to reduce undesired immune responsiveness to a target antigen. A tolerogenic DNA construct is a DNA construct encoding a tolerogenic peptide that reduces undesired immune responsiveness to a target antigen. A tolerogenic peptide is a peptide that acts to reduce undesired immune responsiveness to a target antigen.

[0039] Tolerance can be induced through immunization protocols designed to activate suppressive immune responses to antigens. Tolerance is classified as central or peripheral, depending on whether it is originally induced in the thymus and bone marrow (central) or in other tissues and lymph nodes (peripheral).

[0040] Immune tolerance encompasses a range of physiological mechanisms by which the body reduces or eliminates immune responses to specific agents. It is used to describe the phenomena underlying the discrimination of self from non-self, suppressing allergic responses, allowing chronic infections instead of rejection and elimination, and preventing attack of the fetus by the maternal immune system.

[0041] Peripheral tolerance develops after T and B cells mature and enter peripheral tissues and lymph nodes. It is mediated by T cells, particularly CD4 T cells, which orchestrate the immune response. + Regulation is established by many partially overlapping mechanisms, primarily involved in the control at the level of helper T cells. Reactivity to a particular antigen can be reduced by the induction of tolerance after repeated exposure or by exposure in certain circumstances. In these cases, naive CD4 T cells are converted into induced Treg cells (iTreg cells) in peripheral tissues or nearby lymphoid tissues (lymph nodes, mucosa-associated lymphoid tissues, etc.). + Helper T cells may differentiate.

[0042] Other regulatory immune cells include T cell subsets that are similar to but phenotypically distinct from Treg cells, including TR1 cells that make IL-10 but do not express Foxp3, TGF-β-secreting TH3 cells, and other less well-characterized cells that help establish a local tolerogenic environment.

[0043] medical condition In some embodiments, the methods of the invention involve treating, isolating a cell population from, or diagnosing an individual who is "at risk" of developing an inflammatory disease or who is in the "early stages" of an inflammatory disease. "At risk" of developing an inflammatory disease includes (1) individuals who are at high risk of developing an inflammatory disease, and (2) individuals who exhibit a "preclinical" disease state but do not meet diagnostic criteria for an inflammatory disease (and therefore are not formally considered to have an inflammatory disease).

[0044] Individuals at "high risk" for developing an inflammatory disease (also referred to as "at risk" for developing one) are those who, compared with the general population, are more likely to develop an inflammatory disease or an inflammation-related disease. Such individuals can be identified based on the presence or possession of one or more of the following: a family history of inflammatory disease; the presence of a particular genetic variant (gene) or combination of genetic variants that predispose an individual to such an inflammatory disease; physical examination findings, laboratory test results, imaging findings, marker test results (also referred to as "biomarker" test results) associated with the development of an inflammatory disease, or the presence of a marker test result associated with the development of a metabolic disease; the presence of clinical signs associated with an inflammatory disease; the presence of certain symptoms associated with an inflammatory disease (although the individual is often asymptomatic); markers of inflammation (also referred to as "biomarkers"); and other findings that indicate an individual's high lifetime risk of developing an inflammatory disease or an inflammation-related disease. Most individuals at high risk for developing an inflammatory disease or an inflammation-related disease are asymptomatic and do not experience any symptoms associated with the disease they are at high risk of developing.

[0045] Individuals at high risk for developing inflammatory or inflammation-related diseases include, but are not limited to, individuals exhibiting a "preclinical disease state." A predisease state can be diagnosed based on onset symptoms, physical findings, laboratory test results, imaging results, and other findings that result in the individual meeting diagnostic criteria for an inflammatory disease and therefore being formally diagnosed. An individual with "preclinical disease" exhibits findings suggesting that the individual is in the process of developing an inflammatory disease, but does not exhibit findings, including symptoms, clinical findings, laboratory test results, and / or imaging results, necessary to meet the diagnostic criteria for a formal diagnosis of an inflammatory disease. In some embodiments, individuals exhibiting a preclinical disease state possess a genetic variant or combination of genetic variants that places them at higher risk for developing the disease compared to individuals who do not possess that genetic variant or combination of genetic variants. In some embodiments, these individuals have laboratory results, physical findings, symptoms, or imaging findings that place them at higher risk for developing an inflammatory disease. In some embodiments, individuals with a preclinical disease state are asymptomatic. In some embodiments, individuals in a preclinical disease state exhibit increased or decreased expression levels of particular genes, particular proteins, inflammatory markers, metabolic markers, and other markers.

[0046] In certain embodiments, the present invention relates to the treatment of individuals with established inflammatory or inflammation-related diseases. Inflammatory diseases can be diagnosed based on an individual exhibiting symptoms, signs, clinical features, laboratory test results, imaging test results, biomarker results, and other findings that allow a physician to formally diagnose the individual as having an inflammatory disease, including CD4 activation by the cross-reactive antigen peptides disclosed herein. + This may include detection of T cells.

[0047] In some embodiments, an established inflammatory disease is an inflammatory disease in which an individual has been formally diagnosed by a doctor for more than six months. In established inflammatory diseases, the signs or symptoms of the disease may be more severe, for example, compared to the symptoms of an individual diagnosed with an early stage inflammatory disease. In established inflammatory diseases, the disease process may cause tissue or organ damage. As described herein, in certain embodiments, determining inflammation in an individual with established disease may include analyzing the individual for the presence of at least one marker indicating the presence of inflammation.

[0048] Inflammatory diseases are considered to be diseases that exhibit clinical symptoms (abnormal clinical markers) such as visible inflammation including pain, swelling, warmth, and redness, and in the context of the present invention, are diseases that exhibit antigen-specific pathological CD4 + T cells are involved. Inflammatory diseases include, but are not limited to, autoimmune diseases, and can further include diseases that have a specific T cell-mediated component.

[0049] Inflammatory demyelinating diseases of the central nervous system are of particular interest, including, but not limited to, multiple sclerosis (MS), neuromyelitis optica (NO), and experimental acquired encephalitis (EAE). Demyelinating diseases can be initiated by peptides of myelin-associated proteins, such as MOG, MBP, and MAG. Demyelinating inflammatory diseases of the peripheral nervous system include subtypes of acute inflammatory demyelinating polyradiculoneuropathy, acute motor axonal neuropathy, acute motor and sensory axonal neuropathy, Miller-Fisher syndrome, and Guillain-Barré syndrome (GBS) with acute pandemiosis; subtypes of classic chronic inflammatory demyelinating polyneuropathy (CIDP), CIDP with diabetes, CIDP / monoclonal gammopathy of undetermined significance (MGUS), sensory CIDP, multifocal motor neuropathy (MMN), multifocal acquired demyelinating sensory and motor neuropathy, or Lewis-Sumner syndrome, multifocal acquired sensory and motor neuropathy, and distal acquired demyelinating sensory neuropathy. Although not traditionally classified as an inflammatory disease, ALS has been found to have increased numbers of CD49e macrophages and may be treated by the methods described herein.

[0050] Multiple sclerosis is characterized by various symptoms and signs of CNS dysfunction, accompanied by remissions and recurrent exacerbations. Classifications of interest for analysis by the methods of the present invention include relapsing-remitting MS (RRMS), primary progressive MS (PPMS), and secondary progressive MS (SPMS). The most common symptoms are paresthesia in one or more limbs, trunk, or one side of the face; weakness or clumsiness in the legs or hands; or visual disturbances, such as partial blindness and pain in one eye (retrobulbar optic neuritis), dim vision, or a scotoma. Other common early symptoms include ophthalmoplegia resulting in double vision (diplopia), transient weakness in one or more limbs, slight stiffness or unusual fatigue in the limbs, minor gait disturbances, difficulty with bladder control, dizziness, and mild emotional disturbances, all of which indicate scattered CNS involvement and often occur months or years before the disease is recognized. Excessive fever can exacerbate symptoms and signs.

[0051] Neuromyelitis optica (NMO), or Devic's disease, is an autoimmune inflammatory disorder of the optic nerve and spinal cord. Although inflammation can affect the brain, this disorder differs from multiple sclerosis in that it has a different pattern of response to therapy, possibly a different pattern of autoantigens, and the involvement of different lymphocyte subsets.

[0052] The primary symptom of Devic's disease is loss of vision and spinal cord function. In other etiologies of optic neuritis, visual field defects or loss of color vision can occur in isolation or before formal vision loss, but visual impairment usually manifests as decreased visual acuity. Spinal cord dysfunction can lead to muscle weakness, reduced sensation, or loss of bladder and bowel control. Damage to the spinal cord can range from inflammatory demyelination to necrotic damage to white and gray matter. Inflammatory lesions in Devic's disease have been classified as type II lesions (complement-mediated demyelination), but their prominent perivascular distribution differs from MS pattern II lesions. Thus, the pattern of inflammation is often quite different from that seen in MS.

[0053] Identification of cross-reactive antigenic peptides T cells involved in autoimmune responses can be isolated from patients. Positive immunoselection utilizes, for example, reagents that selectively bind to CD3, CD4, etc. on the surface of T lymphocytes. Negative immunoselection is optionally performed to deplete cells of other lineages, for example, B cell markers, monocyte markers, etc. Size, for example, forward scatter, can be used to gate out blood cells other than lymphocytes. In some embodiments, two, three, four, five, or more negative immunoselection reagents are used, for example, in a cocktail or in separate negative selections. In some embodiments, myeloid cells, B cells, CD8 + A lineage cocktail containing reagents for each negative selection, such as T cells, is used. When negative selection is used, it is often performed before positive selection to deplete the cell population of undesired cells. Positive selection is then performed.

[0054] A specific binding member, usually an antibody or MHC / peptide tetramer, is added to the cell suspension and incubated for a period of time sufficient to bind to the available antigen. Incubation is typically at least about 2 minutes and can be less than about 30 minutes. It is desirable to have a sufficient concentration of antibody in the reaction mixture so that the efficiency of separation is not limited by a lack of reagent. The appropriate concentration can be determined by titration.

[0055] The medium in which the cells are isolated can be any medium that maintains cell viability. Various media are commercially available and can be used depending on the nature of the cells, including Dulbecco's Modified Eagle's Medium (dMEM), Hank's Balanced Salt Solution (HBSS), Dulbecco's Phosphate Buffered Saline (dPBS), RPMI, Iscove's Medium, PBS with 5 mM EDTA, etc. Cells may be placed in culture, formulated for therapy, or frozen.

[0056] The resulting compositions have a variety of uses in clinical therapy, research, development, and commercial purposes. For therapeutic purposes, for example, therapeutically effective doses may be administered, optionally after expansion in culture, to selectively suppress unwanted pathogenic T cell responses.

[0057] Expansion or activation in culture may utilize cytokines and / or antigen-presenting cells (APCs). Contacting may be performed in any suitable culture medium. If present, the APCs may carry suitable peptide antigens or proteins, which are then presented on the cell surface. The ratio of T cells to APCs, if present, may range from about 1:20 to about 20:1 and is not critical as long as the number of APCs is not limiting. A period of up to 8 days, 10 days, 12 days, or 14 days may be sufficient (see, e.g., Dudley et al., JCO 2005;23(10):2346-2357). T cells primed in this manner may be used for any desired purpose, including experimental purposes related to antigen specificity determination, cytokine profiling, etc., and for in vivo delivery.

[0058] Cytokines useful for in vitro expansion include CD8 + The cytokines may include, but are not limited to, one or more cytokines that enhance T cell proliferation, including, but not limited to, type I IFN (IFNα and IFNβ), IL-1, IL-2, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21, IL-25, IL-27, and IL-33. Cells may be cultured in a conventional nutrient medium. Commercially available media, such as Ham's F10 (Sigma), minimal essential medium (MEM), Sigma), RPMI1640 (Sigma), and Dulbecco's modified Eagle's medium (DMEM), Sigma, are suitable for cell culture. Any of these media may be supplemented, as needed, with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides (such as adenosine and thymidine), antibiotics, trace elements, and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. Culture conditions, such as temperature, pH, and the like, are those conventionally used with the host cell selected for expression and will be apparent to those skilled in the art.

[0059] Ex vivo T cell activation can be achieved by established procedures in the art, including cell-based T cell activation, antibody-based activation, or activation using various bead-based activation reagents. Cell-based T cell activation can be achieved by exposing T cells to antigen-presenting cells such as dendritic cells or artificial antigen-presenting cells such as irradiated K562 cells. Antibody-based activation of T cell surface CD3 molecules with soluble anti-CD3 monoclonal antibodies also supports T cell activation in the presence of IL-2.

[0060] T cells can be cultured in contact with a surface that provides an agent that stimulates CD3 TCR complex-associated signals (e.g., an anti-CD3 antibody) and an agent that stimulates costimulatory molecules on the surface of the T cells (e.g., an anti-CD28 antibody). Bead-based activation of T cells can be achieved using commercially available T cell activation reagents, including, but not limited to, Invitrogen® CTS Dynabeads® CD3 / 28 (Life Technologies, Inc. Carlsbad CA) or Miltenyi MACS® GMP ExpAct Treg beads or Miltenyi MACS GMP TransAct™ CD3 / 28 beads (Miltenyi Biotec, Inc.). Conditions suitable for T cell culture are well known in the art (Lin, et al. (2009) Cytotherapy 11(7):912-922; Smith, et al. (2015) Clinical & Translational Immunology 4:e31 published online 16 January 2015). Target cells are maintained under conditions necessary to support growth, for example, at an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air plus 5% CO2).

[0061] The selected T cells can be used as a source of sequences encoding TCRs that provide antigen-specific disease. The TCR-encoding sequences can be isolated by any convenient method, for example as detailed in the Examples.

[0062] The TCR of interest can be expressed in soluble form and multimerized for use as a selective binding agent.Soluble proteins can be single chain or, more usually, heterodimers.In some embodiments, soluble TCRs are modified by adding a biotin acceptor peptide sequence to the C-terminus of one polypeptide.After biotinylation with the acceptor peptide, the TCR can be multimerized by binding to a biotin binding partner, such as avidin, streptavidin, traptavidin, neutravidin, etc.The biotin binding partner can include a detectable label, such as a fluorophore, mass label, etc., or can be bound to particles, such as paramagnetic particles.Selection of the ligand bound to the TCR can be performed by flow cytometry, magnetic selection, etc., as known in the art.

[0063] TCR multimers are utilized in binding assays to libraries of diverse peptide antigens. The peptide ligands are about 8 to about 20 amino acids in length, typically about 8 to about 18 amino acids, about 8 to about 16 amino acids, about 8 to about 14 amino acids, about 8 to about 12 amino acids, about 10 to about 14 amino acids, or about 10 to about 12 amino acids. It is understood that a completely random library would represent a vast number of possible combinations. In some methods, diversity is limited to residues that anchor the peptide to the MHC binding domain, referred to herein as MHC anchor residues. The location of the anchor residues in the peptide is determined by the specific MHC binding domain. Diversity can also be limited to other positions, as shown by binding studies, e.g., TCR anchors. At least 10 6 Pieces, at least 10 7 pieces, more usually at least 10 8 A number of different peptide ligands are present in the library.

[0064] The MHC proteins used in the library can be derived from any mammalian or avian species. Of particular interest are human HLA proteins. HLA proteins include class II subunits HLA-DPα, HLA-DPβ, HLA-DQα, HLA-DQβ, HLA-DRα, and HLA-DRβ, particularly HLA-DR15.

[0065] The peptide-binding domain of MHC can be expressed in a soluble form of a normal membrane-bound protein. The soluble form is derived from the native form by deleting the transmembrane domain. Advantageously, the protein is truncated to remove both the cytoplasmic and transmembrane domains. In some such embodiments, the binding domain is subjected to mutagenesis and selected for amino acid changes that enhance the solubility of the single-chain polypeptide without altering the peptide-binding contact. Approximately 10 or less, usually about 5 or less, of the amino acids of the transmembrane domain are included, and preferably none of the amino acids of the transmembrane domain are included. The deletions are such that they do not interfere with the domain's ability to bind to peptide ligands.

[0066] The library of diverse sequences is generated and inserted into a vector suitable for the host cell of interest, which may be, but is not limited to, suitable for expression in yeast cells, and the yeast cells may be induced to express the polypeptide library. Upon introduction into the host cell, expression of the library is induced and the cells are maintained for a period of time sufficient to provide cell surface display of the library polypeptides.

[0067] Selection of peptides that bind to the TCR is performed by combining the multimerized TCR with a host cell population expressing the library. One round of selection is performed until the selected population has a signal above background, usually at least three rounds, more usually at least four rounds of selection.

[0068] After the final round of selection, polynucleotides are isolated from the selected host cells and the sequences of the selected peptide ligands are determined, usually by high-throughput sequencing.

[0069] The sequencing platform that can be used in the present disclosure includes but is not limited to pyrosequencing, sequencing by synthesis, single molecule sequencing, second generation sequencing, nanopore sequencing, sequencing by ligation or sequencing by hybridization.Preferred sequencing platform is commercially available from Illumina (RNA-Seq) and Helicos (digital gene expression or "DGE")."Next generation" sequencing methods include, but are not limited to, 1) the methods and apparatus described in Margulies et al., Nature (2005) 437:376-380 (2005), and U.S. Patent Nos. 7,244,559, 7,335,762, 7,211,390, 7,244,567, 7,264,929, and 7,323,305; Lifesciences, 2) Helicos BioSciences Corporation (Cambridge, MA), described in U.S. Application No. 11 / 167046, and U.S. Patent Nos. 7,501,245, 7,491,498, 7,276,720, and U.S. Patent Application Publication Nos. 2009 / 0061439, 2008 / 0087826, 2006 / 0286566, 2006 / 0024711, 2006 / 0024678, 2008 / 0213770, and 2008 / 0103058, 3) Applied Biosystems (e.g., SOLiD Sequencing), 4) Dover Systems (e.g., Polonator G.007 sequencing), 5) Illumina, as described in U.S. Patent Nos. 5,750,341, 6,306,597, and 5,969,119, and 6) Pacific, as described in U.S. Patent Nos. 7,462,452, 7,476,504, 7,405,281, 7,170,050, 7,462,468, 7,476,503, 7,315,019, 7,302,146, 7,313,308, and U.S. Application Publication Nos. 2009 / 0029385, 2009 / 0068655, 2009 / 0024331, and 2008 / 0206764. These methods and devices include, but are not limited to, those commercialized by Biosciences, Inc. All references are incorporated herein by reference. Such methods and devices are provided herein by way of example and are not intended to be limiting.

[0070] As shown in the Examples, the peptide antigens thus identified can be native peptides of the individual or cross-reactive peptides that specifically activate pathogenic T cells. The peptides are useful as screening tools and for identification of therapeutic agents that activate tolerance.

[0071] Peptides, including the cross-reactive peptides disclosed herein, typically contain at least about 8 amino acids, at least about 9 amino acids, at least about 10 amino acids, at least about 11 amino acids, at least about 12 amino acids, at least about 13 amino acids, at least about 15 amino acids, or more, and may be about 8 to about 40 amino acids, about 8 to about 30 amino acids, about 8 to about 25 amino acids, about 8 to about 20 amino acids, or about 8 to about 18 amino acids in length. Peptides may include, for example, the amino acid sequences set forth in any of SEQ ID NOS: 1-4 or 23-75. In addition to the provided sequences, they may also include fusion polypeptides known in the art, in which the fusion partner is other than the native protein sequence. Peptides useful in the present invention also include derivatives, variants, and biologically active fragments of naturally occurring peptides. Peptides may contain, for example, one, two, or three amino acid substitutions. Peptide sequences may be designed sequenced sequences derived from mutagenesis in a diverse peptide library. The specificity of a TCR can be conformational, and therefore the peptide that activates the T cell of interest can have a sequence that is essentially unrelated to the native peptide.

[0072] Peptides can be modified for various purposes, for example, by conjugation to a wide variety of other oligopeptides or proteins. For example, post-translational modifications can be made by prenylation, acetylation, amidation, carboxylation, glycosylation, PEGylation, etc. Such modifications can also include glycosylation modifications, for example, by modifying the glycosylation pattern of a polypeptide during synthesis and processing, or during further processing steps, for example, by exposing the polypeptide to enzymes that affect glycosylation, such as mammalian glycosylation or deglycosylation enzymes. In some embodiments, variants of the present invention include variants with phosphorylated amino acid residues, for example, phosphotyrosine, phosphoserine, or phosphothreonine.

[0073] The ability of a peptide to modulate lymphocyte activity can be determined, for example, by the ability of the peptide to induce a cytotoxic effect on activated pathological lymphocytes, as disclosed in the Examples provided herein.

[0074] In some embodiments, the peptide is provided as a fusion protein, e.g., fused in-frame with a second polypeptide. In some embodiments, the second polypeptide can increase the size of the fusion protein, e.g., to prevent rapid clearance of the fusion protein from circulation. In some other embodiments, the second polypeptide is part or all of the Fc region. In some other embodiments, the second polypeptide is any suitable polypeptide substantially similar to Fc, e.g., providing increased size and / or additional binding or interaction with Ig molecules. These fusion proteins can facilitate purification and exhibit increased in vivo half-life. Fusion proteins with disulfide-bonded dimeric structures (due to IgG) can also be more efficient at binding and neutralizing other molecules than monomeric secreted proteins or protein fragments alone.

[0075] In some other embodiments, the peptide variants of the present invention include variants that are further modified to improve resistance to proteolysis, optimize solubility, or make them more suitable as therapeutic agents.For example, the variants of the present invention further include analogs containing residues other than naturally occurring L-amino acids, such as D-amino acids or non-naturally occurring synthetic amino acids.D-amino acids can be substituted for some or all of the amino acid residues.

[0076] Polypeptides can be prepared by cell-free translation systems or synthetic in vitro synthesis using conventional methods known in the art. Various commercially available synthesis devices are available, such as automated synthesizers from Applied Biosystems, Inc., Foster City, Calif., Beckman, etc. By using a synthesizer, naturally occurring amino acids can be substituted with unnatural amino acids. The specific sequence and preparation method are determined by convenience, economy, required purity, etc.

[0077] Polypeptides can also be isolated and purified according to conventional methods of recombinant synthesis. A lysate can be prepared from the expression host and purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification techniques. In most cases, the composition used will contain at least 20% by weight of the desired product, more usually at least about 75% by weight, preferably at least about 95% by weight, and for therapeutic purposes, usually at least about 99.5% by weight, with respect to contaminants related to the method of product preparation and its purification. Percentages are usually based on total protein.

[0078] Antibodies related to cross-reactive peptides and TCRs that recognize such cross-reactive peptides Antibodies can be generated against cross-reactive peptides, complexes of cross-reactive peptides with human MHC proteins, including but not limited to DR15 proteins, or TCRs that respond to the cross-reactive peptides described herein. Such antibodies can be therapeutic in blocking the activation of pathogenic T cells. As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitope determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains, and usually have specific three-dimensional structural and charge characteristics.

[0079] The term "antibody" is used in the broadest sense and specifically covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity. "Antibodies" (Ab) and "immunoglobulins" (Ig) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules that lack antigen specificity. The latter type of polypeptide is produced, for example, at low levels by the lymphatic system and at increased levels by myelomas.

[0080] As used herein, the term "antibody" refers to a polypeptide containing sufficient standard immunoglobulin sequence elements to confer specific binding to a particular target antigen. As is known in the art, naturally occurring intact antibodies are approximately 150 kD tetrameric entities consisting of two identical heavy chain polypeptides (approximately 50 kD each) and two identical light chain polypeptides (approximately 25 kD each) that associate with each other in what is commonly referred to as a "Y-shaped" structure. Each heavy chain consists of at least four domains (each approximately 110 amino acids long)—an amino-terminal variable (VH) domain (located at the tip of the Y structure), followed by three constant domains, CH1, CH2, and carboxy-terminal CH3 (located at the base of the stem of the Y). A short region known as the "switch" connects the heavy chain variable and constant regions. A "hinge" connects the CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region connect the two heavy chain polypeptides to each other in intact antibodies. Each light chain consists of two domains—an amino-terminal variable (VL) domain followed by a carboxy-terminal constant (CL) domain separated from each other by another "switch." An intact antibody tetramer consists of two heavy- and light-chain dimers in which the heavy and light chains are linked to each other by a single disulfide bond, and two other disulfide bonds link the heavy-chain hinge regions together, connecting the dimers to form a tetramer. Naturally produced antibodies are also typically glycosylated on the CH2 domain. Each domain in a natural antibody has a structure characterized by an "immunoglobulin fold" formed from two beta sheets (e.g., a three-, four-, or five-stranded sheet) packed against each other in a compressed antiparallel beta barrel. Each variable domain contains three hypervariable loops (CDR1, CDR2, and CDR3) known as "complement-determining regions" and four somewhat invariant "framework" regions (FR1, FR2, FR3, and FR4).When a native antibody folds, the FR regions form beta sheets that provide the structural framework for the domain, and the CDR loop regions from both the heavy and light chains converge in three-dimensional space to generate a single hypervariable antigen-binding site located at the tip of a Y-structure.

[0081] The Fc region of a naturally occurring antibody binds to elements of the complement system and also to receptors on effector cells, including effector cells that mediate cytotoxicity, specifically involving, for example, ADCP. As is known in the art, the affinity and / or other binding attributes of the Fc region for an Fc receptor can be modulated through glycosylation or other modifications. In some embodiments, antibodies produced and / or utilized in accordance with the present invention comprise a glycosylated Fc domain, including Fc domains in which such glycosylation has been modified or engineered. For purposes of the present invention, in certain embodiments, any polypeptide or polypeptide complex that comprises a sufficient immunoglobulin domain sequence as found in a natural antibody may be referred to and / or used as an "antibody," regardless of whether such polypeptide is produced naturally (e.g., generated by an organism in response to an antigen) or produced by recombinant engineering, chemical synthesis, or other artificial systems or methodologies. In some embodiments, an antibody is polyclonal; in some embodiments, an antibody is monoclonal.

[0082] In some embodiments, the antibody has constant region sequences characteristic of a murine, rabbit, primate, or human antibody, hi some embodiments, the antibody sequence elements are humanized, primatized, chimeric, etc., as known in the art.

[0083] Furthermore, as used herein, the term "antibody" can, in appropriate embodiments (unless otherwise stated or clear from the context), refer to any of the constructs or formats known or developed in the art for utilizing the structural and functional characteristics of antibodies in alternative presentations. For example, in embodiments, antibodies utilized in accordance with the present invention may be any of a variety of antibody types, including intact IgG, IgE, and IgM, bi- or multispecific antibodies (e.g., Zybodies®, etc.), single chain Fv, polypeptide-Fc fusions, Fab, cameloid antibodies, masked antibodies (e.g., Probodies®), Small Modular ImmunoPharmaceuticals ("SMIPs™"), single chain or tandem diabodies (TandAb®), VHHs, Anticalins®, Nanobodies®, minibodies, BiTEs®, ankyrin repeat proteins or DARPINs®, Avimers®, DARTs, TCR-like antibodies, Adnectins®, Affilins®, Trans-bodies®, Affibodies®, TrimerX®, microproteins, Fynomers®, Centyrins®, and KALBITOR®. In some embodiments, the antibody may lack covalent modifications (e.g., glycan attachment) that it has when produced in nature. In some embodiments, antibodies may contain covalent modifications (e.g., glycans, payloads), such as attachment of detectable moieties, therapeutic moieties, catalytic moieties, or other pendant groups (e.g., polyethylene glycol, etc.).

[0084] Exemplary antibody agents include, but are not limited to, human antibodies, primatized antibodies, chimeric antibodies, bispecific antibodies, humanized antibodies, conjugated antibodies (i.e., antibodies conjugated or fused to other proteins, radiolabels, or cytotoxins), Small Modular ImmunoPharmaceuticals ("SMIPs™"), single-chain antibodies, cameloid antibodies, and antibody fragments. As used herein, the term "antibody agent" also includes intact monoclonal antibodies, polyclonal antibodies, single domain antibodies (e.g., shark single domain antibodies (e.g., IgNARs or fragments thereof)), multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments, so long as they exhibit the desired biological activity. In some embodiments, the term encompasses stapled peptides. In some embodiments, the term encompasses one or more antibody-like binding peptidomimetics. In some embodiments, the term encompasses one or more antibody-like binding scaffold proteins. In some embodiments, the term encompasses monobodies or adnectins.

[0085] In many embodiments, antibody agents are or comprise polypeptides whose amino acid sequences include one or more structural elements recognized by those skilled in the art as complementarity-determining regions (CDRs); in some embodiments, antibody agents are or comprise polypeptides that include at least one CDR (e.g., at least one heavy chain CDR and / or at least one light chain CDR) that is substantially identical to that found in a reference antibody. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that they are sequence-identical or contain 1-5 amino acid substitutions compared to the reference CDRs. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that they exhibit at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDRs. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that they exhibit at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDRs. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that at least one amino acid within the included CDRs is deleted, added, or substituted compared to the reference CDRs, but the included CDRs otherwise have an amino acid sequence that is identical to the amino acid sequence of the reference CDRs. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that 1 to 5 amino acids within the included CDRs are deleted, added, or substituted compared to the reference CDRs, but the included CDRs otherwise have an amino acid sequence that is identical to the amino acid sequence of the reference CDRs. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that at least one amino acid within the included CDRs is substituted compared to the reference CDRs, but the included CDRs otherwise have an amino acid sequence that is identical to the amino acid sequence of the reference CDRs. In some embodiments, the included CDR is substantially identical to the reference CDR in that 1 to 5 amino acids within the included CDR are deleted, added, or substituted compared to the reference CDR, but the included CDR otherwise has an amino acid sequence that is identical to the reference CDR.In some embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art as an immunoglobulin variable domain, hi some embodiments, an antibody agent is a polypeptide protein having a binding domain that is homologous or largely homologous to an immunoglobulin binding domain.

[0086] "Native antibodies and immunoglobulins" are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced interchain disulfide bridges. Each heavy chain contains at one end a variable domain (V H ) followed by several constant domains. Each light chain has a variable domain (V L ) and a constant domain at the other end, with the light-chain constant domain aligned with the first constant domain of the heavy chain and the light-chain variable domain aligned with the variable domain of the heavy chain. Particular amino acid residues are thought to form an interface between the light-chain variable domain and the heavy-chain variable domain (Clothia et al., J. Mol. Biol. 186:651 (1985); Novotny and Haber, Proc. Natl. Acad. Sci. USA 82:4592 (1985)).

[0087] The term "variable" refers to the fact that certain portions of the variable domains differ significantly in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in both the light- and heavy-chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Natural heavy- and light-chain variable domains each contain four FR regions that largely adopt a b-sheet configuration, connected by three CDRs that form loops that connect, and in some cases form part of, the b-sheet structure. The CDRs of each chain are held together in close proximity by the FR regions and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institutes of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

[0088] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, a name reflecting the ability to crystallize readily. Pepsin treatment produces an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0089] An "Fv" is the minimum antibody fragment that contains a complete antigen-recognition and -binding site. In two-chain Fv species, this region consists of a dimer of one heavy-chain variable domain and one light-chain variable domain in tight, non-covalent association. In single-chain Fv species (scFv), one heavy-chain variable domain and one light-chain variable domain can be covalently linked by a flexible peptide linker so that the light and heavy chains can associate in a "dimeric" structure similar to that of two-chain Fv species. In this structure, the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind to antigen, albeit with lower affinity than the entire binding site. For a review of scFvs, see Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0090] Fab fragments also contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residues of the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0091] There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the various classes of immunoglobulins are designated a, d, e, g, and m, respectively. The subunit structures and three-dimensional configurations of the various classes of immunoglobulins are well known. Engineered variants of immunoglobulin subclasses, including those that increase or decrease immune effector function, half-life, or serum stability, are also encompassed by the term.

[0092] As used herein, an "antibody fragment," and all grammatical variants thereof, is defined as a portion of an intact antibody that includes the antigen-binding site or variable region of the intact antibody, which portion does not include the constant heavy chain domains of the Fc region of the intact antibody (i.e., CH2, CH3, and CH4, depending on the antibody isotype). Examples of antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, and Fv fragments, diabodies, (1) single-chain Fv (scFv) molecules, (2) single-chain polypeptides comprising only one light-chain variable domain or comprising the three CDRs of a light-chain variable domain without an associated heavy-chain portion, and (3) single-chain polypeptides comprising only one heavy-chain variable region or comprising the three CDRs of a heavy-chain variable region without an associated light-chain variable region, and any antibody fragment that is a polypeptide having a primary structure consisting of a single uninterrupted sequence of contiguous amino acid residues (referred to herein as "single-chain antibody fragments" or "single-chain polypeptides"), as well as multispecific or multivalent structures formed from antibody fragments. In antibody fragments comprising one or more heavy chains, the heavy chain may contain any of the constant domain sequences found in the non-Fc region of an intact antibody (e.g., CH1 in an IgG isotype), and / or may contain any of the hinge region sequences found in an intact antibody, and / or may contain a leucine zipper sequence fused or positioned to the hinge region sequence or constant domain sequence of the heavy chain.

[0093] As used herein, the term "monoclonal antibody" (mAb) refers to an antibody obtained from a substantially homogeneous population of antibodies; i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site; each mAb is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they may be synthesized by a hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in the present invention may be produced in immortalized B cells or hybridomas thereof, or may be produced by recombinant DNA methods.

[0094] An "isolated" antibody is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, the antibody is (1) purified to greater than 75% antibody by weight, and most preferably greater than 80%, 90%, or 99% antibody by weight, as determined by the Lowry method, or (2) purified to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue, or preferably silver stain. Isolated antibody includes the antibody in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

[0095] The terms "specific binding," "specifically binds," and the like refer to the preferential non-covalent or covalent binding of a molecule relative to other molecules or moieties in a solution or reaction mixture (e.g., an antibody specifically binds to a particular polypeptide or epitope relative to other available polypeptides). In some embodiments, the affinity of one molecule for which it specifically binds another molecule is greater than or equal to 10 -5 M or less (e.g., 10 -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, 10 -12 M or less, 10 -13 M or less, 10 -14 M or less, 10 -15 M or less, or 10 -16 M or less) K d "Affinity" refers to the strength of binding, and increased binding affinity is characterized by a lower K d correlates with.

[0096] As used herein, the term "specific binding member" refers to a member of a specific binding pair (i.e., two molecules, usually two different molecules, in which one of the molecules, e.g., the first specific binding member, specifically binds to the other molecule, e.g., the second specific binding member, via non-covalent means).

[0097] Therapy and Diagnosis The peptides or antibodies disclosed herein can be provided in pharmaceutical compositions suitable for therapeutic use, e.g., human treatment. In some embodiments, the pharmaceutical composition comprises one or more therapeutic entities of the present invention, or pharmaceutically acceptable salts, esters, or solvates thereof. In some other embodiments, the pharmaceutical composition of the present invention comprises one or more therapeutic entities of the present invention in combination with another therapeutic agent.

[0098] Therapeutic entities are often administered as pharmaceutical compositions containing an active therapeutic agent and other pharmaceutically acceptable excipients. The preferred form depends on the intended mode of administration and therapeutic application. Depending on the desired formulation, the composition may also contain a pharmaceutically acceptable non-toxic carrier or diluent, which is defined as a vehicle commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents include distilled water, physiological phosphate-buffered saline, Ringer's solution, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also contain other carriers, or non-toxic, non-therapeutic, non-immunogenic stabilizers, etc.

[0099] In yet some other embodiments, the pharmaceutical compositions may also include large, slowly metabolized macromolecules such as proteins, polysaccharides such as chitosan, polylactic acid, polyglycolic acid and copolymers (such as latex-functionalized Sepharose™, agarose, cellulose), polymeric amino acids, amino acid copolymers, and lipid aggregates (e.g., oil droplets or liposomes).

[0100] Also provided are methods of combination therapy, where the combination may provide additive or synergistic benefits.The combination of peptides or antibodies can be obtained with a second drug selected from one or more of the general classes of drugs commonly used in the non-antigen-specific treatment of autoimmune diseases, including corticosteroids and disease-modifying drugs, or selected from antigen-specific drugs.Corticosteroids, such as prednisone, methylpredisone, prednisolone, solumedrol, etc., have both anti-inflammatory and immunological activity.They can be administered systemically or injected locally.Corticosteroids are useful in early disease as temporary adjunctive therapy while waiting for disease-modifying drugs to exert their effects.Corticosteroids are also useful as chronic adjunctive therapy in patients with severe disease.

[0101] Disease-modifying drugs are also useful in combination therapy. These include methotrexate, leflunomide, etanercept, infliximab, adalimumab, anakinra, rituximab, CTLA4-Ig (abatacept), antimalarials, gold salts, sulfasalazine, d-penicillamine, cyclosporine A, cyclophosphamide, azathioprine, and others. Treatment for MS may include interferon-beta, copaxone, and anti-VLA4, which reduce relapse rates. MS is also treated with immunosuppressants, including methylprednisolone, other steroids, methotrexate, cladribine, and cyclophosphamide.

[0102] Combination therapy can be provided sequentially, stepwise, in a co-administered formulation, or by concomitant administration during the same administration period. "Concomitant administration" of a pharmaceutical composition of the present invention with a known therapeutic agent refers to administration of the drug and peptide at a time such that both the known drug and the composition of the present invention have a therapeutic effect. Such concomitant administration can include simultaneous (i.e., at the same time), prior, or subsequent administration of the drug with respect to administration of the compound of the present invention. Those skilled in the art will have no difficulty in determining the appropriate timing, sequence, and dosage of administration for particular drugs and compositions of the present invention.

[0103] The peptide or antibody can function as an active ingredient in pharmaceutical compositions formulated for the treatment of the above-mentioned various disorders.The active ingredient is present in a therapeutically effective amount, i.e., an amount sufficient when administered to treat diseases or medical conditions mediated by reducing the activity of inflammatory lymphocytes.The composition can also contain various other agents to enhance delivery and effectiveness, for example, to enhance the delivery and stability of the active ingredient.

[0104] Thus, for example, depending on the desired formulation, the composition may also contain a pharmaceutically acceptable non-toxic carrier or diluent, which is defined as a vehicle commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents include distilled water, buffered water, physiological saline, PBS, Ringer's solution, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may contain other carriers, or non-toxic, non-therapeutic, non-immunogenic stabilizers, excipients, etc. The composition may also contain additional substances to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, wetting agents, and detergents. The composition may also contain any of a variety of stabilizers, such as antioxidants.

[0105] The peptides may be complexed with various well-known compounds that enhance the in vivo stability of the polypeptide or otherwise enhance the pharmacological properties of the polypeptide (e.g., increase the half-life of the polypeptide, reduce its toxicity, enhance solubility or uptake). Examples of such modifiers or complexing agents include sulfate, gluconate, citrate, and phosphate. The polypeptides of the composition may also be complexed with molecules that enhance their in vivo attributes. Such molecules include, for example, carbohydrates, polyamines, amino acids, other peptides, ions (e.g., sodium, potassium, calcium, magnesium, manganese), and lipids.

[0106] Further guidance regarding suitable formulations for various types of administration can be found in Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, Pa., 17th ed. (1985). For a brief review of methods for drug delivery, see Langer, Science 249:1527-1533 (1990).

[0107] The pharmaceutical compositions can be administered for prophylactic and / or therapeutic treatment. The toxicological and therapeutic efficacy of the active ingredients can be determined according to standard pharmaceutical procedures in cell cultures and / or experimental animals, e.g., LD 50 (a dose lethal to 50% of the population) and ED 50 This involves determining the dose that is therapeutically effective in 50% of the population. The dose ratio between toxic and therapeutic efficacy is the therapeutic index, which is the LD 50 / ED 50 Compounds that exhibit large therapeutic indices are preferred.

[0108] Data obtained from cell culture and animal studies can be used to formulate various dosages for humans. The dosage of the active ingredient is usually selected to achieve an ED with little or no toxicity. 50 The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.

[0109] The pharmaceutical compositions described herein can be administered in a variety of different ways, including by administering the composition containing a pharmaceutically acceptable carrier via oral, intranasal, rectal, topical, intraperitoneal, intravenous, intramuscular, subcutaneous, subdermal, or transdermal means.

[0110] Formulations suitable for parenteral administration, such as by intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents, solubilizers, thickeners, stabilizers, and preservatives.

[0111] The components used to formulate pharmaceutical compositions are preferably highly pure and substantially free of potentially harmful contaminants (e.g., at least National Food (NF) grade, generally at least analytical grade, and more typically at least pharmaceutical grade). Furthermore, compositions intended for in vivo use are preferably sterile. To the extent that a given compound must be synthesized prior to use, the resulting product is preferably substantially free of any potentially toxic agents, such as any endotoxins, that may be present during the synthesis or purification process. Compositions for parenteral administration are also preferably sterile, substantially isotonic, and made under GMP conditions.

[0112] The peptide composition may be administered in a single dose or in multiple doses, usually over a period of time sufficient to reduce the severity of the inflammatory disease, such as daily, every other day, weekly, semi-weekly, monthly, etc., which may include 1, 2, 3, 4, 6, 10 or more doses.

[0113] Determination of a therapeutically or prophylactically effective amount can be made based on animal data using routine calculation methods. In one embodiment, a therapeutically or prophylactically effective amount contains about 0.1 mg to about 1 g of protein. In another embodiment, an effective amount contains about 1 mg to about 100 mg of protein. In a further embodiment, an effective amount contains about 10 mg to about 50 mg of protein. Effective doses depend, at least in part, on the route of administration. Doses can range from about 0.1 μg / kg of patient body weight to about 1 μg / kg, about 10 μg / kg, or about 100 μg / kg.

[0114] In the method of use, an effective dose of the agent of the present invention is administered alone or in combination with an additional active agent for the treatment of the above conditions. The effective dose can be from about 1 ng / kg body weight to 10 ng / kg body weight, 100 ng / kg body weight, 1 μg / kg body weight, 10 μg / kg body weight, 25 μg / kg body weight, 50 μg / kg body weight, 100 μg / kg body weight, 250 μg / kg body weight, 500 μg / kg body weight, 750 μg / kg body weight, 1 mg / kg body weight, 5 mg / kg body weight, 10 mg / kg body weight, 25 mg / kg body weight, 50 mg / kg body weight, 75 mg / kg body weight, 100 mg / kg body weight, 250 mg / kg body weight, 500 mg / kg body weight, 750 mg / kg body weight, etc. The dosage can be administered multiple times as needed, for example, every 4 hours, every 6 hours, every 8 hours, every 12 hours, every 18 hours, daily, every 2 days, every 3 days, weekly, etc. The medication may be administered orally.

[0115] The composition may be administered in a single dose or in multiple doses, usually over a period of time sufficient to reduce the severity of the inflammatory disease, such as daily, every other day, weekly, semi-weekly, monthly, etc., which may include 1, 2, 3, 4, 6, 10, or more doses.

[0116] Determining a therapeutically or prophylactically effective amount of an agent according to the present methods can be performed based on animal data using routine calculations. The effective dose will depend, at least in part, on the route of administration.

[0117] Peptides and peptide-MHC protein complexes are also useful in methods for characterizing an individual's immune profile, particularly for determining the presence of pathogenic T cells with specificity for these peptides and complexes in individuals suspected of having MS or a related inflammatory condition. The method can include contacting a sample containing T cells from the individual with an immunogenic, cross-reactive peptide or an MHC complex containing the peptide and determining the presence of a T cell response to the peptide or complex. The sample can be any biological sample containing T cells, including peripheral blood, lymph node samples, CSF, etc. T cell responses can be determined by direct binding assays, by determining the presence of T cell receptors associated with specificity for these peptide antigens, by determining the presence of activation markers on T cells, by frequency determinations, by determining the presence of enhanced cytokine production in response to the peptide or complex, and the like, as known in the art.

[0118] Antigen-specific immunotherapy Antigen-specific immunotherapy aims to utilize tolerization, immune deviation, and induction of Tregs to promote autoantigen-specific tolerance. Autoimmune diseases are characterized by the pathogenic CD4 + This may be treated by eliminating immune cells or blocking the immune response directed by autoantigen-specific T cells. Several studies have shown immune deviation following administration of autoantigens, consistent with Treg generation, peptide-specific IL-10, and increased levels of IFNγ, IL-5, IL-13, IL-17, IL-6, tumor necrosis factor-α (TNFα), and FoxP3. Another method for inducing immunological changes is through manipulation of dendritic cells (DCs). DCs are essential for the induction phase of the immune response and are therefore crucial in determining whether the response to an antigen is inflammatory or tolerogenic. DCs can influence whether naive T cells undergo deletion, anergy, or differentiation. DC responses to specific antigens are influenced by the tissue environment and innate stimuli associated with that antigen. Therapies can target DCs to induce tolerance.

[0119] For example, cross-reactive adenoviral peptides can be administered via tolerogenic routes, such as oral or nasal administration of soluble or oligomerized peptides. Alternatively, cross-reactive adenoviral peptides can be used as the basis for modified peptide ligands (APLs). Modified peptide ligands are analogs derived from antigenic peptides that contain amino acid substitutions at TCR contact residues, e.g., substitutions of one, two, or three amino acids. TCR engagement by these modified peptide ligands impairs normal T cell function. Modified peptide ligands can specifically antagonize and inhibit T cell activation induced by cognate antigenic peptides. APLs compete with native peptides for TCR binding but bind to TCRs with lower affinity, thereby functioning as antagonists or partial agonists. Antagonists induce T cell anergy, while partial agonists incompletely activate T cells and can induce immune deviation.

[0120] In some embodiments, the peptide is formulated for immunization, e.g., to generate antigen-specific tolerance by subcutaneous or oral administration of the cross-reactive peptide. In some embodiments, the cross-reactive peptide is formulated for transdermal delivery. In some embodiments, a method for inducing immune tolerance includes transdermal administration of the formulated cross-reactive peptide. An effective dose can be low, e.g., less than about 5 mg, less than about 2.5 mg, less than about 1 mg, less than about 500 μg, or less than about 100 μg. In some embodiments, the cross-reactive peptide is encapsulated in mannosylated liposomes to enhance peptide uptake by dendritic cells.

[0121] Alternatively, an amino acid sequence derived from the TCR of a pathogenic T cell clone is administered to induce T cell-mediated immunoregulation directed at T cells expressing those TCRs. TCR sequences of interest for this purpose include, for example, one or more of the peptide sequences set forth in SEQ ID NOS: 5-22 and 76-83, as shown in Figures 1G and 9. Formulation and administration can be, for example, transdermal, intradermal, intramuscular, etc., and can be administered at doses of, for example, up to 100 mg / kg, up to 50 mg / kg, up to 10 mg / kg, up to 5 mg / kg, up to 1 mg / kg, up to 500 μg / kg, up to 100 μg / kg, or up to 10 μg / kg. Adjuvants for increasing immunogenicity can also be included, including, but not limited to, incomplete Freund's adjuvant and complete Freund's adjuvant.

[0122] As an alternative to peptide vaccination, DNA vaccine can be formulated into a tolerization vector of genetically engineered DNA that encodes one or more of the cross-reactive peptides disclosed herein.The tolerization vector can be formulated and administered by intramuscular injection in a modified plasmid backbone, for example, so that it can cause favorable immunological changes in patients with MS, for example, the number of immunostimulatory CpG motifs can be reduced and the number of immunosuppressive GpG motifs can be increased.Lower doses, for example, less than about 5mg, less than about 2.5mg, less than about 1mg, less than about 500μg, less than about 100μg, may be preferred.

[0123] For example, the DNA vector described in U.S. Patent No. 10,098,935 has been shown to provide tolerization (i.e., induction of antigen-specific tolerance). Such vectors are referred to as tolerization vectors. The vectors can be administered, for example, by local injection, including intramuscular injection, and encode cross-reactive adenoviral peptides or proteins containing peptides, and further comprise a promoter sequence operably linked to the nucleic acid sequence, and a DNA backbone linked to the promoter sequence and the nucleic acid sequence, which contains up to four immunostimulatory CpG motifs. The cross-reactive peptides can be modified by one, two, three or more amino acid residues and altered from naturally occurring polypeptides.

[0124] The present invention has been described in terms of specific embodiments discovered or proposed by the inventors to comprise preferred modes for carrying out the invention. Those skilled in the art will understand, in light of this disclosure, that numerous modifications and variations can be made in the specific embodiments exemplified without departing from the intended scope of the invention. Considerations of biological functional equivalence allow for changes in protein structure without affecting biological activity in kind or amount. All such modifications are intended to be within the scope of the appended claims.

[0125] Example 1 MS patient CD4 + Screening of T cells reveals cross-reactive antigen specificities with adenoviral proteins. Here, we have discovered, using advanced recombinant DNA techniques, that T cell receptors in patients with multiple sclerosis (MS) can react with both a common viral antigen (adenovirus) and a major myelin protein (myelin basic protein). This peptide antigen, and its equivalents in other autoimmune diseases, can be used in antigen-specific tolerance protocols in MS patients, which can be both ameliorating and, in some cases, curative. The same bias used herein This approach may also be successful in finding other causative antigens in other autoimmune diseases.

[0126] We analyzed PBMCs from 18 newly diagnosed MS patients and four healthy controls (HCs). We first performed CytoF to determine whether differences existed in T cell populations between HCs and MS patients. MS patients were found to have a higher frequency of brain-homing activated T cells, which we analyzed by single-cell sorting and paired TCR sequencing. We found that MS CD8 T cells proliferated massively, while MS CD4 T cells showed minimal proliferation. γδ T cells proliferated at the same level as HCs. Because MS is highly associated with DR150101 (20-30% susceptible), we focused on CD4 TCR specificity. To identify antigen specificity, we clustered CD4 TCRs with GLIPH. Using GLIPH, we were able to identify CD4 TCR clusters enriched in DR150101 MS individuals. Nine CD4 TCRs clustered with DR15 were selected for antigen discovery. We generated these nine CD4 TCRs as soluble recombinant proteins and screened the 12-MER and 15-MER DR15 yeast libraries. Among the nine CD4 TCRs, four (MS-1 to MS-4 TCRs) showed enrichment (A647-Myc) and tetramer (PE-TCR Tet) staining.

[0127] We generated MS1-TCR cell lines and cocultured them with T2-DR15 antigen-presenting cell lines loaded with peptides generated in the yeast library to examine activation. CD69 was used as a marker of T cell activation. Most of the library-enriched peptides stimulated the MS-1 TCR cell line. We used the yeast library enrichment data to predict the actual peptides / antigens (self or non-self) for the MS-1 CD4 TCR. Surprisingly, we found that an adenovirus peptide (adeno-ATFTSYRSWYLA) was nearly identical to the enriched peptide, and it also stimulated the MS-1 TCR cell line.

[0128] In addition to enriching the yeast library peptides, we added an additional layer to help the algorithm determine wild-type peptides. Essentially, we created single-point mutations on the top, most enriched library peptides and used the T cell stimulation data of these position-mutated peptides to generate a substitution matrix, which we then used in the algorithm to search for actual peptides from the human proteome. Using this position-mutation / refined substitution matrix data, we were able to identify three self-peptides for the TCR2 / MS-1 TCR.

[0129] To directly detect and identify adeno-specific T cells from MS patients (n = 9) and healthy controls (n = 9), we generated pMHC tetramers, enriched them, and enumerated CD4 T cells specific for adeno-peptides. We were able to detect higher numbers of adeno-specific CD4 T cells in MS patients compared to healthy controls. Furthermore, we single-cell sorted these adeno-specific CD4 T cells, sequenced their TCRs, and generated TCR cell lines (adeno-specific TCR).

[0130] TCR cell lines derived from adenotetramer-selected CD4 TCRs were tested for cross-reactivity with MBP85-99 peptide. MS-1 TCRs were also tested for cross-reactivity with MBP. Adenospecific TCRs were stimulated with adeno peptide and MBP. Some TCR2 / ms-1 TCRs and adeno-TCRs (adeno-TCR1 and 9) cross-react with MBP peptide. Sera from MS (n=28) and HC (n=10) were tested for adenovirus titers in the serum. A subset of MS sera had higher adenovirus levels compared to HC sera. CD3 from 8 MS and 5 HC patients were tested. + T cells were sorted and subjected to TCR and RNA sequencing using a 10x platform. Additionally, T cells derived from the cerebrospinal fluid were sequenced in four of the eight MS patients. Clonal expansion was observed in the CSF of each MS patient, with CD4 + T cells and CD8 +Found in T cells.

[0131] Regarding the generation of yeast peptide-MHC libraries, Garcia and colleagues recently developed a yeast peptide-MHC library system for the identification of αβ TCR ligands. To discover peptide antigens for these TCRs, they designed the DR15 construct and generated two different peptide libraries with this mutation, mutating 12- and 15-aa inserts with limited diversity at the primary MHC-binding anchor residues.

[0132] For human samples, peripheral blood mononuclear cells (PBMCs) were obtained from healthy blood donations from the Stanford Blood Center. Healthy human subjects were males and females aged 22–47 years. PBMCs from multiple sclerosis patients were obtained from the Multiple Sclerosis Center at the University of California, San Francisco (UCSF). The UCSF Human Research Committee approved the protocol, and informed consent was obtained from all participants.

[0133] For the generation of soluble TCRs, soluble TCRs were generated as previously described. TCR-variable mouse-constant human (VmCh) chimeras containing engineered C-domain disulfides were cloned into the pAcGP67a insect expression vector (BD Biosciences, 554756) encoding either a C-terminal acidic GCN4-zipper-biotin acceptor peptide (BAP)-6xHis tag. Each chain also encoded a 3C protease site between the C-terminus of the TCR ectodomain and the GCN4 zipper to allow zipper cleavage. Baculovirus for each TCR construct was generated in SF9 cells via cotransfection of BD Baculogold linearized baculovirus DNA (BD Biosciences, 554739) with Cellfectin II (Life Technologies, 10362-100). TCR α and β chain viruses were co-infected into small volumes (2 mL) of High Five cells at various ratios to find the ratio that ensured a 1:1 α:β stoichiometry.

[0134] To prepare soluble TCRs, 1 L of High Five cells was infected with the appropriate ratio of TCRα and TCRβ viruses for 48 hours at 28°C. The collected culture medium was adjusted to 100 mM Tris-HCl (pH 8.0), 1 mM NiCl2, and 5 mM CaCl2, and the resulting precipitate was removed by centrifugation. The medium was then incubated with Ni-NTA resin (QIAGEN 30250) at room temperature for 3 hours and eluted in 1x HBS + 200 mM imidazole (pH 7.2). The TCRs were then site-specifically biotinylated by adding recombinant BirA ligase, 100 μM biotin, 50 mM bicine pH 8.3, 10 mM ATP, and 10 mM magnesium acetate, followed by overnight incubation at 4°C. The reaction was then purified by size-exclusion chromatography using an AKTA Purifier (GE Healthcare) on a Superdex 200 column (GE Healthcare). Peak fractions were pooled and then tested for biotinylation using an SDS-PAGE gel shift assay. Protein was typically 100% biotinylated.

[0135] For the generation, tag enrichment, staining, and selection of yeast-displayed DR150101 peptide libraries, single-chain trimer (SCT) DR150101 yeast constructs were synthesized as N-terminal fusions to the yeast surface protein Aga2p. Full-length SCT constructs were cloned into the vector pYAL. These constructs contained the Aga2p leader sequence, followed by a 12- or 15-mer peptide sequence, a Gly-Ser(GGGGS)3 linker, a second glycine linker (GGGGS)4, the DR150101 sequence, either a Myc or HA epitope tag, a third glycine linker (GGGGS)3, and the Aga2 protein. Constructs were then electroporated into EBY-100 yeast as previously described and induced for expression in SGCAA pH 4.5 medium at 20 °C for 24–72 h until maximum epitope tag staining was observed (typically 40%–70% of the total population). Full-length yeast constructs were mutagenized as previously described. Briefly, constructs were mutagenized via error-prone PCR (Genemorph II kit, Agilent 200550), ligated into the pYAL vector, and had a final error rate of approximately 4-5 nucleotide substitutions per kbp, as determined by sequencing clones. Yeast libraries were generated by electroporation of competent EBY-100 cells via homologous recombination of the linearized pYAL-cMyc / HA vector. The final library contained approximately 5 x 10 8 The yeast transformants contained

[0136] As previously described, peptide libraries were generated in the same manner as error-prone libraries, except that pMHC constructs were instead randomized along the peptide by using mutagenic primers that allow all 20 amino acids through the NNK codon. The libraries allowed only limited diversity at known MHC anchor residues to maximize the number of correctly folded and displayed pMHC clones in the library. bFor pMHC libraries, the P5 and P9 anchors were restricted to Asn (N) and Met / Ile / Leu (M / I / L) using the AAC and MTS codons, respectively. The resulting PCR product was used as a template for a second PCR reaction, and 50 nucleotide sequences homologous to the vector were added to both ends of the PCR product. 50 μg of this second PCR product and approximately 10 μg of the linearized vector were then purified and used to electroporate yeast to generate each library. Prior to selection of the 12-mer and 15-mer pMHC libraries, each was enriched for its respective epitope tag to maximize the proportion of yeast in the initial pool with correctly folded and displayed pMHC molecules on its surface. To achieve this, each library was enriched for 1 × 10 7 Cells were induced separately in 500 mL of SGCAA at a starting density of 1.4 × 10 cells / mL for 24–72 h at 20°C. Maximum epitope tag staining was observed when approximately 1.4 × 10 cells / mL were cultured. 9 Induced yeast cells were washed once in PBS + 0.5% BSA and 1 mM EDTA (PBE buffer) and resuspended in 5 mL of PBE with 200 μL of Miltenyi streptavidin microbeads (Miltenyi, 130-048-101). The cell and bead mixture was incubated at 4 °C for 1 h with rotation, washed again with PBE, resuspended in 5 mL of PBE, and passed through a cell strainer onto a pre-wetted MACS LD column (Miltenyi, 130-042-901). After the column was completely emptied, it was washed twice with 2 mL of PBE and the flow-through was collected.

[0137] Cells were isolated from the flow-through by centrifugation and resuspended in 5 mL of PBE with 80 μL of anti-cMyc AlexaFluor647 or anti-HA AlexaFluor647 antibody (Cell signaling, 2233 and 3444), respectively, and incubated with rotation at 4 °C for 1 h. Cells were washed, resuspended in 5 mL of PBE, and 220 μL of Miltenyi anti-AlexaFluor647 microbeads (Miltenyi, 130-091-395) were added. The mixture was incubated with rotation at 4 °C for 30 min, protected from light. Cells were then washed, resuspended in 6 mL of PBE, and split equally between two pre-wetted MACS LS columns (Miltenyi, 130-042-401). After completely emptying the columns, each column was washed twice with 3 mL of PBE, and the flow-through was set aside. Cells were eluted from the columns with 5 mL of PBE per column. A small fraction of the eluate (5–20 μL) was pooled, and AlexaFluor 647 staining was compared with that of the flow-through for quantification of tag enrichment. The remaining eluted cells were pooled, collected by centrifugation, and resuspended in a total of 40 mL of SDCAA medium. Cell density was measured at 600 nm using a spectrophotometer. SDCAA was then added to adjust the cell density to below its OD, and the yeast was cultured overnight at 30°C. The cells were passaged for another round of overnight growth in SDCAA.

[0138] For tag elution, the cells were cultured in 500 mL of SGCAA at 20 °C. To stain pMHC with TCR tetramers, biotinylated TCRs were incubated with streptavidin conjugated to AlexaFluor 647, AlexaFluor 488, or phycoerythrin at a 5:1 ratio on ice for 5 minutes to ensure complete tetramer formation. Yeast cells were then stained with 250 nM tetramer plus anti-Myc-AlexaFluor 488 or anti-HA-AlexaFluor 488 antibodies (Cell Signaling, 2279 or 2350, respectively) on ice for 3 hours and washed twice with ice-cold PBE buffer before analysis via flow cytometry (Accuri C6 flow cytometer). All yeast selections and sequencing of the yeast libraries were performed as previously described.

[0139] For peptide-MHC tetramer formation, all tetramers were freshly prepared as described above. Briefly, for tetramerization, the amounts of fluorophore-conjugated streptavidin and pMHC monomer were mixed at a molar ratio of 4:1. One-fifth the amount of fluorophore-conjugated streptavidin was added to the monomer solution every 10 min at room temperature.

[0140] For enrichment of tetramer-positive T cells in cell lines, cells were tetramer-stained for 1 hour at room temperature and washed with FACS buffer. After tetramer enrichment, cells were surface-stained with an antibody cocktail for 20 minutes at 4°C. Stained cells were washed using FACS buffer and analyzed on an LSRII (Becton Dickinson) or single-cell / bulk-sorted on a FACS Aria Fusion SORP (Becton Dickinson). Lentivirally transduced Jurkat TCRαβ cells were analyzed. - / - Cell lines were stained with tetramer at a concentration of 20 nM in FACS buffer with 10 μM biotin at room temperature for 1 hour, followed by surface staining with the appropriate antibody for 20 minutes at 4° C. After surface staining, cells were washed with FACS buffer and analyzed on an LSRII (Becton Dickinson).

[0141] For TCR expression by lentiviral transduction, the TCR α and β constructs were cloned into lentiviral constructs. For TCR expression, the alpha and beta TCR lentiviral constructs were transfected separately into 293X cells. Virus was harvested 72 hours post-transfection and transfected into Jurkat αβ cells. - / - or SKW αβ - / - Cells were transduced. SKW or Jurkat cells were enriched for highest expression of TCRαβ by using Miltenyi anti-APC selection (Miltenyi 130-090-855).

[0142] T cell stimulation assays were performed as previously described. All T cell peptide stimulation experiments were performed in 96-well round-bottom plates in a total volume of 200 μL. T2, K562 cells, or BMDCs were pulsed with 10–100 μg of peptide for 45 minutes, washed once, and seeded (10,000 cells / well). TCR-expressing cell lines (100,000 cells / well) were co-cultured with APCs for 18 hours. At the end of stimulation, cells were harvested, washed, stained for TCRβ, human CD3, and CD69, and analyzed for activation using an LSRII (Becton Dickinson).

[0143] For whole-transcriptome sequencing and data analysis, whole-transcriptome sequencing was performed as previously described, and T cells were bulk-sorted directly into Trizol (Qaigen). RNA was extracted using the RNeasy Plus Micro Kit (Qiagen). After analysis on the 2100 Bioanalyzer, the resulting libraries were sequenced on the HiSeq4000 platform (Illumina). For each sample in the whole-transcriptome sequencing library, 75-base-pair paired-end reads were obtained from the sequencer. Each sample condition was completed in triplicate, except for the WT sample, for which only one sample was generated. Read quality was determined using FastQC 0.11.4. Reads were aligned to the mouse reference genome (NCBI / assembly GRCm38) using TopHat v2.0.13. On average, 90% of reads aligned to the reference genome. Differential gene expression analysis and read count normalization, used as input for heatmaps, were determined via DESeq251. TPM values ​​were calculated with RSEMv1.3.052. Heatmaps were generated with the R package "pheatmap". Gene ontology analysis plots were generated with the R package "enrichplot". Data availability RNA-seq data and yeast p-MHC selection data have been deposited in the Gene Expression Omnibus (GEO) data repository under accession number GSE130975.

[0144] array The T cell receptor sequences from Figure 1G are as follows:

[0145] [Table 1]

[0146] The peptide antigens from Figure 2 are as follows:

[0147] [Table 2-1]

[0148] [Table 2-2]

[0149] The peptide antigens from Figure 4 are as follows:

[0150] [Table 3]

[0151] The T cell receptors from Figure 9 are as follows:

[0152] [Table 4]

[0153] The peptide antigens from Figure 9 are as follows:

[0154] [Table 5]

[0155] Each publication cited herein is hereby incorporated by reference in its entirety for all purposes.

[0156] It is understood that this invention is not limited to the particular methodology, protocols, cell lines, animal species or genera, and reagents described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is limited only by the appended claims.

[0157] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a cell" includes a plurality of such cells, a reference to "the culture" includes one or more cultures and equivalents thereof known to those skilled in the art, and so forth. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0158] cross reference This application is a continuation of U.S. Provisional Patent Application No. 62 / 895,805, filed September 4, 2019. No. 60 / 699,999, filed on Oct. 1, 2003, which is incorporated herein by reference in its entirety.

Claims

1. 1. An isolated cross-reactive peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 1 or a variant thereof containing up to one amino acid substitution or deletion, wherein the isolated cross-reactive peptide is up to 40 amino acids in length.

2. The cross-reactive peptide of claim 1 fused to a non-native protein sequence.

3. 10. A pharmaceutical composition comprising the isolated cross-reactive peptide of claim 1 or 2 and a pharmaceutically acceptable excipient.

4. A protein complex comprising a human MHC protein and a cross-reactive peptide according to claim 1 or 2.

5. The protein complex of claim 4, wherein the MHC protein is a class II MHC protein, and the MHC protein is DR15.

6. 4. The pharmaceutical composition of claim 3 for use in a method for reducing symptoms of multiple sclerosis, the method comprising administering a tolerogenic dose of the pharmaceutical composition.

7. A DNA construct encoding the cross-reactive peptide of claim 1 or 2.

8. 8. The DNA construct of claim 7 for use in a method for reducing symptoms of multiple sclerosis, the method comprising administering a tolerogenic dose of the DNA construct.

9. An isolated T cell receptor (TCR) peptide comprising or consisting of an amino acid sequence set forth in any of SEQ ID NOs: 6, 15, 78-79 or 82-83, which binds to the protein complex of claim 4 or 5 or the immunogenic peptide of claim 1 or 2.

10. 10. The isolated TCR peptide of claim 9 fused to a non-native protein sequence.

11. A pharmaceutical composition comprising the isolated TCR peptide of claim 9 or 10.

12. 12. An isolated TCR peptide according to claim 9 or 10, or a pharmaceutical composition according to claim 11, for use in a method for reducing symptoms of multiple sclerosis, the method comprising administering an immunogenic dose of the TCR peptide or pharmaceutical composition.

13. An antibody that specifically binds to any of the protein complexes described in claims 4 or 5, T cell receptors that bind to the protein complexes described in claims 4 or 5, or cross-reactive peptides described in claims 1 or 2.

14. 1. A method for determining the presence of pathogenic T cells in an individual suspected of having multiple sclerosis, comprising: contacting a sample containing T cells from the individual with the cross-reactive peptide of claim 1 or 2 or the protein complex of claim 4 or 5; determining the presence of a T cell response to said cross-reactive peptide or protein complex; A method comprising:

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