Antibody genetics of radiologically isolated syndrome

Abstract

The present disclosure is directed to human recombinant antibodies and analysis of antibody genetics in subjects having radiologically isolated syndrome to determine risk of developing multiple sclerosis. The present disclosure is further directed to methods of evaluating said antibodies for pathogenic agents or neuroprotective candidates.

Description

[0001] DESCRIPTION ANTIBODY GENETICS OF RADIOLOGICALLY ISOLATED SYNDROME PRIORITY CLAIM This application claims benefit of priority to U.S. Provisional application Serial No. 63 / 683,376, filed, August 15, 2024, the entire contents of which are hereby incorporated by reference. STATEMENT REGARDING FEDERAL GRANT SUPPORT This invention was made with government support under grant no. NS102417 awarded by the National Institutes of Health. The government has certain rights in the invention. REFERENCE TO A SEQUENCE LISTING This application contains a Sequence Listing XML, which has been submitted electronically and is hereby incorporated by reference in its entirety. Said Sequence Listing XML, created on August 12, 2025, is named UTFDP4457WO.xml and is 599,928 bytes in size. BACKGROUND 1. Field of the Disclosure The present disclosure relates generally to the fields of medicine, neurology, neuropathology and immunology. More particularly, the disclosure relates to human antibodies present in subjects having radiologically isolated syndrome (RIS) and their use in identifying subjects at risk of developing multiple sclerosis. The disclosure further relates to the characterization of such antibodies and their derivatives, e.g., for evaluating potential pathogenic or neuroprotective effects. 2. Background Radiologically isolated syndrome (RIS) represents the earliest detectable pre- clinical phase of multiple sclerosis (MS) in select individuals, punctuated by abnormal and incidentally found demyelinating lesions within the brain and / or spinal cord from MRI studies in healthy individuals. People with RIS do not show clinical symptoms or

[0002] 1 4936-8845-3466, v.1 signs of neurological dysfunction compared to people with MS. However, subjects are at a higher risk for a first demyelinating event within 5 years particularly if the subject is of younger age (<37 years), male, or has spinal cord lesions. More commonly, individuals evolve and develop relapsing-remitting MS while approximately 10% experience a clinical course consistent with primary progressive MS. Using the 2009 RIS criteria, the estimated risk for a first clinical event is 34% at 5 years which increases to 51% at 10 years. Subjects presenting with a first clinical demyelinating symptom are termed clinically isolated syndrome (CIS), and have an estimated conversion rate to MS over a median time of 4.3 years is 63%. Early treatments with disease-modifying therapies (DMT) are thought to reduce future demyelinating attacks and consequently, conversion to the diagnosis of MS. Distinguishing which individuals with RIS and CIS convert to a diagnosis of MS is an important aspect of providing optimal care for this vulnerable population. One way to address this is to identify biological markers that would operate as a companion to identify those individuals most likely to develop MS. Several biological markers have been studied in MS, including the presence of unique cerebrospinal-fluid (CSF) restricted oligoclonal bands (OCBs) which can be used to identify CIS subjects that convert to MS diagnosis. In fact, individuals with CIS who present with 2 or more lesions by MRI and are OCB positive have a 78% rate of conversion to MS. More recently, CSF- and serum- derived neurofilament light (NfL) levels were found elevated in people with RIS and CIS. Neurofilaments are discharged in the interstitial fluid due to neuronal injury and are ultimately detected in the vasculature of the blood-brain barrier. Thus, it stands to reason that NfL levels in the serum or CSF could predict clinical conversion to MS or changes in disease activity. Other existing potential biomarkers of disease could be useful in stratifying patients likely to convert to MS. Plasmablasts (PBs), are a subset of highly differentiated, early antibody secreting B cells which represent a significant proportion of the B cell pool in the CSF of MS subjects, and display a correlation of frequency with grey matter disease by MRI. The inventor has previously demonstrated that CIS individuals who advance to a diagnosis of MS display an expansion of PBs expressing antibody heavy chain rearrangements of the variable heavy 4 (VH4) family in the blood are enriched for binding to neurons. The details of antibody genetics and their application to RIS biology remain unknown.

[0003] 2 4936-8845-3466, v.1 SUMMARY OF ASPECTS AND EMBODIMENTS The present disclosure in various aspects and embodiments provides human recombinant antibodies cloned from subjects having radiologically isolated syndrome (RIS). In aspects of the present disclosure, methods are provided to determine a risk of a subject having RIS to develop multiple sclerosis, based upon evaluation of antibody genetics (e.g., somatic hypermutation and / or VH usage of antibodies derived from plasmablasts) and / or propensity of antibodies for binding neurons. In aspects of the present disclosure, antibodies and antigen binding portions of antibodies from plasmablasts of RIS subjects are provided, as reagents or components of a library for evaluating pathogenic and / or neuroprotective properties. In other aspects, methods are provided for developing or making neuroprotective compositions. Other aspects and embodiments will be apparent to the skilled person in view of the present disclosure. In an aspect, the present disclosure provides a recombinant antibody or antibody fragment (i.e., antigen binding portion) comprising clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively. In embodiments, the recombinant antibody or portion thereof is encoded by clone-paired nucleotide sequences from Table 1. In embodiments, the recombinant antibody or an antigen-binding portion thereof (i.e., antibody fragment) is encoded by nucleotide sequences that have at least 70%, 80%, or 90% sequence identity to clone-paired nucleotide sequences from Table 1. In embodiments, the recombinant antibody or antibody fragment is encoded by nucleotide sequences having at least 95% sequence identity to clone-paired nucleotide sequences from Table 1. In embodiments, the recombinant antibody or antibody fragment comprises heavy and light chain variable sequences comprising clone-paired amino acid sequences from Table 2. In embodiments, the recombinant antibody or antibody fragment comprises heavy and light chain variable sequences having at least 70%, 80%, or 90% sequence identity to clone-paired amino acid sequences from Table 2. In embodiments, the recombinant antibody or antibody fragment comprises heavy and light chain variable sequences having at least 95%, or at least 97%, or at least 98%, or at least 99% sequence identity to clone-paired amino acid sequences from Table 2. The recombinant antibody may be of any isotype, including IgG (e.g., IgG1,IgG2, IgG3, IgG4), IgM, IgD, IgA, and IgE. In embodiments, the recombinant antibody or antibody fragment specifically binds neurons, and in embodiments binds mouse and human neurons. For example, the

[0004] 3 4936-8845-3466, v.1 recombinant antibody or antibody fragment may have binding activity for Sy5y cells (a neuroblastoma cell line). In embodiments, the antibody or antibody fragment binds a surface target and / or a cytoplasmic target in neurons. Cellular targets and epitopes (cytoplasmic or otherwise) can be determined using known techniques, including protein arrays and immunoprecipitation studies, for example. In embodiments, the recombinant antibody or antibody fragment is a full monoclonal antibody, or is a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab′)2fragment, or Fv fragment. In embodiments, the recombinant antibody or antibody fragment is a recombinant IgG antibody or antibody fragment comprising a modified Fc. In embodiments, the Fc is modified to alter FcR interactions, alter glycosylation pattern, or to increase circulatory half-life (e.g., as described in detail herein). In embodiments, the recombinant antibody or antibody fragment is conjugated to a detectable label, a toxin, a drug, or a prodrug. In other aspects, the present disclosure provides a cell expressing a recombinant antibody or antibody fragment of the disclosure, including but not limited to Chinese Hamster Ovary (CHO) cells and HEK293 cells. In an aspect, the disclosure provides a method of identifying a subject having radiologically isolated syndrome (RIS) that is at high risk for developing multiple sclerosis as compared to the average for RIS patients in general. The method comprises determining an antibody somatic hypermutation (SHM) frequency of antibodies derived from plasmablasts isolated from an RIS subject. Antibody SHM is defined as antibodies having productive heavy chain rearrangements and with greater than three somatic hypermutations. In embodiments, the VH4 antibodies are evaluated for SHM and used for determining the risk of developing MS. In embodiments, other VH families are evaluated for SHM frequency. Alternatively or in addition, the method comprises determining a frequency of neuron-binding for plasma or serum antibodies isolated from the RIS subjects. Based on the analysis, RIS subjects identified as having high antibody SHM frequency (and optionally for VH4 antibodies) and / or high frequency of neuron- binding of serum or plasma antibodies (e.g., compared to a healthy population) are at high risk of developing multiple sclerosis as compared to the average for RIS patients in general. In embodiments, a high SHM frequency is at least 75% of the antibodies derived from plasmablasts, or at least 80% of antibodies derived from plasmablasts. In embodiments, a high frequency of neuron binding of serum or plasma antibodies (e.g.,

[0005] 4 4936-8845-3466, v.1 IgG antibodies) is 3.5% or greater, or 4% or greater, or 4.5% or greater, which can be determined for example, using flow cytometry. In embodiments, amino acid sequences of antibody variable regions (e.g., variable heavy regions) are determined, and optionally VH usage is determined, from a population of plasmablasts of a subject, such as by sequencing of genomic DNA or mRNA of said population of plasmablasts. In embodiments, determining neuron binding comprises assessing binding to a neuronal cell line, which is optionally Sy5y cells or other neuronal cell line. Other suitable cell lines include those derived from human cortical neurons. Neuron binding frequency can be determined using known tools, such as by flow cytometry. In embodiments, the method further comprises treating the subject with an immunosuppressive therapy or multiple-sclerosis disease-modifying therapy, when the subject is identified as having a high risk of developing multiple sclerosis as compared to the average for RIS patients in general. Such agents include biologics such as interferon, or antibodies or other agents targeting B cells. In embodiments, the immunosuppressive therapy is corticosteroid (e.g., intravenous corticosteroid). In embodiments, the disease modifying therapy is selected from glatiramer acetate, ozanimod, fingolimod, siponimod, ponesimod, ocrelizumab, natalizumab, ofatumumab, alemtuzumab, Interferon-beta, teriflunomide, cladribine, monomethyl fumarate, dimethyl fumarate, diroximel fumarate, among others (e.g., disclosed elsewhere herein). In embodiments, subjects identified as not having a high risk of developing multiple sclerosis are not treated with an immunosuppressive therapy or a multiple sclerosis disease modifying therapy. In embodiments, RIS subjects identified as having a high risk of multiple sclerosis are actively monitored for signs or symptoms of MS. In embodiments, the subject receives frequent MRI scans, such as at least about twice per year or at least about once per year. In embodiments, subjects determined to have a high risk for developing MS are evaluated or monitored for other biomarkers of MS, including but not limited to elevated NfL in CSF or blood. In embodiments, the method for identifying an RIS subject that is at high risk of developing multiple sclerosis can be repeated, for example, to monitor changes in the frequency of antibody SHM (of antibodies derived from plasmablasts) and / or the VH usage, and / or determining a change in the frequency of plasma antibodies that bind

[0006] 5 4936-8845-3466, v.1 neurons. For example, the method can be repeated at least once annually or at least once every other year. In other aspects, the disclosure provides a method for making a pharmaceutical composition for providing neuroprotection. The method comprises cloning and expressing recombinant antibodies or antigen binding portions thereof from plasmablasts of one or more RIS subjects, and identifying a recombinant antibody or antigen binding portion thereof that reduces or inhibits apoptosis in neurons (e.g., in stressed neurons), and / or reduces or ameliorates neurodegenerative disease or neuronal loss in an animal model. For example, the method can comprise evaluating at least 50 candidate antibodies, or at least 100 candidate antibodies, or at least 200 candidate antibodies, or at least 500 candidate antibodies, or more. The method further comprises formulating the identified neuroprotective antibody or antigen binding portion for delivery to a human or animal. In embodiments, the candidate antibodies tested include one or more shown in Tables 2 and Tables 3 and 4, and optionally variants of these antibodies as described herein (e.g., having sequence identity thereto) or generated by introducing from one to four (e.g., 1, 2, 3, or 4) amino acid substitutions in one or more of the six CDRs, and / or introducing 1, 2, 3, 4, 5, or 6 or more amino acid substitutions, deletions, or insertions within the framework regions of the VH and / or VL sequences. In embodiments, the antibody variant is prepared or further prepared by modifying the Fc domain, including as described herein. In some embodiments, the method of preparing the variant comprises screening such antibody variants by measuring binding against a neuronal target or epitope. In embodiments, the candidate antibody or antigen binding portion that is evaluated has high SHM and / or has neuron binding activity. For example, the antibody or antigen binding portion has at least three or at least four somatic hypermutations. In embodiments, the antibody is a VH4 antibody. In embodiments, the VH4 antibody satisfies the antibody gene signature (AGS) codons described in U.S. Patent 8,394,583, which is hereby incorporated by reference. In some embodiments, the antibody or antigen binding portion that is selected binds to a target of a neuronal cell line, which in some embodiments is present in the cytoplasm. In some embodiments, the cytoplasmic target may be a heterogeneous nuclear ribonucleoprotein (hnRNP) present in stress granules. In embodiments, the one or more hnRNP comprises an RNA recognition motif 2 (RRM2), which in embodiments acts as an epitope or partial epitope for the antibody. In embodiments, the hnRNP comprises

[0007] 6 4936-8845-3466, v.1 hnRNP A / B or hnRNP A1. In embodiments, the one or more hnRNP comprises hnRNP A2 / B1, hnRNP C1 / C2, hnRNP A1, hnRNP A3, and hnRNP D0. See PCT / US2024 / 036930, which is hereby incorporated by reference in its entirety. In embodiments, the method comprises measuring binding between the antibody or antigen binding portion thereof and the target or epitope thereof by one or more in vitro binding assays, including but not limited to ELISA, surface plasmon resonance (SPR), and biolayer interferometry (BLI). In some embodiments, the antibody binds a cytoplasmic target (e.g., including but not limited to an hnRNP target, such as hnRNP A1, hnRNP A / B, or hnRNPA2 / B1, or RRM2 epitope thereof) according to the in vitro binding assay. For example, the antibody may bind the neuronal target or epitope with an affinity of less than or equal to 1 x 10-7M, or less than or equal to 1 x 10-8M, less than or equal to 1 x 10-9M, less than or equal to 1 x 10-10M, less than or equal to 1 x 10-11M, or less than or equal to 1 x 10-12M. In embodiments, inhibition of apoptosis in neurons is determined in vitro using a mouse or human neuronal cell line. Exemplary neuronal cell lines include but are not limited to Sy5y cells and cells derived human cortical neurons. In embodiments, reduction or amelioration of neurodegenerative disease or neuronal loss is determined in an animal model, which is optionally an experimental autoimmune encephalomyelitis (EAE) mouse model. The EAE mouse model can be employed to assess neuroprotection. The EAE model is described in Robinson, AP, et al. “The experimental autoimmune encephalomyelitis (EAE) model of MS: utility for understanding disease pathophysiology and treatment”, Handb Clin Neurol. 2014; 122: 173-189. For example, EAE can be induced in SJL / j female mice by immunization with 100µg PLP139-151 with 2 µg complete Freund’s adjuvant (CFA). The first attack typically emerges around day 10 and resolves around day 17. Mice that have a confirmed recovery are administered the candidate antibody or control antibody. Mice are scored for EAE for 20 days or until they develop and recover from a subsequent attack. Immunohistochemistry can be used to evaluate demyelination and inflammation in treated and control mice. In embodiments, an in vitro neurotoxicity study is conducted to confirm that the candidate antibody has no appreciable neurotoxicity. Such studies can optionally be conducted in Sy5y cells. Candidate antibodies can be compared to known neurotoxic antibodies that induce apoptosis in the neuronal cell line, such as DS14 antibody.

[0008] 7 4936-8845-3466, v.1 In embodiments, the method further comprises quantifying internalization of the antibody or antigen binding portion in neurons in vitro or using an in vivo animal model. For example, for in vitro analysis the Sy5y human neuroblastoma cell line can be used or alternatively cells derived from human cortical neurons, among other available neuronal cell lines. For example, neuronal cells can be cultured to confluency, incubated with either a non-specific negative control antibody or the candidate antibody, and then fixed, stained, and imaged using fluorescence confocal microscopy at various time points to visualize internalization. In embodiments, the antibody or antigen binding portion thereof is formulated so as to be expressed in neurons via gene or mRNA delivery. Viral vectors and lipid nanoparticles, for example, for delivery according to these embodiments are known in the art. In embodiments, the antibody or antigen binding portion thereof is formulated for parenteral administration or intranasal administration. Pharmaceutically acceptable carriers that can be used in accordance with this disclosure include those known in the art and described herein. In some embodiments, the pharmaceutically acceptable carrier is a physiological solution, e.g., saline or other pharmacologically acceptable solvent or a buffered solution, and may optionally comprise a surfactant. Pharmaceutically acceptable carriers include water, saline, and glycerol. In some embodiments, the formulation may comprise fixed oils, polyethylene glycol, propylene glycol or other solvents. Neuroprotective compositions prepared according to this process can be used for treating neurodegenerative diseases, including but not limited to Multiple Sclerosis (MS), Alzheimer's disease (AD), Parkinson's disease (PD), Amyotrophic Lateral Sclerosis (ALS), Huntington's disease (HD), ischemic stroke, frontotemporal dementia (FTD), Schizophrenia, Progressive supranuclear palsy, Friedreich ataxia, Lewy body disease, Spinal muscular atrophy, and traumatic injury, and other conditions characterized by the loss of neurons. The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The word “about” means plus or minus 10% of the stated number. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein. Other

[0009] 8 4936-8845-3466, v.1 objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

[0010] 9 4936-8845-3466, v.1 BRIEF DESCRIPTION OF THE DRAWINGS The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. FIG. 1. Plasmablasts are not expanded in RIS patients. Traditional flow cytometry was performed to obtain the frequency of PBs in the blood of HC (n=20), RIS patients (n=30), and CIS patients (n=20). The averages of each group are depicted as a horizontal line in the graph and the values are provided below the X-axis. The average mean of the HC was 2.69, the RIS mean was 3.43, and CIS was 5.46. The ANOVA p- value for this cohort was 0.0184. Each data point represents an individual research participant. FIG. 2. PBs from RIS patients display a reduced drive towards antigen-driven selection. Antibody heavy chain sequences were obtained from sorted PBs of the cohort and the percentage of sequences with 3 or more SHM was calculated to obtain a frequency per subject. The averages of each group are depicted as a horizontal line in the graph and the values are provided below the X-axis. The average mean of the HC was 77.0%, RIS had a mean of 77.6%, and CIS was 86.3%. The ANOVA P-value for this cohort was 0.005. Each data point represents an individual research participant. FIGS. 3A-D. PBs in RIS patients are enriched for VH4 use compared to CIS patients. Variable heavy chain family usage for families 1-7 was calculated for each Each data point depicts an individual research participant. The white bars in all 4 panels represent the HC (n=20), the middle light grey represents the RIS (n=30), and the dark gray represents the CIS group (n=20). FIG.3 A depicts VH family usage for all sequences in the database. FIG. 3B depicts VH4 family usage for all sequences in the database, where each data point is an individual subject value. FIG. 3C depicts VH4 family usage for those sequences with high SHM (>3SHM), where each data point is an individual subject value. Panel D depicts VH4 family usage when each subject’s repertoire is restricted to the top 5 clones. FIGS. 4A-B. VH4+ antibodies from RIS patients are enriched for reactivity to human neurons. FIG. 3A compares the percentage of RIS plasma IgG binding to Sy5y cells in the VH3 (n=11) and VH4 family (n=52). The percentage was increased in the VH4 group with an average of 13.06, while the average of VH3 was 3.66. Each data

[0011] 10 4936-8845-3466, v.1 point represents an individual rhAb. FIG. 3B illustrates typical intracellular binding patterns observed by anti-neuronal rhAbs from the RIS cohort. The top left line indicates the rhAb name, the middle-left line indicates the MFI of staining in the region of interest, and the bottom left line indicates the VH4 gene usage, the JH segment, and the percent homology to the germline. FIGS. 5A-C. RIS patients display a higher frequency of neuron-binding plasma IgG. FIG 5A depicts the gating strategy and example of a purified IgG plasma source with no binding to SH-Sy5y. FIG. 5B depicts the gating strategy and an example of a purified IgG plasma source with binding to SH-Sy5y. FIG.5C depicts the percentage of SH-Sy5y cells bound by purified IgG from each subject in the cohort. Each data point represents an individual research participant. The number of data points per cohort and averages of each group are provided below the X-axis. The ANOVA p-value for this cohort was 0.0184. FIG.6. JH gene frequency, CDR3 length and CDR3 charge analysis. FIG.7. VH family and JH gene pairing analysis. FIG.8. Replacement Frequency Analysis. FIG. 9. Gating strategy SH-for Sy5y-FLO with rhAbs. The top panels are an example of a VH3+ rhAb from an RIS patient that does not bind to SH-Sy5y cells. The bottom panels are an example of a VH4+ rhAb from an RIS patient that binds to SH- Sy5Y cells. FIG.10. ICC on SH-Sy5Y with rhAbs.

[0012] 11 4936-8845-3466, v.1 DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS Radiologically isolated syndrome (RIS) is a neurological condition in people with abnormal and incidentally found demyelinating lesions on brain and / or spinal cord magnetic resonance imaging (MRI) studies. Not all individuals experience a first clinical event leading to a diagnosis of multiple sclerosis (MS). Elucidating the immune profile of people with RIS who will advance to an MS diagnosis is critical to the understanding of disease pathogenesis, particularly since the expected rate of a clinical event at 5 years is 34% and increases to 51% at 10 years with approximately 11% of converters evolving to a primary progressive MS clinical course. The inventor’s lab previously showed that plasmablasts (PBs), the earliest B cell subtype producing antibodies, are expanded in people diagnosed with clinically isolated syndrome (CIS). These PBs are enriched for antibody variable heavy chain family 4 (VH4) gene expression and produce antibodies that target neurons. This disclosure examines PB frequency, immunoglobulin selection, and antibody reactivity in people with RIS compared to those with CIS and healthy controls. Comparing features of B cell dysregulation between RIS and CIS groups may elucidate the underlying initial immune mechanisms associated with central nervous system inflammation, independent of clinical episodes. The inventor compared the PB frequency in the blood using flow cytometry and antibody genetics of bulk-sorted PBs using next generation sequencing of 30 people with RIS and compared them to 20 people with CIS along with 20 healthy controls (HC). They purified bulk IgG from plasma samples of the entire cohort and cloned antibodies from individually sorted PBs from those with RIS and controls. They used the purified IgG from the entire cohort and the cloned antibodies from individually sorted RIS PBs in a flow cytometry assay using a human neuron cell line to evaluate reactivity to neurons. PBs were not expanded in RIS individuals and displayed a low drive towards somatic hypermutation (SHM) accumulation in comparison to CIS individuals. The frequency of PBs using VH4 antibody genes was higher in RIS compared to CIS, but similar to HC. Purified plasma IgG from RIS individuals displayed a higher frequency of reactivity to a human neuron cell line compared to CIS and HC. VH4+ antibodies from RIS PBs displayed a higher frequency of reactivity to a human neuron cell line compared to non-VH4+ antibodies. Features of B cell responses including PB expansion, SHM

[0013] 12 4936-8845-3466, v.1 drive, VH4 antibody gene over-usage, neuron reactivity by purified IgG and individually cloned antibodies from PBs may distinguish RIS from CIS. These and other aspects of the disclosure are described in detail below. I. Radiologically Isolated Syndrome, Clinically Isolated Syndrome and Multiple Sclerosis A. Radiologically Isolated Syndrome (RIS) Radiologically isolated syndrome (RIS) is a clinical situation in which a person has white matter lesions suggestive of multiple sclerosis (MS), as shown on an MRI scan that was done for reasons unrelated to MS symptoms. The nerve lesions in these people show dissemination in space with an otherwise normal neurological examination and without historical accounts of typical MS symptoms. MRI findings that are consistent with multiple sclerosis have been observed in healthy people who underwent MRI scanning, and 50% go on to develop symptomatic MS, sometimes with a primary progressive course. This condition was first characterized in 2009. RIS is generally discovered when an MRI scan is performed for other reasons. The most common symptom that led to the incidental discovery of RIS is headache. Other common reasons are trauma, psychiatric disorders, and endocrinological disorders. The criteria for an RIS diagnosis are as follows: (1) the presence of incidental MRI findings in the CNS white matter, including ovoid and well-circumscribed homogeneous foci, with or without involvement of the corpus callosum, T2 hyperintensities larger than 3 mm in diameter, which fulfill at least 3 of the 4 Barkhof MRI criteria for DIS, and CNS abnormalities not consistent with a vascular condition; (2) no historical accounts of clinical symptoms consistent with neurological dysfunction; (3) MRI anomalies not accounting for apparent impairment in social, occupational, or generalized areas of functioning; (4) MRI anomalies not due to substance abuse, such as recreational drug use, toxic exposure, or a prior known medical condition; (5) exclusion of a differential diagnosis of leukoaraiosis, or extensive white matter pathology excluding the corpus callosum; and (6) MRI anomalies of the CNS not accounted for by another disease. Currently, routine clinical follow-up and MRI neuroimaging surveillance is the standard by which patients are observed. While treatment of MS disease modifying therapies have been given to some individuals with RIS, the majority opt for active

[0014] 13 4936-8845-3466, v.1 surveillance and the appearance of clinical symptoms before commencing treatment, as treatment is considered controversial. In a 5-year study, clinical events, which refers to the first symptoms of exacerbations, clinical attacks, flare ups, or severe symptoms, indicative of MS, appeared in 34% of individuals. Of those who developed symptoms, 9.6% fulfilled criteria for primary progressive multiple sclerosis (PPMS). Due to the incidental nature of RIS, exact figures on prevalence is unknown, though it has been suggested that RIS is the most common type of asymptomatic MS. The prevalence may be higher in relatives of patients with MS. One study, at a university hospital that is located in a high region of MS disease incidence, put the disease prevalence at approximately 1 in 2000. An earlier study in 1961 of 15,644 autopsies found 12 cases (0.08%) of unexpected MS findings without a previous history of MS symptoms. The mean age of first indication of RIS from 451 patients is 37.2 years. Though rare, some children that have had MRI scans for reasons unrelated to MS have shown signs of RIS. The most common reason for an initial MRI in these children was a headache. The first occurrence of a clinical event characteristic of MS in nearly half of the children examined was 2 years, though in a majority of cases, “radiologic evolution,” i.e., the increase in the number of size of lesions as detected in subsequent MRI, developed after one year. The presence of oligoclonal bands in the CSF and spinal cord lesions were associated with an increased risk of a first clinical event characteristic of MS. It was found that children with RIS had a substantial risk of subsequent clinical symptoms and / or radiologic evolution. B. Clinically Isolated Syndrome (CIS) Clinically isolated syndrome (CIS) is a clinical situation of an individual's first neurological episode, caused by inflammation or demyelination of nerve tissue. An episode may be monofocal, in which symptoms present at a single site in the central nervous system, or multifocal, in which multiple sites exhibit symptoms. CIS with enough paraclinical evidence can be considered as a clinical stage of multiple sclerosis (MS). It can also be retrospectively diagnosed as a kind of MS when more evidence is available. Brain lesions associated with a clinically isolated syndrome may be indicative of several neurological diseases, like multiple sclerosis (MS) or neuromyelitis optica. In order for such a diagnosis, multiple sites in the central nervous system must present

[0015] 14 4936-8845-3466, v.1 lesions, typically over multiple episodes, and for which no other diagnosis is likely. A clinically definitive diagnosis of MS is made once an MRI detects lesions in the brain, consistent with those typical of MS. Other diagnostics include cerebrospinal fluid analysis and evoked response testing. Currently it is considered that the best predictor of future development of clinical multiple sclerosis is the number of T2 lesions visualized by magnetic resonance imaging during the CIS and their size. It is normal to evaluate diagnostic criteria against the “time to conversion to definite.” In 2001, the International Panel on the Diagnosis of multiple sclerosis issued the McDonald criteria, a revision of the previous diagnostic procedures to detect MS, known as the Poser criteria. “While maintaining the basic requirements of dissemination in time and space, the McDonald criteria provided specific guidelines for using findings on MRI and cerebrospinal fluid analysis to provide evidence of the second attack in those individuals who have had a single demyelinating episode and thereby confirm the diagnosis more quickly.” Further revisions were issued in 2005. C. Multiple Sclerosis (MS) Multiple sclerosis (MS) is an autoimmune disease in which the insulating covers of nerve cells in the brain and spinal cord are damaged. This damage disrupts the ability of parts of the nervous system to transmit signals, resulting in a range of signs and symptoms, including physical, mental, and sometimes psychiatric problems. Specific symptoms can include double vision, vision loss, eye pain, muscle weakness, and loss of sensation or coordination. MS takes several forms, with new symptoms either occurring in isolated attacks (relapsing forms) or building up over time (progressive forms). In the relapsing forms of MS, between attacks, symptoms may disappear completely, although some permanent neurological problems often remain, especially as the disease advances. In the progressive forms of MS, bodily function slowly deteriorates and disability worsens once symptoms manifest and will steadily continue to do so if the disease is left untreated. While the cause is unclear, the underlying mechanism is thought to be either destruction by the immune system or failure of the myelin-producing cells. Proposed causes for this include immune dysregulation, genetics, and environmental factors, such as viral infections. MS is usually diagnosed based on the presenting signs and symptoms and the results of supporting medical tests.

[0016] 15 4936-8845-3466, v.1 No cure for multiple sclerosis is known. Current treatments are aimed at mitigating inflammation and resulting symptoms from acute flares and prevention of further attacks with disease-modifying medications. Physical therapy and occupational therapy, along with patient-centered symptom management, can help with people's ability to function. The long-term outcome is difficult to predict; better outcomes are more often seen in women, those who develop the disease early in life, those with a relapsing course, and those who initially experienced few attacks. Multiple sclerosis is the most common immune-mediated disorder affecting the central nervous system. Nearly one million people in the United States had MS in 2022, and in 2020, about 2.8 million people were affected globally, with rates varying widely in different regions and among different populations. The disease usually begins between the ages of 20 and 50 and is twice as common in women as in men. MS was first described in 1868 by French neurologist Jean-Martin Charcot. The name “multiple sclerosis” is short for multiple cerebro-spinal sclerosis, which refers to the numerous glial scars (or sclerae – essentially plaques or lesions) that develop on the white matter of the brain and spinal cord. As MS lesions can affect any part of the central nervous system, a person with MS can have almost any neurological symptom or sign referable to the central nervous system. Fatigue is one of the most common symptoms of MS. Some 65% of people with MS experience fatigue symptomatology, and of these some 15–40% report fatigue as their most disabling MS symptom. Autonomic, visual, motor, and sensory problems are also among the most common symptoms. Some 60% or more of MS patients find their symptoms, particularly including fatigue, are affected by changes in their body heat. The specific symptoms are determined by the locations of the lesions within the nervous system, and may include focal loss of sensitivity and / or changes in sensation in the limbs, such as feeling tingling, pins and needles, or numbness; limb motor weakness / pain, blurred vision, pronounced reflexes, muscle spasms, difficulty with ambulation, difficulties with coordination and balance (ataxia); problems with speech or swallowing, visual problems (optic neuritis manifesting as eye pain & vision loss, or nystagmus manifesting as double vision), fatigue, and bladder and bowel difficulties (such as urinary and / or fecal incontinence or retention), among others. When multiple sclerosis is more advanced, walking difficulties can occur and the risk of falling increases.

[0017] 16 4936-8845-3466, v.1 Difficulties thinking and emotional problems such as depression or unstable mood are also common. The primary deficit in cognitive function that people with MS experience is slowed information-processing speed, with memory also commonly affected, and executive function less commonly. Intelligence, language, and semantic memory are usually preserved, and the level of cognitive impairment varies considerably between people with MS. Uhthoff's phenomenon, a worsening of symptoms due to exposure to higher-than- usual temperatures, and Lhermitte's sign, an electrical sensation that runs down the back when bending the neck, are particularly characteristic of MS, although may not always be present. Another presenting manifestation that is rare but highly suggestive of a demyelinating process such as MS is bilateral internuclear ophthalmoplegia, where the patient experiences double vision when attempting to move their gaze to the right & left. MS may have a prodromal phase in the years leading up to MS manifestation, characterized by psychiatric issues, cognitive impairment, and increased use of healthcare. Onset of conditions begins in 85% of cases as a clinically isolated syndrome (CIS) over a number of days with 45% having motor or sensory problems, 20% having optic neuritis, and 10% having symptoms related to brainstem dysfunction, while the remaining 25% have more than one of the previous difficulties. Regarding optic neuritis as the most common presenting symptom, people with MS notice sub-acute loss of vision, often associated with pain worsening on eye movement, and reduced color vision. Early diagnosis of MS associated optic neuritis helps timely initiation of MS targeted treatments; however, for correct choice of treatment, it is crucial to adhere to established diagnostic criteria for optic neuritis due to the broad range of alternative causes, such as neuromyelitis optica spectrum disorder (NMOSD), and other autoimmune or infectious conditions. The course of symptoms occurs in two main patterns initially: either as episodes of sudden worsening that last a few days to months (called relapses, exacerbations, bouts, attacks, or flare-ups) followed by improvement (85% of cases) or as a gradual worsening over time without periods of recovery (10–15% of cases). A combination of these two patterns may also occur or people may start in a relapsing and remitting course that then becomes progressive later on. Relapses are usually not predictable, occurring without warning. Exacerbations rarely occur more frequently than twice per year. Some relapses, however, are preceded by common triggers and they occur more frequently during spring and summer. Similarly,

[0018] 17 4936-8845-3466, v.1 viral infections such as the common cold, influenza, or gastroenteritis increase their risk. Stress may also trigger an attack. Multiple sclerosis is typically diagnosed based on the presenting signs and symptoms, in combination with supporting medical imaging and laboratory testing. It can be difficult to confirm, especially early on, since the signs and symptoms may be similar to those of other medical problems. The McDonald criteria, which focus on clinical, laboratory, and radiologic evidence of lesions at different times and in different areas, is the most commonly used method of diagnosis with the Schumacher and Poser criteria being of mostly historical significance. The McDonald criteria states that patients with multiple sclerosis should have lesions which are disseminated in time (DIT) and disseminated in space (DIS), i.e., lesions which have appeared in different areas in the brain and at different times. Below is an abbreviated outline of the 2017 McDonald Criteria for diagnosis of MS: At least 2 clinical attacks with MRI showing 2 or more lesions characteristic of MS. At least 2 clinical attacks with MRI showing 1 lesion characteristic of MS with clear historical evidence of a previous attack involving a lesion at a distinct location in the CNS. At least 2 clinical attacks with MRI showing 1 lesion characteristic of MS, with DIT established by an additional clinical attack at a distinct CNS site or by MRI showing an old MS lesion. 1 clinical attack with MRI showing at least 2 lesions characteristic of MS, with DIT established by an additional attack, by MRI showing old MS lesion(s), or presence of oligoclonal bands in CSF. 1 clinical attack with MRI showing 1 lesion characteristic of MS, with DIS established by an additional attack at a different CNS site or by MRI showing old MS lesion(s), and DIT established by an additional attack, by MRI showing old MS lesion(s), or presence of oligoclonal bands in CSF. As of 2017, no single test (including biopsy) can provide a definitive diagnosis. Although no cure for multiple sclerosis has been found, several therapies have proven helpful. Several effective treatments can decrease the number of attacks and the rate of progression. The primary aims of therapy are returning function after an attack, preventing new attacks, and preventing disability. Starting medications is generally

[0019] 18 4936-8845-3466, v.1 recommended in people after the first attack when more than two lesions are seen on MRI. The first approved medications used to treat MS were modestly effective, though were poorly tolerated and had many adverse effects. Several treatment options with better safety and tolerability profiles have been introduced, improving the prognosis of MS. As with any medical treatment, medications used in the management of MS have several adverse effects. Alternative treatments are pursued by some people, despite the shortage of supporting evidence of efficacy. During symptomatic attacks, administration of high doses of intravenous corticosteroids, such as methylprednisolone, is the usual therapy, with oral corticosteroids seeming to have a similar efficacy and safety profile. Although effective in the short term for relieving symptoms, corticosteroid treatments do not appear to have a significant impact on long-term recovery. The long-term benefit is unclear in optic neuritis as of 2020. The consequences of severe attacks that do not respond to corticosteroids might be treatable by plasmapheresis. Multiple disease-modifying medications were approved by regulatory agencies for RRMS; they are modestly effective at decreasing the number of attacks. Interferons and glatiramer acetate are first-line treatments and are roughly equivalent, reducing relapses by approximately 30%. Early initiated long-term therapy is safe and improves outcomes. Treatment of CIS with interferons decreases the chance of progressing to clinical MS. Efficacy of interferons and glatiramer acetate in children has been estimated to be roughly equivalent to that of adults. The role of some newer agents such as fingolimod, teriflunomide, and dimethyl fumarate, is not yet entirely clear. Making firm conclusions about the best treatment is difficult, especially regarding the long‐term benefit and safety of early treatment, given the lack of studies directly comparing disease- modifying therapies or long-term monitoring of patient outcomes. The relative effectiveness of different treatments is unclear, as most have only been compared to placebo or a small number of other therapies. Direct comparisons of interferons and glatiramer acetate indicate similar effects or only small differences in effects on relapse rate, disease progression, and MRI measures. There is high confidence that natalizumab, cladribine, or alemtuzumab are decreasing relapses over a period of two years for people with RRMS. Natalizumab and interferon beta-1a (Rebif) may

[0020] 19 4936-8845-3466, v.1 reduce relapses compared to both placebo and interferon beta-1a (Avonex) while Interferon beta-1b (Betaseron), glatiramer acetate, and mitoxantrone may also prevent relapses. Evidence on relative effectiveness in reducing disability progression is unclear. There is moderate confidence that a two-year treatment with natalizumab slows disability progression for people with RRMS. All medications are associated with adverse effects that may influence their risk to benefit profiles. Ublituximab was approved for medical use in the United States in December 2022. In 2011, mitoxantrone was the first medication approved for secondary progressive MS. In this population, tentative evidence supports mitoxantrone moderately slowing the progression of the disease and decreasing rates of relapses over two years. New approved medications continue to emerge in modern medicine. In March 2017, the FDA approved ocrelizumab as a treatment for primary progressive MS in adults, the first drug to gain that approval, with requirements for several Phase IV clinical trials. It is also used for the treatment of relapsing forms of multiple sclerosis, including clinically isolated syndrome, relapsing-remitting disease, and active secondary progressive disease in adults. According to a 2021 Cochrane review, ocrelizumab may reduce worsening of symptoms for primary progressive MS and probably increases unwanted effects but makes little or no difference to the number of serious unwanted effects. In 2019, siponimod and cladribine were approved in the United States for the treatment of secondary progressive multiple sclerosis (SPMS). Subsequently, ozanimod was approved in 2020, and ponesimod was approved in 2021, which were both approved for management of CIS, relapsing MS, and SPMS in the U.S., and RRMS in Europe. The spasticity associated with MS can be difficult to manage because of the progressive and fluctuating course of the disease. Although there is no firm conclusion on the efficacy in reducing spasticity, PT interventions can be a safe and beneficial option for patients with multiple sclerosis. Physical therapy including vibration interventions, electrical stimulation, exercise therapy, standing therapy, and radial shock wave therapy (RSWT), were beneficial for limiting spasticity, helping limit excitability, or increasing range of motion. Over 50% of people with MS may use complementary and alternative medicine, although percentages vary depending on how alternative medicine is defined. Regarding the characteristics of users, they are more frequently women, have had MS for a longer time, tend to be more disabled and have lower levels of satisfaction with conventional healthcare. The evidence for the effectiveness of such treatments in most cases is weak

[0021] 20 4936-8845-3466, v.1 or absent. Treatments of unproven benefit used by people with MS include dietary supplementation and regimens, vitamin D, relaxation techniques such as yoga, herbal medicine (including medical cannabis), hyperbaric oxygen therapy, self-infection with hookworms, reflexology, acupuncture, and mindfulness. Evidence suggests vitamin D supplementation, irrespective of the form and dose, provides no benefit for people with MS; this includes for measures such as relapse recurrence, disability, and MRI lesions while effects on health‐related quality of life and fatigue are unclear. There is insufficient evidence supporting high-dose biotin and some evidence for increased disease activity and higher risk of relapse with its use. A recent review of the effectiveness of cannabis and cannabinoids found that compared with placebo nabiximols probably reduce the severity of spasticity in the short term. II. Antibodies and Production Thereof The present disclosure provides antibodies produced by plasmablasts isolated from RIS subjects. The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. An IgM antibody consists of 5 basic heterotetramer units along with an additional polypeptide called J chain, and therefore contain 10 antigen binding sites, while secreted IgA antibodies can polymerize to form polyvalent assemblages comprising 2-5 of the basic 4-chain units along with J chain. In the case of IgGs, the 4-chain unit is generally about 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has at the N-terminus, a variable region (VH) followed by constant domains (CH). Each L chain has at the N-terminus, a variable region (VL) followed by a constant domain (CL) at its other end. The VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (CH1). Particular amino acid residues form an interface between the light chain and heavy chain variable regions. The pairing of a VH and VLtogether forms a single antigen-binding site. The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda based on the amino acid sequences of their constant domains (CL). Depending on the amino acid sequence of the constant domain of their heavy chains (CH), immunoglobulins can be assigned to different classes or

[0022] 21 4936-8845-3466, v.1 isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated alpha, delta, epsilon, gamma and mu, respectively. The gamma and alpha classes are further divided into subclasses on the basis of relatively minor differences in CHsequence and function, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The term "variable" refers to the fact that certain segments of the V domains differ extensively in sequence among antibodies. The V domain mediates antigen binding and defines specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the 110-amino acid span of the variable regions. Instead, the V regions contain relatively invariant stretches called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability called “hypervariable regions”. The variable regions of native heavy and light chains each comprise four FRs, largely adopting a beta-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions 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, 5th Ed. Public Health Service, 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 cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), antibody-dependent neutrophil phagocytosis (ADNP), and antibody-dependent complement deposition (ADCD). The term "hypervariable region" when used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region generally comprises amino acid residues from a "complementarity determining region" or "CDR" (e.g., around about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the VL, and around about 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the VHwhen numbered in accordance with the Kabat numbering system; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)); and / or those residues from a "hypervariable loop" (e.g., residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the VL, and 26-32 (H1), 52-56 (H2) and 95- 101 (H3) in the VH when numbered in accordance with the Chothia numbering system; Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); and / or those residues from a

[0023] 22 4936-8845-3466, v.1 "hypervariable loop" / CDR (e.g., residues 27-38 (L1), 56-65 (L2) and 105-120 (L3) in the VL, and 27-38 (H1), 56-65 (H2) and 105-120 (H3) in the VHwhen numbered in accordance with the IMGT numbering system; Lefranc, M. P. et al. Nucl. Acids Res. 27:209-212 (1999), Ruiz, M. et al. Nucl. Acids Res.28:219-221 (2000)). Optionally the antibody has symmetrical insertions at one or more of the following points 28, 36 (L1), 63, 74-75 (L2) and 123 (L3) in the VL, and 28, 36 (H1), 63, 74-75 (H2) and 123 (H3) in the VH when numbered in accordance with AHo; Honneger, A. and Plunkthun, A. J. Mol. Biol.309:657-670 (2001)). By "germline nucleic acid residue" is meant the nucleic acid residue that naturally occurs in a germline gene encoding a constant or variable region. "Germline gene" is the DNA found in a germ cell (i.e., a cell destined to become an egg or in the sperm). A "germline mutation" refers to a heritable change in a particular DNA that has occurred in a germ cell or the zygote at the single-cell stage, and when transmitted to offspring, such a mutation is incorporated in every cell of the body. A germline mutation is in contrast to a somatic mutation which is acquired in a single body cell. In some cases, nucleotides in a germline DNA sequence encoding for a variable region are mutated (i.e., a somatic mutation) and replaced with a different nucleotide. The term "monoclonal antibody" as used herein refers to an antibody population with the same antigenic binding site, in contrast to polyclonal antibodies that include different antibodies directed against different determinants (epitopes). In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies useful in the present disclosure may be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells, e.g., after single cell sorting of an antigen specific B cell (e.g., an antigen specific plasmablast) or capture of linked heavy and light chains from single cells in a bulk sorted antigen specific collection. The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), for example. A. General Methods It will be understood that antibodies binding to neurons (including from RIS subjects, and including VH4 antibodies) will have several applications. These include

[0024] 23 4936-8845-3466, v.1 applications as laboratory reagents for evaluating MS pathogenesis, and / or as library candidates for identifying therapeutic targets (e.g., for inhibition) and / or agents for inducing neuroprotection. In these contexts, one may link such antibodies to detectable labels, use them as capture agents or competitors in competitive assays, or use them individually without additional agents being attached thereto. The antibodies may be mutated or modified, as discussed further below. Methods for preparing and characterizing antibodies are well known in the art (see, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; U.S. Patent 4,196,265). Somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells, including plasmablasts), can be selected for and in certain cases used in mAb generating protocol. In embodiments, plasmablasts (CD19+, CD27+, CD38+) are isolated from PBMCs of an RIS subject. Nucleotide and amino acid sequences of antibodies produced by the plasmablasts can be determined by genomic or mRNA sequencing using known techniques. A molecular cloning approach may be used to generate the human monoclonal antibodies. Single B cells can be sorted physically using paramagnetic bead selection or flow cytometric sorting, then RNA can be isolated from the single cells and antibody genes amplified by RT-PCR. Various single-cell RNA-seq methods are available to obtain antibody variable genes from single cells. Alternatively, antigen-specific bulk sorted populations of cells can be segregated into microvesicles and the matched heavy and light chain variable genes recovered from single cells using physical linkage of heavy and light chain amplicons, or common barcoding of heavy and light chain genes from a vesicle. Matched heavy and light chain genes from single cells also can be obtained from populations of antigen specific B cells by treating cells with cell-penetrating nanoparticles bearing RT-PCR primers and barcodes for marking transcripts with one barcode per cell. The antibody variable genes also can be isolated by RNA extraction of a hybridoma line and the antibody genes obtained by RT-PCR and cloned into an immunoglobulin expression vector. B. Antibodies of the Present Disclosure Antibodies according to the present disclosure may be defined, in the first instance, by their binding specificity and target antigen. In embodiments, antibodies disclosed herein bind to targets and / or epitopes specific to neurons, and in various embodiments, surface targets or cytoplasmic targets. In some embodiments, the antibody binds to an epitope described in PCT / US2024 / 036930, which is hereby incorporated by

[0025] 24 4936-8845-3466, v.1 reference in its entirety. In some embodiments, the cytoplasmic target is a heterogeneous nuclear ribonucleoprotein (hnRNP), which in embodiments is present in stress granules. In embodiments, the one or more hnRNP comprises an RNA recognition motif 2 (RRM2). In embodiments, the hnRNP comprises hnRNP A / B or hnRNP A1. In embodiments, the one or more hnRNP comprises hnRNP A2 / B1, hnRNP C1 / C2, hnRNP A1, hnRNP A3, and hnRNP D0. In some embodiments, the epitope to which a given antibody bind may consist of a single contiguous sequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) amino acids located within the antigen molecule (e.g., a linear epitope in a domain). Alternatively, the epitope may consist of a plurality of non-contiguous amino acids (or amino acid sequences) located within the antigen molecule (e.g., a conformational epitope). Various techniques known to persons of ordinary skill in the art can be used to determine whether an antibody “interacts with one or more amino acids” within a polypeptide or protein. Exemplary techniques include, for example, routine cross- blocking assays, such as that described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, N.Y.). Cross-blocking can be measured in various binding assays such as ELISA, biolayer interferometry, or surface plasmon resonance. Other methods include alanine scanning mutational analysis, peptide blot analysis (Reineke, Methods Mol. Biol. 248: 443-63, 2004), peptide cleavage analysis, high- resolution electron microscopy techniques using single particle reconstruction, cryoEM, or tomography, crystallographic studies and NMR analysis. In addition, methods such as epitope excision, epitope extraction and chemical modification of antigens can be employed (Tomer Prot. Sci. 9: 487-496, 2000). Another method that can be used to identify the amino acids within a polypeptide with which an antibody interacts is hydrogen / deuterium exchange detected by mass spectrometry. In general terms, the hydrogen / deuterium exchange method involves deuterium-labeling the protein of interest, followed by binding the antibody to the deuterium-labeled protein. Next, the protein / antibody complex is transferred to water and exchangeable protons within amino acids that are protected by the antibody complex undergo deuterium-to-hydrogen back- exchange at a slower rate than exchangeable protons within amino acids that are not part of the interface. As a result, amino acids that form part of the protein / antibody interface may retain deuterium and therefore exhibit relatively higher mass compared to amino acids not included in the interface. After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry analysis, thereby revealing the

[0026] 25 4936-8845-3466, v.1 deuterium-labeled residues which correspond to the specific amino acids with which the antibody interacts. See, e.g., Ehring, Analytical Biochemistry 267: 252-259 (1999); Engen and Smith, Anal. Chem.73: 256A-265A (2001). The term “epitope” refers to a site on an antigen to which B cells respond. B-cell epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Modification-Assisted Profiling (MAP), also known as Antigen Structure-based Antibody Profiling (ASAP) is a method that categorizes large numbers of monoclonal antibodies (mAbs) directed against the same antigen according to the similarities of the binding profile of each antibody to chemically or enzymatically modified antigen surfaces (see U.S. Patent Publication 2004 / 0101920, herein specifically incorporated by reference in its entirety). Each category may reflect a unique epitope either distinctly different from or partially overlapping with epitope represented by another category. This technology allows rapid filtering of genetically identical antibodies, such that characterization can be focused on genetically distinct antibodies. When applied to hybridoma screening, MAP may facilitate identification of rare hybridoma clones that produce mAbs having the desired characteristics. MAP may be used to sort the antibodies of the disclosure into groups of antibodies binding different epitopes. The present disclosure includes antibodies that may bind to the same epitope, or a portion of the epitope. Likewise, the present disclosure also includes antibodies that compete for binding to a target or a fragment thereof with any of the specific exemplary antibodies described herein. One can easily determine whether an antibody binds to the same epitope as, or competes for binding with, a reference antibody by using routine methods known in the art. For example, to determine if a test antibody binds to the same epitope as a reference, the reference antibody is allowed to bind to target under saturating conditions. Next, the ability of a test antibody to bind to the target molecule is assessed. If the test antibody is able to bind to the target molecule following saturation binding with the reference antibody, it can be concluded that the test antibody binds to a different epitope than the reference antibody. On the other hand, if the test antibody is not able to bind to the target molecule following saturation binding with the reference antibody, then

[0027] 26 4936-8845-3466, v.1 the test antibody may bind to the same epitope as the epitope bound by the reference antibody. To determine if an antibody competes for binding with a reference antibody, the above-described binding methodology is performed in two orientations: In a first orientation, the reference antibody is allowed to bind to the antigen under saturating conditions followed by assessment of binding of the test antibody to the target. In a second orientation, the test antibody is allowed to bind to the target molecule under saturating conditions followed by assessment of binding of the reference antibody to the target molecule. If, in both orientations, only the first (saturating) antibody is capable of binding to target, then it is concluded that the test antibody and the reference antibody compete for binding to target. As will be appreciated by a person of ordinary skill in the art, an antibody that competes for binding with a reference antibody may not necessarily bind to the identical epitope as the reference antibody but may sterically block binding of the reference antibody by binding an overlapping or adjacent epitope. Two antibodies bind to the same or overlapping epitope if each competitively inhibits (blocks) binding of the other to the antigen. That is, a 1-, 5-, 10-, 20- or 100-fold excess of one antibody inhibits binding of the other by at least 50% but preferably 75%, 90% or even 99% as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res.199050:1495-1502). Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Additional routine experimentation (e.g., peptide mutation and binding analyses) can then be carried out to confirm whether the observed lack of binding of the test antibody is in fact due to binding to the same epitope as the reference antibody or if steric blocking (or another phenomenon) is responsible for the lack of observed binding. Experiments of this sort can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry or any other quantitative or qualitative antibody-binding assay available in the art. Structural studies with EM or crystallography also can demonstrate whether or not two antibodies that compete for binding recognize the same epitope. In an aspect, there are provided monoclonal antibodies (e.g., recombinant antibodies) having clone-paired CDRs from the heavy and light chains as illustrated in Tables 3 and 4, respectively. Such recombinant antibodies may be produced by the clones

[0028] 27 4936-8845-3466, v.1 discussed below in the Examples section using methods described herein. In embodiments, the recombinant antibodies are encoded by clone-paired nucleotide sequences shown in Table 1. In embodiments, the recombinant antibodies comprise variable regions comprising clone-paired amino acid sequences shown in Table 2. The antibodies may be defined by their variable sequence, which include additional “framework” regions. Furthermore, the antibody sequences may vary from these sequences, optionally using methods discussed in greater detail below. For example, nucleic acid sequences may vary from those set forth in Table 1, for example, may have at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to nucleotide sequences set forth in Table 1. The nucleic acids may vary from those set forth in Table 1 by virtue of the ability to hybridize under high stringency conditions, as exemplified by low salt and / or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCl at temperatures of about 50°C to about 70°C. In embodiments, the variable region amino acid sequence may vary from those set forth in Table 2, for example, may have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In embodiments, the amino acids may vary from those set out above by permitting conservative substitutions (discussed below). In embodiments, the variable region amino acid sequence has a set of variable heavy CDRs and variable light CDRs shown in Tables 3 and 4. Each of the foregoing applies to the nucleic acid sequences and the amino acid sequences. When comparing polynucleotide and polypeptide sequences, two sequences are said to be "identical" if the sequence of nucleotides or amino acids in the two sequences is the same when aligned for maximum correspondence. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. A "comparison window" as used herein, refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, 40 to about 50, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Optimal alignment of sequences for comparison may be conducted by the local identity algorithm of Smith and Waterman (1981) Add. APL. Math 2:482, by the identity alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity methods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA

[0029] 28 4936-8845-3466, v.1 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by inspection. One particular example of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. BLAST and BLAST 2.0 can be used, for example, with the parameters described herein, to determine percent sequence identity for the polynucleotides and polypeptides of the disclosure. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. The rearranged nature of an antibody sequence and the variable length of each gene requires multiple rounds of BLAST searches for a single antibody sequence. The sequence analysis tool IgBLAST (world-wide-web at ncbi.nlm.nih.gov / igblast / ) identifies matches to the germline V, D and J genes, details at rearrangement junctions, the delineation of Ig V domain framework regions and complementarity determining regions. IgBLAST can analyze nucleotide or protein sequences and can process sequences in batches and allows searches against the germline gene databases and other sequence databases simultaneously to minimize the chance of missing possibly the best matching germline V gene. Yet another way of defining an antibody is as a “derivative” of any of the below- described antibodies and their antigen-binding fragments. The term “derivative” refers to an antibody or antigen-binding fragment thereof that immunospecifically binds to an antigen, but which comprises, one, two, three, four, five or more amino acid substitutions, additions, deletions or modifications relative to a “parental” (or wild-type) molecule. Such amino acid substitutions or additions may introduce naturally occurring (i.e., DNA- encoded) or non-naturally occurring amino acid residues. The term “derivative” encompasses, for example, as variants having altered CH1, hinge, CH2, CH3 or CH4 regions, so as to form, for example, antibodies, etc., having variant Fc regions that exhibit enhanced or impaired effector or binding characteristics. The term “derivative” additionally encompasses non-amino acid modifications, for example, amino acids that may be glycosylated (e.g., have altered mannose, 2-N-acetylglucosamine, galactose, fucose, glucose, sialic acid, 5-N-acetylneuraminic acid, 5-glycolneuraminic acid, etc. content), acetylated, pegylated, phosphorylated, amidated, derivatized by known protecting / blocking groups, proteolytic cleavage, linked to a cellular ligand or other

[0030] 29 4936-8845-3466, v.1 protein, etc. In some embodiments, the altered carbohydrate modifications modulate one or more of the following: solubilization of the antibody, facilitation of subcellular transport and secretion of the antibody, promotion of antibody assembly, conformational integrity, and antibody-mediated effector function. In a specific embodiment, the altered carbohydrate modifications enhance antibody mediated effector function relative to the antibody lacking the carbohydrate modification. Carbohydrate modifications that lead to altered antibody mediated effector function are well known in the art (for example, see Shields, R. L. et al. (2002) “Lack Of Fucose On Human IgG N-Linked Oligosaccharide Improves Binding To Human Fcgamma RIII And Antibody-Dependent Cellular Toxicity,” J. Biol. Chem. 277(30): 26733-26740; Davies J. et al. (2001) “Expression Of GnTIII In A Recombinant Anti-CD20 CHO Production Cell Line: Expression Of Antibodies With Altered Glycoforms Leads To An Increase In ADCC Through Higher Affinity For FC Gamma RIII,” Biotechnology & Bioengineering 74(4): 288-294). Methods of altering carbohydrate contents are known to those skilled in the art, see, e.g., Wallick, S. C. et al. (1988), J. Exp. Med.168(3): 1099-1109; Tao, M. H. et al. (1989), J. Immunol.143(8): 2595-2601; Routledge, E. G. et al. (1995), Transplantation 60(8):847- 53; Elliott, S. et al. (2003), Nature Biotechnol.21:414-21; Shields, R. L. et al. (2002), J. Biol. Chem.277(30): 26733-26740). A derivative antibody or antibody fragment can be generated with an engineered sequence or glycosylation state to confer preferred levels of activity in antibody dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), antibody-dependent neutrophil phagocytosis (ADNP), or antibody-dependent complement deposition (ADCD) functions as measured by bead-based or cell-based assays or in vivo studies in animal models. A derivative antibody or antibody fragment may be modified by chemical modifications using techniques known to those of skill in the art, including, but not limited to, specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, etc. In one embodiment, an antibody derivative will possess a similar or identical function as the parental antibody. In another embodiment, an antibody derivative will exhibit an altered activity relative to the parental antibody. For example, a derivative antibody (or fragment thereof) can bind to its epitope more tightly or be more resistant to proteolysis than the parental antibody.

[0031] 30 4936-8845-3466, v.1 C. Engineering of Antibody Sequences In various embodiments, one may choose to engineer sequences of the identified antibodies for a variety of reasons, such as improved expression, improved cross- reactivity or diminished off-target binding. Modified antibodies may be made by any technique known to those of skill in the art, including expression through standard molecular biological techniques, or the chemical synthesis of polypeptides. Methods for recombinant expression are addressed elsewhere in this document. The following is a general discussion of relevant goals techniques for antibody engineering. Hybridomas may be cultured, then cells lysed, and total RNA extracted. Random hexamers may be used with RT to generate cDNA copies of RNA, and then PCR performed using a multiplex mixture of PCR primers expected to amplify all human variable gene sequences. PCR products can be cloned into pGEM-T Easy vector, then sequenced by automated DNA sequencing using standard vector primers. Assay of binding and neutralization may be performed using antibodies collected from hybridoma supernatants and purified by FPLC, using Protein G columns. Recombinant full-length antibodies can be generated by subcloning heavy and light chain Fv DNAs from the cloning vector into an IgG plasmid vector (for example), transfected into 293 (e.g., Freestyle) cells or CHO cells, and antibodies can be collected and purified from the 293 or CHO cell supernatant. Other appropriate host cells systems include bacteria, such as E. coli, insect cells (S2, Sf9, Sf29, High Five), plant cells (e.g., tobacco, with or without engineering for human-like glycans), algae, or in a variety of non-human transgenic contexts, such as mice, rats, goats or cows. Expression of nucleic acids encoding antibodies for the purpose of subsequent antibody purification is also contemplated. Antibody coding sequences can be RNA, such as native RNA or modified RNA. Modified RNA contemplates certain chemical modifications that confer increased stability and low immunogenicity to mRNAs, thereby facilitating expression of therapeutically important proteins. For instance, N1- methyl-pseudouridine (N1mΨ) outperforms several other nucleoside modifications and their combinations in terms of translation capacity. In addition to turning off the immune / eIF2α phosphorylation-dependent inhibition of translation, incorporated N1mΨ nucleotides dramatically alter the dynamics of the translation process by increasing ribosome pausing and density on the mRNA. Increased ribosome loading of modified mRNAs renders them more permissive for initiation by favoring either ribosome recycling on the same mRNA or de novo ribosome recruitment. Such modifications

[0032] 31 4936-8845-3466, v.1 could be used to enhance antibody expression in vivo following inoculation with RNA. The RNA, whether native or modified, may be delivered as naked RNA or in a delivery vehicle, such as a lipid nanoparticle. Alternatively, DNA encoding the antibody may be employed for the same purposes. The DNA is included in an expression cassette comprising a promoter active in the host cell for which it is designed. The expression cassette is advantageously included in a replicable vector, such as a conventional plasmid or minivector. Vectors include viral vectors, such as poxviruses, adenoviruses, herpesviruses, adeno-associated viruses, and lentiviruses are contemplated. Replicons encoding antibody genes such as alphavirus replicons based on VEE virus or Sindbis virus are also contemplated. Delivery of such vectors can be performed by needle through intramuscular, subcutaneous, or intradermal routes, or by transcutaneous electroporation when in vivo expression is desired. Antibody molecules will comprise fragments (such as F(ab′), F(ab′)2) that are produced, for example, by the proteolytic cleavage of the mAbs, or single-chain immunoglobulins producible, for example, via recombinant means. F(ab′) antibody derivatives are monovalent, while F(ab′)2 antibody derivatives are bivalent. In one embodiment, such fragments can be combined with one another, or with other antibody fragments or receptor ligands to form “chimeric” binding molecules. Significantly, such chimeric molecules may contain substituents capable of binding to different epitopes of the same molecule. In related embodiments, the antibody is a derivative of the disclosed antibodies, e.g., an antibody comprising the CDR sequences identical to those in the disclosed antibodies (e.g., a chimeric, or CDR-grafted antibody). Alternatively, one may wish to make modifications, such as introducing conservative changes into an antibody molecule. In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.

[0033] 32 4936-8845-3466, v.1 It also is understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Patent 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. As detailed in U.S. Patent 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: basic amino acids: arginine (+3.0), lysine (+3.0), and histidine (-0.5); acidic amino acids: aspartate (+3.0 ± 1), glutamate (+3.0 ± 1), asparagine (+0.2), and glutamine (+0.2); hydrophilic, nonionic amino acids: serine (+0.3), asparagine (+0.2), glutamine (+0.2), and threonine (-0.4), sulfur containing amino acids: cysteine (-1.0) and methionine (-1.3); hydrophobic, nonaromatic amino acids: valine (-1.5), leucine (-1.8), isoleucine (-1.8), proline (-0.5 ± 1), alanine (-0.5), and glycine (0); hydrophobic, aromatic amino acids: tryptophan (-3.4), phenylalanine (-2.5), and tyrosine (-2.3). It is understood that an amino acid can be substituted for another having a similar hydrophilicity and produce a biologically or immunologically modified protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ± 2 is preferred, those that are within ± 1 are particularly preferred, and those within ± 0.5 are even more particularly preferred. As outlined above, amino acid substitutions generally are based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take into consideration the various foregoing characteristics are well known to those of skill in the art and include arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine. The present disclosure also contemplates isotype modification. By modifying the Fc region to have a different isotype, different functionalities can be achieved. For example, changing to IgG1 can increase antibody dependent cell cytotoxicity, switching to class A can improve tissue distribution, and switching to class M can improve valency. Alternatively or additionally, it may be useful to combine amino acid modifications with one or more further amino acid modifications that alter C1q binding and / or the complement dependent cytotoxicity (CDC) function of the Fc region of an IL- 23p19 binding molecule. The binding polypeptide of particular interest may be one that binds to C1q and displays complement dependent cytotoxicity. Polypeptides with pre- existing C1q binding activity, optionally further having the ability to mediate CDC may

[0034] 33 4936-8845-3466, v.1 be modified such that one or both of these activities are enhanced. Amino acid modifications that alter C1q and / or modify its complement dependent cytotoxicity function are described, for example, in WO / 0042072, which is hereby incorporated by reference. One can design an Fc region of an antibody with altered effector function, e.g., by modifying C1q binding and / or FcγR binding and thereby changing CDC activity and / or ADCC activity. “Effector functions” are responsible for activating or diminishing a biological activity (e.g., in a subject). Examples of effector functions include, but are not limited to: C1q binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions may require the Fc region to be combined with a binding domain (e.g., an antibody variable domain) and can be assessed using various assays (e.g., Fc binding assays, ADCC assays, CDC assays, etc.). For example, one can generate a variant Fc region of an antibody with improved C1q binding and improved FcγRIII binding (e.g., having both improved ADCC activity and improved CDC activity). Alternatively, if it is desired that effector function be reduced or ablated, a variant Fc region can be engineered with reduced CDC activity and / or reduced ADCC activity. In other embodiments, only one of these activities may be increased, and, optionally, also the other activity reduced (e.g., to generate an Fc region variant with improved ADCC activity, but reduced CDC activity and vice versa). FcRn binding. Fc mutations can also be introduced and engineered to alter their interaction with the neonatal Fc receptor (FcRn) and improve their pharmacokinetic properties. A collection of human Fc variants with improved binding to the FcRn have been described (Shields et al., (2001). High resolution mapping of the binding site on human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn and design of IgG1 variants with improved binding to the FcγR, (J. Biol. Chem. 276:6591-6604). A number of methods are known that can result in increased half-life (Kuo and Aveson, (2011)), including amino acid modifications may be generated through techniques including alanine scanning mutagenesis, random mutagenesis and screening to assess the binding to the neonatal Fc receptor (FcRn) and / or the in vivo behavior. Computational strategies followed by mutagenesis may also be used to select one of amino acid mutations to mutate.

[0035] 34 4936-8845-3466, v.1 The present disclosure therefore provides a variant of an antibody described herein with optimized binding to FcRn. In a particular embodiment, the variant comprises at least one amino acid modification in the Fc region (e.g., of an IgG1 antibody), wherein said modification is selected from the group consisting of 226, 227, 228, 230, 231, 233, 234, 239, 241, 243, 246, 250, 252, 256, 259, 264, 265, 267, 269, 270, 276, 284, 285, 288, 289, 290, 291, 292, 294, 297, 298, 299, 301, 302, 303, 305, 307, 308, 309, 311, 315, 317, 320, 322, 325, 327, 330, 332, 334, 335, 338, 340, 342, 343, 345, 347, 350, 352, 354, 355, 356, 359, 360, 361, 362, 369, 370, 371, 375, 378, 380, 382, 384, 385, 386, 387, 389, 390, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401403, 404, 408, 411, 412, 414, 415, 416, 418, 419, 420, 421, 422, 424, 426, 428, 433, 434, 438, 439, 440, 443, 444, 445, 446 and 447 of the Fc region as compared to said parent polypeptide, wherein the numbering of the amino acids in the Fc region is that of the EU index in Kabat. In a further aspect of the disclosure the modifications are M252Y / S254T / T256E. Derivatized antibodies may be used to alter the half-lives (e.g., serum half-lives) of parental antibodies in a mammal, particularly a human. Such alterations may result in a half-life of greater than 15 days, preferably greater than 20 days, greater than 25 days, greater than 30 days, greater than 35 days, greater than 40 days, greater than 45 days, greater than 2 months, greater than 3 months, greater than 4 months, or greater than 5 months. The increased half-lives of the antibodies of the present disclosure or fragments thereof in a mammal, preferably a human, results in a higher serum titer of said antibodies or antibody fragments in the mammal, and thus reduces the frequency of the administration of said antibodies or antibody fragments and / or reduces the concentration of said antibodies or antibody fragments to be administered. Antibodies or fragments thereof having increased in vivo half-lives can be generated by techniques known to those of skill in the art. For example, antibodies or fragments thereof with increased in vivo half-lives can be generated by modifying (e.g., substituting, deleting or adding) amino acid residues identified as involved in the interaction between the Fc domain and the FcRn receptor. Altered Glycosylation. A particular embodiment of the present disclosure is an isolated monoclonal antibody, or antigen binding fragment thereof, containing a substantially homogeneous glycan without sialic acid, galactose, or fucose. The monoclonal antibody comprises a heavy chain variable region and a light chain variable region, both of which may be attached to heavy chain or light chain constant regions

[0036] 35 4936-8845-3466, v.1 respectively. The aforementioned substantially homogeneous glycan may be covalently attached to the heavy chain constant region. The isolated monoclonal antibody, or antigen binding fragment thereof, may comprise a substantially homogenous composition represented by the GNGN or G1 / G2 glycoform, which exhibits increased binding affinity for Fc gamma RI and Fc gamma RIII compared to the same antibody without the substantially homogeneous GNGN glycoform and with G0, G1F, G2F, GNF, GNGNF or GNGNFX containing glycoforms. In embodiments, the antibody dissociates from Fc gamma RI with a Kd of 1 x 10-8M or less and from Fc gamma RIII with a Kd of 1 x 10-7M or less. Glycosylation of an Fc region is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. O-linked glycosylation refers to the attachment of one of the sugars N- acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used. The recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain peptide sequences are asparagine-X-serine and asparagine-X- threonine, where X is any amino acid except proline. Thus, the presence of either of these peptide sequences in a polypeptide creates a potential glycosylation site. The glycosylation pattern may be altered, for example, by deleting one or more glycosylation site(s) found in the polypeptide, and / or adding one or more glycosylation site(s) that are not present in the polypeptide. Addition of glycosylation sites to the Fc region of an antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N- linked glycosylation sites). An exemplary glycosylation variant has an amino acid substitution of residue Asn 297 of the heavy chain. The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original polypeptide (for O-linked glycosylation sites). Additionally, a change of Asn 297 to Ala can remove one of the glycosylation sites. In certain embodiments, the antibody is expressed in cells that express beta (1,4)- N-acetylglucosaminyltransferase III (GnT III), such that GnT III adds GlcNAc to the IL- 23p19 antibody. Methods for producing antibodies in such a fashion are provided in WO / 9954342, WO / 03011878, patent publication 20030003097A1, and Umana et al., Nature Biotechnology, 17:176-180, February 1999.

[0037] 36 4936-8845-3466, v.1 Elimination of monoclonal antibody protein sequence liabilities. It is possible to engineer the antibody variable gene sequences obtained from human B cells to enhance their manufacturability and safety. Potential protein sequence liabilities can be identified by searching for sequence motifs associated with sites containing: 1) Unpaired Cys residues, 2) N-linked glycosylation, 3) Asn deamidation, 4) Asp isomerization, 5) SYE truncation, 6) Met oxidation, 7) Trp oxidation, 8) N-terminal glutamate, 9) Integrin binding, 10) CD11c / CD18 binding, or 11) Fragmentation Such motifs can be eliminated by altering the synthetic gene for the cDNA encoding recombinant antibodies. Protein engineering efforts in the field of development of therapeutic antibodies clearly reveal that certain sequences or residues are associated with solubility differences (Fernandez-Escamilla et al., Nature Biotech., 22 (10), 1302-1306, 2004; Chennamsetty et al., PNAS, 106 (29), 11937-11942, 2009; Voynov et al., Biocon. Chem., 21 (2), 385- 392, 2010) Evidence from solubility-altering mutations in the literature indicate that some hydrophilic residues such as aspartic acid, glutamic acid, and serine contribute significantly more favorably to protein solubility than other hydrophilic residues, such as asparagine, glutamine, threonine, lysine, and arginine. Stability. Antibodies can be engineered for enhanced biophysical properties. One can use elevated temperature to unfold antibodies to determine relative stability, using average apparent melting temperatures. Differential Scanning Calorimetry (DSC) measures the heat capacity, Cp, of a molecule (the heat required to warm it, per degree) as a function of temperature. One can use DSC to study the thermal stability of antibodies. DSC data for mAbs is particularly interesting because it sometimes resolves the unfolding of individual domains within the mAb structure, producing up to three peaks in the thermogram (from unfolding of the Fab, CH2, and CH3 domains). Typically unfolding of the Fab domain produces the strongest peak. The DSC profiles and relative

[0038] 37 4936-8845-3466, v.1 stability of the Fc portion show characteristic differences for the human IgG1, IgG2, IgG3, and IgG4subclasses (Garber and Demarest, Biochem. Biophys. Res. Commun.355, 751- 757, 2007). One also can determine average apparent melting temperature using circular dichroism (CD), performed with a CD spectrometer. Far-UV CD spectra will be measured for antibodies in the range of 200 to 260 nm at increments of 0.5 nm. The final spectra can be determined as averages of 20 accumulations. Residue ellipticity values can be calculated after background subtraction. Thermal unfolding of antibodies (0.1 mg / mL) can be monitored at 235 nm from 25-95 °C and a heating rate of 1 °C / min. One can use dynamic light scattering (DLS) to assess the propensity for aggregation. DLS is used to characterize the size of various particles including proteins. If the system is not dispersed in size, the mean effective diameter of the particles can be determined. This measurement depends on the size of the particle core, the size of surface structures, and particle concentration. Since DLS essentially measures fluctuations in scattered light intensity due to particles, the diffusion coefficient of the particles can be determined. DLS software in commercial DLA instruments displays the particle population at different diameters. Stability studies can be done conveniently using DLS. DLS measurements of a sample can show whether the particles aggregate over time or with temperature variation by determining whether the hydrodynamic radius of the particle increases. If particles aggregate, one can see a larger population of particles with a larger radius. Stability depending on temperature can be analyzed by controlling the temperature in situ. Capillary electrophoresis (CE) techniques include proven methodologies for determining features of antibody stability. One can use an iCE approach to resolve antibody protein charge variants due to deamidation, C-terminal lysines, sialylation, oxidation, glycosylation, and any other change to the protein that can result in a change in pI of the protein. Each of the expressed antibody proteins can be evaluated by high throughput, free solution isoelectric focusing (IEF) in a capillary column (cIEF), using a Protein Simple Maurice instrument. Whole-column UV absorption detection can be performed every 30 seconds for real time monitoring of molecules focusing at the isoelectric points (pIs). This approach combines the high resolution of traditional gel IEF with the advantages of quantitation and automation found in column-based separations while eliminating the need for a mobilization step. The technique yields reproducible, quantitative analysis of identity, purity, and heterogeneity profiles for the expressed antibodies. The results identify charge

[0039] 38 4936-8845-3466, v.1 heterogeneity and molecular sizing on the antibodies, with both absorbance and native fluorescence detection modes and with sensitivity of detection down to 0.7 µg / mL. Solubility. One can determine the intrinsic solubility score of antibody sequences. The intrinsic solubility scores can be calculated using CamSol Intrinsic (Sormanni et al., J Mol Biol 427, 478-490, 2015). The amino acid sequences for residues 95-102 (Kabat numbering) in HCDR3 of each antibody fragment such as a scFv can be evaluated via the online program to calculate the solubility scores. One also can determine solubility using laboratory techniques. Various techniques exist, including addition of lyophilized protein to a solution until the solution becomes saturated and the solubility limit is reached, or concentration by ultrafiltration in a microconcentrator with a suitable molecular weight cut-off. The most straightforward method is induction of amorphous precipitation, which measures protein solubility using a method involving protein precipitation using ammonium sulfate (Trevino et al., J Mol Biol, 366: 449-460, 2007). Ammonium sulfate precipitation gives quick and accurate information on relative solubility values. Ammonium sulfate precipitation produces precipitated solutions with well-defined aqueous and solid phases and requires relatively small amounts of protein. Solubility measurements performed using induction of amorphous precipitation by ammonium sulfate also can be done easily at different pH values. Protein solubility is highly pH dependent, and pH is considered the most important extrinsic factor that affects solubility. D. Single Chain Antibodies A single chain variable fragment (scFv) is a fusion of the variable regions of the heavy and light chains of immunoglobulins, linked together with a short (usually serine, glycine) linker. This chimeric molecule retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of a linker peptide. This modification usually leaves the specificity unaltered. These molecules were created historically to facilitate phage display where it is highly convenient to express the antigen binding domain as a single peptide. Alternatively, scFv can be created directly from subcloned heavy and light chains derived from a hybridoma or B cell. Single chain variable fragments lack the constant Fc region found in complete antibody molecules, and thus, the common binding sites (e.g., protein A / G) used to purify antibodies. These fragments can often be purified / immobilized using Protein L since Protein L interacts with the variable region of kappa light chains.

[0040] 39 4936-8845-3466, v.1 Flexible linkers generally are comprised of helix- and turn-promoting amino acid residues such as alanine, serine, and glycine. However, other residues can function as well. Tang et al. (1996) used phage display as a means of rapidly selecting tailored linkers for single-chain antibodies (scFvs) from protein linker libraries. A random linker library was constructed in which the genes for the heavy and light chain variable domains were linked by a segment encoding an 18-amino acid polypeptide of variable composition. The scFv repertoire (approx. 5 × 106different members) was displayed on filamentous phage and subjected to affinity selection with hapten. The population of selected variants exhibited significant increases in binding activity but retained considerable sequence diversity. Screening 1,054 individual variants subsequently yielded a catalytically active scFv that was produced efficiently in soluble form. Sequence analysis revealed a conserved proline in the linker two residues after the VH C terminus and an abundance of arginine and proline residues at other positions as the only common features of the selected tethers. The recombinant antibodies of the present disclosure may also involve sequences or moieties that permit dimerization or multimerization of the receptors. Such sequences include those derived from IgA, which permit formation of multimers in conjunction with the J-chain. Another multimerization domain is the Gal4 dimerization domain. In other embodiments, the chains may be modified with agents such as biotin / avidin, which permit the combination of two antibodies. In a separate embodiment, a single-chain antibody can be created by joining receptor light and heavy chains using a non-peptide linker or chemical unit. Generally, the light and heavy chains will be produced in distinct cells, purified, and subsequently linked together in an appropriate fashion (i.e., the N-terminus of the heavy chain being attached to the C-terminus of the light chain via an appropriate chemical bridge). Cross-linking reagents are used to form molecular bridges that tie functional groups of two different molecules, e.g., a stabilizing and coagulating agent. However, it is contemplated that dimers or multimers of the same analog or heteromeric complexes comprised of different analogs can be created. To link two different compounds in a stepwise manner, hetero-bifunctional cross-linkers can be used that eliminate unwanted homopolymer formation. An exemplary hetero-bifunctional cross-linker contains two reactive groups: one reacting with primary amine group (e.g., N-hydroxy succinimide) and the other reacting with a thiol group (e.g., pyridyl disulfide, maleimides, halogens, etc.). Through the

[0041] 40 4936-8845-3466, v.1 primary amine reactive group, the cross-linker may react with the lysine residue(s) of one protein (e.g., the selected antibody or fragment) and through the thiol reactive group, the cross-linker, already tied up to the first protein, reacts with the cysteine residue (free sulfhydryl group) of the other protein (e.g., the selective agent). It is preferred that a cross-linker having reasonable stability in blood will be employed. Numerous types of disulfide bond-containing linkers are known that can be successfully employed to conjugate targeting and therapeutic / preventative agents. Linkers that contain a disulfide bond that is sterically hindered may prove to give greater stability in vivo, preventing release of the targeting peptide prior to reaching the site of action. These linkers are thus one group of linking agents. Another cross-linking reagent is SMPT, which is a bifunctional cross-linker containing a disulfide bond that is “sterically hindered” by an adjacent benzene ring and methyl groups. It is believed that steric hindrance of the disulfide bond serves a function of protecting the bond from attack by thiolate anions such as glutathione which can be present in tissues and blood, and thereby help in preventing decoupling of the conjugate prior to the delivery of the attached agent to the target site. The SMPT cross-linking reagent, as with many other known cross-linking reagents, lends the ability to cross-link functional groups such as the SH of cysteine or primary amines (e.g., the epsilon amino group of lysine). Another possible type of cross- linker includes the hetero-bifunctional photoreactive phenylazides containing a cleavable disulfide bond such as sulfosuccinimidyl-2-(p-azido salicylamido) ethyl-1,3′- dithiopropionate. The N-hydroxy-succinimidyl group reacts with primary amino groups and the phenylazide (upon photolysis) reacts non-selectively with any amino acid residue. In addition to hindered cross-linkers, non-hindered linkers also can be employed in accordance herewith. Other useful cross-linkers, not considered to contain or generate a protected disulfide, include SATA, SPDP and 2-iminothiolane (Wawrzynczak & Thorpe, 1987). The use of such cross-linkers is well understood in the art. Another embodiment involves the use of flexible linkers. U.S. Patent 4,680,338 describes bifunctional linkers useful for producing conjugates of ligands with amine-containing polymers and / or proteins, especially for forming antibody conjugates with chelators, drugs, enzymes, detectable labels and the like. U.S. Patents 5,141,648 and 5,563,250 disclose cleavable conjugates containing a labile bond that is cleavable under a variety of mild conditions. This linker is particularly

[0042] 41 4936-8845-3466, v.1 useful in that the agent of interest may be bonded directly to the linker, with cleavage resulting in release of the active agent. Particular uses include adding a free amino or free sulfhydryl group to a protein, such as an antibody, or a drug. U.S. Patent 5,856,456 provides peptide linkers for use in connecting polypeptide constituents to make fusion proteins, e.g., single chain antibodies. The linker is up to about 50 amino acids in length, contains at least one occurrence of a charged amino acid (preferably arginine or lysine) followed by a proline, and is characterized by greater stability and reduced aggregation. U.S. Patent 5,880,270 discloses aminooxy-containing linkers useful in a variety of immunodiagnostic and separative techniques. E. Multispecific Antibodies In certain embodiments, antibodies of the present disclosure are used to prepare bispecific or multispecific antibodies. Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind two different epitopes of a single antigen. Other such antibodies may combine a first antigen binding site with a binding site for a second antigen. Bispecific antibodies may also be used to localize cytotoxic agents to target cells. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab′)2bispecific antibodies). Methods for making bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et al., Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93 / 08829, and in Traunecker et al., EMBO J., 10:3655-3659 (1991). According to a different approach, antibody variable regions with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. Preferably, the fusion is with an Ig heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light chain

[0043] 42 4936-8845-3466, v.1 bonding, present in at least one of the fusions. DNA encoding the immunoglobulin heavy chain fusions and, if desired, DNA encoding the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host cell. This provides for greater flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yield of the desired bispecific antibody. It is, however, possible to insert the coding sequences for two or all three polypeptide chains into a single expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios have no significant effect on the yield of the desired chain combination. In a particular embodiment of this approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in WO 94 / 04690. According to another approach described in U.S. Patent 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers that are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers. Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science, 229: 81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')2fragments. These fragments are reduced in the presence of the dithiol complexing agent, sodium arsenite, to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab'

[0044] 43 4936-8845-3466, v.1 fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes. Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described (Merchant et al., Nat. Biotechnol.16, 677–681 (1998). doi:10.1038 / nbt0798-677pmid:9661204). For example, bispecific antibodies have been produced using leucine zippers (Kostelny et al., J. Immunol., 148(5):1547-1553, 1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re- oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a VH connected to a VLby a linker that is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VLand VHdomains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol., 152:5368 (1994). In a particular embodiment, a bispecific or multispecific antibody may be formed as a DOCK-AND-LOCK™ (DNL™) complex (see, e.g., U.S. Patents 7,521,056; 7,527,787; 7,534,866; 7,550,143 and 7,666,400, the Examples section of each of which is incorporated herein by reference.) Generally, the technique takes advantage of the specific and high-affinity binding interactions that occur between a dimerization and docking domain (DDD) sequence of the regulatory (R) subunits of cAMP-dependent protein kinase (PKA) and an anchor domain (AD) sequence derived from any of a variety of AKAP proteins (Baillie et al., FEBS Letters. 2005; 579: 3264; Wong and Scott, Nat. Rev. Mol. Cell Biol. 2004; 5: 959). The DDD and AD peptides may be attached to any protein, peptide, or other molecule. Because the DDD sequences spontaneously dimerize

[0045] 44 4936-8845-3466, v.1 and bind to the AD sequence, the technique allows the formation of complexes between any selected molecules that may be attached to DDD or AD sequences. Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared (Tutt et al., J. Immunol. 147: 60, 1991; Xu et al., Science, 358(6359):85-90, 2017). A multivalent antibody may be internalized (and / or catabolized) faster than a bivalent antibody by a cell expressing an antigen to which the antibodies bind. The antibodies of the present disclosure can be multivalent antibodies with three or more antigen binding sites (e.g., tetravalent antibodies), which can be readily produced by recombinant expression of nucleic acid encoding the polypeptide chains of the antibody. The multivalent antibody can comprise a dimerization domain and three or more antigen binding sites. The preferred dimerization domain comprises (or consists of) an Fc region or a hinge region. In this scenario, the antibody will comprise an Fc region and three or more antigen binding sites amino-terminal to the Fc region. The preferred multivalent antibody herein comprises (or consists of) three to about eight, but preferably four, antigen binding sites. The multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain(s) comprise two or more variable regions. For instance, the polypeptide chain(s) may comprise VD1-(X1)n-VD2-(X2)n-Fc, wherein VD1 is a first variable region, VD2 is a second variable region, Fc is one polypeptide chain of an Fc region, X1 and X2 represent an amino acid or polypeptide, and n is 0 or 1. For instance, the polypeptide chain(s) may comprise: VH-CH1-flexible linker-VH-CH1-Fc region chain; or VH-CH1- VH-CH1-Fc region chain. The multivalent antibody herein preferably further comprises at least two (and preferably four) light chain variable region polypeptides. The multivalent antibody herein may, for instance, comprise from about two to about eight light chain variable region polypeptides. The light chain variable region polypeptides contemplated here comprise a light chain variable region and, optionally, further comprise a CL domain. Charge modifications are particularly useful in the context of a multi-specific antibody, where amino acid substitutions in Fab molecules result in reducing the mispairing of light chains with non-matching heavy chains (Bence-Jones-type side products), which can occur in the production of Fab-based bi- / multi-specific antigen binding molecules with a VH / VL exchange in one (or more, in case of molecules comprising more than two antigen-binding Fab molecules) of their binding arms (see

[0046] 45 4936-8845-3466, v.1 also PCT publication no. WO 2015 / 150447, particularly the examples therein, incorporated herein by reference in its entirety). F. Purification In certain embodiments, the antibodies of the present disclosure may be purified. The term “purified,” as used herein, is intended to refer to a composition, isolatable from other components, wherein the protein is purified to any degree relative to its naturally obtainable state. A purified protein therefore also refers to a protein, free from the environment in which it may naturally occur. Where the term “substantially purified” is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the proteins in the composition. Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the cellular milieu to polypeptide and non-polypeptide fractions. Having separated the polypeptide from other proteins, the polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing. Other methods for protein purification include precipitation with ammonium sulfate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; gel filtration, reverse phase, hydroxylapatite and affinity chromatography; and combinations of such and other techniques. In purifying an antibody, it may be desirable to express the polypeptide in a prokaryotic or eukaryotic expression system and extract the protein using denaturing conditions. The polypeptide may be purified from other cellular components using an affinity column, which binds to a tagged portion of the polypeptide. As is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified protein or peptide. Commonly, complete antibodies are fractionated utilizing agents (i.e., protein A) that bind the Fc portion of the antibody. Alternatively, antigens may be used to simultaneously purify and select appropriate antibodies. Such methods often utilize the selection agent bound to a support, such as a column, filter, or bead. The antibodies are

[0047] 46 4936-8845-3466, v.1 bound to a support, contaminants removed (e.g., washed away), and the antibodies released by applying conditions (salt, heat, etc.). Various methods for quantifying the degree of purification of the protein or peptide will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptides within a fraction by SDS / PAGE analysis. Another method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity. The actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the expressed protein or peptide exhibits a detectable activity. It is known that the migration of a polypeptide can vary, sometimes significantly, with different conditions of SDS / PAGE (Capaldi et al., 1977). It will therefore be appreciated that under differing electrophoresis conditions, the apparent molecular weights of purified or partially purified expression products may vary. III. Antibody Conjugates Antibodies of the present disclosure may be linked to at least one agent to form an antibody conjugate. For example, it is conventional to link or covalently bind or complex at least one desired molecule or moiety. Such a molecule or moiety may be, but is not limited to, at least one effector or reporter molecule. Effector molecules comprise molecules having a desired activity, e.g., cytotoxic activity. Non-limiting examples of effector molecules which have been attached to antibodies include toxins, anti-tumor agents, therapeutic enzymes, radionuclides, antiviral agents, chelating agents, cytokines, growth factors, and oligo- or polynucleotides. By contrast, a reporter molecule is defined as any moiety which may be detected using an assay. Non-limiting examples of reporter molecules which have been conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, photoaffinity molecules, colored particles or ligands, such as biotin. Antibody conjugates can be useful as diagnostic agents or laboratory tools. Antibody diagnostics generally fall within two classes, those for use in in vitro diagnostics, such as in a variety of immunoassays, and those for use in vivo diagnostic protocols, generally known as "antibody-directed imaging." Many appropriate imaging agents are known in the art, as are methods for their attachment to antibodies (see, for

[0048] 47 4936-8845-3466, v.1 e.g., U.S. Patents 5,021,236, 4,938,948, and 4,472,509). The imaging moieties used can be paramagnetic ions, radioactive isotopes, fluorochromes, NMR-detectable substances, and X-ray imaging agents. In the case of paramagnetic ions, one might mention by way of example ions such as chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and / or erbium (III), with gadolinium being particularly preferred. Ions useful in other contexts, such as X-ray imaging, include but are not limited to lanthanum (III), gold (III), lead (II), and especially bismuth (III). In the case of radioactive isotopes for therapeutic and / or diagnostic application, one might mention astatine211,14carbon,51chromium,36chlorine,57cobalt,58cobalt, copper67,152Eu, gallium67,3hydrogen, iodine123, iodine125, iodine131, indium111,59iron,32phosphorus, rhenium186, rhenium188,75selenium,35sulphur, technicium99mand / or yttrium90.125I is often preferred for use in certain embodiments, and technicium99mand / or indium111are also often preferred due to their low energy and suitability for long range detection. Radioactively labeled monoclonal antibodies of the present disclosure may be produced according to well-known methods in the art. For instance, monoclonal antibodies can be iodinated by contact with sodium and / or potassium iodide and a chemical oxidizing agent such as sodium hypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase. Monoclonal antibodies according to the disclosure may be labeled with technetium99mby ligand exchange process, for example, by reducing pertechnate with stannous solution, chelating the reduced technetium onto a Sephadex column and applying the antibody to this column. Alternatively, direct labeling techniques may be used, e.g., by incubating pertechnate, a reducing agent such as SNCl2, a buffer solution such as sodium-potassium phthalate solution, and the antibody. Intermediary functional groups which are often used to bind radioisotopes which exist as metallic ions to antibody are diethylenetriaminepentaacetic acid (DTPA) or ethylene diaminetetracetic acid (EDTA). Among the fluorescent labels contemplated for use as conjugates include Alexa 350, Alexa 430, AMCA, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY-FL, BODIPY- R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and / or Texas Red.

[0049] 48 4936-8845-3466, v.1 Additional types of antibodies contemplated in the present disclosure are those intended primarily for use in vitro, where the antibody is linked to a secondary binding ligand and / or to an enzyme (an enzyme tag) that will generate a colored product upon contact with a chromogenic substrate. Examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase or glucose oxidase. Preferred secondary binding ligands are biotin and avidin and streptavidin compounds. The use of such labels is well known to those of skill in the art and are described, for example, in U.S. Patents 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149 and 4,366,241. Yet another known method of site-specific attachment of molecules to antibodies comprises the reaction of antibodies with hapten-based affinity labels. Essentially, hapten-based affinity labels react with amino acids in the antigen binding site, thereby destroying this site and blocking specific antigen reaction. However, this may not be advantageous since it results in loss of antigen binding by the antibody conjugate. Molecules containing azido groups may also be used to form covalent bonds to proteins through reactive nitrene intermediates that are generated by low intensity ultraviolet light (Potter and Haley, 1983). In particular, 2- and 8-azido analogues of purine nucleotides have been used as site-directed photoprobes to identify nucleotide binding proteins in crude cell extracts (Owens & Haley, 1987; Atherton et al., 1985). The 2- and 8-azido nucleotides have also been used to map nucleotide binding domains of purified proteins (Khatoon et al., 1989; King et al., 1989; Dholakia et al., 1989) and may be used as antibody binding agents. Several methods are known in the art for the attachment or conjugation of an antibody to its conjugate moiety. Some attachment methods involve the use of a metal chelate complex employing, for example, an organic chelating agent such a diethylenetriaminepentaacetic acid anhydride (DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and / or tetrachloro-3α-6α-diphenylglycouril-3 attached to the antibody (U.S. Patents 4,472,509 and 4,938,948). Monoclonal antibodies may also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with an isothiocyanate. In U.S. Patent 4,938,948, imaging of breast tumors is achieved using monoclonal antibodies and the detectable imaging

[0050] 49 4936-8845-3466, v.1 moieties are bound to the antibody using linkers such as methyl-p-hydroxybenzimidate or N-succinimidyl-3-(4-hydroxyphenyl)propionate. In other embodiments, derivatization of immunoglobulins by selectively introducing sulfhydryl groups in the Fc region of an immunoglobulin, using reaction conditions that do not alter the antibody combining site are contemplated. Antibody conjugates produced according to this methodology are disclosed to exhibit improved longevity, specificity, and sensitivity (U.S. Patent 5,196,066, incorporated herein by reference). Site-specific attachment of effector or reporter molecules, wherein the reporter or effector molecule is conjugated to a carbohydrate residue in the Fc region have also been disclosed in the literature (O’Shannessy et al., 1987). This approach has been reported to produce diagnostically and therapeutically promising antibodies which are currently in clinical evaluation. IV. Immunodetection Methods In still further embodiments, the present disclosure concerns immunodetection methods for binding, detecting, purifying, quantifying and otherwise generally detecting antigens. The methods may also be used to screen various antibodies for appropriate / desired reactivity profiles. A wide variety of assay formats are contemplated, but specifically those that would be used to detect neuron binding. Some immunodetection methods include enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay, bioluminescent assay, and Western blot to mention a few. In particular, a competitive assay for the detection of neuron binding antibodies is provided. The steps of various useful immunodetection methods have been described in the scientific literature, such as, e.g., Doolittle and Ben-Zeev (1999), Gulbis and Galand (1993), De Jager et al. (1993), and Nakamura et al. (1987). In general, the immunobinding methods include contacting a neuron with a first antibody in accordance with the present disclosure, as the case may be, under conditions effective to allow the formation of immunocomplexes. The immunobinding methods also include methods for detecting and quantifying the amount of neuronal binding. Here, one would provide a neuron or neuronal antibody and contact this target with an antibody or components thereof, followed by detecting and quantifying the amount of immune complexes formed under the specific conditions. Contacting the target with the antibody under effective conditions and for a period

[0051] 50 4936-8845-3466, v.1 sufficient to allow the formation of immune complexes (primary immune complexes) is generally a matter of simply adding the antibody composition to the target and incubating the mixture for a period of time long enough for the antibodies to form immune complexes with, i.e., to bind to the neuron or neuronal antigen. After this time, the bound complex, such as a tissue section, ELISA plate, dot blot or Western blot, will generally be washed to remove any non-specifically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected. In general, the detection of immunocomplex formation is well known in the art and may be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or marker, such as any of those radioactive, fluorescent, biological and enzymatic tags. Patents concerning the use of such labels include U.S. Patents 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149 and 4,366,241. Of course, one may find additional advantages using a secondary binding ligand such as a second antibody and / or a biotin / avidin ligand binding arrangement, as is known in the art. The antibody employed in the detection may itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of the primary immune complexes in the composition to be determined. Alternatively, the first antibody that becomes bound within the primary immune complexes may be detected by means of a second binding ligand that has binding affinity for the antibody. In these cases, the second binding ligand may be linked to a detectable label. The second binding ligand is itself often an antibody, which may thus be termed a “secondary” antibody. The primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under effective conditions and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes are then generally washed to remove any non-specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected. Further methods include the detection of primary immune complexes by a two- step approach. A second binding ligand, such as an antibody that has binding affinity for the antibody, is used to form secondary immune complexes, as described above. After washing, the secondary immune complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under effective conditions and for a period sufficient to allow the formation of immune complexes (tertiary immune complexes). The third ligand or antibody is linked to a detectable label,

[0052] 51 4936-8845-3466, v.1 allowing detection of the tertiary immune complexes thus formed. This system may provide signal amplification if this is desired. One method of immunodetection uses two different antibodies. A first biotinylated antibody is used to detect the target antigen, and a second antibody is then used to detect the biotin attached to the complexed biotin. In that method, the sample to be tested is first incubated in a solution containing the first step antibody. If the target antigen is present, some of the antibody binds to the antigen to form a biotinylated antibody / antigen complex. The antibody / antigen complex is then amplified by incubation in successive solutions of streptavidin (or avidin), biotinylated DNA, and / or complementary biotinylated DNA, with each step adding additional biotin sites to the antibody / antigen complex. The amplification steps are repeated until a suitable level of amplification is achieved, at which point the sample is incubated in a solution containing the second step antibody against biotin. This second step antibody is labeled, for example, with an enzyme that can be used to detect the presence of the antibody / antigen complex by histoenzymology using a chromogen substrate. With suitable amplification, a conjugate can be produced which is macroscopically visible. Another known method of immunodetection takes advantage of the immuno- PCR (Polymerase Chain Reaction) methodology. The PCR method is like the Cantor method up to the incubation with biotinylated DNA, however, instead of using multiple rounds of streptavidin and biotinylated DNA incubation, the DNA / biotin / streptavidin / antibody complex is washed out with a low pH or high salt buffer that releases the antibody. The resulting wash solution is then used to carry out a PCR reaction with suitable primers with appropriate controls. At least in theory, the enormous amplification capability and specificity of PCR can be utilized to detect a single antigen molecule. A. ELISAs and Western Blots Immunoassays, in their most simple and direct sense, are binding assays. Certain preferred immunoassays are the various types of enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and other approaches may also be used.

[0053] 52 4936-8845-3466, v.1 In one exemplary ELISA, the antibodies of the disclosure are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition containing a neuron or neuronal antigen is added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound antigen may be detected. Detection may be achieved by the addition of another anti-neuron antibody that is linked to a detectable label. This type of ELISA is a simple “sandwich ELISA.” In another exemplary ELISA, the neuron or neuronal antigen can be immobilized onto the well surface and then contacted with the anti-neuron antibodies of the disclosure. After binding and washing to remove non-specifically bound immune complexes, the bound anti-neuron antibodies are detected. Where the initial anti-neuron antibodies are linked to a detectable label, the immune complexes may be detected directly. Again, the immune complexes may be detected using a second antibody that has binding affinity for the first anti-neuron antibody, with the second antibody being linked to a detectable label. Irrespective of the format employed, ELISAs have certain features in common, such as coating, incubating and binding, washing to remove non-specifically bound species, and detecting the bound immune complexes. These are described below. In coating a plate with either antigen or antibody, one will generally incubate the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period of hours. The wells of the plate will then be washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then “coated” with a nonspecific protein that is antigenically neutral with regard to the test antisera. These include bovine serum albumin (BSA), casein or solutions of milk powder. The coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface. In ELISAs, it is probably more customary to use a secondary or tertiary detection means rather than a direct procedure. Thus, after binding of a protein or antibody to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the biological sample to be tested under conditions effective to allow immune complex (antigen / antibody) formation. Detection of the immune complex then requires a labeled secondary binding ligand or antibody, and a secondary binding ligand or antibody in conjunction with a labeled tertiary antibody or a third binding ligand.

[0054] 53 4936-8845-3466, v.1 “Under conditions effective to allow immune complex (antigen / antibody) formation” means that the conditions preferably include diluting the antigens and / or antibodies with solutions such as BSA, bovine gamma globulin (BGG) or phosphate buffered saline (PBS) / Tween. These added agents also tend to assist in the reduction of nonspecific background. The “suitable” conditions also mean that the incubation is at a temperature or for a period of time sufficient to allow effective binding. Incubation steps are typically from about 1 to 2 to 4 hours or so, at temperatures preferably on the order of 25°C to 27°C or may be overnight at about 4°C or so. Following all incubation steps in an ELISA, the contacted surface is washed so as to remove non-complexed material. A preferred washing procedure includes washing with a solution such as PBS / Tween, or borate buffer. Following the formation of specific immune complexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minute amounts of immune complexes may be determined. To provide a detecting means, the second or third antibody will have an associated label to allow detection. Preferably, this will be an enzyme that will generate color development upon incubating with an appropriate chromogenic substrate. Thus, for example, one will desire to contact or incubate the first and second immune complex with a urease, glucose oxidase, alkaline phosphatase, or hydrogen peroxidase-conjugated antibody for a period of time and under conditions that favor the development of further immune complex formation (e.g., incubation for 2 hours at room temperature in a PBS- containing solution such as PBS-Tween). After incubation with the labeled antibody, and subsequent to washing to remove unbound material, the amount of label is quantified, e.g., by incubation with a chromogenic substrate such as urea, or bromocresol purple, or 2,2'-azino-di-(3-ethyl- benzthiazoline-6-sulfonic acid (ABTS), or H2O2, in the case of peroxidase as the enzyme label. Quantification is then achieved by measuring the degree of color generated, e.g., using a visible spectra spectrophotometer. In another embodiment, the present disclosure contemplates the use of competitive formats. This is particularly useful in the detection of human anti-neuron antibodies in a sample. In competition-based assays, an unknown amount of antibody is determined by its ability to displace a known amount of labeled antibody. Thus, the

[0055] 54 4936-8845-3466, v.1 quantifiable loss of a signal is an indication of the amount of unknown antibody or analyte in a sample. Here, the inventor proposes the use of labeled anti-neuron antibodies to determine the amount of human anti-neuron antibodies in a sample. The basic format would include contacting a known amount of labeled antibody with a sample containing human antibodies. After binding of the labeled antibody to a support having a neuron or neuronal antigen affixed thereto, the sample is added and incubated under conditions permitting any unlabeled antibody in the sample to compete with, and hence displace, the labeled monoclonal antibody. By measuring either the lost label or the label remaining (and subtracting that from the original amount of bound label), one can determine how much non-labeled antibody is bound to the support, and thus how much antibody was present in the sample. The Western blot (alternatively, protein immunoblot) is an analytical technique used to detect specific proteins in each sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native / non- denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein. Samples may be taken from whole tissue or from cell culture. In most cases, solid tissues are first broken down mechanically using a blender (for larger sample volumes), using a homogenizer (smaller volumes), or by sonication. Cells may also be broken open by one of the above mechanical methods. Assorted detergents, salts, and buffers may be employed to encourage lysis of cells and to solubilize proteins. Protease and phosphatase inhibitors are often added to prevent the digestion of the sample by its own enzymes. Tissue preparation is often done at cold temperatures to avoid protein denaturing. The proteins of the sample are separated using gel electrophoresis. Separation of proteins may be by isoelectric point (pI), molecular weight, electric charge, or a combination of these factors. The nature of the separation depends on the treatment of the sample and the nature of the gel. This is a very useful way to determine a protein. It is also possible to use a two-dimensional (2-D) gel which spreads the proteins from a single sample out in two dimensions. Proteins are separated according to isoelectric point (pH at which they have neutral net charge) in the first dimension, and according to their molecular weight in the second dimension.

[0056] 55 4936-8845-3466, v.1 In order to make the proteins accessible to antibody detection, they are moved from within the gel onto a membrane made of nitrocellulose or polyvinylidene difluoride (PVDF). The membrane is placed on top of the gel, and a stack of filter papers placed on top of that. The entire stack is placed in a buffer solution which moves up the paper by capillary action, bringing the proteins with it. Another method for transferring the proteins is called electroblotting and uses an electric current to pull proteins from the gel into the PVDF or nitrocellulose membrane. The proteins move from within the gel onto the membrane while maintaining the organization they had within the gel. As a result of this blotting process, the proteins are exposed on a thin surface layer for detection (see below). Both varieties of membrane are chosen for their non-specific protein binding properties (i.e., binds all proteins equally well). Protein binding is based upon hydrophobic interactions, as well as charged interactions between the membrane and protein. Nitrocellulose membranes are cheaper than PVDF but are far more fragile and do not stand up well to repeated probings. The uniformity and overall effectiveness of transfer of protein from the gel to the membrane can be checked by staining the membrane with Coomassie Brilliant Blue or Ponceau S dyes. Once transferred, proteins are detected using labeled primary antibodies, or unlabeled primary antibodies followed by indirect detection using labeled protein A or secondary labeled antibodies binding to the Fc region of the primary antibodies. B. Flow Cytometry Flow cytometry is a technique used to detect and measure the physical and chemical characteristics of a population of cells or particles. In this process, a sample containing cells or particles is suspended in a fluid and injected into the flow cytometer instrument. The sample is focused to ideally flow one cell at a time through a laser beam, where the light scattered is characteristic to the cells and their components. Cells are often labeled with fluorescent markers so light is absorbed and then emitted in a band of wavelengths. Tens of thousands of cells can be quickly examined, and the data gathered are processed by a computer. Flow cytometry is routinely used in basic research, clinical practice, and clinical trials. Uses for flow cytometry include cell counting, cell sorting, determining cell characteristics and function, detecting microorganisms, biomarker detection, protein engineering detection, diagnosis of health disorders such as blood cancers, and measuring genome size.

[0057] 56 4936-8845-3466, v.1 A flow cytometry analyzer is an instrument that provides quantifiable data from a sample. Other instruments using flow cytometry include cell sorters which physically separate and thereby purify cells of interest based on their optical properties. Modern flow cytometers are able to analyze many thousands of particles per second, in "real time" and, if configured as cell sorters, can actively separate and isolate particles with specified optical properties at similar rates. A flow cytometer is similar to a microscope, except that, instead of producing an image of the cell, flow cytometry offers high-throughput, automated quantification of specified optical parameters on a cell-by-cell basis. To analyze solid tissues, a single-cell suspension must first be prepared. A flow cytometer has five main components: a flow cell, a measuring system, a detector, an amplification system, and a computer for analysis of the signals. The flow cell has a liquid stream (sheath fluid), which carries and aligns the cells so that they pass single file through the light beam for sensing. The measuring system commonly uses measurement of impedance (or conductivity) and optical systems – lamps (mercury, xenon); high-power water-cooled lasers (argon, krypton, dye laser); low- power air-cooled lasers (argon (488 nm), red-HeNe (633 nm), green-HeNe, HeCd (UV)); diode lasers (blue, green, red, violet) resulting in light signals. The detector and analog-to-digital conversion (ADC) system converts analog measurements of forward- scattered light (FSC) and side-scattered light (SSC) as well as dye-specific fluorescence signals into digital signals that can be processed by a computer. The amplification system can be linear or logarithmic. The process of collecting data from samples using the flow cytometer is termed "acquisition". Acquisition is mediated by a computer physically connected to the flow cytometer, and the software which handles the digital interface with the cytometer. The software is capable of adjusting parameters (e.g., voltage, compensation) for the sample being tested, and also assists in displaying initial sample information while acquiring sample data to ensure that parameters are set correctly. Early flow cytometers were, in general, experimental devices, but technological advances have enabled widespread applications for use in a variety of both clinical and research purposes. Due to these developments, a considerable market for instrumentation, analysis software, as well as the reagents used in acquisition such as fluorescently labeled antibodies have been developed. Modern instruments usually have multiple lasers and fluorescence detectors. Increasing the number of lasers and detectors allows for multiple antibody labeling, and

[0058] 57 4936-8845-3466, v.1 can more precisely identify a target population by their phenotypic markers. Certain instruments can even take digital images of individual cells, allowing for the analysis of fluorescent signal location within or on the surface of cells. Cell sorting is a method to purify cell populations based on the presence or absence of specific physical characteristics. In flow cytometers with sorting capabilities, the instrument detects cells using parameters including cell size, morphology, and protein expression, and then droplet technology to sort cells and recover the subsets for post- experimental use. Flow cytometry cell sorters have a collection system unlike flow cytometry analyzers. The collection process starts when a sample is injected into a stream of sheath fluid that passes through the flow cell and laser intercepts. The stream then carries the cell through a vibrating nozzle which generates droplets with most containing either one cell or no cells. An electrical charging ring is placed just at the point where the stream breaks into droplets and a charge is placed on the ring based immediately prior to fluorescence intensity being measured; the opposite charge is trapped on the droplet as it breaks from the stream and the droplets are therefore charged. The charged droplets then fall through an electrostatic deflection system that diverts droplets into containers based on their charge. In some systems, the charge is applied directly to the stream, and the droplet breaking off retains charge of the same sign as the stream. The stream is then returned to neutral after the droplet breaks off. After collecting, these cells can be further cultured, manipulated, and studied. Flow cytometry uses the light properties scattered from cells or particles for identification or quantitative measurement of physical properties. Labels, dyes, and stains can be used for multi-parametric analysis (understand more properties about a cell). Immunophenotyping is the analysis of heterogeneous populations of cells using labeled antibodies and other fluorophore containing reagents such as dyes and stains. A wide range of fluorophores can be used as labels in flow cytometry. Fluorophores are typically attached to an antibody that recognizes a target feature on or in the cell; they may also be attached to a chemical entity with affinity for the cell membrane or another cellular structure. Each fluorophore has a characteristic peak excitation and emission wavelength, and the emission spectra often overlap. Consequently, the combination of labels which can be used depends on the wavelength of the lamp(s) or laser(s) used to excite the fluorochromes and on the detectors available. Flow cytometry uses fluorescence as a quantitative tool; the utmost sensitivity

[0059] 58 4936-8845-3466, v.1 of flow cytometry is unmatched by other fluorescent detection platforms such as confocal microscopy. Absolute fluorescence sensitivity is generally lower in confocal microscopy because out-of-focus signals are rejected by the confocal optical system and because the image is built up serially from individual measurements at every location across the cell, reducing the amount of time available to collect signal. C. Lateral Flow Assays Lateral flow assays, also known as lateral flow immunochromatographic assays, are simple devices intended to detect the presence (or absence) of a target analyte in sample (matrix) without the need for specialized and costly equipment, though many laboratory-based applications exist that are supported by reading equipment. Typically, these tests are used as low resources medical diagnostics, either for home testing, point of care testing, or laboratory use. Widely spread and well-known applications are home pregnancy tests and COVID-19 tests. The technology is based on a series of capillary beds, such as pieces of porous paper or sintered polymer. Each of these elements has the capacity to transport fluid (e.g., urine) spontaneously. The first element (the sample pad) acts as a sponge and holds an excess of sample fluid. Once soaked, the fluid migrates to the second element (conjugate pad) in which the manufacturer has stored the so-called conjugate, a dried format of bio- active particles (see below) in a salt-sugar matrix that contains everything to guarantee an optimized chemical reaction between the target molecule (e.g., an antigen) and its chemical partner (e.g., antibody) that has been immobilized on the particle's surface. While the sample fluid dissolves the salt-sugar matrix, it also dissolves the particles and in one combined transport action the sample and conjugate mix while flowing through the porous structure. In this way, the analyte binds to the particles while migrating further through the third capillary bed. This material has one or more areas (often called stripes) where a third molecule has been immobilized by the manufacturer. By the time the sample-conjugate mix reaches these strips, analyte has been bound on the particle and the third 'capture' molecule binds the complex. After a while, when more and more fluid has passed the stripes, particles accumulate and the stripe-area changes color. Typically, there are at least two stripes: one (the control) that captures any particle and thereby shows that reaction conditions and technology worked fine, the second contains a specific capture molecule and only captures those particles onto which an analyte molecule has been immobilized. After passing these reaction zones, the fluid enters the

[0060] 59 4936-8845-3466, v.1 final porous material – the wick – that simply acts as a waste container. Lateral Flow Tests can operate as either competitive or sandwich assays. Lateral flow assays are disclosed in U.S. Patent 6,485,982. D. Immunohistochemistry The antibodies of the present disclosure may also be used in conjunction with both fresh-frozen and / or formalin-fixed, paraffin-embedded tissue blocks prepared for study by immunohistochemistry (IHC). The method of preparing tissue blocks from these particulate specimens has been successfully used in previous IHC studies of various prognostic factors and is well known to those of skill in the art (Brown et al., 1990; Abbondanzo et al., 1990; Allred et al., 1990). Briefly, frozen-sections may be prepared by rehydrating 50 ng of frozen “pulverized” tissue at room temperature in phosphate buffered saline (PBS) in small plastic capsules; pelleting the particles by centrifugation; resuspending them in a viscous embedding medium (OCT); inverting the capsule and / or pelleting again by centrifugation; snap-freezing in -70°C isopentane; cutting the plastic capsule and / or removing the frozen cylinder of tissue; securing the tissue cylinder on a cryostat microtome chuck; and / or cutting 25-50 serial sections from the capsule. Alternatively, whole frozen tissue samples may be used for serial section cuttings. Permanent-sections may be prepared by a similar method involving rehydration of the 50 mg sample in a plastic microfuge tube; pelleting; resuspending in 10% formalin for 4 hours fixation; washing / pelleting; resuspending in warm 2.5% agar; pelleting; cooling in ice water to harden the agar; removing the tissue / agar block from the tube; infiltrating and / or embedding the block in paraffin; and / or cutting up to 50 serial permanent sections. Again, whole tissue samples may be substituted. E. Immunodetection Kits In still further embodiments, the present disclosure concerns immunodetection kits for use with the immunodetection methods described above. As the antibodies may be used to detect neuron binding, the antibodies may be included in the kit. The immunodetection kits will thus comprise, in suitable container means, a first antibody that binds to a neuron, and optionally an immunodetection reagent. In certain embodiments, the antibody may be pre-bound to a solid support, such as a column matrix and / or well of a microtiter plate. The immunodetection reagents of

[0061] 60 4936-8845-3466, v.1 the kit may take any one of a variety of forms, including those detectable labels that are associated with or linked to the given antibody. Detectable labels that are associated with or attached to a secondary binding ligand are also contemplated. Exemplary secondary ligands are those secondary antibodies that have binding affinity for the first antibody. Further suitable immunodetection reagents for use in the present kits include the two-component reagent that comprises a secondary antibody that has binding affinity for the first antibody, along with a third antibody that has binding affinity for the second antibody, the third antibody being linked to a detectable label. As noted above, several exemplary labels are known in the art and all such labels may be employed in connection with the present disclosure. The kits may further comprise a suitably aliquoted composition of the antibody, whether labeled or unlabeled, as may be used to prepare a standard curve for a detection assay. The kits may contain antibody-label conjugates either in fully conjugated form, in the form of intermediates, or as separate moieties to be conjugated by the user of the kit. The components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the antibody may be placed, or preferably, suitably aliquoted. The kits of the present disclosure will also typically include a means for containing the antibody, antigen, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow- molded plastic containers into which the desired vials are retained. V. Examples The following examples are included to demonstrate preferred embodiments. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of embodiments, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

[0062] 61 4936-8845-3466, v.1 Example 1 - Materials and Methods Human blood acquisition and processing. All subjects and / or their legally authorized study partners signed the written informed consent approved by the Institutional Review Board of the UT Southwestern Medical Center (UTSW), in accordance with the Federal-wide Assurance on file with the Department of Health and Human Services (USA). Samples were collected and processed through the Neuroscience Biorepository at UTSW under authorization by the IRB. Individuals fulfilling 2009 RIS Criteria (n=30) were also enrolled in the ARISE trial. Individuals experiencing a first demyelinating event placing them at risk for evolution to MS were categorized as clinically isolated syndrome (CIS) (n=20). Twenty healthy controls (HC) were also included that were gender and age matched to the cohort. None of the subjects in the cohort were on immune-modulating treatment at the time of sample collection and the CIS cohort was sampled at the time of the first demyelinating event. After collection, PBMCs were isolated from participants’ blood by Ficoll centrifugation and stored at - 140˚C before analysis. Flow Cytometry for Immune Profiling B cell subsets. Patients’ PBMC cells were removed from cryostasis and stained for CD19, CD27, and CD38 according to standard protocol as previously described. A BD FACSAria V cell sorter was used to sort CD19+, CD27++, and CD38+ plasmablast B cells. Single plasmablast B cells were sorted into a 96-well plate. BCR library preparation and sequencing. Genomic DNA (gDNA) was isolated from sorted plasmablast B cells using DNeasy Blood and Tissue kit (Qiagen). The PCR product was amplified using Qiagen Multiplex PCR kit (Qiagen). The inventor used B-cell receptor gene rearrangement primer sets and conditions previously published by others. Nextera UD Index Primers (IDT) were added to the amplicons and were purified using the AMPure XP beads (Beckman Coulter). Barcoded libraries were pooled and loaded onto an Illumina MiSeq instrument using 2 × 300 bp paired end kits (Illumina) at UTSW genomics sequencing core. Antibody Sequence Analysis. Sequences were analyzed using the VDJServer analysis portal. V, D, J gene calls, FR, and CDR regions were annotated using IgBlast v1.14.1 and the VDJServer IMGT 2019.01.23 germline database. Unproductive antibody rearrangements and truncated sequence reads (did not extend from the beginning of CDR1 to the first two codons of the J gene) were filtered out. CDR3 charge properties

[0063] 62 4936-8845-3466, v.1 and mutational analysis was performed using Alakazam in the Immcantation suite v4.2.0 GraphPad Prism software was used to determine the statistical significance of differences between groups and build graphs for figures. Non-parametric ANOVA was used with a post-hoc analysis to do a pairwise comparison of patient groups with the healthy controls using the Dunnett multiple comparison method. Replacement frequency in CDR:FR was analyzed excluding near-zero ratios. Recombinant human Ab (rhAb) cloning, expression, and purification. The variable domains of the recombinant antibodies of heavy chains and light chains were synthesized (Integrated DNA Technologies) and cloned into IgG1 or IgK backbones provided by Michel Nussenzweig at Rockefeller University as previously described. The FreeStyle CHO-S cells (Invitrogen) were maintained in FreeStyle CHO expression media (Life technologies). The plasmids encoding the rhAbs were transiently transfected into CHO-S cells with FreeStyle Max reagent (Invitrogen). Supernatants from these cultures were collected on day 6. The cell pellets were spun down and supernatants were passed through 0.2-μm filters and subjected to rhAb purification on the NGC Quest 10 system (BioRad). The concentrations of the rhAbs were determined by sandwich ELISA as done previously. Purification of IgG from plasma. The inventor employed protein G chromatography (Cytiva Life Sciences) following manufacturer recommendations to purify bulk IgG from plasma samples. The inventor quantified pure IgG preparations with an in-house IgG ELISA to facilitate normalization across all samples for assays. Determination of neuron-binding frequency of rhAb or purified IgG by flow cytometry. The human neuroblastoma cell line, SH-Sy5y, were incubated with 25 µg / ml of rhAb or patient-derived IgG for 20 minutes before incubation with a secondary antibody (anti-human IgG Fc; clone M1310G05) for 15 minutes. Cells were subsequently fixed with 2% paraformaldehyde before being analyzed using a Becton Dickinson Fortessa Flow Cytometer. The percent frequency of SH-SY5Y cells bound by rhAb or plasma-derived purified IgG was calculated using FlowJo software. The inventor determined the bi-modal distribution of sample data for the binding SH-Sy5y by flow cytometry using the frequency distribution analysis function in GraphPad PRISM. The significance between data clusters was determined by the Mann-Whitney T-Test (α=0.05). P values <0.05 were considered statistically significant. Determination of neuron binding frequency of rhAbs by Immunocytochemistry. Cells of the human neuroblastoma cell line, SH-Sy5y, were

[0064] 63 4936-8845-3466, v.1 plated on laminin-coated coverslips (Sigma L2020) and rested overnight in an 37C / 5% CO2 incubator. The next day, cells were fixed with ice-cold 4% PFA for 10min, then washed with PBS for 5min. Cells were permeabilized with 0.2% Triton X-100 + 2mg / mL BSA (Sigma A9647) for 10min, then blocked with 0.1% Triton X-100 + 1% Goat Serum + 3% BSA for 2hr. Cells were incubated overnight at 4C with primary antibodies diluted in blocking buffer: rhAbs (20 μg / mL). The next day, cells were washed 4x3min with 0.05% Triton X-100 + 1% Goat Serum + 1% BSA, then incubated for 1hr at RT with secondary antibodies diluted in blocking buffer: Goat anti-Human conj AlexaFluor 488. The washes were repeated, then cells were counterstained with DAPI and washed twice with PBS. Coverslips were mounted on glass slides with Fluoromount G, then visualized on a Zeiss Axioscan 7 fluorescent microscope with a 20x magnification. Quantification of autoreactivity by rhAbs. Autoreactivity was quantified using Fiji. Cells were identified by using the Threshold function to detect nuclei and were added to the Region of Interest (ROI) manager. The mean fluorescence intensity (MFI) of positively stained cells was measured, and the MFI of the background (regions where no cells were detected) was subtracted from this value. Example 2 – Results Plasmablasts are not expanded in RIS patients. The inventor previously demonstrated that a high frequency of PBs indicates ongoing active inflammation prominent in individuals subsequently diagnosed with MS. Others have demonstrated that the frequency of PBs in the CSF of untreated early and established MS patients correlates with increased inflammation by MRI. PB frequencies are also elevated in the blood when the first clinical attack occurs and will continue to rise if the subject remains untreated. To determine if PBs are expanded in people with RIS who do not display clinical manifestations of disease, the frequency of PB (CD19+CD27++CD38+) in the blood was determined by flow cytometry. FIG.1 shows people with RIS have an average frequency of 3.43% PBs in blood, which is similar to the average frequency of PBs in HCs (2.69%). In contrast, CIS subjects, who manifest a first clinical event, but subsequently evolve to MS diagnosis after a second attack, have an average frequency of 5.46% PBs in the blood, a finding significantly higher when compared to HCs (p=0.02). PBs from RIS patients display a reduced drive towards antigen-driven selection. Lack of PB expansion in the RIS cohort compared to HCs may represent an

[0065] 64 4936-8845-3466, v.1 early indicator of reduced antigen driven selection. To investigate this, the inventor used a known feature of B cell biology in which PBs with high (3 or more) somatic hypermutation (SHM) events have undergone a robust drive towards antigen-driven selection. The HC and RIS cohorts had similar frequencies of PBs expressing productive antibody heavy chain rearrangements with 3 or more SHM (>3SHM) (FIG.2). In contrast, the CIS cohort had an average of 86% PBs expressing productive antibody heavy chain rearrangements with >3SHM, which was significantly greater than both the HC (76%, p=0.004) and RIS (77%, p=0.02) cohort frequencies. Of note, the RIS cohort was statistically verified to display a bi-modal population with the upper population (n=21) at high SHM frequency and the lower population (n=9) at a statistically lower SHM frequency (88.51 vs 52.19; p<0.0001). PBs in RIS patients are enriched for VH4 use compared to CIS patients. The RIS cohort displayed a similar frequency of PBs as HC (FIG. 1) and a reduced drive towards antigen-driven selection compared to CIS (FIG.2). These features may manifest as discordance in antibody gene usage. To address this, the inventor calculated the frequency of Variable Heavy (VH) chain family usage in bulk-sorted PBs of all subjects in the 3 cohorts. The VH family genes are divided into 7 families according to gene homology. The RIS cohort displayed a similar frequency of usage for six of the 7 VH families (FIG.3A). The RIS cohort displayed an expansion of VH family 4 (VH4) usage compared to the CIS cohort (p=0.04) (FIG.3B). VH4 expansion was further emphasized in the RIS cohort when only those sequences with >3SHM were considered (FIG.3C) or when only the top 5 clones of each subject were included in the analysis (FIG.3D). Other immunogenetics features comparing RIS to the HC and CIS cohorts are provided in Supplemental FIGS.1-3. VH4+ antibodies from RIS patients are enriched for reactivity to human neurons. VH4+ antibodies expressed by PBs from CIS patients have a propensity to bind neurons. To determine the frequency of PBs expressing neuron-binding antibodies in the RIS cohort, the inventor cloned 63 antibodies expressed by individual PBs sorted from the blood of people with RIS. The rhAbs cloned were from 12 RIS subjects that displayed high PB frequency and ranged from 5-8 rhAbs cloned per subject (Table A). All 6 major VH4 genes were included in the RIS rhAb cohort with representation of all 6 JH segments. 52 of the rhAbs were VH4+ with an average homology of 95% (range 79-100%) For comparison, 11 VH3+ rhAbs were cloned from the RIS cohort with an average homology of 93% (range 83-99%). The inventor tested the ability of the rhAbs to bind a human

[0066] 65 4936-8845-3466, v.1 neuroblastoma cell line, SH-Sy5y, using flow cytometry (FIGS. 4A-B). The gating strategy for this experiment is included in Supplemental FIG.4. The average percentage of VH3+ rhAbs binding to SH-Sy5y was 3.66% (FIG. 4A) whereas the average percentage of VH4+ rhAbs binding to SH-Sy5y was 13.06% (FIG.4A). Of the top 20% rhAbs that bound SH-Sy5y in this assay, all 12 utilized VH4 genes with a propensity to pair with JH6 genes, but otherwise used all VH4 genes with SHM accumulation across the spectrum (Table B). The inventor used immunocytochemistry (ICC) of SH-Sy5y cells to query the staining patterns of the RIS rhAbs. Four examples are presented in FIG.4B demonstrating the majority of RIS rhAbs bind targets residing in the cytoplasm of SH- Sy5y cells. RIS patients display higher frequency of neuron-binding plasma IgG. Finally, the inventor asked whether antibody pools from RIS would also display autoreactivity towards neurons, as she had demonstrated with individually cloned rhAbs. To do this, the inventor purified bulk IgG from cohort plasma samples using protein G chromatography and used flow cytometry to determine the frequency of SH-Sy5y cells bound by the IgG purified from each subject (FIGS. 5A-C). RIS patients displayed the highest frequency of plasma purified IgG bound to SH-Sy5y cells with an average of 4.4% (n=30). This frequency was statistically higher than either HC (3.08%, p=0.001) or CIS (3.44%, p=0.002). The frequency of plasma purified IgG binding to SH-Sy5y cells from only those RIS patients the inventor cloned rhAbs from was similar to the average of the RIS cohort as a whole (4.4%). All RIS patients from which rhAbs were cloned displayed high frequencies of SH-Sy5y binding by IgG purified from plasma, but not all rhAbs displayed high frequencies of SH-Sy5y binding.

[0067] 66 4936-8845-3466, v.1 TABLE A: Cloning Summary by RIS subject Total cloned Total VH4 (%) AVE Homology

[0068] 67 4936-8845-3466, v.1 Table B: Immunogenetics of Top 20% rhAbs that bind Sy5y by flow cytometry e 6 4 4

[0069] 68 4936-8845-3466, v.1 SUPPLEMENTAL Table 1: Features of Autoreactivity in RIS Patient Code %PB %SHM %VH4 %Sy5y by IgG FI FI 7 FI FI 1

[0070] 69 4936-8845-3466, v.1 SUPPLEMENTAL Table 2: rhAb features from RIS patients Patient rhAb Name VH:JH %HOMOLOGY % binding 5 0 5 0 0 4 5 9 4 1 0 4 5 2 5 0 0 5 6 7 0 1 0 2 8 0 5 7 2 9 4 7 0 1 0 0 8 0 9 4936-8845-3466, v.1 SUPPLEMENTAL Table 2: rhAb features from RIS patients Patient rhAb Name VH:JH %HOMOLOGY % binding 4 0 9 5 6 8 4 5 6 1 1 3 8 7 0 9 4 5 2 0 9 2 5 7

[0071] 71 4936-8845-3466, v.1 Example 3 – Discussion The ability to stratify individuals with RIS according to their risk for a first neurological event represents an important advancement, offering valuable guidance for both counseling and treatment recommendations, especially with the anticipated inclusion of “asymptomatic MS” in the updated 2024 McDonald MS criteria. Currently, baseline demographic, para-clinical, and radiological risk factors are available to guide healthcare providers.28Recent data also indicated elevated serum neurofilament light chain (sNFL) levels within individuals prior to a diagnosis of MS. However, MRI features from these individuals were unknown and would have provided valuable context for these findings. Still, RIS represents an earlier manifestation of CNS inflammation than CIS within the demyelinating disease spectrum. In addition, the recent success of clinical trials in the treatment of RIS to prevent advancement to MS diagnosis raises important questions regarding the underlying pathobiology. The inventor’s focus was to compare features of B cell dysregulation between people with RIS and CIS which may elucidate the underlying, initial immune mechanisms associated with CNS inflammation prior to the first demyelinating event. PBs and the antibodies they produce are a central component of the neuropathology of MS. This is evidenced by CSF expansion in people with CIS who evolve to MS; the strong correlation between PB or antibody levels respectively, with brain gray matter atrophy, disability among MS patients, and the success of disease- modifying therapies that target B cells, including PBs. RIS subjects presented here display the ability to suppress both antigen-driven selection and expansion of PBs. These data, combined with these previously published observations regarding B cell features in the context of MS pathology, infer that the RIS cohort is unlikely to evolve to MS. Nevertheless, of the 9 people with RIS who had suppression of both antigen-driven selection and expansion of PBs, 67% of them had spinal cord lesions. Of note, the strongest predictor for a clinical relapse is the presence of spinal cord lesions with an odds ratio of 128 for a first attack. Yet, none of the 9 RIS patients who displayed suppression of both antigen-driven selection and PB expansion had relapses over a period of 96 weeks. B cell depletion is an efficacious treatment approach for the suppression of disease evolution. Thus, it is possible that RIS subjects, displaying PB suppression by flow cytometry and PB suppression of antigen-driven selection by immunogenetics analysis of SHM accumulation, would be less likely to advance to MS diagnosis.

[0072] 72 4936-8845-3466, v.1 Therefore, B cell depletion treatment is unlikely to benefit RIS individuals who display natural B cell suppression. Capturing additional biomarkers of neurodegeneration such as sNFL and glial fibrillary acidic protein (GFAP) may further elucidate the relationship between the B cell profile and disease progression in RIS subjects. Furthermore, elucidation of the pathway to suppress PB in these RIS subjects may reveal a new mechanism that slows or prevents advancement to MS. Individuals with CIS advancing to MS and individuals diagnosed with MS typically display over-use of antibody variable heavy chain genes from family 4 (VH4). Based on these data, the inventor concluded that high frequencies of VH4+ PBs is a feature associated with advancing neuroinflammation leading to a diagnosis of MS. Thus, she predicted that PBs from RIS subjects, who lack clinical symptoms of demyelination, would not display over-use of the VH4 family genes in the PB heavy chain gene rearrangement repertoire. Instead, the inventor observed that the frequency of VH4+ PB was significantly higher in the RIS cohort compared to the CIS cohort, regardless of how the data was analyzed. One explanation for this finding may be that those with RIS have not experienced sufficient neuroinflammation at the blood brain barrier (BBB) to accommodate highly active lymphocyte trafficking resulting in elevations of VH4+ PBs in the blood of RIS subjects compared to people with CIS. Indeed, data from ARISE indicated that only 10% of RIS subjects had gadolinium enhancement on their baseline scan compared to 25% of MS subjects with gadolinium enhancement on their baseline scan in the OPERA I and II studies. The RIS cohort presented here had 4 RIS subjects with gadolinium enhancement suggesting that the BBB of the vast majority of RIS subjects remained intact. As this is the case, the over-use of VH4 in the RIS cohort compared to CIS may be attributable to distinctions in BBB integrity between RIS and CIS subjects. A similar conclusion could be made concerning the enrichment of purified IgG from RIS plasma to bind SY5Y cells. Biomarkers of BBB breakdown are beginning to emerge across neurodegenerative conditions and may be elucidative in the context of RIS and CIS. The inventor previously documented that VH4+ PBs from CIS individuals who advance to MS diagnosis are enriched for autoreactivity towards neurons. To examine the autoreactive nature of VH4+ PBs in the RIS cohort, the inventor cloned 52 VH4+ rhAbs and 11 VH3+ rhAbs as controls and demonstrated by flow cytometry that antibodies expressed by VH4+ PB have an increased frequency of SH-Sy5y binding compared to antibodies expressed by VH3+ PB. The frequency of anti-neuronal binding

[0073] 73 4936-8845-3466, v.1 antibodies among the RIS cohort was 44% by either flow cytometry or ICC. By comparison, of the 30 VH4+ PB-derived antibodies the inventor cloned from people with CIS, 60% bound to SH-Sy5y cells by ICC. Thus, the frequency of VH4+ PBs producing anti-neuronal antibodies was higher in the CIS cohort compared to RIS (60% vs 44%). One limitation of this comparison is that the antibodies the inventor cloned from the CIS cohort were enriched for high SHM, whereas the antibodies she cloned from the RIS cohort were a mixture of high and low SHM. When the RIS antibody panel is restricted to the antibodies cloned with high SHM, the frequency of anti-neuronal binding is increased to 52%, which is more in line with the frequency observed in antibodies cloned from those with CIS. In addition, the staining patterns of rhAbs from RIS and CIS patients are similar in that they tend to bind intracellular antigen targets rather than surface antigen targets. Current dogma has been that the cellular localization of the target antigen determines the pathological potential of the autoantibody, such that most pathological autoantibodies bind extracellular targets, not intracellular targets. However, more recent examples indicate that some autoantibodies recognizing intracellular targets can be pathogenic. Thus, the lack of PB expansion in RIS subjects may serve as a protective mechanism, limiting the expansion of the VH4+ PB pool which harbors anti-neuronal binding properties with pathogenic potential. A detailed longitudinal analysis of the B cell profile in the RIS cohort would be necessary to elucidate this potential protective mechanism. Future studies should also focus on determining the impact of these VH4+ anti-neuronal binding antibodies expressed by rare PBs in the blood of RIS subjects on neuronal health.

[0074] 74 4936-8845-3466, v.1 S NOIGERELBAIRAVYDOBITNAROFSECNEUQESEDI ToiTTTAGA TATG TAGG C TA T TG GT TC C g AG TT GTG A AGT GTGT ATG A GC A O e C G G T CC CG C T CT GAGA T GGC ERAGAGG CTAA CG G G CT C CCTCTC C A e TCGATG T GACCG A G G TTTATC GACA TA A TCCTCC GTGA CACC c GGGCTC AA GCCT CTTTG GGGCGG GCC TTTA GCAAA G TG TCATL C ne TT CTGTT CACTC TT CAC TTA C C CACTG T TGTGT ATG C CCCGC G U u GCC q GAATTG AG ACCAT C GCG C GCAT ACGTC T GCGCA TG ACC TGANe C GA CTC ACAG GT T C ACAT G GC G AC GT AAAC TAT TC CA GT ACT CCTGGGA C GCTCC CC CTCCTC CAA T CTCC C CAAA CTC C CGCG– SelACGATT C TTCAA A ACGCA G TC TTCAC C T ATGTGT G TCGTG T1b ACA ACG TCA CGCG AAG TT GCG A G CG AG AC T GT C A C C GCC CGC A E aiCC T G GT CGGC G G CGT CCGA A GT TCGC TGC A A TGC TGC C A GA G G LrG a TGGAGC G G G A GAGTC TGAT G GT CC GAGTG TT G T GG CCGG G GAGTAB V C T G G G T T C A C A C T A G C A T C A C T C G C T C G T A G G CA Tniyty y a v h vth vth h aegilaegilaegi C h h hl.O QN1 2 3 4 5 6 E SDI1 e -.v n 1 - l 0 1 -,0 1 6 oS1S2 0 S 3 64 CI I IR R R3-548 8-63 94 GGA TG GCAGT ATGATC GTTTAAT AGGGTA GTGT TGGAGA ACC TAGGC CCGCC C AA TC CC T CCGAGTG TTTCC AT A C GCTC GGC TA GAGCA AGTT ATG GACTTTGAATCCG GCCA C C AATTCT A C C C C GG T G G C C A G T T G C G AC G A G AA TCA T AG G AGGCA TA C C T TGG TA CAG TGGT A CT G TC C T CAG GC C AG TGATC CAG TTG T A ACAGCT GCGCGA TC ACAATTGC A A GCTC ACA C GCGC T TT ATA AAA CAC CCAGG GCAA ACTC GC C AC GC G AGG CGC A A A GG AA CA T GTT CA C CA CA A CA CCATCG CC T A TG T C C CA C C C ATG C C GG GC G GTCC C GCC CCG C T A G T A GCAA ATGGC G G TT GCGGATGTG CG TTCAG ATGGC CA G C AC AGG C C ACGG CG GTAC A C C GG C TT GG GAGGTATCCGACG T TG G CCGG TGATGG CCGGGT TGG TGATTC CACC TG CA GC T C A AA G T ACAG G GGGA GC CGG TTG G C TGTTCTAC GGATATTCCGTGAATC TTCTCG A C T T G C G T G C G C G C T T C A A T C G C T C G T A A C A C G G G A T yvtyvtyvty a h h h v e hgilae hgilae hgilae h 7 8 9 01 11 21 31 1 - -.v 1 1 - -,0 0 1 1 6 SI4SI5 0 SI6 0 SI7 64 R R R R3-548 8-63 94 AAA GGGAGGGGTGAGATCA CAG GTA AGCATT GAGACA CCGGTTCAT GTCTGG ATATAC CCGTCG TATTCCACC CTATAATGCT TGCTAGCTGTCAGACAAACCACGACAGATGGTTTATACT AGA GAAGCCCACTTCTAAGCCAAG TGGAAC GGCT CTG AAA TCCCACTTGA GTGCCT AGGAGC A GTGCA T AGATGC GGATTT AGTGATT TGCCTTT CCAA T CCGCT TT CC TCCAT T G CCGGTT T C T GCC T C GTTA CTA CCAAGC TC TGACTTCAATTAT GACCTT AAGATGC G A CC AAG AA GACCTG CT CCA TTG GCTTC TCTTTT G CC A T TC G G G TCT G C CTTG T CC GA GAAT CA AC TG TCG C CCT T G C T T TTT TACAGCACAACGCGC CTA C ACG C AG G G GA ACAAC GCGATTG CTC AACACGT CAG G GCAC AC GCG GGT C A GG ACAGT A ACCATCC CAA TTC AT C CG T AA CGTTC CAT TGCCATCA CCATGA C C GCCAC CA C C CCC CCA T CCAAAA ATCCCCT CG CA C CCATTTCGC TGGCG TC A TA T G A TCGAA GTC TCGC TA GAC TG GT A GGGCT TCC GA ATTC T GCGA GAAC G C CG AC T CTCGTAC A T CGCG C C G A G G C T G C A T CGG C CC C G G G CC C CTG GAT G G CACG G GA TC GAT G G AC G GG GT G A T CG T A C C TG A T GTC G G G G TC GTAGAAC T CTCA GAACA TTAGGT GGGTCTTGATT GAACT T C A T G C G C G A C T G A T T C G C C G C T A G C A C G A C T C T C A T A t y h vty h vty h vtg agaga h ile i hle i hle hgil41 51 61 71 81 91 02 1 - -.v 1 -,0 3 3 6 SI8 0 SI1 0 SI2 64 R R R3-548 8-63 94 T CCACCGCCAAGGGACACTGCCGGCCCCTCTTTGCACCGCGGCATGACAGGGTGCGGAG TTGCCT CTAACGTCGCAACCCTGCTACGGGAGGTG GTGCAGTTA TGG AAACTGGTGAAACTCTCGATTCACCTGTGACACGGGGGTTGCGT TGGAACC G CCTG TCCTTGTGAATG CCGC AA TCATC AC C GGGA TCGGC G A CT GGA TC A C C CGG CG TCTAT AGTC AGGATA GTGA A GACC G CA A G CAA GGG TTCG ACC GGGGT ACTC ATTCGGCGTTCGT G ATCCTGTA T GGGCTC C GC T TT CTCA G TATG GCTTTTA T GCCATATGGTGTAGCAGGA CGCT ACAAC TT GCAT GCC AG CCCGC G G ACCGG T GTGCG C AT GGCTC CACTG C CAGA ACA G T GTGAT ATGCGAGG CC G CGTT CTGG GTCA GC CGC TTCCAACTGA CTCCT GT CCTATG A CACTT GC CTGCAC ACAA CATCC GC CAGAAC TCCATGG TGC CGGA GC CTCCAG CTT GCACG TG CCTTT CACC GCCAC A CCA ACT GCATGGT T AT ACGATC TGG AT A TTC AT T TACAACC GA G AAG T AGT ACGCAG GT GGC GTCG T CG T CGC GGT C CGA AGC GT AC CGCCGG AG CGGTGA GA T C AG GAC A CCGG TGCACGTTCAAC C CGG C GAGTTT TGCGT GGGATG TGGGG GCGGGCTT T CCTAT TGAACG GAGTA TAGCTGA GAATTT TTACGA C G C G A A T G G T T C C T A G G C T A G G C C G A T C A T C A T A C G C T C C yvty a h vty y h vtv egi aegi a h gi a hlhle hle h 12 22 32 42 52 62 72 1 -.v 3 -3 -3 -,0 0 0 90 6 SI3SI4SI5SI1 64 R R R R3-548 8-63 94 AGG GAGG GTGAGG AACACC AGGTAGCACTGGAA CAC CGGTTATCCGTTGGATAT ACGCCGTCTAACCCCCCGCTGTTTGCTGCGGTGGCTTTGGGGAA ACGTACGCCAAATTGTTTATATTGGAAATCGC TCATCGATCCGGAAGGCAACACGTT ATGTAGCCCTATA C GT T TGAC A G GCAA TTG C A CTTTGCG A ACTTGG C TGT GG T TCT TCGC TGGCT TCT TG TTGTTATTCCA AA AT T C TGTAAA TTATTGAACC TGGT TC CCG TG G GCCT AGG TCCTT AT CCGCGG G TTCC AT TGTT C CCCGCCTT ACGC ACTTT ATA T G TGT A GACG G CTCA GAA CCTA AG TTCC G T G T AT T GG ATTATAT C GAC AC GG TAC G AT C CTGGC C GC G A A GC GTG CTCA CAC GGC GG G CC A T T AGA TT A G TTAACAAT GCGGAG CCACG G AATC C A TG GGTC C C GACTCT GC T TGAT A TC ACCAG C GCGA CGC ACAA ACTCT C GCGAGTACAG G ACAACCACAAG GT CCTCAA CACGT T CT C CAGTTG CACAA TCATC TTC CATCC CC C T CCG T CG C C CACTTCG ACAC G G AGG TCTGA CC ATCA TA GCCG G TTC ACC T G ATATAG GC TTCGTCGC G AAG TT CTA ACGGT G AACGATA GC CGAGGA C CG T T C GTGA AGC G TG C C A GG TC C A GTTG C G A T GAGTTT G T G CC CATG GT GGT GCGCTC TGA CCCGCATG CGA GCGCTC TG AACT T AGCG AAGAT TT A T A CATT GT A A AG C G G G C G ATT A GC G ACT T C A A T C T A G G G T A C G A C G A C T G T C A A T C G A C T T T C A T A t y y h vtgila h vtytegila h v h egilaegi h h hl82 92 03 13 23 33 43 1 -.v 9 -9 -9,0 0 6 SI2SI3 0 SI4 64 R R R3-548 8-63 94 TG CAGCCCC TAC CGGCAACGGGAGAACCCTGTTGCCCCC CGCTATGAACGG GCTTCGCAG ATGCCGAAGTGGCGTA CTA CCACTC ACCCGATGGTAGGGCGTGTGGG ATA CGG AGGTAAACCCGTTTAACCTGCCATTCCA TGATTG TAGGGGAGG TCCG TTC TCCATTTAGG TG TGGCGCCTGAGTTAAG GCCAC ATACGC GCCAC ATACGCCC AC GT T GGTCCTC AC TCCGAG AC TCGGC AA CCGAA AT TCGGC GAA AA CC GATAA T GTTG AT G T GTG C AGGGCG GTG AGGTGC GGG A TGGGT GCTACG GCAC TCT C CT T G GTGC GC C CT T AC GG CG GAGTG GGT CTTC G CACACGT GT CAGTG CC A ACCGG TT A GCGAGA CC G C ACCGA GCGAGA CGCCAA GTTCACCAG TGCGA CG TC ACT CGT GCGTT TTCT ACT CGT GCGCT TTCTCA ATACACG CAG T CA TG CA C CA TG CA CCCGCAG CCGCCCTTC CGC CAA CGC CAA G G T A CAAC G GTG CCGTTC G GTG CC TTCAT CTGTC G AT T C TC T AT ATT TC ATG TACG G CTAACGATT AG ACG C AGT ACGAT A GCG CG A A CG A ACCCACTAGAGCTCCGCT AC GACGT C CCGCTT GACGT C CCG TTG ATGA G TA G G T GCG G GA G GC C C T G C CT T G C G T GG GCG GGTTGGCCGAAGATTTGAGG GAGAA TTACTC GAGAA TT A ACTC C G G A C C T A C G A C G A C T C T A G G C C G A T G C T A G G C C G A T G C yvty a h vty h vty h v egi aegi aegi a hlhlhle h 53 63 73 83 93 04 14 1 - -.v 9 3 -3 -3,0 1 6 SI5 1 1 SI1SI2SI3 64 R R R R3-548 8-63 94 ATAGCCGGGGAGGAAAGCGACACTCCGTAGGCGTCATACTTACTGG GAGTTCCCCCCCCG AAATCCTGGC AGGAGCCTACGCTACCGTGGGAAGCCA GCGCATGATAGGCTTCTGG ATACGG AGTTCGAT GGAAAAGCACCCGTGATGCAACCCAGCTTGCC T T G T G GTCGGC GCA AA CC AAC TAGCA CCGCG GCTG TAGCA CT CGAGA GCTCTAGGGTG AAG GAGGC AA G GAGGC AA GA CGC C T T GC AG TCT CAAG GCTAA T A G GT G G C T A CA CA C G C TG TTG C CA T C G T GT C CGT TGATT CCCGC GA GCCCACCG GCG G ACA GC CAC A A T GC A A A CTGA GAG CAGG G T CAGG GCTG CAGACACTT GC CATCTC A T C CAA T GC C C A CAGAGC CAATT GC C CAG CAAAAG CGCG CTC CGGC A T CGGC AGTACC TGTCGTG CC A ATATAA G A C TGA A CC C C G A CC T ATATGA CA A TA GCGCC AGCGAGT A ACGAT TT CGCCTC T ACGGT T G CGCCTT A T ACGT GCCT CGCA CGACG C T CCGC G AGT C CCGAC G T G CG G G A GGAAA GC G G CC T G AAC GGG GTTG G GCGCTG T G GCGG T T A CG TGATGAGA TA G GA TTG T CGT GACGTTGG G A T A T CG T A TTACGGGACA T A G G C C G A C T C T G C C T T C G C T C G T G C C T G C G C T C G T G C G t y y y h vtgila h vth vth e hgilae hgilae hgil24 34 44 54 64 74 84 1 - - -.v 3,1 31 51 6 SI4SI5SI1 64 R R R3-548 8-63 94 AC T C A GT CAT CTC TT CT A G CCGGGG AA TCAC CCACC CT TT AA C CAT CCG C TGGA CCAC C A GT AG CT TT TA AG A TT CA AG AT A G GC TTC TAC TGTA T TACGC TTC CTG TGT A AAGG TTTC CTG CT GC CG A AAC GC C AT CA GC A TTC T CT CCG TCCT CCG G TCCTA TA C GC TCTC GGGA TGC GAG T C AGTACG GTC T AA GCG GTTG G G T G A T T G CA ATT T C TCA GGGTG C C AG TG C GGC GGGTT C G CAG TTG CCGG T A GAC TAG G GAGA CCGA T ACG TA GGG CCGA ACG CCC ACACG GC GCGCTA AC ACCAG GC GCGATG AC G ACCAG TTA C TGC CACAG G CGT CATTCC CCCATAC CACGT GCCGC CATC CCCTC AAG CACGT GCCGC GCC TCCGC TG TG CT A TA TG T A ACA T CATGA A ACGAT T CAG CGCGTA A T ACGAT AT TC G C CTG GGG GACTA CCGCTG GAGCT CCGCTC G AGCT CGG TGGCT GCGAGG TGGTA A G GCGCGT T GTA TCG A TA TT TT AGA TT AT GAGA G G A AC GA T A C GA T G A A T A A G T C G A T G C T A C G A C G A C G A T T C G A yvtyvtyta h egi a h v egi a h gi hlhle hl94 05 15 25 35 45 1 -5 - -.v,1 5 5 6 SI2 1 SI3 1 SI4 64 R R R3-548 8-63 94 T CGGCGACGGAACGCACGGCAGCGCCATGATACCCCCCGGCACCACGTGTACTGCGGCTTACCAAGCGAAGGAGCCTAGCA ACCTCGCAG CGAGGTTATGAGGGATGCGG GAAACCTATATCCCCGTAAGACGCGTG TAGGA AGGATGCAGCTGCCGTTTGATACCG T C C TTTAC CCG T C C AA GGGG C CC GGTGG A GAGA CTGC CGCATTG CTACG GAC G TT TTA TA CTTTC GCCC AG TG CTC T TGCC GT AA G CAT T GAGCGTGGTG TAGTG C T CTCTC CCC G AAAGG TG TATAG CCTTT C TCCA G TGAA TATGC C TA CTGGA GC GTG CGAC TTAACC GTG GAGC C A C GATC C CG GGGG C ACG AATC C CTA AC GCGC CTG AAG GC AT AC GC G TGCG ACAA GCG A ACAGC G G G A CAG G G GGCATTCT AAC CATCT GCAGC TTA C CCCAC C TCATC TC AG C CACGT C CAGA CTCCCCTA T ATA GCCAACCACTTGATCA CCCATA GCCGC CCTTCC TG TC A T CA G A T A C T C G T GTAT T C TC TAT TT TT G AT ATAACG A CGC ACGGG CTCG ACGA C CTA ACA CGC T GA C T A CG T G T TTCC T ACGACCGAC CTGGCGG CCGCTC GAGCA CCGAATGCG GT TGG CG C TAC C CGG T GT CGCTG T A T A CG G GA G G G A GT CTC GAACATTCCGTGGGTTTTAATG GAAGT TTGCGG C G A T G A T C G T T C G C T G G T A A C A C G A C G G T A C G A C G A A T T yvtytyty a h v h v h v e hgilae hgilae hgilae h 55 65 75 85 95 06 16 1 - - -.v 5 7 7 -7,1 1 6 SI5 1 1 SI1SI2SI3 64 R R R R3-548 8-63 94 A GCGGGGGGGTAACCCCGCA AGTCA GGT AGA CCTGGTAGCCGTCAGACTTCCGGCGACTCACCCCCCGGAAATCTTAC GTA AC CTCAGGCAACCC GGGAGCTGATAGGCTTCAGAGACTGCGAATCA CAT GGAAA CGG GACCG GATTGCAACCAAGCTTGTGC CCGTT GCCAT AGGGCCG GC AACAA GT C AG C C G GGCTGGC T GCT AAGCGCTAGA C TCGTTGT GCAC CAT TCA G GGTT GCG TGGACCGAC TG TATTACACA G C A TGGAT CT C G AC CCG TGGAGG C CAGTAACACG GCGGGGC ACAC GA GT CGC C CTC ACCGG T A GCTT CTC C ACAG ACACCAG GATG CA C G GC CTG C CA CC GCACGT GC CATCCGA CAAGT GC A A CAACTA CACTT GC CAATTAC A CTCCTGCCGC CTAA CGTG A A GCG GATT C GATCTGA CC A ATGTTAG TCACT CC TG GC ATG TG CGTG CC TGCTG GCG AGCCGCTAGAGCT ACGAT ACCGCA CC ACGCTCG T CGAGT A A T A ACGCTG CGATG GTTGGTA CCGCTC G GCGCGGG G A CCGGG CGT AA GG TGAT G TT GA GCACGA TGC CC GG GCACGC TA G TCAACGAAGAT TTAATGGACAT ACTTCTCC GAGTA TTGCCC GCCCTAT T A C G A C G A C G G T G C G T G C G C G A C T A G G C C G G G C C T G G T T C t y y h vtg h vty h vth ilae hgilae hgilae hgil26 36 46 56 66 76 86 1 - - -.v 7,1 71 71 6 SI4SI5SI6 64 R R R3-548 8-63 94 T CACCGTCGGAGAGAGACC CTGCGG ACCTCGTGACAACCC GTGGCACCAGCC TTT ACGGGC CTGCCT CAA AGCCGAAAGTCGCCTACCAGACGT TCGAACTCTTGGGAAAGGTGGGGAATCCGGTATACCCA CTG TATCCC GTGGCTTTGGAGAAT TACCTCTGCCGTTGTAGT TT AAAT GC TG A GAA GTGCCATGAGAG G A ACGA CCC G TCC A C CC GG TTTA CG TCTA G CG CTA GCG A GTTG AGGG T CC GCCCAAAGC TTTTGAGCA A GGGT G A TC A C G T T CGC A GC C GGGT GTA C CCTTT C A TGG T CT TT A T C GA TT A CTCAATTCATTCA A AAGCA GT CACG G G GT A A G G AC CA C G GTC GAT A AG CGCT A G C CGCT AC GT CGCACGC A C ACC GC T G ACAG GC C TA AG GT T TCAG G CACGT CAT CG C AAC G CG AA G T T TCTC C CAA CT CCAAAT CC TT CCCATGCC T T GCCG CC TTTGA C CC T CG GG CT GATC CC TCT A TG C CAA TATGC CA C GCA AAAT CGTA A AT TC TA G C TG AA AC CTTAC GTT CG ACG GCT CAC CTCG T C TAC G A C C C AAG G CT ATCCC A G A CCG C G G G C G T G TA C CC A G A GA G GTG TTCCGGCTCGC CAC TGG GCGAG T CG GCGG TGAT GA TGCTC GA GAT TACTA A GGG T C G A GTGCT A T T TTA GG ACATGTGATTA C G A A G A T A C G A C G A T G G T A A C A C G C T C G T G C G T G C T A A C A yvtyvtyvty a h a h a h v e hgile hgile hgilae h 96 07 17 27 37 47 57 1 - - - -.v 81 8 8 8,6 SI1 1 SI2 1 1 SI3SI4 64 R R R R3-548 8-63 94 AT CGGACG AGA TCGGATCGG TTCTCCACCGCAGTCTTGGTTGCAGCTGCCCGACTATCTCCACCAGTAGCGGTGCCTGAA GAC ACTTTA ACCTTT TGAGACGCGGTT CTC TACTGTATCTACCGGTTAAGGGTATTCCATCTGTAACGTGGCAGTCGGATGCAACCCTAGCTTT GCGTCT AAGCTCAGCGCA ATGC CG TCTATA T GCCTCTCAA CCG TCTA C T CGTT AT GGG T CTAGTCGC AAGA CT GG CC TTGAAAG AATT AA TAGCC ACCTGCC AAGATC C GTTGT CGG T T GC GCC TCC ATTTGCT C ATCT C C TG T AAT CACCC GG AATCTGACGGA GCA GACAAC T GCGC T TTCCCA AGT T GCT G G GCACTCA CGGGT GTGAAG CC CACGACC G CGT A T GC CA T CGAT A A G CC T A CAGCTCCT GT C TA A CCCAGGC GC CACAA CAACGTCATCTGC C C AC CAGCG ACGTGCTCGGA C AGC CTCGCA C ACGA AG TCAA CCA TT ATC CCC GGCCAA ATGGCCTTCGTCCA ATCATC C CGA GCCGC C TCT ACGATG AAGCGG G TTGA GAG T GTCT CGTA G GT AA CGG C AA G CCGCGGAC G C CG TG G CGG CGG A TGCCG GAGAC CC G G GAGCTCAAC A GAGTGCGTT CACC GA GAG TG G G G CTGGTTGC T G CCCA A A TG T C G G AG TT TGA CAG CC T C ATGAACATTCGGT GAAGT T G G T T C C T A G A C T G A C G G C G C G C G T C A T A C G C C C C T A C G A t y h vtytytgila h v egi a h v hlegi a h hlegi hl67 77 87 97 08 18 28 1 - -.v 8 -,1 9 9 6 SI5 1 SI1 1 SI2 64 R R R3-548 8-63 94 AC GGC TCGG A GTGAA CCGACC A GAG A GGGC T GCCCG C ACGCTC TCCTA TGC CCGAA GGG AT C G A A ACT ATTCT GT T AG C CGG GCAC CAT TTG T TC A GCTTG TCCAAG GCCAC TCTATGA CTC CT CCGC GG G GCTG CTTTC AT GGCTTC CTGGC GGG AGTC TCT CCCGG T GTATCG CAG T GC TGAT AG CCGA T ACG TGAGG CAG A A C C ACG GC C A AC GC TC ACTGT G G AG AAC G T CCAG GC TTC ATG C C TT AC GC C C A T GC TCT CA T CTG A CATCC C G G ACG T T CAA GCGCG CCTATC CGAA CAAATTT C C CCGC CA CCCAAC CTC TCGTG A GAGT ATCCA ACCCTC G G TTCCT GT AC TATG G G CC TCT CGA T TA ATG TA GAT GT C A G CGC A AT G T CG AC CAG CTC GAC CGT CAGA CTG GACGA CCGACC GAGCT A CCGCTC TGC TG GG GG TG TG T CG GT T GT C T GT GA T GCC GT G G GA G GAG TTT GG A T GTCG GAA A T ACGG GAA T TTACTG A C C T A G G C C G A A C T T C A G T C G C T C C T A C G A C G A T G C yvty y y a h vth vth v e hgilae hgilae hgilae h38 48 58 68 78 88 98 1 - -.v 9 9 - -,1 1 9 9 6 SI3SI4 1 SI5 1 SI6 64 R R R R3-548 8-63 94 ATAGCCGCGGAGGATAAGCGACACTCTCGTTAGG GTC CATACTTACGGTGGA CTTCCC CCCTCGATA TCC GGTGGCTTATCCCACGCCACCCGTGGGAGAGAC CCG AGCT TAA CGGCTTT T GAGTACGGGTATTCGATGAGTAAAGCCACCCGTTGATGCAATCCCAGCTTTGTCC CG GGTGAA CG A GAG CT T C CCTT C GTTCC AGG TAGG T C GCCTTG CGG C GGGA TCGA GGGCACC G T TCACG A TTCT AT G ATTATC GTGACCTCA G GGTT G GCC C T TAGG GGGCCC G A CTCG AC T T GGGCGG GC TACC CGC TGGAG C A TTAAT C A TACTC C CATCC G T TTC CCG G C CTC CAAGC TC T CC TACCG G T C CACT G G CT C CT G GTCT CCG CTGA CCCCA A CA CGT A A T CGCTC A CGGA C T GC TA AC C GT AAG ACTC GT TT AC ACA T CAAT C CACGC C T TG ACAT T A AGG CGCG GATT CCG CCCC C T CCCAT C CTGGTCGTG CC T G A CT ATGC G TCTGC ACGCTT C GCCG TTC T CT A GCAG C GC CGCGCGAGT C A ACGCATG CT GAG GC CGCTT T GTC A CG G AA CTC CGCGA AA AG CG GTGA GT CC AGC AGCA CGGT C A CGG TG AA T GC G AC G GGTT GAG G G GT TG T A GA C GT GG C TA T T A C T CGT GT TGC G A CC GTG GGA A T CC GAA G TGA GA GAATT TGATTG GACGG T A G G C C G G G C C T A C G C C T A G G G T C A A T C T A A C A T A G C A t y h vtyvtytgila h egi a h v hlegi a h hlegi hl09 19 29 39 49 59 69 1 - -.v 0 -,2 0 0 6 SI1 2 SI2 2 SI3 64 R R R3-548 8-63 94 G AGG TC TT TTC AT CCCGAAA GGGGTCA CTT GG CCGAAT GGG TCCC AC CCGCG GAAC C A GGCCAAG C GT AA GTG C A T TG T T TA C T GC GG TC CG GC G CG TTCG G GG CTA GAGT C G GGA TA GGATG ATC A C A C C GC CAT T TT AGAC A C TT AG C A G G TTA AAGAGGCG C C G GCT ACC G GCGCTC ACCGG GCGATA ATT ACAG ACGCTTCCATCAT GCGTCC A ACC G GCG G TCTC CAG CCG TCCCACAAGCC C G ACCATTAG CATG C G CACTTT CTG GA AG CACCC TGTTGGC G G CGCCCT C GT CC C TA T G T GAA TC TC A TCGTA GATGTGAGGGA ATG TCG ACG AT GTTC C C C CA C C ACGCAG CG AC A T GA CCGTCCG TG GG TTC ATG T T T G T C TG G CACTA A CCGC C TTC TACGGGCGAG TCAACGGCGTCA G TGCGT A T GCGCTT ACA GGGTATTACTC AGCCCATGTTACTC GACGT TTAAGG G G T T A A C T C G A T G T T G G T T C C G A C G C T A G G G C G A C T C yvty a h vtyvtyvegi a h hlegi a h hlegi a hle h7 0 1 2 39 89 99 01 01 01 01 1 - - - -.v 0 0 2,2 2 2 2 6 SI4SI5SI1 2 SI2 64 R R R R3-548 8-63 94 AG GTGCGCGAAA ACCGTACCTAGATCTCTACTTGGGTTCCCCCGAACTGTGCAATCCCTA ACTCGGAGCCGGCAAT TGTGTTGGTAGGTATCATAGAAGCACCG TGTTT A C CT AT GACC CCTATT TGTTA AT CC TGACT CAGCC CC TG T T GCCC AT CAGCCCAACAA TACAC AACAG TGT A A TG TGTATC TC C GC T CCTC CCGGGC CCTCG CCGGTCACTGCTC CC G TTGTG TGGC AGCTCT C TTTCGT CAT C TCTT TAAG AGGCC TTTACGGTCT TTCC TG T ATTCAT T G TCTT TG GCTCC ATACG TTTAT TG G AG GTC T ATAC CTCTA C AAGC T CAC CCGA C T CG CCGAACGTGGC C AAGCA GC C GTGGA AGGGGA CCT TACC G TTC A GGGAG A T T C C A GC AC TC G CA C G CC T CTACT G GTG C C AG G A G T T CCCAG TG TA G CAAGCACCGG T GCATT A GAGG GG ACCGG GC CT ACAAT GAG T GC CTGACCGG GCGA G ACCGG GCG G ACTC GCG CCATG CAG TT CATGT CATTCG ACAA TTCAGCCTAT CCTCCC CCT CAAA C CC T CACAACTCGC ATCTAC G G CC TCGC ATGTT A A G TT G C T CC ATGTTCCGGTG G ACAATT TC CGATG T T ACGCAC CGCGG AT GA ACGCAAGAAGT CCGAAA GAAGA CCG TC A CCG TGTGCG CGCTA T CGT C C GG G GT TG T C C GGGAC A G G G GA ACA G GA GT TTGCGG GACGG TTACTG GAATA TTACTG T A G T C C G G A T G T A G T A C G A T G G T C A A T C G A T G A t y y y h vth vtv gilaegi a h hlegi a hle h 40 50 6 7 8 9 1 1 01 01 01 01 1 -.v 2 - 2 2 -2,6 SI3 2 SI4 2 SI5 64 R R R3-548 8-63 94 A GCGGG GGGTAGCCACGCACAGTAC GTG AAG CTCGGTAGCCGTCAGA TCT CCGGCGCTA CACCCCCCGGGAATCTGCC AGTCACTCAGGCAACCCGGG AGCTGATAG CTGATC GAG CTACGGAA CATTCAGGAAA CGG GACCG GATTGCAACCAA CTGT T C TGGCCTG ATACGG T G GTTAC T AG TAAGATT C T G G CCTCATGAGGT AGTG TCCTCCTC GAAGTTGC CC GGT TTC C C CTA CCGCAC AGTTCCTACCGCT T AA TTCCTCCCC CA A TGG TA C GATTC AAG CGTTCGAA TTGA GG T T GT G TCTGC A CGCCAGT C T C C G G G C GAC GGGTGTCTGGCGG CCCCGA A T CA GC TTA TCCCGAGTT C TC G CACACG GCGAGA ACAA GC AAC GCGG AGTACACGGCGCTCACAGA CCAG GCT TCG ACTCC GCG T C GACCATGCGAGTACAACCACGT CAT CA AC CAT CCCACG A AG ATC CCGC CAAG C A CTA CCA CCG ACG C C CACGTCTGA CC T ATG TT A GCCG TTT T CC A ATATTCG ACC ATCTGAATGTCCGC TC G CTA ACGATT G GT ACGAT CT GCTCAT GTCG A C C ACC CAC GC GCT CTG A GA G G C GGGA CCGC G GT CCGCTTGAGCGCC GGC AC G TA G TG A G C CGGT G A C CG GAT G C G T C T GA GTGAT ACT GTG T G GTG TG TCT GTCGAA T T AC C GAATA T AA GGAA T CGGAACA T A C G A C G A T G A T C A A A C G A C G T T A C G G C G G G A G T C A T T t y h vty g h vty h vth ilae hgilae hgilae hgil01 11 21 31 41 5 6 1 1 1 1 1 11 11 1 - -.v 2 -,2 3 3 6 SI6 3 SI1 3 SI2 64 R R R3-548 8-63 94 CC CTC GCCAAC CGGGAGAAGCCG TCCCTGCTCCCCTCTGGGCAGCAGCGTATTAGGCGAATGTCGCATAGGAGGCATCCCACACCAGGGGGAGGGGCTGTGTGAAACA GTG AGACAC TTCATGCTTCCCTTGCAAGA ACGGAGT A GG G GGTGTACTTC ACG TGCTGGTTTACGC AAG GAGTCGCTCGA CAGC A TAGT T TTCCGAG CAGC G A TAG TCG TC GC ACCTC T A TATTC CAAGGGTTTCGGTCAG C CCA G CCT TTAGTCA C T C G CA G CACA CTT TCTT GCATT ATGTTC AACA TGA TGCAC C TGGTA C T TGG GGCGT ATCC AGC T A TTACA T GCGCACC G T ACG T GCGATCTCAGA GC GTGCA CTT CGC CAGG G TT CAGG GCT AGA G C CG GCGA ACAA T GC C GAA TT GC A ACAAC GC A TCCGCT G C G T CAGAG C A CAGCTG C TC ACT ATC CAGC A TC CGGCC AA A C CAATGGCT TTGC A CC C GG AA CC TTGCCAA CC CA AC CT C A ATATG TCA G TACTC TC TGGCAC CATGT G CT G CA GCCT A GCA T GT A G CACCGGC C GT G GATT AC G CCCGCCCCG T GTCG TAC C CCCG CCGGCC A G AC A G GGGTGAC G CTGCAAGGCGGGTG TTGAAAGGCGACA G C TG GA CC GT CG GT GGG A GT TTC G ACGTGTTAGGA A C GC CG TTCT T G CG C TG C CG T G ACA CA C T A C G A T G A C T T C G C T C T T G C C T C C G C C G G T C A T T C G G G A T yvtyvtyty a h h v h v e hgilaegi a hlegi a hle h 71 8 1 1 9 1 1 0 1 2 1 1 2 2 1 2 3 1 21 1 - - -.v 3 3 3 -3,3 3 6 SI3 3 3 SI4SI5SI6 64 R R R R3-548 8-63 94 AGGGGGAGTGAGGCAACCTAGGAGTACCTGG AAACCCTGG ATT GCCGTT AAGTATGCGGTCTACCCTCCTGCCATTTGACTGCGGTGGCTTTCGGGAAAACCACGCCAGATTGTT ATTATG GAGAGCCGC ATCCATATCCGGAATGGCACGCGTT ATGTAGC CTCATATGGGTTTTC C G CTT A G GC GTC C A GCG GAG C A GAGT C ATTA ATCT GGG A TAC G ATGC GACCCGCCT TC T C GC G TCG T GCAAG TC C G TTCTGAG C GGC C GAAA CTG A A TA GGTGG T AACC CACCA GGGAGA CC ATT CTTTCCAAG TT T C C GG TT C C CGC TT G CA TGACAAC C GCGATT CA C G T ACCAG GCATG GGTT AC CAGT G GGG G C ATC C ACAACACTC GC CAG A C GC C A AT GCG TACAGCCACAT T CAGC TC T GAGT AA T TCC A G AAA ATG CC GA CATCT CC GC CACTA C CGCCGT CC GG A A TTT GCGGC CCCTCG GCGAA CCAATC GCTCCTTC T C C A T A T TCA G A TGATG A AAAC TG CT A TTG T CCGC AC C C C AC T C C CT ACGAAA CGCGTGAGGA TC CCGGC TCC GGGT CCGATA G TTC T AC A GCAAC G T G GAA CCGC C G GGTGACA GCG TG ATA T GA GCCCTA GC C T T AC TGA GC G TG GCGCGTTGCGA A T AG T TT ACGTG TTAATC GAGTC T C A A T C G C C G G T G G T T C C G A A C G T G C C T T C G A C G C T C A C A t y y y h vtgila h vtegila h vtegi a h egi h hlhl42 5 6 7 8 9 0 1 21 21 21 21 21 31 1 - - -.v 4 4,3 3 4 6 SI1SI2 3 SI3 64 R R R3-548 8-63 94 TA CGCC T GC GAGA AG GACGCCT CGGCACTCGTGACAACCC GTGAGCACCGCC TTT CGA GCGTGC CCTACA AAG CGA GAACGTCTC CCAAA CTGAA GGAAT AAGC TAG C GACAAC CT TAGGC TGTCT CT GT TCATT CTGAAGCC G ACT TCGA G CCT TCTAA T G TAA GA TCGAC AGGGACG GGGG T TTTA GAATACT AC AAATA CCG TG A ACGTTGGGAAC CC AGGC C ACG TGACA CC G GA ATTACT CGACT TC C CG GTAAA CACAATG AA TCAT AG T G ATCAGGGG CC GCC GTGA T G G GGGC C TTCG G GAGTCG GATGAAATCCTGCA TAT TT ACTG TCGCT T TTA ACC GA TA A GT CATATA AC AC GGGCCGTA TT ATGCC G CTT AATC 4CCC ATT A CG T CGC TCAT T A A CCTCGC CGCGA CAG 9 CGTTTGT CA AA TCC TA ATAGT CATC TG GCGA TCA GACACA ATA G ATA GC AT GGCGT C CG GG CTTGAT GCAAGTGCCA CA TTC A GATAAG CAGCA C AAGC T AATC A TTCGGAA GT GAT AC TG G CAG GGCCGC GCTA CCA AGG CGGTAA CT C GTA T CGTCGATA G TGGC ACTGAC TG CGCCTACTGCGA T TCG TAT T T AAC AGAAG G CAGG AATCTCCGCGC CT TGG CCCCTC CC AA T CCT G CCC CTTTTTG GC A TC TGTCGT A ACGTGTG TTGA T T TAAA TG TCATTCA AACTGT TT A TGCC ACGGGA TGC ACC GTTAACC CGC TCTAA CGG TCCCGATAG A GACCC ATGGGA GAC CGAGG G T T TATG G TTGG TGC CCTTT GCCGG C TT TGGTGA CATGC CT C G TT T CACTG TCACT CTCTG G ACGA G GTCG A CAGAC ACAGGCGGG ACAGC GCGCAA A ACCAGATCCCTCCG CAAAC T C CCTTTC CA CATC CATCC CATCTAA GCTCT CT A GCCA TTCA ACGCTG TTCGAACAATC GCGAT AAGC G G T TCCGATTC AC G C CG G A A G AC G G CGG TG G C A CG C C GG CTC TG GA GAGTGG TGGTTTGCGT AGTC T ATTT AA C G G G CA C G A A T C T C A C A C T A A C C T C A T T yvty a h vth egilaegi h hl13 23 3 4 1 1 31 31 1 -.v 4 -,3 43 6 SI4SI5 64 R R3-548 8-63 TABLE 2 – PROTEIN SEQUENCES FOR ANTIBODY VARIABLE REGIONS Clone SEQ ID NO. Chain Variable Sequence Region ATGVHSQVQLQESGPGLVKPSGTLSLTCGVSGGSITNNNWWSWVRQSPGKGLEWIGEIYHS YY Q G S A S V R S Q Y SG Q ES S G S G S ET SG M S TG V S TT A G

[0075] 954936-8845-3466, v. 1 ATGVHSQLQLQESGPGLVKPSETLSLTCTVSGGSISSSSSYWGWIRQPPEKGLEWIGSIYYSG 155heavySTSTSPSLESRVTISVDTSKNQFSLKLNSVTAADTAVYYCARHRHYYESSGFRRPSDSFSIWGQ GTMVTVSSASTSX RIS03-3 LG G I R G Q ES G A S GS G A G S G SS T S T A N A R N A R N A R

[0076] 964936-8845-3466, v. 1 ATGVHSQVQLQESGPGLVKPSETLSLTCTVSGGSISTYYWSWIRQPPGKGLEWIGYIYYSGST 177heavyNYNPSLKSQVTISVDTSKNQFSLKLSSVTAADTAMYYCAKESWNYYGSGSTFDSWGQGTLVT VSSASTSX RIS13-4 Y G P Y G G S Y T AA S G A T G A S D S G SA S S G RA R V A S G Y

[0077] 974936-8845-3466, v. 1 ATGVHSQLQLQESGPGLVKPSETLSLTCTVSGGSVSSTAYYWGWIRQPPGKGLEWVGTISYT 199heavyGSAYYNPSLRSRLTISVDTSKNQFSLKLSSVTAADTAVYYCARHLDSSGYYYVRSFDYWGQGT LVTVSSASTSX RIS17-5 R N VS LG T SA A S VT S G S Y G GT L Y GT Y A S V N T T A E S R S W Q

[0078] 984936-8845-3466, v. 1 ATGVHSQVQLQESGPGLVKPSQTRSFTCTVSGGSISSGDYYWSWIRQPPGKGLEWIGYIYYS 221heavyGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGSYGPYFDYWGQGTLVTVSS ASTSX RIS19-5 RA S S R N VS A GT LV ES N S R G Y S S Q Y T G Q R GS SS R N L R S Q S

[0079] 994936-8845-3466, v. 1 ATGVHSQVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGS 243heavyTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASLRFSVSVGYYYYGMDVWGQGTT VTVSSASTSX RIS22-5 A S M S T S AT S S G G S Y S AS Y VA GI T S G V Q GI T LG T S Y T T ES

[0080] 1004936-8845-3466, v. 1 ATGVHSQVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWNWIRQPPGKGLEWIGYIYYSGST 265heavyNYNPSLKSRVTISVDTSKTQFSLKLSSVTAADTAVYYCAAGGSQFLPPLHWGQGTLVTVSSAST SX RIS34-4 S D W QS

[0081] 1014936-8845-3466, v. 1 N 04 04 04 04 04 04 04 14 14 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 DI4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 ITS G W H HV G Y Y N Y W K S Y Y N N Y N S N Y Y Y Y Y S Y Y S Y Y S G N AF G Y S N G G S G D Y S N G Y 1 G Y N Y SS S Y S G S S S S S WS Y GS Y GS Y GS Y D S S S S YS SS RTIN S SINF K G N FIYVS S SF YFSIS S V N SIS SIN S D G S F S G S G G GIS SISISD ISLIWSIHF SF G D T V T LISS D GISS S S D G DISS S G LS T V F S C G Q G Q G Q G Q G Q G Q G Q G Q G Q G Q G Q A Q niayvyathvyathvyathvyathvyathvyathvyathvyat vy y yat vat vat vath C e h gile h gile h gile h gile h gile h gile h gile h h gile h h gile h h gile h h gile h h gil1..v,O 9 N 6 0 2 7 1 2 7 2 2 7 3 2 7 4 2 7 5 2 7 6 2 7 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 66 2 72 72 72 82 82 82 82 82 82 82 82 82 82 92 92 92 4 3D-I548 e - n 1 - 0 1 -8- 0 1 - 0 1 - 0 1 - 0 1 - 0 1 - - - - - 6 0 10 30 30 30 3 3 ol I1 2 3 4 5 6 7 8 1 2 3 0494 CSRISRISRISRISRISRISRISRISRISRISRISR Y Y Y Y N L K NK NK A S Y Y Y N YY A Y N Y Y D F Y Y YY Y Y N D Y D Y D Y N Y N S S N S N G Y S S S D V Y Y G N Y G N D S D S D SYR G Y G N Y N S WD W S HSDIS RV S N S S S S S S S STSY S SY S SY S G S S S S FNIG T S F S FS MAFIRS GISS TF LR GISS S GISIS F S F S F SSILSILISFSIT F SSIS D SY V S S G V S S G V S S G V S S G V S S G VS SA V S S G V S S G V S S G VS G Q G Q G Q G Q G Q G Q G Q G Q G Q G Q G Q G Q G H G Q G Q yvyvyvyvyvyvyvyvy y y y y y yath atatatatatatat vat vat vat vat vat vat vate g e h h g e h h g e h h g e h h g e h h g e h h g e h h g e h h e h h e h h e h h e h h e h h e h hililililililililgilgilgilgilgilgilh gil1.v,39 4 2 9 5 2 9 6 2 9 7 2 9 8 2 9 9 2 9 0 2 0 1 3 0 2 3 0 3 3 0 4 3 0 5 3 0 6 3 0 7 3 0 8 3 0 9 3 0 0 6 3 1 1 3 1 2 3 1 3 3 1 4 3 1 5 3 1 6 3 1 7 3 1 8 9 0 1 2 6 3 13 13 23 23 23 4 3-548 - - - - - - - - - - - - - -8- 3 9 9 9 9 9 3 - 6 0 3 3 3 3 5 5 5 5 3 IS5 0 IS1 0 IS2 0 I 3 04 05 1 1 1 2 1 3 1 4 1 5 1 1 1 2 1 3 1 494 R R RSRISRISRISRISRISRISRISRISRISRISRISR Y Y Y Y N Y D R N Y N Y N Y K N R Y Y Y G Q Q N Y S Y G Y Y N N G N YS Y Y A Y F N AT Y Y Y D N D D T N G S G G N Y Y P S Y G N Y V S Y Y S S N Y F S Y F S H Y G S V T S N F S S S S N F S G S S S S SF YL V S S S S K SSIH S GS Y S S S N D S S WS N N SS LSIS F SSIYL S H A YL V SSIR S R GISS SISA G S V S V S V S V S L S V SISS V FT LL F V SIVS S G S D S G S G S Y S S G G G S F SITS G S G V FTF VS G Q G Q S Q G Q S Q G Q G Q G S Q G Q G Q G Q G Q G Q G S E G Q yvy y y y y y y y y y y y y yat vat vat vat vat vat vat vat vat vat v t v t v t v t v te h h g e h h g e h h g e h h e h h e h h e h h e h h e h h e h a h e h a h e h a h e h a hle h a hle h ilililgilgilgilgilgilgilgilgilgilgigih gil1.v,32 4 3 2 5 3 2 6 3 2 7 3 2 8 3 2 9 3 2 0 3 3 1 3 3 2 3 3 3 3 3 4 3 3 5 3 3 6 6 3 3 7 3 3 8 3 3 9 3 3 0 3 4 1 3 4 2 3 4 3 3 4 4 3 4 5 6 7 8 9 0 1 2 6 3 43 43 43 43 43 53 53 53 4 3-548 - - - - - - - - - - - -8- 51 7 7 7 7 7 7 8 8 8 8 8 -9 -9 -9 63 IS5 1 IS1 1 IS2 1 3 1 4 1 5 1 6 1 1 1 2 1 3 1 4 1 5 1 1 1 2 1 394 R R RISRISRISRISRISRISRISRISRISRISRISRISR Y Y T A Y Y D Y S Y S Y N F Y S D Y W Y G YY H Y Y Y Y Y Y Y Y Y Y Y Y D D GS N G S G Y SL A D N YS Y G DS SS N G N Y G N G G N G A YS Y D NSIS SF S S R R R S S S S SF Y M S S GSIS S WS S S S SSIS FTFRIS S S RSIN F G F S FISS V S S FS S S S FS FS S FSISS S A G VS G VS SY VS SF VS NL VS TF G G LS G VT G VS G VS GISS G VS G G G VS G Q G Q G Q G Q G Q G Q G Q G Q G Q G Q G Q G Q G Q G Q G Q yvatyhvatyhvyathvyathvyathvyathvyathvyathvyathvyat vyat vyat vyat vyat vate h gile h gile h gile h gile h gile h gile h gile h gile h gile h h gile h h gile h h gile h h gile h h gile h h gil1.v,35 4 3 5 5 3 5 6 3 5 7 3 5 8 3 5 9 3 5 0 3 6 1 3 6 2 3 6 3 3 6 4 3 6 5 3 6 6 3 6 7 3 6 8 3 6 9 3 6 0 3 7 1 3 7 2 3 7 3 3 7 4 3 7 5 6 3 7 6 3 7 7 3 7 8 3 7 9 3 7 0 3 8 1 3 8 2 6 3 83 4 3-548 -9 - - - - - - - - - - - - - -8-6 1 91 91 0 0 0 0 0 2 2 2 2 2 2 3 3 IS4IS5IS6 2 IS1 22 23 24 25 21 22 23 24 25 26 3194 R R R RISRISRISRISRISRISRISRISRISRISRISR TS T G G TSTIT T TS T TI6 PS 01 S G G T N S S S G S T S S G G G S G G D Y S R M A H A S A S S S S T S S A S S S S AIFWS Y T WIYA T AIYA Y AIYG Y Y A Y LIYWIYGIYA Y S AISA V yvatyhvatyhvatyhvyathvyat vyat vyat vyat vyat vate g e g e g e g e hg e hg e h e h e h e h hilhilhilhilhilhilh gilh gilh gilh gil71 81 91 02 12 22 32 42 52 62 7 8 9 0 1 2 3 4 5 6 5 5 5 5 5 5 5 5 5 5 25 25 25 35 35 35 35 35 35 35 Y Y N Y Y Y K N N Y R N K Y WN Y N YY YY Y N S N N TS N N S Y G V Y N YY NS Y Y S G E S G S G S S Y Y Y S Y S SY SY S Y S WR H N RY S Y S Y S Y ITS SAIS SIS LNIS LRIS S SSIS SISV LSIS LSIN S S G LS G L S S G V G G D LS G LS GIS SIS S N S L R GISY TFISS G Q G Q G Q G S L G Q G Q G Q G Q G Q G Q yvy y y y y y y y yat vat vat v t v t v t v t v t v t v te h h gile h h gile h h g a ile h h g a h a h a h a h a h a h ile h gile h gile h gile h gile h gile h gil1.v,38 4 5 6 7 8 9 0 1 2 3 4 5 66 3 83 83 83 83 83 83 93 93 93 93 93 9 6 7 8 9 0 1 2 3 93 93 93 93 04 04 04 4 3-548 - - - - - - - - -8- 3 3 3 3 3 4 4 4 4 - 6 3 3 3 3 3 3 3 3 3 4 3 IS2IS3IS4IS5 6 1 2 3 4 3594 R R R RISRISRISRISRISRISR * * * * * * * * * * * * * * * * * All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.

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[0089] 1144936-8845-3466, v. 1

Claims

WHAT IS CLAIMED IS:

1. A recombinant antibody or antibody fragment comprising clone-paired heavy and light chain CDR sequences from Table 3.

2. The recombinant antibody or antibody fragment of claim 1, wherein said antibody or antibody fragment is encoded by clone-paired nucleotide sequences from Table 1.

3. The recombinant antibody or antibody of fragment claim 1, wherein said antibody or antibody fragment is encoded by nucleotide sequences having at least 70%, 80%, or 90% sequence identity to clone-paired nucleotide sequences from Table 1.

4. The recombinant antibody or antibody fragment of claim 1, wherein said antibody or antibody fragment is encoded by nucleotide sequences having at least 95% sequence identity to clone-paired nucleotide sequences from Table 1.

5. The recombinant antibody or antibody fragment of claim 1, wherein said antibody or antibody fragment comprises heavy and light chain variable sequences comprising clone-paired amino acid sequences from Table 2.

6. The recombinant antibody or antibody fragment of claim 1, wherein said antibody or antibody fragment comprises heavy and light chain variable sequences having at least 70%, 80%, or 90% sequence identity to clone-paired amino acid sequences from Table 2.

7. The recombinant antibody or antibody fragment of claim 1, wherein said antibody or antibody fragment comprises heavy and light chain variable sequences having at least 95% sequence identity to clone-paired amino acid sequences from Table 2.

8. The recombinant antibody or antibody fragment of any one of claims 1 to 7, wherein the antibody or antibody fragment binds neurons.

9. The recombinant antibody or antibody fragment of claim 8, wherein the antibody or antibody fragment binds a cytoplasmic target in neurons.1154936-8845-3466, v.

110. The recombinant antibody or antibody fragment of any one of claims 1 to 9, wherein the antibody fragment is a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab′)2 fragment, or Fv fragment.

11. The recombinant antibody or antibody fragment of any one of claims 1 to 10, wherein said antibody is a recombinant IgG antibody or antibody fragment comprising a modified Fc.

12. The recombinant antibody or antibody fragment of claim 11, wherein the Fc is modified to alter FcR interactions, alter glycosylation pattern, or to increase circulatory half-life.

13. The recombinant antibody or antibody fragment of any one of claims 1 to 12, wherein said antibody or antibody fragment is conjugated to a detectable label, a toxin, a drug, or a prodrug.

14. A cell expressing a recombinant antibody or antibody fragment of any one of claims 1 to 12.

15. A method of identifying a subject having radiologically isolated syndrome (RIS) that is at high risk for developing multiple sclerosis: (a) determining an antibody somatic hypermutation (SHM) frequency of plasmablasts isolated from an RIS subject; and / or determining a frequency of neuron-binding for plasma antibodies isolated from the RIS subject; and (b) identifying whether the RIS subject has high antibody SHM frequency and / or high frequency of neuron-binding of antibodies compared to a healthy population as at high risk of developing multiple sclerosis.

16. The method of claim 15, wherein SHM frequency for VH4 antibodies is determined.

17. The method of claim 15 or claim 16, wherein a high SHM frequency is at least 75% of the antibodies from isolated plasmablasts, or at least 80% of antibodies from isolated plasmablasts.

18. The method of any one of claims 15 to 17, wherein a high frequency of neuron binding of antibodies is greater than 3.5% or greater than 4%, or greater than 4.5%.1164936-8845-3466, v.

119. The method of any one of claims 15 to 18, wherein amino acid sequences of antibody variable regions are determined, and optionally VH usage is determined, from a population of plasmablasts, such as by sequencing of genomic DNA or mRNA of said population.

20. The method of any one of claims 15 to 19, wherein determining neuron binding comprises assessing binding to a neuronal cell line.

21. The method of any one of claims 15 to 20, further comprising treating a subject with an immunosuppressive therapy or multiple-sclerosis disease-modifying therapy, when the subject is identified as having a high risk of developing multiple sclerosis.

22. The method of claim 21, wherein a subject identified as not having a high risk of developing multiple sclerosis is not treated with an immunosuppressive therapy or a multiple sclerosis disease modifying therapy.

23. The method of any one of claims 15 to 22, wherein an RIS subject identified as having a high risk of multiple sclerosis is actively monitored for signs or symptoms of MS.

24. The method of claim 23, wherein the subject receives frequent MRI scans.

25. The method of any one of claims 15 to 24, further comprising performing steps (a) and (b) at least a second time to determine a change in the frequency of antibody SHM of plasmablasts and / or VH usage, or determining a change in the frequency of neuron binding, optionally where steps (a) and (b) are repeated at least once annually or at least once every other year.

26. A method for making a pharmaceutical composition for providing neuroprotection, comprising: cloning and expressing recombinant antibodies or antigen binding portions thereof that are derived from plasmablasts of one or more RIS subjects, identifying a recombinant antibody or antigen binding portion thereof that reduces or inhibits apoptosis in neurons, and / or reduces or ameliorates neurodegenerative disease or neuronal loss in an animal model, and formulating the identified antibody or antigen binding portion for delivery to a human or animal.1174936-8845-3466, v.

127. The method of claim 26, wherein the antibody or antigen binding portion has high SHM and / or has neuron binding activity.

28. The method of claim 27, wherein the antibody or antigen binding portion binds to a surface target or a cytoplasmic target of a neuronal cell line.

29. The method of any one of claims 26 to 28, comprises measuring binding between the antibody or antigen binding portion thereof and the cytoplasmic target or epitope thereof by one or more of ELISA, surface plasmon resonance (SPR), and biolayer interferometry (BLI).

30. The method of any one of claims 26 to 29, wherein inhibition of apoptosis in neurons is determined in vitro using a mouse or human neuronal cell line.

31. The method of any one of claims 26 to 30, wherein reduction or amelioration of neurodegenerative disease or neuronal loss is determined in an animal model, which is optionally an EAE mouse model.

32. The method of any one of claims 26 to 31, further comprising quantifying internalization of the antibody or antigen binding portion in neurons in vitro or using an in vivo animal model.

33. The method of any one of claims 26 to 32, wherein the antibody or antigen binding portion thereof is formulated so as to be expressed in neurons via gene or mRNA delivery.

34. The method of any one of claims 26 to 33, comprising formulating the antibody or antigen binding portion thereof for parenteral administration or intranasal administration.1184936-8845-3466, v. 1

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