Astrocytic monomeric, oligomeric, or aggregate breakdown products of glial fibrillary acidic protein

Abstract

A method of diagnosing a disease in a subject is provided that includes sampling from the subject and the detection of at least one of astrocytic monomeric-, oligomeric-, or aggregate of a GFAP breakdown product (G*) in the subject as a ratio of at least different two molecular weight G*s or a ratio of the G* relative to intact GFAP from the subject. A diagnosis of the disease or severity thereof in the subject is provided based on the detection. A composition is provided that includes an antibody or an oligonucleotide that binds with a degree of specificity to an N-terminal of GBDP38K, a C-terminal neoepitope of GBDP38K, or a conserved core domain of GBDP38K and GFAP.A method of treating a disease in a subject is also provided.

Description

ASTROCYTIC MONOMERIC, OLIGOMERIC, OR AGGREGATE BREAKDOWNPRODUCTS OF GLIAL FIBRILLARY ACIDIC PROTEINFIELD OF THE INVENTION

[0001] The present invention in general relates to neuro injury, neuro disease, and neuro repair and in particular to a glial fibrillary acidic protein (GFAP) proteoforms, including GFAP breakdown products (G*), as diagnostic biomarkers or therapeutic targets.BACKGROUND OF THE INVENTION

[0002] Traumatic brain injury (TBI) occurs when external forces, through direct impact or acceleration, traumatically injure the brain often through falls, vehicle accidents, and violence. TBI can be characterized by its severity, from mild to severe, and the effects of such injury can be physical, cognitive, social, emotional, or behavioral and clinical outcomes range from complete recovery to permanent disability and death. TBI is a leading cause of mortality and morbidity around the world with a broad spectrum of symptoms and disabilities. There are approximately 1.7-2.0 million incidents of TBI annually. Among all ages, unintentional injuries are the fourth leading cause of death, with over 136,000 lives lost annually. Millions of others suffer a non-fatal injury each year. Injury can also manifest in the form of neurodegenerative and neurological disease(s). For example, TBI is also a risk factor for Parkinson disease (PD), Alzheimer’s disease, chronic traumatic encephalopathy (CTE), epilepsy, Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS), frontal temporal dementia and other forms of dementia. However, to date, there are still no FDA-approved therapies to treat any forms of TBI, including treatment for repair.

[0003] More common methods and areas of continuing research for clinical diagnosis of injury, disease, and repair usually involve an examination and assigning a score to an individual (e.g., Glasgow Coma Score or GCS for TBI and expanded disability status scale or EDSS for MS). These methods are of limited value and often preclude a nuanced diagnosis due to the subjectivity of the testing, and the ability of a patient to knowingly alter their true response to achieve a desired result. Neuroimaging, such as computed tomography (CT) and magnetic resonance imaging (MRI) are also widely used to help determine the scope of injury or disease and potential for intervention for repair. However, these tests are both costly and time consuming, as well as frustrated by the same problems of having an inability to distinguish one injury, disease, and repair type from another. In addition, individuals with only mild or moderate injury or disease may be unaware damage has occurred and fail to seek treatment forrepair. Likewise, individuals (and their physicians) might be unaware that their injury or disease is beginning to become refractory or responsive to a current repair treatment and thus should or shouldn’t seek new treatment options, respectively. It should be appreciated that repeated mild to moderate injuries (e.g., repetitive TBI) or diseases (e.g., multiple relapses in MS) can have a cumulative effect and result in a prognosis of poor repair and poor clinical outcome.

[0004] Early clinical diagnosis of injury, disease, and repair continues to be an area requiring further development. Early diagnosis can minimize injury and disease and maximize repair by facilitating earlier intervention. Much emphasis is being placed on developing biomarkers as early indicators of CNS injury, disease and repair. For example, upon injury to or disease activity in the brain, otherwise isolated brain-derived proteins are released into the interstitial fluid of the brain and eventually cross the blood brain barrier where they can be more easily measured as peripheral biomarkers of injury, disease, and repair. Identifying specific proteins and measuring the concentrations (levels) that enter circulation before or after the onset of clinically observable injury, disease, or repair can provide an effective early means of detecting the phase, severity and type of the injury, disease or repair, as well as provide various clinical and medical utilities as described below.

[0005] There has been progress in understanding the cascades of brain proteins the cross the blood brain barrier (BBB) into the blood as biofluid biomarkers after TBI (1-10) and therapeutic targets for brain injury, these have still not become the standard of care. Recent findings suggest that therapeutic reduction of biofluid biomarkers, particularly pharmacodynamic (PD) biomarkers for tracking patient response, as a promising new drug development path for TBI treatment.(l 1-13).

[0006] Astrocytes are a major cell type in the central nervous system (CNS) and the peripheral nervous system (PNS). Astrocyte stress, compromise in structural integrity, injury, or death in the CNS, including the brain and spinal cord, as well as the PNS, can be a result of mechanical forces, or neurochemical and other unfavorable brain macro- or micro- environmental factors (e.g oxygen, nutrient deprivation), or endogenously or exogenously derived neurotoxin exposures.

[0007] Glial fibrillary acidic protein (GFAP) is the major component of the cytoskeleton of astrocytes-with a guiding role in elastic retraction and extension. (17) The sequence of cellular and molecular events known to occur in TBI include: TBI induces the release of an early, high concentration wave of glutamate into the extracellular fluid that exceeds the buffering potential of glutamate transporters such as astroglial GLT-1. High glutamateconcentrations in turn cause the degeneration of neurons and astroglia. Under these TBI conditions, calpain cleaves GFAP and generates GFAP breakdown products (collectively denoted as G* or G*s, or singularly denoted as G*). Of particular note is a 38 kDa G* (GBDP38K) in the injured cortex during the first hours to 30-90 days post-injury (dpi) in mouse TBI models (18, 19). This is shown in prior art FIGs. 3A-3F. In parallel, astroglial GBDP38K is released into the extracellular fluid and other biofluids (CSF, blood) to more than 28 dpi with a peak at 1-2 dpi.( 15 , 16, 20) Other protein “debris” is also released from neuronal cell bodies (e.g., ubiquitin C-terminal hydrolase 1 ) and injured axons (e.g., neurofilament light / NFL, microtubule associated protein tau / tau, and phosphorylated tau / p-tau). (21-24) In turn, tau and other protein debris exacerbate glutamate cytotoxicity.(25) Clearance of accumulating debris is performed by cellular phagocytosis (e.g., FcyR + microglia) and by the expression and perivascular localization of the astroglial water channel aquaporin-4 in the glymphatic system.(14, 26)

[0008] After TBI, injured astrocytes release large amounts of GFAP and G*s into the interstitial fluid, from whence these proteins enter the blood. This process is initiated by the TBl-induced disruption of calcium homeostasis, followed by wide-spread activation of calcium-dependent proteases, including calpain (135) and release of an early, high concentration wave of glutamate into the extracellular fluid that exceeds the buffering potential of glutamate transporters. High glutamate concentrations in turn cause the degeneration of neurons and astroglia.

[0009] GFAP, mainly in the form of GBDP38K and other larger, less abundant G*s which share the N-terminal neo-epitope and core of GBDP38K), is the most abundant protein released into circulation after TBI.(27, 28)

[0010] The brain may mount a self-protective immuno-response after TBI in some patients, in which excess amounts of extracellular GFAP and G*s prime T- and B-cells to generate anti-GDBP antibodies (Abs).(34) Such autoAbs, in the form of IgM first, then IgG, are observed beginning within 5 dpi, but do not peak until 14 dpi. Auto Ab binding to GBDP debris (i.e., opsonization) can trigger activation of microglia, the primary immune effector cells of the CNS, and macrophages infiltrating through the compromised blood-brain-barrier (BBB), via binding of the Ab to immune cell surface FcyR (Immunoglobulin Fc-gamma receptor). In this scenario, activated phagocytes (microglia and macrophages) are likely beneficial, as they “devour” harmful protein debris, and pro-phagocytic opsonins that signal “eat me” (e.g., calreticulin).(35, 36) These activated phagocytes may also promote this beneficial opsonization / phagocytosis process. In contrast, checkpoint inhibitors, such as CD47, onhealthy myelin signal “don’t eat me” to “brake” harmful opsonization / phagocytosis of healthy cells. (37) Importantly, GBDP autoAbs are observed in only a subpopulation of TBI subjects and their titers can vary. (38)

[0011] GFAP autoAbs are found in a number of clinical conditions in addition to TBI. (i) Clinical and veterinary studies have identified a role for GFAP autoAbs in the pathology of a distinctive, corticosteroid-responsive, sometimes paraneoplastic autoimmune meningoencephalitis in canines. (39-41) (ii) GFAP autoAbs are present in some cases of autism, Tourette Syndrome or autoimmune astrocytopathy, but the authors concluded that they are unlikely to play a pathogenic role.(42, 43) On the contrary, several lines of evidence suggest that GFAP autoAb or Ab immunotherapy might be neuroprotective. (i) In a cell-based model of glaucoma, GFAP Abs had protective effects on retinal ganglion cells subjected to oxidative stress. 44 (ii) GFAP Abs have protective effects on neuroretinal cells in culture. (45) (iii) Levels of GFAP autoAbs in stroke patients with favorable outcomes were significantly higher than in those with poor outcomes. (46) As a result, the role of GFAP autoAb and Ab treatments in other CNS indications is unclear with both potentially harmful and beneficial effects having been noted in the literature. The role of GFAP and G*s in other diseases remains unclear in the literature.

[0012] A 2013 TRACK-TBI paper entitled “GFAP-BDP as an Acute Diagnostic Marker in Traumatic Brain Injury: Results from the Prospective Transforming Research and Clinical Knowledge in Traumatic Brain Injury Study”, (150) where characterization of the mouse monoclonal antibody (mAb) for capture and rabbit polyclonal antibody (pAb) for detection used for the sandwich ELISAs employed were described in a 2012 paper to be non-specific for GBDP38K151. In other words, the TRACK-TBI paper was actually using capture and detection antibodies that BOTH recognized the conserved core domain shared by GBDP38K, GBDP44K, and GFAP (not the N — terminal neoepitope of GBDP38K)(see multiple bands in the western blot shown in Figure 2 in 151). This inadvertent error was made again in a 2015 paper entitled “Measurement of the glial fibrillary acidic protein and its breakdown products GFAP-BDP biomarker for the detection of traumatic brain injury compared to computed tomography and magnetic resonance imaging”.(152) Indeed, to the best of our knowledge, all commercial GFAP assays for TBI and other CNS diseases use antibodies that recognize the conserved core domain shared by GBDP38K, GBDP44K, and GFAP (not the N — terminal neoepitope of GBDP38K).

[0013] Thus, there is an unmet need to detect G*s as biomarkers in subject fluids, or in subject tissues to classify and distinguish injury, disease, and repair for individual patients.There also remains an unmet need for clinical intervention through the use of an in vitro diagnostic device to identify G*s so that subject results may be obtained rapidly in any medical setting to direct the proper course of treatment for repair of subjects with an injury or disease; something currently not provided by either CT or MRI scans. There also exists a need for therapeutics that target G*s as a method to treat the conditions responsible for the elevated levels of G*s in the subject.SUMMARY OF THE INVENTION

[0014] A method of diagnosing a disease in a subject is provided that includes sampling from the subject and the detection of at least one of astrocytic monomeric-, oligomeric-, or aggregate of a GFAP breakdown product (G*) in the subject as a ratio of at least different two molecular weight G*s or a ratio of the G* relative to intact GFAP from the subject. A diagnosis of the disease or severity thereof in the subject is provided based on the detection.

[0015] A composition is provided that includes an antibody or an oligonucleotide that binds with a degree of specificity to an N-terminal of GBDP38K, a C-terminal neoepitope of GBDP38K, or a conserved core domain of GBDP38K and GFAP.

[0016] A method of treating a disease in a subject is also provided that includes sampling a biofluid from the subject and the detection of the abnormal presence of at least one of monomeric-, oligomeric-, or aggregate containing of a glial fibrillary acidic protein breakdown product (G*) in the biofluid from the subject. A therapeutic is administered to the subject that selectively binds to the at least one of astrocytic monomeric, oligomeric, or aggregate G*s to treat the disease in response to the detection.BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The subject matter that is regarded as the invention is particularly pointed out and distinctly claimed in the claims at the conclusion of the specification. The foregoing and other objects, features, and advantages of the invention are apparent from the following detailed description taken in conjunction with the accompanying drawings wherein:

[0018] FIGs. 1A-1D are representative illustrations of distinct epitopes of GBDP38K, GBDP44K, and GFAP (50K) targeted by our therapeutic and diagnostic monoclonal antibodies (mAbs), where FIG. 1A shows mAbG* targets the N-terminal neoepitope of monomeric GBDP38K (yellow), mAbG# targets the conserved core of monomeric GBDP38K shared by intact GFAP (and GBDP44K) (including epitopes shown in pink and purple), and mAbG targets the N-terminus of monomeric GFAP alone (gray), where FIG. IB shows the sameepitopes are also targeted in parallel and antiparallel heterodimers (not shown) and homodimers (shown) where a disulfide bond is formed with the thiol (-SH) functional group of a single cysteine in each monomer, where the monomer can be GBDP38K, GBDP44K, or GFAP, where FIGs. 1C and ID show the same epitopes can also be targeted in dimers and higher order oligomeric and aggregate structures where disulfide bonds may or may not be present because the IB domain enables non-covalent coil-coil interactions for the formation of tetramers and other species, where FIG. 1C shows as an example, a homodimer in a parallel, symmetric orientation without a disulfide bond is shown, and where FIG. I D shows one of many possible examples of higher order structures, only the N-terminal neoepitope of monomeric GBDP38K is shown (blue)-corresponding to the yellow epitope shown in FIGs. 1 A and IB;

[0019] FIG. 2 is a schematic of how therapeutic mAbs can be used as tools to probe the mechanisms and markers involved in TBI and PTE by neutralizing cellular and released astrocytic neurotoxins and harnessing microglia to accelerate phagocytosis of mAb-GFAP / G* complexes;

[0020] FIGs. 3A-3F are plots of representative reducing immunoblots that show the formation and release of different GFAP / G*s in mouse TBI brain tissue (0-3m post-injury), where GFAP at 50K is the most abundant GFAP proteoform in mouse TBI, followed by GBDP44K and GBDP38K (least abundant comprising < 5% of the total GFAP / G*species). Immunoblots were performed with diagnostic mAb cocktail#2 that does NOT recognize GBDP38K but does recognize other G*s (~40K), and GFAP (50K) to show results for FIG. 3A GFAP and FIG. 3A GBDP44K, FIG. 3C shows results from an diagnostic mAb that only recognizes GBDP38K, relative levels are also shown by FIG. 3D pooled data for panels A-C and zoom views of E) GBDP44K in panel B, and F) GBDP38K in panel C. One way ANOVA Dunnet’s multiple comparison test, *p< 0.05.;

[0021] FIGs. 4A-4D show assays with diagnostic mAbs for GBDP38K formation and GFAP / GBDP38K release in human brain tissue and biofluids, where FIG. 4A shows immunoblots testing the specificity of mAbG#-WT (left) and mAbG*-WT (right) for the conserved Core domain epitope of GBDP38K and intact GFAP and the N-terminal neo-epitope of GBDP38K, respectively, Novel sandwich ELIS As for trajectory analysis of GBDP38K and GFAP are shown in FIG. 4B CSF (n=25, median +IQR) and FIGs. 4C and 4D for serum (n=30, median + IQR) from severe TBI patients (1-10 dpi), where FIG. 4D shows that the GBDP38K7GFAP ratio peaks at 2-5 dpi;

[0022] FIGs. 5A and 5B show the cytotoxic effects of recombinant intact human GFAP (rhGFAP) and calpain-generated G*s on rat cerebrocortical (CTX) neurons (red=GFAP / G*s;blue=DAPI stain for DNA). CTX culture in 96-well plates were treated with lOOng of protein in 100 LIL media for 16h. Cell viability is assessed by the mitochondrial uptake and reduction of dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) and lactate dehydrogenase release into culture medium test (n=8). (* p< 0.05; ** p < 0.01), where FIG. 5A shows cellular epitopes (intact GFAP) and released epitopes (G*s, including but not limited to GBDP38K) generate increasing levels of neurotoxic reactive astrocytes following injury with concomitant decreases in mitochondrial function (green arrows), and where FIG. 5B shows GFAP proteolysis in rat primary glial culture after cytotoxin-or LPS challenge (200 ng / mL) stimulation for 24 h. Data shown is representative of n=3. A232: A23187, STS; staurosporine, SNI; calpain inhibitor SNJ1945; pan caspase inhibitor, Z-D-DCB;

[0023] FIGs. 6A-6E show active GFAP / GBDP38K protein immunization before TBI induced Ab production and provided neuroprotection after TBI in mice., where FIG. 6A shows titers of anti-GFAP IgM and IgG levels after immunization. Compared to DO preimmunization (** p<0.01 ; ***p<0.001 ), pre TBI GFAP immunization., where FIG. 6B shows attenuated TBI- induced cortical elevations of GFAP., where FIG. 6C shows reduced the P-Tau / -tau level ratio, and FIG. 6D shows improved neurobehavioral performance at 20 dpi in Morris water maze test [mean + SEM (n=20) **p = 0.017], where FIG. 6E shows Immuno-histochemistry shows that TBI drastically induces GFAP in the ipsilateral hippocampus (IH) (orange arrow) compared to contralateral hippo (CH) in non-immunized mice, yet there is a striking reduction in GFAP levels at 7 dpi in immunized mice (red arrow) (n=4).;

[0024] FIGs. 7A-7E depict a passive anti-GBDP38K mAh immunotherapy after TBI provided neuroprotection after TBI for C57BL / 6 TBI mouse studies, where FIG. 7A is a plot of suppression of TBI-induced GFAP (cellular epitope) and G*s (released epitope, including but not limited to GBDP38K) release in mouse cortex assessed with polyclonal antibodies (pAbs), reported as % change compared to untreated TBI mice, where FIG. 7B is a plot showing reduced ptau / total-tau ratio in cortex- reported as a % change compared to naive (baseline / BL), (C) reduced tau levels in serum, and FIGs. 7D and 7E are schematic showing improved spatial memory in the Y-maze test (as evidenced by more time spent in the novel arm in the probe trail stage). * = p <0.05; ** = p <0.01; *** = p <0.001;

[0025] FIGs. 8 A and 8B show the generation of GBDP38K specific mAbs by targeting its N- or C-terminal neo-epitopes, where FIG. 8A depicts calpain cleavage of GFAP at N59 *60A and T383*384F, producing (NH2)-AGFKET... and . . ,ITIPVQT-(CO2H), using synthetic peptides that mimic and / or span these neo-epitopes and extended version, the mAbs generated are highly specific to GBDP38K., where FIG. 8B depicts the feasibility of generating anti-G*mAbs, where rhGFAP was untreated or digested with calpain-1 or caspase-6: (left) Immunoblot with anti-total G* mAb (BD#60341D); (right) Immunoblot with new rabbit anti- GBDP neo-N-terminal polyclonal Ab (pAb) only recognizes calpain-generated GBDP38K (and minor GBDP40K), not intact GFAP or other caspase- generated fragments;

[0026] FIGs. 9A-9C are confirmatory data is a second mouse model, where FIG. 9A is the rigor and reproducibility of the data with a second mouse species (CD1) in a second laboratory (IRFMN) and FIGs. 9B and 9C show the impact of effector function on sensorimotor measures of efficacy, where FIG. 9 A provides the findings from CD1 TBI mouse studies performed at IRFMN, akin to the findings from the C57BL / 6 TBI mouse studies shown in FIGS. 8C and 8D, show that treatment with a mixture of mAbG* and mAbG# showed a statistically significant > 10-20% improvement in spatial memory in the Y-maze test compared to isotype controls (ICs) and sham, respectively, the impact of effector function on sensorimotor measures was tested with the Simple Neuroassessment of Asymmetric impairment (SNAP) test following treatment of CD1 TBI mice with (B) mAbG* variants recognizing the N-terminus of GBDP38K or mAbG# variants recognizing the conserved core domain of GFAP and GBDP38K, the variants have different mutations in the Fc domain for modulating effector function (i.e. , mouse lgG2a ICs, symmetric and asymmetric GSI mutants for enhanced effector function, and LALPG mutations for reduced effector function), as evidence that inventive MoA, mAbG*-Sym and mAbG*-Asym mutants with enhanced effector function showed a statistically significant > 10- 20% improvement in SNAP test measures compared to ICs and mAbG* -LALPG mutants, with mAbG*-WT mAbs functioning with intermediate effect therebetween, a similar trend, albeit less pronounced, was observed for various mAbG# mutants, where FIG. 9C is a plot of longitudinal assessments for the efficacy of mAbG*-Asym and mAbG#-Asym treatments vs. mouse IgG2a IC, with mAbG*-Asym demonstrating the highest efficacy, one way ANOVA Dunnett’ s multiple comparison test (all vs IC); two way ANOVA Tukey’s multiple comparison test. * = p<0.05, ** = p<0.01, and *** = p<0.001 (n=8-12 per group);

[0027] FIGs. 10A-10C depict changes in astrocyte pathology after chronic human TBI revealed by immunofluorescence staining, where FIG. 10A is a micrograph of representative staining of the conserved core domain of GBDP38K that is shared by intact GFAP (i.e., Total GFAP), where thickened processes in the chronic TBI case are observed, where FIG. 10B is a micrograph of representative staining of the N-terminal neo-epitope of GBDP38K alone, where increased staining of fragmented processes is observed in TBI compared to control, where FIG. 10C is a a plot of quantification of the N-terminal GBDP38K stain B) to Total GFAP stain A) area ratio, expressed as a percent (%), in age-sex matched TBI and control cases (*p = 0.0150,Welch’s t-test, n=l l per group). Primary mouse antibodies for mAbG# and mAbG* were sourced from Gryphon Bio, and mouse secondary antibodies, were employed for FIGs. 10A and 10B, respectively;

[0028] FIG. 11 shows representative immunofluorescence images showing nuclei (blue), astrocyte (magenta) and GBDP38K (green) in TBI mice 3 weeks after injury, where in the left panel the presence of GBDP38K aggregates (white arrows) are clearly visible in the cortical area close to the contusion site while no GBDP38K signal was detect in sham mice (right panel);

[0029] FIG. 12 are volcano plots showing the statistical significance vs. differential abundance of proteins observed by LC-MS / MS-based proteomics of naive (naive 2), TBI (TBInoPTE) and TBI-induced PTE (TBIPTE) mouse brain tissue specimens. GFAP / G*s (P03995) were clearly observed at statistically significant (p <0.001) down-regulated levels in naive mice vs. TBI mice (blue box), while GFAP / G*s (P03995) were clearly observed at statistically significant (p < 0.001) up-regulated levels in TBI vs. PTE mice (red box);

[0030] FIGs. 13 A and 13B are electrophoretic gels showing characterization of the binding specificity, where FIG. 13A shows mAbG#, which binds the conserved core of G*s and GFAP, where FIG. 13B shows mAbG*, which binds the N-terminal neoepitope of GBDP38K, by immunoblottng, using recombinant human intact GFAP and also the calpain- generated GBDP38K, oligomers of both are also observed.2 are a series of graphs showing the efficacy of temporal pharmacodynamic (PD) biomarker-powered precision medicines for targeting SV2A;

[0031] FIGs. 14A and 14B depict representative non-reducing immunoblots showing the presence of aggregates (oligomers) in mouse TBI brain tissue (0-3m post-injury) where GBDP38K (and aggregates thereof) is the most abundant GFAP / G* species in mouse TBI, where FIG. 12A shows results from an anti-GBDP38K mAb that only recognizes GBDP38K (and aggregates thereof) and , where FIG. 12B shows results from an anti-GFAP / G* mAb cocktail#2 that does NOT recognize GBDP38K (and aggregates thereof) but does recognize other G*s (~40K), and GFAP (50K) (and aggregates thereof);

[0032] FIGs. 15A and 15B depict representative non-reducing immunoblots showing the formation and release of different GFAP / G*s and aggregates (oligomers) in human TBI CSF (0-48h post-injury), where GBDP38K (and aggregates thereof) is the most abundant GFAP / GBDP species in human TBI, where FIG. 15A shows results from an anti-GFAP / G* mAb cocktail#! that does recognize GBDP38K and its aggregates as well as other G*s (~40K)such as GBDP44K, and GFAP (50K) and FIG. 15B shows results from an anti-GBDP38KmAb that only recognizes GBDP38K and aggregates thereof;

[0033] FIGs. 16A-16D are direct ELISA characterization of mAbG* and mAbG#: FIG. 16A, 16B, and various mAbG* and mAbG# clones bind to the conserved core domain of G*s and GFAP, FIG. 16C, 16D, however, only mAbG* clones that bind specifically to the N- terminal neoepitope of GBDP38K are not recognized by the anti- GFAP (5 OK) detection antibody. mAbG* clones 13G9, 2G5-1A12, 2G5-2D4, and 2C3 are clones are calpain- generated GBDP38K-specific, in contrast, the anti-GFAP mAh cocktail of three conserved core binding mAbG# clones (BD 556330) is not GBDP38K specific;

[0034] FIG. 17 is a schematic showing increasing GFAP, together with GBDP (novel disease specific N / C-terminal or core biomarkers i.e. G, G*,N*, C* or combination thereof) are detected in biofluids with increasing astrocytic dysfunction or death;

[0035] FIG. 18 is a schematic summarizing GFAP assembly, oligomerization, aggregation, and consequential damage to CNS cells;

[0036] FIGs. 19A-19C show Glial fibrillary acidic protein (GFAP) structure and assembly, where FIG. 19A is a linear structure, functional domains, and key modifications, where FIG. 19B is a 3D GFAP structure, where FIG. 19C is a proposed GFAP dimer and tetramer assembly and oligomerization model;

[0037] FIG. 20 depicts isoforms and a specific fragment generated in activated or damaged astrocytes that is highly toxic and aggregation prone fragment (orange), colored vertical bars indicate few representative examples from our monoclonal antibody (mAb) collection that are strategically designed to specifically target epitopes so as to recognize all or most proteoforms or discriminate between various fragmentation or covalently modified products, core protein and disease associated proteoforms such as GBDP38K, the N-terminal neoepitope of GBDP38K is denoted as G*, while the N-terminal and C-terminal epitopes unique to GFAP are denoted as N* and C*, respectively, the conserved core domain is denoted as G., ratios of these proteoforms provide various contexts of use for biomarker-based biofluid or tissue tests, including but not limited to those shown above;

[0038] FIG. 21 depicts personalized trajectories of blood biomarkers and their ratios reveal brain health status, identify periods of risk for secondary monitoring and administration of disease modifying therapies (DMTs), as well as provide readout for recovery, longitudinal monitoring of TBI patients (2.5 million in the US alone) and routine screening for >65 year olds is a major current unmet need (with an expected 7.5 million over age 65 individuals with undiagnosed mild cognitive impairment / MCI in the US alone), the N-terminal neoepitope ofGBDP38K is denoted as G*, while the N-terminal and C-terminal epitopes unique to GFAP are denoted as N* and C*, respectively. The conserved core domain is denoted as GFAP. Ratios of these proteoforms provide various contexts of use for biomarker-based biofluid or tissue tests, including but not limited to those shown above;

[0039] FIG. 22 are schematic plots dependence of toxic burden (redline) in the brain on diurnal clearance capacity (blue) and age(x-axis). and is sharply impacted by TBI, increasing levels of proteinaceous aggregates comprised of GBDP38K and other proteoforms of GFAP, as well as other proteins (e.g., vimentin) comprise toxic burden in the brain;

[0040] FIGs. 23A-23C are plots of. GFAP proteoforms (GZ G* / N* / C*) and calculated ratios (black) in brain and biofluids identifies treatment windows by sampling accessible biofluids like blood, the N-terminal neoepitope of GBDP38K is denoted as G* in the context of these plots and those of FIG. 24, while the N-terminal and C-terminal epitopes unique to GFAP are denoted as N* and C*, respectively, he conserved core domain is denoted as G, ratios of these proteoforms provide various contexts of use for biomarker-based biofluid or tissue tests, including but not limited to those shown above, where FIG. 23A is for a healthy subject, FIG. 23B is for GFAP activation and cleavage, and FIG. 23C is for sustained GFAP activation, cleavage and aggregation in the brain;

[0041] FIG. 24 is a schematic showing changes in the absolute level of GFAP proteoform monomers (e.g., G / N* / G*) (shown as green strings) or their ratio (e.g., G / G*) in biofluid (e.g., blood) indicates changes in GFAP proteoforms in brain tissue-suggesting changes in oligomer / aggregate formation in the CNS, therapeutic monoclonal antibodies (mAbs) (Y- shaped structures) can bind and reduce oligomer / aggregate formation through multiple mechanisms of action, the N-terminal neoepitope of GBDP38K is denoted as G*, while the N- terminal and C-terminal epitopes unique to GFAP are denoted as N* and C*, respectively, the conserved core domain is denoted as G, ratios of these proteoforms provide various contexts of use for biomarker-based biofluid or tissue tests;

[0042] FIG. 25 are schematic of peptide cyclization to generate antibodies recognizing the N-terminal or C-terminal neoepitopes of G*s such as GBDP38K;

[0043] FIG. 26 is a plot of G* interassay precision as a function of added G* ;

[0044] FIG. 27 is a comparative plot of G* relative to GFAP for cerebrospinal fluid(CSF)sampling fluid obtained from a subject suffering from traumatic brain injury (TBI);

[0045] FIGs. 28A-28C are plots for CSF samples for GFAP (FIG. 28A), G* (FIG. 28B) and a ratio therebetween (FIG. 28C) for pooled healthy cohort, pooled TBI and subject CSF groups thereby providing evidence for clinical utility as a companion diagnostic;

[0046] FIGs. 29 A and 29B are plots of GFAP and G* as a function of post- injury time (hr) for CSF (FIG. 29 A) and serum (FIG. 29B) samples from TBI subjects thereby showing G* has a uniquely longer trajectory than GFAP with greater potential for patient monitoring in an intensive care setting and beyond;

[0047] FIGs. 30A and 30B are time block plots for serum GFAP (FIG. 30A) and G*(FIG. 30B) from TBI subjects showing the longer trajectory for G* with the oval marked regions to highlight that G* is more resistant to decay in the 72h to 96h time window, the control (H.C.) is also noted; and

[0048] FIGs. 31A and 31B are biomarker levels in pediatric TBI subject samples for GFAP (FIG. 31A) and G* (FIG. 31B) for Glasgow Outcome Score Extended (GOSE) for low and high value groupings showing the longer trajectory for G* and that sustained G* levels by day 3 inform on poor outcome by GOSE.DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0049] The present invention has utility as a method of detecting glial fibrillary acidic protein (GFAP) and breakdown products thereof (G*s) for the diagnosis or treatment or combination thereof of conditions that cause astroglial release of GFAP proteoforms into extracellular fluid and ultimately across the blood brain barrier (BBB). It is noted that surprisingly useful correlations are obtained by examining ratios of various G*s relative to intact GFAP, or relative to other G*s, as measured in a biofluid from the subject, different fluids, or as a function of time relative to an injury, condition, or disease state that induced GFAP proteoforms release across the BBB.

[0050] The present invention provides methods of exploiting the fact that astrocyte compromise or injury conditions, such as after traumatic brain injury (TBI), results in GFAP (alpha-isoform, 50K) undergoes significant post-translational modification. Namely, GFAP is truncated at both the N- and C-terminus by intracellular calcium-activated proteases such as calpain to G*s of molecular weight 22-48kDa, including dominant species GBDP38K and GBDP44K. The present invention is based on the surprising result that these released G*s and the associated intact form of GFAP have an enhanced tendency to form atypical oligomeric forms and / or aggregates. Such released monomeric-, oligomeric and / or aggregate-G*s and GFAP proteoforms, as well as other proteins (e.g., vimentin), become toxic to neurons and glia in the nerve tissue (i.e., the brain microenvironment or milieu). The ratios of these monomeric - , oligomeric and / or aggregate-G*s and GFAP proteoforms, as well as other proteins are exploited in the present invention to provide detection, severity, temporal progress and eventreatment of astrocyte compromise, regardless of the basis of such compromise. Besides, TBI, the present invention is efficacious in detecting and even treatment of post-traumatic epilepsy (PTE), Alexander disease (AxD), mild cognitive impairment (MCI), Alzheimer’s disease (AD), multiple sclerosis, Parkinson’s disease, Lewy Body Dementia, frontotemporal dementia, stroke, or vanishing white matter disease.

[0051] hi some inventive embodiments, GFAP and G*s released into the intracellular compartment (i.e. brain interstitial fluid) which ultimately equilibrates with the cerebrospinal fluid (CSF) and also released into the circulating blood via the glymphatic drainage, compromised brain-blood barrier pathways, and other pathways provides temporal detection information as to the extent the astrocytic damage.

[0052] In some inventive embodiments, methods are provided based on the surprising results that: (i) examining and interrogating the unique patterns of astrocytic G*s, alone, or in combination with GFAP provides invaluable clinical data. The G*s and GFAP proteoforms in the form of GBDP22-24K, GBDP38K, GBDP44K, monomeric-, oligomeric-, and / or aggregate- GFAP alone or with G*s; (ii) and relative abundance or ratio of any of the aforementioned can serve as astrocyte compromise / injury biomarkers and / or as a mechanistic biomarkers for CNS or PNS injury, disease or degeneration themselves; (iii) the same proteoforms can serve as therapeutic targets for attenuating astrocyte compromise / injury, CNS or PNS injury, disease or degeneration.

[0053] The present invention has application in the detection of TBI, Post-Traumatic Epilepsy (PTE), Alexander’s disease (AxD), Alzheimer’s disease (AD), multiple sclerosis (MS), stroke, Parkinson’s disease, Lewy body dementia, frontotemporal dementia, vanishing white matter disease, epilepsy, post-traumatic headache, mild cognitive impairment (MCI), amyotrophic lateral sclerosis (ALS), and peripheral neuropathy. In some inventive embodiments, the utility of assessing (with diagnostic antibodies) and removing (with therapeutic antibodies) increased levels of GFAP and related G*s, that are upregulated in disease states upon astrocyte activation from injury, infection, inflammation or due to mutations.

[0054] According to the present invention, excessive G*s, predominantly generated upon enzymatic cleavage of GFAP by intracellular or circulating calpains (and caspases), correlate with astrocyte dysfunction and / or death-which in turn promotes oligodendrocyte and neuronal damage and / or disintegration within the CNS, can promote overall brain health if cleared from the CNS milieu. This can be achieved by targeting GBDP22-24K, GBDP38K, GBDP44K, monomeric-, oligomeric-, and / or aggregated GFAP using monoclonal antibodies (mAbs),bispecific mAbs (bsMAbs), or other means (including oligonucleotides such as DNA or RNA aptamers or mRNA knockdown strategies such as siRNA). Data is provided herein as to the beneficial effects of targeting therapies after TBI as model for astrocyte compromise. Novel therapeutics are provided for the same. To minimize the induced toxicity (of GBDP22-24K, GBDP38K, GBDP44K, monomeric-, oligomeric-, and / or aggregated GFAP), novel mAbs operative as therapeutics are provided to target GBDP22-24K, GBDP38K, GBDP44K, monomeric-, oligomeric-, and / or aggregated GFAP for phagocytic destruction and / or removal or clearance from the CNS. An inventive mAh is designed to optionally exclude the major uncleaved GFAP isoform at 50kDa (50K) to avoid targeting healthy astrocytes but specifically bind aggregates or toxic G*s. In some inventive embodiments, the highly toxic breakdown products of GBDP38K or GBDP44K are targeted. Unlike intact GFAP, possibly not generated during normal aging, mAbs targeting GBDP38K and GBDP44K are ideal for extended use. An inventive diagnostic mAb-based assays for GBDP22-24K, GBDP38K, GBDP44K, monomeric-, oligomeric-, and / or aggregated GFAP in biofluids provide a companion diagnostic for our therapeutic mAb and directly identify a therapeutic window as well as assess the ability of the therapeutic mAbs in reducing the toxic load of the mAb target G*.

[0055] An in vitro diagnostic device is provided for detecting an aforementioned condition or disease associated with astrocyte compromise in a subject. The device includes a sample chamber for holding a biological sample collected from the subject, an assay module in fluid communication with the sample chamber, and a user interface. The user interface relates the amount of the one or more biomarkers measured in the assay module to detecting an injury, disease, or repair in the subject or the severity of injury, disease, or repair in the subject.

[0056] It is to be understood that in instances where a range of values are provided that the range is intended to encompass not only the end point values of the range but also intermediate values of the range as explicitly being included within the range and varying by the last significant figure of the range. By way of example, a recited range of from 1 to 4 is intended to include 1-2, 1-3, 2-4, 3-4, and 1-4.

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0058] Unless indicated otherwise, explicitly or by context, the following terms are used herein as set forth below.

[0059] As used in the description of the invention and the appended claims, the singular forms “a,” “an’- and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0060] Also as used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0061] As used herein, the breakdown products of GFAP, G*s-including natural peptides derived from the sequence of GFAP and for example, the central core breakdown product GFAP38K (with residue range about 79-383 in GFAP-a), the N-terminal head region with residue range about 1-72 in GFAP-a, and the C-terminal tail region with residue range about 378-432 in GFAP-a, i.e., the truncated forms of GFAP (G*) with apparent molecular weights of about 44 kDa, 42 kDa, 40 kDa, 38kDa and 22-24 kDa.

[0062] AS used herein, the term “conserved core” refers to the domain shared by GBDP38K, GBDP44K, and GFAP proteoforms.

[0063] As used herein, the term “immunization” refers to any passive or active method of introducing or producing antibodies specific to a particular antigen. For example, immunization for GFAP includes administration of mAbs or bsMAbs that specifically recognize GFAP or an epitope or hapten of GFAP to a subject, or an aptamer that binds to GFAP; such types of immunization relate to a passive immunization. Immunization also includes administration of GFAP protein or a peptide derived from GFAP to the subject in order to stimulate the immune system of the subject to produce antibodies that specifically recognize GFAP, an active immunization. Both active and passive immunization is included in the term “immunization” and all of its cognates, unless stated otherwise.

[0064] As used herein, the term “GFAP antibody (“anti-GFAP antibody”) or a fragment thereof’ refers to an intact anti-GFAP proteoform antibody, monoclonal or polyclonal, monospecific or bi- or tri-specific, or a combination of fragmented heavy and light chains of immunoglobulin or single chain fusion protein containing heavy-light chain plus light brain variable fragments. Any type of antibody is included within the term if it specifically binds to GFAP or a fragment or breakdown product of GFAP.

[0065] As used herein, the term “therapeutically effective amount” refers to an amount of a compound or composition that, when administered to a subject for treating a disease or disorder, or at least one of the clinical symptoms of a disease or disorder, is sufficient to affect such disease, disorder, or symptom. A “therapeutically effective amount” includes an amountthat ameliorates, reduces or cures the disease, disorder, or symptom and may vary depending, for example, on the compound, the disease, disorder, and / or symptoms of the disease or disorder, severity of the disease, disorder, and / or symptoms of the disease or disorder, the age, weight, and / or health of the subject to be treated, the capacity of the individual’s immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor’s assessment of the medical situation, and other relevant factors. A therapeutically effective amount can be a single dose or a series of doses administered to a subject in need thereof. An appropriate amount in any given instance may be readily ascertained by those skilled in the art or can be determined by routine experimentation.

[0066] The present invention also provides clinical treatment with precision medicines for the same therapeutic targets as a subset of the temporal biomarkers. If the clinical treatment is with a precision medicine for at least one of the same therapeutic targets as a subset of the temporal biomarkers and involves opsonization, stabilization or destabilization, binding, and / or accelerated clearance or phagocytosis of brain debris or decelerated generation of brain debris, then accelerated clearance of these proteins and other temporal biomarkers described herein are then reflected by modulated levels in the blood / CSF / lymphatic fluid and / or inversely modulated levels in the brain.

[0067] The present invention provides for the detection of injury, disease, or repair in a subject. An injury, disease, or repair may be an abnormal injury, disease, or repair such as that caused by genetic disorder, injury, or disease to nervous tissue. As such, it is a further object of the present invention to provide a means for detecting or diagnosing an abnormal injury, disease, or repair in a subject.

[0068] As used herein, “proteoform” is intended to define any variant of a genetically encoded GFAP intact protein of G* thereof; the variants associated with, for example, post- translational modification, that can give rise to these variants which may have altered structure (primary through tertiary), function, interactions, or solubility.

[0069] In one embodiment, the invention includes a diagnostic kit for use in screening serum containing antigens of the biomarkers of the invention. The diagnostic kit in this embodiment includes a substantially isolated antibody or aptamer specifically immunoreactive with peptide or polynucleotide antigens, and visually detectable labels associated with the binding of the polynucleotide or peptide antigen to the antibody or aptamer. In one embodiment, the antibody or aptamer is attached to a solid support. Antibodies or aptamers used in the inventive kit are those raised against any one of the biomarkers used herein for temporal data. In one embodiment, the antibody is a monoclonal or polyclonal antibody oraptamer raised against the rat, rabbit or human forms of the biomarker. The detection reagent of the kit includes a second, labeled monoclonal or polyclonal antibody or aptamer. Alternatively, or in addition thereto, the detection reagent includes a labeled, competing antigen.

[0070] In one diagnostic configuration, test serum is reacted with a solid phase reagent having a surface-bound antigen obtained by the methods of the present invention. After binding with specific antigen antibody or aptamer to the reagent and removing unbound serum components by washing, the reagent is reacted with reporter-labeled anti-human antibody or aptamer to bind reporter to the reagent in proportion to the amount of bound anti-antigen antibody or aptamer on the solid support. The reagent is again washed to remove unbound labeled antibody or aptamer, and the amount of reporter associated with the reagent is determined. Typically, the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric, luminescent or colorimetric substrate.

[0071] The solid surface reagent in the above assay is prepared by known techniques for attaching protein or oligonucleotide material to solid support material, such as polymeric beads, dip sticks, 96-well plate or filter material. These attachment methods generally include nonspecific adsorption of the protein oligonucleotide to the support or covalent attachment of the protein or oligonucleotide, typically through a free amine group, to a chemically reactive group on the solid support, such as an activated carboxyl, hydroxyl, or aldehyde group. Alternatively, streptavidin coated plates can be used in conjunction with biotinylated antigen(s).

[0072] In some embodiments, a standard or control information is provided so that the test sample can be compared with the control information standard to determine if the test amount of a marker detected in a sample is a diagnostic amount consistent with a diagnosis of injury, disease, or repair, including type, phase, amplitude (severity), subcellular localization, brain disorder and / or effect of treatment on the patient.

[0073] The inventive method provides the ability to detect and monitor levels of those temporal protein biomarkers or autoantibodies thereto which are released into the body after neurotoxicity or CNS or PNS injury, disease, or repair to provide enhanced diagnostic capability by allowing clinicians (i) to determine the type, phase and amplitude (severity) of injury or disease or repair in various patients, (ii) to monitor patients for signs of secondary CNS or PNS injuries, diseases or repairs that may elicit these cellular changes and (iii) to continually monitor the progress of the injury, disease, or repair and the effects of therapy by examination of these temporal biomarkers in biological fluids (synonymously referred to herein as “biofluids’'), such as blood, plasma, serum, CSF, urine, saliva or sweat. Unlike other organ-based diseases where rapid diagnostics by surrogate biomarkers prove invaluable to the course of action taken to treat the disease, no such rapid, definitive diagnostic tests exist for injury, disease, or repair states such as traumatic or ischemic injury that might provide physicians with quantifiable temporal biomarkers to help determine the degree of the injury, disease or repair; the anatomical and cellular pathology of the injury, disease or repair; and the implementation of appropriate medical management and treatment.

[0074] A biological sample operative herein includes cells, tissues, cerebral spinal fluid (CSF), whole blood, serum, plasma, cytosolic fluid, urine, feces, stomach fluids, digestive fluids, saliva, nasal or other airway fluid, vaginal fluids, semen, or other biological fluid recognized in the art. It should be appreciated that after injury or disease of the CNS or PNS (such as TBI), the neural cell membrane is compromised, leading to the efflux of neural proteins first into the extracellular fluid, and to the cerebrospinal fluid. Eventually the neural proteins efflux to the circulating blood (as assisted by the compromised blood brain barrier for brain injuries or diseases) and, through normal bodily function (such as impurity removal from the kidneys), the neural proteins migrate to other biological fluids such as urine, sweat, and saliva. Thus, other suitable biological samples include, but are not limited to such cells or fluid secreted from these cells. It should also be appreciated that obtaining biological fluids such as cerebrospinal fluid, blood, plasma, serum, saliva, and urine, from a subject is typically much less invasive and traumatizing than obtaining a solid tissue biopsy sample. Thus, biofluids, are preferred for use in the invention.

[0075] Biological samples of CSF, blood, urine, and saliva are collected using normal collection techniques. For example, and not to limit the sample collection to the procedures contained herein, CSF Lumbar Puncture (LP) a 20-gauge introducer needle is inserted, and an amount of CSF is withdrawn. For blood, the samples may be collected by venipuncture in Vacutainer tubes and being amenable to being spun down and separated into serum and plasma. For urine and saliva, samples that are collected avoiding the introduction of contaminants into the specimen are preferred. All biological samples may be stored in aliquots at -80 °C for later assay. Surgical techniques for obtaining solid tissue samples are well known in the art. Any suitable biological samples can be obtained from a subject to detect markers. It should be appreciated that the methods employed herein may be identically reproduced for any biological fluid to detect a marker or markers in a sample.

[0076] After insult, the damaged tissue, organs, or nerve cells in in vitro culture or in situ in a subject express altered levels or activities of one or more proteins than do such cells notsubjected to the insult. Thus, samples that contain nerve cells, e.g., a biopsy of CNS or PNS tissue are illustratively suitable biological samples for use in the invention.

[0077] A subject illustratively includes a dog, a cat, a horse, a cow, a pig, a sheep, a goat, a chicken, non-human primate, a human, a rat, and a mouse. Subjects who most benefit from the present invention are those suspected of having or at risk for developing abnormal injury, disease, or repair, such as victims of the injuries or diseases such as those aforementioned herein.

[0078] Baseline levels of several biomarkers are those levels obtained in the target biological sample in the species of desired subject in the absence of a known injury, disease, or repair. These levels need not be expressed in hard concentrations but may instead be known from parallel control experiments and expressed in terms of fluorescent units, density units, and the like. Typically, baselines are determined from subjects where there is an absence of a biomarker or present in biological samples at a negligible amount. However, some proteins may be expressed less in an injured, diseased or repaired patient or before any clinical measures of injury, disease, or repair . Determining the baseline levels of protein biomarkers in a particular species is well within the skill of the art.

[0079] To provide correlations between an injury, disease, or repair and measured quantities of the temporal biomarkers, biological samples are collected from subjects in need of measurement for these biomarkers to assess injury, disease, or repair. Detected levels of a given temporal biomarker are optionally correlated with CT scan results as well as GCS scoring.

[0080] The biomarkers of the invention can be detected in a sample by a variety of conventional methods. For example, immunoassays, include but are not limited to competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, magnetic immunoassays, radioisotope immunoassay, fluorescent immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, fluorescent immunoassays, chemiluminescent immunoassays, phosphorescent immunoassays, anodic stripping voltammetry immunoassay, and the like. Inventive in vitro diagnostic devices may also include any known devices currently available that utilize ion-selective electrode potentiometry, microfluids technology, fluorescence or chemiluminescence, or reflection technology that optically interprets color changes on a protein test strip. Such assays are routine and well known in the art. Exemplary immunoassays are described briefly below (but are not intended by way of limitation). It shouldbe appreciated, that at present, none of the existing technologies present a method of detecting or measuring any of the ailments disclosed herein, nor does there exist any methods of using such in vitro diagnostic devices to detect any of the disclosed biomarkers to detect their associated injuries.

[0081] An exemplary process for detecting the presence or absence of a biomarker, alone or in combination, in a biological sample involves obtaining a biological sample from a subject, such as a human, contacting the biological sample with a compound or an agent capable of detecting of the marker being analyzed, illustratively including an antibody, and analyzing binding of the compound or agent to the sample after washing. Those samples having specifically bound compound or agent express the marker being analyzed.

[0082] For example, in vitro techniques for detection of a marker illustratively include enzyme linked immunosorbent assays (ELIS As), radioimmunoassay, radioassay, western blot, Southern blot, northern blot, immunoprecipitation, immunofluorescence, mass spectrometry, RT-PCR, PCR, liquid chromatography, high performance liquid chromatography, enzyme activity assay, cellular assay, positron emission tomography, mass spectroscopy, combinations thereof, or other technique known in the art. Furthermore, in vivo techniques for detection of a marker include introducing a labeled agent that specifically binds the marker into a biological sample or test subject. For example, the agent can be labeled with a radioactive marker whose presence and location in a biological sample or test subject can be detected by standard imaging techniques. In some inventive embodiments a first temporal biomarker early, intermediate, and late specific binding agent and other agents specifically binding at least one additional temporal biomarker are bound to a substrate. It is appreciated that a bound agent assay is readily formed with the agents bound with spatial overlap, with detection occurring through discernibly different detection of each temporal biomarkers. A color intensity-based quantification of each of the spatially overlapping bound biomarkers is representative of such techniques.

[0083] A preferred agent for detecting a temporal biomarker is an antibody capable of binding to the biomarker being analyzed. More preferably, the antibody is conjugated with a detectable label. Such antibodies can be polyclonal or monoclonal. An intact antibody, a fragment thereof (e.g., Fab or F(ab’)2), or an engineered variant thereof (e.g., sFv) or an aptamer or bi- / tri-specific aptamer can also be used. Such antibodies can be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.

[0084] An antibody or aptamer is labeled in some inventive embodiments. A person of ordinary skill in the art recognizes numerous labels operable herein. Labels illustratively include, fluorescent labels, biotin, peroxidase, radionucleotides, or other label known in the art.Alternatively, a detection species of another antibody or aptamer or other compound known to the art is used as form detection of a biomarker bound by an antibody or aptamer.

[0085] Antibody- and aptamer-based assays operative herein include western blotting immunosorbent assays (e.g., ELISA and RIA) and immunoprecipitation assays. As one example, the biological sample or a portion thereof is immobilized on a substrate, such as a membrane made of nitrocellulose or PVDF; or a rigid substrate made of polystyrene or other plastic polymer such as a microtiter plate, and the substrate is contacted with an antibody or aptamer that specifically binds a temporal biomarker under conditions that allow binding of antibody to the biomarker being analyzed. After washing, the presence of the antibody on the substrate indicates that the sample contained the marker being assessed. If the antibody is directly conjugated with a detectable label, such as an enzyme, fluorophore, or radioisotope, the presence of the label is optionally detected by examining the substrate for the detectable label. Alternatively, a detectably labeled secondary antibody that binds the marker- specific antibody or aptamer is added to the substrate. The presence of detectable label on the substrate after washing indicates that the sample contained the biomarker.

[0086] Numerous permutations of these basic immunoassays are also operative in the invention. These include the biomarker-specific antibody, as opposed to the sample being immobilized on a substrate, and the substrate is contacted with a biomarker conjugated with a detectable label under conditions that cause binding of antibody to the labeled marker. The substrate is then contacted with a sample under conditions that allow binding of the marker being analyzed to the antibody. A reduction in the amount of detectable label on the substrate after washing indicates that the sample contained the marker.

[0087] A myriad of detectable labels that are operative in a diagnostic assay for biomarker expression are known in the art. Agents used in methods for detecting a biomarker are conjugated to a detectable label, e.g., an enzyme such as horseradish peroxidase. Agents labeled with horseradish peroxidase may be detected by adding an appropriate substrate that produces a color change in the presence of horseradish peroxidase. Several other detectable labels that may be used are known. Common examples of these detectable labels include alkaline phosphatase, horseradish peroxidase, fluorescent compounds, luminescent compounds, colloidal gold, magnetic particles, biotin, radioisotopes, and other enzymes. It is appreciated that a primary / secondary antibody or aptamer system is optionally used to detect one or more biomarkers. A primary antibody or aptamer that specifically recognizes one or more biomarkers is exposed to a biological sample that may contain the biomarker of interest. A secondary antibody or aptamer with an appropriate label that recognizes the species oriso type of the primary antibody or aptamer is then contacted with the sample such that specific detection of the one or more biomarkers in the sample is achieved.

[0088] The present invention provides a step of comparing the quantity of one or more temporal biomarkers to normal levels to determine the injury, disease, or repair of the subject. It is appreciated that selection of the temporal biomarkers or even additional biomarkers allows one to identify the types of cells implicated in an abnormal organ or physical condition as well as the nature of cell death in the case of an axonal injury marker. The practice of an inventive process provides a test which can help a physician determine suitable therapeutics to administer for optimal benefit of the subject.

[0089] The results of such a test using an in vitro diagnostic device can help a physician determine whether the administration of a particular therapeutic or treatment regimen may be effective and provide a rapid clinical intervention to the injury or disorder to enhance a patient’ s recovery.

[0090] It is appreciated that other reagents such as assay grade water, buffering agents, membranes, assay plates, secondary antibodies or aptamers, salts, and other ancillary reagents are available from vendors known to those of skill in the art.

[0091] Methods involving conventional biological techniques are described herein. Such techniques are generally known in the art and are described in detail in methodology treatises such as Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1992 (with periodic updates). Immunological methods (e.g., preparation of antigen- specific antibodies, immunoprecipitation, and immunoblotting) are described, e.g., in Current Protocols in Immunology, ed. Coligan et al., John Wiley & Sons, New York, 1991; and Methods of Immunological Analysis, ed. Masseyeff et al., John Wiley & Sons, New York, 1992.

[0092] Referring now to the data presented in the drawings, severe TBI patients show that the GBDP38K / GFAP ratio peaks at 2-5 dpi in serum (FIG. 4D) due to the shallower trajectory of GBDP38K vs. GFAP (FIGs. 4B,C), albeit sustained elevation of GFAP proteoforms can last years after TBI.(23, 29) Akin to mouse brain tissue (FIGs. 3A-3D), levels of GBDP38K are ~ 5% of GFAP in human CSF (FIG. 4B). However, serum levels are similar (FIG. 4C) - suggesting GBDP38K is far more dominant in blood.

[0093] Rosenthal fibers are a prominent feature of Alexander disease (AxD), which is caused by GFAP mutations (30, 31) are shown to increase GFAP aggregation. (32, 33) Caspase-cleaved G*s has also been shown to form filamentous aggregates. The presentinvention extends there finds to show that proteolytic processing of GFAPs into GBDP38K that is more prone to aggregate formation, and thus becomes neurotoxic.

[0094] Monoclonal antibodies (mAbs) are detailed herein that target distinct epitopes of the 38 kDa glial fibrillary acidic protein (G*) breakdown product (GBDP38K), spanning amino acid residues 60-383 of the intact protein (GFAP, 50K) that are functional for diagnostic and therapeutic applications in TBI and the aforementioned indications. mAbG* specifically binds the N-terminal neoepitope of GBDP38K, while mAbG# binds the conserved core shared by GBDP38K, GBDP44K, and intact GFAP (50K), as shown in FIGs. 1 A-1 D. Other mAbs bind the N-terminal epitope of GFAP alone or the epitopes spanning the N- or C-terminal epitopes of GBDP38K or GBDP44K. The present invention shows that mAbG* and mAbG#, and bispecific and trispecific variants of these constructs, reduce neurodegeneration and neuroinflammation, thus accelerating brain repair and improving cognition and other outcome measures in TBI patients. It is appreciated that mAbs used herein are readily tailored to modify stability properties. Conventional techniques for doing so illustratively include imparting asymmetry, modifying glycosylation, including of synthetic aminoacids, or sugars, and combinations thereof.

[0095] Anti-GBDP38K mAb immunotherapy is efficacious in two complementary mouse models of TBI. In some inventive embodiments, target engagement (reduced levels of GBDP38K and GFAP), in combination with subacute temporal PD biomarkers (e.g., NFL, tau, and p-tau) can be used to track treatment response and adjust dosing for successful translation from the laboratory to the clinic and to aid in meaningful benefit-risk decision-making in treating a subject, such as a human. Anti-GBDP38K mAbs are readily administered by intravenous infusion to maximize brain exposure, with subacute temporal PD biomarkers to track treatment response and adjust dosing. In still other inventive embodiments, a therapeutic mAb is administered via the in tracerebro ventricular or intrathecal route.

[0096] Preliminary Data in TBI Models. High concentrations of recombinant intact human GFAP (rhGFAP) and calpain-generated G*s, like glutamate, are toxic in rat brain cortical cell models of TBI cytotoxic effects (FIGs. 5A and 5B), which may involve increased aggregation of GBDP38K. Mice were pre-immunized with complete Freund’s adjuvant and intact GFAP plus GBDP38K followed by assessment of GFAP and GBDP38K (and GBDP44K) IgG (and IgM) auto Ab titers prior to CCI. We observed elevated antigen-specific IgM levels first (20 dpi) followed by IgG (30-50 dpi) (FIG. 6A). Complementary temporal diagnostic and cognitive assessment readouts were performed for individual subjects. GFAP / GBDP33K immunization attenuated the TBI-induced increases in cortical expression of GFAP, p-NF-H, tau and p-tau.GFAP / GBDP33K immunization also reduced the increases in cortex GFAP levels seen at 20 dpi and in cortex pNF-H, tau, and p-tau levels seen at 50 dpi (FIG. 6C). Significantly, GFAP immunization significantly alleviated anxiety behavior (elevated plus maze test-data not shown) and improved cognitive performance (Morris water maze test) at 20 dpi too (FIG. 6D). The IHC results shown in FIG. 6E clearly show that active GFAP immunotherapy attenuated GFAP levels in the injured hippocampus in mice. Passive anti-GBDP38K mAb immunotherapy after TBI (FIG. 7A-7E) has neuroprotective effects very similar to those observed with active GFAP / GBDP38K protein immunization before TBI (FIGs. 6A-6E). In both cases, we observed reduced brain GFAP / GBDP38K levels with robust neuroprotective effects, including reduced levels of TBI biomarkers in the cortex, alleviation of anxiety behavior, and improved cognitive performance. To test the potential of anti-G* mAb immunotherapy we first treated mice with a commercial anti-GFAP mAb cocktail (BD cat#566330) that, akin to mAbG#, recognizes the TBI-released GBDP38K Core (shared by intact GFAP) for once-a-week retro-orbital IV injections in mice with 25 pg per mouse for four weeks (0, 7, 14, 21, 28 dpi, with the first dose immediately post-injury). This immunotherapy suppressed TBI-induced GBDP38K (and GFAP) release in mouse cortex and hippocampus (FIG. 7A), reduced the p-tau / total-tau ratio in cortex (FIG. 7B) and hippocampus, reduced tau levels in serum (FIG. 7C), and improved spatial memory in the Y-maze test (FIG. 7D,E). Additional striking preliminary in vivo data providing insights on MoA is presented in the Innovation section below.

[0097] In some inventive embodiments, brain targeting agents are conjugated to the mAbs through cleavable and non-cleavable linkers include: (i) a transferrin receptor (TfR)-binding DNA oligonucleotide (oligo) for receptor- mediated transcytosis by vascular endothelial cells, as known for a TfR- / tau-binding bispecific oligo47; and (ii) Angiopep-2, which has been successfully used for the peptide ANG1005 (Phase 3, NCT03613181) conjugated to the anticancer drug Paclitaxel to improve CNS exposure as much as ~ 100-fold. (48-51)

[0098] The inventive mAbs, even without brain targeting agents, are already entering the brain at therapeutically effective concentrations. Even with mild TBI, BBB disruption can persist for prolonged periods at the site of focal injury(52) and this fact has already been exploited by TBI nanomedicines for brain delivery without brain targeting agents. (53) Shortterm dosing with the inventive mAb may have long-term effects, due to the long circulating half-life of mAbs arising from FcRn recycling (e.g., 4-8 d in mice(54) and 7-28 d in humans(55)), such that a single intravenous dose of anti-GBDP mAb 1-2 days post-injury might improve outcome. According to the present invention, passive anti-GBDP38K mAbimmunotherapy after TBI has neuroprotective effects very similar to those seen with active GFAP / GBDP38K protein immunization before TBI. As an inventive mAh immunotherapy has already succeeded in producing neuroprotective effects, a sufficient amount of mAb does indeed cross the BBB in the TBI context.

[0099] The dominant calpain cleavages that yield GBDP38K are provided in FIGs. 8B and 8B. Techniques for the formation of neo-epitope-based anti-GBDP mAbs are known to the art.(56-60). Inventive mAbs are based on several possible cleavages at the C-terminal (the primary one being T383-F384) and a single dominant cleavage at N-terminal between N50*A60, which produces a neo-N-terminal GBDP38K (NH2-A60GFKETRASER...) (SEQ ID NO 1) (shared by larger G*s). FIGs. 8A-8B show how we can harness the TBI-released GBDP38K Core (shared by intact GFAP) or one or more N-terminal or C-terminal neoepitopes specific to GBDP38K to generate anti-GBDP mAbs. Calpain cleaves GFAP at N59*60A and T383*384F, producing (NH2)-AGFKETRASER. . . (SEQ ID NO 1) and . ..ENRITIPVQT-(COOH) (SEQ ID NO 2), respectively (FIG 8A). By making short synthetic peptides (8-10 residues) that mimic or span these neo-epitopes or extended forms, including the polar NH2 or COOH groups, we can select anti-GBDP mAbs that are highly specific to GBDP38K. The feasibility of generating anti-GBDP mAbs is shown FIG. 8B, where rhGFAP was untreated or digested with calpain- 1 or caspase-6: (left) Immunoblot for a mAb cocktail (BD#60341D) targeting the GBDP38K Core shared by intact GFAP; (right) Immunoblot for our rabbit anti-GBDP polyclonal Ab (pAb) targeting the N-terminal neo-epitope specific to GBDP38K only recognizes calpain-generated GBDP38K (and minor levels of GBDP40K), not intact GFAP or other caspase- generated fragments. More than 25 anti-GBDP38K and anti- GFAP mAbs have been generated, with the neo N-terminal peptide described above and intact GFAP as antigens (FIG. 25), with hybridoma technology, and characterized the mAbs with binding and epitope mapping assays. Binding studies with the Octet system revealed that mAbG* has a dissociation constant (KD) of < 10 pM for human GBDP38K with no binding to rhGFAP-as expected. In contrast, mAbG# has a KD < 10 pM for both GBDP38K and intact GFAP-as expected. In some inventive embodiments, mAbG* binds specifically to recombinant mouse, monkey, and human GBDP38K (overexpressed in E. coli with a cleavable SUMO-His tag on the N-terminus) with a KD < 10 pM and not to intact hGFAP. 16 anti-GBDP38K and GFAP mAbs have been characterized with microglial uptake (phagocytosis) assays. Phagocytosis studies with this system revealed that mAbG* improves microglial uptake of GBDP38K beads by > 150% relative to isotype and vehicle controls, as expected becausemAbG* binds the N-terminal neoepitope of GBDP38K alone. In contrast, mAbG# improves microglial uptake of GBDP38K and GFAP beads by > 150%, as expected.

[0100] GBDP38K is a novel brain target that is abundant and neurotoxic in the extracellular space of subjects suffering any of the aforementioned conditions. Beneficial opsonization of G*s such as GBDP38K with anti-GBDP mAbs: (i) block harmful neurotoxic aggregate formation / accumulation and / or (ii) accelerate beneficial phagocytosis of opsonized neurotoxic aggregates by activated FcyR+ phagocytes. The inventive beneficial opsonization and accelerated phagocytosis potentially has parallels to CD47+ cancer cells with anti-CD47 mAbs that Weissman’s group has pioneered for oncology indications, (35, 36, 63, 64) where binding of the CD47 checkpoint inhibitor with anti-CD47 mAbs led to an approximately 10X acceleration of CD47+ cancer cell phagocytosis relative to normal IgG mAb isotype controls.

[0101] In vivo data in severe focal TBI (FIGs. 9A-9C), the findings of FIGs. 7C,D are extended by illustrating (FIG.9A) the rigor and reproducibility of the data with a second mouse species (CD1) in a second laboratory (IRFMN) and (FIGs. 9B,C) the impact of effector function on sensorimotor measures of efficacy. For the same constructs, target engagement (reduction) and PD biomarker reduction were observed by sandwich ELlSAs and neuropathology (lesion volume, neuronal preservation, IHC) further supporting our previous observations for target engagement and PD biomarker reduction (FIGs.9A,B). The Asym variants for both mAbG* and mAbG#, where different mutations are introduced into paired Fc chains for enhanced effector function without a loss in thermal stability, (65-67) consistently outperformed the other variants. Without intending to be bound by a particular theory, FcyR- dependent are more potent than FcyR-independent mechanisms of action.

[0102] Anti-GBDP38K mAbs appear to have a high therapeutic index. Mabs often target cell-surface proteins to remove harmful cells from circulation, although the same cell surface proteins can also be found on healthy cells (e.g., CD20 is found on B-cells and CD47 on red blood cells). In contrast, anti-G* mAbs target a normally intracellular brain protein released into the extracellular fluid of the brain; GBDP38K is not believed to be expressed on the surfaces of healthy astroglia. Considering the above evidence and potential risks of active GFAP immunization, in some inventive embodiments, TBI subjects treated with anti-G* mAbs targeting GBDP38K benefit from a high therapeutic index. It is noted that GBDP38K is generated only after TBI, and unlike intact GFAP, GBDP38K has no known normal brain function, but instead is prone to aggregate and become neurotoxic (like mutated GFAP in AxD).(33) Furthermore, GBDP38K is the most abundant GBDP in human TBI patients (FIGs. 4A-4D) and is released from injured or dying astrocytes in the extracellular space, making itan ideal target for mAb-directed opsonization and phagocytosis. According to some inventive embodiments, active GFAP / GBDP38K protein immunization before TBI is safe and efficacious in mouse models of TBI. A cadre of 60 mice immunized with intact GFAP and GBDP38K protein and confirmed strong Ab responses (FIGs. 6A-6E) without any observable meningoencephalitis or astrocytopathy.

[0103] The inventive mAb immunotherapy is safe and efficacious in mouse models of TBI. In some inventive embodiments, mAb is administered for a short duration (e.g., no more than 28 dpi), for example, since GBDP38K production is maximal during the first 48-120h (2- 5 dpi) (FIG. 4D). This short treatment duration further reduces the risk of patients developing chronic neurodegenerative conditions like autoimmune meningoencephalitis. No adverse events have been observed in any mice treated with mAbs at 5 mg / kg for 28 days.

[0104] Therapeutic mAbs in some inventive embodiments rely on diagnostic assays for temporal blood biomarkers for enrichment and tracking of responding patients. GBDP38K is distinguished from intact GFAP using a sandwich ELISA, data for which is shown in FIGs. 2A-2d to distinctly track GBDP38K and GFAP target engagement as well as therapeutic response over the course of treatment. In some inventive embodiments, subacute temporal biomarkers responsive to anti-GBDP38K mAb immunotherapy are provided; these illustratively include diffuse axonal injury (DAI) markers (e.g., NFL and phosphorylated neurofilament heavy / p-NFH), a synaptic injury marker (synapsin-1), and the neurodegeneration temporal PD biomarkers tau and p-tau, markers that are sustained at detectable levels in the subacute phase (2 -28 dpi and beyond). (22, 29) The data on serum NFL includes a pig FPI study with NFL, three different rodent models from the OBTT consortium using p-NFH,20 and human TBI data that show elevated p-NFH one day after severe TBI.(33) For tau, data include elevated serum total-tau in rat CCI and p-tau following repeated close head injury. Synapsin-1 was found to be a proteolytic biomarker in a rat CCI study.(70, 71) Quantification of these biomarkers with traditional ELISA is conventional to the art.

[0105] In some inventive embodiments, 1-28 dpi is a critical therapeutic window for therapeutic mAbs to attenuate nerve fiber damage (assessed by NF-L and / or p-NFH levels) and neurodegeneration (assessed by tau and p-tau) via beneficial opsonization and accelerated phagocytosis of GDBP38K+ brain cell debris. In still other inventive embodiments, the maximum brain exposure to the mAb for greater than 4 dpi, with greater than 1.0% brain penetrance and circulation ' / 2-lile greater than 2 d, when the BBB is most compromised, is an important PK / PD consideration.

[0106] Therapeutic and diagnostic mAbs bind GBDP22-24K, GBDP38K, GBDP44K, monomeric-, oligomeric-, and / or aggregates GFAP in TBI, PTE, AxD, AD, and other aforementioned disorders. Abnormal aggregates of misfolded proteins are a hallmark of most neurodegenerative diseases collectively known as proteinopathies. Well-known examples of these aggregates include amyloid-beta plaques and tau tangles in Alzheimer's disease (AD) (68), synuclein deposits in Parkinson's disease and Lewy Body Dementia, and TDP-43 in ALS. Aggregates exist in a wide range of sizes and conformations, from low molecular weight, soluble oligomers to high molecular weight, insoluble plaques sized 500 square microns or more (in the case of amyloid-beta) (72, 73) Individual protein molecules within the aggregates typically bear abnormal accumulations of post-translational modifications (e.g., phosphorylation, ubiquitination) and exist in a variety of truncated as well as full-length forms. (74-78) Aggregates can occur inside cells, outside, or both, depending on the protein involved and toxic both inside and external to cells. Such external aggregates include soluble aggregates, oligomers and protofibrils. (75, 79-81)

[0107] Aggregates in TBI. GFAP aggregate formation in TBI can have implications for the detection and evaluation of TBI, as well as for the development of neurodegenerative diseases. GBDP38K is directly observed as aggregates by immunofluorescence of human TBI brain tissue (FIGs. 10A-10C) and mouse TBI brain tissue (FIG. 11). Liquid chromatographytandem mass spectrometry (LC-MS / MS)-proteomic analysis of naive, TBI, and PTE mouse brain tissue specimens showed several proteins up-regulated post-TBEPTE vs. naive controls (FIG. 12). GFAP / G*s (P03995) levels were clearly up-regulated (p <0.001) in TBI vs. naive mice, and further significant up-regulation was found in PTE vs TBI (no PTE) mice (p <0.001 ). However, like most proteomics experiments, sequence coverage from the peptides observed by LC-MS / MS was insufficient to determine whether intact GFAP or its G*s were differentially regulated. Consequently, the major forms of GFAP / G*s in human and mouse TBI / PTE with non-reducing and reducing immunoblots using the above described diagnostic mAbs (FIGs. 13-15). Different GFAP / G*s (and aggregates thereof) were clearly observed in mouse TBI (FIGs. 14A and 14B) vs. human TBI (FIG.12-2), where the most abundant GFAP / GBDP species is GBDP38K (and aggregates thereof) in human TBI CSF (FIGs. 15A and 15B), and the most abundant GFAP / G* species is GFAP at 50K and GBDP44K (and aggregates thereof) in mouse TBI, albeit GBDP38K and other G*s were observed. Indeed, GBDP38K (and aggregates thereof) comprise less than 5% of the total GFAP / G* species in mouse TBI brain tissue (FIG. 1) and human CSF (FIGs. 4A-4D). An inventive mAb clone, known as mAbG*, binds to both monomers and aggregates of GBDP38K (FIGs. 1A-1D).

[0108] GFAP is used as a biomarker to aid in the evaluation of mild traumatic brain injury (TBI). TBI causes protease-mediated GFAP-breakdown (BDP) product formation in injured glial cells and subsequent release of such GFAP aggregates. Another study found that GFAP levels were elevated in participants with moderate and severe TBI compared to control participants, and that this initial increase was followed by a reduction over the following 6 months after injury, but an increase over the subsequent years. Protein aggregates can contribute to the extraordinary long-term stability of GFAP, and these aggregates can last for several years at ambient temperature. The formation of protein aggregates can cause a hook effect, which can affect the detection of GFAP in blood. GFAP aggregate formation in TBI can have several implications. GFAP is used as a biomarker to aid in the evaluation of mild TBI. A key feature of TBI is the increase in GFAP immunostaining, indicating activation and / or proliferation of astrocytes. According to one study, TBI causes protease-mediated GFAP- breakdown product (G*) formation in injured glial cells and subsequent release of such GFAP proteoform aggregates. Protein aggregates can contribute to the extraordinary long-term stability of GFAP, and these aggregates can last for millennia at ambient temperature. The formation of protein aggregates can cause a hook effect, which can affect the detection of GFAP in blood. Tau and Ap aggregates have also been associated with TBI, and the mechanism by which TBI induces the formation of protein deposits of tau and A is still unknown but is strongly related to the development of CTE and AD. Some researchers believe GFAP aggregates are toxic to astrocytes and might contribute to the astroglial degeneration and the subsequent white matter.

[0109] In GFAP knockout (GFAP-KO) animals, the response to traumatic brain injury (TBI) or neurotoxicity has been studied. One study found that GFAP-KO mice showed enhanced neurite sprouting. Another study involving Sigma-1 receptor knockout (S IR- / -) mice, which presented a significantly reduced GFAP expression in Bergmann glial cells in the cerebellum compared to wild-type (WT) mice, found that neurological deficits and motor coordination impairment were less pronounced in SIR- / - mice with TBI than in WT mice with TBI 24 hours after injury. TBI-induced short-term memory impairments were present in WT but not SIR- / - mice 7 months after injury. Compared to WT animals, S IR- / - mice exhibited better motor coordination and less pronounced despair behavior for up to 12 months postinjury.

[0110] Aggregates in AxD. In Alexander’s disease (AxD), a leukodystrophy caused by heterozygous mutations in GFAP, astrocytic expression of mutant GFAP proteins results in a profound loss of white matter. Histological hallmarks of AxD are the emergence ofperivascular Rosenthal fibers consisting of ubiquinated aggregates of GFAP, vimentin, small heat shock proteins ap-crystallin and Hsp27, and plectin (83, 84) Sequence analysis of DNA samples from patients representing different Alexander disease phenotypes revealed that most cases are associated with non-conservative mutations in the coding region of GFAP85. Pathogenic variants have been identified in 87 of the 432 amino acids of the major isoform, GFAPa, and are distributed throughout the protein (though few in the nonhelical head domain), suggesting that the disorder is caused by a global property of the protein rather than alteration of a local interaction site underscoring the potential for a common therapeutic solution. The pathological hallmark of all forms of Alexander disease is the presence of Rosenthal fibers both as cytoplasmic inclusions and in perivascular spaces. Overexpressed mutant GFAP (R239C) tended to aggregate into inclusions, which were associated with components of the proteasome system, such as proteasome 20 S core complexes, ubiquitinated proteins, and small heat shock proteins, cells and of Rosenthal fibers in AxD brains. Since, overexpression of normal human GFAP in astrocytes of transgenic mice is fatal and accompanied by hypertrophic astrocytes, that up-regulate small heat-shock proteins, and contain inclusion bodies identical histologically and antigenically to the Rosenthal fibers. (86) These results suggest that upregulated GFAP may directly contribute to aggregate formation either due to genetic predisposition or chronic activation.

[0111] Aggregates in AD. AD is an aging associated neurodegenerative disease characterized by the presence of extracellular amyloid plaques and intracellular Tau tangles, both distinct and toxic protein aggregates that contain amyloid beta(Ab) fragments and Tau respectively in addition to a large number of other proteins. (87) GFAP expression and protein was found to be increased with Tau accumulation and soluble Ap42 aggregates formed at different stages of plaque aggregation process (88, 89) and may be a driver or a consequence of ongoing pathology. While the large Ab42 aggregates induce an inflammatory response in astrocytes, the small soluble aggregates cause lipid bilayer disruption, thus promoting the cascade of GFAP upregulation and aggregation and its independent neurotoxic effects. Interestingly, proteomic identification of proteins in sarkosyl-insoluble aggregates from AD brains revealed the presence of GFAP aggregates in AD brains. (89) Despite reactive astrocytosis surrounding the plaques, that has been consistently observed in AD, chronically elevated glutamate concentrations that are neurotoxic have been observed in plaques in AD models, suggesting compromised or sub-optimal glutamate detoxification by chronically activated astrocytes and / or direct impact of GFAP aggregates around AD plaques that may aggravate the disease pathology. It has been reported that activated astrocytes (and by extensionGFAP) may be central to neurodegeneration and cognitive impairment downstream of Tau protein. Ap was associated with increased plasma phosphorylated tau only in individuals positive for astrocyte reactivity Recent study by Ganne et al, suggest that GFAP phosphorylation plays a key role in neuropathic aggregate accrual in Alzheimer's disease. (90) Pyroglutamate-3 A (pGlu-3 AP) is an N-terminally truncated and post-translationally modified Ap species found in Alzheimer’s disease (AD) brain. Its increased peptide aggregation propensity and toxicity make it an attractive emerging treatment strategy for AD. Donanemab, a humanized immunoglobulin G1 monoclonal antibody that specifically targets N-terminally truncated pyroglutamate-modified amyloid P (AppE), showed a substantial reduction in Alzheimer's disease-associated cerebral amyloid-plaque load, measured by amyloid positron emission tomography (PET), in the intervention group compared with the placebo group. (91) With Donanemab, treatment, significant reductions in plasma biomarkers phosphor-Tau217 and GFAP were observed in patients with early symptomatic Alzheimer disease compared with placebo.

[0112] Aggregates in Aging The current findings demonstrate that plasma GFAP levels are elevated in cognitively normal older adults at risk of AD. These observations suggest that astrocytic damage or activation begins from the pre-symptomatic stage of AD and is associated with brain Ap load. Observations from the present study highlight the potential of plasma GFAP to contribute to a diagnostic blood biomarker panel (along with plasma Api-42 / Api- 40 ratios) for cognitively normal older adults at risk for dementia.

[0113] Aggregates Other CNS Disorders. In amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disease that affects nerve cells in the brain and spinal cord and in Frontotemporal dementia (FTD), astrocytes play a significant role in the onset and progression of the disease. In both ALS and FTD, astrocytes not only have a reactive and inflammatory phenotype but also show impaired protective functions. This complex role of astrocytes in these neurodegenerative diseases is still being explored, and further research is needed to fully understand their contribution to disease progression. These astrocytes become dysfunctional and adopt an atypical, neurotoxic phenotype, promoting neuroinflammation and motor neuron degeneration. GFAP, is usually upregulated in response to reactive astrogliosis, a process that occurs in response to injury in the central nervous system. In ALS, reactive GFAP-astrocytes have been found in the ventral horn of patients, with elevated appearance in the cerebrospinal fluid (CSF) relative to other neurologic diseases. (92) Furthermore, serum levels of GFAP have been found to be increased in ALS patients, suggesting astrocyte involvement in ALS pathophysiology. (93) In FTD, astrocytes also play a crucial role. Asubpopulation of GFAP(+) astrocytes has been observed to degenerate in FTD, correlating inversely with cerebral blood flow. This suggests that astrocytes may be affected by or perhaps have a causal role in the disturbances of cerebral perfusion in FTD. Additionally, plasma GFAP levels have been found to correlate with a higher burden of cognitive impairment during FTD, suggesting its potential as a progression biomarker. (94) In both ALS and FTD, astrocytes not only have a reactive and inflammatory phenotype but also show impaired protective functions. This complex role of astrocytes in these neurodegenerative diseases is still being explored. (94, 95) However removal of toxic GFAP fragments found in degenerating astrocytes are therefore beneficial in slowing disease progression where no drugs exist or in combination with disease specific therapies. Furthermore, in ALS, FTD and several other neurological disorders TAR DNA-binding protein 43 (TDP-43) is known to mislocalize and aggregate in neurons and astrocytes. (96). Recent findings on the sequence and structural determinants of its aggregation and neurotoxicity have revealed a cleavage dependent seeding function of TDP-43 fibril that is boosted by the amyloid core (97) suggesting crosstalk between aggregation prone proteins may synergize to exacerbate neurodegeneration.

[0114] With GFAP emerging as a blood biomarker in several neurological and spinal cord disorders and its likely involvement in contributing to aggregate formation in proteinopathies opens a therapeutic window prior to progression to debilitating neuropathy.

[0115] Misfolding and aggregation of GFAP and G*s, which can lead to loss of a protein’s normal functions as well as gain of toxic functions, might be important for TBI, AxD, AD, and other Aging and other CNS disorders.

[0116] Post-traumatic epilepsy (PTE) is a common and serious complication of TBI. PTE accounts for 5% of all epilepsies and 10-20% of the acquired forms (98, 99) with approximately 20,000 new PTE patients yearly in the U.S. (100) A population-based study showed that the 30-year cumulative incidence of PTE is 1.2% for mild, 4.2% for moderate, and 16.7% for severe TBI. (101) The latency between TBI and PTE onset in high-risk patients offers a therapeutic window for intervention to prevent or improve the disease course. However, there are still no anti-epileptogenic DMTs for PTE, and anti-seizure drugs, while preventing early symptomatic seizures after TBI, do not prevent PTE. (102-105) Progress towards effective DMTs for TBI and PTE have been hampered by the limited knowledge of the mechanisms and markers of TBI and TBI-induced epileptogenesis, respectively, and by the lack of validated therapeutic targets.

[0117] Astrocytes and TBI / PTE. Reactive astrogliosis is the term that describes the morphological and functional changes in astroglial cells / astrocytes responding to centralnervous system (CNS) injury and neurological diseases, and is a common feature of many neurodegenerative conditions, including TBI and PTE. (106-108) Reactive astrocytes can be either neurotoxic or neuroprotective, and the two subtypes have been termed Al and A2 astrocytes, respectively. (109) Note that this binary terminology is a major simplification of astrocyte diversity (110) but is nonetheless useful for the purposes of this proposal. Al astrocytes predominate in neuroinflammatory conditions (including TBI) and are themselves proinflammatory, whereas A2 astrocytes predominate in stroke and are anti-inflammatory (1 1 1 , 1 12) There is strong evidence that reactive astrocytes contribute to TBI-associated neurodegeneration and PTE. Increased levels of 1H-MRS myo-Inositol (mln), a metabolite reflecting astrocyte activation, are present after TBI and correlate to neuronal damage and worse outcome. (113) Furthermore, we showed that high mln levels predict epilepsy development evoked by de novo status epilepticus in rodents (114), and are associated with PTE in mice. (116) Reactive astrogliosis (together with microgliosis) flares up both at the primary injury site and in distant brain regions after a TBI and can persist for years in both humans and rodent models. (116, 117) Reactive astrocytes may contribute to neural dysfunction and or epileptogenesis in a variety of ways, including (1) their reduced gap junction coupling, which disrupts circuit function and can contribute to neuronal death, (2) hypertrophy, which can lead to the formation of abnormal neural circuits, and (3) loss of aquaporin function, which is associated with a dysregulation of water and potassium homeostasis that in turn affects seizure susceptibility (106, 118). Increased levels of Al -associated cytokines (IL1 and TNF) have been correlated with increased seizure susceptibility in general, and the development of PTE in particular. (106, 1119-124) In addition, complement protein 3 (C3), which is selectively expressed by astrocytes of the Al type, is induced by both TBI and experimental seizure (125- 128), and higher C3 levels have been linked to higher seizure frequency and susceptibility in animal models and in temporal lobe epilepsy patients. (131- 134)

[0118] Without intending to be bound to a particular theory, under experimental neurotrauma conditions, calpain cleaves GFAP and generates G*s, particularly GBDP38K with a lower MW limit of 38 kDa, in the injured cortex during the first hours to 28 days post injury (dpi) in mouse TBI models. In parallel, GFAP / G*s are released into the extracellular fluid and other biofluids (CSF, blood) for 2 to >28 dpi, with a peak at 2 dpi 18-20. Other protein “debris” are also released from neuronal cell bodies (ubiquitin C-terminal hydrolase 1 / UCH-L1) and injured axons (neurofilament-heavy / pNF-H and light / NF-L, tau and phosphorylated tau or p p- p-tau). (21, 25,133-135).

[0119] Clinical AD Evidence. Given that a precipitous drop in CSF Ab42 levels, a marker of parenchymal Ab deposition in brain tissue, is one of the earliest steps in the well-established pathological progression of AD (136), the precipitous drop in levels of G*s observed in TBI might reflect deposition of G*s in brain tissue (FIG. 24). Evidence from cross-sectional and longitudinal tau-positron emission tomography analyses revealed an AD-like pattern of tau tangle accumulation as a function of Ap only in cognitively unimpaired individuals with high levels of serum GFAP-suggesting astrocyte reactivity as an important upstream event linking A with initial tau pathology. (137) This finding is echoed by other papers suggesting GFAP as a biomarker for AD and other proteinopathies. (138)

[0120] GFAP / G*s as novel therapeutic targets for TBI / PTE and other CNS diseases, including brain cancers and the CNS diseases described above. Evidence from our past research and published data suggest that astrocyte derived GFAP and GBDP38K, may contribute to the worsening of TBI and the development of PTE, (17, 111, 123, 124, 139-141) GFAP is not traditionally listed among aggregation-prone proteins associated with neurodegenerative diseases. However, there is a rare hereditary leukodystrophy involving destruction of the myelin, Alexander’s disease (AxD), caused by mutations in the GFAP gene, in which mutant GFAP protein monomers form intracellular aggregates (called Rosenthal fibers) are directly toxic to glia (142, 143) Moreover, a recent study in astrocytes generated from human induced pluripotent stem cells (iPSCs) showed that AxD mutations increase both the sensitivity of GFAP to cleavage by caspase (another calcium-dependent enzyme like calpain) and its propensity to aggregate. (144)

[0121] Abnormal aggregates of GFAP / G*s might be promising therapeutic targets for TBI, AxD, AD, and other Aging and other CNS disorders.

[0122] GFAP / G* autoantibodies are beneficial. GFAP / G* autoantibodies (autoAbs) in TBI. The brain may mount a self-protective immune response after TBI in some patients, in which excess amounts of extracellular GFAP / G*s prime T- and B-cells to generate anti- GFAP / GBDP autoAbs. Such autoAbs are observed in a subpopulation of TBI subjects from 5 dpi and peak at 14 dpi. Auto Ab binding to GFAP / GBDP debris (i.e., opsonization) can trigger the activation of microglia / recruited macrophages infiltrating through the compromised bloodbrain barrier (BBB), via binding of the Ab to immune cell surface FcyR. In this scenario, activated phagocytes (microglia and macrophages) are likely beneficial, as they “devour” harmful protein debris. ( 35-37). Importantly, anti-GFAP / G* autoAbs are observed in only a subpopulation of TBI subjects and their titers can vary 38. Anti-GFAP / G* autoAbs and mAb treatments in other CNS indications. Anti-GFAP / G* autoAbs are found in several clinicalconditions (39-43) in addition to TBI / PTE. (38) However, several lines of evidence suggest that anti-GFAP / G* auto Ab or mAb immunotherapy might be neuroprotective.(69) (i) In a cellbased model of glaucoma, anti-GFAP Abs had protective effects on retinal ganglion cells subjected to oxidative stress. (44, 45) (ii) Levels of anti-GFAP / G*P autoAbs in stroke patients with favorable outcomes were significantly higher than in those with poor outcomes. (46)

[0123] The mechanism of action for an inventive mAb is, without intending to be bound to a particular theory, is opsonization of toxic GFAP / G* debris from astrocytes, followed by engagement via the Fab domain of immunoglobulin Fc-gamma receptors (FcyRs) on microglia (FIG. 2). An inventive mAb is designed to harness microglia to accelerate protein debris clearance from the brain and rebalance reactive astrocytes from a toxic to a neuroprotective / quiescent state. In some inventive embodiments, an inventive acute, subacute, and chronic temporal blood biomarker panel for longitudinal monitoring of PD biomarkers and target engagement.

[0124] Diagnostic inventive mAbs for GFAP / G* aggregates are provided for the targeting GFAP / G* aggregates for TBI, AxD, AD, and other Aging and other aforementioned disorders. As illustrated in FIGs.20-21, there are numerous contexts of use for diagnostic tests based on mAbs for measuring GFAP proteoforms in blood and brain tissue (FIGs. 19A and 19B). Personalized trajectories of blood biomarkers and their ratios reveal brain health status, identify periods of risk for secondary monitoring and administration of disease modifying therapies (DMTs) (FIG. 22), as well as provide readout for recovery.

[0125] As shown in FIG. 21 , toxic burden (redline) in the brain is dependent on diurnal clearance capacity (blue) and age (x-axis) and is sharply impacted by TBI. Increasing proteinaceous aggregates that comprise toxic burden in the brain.

[0126] According to some inventive embodiments, antibodies to specifically bind to the N- or C-terminus of GBDP38K, GBDP44K, and GFAP, are provided to bind to a region spanning these termini.

[0127] In some inventive embodiments, GBDP22-24K, GBDP38K, GBDP44K, monomeric, oligomeric-, and / or aggregate- GFAP provide both age-dependent and ageindependent information on brain health, injury, and disease. G*s offer the intriguing possibility of having distinct age-related time profiles from GFAP, where we and others have observed dramatic increases in GFAP levels with age. Since G*s are generated by intracellular cleavage by calcium activated proteases like calpain following astrocytic injury, G*s do not show an age-dependent trajectory like GFAP. This is important because it allows for theestablishment of cutoff values for diagnostic and therapeutic applications that don’t need to be normalized through a z-score derived from a large normative database of health aging controls.

[0128] Detrimental effects of GFAP overexpression: GFAP is an intermediate filament (IF) protein and is important for maintaining structural integrity of cells. It turns over rapidly within cells and supports dynamic changes in cell shape required to support functions in response to stimuli. Excessive overexpression of GFAP results in stiffening of the fibers, reduction in elongated processes and reduce motility, thus compromising astrocyte functions. In vitro, overexpression of GFAP can lead to the formation of protein aggregates, which have a toxic effect on neighboring neurons. Both mutant and wild type GFAP overexpressing astrocytes promote formation of cytoplasmic inclusions, disrupts the cytoskeleton, decrease cell proliferation increase cell death, reduce proteasomal function and compromise associated resistance to stress. Thus, excessive GFAP can trigger a feed forward cycle of CNS damage either directly by the toxic aggregates or fragments that are released during normal turnover or upon death of the activated astrocytes. Moreover, with fewer and compromised astrocytes, the natural clearance processes that are astrocyte mediated (including microglia recruitment and sleep dependent glymphatic clearance) are also adversely affected, exaggerating the CNS toxic burden and subsequent damage.

[0129] Structure of GFAP / G* monomers, oligomers, and aggregates. GFAP was originally discovered nearly half a century ago as a component of aggregates in disease brains. Since then, GFAPa has been established as a reliable marker of astrocytes with respect to both, their proliferation and activation within the CNS (FIG. 17-18).

[0130] According to some inventive embodiments, excessive monomers or oligomers of GFAP and / or its modified toxic G*s products are targeted. In still other embodiments, these proteoforms as biomarkers are monitored for diagnostic tests and in still other embodiments, clearing of these proteoforms as therapeutic targets mitigates the deleterious effects of activated / dysfunctional astrocytes, without impacting its normal functions.

[0131] GFAP forms the cytoplasmic intermediate filament network of mature astrocytes together with vimentin. Each monomer is mainly a-helical central domain (rod), flanked by intrinsically disordered head and tail domains (FIG. 19). (150) The GFAP monomer structure is integrated into the filament by assembly of a multimeric unit length fragment. Like other type III intermediate filament proteins, assembly of GFAP into filaments is considered to proceed through the association of monomers into parallel dimers by non-covalent interactions between the IB helix to form a coiled structure. The antiparallel association of dimers intotetramers forming the coiled coil, and the gathering of several tetramers into unit length filaments that elongate by head to tail engagement.

[0132] The sole cysteine in human GFAP, present at position 294 and conserved in animals ranging from fish to mice and humans. The cysteine can function as a redox sensor and is susceptible to lipoxidation in cultured cells via prostaglandins. Interestingly, mutating this cysteine to serine interferes with the ability of GFAP to assemble into stable cytoskeletal network, suggesting that its lipoxidation would have a similar effect. (151) Conversely, a cysteine-cysteine bridge between monomers within the assembled unit length fragment could stabilize the intermediate filament, reducing filament turnover and dynamics while promoting rigidity of the cell. Supporting this idea, mutations in GFAP that cause the gain of a cysteine (e.g. R239C or R79C), also cause Alexander’s disease, and protein aggregation. Although a cysteine-cysteine bridge has never been described for GFAP, we have found evidence for a crosslinked dimer under certain conditions. We propose that oxidized environment and / or certain mutations may promote cysteine-based crosslinking of GFAP monomers forming stable structures that are resistant to removal mechanisms. This, in turn, may promote damage and dysfunction of astrocytes internally as well as of other CNS cells externally either upon release after death / damage of astrocytes or due to their aberrant function.

[0133] More than ten isoforms of GFAP have been identified that arise from alternative splicing and differ mainly in the C-terminal domain. (152) The most abundant and thoroughly studied isoform is GFAPa while some forms and or cleavage products are likely to be preferentially generated in particular disease states their relevance or contribution to disease progression has never been described. We have identified an aggregation prone fragment of GFAP called GBDP38K (FIG. 20).

[0134] FIG. 21 depicts personalized trajectories of blood biomarkers and their ratios reveal brain health status, identify periods of risk for secondary monitoring and administration of disease modifying therapies (DMTs), as well as provide readout for recovery, longitudinal monitoring of TBI patients (2.5 million in the US alone) and routine screening for >65 year olds is a major current unmet need (with an expected 7.5 million over age 65 individuals with undiagnosed mild cognitive impairment / MCI in the US alone), the N-terminal neoepitope of GBDP38K is denoted as G*, while the N-terminal and C-terminal epitopes unique to GFAP are denoted as N* and C*, respectively. The conserved core domain is denoted as GFAP. Ratios of these proteoforms provide various contexts of use for biomarker-based biofluid or tissue tests, including but not limited to those shown above;

[0135] FIG. 22 are schematic plots dependence of toxic burden (redline) in the brain on diurnal clearance capacity (blue) and age(x-axis). and is sharply impacted by TBI, increasing levels of proteinaceous aggregates comprised of GBDP38K and other proteoforms of GFAP, as well as other proteins (e.g., vimentin) comprise toxic burden in the brain.

[0136] FIGs. 23A-23C are plots of. GFAP proteoforms (GZ G* / N* / C*) and calculated ratios (black) in brain and biofluids identifies treatment windows by sampling accessible biofluids like blood, the N-terminal neoepitope of GBDP38K is denoted as G* in the context of these plots and those of FIG. 24, while the N-terminal and C-terminal epitopes unique to GFAP are denoted as N* and C*, respectively, he conserved core domain is denoted as G, ratios of these proteoforms provide various contexts of use for biomarker-based biofluid or tissue tests, including but not limited to those shown above, where FIG. 23A is for a healthy subject, FIG. 23B is for GFAP activation and cleavage, and FIG. 23C is for sustained GFAP activation, cleavage and aggregation in the brain.

[0137] FIG. 24 is a schematic showing changes in the absolute level of GFAP proteoform monomers (e.g., G / N* / G*) (shown as green strings) or their ratio (e.g., G / G*) in biofluid (e.g., blood) indicates changes in GFAP proteoforms in brain tissue-suggesting changes in oligomer / aggregate formation in the CNS, therapeutic monoclonal antibodies (mAbs) (Y- shaped structures) can bind and reduce oligomer / aggregate formation through multiple mechanisms of action, the N-terminal neoepitope of GBDP38K is denoted as G*, while the N- terminal and C-terminal epitopes unique to GFAP are denoted as N* and C*, respectively, the conserved core domain is denoted as G, ratios of these proteoforms provide various contexts of use for biomarker-based biofluid or tissue tests.

[0138] FIG. 25 are schematic of peptide cyclization to generate antibodies recognizing the N-terminal or C-terminal neoepitopes of G*s such as GBDP38K.

[0139] FIG. 26 is a plot of G* interassay precision as a function of added G*.

[0140] FIG. 27 is a comparative plot of G* relative to GFAP for cerebrospinal fluid (CSF)sampling fluid obtained from a subject suffering from traumatic brain injury (TBI).

[0141] FIGs. 28A-28C are plots for CSF samples for GFAP (FIG. 28A), G* (FIG. 28B) and a ratio therebetween (FIG. 28C) for pooled healthy cohort, pooled TBI and subject CSF groups thereby providing evidence for clinical utility as a companion diagnostic.

[0142] FIGs. 29A and 29B are plots of GFAP and G* as a function of post-injury time (hr) for CSF (FIG. 29 A) and serum (FIG. 29B) samples from TBI subjects thereby showing G* has a uniquely longer trajectory than GFAP with greater potential for patient monitoring in an intensive care setting and beyond.

[0143] FIGs. 30A and 30B are time block plots for serum GFAP (FIG. 30A) and G*(FIG. 30B) from TBI subjects showing the longer trajectory for G* with the oval marked regions to highlight that G* is more resistant to decay in the 72h to 96h time window, the control (H.C.) is also noted.

[0144] FIGs. 31A and 31B are biomarker levels in pediatric TBI subject samples for GFAP (FIG. 31 A) and G* (FIG. 3 IB) for Glasgow Outcome Score Extended (GOSE) for low and high value groupings showing the longer trajectory for G* and that sustained G* levels by day 3 inform on poor outcome by GOSE.REFERENCES CITED

[0145] 1. Maas, A.I.R. et al. Traumatic brain injury: integrated approaches to improve prevention, clinical care, and research. The Lancet Neurology 16, 987-1048-1048 (2017).

[0146] 2. Kobeissy, F.H. et al. Neuroproteomics and Systems Biology Approach toIdentify Temporal Biomarker Changes Post Experimental Traumatic Brain Injury in Rats. Front Neurol 7, 198 (2016).

[0147] 3. Yang, Z. et al. Temporal Profile and Severity Correlation of a Panel of RatSpinal Cord Injury Protein Biomarkers. Molecular Neurobiology 55, 2174-2184-2184 (2018).

[0148] 4. Yang, Z. et al. Temporal MRI characterization, neurobiochemical and neurobehavioral changes in a mouse repetitive concussive head injury model. Scientific Reports 5, 11178 (2015).

[0149] 5. Zhao, X. et al. Subcellular Localization and Duration of p-Calpain and m-Calpain Activity After Traumatic Brain Injury in the Rat: A Casein Zymography Study. Journal of Cerebral Blood Flow & Metabolism 18, 161-167-167 (1998).

[0150] 6. Evans, T.M. et al. Microwave and magnetic (M2) proteomics of a mouse model of mild traumatic brain injury. Advances in Integrative Medicine 3 (2014).

[0151] 7. Mahesula, S. et al. Immunoenrichment microwave and magnetic proteomics for quantifying CD47 in the experimental autoimmune encephalomyelitis model of multiple sclerosis. ELECTROPHORESIS 33, 3820-3829-3829 (2012).

[0152] 8. Raphael, I. et al. Microwave and magnetic (M2) proteomics of the experimental autoimmune encephalomyelitis animal model of multiple sclerosis. ELECTROPHORESIS 33, 3810-3819-3819 (2012).

[0153] 9. Raphael, I. et al. Microwave & Magnetic (M2) Proteomics Reveals CNS-Specific Protein Expression Waves that Precede Clinical Symptoms of Experimental Autoimmune Encephalomyelitis. Scientific Reports 4, 6210 (2014).

[0154] 10. Raphael, I. et al. Serum Neuroinflammatory Disease-Induced CentralNervous System Proteins Predict Clinical Onset of Experimental Autoimmune Encephalomyelitis. Frontiers in Immunology 8, 812 (2017).

[0155] 11. Korley, F.K. et al. Valproic Acid Treatment Decreases Serum Glial FibrillaryAcidic Protein and Neurofilament Light Chain Levels in Swine Subjected to Traumatic Brain Injury. J Neurotrauma 35, 1185-1191 (2018).

[0156] 12. Korley, F.K. et al. Valproic Acid Treatment Decreases Serum GFAP andUCH-L1 Level in Swine Subjected to Traumatic Brain Injury. Journal of Neurotrauma (2018).

[0157] 13. Karlsson, M. et al. Neuroprotective Effects of Cyclosporine in a Porcine Pre-Clinical Trial of Focal Traumatic Brain Injury. J Neurotrauma (2018).

[0158] 14. Pisani, F. et al. Identification of two major conformational aquaporin-4 epitopes for neuromyelitis optica autoantibody binding. J Biol Chem 286, 9216-9224 (2011).

[0159] 15. Papa, L. et al. Elevated levels of serum glial fibrillary acidic protein breakdown products in mild and moderate traumatic brain injury are associated with intracranial lesions and neurosurgical intervention. Ann Emerg Med 59, 471-483 (2012).

[0160] 16. Yang, Z. et al. Characterization of Calpain and Caspase-6-Generated GlialFibrillary Acidic Protein Breakdown Products Following Traumatic Brain Injury and Astroglial Cell Injury. Int J Mol Sci 23 (2022).

[0161] 17. Li, D. et al. Neurochemical regulation of the expression and function of glial fibrillary acidic protein in astrocytes. Glia 68, 878-897 (2020).

[0162] 18. Mondello, S. et al. Blood-Based Protein Biomarkers for the Management ofTraumatic Brain Injuries in Adults Presenting to Emergency Departments with Mild Brain Injury: A Living Systematic Review and Meta- Analysis. Journal of neurotrauma (2018).

[0163] 19. Korley, F.K. et al. Performance Evaluation of a Multiplex Assay forSimultaneous Detection of Four Clinically Relevant TBI Biomarkers. Journal of Neurotrauma (2018).

[0164] 20. Yang, Z. et al. Serum-based phospho-neurofilament-heavy protein as theranostic biomarker in three models of traumatic brain injury: an Operation Brain Trauma Therapy (OBTT) study. Journal of Neurotrauma (2018).

[0165] 21. Wang, K.K. et al. An update on diagnostic and prognostic biomarkers for traumatic brain injury. Expert Review of Molecular Diagnostics (2018).

[0166] 22. Rubenstein, R. et al. Temporal Profiles of P-Tau, T-Tau, and P-Tau:TauRatios in Cerebrospinal Fluid and Blood from Moderate-Severe Traumatic Brain InjuryPatients and Relationship to 6-12 Month Global Outcomes. J Neurotrauma 41 , 369-392 (2024).

[0167] 23. Robertson, C.S. et al. Serial Measurements of Serum Glial Fibrillary AcidicProtein in Moderate-Severe Traumatic Brain Injury: Potential Utility in Providing Insights into Secondary Insults and Long-Term Outcome. J Neurotrauma 41, 73-90 (2024).

[0168] 24. Frankel, M. et al. Association of Very Early Serum Levels of SIOOB, GlialFibrillary Acidic Protein, Ubiquitin C-Terminal Hydrolase-Ll, and Spectrin Breakdown Product with Outcome in ProTECT III. J Neurotrauma 36, 2863-2871 (2019).

[0169] 25. Bi, M. et al. Tau exacerbates excitotoxic brain damage in an animal model of stroke. Nat Commun 8, 473 (2017).

[0170] 26. Mestre, H. et al. Aquaporin-4-dependent glymphatic solute transport in the rodent brain. Elife 7 (2018).

[0171] 27. Gardner, R.C. et al. Age-Related Differences in Diagnostic Accuracy ofPlasma Glial Fibrillary Acidic Protein and Tau for Identifying Acute Intracranial Trauma on Computed Tomography: A TRACK-TBI Study. Journal of neurotrauma (2018).

[0172] 28. Huie, J.R. et al. Testing A Multivariate Proteomic Panel for TBI BiomarkerDiscovery: A Track-TBI Pilot Study. Journal of Neurotrauma 36, 100-110-110 (2018).

[0173] 29. Newcombe, V.F.J. et al. Post-acute blood biomarkers and disease progression in traumatic brain injury. Brain 145, 2064-2076 (2022).

[0174] 30. Messing, A., Brenner, M., Feany, M.B., Nedergaard, M. & Goldman, J.E.Alexander Disease. The Journal of Neuroscience 32, 5017-5023-5023 (2012).

[0175] 31. Messing, A., Daniels, C.M.L. & Hagemann, T.L. Strategies for treatment inAlexander disease. Neurotherapeutics 7, 507-515-515 (2010).

[0176] 32. Lee, S.H. et al. Aggregation- prone GFAP mutation in Alexander disease validated using a zebrafish model. BMC Neurol 17, 175 (2017).

[0177] 33. Lin, N.H., Yang, A.W., Chang, C.H. & Perng, M.D. Elevated GFAP isoform expression promotes protein aggregation and compromises astrocyte function. FASEB J 35, e21614 (2021).

[0178] 34. Needham, E.J. et al. Complex Autoantibody Responses Occur followingModerate to Severe Traumatic Brain Injury. J Immunol 207, 90-100 (2021).

[0179] 35. Gholamin, S. et al. Disrupting the CD47-SIRPa anti-phagocytic axis by a humanized anti-CD47 antibody is an efficacious treatment for malignant pediatric brain tumors. Science Translational Medicine 9, eaaf2968 (2017).

[0180] 36. Han, M.H. et al. Janus-like opposing roles of CD47 in autoimmune braininflammation in humans and mice. The Journal of Experimental Medicine 209, 1325-1334- 1334 (2012).

[0181] 37. Gitik, M., Liraz-Zaltsman, S., Oldenborg, P.-A., Reichert, F. & Rotshenker,S. Myelin down-regulates myelin phagocytosis by microglia and macrophages through interactions between CD47 on myelin and SIRPa (signal regulatory protein-a) on phagocytes. Journal of Neuroinflammation 8, 1-11-11 (2011).

[0182] 38. Zhang, Z. et al. Human Traumatic Brain Injury Induces AutoantibodyResponse against Glial Fibrillary Acidic Protein and Its Breakdown Products. PLoS ONE 9, e92698 (2014).

[0183] 39. Fang, B. et al. Autoimmune Glial Fibrillary Acidic Protein Astrocytopathy: ANovel Meningoencephalomyelitis. JAMA Neurology (2016).

[0184] 40. Shibuya, M. et al. Autoantibodies against Glial Fibrillary Acidic Protein(GFAP) in Cerebrospinal Fluids from Pug Dogs with Necrotizing Meningoencephalitis. Journal of Veterinary Medical Science 69, 241-245-245 (2007).

[0185] 41. Long, Y. et al. Autoimmune glial fibrillary acidic protein astrocytopathy inChinese patients: a retrospective study. European Journal of Neurology 25, 477-483-483 (2018).

[0186] 42. Kirkman, N.J. et al. How Relevant are GFAP Autoantibodies in Autism andTourette Syndrome? Journal of Autism and Developmental Disorders 38, 333-341-341 (2008).

[0187] 43. Wickel, J. et al. Encephalitis with radial perivascular emphasis: Not necessarily associated with GFAP antibodies. Neurol Neuroimmunol Neuroinflamm 7 (2020).

[0188] 44. Wilding, C. et al. GFAP antibodies show protective effect on oxidatively stressed neuroretinal cells via interaction with ERP57. Journal of Pharmacological Sciences 127, 298-304-304 (2015).

[0189] 45. Bell, K. et al. Neuroprotective effects of antibodies on retinal ganglion cells in an adolescent retina organ culture. Journal of Neurochemistry 139, 256-269-269 (2016).

[0190] 46. Kamchatnov, P.R. et al. Autoantibodies to GFAP (glial fibrillary acidic protein) and to dopamine in patients with acute and chronic cerebrovascular disorders. Health 02, 1366-1371-1371 (2010).

[0191] 47. Li, X. et al. Enhanced in Vivo Blood-Brain Barrier Penetration by CircularTau-Transferrin Receptor Bifunctional Aptamer for Tauopathy Therapy. J Am Chem Soc 142, 3862-3872 (2020).

[0192] 48. Regina, A. et al. Antitumour activity of ANG1005, a conjugate between paclitaxel and the new brain delivery vector Angiopep-2. Br J Pharmacol 155, 185-197 (2008).

[0193] 49. Demeule, M. et al. Involvement of the low-density lipoprotein receptor- related protein in the transcytosis of the brain delivery vector angiopep-2. J Neurochem 106, 1534-1544 (2008).

[0194] 50. Lu, F. et al. Angiopep-2-conjugated poly(ethylene glycol)-co- poly(epsilon- caprolactone) polymersomes for dual-targeting drug delivery to glioma in rats. Int J Nanomedicine 12, 2117-2127 (2017).

[0195] 51. Kumthekar, P. et al. ANG1005, a Brain-Penetrating Peptide-Drug Conjugate,Shows Activity in Patients with Breast Cancer with Leptomeningeal Carcinomatosis and Recurrent Brain Metastases. Clinical Cancer Research 26, 2789-2799 (2020).

[0196] 52. Hay, J.R., Johnson, V.E., Young, A.M., Smith, D.H. & Stewart, W. Blood-Brain Barrier Disruption Is an Early Event That May Persist for Many Years After Traumatic Brain Injury in Humans. J Neuropathol Exp Neurol 74, 1147-1157 (2015).

[0197] 53. Kwon, E.J., Skalak, M., Lo Bu, R. & Bhatia, S.N. Neuron-TargetedNanoparticle for siRNA Delivery to Traumatic Brain Injuries. ACS Nano 10, 7926-7933 (2016).

[0198] 54. Vieira, P. & Rajewsky, K. The half-lives of serum immunoglobulins in adult mice. Eur J Immunol 18, 313-316 (1988).

[0199] 55. Keizer, R.J., Huitema, A.D.R., Schellens, J.H.M. & Beijnen, J.H. ClinicalPharmacokinetics of Therapeutic Monoclonal Antibodies. Clinical Pharmacokinetics 49, 493- 507-507 (2010).

[0200] 56. Nath, R. et al. Development and characterization of antibodies specific to caspase-3-produced alpha II-spectrin 120 kDa breakdown product: marker for neuronal apoptosis. Neurochemistry International 37, 351-361-361 (2000).

[0201] 57. Pike, B.R. et al. Accumulation of Calpain and Caspase-3 ProteolyticFragments of Brain-Derived all-Spectrin in Cerebral Spinal Fluid after Middle Cerebral Artery Occlusion in Rats. Journal of Cerebral Blood Flow & Metabolism 24, 98-106-106 (2003).

[0202] 58. Pineda, J.A. et al. Clinical Significance ofall- Spectrin Breakdown Products inCerebrospinal Fluid after Severe Traumatic Brain Injury. Journal of Neurotrauma 24, 354- 366-366 (2007).

[0203] 59. Liu, M.C. et al. Dual vulnerability of tau to calpains and caspase-3 proteolysisunder neurotoxic and neurodegenerative conditions. ASN Neuro 3, e00051 (2011).

[0204] 60. Zhang, Z. et al. Multiple alphall- spectrin breakdown products distinguish calpain and caspase dominated necrotic and apoptotic cell death pathways. Apoptosis 14, 1289-1298-1298 (2009).

[0205] 61. Lee, S.H. et al. Antibody-Mediated Targeting of Tau In Vivo Does NotRequire Effector Function and Microglial Engagement. Cell Rep 16, 1690-1700 (2016).

[0206] 62. Couch, J. A. et al. Addressing safety liabilities of TfR bispecific antibodies that cross the blood-brain barrier. Sci Trans! Med 5, 183ral 57, 181 -1 12 (2013).

[0207] 63. Tseng, D. et al. Anti-CD47 antibody-mediated phagocytosis of cancer by macrophages primes an effective antitumor T-cell response. Proceedings of the National Academy of Sciences 110, 11103-11108-11108 (2013).

[0208] 64. Weiskopf, K. et al. Eradication of Canine Diffuse Large B-Cell Lymphoma in a Murine Xenograft Model with CD47 Blockade and Anti-CD20. Cancer Immunology Research 4, 1072-1087-1087 (2016).

[0209] 65. Wang, X., Mathieu, M. & Brezski, R.J. IgG Fc engineering to modulate antibody effector functions. Protein Cell 9, 63-73 (2018).

[0210] 66. Richards, J.O. et al. Optimization of antibody binding to FcgammaRIIa enhances macrophage phagocytosis of tumor cells. Mol Cancer Ther 7, 2517-2527 (2008).

[0211] 67. Mimoto, F. et al. Novel asymmetrically engineered antibody Fc variant with superior FcgammaR binding affinity and specificity compared with afucosylated Fc variant. MAbs 5, 229-236 (2013).

[0212] 68. Ramakrishnan, V. et al. Quantitative systems pharmacology model of the amyloid pathway in Alzheimer's disease: Insights into the therapeutic mechanisms of clinical candidates. CPT Pharmacometrics Syst Pharmacol 12, 62-73 (2023).

[0213] 69. Stein, T.D., Fedynyshyn, J.P. & Kalil, R.E. Circulating autoantibodies recognize and bind dying neurons following injury to the brain. J Neuropathol Exp Neurol 61, 1100-1108 (2002).

[0214] 70. Liu, M.C. et al. Comparing calpain- and caspase-3-mediated degradation patterns in traumatic brain injury by differential proteome analysis. Biochem J 394, 715-725 (2006).

[0215] 71. Kobeissy, F.H. et al. Novel Differential Neuroproteomics Analysis ofTraumatic Brain Injury in Rats. Molecular & Cellular Proteomics 5, 1887-1898-1898 (2006).

[0216] 72. Serrano-Pozo, A. et al. Stable size distribution of amyloid plaques over the course of Alzheimer disease. J Neuropathol Exp Neurol 71, 694-701 (2012).

[0217] 73. Selkoe, D.J. & Hardy, J. The amyloid hypothesis of Alzheimer’s disease at 25 years. EMBO Mol Med 8, 595-608 (2016).

[0218] 74. Chiti, F. & Dobson, C.M. Protein Misfolding, Amyloid Formation, andHuman Disease: A Summary of Progress Over the Last Decade. Annu Rev Biochem 86, 27- 68 (2017).

[0219] 75. Sweeney, P. el al. Protein misfolding in neurodegenerative diseases: implications and strategies. Transl Neurodegener 6, 6 (2017).

[0220] 76. Schaffert, L.N. & Carter, W.G. Do Post-Translational ModificationsInfluence Protein Aggregation in Neurodegenerative Diseases: A Systematic Review. Brain Sci 10 (2020).

[0221] 77. Leistner, C. et al. The in-tissue molecular architecture of P-amyloid pathology in the mammalian brain. Nat Commun 14, 2833 (2023).

[0222] 78. Alquezar, C., Arya, S. & Kao, A.W. Tau Post-translational Modifications:Dynamic Transformers of Tau Function, Degradation, and Aggregation. Front Neurol 11, 595532 (2020).

[0223] 79. Kayed, R. et al. Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis. Science 300, 486-489 (2003).

[0224] 80. Yang, T., Li, S„ Xu, H„ Walsh, D.M. & Selkoe, D.J. Large SolubleOligomers of Amyloid -Protein from Alzheimer Brain Are Far Less Neuroactive Than the Smaller Oligomers to Which They Dissociate. J Neurosci 37, 152-163 (2017).

[0225] 81. Kulenkampff, K., Wolf Perez, A.M., Sormanni, P., Habchi, J. & Vendruscolo,M. Quantifying misfolded protein oligomers as drug targets and biomarkers in Alzheimer and Parkinson diseases. Nat Rev Chem 5, 277-294 (2021).

[0226] 82. Zurek, J. in Essentials of Neuroanesthesia, (ed. H. Prabhakar) 587-591(Academic Press, 2017).

[0227] 83. Messing, A., Brenner, M., Feany, M.B., Nedergaard, M. & Goldman, J.E.Alexander disease. J Neurosci 32, 5017-5023 (2012).

[0228] 84. Lanciotti, A. et al. Astrocytes: Emerging Stars in LeukodystrophyPathogenesis. Transl Neurosci 4 (2013).

[0229] 85. Brenner, M. et al. Mutations in GFAP, encoding glial fibrillary acidic protein, are associated with Alexander disease. Nature Genetics 27, 117-120 (2001).

[0230] 86. Messing, A. et al. Fatal encephalopathy with astrocyte inclusions in GFAP transgenic mice. Am J Pathol 152, 391-398 (1998).

[0231] 87. Kepchia, D. et al. Diverse proteins aggregate in mild cognitive impairmentand Alzheimer’s disease brain. Alzheimers Res Ther 12, 75 (2020).

[0232] 88. Ferrari-Souza, J.P. et al. Astrocyte biomarker signatures of amyloid-P and tau pathologies in Alzheimer’s disease. Molecular Psychiatry 27, 4781-4789 (2022).

[0233] 89. De, S. et al. Different soluble aggregates of Abeta42 can give rise to cellular toxicity through different mechanisms. Nat Commun 10, 1541 (2019).

[0234] 90. Ganne, A., Balasubramaniam, M., Griffin, W.S.T., Shmookler Reis, R.J. &Ayyadevara, S. Glial Fibrillary Acidic Protein: A Biomarker and Drug Target for Alzheimer's Disease. Pharmaceutics 14 (2022).

[0235] 91. Mintun, M.A. et al. Donanemab in Early Alzheimer's Disease. N Engl J Med384, 1691-1704 (2021).

[0236] 92. Vaz, S.H., Pinto, S., Sebastiao, A.M. & Brites, D. in Amyotrophic LateralSclerosis, (ed. T. Araki) (Brisbane (AU); 2021).

[0237] 93. Verde, F. et al. Serum levels of glial fibrillary acidic protein in patients with amyotrophic lateral sclerosis. Ann Clin Transl Neurol 10, 118-129 (2023).

[0238] 94. Peteri, U.-K., Niukkanen, M. & Castren, M.L. Astrocytes inNeuropathologies Affecting the Frontal Cortex. Frontiers in Cellular Neuroscience 13 (2019).

[0239] 95. Radford, R.A. et al. The established and emerging roles of astrocytes and microglia in amyotrophic lateral sclerosis and frontotemporal dementia. Front Cell Neurosci 9, 414 (2015).

[0240] 96. Riku, Y. et al. Pathway from TDP-43 -Related Pathology to NeuronalDysfunction in Amyotrophic Lateral Sclerosis and Frontotemporal Lobar Degeneration. Int J Mol Sci 22 (2021).

[0241] 97. Kumar, S.T. et al. Seeding the aggregation of TDP-43 requires post- fibrillization proteolytic cleavage. Nat Neurosci 26, 983-996 (2023).

[0242] 98. Verellen, R.M. & Cavazos, J.E. Post-traumatic epilepsy: an overview.Therapy 7, 527-531 (2010).

[0243] 99. Agrawal, A., Timothy, J., Pandit, L. & Manju, M. Post-traumatic epilepsy: an overview. Clin Neurol Neurosurg 108, 433-439 (2006).

[0244] 100. Klein, P. & Tyrlikova, I. Prevention of epilepsy: Should we be avoiding clinical trials? Epilepsy Behav 72, 188-194 (2017).

[0245] 101. Annegers, J.F., Hauser, W.A., Coan, S.P. & Rocca, W.A. A populationbased study of seizures after traumatic brain injuries. N Engl J Med 338, 20-24 (1998).

[0246] 102. Hung, C. & Chen, J.W. Treatment of post-traumatic epilepsy. Curr TreatOptions Neurol 14, 293-306 (2012).

[0247] 103. Temkin, N.R. Preventing and treating posttraumatic seizures: the human experience. Epilepsia 50 Suppl 2, 10-13 (2009).

[0248] 104. Thompson, K., Pohlmann-Eden, B., Campbell, L.A. & Abel, H.Pharmacological treatments for preventing epilepsy following traumatic head injury. Cochrane Database Syst Rev, CD009900 (2015).

[0249] 105. Klein, P. & Tyrlikova, I. No prevention or cure of epilepsy as yet.Neuropharmacology 168, 107762 (2020).

[0250] 106. Mukherjee, S. et al. Neuroinflammatory mechanisms of post-traumatic epilepsy. J Neuroinflammation 17, 193 (2020).

[0251] 107. Bolte, A.C. & Lukens, J.R. Neuroimmune cleanup crews in brain injury.Trends Immunol 42, 480-494 (2021).

[0252] 108. Sofroniew, M.V. Astrocyte Reactivity: Subtypes, States, and Functions inCNS Innate Immunity. Trends Immunol 41, 758-770 (2020).

[0253] 109. Liddelow, S.A. & Barres, B.A. Reactive Astrocytes: Production, Function, and Therapeutic Potential. Immunity 46, 957-967 (2017).

[0254] 110. Escartin, C. et al. Reactive astrocyte nomenclature, definitions, and future directions. Nat Neurosci 24, 312-325 (2021).

[0255] 111. Liddelow, S.A. et al. Neurotoxic reactive astrocytes are induced by activated microglia. Nature 541, 481-487 (2017).

[0256] 112. Vainchtein, I.D. & Molofsky, A.V. Astrocytes and Microglia: In Sickness and in Health. Trends Neurosci 43, 144-154 (2020).

[0257] 113. Brown, M. et al. Magnetic resonance spectroscopy abnormalities in traumatic brain injury: A meta-analysis. J Neuroradiol 45, 123-129 (2018).

[0258] 114. Di Sapia, R. et al. In-depth characterization of a mouse model of post- traumatic epilepsy for biomarker and drug discovery. Acta Neuropathol Commun 9, 76 (2021).

[0259] 115. Pascente, R. et al. Cognitive deficits and brain myo-Inositol are early biomarkers of epileptogenesis in a rat model of epilepsy. Neurobiol Dis 93, 146-155 (2016).

[0260] 116. Johnson, V.E. et al. Inflammation and white matter degeneration persist for years after a single traumatic brain injury. Brain 136, 28-42 (2013).

[0261] 117. Pischiutta, F. et al. Single severe traumatic brain injury produces progressive pathology with ongoing contralateral white matter damage one year after injury. Exp Neurol 300, 167-178 (2018).

[0262] 1 18. Sun, L. et al. The Role of Neuroinflammation in Post-traumatic Epilepsy.Front Neurol 12, 646152 (2021).

[0263] 119. Ravizza, T. & Vezzani, A. Pharmacological targeting of brain inflammation in epilepsy: Therapeutic perspectives from experimental and clinical studies. Epilepsia Open 3, 133-142 (2018).

[0264] 120. Verhoog, Q.P., Holtman, L., Aronica, E. & van Vliet, E.A. Astrocytes asGuardians of Neuronal Excitability: Mechanisms Underlying Epileptogenesis. Front Neurol 11, 591690 (2020).

[0265] 121. Webster, K.M. et al. Inflammation in epileptogenesis after traumatic brain injury. J Neuroinflammation 14, 10 (2017).

[0266] 122. Sofroniew, M.V. Astrocyte barriers to neurotoxic inflammation. Nat RevNeurosci 16, 249-263 (2015).

[0267] 123. Devinsky, O., Vezzani, A., Najjar, S., De Lanerolle, N.C. & Rogawski,M.A. Glia and epilepsy: excitability and inflammation. Trends Neurosci 36, 174-184 (2013).

[0268] 124 Ravizza, T. et al. The IL-lbeta system in epilepsy-associated malformations of cortical development. Neurobiol Dis 24, 128-143 (2006).

[0269] 125. Aronica, E. et al. Complement activation in experimental and human temporal lobe epilepsy. Neurobiol Dis 26, 497-51 1 (2007).

[0270] 126. Wyatt, S.K., Witt, T., Barbara, N.M., Cohen-Gadol, A.A. & Brewster, A.L.Enhanced classical complement pathway activation and altered phagocytosis signaling molecules in human epilepsy. Exp Neurol 295, 184-193 (2017).

[0271] 127. Andoh, M., Ikegaya, Y. & Koyama, R. Synaptic Pruning by Microglia inEpilepsy. J Clin Med 8 (2019).

[0272] 128. Ziabska, K., Ziemka-Nalecz, M., Pawelec, P., Sypecka, J. & Zalewska, T.Aberrant Complement System Activation in Neurological Disorders. Int J Mol Sci 22 (2021).

[0273] 129. Gavriilaki, M., Kimiskidis, V.K. & Gavriilaki, E. Precision Medicine inNeurology: The Inspirational Paradigm of Complement Therapeutics. Pharmaceuticals (Basel) 13 (2020).

[0274] 130. Koenig, J. & Dulla, C. Complements to the Chef: Are Microglia EatingNeurons in the Epileptic Brain? Epilepsy Curr 18, 128-130 (2018).

[0275] 131. Jamali, S. et al. Functional variant in complement C3 gene promoter and genetic susceptibility to temporal lobe epilepsy and febrile seizures. PLoS One 5 (2010).

[0276] 132. McGinn, M.J. et al. Biochemical, structural, and biomarker evidence for calpain-mediated cytoskeletal change after diffuse brain injury uncomplicated by contusion. J Neuropathol Exp Neurol 68, 241-249 (2009).

[0277] 133. Haskins, W.E. et al. Rapid discovery of putative protein biomarkers of traumatic brain injury by SDS-PAGE-capillary liquid chromatography-tandem mass spectrometry. J Neurotrauma 22, 629-644 (2005).

[0278] 134. Wang, K.K.W., Kobeissy, F.H., Shakkour, Z. & Tyndall, J.A. Thorough overview of ubiquitin C-terminal hydrolase-Ll and glial fibrillary acidic protein as tandem biomarkers recently cleared by US Food and Drug Administration for the evaluation of intracranial injuries among patients with traumatic brain injury. Acute Med Surg 8, e622 (2021).

[0279] 135. Bazarian, J.J. et al. Serum GFAP and UCH-L1 for prediction of absence of intracranial injuries on head CT (ALERT-TBI): a multicentre observational study. The Lancet Neurology 17 (2018).

[0280] 136. Hahn, C. & Lee, C.U. A Brief Review of Paradigm Shifts in Prevention ofAlzheimer's Disease: From Cognitive Reserve to Precision Medicine. Front Psychiatry 10, 786 (2019).

[0281] 137. Bellaver, B. et al. Astrocyte reactivity influences amyloid-beta effects on tau pathology in preclinical Alzheimer's disease. Nat Med (2023).

[0282] 138. Abdelhak, A. et al. Blood GFAP as an emerging biomarker in brain and spinal cord disorders. Nat Rev Neurol 18, 158-172 (2022).

[0283] 139. Klein, P. et al. Commonalities in epileptogenic processes from different acute brain insults: Do they translate? Epilepsia 59, 37-66 (2018).

[0284] 140. Xu, S. et al. Role of Astrocytes in Post-traumatic Epilepsy. Front Neurol 10,1149 (2019).

[0285] 141. Li, T. et al. Microglia induce the transformation of A1 / A2 reactive astrocytes via the CXCR7 / PI3K / Akt pathway in chronic post-surgical pain. J Neuroinflammation 17, 21 1 (2020).

[0286] 142. Heaven, M.R., Wilson, L., Barnes, S. & Brenner, M. Relative stabilities of wild-type and mutant glial fibrillary acidic protein in patients with Alexander disease. J Biol Chem 294, 15604-15612 (2019).

[0287] 143. Jones, J.R. et al. Mutations in GFAP Disrupt the Distribution and Function of Organelles in Human Astrocytes. Cell Rep 25, 947-958 e944 (2018).

[0288] 144. Battaglia, R.A. et al. Site-specific phosphorylation and caspase cleavage ofGFAP are new markers of Alexander disease severity. Elife 8 (2019).

[0289] 145. Chen, M.H., Hagemann, T.L., Quinlan, R.A., Messing, A. & Perng, M.D.Caspase cleavage of GFAP produces an assembly-compromised proteolytic fragment thatpromotes filament aggregation. ASN Neuro 5, e00125 (2013).

[0290] 146. Geerts, H. et al. A combined physiologically-based pharmacokinetic and quantitative systems pharmacology model for modeling amyloid aggregation in Alzheimer's disease. CPT Pharmacometrics Syst Pharmacol 12, 444-461 (2023).

[0291] 147. Okonkwo, D.O. et al. GFAP-BDP as an acute diagnostic marker in traumatic brain injury: results from the prospective transforming research and clinical knowledge in traumatic brain injury study. J Neurotrauma 30, 1490-1497 (2013).

[0292] 148. Zoltewicz, J.S. et al. Characterization of Antibodies that Detect HumanGFAP after Traumatic Brain Injury. Biomark Insights 7, 71-79 (2012).

[0293] 149. McMahon, P.J. et al. Measurement of the glial fibrillary acidic protein and its breakdown products GFAP-BDP biomarker for the detection of traumatic brain injury compared to computed tomography and magnetic resonance imaging. J Neurotrauma 32, 527-533 (2015).

[0294] 150. Yang, Z. & Wang, K.K. Glial fibrillary acidic protein: from intermediate filament assembly and gliosis to neurobiomarker. Trends Neurosci 38, 364-374 (2015).

[0295] 151. Viedma-Poyatos, A., Gonzalez-Jimenez, P., Pajares, M.A. & Perez-Sala, D.Alexander disease GFAP R239C mutant shows increased susceptibility to lipoxidation and elicits mitochondrial dysfunction and oxidative stress. Redox Biology 55, 102415 (2022).

[0296] 152. McKeon, A. & Benarroch, E.E. Glial fibrillary acid protein: Functions and involvement in disease. Neurology 90, 925-930 (2018).

[0297] Patent documents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. These documents and publications are incorporated herein by reference to the same extent as if each individual document or publication was specifically and individually incorporated herein by reference.

[0298] The foregoing description is illustrative of particular embodiments of the invention but is not meant to be a limitation upon the practice thereof. The following claims, including all equivalents thereof, are intended to define the scope of the invention.

Claims

CLAIMS1. A method of diagnosing a disease in a subject comprising: sampling from the subject; and detecting at least one of astrocytic monomeric-, oligomeric-, or aggregate of a G* in the subject as a ratio of at least different two molecular weight G*s or a ratio of the G* relative to intact GFAP from the subject; providing a diagnosis of the disease or severity thereof in the subject based on the detecting.

2. The method of claim 1 wherein the G* comprises at least one of GBDP22-24K, GBDP38K, GBDP44K, or combinations thereof.

3. The method of claim 1 wherein the disease is traumatic brain injury (TBI), post- traumatic epilepsy (PTE), Alexander disease (AxD), mild cognitive impairment (MCI) Alzheimer’s disease (AD), multiple sclerosis, stroke, brain cancer, Parkinson’s disease, Lewy Body Dementia, frontotemporal dementia, or vanishing white matter disease.

4. The method of claim 1 further comprising in vivo or in vitro tissue imaging to detect the at least one of astrocytic monomeric-, oligomeric-, or aggregate of a G* in the subject as a ratio of at least different two molecular weight G*s or a ratio of one from of a GBDP relative to intact GFAP.

5. The method of any one of claims 1 to 4 wherein the G* comprises at least one of GBDP22-24K, GBDP38K, GBDP44K, or combinations thereof.

6. The method of claim 1 further comprising measuring the G* from at least two biofluids, at least two tissues, or at least one biofluid and at least one tissue; and determining a ratio of the GBDP in the at least two biofluid, the at least two tissues, or the at least one biofluid and the at least one tissue.

7. The method of claim 6 wherein the at least two biofluids are brain interstitial fluid and circulating blood.

8. The method of any one of claims 1 to 4 wherein measuring the G* concentrations occurs twice daily corresponding approximately to morning and evening wherein a difference in moming / evening values is used as a surrogate measure for brain health, glymphatic clearance, or a combination thereof.

9. The method of claim 1 wherein measuring the G* concentrations occurs for reduced and non-reduced samples, and a difference in reduced / non-reduced values is used as an indirect measure of a reducible oligomer or aggregate and as a surrogate measure for brain health and / or glymphatic clearance.

10. The method of claim 9 wherein the reducible oligomer or aggregate includes a G* containing an inter- or intra-molecular disulfide bond.

11. The method of any one of claims 1 to 4 wherein a biofluid is sampled and is one or more of cerebral spinal fluid, capillary blood, plasma, serum whole blood, or saliva.

12. The method of any one of claims 1 to 4 wherein a biofluid is sampled and collected as a dried spot.

13. The method of claim 1 wherein the at least one of astrocytic monomeric-, oligomeric-, or aggregate of a G* in the subject as a ratio of at least different two molecular weight G*s or a ratio of the G* relative to intact GFAP is measured with an antibody- or an oligonucleotide- based assay.

14. The method of claim 13 wherein the assay binds to an N- or C-terminus of the G* or a region spanning the N- and the C-termini of the G* and intact GFAP.

15. The method of claim 13 wherein the assay binds to a conserved core of GBDP38K and intact GFAP, a region spanning the N- and the C-termini of the G*.

16. The method of 13 wherein the assay shows reduced oligomerization and / or aggregation of a bound complex inclusive of the G*.

17. The method of 13 wherein the assay shows increased cellular phagocytosis of a bound complex inclusive of G*.

18. The method of claim 13 wherein the assay provides both age-dependent and ageindependent information as to at least one of brain health, brain injury, or the disease.

19. The method of claim 13 wherein the assay further comprises information about the presence and / or concentration of at least one protein of Api -40, Api-42, IL6, CypD (PPIF), NFL, WAVE1, WAVE3, VAMP5, MOG, OMG, tau, ptau 181, ptau 202, ptau 205, ptau 217, ptau 231, ptau 262, ptau 396, ptau 404, ptau 433, or ptau 435.

20. The method of claim 13 wherein the assay is a sandwich ELISA that binds at least one epitope of the G*.

21. A composition comprising an antibody or an oligonucleotide that binds with a degree of specificity to an N-terminal of GBDP38K, a C-terminal neoepitope of GBDP38K, or a conserved core domain of GBDP38K and GFAP.

22. The composition of claim 21 wherein the N-terminal neoepitope is bound and is one of NH2- AgoGFKETRASER or C-terminally extended forms.

23. The composition of claim 21 wherein the C-terminal neoepitope is bound and is one of the ENRITIPVQT-COOH or N-terminally extended forms.

24. The composition of any one of claims 21 to 23 further comprising binding sites for monomeric-, oligomeric-, or aggregated-forms including at least one of GBDP22-24K, GBDP38K, GBDP44K, GFAP, or a combination thereof.

25. The composition of any one of claims 21 to 23 further comprising binding sites for at least one protein of A 1-4O, A 1-42, IL6, CypD (PPIF), NFL, WAVE1, WAVE3, VAMP5, MOG, OMG, tau, ptau 181, ptau 202, ptau 205, ptau 217, ptau 231, ptau 262, ptau 396, ptau 404, ptau 433, or ptau 435.

26. A method of treating a disease in a subject comprising:sampling a biofluid from the subject; and detecting the abnormal presence of at least one of monomeric-, oligomeric-, or aggregate containing of a glial fibrillary acidic protein breakdown product (G*) in the biofluid from the subject; and administering a therapeutic to the subject that selectively binds to the at least one of astrocytic monomeric, oligomeric, or aggregate G*s to treat the disease in response to the detection.

27. The method of claim 26 wherein the therapeutic is a mono-, bi-, or tri-specific, afucosylated and / or mutated effector or effectorless monoclonal antibody, an oligonucleotide, or other anti- GF AP proteoform antibody modalities.

28. The method of claim 26 wherein the disease is traumatic brain injury (TBI), post- traumatic epilepsy (PTE), Alexander disease (AxD), mild cognitive impairment (MCI), Alzheimer’s disease (AD), multiple sclerosis, Parkinson’s disease, Lewy Body Dementia, frontotemporal dementia, or vanishing white matter disease, epilepsy, stroke, brain cancer, post-traumatic headache, mild cognitive impairment, amyotrophic lateral sclerosis (ALS), or peripheral neuropathy.

29. The method of claim 26 wherein the therapeutic increases glymphatic clearance of a bound complex inclusive of the G*.

30. The method of claim 26 wherein the therapeutic reduces oligomerization and / or aggregation of a bound complex inclusive of the G*.

31. The method of claim 26 wherein the therapeutic increases cellular phagocytosis of a bound complex inclusive of the G*.

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