Compositions and methods for immunotherapy of neurodegenerative disorders

Abstract

Embodiments are directed to a toxic tau conformation-specific monoclonal antibodies as well as an α-Syn monoclonal antibodies. Certain compositions are include antibodies loaded into or conjugated to micelles as a carrier for improved intracellular delivery in the brain. In certain aspects the intranasal route for delivery was used, leveraging the direct nose- to-brain anatomic pathway, recognized as a viable, non-invasive, safe approach for effective drug-delivery in the brain. In certain other aspects the monoclonal antibodies are used as combination therapies.

Description

FJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A COMPOSITIONS AND METHODS FOR IMMUNOTHERAPY OF NEURODEGENERATIVE DISORDERS RELATED APPLICATIONS

[0001] This Application is an International Application claiming priority to US Provisional Patent applications 63 / 649,367 filed 5 / 19 / 2024 and 63 / 782,482 filed 4 / 2 / 2025, each of which is incorporated herein by reference in their entirety. STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

[0002] None. REFERENCE TO SEQUENCE LISTING

[0003] A sequence listing required by 37 CFR 1.821-1.825 is being submitted electronically with this application. The sequence listing is incorporated herein by reference. The sequence listing that is contained in the file named "UTMBP0419WO" which is 72 KB (as measured in Microsoft Windows®) and was created on 5 / 18 / 2025. FIELD

[0004] Embodiments are directed generally to the field of medicine, more particularly to the treatment of neurodegenerative diseases using antibodies. BACKGROUND

[0005] Neurodegenerative disorders related to tau and synuclein proteins are a group of progressive, incurable conditions characterized by the accumulation of abnormal protein aggregates in the brain, leading to neuronal dysfunction and death. Tauopathies, such as Alzheimer’s disease and frontotemporal dementia, involve the misfolding and aggregation of tau, a microtubule-associated protein, into neurofibrillary tangles, disrupting neuronal stability and transport. Synucleinopathies, including Parkinson’s disease, dementia with Lewy bodies, and multiple system atrophy, are marked by the accumulation of misfolded α-synuclein into Lewy bodies or glial inclusions, impairing synaptic function and promoting neurotoxicity. These disorders share common features, including protein misfolding, neuroinflammation, andFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A progressive cognitive or motor deficits, but differ in clinical presentation, affected brain regions, and specific protein pathology. Research continues to explore shared mechanisms and potential therapies targeting protein aggregation and clearance.

[0006] Previous studies using tau immunotherapy in AD mouse models demonstrated the ability of anti-tau monoclonal antibodies (MABs) to halt tauopathy progression and even improve cognitive and motor functions in some cases (Boutajangout et al., J Neurochem 118, 658-667, 2011; Chai et al., J Biol Chem 286, 34457-34467, 2011; Yanamandra et al., Neuron 80, 402-414, 2013). However, conventional MABs primarily target extracellular tau. As a result, intracellular pathological tau aggregates remain largely unaffected in current immunotherapies. Despite this limitation, several clinical trials were launched to test the efficacy of anti-tau MABs based on promising results in mice (Congdon et al., Nat Rev Neurol 14, 399-415, 2018; Jadhav, Acta Neuropathol Commun, 7, 22, 2019).

[0007] Disappointingly, trials with the MABs Tilavonemab and Gosuranemab, failed to demonstrate improvements in patients with tauopathies (Ji et al., Drugs, 81, 1135-1152, 2021); suggesting that solely targeting extracellular tau may not be efficacious for treating patients with tauopathies (Congdon et al., Semin Cell Dev Biol 126, 125-137, 2022). Consistent with these observations, recent studies suggest that intracellular tau aggregates and synaptic tau seeds play a more significant role in the disease than their extracellular counterparts (Colom-Cadena et al., Neuron, 2023; Meisl et al., Sci Adv, 7, eabh1448, 2021). Indeed, tau pathology can propagate via exosomes or nanotubes, protecting tau seeds from therapeutic antibodies (Leroux et al., Mol Ther, 30, 782-797, 2022). Thus, to effectively attenuate tauopathy progression and cognitive decline, a strong rationale exists for developing immunotherapies that target intracellular tau and block seeding activity.

[0008] Several challenges exist to develop safe and effective tau immunotherapies, including (1) identifying anti-tau and / or anti-synuclein MABs that selectively recognize pathological, misfolded, and toxic tau or synuclein conformations rather than the more abundant physiological tau or synuclein; (2) efficiently reaching intracellular compartments in the brain to remove pathological tau or synuclein; (3) effectively neutralizing or clearing pathological tau seeds or synuclein; and (4) removing pathological tau or synuclein without triggering deleterious inflammatory responses.FJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A SUMMARY

[0009] Solutions to problems presented above were generated by developing various monoclonal antibodies to tau proteins or synuclein proteins, i.e., Toxic Tau Conformation Monoclonal antibodies (TTCMs), Tau Oligomer Monoclonal Antibodies (TOMAs) and α- Synuclein Antibodies. Monoclonal antibodies can be loaded into micelles or other delivery vehicles for improved intracellular delivery, in particular in the brain. The antibodies can be delivered individually or in various combinations with other antibodies. In certain aspects, an intranasal route is used for delivery, leveraging the direct nose-to-brain anatomic pathway, recognized as a viable, non-invasive, safe approach for effective drug-delivery in the brain (Wang et al., J Pharmacol Exp The,r 370, 593-601, 2019). Intranasal delivery offers several advantages over intravenous (i.v.) injection, including increased bioavailability in the brain and reduced systemic exposure to the drug, limiting adverse side effects. In contrast, conventional immunotherapy approaches for AD require long-term intravenous (i.v.) administration of MABs at high doses to achieve therapeutic concentrations due to their poor bioavailability and limited brain uptake. This increases the likelihood of side effects and drug toxicity (Wang et al., J Pharmacol Exp The,r 370, 593-601, 2019).

[0010] In certain aspects one or more TTCM antibody; one or more TOMA antibody; one or more α-Synuclein antibody; a combination of one or more TTCM antibody and one or more TOMA antibody; a combination of one or more TTCM antibody and one or more α-Synuclein antibody; a combination of one or more TOMA antibody and one or more α-Synuclein antibody; or a combination of one or more TTCM antibody, one or more TOMA antibody, and one or more α-Synuclein antibody can be used in the same or separate delivery vehicles.

[0011] Toxic Tau Conformation Monoclonal antibodies (TTCM). In certain aspects, anti-Tau antibodies are conformation specific and bind toxic Tau (Toxic Tau Conformation Monoclonals – TTCM) and include methods of using TTCMs. In one embodiment, a TTCM1 antibody has a heavy chain amino acid sequence comprising an amino acid sequence set forth in SEQ ID NO:1 and a light chain amino acid sequence comprising an amino acid sequence set forth in SEQ ID NO:2. In some embodiments, the antibodies, or antigen binding portions thereof, comprise a heavy chain CDR1 domain comprising the amino acid sequence forth in SEQ ID NO:3, a heavy chain CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO:4, a heavy chain CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO:5, andFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A optionally a constant region having the amino acid sequence set forth in SEQ ID NO:6; and / or a light chain CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO:7, a light chain CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO:8, a light chain CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO:9, and optionally a constant region having an amino acid sequence set forth in SEQ ID NO:10.

[0012] In one embodiment, a TTCM2 antibody has a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:11, and a light chain comprising the amino acid sequence set forth in SEQ ID NO:12. In some embodiments, the antibodies, or antigen binding portions thereof, comprise a heavy chain CDR1 domain comprising an amino acid sequence set forth in SEQ ID NO:13, a heavy chain CDR2 domain comprising an amino acid sequence set forth in SEQ ID NO:14, a heavy chain CDR3 domain comprising an amino acid sequence set forth in SEQ ID NO:15, and an optional constant region having an amino acid sequence set forth in SEQ ID NO:16; and a light chain CDR1 domain comprising an amino acid sequence set forth in SEQ ID NO:17, a light chain CDR2 domain comprising an amino acid sequence set forth in SEQ ID NO:18, a light chain CDR3 domain comprising an amino acid sequence set forth in SEQ ID NO:19, and an optional constant region having an amino acid sequence set forth in SEQ ID NO:20.

[0013] Tau Oligomer Monoclonal Antibody (TOMA). In one embodiment, a TOMA1 antibody has a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO:21 and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO:22. In some embodiments, the antibodies, or antigen binding portions thereof, comprise a heavy chain CDR1 domain comprising an amino acid sequence set forth in SEQ ID NO:23, a heavy chain CDR2 domain comprising an amino acid sequence set forth in SEQ ID NO:24, a heavy chain CDR3 domain comprising an amino acid sequence set forth in SEQ ID NO:25; and a light chain CDR1 domain comprising an amino acid sequence set forth in SEQ ID NO:26, a light chain CDR2 domain comprising an amino acid sequence set forth in SEQ ID NO:27, a light chain CDR3 domain comprising an amino acid sequence set forth in SEQ ID NO:28.

[0014] In certain embodiments, a TOMA2 antibody has a heavy chain comprising an amino acid sequence set forth in SEQ ID NO:29 and a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO:30. In some embodiments, the antibodies, or antigen binding portions thereof, comprise a heavy chain CDR1 domain comprising an amino acidFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A sequence set forth in SEQ ID NO:31, a heavy chain CDR2 domain comprising an amino acid sequence set forth SEQ ID NO:32, a heavy chain CDR3 domain comprising an amino acid sequence set forth in SEQ ID NO: 33; and a light chain CDR1 domain comprising an amino acid sequence set forth in SEQ ID NO:34, a light chain CDR2 domain comprising an amino acid sequence set forth in SEQ ID NO:35, a light chain CDR3 domain comprising an amino acid sequence set forth in SEQ ID NO:36.

[0015] In certain embodiments, a tau oligomer monoclonal antibody (TOMA), for example a TOMA3 antibody, has a heavy chain comprising an amino acid sequence set forth in SEQ ID NO:37 and a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO:38. In some embodiments, the antibodies, or antigen binding portions thereof, comprise a heavy chain CDR1 domain comprising an amino acid sequence set forth in SEQ ID NO:39, a heavy chain CDR2 domain comprising an amino acid sequence set forth in SEQ ID NO:40, a heavy chain CDR3 domain comprising an amino acid sequence set forth in SEQ ID NO:41, and a light chain CDR1 domain comprising an amino acid sequence set forth in SEQ ID NO:42, a light chain CDR2 domain comprising an amino acid sequence set forth in SEQ ID NO:43, a light chain CDR3 domain comprising an amino acid sequence set forth in SEQ ID NO:44.

[0016] In certain embodiments, a TOMA4 antibody has a heavy chain comprising an amino acid sequence set forth in SEQ ID NO:45 and a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO:46. In some embodiments, the antibodies, or antigen binding portions thereof, comprise a heavy chain CDR1 domain comprising an amino acid sequence set forth in SEQ ID NO:47, a heavy chain CDR2 domain comprising an amino acid sequence set forth in SEQ ID NO:48, a heavy chain CDR3 domain comprising an amino acid sequence set forth in SEQ ID NO:49, and a light chain CDR1 domain comprising an amino acid sequence set forth in SEQ ID NO:50, a light chain CDR2 domain comprising an amino acid sequence set forth in SEQ ID NO:51, a light chain CDR3 domain comprising an amino acid sequence set forth in SEQ ID NO:52.

[0017] α-Syn Oligomers Conformation-Specific Monoclonal Antibodies. Certain embodiments are directed to one or more alpha synuclein oligomer antibody and uses thereof comprising: (i) a first complementarity determining region (CDR) set, first CDR 1 having an amino acid sequence set forth in SEQ ID NO: 54, first CDR 2 having an amino acid sequence set forth in SEQ ID NO:55, first CDR 3 having an amino acid sequence set forth in SEQ ID NO:56FJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A in combination with a second CDR set with a second CDR1 having an amino acid sequence set forth in SEQ ID NO:58, second CDR 2 having an amino acid sequence set forth in SEQ ID NO:59, and second CDR 3 having an amino acid sequence set forth in SEQ ID NO:60); (ii) a first complementarity determining region (CDR) set, first CDR 1 having an amino acid sequence set forth in SEQ ID NO:62, first CDR 2 having an amino acid sequence set forth in SEQ ID NO:63, first CDR 3 having an amino acid sequence set forth in SEQ ID NO:64 in combination with a second CDR set with a second CDR1 having an amino acid sequence set forth in SEQ ID NO:66, second CDR 2 having an amino acid sequence set forth in SEQ ID NO:67, and second CDR 3 having an amino acid sequence set forth SEQ ID NO:68; or (iii) a first complementarity determining region (CDR) set, first CDR 1 having an amino acid sequence set forth in SEQ ID NO:70, first CDR 2 having an amino acid sequence set forth in SEQ ID NO:71, first CDR 3 having an amino acid sequence set forth in SEQ ID NO:72 in combination with a second CDR set with a second CDR1 having an amino acid sequence set forth in SEQ ID NO:74, second CDR 2 having an amino acid sequence set forth in SEQ ID NO:75, and second CDR 3 having an amino acid sequence set forth in SEQ ID NO:76. In certain aspects the (i) the first and second CDR sets are comprised in a heavy chain amino acid sequence of SEQ ID NO:53 and a light chain amino acid sequence of SEQ ID NO:57, respectively; (ii) the first and second CDR sets are comprised in a heavy chain amino acid sequence of SEQ ID NO:61 and a light chain amino acid sequence of SEQ ID NO:65, respectively; or (iii) the first and second CDR sets are comprised in a heavy chain amino acid sequence of SEQ ID NO:69 and a light chain amino acid sequence of SEQ ID NO:73.

[0018] In certain aspects one or more antibody described herein is formulated in a therapeutic composition. The therapeutic compositions and comprise a micellar compositions encapsulating one or more antibodies.

[0019] Other aspects are directed to an immunotherapeutic composition comprising one or more of an antibody or an antibody derivative / variant described herein. The immunotherapy composition can be a micellar composition.

[0020] Other aspects are directed to methods of treating a subject diagnosed with or at risk of having or developing a neurodegenerative condition comprising administering one or more antibody or antibody fragment described herein. The neurodegenerative condition can be aFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A tauopathy or a synucleinopathy. In certain aspects a composition (e.g., a therapeutic composition or antibody composition) is administered intranasally.

[0021] The invention also provides, in certain embodiments, isolated nucleic acids encoding the antibodies, or antigen binding portions, and variants thereof described herein.

[0022] In some embodiments of the invention, the antibodies, or antigen binding portions thereof, can include a heavy chain and / or light chain immunoglobulin constant domain selected from the group consisting of a human IgG constant domain, a human IgM constant domain, a human IgE constant domain, or a human IgA constant domain. In some embodiments, the IgG constant domain is selected from the group consisting of an IgG1 constant domain, an IgG2 constant domain, an IgG3 constant domain, and an IgG4 constant domain.

[0023] In other embodiments of the invention, the antibodies, or antigen binding portions thereof, can be a Fab, a Fab′, a F(ab′)2, a Fv, a disulfide linked Fv, an scFv, a single domain antibody, and a diabody.

[0024] In order that the invention may be more readily understood, certain terms are first defined. In addition, it should be noted that whenever a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also intended to be part of this invention.

[0025] The terms “specific binding” or “specifically binding”, as used herein, in reference to the interaction of an antibody with a target, mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the target; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally.

[0026] The term “antibody” broadly refers to an immunoglobulin (Ig) molecule, generally comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivative thereof, that retains the essential target binding features of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art. Non-limiting embodiments of which are discussed below. In a full-length antibody, each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chainFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy- terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY) and class (e.g., IgG1, IgG2, IgG 3, IgG4, IgA1 and IgA2) or subclass.

[0027] The term “antigen binding portion” of an antibody (or simply “antibody portion”), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Examples of binding fragments encompassed within the term “antigen binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546, Winter et al., PCT publication WO 90 / 05144 A1 herein incorporated by reference), which comprises a single variable domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term “antigen binding portion” of an antibody. In certain embodiments of the invention, scFv molecules may be incorporated into a fusion protein.

[0028] The term “antibody construct” as used herein refers to a polypeptide comprising one or more the antigen binding portions of the invention linked to a linker polypeptide or an immunoglobulin constant domain.

[0029] The term “humanized antibody” refers to antibodies which comprise heavy and light chain variable region sequences from a nonhuman species (e.g., a mouse) but in which at least a portion of the VH and / or VL sequence has been altered to be more “human-like”, i.e., more similar to human germline variable sequences. In particular, the term “humanized antibody” is anFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A antibody or a variant, derivative, analog or fragment thereof which immunospecifically binds to an antigen of interest and which comprises a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementary determining region (CDR) having substantially the amino acid sequence of a non-human antibody. As used herein, the term “substantially” in the context of a CDR refers to a CDR having an amino acid sequence at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of a non-human antibody CDR. A humanized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab′, F(ab)2, FabC, Fv) in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. Preferably, a humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. In some embodiments, a humanized antibody contains both the light chain as well as at least the variable domain of a heavy chain. The antibody also may include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, a humanized antibody only contains a humanized light chain. In other embodiments, a humanized antibody only contains a humanized heavy chain. In specific embodiments, a humanized antibody only contains a humanized variable domain of a light chain and / or humanized heavy chain.

[0030] The humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including without limitation IgG1, IgG2, IgG3 and IgG4. The humanized antibody may comprise sequences from more than one class or isotype, and particular constant domains may be selected to optimize desired effector functions using techniques well-known in the art.

[0031] The terms “Kabat numbering,” “Kabat definitions,” and “Kabat labeling” are used interchangeably herein. These terms, which are recognized in the art, refer to a system of numbering amino acid residues which are more variable (i.e., hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody, or an antigen binding portion thereof (Kabat et al. (1971) Ann. NY Acad, Sci. 190:382-391 and, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No.91-3242). For the heavy chain variable region, the hypervariable region ranges from amino acid positions 31 to 35 for CDR1, amino acidFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A positions 50 to 65 for CDR2, and amino acid positions 95 to 102 for CDR3. For the light chain variable region, the hypervariable region ranges from amino acid positions 24 to 34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acid positions 89 to 97 for CDR3.

[0032] As used herein, the term “CDR” refers to the complementarity determining region within antibody variable sequences. There are three CDRs in each of the variable regions of the heavy chain (HC) and the light chain (LC), which are designated CDR1, CDR2 and CDR3 (or specifically HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3), for each of the variable regions. The term “CDR set” as used herein refers to a group of three CDRs that occur in a single variable region capable of binding the antigen.

[0033] As used herein, the term “framework” or “framework sequence” refers to the remaining sequences of a variable region minus the CDRs. Because the exact definition of a CDR sequence can be determined by different systems, the meaning of a framework sequence is subject to correspondingly different interpretations. The six CDRs (CDR-L1, CDR-L2, and CDR-L3 of light chain and CDR-H1, CDR-H2, and CDR-H3 of heavy chain) also divide the framework regions on the light chain and the heavy chain into four sub-regions (FR1, FR2, FR3 and FR4) on each chain, in which CDR1 is positioned between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. Without specifying the particular sub-regions as FR1, FR2, FR3 or FR4, a framework region, as referred by others, represents the combined FR's within the variable region of a single, naturally occurring immunoglobulin chain. As used herein, a FR represents one of the four sub-regions, and FRs represents two or more of the four sub-regions constituting a framework region.

[0034] The framework and CDR regions of a humanized antibody need not correspond precisely to the parental sequences, e.g., the donor antibody CDR or the consensus framework may be mutagenized by substitution, insertion and / or deletion of at least one amino acid residue so that the CDR or framework residue at that site does not correspond to either the donor antibody or the consensus framework. In a preferred embodiment, such mutations, however, will not be extensive. Usually, at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% of the humanized antibody residues will correspond to those of the parental FR and CDR sequences. As used herein, the term “consensus framework” refers to the framework region in the consensus immunoglobulin sequence. As used herein, the term “consensus immunoglobulin sequence” refers to the sequence formed from the most frequentlyFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A occurring amino acids (or nucleotides) in a family of related immunoglobulin sequences (See e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987). In a family of immunoglobulins, each position in the consensus sequence is occupied by the amino acid occurring most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence.

[0035] “Percent (%) amino acid sequence identity” with respect to a peptide or polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. In one embodiment, the invention includes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 31, 35-40, or 50 to 85.

[0036] The term “administering” as used herein is meant to refer to the delivery of a substance to achieve a therapeutic objective. Modes of administration may be parenteral, enteral and topical. Parenteral administration is usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.

[0037] As used herein, the term “effective amount” or “therapeutically effective amount” refers to the amount of a drug which is sufficient to reduce or ameliorate the severity and / or duration of a disorder or one or more symptoms thereof, prevent the advancement of a disorder, cause regression of a disorder, prevent the recurrence, development, onset or progression of one or more symptoms associated with a disorder, detect a disorder, or enhance or improve theFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A prophylactic or therapeutic effect(s) of another therapy (e.g., prophylactic or therapeutic agent). The effective amount may, for example, improve overall survival (OS).

[0038] Various aspects of the invention are described in further detail in the following subsections.

[0039] Other embodiments of the invention are discussed throughout this application. Any embodiment discussed with respect to one aspect of the invention applies to other aspects of the invention as well and vice versa. Each embodiment described herein is understood to be embodiments of the invention that are applicable to all aspects of the invention. It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions and kits of the invention can be used to achieve methods of the invention.

[0040] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0041] Throughout this application, the term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0042] The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.”

[0043] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0044] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains”, “containing,” “characterized by” or any other variation thereof, are intended to encompass a non-exclusive inclusion, subject to any limitation explicitly indicated otherwise, of the recited components. For example, a chemical composition and / or method that “comprises” a list of elements (e.g., components or features or steps) is not necessarily limited to only those elements (or components or features or steps), but may include other elements (orFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A components or features or steps) not expressly listed or inherent to the chemical composition and / or method.

[0045] As used herein, the transitional phrases “consists of” and “consisting of” exclude any element, step, or component not specified. For example, “consists of” or “consisting of” used in a claim would limit the claim to the components, materials or steps specifically recited in the claim except for impurities ordinarily associated therewith (i.e., impurities within a given component). When the phrase “consists of” or “consisting of” appears in a clause of the body of a claim, rather than immediately following the preamble, the phrase “consists of” or “consisting of” limits only the elements (or components or steps) set forth in that clause; other elements (or components) are not excluded from the claim as a whole.

[0046] As used herein, the transitional phrases “consists essentially of” and “consisting essentially of” are used to define a chemical composition and / or method that includes materials, steps, features, components, or elements, in addition to those literally disclosed, provided that these additional materials, steps, features, components, or elements do not materially affect the basic and novel characteristic(s) of the claimed invention. The term “consisting essentially of” occupies a middle ground between “comprising” and “consisting of”.

[0047] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. DESCRIPTION OF THE DRAWINGS

[0048] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of the specification embodiments presented herein.

[0049] FIG. 1A-1G. TTCM2 recognizes pathological tau in brain tissue from patients with AD, DLB, and PSP, but not NDCs, and it also inhibits tau-seeding activity of AD-BDTOs. (A) Immunofluorescence staining with AT8 antibody, TTCM2 antibody, and DAPI in frontal cortexFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A from AD, DLB, and PSP patients and NDCs. (B) Mean Relative Fluorescence Intensities (RFI) of TTCM2 staining in brain tissue from AD (n = 8), DLB (n = 6), and PSP (n = 6) and NDCs (n = 8) and the analysis employed one-way ANOVA, Tukey’s post hoc analysis. (C) Pearson’s correlation coefficient analysis assessing colocalization between TTCM2 and AT8. Graphs show the mean ± SEM; ***P < 0.001, ****P < 0.0001. (D) ELISAs were used to measure immunoreactivity of TTCM2 against brain homogenates from AD, DLB, and PSP patients and tauopathy mouse model, including 3- and 18-month-old hTau mice (n= 3 / group). Brain homogenates from Tau-knockout mice were used as a control (n= 3 mice / group). Recombinant tau monomers (TauM), Tau oligomers (TauO), and Tau fibrils (TauF) were also assessed for immunoreactivity with TTCM2. Graphs show the mean ± SEM; **P < 0.01, ***P < 0.001, ns=non-significant. (E) Seeding activity of AD-BDTOs isolated from PBS-soluble fractions by immunoprecipitation with T22 antibody analyzed using tau biosensor cells ± TTCM2. (F) Representative images showing seeding activity of AD-BDTOs and its inhibition by TTCM2; the untreated control is shown at the left. DAPI counterstain was used to detect total nuclei. Scale bar, 20 μm. (G) Graph showing dose-dependent effect of TTCM2 on AD-BDTO seeding activity in biosensor cells. Lipofectamine 2000 transfection reagent was used to deliver the AD-BDTO: TTCM2 immune complexes to cells. The 1:4 ratio of AD-BDTO to TTCM2 was sufficient to neutralize tau seeding activity. Percentage of FRET-positive cells was determined by dividing the number of FRET-positive cells by total DAPI-positive cells representative of n= 4 experiments. Significance was determined by one-way ANOVA followed by Tukey’s test. Graphs show the mean ± SEM; **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0050] FIG. 2A-2D. Intranasally administered TTCM2-ms rapidly distributed in the brain of tauopathy mice. (A) Schematic diagram showing the experiment design. Intranasal administration of AF568-labeled TTCM2-ms (15 µL / nostril; 30 µL / mouse) was performed on lightly anesthetized animals, and mice were euthanized 3 h after treatment. Following perfusion, brain tissues were extracted, and unfixed tissues were immediately analyzed for ex-vivo fluorescence imaging and immunofluorescence microscopy. (B) Representative ex-vivo fluorescence images of whole brain (upper panel) and brain slices (lower panel) of hTau and WT mice intranasally treated with TTCM2-ms or IgG-ms. Graph showing quantification of high radiant efficiency of TTCM2-ms and IgG-ms in hTau mice and WT control mice. The scale indicates the minimum and maximum radiant efficiency in the brain tissues. Untreated WT miceFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A were used as a baseline. The graph represents the mean ± SEM data from three mice: ***P < 0.001, and ns=non-significant. (C) Representative immunofluorescence images depicting the neuronal postsynaptic marker, PSD95 and distribution of TTCM2-ms in various brain regions, including the olfactory bulb (Ob), hippocampus (Hp), cortex (Cx), cerebellum (Cb), and thalamus (Thl). Scale bars, 20 µM. The data are representative of three mice. (D) Representative immunofluorescence images showing the target engagement by TTCM2 in the hippocampus of hTau tauopathy mice following intranasal delivery, with TTCM2 colocalized with AT8-positive tau aggregates. Arrows highlight areas of colocalization between AT8 and TTCM2. Additionally, the scatter plot illustrates significant colocalization between TTCM2 and AT8, as indicated by Pearson’s correlation coefficient of 0.83.

[0051] FIG. 3A-3L. Intranasal TTCM2-ms treatment ameliorates cognitive impairments and enhances levels of pre-synaptic and synaptic markers in the brain of hTau mice. (A) Experimental design: 15-month-old hTau mice were intranasally treated with a single dose of TTCM2-ms or IgG-ms. After 2-weeks, animals were subjected to the NOR and Y-maze test, followed by brain pathology analysis. (B to F) NOR test measuring impact of intranasal TTCM2- ms treatment on memory in hTau mice. (B) Heat map indicating time spent exploring old or novel objects for representative TTCM2-ms and IgG-treated mice. Discrimination index (C), preference for novel object quadrant (D), number of entries to novel-objects quadrant zone (E), and time spent in novel-objects quadrant zone (F) for TTCM-ms vs. IgG-treated hTau mice (n = 6 / group). Significance was determined by unpaired, two-tailed Student’s t-test. (G and H) Y- maze spontaneous alternation test; the percentage of spontaneous alterations was measured before and 2 weeks after TTCM2-ms or IgG treatment (n =6 / group). Graphs show the mean ± SEM; significance was determined by unpaired, two-tailed Student’s t-test. (I to L) Immunoblot analysis showing the impact of intranasal TTCM2-ms or IgG on the levels of PSD95 (J), Synaptophysin (K), and NeuN (L) in the hippocampus of aged hTau mice (n =6 / group). β-actin is the loading control. The graphs show the mean ± SEM, and significance was determined using an unpaired, two-tailed Student’s t-test *P < 0.05, **P < 0.01, **P < 0.01, and ns= non- significant.

[0052] FIG. 4A-4L. Intranasal TTCM2-ms treatment reduces tau pathology in the brain of tauopathy mice. (A and B) Representative images of Thioflavin-S staining and quantification of NFTs per region-of-interest (ROI) in brains from 15-month-old hTau mice intranasally treatedFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A with IgG-ms or TTCM2-ms. Scale bar: 20 μm. The graph shows the mean ± SEM (n= 6 / group). Significance was determined by unpaired, two-tailed Student’s t-test. (C to F) Representative immunofluorescence images showing staining and quantification of phosphorylated tau detected by AT8 (S202, T205) (D), perimeter of AT8 positive tau aggregates and (E), number of AT8 positive cells from random fields (F) in brains from 15-month-old hTau mice intranasally treated with IgG or TTCM2-ms (n=6 mice / group). (G to L) Representative immunofluorescence images showing staining and quantification of phosphorylated tau detected by AT100 (T212, S214) (G to I and Fig. 9C) and AT180 (T231) (J to L and Fig. 9D) along with quantification of RFI, and perimeter of tau inclusions in brains from 15-month-old hTau mice intranasally treated with IgG or TTCM2-ms (scale bars, 20 μm). The graphs show the mean ± SEM (n =6 mice / group), significance was determined by unpaired, two-tailed Student’s t-test (*P < 0.05, and **P < 0.01).

[0053] FIG. 5A-5L. Cytosolic Fc receptor TRIM21 is linked to TTCM2-ms antibody- mediated clearance of intracellular tau aggregates. (A) Immunofluorescence analysis of primary cortical neurons treated with AD-BDTOs for 24 h to establish intracellular tau aggregates. Subsequently, the cells were washed and treated with IgG-ms or TTCM2-ms for 0–60 min. Intracellular TRIM21-tau puncta are indicated by arrows. (B to E) Quantification of total tau (tau5) (B), TauO (T22) (C), size of the tau aggregates (D), and number of TRIM21-positive tau puncta (E) at 0-, 30-, and 60 min. Graphs represent the mean ± SEM from four experiments. Significance was determined by one-way ANOVA followed by Tukey’s test. (F to I) Immunoblot analysis and quantification of TRIM21 and tau aggregates levels in cell lysates from the cells treated with control or TRIM21 siRNAs followed by exposure to AD-BDTOs: TTCM2 at 1:4 ratio. β-actin is the loading control. (J to L) Immunofluorescence analysis quantification of AD-BDTOs in cells treated with TRIM21-siRNA or control siRNA followed by exposure to AD-BDTOs: TTCM2 at 1:4 ratio. The graphs show the mean ± SEM from four experiments. Significance was determined by unpaired, two-tailed Student’s t-test. *P < 0.05, ***P < 0.001, and ****P < 0.0001.

[0054] FIG.6A-6K. TTCM2-ms efficiently internalized and selectively clear seed-competent tau from the brains of aged hTau mice. (A to D) Internalization of TTCM2-ms at 4°C and 37°C. Cells expressing hTau were exposed to AF568–labeled TTCM2 or TTCM2-ms for 30 min, followed by washing to remove unbound / extracellular antibodies. Relative red fluorescence intensities (RFI) measured at 4°C (A and B) and 37°C (C and D) to detect binding andFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A internalization, respectively. The graphs show the mean ± SEM n=8-10 ROIs from 3 experiments. Significance was determined by unpaired, two-tailed Student’s t-test. (E) Experimental design to assess clearance of intracellular tau aggregates. Tau biosensor cells incubated with AD-BDTOs for 24 h to establish intracellular tau aggregates were treated with or without TTCM2-ms antibody for 3 h. (F) Representative images showing size of tau aggregates and percent FRET positivity in AD-BDTO–treated cells ± TTCM2-ms. (G) Percentage of FRET- positive cells and (H) size of tau aggregates in AD-BDTO–treated cells ± TTCM2-ms. Significance was determined by unpaired, two-tailed Student’s t-test. Graphs represent the mean ± SEM from four experiments. (I) Schematic showing experimental strategy for detecting tau- seeding activity in brain homogenates of tauopathy mice intranasally treated with IgG or TTCM2-ms. (J) Representative images showing tau-seeding activity in IgG- and TTCM2-ms– treated mice; untreated control is shown at left. DAPI counterstain was used to detect total nuclei. Scale bar, 20 μm. (K) Percentage of FRET-positive cells in TTCM2-ms– vs. IgG-ms- treated mice (n=4 brain homogenates / group). Significance was determined by one-way ANOVA followed by Tukey’s test. Graph represents the mean ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0055] FIG. 7A-7E. TTCM2-ms treatment promotes the clearance of tau aggregates from synaptic compartments of neurons in the brains of tauopathy mice. (A) Immunostaining for PSD95 and Tau HT7 and (B) quantification of RFI of tau in PSD95-positive compartments in brain tissue from 15-month-old hTau mice intranasally treated with TTCM2-ms or IgG-ms (n=6 mice / group). Scale bar, 20 μm. (C) RFI of total tau (Tau HT7). (D) Pearson correlation coefficient analysis assessing colocalization between HT7 tau and PSD95. (E) Number of PSD95 puncta / ROI in the brain of 15-month-old hTau mice intranasally treated with TTCM2-ms or IgG control. Graphs show the mean ± SEM, n=6 mice / group. Significance was determined by unpaired, two-tailed Student’s t-test (**P < 0.01, and ****P < 0.0001).

[0056] FIG. 8A-8G. (A) Representative images of TTCM2 and AT8 immunohistochemical (IHC) staining in fixed frontal cortex sections from AD, DLB, PSP and NDC brain tissues. TTCM2 staining exhibited a strong immunoreactivity in AD, DLB and PSP cases and no immunoreactivity observed in NDC brain tissues. TTCM2 preferentially stained neuritic and intracellular / perinuclear tau inclusions, indicated by arrows. Scale bar: 20 μm. (B) Dot blot assay demonstrates the immunoreactivity of TTCM2 immunoreactivity against T22-affinity purifiedFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A brain-derived tau oligomers (BDTOs) from patients with AD, PSP, and DLB, as well as recombinant tau oligomers. TTCM2 does not detect Aβ oligomers or αSynuclein oligomers, indicating TTCM2 selectively recognize pathologically relevant toxic TauO. Phosphate-buffered saline (PBS) was used as a negative control, and anti-rabbit (AR) and anti-mouse (AM) HRP- conjugates alone were used to confirm the absence of non-specific binding. (C) TTCM2 neutralizes TauO toxicity in SH-SY5Y cells in a dose-dependent manner. TauO (0.5 μM) were preincubated with TTCM2 at different ratios (1:0, 1:1, 1:2, 1:4 and 1:8) and exposed to cells for 24 h. PBS was used as a negative control. The cytotoxicity was measured by MTT assay. Graph shows mean ± SEM from three experiments. Significance was determined by one-way ANOVA followed by Tukey’s test (*P < 0.05, ***P < 0.001, and ****P < 0.0001). (D-G) TTCM2’s immunoreactivity is dependent on the pathological conformation of tau. Dot blot assay of tau monomer (TauM), Tau Oligomers (TauO) and Tau Fibrils (TauF) immobilized onto nitrocellulose membrane non-denatured (ND) or denatured (D) by urea and heat treatment. TTCM2 detected TauM, TauO and TauF with reduced immunoreactivity after denaturation. This indicates that the immunoreactivity of TTCM2 is dependent on the pathological conformation of tau (D-E). Additionally, the Tau13 antibody, which detects total tau, showed similar levels of tau immunoreactivities across the different conditions (F-G). Significance was determined by one- way ANOVA followed by Tukey’s test. Graphs show the mean ± SEM from three experiments *P < 0.05, ****P < 0.0001, ns=non-significant.

[0057] FIG. 9A-9D. (A) Representative immunoflourescence staining showing NeuN- positive neurons in brain sections from IgG and TTCM2-ms-treated hTau mice. White arrows indicate loss of NeuN-positive neurons in the IgG group, while yellow arrows indicate abundant NeuN-positive neurons in the TTCM2 group. (B) Quantification of NeuN positive neurons in brain sections from IgG and TTCM2-ms-treated hTau mice. Student t test was used to determine statistical difference between the groups (* P<0.05 and **P < 0.01). (C and D) Number of AT180- and AT100-positive cells in the brain of aged hTau mice treated with TTCM2-ms or IgG-ms. Significance was determined by unpaired, two-tailed Student’s t-test (*P < 0.05).

[0058] FIG. 10. Immunoblot analysis and quantification showing that TTCM2-ms treatment reduces pathological tau aggregates detected by Tau13, T22, AT8, AT100, AT180 and T18 antibodies from the brain of aged hTau mice. Significance was determined by unpaired, two- tailed Student’s t-test (* P<0.05 and ns=non-significant).FJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A

[0059] FIG. 11A-11F. (A) Representative immunoflourescence images demonstrate the colocalization of TTCM2, TRIM21 and pathological tau in brain sections of hTau mice intranasally treated with TTCM2-ms. Arrows indicate intracellular complexes formed by the TTCM2 antibody and pathological tau detected by AT8, with most of these complexes colocalizing with TRIM21 puncta. Scale bar: 20 μm; ROI zoom scale bar: 20 μm. (B) The number of detectable TRIM21 positive tau puncta per ROI in the brain section of hTau mice 3 hours after intranasal delivery in the presence or absence of TTCM2. (C) Representative Immunofluorescence images showing AT8-positve tau aggregates, and TRIM21 in brain sections of hTau mice treated intranasally with either IgG or TTCM2-ms for 3h and 24h. (D) Quantification of AT8-positive tau aggregates per ROI. (E) Quantification of tau positive- TRIM21 puncta per ROI. (F) Perimeter of AT8 positive tau aggregates at 3h and 24 h after intranasal treatment with TTCM2-ms or IgG-ms. One-way ANOVA, Tukey’s post hoc analysis used to determine statistical significance. Graphs show the mean ± SEM (n=6 mice / group); *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns=non-significant.

[0060] FIG. 12A-12F. (A) Immunoblot analysis of sarkosyl-insoluble fractions prepared from the brain homogenates of hTau mice intranasally treated with TTCM2-ms or IgG-ms. Each lane represents individual animal. Densitometric quantification of tau in sarkosyl-insoluble fractions by using antibodies, AT8 (B), HT7 (C) and Tau5 (D), normalized to total protein concentration. Student t test was used to determine statistical difference between the groups, * P<0.05. (E) TTCM2 reactivity with Tau-RD expressed by biosensor cells as shown by colocalization analysis. (F) Immunoblot analysis of TauRD from cell lysates of biosensor cells. Cell lysates were subjected to non-denatured (ND) or denatured (D) conditions by urea and heat treatment followed by immunoblotting with TTCM2. TTCM2 detected Tau RD monomers and aggregates in non-denatured conformations while reduced immunoreactivity after denaturation. The data indicates that immunoreactivity of TTCM2 is dependent on the pathological conformation of TauRD.

[0061] FIG. 13A-13D. (A) Immunostaining for synaptophysin 1 and T22-positive TauO showing that TTCM2-ms treatment promotes clearance TauO from synaptic compartments. White arrows indicate the colocalization between T22-positive TauO and synaptophysin in the IgG group, suggesting the presence of TauO in synapses. In contrast, arrows indicate no colocalization between TauO and synaptophysin 1, indicating clearance of TauO from synapsesFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A in the TTCM2 group. Scale bar, 20 μm. (B) Quantification of RFI of T22-positive TauO in synaptophysin 1-positive compartments in brain tissue from 15-month-old hTau mice intranasally treated with TTCM2-ms or IgG-ms control (n=6 mice / group). (C) Pearson correlation coefficient analysis assessing colocalization between T22-positive TauO and synaptophysin 1. (D) Number of synaptophysin 1 puncta per ROI in the brain of 15-month-old hTau mice intranasally treated with TTCM2-ms or IgG control. Graphs show the mean ± SEM, n=6 mice / group. Significance was determined by unpaired, two-tailed Student’s t-test (**P < 0.01).

[0062] FIG.14A-14C. Tau aggregates from IgG- and TTCM2-ms–treated mice show distinct conformations, as detected by differential proteolytic stability. Brain homogenates from IgG- and TTCM2-ms–treated hTau mice were incubated with proteinase K (PK) at various concentrations for 1 h. The proteolyzed products were then assayed by immunoblots with sequence-specific tau antibodies, including Tau 5 (A), RD4 (B), and Tau46 (C).

[0063] FIG.15A-15B. (A) Immunostaining images showing differential PK sensitivity of tau aggregates in brain homogenates from IgG-ms- and TTCM2-ms–treated mice; staining was performed with Tau5 and DAPI, and (B) RFI was measured with ImageJ.

[0064] FIG. 16A-16C. Synthesis of micelles. (A) Synthesis scheme and (B and C) synthesis setup to yield product.

[0065] FIG. 17. Fourier Transform Infrared Spectroscopy analysis indicating carboxylated Pluronic F-127, referred to as “functionalized F127,” and the characteristic C=O stretch at 1730 cm-1.

[0066] FIG. 18A-18C. Size distribution of toxic tau conformation–specific monoclonal- antibody-2 (TTCM2)-loaded micelles (TTCM2-ms); (A) concentration / size and (B) average concentration.

[0067] FIG. 19. Schematic of experimental overview. Three different α-Syn oligomeric polymorphs were systematically characterized biochemically, biophysically, and evaluated by cellular spreading. In addition to traditional methods, we used 3 novel α-Syn toxic conformation antibodies (Anti-Syns).

[0068] FIG. 20A-20I. Biochemical characterization of α-Syn oligomeric polymorphs. α-Syn oligomeric polymorphs were characterized by dot blotting, western blotting (middle) and Indirect enzyme-linked immunosorbent assay (ELISA) (bottom). α-Syn monomer, different α-SynFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A oligomer preparations, α-Syn fibrils, and amyloidogenic proteins, tau and amyloid β, were characterized with primary antibodies: Anti-Syn1 (A, E, I), Anti-Syn2 (B, F, J), Anti-Syn3 (C, G, K). Results revealed the selectivity of the Anti-Syns for α-synuclein, confirmed by total Syn antibody, LB509 (D, H, I). Analyses confirm differences in immunoreactivity for α-Syn oligomeric conformers. Indirect ELISA: Data represented as mean ± SD.

[0069] FIG. 21. Isothermal Titration Calorimetry (ITC) confirms distinct binding profiles of Anti-Syns and α-Syn oligomers. Integrated binding curves of the isothermal titration calorimetry (ITC) experiment of 2 μM α-Syn oligomer titrated with 8 μM Anti-Syn at 25 °C. A one set of sites binding model was used for all experiments. Binding curves were fitted with a nonlinear regression model. Thermodynamic and stoichiometric parameters obtained from the fitting of the binding curve are shown.

[0070] FIG. 22. Anti-Syn immunodepletion of α-Syn oligomeric polymorphs reduce α-Syn seeding and endogenous aggregation. Primary neurons were treated with α-Syn oligomer or α- Syn oligomer preincubated with a Anti-Syn for 30 mins at RT. Immunocytochemistry was done following 24hr incubation. Syn10842, total α-Syn polyclonal antibody (rabbit), LB509, total α- Syn monoclonal antibody (mouse), and BIIITubulin, neuronal marker, were used to stain cells. White arrowheads indicate colocalization of Anti-Syn10842 and Anti-LB509 in neurons. Quantification of average fluorescence intensity of α-Syn aggregates calculated from five different regions of interest (ROI). Bar graph showed as mean ± SD (****p < 0.0001). Scale bar = 10 µm. Statistical analyses were calculated by one way ANOVA and Brown-Forsythe test.

[0071] FIG. 23A-23L. α-Syn oligomeric polymorphs are cytotoxic and are differentially neutralized by Anti-Syns. Anti-Syns inhibit cytotoxicity exerted by α-synuclein oligomers in human neuroblastoma SH-SY5Y cells (A-F) and primary cortical neurons isolated from mice overexpressing human α-Syn (G-L). α-Syn oligomers (0.5 µM) were preincubated with a Anti- Syn (2 µM) at a ratio of 1:4 for 30 mins at RT and added to the cells for 24 hours. Cytotoxicity was analyzed by LDH and MTS cell-based assays. Bars and error bars represent means and standard deviations, respectively (***P<0.001).

[0072] FIG. 24A-24D. Dot blot quantification. α-Syn monomer, different α-Syn oligomer preparations, α-Syn fibrils, and amyloidogenic proteins, tau and amyloid β were characterized by dot blotting with primary antibodies: Anti-Syn1 (A), Anti-Syn2 (B), Anti-Syn3 (C). Dot blotFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A quantification reveals the immunoreactivity and selectivity of the Anti-Syns for α-synuclein, confirmed by total Syn antibody, LB509 (D).

[0073] FIG.25A-25C. Proteolytic digestion profiles of α-Syn oligomeric polymorphs. Silver staining images of α-Syn oligomers, Syn O aCSF (A), Syn O DA (B), Syn O DHA (C) digested with 1, 1.5, and 2 μg / mL proteinase K (PK) enzyme.

[0074] FIG. 26. Atomic Force Microscopy of Syn O aCSF and Syn Fibrils. Representative AFM images of Syn O aCSF and Syn Fibrils. Syn O aCSF exhibit spherical structures while Syn Fibrils exhibit protofilaments.

[0075] FIG. 27A – 27B. Isothermal Titration Calorimetry (ITC) Binding profiles of Anti- Syns and monomeric and fibrillar α-Syn. Integrated binding curves of the isothermal titration calorimetry (ITC) experiment of 2 μM α-Syn monomer (A) or fibrils (B) titrated with 8 μM Anti- Syn at 25 °C. The binding curve of each experiment fitted with an n independent binding site model is shown in the graph. Thermodynamic and stoichiometric parameters obtained from the fitting of the binding curve are shown.

[0076] FIG. 28A-28C. Fitting of α-Syn oligomer:Anti-Syn ITC data. Primary ITC injection data generated through MicroCal PEAQ-ITC analysis software (A). Outliers were determined and excluded utilizing analysis software. Data points were then extracted and graphed in GraphPad (B). A Gaussian nonlinear fit model was used to fit the integrated binding curves utilizing GraphPad (C).

[0077] FIG. 29A-29C. Fitting of α-Syn monomer:Anti-Syn ITC data. Primary ITC injection data generated through MicroCal PEAQ-ITC analysis software (A). Outliers were determined and excluded utilizing analysis software. Data points were then extracted and graphed in GraphPad (B). A Gaussian nonlinear fit model was used to fit the integrated binding curves utilizing GraphPad (C).

[0078] FIG. 30A-30C. Fitting of α-Syn monomer:Anti-Syn ITC data. Primary ITC injection data generated through MicroCal PEAQ-ITC analysis software (A). Outliers were determined and excluded utilizing analysis software. Data points were then extracted and graphed in GraphPad (B). A Gaussian nonlinear fit model was used to fit the integrated binding curves utilizing GraphPad (C).

[0079] FIG. 31A-31X. Anti-Syn neutralization of α-Syn oligomeric polymorphs affect α-Syn endogenous aggregation. Primary cortical neurons were treated with α-Syn oligomer or α-SynFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A oligomer preincubated with a Anti-Syn for 30 mins at RT. Immunocytochemistry was done following 24hr incubation. Syn10842, total α-Syn polyclonal antibody (rabbit), LB509, total α- Syn monoclonal antibody (mouse), and BIIITubulin, neuronal marker, primary antibodies were used to stain cells. The colocalization profiles for individual selected neurons are shown above. Region of interest (ROI) marked in white line indicating colocalization of anti-Syn LB509, anti- Syn10842, and anti-BIITubulin in neurons. DESCRIPTION

[0080] The following discussion is directed to various embodiments of the invention. The term “invention” is not intended to refer to any particular embodiment(s) or otherwise limit the scope of the disclosure. Although one or more of these embodiments may be preferred, the embodiments disclosed should not be interpreted, or otherwise used, as limiting the scope of the disclosure, including the claims. In addition, one skilled in the art will understand that the following description has broad application, and the discussion of any embodiment is meant only to be an example of that embodiment and not intended to imply that the scope of the disclosure, including the claims, is limited to that embodiment.

[0081] Neurodegenerative disorders related to tau and synuclein proteins are a group of progressive, incurable conditions characterized by the accumulation of abnormal protein aggregates in the brain, leading to neuronal dysfunction and death. Tauopathies, such as Alzheimer’s disease and frontotemporal dementia, involve the misfolding and aggregation of tau, a microtubule-associated protein, into neurofibrillary tangles, disrupting neuronal stability and transport. Synucleinopathies, including Parkinson’s disease, dementia with Lewy bodies, and multiple system atrophy, are marked by the accumulation of misfolded alpha-synuclein into Lewy bodies or glial inclusions, impairing synaptic function and promoting neurotoxicity. These disorders share common features, including protein misfolding, neuroinflammation, and progressive cognitive or motor deficits, but differ in clinical presentation, affected brain regions, and specific protein pathology. Research continues to explore shared mechanisms and potential therapies targeting protein aggregation and clearance. The formation of distinct polymorphs of amyloidogenic proteins tau, amyloid β, and α-Syn present a new challenge for developing diagnostics and therapeutics (Tycko et al., Neuron, 86, 632-45, 2015; Sengupta et al., MolecularFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A neurobiology, 57, 2741-2765, 2020; Sidhu et al., ACS chemical neuroscience, 8, 538-547, 2017; Li and Liu, Nat Chem Biol, 17, 237–245, 2021). I. Immunotherapy of Neurodegenerative Tauopathies

[0082] Neurodegenerative tauopathies, including Alzheimer's disease (AD), are characterized by the misfolding and aggregation of the microtubule (MT)-associated protein tau. Normally, tau binds to and stabilizes MTs, thereby maintaining the network of MTs essential for axonal transport in neurons. In AD, tau becomes sequestered into aggregates, known as neurofibrillary tangles (NFTs) and neuropil threads, resulting in reduced MT-binding. This loss of tau function is believed to lead to MT destabilization and consequent axonal transport deficits, which could result in neuronal dysfunction and death. Other neurodegenerative diseases where MT function may be compromised include frontotemporal lobar degeneration, multiple sclerosis, Parkinson's disease, Pick’s Disease, amyotrophic lateral sclerosis, schizophrenia, Huntington's disease, multiple sclerosis, and traumatic brain injury (TBI), especially repetitive TBI (rTBI) such as that due to dementia pugilistica and recurrent football concussions and military closed head injuries, which also is known as chronic traumatic encephalopathy (CTE).

[0083] The primary pathological hallmark of neurodegenerative tauopathies is the formation of intracellular tau aggregates. In the brains of patients afflicted with tauopathies, a significant proportion of pathological tau localizes within the intracellular and synaptic compartments of neuronal cells (Congdon et al., Semin Cell Dev Biol 126, 125-137, 2022; Albert et al., Brain, 142, 1736-1750, 2019; Braak et al., Neurobiol Aging, 16, 271-278; discussion 278-284, 1995). These neurons release tau seeds into the extracellular space, where they enter neighboring cells and facilitate tau aggregation, thereby promoting tauopathy progression (Guo and Lee, Nat Med 20, 130-138, 2014; Braak et al., Neurobiol Aging, 16, 271-278; discussion 278-284, 1995; Frost et al., J Biol Chem, 284, 12845-12852, 2009; Pooler et al., EMBO Rep, 14, 389-394, 2013). Distribution of pathological tau strongly correlates with clinical severity and neurodegeneration in AD patients (Arriagada et al., Neurology 42, 631-639, 1992, Brier et al., Sci Transl Med 8, 338ra366, 2016).

[0084] One promising approach to treating tauopathies is to specifically target and remove pathological tau aggregates from the brain using immunotherapy. However, therapeutic antibodies face challenges in reaching the brain due to the blood-brain barrier (BBB) and poorFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A internalization by neurons. Given that pathological tau aggregates are prevalent within intracellular compartments of neurons, the ability of antibodies to effectively target and eliminate pathological tau aggregates is impeded (Congdon et al., Semin Cell Dev Biol 126, 125-137, 2022; Han et al., J Neuropathol Exp Neurol 76, 44-51, 2017). These limitations present a significant barrier to advancing effective therapies for neurodegenerative tauopathies (Congdon et al., Semin Cell Dev Biol 126, 125-137, 2022; Han et al., J Neuropathol Exp Neurol 76, 44-51, 2017).

[0085] Toxic Tau Conformational Monoclonal antibodies (TTCM) have been developed. Studies have shown: (1) TTCM2 specifically recognized pathological tau in the brain tissues from patients with tauopathies, including AD, DLB, and PSP, and it effectively neutralized the seeding activity of AD-BDTOs. (2) Upon intranasal administration, TTCM2-ms rapidly and efficiently distributed across various brain regions in hTau mice and engaged with pathological tau in intracellular compartments of neuronal cells in vivo. (3) TTCM2-ms treatment promoted clearance of pathological tau aggregates, including NFTs, phosphorylated, and oligomeric tau from brains of aged hTau mice, leading to improved cognitive functions. (4) TTCM2-ms readily colocalized with TRIM21 and pathological tau complexes and facilitated clearance of intracellular tau aggregates. (5) TTCM2-ms effectively cleared seed-competent, intracellular, and synaptic tau from the brains of aged hTau mice. Together, these findings have highlighted a crucial role for seed-competent pathological tau conformations in progression and cognitive decline and further support the use of intranasal administration of TTCM2-ms as a promising strategy for rapidly and effectively removing pathological tau seeds and aggregates from the brain.

[0086] Intranasal delivery can be used, which allows direct delivery of antibodies into the brain via the olfactory and trigeminal nerves, bypassing the BBB (Lochhead et al., Adv Drug Deliv Rev, 64, 614-628, 2012). It was observed that upon intranasal treatment, TTCM2-ms efficiently reached various regions of the brain, including the olfactory bulb, cortex, hippocampus, cerebellum, and thalamus, as indicated by ex-vivo fluorescence imaging, demonstrating successful delivery to the brain. Notably, it was also found that intranasally administered TTCM2-ms colocalized with the synaptic compartments. In some instances, whole cellular bodies were stained by TTCM2-ms, indicating delivery to the intracellular and synaptic compartments. These observations align with numerous studies consistently showing the presence of pathological tau in various cellular regions, including the dendritic, somatic, andFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A axonal compartments of neuronal cells (Montalbano et al., Brain Pathol, e13112, 2022; Braak et al., Cereb Cortex, 28, 3372-3384, 2018; Brady et al., Neurobiol Aging, 16, 479-486, 1995; Zempel et al., Mol Neurodegener, 10, 68, 2015). Clinical and preclinical studies have shown that intranasal drug delivery can effectively and safely be used for the treatment of brain pathologies (Craft et al., JAMA Neurol, 77, 1099-1109, 2020; Benedict et al., Psychoneuroendocrinology, 29, 1326-1334, 2004; Dhamoon et al., Neurology, 72, 292-293; author reply 293-294, 2009; Hallschmid et al., CNS Drugs, 35, 21-37, 2021; Pietrowsky et al., Biol Psychiatry, 39, 332-340, 1996). Evidence supports the feasibility of direct delivery of MABs through the nose-to-brain pathway in patients with tauopathies, potentially for diagnostic and therapeutic purposes. TTCM2-ms demonstrates rapid and efficient distribution in various brain regions upon intranasal administration. Furthermore, TTCM2-ms efficiently internalized into intracellular compartments, effectively cleared intracellular tau pathology in older tauopathy mice with established disease (Polydoro et al., J Neurosci, 29, 10741-10749, 2009; Bittar et al., J Alzheimers Dis, 90, 1103- 1122, 2022). Additionally, TTCM2 specifically detects pathological tau aggregates in brain tissues from patients with AD, DLB, and PSP.

[0087] The impact of intranasal TTCM2-ms treatment on hippocampus-dependent learning and memory in 15-month-old hTau mice were investigated using the Y-maze and NOR tests. Remarkably, a single dose of intranasal TTCM2-ms significantly improved cognitive functions in aged tauopathy mice. The beneficial effects of TTCM2-ms treatment were associated with decreased levels of NFT deposition and reduced levels of phosphorylated, oligomeric, and misfolded tau aggregates, whereas monomeric tau levels remained unaltered. The findings are consistent with previous studies showing that inhibiting tau-seeding activity and clearing synaptic tau aggregates leads to reduced tau pathology and improved cognitive functions in various AD mouse models (Yanamandra et al., Neuron 80, 402-414, 2013; Schaler et al., Sci Transl Med, 13, 2021; Albert et al., Brain, 142, 1736-1750, 2019; Yanamandra et al., Ann Clin Transl Neurol, 2, 278-288, 2015; Herline et al., Alzheimers Res Ther, 10, 54 2018; Castillo- Carranza et al., J Neurosci, 35, 4857-4868, 2015; Boutajangout et al., J Neurosci, 30, 16559- 16566, 2010). Moreover, the observations are supported by prior studies showing that immunotherapy with conformation-specific tau MABs efficiently reduces tau pathology and neuronal loss and rescues cognitive impairments in different AD animal models (Chai et al., J Biol Chem 286, 34457-34467, 2011; Congdon et al., Nat Rev Neurol 14, 399-415, 2018; Jadhav,FJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A Acta Neuropathol Commun, 7, 22, 2019; Castillo-Carranza et al., J Neurosci, 35, 4857-4868, 2015; Gibbons et al., Mol Neurodegener, 15, 64, 2020; Jicha et al., J Neurosci Res, 48, 128-132, 1997; Schroeder et al., Alzheimers Res Ther, 9, 46, 2017). Thus, targeting seed-competent, toxic tau conformations via intranasal delivery of TTCM2-ms represents a promising therapeutic strategy for tauopathies.

[0088] Studies have revealed that a single intranasal dose of TTCM2-ms efficiently enter the brain and distributed across various brain regions and targeted pathological tau in cytosolic and synaptic compartments. TRIM21 recognized the complexes formed by TTCM2-ms and pathological tau in the cytosol. This distinctive property facilitated clearance of intracellular tau aggregates from the brain, leading to improved cognitive functions in aged tauopathy mice. This work establishes a rapid and efficient approach for clearance of intracellular tau pathology-a significant challenge in the field of tau immunotherapy. Furthermore, TTCM2-ms inhibited tau toxicity and seeding activity, and eliminated seed-competent tau from the brain. These findings are consistent with a recent study demonstrating TRIM21 is required for effective tau immunotherapy and clearance of tau pathology in mouse models (Mukadam et al., Science, 379, 1336-1341, 2023). Studies provide important mechanistic insights and strategies for optimizing tau immunotherapy approaches in neurodegenerative tauopathies. II. Immunotherapy of Neurodegenerative Synucleinopathies

[0089] Certain embodiments are directed to treatment of synucleinopathies. Neurodegenerative synucleinopathies, including Parkinson’s disease, dementia with Lewy bodies, and multiple system atrophy are characterized by the misfolding and aggregation of α- Syn. The symptoms vary significantly among the diseases but include motor dysfunction, cognitive decline, autonomic disturbances, and sleep disorders. α-Syn is a 140 amino acid neuronal protein that is the major component of Lewy bodies, a pathological hallmark of synucleinopathies (MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSK TKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQL GKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA, SEQ ID NO:77) (Liu et al., Frontiers in cellular neuroscience, 15, 633727, 2021). Investigating the therapeutic targeting of polymorphic α-Syn oligomers and its effects on α-Syn endogenous aggregation and mediated toxicity provides insight into the design and molecular recognition needed for antibodies toFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A effectively target biologically relevant amyloidogenic aggregates (Sengupta et al., Molecular neurobiology, 57, 2741-2765, 2020; Fields et al., Frontiers in molecular neuroscience, 12, 299, 2019; Games et al., The Journal of neuroscience: the official journal of the Society for Neuroscience, 34, 9441-54, 2014; Spencer et al., Acta neuropathologica communications, 5, 7, 2017; Du et al., International journal of molecular sciences, 21, 8645, 2020; Lorenzen et al., Journal of the American Chemical Society, 136, 3859-68, 2014).

[0090] α-Syn is conformationally dynamic, and this often poses a challenge for developing effective antibodies (Gurnev et al., Biophysical journal, 106, 556-65, 2014). Dot blotting and western blotting were used to evaluate protein recognition with both denaturing and non- denaturing conditions. Both immunoblotting conditions in addition to the ELISA results showed differences in α -Syn oligomeric strain immunoreactivity. These differences provide evidence for biological relevance of polymorph-specific antibody interactions. Vaikath et al. showed the detection of distinct α-Syn micro-aggregates and thin neurites in synucleinopathy brain tissue utilizing conformation specific antibodies (Vaikath et al., Neurobiology of disease, 79, 81-99, 2015). Furthermore, Choi et al recently demonstrated α-Syn conformation-specific antibodies promoted phagocytosis of extracellular α-synuclein aggregates (Choi et al., Experimental neurobiology, 31, 29-41, 2022). This application further supports these findings while considering physiological conditions that contribute to the oligomerization and heterogeneity observed in neurodegenerative diseases with α-Syn pathology.

[0091] Highly sensitive isothermal titration calorimetry was used to characterize the thermodynamic binding interactions between Anti-Syns and different α-Syn species. Isothermal titration calorimetry provides a label-free method for characterizing biomolecule binding reactions, giving insight to functionality. Antibodies binding in the low nanomolar range (10-9) to picomolar (10-12) range are considered high affinity antibodies (Landry et al., Journal of immunological methods, 417, 86-96, 2015). Each Anti-Syn exhibited a distinct binding profile to oligomeric α-Syn. Anti-Syn1 and Anti-Syn2 differed in terms of binding and stoichiometry, but still exhibited high affinity binding to oligomeric α-Syn. Studies show differential binding and entropy in a species-specific manner. While there are minimal antibodies targeting aggregated α- Syn being considered for therapeutic treatment, study shows the biological relevance of polymorphic aggregates. In addition, studies have shown antibody subtype correlates with differences in thermodynamic binding parameters (Dam et al., The Journal of biologicalFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A chemistry, 283, 31366-70, 2008; Janda et al., The Journal of biological chemistry, 287, 35409- 35417, 2012; Janda et al., Frontiers in microbiology, 7, 22, 2016). Anti-Syn consists of a IgG2a / Lambda isotype, while Anti-Syn2 and Anti-Syn3 consist of a IgG1 / Kappa isotype, which may explain Anti-Syn1's high affinity to α-Syn oligomer compared to Anti-Syn2 and Anti-Syn3. Differences in antibody subtype and subclass may affect α-Syn species-specific binding which provides insight to both antibody and species functionality (Vidarsson et al., Frontiers in immunology, 5, 520, 2014; Yu et al., Journal of hematology & oncology, 13, 45, 2020; Irani et al., Molecular immunology, 67, 171-82, 2015).

[0092] Antibody binding exhibits minor variability due to technique-specific conditions (Thomas and Balthasar, Antibodies, 8, 56, 2019; Pillai-Kastoori et al., The Journal of biological chemistry, 295, 926-939, 2020). Therefore, a combination of different methods was used to conduct a thorough investigation of α-Syn oligomeric polymorph immunoreactivity and binding. In contrast to western blots, proteins are not electrophoretically separated by size in dot blots. Limitations of this feature include difficult detection of non-specific binding, and the method does not give any information regarding molecular weight, an important characteristic for distinct protein aggregates. Western blotting involves separation and denaturation which may affect antigenicity and ultimately antibody reactivity. Providing both immunoblotting techniques accounts for limitations of both techniques. In terms of oligomer versus fibril Anti-Syn selectivity, there is a complex dynamic equilibrium amongst oligomeric and various soluble and insoluble higher-order oligomers and protofibrils (Bonito-Oliva et al., Journal of cellular and molecular medicine, 23, 2103-2114, 2019). For this reason, conformation-specific antibodies may preferentially bind aggregated forms of α-Syn.

[0093] The highly sensitive ITC was used to biophysically characterize Anti-Syn binding and selectivity for monomeric, oligomeric, and fibrillar α-Syn species. Anti-Syns did not exhibit binding to α-Syn fibrils in ITC experiments. ITC results exhibit differences compared to immunoblotting and ELISA results, specifically Anti-Syn3 did not exhibit binding to α-Syn species in ITC experiments. Protein aggregation is dynamic, and any conformational changes can impact the measured binding interaction (Dam et al., The Journal of biological chemistry, 283, 31366-70, 2008; Janda et al., The Journal of biological chemistry, 287, 35409-35417, 2012). ITC is conducted in solution; however, oligomers are hydrophobic and bind to surface. Entropic changes due to buffer solution can affect the binding of aggregated α-Syn, therefore unmodifiedFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A α-Syn oligomer was used (Lee et al., Nano letters, 18, 7494-7501, 2018; Jamadagni et al., Langmuir: the ACS journal of surfaces and colloids 25, 13092-9, 2009). This may explain differences observed in biophysical experiments (Kayser et al., mAbs, 3, 408-11, 2011; Al Qaraghuli et al., Sci Rep, 10, 13696, 2020; Linkuvienė et al., Analytical biochemistry, 515, 61- 64, 2016; Bharathi, and K S J Rao. Biochemical and biophysical research communications 359, 115-20, 2007). Conformational epitopes might be preferred for applications involving protein targets in their native state while linear epitopes might be preferred for applications involving protein denaturation (Choi et al., Experimental neurobiology, 31, 29-41, 2022). Commercial total α-Syn antibody, LB509, exhibited increased immunoreactivity to all α-Syn species compared to Anti-Syns and served as a positive control for all biochemistry experiments. These differences reveal considerations for utilizing conformation specific antibodies for polymorphic amyloidogenic aggregates. The effects of antibody selectivity on cytotoxicity and α-Syn propagation in the cell culture experiments were investigated.

[0094] Amyloid polymorphs including α-Syn act as seeds in recipient cells and recruit endogenous protein into aggregation ultimately resulting in cell death (Sengupta et al., Molecular neurobiology, 57, 2741-2765, 2020; Oueslati et al., Experimental neurobiology, 23, 324-36, 2014). To further investigate the effects of polymorphic α-Syn oligomers, α-Syn oligomeric polymorphs were immunodepleted in primary cortical neurons overexpressing human α-Syn and changes in α-Syn endogenous aggregation and neurotoxicity were examined. It is shown when α- Syn oligomeric polymorphs are exposed to human neuroblastoma, SH-SY5Y, cells they act as potent seeds of α-Syn endogenous aggregation and cytotoxicity in a dose-dependent manner (Sengupta et al., Molecular neurobiology, 57, 2741-2765, 2020). The selected dose and time of incubation were corroborated by other studies (Sengupta et al., Molecular neurobiology, 57, 2741-2765, 2020; Sengupta et al., Biological psychiatry, 78, 672-83, 2015; Castillo-Carranza et al., Biological psychiatry, 84, 499-508, 2018; Lo Cascio et al., The Journal of biological chemistry, 295, 14807-14825, 2020). When the effects of α-Syn oligomeric polymorph immunodepletion is examined in primary neurons, differential reduction of α-Syn endogenous aggregation in a polymorph-specific manner is observed. SynODA and SynODHA were more potent seeds of endogenous aggregation compared to SynOaCSF. Furthermore, neurons treated with SynOaCSF and SynODHA exhibited morphological defects and decreased dendrite growth. While oligomers are the most toxic species, physiological conditions, protein-proteinFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A interactions, and posttranslational modifications have been implicated in increased oligomerization and internalization of amyloid oligomers (Fecchio et al., PloS one, 8, e82732, 2013; Mor et al., Nature neuroscience, 20, 1560-1568, 2017; Park et al., Journal of clinical neurology, 7, 215-22, 2011; Montalbano et al., Nat Commun, 11, 4305, 2020; Puangmalai et al., Cell Death Dis, 11, 314, 2020; Puangmalai et al., The Journal of biological chemistry, 298, 101766, 2022).

[0095] Neurotoxicity is a key pathological process that occurs in synucleinopathies, and there are currently no disease-modifying treatments that can neutralize this cellular toxicity. When α-Syn oligomeric polymorphs are immunodepleted by each Anti-Syn, a reduction in neurotoxicity is observed in primary cortical neurons isolated from mice overexpressing human α-Syn. This reduction in toxicity is also shown in SHSY-5Y cells. Differential reduction of toxicity following Anti-Syn immunodepletion suggest polymorph-specific mediated cytotoxicity. While significance of differences varies for the assays, the values of toxicity and viability agree for each polymorph. The best way to target conformationally dynamic amyloidogenic aggregates remains unclear; however, antibodies that target oligomeric polymorphisms halt oligomer propagation and reduce neurotoxicity that may be important for inducing a protective response to protein aggregation (Dehay et al., The Lancet. Neurology, 14, 855-866, 2015; Fields et al., Frontiers in molecular neuroscience, 12, 299, 2019). In vitro studies lay the foundation for investigation into the role of polymorphic oligomers and their correlation with the distinct pathology and behavioral symptoms reflected in Parkinson’s disease, Dementia with Lewy Bodies, and Alzheimer’s disease.

[0096] Identifying the biological properties of amyloidogenic polymorphs is essential to improving the design of immunotherapeutic approaches targeting conformationally distinct proteinaceous aggregates. Amyloidogenic polymorphisms may reflect the variability in behavioral phenotypes and pathological implications observed in neurodegenerative diseases (Wang et al., Biomedicine & pharmacotherapy, 115, 108843, 2019; Gómez-Benito et al., Frontiers in pharmacology, 11, 356, 2020). While multiple antibodies that target α-Syn are being developed and investigated, these antibodies target all forms of α-Syn rather than specific aggregates or polymorphisms of α-Syn aggregates (Brys et al., Movement disorders: official journal of the Movement Disorder Society, 34, 1154-1163, 2019; Wang et al., Biomedicine & pharmacotherapy, 115, 108843, 2019; Jankovic et al., JAMA neurology, 75, 1206-1214, 2018).FJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A Overlapping proteinopathies have been observed in multiple neurodegenerative diseases and oligomeric polymorphisms may contribute to these protein-protein interactions (Sengupta et al., Molecular neurobiology, 57, 2741-2765, 2020; Ciccocioppo et al., Neural regeneration research, 15, 850-856, 2020; Marsh, Neurological research and practice, 1, 35, 2019; Castillo- Carranza et al., Biological psychiatry, 84, 499-508, 2018; Sengupta et al., Biological psychiatry, 78, 672-83, 2015). It is important to further optimize the efficacy of utilizing antibodies to target the conformational heterogeneity of amyloidogenic oligomers. III. Polypeptide compositions A. Toxic Tau Conformational Monoclonal (TTCM) antibody

[0097] In certain aspects, the present invention provides for anti-Tau antibodies that specifically bind to Tau.

[0098] Certain aspects are directed to a TTCM1 antibody having a heavy chain comprising the amino acid sequence MDSRLNLVFLVLILKGVQCDVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMH1WVR QAPEKGLEWVAYINGGGSTIYYADTVKG2RFTISRDNPKNTLFLQMTSLRSEDTAMYYCT REIKTAMDY3WGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVT VTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIV PRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDV EVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGR PKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTD GSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (1=CDR1, 2=CDR2, 3=CDR3) set forth in SEQ ID NO:1 and a light chain comprising the amino acid sequence MKLPVRLLVLMFWIPASSSDDVMIQTPLSLPVSLGDQASISCRSSQSLVHSNGYTYLH1W YLQKPGQSPKLLIYKVSNRFS2GVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTLVP PT3FGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSER QNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (1=CDR1, 2=CDR2, 3=CDR3) in SEQ ID NO:2. In some embodiments, the antibodies, or antigen binding portions thereof, comprise a heavy chain CDR1 domain comprising the amino acid sequence SFGMH set forth in SEQ ID NO:3, a heavy chain CDR2 domain comprising theFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A amino acid sequence YINGGGSTIYYADTVKG set forth in SEQ ID NO:4, a heavy chain CDR3 domain comprising the amino acid sequence EIKTAMDY set forth in SEQ ID NO:5, and constant region having the amino acid sequence AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQS DLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIF PPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSV SELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDK VSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAG NTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO:6); and a light chain CDR1 domain comprising the amino acid sequence RSSQSLVHSNGYTYLH set forth in SEQ ID NO:7, a light chain CDR2 domain comprising the amino acid sequence KVSNRFS set forth in SEQ ID NO:8, a light chain CDR3 domain comprising the amino acid sequence SQSTLVPPT set forth in SEQ ID NO:9, and constant region having the amino acid sequence RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQ DSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO:10.

[0099] In one embodiment, the invention features a TTCM2 antibody having a heavy chain comprising the amino acid sequence MKCSWVIFFLMALVIGINSEVQLQQSGAELVRSGASVKLSCTASGFNIKDYYMQ1WVKQ RPEQGLEWIGWIDPENGDADYAPNFQG2KATMTADTSSNTAYLQLNSLTSEDTAVYYCS TWGGSVYV3WGRGTTLTVSSAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTV TWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEP SGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPD VQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPS PIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENY KDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK(1=C DR1, 2=CDR2, 3=CDR3) set forth in SEQ ID NO:11, and a light chain comprising the amino acid sequence MSPAQFLFLLVFWIRETNGDVVMTQTPLTLSVTIGQPASISCKSSQSLLYSDGKTYLN1WL LQRPGQSPKRLIYLVSKLDS2GVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFP QT3FGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSER QNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNECFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A (1=CDR1, 2=CDR2, 3=CDR3) in SEQ ID NO:12. In some embodiments, the antibodies, or antigen binding portions thereof, comprise a heavy chain CDR1 domain comprising the amino acid sequence DYYMQ (SEQ ID NO:13), a heavy chain CDR2 domain comprising the amino acid sequence WIDPENGDADYAPNFQG (SEQ ID NO:14), a heavy chain CDR3 domain comprising the amino acid sequence WGGSVYV (SEQ ID NO:15), and a constant region having the amino acid sequence AKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSG LYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAP NLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTH REDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPP PAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNM KTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK (SEQ ID NO:16); and a light chain CDR1 domain comprising the amino acid sequence KSSQSLLYSDGKTYLN (SEQ ID NO:17), a light chain CDR2 domain comprising the amino acid sequence LVSKLDS (SEQ ID NO:18), a light chain CDR3 domain comprising the amino acid sequence WQGTHFPQT (SEQ ID NO:19), and a constant region having the amino acid sequence RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQ DSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO:20). B. Tau Oligomer Monoclonal Antibodies (TOMA)

[0100] In one embodiment, the invention features a TOMA1 antibody having a heavy chain variable region comprising the amino acid sequence MAWVWTLLFLMAAAQSIQAQIQLVQSGPELKKPGETVKISCKASGYTFTDYSMH1WVK QAPGKGLKWMGWINTETGEPTYADDFKG2RFAFSLETSASTAYLQINNLKSEDTATYFC ASYGYDGYAMDY3WGQGTSVTVSS (1=CDR1, 2=CDR2, 3=CDR3) (SEQ ID NO:21) and a light chain variable region comprising the amino acid sequence MSVPTQVLGLLLLWLTDARCDIQMTQSPASLSVSVGETVTITCRASENIYSNLA1WYQQK QGKSPQLLVYAATNLAD2GVPSRFSGSGSGTQYSLKINSLQSEDFGSYYCQHFWGTPWT3FGGGTKLEIK (1=CDR1, 2=CDR2, 3=CDR3) (SEQ ID NO:22). In some embodiments, the antibodies, or antigen binding portions thereof, comprise a heavy chain CDR1 domain comprising the amino acid sequence GYTFTDYSMH (SEQ ID NO:23), a heavy chain CDR2FJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A domain comprising the amino acid sequence WINTETGEPTYADDFKG (SEQ ID NO:24), a heavy chain CDR3 domain comprising the amino acid sequence MDY (SEQ ID NO:25); and a light chain CDR1 domain comprising the amino acid sequence RASENIYSNLA (SEQ ID NO: 26), a light chain CDR2 domain comprising the amino acid sequence AATNLAD (SEQ ID NO:27), a light chain CDR3 domain comprising the amino acid sequence QHFWGTPWT (SEQ ID NO:28).

[0101] In one embodiment, the invention features a TOMA2 antibody having a heavy chain comprising the amino acid sequence MNFGLRLIFLVLTLKGVKCEVQLVESGGGLVKPGKSLKLSCAASGFAFSTYDMS1WVRQ TPEKRLEWVAYISNGGGRTYYLDTVKG2RFTISRDNAKNTLSLQMSSLKSEDTAMYYCA RHRRVRRGSYYAMDY3WGQGTSVTVSS (1=CDR1, 2=CDR2, 3=CDR3) (SEQ ID NO:29) and a light chain variable domain comprising the amino acid sequence METDTLLLWVLLLWVPGSTGDIVLTQSPASLVVSLGQRATISCRASKSVSTSGYSYMH1W YQQKPGQPPKLLIYLASNLES2GVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSREL PFT3FGSGTKLEIK (1=CDR1, 2=CDR2, 3=CDR3) (SEQ ID NO:30). In some embodiments, the antibodies, or antigen binding portions thereof, comprise a heavy chain CDR1 domain comprising the amino acid sequence GFAFSTYDMS set forth in SEQ ID NO:31, a heavy chain CDR2 domain comprising the amino acid sequence YISNGGGRTYYLDTVKG SEQ ID NO:32, a heavy chain CDR3 domain comprising the amino acid sequence MDY set forth in SEQ ID NO: 33; and a light chain CDR1 domain comprising the amino acid sequence RASKSVSTSGYSYMH (SEQ ID NO:34), a light chain CDR2 domain comprising the amino acid sequence LASNLES (SEQ ID NO:35), a light chain CDR3 domain comprising the amino acid sequence QHSRELPFT set forth in SEQ ID NO:36.

[0102] In one embodiment, the invention features a TOMA3 antibody having a heavy chain comprising the amino acid sequence MAWVWTLLFLMAAAQSIQAQIQLVQSGPELKKPGETVKISCKASGYTFTDYSMH1WVK QAPGKGLKWMGWINTETGEPTYADDFKG2RFAFSLETSASTAYLQINNLKSEDTATYFC ASYGYDGYAMDY3WGQGTSVTVSS (1=CDR1, 2=CDR2, 3=CDR3) (SEQ ID NO:37) and a light chain variable domain comprising the amino acid sequence MSVPTQVLGLLLLWLTDARCDIQMTQSPASLSVSVGETVTITCRASENIYSNLA1WYQQK QGKSPQLLVYAATNLAD2GVPSRFSGSGSGTQYSLKINSLQSEDFGSYYCQHFWGTPWT3FJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A FGGGTKLEIK (1=CDR1, 2=CDR2, 3=CDR3) (SEQ ID NO:38). In some embodiments, the antibodies, or antigen binding portions thereof, comprise a heavy chain CDR1 domain comprising the amino acid sequence GYTFTDYSMH set forth in SEQ ID NO:39, a heavy chain CDR2 domain comprising the amino acid sequence WINTETGEPTYADDFKG set forth in SEQ ID NO:40, a heavy chain CDR3 domain comprising the amino acid sequence MDY set forth in SEQ ID NO:41, and a light chain CDR1 domain comprising the amino acid sequence RASENIYSNLA (SEQ ID NO:42), a light chain CDR2 domain comprising the amino acid sequence AATNLAD (SEQ ID NO:43), a light chain CDR3 domain comprising the amino acid sequence QHFWGTPWT (SEQ ID NO:44).

[0103] In one embodiment, the invention features a TOMA4 antibody having a heavy chain comprising the amino acid sequence MEWSWIFLFLLSGTAGVHSEVQLQQSGPELVKPGASVRMSCKASGYTFTSYVMH1WMK QKPGQGLEWIGYINPYNDGTKYNEKFKG2KATLTSDKSSSTAYMEISSLTSEDSAVYYCA RPPYGTWFAY3WGQGTLVTVSA (1=CDR1, 2=CDR2, 3=CDR3) (SEQ ID NO:45) and a light chain variable domain comprising the amino acid sequence MDFQVQIFSFLLISASVIMSRGQIVLTQSPAIMSASLGEEITLTCSASSSVSYMH1WYQQKS GTSPKLLIYSTSNLAS2GVPTRFSGSGSGTFYSLTISSVEAEDAADYYCHQWSSWT3FGGG TKLEIK (1=CDR1, 2=CDR2, 3=CDR3) (SEQ ID NO:46). In some embodiments, the antibodies, or antigen binding portions thereof, comprise a heavy chain CDR1 domain comprising the amino acid sequence GYTFTSYVMH (SEQ ID NO:47), a heavy chain CDR2 domain comprising the amino acid sequence YINPYNDGTKYNEKFKG (SEQ ID NO:48), a heavy chain CDR3 domain comprising the amino acid sequence PPYGTWFAY (SEQ ID NO:49), and a light chain CDR1 domain comprising the amino acid sequence SASSSVSYMH (SEQ ID NO:50), a light chain CDR2 domain comprising the amino acid sequence STSNLAS (SEQ ID NO:51), a light chain CDR3 domain comprising the amino acid sequence HQWSSWT (SEQ ID NO:52). C. Anti-Synuclein antibodies (α-Syn antibodies)

[0104] Biochemical and biophysical methods were employed to investigate the biological and immunoreactive properties of three α-Syn oligomeric polymorphs utilizing established methods and three α-Syn Toxic Conformation Monoclonal Antibodies, Anti-Syns. Initial epitopeFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A mapping data showed Anti-Syn binding sites are discontinuous, nonoverlapping sequence patterns further suggesting conformational epitopes. In certain aspects, the present invention provides for anti-α-Syn oligomer antibodies (anti-Syns) that specifically bind to α-Syn oligomers.

[0105] A Anti-Syn 1 antibody has a heavy chain comprising the amino acid sequence MVLGLKWVFFVVFYQGVHCEVQLVESGGGLVQPKGSLKLSCAASGFTFNPYGMH1WV RQAPGKGLEWVARIRSKSSNYATYYADSVKD2RFTISRDDSQSMVYLQMNNLKTEDTAM YYCVRGGGSSGYGFAY3WGQGTLVTVSAAKTTAPSVYPLAPVCGGTTGSSVTLGCLVK GYFPEPVTLTWNSGSLSSGVHTFPALLQSGLYTLSSSVTVTSNTWPSQTITCNVAHPASS TKVDKKIEPRVPITQNPCPPLKECPPCAAPDLLGGPSVFIFPPKIKDVLMISLSPMVTCVVV DVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCK VNNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGFLPAEIAVDWTS NGRTEQNYKNTATVLDSDGSYFMYSKLRVQKSTWERGSLFACSVVHEGLHNHLTTKTI SRSLGK (1=CDR1, 2=CDR2, 3=CDR3) set forth in SEQ ID NO: 53. In some embodiments, the antibodies, or antigen binding portions thereof, comprise a heavy chain CDR1 domain comprising the amino acid sequence PYGMH set forth in SEQ ID NO:54, a heavy chain CDR2 domain comprising the amino acid sequence RIRSKSSNYATYYADSVKD set forth in SEQ ID NO:55, and a heavy chain CDR3 domain comprising the amino acid sequence GGGSSGYGFAY set forth in SEQ ID NO:56. In certain aspects a binding moiety comprises a CDR set of SEQ ID NO:53, 54, and 56.

[0106] The Anti-Syn 1 antibody has a light chain comprising the amino acid sequence MAWTSLILSLLALCSGASSQAVVTQESALTTSPGGTVILTCRSSTGAVTTSNYAN1WVQE KPDHLFTGLIGGTSNRAP2GVPVRFSGSLIGDKAALTITGAQTEDDAMYFCALWYSTHWV3FGGGTKLTVLGQPKSSPSVTLFPPSSEELETNKATLVCTITDFYPGVVTVDWKVDGTPV TQGMETTQPSKQSNNKYMASSYLTLTARAWERHSSYSCQVTHEGHTVEKSLSRADCS (1=CDR1, 2=CDR2, 3=CDR3) set forth in SEQ ID NO:57. In some embodiments, the antibodies, or antigen binding portions thereof, comprise a light chain CDR1 domain comprising the amino acid sequence RSSTGAVTTSNYAN set forth in SEQ ID NO:58, a light chain CDR2 domain comprising the amino acid sequence GTSNRAP set forth in SEQ ID NO:59, and a light chain CDR3 domain comprising the amino acid sequence ALWYSTHWV set forth in SEQ ID NO:60. In certain aspects a binding moiety comprises a CDR set of SEQ ID NO:58, 59, and 60.FJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A

[0107] A Anti-Syn 2 antibody has a heavy chain comprising the amino acid sequence MGWSCIILILVAAATGVHSQAHLQQPGAELVKPGASVKMSCKASGYTFTNYWIT1WVK QRPGQGLEWIGDIYPGSGSTNFNEKFKN2KATLTVDISSSTAYMHLSSLTSEDSAVYYCA GGQTTFAS3WGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTV TWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVP RDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVE VHTAQTKPREEQINSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRP KAPQVYTIPPPKEQMAKDKVSLTCMITNFFPEDITVEWQWNGQPAENYKNTQPIMDTD GSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (1=CDR1, 2=CDR2, 3=CDR3) set forth in SEQ ID NO:61. In some embodiments, the antibodies, or antigen binding portions thereof, comprise a heavy chain CDR1 domain comprising the amino acid sequence NYWIT set forth in SEQ ID NO:62, a heavy chain CDR2 domain comprising the amino acid sequence DIYPGSGSTNFNEKFKN set forth in SEQ ID NO:63, and a heavy chain CDR3 domain comprising the amino acid sequence GQTTFAS set forth in SEQ ID NO:64. In certain aspects a binding moiety comprises a CDR set of SEQ ID NO:62, 63, and 64.

[0108] The Anti-Syn 2 antibody has a light chain comprising the amino acid sequence MKLPVRLLVLMFWIPASSSDVLMTQTPLSLPVSLGDQASISCRSSQNIVHSNGNTYLD1W YLQKPGQSPRLLIYKVSNRFS2GVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHV PRT3FGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSE RQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNE C (1=CDR1, 2=CDR2, 3=CDR3) set forth in SEQ ID NO:65. In some embodiments, the antibodies, or antigen binding portions thereof, comprise a light chain CDR1 domain comprising the amino acid sequence RSSQNIVHSNGNTYLD set forth in SEQ ID NO:66, a light chain CDR2 domain comprising the amino acid sequence KVSNRFS set forth in SEQ ID NO:67, and a light chain CDR3 domain comprising the amino acid sequence FQGSHVPRT set forth in SEQ ID NO:68. In certain aspects a binding moiety comprises a CDR set of SEQ ID NO:66, 67, and 68.

[0109] A Anti-Syn 3 antibody has a heavy chain comprising the amino acid sequence MNFGLSLIFLVLVLKGVQCEVQLVESGGGLVKPGGSLKLSCAASGFTFSSYAMS1WVRQ TPEKRLEWVATISNGGSYTFYADNVKG2RFTISRDNAKNNLYLQMSHLKSEDTAMYYCS RAGAGY3WGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A NSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRD CGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVH TAQTKPREEQINSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAP QVYTIPPPKEQMAKDKVSLTCMITNFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYF VYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (1=CDR1, 2=CDR2, 3=CDR3) set forth in SEQ ID NO:69. In some embodiments, the antibodies, or antigen binding portions thereof, comprise a heavy chain CDR1 domain comprising the amino acid sequence SYAMS set forth in SEQ ID NO:70, a heavy chain CDR2 domain comprising the amino acid sequence TISNGGSYTFYADNVKG set forth in SEQ ID NO:71, and a heavy chain CDR3 domain comprising the amino acid sequence AGAGY set forth in SEQ ID NO:72. In certain aspects a binding moiety comprises a CDR set of SEQ ID NO:70, 71, and 72.

[0110] The Anti-Syn 3 antibody has a light chain comprising the amino acid sequence MMSPAQFLFLLVLWIRESNGDVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLN1WVFQRPGQSPKRLIYLVSKMDS2GVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGT HFPRT3FGGGTKLEINRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDG SERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNR NEC (1=CDR1, 2=CDR2, 3=CDR3) set forth in SEQ ID NO:73. In some embodiments, the antibodies, or antigen binding portions thereof, comprise a light chain CDR1 domain comprising the amino acid sequence KSSQSLLDSDGKTYLN set forth in SEQ ID NO:74, a light chain CDR2 domain comprising the amino acid sequence LVSKMDS set forth in SEQ ID NO:75, and a light chain CDR3 domain comprising the amino acid sequence WQGTHFPRT set forth in SEQ ID NO:76. In certain aspects a binding moiety comprises a CDR set of SEQ ID NO:73, 74, and 75. D. Functional Equivalents

[0111] Modifications and / or changes may be made in the amino acid composition of polypeptides (e.g., TTCM, TOMA, and / or α-Syn antibodies), and thus the present invention contemplates variation in sequences of the polypeptides, and nucleic acids coding therefor, where they are nonetheless able retain substantial activity with respect to the therapeutic, preventative, and curative aspects of the present invention.FJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A

[0112] The biological functional equivalent may comprise a polynucleotide that has been engineered to contain distinct sequences while at the same time retaining the capacity to encode a polypeptide. This can be accomplished through the degeneracy of the genetic code, i.e., the presence of multiple codons, which encode for the same amino acids. In one example, one of skill in the art may wish to introduce a restriction enzyme recognition sequence into a polynucleotide while not disturbing the ability of that polynucleotide to encode a protein.

[0113] In another example, a polynucleotide may encode a biological functional equivalent with more significant changes. Certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies, binding sites on substrate molecules, receptors, and such like. In certain aspects 1, 2, 3, 4 or more amino acids can be substituted as long as the equivalent maintains the binding specificity of the parent antibody. So-called “conservative” changes do not disrupt the biological activity of the protein, as the structural change is not one that impinges on the protein's ability to carry out its designed function. It is thus contemplated by the inventors that various changes may be made in the sequence of genes and proteins disclosed herein, while still fulfilling the goals of the present invention.

[0114] In terms of functional equivalents, it is well understood by the skilled artisan that, inherent in the definition of a “biologically functional equivalent” protein and / or polynucleotide, is the concept that there is a limit to the number of changes that may be made within a defined portion of the molecule while retaining a molecule with an acceptable level of equivalent biological activity. Biologically functional equivalents are thus defined herein as those proteins (and polynucleotides) in selected amino acids (or nucleotides) may be substituted. In certain aspects, a polypeptide is 80, 85, 90, 92, 94, 96, 98, or 100% identical to the wildtype form of the polypeptide. In certain aspects, polypeptide(s) 80, 85, 90, 92, 94, 96, 98, or 100% identical to SEQ ID NO:1, 2, 11, 12, 21, 22, 29, 30, 37, 38, 45, 46, 53, 57, 61, 65, 69, or 73 are used or nucleic acids encoding the same. Function of a polypeptide can be determined by using various assays know to detect the activity of the polypeptide of interest.

[0115] Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and / or the like. An analysis of the size, shape and / or type of the amino acid side-chain substituents reveals that arginine, lysine, and / or histidine are all positively charged residues; thatFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A alanine, glycine, and / or serine are all a similar size; and / or that phenylalanine, tryptophan, and / or tyrosine all have a generally similar shape. Therefore, based upon these considerations, arginine, lysine, and / or histidine; alanine, glycine, and / or serine; and / or phenylalanine, tryptophan, and / or tyrosine are defined herein as biologically functional equivalents.

[0116] To effect more quantitative changes, the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and / or charge characteristics, these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and / or arginine (−4.5).

[0117] The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte & Doolittle, 1982, incorporated herein by reference). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index and / or score and / or still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those that are within ±1 are particularly preferred, and / or those within ±0.5 are even more particularly preferred.

[0118] It also is understood that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. As detailed in U.S. Patent 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). In making changes based upon similar hydrophilicity values, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those that are within ±1 are particularly preferred, and / or those within ±0.5 are even more particularly preferred. E. Proteolysis Targeting Chimeras (PROTACS)

[0119] PROTACs are a class of biotechnology molecules designed to degrade specific proteins within cells by hijacking the body’s natural protein disposal system, the ubiquitin-FJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A proteasome pathway. They are heterobifunctional molecules, meaning they have two distinct binding regions connected by a linker: Target Protein Ligand on one end binds to a specific protein of interest (the disease-causing or unwanted protein). E3 Ligase Ligand on the other end recruits an E3 ubiquitin ligase, an enzyme that tags proteins for degradation. Linker is a chemical bridge that holds the two ligands together, ensuring precise spatial orientation for effective interaction. The antibodies described herein can be used to target tau or synuclein and can be coupled to a E3 ligase ligand.

[0120] E3 ligase ligands. E3 ubiquitin ligases (of which over 600 are known in humans) confer substrate specificity for ubiquitination. There are known ligands that bind to these ligases. As described herein, an E3 ubiquitin ligase binding group is a peptide or small molecule that can bind an E3 ubiquitin ligase. Specific E3 ubiquitin ligases include, but are not limited to: von Hippel-Lindau (VHL); cereblon; XIAP; E3A; MDM2; Anaphase-promoting complex (APC); UBR5 (EDD1); SOCS / BC-box / eloBC / CUL5 / RING; LNXp80; CBX4; CBLL1; HACE1; HECTD1; HECTD2; HECTD3; HECW1; HECW2; HERC1; HERC2; HERC3; HERC4; HUWE1; ITCH; EDD4; NEDD4L; PPIL2; PRPF19; PIAS1; PIAS2; PIAS3; PIAS4; RANBP2; R4; RBX1; SMURF1; SMURF2; STUB1; TOPORS; TRIP12; UBE3A; UBE3B; UBE3C; UBE4A; UBE4B; UBOX5; UBR5; WWPl; WWP2; Parkin; A20 / TNFAIP3; AMFR / gp78; ARA54; beta-TrCP1 / BTRC; BRCA1; CBL; CHIP / STUB1; E6; E6AP / UBE3A; F-box protein 15 / FBX015; FBXW7 / Cdc4; GRAIL / RNF 128; HOIP / RNF31; cIAP-1 / HIAP-2; cIAP-2 / HIAP-l; cIAP (pan); ITCH / AIP4; KAPl; MARCH8;; Mind Bomb 1 / MIB1; Mind Bomb 2 / MIB2; MuRF1 / TRFM63 ; DFIP 1; EDD4; NleL; Parkin; R F2; R F4; RNF8; R F 168; R F43; SART1; Skp2; SMURF2; TRAF-1; TRAF-2; TRAF-3; TRAF-4; TRAF-5; TRAF-6; TRFM5; TRFM21; TRFM32; UBR5; and ZRF3.

[0121] Ubiquitination is a post-translational modification process where ubiquitin, a small regulatory protein, is attached to a substrate protein. This process plays crucial roles in various cellular pathways, primarily targeting proteins for degradation, but also affecting protein localization, activity, and interactions. Ubiquitin is a highly conserved protein of 76 amino acids in eukaryotes. It is attached to proteins via an isopeptide bond between the C-terminal glycine of ubiquitin and the ε-amino group of lysine residues on the target protein. Ubiquitin is first activated by an E1 ubiquitin-activating enzyme, which forms a thioester bond with ubiquitin in an ATP-dependent reaction. The activated ubiquitin is then transferred to an E2 ubiquitin-FJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A conjugating enzyme. Finally, an E3 ubiquitin ligase recognizes the target protein and facilitates the transfer of ubiquitin from the E2 enzyme to the substrate protein. There are hundreds of E3 ligases, providing specificity to the ubiquitination process.

[0122] A “linker” refers to the molecular segment or chain that connects two functional or binding moieties within the same molecule. The primary function of a linker is to physically join two different functional groups or binding domains, allowing them to work in concert or to target two different entities simultaneously. It can provide the necessary distance between the two moieties to ensure that each can interact effectively with its target without steric hindrance. In certain aspects, the LNK (linker) comprises a covalent attachment of the TBM to UBM. In some embodiments, the LNK may be attached to a terminal nucleotide or a nucleotide in the middle of an oligonucleotide sequence. In some embodiments, the LNK may be attached to the 5’ or 3’ or 2’ sugar moiety of a terminal nucleotide or the nucleotide in the middle of the sequence. In some embodiments, the LNK may be attached to the sugar mimetics of a terminal nucleotide or the nucleotide in the middle of the sequence. In some embodiments, the LNK may be attached to the modified nucleobase of a terminal nucleotide or the nucleotide in the middle of the sequence.

[0123] In some embodiments, the LNK is a group comprised of one or morecovalentlyconnected structural units of A (e.g., -A1. . . Aq-), wherein A1is coupled to aTBM, and q is aninteger greater than or equal to 0. In certain embodiments, q is an integer greater than or equal to1.In certain embodiments, e.g., wherein q is greater than 2, Aqis a group that is connected to aUBM or an E3 ligase ligand, and A1and Aqare connected via structuralunits of A (number ofsuch structural units of A : q-2).In certain embodiments, e.g., wherein q is 2, Aqis a group that isconnected to A1,and to a UBM or an E3 ligase ligand. In certain embodiments, e.g., wherein q is1, the structure of theLNK is -A1-, and A1is a group that is connected to a UBM or an E3 ligaseligand and an TMB.In additional embodiments, q is an integer from 1 to 40, 1 to 30, 1 to 20, or 1to 10.

[0124] In certain embodiments, A1 to Aq are, each independently selected from a bond, CRL1RL2, O, S, SO, SO2, NRL3, SO2NRL3, SONRL3, CONRL3, NRL3CONRL4, NRL3SO2NRL4, CO,CRL1=CRL2, C≡C, SiRL1CRL2, P(O)ORL1, P(O)ORL1, NRL3C(=NCN)NRL4,NRL3C(=NCN),NRL3C (=CNO)NRL4, C3-11cycloalkyl optionally substituted with 0-6 RL1and / or RL2groups, C3-11heteocyclyl optionally substituted with 0-6 RL1and / or RL2groups, aryl optionally substituted with 0-6 RL1and / or RL2groups, heteroaryl optionally substituted with 0-6 RL1and / or RL2groups,FJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A wherein RL1or RL2, each independently, can be linked to other A groups to form cycloalkyl and / or hetero cyclyl moeity which can be further substituted with 0-4 RL5groups. In some cases,RL1, RL2, RL3, RL4and RL5are,each independently, H, halo, Cl - galkyl, OC1-8alkyl, SC1-8alkyl,NHC1-8alkyl, N(C1- 8alkyl )2, C3-11cycloalkyl, aryl, heteroaryl, C3-11heterocyclyl, OC1-8cycloalkyl, S C1- 8cycloalkyl, NH C1-8cycloalkyl, N(C1-8cycloalkyl)2, N (C1-8cycloalkyl ) (C1-8alkyl ), OH, NH2, SH, SO2C1-8alkyl, P (O) (OC1-8alkyl ) (C1-8alkyl ), P(O) (O C1-8alkyl )2, CC - C1-8alkyl, CCH, CH=CH (C1-8alkyl), C (C1-8alkyl )=CH (C1-8alkyl ), C(C1-8alkyl ) = C (C1-8alkyl)2, Si(OH)3, Si (C1-8alkyl )3, Si (OH) (C1-8alkyl )2, CO C1-8alkyl, CO2H , halogen, CN, CF3, CHF2, CH2F, NO2, SF5, SO2NHC1-8alkyl, SO2N(C1-8alkyl)2, SONHC1-8alkyl, SON(C1-8alkyl)2, CONHC1-8alkyl, CON(C1-8alkyl)2, N(C1-8alkyl)CONH(C1-8alkyl), N(C1- 8alkyl)CON(C1-8alkyl)2, NHCONH(C1-8alkyl), NHCON (C1-8alkyl)2, NHCONH2, N(C1-8alkyl)SONH(C1-8alkyl), N(C1-8alkyl) SO2N(C1-8alkyl)2, NHSONH(C1-8alkyl ), NHSON(C1-8alkyl )2, or NHSO2NH2.

[0125] In some embodiments, the linker may be an alkylene chain or a bivalent alkylene chain, either of which may be interrupted by, and / or terminate (at either or both termini) in - P(O)(OH)O-, -O-PO(OH)-O-, -O-, -S-, -N(R')-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, - C(NOR')-, C(O)N(R')-, -C(O)N(R')C(O)-, -C(O)N(R)C(O)N(R')-, -N(R)C(O)-, -N(R)C(O)N(R)-,-N(R)C(O)O-, -OC(O)N(R)-, -C(NR)-, -N(R')C(NR')-, -C(NR')N(R)-, -N(R')C(NR)N(R')-, -S(O)2- -OS(O)-, -S(O)O- -S(O)-, -OS(O)2- , - S(O)2O-, -N(R)S(O)2-, -S(O)2N(R)-, -N(R')S(O)-, -S(O)N(R')-, -N(R)S(O)2N(R')-, -N(R)S(O)N(R)-, C1-C12 carbocyclene, 3- to 12-memberedheterocyclene, 5- to 12- membered heteroarylene or any combination thereof, wherein R is H or C1-C12 alkyl, wherein the interrupting and the one or both terminating groups may be the same or different.

[0126] In some embodiments, the linker may be a polyethylene glycol chain which may terminate (at either or both termini) in -P(O)(OH)O-, -O-PO(OH)-O-, -S-, -N(R')-, - C(O)-, - C(O)O-, -OC(O)-, - OC(O)O -, -C(NOR)-, -C(O)N(R')-, -C(O)N(R)C(O)-, -C(O)N(R)C(O)N(R')-, -N(R)C(O)-, -N(R')C(O)N(R)-, -N(R)C(O)O-, -OC(O)N(R)-, -C(NR')-, -N(R)C(NR')-, -C(NR')N(R)-, -N(R)C(NR')N(R)-, -S(O)2- , -OS(O)-, -S(O)O-, -S(O)-, -OS(O)2-, - S(O)2O-, -N(R)S(O)2-, -S(O)2N(R)-, -N(R')S(O)-, -S(O)N(R)-, -N(R)S(O)2N(R')-, -N(R')S(O)N(R')-, C3-12 carbocyclene, 3-to 12-memberedheterocyclene, 5-to 12-memberedheteroarylene or any combination thereof, wherein R is H or C1 C6 alkyl, wherein the one or both terminating groups may be the same or different.FJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A IV. Treatment of Neurodegenerative Diseases

[0127] In the last decade, tau immunotherapy has progressed from proof-of-concept experiments to several clinical trials (Ji et al., Drugs, 81, 1135-1152, 2021; Bittar et al., Neurobiol Dis, 134, 104707, 2020). However, MABs that exclusively target extracellular tau have failed clinical testing. This outcome is unsurprising, considering that the majority of pathological tau resides within the intracellular and synaptic compartments of neurons, with only a small fraction found extracellularly (Han et al., J Neuropathol Exp Neurol 76, 44-51, 2017; Sigurdsson, Prog Mol Biol Transl Sci, 168, 205-217, 2019). Thus, effective clearance of intracellular tau aggregates is necessary for achieving clinical benefits. Indeed, studies in mouse models have revealed that conventionally delivered tau immunotherapy primarily focuses on reducing the deposition and spread of tau pathology in young-to-middle-aged tauopathy mice. However, its effectiveness diminishes in aged animals with well-established tau pathology (Lee et al., Cell Rep, 16, 1690-1700, 2016). Moreover, due to the BBB, only a minimal fraction (0.1- 1%) of therapeutic MABs delivered via conventional routes reach the brain from the circulation (Wang et al., Clin Pharmacol Ther, 84, 548-558, 2008; Shah et al., Mabs, 5, 297-305, 2013).

[0128] Certain embodiments are directed to the treatment of various neurodegenerative disease including synucleinopathies and tauopathies by administering one or more antibody or antibody fragment described herein. Addressing these conditions can include multifaceted treatments, focusing on both symptom management and disease modification. Current therapeutic strategies encompass pharmacological interventions, such as medications to manage motor and non-motor symptoms, alongside emerging treatments like gene therapy, immunotherapy, and small molecule inhibitors aimed at reducing pathological alpha-synuclein or tau accumulation. Additionally, lifestyle modifications, physical therapy, and supportive care play critical roles in enhancing patients' quality of life.

[0129] Neurodegenerative diseases in general include Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Amyotrophic Lateral Sclerosis (ALS), Multiple Sclerosis (MS), Frontotemporal Dementia (FTD), Lewy Body Dementia, Creutzfeldt-Jakob Disease, Progressive Supranuclear Palsy (PSP), and Spinocerebellar Ataxia to name a few.FJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A

[0130] Tauopathies are a group of neurodegenerative diseases characterized by the accumulation of abnormal tau protein in the brain. A list of some well-known tauopathies includes Alzheimer’s Disease (AD), Progressive Supranuclear Palsy (PSP), Corticobasal Degeneration (CBD), Frontotemporal Dementia (FTD), Chronic Traumatic Encephalopathy (CTE), Primary Age-Related Tauopathy (PART), Argyrophilic Grain Disease (AGD), Globular Glial Tauopathy (GGT), and Tangle-Predominant Senile Dementia to name a few. These conditions vary in symptoms and brain regions affected, but they all share the hallmark of tau protein dysfunction.

[0131] Synucleinopathies are a group of neurodegenerative disorders characterized by the abnormal accumulation of alpha-synuclein protein aggregates, known as Lewy bodies or Lewy neurites, in the brain and nervous system. These aggregates disrupt neuronal function, leading to progressive motor and cognitive impairments. The primary synucleinopathies include Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). Parkinson's disease is marked by tremors, rigidity, and bradykinesia, often with later cognitive decline. Dementia with Lewy bodies features early cognitive impairment, visual hallucinations, and fluctuating attention alongside motor symptoms. Multiple system atrophy presents with autonomic dysfunction, parkinsonism, and cerebellar ataxia. These disorders share pathological features but differ in clinical presentation, progression, and affected brain regions.

[0132] The pathologies of these disorders may share common features but also have distinct characteristics depending on the specific disease. The hallmark of all synucleinopathies is the presence of alpha-synuclein in abnormal forms, either as Lewy bodies or glial cytoplasmic inclusions, depending on the disease. As the disease develops there is a progressive loss of neurons in affected brain regions, eventually leading to the clinical symptoms. In addition, there is activation of microglia and astrocytes, indicating an inflammatory response to the pathological changes. Diagnostic practices for synucleinopathies and other neurodegenerative diseases can include imaging techniques such as magnetic resonance imaging (MRI), dopamine transporter imaging (DaTscan), fluorodeoxyglucose positron emission tomography (FDG-PET), and MIBG myocardial scintigraphy. Laboratory tests can also be used, these include cerebrospinal fluid (CSF) analysis and genetic testing. Diagnosis often involves a combination of these techniques, however, the gold standard for diagnosing synucleinopathies involves finding Lewy bodies or glial cytoplasmic inclusions in brain tissue after death.FJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A A. Combination Therapy

[0133] Neurodegenerative disorders, such as Alzheimer’s and Parkinson’s diseases, present complex challenges due to their multifactorial pathologies, including protein misfolding, aggregation, and neuroinflammation. Combining antibody therapies targeting distinct disease- related proteins offers a promising strategy to enhance therapeutic efficacy. By integrating antibodies such as TTCM (targeting toxic tau protein), TOMA (targeting oligomeric tau), and α- Syn (targeting alpha-synuclein), a synergistic approach can address multiple pathological hallmarks simultaneously. This combinatorial strategy aims to mitigate tau and alpha-synuclein aggregation, reduce neurotoxic oligomers, and promote clearance of misfolded proteins, potentially leading to improved clinical outcomes for patients with neurodegenerative disorders.

[0134] A promising combination therapy for neurodegenerative disorders targets the dual pathologies of alpha-synuclein and tau protein aggregates. This dual approach aims to address the synergistic interplay between alpha-synuclein and tau pathologies, potentially slowing disease progression more effectively than monotherapies, while enhancing neuroprotection and preserving cognitive function in affected patients. B. Antibody Delivery

[0135] Antibody delivery methods are designed to optimize the stability, bioavailability, and efficacy of antibodies while minimizing side effects and ensuring precise targeting within the body. Methods of delivering antibodies include but are not limited to intravenous infusion, subcutaneous injection, and intranasal administration.

[0136] Lipid-Mediated Delivery. In a further embodiment, a nucleic acid or polypeptide may be entrapped in a lipid particle such as, for example, a liposome or micelle. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, In: Liver Diseases, Targeted Diagnosis and Therapy Using Specific Receptors and Ligands, Wu et al. (Eds.), Marcel Dekker, NY, 87-104, 1991).

[0137] In certain embodiments of the invention, a lipid particle may be complexed with one or more antibody or antibody fragment.FJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A

[0138] Conjugated micelles (ms). Antibody-conjugated micelles can be prepared as described previously (Fischer et al., Biomacromolecules 10, 2408-2417, 2009; Liu et al., Theranostics 2, 705-713, 2012; Sahu et al., J Biomater Appl 25, 619-639, 2011; Song et al., Biomaterials 31, 2302-2312, 2010). In certain aspects, micelles are synthesized using poly(ethylene oxide)–poly(propylene oxide)–poly (ethylene oxide) (PEO–PPO–PEO) copolymers and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) chemistry to modify the hydroxyl end of Pluronic F-127 to a carboxyl group and facilitate conjugation of MABs. This conjugation is achieved via maleic anhydride in the presence of pyridine and toluene under an inert atmosphere of nitrogen gas (N2).

[0139] The term “poloxamer” or “PEO-PPO-PEO” is used herein to mean nonionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) (PPO) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)) (PEO). These molecules are also sold under the trade name PLURONIC(S)®. Poloxamers are described in U.S. Pat. No.3,740,421.

[0140] The term “PF-127” is used herein to refer to PLURONIC® F-127 (PEO100-PPO65- PEO100, Mw=12,600).

[0141] The term “micelle” is used herein to refer to a structure formed by an amphipathic molecule. In an aqueous solution, the hydrophilic portions of the molecules are on the outside of the structure, in contact with water molecules, while the hydrophobic portions of the molecules are within the structure, sequestered away from water. A “filamentous micelle” as used herein refers to a micelle that has a length that is measurably different than its width, e.g., the micelle is ovate or rod-shaped.

[0142] The term “mixed micelle” is used herein to refer to a micelle comprising more than one amphipathic molecule. In one embodiment, a mixed micelle comprises PEO-PHB-PEO and PEO-PPO-PEO molecules, i.e., a PEO-PHB-PEO:PEO-PPO-PEO mixed micelle.

[0143] Micelle components are members of a family of molecules that include an A block and a B block with the general formula ABA. The A polymer block comprises a poly(alkylene oxide). In one preferred embodiment of the invention, the A polymer block is poly(ethylene oxide) (PEO) and the B polymer block is poly(propylene oxide) (PPO), and the copolymer is the triblock ABA copolymer PEO-PPO-PEO.FJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A

[0144] A hydrophilic poly(alkylene oxide) end segment structure (the A block polymer) is used in the triblock copolymers of the present invention. The poly(alkylene oxide) is suitably selected from poly(ethylene oxide), poly(tetramethylene oxide) and poly(tetrahydrofuran). A preferred poly(alkylene oxide) for use in the present invention is poly(ethylene oxide) (PEO) or a derivative thereof, and most preferably is PEO. PEO is also referred to as poly(ethylene glycol), and as used herein the term poly(ethylene oxide) (and the abbreviation PEO) is intended to also refer to poly(ethylene glycol) (PEG).

[0145] The poly(alkylene oxide) may have different forms and different end groups. For example, in the case of PEO, the PEO derivatives may have different structures, e.g. star-shaped PEO, comb-like PEO, etc. The poly(ethylene oxide) may be in the form of modified molecules, e.g. PEGylated polysaccharides, PEGylated poly(amino acid)s, PEGylated proteins, etc. In addition, a polyamine derivative of PEO, e.g. PEGylated poly(ethylene imine) or PEGylated polylysine, may be used.

[0146] The relative molecular mass (Mr) range of PEO or other poly(alkylene oxide) utilized as the A block polymer in the copolymers of the present invention is suitably 500 to 20,000, and preferably is 2,000 to 10,000.

[0147] Suitable hydrophobic B polymer blocks for use in the present invention are poly(propylene oxide) (PPO) or poly(hydroxyalkanoate)s. Examples of suitable B polymers for use in the present invention include: poly(propylene oxide) (PPO); poly[(R)-3-hydroxybutyrate] (PHB), also referred to as poly[(R)-3-hydroxybutryic acid] or poly(.beta.-hydroxy acid); poly[(R)-4-hydroxybutyrate] (PGHB); poly[(R)-3-hydroxyvalerate] (PHV); poly[(R)-3- hydroxybutyrate]-co-poly[(R)-3-hydroxyvalerate] (PHB / HV); poly[(R)-3-hydroxyhexanoate] (PHHx); poly[(R)-3-hydroxyheptanoate] (PHHp); (S) enantiomers of each of the above (R) enantiomers; racemic mixtures of the above (S) and (R) enantiomers; and mixtures of the above poly(hydroxyalkanoate)s. Preferred poly(hydroxyalkanoate)s are poly(β-hydroxy alkanoate)s, and more specifically are poly[(R)-3-hydroxybutyrate] (PHB) and related poly[(R)-3- hydroxyalkanoate]s. A most preferred poly(hydroxyalkanoate) for use in the present invention is PHB.

[0148] Poloxamers, trade name PLURONICS™, are a series of water-soluble block copolymers, composed of two polyoxyethylene blocks separated by a polyoxypropylene block. Poloxamers have the general structure of PEO-PPO-PEO. The ability of poloxamers to formFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A micelles and gels makes them an important class of surfactant, that find widespread use in industrial applications such as detergency, dispersion stabilization, foaming, emulsification, lubrication and formation of cosmetics and inks. The amphiphilic property of poloxamers is the reason for their ability to create micelles above the CMC (critical micellization concentration) and the CMT (critical micellization temperature). The micellization of block copolymers, as in the case of conventional surfactants, obeys the closed association model, which assumes equilibrium between molecularly disposed copolymer (unimer) and multimolecular aggregates (micelles). In the case of poloxamers, when micellization occurs the degree of structuring of the water molecules decrease. The hydrogen bonding structure in the water is restored and the water entropy increases, overcoming the entropy loss due to the localization of the hydrophobic chains in the micelles. The structure of the poloxamer micelles in water has been investigated in many studies. In general, the unimer size is found to be approximately 1 nm and the micelle size 10 nm, independent of copolymer concentration.

[0149] The water solution block copolymers of poly(ethylene ocide)-poly(propylene oxide)- poly(ethylene oxide) which are commercially available (ICI company) and (Basf company). Some examples of poloxamers or PLURONICS for use in the invention are: F-127, F-108, F-98, F-88, F-68, F-87, F-77, P-105, P-85, P-75, P-65, P-104, P-94, P-84, L-64, L-63, L-121, and L- 122.

[0150] Micelles self-assemble by combining a desired formulation of components at concentrations above the critical micellar concentration. For most purposes micelles are loaded or conjugated with an agent of interest, e.g., an antibody, an imaging agent or a therapeutic agent. Micelles can have a size between about 10 and about 120 nm

[0151] The micelles of the present invention may be administered to a subject as a pharmaceutical composition or formulation. In particular embodiments pharmaceutical compositions of the present invention may be in any form which allows for the composition to be administered to a subject. For example, the composition may be in the form of a solid, liquid or gas (aerosol). Typical routes of administration include, without limitation, oral, topical, parenteral, sublingual, rectal, vaginal, and intranasal.

[0152] Routes and frequency of administration of the micelles described herein, as well as dosage, will vary from individual to individual, and may be readily established using standard techniques. Preferably, between 1 and 100 doses may be administered over a 52-week period. AFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A suitable dose is an amount of micelle that, when administered as described above, has a beneficial therapeutic effect.

[0153] Kits for administering the micelles may be prepared containing a composition or formulation of the micelle comprising a therapeutic agent or an imaging agent, together with the customary items for administering the therapeutic ingredient. V. Pharmaceutical Compositions

[0154] In light of the current specification, the determination of an appropriate treatment regimen (e.g., dosage, frequency of administration, systemic vs. local, etc.) is within the skill of the art. For administration, the components described herein will be formulated in a unit dosage form (solution, suspension, emulsion, etc.) in association with a pharmaceutically acceptable carrier. Such vehicles are usually nontoxic and non-therapeutic. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and Hank's solution. Non-aqueous vehicles such as fixed oils and ethyl oleate may also be used. A preferred vehicle is 5% (w / w) human albumin in saline. The vehicle may contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives.

[0155] The therapeutic compositions described herein, as well as their biological equivalents, can be administered independently or in combination by any suitable route. Examples of parenteral administration include intranasal, intravenous, intraarterial, intramuscular, intraperitoneal, and the like. The routes of administration described herein are merely an example and in no way limiting.

[0156] The dose of the therapeutic compositions administered to an animal, particularly in a human, in accordance with embodiments of the invention, should be sufficient to result in a desired response in the subject over a reasonable time frame. It is known that the dosage of therapeutic compositions depends upon a variety of factors, including the strength of the therapeutic composition employed, the age, species, condition or disease state, and the body weight of the animal.

[0157] Antibody administration can include administration of varied dosages and regimens tailored to therapeutic goals, antibody type, and patient factors. Intranasal administration, for example, delivers antibodies directly to mucosal surfaces. Dosages typically range from micrograms to milligrams per dose, with regimens varying from single-dose applications toFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A repeated daily or weekly administrations over days or weeks, depending on the condition’s severity and antibody half-life. For example, intranasal monoclonal antibodies can be administered at doses of 0.5–5 mg per nostril, administered once or twice daily for 3–7 days, ensuring high local concentrations in the nasal mucosa. Factors like formulation (liquid or aerosol), delivery device (spray or nebulizer), and patient-specific variables (e.g., mucosal inflammation) influence dosing precision. Safety monitoring is critical, as high doses may cause local irritation or systemic absorption, necessitating individualized regimens based on clinical trials and pharmacokinetic data.

[0158] Moreover, dose and dosage regimens will depend mainly on the type of biological damage to the host, the type of subject, the history of the subject, and the type of therapeutic composition being administered. The size of the dose will be determined by the route, timing and frequency of administration as well as the existence, nature and extent of any adverse side effects that might accompany the administration of a particular therapeutic composition and the desired physiological effect. It is also known that various conditions or disease states, in particular, chronic conditions or disease states, may require prolonged treatment involving multiple administrations.

[0159] Therefore, the amount of the therapeutic composition must be effective to achieve an enhanced therapeutic index. If multiple doses are employed, the frequency of administration will depend, for example, on the type of subject. One skilled in the art can ascertain upon routine experimentation the appropriate route and frequency of administration in a given subject that are most effective in any specific case. Suitable doses and dosage regimens can be determined by conventionally known range-finding techniques. Generally, treatment is initiated with smaller dosages, which are less than the optimal dose of the compound. Thereafter, the dosage is increased by small increments until the optimal effect under the circumstances is obtained.

[0160] The therapeutic compositions for use in embodiments of the invention generally include carriers. These carriers may be any of those conventionally used and are limited only by the route of administration and chemical and physical considerations, such as solubility and reactivity with the therapeutic agent. In addition, the therapeutic composition may be formulated as polymeric compositions, inclusion complexes, such as cyclodextrin inclusion complexes, liposomes, micelles, microspheres, microcapsules, and the like, without limitation.FJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A

[0161] The pharmaceutically acceptable excipients described herein, for example, vehicles, adjuvants, carriers, or diluents, are well known and readily available. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert with respect to the therapeutic composition and one that has no detrimental side effects or toxicity under the conditions of use.

[0162] The choice of excipient will be determined, in part, by the therapeutic composition, as well as by the method used to administer the composition. Accordingly, there are a wide variety of suitable formulations of the pharmaceutical composition used in the embodiments of the invention. For example, the non-limiting formulations can be, but not limited to, liquid solutions, including suspensions and emulsions, capsules, sachets, tablets, lozenges, and the like. The solutions can include oils, fatty acids, including detergents and the like, as well as other well- known and common ingredients in such compositions, without limitation. VI. Kits

[0163] Certain aspects provide a therapeutic kit designed for the targeted treatment of neurodegenerative diseases comprising a combination of therapeutic antibodies and / or micellar agents. The kit includes a first component comprising one or more monoclonal antibodies specifically engineered to bind to disease-associated antigens, thereby facilitating precise immune-mediated targeting of pathological cells. The second component comprises a micellar agent, formulated as a biocompatible nanoparticle carrier, which encapsulates therapeutic payloads such as chemotherapeutic drugs, immunomodulators, or nucleic acids, enhancing their solubility, stability, and targeted delivery to diseased tissues. The micellar agents are designed to synergistically enhance the therapeutic efficacy of the antibodies by improving cellular uptake and localized drug release at the target site, while minimizing systemic toxicity. The kit further includes instructions for sequential or simultaneous administration of the antibodies and micellar agents, tailored to specific disease profiles, offering a versatile and highly effective platform for personalized medicine. This innovative combination addresses the limitations of conventional therapies by integrating the specificity of antibody-based targeting with the enhanced delivery capabilities of micellar nanotechnology, thereby improving therapeutic outcomes in conditions such as cancer, autoimmune disorders, and infectious diseases.FJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A

[0164] In another aspect, the present invention provides kits for treating tauopathies. In one embodiment, the kit comprises a delivery device loaded with a therapeutic. In a further embodiment, such a kit can comprise instructions for suitable operational parameters in the form of a label or separate insert. In yet another embodiment, the kit can comprise one or more containers loaded with a therapeutic. VII. Examples

[0165] The following examples as well as the figures are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples or figures represent techniques discovered by the inventors to function well in the practice of the invention and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. EXAMPLE 1 NASAL IMMUNOTHERAPY EFFECTIVELY CLEARS INTRACELLULAR TAU PATHOLOGY VIA TRIM21 AND IMPROVES COGNITIVE FUNCTIONS IN AGED TAUOPATHY MICE A. RESULTS

[0166] TTCM2 recognizes disease-relevant tau aggregates in brain tissue from patients with tauopathies and inhibits tau-seeding activity of AD-BDTOs in vitro.

[0167] To determine whether TTCM2 specifically recognizes disease-relevant pathological tau, we performed double-label immunostaining using TTCM2 and AT8 antibodies on brain tissue from patients with AD (n = 8), DLB (n = 6), and PSP (n = 6) and age-matched non- demented controls (NDCs; n = 8) (Puangmalai et al., Cell Death Dis 11, 314, 2020; Gaikwad et al., Cell Rep 36, 109419, 2021; Montalbano et al., Brain Pathol, e13112, 2022). The AT8 antibody recognizes tau phosphorylation at Ser202 / Thr205 and is widely regarded as a reliable marker to detect pathological NFTs. AT8 immunoreactivity is primarily found in intraneuronal NFTs (iNFTs), extraneuronal NFTs (eNFTs), and pretangle phospho-tau aggregates (pre-NFTs) (Augustinack et al., Acta Neuropathol, 103, 26-35, 2002). Our results revealed strong TTCM2 (red) immunoreactivity in brain sections from patients with AD, DLB and PSP and relativelyFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A sparse staining in the NDCs (FIG.1A and 1B). Correlation analysis of immunostaining for AT8 and TTCM2 demonstrated significant colocalization between TTCM2 and AT8 in all human tauopathy brain tissues examined (FIG. 1C). TTCM2 primarily stained iNFTs and pre-NFTs (perinuclear staining) but not eNFTs (FIG.1A, 1B and 1C). Additionally, immunohistochemistry staining revealed that TTCM2 exhibits a very strong immunoreactivity to pathological tau present in neuropil threads, iNFTs and pre-NFTs in AD, DLB and PSP cases (FIG. 8A). However, TTCM2 exhibited little or no immunoreactivity in brain sections from NDC cases (FIG.8A).

[0168] To further validate these data, we performed enzyme-linked immunosorbent assays (ELISAs) using brain homogenates from human and murine tauopathy brain tissues, as well as different forms of tau, including tau monomers (TauM), tau oligomers (TauO) and tau fibrils (TauF) (Lasagna-Reeves et al., Mol Neurodegener, 6, 39, 2011; Lasagna-Reeves et al., Sci Rep, 2, 700, 2012). The results confirmed strong TTCM2 immunoreactivity in brain homogenates from AD, DLB, and PSP compared to NDC (FIG. 1D). Moreover, we observed enhanced TTCM2 immunoreactivity in 18-month-old hTau mice relative to 3-month-old hTau mice; with no reactivity detected in tau-knockout mice (FIG. 1D). Additionally, we found that TTCM2 exhibit the highest reactivity with TauO relative to TauF and TauM (FIG.1D).

[0169] To assess the binding specificity of TTCM2 with TauO, we performed dot blot assays with TTCM2 and Tau5 antibodies and various tau aggregates, including T22 affinity-purified brain-derived tau oligomers (BDTOs) from AD, DLB, and PSP patients; recombinant Aβ, αSynuclein, and TauO. Results indicate strong immunoreactivity for TTCM2 against BDTOs from AD, DLB, and PSP patients and recombinant TauO. However, TTCM2 did not detect Aβ or αSynuclein oligomers (FIG. 8B), suggesting that TTCM2 specifically detects TauO. We and others have demonstrated that TauO are highly toxic in vitro and in vivo (Lasagna-Reeves et al., Mol Neurodegener, 6, 39, 2011; Lasagna-Reeves et al., Biochemistry, 49, 10039-10041, 2010; Niewiadomska et al., Life, (Basel) 11, 2021; Shafiei et al., Frontiers in aging neuroscience, 9, 83, 2017). We found that TTCM2 effectively neutralizes TauO toxicity in SH-SY5Y neuroblastoma cells in a dose-dependent manner (FIG. 8C). Given that TTCM2 detected abnormal TauM, it is worth noting that while TauO and TauF are well-established pathological forms of tau in neurodegenerative diseases (Gaikwad et al., Cell Rep 36, 109419, 2021; Bittar et al., Brain communications, 1, fcz004, 2019; Goedert et al., Annu Rev Neurosci, 40, 189-210,FJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A 2017; Kayed et al., Hum Vaccin, 6, 931-935, 2010; Lamontagne-Kam et al., Front Neurosci, 17, 1219299, 2023; Lippens et al., J Biol Chem, 294, 9316-9325, 2019; Maeda et al., Adv Exp Med Biol, 1184, 373-380, 2019; Xu et al., Acta Neuropathol, 141, 193-215, 2021), recent studies indicated that TauM can also exist in abnormal or pathological forms that initiate seeding and spreading of tau pathology (Goedert et al., Alzheimers Dement, 12, 1040-1050, 2016; Mirbaha et al., Elife, 7, 2018; Mirbaha et al., J Biol Chem, 298, 102163, 2022; Sharma et al., Elife, 7, 2018). The ability to detect the monomeric form of tau is a characteristic feature of several tau conformational antibodies (Mirbaha et al., Elife, 7, 2018; Mirbaha et al., J Biol Chem, 298, 102163, 2022; Sharma et al., Elife, 7, 2018; Hallinan et al., J Neurosci, 39, 9623-9632, 2019; Hitt et al., J Biol Chem, 299, 105252, 2023; Michel et sl., J Biol Chem, 289, 956-967, 2014).

[0170] Next, to examine the tau conformational specificity of TTCM2, we used dot blot assays to measure its immunoreactivity in different conformations of tau protein, including non- denatured (ND) as well as denatured (D) states, by chemical and thermal treatment of TauM, TauO and TauF. We found that TTCM2 shows stronger reactivity with ND forms of tau, and when tau loses its natural structure (upon chemical and thermal denaturation), TTCM2's reactivity with TauM and TauO significantly decreases (FIG.8D and 8E). Interestingly, TTCM2 demonstrated the greatest differences in reactivity with TauO compared to TauM or TauF (FIG. 8D and 8E). Conversely, the tau 13 antibody, which detects total tau, showed no differences in immunoreactivities between ND and D tau forms (FIG. 8F and 8G). This data indicates that TTCM2 is a tau conformational antibody with strong immunoreactivity to misfolded and toxic TauO (FIG.1D and FIG.8A-8G).

[0171] An important aspect of tauopathy is the presence of seed-competent toxic tau conformations in the brain at early stages of disease (Furman et al., Acta Neuropathol 133, 91- 100, 2017; DeVos et al., Front Neurosci, 12, 267, 2018). These pathological tau conformations act as seeds that recruit and misfold endogenous naïve tau monomers and propagate tau pathology across the brain (Ferrer et al., Brain Pathol, 30, 298-318, 2020). Tau-seeding activity is clinically relevant, correlating with disease progression and cognitive decline in AD patients (Furman et al., Acta Neuropathol 133, 91-100, 2017; Dujardin et al., Nat Med, 26, 1256-1263, 2020). Several studies indicate that TauOs isolated from human tauopathies exhibit potent tau- seeding activities (Dujardin et al., Nat Med, 26, 1256-1263, 2020; Colom-Cadena et al., Neuron, 111, 2170-2183 e2176, 2023; Miyoshi et al., Lab Invest, 101, 1605-1617, 2021; Puangmalai etFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A al., J Biol Chem, 298, 101766, 2022; Usenovic et al., J Neurosci, 35, 14234-14250, 2015). We previously demonstrated that BDTOs from AD patients are highly neurotoxic, propagate tau pathology across brain-regions, and induce cognitive dysfunction in mouse models (Lasagna- Reeves et al., Mol Neurodegener, 6, 39, 2011; Gerson et al., Front Neurol, 4, 93, 2013). These observations suggest that blocking tau-seeding activity via immunotherapy may be beneficial for halting disease progression. Tau biosensor cells are widely used to quantify seed-competent tau in human and rodent biofluids, including cerebrospinal fluid and lysates from postmortem AD brains (Dujardin et al., Nat Med, 26, 1256-1263, 2020).

[0172] Here, we used a biosensor cell line that constitutively expresses human tau repeat domain (TauRD) with P301S mutation (Holmes et al., Proc Natl Acad Sci U S A 111, E4376- 4385, 2014). These cells form intracellular aggregates when exposed to bioactive tau seeds. We investigated whether TTCM2 treatment inhibits seeding activity of AD-BDTOs in tau biosensor cells. Lipofectamine 2000 transfection reagent was used to deliver the AD-BDTOs: TTCM2 immune complexes to the cells (FIG. 1E). Results show that at nanomolar concentrations, AD- BDTOs profoundly induced tau aggregation in biosensor cells, and this seeding activity was significantly inhibited by TTCM2-ms treatment (FIG. 1F and 1G). The optimal ratio of tau to TTCM2 for inhibiting seeding activity was 1:4, which reduced the seeding activity by 70% compared to AD-BDTO alone. Higher ratios of tau to TTCM2 (such as 1:8) did not show any significant improvement over 1:4 ratio suggesting plateau in the inhibition of seeding activity, and that a 1:4 ratio is the optimal stoichiometry of AD-BDTOs to TTCM2 for neutralizing tau seeding. Together, these findings demonstrate that TTCM2 is a tau conformation-specific antibody capable of recognizing disease-relevant tau aggregates in brain tissue from patients with AD, DLB, and PSP. Moreover, TTCM2 effectively neutralized the toxicity of TauO and blocked tau-seeding activity of AD-BDTOs.

[0173] Intranasally administered TTCM2-ms rapidly distributed to various brain regions, effectively entered neuronal intracellular compartments, and engaged with pathological tau in aged hTau tauopathy mice.

[0174] The BBB prevents efficient transport of therapeutic tau MABs from circulation to the brain. Intranasal drug delivery can bypass the BBB and thus is amenable for direct delivery of brain-targeted therapeutic drugs (Wang et al., J Pharmacol Exp The,r 370, 593-601, 2019; Jiang et al., Photochem Photobiol, 65, 701-706, 1997). However, despite efficient delivery to the brain,FJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A intranasally delivered tau MABs are poorly internalized by neurons, resulting in intact intracellular tau and immunotherapy failure (Congdon et al., Semin Cell Dev Biol 126, 125-137, 2022). Here, to investigate their therapeutic potential, we assessed the brain and cellular distribution of TTCM2-ms after intranasal administration to hTau mice—a transgenic mouse model of human tauopathy, which expresses all six isoforms of human tau protein and exhibits cognitive dysfunction and tau pathology in an age-dependent manner (Polydoro et al., J Neurosci, 29, 10741-10749, 2009). Aged hTau and wild-type (WT) mice were intranasally treated with Alexa Fluor (AF) 568–labeled TTCM2-ms or IgG and euthanized 3 h after treatment. Following perfusion, brain tissues were extracted, and unfixed tissues were immediately analyzed by ex-vivo fluorescence imaging and immunofluorescence microscopy (FIG. 2A). Imaging results indicate that TTCM2-ms readily reached the brain, exhibiting a radiant efficiency that is significantly increased in hTau mice relative to WT control mice (FIG. 2B). In contrast, radiant efficiency of IgG retention was similar in hTau mice and WT mice (FIG. 2B), suggesting that TTCM2 antibody has higher affinity and specificity to recognize and target the pathological tau present in the brain of tauopathy mice. It is important to note that the intranasal delivery approach bypasses the BBB and delivers drugs to the brain within minutes (De Rosa et al., Proc Natl Acad Sci U S A, 102, 3811-3816, 2005; Hanson et al., BMC Neurosci, 9 Suppl 3, S5, 2008). Thus, regardless of the BBB integrity, intranasally administered TTCM2- ms and IgG-ms are efficiently delivered in the brain of hTau mice. Given the higher pathological tau loads in the aged hTau tauopathy mice relative to wild-type controls (Polydoro et al., J Neurosci, 29, 10741-10749, 2009; Gaikwad et al., Alzheimers Dement, 2023; Nilson et al., J Alzheimers Dis, 55, 1083-1099, 2017), we observed an increased retention of TTCM2 in tauopathy mice (FIG. 2B). We then evaluated TTCM2-ms distribution in hTau mouse brain sections using the neuronal postsynaptic marker, postsynaptic density protein 95 (PSD95) (FIG. 2C). We found that 3 h post-intranasal administration, TTCM2-ms (red) followed the nose-to- brain drug-delivery route and was distributed in various brain regions, including the olfactory bulb (Ob), hippocampus (Hp), cortex (Cx), cerebellum (Cb), and thalamus (Thl) (FIG. 2C). Additionally, we observed that TTCM2 can reach into neuronal intracellular compartments as shown by colocalization between TTCM2 (red) with PSD95 (green) (FIG. 2C). Subsequently, we explored the target engagement of intranasally delivered TTCM2-ms in the brain of hTau mice by staining brain tissues with AT8 antibody (green) and determined its colocalization withFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A TTCM2-ms (red). Immunoflourescence microscopy and Pearson’s correlation coefficient (PCC) analysis revealed a significant colocalization between TTCM2-ms and AT8-positive pathological tau aggregates (FIG.2D). Scatter plot demonstrated a significant colocalization between TTCM2 and AT8, as indicated by the PCC of 0.83. Therefore, our findings indicate that intranasally administered TTCM2-ms rapidly distributed across the brains of hTau mice, entered the intracellular compartments of neuronal cells and engaged with pathological tau in vivo.

[0175] A single intranasal TTCM2-ms treatment ameliorates tau pathology and cognitive decline in aged tauopathy mice. We next determined whether intranasal administration of TTCM2-ms ameliorates tau pathology and cognitive decline in aged hTau mice. Beginning at 15 months of age, the time when these animals show advanced-stage tau pathology and cognitive impairment (Gaikwad et al., Cell Rep 36, 109419, 2021; Polydoro et al., J Neurosci, 29, 10741- 10749, 2009), both male and female hTau mice were treated with a single dose of intranasal TTCM2-ms or IgG-ms. Hippocampus-dependent spatial memory and exploration potential of aged hTau mice were measured by Y-maze test immediately before and at 2-weeks post- intranasal administration. Mice were also subjected to the novel object recognition (NOR) test at 2-weeks post-intranasal TTCM2-ms or IgG-ms treatment. After behavior analyses, mice were euthanized, and brains were processed for biochemical and pathological analyses (FIG. 3A). Compared to IgG-ms-treated hTau mice, TTCM2-ms–treated hTau mice exhibited a significantly enhanced discrimination index, indicative of a greater tendency to explore a novel object than a familiar one (FIG. 3B to 3F). Moreover, TTCM2-ms–treated hTau mice performed significantly better in the Y-maze test, suggesting TTCM2-ms treatment significantly alleviates short-term memory loss in mice with established tauopathy (FIG. 3G and 3H). Consistent with these findings, compared to IgG-ms-treated hTau mice, TTCM2-ms–treated hTau mice showed increased levels of neuronal markers, including PSD95, synaptophysin, and neuronal nuclear protein (NeuN) in the hippocampus—the region associated with memory formation and cognitive functions (FIG.3I to 3L and FIG.9A and 9B).

[0176] A distinctive characteristic of hTau mice is the development of disease-relevant tau pathology, including the appearance of NFTs and phosphorylated tau aggregates by 12 months of age (Gaikwad et al., Cell Rep 36, 109419, 2021; Polydoro et al., J Neurosci, 29, 10741-10749, 2009). To determine whether these pathologic features are affected by TTCM2-ms treatment, we assessed NFT levels using thioflavin-S staining and measured phosphorylated tau byFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A immunofluorescence staining with pathological tau-specific antibodies, including AT8 (anti- pTau S202, T205), AT100 (anti-pTau T212, S214), and AT180 (anti-pTau T231) (Gaikwad et al., Cell Rep 36, 109419, 2021; Smith et al., Acta Neuropathol Commun, 11, 172, 2023), in brain sections from IgG-ms- and TTCM2-ms–treated hTau mice. Results show that NFT deposition was significantly reduced following intranasal TTCM2-ms treatment (FIG. 4A and 4B). Consistent with this finding, intranasal TTCM2-ms treatment reduced AT8, AT100, and AT180 positive pathological tau species, all of which are highly relevant for human tauopathies (FIG. 4C to 4L, and FIG. 9C and 9D). Immunoblot analyses revealed reduced levels of high- molecular-weight tau aggregates (detected by Tau13), oligomeric tau (detected by T22), phosphorylated tau (detected by AT8, AT100, and AT180), and misfolded tau conformations (detected by TTC18) in brains of TTCM2-ms relative to IgG-ms-treated mice (FIG. 10). Conversely, levels of monomeric tau (~50 kDa; detected by Tau 13) were unaltered by TTCM2- ms treatment (FIG.10). Together, these results suggest intranasal TTCM2-ms treatment removes pathological tau aggregates from the brain and ameliorates cognitive impairments in hTau mice.

[0177] TTCM2-ms clears intracellular tau aggregates via cytosolic Fc receptor TRIM21. TRIM21 is a cytosolic Fc receptor and E3 ubiquitin ligase that recognizes intracellular antibody- bound proteins (e.g. antigens and pathogens) and triggers their clearance by the ubiquitin– proteasome degradation pathway (Fletcher et al., Proc Natl Acad Sci U S A, 112, 10014-10019, 2015). During this process, TRIM21 and the antibody are also degraded (Fletcher et al., Proc Natl Acad Sci U S A, 112, 10014-10019, 2015). TRIM21 is widely expressed and active in different cell types, including neurons (Zhang et al., J Neurosci, 34, 11929-11947, 2014; Uhlen et al., Science, 347, 1260419, 2015). TRIM21 exhibits broad antibody isotype specificity, with the ability to bind IgG, IgM, and IgA (Foss et al., Immunol Rev, 268, 328-339, 2015), thereby TRIM21 promotes degradation of antibody-bound pathological tau aggregates (McEwan et al., Proc Natl Acad Sci U S A, 114, 574-579, 2017). Therefore, we assessed whether TRIM21 is involved in the clearance of intracellular pathological tau from neurons by TTCM2-ms. We established primary neuronal cultures from hTau mice and exposed them to tau seeds (AD- BDTOs) for 24 h to induce intracellular tau pathology. Cultures were then treated with either TTCM2-ms or IgG-ms at different time points. We observed recruitment of bright TRIM21 foci to the TTCM2-ms–tau complex after 30 min of seeding, and both TRIM21 and TTCM2-ms–tau complex were cleared after 1 h, as evidenced by the decreased fluorescence intensity of total tauFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A (Tau5; green) (FIG. 5A and 5B) and tau oligomers (T22; red) (FIG. 5C), as well as the reduced size of tau aggregates (FIG.5D) and number of TRIM21 puncta (TRIM21; magenta) (FIG.5E). These data suggest that TTCM2-ms–tau aggregate complex rapidly promotes recruitment of TRIM21 and clears antibody-bound tau aggregates. The number of TRIM21 puncta per cell in IgG- and TTCM2-treated cells were similar at 0 min (mean IgG=154 vs TTCM2=147), but significantly increased by TTCM2 at 30 min (mean IgG=193 vs TTCM2=267) and decreased significantly at 60 min (mean IgG=172 vs TTCM2=38), indicating clearance of TRIM21 and antibody-bound tau aggregates upon TTCM2 treatment. To further validate and extend these findings, we investigated the ability of TTCM2-ms to induce intracellular tau-positive TRIM21 puncta and their subsequent clearance in vivo after intranasal administration. To this end, aged hTau mice received intranasal treatment with AF568–labeled TTCM2-ms and were euthanized at 3 h and 24 h post-treatment. Brain tissues were then examined using immunofluorescence microscopy. The analyses revealed that at 3 h post-treatment, TTCM2-ms readily colocalized with intracellular TRIM21 and tau aggregates (FIG. 11A). Additionally, the presence of TTCM2-ms led to a significant increase in the number of puncta for intracellular TRIM21- positive tau aggregates, indicating that upon intranasal delivery, TTCM2-ms can reach the intracellular compartment and form a complex with intracellular tau aggregates and TRIM21 (FIG. 11B). Furthermore, we observed that TTCM2-ms reduced pathological tau aggregates detected by AT8 at 24 h but not at 3 h post-treatment (FIG.11C and 11D). Tau positive TRIM21 puncta were significantly increased at 3h and decreased significantly at 24 h after TTCM2-ms treatment (FIG. 11B and 11E). No significant changes were observed in IgG-ms treated mice (FIG. 11C to 11F). Notably, TTCM2-ms also reduced sarcosyl-insoluble HT7+human tau protein and AT8+pathologically relevant-phosphorylated tau aggregates (FIG. 12A to 12C). These observations indicate that upon intranasal delivery, TTCM2-ms readily colocalized with intracellular antibody receptor TRIM21 and tau aggregates, resulting in the clearance of pathological tau aggregates.

[0178] To examine the role of TRIM21 in TTCM2-ms–mediated clearance of pathological tau in tau biosensor cells, we conducted TRIM21 silencing using small-interfering RNA (siRNA). To this end, cells were treated with preincubated AD-BDTO and TTCM2-ms, and tau aggregate levels were examined by immunoblots and immunofluorescence microscopy. Immunoblot analyses showed that TRIM21 siRNA significantly reduced TRIM21 protein levelsFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A compared to control siRNA (FIG. 5F and 5G). In control siRNA-treated cells, TTCM2-ms effectively cleared tau aggregates, whereas tau clearance was notably impaired in TRIM21- siRNA-treated cells (FIG. 5F to 5I). These data indicate that TRIM21 is essential for TTCM2- ms–mediated clearance of intracellular tau aggregates. TauRD, expressed by tau biosensor cells, contains aggregation-competent residues that form pathological tau aggregates (Sanders et al., Neuron 82, 1271-1288, 2014; Ballatore et al., Nat Rev Neurosci, 8, 663-672, 2007; Lee et al., Annu Rev Neurosci, 24, 1121-1159, 2001; Mocanu et al., J Neurosci, 28, 737-748, 2008; Stohr et al., Nat Chem, 9, 874-881, 2017; Wischik et al., Proc Natl Acad Sci U S A, 85, 4506-4510, 1988; Roberts et al., Acta Neuropathol Commun, 8, 13, 2020). To understand the nature of TTCM2’s immunoreactivity with TauRD, specifically whether it is conformation-dependent, like full- length tau, we conducted immunoblot assays using cell lysates from tau biosensor cells and control untransfected HEK cells. These cell lysates were subjected to non-denatured or denatured conditions by urea and heat treatment, followed by immunoblotting with TTCM2. We observed that TTCM2 exhibit strong immunoreactivity with TauRD aggregates in non-denatured conformations but shows weak immunoreactivity after denaturation (FIG. 12E). No immunoreactivity was observed in control HEK cells (FIG. 12E), indicating TTCM2’s immunoreactivity with TauRD is both conformation-dependent and specific. Collectively, our data suggests that upon intranasal administration, TTCM2-ms form complexes with the cytosolic Fc receptor TRIM21 and tau aggregates and promote clearance of tau pathology. Importantly, our findings align with previous studies that have established the crucial role of TRIM21 in effective tau immunotherapy (McEwan et al., Proc Natl Acad Sci U S A, 114, 574-579, 2017; Mukadam et al., Science, 379, 1336-1341, 2023).

[0179] TTCM2-ms efficiently internalize into intracellular compartments and clear seed- competent, intracellular tau from the brain of aged hTau mice. Given that pathological tau aggregates are primarily intraneuronal in AD and other tauopathies, therapeutic tau antibodies should be able to efficiently reach the intracellular compartments (Congdon et al., EBioMedicine, 42, 157-173, 2019). We therefore measured the internalization efficiency of TTCM2-ms by treating hTau-expressing cells (green) with AF568–labeled TTCM2 (red) with or without ms at 4°C or 37°C and analyzed by fluorescence microscopy. We observed significantly increased binding and internalization of TTCM2-ms relative to TTCM2 in phosphate-buffered saline (PBS), which display only marginal binding / internalization (FIG.6A to 6D).FJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A

[0180] We next determined whether TTCM2-ms treatment clears established intracellular tau aggregates from tau biosensor cells (Holmes et al., Proc Natl Acad Sci U S A 111, E4376-4385, 2014). To this end, cells were first treated with AD-BDTO seeds for 24 h to establish intracellular tau aggregates and then incubated with or without TTCM2-ms for 3 h. Fluorescence microscopy was used to assess tau clearance (FIG. 6E). Results indicate that intracellular tau aggregates were formed in AD-BDTO-treated cells. However, TTCM2-ms treatment reduced the number of tau aggregate-positive cells and decreased the size of the intracellular tau aggregates (FIG. 6F to 6H), suggesting clearance of established intracellular tau aggregates. We further hypothesized that TTCM2-ms–mediated removal of seed-competent tau conformers may be associated with reduced tau pathology in the brain of aged hTau mice. To test this possibility, we measured seeding activities of brain homogenates from IgG-ms- and TTCM2-ms–treated hTau mice (FIG. 6I). We found that brain homogenates from IgG-ms-treated control mice exhibited enhanced seeding activity, as evidenced by the formation of tau aggregates in tau biosensor cells. Conversely, brain homogenates from TTCM2-ms–treated mice displayed a significant reduction in tau-seeding activity (FIG. 6J and 6K), suggesting that TTCM2-ms treatment effectively removes seed-competent tau aggregates from the brains of hTau mice with tauopathy.

[0181] TTCM2-ms treatment clears intracellular and synaptic tau aggregates from the brain of tauopathy mice. Pathological tau aggregates deposited in neuronal synapses and intracellular compartments contribute to AD pathogenesis. Tau aggregates isolated from synaptic compartments of mouse and human AD brains exhibit tau-seeding activity, which promotes tauopathy progression and cognitive deficits (DeVos et al., Front Neurosci, 12, 267, 2018; Schaler et al., Sci Transl Med, 13, 2021; Bejanin et al., Brain, 140, 3286-3300, 2017). An absence of tau oligomers in synapses is linked to better cognitive function in individuals with high AD neuropathology (Perez-Nievas et al., Brain, 136, 2510-2526, 2013; Singh et al., J Alzheimers Dis, 78, 1661-1678, 2020), suggesting that pathological tau in synapses may preferentially cause synaptic loss and cognitive decline. Therefore, we examined whether TTCM2-ms can remove synaptic tau aggregates from the brain of aged tauopathy mice. To this end, brain sections from IgG-ms- and TTCM2-ms–treated hTau mice were stained with anti-tau antibody (tau HT7) and a synaptic marker (anti-PSD95 antibody), followed by microscopic analysis. As evidenced by HT7 Tau and PSD95 colocalization, TTCM2-ms-treated mice exhibited significantly decreased levels of total tau aggregates (Tau HT7, red) and withinFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A PSD95-positive synaptic compartments (PSD95, green) in the brains of aged hTau mice compared to IgG-ms treated mice (FIG. 7A to 7D). We also found that the number of PSD95 puncta were significantly increased in the brain of TTCM2-ms-treated mice compared to IgG- ms-treated mice (FIG. 7A and 7E). Additionally, we found reduced levels of T22-antibody- positive TauO (green) in Synaptophysin 1-positive synaptic compartments (Synaptophysin 1; red) in TTCM2-ms–treated hTau mice (FIG. 13A to 13D). Furthermore, the number of Synaptophysin 1 puncta were significantly increased in the brains of TTCM2-ms-treated mice compared to IgG-ms-treated mice (FIG. 13A to 13D). These findings suggest that TTCM2-ms effectively clears pathological tau aggregates from intracellular and synaptic compartments of neurons in the brains of hTau mice.

[0182] Cryo-electron microscopy studies have shown that tau forms distinct pathological tau structures known as tau strains / polymorphs (Arakhamia et al., Cell, 180, 633-644 e612, 2020; Shi et al., Nature, 598, 359-363, 2021). This diversity of tau strains / polymorphs drives specific neuropathological features in AD, PD, PSP, and CBD (Arakhamia et al., Cell, 180, 633-644 e612, 2020; Shi et al., Nature, 598, 359-363, 2021). Tau aggregates exhibit protease K (PK)- resistant cores, and their distinct strains / polymorphs can be distinguished by their PK-resistant patterns (Sanders et al., Neuron 82, 1271-1288, 2014; Legname et al., Proc Natl Acad Sci U S A, 102, 2168-2173, 2005; Narasimhan et al., J Neurosci, 37, 11406-11423, 2017; Vaquer-Alicea et al., Acta Neuropathol, 142, 57-71, 2021). We used PK-digestion analysis to investigate structural differences of tau aggregates within brain homogenates of IgG-ms- and TTCM2-ms–treated mice. Homogenates were treated with PK at various concentrations (0–20 µg / mL) for 1 h, and digestion patterns were evaluated by immunoblot using different sequence-specific tau antibodies, including Tau5 (recognizes the middle region), RD4 (recognizes the four-repeat isoform), and Tau46 (recognizes the C-terminal sequence). We found that tau aggregates in brain homogenates from IgG-ms treated mice were resistant to PK-digestion, an indicator of compacted tau structures (Martinez et al., Nat Neurosci, 25, 1597-1607, 2022), while tau aggregates in brain homogenates from TTCM2-ms–treated mice were sensitive to PK-digestion, an indicator of relaxed tau structure (Martinez et al., Nat Neurosci, 25, 1597-1607, 2022) (FIG. 14A to 14C). Immunofluorescence staining analyses further validated the differential PK sensitivity of tau in IgG- vs. TTCM2-ms–treated mice (FIG.15A to 15B). The distinct pattern of PK-digestion observed in this study may indicate the existence of different tauFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A strains / polymorphs in the brains of aged hTau tauopathy mice. This finding is consistent with previous studies that have reported the presence of diverse tau strains / polymorphs in both mouse and human tauopathies (Sanders et al., Neuron 82, 1271-1288, 2014; Dujardin et al., Nat Med, 26, 1256-1263, 2020; Vaquer-Alicea et al., Acta Neuropathol, 142, 57-71, 2021; Bittar et al., J Alzheimers Dis, 90, 1103-1122, 2022; Bittar et al., NPJ Vaccines, 3, 9, 2018). Thus, understanding and targeting these tau strains / polymorphs could contribute to the development of effective immunotherapy strategies. B. MATERIALS AND METHODS

[0183] Study design. The goals of the study were to develop TTCM2-ms to rapidly clear toxic tau conformations from the brain of aged hTau mice and analyze whether intranasal treatment with TTCM2-ms effectively clears intracellular, synaptic, and seed-competent tau and its impact on cognitive functions. This study employed rigorous scientific methods, statistical analyses, and a focus on reproducibility to ensure the validity and reliability of the findings. No power analysis was performed to determine the sample size. Sample sizes were chosen based on previous experience with tau immunotherapeutic studies using the same hTau mouse line and human postmortem brain tissues. A randomized experimental design was used, where aged hTau mice were randomly assigned to different experimental groups. No samples or data points were excluded from the analyses. Blinded investigators conducted most assays, including animal treatments, behavioral testing, tau seeding activities, quantification of immunofluorescence images, and immunoblot analyses. Using primary neuronal cultures from hTau mice and Tau biosensor cells, we tested the effects of TTCM2-ms on intracellular tau pathology. We determined the mechanism underlying rapid clearance of intracellular tau aggregates by TTCM2- ms. All animal experiments were conducted following the guidelines established by the National Institutes of Health (NIH) and received approval from the Institutional Animal Care and Use Committee at the UTMB Galveston. Detailed methodologies for both in vitro and in vivo experiments can be found in the Supplementary Materials and Methods.

[0184] Production of TTCM2. TTCM2 was produced by GenScript Biotech, as described previously (Montalbano et al., Brain Pathol, e13112, 2022). Briefly, tau oligomers were prepared using human WT tau protein [(2N4R) tau-441)] monomers expressed and purified from Escherichia coli BL21(DE3) cultures (Agilent; #200131). Purified monomeric tau was dissolvedFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A in PBS at 0.5 mg / ml and stirred overnight at room temperature in a fume hood. Tau oligomers were then purified using fast protein liquid chromatography (Superdex 200 HR 10 / 30 column; GE Healthcare) and characterized as described (Sengupta et al., Methods Mol Biol 1779, 113- 146, 2018). Purified tau oligomers were mixed with Freund’s complete adjuvant and used to immunize 8–12-week-old BALB / c mice via intraperitoneal injection (100-μl antigen–adjuvant mixture at 1:1 ratio; 20 μg / mouse) followed by booster doses at different time points. TTCM2 hybridoma clones were isolated, and the antibody was purified from culture medium by affinity chromatography, followed by high-performance liquid chromatography, as described (Castillo- Carranza et al., J Neurosci 34, 4260-4272, 2014). Immunoreactivity and specificity of TTCM2 antibodies were assessed by western blot, dot blots assays, and ELISAs with oligomeric and monomeric tau. Aβ oligomers and α-synuclein oligomers were used to rule out cross-reactivity between TTCM2 and other amyloid oligomers.

[0185] Formulation of TTCM2-conjugated micelles (TTCM2-ms). Antibody-conjugated micelles were prepared as described previously (Fischer et al., Biomacromolecules 10, 2408- 2417, 2009; Liu et al., Theranostics 2, 705-713, 2012; Sahu et al., J Biomater Appl 25, 619-639, 2011; Song et al., Biomaterials 31, 2302-2312, 2010). Micelles were synthesized using poly(ethylene oxide)–poly(propylene oxide)–poly (ethylene oxide) (PEO–PPO–PEO) copolymers and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) chemistry to modify the hydroxyl end of Pluronic F-127 to a carboxyl group and facilitate conjugation of MABs. This conjugation was achieved via maleic anhydride in the presence of pyridine and toluene under an inert atmosphere of nitrogen gas (N2). Micelle synthesis scheme and experimental setup for synthesis are shown in FIG. 16. Fourier Transform Infrared Spectroscopy analysis was performed to confirm the successful transformation of hydroxyl groups to carboxylic acids via the appearance of the C=O stretch at ~1730 cm-1 (FIG.17). This carboxylated Pluronic F-127 is referred to as “functionalized F-127”. A 4% solution of antibody-loaded micelles was prepared in deionized water, and size distribution analysis by NanoSight revealed that they are 115 ± 5.6 nm (FIG. 18A-18C). Dot blot assays were then performed to validate reactivity of antibody- loaded micelles (TTCM2-ms), which were used in subsequent experiments.

[0186] Primary neuronal culture. Primary cortical neuronal cultures were prepared from hTau mice (Jackson Laboratory; stock #004808) at embryonic days 13–16, as described previously (Puangmalai et al., J Biol Chem 298, 101766, 2022). Briefly, cells were dissociatedFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A from cortical tissues by treatment with Accutase and gentle trituration with fire-polished glass pasture pipets. Cells were seeded onto poly-L-lysine–coated coverslips or six-well plates. Neuronal culture medium was prepared with Neurobasal Medium (Gibco; 12348017), B-27™ plus supplement (Gibco; A3582801), GlutaMax (Gibco; 35050–061), and 1× Antibiotic– Antimycotic solution (Gibco; 15240062). After every 3–4 days, half the medium was replaced with fresh culture medium, and at days 10–13 of in vitro culture, cells were used for the indicated experiments.

[0187] TRIM21 silencing. To determine the role of TRIM21 in TTCM2-ms–mediated clearance of tau aggregates, tau biosensor cells were transfected with 200 nM of control or TRIM21 siRNA (Accell SMARTpool siRNA, Dharmacon). After 48 h, cells were treated with AD-BDTOs (0.25 μM) and TTCM2-ms (1μM) at a 1:4 ratio. All cells were preincubated at 37°C for 1 h and treated for 3 h, after which the cells were washed with PBS and analyzed by either immunoblot or immunofluorescence analysis.

[0188] Tau-seeding activity. Tau-seeding activity was measured using a tau biosensor cell line (ATCC #CRL-3275), as described previously (Puangmalai et al., J Biol Chem 298, 101766, 2022). Briefly, tau biosensor cells were cultured in Dulbecco’s Modified Eagle Medium containing 10% fetal bovine serum and penicillin–streptomycin (100 μg / ml) in a humidified atmosphere equipped with 5% CO2 and maintained at 37°C. To assess tau-seeding activity, cells were grown on poly-L-lysine–coated coverslips in 24-well plates at a density of 105 cells / well. Cells were then exposed to Lipofectamine 2000 Transfection Reagent (Thermo; #11668030) containing BDTOs (100 nM) or brain homogenates (2.5-μg total protein in PBS) in the presence or absence of TTCM2-ms (500 nM) for 24 h. Following three washes with PBS, cells were fixed with formaldehyde solution and mounted using ProLong Gold Antifade Mountant. Cells were examined under a fluorescence microscope (Keyence BZ-X710), and each condition was assessed in triplicate.

[0189] Human brain tissue processing. Postmortem brain tissue from AD, DLB, PSP, and age matched NDC subjects was provided as frozen blocks by the Oregon Brain Bank and University of Kentucky Alzheimer’s Disease Center Tissue Bank. The tissue processing protocol was approved by the Institutional Ethics Committee at UTMB. Briefly, brain tissues were sectioned (12 μm) using a freezing microtome, and sections were fixed in ice-chilled methanol for 30 min, followed by treatment with TrueBlack Lipofuscin Autofluorescence QuencherFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A (Biotium; #23007) for 15 min. After three washes with PBS, sections were treated with blocking solution containing goat serum (5%) and bovine serum albumin (5%) prepared in PBS for 1 h, followed by incubation with TTCM2-ms antibody (1:100), Tau5 antibody (1:300, BioLegend; #806402), and NeuN antibody (1:250, Abcam; #ab190195 Alexa Fluor 488) overnight at 4°C. After primary incubation, sections were washed three times with PBS and probed with respective secondary antibodies. Slides were then mounted with Prolong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific; #P36931) and examined with a Keyence BZ-X700 or confocal microscope, as described previously (Montalbano et al., Brain Pathol, e13112, 2022; Gaikwad et al., Cell Rep 36, 109419, 2021).

[0190] Animal studies. Male and female hTau mice (#004808) and C57BL / 6J mice (#000664) were purchased from The Jackson Laboratory and maintained at the University of Texas Medical Branch (UTMB) animal facility. All offspring were genotyped by PCR analysis using DNA extracted from ear-punch tissues. The hTau mice express all six isoforms of the human tau protein but not mouse tau (Polydoro et al., J Neurosci 29, 10741-10749, 2009) and develop age-dependent tau pathology at 12 months of age, showing impaired cognitive and synaptic functions and progressive tauopathy (Polydoro et al., J Neurosci 29, 10741-10749, 2009). Beginning at 15 months of age, both male and female hTau mice were randomly assigned to the control or treatment groups. Animals were intranasally administered a single dose of TTCM2-ms or control IgG, as described previously (Hanson et al., J Vis Exp, 2013; Rodriguez et al., Sci Rep 7, 1862, 2017). Briefly, TTCM2-ms or IgG antibodies in micelles were prewarmed for 1 h at 37°C prior to use in order to achieve critical micellization temperature. Animals were anesthetized by intraperitoneal injection of Xylazine–Ketamine mixture in PBS (17 and 80 mg / kg body weight, respectively), and intranasal administration was performed on lightly anesthetized mice. Each mouse was kept on a sterile surgical pad and slightly stretched to hold the scruff. With a firm grip on the scruff, the mouse was turned on its back while still allowing the animal to breathe and be comfortable. The tip of a pipettor containing the sample was then placed near the left nostril of the mouse at a 45-degree angle, and 5-μL aliquots of sample were administered in 2–3 sec intervals, for a total of 15 μL / nostril (30 μL / mouse). Each treated animal was kept restrained on its back until the material disappeared into the nares, and then it was returned to its cage. This process was repeated for each mouse in the study. Mice were housed under a 12 h:12 h light:dark cycle in pathogen-free conditioning cages with free access to waterFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A and food. At 2-weeks post-intranasal administration, the animals were examined for cognitive function and neuropathology. Mice were then euthanized, followed by perfusion and brain extraction. The right brain hemisphere was used for cryosectioning and immunofluorescence staining, and hippocampus from the left hemisphere was used for biochemical analyses.

[0191] Ex-vivo fluorescence imaging was performed to detect TTCM2-ms in the brain. Briefly, 15-month-old hTau and WT control mice were intranasally administered 30-μg AF-568– labeled TTCM2-ms or IgG-loaded micelles, as described above. After 3 h, the animals were euthanized; following perfusion with PBS, brain tissues were extracted and immediately imaged using the IVIS Spectrum Imaging System (PerkinElmer). All animal experiments were performed in compliance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals under protocols approved by the Institutional Animal Care and Use Committee at the University of Texas Medical Branch, Galveston.

[0192] NOR and Y-Maze tests. NOR testing was performed to assess memory deficits in hTau mice, as previously described (Polydoro et al., J Neurosci 29, 10741-10749, 2009; Gerson et al., Journal of neurotrauma 33, 2034-2043, 2016). Briefly, mice from each group were acclimatized to the NOR arena (white open field, 60-cm height, and 55-cm diameter) for 15 min. After acclimatization, mice were removed and familiarized with two identical objects for 15 min. Mice were then tested for 15 min with one familiar object (presented during familiarization) and one novel object (different in shape and color but sharing a common size and volume). After each trial, the apparatus was wiped using 70% ethanol. The time spent exploring each object during the 15-min test was recorded using ANY-Maze software. Differences in learning and memory were expressed as a discrimination index and novelty preference, calculated by the following formula: Discrimination index =(T (Novel)−T (familiar)) / (T (Novel)+T (familiar).

[0193] The Y-maze test was used to evaluate short-term working memory, as previously described (Gerson et al., Journal of neurotrauma 33, 2034-2043, 2016). Briefly, mice (n = 6–8 per group) were placed at the center of the Y-maze (San Diego Instruments) and allowed to explore for one 8-min trial. The total number of arm entries and sequences of arm entries were recorded. One successful spontaneous alternation was recorded when an animal consecutively entered three different arms of the maze, and the percent spontaneous alternation over an 8-min trial was calculated using the following formula: % ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^=(#^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^) / (total number of arm entries−2) ^^^^ 100.FJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A

[0194] Immunoblotting. Relative protein levels were measured by immunoblotting, as described previously (Gaikwad et al., Cell Rep 36, 109419, 2021; Puangmalai et al., Cell Death Dis 11, 314, 2020)(9, 14). Briefly, mouse brain tissues were homogenized with a TissueLyser LT (QIAGEN) at 50 pulses / s for 30 s in PBS containing 2% Protease / Phosphatase Inhibitors Cocktail (Sigma; #P8340) at 1:3 ratio of brain:PBS (w / v). Samples were centrifuged at 10,000× g for 10 min at 4°C, and supernatants were collected and stored in aliquots at ‒80°C until use. Cell lysates were prepared in ice-cold RIPA buffer (CST; #9806) containing 2% Protease / Phosphatase Inhibitor Cocktail (Sigma; #P8340). After centrifugation for 10 min at 13,000× g and 4°C, the supernatant was collected and quantified using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific; #23225). An equal amount of protein (10 μg) from each sample was loaded and resolved with Pre-Cast NuPAGE 4–12% Bis-Tris Gels, followed by transfer to nitrocellulose membranes. After blocking with 10% nonfat dried milk for 1 h, membranes were probed with the indicated primary antibodies. Monoclonal anti-β- Actin−Peroxidase antibody (1: 25000; Sigma #A3854) was used as a loading control, and horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse IgG (1:10,000; GE Healthcare) were used as secondary antibodies. The signal was developed using WesternBright ECL HRP Substrate (Advanstra #K-12045-D50), and Image J (NIH) was used for densitometry analysis and normalization against the respective loading controls.

[0195] Dot blot analysis. Protein concentrations were determined by Pierce™ BCA Protein Assay Kit, and equal amounts of protein were applied on nitrocellulose membranes, as described previously (Yang et al., J Biol Chem 280, 5892-5901, 2005). After air drying, the membranes were blocked with 10% nonfat dried milk in 1× PBS with Tween 20 (PBST) overnight at 4°C. Following one wash with PBST, the membranes were incubated with indicated antibodies for 1 h at room temperature. Membranes were washed three times and incubated with HRP-conjugated anti-mouse IgG or HRP-conjugated anti-rabbit IgG for 1 h at room temperature. After three washes, the signal was developed using the ECL Plus Chemiluminescence Kit and acquired on X-ray films; quantification was performed using ImageJ.

[0196] ELISA. ELISAs were performed as previously described (Lo Cascio et al., Sci Rep 9, 19011, 2019). Briefly, ELISA plates (Nunc; #442404) were coated with samples in 50-μl sodium bicarbonate buffer (pH 9.6) at 4°C overnight. After two washes with PBST, plates were blocked with 100-μl 10% milk for 2 h at 37°C. Plates were then probed with 100-μl TTCM2-ms antibodyFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A (1:100 dilution) for 2 h at room temperature. After three washes, HRP-conjugated anti-mouse IgG (1:1000 dilution) was added, and plates were incubated for 1 h at room temperature. Plates were then washed five times, and 100 μl of 3,3,5,5-tetramethylbenzidine (TMB + Substrate- Chromogen, S1599, Dako) was added; after 15 min, the reaction was stopped with 50-μl 1-M HCl. Absorbance was measured using plate reader (POLARstar OMEGA), and optical density was plotted.

[0197] Proteolytic digestion using proteinase K (PK) enzyme. Tau strains / polymorphism assays were performed with PK-digestion, as described previously (Sengupta et al., Mol Neurobiol 57, 2741-2765, 2020). Briefly, brain homogenates from IgG- and TTCM2-ms–treated hTau mice (100–120 μg) were treated with various PK concentrations (0–20 μg / ml) in Tris- buffered saline and incubated at 37°C for 1 h. Proteolysis was terminated by addition of 4× LDS sample buffer and boiling at 95°C for 10 min, and the samples were stored at ‒80°C. Immunoblot analysis using sequence-specific antibodies, including Tau5 (1:5000), anti-Tau RD4 (clone 1E1 / A6, 1:1000), and Tau46 (1:5000), was performed to visualize the differences in PK- sensitivity.

[0198] Statistical analysis. All in-vitro experiments were performed at least three times. Data shown represent the means ± standard error of the mean (SEM), analyzed using GraphPad Prism 9. Statistical analyses included the unpaired, two-tailed Student’s t-test or one-way analysis of variance (ANOVA) followed by Tukey’s test. Differences were considered statistically significant if P < 0.05. Details of statistical analyses performed for each experiment are described in the figure legends. List of Key resources used in this study. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Tau5 antibody BioLegend Cat#806402 Tau13 antibody BioLegend MMS-520R T22 antibody In-house1N / A TTCM2 In-house2N / A Tau RD4 (clone 1E1 / A6) Millipore Sigma Cat#05-804 Tau46 abcam Cat#Ab22261 AT8, Phospho-Tau (Ser202, Thermo Cat#MN1020; Thr205) RRID:AB_223647 Antibody AT180 (T231) Invitrogen Cat# MN1040 AT100 (T212, S214) Invitrogen Cat# MN1060FJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A T18 (misfolded tau) In-house3N / A TRIM21 antibody Proteintech Cat#12108-1-AP HT7 antibody Invitrogen Cat#MN1000 Synaptophysin 1 antibody abcam Cat#ab8049 Anti-NeuN Antibody, clone A60 Chemicon Cat#MAB377; antibody RRID:AB_2298772 Alexa fluor488 anti-NeuN Abcam Cat#ab190195, antibody RRID:AB_2716282 Anti-PSD95 antibody Abcam Cat#ab18258; RRID:AB_444362 Goat anti-Rabbit IgG (H+L) Invitrogen Cat#A-11011; CrossAdsorbed Secondary RRID:AB_143157 Antibody, Alexa Fluor 568 Goat anti-Rabbit IgG (H+L) Invitrogen Cat#A-11034; Highly CrossAdsorbed Secondary RRID:AB_2576217 Antibody, Alexa Fluor 488 Goat anti-Mouse IgG (H+L) Invitrogen Cat#A-21052; CrossAdsorbed Secondary RRID:AB_2535719 Antibody, Alexa Fluor 633 HRP-conjugated anti-rabbit IgG GE Healthcare NA934-1ML; RRID:AB_772206 HRP-conjugated anti-mouse IgG GE Healthcare NA931-1ML; RRID:AB_772210 Anti-GAPDH antibody Abcam Cat#ab9485; RRID:AB_307275 Monoclonal Anti-b-Actin– Sigma Cat#A3854; Peroxidase antibody RRID:AB_262011 biotinylated horse anti-rabbit or Vector Laboratories PK-4001, PK-4002 mouse IgG Biological samples Human brain tissues from The Institute for Brain Aging N / A NDC, AD, DLB and PSP cases and Dementia at UC Irvine4Escherichia coli BL21(DE3) Agilent Cat# 200131 competent cells Reagents PBS Corning Cat#46-013-CM RIPA buffer Cell Signaling Technology Cat#9806 Thioflavine S Sigma Cat# T1892 Bovine serum albumin (BSA) Sigma Cat# A4161 Triton X-100 Sigma Cat# T8787 Protease inhibitor cocktail Sigma Cat#p8340-1ml HBSS GIBCO Cat#14175-095 Formaldehyde solution Sigma Cat#F8775-500ML ProLong Gold Antifade reagent Thermo Cat#P36935 Fetal Bovine Serum GIBCO Cat#1600-044 DMEM GIBCO Cat# 11960-044FJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A Antibiotic-Antimycotic (100X) GIBCO Cat# 15240096 Neurobasal medium GIBCO Cat# 12348017 B27TM plus supplement GIBCO Cat# A3582801 GlutaMax GIBCO Cat# 35050-061 Accell Human TRIM21 (6737) Dharmacon E-006563-00-0005 siRNA SMARTpool Poly-L-lysine solution Sigma Cat#P4832 Lipofectamine 2000 Transfection Thermo Cat#11668030 reagent VECTASTAIN® Elite® ABC- Vector Laboratories Cat#PK-6100 HRP Kit Hematoxylin Vector Laboratories Cat#H-3401 Cytotoxicity Detection kit PLUS Roche Applied Science Cat#04744926001 Critical commercial assays Cation exchange chromatography column Superdex 200 Increase 10 / 300 Cytiva Cat# 29018183 GL WesternBright ECL HRP Cytiva Cat# 28990944 substrate TrueBlack Lipofuscin Advanstra Cat#K-12045-D50 Autofluorescence Quencher BCA protein assay kit Biotium Cat#23007 AF568 Protein Labeling kit Pierce Ca#23225 Experimental models: Invitrogen Cat# A10238 Organisms / strains Mouse: hTau Tau biosensor cell line Jackson Laboratory Stock#004808 Software and algorithms ATCC Cat# CRL-3275 ImageJ NIH imagej.net / ij / BZ-X Analyzer Keyence www.keyence.com / ANY-maze Behavioral tracking ANY-maze www.any-maze.com / software GraphPad Prism 9 Prism - GraphPad www.graphpad.com / 1(Gaikwad et al., Cell Rep 36, 109419, 2021),2(Montalbano et al., Brain Pathol 33, e13112, 2023),3(Puangmalai et al., J Biol Chem 298, 101766, 2022),4(Puangmalai et al., J Biol Chem 298, 101766, 2022; Gaikwad et al., Cell Rep 36, 109419, 2021; Puangmalai et al., Cell Death Dis 11, 314, 2020; Montalbano et al., Brain Pathol 33, e13112, 2023) Example 2 Polymorphic Alpha Synuclein Oligomers - Characterization and Differential Detection with Novel Corresponding AntibodiesFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A A. Results

[0199] Biochemical Characterization of α-Syn Oligomeric Strains / Anti-Syns. It has previously been shown how α-Syn oligomeric conformers differ by aggregate size, conformation, and hydrophobicity (Sengupta et al., Molecular neurobiology, 57, 2741-2765, 2020 ). To further investigate if these oligomeric polymorphs have biological relevance, standard biochemical characterization was performed utilizing dot blot (FIG. 20A-20D), western blot (FIG. 20E-20H) and indirect ELISA (FIG. 20I-20L). To test selectivity of Anti-Syns, α-Syn monomer, α-Syn oligomeric polymorphs (SynODA, SynODHA, SynOaCSF), α-Syn fibrils, tau, and amyloid-β were used. Each Anti-Syn exhibits selectivity for α-Syn species and distinct immunoreactivity for each oligomeric polymorph (FIG. 20). Dot blotting revealed distinct intensities detected by Anti-Syns for each α-Syn oligomeric species compared to detection by total α-Syn commercial antibody, LB509 (FIG. 20D). Dot blot quantification is provided (FIG. 24). Anti-Syn1 differentially detected all α-Syn species with the highest intensity for α-Syn oligomeric polymorphs and weakest intensity for α-Syn monomer (FIG. 20A). Anti-Syn2 differentially detected all α-Syn species with low immunoreactivity for α-Syn monomer (FIG. 20B). Anti-Syn3 detected SynOaCSF and SynODHA, but did not detect α-Syn monomer, SynODA, or α-Syn fibrils (FIG. 20C). Overall Anti-Syns selectively detect all α-Syn species with a higher selectivity for aggregated α-Syn. For more biochemical characterization we performed western blotting, which revealed detection of different populations of aggregates amongst α-Syn oligomeric polymorphs. (FIG. 20E-20H). Anti-Syns differentially detect monomeric species around 14 kDa, oligomeric species ranging from 25 kDA to 75 kDA and higher molecular weight aggregates above 250 kDa (FIG. 20E-20H). While Anti-Syn2 strongly detected α-Syn monomer (FIG. 20F), Anti-Syn1 (FIG. 20E) and Anti-Syn3 (FIG. 20G) show least immunoreactivity to α-Syn monomer. SynODHA did not exhibit immunoreactivity to Anti- Syn1 or Anti-Syn2 but exhibited strong immunoreactivity to Anti-Syn3. All α-Syn species were detected and confirmed by total α-Syn antibody, LB509 (FIG. 20H). Next, indirect ELISA was performed and like immunoblotting results, differences in immunoreactivity were also observed (FIG. 20I-20L). Anti-Syn1 exhibited the least binding to α-Syn species (FIG. 20I) in agreement with western blotting (FIG. 20E). Anti-Syn2 exhibited binding to α-Syn monomer (FIG. 20J) similarly shown in western blotting (FIG. 20F). α-Syn immunoreactivity detected by Anti-Syn3 (FIG.20K) was not as distinct among all α-Syn species compared to dot blotting (FIG.20C).FJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A

[0200] α-Syn Oligomeric Polymorphs Reveal Distinct Sensitivity to Proteinase K. α-Syn oligomers are different in their aggregate size, hydrophobicity, and biological properties (Sengupta et al.2020). To evaluate the conformational differences between the three oligomeric polymorphs as well as their stability as oligomers, their sensitivity for proteinase K (PK) enzyme digestion was measured. PK digestion has long been used in classifying strains of prion fibrils (Groveman et al., acta neuropathol commun 6, 7, 2018; Fecchio et al., Molecules 23 1531, 2018). Nevertheless, this method has been extended and widely used for identifying amyloid strains, of amyloid-β, α-Syn, and tau fibrils (Shamoto-Nagai et al., Journal of clinical biochemistry and nutrition 62, 207-212, 2018). SynODA, SynODHA, and SynOaCSF were treated with increasing concentrations of PK enzyme (0–2 μg / mL). All the digested samples were then run in SDS-PAGE followed by silver staining (FIG. 25). The pattern of fragments generated by PK digestion provides information on the stability of the oligomers, as well as its core. It was observed that SynODA was resistant to PK, thus indicating a stable core of these oligomers. In contrast, SynODHA and SynOaCSF were sensitive to PK showing two distinct fragmentation patterns. These results suggest each oligomeric polymorph exhibit conformational differences.

[0201] Atomic Force Microscopy of SynOaCSF and Syn Fibrils. Due to the similar immunoreactivity exhibited by SynOaCSF and Syn Fibrils observed in biochemical studies (FIG. 20), atomic force microscopy was utilized to observe structural properties of each sample (FIG. 26). Representative AFM images of Syn O aCSF exhibits spherical structures. In contrast, Syn Fibrils exhibit protofilaments. These results confirm structural differences amongst SynOaCSF and Syn Fibrils.

[0202] Biophysical Characterization of α-Syn Oligomeric Strains / anti-Syn. We utilized the highly sensitive method, Isothermal Titration Calorimetry (ITC), to quantify the binding interactions between Anti-Syns and oligomeric α-Syn (FIG. 21). The buffer and pH for ITC experiments were assigned and optimized based on a previously published protocol (Dong et al., Protein science: a publication of the Protein Society 28, 1690-1702, 2019). Unmodified α-Syn oligomers were utilized instead of polymorphs due to the significance of buffer sensitivity when conducting ITC (Tesmar et al., J Therm Anal Calorim 126, 97–102, 2016). Each Anti-Syn exhibited a distinct binding profile for α-Syn oligomers. Anti-Syn1 bound oligomeric and monomeric α-Syn but not fibrillar. Results suggest Anti-Syn1 bound α-Syn oligomer at very highFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A affinity with a KDof 1.00 pM ± 26.5 nM (FIG.21). Anti-Syn1 bound α-Syn monomer at a lower affinity with a KDof 5.31 ± 45.1 nM (FIG.27A). Anti-Syn2 selectively bound α-Syn oligomer at high affinity with a KDof 379 ± 850 nM (FIG.21) and did not bind monomeric or fibrillar α-Syn (FIG.27A, 27B). Lastly, Anti-Syn3 did not bind α-Syn species. Anti-Syn1 exhibited the highest affinity to α-Syn oligomers but also bound monomeric α-Syn at a lower affinity. Anti-Syn2 exhibited lower affinity to oligomeric α-Syn than Anti-Syn1 but did not bind monomeric or fibrillar α-Syn. Anti-Syn3 does not demonstrate binding to α-Syn, which is not in agreement with previous results. These differences in binding affinity provide insight into the functionality of Anti-Syns and the effects of conformation-specific antibodies.

[0203] Anti-Syns Differentially Reduce α-Syn Oligomeric Seeding in Primary Neurons. One of the key phenomena in amyloid strains is that strains act as seeds in the recipient cells, thus recruiting endogenous protein into aggregation and mediating the degeneration of cells (Sengupta et al., Molecular neurobiology, 57, 2741-2765, 2020; Oueslati et al., Experimental neurobiology, 23, 324-36, 2014). To evaluate the seeding propensity of each α-Syn oligomeric polymorph and the ability of Anti-Syns to reduce α-Syn propagation in primary neurons, cell- based antibody neutralization assays were performed. This was followed by immunocytochemistry utilizing, neuronal marker βIIITubulin (1:1000; Abcam78078), total α- Syn antibody LB509 (1:5000; Abcam 27766), and total α-Syn rabbit polyclonal antibody Syn10842 (1:1000; ThermoFisher 10842-1-AP) to eliminate cross reactivity with Anti-Syn mouse monoclonal antibodies. Immunocytochemistry was used to visualize the effects of Anti- Syn neutralization of α-Syn oligomeric polymorphs in primary cortical neurons isolated from mice expressing human α-Syn (FIG.22). The average fluorescent intensity of α-Syn detected by Syn10842 was quantified (FIG. 22C, 22F, 22I) and LB509 (FIG. 22B, 22E, 22H) to evaluate total α-Syn propagation. SynODA exhibited the highest seeding propensity amongst the three polymorphs (FIG.22A, 22D, 22G). SynOaCSF (FIG.22A-22C), SynODA (FIG.22D-22F), and SynODHA (FIG. 22G-22I) were all differentially immunodepleted by all three Anti-Syns. Anti- Syn3-immunodepleted neurons exhibiting the highest reduction in total α-Syn. The colocalization profiles for individual regions of interest, specifically neuronal projections from to one cell body to another, were also quantified and results were consistent with these findings (FIG. 31). The inventor next sought out to determine if cells exposed to toxic α-Syn oligomeric conformers is differentially reduced when immunodepleted by Anti-Syns.FJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A Anti-Syns Differentially Reduce α-Syn Oligomer-Mediated Neurotoxicity. Previous reports observed that exogenously added α-Syn oligomers cause cellular toxicity either by seeding endogenous protein or by acting on cellular membranes (Danzer et al., The Journal of neuroscience: the official journal of the Society for Neuroscience 27, 9220-32, 2007; Danzer et al., Journal of neurochemistry 111, 192-203, 2009). Studies have shown SHSY-5Y cells respond to α-Syn fibrillar seed-induced disruption of protein homeostasis predominantly by secreting α- Syn aggregates (Sang et al., Communications biology 4, 613, 2021; Trinkaus et al., Nature communications 12, 2110, 2021). Furthermore, results reveal that α-Syn oligomeric conformers recruit cytosolic α-Syn aggregates and exhibit distinct seeding potencies and cytotoxicity (Sengupta et al., Molecular neurobiology, 57, 2741-2765, 2020). The inventor sought to assess the cytotoxic effects of immunodepleting α-Syn oligomeric strains in SH-SY5Y cells (FIG.23A- 23F) and primary cortical neurons isolated from mice overexpressing human α-Syn (FIG. 23G- 23L). Cytotoxicity in both SHSY-5Y cells and primary neurons was measured by LDH (FIG. 23A-23C, 23G-23I) and cell viability was measured by MTS cell-based assays (FIG. 23D-23F, 23J-23L). α-Syn oligomeric polymorphs exhibit cytotoxicity in SH-SY5Y cell and primary cortical neurons. Anti-Syns differentially reduce α-Syn oligomer-mediated cytotoxicity in SHSY-5Y cells and primary cortical neurons. SHSY-5Y cells treated with α-Syn oligomeric conformers exhibited higher cytotoxicity than primary neurons. Anti-Syn immunodepletion caused reduction in cytotoxicity and an increase in cell viability for each oligomeric conformer in both SHSY-5Y cells and primary neurons with varying significance. These results suggest that Anti-Syns bind in a polymorph-specific manner and that α-Syn oligomeric polymorphs mediate distinct neurotoxicity that can be reduced by immunodepletion. B. Materials and Methods

[0204] Generation of α-Syn monomers, oligomers, and fibrils. Recombinant α-Syn monomer, oligomers, and fibrils were prepared following our published methods (Sengupta et al., Molecular neurobiology, 57, 2741-2765, 2020; Sengupta et al., Biological psychiatry, 78, 672-83, 2015; Castillo-Carranza et al., Biological psychiatry, 84, 499-508, 2018).

[0205] Generation of Anti-Syns. Briefly, the cell lines for the antibodies were developed utilizing α-Syn oligomer antigen prepared in our lab (Genscript Biotech, NJ, USA). Several cell lines were evaluated for their immunoreactivity using different preparations of α-Syn oligomersFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A and α-Syn monomer by dot blot, filter trap assay, and Enzyme-Linked Immunosorbent Assay (ELISA). Finally, the three Anti-Syn clones were selected for production.

[0206] Anti-Syn Screening. Indirect ELISA. Anti-syn oligomer antibody response was determined by screening serial dilutions of animal sera using an Indirect enzyme-linked immunosorbent assay (ELISA) as previously published (Lasagna-Reeves et al., Biochemistry 49, 10039-41, 2010; Lo Cascio et al., The Journal of biological chemistry, 295, 14807-14825, 2020). Briefly, 96-well plates (Nunc Immobilizer, Amino Plates and Modules, 436006, Thermo Fisher Scientific) were previously coated with 1 μl α-Syn oligomers, Aβ oligomers, or tau oligomers in 50 μl of 1× PBS, pH 7.4, as coating buffer. After washing three times with TBS-T, plates were blocked for 2 h at room temperature with 10% nonfat milk in TBS-T. Plates were then washed three times with TBS-T and probed with 100 μl of primary antibodies for 1 h at room temperature (RT): commercial antibodies, LB509 (1:5000;Abcam 27766), Syn211 (1:5000;Abcam 80627)), sequence-specific α-Syn antibodies Syn 33 (1:1000;SigmaAldrich ABN2265), MJFR (1:1000;Abcam 209538), and F8H7 (1:1000). Plates were then washed three times with TBS-T and incubated with 100 μl of HRP-conjugated anti-rabbit or anti-mouse IgG, diluted 1:10,000 in 5% nonfat milk in TBS-T, for 1 h at room temperature. Plates were washed three times with TBS-T and developed with 3,3,5,5-tetramethylbenzidine (S1599, Dako). The reaction was stopped using 100 μl of 1M HCl, and absorbance was read at 450 nm using a POLARstar OMEGA plate reader. All experiments were performed in triplicate.

[0207] Dot Blotting. Dot blot was also used to test Anti-Syn specificity. Each strip had seven protein dots: dot #1 (α-Syn monomer), dots #2–4 (α-Syn oligomers from different preparations), dot #5 (α-Syn fibrils), dot #6 (tau oligomer), and dot #7 (Aβ oligomer).1 μl of each sample was dotted on nitrocellulose membrane. Next, membranes were blocked with 10% nonfat milk in TBS-T overnight at 4 °C. After blocking, membranes were probed with previously listed antibodies in 5% nonfat milk for 1 h at RT. This was followed by incubation with HRP- conjugated IgG anti-mouse or anti-rabbit (1:6000, GE Healthcare) secondary antibody to detect primary antibodies. ECL plus (GE Healthcare) was used for signal detection.

[0208] Western Blotting. 1 μg / µL of each sample (α-Syn monomer, α-Syn oligomers, α-Syn fibrils, tau oligomer, and Aβ oligomer was resolved on a precast NuPAGE 4–12% BisTris gel for SDS-PAGE (NP0335BOX, Invitrogen) and transferred to nitrocellulose membranes. Next, membranes were blocked with 10% nonfat milk in TBS-T overnight at 4 °C. After blocking,FJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A membranes were probed with previously listed antibodies, diluted in 5% nonfat milk for 1 h at RT. Membranes were then incubated with HRP-conjugated IgG anti-mouse (1:6000, GE Healthcare) secondary antibody to detect α-Syn antibodies. ECL plus (GE Healthcare) was used for signal detection. Finally, the selected clones (Anti-Syns) were tested using human and mouse brains.

[0209] Indirect ELISA. ELISA plates were coated with 1ug / well of α-Syn monomer, α-Syn oligomeric polymorphs, and tau oligomer. 0.1 M sodium bicarbonate, pH 9.6, was used as a coating buffer followed by overnight incubation with primary antibodies: Anti-Syn1 (1:4000), Anti-Syn2 (1:1000), Anti-Syn3 (1:1000), and total α-Syn commercial antibody LB509 (1:5000; Abcam 27766) at 4 °C. Plates were then washed three times with TBS-T and incubated with 100 μl of HRP-conjugated anti-mouse IgG, diluted in 5% nonfat milk in TBS-T, for 1 h at room temperature. Plates were washed three times with TBS-T and developed with 3,3,5,5- tetramethylbenzidine (S1599, Dako). The reaction was stopped using 100 μl of 1 M HCl, and absorbance was read at 450 nm using a POLARstar OMEGA plate reader. All experiments were performed in triplicate.

[0210] Dot Blotting. 1 μl of each sample, dot #1 (α-Syn monomer), dots #2–4 (α-Syn oligomeric polymorphs), dot #5 (α-Syn fibrils), dot #6 (tau oligomers), and dot #7 / #8 (Aβ40 / 42 oligomers) was dotted on nitrocellulose membrane and let dry for 1 hour at RT. Next, membranes were blocked with 10% nonfat milk in TBS-T overnight at 4 °C. After blocking, membranes were probed with primary antibodies Anti-Syn1 (1:4000), Anti-Syn2 (1:1000), Anti- Syn3 (1:1000), and total α-Syn commercial antibody LB509 (1:5000; Abcam 27766) in 5% nonfat milk for 1 h at RT followed by incubation with HRP-conjugated IgG anti-mouse (1:6000, GE Healthcare) secondary antibody to detect α-Syn antibodies. ECL plus (GE Healthcare) was used for signal detection.

[0211] Western Blotting. For western blotting, 1 μg of each sample (α-Syn monomer, α-Syn oligomeric polymorphs, (α-Syn fibril, tau oligomer, and Aβ40 / 42 oligomers) were loaded on precast NuPAGE 4-12% Bis-Tris gels (Invitrogen) for SDS-PAGE analysis. Gels were subsequently transferred onto nitrocellulose membranes. Membranes were blocked with 10% nonfat dry milk at 4 °C overnight. This was followed by incubation with primary antibody followed by secondary antibody incubation. After antibody binding, the membrane is incubated with chemiluminescent substrate and imaged. Primary antibodies for each experiment includeFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A Anti-Syn1 (1:4000), Anti-Syn2 (1:1000), Anti-Syn3 (1:1000), and total α-Syn commercial antibody LB509 (1:5000; Abcam 27766). HRP-conjugated, anti-mouse IgG (1:6000, GE Healthcare) was used to detect each Anti-Syn and LB509. ECL plus (GE Healthcare) was used to visualize the bands.

[0212] Proteolytic Digestion of α-Syn Oligomers by Proteinase K Enzyme. Different oligomer preparations of α-Syn (10–12 μg) were treated with different concentrations of proteinase K enzyme (Sigma) ranging from 1 to 2 μg / mL in the presence of 1× PBS buffer and incubated at 37 °C for 30 min. At the end of incubation time, 1× LDS sample buffer (Invitrogen) was added and heated at 95 °C for 10 min. Samples were immediately transferred onto ice to stop the cleavage reaction followed by loading the digestion products into 4–12% Bis-Tris precast gels (Invitrogen) for SDS-PAGE gel electrophoresis. Gels with digested samples were processed for silver staining (Pierce Silver Stain Kit, Thermo Scientific; 24,612) to visualize the fragments following manufacturer’s instructions (Sengupta et al., Molecular neurobiology, 57, 2741-2765, 2020).

[0213] Atomic Force Microscopy. SynOaCSF and fibrillar α-Syn were analyzed by AFM using a non-contact tapping method with a Multimode 8 AFM machine (Bruker, Billerica MA). Briefly, 3–4μl of each sample was applied onto a fresh-cleaved mica surface and allowed to adsorb at RT overnight. Mica was then washed with 200μl of deionized water and air-dried. Images were taken from 5 different areas on the mica surface (Sengupta et al., Molecular neurobiology, 57, 2741-2765, 2020).

[0214] Isothermal Titration Calorimetry (ITC). ITC measurements were made using a MicroCal PEAQ-ITC (Linkuvienė et al., Analytical biochemistry, 515, 61-64, 2016 ). 8 μM of either Anti-Syn1, 2, or 3 was titrated into 2 μM α‐synuclein monomer, unmodified oligomer, or fibril using an initial .4‐μL injection followed by 2‐μL injections at intervals of 2 min, with a stirring rate of 1000 rpm. The temperature was maintained at 25°C. Protein samples were prepared in phosphate buffer at a pH of 7.4. Titrations of ligand into buffer were measured and used for background subtraction before fitting the data. A one set of sites binding model was used for all experiments and binding curves were fit with a Gaussian nonlinear regression model on Prism 9.4 (GraphPad Software). (Rovere, Methods in molecular biology 123-143, 2019; Nuscher et al., The Journal of biological chemistry 279, 21966-75, 2004; Dong et al., Protein science: a publication of the Protein Society 28, 1690-1702, 2019).FJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A

[0215] Cell Treatment with α-Syn Oligomers. Human neuroblastoma, SH-SY5Y, cells were cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented 10% fetal bovine serum (Gibco) and 1% penicillin / streptomycin (Gibco). α-Syn oligomeric polymorphs (0.5 μM), generated as previously published (Sengupta et al., Molecular neurobiology, 57, 2741-2765, 2020), were incubated with each Anti-Syn (2 μM) for 30 minutes at RT. Cells were then exposed to SynO polymorphs or Anti-Syn-immunodepleted SynO polymorphs for 24 hr.

[0216] Primary Cortical Neuron Culture and Treatment. C57BL / 6 transgenic mice expressing human α-Syn (Jackson Laboratory, 017682) were used for primary cortical neuron isolation. Primary cortical neuronal cells from mice during embryonic days 16–18 was isolated using Accutase solution (Sigma, A6964) and maintained as previously published (Sengupta et al., Molecular neurobiology, 57, 2741-2765, 2020). Briefly, neuronal cells were plated on poly- D-lysine-coated glass coverslips (Corning, Inc.) at a density of 2 × 105 cells / mL in a 24- well plate containing neurobasal medium (Gibco, 12348017) supplemented with 2% B-27, 0.5 mM GlutaMax (Gibco, 35050-061), 10,000 units / mL penicillin, 10,000 μg / mL streptomycin, and 25 μg / mL amphotericin B supplement. Media changes were performed every 3–5 days by replacing 50% culture media with fresh media. Cells were grown for 10–13 days in vitro (DIV) before experiments. Primary cortical neurons grown on coverslips in 24-well plates. α-Syn oligomeric polymorphs (0.5 μM), generated as previously published (Sengupta et al., Molecular neurobiology, 57, 2741-2765, 2020), were incubated with each Anti-Syn (2 μM) for 30 minutes at RT. Primary cortical neurons were then exposed for 24 hours (Puangmalai et al., Cell Death Dis 11, 314, 2020; Puangmalai et al., The Journal of biological chemistry 298, 101766, 2022). The procedures involving experimentation on animal subjects are done in accordance with UTMB’s guidelines.

[0217] Immunocytochemistry and Image Analysis. Following the 24 hr incubation, cells were washed 3 times with 1× PBS and fixed with 4% formaldehyde solution for 15 min at RT. Cells were then washed 3 times with 1× PBS followed by permeabilizing with 0.25% Triton X-100 in PBS for 10 min at RT. Cells were blocked in 5% goat serum for 30 min at RT and incubated with primary antibodies: βIIITubulin (1:1000; Abcam78078), Syn10842 (1:1000;ThermFisher 10842-1-AP), and total α-Syn antibody LB509 (1:5000; Abcam 27766) at 4 °C overnight. The next day, cells were washed and incubated with Alexa-conjugated secondary antibodies (1:000;FJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A Life Technologies) for 1 h at RT in the dark. After three washes, cells were mounted with Prolong Gold antifade reagent with DAPI. Each treatment condition was performed in 3 replicates and were randomly imaged at five different regions of interest. Images were captured with a Keyence BZ-800 Microscope and analyzed using BZ-X Analyzer. A Nikon 60X objective was used for image acquisition. To eliminate species cross-reactivity, fluorescent intensity of total α-Syn was quantified by total α-Syn anti-rabbit polyclonal, Syn10842 (1:1000; Thermo Fisher 10842-1-AP). All images were analyzed by ImageJ (NIH) software. Statistical significance is measured by using two-way ANOVA with Bonferroni post hoc analysis. **p < 0.01, ****p < 0.0001. Scale bar 10 μm.

[0218] Cell Toxicity and Viability Assays. Cytotoxicity was determined by measuring lactate dehydrogenase (LDH) release using Cytotoxicity Detection kit PLUS (Roche, 04744926001) and cell viability was measured by CellTiter 96® Aqueous Non-Radioactive Cell Proliferation Assay (MTT) (Promega, G5421) following manufacturers’ instructions as previously described. In brief, following strain and immunodepletion treatment for 24 h, cells were assayed with LDH or MTT for cytotoxicity and cell viability assays respectively. For both assays, absorbance was measured at 490 nm with a Polar Star Omega plate reader (BMG Labtech). Each experimental condition was performed in triplicates in three different independent assays. For the MTS assay, the percentage of viable cells was calculated as ((ODtreated -ODuntreated control) / ODuntreated control) × 100. For LDH assay, the percentage of affected cells was calculated following the formula provided by the manufacturer.

[0219] Statistical Analysis. All experiments were repeated at least three times. Statistical analyses were performed using Prism 9.4 (GraphPad Software) through unpaired two-tailed Student’s t test or one-way analysis of variance (ANOVA) according to group. Results are considered statistically significant at p < 0.01.

Claims

FJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A CLAIMS 1. A method of treating a neurodegenerative disorder comprising administering one or more TTCM antibody; one or more TOMA antibody; one or more α-Synuclein antibody; a combination of one or more TTCM antibody and one or more TOMA antibody; a combination of one or more TTCM antibody and one or more α-Synuclein antibody; a combination of one or more TOMA antibody and one or more α-Synuclein antibody; or a combination of one or more TTCM antibody, one or more TOMA antibody, and one or more α-Synuclein antibody.

2. The method of claim 1, wherein the antibody or antibodies are selected from antibodies or antibody fragments having: (i) a variable heavy chain complementarity determining region (CDR) 1 having an amino acid sequence of SEQ ID NO:13, CDR 2 having an amino acid sequence of SEQ ID NO:14, CDR 3 having an amino acid sequence of SEQ ID NO:15; (ii) a variable light chain complementarity determining region (CDR) 1 having an amino acid sequence of SEQ ID NO:17, CDR 2 having an amino acid sequence of SEQ ID NO:18, CDR 3 having an amino acid sequence of SEQ ID NO:19; (iii) a first complementarity determining region (CDR) set, first CDR 1 having an amino acid sequence (SEQ ID NO:54), first CDR 2 having an amino acid sequence (SEQ ID NO:55), first CDR 3 having an amino acid sequence (SEQ ID NO:56) in combination with a second CDR set with a second CDR1 having an amino acid sequence (SEQ ID NO:58), second CDR 2 having an amino acid sequence (SEQ ID NO:59), and second CDR 3 having an amino acid sequence (SEQ ID NO:60); (iv) a first complementarity determining region (CDR) set, first CDR 1 having an amino acid sequence (SEQ ID NO:62), first CDR 2 having an amino acid sequence (SEQ ID NO:63), first CDR 3 having an amino acid sequence (SEQ ID NO:64) in combination with a second CDR set with a second CDR1 having an amino acid sequence (SEQ ID NO:66), second CDR 2 having an amino acid sequence (SEQ ID NO:67), and second CDR 3 having an amino acid sequence (SEQ ID NO:68); or (v) a first complementarity determining region (CDR) set, first CDR 1 having an amino acid sequence (SEQ ID NO:70), first CDR 2 having an amino acid sequence (SEQ ID NO:71), first CDR 3 having an amino acid sequence (SEQ ID NO:72) in combination with a second CDR setFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A with a second CDR1 having an amino acid sequence (SEQ ID NO:74), second CDR 2 having an amino acid sequence (SEQ ID NO:75), and second CDR 3 having an amino acid sequence (SEQ ID NO:76).

3. The method of claim 1, wherein one or more antibodies are comprised in a delivery vehicle.

4. The method of claim 3, wherein one or more antibodies are in one delivery vehicle.

5. The method of claim 3, wherein one or more antibodies are in separate delivery vehicles.

6. The method of claim 1, wherein one or more antibodies are in a micellar delivery vehicle.

7. The method of claim 1, wherein the one or more antibodies is administered by intranasal administration.

8. A tau toxic antibody comprising: (i) a variable heavy chain complementarity determining region (CDR) 1 having an amino acid sequence of SEQ ID NO:13, CDR 2 having an amino acid sequence of SEQ ID NO:14, CDR 3 having an amino acid sequence of SEQ ID NO:15; and (ii) a variable light chain complementarity determining region (CDR) 1 having an amino acid sequence of SEQ ID NO:17, CDR 2 having an amino acid sequence of SEQ ID NO:18, CDR 3 having an amino acid sequence of SEQ ID NO:

19.

9. The antibody according to claim 8, wherein the variable heavy chain region has an amino acid sequence of SEQ ID NO:

11.

10. The antibody according to claim 8, wherein the variable light chain region has an amino acid sequence of SEQ ID NO:12.FJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A 11. The antibody according to claim 8, further comprising a heavy chain constant region having an amino acid sequence of SEQ ID NO:

16.

12. The antibody according to claim 8, further comprising a light chain constant region having an amino acid sequence of SEQ ID NO:

20.

13. An immunotherapy composition comprising an antibody of claim 8 and a pharmaceutically acceptable composition.

14. The composition of claim 13, wherein the pharmaceutically acceptable composition is a micellar composition.

15. A method of treating a tauopathy comprising administering a composition of claim 13 to a subject having or at risk of developing a tauopathy.

16. The method of claim 15, wherein administering is intranasal administering.

17. An alpha synuclein oligomer antibody comprising: (i) a first complementarity determining region (CDR) set, first CDR 1 having an amino acid sequence (SEQ ID NO:54), first CDR 2 having an amino acid sequence (SEQ ID NO:55), first CDR 3 having an amino acid sequence (SEQ ID NO:56) in combination with a second CDR set with a second CDR1 having an amino acid sequence (SEQ ID NO:58), second CDR 2 having an amino acid sequence (SEQ ID NO:59), and second CDR 3 having an amino acid sequence (SEQ ID NO:60); (ii) a first complementarity determining region (CDR) set, first CDR 1 having an amino acid sequence (SEQ ID NO:62), first CDR 2 having an amino acid sequence (SEQ ID NO:63), first CDR 3 having an amino acid sequence (SEQ ID NO:64) in combination with a second CDR set with a second CDR1 having an amino acid sequence (SEQ ID NO:66), second CDR 2 having an amino acid sequence (SEQ ID NO:67), and second CDR 3 having an amino acid sequence (SEQ ID NO:68); orFJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A (iii) a first complementarity determining region (CDR) set, first CDR 1 having an amino acid sequence (SEQ ID NO:70), first CDR 2 having an amino acid sequence (SEQ ID NO:71), first CDR 3 having an amino acid sequence (SEQ ID NO:72) in combination with a second CDR set with a second CDR1 having an amino acid sequence (SEQ ID NO:74), second CDR 2 having an amino acid sequence (SEQ ID NO:75), and second CDR 3 having an amino acid sequence (SEQ ID NO:76).

18. The antibody of claim 17, wherein (i) the first and second CDR sets are comprised in a heavy chain amino acid sequence of SEQ ID NO:53 and a light chain amino acid sequence of SEQ ID NO:57, respectively; (ii) the first and second CDR sets are comprised in a heavy chain amino acid sequence of SEQ ID NO:61 and a light chain amino acid sequence of SEQ ID NO:65, respectively; or (iii) the first and second CDR sets are comprised in a heavy chain amino acid sequence of SEQ ID NO:69 and a light chain amino acid sequence of SEQ ID NO:

73.

19. The antibody according to claim 17 or claim 18, wherein the antibody is formulated in a therapeutic composition.

20. An immunotherapy composition comprising one or more of an antibody of claim 17 or claim 18.

21. The composition of claim 20, wherein the immunotherapy composition is a micellar composition.

22. A method of treating a subject diagnosed with or at risk of having or developing a neurodegenerative condition comprising administering one or more antibody of claim 17.

23. The method of claim 22, wherein the neurodegenerative condition is a tauopathy or a synucleinopathy.

24. The method of claim 23, wherein administering is through an intranasal route.FJ ref. UTMB-P0419WO / Client ref. KAYED-RA-24A 25. The method of claim 22, further comprising administering a second therapy or therapeutic antibody.

26. The method of claim 25, wherein the second therapeutic antibody is a tau binding antibody.

27. The method of claim 26, wherein the tau binding antibody is a conformation specific antibody.

Images