Attorney Docket No.06527-2503253 ANTI-TAU PROTEIN BINDING REAGENTS CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to United States Provisional Patent Application Nos.63/693,956 filed September 12, 2024, and 63/824,441, filed June 16, 2025, the disclosures of which are hereby incorporated by reference in their entireties. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH [0002] This invention was made with government support under AG083874 awarded by the National Institutes of Health. The government has certain rights in the invention. REFERENCE TO A SEQUENCING LISTING [0003] The Sequence Listing associated with this application is filed in electronic format via Patent Center and is hereby incorporated by reference into the specification in its entirety. The name of the XML file containing the Sequence Listing is 2503253.xml. The size of the XML file is 330,049 bytes and the XML file was created on September 10, 2025. BACKGROUND OF THE INVENTION Field of the Invention [0004] Provided herein are binding reagents, such as antibodies, and uses thereof. Description of Related Art [0005] The polymerization of highly phosphorylated and often truncated tau protein into fibrillar intracellular brain aggregates is a defining feature of several neurodegenerative diseases including Alzheimer's disease (AD). In AD, the severity of tau pathology in neurofibrillary tangles (NFTs), assessed according to the Braak staging for NFTs, is a stronger correlate and predictor of cognitive outcomes than amyloid β (Aβ) plaques, another principal neuropathological feature of AD. Positive tau-immunohistochemical staining for NFTs is required for neuropathological diagnosis of AD at autopsy, while in vivo neuroimaging of insoluble tau aggregate deposits by tau positron emission tomography (PET) facilitates patients' clinical diagnosis. Methods for characterization of tau proteins and aggregates, including their post-translation modifications, are useful. Biomarker methods for quantifying tau 1 6778060.DOCX
Attorney Docket No.06527-2503253 aggregates, including early-stage pre-fibrillar tau aggregates, and their associated modifications, in biofluids are lacking. [0006] Converging evidence indicates that physiological buffer-soluble, low-order tau aggregates (e.g., oligomers, protofibrils) that form in the initial stages of the tau aggregation cascade and constitute the building blocks for the higher-order fibril and NFT structures, are highly efficient in seeding and propagating tau toxicity. These findings indicate that soluble tau assemblies (STAs) may have utility as early diagnostic and prognostic biomarkers and may be ideal targets for the development of anti-tau therapeutics. Yet, little is known about the biochemical features of soluble tau assemblies. [0007] There is a great need for useful antigen binding molecules, such as antibodies, for use in characterizing both soluble and in situ tau proteins and aggregates. SUMMARY OF THE INVENTION [0008] Provided herein are tau protein binding reagents and uses thereof. [0009] Provided herein is an antibody that binds to a tau protein, including a heavy chain having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to a sequence selected from SEQ ID NO: 4 SEQ ID NO: 8, and SEQ ID NO: 12; SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 60, SEQ ID NO: 64, SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 76, SEQ ID NO: 80, SEQ ID NO: 84, SEQ ID NO: 88, SEQ ID NO: 92, SEQ ID NO: 96, SEQ ID NO: 100, SEQ ID NO: 104, SEQ ID NO: 108, SEQ ID NO: 112, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 122, SEQ ID NO: 126, SEQ ID NO: 130, SEQ ID NO: 144, SEQ ID NO: 148, SEQ ID NO: 152, SEQ ID NO: 156, SEQ ID NO: 160, SEQ ID NO: 164, SEQ ID NO: 168, SEQ ID NO: 172, SEQ ID NO: 176, SEQ ID NO: 180, SEQ ID NO: 184, SEQ ID NO: 188, SEQ ID NO: 192, SEQ ID NO: 196, SEQ ID NO: 200, and SEQ ID NO: 204; and a light chain having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to a sequence selected from SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 38, SEQ ID NO: 42, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO:62, SEQ ID NO: 66, 2 6778060.DOCX
Attorney Docket No.06527-2503253 SEQ ID NO: 70, SEQ ID NO: 74, SEQ ID NO: 78, SEQ ID NO: 82, SEQ ID NO: 86, SEQ ID NO: 90, SEQ ID NO: 94, SEQ ID NO: 98, SEQ ID NO:102, SEQ ID NO: 106, SEQ ID NO:110, SEQ ID NO:114, SEQ ID NO: 120, SEQ ID NO: 124, SEQ ID NO: 128, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 146, SEQ ID NO: 150, SEQ ID NO: 154, SEQ ID NO: 158, SEQ ID NO: 162, SEQ ID NO: 166, SEQ ID NO: 170, SEQ ID NO: 174, SEQ ID NO: 178, SEQ ID NO: 182, SEQ ID NO: 186, SEQ ID NO: 190, SEQ ID NO: 194, SEQ ID NO: 198, and SEQ ID NO: 202. [0010] Also provided here is a composition including an antibody as described herein and a pharmaceutically-acceptable excipient. [0011] Also provided herein is a method of determining whether a patient has or is at risk of developing a tauopathy, including contacting a sample obtained from the patient with an antibody as described herein. [0012] Also provided herein a method of determining whether a patient has or is at risk of developing a tauopathy, including obtaining a sample from a patient suspected of having or at risk of developing a tauopathy; incubating the sample with an antibody as described herein; and detecting binding between the antibody and a tau protein in the sample, wherein detecting binding between the first binding reagent and the tau protein indicates that the patient has or is at risk of developing a tauopathy. [0013] Also provided herein is a method of preventing and/or treating a patient having a tauopathy, including administering to the patient an amount of an antibody as described herein effective to prevent and/or treat the disease in the patient. [0014] Further non-limiting embodiments are set forth in the following numbered clauses: [0015] 1. An antibody that binds to a tau protein, comprising: a heavy chain having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to a sequence selected from SEQ ID NO: 4 SEQ ID NO: 8, and SEQ ID NO: 12; SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 60, SEQ ID NO: 64, SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 76, SEQ ID NO: 80, SEQ ID NO: 84, SEQ ID NO: 88, SEQ ID NO: 92, SEQ ID NO: 96, SEQ ID NO: 100, SEQ ID NO: 104, SEQ ID NO: 108, SEQ ID NO: 112, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 122, SEQ ID NO: 126, SEQ ID NO: 130, SEQ ID NO: 144, SEQ ID NO: 148, SEQ ID NO: 152, SEQ ID 3 6778060.DOCX
Attorney Docket No.06527-2503253 NO: 156, SEQ ID NO: 160, SEQ ID NO: 164, SEQ ID NO: 168, SEQ ID NO: 172, SEQ ID NO: 176, SEQ ID NO: 180, SEQ ID NO: 184, SEQ ID NO: 188, SEQ ID NO: 192, SEQ ID NO: 196, SEQ ID NO: 200, and SEQ ID NO: 204; and a light chain having an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to a sequence selected from SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 38, SEQ ID NO: 42, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO:62, SEQ ID NO: 66, SEQ ID NO: 70, SEQ ID NO: 74, SEQ ID NO: 78, SEQ ID NO: 82, SEQ ID NO: 86, SEQ ID NO: 90, SEQ ID NO: 94, SEQ ID NO: 98, SEQ ID NO:102, SEQ ID NO: 106, SEQ ID NO:110, SEQ ID NO:114, SEQ ID NO: 120, SEQ ID NO: 124, SEQ ID NO: 128, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 146, SEQ ID NO: 150, SEQ ID NO: 154, SEQ ID NO: 158, SEQ ID NO: 162, SEQ ID NO: 166, SEQ ID NO: 170, SEQ ID NO: 174, SEQ ID NO: 178, SEQ ID NO: 182, SEQ ID NO: 186, SEQ ID NO: 190, SEQ ID NO: 194, SEQ ID NO: 198, and SEQ ID NO: 202. [0016] 2. The antibody of clause 1, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 4; and the light chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 2. [0017] 3. The antibody of clause 1 or clause 2, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 8; and the light chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 6. [0018] 4. The antibody of any of clauses 1-3, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 12; and the light chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 10. [0019] 5. The antibody of any of clauses 1-4, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 16; and the light chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 14. 4 6778060.DOCX
Attorney Docket No.06527-2503253 [0020] 6. The antibody of any of clauses 1-5, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 20; and the light chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 18. [0021] 7. The antibody of any of clauses 1-6, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 24; and the light chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 22. [0022] 8. The antibody of any of clauses 1-7, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 28; and the light chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 26. [0023] 9. The antibody of any of clauses 1-8, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 32; and the light chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 30. [0024] 10. The antibody of any of clauses 1-9, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 36; and the light chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 34. [0025] 11. The antibody of any of clauses 1-10, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 40; and the light chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 38. [0026] 12. The antibody of any of clauses 1-11, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 44; and the light chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 42. [0027] 13. The antibody of any of clauses 1-12, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 48; and the light chain has an amino acid sequence 5 6778060.DOCX
Attorney Docket No.06527-2503253 having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 46. [0028] 14. The antibody of any of clauses 1-13, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 52; and the light chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 50. [0029] 15. The antibody of any of clauses 1-14, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 56; and the light chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 54. [0030] 16. The antibody of any of clauses 1-15, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 60; and the light chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 58. [0031] 17. The antibody of any of clauses 1-16, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 64; and the light chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 62. [0032] 18. The antibody of any of clauses 1-17, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 68; and the light chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 66. [0033] 19. The antibody of any of clauses 1-18, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 72; and the light chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 70. [0034] 20. The antibody of any of clauses 1-19, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% 6 6778060.DOCX
Attorney Docket No.06527-2503253 sequence identity, to SEQ ID NO: 76; and the light chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 74. [0035] 21. The antibody of any of clauses 1-20, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 80; and the light chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 78. [0036] 22. The antibody of any of clauses 1-21, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 84; and the light chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 82. [0037] 23. The antibody of any of clauses 1-22, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 88 and the light chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 86. [0038] 24. The antibody of any of clauses 1-23, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 92; and the light chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 90. [0039] 25. The antibody of any of clauses 1-24, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 96; and the light chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 94. [0040] 26. The antibody of any of clauses 1-25, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 100; and the light chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 98. 7 6778060.DOCX
Attorney Docket No.06527-2503253 [0041] 27. The antibody of any of clauses 1-26, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 104; and the light chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 102. [0042] 28. The antibody of any of clauses 1-27, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 108; and the light chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 106. [0043] 29. The antibody of any of clauses 1-28, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 112; and the light chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 110. [0044] 30. The antibody of any of clauses 1-29, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 116; and the light chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 114. [0045] 31. The antibody of any of clauses 1-30, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 118. [0046] 32. The antibody of any of clauses 1-31, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 122; and the light chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 120. [0047] 33. The antibody of any of clauses 1-32, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 126; and the light chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 124. 8 6778060.DOCX
Attorney Docket No.06527-2503253 [0048] 34. The antibody of any of clauses 1-33, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 130; and the light chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 128. [0049] 35. The antibody of any of clauses 1-34, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 144; and the light chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, or SEQ ID NO: 142. [0050] 36. The antibody of any of clauses 1-35, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 148; and the light chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 146. [0051] 37. The antibody of any of clauses 1-36, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 152; and the light chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 150. [0052] 38. The antibody of any of clauses 1-37, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 156; and the light chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 154. [0053] 39. The antibody of any of clauses 1-38, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 160; and the light chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 158. [0054] 40. The antibody of any of clauses 1-39, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 164; and the light chain has an amino acid sequence 9 6778060.DOCX
Attorney Docket No.06527-2503253 having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 162. [0055] 41. The antibody of any of clauses 1-40, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 168; and the light chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 166. [0056] 42. The antibody of any of clauses 1-41, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 172; and the light chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 170. [0057] 43. The antibody of any of clauses 1-42, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 176; and the light chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 174. [0058] 44. The antibody of any of clauses 1-43, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 180; and the light chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 178. [0059] 45. The antibody of any of clauses 1-44, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 184; and the light chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 182. [0060] 46. The antibody of any of clauses 1-45, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 188; and the light chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 186. [0061] 47. The antibody of any of clauses 1-46, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% 10 6778060.DOCX
Attorney Docket No.06527-2503253 sequence identity, to SEQ ID NO: 192; and the light chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 190. [0062] 48. The antibody of any of clauses 1-47, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 196; and the light chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 194. [0063] 49. The antibody of any of clauses 1-48, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 200; and the light chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 198. [0064] 50. The antibody of any of clauses 1-49, wherein: the heavy chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 204; and the light chain has an amino acid sequence having at least 90% sequence identity, optionally 100% sequence identity, to SEQ ID NO: 202. [0065] 51. A composition comprising an antibody of any of clauses 1-50 and a pharmaceutically-acceptable excipient. [0066] 52. A method of determining whether a patient has or is at risk of developing a tauopathy, comprising contacting a sample obtained from the patient with an antibody of any of clauses 1-50. [0067] 53. A method of determining whether a patient has or is at risk of developing a tauopathy, comprising: obtaining a sample from a patient suspected of having or at risk of developing a tauopathy; incubating the sample with an antibody of any of clauses 1-50; and detecting binding between the antibody and a tau protein in the sample, wherein detecting binding between the first binding reagent and the tau protein indicates that the patient has or is at risk of developing a tauopathy. [0068] 54. The method of clause 53, wherein the sample is a blood sample. [0069] 55. The method of clause 53 or clause 54, wherein the sample is a cerebrospinal fluid (CSF) sample. [0070] 56. The method of any of clauses 53-55, wherein the sample is a tissue sample. 11 6778060.DOCX
Attorney Docket No.06527-2503253 [0071] 57. The method of any of clauses 53-56, wherein the tissue sample is a central nervous system (CNS) tissue sample. [0072] 58. The method of any of clauses 53-57, the presence of a pre-stage NFT in the patient sample. [0073] 59. The method of any of clauses 53-58, wherein the tauopathy is Alzheimer’s Disease. [0074] 60. A method of preventing and/or treating a patient having or at risk of developing a tauopathy, comprising administering to the patient an amount of an antibody of any of clauses 1-50 effective to prevent and/or treat the disease in the patient. [0075] 61. The method of clause 60, wherein the antibody is conjugated to a therapeutic composition. [0076] 62. The method of clause 60 or clause 61, wherein the therapeutic composition comprises one or more of a cholinesterase inhibitor, a glutamate receptor modulator, lecanemab, and/or donanemab. BRIEF DESCRIPTION OF THE DRAWINGS [0077] FIGS. 1-13 show results of enzyme-linked immunosorbent assay experiments for non-limiting embodiments of antigen binding reagents as described herein. [0078] FIG. 14 shows antigens (SEQ ID NOS: 206-216) used to generate antibodies according to non-limiting embodiments described herein. DESCRIPTION OF THE INVENTION [0079] The use of numerical values in the various ranges specified in this application, unless expressly indicated otherwise, are stated as approximations as though the minimum and maximum values within the stated ranges are both preceded by the word "about". In this manner, slight variations above and below the stated ranges can be used to achieve substantially the same results as values within the ranges. Also, unless indicated otherwise, the disclosure of these ranges is intended as a continuous range including every value between the minimum and maximum values. For definitions provided herein, those definitions refer to word forms, cognates and grammatical variants of those words or phrases. As used herein "a" and "an" refer to one or more. 12 6778060.DOCX
Attorney Docket No.06527-2503253 [0080] As used herein, the term "comprising" is open-ended and may be synonymous with "including", "containing", or "characterized by". As used herein, embodiments "comprising" one or more stated elements or steps also include but are not limited to embodiments "consisting essentially of" and "consisting of" these stated elements or steps. [0081] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. It is to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Unless otherwise indicated, polymer molecular weight is expressed as number-average molecular weight (Mn). Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. [0082] The term “contacting” refers to placement in direct physical association; includes both in solid and liquid form. “Contacting” is often used interchangeably with “exposed.” In some cases, “contacting” includes transfecting, such as transfecting a nucleic acid molecule into a cell. In other examples, “contacting” refers to incubating a molecule (such as an antibody) with a biological sample. [0083] An “isolated” or “purified” biological component (such as a nucleic acid, peptide, protein, protein complex, or particle) refers to a component that has been substantially separated, produced apart from, or purified away from other components in a preparation or other biological components in the cell of the organism in which the component occurs, that is, other chromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acids, peptides and proteins that have been “isolated” or “purified”, thus, include, for example and without limitation, nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids or proteins. The term “isolated” or “purified” does not require absolute purity; rather, it is intended as a relative term. Thus, for example, an isolated biological component is one in which the biological component is more enriched than the biological component is in its natural environment within a cell, or other production vessel. A preparation may be purified such that the biological 13 6778060.DOCX
Attorney Docket No.06527-2503253 component represents at least 50%, such as at least 70%, at least 90%, at least 95%, or greater, of the total biological component content of the preparation. [0084] A nucleic acid molecule (a nucleic acid) refers to a polymeric form of nucleotides, which may include both sense and anti-sense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. A nucleotide refers to a ribonucleotide, deoxynucleotide or a modified form of either type of nucleotide. The term “nucleic acid molecule” as used herein is synonymous with “nucleic acid” and “polynucleotide.” The term includes single- and double-stranded forms of DNA. A polynucleotide may include either or both naturally occurring and modified nucleotides linked together by naturally occurring and/or non-naturally occurring nucleotide linkages. [0085] A first nucleic acid is said to be operably linked to a second nucleic acid when the first nucleic acid is placed in a functional relationship with the second nucleic acid. Generally, operably linked DNA sequences are contiguous (e.g., in cis) and, where the sequences act to join two protein coding regions, in the same reading frame (e.g., open reading frame or ORF), for example to produce a fusion protein. Operably linked nucleic acids include a first nucleic acid contiguous with the 5′ or 3′ end of a second nucleic acid. In other examples, a second nucleic acid is operably linked to a first nucleic acid when it is embedded within the first nucleic acid, for example, where the nucleic acid construct includes (in order) a portion of the first nucleic acid, the second nucleic acid, and the remainder of the first nucleic acid. [0086] A “codon-optimized” nucleic acid refers to a nucleic acid sequence that has been altered such that the codons are optimal for expression in a particular system (such as a particular species of group of species). For example, a nucleic acid sequence can be optimized for expression in yeast cells. Codon optimization does not alter the amino acid sequence of the encoded protein. [0087] A conservative substitution is a substitution of one amino acid residue in a protein sequence for a different amino acid residue having similar biochemical properties. Typically, conservative substitutions have little to no impact on the activity of a resulting polypeptide. For example, an antigen binding molecule or antibody polypeptide sequence may include one or more conservative substitutions (for example 1-10, 2-5, or 10-20, or no more than 2, 5, 10, 20, 30, 40, or 50 substitutions) yet retains the affinity or avidity of a given antigen binding molecule such as those described herein for binding to, among other possibilities, human immunodeficiency 14 6778060.DOCX
Attorney Docket No.06527-2503253 virus (HIV). A polypeptide can be produced to contain one or more conservative substitutions by manipulating the nucleotide sequence that encodes that polypeptide using, for example, standard procedures such as site-directed mutagenesis or PCR. Methods are provided herein to ascertain proper expression of any sequence. [0088] A polypeptide is a polymer in which the monomers are amino acid residues which are joined together through amide bonds. When the amino acids are alpha- amino acids, either the L-optical isomer or the D-optical isomer can be used. The terms “polypeptide”, “peptide”, or “protein” as used herein are intended to encompass any amino acid sequence and include proteins and modified sequences such as glycoproteins. The term “polypeptide” is specifically intended to cover naturally occurring proteins, as well as those which are synthetically produced such as by recombinant or chemical synthesis methods. The term “residue” or “amino acid residue” includes reference to an amino acid that is incorporated into a protein, polypeptide, or peptide. [0089] Conservative amino acid substitutions are those substitutions that, when made, least or minimally interfere with the properties of the original protein, that is, in the context of the end-use, the structure and function of the protein is conserved and not significantly changed by such substitutions, and may be identified by use of matrices, such as the BLOSUM series of matrices, and other matrices. Conservative substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in protein properties will be non-conservative, for instance changes in which (a) a hydrophilic residue, for example, seryl or threonyl, is substituted for (or by) a hydrophobic residue, for example, leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, for example, lysyl, arginyl, or histadyl, is substituted for (or by) an electronegative residue, for example, glutamyl or aspartyl; or (d) a residue having a bulky side chain, for example, phenylalanine, is substituted for (or by) one not having a side chain, for example, glycine. In terms of antibody structure, conservative substitutions may be relatively freely made to framework amino acids and constant region amino acids, such as to humanize an antigen binding molecule. 15 6778060.DOCX
Attorney Docket No.06527-2503253 [0090] As used herein, the term “epitope” refers to a physical structure or moiety of a molecule that interacts with an antibody or antibody binding reagent. In terms of proteins or polypeptides, the primary amino acid sequence can define an epitope, but secondary and tertiary protein structure, as well as post-translational modifications, can define an epitope. For example, HIV protein and protein fragments, including isoforms and post-transcriptionally-modified polypeptides for use in the immunodetection methods, devices, and kits may be produced in mammalian cells, such as HEK293 cells, to produce a protein with mammalian post-translational modifications. Portions of a natural protein can contain an epitope present in the complete natural protein and typically react to antibodies raised to the natural protein. [0091] A recombinant nucleic acid refers to a nucleic acid molecule (or protein or virus) that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids. The term recombinant includes nucleic acids and proteins that have been altered solely by addition, substitution, or deletion of a portion of a natural nucleic acid molecule or protein. [0092] “Sequence identity” refers to the similarity between nucleic acid or amino acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity may be measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs, orthologs, isoforms, or variants of a polypeptide often possess a relatively high degree of sequence identity when aligned using standard methods. Methods of alignment of sequences for comparison are well- known in the art. Various programs and alignment algorithms are described in the art (see, e.g., Chao J, et al. Developments in Algorithms for Sequence Alignment: A Review. Biomolecules.2022 Apr 6;12(4):546). [0093] Once aligned, the number of matches may be determined by counting the number of positions where an identical nucleotide or amino acid residue is present in both sequences. The percent sequence identity may be determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence, or by an articulated length (such as 100 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100. For example, a peptide sequence that has 1166 16 6778060.DOCX
Attorney Docket No.06527-2503253 matches when aligned with a test sequence having 1554 amino acids is 75.0 percent identical to the test sequence (1166÷1554*100=75.0). The percent sequence identity value may be rounded to the nearest tenth. For example, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. The length value will always be an integer. [0094] Homologs and variants of a polypeptide are typically characterized by possession of at least about 75%, for example, at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity counted over the full-length alignment with the amino acid sequence of interest. Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When less than the entire sequence is being compared for sequence identity, homologs and variants may typically possess at least 80% sequence identity over short windows of 10-20 amino acids, and may possess sequence identities of at least 85% or at least 90% or 95% depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available at the NCBI website on the internet. One of skill in the art will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided. [0095] For sequence comparison of nucleic acid sequences, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences may be entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters may be used. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, for example and without limitation, by the local homology algorithm of Smith & Waterman, by the homology alignment algorithm of Needleman & Wunsch, by the search for similarity method of Pearson & Lipman, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, Wis.), or by manual alignment and visual inspection. One example of a useful algorithm is PILEUP. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle. Using PILEUP, 17 6778060.DOCX
Attorney Docket No.06527-2503253 a reference sequence may be compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps. PILEUP can be obtained from the GCG sequence analysis software package (see, e.g., Chao J, et al. Biomolecules.2022 Apr 6;12(4):546). [0096] Another example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (ncbi.nlm.nih.gov). The BLASTN program may be used for nucleotide sequences. The BLASTP program may be used for amino acid sequences. [0097] As used herein, reference to “at least 70% identity” (or similar language) may refer to “at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity” to a specified reference sequence. As used herein, reference to “at least 90% identity” (or similar language) may refer to “at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity” to a specified reference sequence. [0098] Complementary refers to the ability of polynucleotides (nucleic acids) to hybridize to one another, forming inter-strand base pairs. Base pairs are formed by hydrogen bonding between nucleotide units in polynucleotide strands that are typically in antiparallel orientation. Complementary polynucleotide strands can base pair (hybridize) in the Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes. In RNA as opposed to DNA, uracil rather than thymine is the base that is complementary to adenosine. Two sequences comprising complementary sequences can hybridize if they form duplexes under specified conditions, such as in water, saline (e.g., normal saline, or 0.9% w/v saline) or phosphate-buffered saline), or under other stringency conditions, such as, for example and without limitation, 0.1X SSC (saline sodium citrate) to 10X SSC, where 1X SSC is 0.15M NaCl and 0.015M sodium citrate in water. Hybridization of complementary sequences is dictated, e.g., by the nucleobase content of the strands, the presence of mismatches, the length of complementary sequences, salt concentration, temperature, with the melting temperature (Tm) lowering with shorter complementary sequences, increased mismatches, and increased stringency. 18 6778060.DOCX
Attorney Docket No.06527-2503253 Perfectly matched sequences are said to be “fully complementary”, though one sequence (e.g., a target sequence in an mRNA) may be longer than the other. [0099] A vector is a nucleic acid molecule allowing insertion of foreign nucleic acid without disrupting the ability of the vector to replicate and/or integrate in a host cell. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. An insertional vector is capable of inserting itself into a host nucleic acid. A vector can also include one or more selectable marker genes and other genetic elements. An expression vector is a vector that contains the necessary regulatory sequences to allow transcription and translation of inserted gene or genes. [00100] By “expression” or “gene expression,” it is meant the overall flow of information from a gene. A “gene” is a sequence of DNA or RNA which codes for a molecule, such as a protein or a functional RNA, such as an ncRNA that has a function. A “gene” is a functional genetic unit for producing its gene product, such as RNA or a protein in a cell, or other expression system encoded on a nucleic acid and generally comprising: a transcriptional control sequence, such as a promoter and other cis- acting elements, such as transcriptional response elements (TREs) and/or enhancers; an expressed sequence that typically encodes a protein (referred to as an open- reading frame or ORF) or functional/structural RNA; and a polyadenylation sequence). A gene produces a gene product (typically a protein, optionally post-translationally modified, or a functional/structural RNA) when transcribed. By “expression of genes under transcriptional control of,” or alternately “subject to control by” a designated sequence such as a promotor, it is meant gene expression from a gene containing the designated sequence operably linked (functionally attached, typically in cis) to the gene. A gene that is “under transcriptional control” of an inducible promotor or transcription control element, is a gene that is transcribed at detectably different levels in the presence of a transcription factor, e.g., in specific cell types or conditions. A “gene for expression of” a stated gene product is a gene capable of expressing that stated gene product when placed in a suitable environment, that is, for example, when transformed, transfected, transduced, etc. into a cell, and subjected to suitable conditions for expression. In the case of a constitutive promoter “suitable conditions” means that the gene typically need only be introduced into a host cell. In the case of an inducible promoter, “suitable conditions” means when factors that regulate transcription, such as DNA-binding proteins, are present or absent, for example, an amount of the respective inducer is available to the expression system (e.g., cell), or 19 6778060.DOCX
Attorney Docket No.06527-2503253 factors causing suppression of a gene are unavailable or displaced - effective to cause expression of the gene. [00101] In further detail, transcription is the process by which the DNA gene sequence is transcribed into RNA. The steps include transcript initiation, transcript elongation, and transcript termination. The molecular machinery of transcription includes but is not limited to: RNA polymerase, general transcription factors, enhancers, and promoter DNA, and RNA transcript. Transcription factors (TFs) are proteins that control the rate of transcription of genetic information from DNA to RNA, by binding to a specific DNA sequence (e.g., the promoter region). The function of TFs is to regulate genes in order to make sure that they are expressed in the right cell at the right time and in the right amount throughout the life of the cell and the organism. The promoter region of a gene is a region of DNA that initiates transcription of that particular gene. Promoters are located near the transcription start sites of genes, on the same strand, and often, but not exclusively, are upstream (towards the 5' region of the sense strand) on the DNA. Promoters can be about 100–1000 base pairs long. Additional sequences and non-coding elements can affect transcription rates. If the cell has a nucleus (eukaryotes), the RNA is further processed. This includes polyadenylation, capping, and splicing. Polyadenylation refers to the addition of a poly(A) tail to a messenger RNA. The poly(A) tail consists of multiple adenosine monophosphates; in other words, it is a stretch of RNA that has only adenine bases. In eukaryotes, polyadenylation is part of the process that produces mature messenger RNA (mRNA) for translation. Capping refers to the process wherein the 5’ end of the pre-mRNA has a specially altered nucleotide. In eukaryotes, the 5’ cap (cap-0), found on the 5’ end of an mRNA molecule, consists of a guanine nucleotide connected to mRNA via an unusual 5’ to 5’ triphosphate linkage. During RNA splicing, pre-mRNA is edited. Specifically, during this process introns are removed, and exons are joined together. The resultant product is known as mature mRNA. The RNA may remain in the nucleus or exit to the cytoplasm through the nuclear pore complex. [00102] Gene expression involves various steps, including transcription, post- transcriptional RNA modification, translation, and post-translational modification of a protein. Expression of a gene may also include reduction of the total amount of the protein product, such as by cleavage, sequestration, binding, or other means of decreasing the function or amount of a protein product. 20 6778060.DOCX
Attorney Docket No.06527-2503253 [00103] Nucleic acids and vectors encoding the described fusion proteins may be provided. In some non-limiting examples, disclosed is a recombinant vector, such as a yeast plasmid, that expresses the disclosed fusion proteins. One of skill in the art can readily use the genetic code to construct a variety of functionally equivalent nucleic acids, such as nucleic acids which differ in sequence, but which encode the same protein sequence due to codon degeneracy. In some embodiments, the polynucleotide is codon-optimized for expression in mammalian cells. [00104] Exemplary nucleic acids may be prepared by cloning techniques, e.g., as are broadly-known and implemented either commercially, or in the art. Multiple textbooks and reference manuals describe and provide examples of useful and appropriate cloning and sequencing techniques, and instructions sufficient to direct persons of skill through such techniques are known. Commercial and public product information from manufacturers of biological reagents and experimental equipment also provide useful information. Such manufacturers include the SIGMA Chemical Company (Saint Louis, Mo.), R&D Systems (Minneapolis, Minn.), Pharmacia Amersham (Piscataway, N.J.), CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersburg, Md.), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen (Carlsbad, Calif.), Addgene, and Applied Biosystems (Foster City, Calif.), as well as many other commercial sources. [00105] Nucleic acids can also be prepared by amplification methods. Amplification methods include polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the self-sustained sequence replication system (3SR). A wide variety of cloning methods, host cells, and in vitro amplification methodologies are well known to persons of skill. [00106] Provided herein are antigen binding molecules, e.g., antibody compounds comprising an antibody domain targeting a protein, for example a tau protein (including, without limitation, recombinant proteins and/or phosphorylated proteins) and epitopes, and methods of use of those compounds. The term “antigen binding molecule”, for ease of reference and unless otherwise specified, refers to an immunoglobulin, derivatives thereof which maintain specific binding ability, and proteins having an antigen-binding domain which is homologous or largely homologous to an immunoglobulin binding domain, and complexes thereof, which are 21 6778060.DOCX
Attorney Docket No.06527-2503253 typically covalently linked, as in immunoglobulin (see, e.g., Chailyan A, Marcatili P, Tramontano A. The association of heavy and light chain variable domains in antibodies: implications for antigen specificity. FEBS J. 2011 Aug;278(16):2858-66, and US Patent No. 11,578,428 B2, and U.S. Patent Publication No. 2024/0158529, showing typical antibody structures, including humanized antibodies). As such, the antigen binding molecule operates as a ligand for its cognate antigen, which can be virtually any polypeptide or protein. Natural antibodies typically comprise two heavy chains and two light chains and are bi-valent. The interaction between the variable regions of heavy and light chain forms a binding site (e.g., a paratope, defined by a set of CDRs) capable of specifically binding an antigen. The term “VH” refers to a heavy chain variable region of an antibody. The term “VL” refers to a light chain variable region of an antibody. Antibodies may be derived from natural sources, or partly or wholly synthetically produced, and may be “humanized” to reduce immunogenicity, as is known in the related arts. An antibody may be monoclonal or polyclonal. An antibody may be a member of any immunoglobulin class, including, for example and without limitation, any of the human classes: IgG, IgM, IgA, IgD, and IgE. [00107] An antigen binding molecule or complexes thereof may be, for example and without limitation, a monoclonal antibody, including fragments, derivatives, or analogs thereof, or complexes thereof, including without limitation: Fab, Fab′, Fv fragments, single chain Fv (scFv) fragments, dsFv, Fab1 fragments, F(ab′)2 fragments, single domain antibodies, camelized (camelid) antibodies and antibody fragments, humanized antibodies and antibody fragments, and multivalent versions of the foregoing; multivalent binding reagents including without limitation: monospecific or bispecific antibodies, such as disulfide stabilized Fv fragments, scFv tandems ((ScFv)2 fragments), diabodies, triabodies, tetrabodies, which typically are covalently linked or otherwise stabilized (e.g., leucine zipper or helix stabilized) scFv fragments, bi-specific T-cell engager (BiTE, e.g., a DbTE), di-scFv (dimeric single-chain variable fragment), single-domain antibody (sdAb), or antibody binding domain fragments. Antibody fragments also include miniaturized antibodies or other engineered binding reagents that exploit the modular nature of antibody structure, comprising, often as a single chain, one or more antigen-binding or epitope-binding sequences (e.g., paratope) and, at a minimum, any other amino acid sequences needed to ensure appropriate specificity, delivery, and stability of the composition. 22 6778060.DOCX
Attorney Docket No.06527-2503253 [00108] scFv molecules may be manufactured using any suitable technology. Typically, recombinant cells comprising genes for expressing scFv-containing polypeptides are engineered, e.g., according to decades-old methods using any of a variety of publicly- and commercially-available expression systems. Huston J. S., M. Mudgett-Hunter, M. S. Tai et al., “Protein engineering of single-chain Fv analogs and fusion proteins, ”Methods in Enzymology, vol.203, pp.46–88, 1991; Ahmad ZA, Yeap SK, Ali AM, Ho WY, Alitheen NB, Hamid M. scFv antibody: principles and clinical application. Clin Dev Immunol. 2012;2012:980250; Gąciarz A, Ruddock LW. Complementarity determining regions and frameworks contribute to the disulfide bond independent folding of intrinsically stable scFv. PLoS One. 2017 Dec 18;12(12):e0189964; Sandomenico A, Sivaccumar JP, Ruvo M. Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments. Int J Mol Sci.2020 Aug 31;21(17):6324; Petrus MLC, Kiefer LA, Puri P, Heemskerk E, Seaman MS, Barouch DH, Arias S, van Wezel GP, Havenga M. A microbial expression system for high-level production of scFv HIV-neutralizing antibody fragments in Escherichia coli. Appl Microbiol Biotechnol.2019 Nov;103(21-22):8875- 8888; and Toleikis L, Frenzel A. Cloning single-chain antibody fragments (ScFv) from hybridoma cells. Methods Mol Biol. 2012;907:59-71; see, also, www.kbdna.com/cloning-scfv [00109] The antigen binding molecules described herein, comprise, at their core paratopes formed from VL and VH polypeptides, that are defined by three CDR’s (typically loops), CDR1, CDR2, and CDR3, which for VH peptides may be termed HCDR1, HCDR2, and HCDR3, respectively, and which for VL peptides may be termed LCDR1, LCDR2, and LCDR3, respectively, each of which are flanked by, and separated by framework (e.g., joining or scaffold) amino acid sequences that space apart and support the CDRs, and which may differ from antibody-to-antibody, and which may be “humanized” to minimize antigenicity when administered to a human patient. In nature, HCDR3 and LCDR3 are typically the most variable of the CDRs, contributing significantly to antibody specificity. Various methods may be used to identify the precise limits of each CDR, but the sequences provided herein can be evaluated by any suitable method to determine the CDRs. [00110] Exemplary sequences of antibody heavy and light chains are provided in the attached sequence listing, which is incorporated herein by reference in its entirety. Antibody constant and variable regions, including CDR sequences and framework 23 6778060.DOCX
Attorney Docket No.06527-2503253 sequences can be readily ascertained from the sequences provided in in the attached sequence listing. Reference to a CDR herein may refer to a Kabat CDR numbering scheme or any other applicable CDR numbering scheme, including but not limited to Chothia, Martin (enhanced Chothia), Gelfand, IMGT, Honneger, or any other numbering scheme (see, e.g., Dondelinger M, et al. Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface/Residue Definition. Front Immunol. 2018 Oct 16;9:2278). For example and without limitation, antibody sequence annotation, including definition of CDR sequences may be conducted using tools described and provided in abYsis (abysis.com), or Abnum (www.bioinf.org.uk/abs/abnum/). Other methods of CDR identification are known in the art (see, e.g., Kunik V, Ashkenazi S, Ofran Y. Paratome: an online tool for systematic identification of antigen-binding regions in antibodies based on sequence or structure. Nucleic Acids Res. 2012 Jul;40(Web Server issue):W521-4; Adolf- Bryfogle J, Xu Q, North B, Lehmann A, Dunbrack RL Jr. PyIgClassify: a database of antibody CDR structural classifications. Nucleic Acids Res. 2015 Jan;43(Database issue):D432-8), and assorted online tools and applications as are broadly-available. As such, an amino acid sequence of a VH or VL may be provided or determined, comprising CDRs, and one of ordinary skill can determine the precise metes and bounds of CDRs within that antibody sequence without undue experimentation. Framework sequences may be optimized (see, e.g., Gopal R, Fitzpatrick E, Pentakota N, Jayaraman A, Tharakaraman K, Capila I. Optimizing Antibody Affinity and Developability Using a Framework-CDR Shuffling Approach-Application to an Anti- SARS-CoV-2 Antibody. Viruses.2022 Nov 30;14(12):2694) and/or humanized based on knowledge of amino acid sequences of the CDRs, e.g., LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 of antibodies described herein. [00111] Antibodies may be produced by any effective method, such as by hybridoma or it may be recombinantly or synthetically produced. In the context of the present disclosure and for ease of reference, “antibodies” or “antibody” may refer to both natural antibodies as well as protein antibody analogs, antibody fragments, and derivatives, any of which comprising VL and/or VH sequences and/or CDRs (e.g., all three CDRs of any VH or VL region, defining a paratope as described herein) according to any example, aspect, or embodiment described herein. The antibody or antibodies may be synthetic, in that they do not comprise a naturally-occurring sequence, such as certain antigen binding molecules, including engineered versions 24 6778060.DOCX
Attorney Docket No.06527-2503253 and derivatives thereof, such as scFv versions thereof, humanized versions thereof, BiTEs, and/or sequence derivatives thereof, including without limitation, proteins comprising the CDRs (e.g., one or more, or all three CDRs) of the antibodies provided herein. [00112] Nanobodies, which may be referred to a VHH antibodies or single-domain antibodies, may be constructed using CDR sequences, such as CDRs of the antibodies described herein. Nanobodies may be created by grafting of the complementarity determining regions (CDRs) from already existing, non-camelid antibodies to VHH frameworks, followed by affinity maturation using synthetic phage libraries (see, e.g., Wagner HJ, Wehrle S, Weiss E, Cavallari M, Weber W. A Two- Step Approach for the Design and Generation of Nanobodies. Int J Mol Sci.2018 Nov 2;19(11):3444). A VH, alone, may be capable of defining an antigen-binding site (e.g., paratope) with sufficient strength to be pharmacologically-useful, and can be referred to as a nanobody (see, e.g., Wesolowski J, Alzogaray V, Reyelt J, Unger M, Juarez K, Urrutia M, Cauerhff A, Danquah W, Rissiek B, Scheuplein F, Schwarz N, Adriouch S, Boyer O, Seman M, Licea A, Serreze DV, Goldbaum FA, Haag F, Koch-Nolte F. Single domain antibodies: promising experimental and therapeutic tools in infection and immunity. Med Microbiol Immunol. 2009 Aug;198(3):157-74, providing structure and sequences of various nanobodies and Bever CS, Dong JX, Vasylieva N, Barnych B, Cui Y, Xu ZL, Hammock BD, Gee SJ. VHH antibodies: emerging reagents for the analysis of environmental chemicals. Anal Bioanal Chem. 2016 Sep;408(22):5985- 6002). Single-domain antigen binding molecules originally were camelid antibodies, which naturally comprise only heavy chains (see, e.g., Mitchell LS, Colwell LJ. Comparative analysis of nanobody sequence and structure data. Proteins. 2018 Jul;86(7):697-706). More recently other single-chain antigen-binding molecules have been developed. Construction and humanization of single-domain antigen binding molecules is broadly-known (see, e.g., Valdés-Tresanco MS, Molina-Zapata A, Pose AG, Moreno E. Structural Insights into the Design of Synthetic Nanobody Libraries. Molecules.2022 Mar 28;27(7):2198; Wu Y, Jiang S, Ying T. Single-Domain Antibodies As Therapeutics against Human Viral Diseases. Front Immunol.2017 Dec 13;8:1802; Hoey RJ, Eom H, Horn JR. Structure and development of single domain antibodies as modules for therapeutics and diagnostics. Exp Biol Med (Maywood). 2019 Dec;244(17):1568-1576; Rossotti MA, Bélanger K, Henry KA, Tanha J. Immunogenicity and humanization of single-domain antibodies. FEBS J. 2022 25 6778060.DOCX
Attorney Docket No.06527-2503253 Jul;289(14):4304-4327; and Khodabakhsh F, Behdani M, Rami A, Kazemi-Lomedasht F. Single-Domain Antibodies or Nanobodies: A Class of Next-Generation Antibodies. Int Rev Immunol. 2018;37(6):316-322). Multimerization methods are broadly-known, too (see, e.g., Miller A, Carr S, Rabbitts T, Ali H. Multimeric antibodies with increased valency surpassing functional affinity and potency thresholds using novel formats. MAbs. 2020 Jan-Dec;12(1):1752529), for example to produce bi-specific antibody binding molecules, such as bi-specific T-cell engagers (e.g., BiTEs), discussed in further detail, below. Nanobody construction has been commercialized, e.g. in Crescendo Biologics’ Humabody platform (see, Teng Y, et al., Diverse human VH antibody fragments with bio-therapeutic properties from the Crescendo Mouse. N Biotechnol. 2020 Mar 25;55:65-76, US 11,547,099 B2, and WO 2016/062988 for exemplary constructs, transgenic mice, and methods for producing VH nanobodies). The term “antigen-binding molecule” is used interchangeably with “antibody” herein. [00113] An antibody-drug conjugate (ADC) may be provided. An antigen binding molecule according to any aspect, embodiment, or example provided described herein may be linked to a payload (e.g., a cargo or warhead) that causes a desired physiological effect, such as killing a cell expressing a binding partner to the antigen binding molecule. The antigen binding molecule, e.g. an antibody, can be effectively covalently-linked to other moieties, for example by their Fc sequences yet retain significant antigen-binding capacity. ADCs comprise an antigen binding moiety (a linked antigen-binding molecule), a linker that may be cleavable or non-cleavable, and the payload moiety. A “moiety” is a chemical group or entity, often functional, that forms part of a larger molecule. Linkers may be used to join a payload moiety to the antigen binding molecule, and choice of linkers can depend on how the APC is handled by the cell, and how the payload becomes effective on processing by a cell, or by release due to chemical lability of the linker. Non-cleavable linkers include, without limitation, alkyl moieties, and thioether moieties (e.g., Succinimidyl 4-(N- maleimidomethyl)cyclohexane-1-carboxylate (SMCC)). Cleavable or labile linkers may include, for example and without limitation, acid-labile linkers (hydrolysable in lysosomes or endosomes), Lysosomal protease–sensitive linkers (e.g., peptide-based linkers), β-glucuronide linkers, and glutathione-sensitive disulfide linkers, with examples including, without limitation: ester-, hydrazone-, Valine-citrulline (v-c)-, Valine-alanine (v-a)-, and phenylalanine-lysine (p-l)-containing linkers. (see, e.g., Khongorzul P, Ling CJ, Khan FU, Ihsan AU, Zhang J. Antibody-Drug Conjugates: A 26 6778060.DOCX
Attorney Docket No.06527-2503253 Comprehensive Review. Mol Cancer Res.2020 Jan;18(1):3-19, describing exemplary linking methods and suitable cytotoxic payloads or warheads). Examples of payloads include, without limitation: microtubule-disrupting agents, such as auristatin, maytansinoids, eribulin (e.g., eribulin mesylate), tubulysins, cryptophycins, and EG5 inhibitors; DNA-damaging agents, such as, without limitation calicheamicin, duocarmycins, doxorubicin, enediyne, topoisomerase I inhibitors, and Pyrrolo[2,1- c][1,4] benzodiazepines; RNA-targeting payloads, such as thailanstatins and amatoxins; immune payloads, such as Toll-like receptor agonists, STING agonists, glucocorticoid receptor modulators; and other payloads, such as Bcl-xL inhibitors, NAMPT inhibitors, and proteasome inhibitors such as carmaphycins. Design and optimization considerations for production of ADCs are provided in Khongorzul P, et al. (Khongorzul P, Ling CJ, Khan FU, Ihsan AU, Zhang J. Antibody-Drug Conjugates: A Comprehensive Review. Mol Cancer Res. 2020 Jan;18(1):3-19, describing exemplary linking methods and suitable cytotoxic payloads or warheads, and see, e.g., Gogia P, Ashraf H, Bhasin S, Xu Y. Antibody-Drug Conjugates: A Review of Approved Drugs and Their Clinical Level of Evidence. Cancers (Basel).2023 Jul 30;15(15):3886; Baah S, Laws M, Rahman KM. Antibody-Drug Conjugates-A Tutorial Review. Molecules. 2021 May 15;26(10):2943; and Wang Z, Li H, Gou L, Li W, Wang Y. Antibody-drug conjugates: Recent advances in payloads. Acta Pharm Sin B. 2023 Oct;13(10):4025-4059). ADCs with multiple payloads, PROTAC-guided ADCs, ADCs with peptide-drug-conjugates, and ADCs with photo-reactive payloads also may be produced. As such a person of ordinary skill can produce, based on the teachings herein and without undue experimentation, an ADC comprising an antigen binding molecule as described herein linked to a cytotoxic payload via a chemical linker. In non-limiting embodiments, an antibody as disclosed herein may be linked and/or bound to a cholinesterase inhibitor (e.g., donepezil/Aricept), a glutamate receptor modulators (e.g., memantine), and/or anti-Aβ monoclonal antibodies (e.g., lecanemab or donanemab). [00114] As used herein, the “treatment” or “treating” of a patient means administration to a patient by any suitable dosage regimen, procedure and/or administration route of a composition, device, or structure (e.g., an antigen binding molecule or antibody as described herein) with the object of achieving a desirable clinical/medical end-point, including but not limited to, any suitable treatment for a condition associated with a protein, including a tau protein, and also includes 27 6778060.DOCX
Attorney Docket No.06527-2503253 monitoring the patient by any useful method, including by use of an antigen binding molecule described herein. In non-limiting embodiments, the condition is a tauopathy, such as Alzheimer’s Disease. “Treatment” or “treating” as used herein may also refer to prevention, for example, through administration of the compositions described herein as a vaccine, for example to a patient who may be at risk of developing a condition or disease, such as a tauopathy. [00115] A "therapeutically effective amount" refers to an amount of a drug product or active agent effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. An “amount effective” for treatment of a condition is an amount of an active agent or dosage form, such as a single dose or multiple doses, effective to achieve a determinable end-point. The “amount effective” is preferably safe - at least to the extent the benefits of treatment outweighs the detriments, and/or the detriments are acceptable to one of ordinary skill and/or to an appropriate regulatory agency, such as the U.S. Food and Drug Administration. A therapeutically effective amount of an active agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the active agent to elicit a desired response in the individual. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount may be less than the therapeutically effective amount. [00116] Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the composition may be administered continuously or in a pulsed fashion with doses or partial doses being administered at regular intervals, for example, every 10, 15, 20, 30, 45, 60, 90, or 120 minutes, every 2 through 12 hours daily, or every other day, etc., be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. In some instances, it may be especially advantageous to formulate compositions, such as parenteral or inhaled compositions, in dosage unit form for ease of administration and uniformity of dosage. The specification for the dosage unit forms are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic or prophylactic 28 6778060.DOCX
Attorney Docket No.06527-2503253 effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals. [00117] An “effective amount” or “amount effective” to achieve a desirable therapeutic, pharmacological, medicinal, or physiological effect is any amount that achieves the stated purpose, for example, an amount of an active agent (antigen binding molecule or antibody) described herein effective to treat a condition described herein. Based on the teachings provided herein, one of ordinary skill can readily ascertain effective amounts of the elements of the described dosage form and produce a safe and effective dosage form and drug product. Examples of an effective amount of an active agent compounded in a delivery vehicle includes from 1 µg/ml (micrograms per milliliter) to 100 mg/ml of solution, including any increment therebetween, such as from 1 µg/ml to 1 mg/ml (milligram/milliliter), all values and subranges therebetween inclusive. [00118] Drug products, or pharmaceutical compositions comprising an active agent (e.g., drug), may be prepared by any method known in the pharmaceutical arts, for example, by bringing into association the active ingredient with the carrier(s) or excipient(s). As used herein, a “pharmaceutically acceptable excipient”, “carrier”, or “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable excipients include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations thereof. In many cases, it may be preferable to include isotonic agents, for example, sugars, polyalcohol’s such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives, or buffers, which enhance the shelf life or effectiveness of the active agent. In certain aspects, the active compound may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used in delivery systems, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are broadly-known to those skilled in the art. The preferred form may depend on the intended mode of administration and 29 6778060.DOCX
Attorney Docket No.06527-2503253 therapeutic application, which will in turn dictate the types of carriers/excipients. Suitable forms include, but are not limited to, liquid, semi-solid, and solid dosage forms. [00119] Pharmaceutical formulations adapted for oral administration may be presented, for example and without limitation, in capsules, tablets, oral solutions, or the like, and include suitable carriers and coatings as are broadly-known in the pharmaceutical arts. [00120] Pharmaceutical formulations adapted for parenteral administration may be presented, for example and without limitation, in syringes, vials, bottles, IV/infusion bags, or the like, as are broadly-known to those of ordinary skill. Excipients include, for example and without limitation, water, saline, PBS, lactated Ringers, or any other injectable carriers. Suitable emulsifiers, lipids, surfactants, or the like may be utilized to maintain an active agent in solution. [00121] Pharmaceutical formulations adapted for transdermal administration may be presented, for example and without limitation, as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time or electrodes for iontophoretic delivery. [00122] Pharmaceutical formulations adapted for topical administration may be formulated, for example and without limitation, as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols, or oils. [00123] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. For example, sterile injectable solutions can be prepared by incorporating the active agent in the required amount in an appropriate solvent with suitable carrier(s), followed by filter-sterilization. An appropriate fluidity of a solution can be maintained, for example, by the use of a rheology modifier. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin. [00124] The phrase “pharmaceutically-acceptable carrier” as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium, zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. 30 6778060.DOCX
Attorney Docket No.06527-2503253 [00125] The therapeutic agents described herein can be administered by any effective route. Examples of delivery routes include, without limitation: topical, for example, epicutaneous, inhalational, enema, ocular, otic, and intranasal delivery; enteral, for example, orally, by gastric feeding tube, and rectally; and parenteral, such as, intravenous, intraarterial, intrathecally, intramuscular, intracardiac, subcutaneous, intraosseous, intradermal, intrathecal, intraperitoneal, transdermal, iontophoretic, transmucosal, epidural, and intravitreal, with intrathecal and oral approaches being preferred in many instances. Suitable dosage forms may include single-dose, or multiple-dose vials or other containers, such as medical syringes, containing a composition comprising the therapeutic agent useful for treatment of graft rejection as described herein. [00126] Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the therapeutic agent may be administered continuously or in a pulsed fashion with doses or partial doses being administered at regular intervals, for example, every 10, 15, 20, 30, 45, 60, 90, or 120 minutes, every 2 through 12 hours daily, or every other day, etc., be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. In some instances, it may be especially advantageous to formulate therapeutic agents in dosage unit form for ease of administration and uniformity of dosage. The specification for the dosage unit forms may be dictated by and directly dependent on (a) the unique characteristics of the therapeutic agent and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such a therapeutic agent for the treatment of sensitivity in individuals. [00127] Provided herein are antigen binding reagents for binding tau proteins. The term "binding reagent", for ease of reference and unless otherwise specified, is used interchangeably herein with “antigen-binding reagent,” antigen-binding molecule,” and like terms, and may refer to an immunoglobulin, and/or fragments or derivatives thereof which maintain specific binding ability, and proteins having an antigen-binding domain which is homologous or largely homologous to an immunoglobulin binding domain, and complexes thereof, which are typically covalently linked, as in immunoglobulin (see, e.g., Chailyan et al. The association of heavy and light chain variable domains in antibodies: implications for antigen specificity. FEBS J. 2011 31 6778060.DOCX
Attorney Docket No.06527-2503253 Aug;278(16):2858-66, U.S. Patent No. 11,578,428, and U.S. Patent Publication No. 2024/0158529, showing typical antibody structures, including humanized antibodies, each of which is incorporated herein by reference in its entirety). An antigen-binding molecule (a term used interchangeably herein with “binding reagent” and “antibody”) may comprise a nucleic acid, as in the case of an aptamer. As such, the binding reagent operates as a ligand for its cognate antigen, which can be virtually any polypeptide or protein. Natural antibodies typically comprise two heavy chains and two light chains and are bi-valent. The interaction between the variable regions of heavy and light chain forms a binding site (e.g., a paratope, defined by a set of CDRs) capable of specifically binding an antigen. The term "VH" refers to a heavy chain variable region of an antibody. The term "VL" refers to a light chain variable region of an antibody. Antibodies may be derived from natural sources, or partly or wholly synthetically produced, and may be "humanized" to reduce immunogenicity, as is known in the related arts. An antibody may be monoclonal or polyclonal. An antibody may be a member of any immunoglobulin class, including, for example and without limitation, any of the human classes: IgG, IgM, IgA, IgD, and IgE. [00128] Antigen-binding molecules bind specifically to a target, e.g., an epitope and are therefore "target-specific". By "target-specific" or reference to the ability of one compound to bind another target compound specifically, it is meant that the compound binds to the target compound to the exclusion of others in a given reaction system, e.g., in vitro, or in vivo, to acceptable tolerances, permitting a sufficiently specific diagnostic or therapeutic effect according to the standards of a person of skill in the art, a medical community, and/or a regulatory authority, such as the U.S. Food and Drug Agency (FDA), in aspects, in the context of use of antibodies or binding reagents described herein for detection of tau proteins in blood and/or CNS tissue samples according to various aspects, embodiments, or examples of the methods provided herein. [00129] The binding reagent may optionally be a single chain antibody fragment. Alternatively, the binding reagent may comprise multiple chains which are linked together, for instance, by disulfide linkages. The binding reagent may also optionally be a multi-molecular complex. A functional binding reagent may consist of at least about 50 amino acids or at least about 200 amino acids. Binding reagent also includes miniaturized antibodies or other engineered binding reagents, such as scFvs, that exploit the modular nature of antibody structure, comprising, often as a single chain, 32 6778060.DOCX
Attorney Docket No.06527-2503253 one or more antigen-binding or epitope-binding (e.g., paratope) sequences and, at a minimum, any other amino acid sequences needed to ensure appropriate specificity, delivery, and stability of the composition. [00130] A binding reagent or complexes thereof may be, for example and without limitation, a monoclonal antibody, a polyclonal antibody, including fragments, derivatives, or analogs thereof, or complexes thereof, including without limitation: Fab, Fab’, Fv fragments, single chain Fv (scFv) fragments, dsFv, Fab1 fragments, F(ab’)2 fragments, single domain antibodies, humanized antibodies and antibody fragments, and multivalent versions of the foregoing; multivalent binding reagents including without limitation: monospecific or bispecific antibodies, such as disulfide stabilized Fv fragments, scFv tandems ((ScFv)2 fragments), diabodies, triabodies, tetrabodies, which typically are covalently linked or otherwise stabilized (e.g., leucine zipper or helix stabilized) scFv fragments, bi-specific T-cell engager (BiTE, e.g., a DbTE), di-scFv (dimeric single-chain variable fragment), single-domain antibody (sdAb), or antibody binding domain fragments. Antibody fragments also include miniaturized antibodies or other engineered binding reagents that exploit the modular nature of antibody structure, comprising, often as a single chain, one or more antigen-binding or epitope- binding sequences (e.g., paratope) and, at a minimum, any other amino acid sequences needed to ensure appropriate specificity, delivery, and stability of the composition. [00131] scFv molecules may be manufactured using any suitable technology. Typically, recombinant cells comprising genes for expressing scFv-containing polypeptides are engineered, e.g., according to decades-old methods using any of a variety of publicly- and commercially available expression systems. Huston J. S., M. Mudgett-Hunter, M. S. Tai et al., "Protein engineering of single-chain Fv analogs and fusion proteins, "Methods in Enzymology, vol.203, pp.46-88, 1991; Ahmad ZA, Yeap SK, Ali AM, Ho WY, Alitheen NB, Hamid M. scFv antibody: principles and clinical application. Clin Dev Immunol. 2012;2012:980250; Gaciarz A, Ruddock LW. Complementarity determining regions and frameworks contribute to the disulfide bond independent folding of intrinsically stable scFv. PLoS One. 2017 Dec 18;12(12):e0189964; Sandomenico A, Sivaccumar JP, Ruvo M. Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments. Int J Mol Sci.2020 Aug 31;21(17):6324; Petrus MLC, Kiefer LA, Puri P, Heemskerk E, Seaman MS, Barouch DH, Arias S, van Wezel GP, Havenga M. A microbial 33 6778060.DOCX
Attorney Docket No.06527-2503253 expression system for high-level production of scFv HIV-neutralizing antibody fragments in Escherichia coli. Appl Microbiol Biotechnol.2019 Nov;103(21-22):8875- 8888; and Toleikis L, Frenzel A. Cloning single-chain antibody fragments (ScFv) from hybridoma cells. Methods Mol Biol. 2012;907:59-71; see, also,
of binding reagents are described specifically herein, any derivative of such binding reagents reactive with the specified antigen (e.g., peptides comprising tau epitopes, including human tau epitopes, including phosphorylated epitopes) may be utilized in the specified assays. Effective binding may be evaluated in an affinity assay, such as an ELISA assay, bilayer interferometry (e.g., BLItz, see, e.g., Müller-Esparza H, Osorio-Valeriano M, Steube N, Thanbichler M, Randau L. Bio-Layer Interferometry Analysis of the Target Binding Activity of CRISPR-Cas Effector Complexes. Front Mol Biosci. 2020 May 27;7:98), single- molecule array (e.g., SIMOA), surface plasmon resonance (SPR), oblique-incidence reflectivity difference (OI-RD) binding affinity, or cell binding assay, among other antibody specificity, affinity, and/or avidity assay methods. Binding reagents with high binding affinities may bind to their corresponding antigen with a KD of 10 µM or less, 500 nM or less, 100nM or less, 75nM or less, 50nM or less, or 25 nM or less. [00133] In non-limiting embodiments, the antigen binding molecule may be an antibody, or fragment or scFv thereof, having a heavy chain having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 99% or greater, and/or 100% sequence identity to SEQ ID NO: 4 SEQ ID NO: 8, and SEQ ID NO: 12; SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 60, SEQ ID NO: 64, SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 76, SEQ ID NO: 80, SEQ ID NO: 84, SEQ ID NO: 88, SEQ ID NO: 92, SEQ ID NO: 96, SEQ ID NO: 100, SEQ ID NO: 104, SEQ ID NO: 108, SEQ ID NO: 112, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 122, SEQ ID NO: 126, SEQ ID NO: 130, SEQ ID NO: 144, SEQ ID NO: 148, SEQ ID NO: 152, SEQ ID NO: 156, SEQ ID NO: 160, SEQ ID NO: 164, SEQ ID NO: 168, SEQ ID NO: 172, SEQ ID NO: 176, SEQ ID NO: 180, SEQ ID NO: 184, SEQ ID NO: 188, SEQ ID NO: 192, SEQ ID NO: 196, SEQ ID NO: 200, and/or SEQ ID NO: 204. In non-limiting embodiments, the antigen binding molecule may be an antibody, or fragment or scFv thereof, having a light chain having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 34 6778060.DOCX
Attorney Docket No.06527-2503253 90% or greater, 95% or greater, 99% or greater, and/or 100% sequence identity to SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 38, SEQ ID NO: 42, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO:62, SEQ ID NO: 66, SEQ ID NO: 70, SEQ ID NO: 74, SEQ ID NO: 78, SEQ ID NO: 82, SEQ ID NO: 86, SEQ ID NO: 90, SEQ ID NO: 94, SEQ ID NO: 98, SEQ ID NO:102, SEQ ID NO: 106, SEQ ID NO:110, SEQ ID NO:114, SEQ ID NO: 120, SEQ ID NO: 124, SEQ ID NO: 128, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 146, SEQ ID NO: 150, SEQ ID NO: 154, SEQ ID NO: 158, SEQ ID NO: 162, SEQ ID NO: 166, SEQ ID NO: 170, SEQ ID NO: 174, SEQ ID NO: 178, SEQ ID NO: 182, SEQ ID NO: 186, SEQ ID NO: 190, SEQ ID NO: 194, SEQ ID NO: 198, and/or SEQ ID NO: 202. In non- limiting embodiments, the sequence of the heavy and/or light chain may be varied, so long as the antibody is capable of binding a tau protein, including phosphorylated amino acids and/or regions, specifically brain-derived tau (tau-441; UnoProt ID P10636-8) phosphorylated positions 181, 202, 205, 217, 356, and tau-441 regions 111 to 130, and big-tau (UniProt ID P10636-1) regions 216-232. [00134] In non-limiting embodiments, the antigen binding molecule may be a humanized antibody having a heavy chain and/or light chain have a sequence substantially similar to those disclosed herein. Methods for humanizing an antibody are known to those of skill in the art. [00135] Also provided herein are methods that allow for highly sensitive and accurate detection of tau proteins, including phosphorylated tau proteins, which may allow for diagnosis of tauopathies, such as Alzheimer’s Disease (AD) or pre-AD, in a patient. Such methods allow for earlier diagnoses, and thus earlier implementation of treatments and/or other interventions, potentially slowing progression and/or increasing survival rates. [00136] In non-limiting embodiments a method may include detecting a tau protein in a patient sample. The sample may be obtained directly from a patient and/or may be obtained from a sample repository. The sample may be a tissue sample, a fluid sample, and/or any other suitable sample obtainable from a patient and likely to have a tau protein therein. In non-limiting embodiments, the sample is from a patient’s CNS (e.g., neural tissue (including one or more neurons, glial cells (e.g., astrocytes) from one or more of hippocampus, entorhinal cortex, or basal forebrain tissue, and/or the 35 6778060.DOCX
Attorney Docket No.06527-2503253 like, cerebral-spinal fluid (CSF), and/or any other neural sample). In non-limiting embodiments, the sample may be a blood sample, a plasma sample, and/or the like. In non-limiting embodiments, the patient has been diagnosed with a tauopathy. In non-limiting embodiments, the patient is at risk of developing a tauopathy, based on genetics, one or more co-morbidities, age, family history, and/or the like. [00137] A method may include, in non-limiting embodiments, incubating the sample with a composition that includes a first binding reagent. [00138] Continuing with the method, in non-limiting embodiments the first binding reagent is specific to a region of the tau protein. As used herein a “tau protein” means a protein having, in a human, an amino acid sequence of SEQ ID NO: 205, or a sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, and/or 99% or greater sequence identity to SEQ ID NO: 205. In non-limiting embodiments, the binding reagent is sensitive to one or more specific positions of the tau protein, including positions 216 to 232 of big-tau (UniProt ID P10636-1). By region it is meant a region encompassing 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more amino acids, all values and subranges therebetween inclusive. In non-limiting embodiments, the first binding reagent may be specific to a phosphorylated amino acid. In non-limiting embodiments the first binding reagent may be an IgG. In non-limiting embodiments, the first binding reagent may be diluted prior to incubation with the sample, for example, diluted 1:250. Dilutions of the first binding reagent for optimal signal to noise ratio may be optimized as is known in the art. [00139] In non-limiting embodiments, the method may include detecting binding between the first binding reagent and the tau protein, where such detecting indicates the presence of a tauopathy (or a likelihood of developing a tauopathy). Suitable methods of detecting binding may include, for example and without limitation, immunoprecipitation assays, including binding reagents that are themselves bound to a substrate, such as a bead, as are known in the art. In non-limiting embodiments the binding reagent (e.g., antibody or fragment thereof) may be conjugated to a substrate that may, for example, increase the ability to isolate the bound protein. In non-limiting embodiments, the substrate may be a bead, for example an agarose bead or a magnetic bead. Suitable substrates may be modified as is known in the art, for example with coatings that properly configure the binding reagent, that improve signaling during various blotting procedures, and/or that improve adhesion. 36 6778060.DOCX
Attorney Docket No.06527-2503253 [00140] In non-limiting embodiments, the method further includes washing the immunoprecipitated tau protein. Washing may be performed with any suitable solvent and/or buffer. In non-limiting embodiments, the washing is conducted with saline, for example a buffered saline such as phosphate-buffered saline (PBS). [00141] In non-limiting embodiments, the method further includes eluting the washed, immunoprecipitated tau peptide (and substrate, e.g., beads), thereby generating free a protein/binding reagent complex (e.g., free of the beads). In non- limiting embodiments, the elution may be conducted with one or more solutions, for example, a glycine-containing buffer (e.g., a glycine elution buffer). [00142] In non-limiting embodiments the first binding reagent is an antibody, for example those disclosed herein, a fragment thereof, and/or an scFv that binds to a region of the tau protein and/or to a phosphorylated equivalent thereof. In non-limiting embodiments, the first binding reagent may be an antibody having a heavy chain having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 99% or greater, and/or 100% sequence identity to SEQ ID NO: 4 SEQ ID NO: 8, and SEQ ID NO: 12; SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 60, SEQ ID NO: 64, SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 76, SEQ ID NO: 80, SEQ ID NO: 84, SEQ ID NO: 88, SEQ ID NO: 92, SEQ ID NO: 96, SEQ ID NO: 100, SEQ ID NO: 104, SEQ ID NO: 108, SEQ ID NO: 112, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 122, SEQ ID NO: 126, SEQ ID NO: 130, SEQ ID NO: 144, SEQ ID NO: 148, SEQ ID NO: 152, SEQ ID NO: 156, SEQ ID NO: 160, SEQ ID NO: 164, SEQ ID NO: 168, SEQ ID NO: 172, SEQ ID NO: 176, SEQ ID NO: 180, SEQ ID NO: 184, SEQ ID NO: 188, SEQ ID NO: 192, SEQ ID NO: 196, SEQ ID NO: 200, and/or SEQ ID NO: 204. In non-limiting embodiments, the first binding reagent may be an antibody having a light chain having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 99% or greater, and/or 100% sequence identity to SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 38, SEQ ID NO: 42, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO:62, SEQ ID NO: 66, SEQ ID NO: 70, SEQ ID NO: 74, SEQ ID NO: 78, SEQ ID NO: 82, SEQ ID NO: 86, SEQ ID NO: 90, SEQ ID NO: 94, SEQ ID NO: 98, SEQ ID NO:102, SEQ ID NO: 106, SEQ ID NO:110, SEQ ID NO:114, SEQ ID NO: 120, SEQ 37 6778060.DOCX
Attorney Docket No.06527-2503253 ID NO: 124, SEQ ID NO: 128, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 146, SEQ ID NO: 150, SEQ ID NO: 154, SEQ ID NO: 158, SEQ ID NO: 162, SEQ ID NO: 166, SEQ ID NO: 170, SEQ ID NO: 174, SEQ ID NO: 178, SEQ ID NO: 182, SEQ ID NO: 186, SEQ ID NO: 190, SEQ ID NO: 194, SEQ ID NO: 198, and/or SEQ ID NO: 202. [00143] In non-limiting embodiments, the method may include an eluting step. In non-limiting embodiments, following elution with a suitable solution, the protein and binding reagent complex can be used in an assay. For example, and without limitation, such an assay may include a mass spectrometry assay, a chromatography assay, a binding assay (e.g., a Western Blot), and/or any assay known to those of skill in the art useful for identification and/or quantification of proteins. [00144] In non-limiting embodiments, the immunoprecipitated protein may be stained, for example with a dye, for example with a dye that allows for localization of binding between the first binding reagent and the tau protein. In such embodiments, the binding reagent may be labeled and the signal from that label may be compared to the staining from the dye. [00145] In non-limiting embodiments, the patient sample may be from a human patient (though, as noted above, other species are contemplated and fall within the scope of this disclosure). In non-limiting embodiments, the patient sample is from a living patient. In non-limiting embodiments, the patient sample is from a living patient and, when a tau protein is detected based on the methods described herein, the patient may be treated for early-stage tauopathy, for example early-stage AD, for example, with therapeutic agents such as cholinesterase inhibitors (e.g., donepezil/Aricept), glutamate receptor modulators (e.g., memantine), anti-Aβ monoclonal antibodies (e.g., lecanemab or donanemab), and/or cognitive/behavioral interventions, such as cognitive rehabilitation, exercise, and/or the like. [00146] Also provided herein are methods of generating an antibody, for example a recombinant antibody and/or a variant thereof, with the nucleic acids disclosed herein. For example, and without limitation, antibodies, recombinant antibodies, and/or variants of the foregoing may be constructed based on a heavy chain DNA sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 99% or greater, and/or 100% sequence identity to SEQ ID NO: 3 SEQ ID NO: 7, and SEQ ID NO: 11; SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 39, 38 6778060.DOCX
Attorney Docket No.06527-2503253 SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 55, SEQ ID NO: 59, SEQ ID NO: 63, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 75, SEQ ID NO: 79, SEQ ID NO: 83, SEQ ID NO: 87, SEQ ID NO: 91, SEQ ID NO: 95, SEQ ID NO: 99, SEQ ID NO: 103, SEQ ID NO: 107, SEQ ID NO: 111, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 121, SEQ ID NO: 125, SEQ ID NO: 129, SEQ ID NO: 143, SEQ ID NO: 147, SEQ ID NO: 151, SEQ ID NO: 155, SEQ ID NO: 159, SEQ ID NO: 163, SEQ ID NO: 167, SEQ ID NO: 171, SEQ ID NO: 175, SEQ ID NO: 179, SEQ ID NO: 183, SEQ ID NO: 187, SEQ ID NO: 191, SEQ ID NO: 195, SEQ ID NO: 199, and/or SEQ ID NO: 203. In non-limiting embodiments, the first binding reagent may be an antibody constructed based on a light chain DNA sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 99% or greater, and/or 100% sequence identity to SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO: 57, SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 69, SEQ ID NO: 73, SEQ ID NO: 77, SEQ ID NO: 81, SEQ ID NO: 85, SEQ ID NO: 89, SEQ ID NO: 93, SEQ ID NO: 97, SEQ ID NO:101, SEQ ID NO: 105, SEQ ID NO: 109, SEQ ID NO: 113, SEQ ID NO: 119, SEQ ID NO: 123, SEQ ID NO: 127, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 145, SEQ ID NO: 149, SEQ ID NO: 153, SEQ ID NO: 157, SEQ ID NO: 161, SEQ ID NO: 165, SEQ ID NO: 169, SEQ ID NO: 173, SEQ ID NO: 177, SEQ ID NO: 181, SEQ ID NO: 185, SEQ ID NO: 189, SEQ ID NO: 193, SEQ ID NO: 197, and/or SEQ ID NO: 201. [00147] In non-limiting embodiments, a B-cell may be engineered by introducing a nucleic acid as described above into the genome thereof, to allow for expression and/or production of antibodies or fragments thereof. Methods for genetically engineering cells, such as B cells, are known to those of skill in the art, and can include use of vectors, such as viral vectors, electroporation and/or nanoporation, CRISPR- Cas9 methods, and other techniques known to those of skill in the art. In non-limiting embodiments, a B cell so engineered may be introduced to a patient. In non-limiting embodiments, the B cell may be autologous to the patient. Example Monoclonal antibodies were prepared by immunization of mice with antigen as shown in FIG.14. 39 6778060.DOCX
Attorney Docket No.06527-2503253 [00148] Briefly, antigens, conjugated to virus-like particle (VLP) reagents, were used to immunize female Balb/c mice through subcutaneous injections at multiple abdominal sites. [00149] Total RNA was isolated from hybridoma cells following the technical manual of the TriPure Isolation Reagent. Reverse transcription was performed according to the technical manual of RT reagent Kit from Sino Biological. Primers were annealed to template after RNA secondary structure was denatured. With the involvement of reverse transcriptase , total RNA was then reverse-transcribed into cDNA. Antibody fragments of heavy chain and light chain were amplified by PCR with specific primers. Amplified antibody fragments were cloned into the expression vector or commercial vector. The consensus sequence was provided. [00150] Indirect ELISA was employed to determine the titer in blood serum samples collected from the immunized mice. Once the titer reached a satisfactory level, the animals received a final booster injection. Three to four days post-boost, mouse splenocytes were harvested and fused with myeloma cells using electrofusion to generate hybridoma cells. Two to three rounds of limiting dilution and screening were applied to the primary fusion cells until a stable monoclonal cell line was established. Indirect ELISA was used to identify antigen-specific clones. Protein A affinity was utilized to purify the antibodies from the hybridoma cell culture supernatant. Antibodies were tested for their ability to bind Tau441 (amino acids 1- 441 of Tau), phosphorylated tau (pTau441), or peripheral Tau (PNS-tau) using a sandwich ELISA assay. Briefly, antibodies were used to coat the ELISA wells. Recombinant Tau441, pTau441, or PNS-tau at various concentrations were added, followed by the addition of biotinylated anti-tau mouse mAb, clone Tau 12 (specific to Tau441 amino acids 6-18) to enable immunocomplex formation. Streptavidin-HRP was then added to bind the biotinylated antibody. The level of the immunocomplex was quantified by measuring the optical density (OD) at 450 nm, which reflected the colorimetric change of TMB (3,3′, 5,5′-tetramethylbenzidine dihydrochloride) due to the enzymatic activity of streptavidin-HRP. ELISA validation results are provided in FIGS. 1-13. Clones for each antigen were selected based on their specific recognition of the target antigen, as determined by OD450nm measurements. [00151] Having described this invention above, it will be understood to those of ordinary skill in the art that the same can be performed within a wide and equivalent range of conditions, formulations and other parameters without affecting the scope of 40 6778060.DOCX
Attorney Docket No.06527-2503253 the invention or any embodiment thereof. Any document incorporated herein by reference is only done so to the extent of its technical disclosure and to the extent it is consistent with the present document and the disclosure provided herein. 41 6778060.DOCX