EP-4739705-A1 - TARGETING HETEROGENEOUS NUCLEAR RIBONUCLEOPROTEINS FOR NEUROPROTECTION

Abstract

The disclosure relates to compositions and methods for providing neuroprotection in a subject and for treating neurodegenerative diseases and traumatic brain or spinal injuries. In embodiments, the disclosure provides agents that bind to cytoplasmic heterogeneous nuclear ribonucleoprotein (hnRNP) in neurons and that can inhibit neuronal cell death.

Inventors

• MONSON, NANCY

Assignees

• The Board of Regents of the University of Texas System

Dates

Publication Date

20260513

Application Date

20240705

Claims (1)

1. WHAT IS CLAIMED: 1. A method for neuroprotection, comprising administering to a subject in need thereof an effective amount of an agent that binds to cytoplasmic heterogeneous nuclear ribonucleoprotein (hnRNP) in neurons, to thereby inhibit neuronal cell death. 2. The method of claim 1, wherein the hnRNP comprises hnRNP A1 or hnRNP A/B. 3. The method of claim 1, wherein the hnRNP comprises one or more of hnRNP A2/B1, hnRNP C1/C2, hnRNP A1, hnRNP A3, and hnRNP D0. 4. The method of any one of claims 1 to 3, wherein the agent binds an RRM2 epitope in Table 1. 5. The method of any one of claims 1 to 4, wherein the agent is an antibody or antigen binding fragment or portion thereof. 6. The method of claim 5, wherein the agent is an IgG monoclonal antibody. 7. The method of claim 5, wherein the agent is a scFv. 8. The method of claim 5, wherein the agent is a F(ab')2. 9. The method of any one of claims 1 to 8, wherein the agent is a polypeptide expressed in neurons via delivered nucleic acid. 10. The method of claim 9, wherein the nucleic acid is DNA or mRNA. 11. The method of claim 9 or 10, wherein the nucleic acid is delivered by lipid nanoparticle or are encoded in a viral vector. 12. The method of any one of claims 1 to 11, wherein the agent is a TGM-010 antibody or antigen-binding fragment or portion thereof, or a variant thereof. 13. The method of claim 12, wherein the antibody or antigen-binding fragment or portion thereof comprises the following complementarity determining regions (CDRs) of heavy chain CDR1 of SEQ ID NO: 2, heavy chain CDR2 of SEQ ID NO: 3, heavy chain CDR3 of SEQ ID NO: 4, light chain CDR1 of SEQ ID NO: 6, light chain CDR2 of SEQ ID NO: 7, and light chain CDR3 of SEQ ID NO: 8. 14. The method of claim 13, wherein the antibody or antigen-binding fragment or portion thereof comprises a heavy chain variable domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1, and a light chain DB1/ 148791292.1 40 4 879-8182-7022, v. 1 variable domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 5. 15. The method of claim 13, wherein the antibody or antigen-binding fragment or portion thereof comprises a heavy chain variable domain comprising the amino acid sequence having of SEQ ID NO: 1, and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 5. 16. The method of any one of claims 1 to 15, wherein the subject has or is at risk of a neurodegenerative disease or traumatic injury. 17. The method of claim 16, wherein the neurodegenerative disease is Multiple Sclerosis (MS), clinically isolated syndrome (CIS), Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), frontotemporal dementia (FTD), Amyotrophic Lateral Sclerosis (ALS), schizophrenia, Progressive supranuclear palsy, Friedreich ataxia, Lewy body disease, or Spinal muscular atrophy. 18. The method of any one of claims 1 to 15, wherein the subject is at risk or experiencing or recovering from ischemic or hemorrhagic stroke, traumatic brain injury, or spinal injury. 19. The method of any one of claims 1 to 18, wherein the agent is administered systemically. 20. The method of any one of claims 1 to 18, wherein the agent is administered parenterally, optionally by subcutaneous administration, intravenous administration, intracerebroventricular administration, intraparenchymal administration, or intrathecal administration. 21. The method of any one of claims 1 to 18, wherein the agent is administered intranasally. 22. The method of claim 20 or 21, wherein the agent is administered once, or is administered a plurality of times optionally at a frequency of from about daily to about monthly. 23. The method of claim 22, wherein the agent is administered upon symptoms of neuronal injury or attack of a neurodegenerative disease, or during or after recovery from the injury or attack. DB1/ 148791292.1 41 4 879-8182-7022, v. 1 24. A method for neuroprotection, comprising administering to a subject in need thereof an effective amount of an antibody, or an antigen-binding portion thereof, having the following complementarity determining regions (CDRs): heavy chain CDR1 of SEQ ID NO: 2, heavy chain CDR2 of SEQ ID NO: 3, heavy chain CDR3 of SEQ ID NO: 4, light chain CDR1 of SEQ ID NO: 6, light chain CDR2 of SEQ ID NO: 7, and light chain CDR3 of SEQ ID NO: 8. 25. The method of claim 24, wherein the antibody, or an antigen-binding portion thereof, binds one or more cytoplasmic heterogeneous nuclear ribonucleoprotein (hnRNP) in neurons. 26. The method of claim 25, wherein the hnRNP comprises hnRNP A/B. 27. The method of claim 25, wherein the hnRNP comprises hnRNP A1. 28. The method of any one of claims 24 to 27, wherein the antibody is an IgG antibody, optionally selected from IgG1, IgG2, IgG3, and IgG4. 29. The method of any one of claims 24 to 28, wherein the antibody is a scFv or F(ab')2. 30. The method of any one of claims 24 to 29, wherein the antibody or antigen binding fragment thereof is expressed in neurons via DNA or mRNA delivery. 31. The method of claim 30, wherein the antibody or antigen binding fragment thereof is delivered using lipid nanoparticles. 32. The method of claim 30, wherein the antibody or antigen binding fragment thereof is encoded in a viral vector. 33. The method of any one of claims 24 to 32, wherein the antibody or antigen- binding fragment thereof comprises a heavy chain variable domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1, and a light chain variable domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 5. 34. The method of claim 33, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1, and a light chain variable domain comprising an amino acid sequence having at least 95% sequence identity to SEQ ID DB1/ 148791292.1 42 4 879-8182-7022, v. 1 NO: 5. 35. The method of claim 33, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence having of SEQ ID NO: 1, and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 5. 36. The method of any one of claims 24 to 35, wherein the subject has or is at risk of a neurodegenerative disease or acute or traumatic injury. 37. The method of claim 36, wherein the neurodegenerative disease is Multiple Sclerosis (MS), clinically isolated syndrome (CIS), Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), Frontotemporal dementia (FTD), Amyotrophic Lateral Sclerosis (ALS), Schizophrenia, Progressive supranuclear palsy, Friedreich ataxia, Lewy body disease, or Spinal muscular atrophy. 38. The method of claim 36, wherein the subject is at risk, experiencing, or recovering from ischemic or hemorrhagic stroke, traumatic brain injury, or spinal injury. 39. The method of any one of claims 24 to 38, wherein the antibody or antigen binding fragment thereof is administered parenterally. 40. The method of claim 39, wherein the antibody or antigen binding fragment thereof is administered by subcutaneous administration, intravenous administration, intracerebroventricular administration, intraparenchymal administration, or intrathecal administration. 41. The method of any one of claims 24 to 38, wherein the antibody or antigen binding fragment thereof, or DNA or mRNA encoding the same, is administered intranasally. 42. The method of claim 40 or 41, wherein the antibody or antigen binding fragment thereof is administered at a frequency of from about daily to about monthly. 43. The method of any one of claims 34 to 42, wherein the antibody or antigen binding fragment thereof is administered upon symptoms of neuronal injury or attack of neurodegenerative disease, or during or after recovery from the injury or attack. 44. A pharmaceutical composition comprising a monoclonal antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1, and DB1/ 148791292.1 43 4 879-8182-7022, v. 1 a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5, and a pharmaceutically acceptable carrier; the monoclonal antibody further comprising one or more selected from: a moiety that enhances cell penetration, a modification that attenuates Fc-mediated effector functions, and an Fc modification that improves pharmacodynamic (PD) properties. 45. The pharmaceutical composition of claim 44, wherein the monoclonal antibody comprises one or more modifications that increase circulating or serum half-life, increase blood-brain barrier (BBB) penetration, increase neuronal uptake, and reduce off-target or non-specific uptake. 47. A pharmaceutical composition comprising an antigen binding fragment of an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5, or a polynucleotide encoding the same, and a pharmaceutically acceptable carrier. 48. The pharmaceutical composition of claim 47, wherein the fragment is a scFv or F(ab')2. 49. The pharmaceutical composition of claim 47 or 48, wherein the fragment further comprises a moiety that enhances cell penetration. 50. The pharmaceutical composition of claim 49, wherein the moiety comprises a cell penetrating peptide. 51. The pharmaceutical composition of claim 47, comprising a polynucleotide encoding a scFv, optionally further comprising a peptide for enhancing cell penetration. 52. The pharmaceutical composition of claim 51, wherein the polynucleotide is DNA or mRNA. 53. The pharmaceutical composition of claim 51, wherein the polynucleotide is encapsulated by lipid nanoparticles. 54. The pharmaceutical composition of claim 52, wherein the DNA or mRNA is encoded or expressed from a viral vector. 55. A method for making a pharmaceutical composition for providing neuroprotection, comprising: DB1/ 148791292.1 44 4 879-8182-7022, v. 1 identifying an agent that binds to one or more cytoplasmic heterogeneous nuclear ribonucleoprotein (hnRNP) in stress granules or an RRM2 epitope thereof, determining whether the agent reduces or inhibits apoptosis in neurons, and/or reduces or ameliorates neurodegenerative disease or neuronal loss in an animal model, and formulating the agent for delivery to a human or animal. 56. The method of claim 55, wherein the one or more hnRNP comprises an RNA recognition motif 2 (RRM2). 57. The method of claim 55 or 56, wherein the hnRNP comprises hnRNP A/B or hnRNP A1. 58. The method of claim 55 or 56, wherein the one or more hnRNP comprises hnRNP A2/B1, hnRNP C1/C2, hnRNP A1, hnRNP A3, and hnRNP D0. 59. The method of any one of claims 55 to 58, wherein the agent blocks the binding of TGM-010 to said hnRNP or an RRM2 epitope, as measured by an in vitro binding assay. 60. The method of any one of claims 55 to 59, wherein the agent binds the hnRNP or RRM2 epitope with an affinity of less than or equal to 1 x 10 -7 M, or less than or equal to 1 x 10 -8 M, less than or equal to 1 x 10 -9 M, less than or equal to 1 x 10 -10 M, less than or equal to 1 x 10 -11 M, or less than or equal to 1 x 10 -12 M. 61. The method of claim 59 or 60, wherein the agent is an antibody or antigen binding fragment thereof. 62. The method of claim 59 or 60, wherein the agent is a peptide, an aptamer, an adnectin, or DARPin. 63. The method of claim 59 or 60, wherein the agent is selected from a single-domain antibody, a recombinant heavy-chain-only antibody (VHH), a single-chain antibody, single chain variable fragment (scFv), a shark heavy-chain-only antibody (VNAR), a Tetranectin, an Affibody, a Transbody, an Anticalin, an Affilin, a Microbody, a phylomer, a stradobody, a fynomer, an avimer, a triomab, a Fv, a Fab, a Fab', and a F(ab')2. 64. The method of claim 59 or 60, wherein the agent is an antibody, and optionally a DB1/ 148791292.1 45 4 879-8182-7022, v. 1 human antibody, and is optionally a TGM-010 variant. 65. The method of claim 64, wherein the agent is a VH4 antibody that binds selectively to neurons or selectively to neurons and astrocytes. 66. The method of claim 64, wherein the TGM-010 variant is prepared by introducing from one to four amino acid substitutions in one or more of the TGM-010 CDRs (SEQ ID NOs: 2-5 or 6-8), and/or introducing from 1 to 6 amino acid substitutions, deletions, or insertions within the framework regions of the TGM-010 VH (SEQ ID NO: 1) and/or VL (SEQ ID NO: 5) chains. 67. The method of claim 64, wherein the TGM-010 variant is prepared by modifying the Fc domain. 68. The method of any one of claims 55 to 67, comprising screening candidate agents for binding against one or more peptides from an RRM2 domain epitope. 69. The method of claim 68, wherein the RRM2 domain epitope comprises at least 20 amino acids, or at least 25 amino acids, or at least 40 amino acids. 70. The method of claim 69, wherein the RRM2 domain epitope comprises an amino acid sequence of Table 1 or a portion thereof. 71. The method of claim 69, wherein the RRM2 domain epitope has at least about 80%, at least about 90%, or at least about 95%, or 100% amino acid sequence identity to at least 20 amino acids, or at least 25 amino acids, or at least 40 amino acids of an amino acid sequence listed in Table 1. 72. The method of any one of claims 66 to 71, further comprises measuring binding between candidate binding agents and RRM2 epitope by ELISA, surface plasmon resonance (SPR), and biolayer interferometry (BLI). 73. The method of any one of claims 55 to 72, wherein inhibition of apoptosis in neurons is determined in vitro using a mouse or human neuronal cell line. 74. The method of any one of claims 55 to 73, wherein inhibition of apoptosis in neurons is determined in vitro. 75. The method of any one of claims 55 to 74, wherein reduction or amelioration of neurodegenerative disease or neuronal loss is determined in an animal model, which is optionally an EAE mouse model. DB1/ 148791292.1 46 4 879-8182-7022, v. 1 75. The method of any one of claims 55 to 74, further comprising quantifying internalization in neurons in vitro or using an in vivo animal model. 76. The method of any one of claims 55 to 75, wherein the agent is a polypeptide and is formulated so as to be expressed in neurons via gene or mRNA delivery. 77. The method of any one of claims 55 to 75, comprising formulating the agent for parenteral administration or intranasal administration. DB1/ 148791292.1 47 4 879-8182-7022, v. 1

Description

DESCRIPTION TARGETING HETEROGENEOUS NUCLEAR RIBONUCLEOPROTEINS FOR NEUROPROTECTION REFERENCE TO A SEQUENCE LISTING This application contains a Sequence Listing XML, which has been submitted electronically and is hereby incorporated by reference in its entirety. Said XML Sequence Listing, created on July 5, 2024, is named UTFDP4109WO.xml and is 33,844 bytes in size. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Application No. 63/512,412, filed July 7, 2023, the entire contents of which are incorporated by reference. BACKGROUND I. Field The present disclosure relates to the fields of medicine, cell biology, neurology and neurodegenerative disease. More specifically, it relates to agents that bind to cytoplasmic heterogeneous nuclear ribonucleoprotein (hnRNP) in neurons and which can inhibit neuronal cell death, as well as methods of making such agents and uses therefor. II. Related Art Neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), Multiple Sclerosis (MS), Amyotrophic Lateral Sclerosis (ALS), Huntington's disease (HD), ischemic stroke, and others, are characterized by the loss of neurons. Affected neurons exhibit neuronal stress responses including ER stress, oxidative stress, mitochondrial dysfunction, impairment of the proteasome and protein aggregation. These stress responses cause disruption of normal neuronal function and can eventually lead to cell death. Neurons can also mount pro-survival responses to protect themselves from these deleterious effects. See Gorman AM, “Neuronal cell death in neurodegenerative diseases: recurring themes around protein handling,” J Cell Mol Med.2008 Dec; 12(6a): 2263–2280. DB1/ 148791292.1 1 4879-8182-7022, v.1 RNA-binding proteins (RBPs) are a diverse class of intracellular proteins that have attracted attention specifically due to their roles in the progression of neurodegenerative diseases. Of the RBPs associated with diseases marked by neuronal loss is the heterogenous nuclear ribonucleoprotein “hnRNP” family of proteins. hnRNPs are canonically central to cellular RNA metabolism. hnRNP proteins are commonly known by a variety of names, including for example, Auf1 (hnRNP D), PCBP1 and 2 (hnRNP A1 and A2), RMBX (hnRNP G), PTB-1 (hnRNP I), FUS (hnRNP P2), and SYNCRIP (hnRNP Q). A core subset of six hnRNPs (A1, A2, B1, B2, C1, and C2) have been found to be expressed and consistently tightly associated with hnRNAs. See Thibault PA, et al., “hnRNP A/B Proteins: An Encyclopedic Assessment of Their Roles in Homeostasis and Disease,” Biology, Vol.10, No.712 (2021). Structurally, the hnRNP A/B subgroup, comprising hnRNPs A1, A2/B1, A3, and A0, share two N-terminal RNA recognition motifs (RRMs) and a C-terminal glycine-rich domain, referred to as a low- complexity domain (LCD), which includes an RGG motif (arginine-glycine-glycine), an M9 nuclear localization sequence, and an intrinsically-disordered core prion-like domain (PrLD). hnRNP A/B do not demonstrate appreciable enzymatic activity, rather, they function more like chaperone or scaffold proteins which promote, protect, and traffic RNAs, including during transcription, splicing, and translation. In particular, hnRNP A1 and A2/B1 are the most abundant proteins of the 40S ribonucleoprotein complex (see Thibault, 2021). hnRNP A/B are highly expressed in central nervous system (CNS) tissue. While hnRNP A/B generally localize to the nucleus, under stressed conditions hnRNP A/B translocate to the cytoplasm, forming RNA:protein granules (e.g., stress granules, (SG)). SGs and markers thereof have been found to be associated with a variety of neurodegenerative disease and pathologies marked by neuronal cell death. There remains a need for compositions and methods that reduce neuronal loss during neuronal injury, including to stop or slow progression of neurodegenerative diseases. Moreover, there remains a need for neuroprotective agents that target the hnRNP family of proteins and their role in neurol cell death. DB1/ 148791292.1 2 4879-8182-7022, v.1 SUMMARY OF THE DISCLOSURE In accordance with the present disclosure, there is provided a method for neuroprotection, comprising administering to a subject in need thereof an effective amount of an agent that binds to cytoplasmic heterogeneous nuclear ribonucleoprotein (hnRNP) in neurons, to thereby inhibit neuronal cell death. The hnRNP may comprise hnRNP A/B or may comprise hnRNP A2/B1, hnRNP C1/C2, hnRNP A1, hnRNP A3, and hnRNP D0. In embodiments, the hnRNP localizes in SGs. TGM-010, as described herein, is an antibody that binds neurons, and specifically interacts with cytoplasmic hnRNP targets. It is believed that TGM-010 functions to bind hnRNPs and prevent them from contributing to the SG, or removing them from the SG, reducing SG composition, size, and formation. Accordingly, the agent may be TGM-010 or an antigen-binding fragment or portion thereof, or another ag