Targeting heteronuclear ribonucleoproteins for neuroprotection

Abstract

This disclosure relates to compositions and methods for providing neuroprotection in subjects and for treating neurodegenerative diseases and traumatic brain injury or spinal cord injury. In embodiments, this disclosure provides active ingredients that can bind to intracytoplasmic heteronuclear ribonucleoproteins (hnRNPs) in neurons and inhibit neuronal cell death.

Description

[Technical Field]

[0001] Sequence listing reference This application includes an electronically filed sequence listing XML, which is incorporated herein by reference in its entirety. The XML sequence listing, created on July 5, 2024, is named UTFDP4109WO.xml and has a size of 33,844 bytes.

[0002] Cross-reference of related applications This application claims the benefits and priority of U.S. Provisional Application No. 63 / 512,412, filed on 7 July 2023, the entire contents of which are incorporated by reference.

[0003] background I. Field This disclosure relates to the fields of medicine, cell biology, neurology, and neurodegenerative diseases. More specifically, this disclosure relates to active agents that can bind to cytoplasmic heteronuclear ribonucleoproteins (hnRNPs) in neurons and inhibit neuronal cell death, as well as methods for producing such active agents and their use. [Background technology]

[0004] II. Related Technologies Neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), and ischemic stroke, are characterized by the loss of neurons. Affected neurons exhibit neuronal stress responses, including ER stress, oxidative stress, mitochondrial dysfunction, proteasome dysfunction, and protein aggregation. These stress responses can lead to the disruption of normal neuronal function and ultimately to cell death. Neurons can also initiate survival-promoting responses to protect themselves from these harmful 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 (Non-patent Literature 1).

[0005] RNA-binding proteins (RBPs) are a diverse class of intracellular proteins that have attracted particular attention due to their role in the progression of neurodegenerative diseases. Among the RBPs associated with diseases characterized by neuronal loss are proteins of the heteronuclear ribonucleoprotein "hnRNP" family. hnRNPs are typically central to cellular RNA metabolism. hnRNP proteins are commonly known by various 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) are expressed and consistently shown to be closely associated with hnRNA. 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) (Non-Patent Literature 2). The hnRNP A / B subgroup, including hnRNP A1, A2 / B1, A3, and A0, structurally share two N-terminal RNA recognition motifs (RRMs) and a C-terminal glycine-rich domain called a low-complexity domain (LCD), the LCD containing an RGG motif (arginine-glycine-glycine), an M9 nuclear localization sequence, and a prion-like domain (PrLD) which is an intrinsically disordered core.

[0006] hnRNP A / B do not exhibit clear enzymatic activity; rather, they function as chaperone or scaffold proteins that promote, protect, and transport RNA, including during transcription, splicing, and translation. In particular, hnRNP A1 and A2 / B1 are the most abundant proteins in the 40S ribonucleoprotein complex (see Thibault, 2021). hnRNP A / B are highly expressed in central nervous system (CNS) tissues. While generally localized to the nucleus, under stress conditions, hnRNP A / B move to the cytoplasm and form RNA:protein granules (e.g., stress granules (SGs)). SGs and their markers have been shown to be associated with various neurodegenerative diseases and lesions characterized by neuronal cell death.

[0007] Compositions and methods to reduce neuronal loss during neuronal injury, including halting or slowing the progression of neurodegenerative diseases, remain in demand. Furthermore, neuroprotective agents targeting the hnRNP family of proteins and their roles in neuronal cell death are still needed. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Gorman AM, “Neuronal cell death in neurodegenerative diseases: recurring themes around protein handling,” J Cell Mol Med.2008 Dec;12(6a):2263-2280 [Non-Patent Document 2] Thibault PA, et al., “hnRNP A / B Proteins: An Encyclopedic Assessment of Their Roles in Homeostasis and Disease,” Biology, Vol. 10, No. 712 (2021) [Overview of the Initiative]

[0009] Summary of Disclosure This disclosure provides a method for neuroprotection comprising administering an effective amount of an active substance to a subject in need of it, which binds to cytoplasmic heteronuclear ribonucleoproteins (hnRNPs) in neurons and thereby inhibits neuronal cell death. The hnRNPs may comprise hnRNP A / B, or hnRNP A2 / B1, hnRNP C1 / C2, hnRNP A1, hnRNP A3, and hnRNP D0. In embodiments, the hnRNPs are localized to SGs.

[0010] As described herein, TGM-010 is an antibody that binds to neurons and specifically interacts with intracytoplasmic hnRNP targets. TGM-010 is thought to function by binding to hnRNP and preventing its contribution to SGs, or by removing them from SGs, thereby reducing the composition, size, and formation of SGs. Therefore, the active agent may be TGM-010 or its antigen-binding fragment or moiety, or another active agent having similar hnRNP-binding properties, for example, an active agent that competes with the binding of TGM-010 to hnRNP in an in vitro binding assay. The active agent may be a monoclonal antibody (which may be a human antibody), a single-chain variable fragment (scFv), or an antibody or its antigen-binding fragment containing a single-chain antibody. In some embodiments, the active agent is selected from Fab, Fab', and F(ab')2. The active agent may be an IgG monoclonal antibody, which can be modified for improved pharmacodynamics (e.g., circulating half-life), blood-brain barrier (BBB) ​​penetration, or neuronal entry. In embodiments, the active agent may further comprise a cell membrane-permeable peptide to enhance intracellular translocation. The active agent may be encapsulated in liposomes, lipid nanoparticles, or polymer particles, or conjugated on their surface. In embodiments, the active agent is a polypeptide (e.g., scFv) expressed in neurons via gene or mRNA delivery. In embodiments, the active agent translocates into neurons and reduces intracellular SGs that contribute to neuronal apoptosis.

[0011] The subjects requiring this assistance may have neurodegenerative diseases such as multiple sclerosis (MS), clinically isolated syndrome (CIS), Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), schizophrenia, progressive supranuclear palsy, Friedreich's ataxia, Lewy body dementia, or spinal muscular atrophy, or may have traumatic injuries. In embodiments, subjects may have experienced or recovered from ischemic or hemorrhagic stroke, traumatic brain injury, or spinal cord injury. In embodiments, subjects may be at risk of developing neurodegenerative diseases.

[0012] In embodiments, the active ingredient is administered systemically. The active ingredient may be administered parenterally via several routes, including subcutaneous, intravenous, or intrathecal administration. In embodiments, the active ingredient may be administered intranasally to facilitate administration to the brain. The active ingredient may be administered as a single dose, or as multiple doses administered at a frequency ranging from nearly daily to nearly monthly, such as nearly daily, nearly twice a week, nearly weekly, nearly twice a month (e.g., once every two weeks), or nearly monthly. Therapy may involve methods to enhance blood-brain barrier penetration, including high-frequency ultrasound. Therapy may be initiated at the time of symptoms of neuronal injury or an attack of neurodegenerative disease, or during or after recovery from such injury or attack. In embodiments, the therapy is initiated in subjects at risk of neuronal injury or neurodegenerative disease, for example, subjects considered to be at risk of developing neurodegenerative disease.

[0013] In aspects and embodiments, the Disclosure provides compositions and methods for neuroprotection comprising an antibody (or its use) or its antigen-binding moiety 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. The antibody may be an IgG antibody, which can be modified for improved pharmacodynamics (e.g., circulating half-life), blood-brain barrier (BBB) ​​permeability, or neuronal entry. The antibody may also be an scFv. The antibody or its antigen-binding fragment may further comprise a cell membrane permeable peptide to improve cell entry. The antibody or its antigen-binding fragment may be encapsulated in liposomes, lipid nanoparticles, or polymer particles, or conjugated on their surface. The antibody or its antigen-binding fragment may be expressed in neurons via gene or mRNA delivery.

[0014] The composition may be administered to subjects with neurodegenerative diseases such as multiple sclerosis (MS), clinical isolation syndrome (CIS), Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), schizophrenia, progressive supranuclear palsy, Friedreich's ataxia, Lewy body dementia, or spinal muscular atrophy, or to subjects with acute or traumatic injury. Subjects may have experienced or recovered from ischemic or hemorrhagic stroke, traumatic brain injury, or spinal cord injury. In the embodiments, subjects are at risk of developing neurodegenerative diseases.

[0015] An antibody or an antigen-binding fragment thereof, or a gene or mRNA encoding the same, can be administered systemically or parenterally. The antibody or an antigen-binding fragment thereof can be administered, for example, by subcutaneous administration, intravenous administration, or intrathecal administration. An antibody or an antigen-binding fragment thereof, or a gene or mRNA encoding the same, can be administered intranasally. The agent can be administered once or can be administered in multiple doses at a frequency of approximately daily to approximately monthly, such as approximately daily, approximately twice a week, approximately weekly, approximately twice a month (e.g., once every two weeks), or approximately monthly. The therapy may involve a method of enhancing blood-brain barrier penetration, including high-frequency ultrasound. The therapy may be initiated when there is a suspicion of future neurodegenerative disease diagnosis, during the symptoms of neuronal injury or during the onset of a neurodegenerative disease, or during or after recovery from the injury or the onset.

[0016] In aspects and embodiments, the present disclosure provides monoclonal antibodies (and their use in methods of neuroprotection), which antibodies comprise a heavy-chain variable region comprising the amino acid sequence of SEQ ID NO: 1, and a light chain comprising the amino acid sequence of SEQ ID NO: 5, as well as variants thereof. The antibody may be an IgG antibody (e.g., IgG1), which can be modified for improved pharmacokinetics (e.g., circulating half-life), blood-brain barrier (BBB) penetration, or neuronal entry. In embodiments, the monoclonal antibody may further comprise a cell-penetration enhancing moiety, such as a cell-penetrating peptide.

[0017] In aspects and embodiments, the present disclosure provides an antigen-binding fragment of an antibody (and its use in methods of neuroprotection), which antigen-binding fragment comprises a heavy-chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 5. The fragment may be a scFv or F(ab’)2. The fragment may further comprise a cell-penetration enhancing moiety, such as a cell-penetrating peptide.

[0018] In aspects and embodiments, the present disclosure provides a polynucleotide encoding an antibody or antigen-binding fragment described herein (and its use in methods for neuroprotection).

[0019] Also provided is a pharmaceutical composition comprising an effective amount of an antibody, an antigen-binding fragment, or a polynucleotide described herein, and a pharmaceutically acceptable carrier. Such pharmaceutical compositions are useful in methods for neuroprotection in a subject that needs it.

[0020] Also provided is a method for making a pharmaceutical composition for providing neuroprotection, the method comprising: (a) identifying an agent that binds to cytoplasmic hnRNP in stress granules (or an epitope thereof, such as the RRM2 epitope); (b) evaluating whether the agent inhibits apoptosis in neurons in vitro and / or determining whether the agent reduces or ameliorates the symptoms of a disease (such as a neurodegenerative disease or neuronal loss) in an animal model. If the agent inhibits apoptosis in neurons and / or reduces or ameliorates the symptoms of a disease in an animal model, the agent is formulated for delivery to a human or an animal. The hnRNP may include the hnRNP A / B subgroup, and an agent that binds to its RRM2 epitope may be selected. In embodiments, the hnRNP may also include hnRNP A2 / B1, hnRNP C1 / C2, hnRNP A1, hnRNP A3, and hnRNP D0, and an agent that binds to its RRM2 epitope may be selected. The agent may be formulated for systemic or parenteral administration, or for intranasal administration.

[0021] The activator may be an antibody or its antigen-binding fragment, or a peptide, aptamer, adonectin, or DARPin. The activator may be selected from single-domain antibodies, recombinant heavy-chain-only antibodies (VHH), single-chain antibodies, single-chain variable fragments (scFv), shark heavy-chain-only antibodies (VNAR), tetranectin, afibody, transbody, antikalin, affin, microbody, phylomer, stradobody, fynomer, avimer, triomab, Fv, Fab, Fab', and F(ab')2. The activator may be a monoclonal antibody. The activator may be an scFv. The activator may be F(ab')2. The activator may compete for the binding of TGM-010 to an hnRNP or RRM2 epitope using an in vitro binding assay. In embodiments, the activator is a TGM-010 variant.

[0022] The active agent may be a polypeptide delivered to an animal or human as a nucleic acid encoding the polypeptide, which is then expressed in neurons via gene or mRNA delivery. Gene or mRNA delivery may be via lipid nanoparticles or viral vectors.

[0023] Other purposes, features, and advantages of this disclosure will become apparent from the following detailed description. However, since various changes and modifications within the purpose and scope of this disclosure will be apparent to those skilled in the art from the detailed description, it should be understood that the detailed description and specific examples are provided only as illustrative examples, while illustrating specific embodiments of this disclosure. [Brief explanation of the drawing]

[0024] [Figure 1] Binding of TGM-010 to mouse brain tissue by immunofluorescence staining. [Figure 2] Immunofluorescence staining of TGM-010 binding in mouse cortical hippocampal neurons and human motor neurons. [Figure 3]Binding of TGM-010 (top) and control antibody (bottom) in primary mouse neural progenitor cells, as shown by flow cytometry. [Figure 4] Binding ability of TGM-010 in human cortical neurons derived from ALS (C9orf72). [Figure 5] Random antibody administration of TGM-010 to mice (EAE model). The figure shows data on cumulative EAE scores. [Figure 6] Random administration of TGM-010 antibodies to mice (experimental autoimmune encephalitis, "EAE", model) via intraperitoneal (IP) injection over two weeks, starting one day after recovery from the first seizure. The figure shows data for cumulative EAE scores. [Figure 7] Identification of potential targets of TGM-010 using human protein arrays. [Figure 8] Immunoprecipitation assay using TGM-010 (TGM10) or the control antibody JRR67 against Sy5y cell lysates, as shown on an SDS-PAGE gel. Cell lysate proteins were prepared and used for immunoprecipitation. [Figure 9] Mass spectrometry of the 37 / 40 kDa double line, 100 kDa band, and 50 kDa double line digested with trypsin, as shown in Figure 8. [Figure 10] Antibody cytotoxicity analysis of TGM-010 compared to the positive control antibody "DS14". [Figure 11A] RRM2 alignment from hnRNP A2B1 (SEQ ID NO: 9), A1 (SEQ ID NO: 10), and AB (SEQ ID NO: 11). The lower side shows the positions of peptides 1-8 (SEQ ID NO: 12), 1-9 (SEQ ID NO: 13), 1-10 (SEQ ID NO: 14), 1-11 (SEQ ID NO: 15), 3-8 (SEQ ID NO: 16), 3-9 (SEQ ID NO: 17), 3-10 (SEQ ID NO: 18), 3-11 (SEQ ID NO: 19), 6-11 (SEQ ID NO: 20), 6-12 (SEQ ID NO: 21), 6-13 (SEQ ID NO: 22), and 6-14 (SEQ ID NO: 23). [Figure 11B] Results of an ELISA showing specificity for the RRM2 epitope. [Figure 12A] Confocal fluorescence image of Sy5y human neuroblastoma cells showing internal migration of TGM-010 (scale bar = 20 μm). [Figure 12B] Time-resolved quantification of TGM-010 internal transfer, measured as the average fluorescence intensity in human neuroblastoma cells. [Figure 12C] Confocal fluorescence image of mouse cortical neurons showing interspecies intracellular migration of TGM-010 (scale bar = 20 μm). [Figure 12D] Time-resolved quantification of TGM-010 internal transfer, measured as average fluorescence intensity in mouse cortical neurons. [Figure 13A] Schematic diagram of pepsin cleavage of full-length IgG TGM-010 to remove the Fc domain. The resulting F(ab')2 product was isolated to evaluate Fc-independent intracellular translocation. [Figure 13B] Sy5y human neuroblastoma cells stained with a control antibody (e.g., JRR67). [Figure 13C] Comparison of full-length (FL) TGM-010 staining and F(ab')2 TGM-010 staining of Sy5y human neuroblastoma cells. This demonstrates that staining is Fc domain independent. [Figure 14] The neuroprotective effect of TGM-010 in Sy5y neuroblastoma cells under cellular stress was measured using a heat shock neurotoxicity assay, which involved confocal microscopy and the detection of cell lysis by caspase-3 / 7 fluorescence-generating substrate. The neuroprotective effect was compared with that of the neurotoxicity control antibody "DS14". [Figure 15A] Fluorescence confocal microscopy images of mouse cortical neurons under heat-induced stress granule (SG) formation. SG formation was visualized using stress granule organizing factor 1 (G3BP1) staining. [Figure 15B] Localization of a negative control nonspecific antibody (JRR67) in mouse cortical neurons associated with heat stress-induced SG formation, visualized by fluorescence imaging. [Figure 15C]Colocalization of TGM-010 with stress granule enrichment in thermally stressed mouse cortical neurons, visualized by fluorescence imaging. [Figure 15D] Quantification of SG colocalization. [Figure 16] Figure 16A. Neuroprotective effect of TGM-010 demonstrated in an experimental autoimmune encephalitis (EAE) mouse model. EAE was induced in SJL / j female mice by immunization using myelin proteolipide protein residues 139-151 (PLP139-151). Mice (n=28) that had recovered from the first demyelinating seizure were randomly and blinded, and administered either TGM-010 (5 μg) or a nonspecific control antibody via intraventricular (ICV) administration into the brain. Mice were scored for the severity of EAE. Figure 16B. Subsequent blinded EAE in vivo studies conducted in mice (n=26) confirmed the neuroprotective effect of TGM-010 administered via systemic intraperitoneal (IP) administration. [Figure 17A] Quantification of TGM-010 staining in neurons compared to the control antibody JRR67. TGM-010 staining was statistically significant compared to JRR67. [Figure 17B] Labeling of anterior, middle, and distal brain sections of mouse cerebral cortex layer 1 24 hours after drug administration. Strong staining of TGM-010 was observed in all parts of the cortex. [Figure 18] Time-resolved quantification of TGM-010 internal transfer into ALS-derived human cortical neurons, measured as the mean fluorescence intensity in human cortical neurons. [Modes for carrying out the invention]

[0025] Detailed explanation In various embodiments, the Disclosure provides compositions and methods for providing neuroprotection to subjects requiring neuroprotection, including use in the treatment of neurodegenerative diseases and traumatic brain or spinal cord injury. In some embodiments, the Disclosure provides agents that bind to intracellular hnRNPs of neurons, including heteronuclear ribonucleoproteins (hnRNPs) involved in SG formation.

[0026] hnRNP particles are RNA-protein complexes present in the cell nucleus during gene transcription and subsequent post-transcriptional modification of newly synthesized RNA (pre-mRNA). The presence of hnRNP bound to pre-mRNA serves as a signal that the pre-mRNA is not yet fully processed and therefore not ready for export to the cytoplasm. Even after splicing occurs, hnRNP remains bound to spliced ​​introns, targeting them for degradation. hnRNP is also essential for the 40S subunit of ribosomes and is therefore important for mRNA translation in the cytoplasm. hnRNP contains a nuclear localization sequence (NLS) and is therefore primarily found in the nucleus, although some hnRNP translocates between the cytoplasm and the nucleus.

[0027] There are approximately 20 major hnRNPs (hnRNP A1 to hnRNP U), and the location and function of each member differ in each cell type. Several structural domains are shared among different family members. See Geuens T, et al., “The hnRNP family: insights into their roles in health and disease,” Hum Genet (2016) 135:851-867. The hnRNP A / B subgroup includes four gene products, A1, A2 / B1, A3, and A0, which are involved in mRNA translation and splicing and are components of the 40S hnRNP complex.

[0028] hnRNP A2 / B1 plays a crucial role in mRNA transport in oligodendrocytes and neurons, and is also important for the precise localization of transcripts containing A2 response elements (A2REs) or A2RE-like sequences, such as myelin basic protein (MBP) or Ca2+ / calmodulin-dependent protein kinase II (CaMKII), regulatory cytoskeleton-related proteins (Arc), and neurogranin (NRGN) mRNA.

[0029] hnRNP A1 has been reported to localize to stress granules (SGs) during cellular stress responses. See Guil S, et al., “hnRNP A1 Relocalization to the Stress Granules Reflects a Role in the Stress Response,” Mol Cell Biol (2006) (15):5744-58. Post-transcriptional regulation of gene expression during various stimuli such as heat shock, oxidative stress, and viral infection is crucial for cell survival. SGs represent cytoplasmic regions where translationally stopped mRNA and numerous RNA-binding proteins form nuclei during stress responses, allowing cells to reprogram gene expression. Therefore, SGs enable mRNA to triage, instructing whether to degrade or translate it. SGs and their dysregulation are thought to play a role in the pathophysiology of neurodegeneration. For example, cytoplasmic localization of hnRNP and its association with stress granules (SGs) are markers of neurodegeneration in MS tissue and may contribute to neurodegeneration in EAE and MS. See Salapa HE, et al, “Dysfunctional RNA-binding protein biology and neurodegeneration in experimental autoimmune encephalomyelitis in female mice.” J Neurosci Res 2020 Apr;98(4):704-717. Abnormal persistence of SG formation is pathogenic to cells and drives apoptosis. The cytotoxic effects of SGs have been observed in conjunction with anticancer drugs. For example, the pankinase inhibitor sorafenib induces SG formation in hepatoma cells due to its cytotoxic effect, and furthermore, 5-fluorouracil and vinca alkaloids induce general anticancer effects associated with increased SG formation.

[0030] Numerous neurodegeneration-associated mutations have been identified in hnRNP, which is known to be associated with stress granules (SGs) that contribute to diseases such as ALS, FTLD, and neuromuscular myopathy. See Protter and Parker, “Principles and Properties of Stress Granules,” Trends Cell Biol. Vol.26, No.9, (2016). Patient biopsies contain aggregates of SG components mixed with sequestered RNA, and pathogenicity models have emerged showing that SG formation leads to a series of events ultimately resulting in cell death.

[0031] As stated in this disclosure, and not intended to be theoretically bound, the human antibody TGM-010 is thought to interfere with hnRNP-SG interaction and / or segregation in neurons, thereby inhibiting the induction of neuronal apoptosis mediated by mechanisms including SG biology. In embodiments, and not intended to be theoretically bound, TGM-010 functions to bind to hnRNP to prevent hnRNP's contribution to SG, or to remove hnRNP from SG, thereby reducing the composition, size, and formation of SG. Thus, and not intended to be theoretically bound, the TGM-010 antibody and its antigen-binding fragment are thought to function to bind to hnRNP and to function as a selective inhibitor of neuronal cell death based on the SG pathway, thereby treating and / or slowing the progression of diseases characterized by neuronal loss via the SG pathway. In some embodiments, TGM-010 and other modalities for inhibiting hnRNP-SG interaction are candidates for neuroprotection in a variety of neurodegenerative conditions, including but not limited to multiple sclerosis (MS), stroke, Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and Huntington's disease. In some embodiments, TGM-010 and other modalities for inhibiting hnRNP-SG interaction are candidates for treating brain or spinal injuries, including stroke, TBI, and spinal cord injury.

[0032] "TGM-010" refers to an antibody having the following complementarity-determining regions (CDRs) and their antigen-binding moieties or fragments: 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. TGM-010 may include a heavy chain variable sequence containing SEQ ID NO: 1 and a light chain sequence containing SEQ ID NO: 5. In some embodiments, the TGM-010 antibody has cell permeability activity that enables binding to cytoplasmic targets such as hnRNP.

[0033] In one embodiment, the Disclosure provides a method for neuroprotection. This method involves administering an effective amount of an active agent to a target requiring it, which binds to intracytoplasmic heteronuclear ribonucleoproteins (hnRNPs) in neurons and thereby inhibits neuronal cell death (including, but not limited to, neuronal apoptosis). In embodiments, the active agent comprises TGM-010 or its antigen-binding moiety or fragment. According to the Disclosure, TGM-010 has been shown to bind to antigens in neurons, exhibit neuroprotection in EAE models, and specifically bind to hnRNPs, including hnRNPs in SGs. Thus, the Disclosure identifies intracytoplasmic hnRNPs as targets for neuroprotection, for example, during abnormal or chronic stress responses. In some embodiments, TGM-010 antibodies and their antibody fragments and variants are capable of permeating neurons in an Fc-independent manner and binding to hnRNPs intracellularly. The Disclosure further identifies TGM-010 and its derivatives (including its fragments and antigen-binding moieties) as candidate neuroprotective agents.

[0034] In the embodiment, the active substance is an antibody or its antigen-binding fragment or portion, comprising a heavy chain variable domain containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1, and a light chain variable domain containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 5. In the embodiment, the antibody or its antigen-binding fragment or portion comprises the six CDR sequences of TGM-010. In the embodiment, the antibody or its antigen-binding fragment or portion comprises a heavy chain variable domain containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 1, and a light chain variable domain containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 5. In the embodiment, the antibody or its antigen-binding fragment or portion comprises a heavy chain variable domain containing an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 1, and a light chain variable domain containing an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 5. In the embodiment, the antibody or its antigen-binding fragment or portion comprises a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 1, and a light chain variable domain containing the amino acid sequence of SEQ ID NO: 5.

[0035] In various embodiments, the hnRNPs targeted by the active agent (e.g., a binding agent) include hnRNP A / B (UniProtKB-Q99729). hnRNP A / B localize to cytoplasmic mRNA granules containing untranslated mRNA. ALS20 type is caused by mutations in the hnRNPA1 gene. Krebs BB and De Mesquita JF, “Amyotrophic Lateral Sclerosis Type 20 - In Silico Analysis and Molecular Dynamics Simulation of hnRNPA1.” PLoS One (2016 Jul 14;11(7):e0158939). Autoantibodies against these proteins have also been detected. Dangli A, et al. “Recognition of subsets of the mammalian A / B-type core heterogeneous nuclear ribonucleoprotein polypeptides by novel autoantibodies.” Biochem.J.320,761-767. Antibodies against hnRNP A1 have been reported to contribute to neuronal loss in animal models of multiple sclerosis (MS). See Libner CD et al., J Comp Neurol 2020 Jul;528(10):1704-1724. Cytoplasmic localization of hnRNP and its association with stress granules (SGs) are markers of neurodegeneration in MS tissue and may contribute to neurodegeneration. See Salapa HE, et al. “Dysfunctional RNA-binding protein biology and neurodegeneration in experimental autoimmune encephalomyelitis in female mice.” J Neurosci Res 2020 Apr;98(4):704-717. Cytoplasmic hnRNP A1 levels were significantly higher in EAE mice compared to naive mice and have been reported to directly correlate with neuronal death and clinical scores in animal models.

[0036] In some embodiments, the hnRNP targeted by the active agent includes hnRNP A / B (UniProtKB-Q99729), hnRNP A2 / B1 (UniProtKB-P22626), hnRNP C1 / C2 (UniProtKB-P07910), hnRNP A1 (UniProtKB-P09651), hnRNP A3 (UniProtKB-P51991), or hnRNP D0 (UniProtKB-Q14103). In some embodiments, the active agent binds to any hnRNP protein family member, including an RNA recognition motif (RRM) (e.g., RRM2) containing, for example, hnRNP A, hnRNP A / B, hnRNP C, hnRNP G, hnRNP I, hnRNP L, hnRNP M, hnRNP P, hnRNP Q, and / or hnRNP R. See Geuens et al., 2016. For example, as described herein, in the embodiments, the antibody and activator bind to hnRNP A1 containing a peptide comprising the amino acid sequence of SEQ ID NO: 24, SEQ ID NO: 16, and / or SEQ ID NO: 19, and / or hnRNP A / B containing a peptide comprising the amino acid sequence of SEQ ID NO: 25, SEQ ID NO: 20, and / or SEQ ID NO: 23. SEQ ID NOs: 24 and 25 have approximately 40% sequence identity with each other. The RRM2 epitopes of the antibodies and activators described herein are summarized in Table 1. In the embodiments, these RRM2 epitopes compete for hnRNP binding by the antibodies and activators described herein.

[0037] (Table 1) Epitope region of the RRM2 domain of the hnRNP protein. All amino acid numbering is based on the standard UniProtKB sequence. TIFF2026522973000001.tif111165

[0038] The activator may optionally be an antibody or its antigen-binding portion, or an antibody mimetic. The term “antibody” includes antibodies containing heavy and light chains, or their antigen-binding fragments or antigen-binding portions. In some embodiments, the antibody is a human antibody. Antibody fragments include F(ab')2, Fab, Fab', Fv, and Fd fragments, as well as single-domain antibodies and single-chain antibodies. Other antibody platforms known to be used include nanobodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR, and bis-scFv (see, for example, Hollinger and Hudson, Nature Biotechnology, 23(9):1126-1136 (2005)). In some embodiments, the activator is a peptide, aptamer, adonectin, or DARPin. In some embodiments, the active agent is selected from single-domain antibodies, recombinant heavy chain-only antibodies (VHH) ("nanobodies"), single-chain antibodies, single-chain variable fragments (scFv), shark heavy chain-only antibodies (VNAR), tetranectin, afibodies, transbodies, antikalin, affilin, microbodies, phylomers, stradobodies, finomers, avimers, or triomabs. Various conjugate active agent platforms that may be used are documented in U.S. Patent and Published Patent Applications, US7,417,130, US2004 / 132094, US5,831,012, US2004 / 023334, US7,250,297, US6,818,418, US2004 / 209243, US7,838,629, US7,186,524, US6,004, See also US Patent Nos. 746, US5,475,096, US2004 / 146938, US2004 / 157209, US6,994,982, US6,794,144, US2010 / 239633, US Patent Nos. 7,803,907, US2010 / 119446, and US7,166,697, the contents of which are incorporated herein by reference in their entirety. See also Storz MAbs. 2011 May-June;3(3):310-317.In embodiments, the binding agent is selected to bind to the hnRNP RRM2 epitope described herein, in accordance with this disclosure. In embodiments, the binding agent has a dissociation constant (K) of less than 1 μM to the hnRNP target (or epitope described herein). D ), or K less than approximately 100 nM D , or K less than approximately 10 nM D , or K less than approximately 1 nM D The binding can be determined using known methods such as ELISA, surface plasmon resonance (SPR, e.g., BIACORE assay), and biolayer interferometry (BLI).

[0039] In some embodiments, the active agent is or derived from a VH4 antibody (e.g., a human antibody) that satisfies the antibody gene signature (AGS) codon described in U.S. Patent No. 8,394,583, which is incorporated herein by reference. In some embodiments, the active agent is an antibody that selectively binds to neurons, or selectively binds to neurons and astrocytes. Exemplary antibodies containing the AGS codon, cloned from CSF B cells isolated from patients with clinical isolation syndrome optic neuritis (ON-CIS), clinical isolation syndrome transverse myelitis (TM-CIS), or clinically clear MS, are described in U.S. Patent Publication 2021 / 0214423, which is incorporated herein by reference in its entirety.

[0040] In some embodiments, the active ingredient is TGM-010, or its derivatives or antigen-binding fragments, as described herein. The TGM-010 antibody may include the heavy chain variable sequence of SEQ ID NO: 1 and the light chain sequence of SEQ ID NO: 5.

[0041] In some embodiments, the agent blocks or competes with the binding of TGM-010 to an hnRNP target (e.g., hnRNP A1, hnRNP A / B, or hnRNPA2 / B1, or their RRM2 epitopes) according to an in vitro binding assay. Any of the known binding assays and equipment can be used to determine whether the agent and TGM-010 bind to the same epitope or overlapping epitopes. For example, the agent can bind to a selected hnRNP (or its RRM2 epitope) with an affinity of 1×10 -7 M or less, or 1×10 -8 M or less, 1×10 -9 M or less, or 1×10 -10 M or less, or 1×10 -11 M or less, or 1×10 -12 M or less. Binding affinity and competition with the binding of TGM-010 can be determined by ELISA or surface plasmon resonance (SPR, e.g., BIACORE assay), biolayer interferometry (BLI), and other techniques known in the art.

[0042] In an exemplary embodiment, the agent is a human or humanized monoclonal antibody, i.e., including heavy and light chains including, but not limited to, TGM-010 and its variants. The antibody can be of any isotype such as IgG, IgD, IgA, or IgM. In some embodiments, the antibody is an IgG antibody (e.g., IgG1, IgG2, IgG3, or IgG4). In some embodiments, the antibody is of the IgG4 isotype. IgG4 is preferred in some embodiments because of its insufficient induction of Fc-mediated effector functions. In an embodiment, the antibody is of the IgG1 isotype, which is optionally modified to reduce or eliminate one or more effector functions and / or increase the circulation half-life and / or increase the penetration of the BBB and / or retention in the brain.

[0043] In some embodiments, the antibody (including, but not limited to, TGM-010) includes an Fc domain (e.g., IgG1, IgG2, or IgG4 isotype). In some embodiments, substitution, insertion, or deletion of one or more amino acids in the Fc domain attenuates the Fc-mediated effector function and / or improves the pharmacokinetic (PK) and / or pharmacodynamic (PD) properties. These properties include increased half-life (e.g., circulating or serum half-life), improved blood-brain barrier (BBB) ​​permeability, and / or reduced off-target or nonspecific uptake. In various embodiments, the antibodies and their antigen-binding fragments described herein do not accumulate clearly in cells containing the BBB, including endothelial cells (ECs), pericytes (PCs), capillary basement membranes, and astrocyte end feet (compared to neurons in the brain).

[0044] In some embodiments, the active agent (including, but not limited to, TGM-010) does not contain an Fc domain. For example, the active agent may be F(ab')2, Fab, Fab', Fv, or Fd fragment. In some embodiments, the active agent is a single-chain variable fragment (scFv), which may be the scFv of TGM-010. The scFv is a fusion protein of the variable regions of the heavy chain (VH) and light chain (VL) of an antibody, linked by a short linker peptide of 10 to about 25 amino acids. These smaller forms may be engineered for parenteral administration routes, such as intranasal administration, to increase tissue or cell permeability in some embodiments, and may also allow for better CNS permeability. In some embodiments, the antigen-binding moiety (e.g., scFv or other antibody mimetic) is polymerized to enhance antigen binding. In some embodiments, the active agent is F(ab')2 (including, but not limited to, TGM-010 F(ab')2).

[0045] In some embodiments, the active agent is modified or formulated to enhance CNS and / or intracellular delivery of the conjugated active agent. For example, the active agent may be modified or formulated to enhance delivery across the blood-brain barrier (BBB). One of the most selective barriers surrounding the CNS is the BBB, formed by endothelial cells via tight junctions (TJs) and adherent junctions (AJs). The BBB excludes large molecules. Furthermore, FcRn expressed at the BBB and the blood-cerebrospinal fluid (CSF) barrier contributes to the efflux of full-length IgG from the brain. Therefore, in some embodiments, smaller conjugated active agents, such as nanobodies and antibody derivatives that do not contain the Fc domain (e.g., those described herein), are used for better CNS permeability and half-life. In other embodiments, the conjugated active agent includes fusions, conjugates, or particle formulations with ligands that facilitate active transport. Active transport of molecules such as proteins and peptides across the blood-brain barrier (BBB) ​​is mediated by three mechanisms: adsorption-mediated transcytosis (AMT), transporter-mediated transcytosis, and receptor-mediated endocytosis (RMT). For example, the human insulin receptor is known to transcytose insulin across the BBB. Antibodies that bind to this receptor have been shown to be transported to the central nervous system (CNS). Therefore, antibodies or their antigen-binding moieties against receptors at the BBB, such as insulin or low-density lipoprotein (LDL) receptors, can be presented by liposomes or other nanoparticles / microparticles to target and induce the encapsulated binding agent across the BBB. In some embodiments, the paratopes or binding moieties for promoting BBB transport or neuronal targeting replace the Fc domain of the antibody or are fused to the Fc domain (or a portion thereof) (e.g., TGM-010 or its variants).

[0046] In some embodiments, the active ingredient is modified or formulated to improve intracellular delivery. Possible approaches to improve cellular delivery include direct intracellular expression, the use of protein transduction domains or their mimetic counterparts, and the use of various nanoparticle carriers (including inorganic nanoparticles, liposomes, polymer particles, and viral envelopes).

[0047] In some embodiments, the active ingredient comprises a fusion to a protein transduction domain (i.e., a cell-penetrating peptide or CPP). Exemplary CPP fusions include HIV1 transactivating (TAT) protein or peptides derived therefrom. In some embodiments, the active ingredient is a cell-penetrating TransMab (Muller et al., 2005) or transbody. See also Muller, S., et al. (2005). “TransMabs: cell-penetrating antibodies, the next generation.” Expert Opin. Biol. Ther. 5, 237-241. Slastnikoval AV, et al., “Targeted Intracellular Delivery of Antibodies: The State of the Art,” Front. Pharmacol., 24 October 2018.

[0048] Cell permeable peptides (CPPs) can be manipulated within or at the end of an antibody sequence (including, but not limited to, TGM-010 and its variants) to improve cell permeability. In various embodiments, the CPP is fused or conjugated at the N-terminus or C-terminus of the heavy and / or light chain, or at the N-terminus and / or C-terminus of scFv or a single-chain antibody. In some embodiments, one or more CPPs are fused immediately before or after the antibody hinge region. Exemplary CPPs include Pep1 (KETWWETWWTEWSQPKKKRKV, SEQ ID NO: 27), TAT (YGKKRRQRRR, SEQ ID NO: 28), PEPth (VKKKKIKAEIKI, SEQ ID NO: 29), aurein1, 2 (GLFDIIKKIAESF, SEQ ID NO: 30), MTS (KGEGAAVLLPVLLAAPG, SEQ ID NO: 31), and GFWFG (SEQ ID NO: 32), as well as their derivatives. See Gaston J, et al., “Intracellular delivery of therapeutic antibodies into specific cells using antibody-peptide fusions.” Scientific Reports (2019) 9:18688. In various embodiments, CCP is one of the known TAT derivatives.

[0049] In some embodiments, the antibody or active agent (including, but not limited to, TGM-010) is conjugated to a phosphorothioate (PS) DNA oligonucleotide to enhance cell penetration. See Herrmann A., et al. “An effective cell-penetrating antibody delivery platform,” JCI Insight 2019;4(14):e127474.

[0050] In some embodiments, the active ingredient may be encapsulated in or conjugated on inorganic particles, liposomes, lipid nanoparticles, or polymer particles. In some embodiments, the polymer particles may include poly(lactic acid)-polyethylene glycol (PLA-PEG) or poly(lactic acid-co-glycolic acid)-polyethylene glycol (PLGA-PEG) copolymers, and one or more polypeptide ligands conjugated to PEG via functional groups such as thioether links. In such embodiments, the neuronal cell targeting agent may be conjugated with PEG as a targeting ligand. The particles may be sized, for example, less than about 200 nm, less than about 150 nm, or less than about 100 nm, to improve blood-brain barrier (BBB) ​​penetration.

[0051] Alternative polymers that can be used include cyclodextrin-containing polymers, cationic cyclodextrin-containing polymers, poly(D,L-lactic acid-co-glycolic acid) (PLGA), poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), and poly(L-lactide). (PLLA), PLGA-b-poly(ethylene glycol)-PLGA (PLGA-bPEG-PLGA), PLLA-bPEG-PLLA, PLGA-PEG-maleimide (PLGA-PEG-mal), PLA-PEG-maleimide, poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co-caprolactone-co-glycolide), poly(D,L-lactide-co-PEO-co-D,L-lactide), poly(D,L-lactide-co-PPO. approx.Polyalkylenes such as poly(D,L-lactide), polyalkylcyanoacrylate, polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), polyethylene glycol, poly-L-glutamic acid, poly(hydroxy acid), polyacid anhydride, poly(esteramide), poly(ester ether), polycarbonate, polyethylene and polypropylene, polyalkylene glycols such as poly(ethylene glycol) (PEG), polyalkylene oxide (PEO), polyalkylene terephthalates such as poly(ethylene terephthalate), polyvinyl alcohol (PVA), polyvinyl ether, polyvinyl esters such as poly(vinyl acetate), poly(vinyl chloride) (PVC), polyvinylpyrrolidone, polysiloxane, polystyrene (PS), polyurethane, derivatized cellulose such as alkylcellulose, hydroxyalkylcellulose, cellulose ether, cellulose ester, nitrocellulose, hydroxypropylcellulose, carboxymethylcellulose, polymethyl methacrylate) (P Acrylic acid polymers such as MA, poly(ethyl (meth)acrylate), poly(butyl (meth)acrylate), poly(isobutyl (meth)acrylate), poly(hexyl (meth)acrylate), poly(isodecyl (meth)acrylate), poly(lauryl (meth)acrylate), poly(phenyl (meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), polyisobutyl acrylate), poly(octadecyl acrylate) (polyacrylic acid), and copolymers and Examples include mixtures thereof, polydioxanone and its copolymers, polyhydroxyalkanoates, polypropylene fumarates, polyoxymethylene, poloxamers, poly(ortho)esters, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), trimethylene carbonate, polyvinylpyrrolidone, polyorthoesters, polyphosphazenes, and polyphosphate esters, their dendrimers and derivatives, as well as blends and / or block copolymers of two or more such polymers.

[0052] In other embodiments, the particles are lipid nanoparticles containing PEG-conjugate lipids. Exemplary PEG lipids are selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. The PEG lipids may be selected from PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-cholesterol, PEG-tocopherol, or PEG-DSPE lipids.

[0053] In some embodiments, the lipid nanoparticles further comprise cationic or ionizable lipids, neutral lipids or phospholipids, and structural lipids. Exemplary structural lipids can be selected from one or more of cholesterol, fecosterol, sitosterol, ergosterol, campesterule, stigmasterol, brassicasterol, tomatidine, urisolic acid, and tocopherol (e.g., alpha-tocopherol). In some embodiments, the structural lipid is cholesterol. In some embodiments, the LNP comprises one or more phospholipids. Exemplary phospholipids are selected from the group consisting of cardiolipin, sterol-modified lipids (lipids modified by a cholesterol moiety bonded to the sn-2 carbon of a glycerol backbone), mixed acylglycerophospholipids, and symmetric acylglycerophospholipids. Examples of the head group of acylglycerophospholipids include phosphatidic acid, lysophosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphoinositides, and phosphatidylserine.Exemplary phospholipids include 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and 1,2-diundecane. Noyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), l-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-difitanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 Selected from PE), 1,2-distearoyl-sn-glycero-3-phosphate ethanolamine, 1,2-dilinoleyl-sn-glycero-3-phosphate ethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphate ethanolamine, l,2-diarachidonoyl-sn-glycero-3-phosphate ethanolamine, 1,2-didocosaenoyl-sn-glycero-3-phosphate ethanolamine, 1,2-dioleyl-sn-glycero-3-phosphate-rac-(l-glycerol) sodium salt (DOPG), and sphingomyelin.

[0054] In some embodiments, the active agent is a polypeptide encoded and expressed by one or more nucleic acids. In some embodiments, the nucleic acid is DNA (e.g., a gene or gene cassette) or mRNA. In some embodiments, the peptide is expressed in neurons via DNA or mRNA delivery. In some embodiments, the active agent is expressed using a neuron-specific promoter such as the synapsin 1 promoter. Exemplary active agents encoded by genes or mRNA are single-chain antibodies or scFv based on TGM-010 or its derivatives as described herein. Gene or mRNA delivery can employ known gene delivery techniques, including the lipid nanoparticles described above or viral vectors (e.g., AAV9, lentiviral vectors, etc.).

[0055] TGM-010 and its variants, as well as other hnRNP-binding agents described herein, may be used in methods for treating subjects having or at risk of having neuronal injury or neurodegenerative disease. In various embodiments, the subject in question has neurodegenerative disease or traumatic injury to one or more tissues of the central nervous system (CNS). In embodiments, the subject is at risk of developing neurodegenerative disease and may be considered, for example, asymptomatic or prodromal. Examples of neurodegenerative diseases include multiple sclerosis (MS), clinical isolation syndrome (CIS), Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), progressive supranuclear palsy, frontotemporal dementia (FTD), schizophrenia, Friedreich's ataxia, Lewy body dementia, and spinal muscular atrophy. In some embodiments, the subject in question has experienced or is recovering from ischemic or hemorrhagic stroke, traumatic brain injury, or spinal cord injury. In this embodiment, the subjects are at risk of developing a neurodegenerative disease (for example, progressing from an asymptomatic or prodromal state).

[0056] In another aspect, the present invention provides an antibody known as TGM-010, its variants, and polynucleotides encoding the same, as well as their use in methods for providing neuroprotection. Illustrative subjects include those suffering from neurodegenerative diseases, strokes, or traumatic brain or spinal cord injuries, as described herein.

[0057] In some embodiments, the TGM-010 antibody or variant is an IgG antibody, such as an IgG4 isotype. In some embodiments, the TGM-010 antibody or variant is an IgG1 isotype, which can be modified as described herein. In some embodiments, the TGM-010 antibody lacks an Fc domain. In some embodiments, derivatives or variants of the TGM-010 antibody are single-chain antibodies, scFvs, or nanobodies, or other antibody forms described herein (such as those lacking an Fc domain). In some embodiments, the TGM-010 antibody derivative may further comprise a cell membrane permeable peptide fusion, as described herein. The permeable peptide fusion may, in some embodiments, be fused at the N-terminus and / or C-terminus.

[0058] TGM-010 derivatives (i.e., TGM-010 variants) include antibodies or a portion thereof having 1 to 4 (e.g., 1, 2, 3, or 4) amino acid substitutions in six CDRs (i.e., collectively). Amino acid substitutions in the CDRs can be selected to increase binding to hnRNP targets such as hnRNP A1 or hnRNP A / B (or their RRM2 epitopes), increase cell permeability or antibody stability, or enhance neuroprotection (e.g., in vitro or in animal models such as EAE model mice). In addition, or alternatively, 1 to 10 amino acid modifications (e.g., substitutions) may be applied to the TGM-010 framework region. For example, 1 to 8 or 1 to 5 amino acid substitutions may be applied within the framework region. Amino acid substitutions in the framework region can be selected from those that increase binding to hnRNP targets (such as hnRNP A / B or hnRNP A1, or their RRM2 epitopes), increase cell permeability, increase antibody stability, or enhance neuroprotection (e.g., in vitro or in animal models such as EAE model mice).

[0059] In some embodiments and settings, a TGM-010 antibody variant that binds to hnRNP (including the RRM2 epitope described herein), including an hnRNP target (e.g., hnRNP A1 and / or hnRNP A / B) that forms the SG described herein, can be employed. In some embodiments and settings, a method for preparing the variant includes introducing 1 to 4 (e.g., 1, 2, 3, or 4) amino acid substitutions into one or more of the six CDRs of TGM-010 (i.e., SEQ ID NOs. 2-8), and / or introducing 1, 2, 3, 4, 5, or 6 or more amino acid substitutions, deletions, or insertions into the framework region of VH (SEQ ID NO. 1) and / or VL (SEQ ID NO. 5). In some embodiments, the TGM-010 variant includes those described herein and is prepared by modifying the Fc domain or further prepared. In some embodiments, a method for preparing the variant includes screening such antibody variants by measuring their binding to one or more peptides derived from the target hnRNP protein. In non-limiting examples, the peptide comprises an RRM2 domain epitope, or at least 20 amino acids of an RRM2 domain epitope, or at least 25 amino acids of an RRM2 domain epitope, or at least 40 amino acids of an RRM2 domain epitope, and includes the RRM2 epitopes of Table 1. In embodiments, the peptide comprises an epitope having at least about 80%, at least about 90%, at least about 95%, or 100% amino acid sequence identity with one or more regions of the amino acid sequences of Table 1, or at least 20 amino acids, or at least 25 amino acids, or at least 40 amino acids of the amino acid sequences listed in Table 1. In embodiments, the method comprises performing one or more techniques for measuring binding between an antibody variant and a peptide, including, for example, ELISA, surface plasmon resonance (SPR, e.g., BIACORE assay), and biolayer interferometry (BLI), and other techniques known in the art.

[0060] In embodiments, the active substance (including but not limited to TGM-010, its variants, and / or antigen-binding fragments or portions) or its pharmaceutical composition is administered systemically. In embodiments, the active substance or composition may be administered parenterally, such as by subcutaneous, intravenous, intraventricular, intraparenchymal, or intrathecal administration. In embodiments, the active substance or composition may be administered intranasally for more direct delivery to the brain. In some embodiments, the active substance or composition is administered as a single dose. In some embodiments, the active substance or composition is administered in multiple doses at a frequency ranging from nearly daily to nearly monthly (e.g., administration schedule), such as nearly daily, nearly twice a week, nearly weekly, nearly twice a month (e.g., once every two weeks), or nearly monthly. In embodiments, the frequency of administration can vary. In embodiments, the active substance or composition is administered as a single dose as needed when the subject experiences symptoms of neurodegenerative disease or neuronal injury. For example, therapy may be initiated at the time of symptoms of neuronal injury or an episode of neurodegenerative disease, or during or after recovery from such injury or episode. In the embodiments, the subjects are at risk of developing a neurodegenerative disease or of disease progression. In the embodiments, the active substance or its pharmaceutical composition is administered to subjects who are asymptomatic or in a prodromal state.

[0061] In some embodiments, the Disclosure provides a pharmaceutical composition comprising an effective amount of the antibody or active agent of the Disclosure, or a polynucleotide of the Disclosure, and a pharmaceutically acceptable carrier. Pharmacochemically acceptable carriers that can be used in accordance with the Disclosure are known in the Art. In some embodiments, the pharmaceutically acceptable carrier is a physiological solution, e.g., saline or other pharmaceutically acceptable solvent or buffer, and may optionally include a surfactant. Pharmacochemically acceptable carriers include water, saline, and glycerol. In some embodiments, the formulation may include a non-volatile oil, polyethylene glycol, propylene glycol, or other solvent.

[0062] The pharmaceutical compositions of this disclosure may, for convenience, be presented in unit dose form containing a predetermined amount of the active substance of this disclosure per dose. Self-injectors, such as "injection pens," are spring-loaded syringes designed to deliver specific doses of drugs. By design, injection pens are intended for ease of use and for self-administration by patients or untrained individuals. Injection pens are designed to overcome the hesitation associated with self-administration of needle-based drug delivery devices. Injection pens also have passive safety mechanisms to keep the needle tip protected before injection and to prevent accidental discharge (injection). The injection depth may be adjustable or fixed and may incorporate a function to remove the needle protection. By pressing a button, the injection needle is automatically inserted into the subcutaneous tissue and the drug is delivered. Some injection pens have visual or auditory indicators to confirm that the entire dose has been delivered upon completion of the injection.

[0063] In some embodiments, the injection pen contains 1 to 10 unit doses, or 1 to 5 unit doses. In some embodiments, the unit dose is about 1.5 mL or less in volume (whether contained or delivered by the injection pen). The injection pen may contain at least 4 unit doses (e.g., at least about 5, at least about 8, or at least about 10 unit doses).

[0064] In other embodiments, the Disclosure provides a method for preparing a pharmaceutical composition for providing neuroprotection. The method includes identifying an active agent (including a variant of TGM-010 as described) that binds to cytoplasmic hnRNP in stress granules, determining whether the active agent reduces or inhibits apoptosis of neurons (e.g., stressed neurons), and / or reduces or improves neurodegenerative disease or neuronal loss in animal models. If the active agent reduces or inhibits apoptosis of neurons (e.g., stressed neurons), and / or reduces or improves neurodegenerative disease or neuronal loss in animal models, the active agent is formulated for delivery to humans or animals.

[0065] In embodiments, the activator competes for the binding of TGM-010 to hnRNP or its RRM2 domain epitope (as already described) according to an in vitro binding assay (as already described). In various embodiments, the activator may employ the binding activator format described herein, including but not limited to antibodies (e.g., scFv or single-chain antibodies), and may include one or more cell membrane permeable peptides (e.g., as described herein). In various embodiments, the binding activator is applied to a selected hnRNP or RRM2 domain epitope (e.g., see Table 1) with a binding of 1 × 10⁻¹⁶. -7 M or less, or 1 × 10 -8 M or less, 1×10 -9 M or less, 1×10 -10 M or less, or 1 × 10 -11 M or less, or 1 × 10 -12 It can bind with an affinity of M or less. Binding affinity and competition for binding with TGM-010 can be determined by ELISA assay or surface plasmon resonance (SPR, e.g., BIACORE assay), biolayer interferometry (BLI), and other techniques known in the art.

[0066] In some embodiments, the active agent is a VH4 antibody that satisfies the antibody gene signature (AGS) codon described in U.S. Patent No. 8,394,583, which is incorporated herein by reference. In some embodiments, the active agent is an antibody that selectively binds to neurons, or selectively binds to neurons and astrocytes.

[0067] In some embodiments, neuroprotection is determined in vitro using mouse and / or human neuronal cell lines (e.g., those described herein) and / or in vivo using a suitable animal model such as an EAE mouse model. In embodiments, internal migration in neurons is quantified in vitro or in vivo using an animal model.

[0068] In some embodiments, the identified active agent is expressed in the target neuron via gene or mRNA delivery as described.

[0069] In some embodiments, the active ingredient is formulated for parenteral administration or non-parenteral administration (e.g., intranasal administration) as described.

[0070] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple subjects unless otherwise explicitly indicated.

[0071] Where used herein, the term "approximately" means ±10% of the stated value.

[0072] Next, embodiments of the present disclosure will be described with reference to the following examples. [Examples]

[0073] The following embodiments are included to demonstrate preferred embodiments. It should be understood by those skilled in the art that the techniques disclosed in the following embodiments are representative of the techniques that the inventors have found to function well in the implementation of the embodiments and can therefore be considered to constitute a preferred mode for implementation. However, those skilled in the art will understand that, in light of this disclosure, many modifications can be made in the specific embodiments disclosed without departing from the spirit and scope of this disclosure, and similar or comparable results can still be obtained.

[0074] Example 1: TGM-010 staining pattern TGM-010 is a recombinant human IgG antibody described in U.S. Patent Publication No. 20210214423, which is incorporated in its entirety herein by reference. TGM-010 is used for clinical isolation syndrome optic neuritis (ON- CISIt contains variable heavy and light chains cloned from a single CSF B cell isolated from a patient having ). TGM-010 is a VH4 antibody that satisfies the antibody gene signature (AGS) codon described in U.S. Patent No. 8,394,583, which is incorporated herein by reference.

[0075] The staining characteristics of TGM-010 in mouse brain sections and cell cultures were evaluated. TGM-010 strongly stains damaged mouse brain tissue (stroke and EAE brain tissue) compared to mouse wild-type brain tissue using diaminobenzidine (DAB) immunohistochemistry. Immunofluorescence (IFC) staining shows that TGM-010 stains neurons in mouse brain tissue but not astrocytes.

[0076] As shown in Figure 2, TGM-010 stains mouse cortical hippocampal neurons and human motor neurons, as indicated by immunofluorescence staining. TGM-010 also stains mouse neural progenitor cells, as indicated by flow cytometry in Figure 3. Therefore, the cellular target of TGM-010 is identified as neurons.

[0077] The binding affinity of TGM-010 was tested in ALS-derived (C9orf72) human cortical neurons. ALS-derived (C9orf72) human cortical neurons were incubated overnight at 4°C with primary antibodies diluted with blocking buffer rhAb (20 μg / mL), anti-ChAT, and anti-MAP2. Choline acetyltransferase (ChAT) was used as a neuronal marker, and microtubule-associated protein 2 (MAP2) was used to visualize neuronal boundaries. Cells were visualized using a confocal fluorescence microscope. As shown in Figure 4, TGM-010 exhibited superior binding compared to non-specific control antibodies, as evidenced by the fluorescence intensity within the boundaries of human cells (scale bar = 20 μm).

[0078] Example 2: TGM-010 prevents recurrence in the EAE mouse model. Neuroprotection by TGM-010 was evaluated using an experimental autoimmune encephalomyelitis (EAE) mouse model. The EAE model is described in Robinson, AP, et al. “The experimental autoimmune encephalomyelitis (EAE) model of MS: utility for understanding disease pathophysiology and treatment”, Handb Clin Neurol. 2014;122:173-189. 100 μg of PLP was administered along with 2 μg of complete Freund's adjuvant (CFA). 139~151 EAEs were induced in SJL / j female mice by immunization using [a specific method]. The first seizure typically occurred around day 10 and resolved around day 17. Mice that had recovered were administered either TGM-010 (5 μg) or a control antibody via intraventricular (ICV) administration. The site of ICV administration was recorded. Mice were scored for EAEs for 20 days, or until the mouse had another seizure and recovered. Immunohistochemistry could be used to assess demyelination and inflammation in treated and control mice. In these tests, TGM-010 was evaluated side-by-side with control antibodies HD10-15 and G11, as well as a control without antibodies (PBS). The TGM-007 (also known as AJL7) antibody, a human recombinant AGS antibody that binds to neurons and astrocytes, was also evaluated.

[0079] As shown in Figure 5, cumulative EAE scores were similar between the groups during the first seizure. After randomization and antibody administration, the cumulative EAE score was significantly reduced in mice treated with TGM-010. The cumulative EAE score data indicates that 6 out of 10 mice treated with TGM-010 showed no signs of disease activation after TGM-010 injection compared to the control group.

[0080] In further experiments, TGM-010 was delivered over two weeks by intraperitoneal (IP) injection, starting one day after recovery from the first seizure. Mice with a confirmed first seizure were randomized and administered either placebo or TGM-010 at either 200 ug or 100 ug via intraperitoneal (IP) injection, for a total of six injections per mouse. Mice were scored for an additional 20 days, and EAE scores tracking disease severity were recorded daily. Cumulative EAE score data showed that TGM-010 injected at 200 ug per dose significantly reduced cumulative EAE scores compared to placebo (HOLO). TGM-010 at 100 ug per dose had a moderate effect on cumulative EAE scores, demonstrating a dose-effect. Cumulative EAE score data showed that the severity of the first seizure was similar in all mice. See Figure 6.

[0081] These studies demonstrate that TGM-010 possesses neuroprotective properties in vivo.

[0082] Example 3: TGM-010 specifically recognizes hnRNP targets, suggesting a potential mechanism of TGM-010 neuroprotection. Potential targets of TGM-010 were identified using a human protein array (CDI Laboratories, Inc.). Nonspecific hits that directly bound to the secondary antibody were excluded from the analysis. Using CDI software, the specificity of each individual sample to a particular protein on the array was quantified based on the Z-score. The Z-score is the average of two spots for a given protein (each protein is printed two times on the array). The Z-score for each spot on the array was calculated according to the algorithm Z = [F635 - 5635(avg)] / F635(std), where F635(avg) and F635(std) are the mean and standard deviation of the F635 values ​​for all spots on the array.

[0083] The results of the protein array analysis are shown in Figure 7. The top hit was heteronucleoprotein A / B (hnRNPAB), with a Z-score of 79.65.

[0084] Protein arrays have limitations as indicators of brain antigen targets, and do not always present proteins in their physiological state. Therefore, the antigen target of TGM-010 was also evaluated by immunoprecipitation.

[0085] For immunoprecipitation testing, Sy5y cell lysates were prepared and used for immunoprecipitation with TGM-010 or the control antibody JRR67. Immunoprecipitated proteins were separated by SDS-PAGE, followed by trypsin digestion of individual bands and mass spectrometry of individual peptides. The SDS-PAGE gel, along with the bands subjected to mass spectrometry, is shown in Figure 8. This includes a double line at approximately 37 / 40 kDa, as well as a 100 kDa band and a double line at 50 kDa. The mass spectrometry results for the 37 / 40 kDa band are shown in Figure 9. Most sequences (6 of the top 10), including the top CDI array hit (heteronuclear ribonucleoprotein A / B), correspond to heteronuclear ribonucleoproteins. Based on these tests, TGM-010 is considered to have high specificity for hnRNPs, particularly the hnRNP A / B family subgroup.

[0086] hnRNP particles are RNA-protein complexes present in the cell nucleus during gene transcription and subsequent post-transcriptional modification of newly synthesized RNA (pre-mRNA). The presence of these proteins bound to pre-mRNA molecules serves as a signal that the pre-mRNA is not yet fully processed and therefore not ready for export to the cytoplasm. Even after splicing occurs, hnRNPs remain bound to spliced ​​introns, targeting these introns for degradation. hnRNPs are also essential for the 40S subunit of ribosomes and are therefore important for mRNA translation in the cytoplasm. hnRNPs have their own nuclear localization sequences (NLS) and are therefore primarily found in the nucleus, although some hnRNPs translocate between the cytoplasm and the nucleus.

[0087] There are approximately 20 major hnRNPs (hnRNP A1 to hnRNP U), and the location and function of each member differ in each cell type. Several structural domains are shared among different family members. See Geuens T, et al., “The hnRNP family: insights into their roles in health and disease,” Hum Genet (2016) 135:851-867. hnRNP A / B comprises four subgroups, A1, A2 / B1, A3, and A0, and are involved in mRNA translation and splicing, and are components of the 40S hnRNP complex. hnRNP A2 / B1 plays a crucial role in mRNA transport in oligodendrocytes and neurons. hnRNP A2 / B1 is important for the precise localization of mRNAs containing A2 response elements (A2REs) or A2RE-like sequences, such as myelin basic protein (MBP) or Ca2+ / calmodulin-dependent protein kinase II (CaMKII), regulated cytoskeleton-related proteins (Arc), and neurogranins (NRGN). hnRNP A1 has been reported to localize to stress granules (SGs) during the cellular stress response. See Guil S, et al., “hnRNP A1 Relocalization to the Stress Granules Reflects a Role in the Stress Response,” Mol Cell Biol (2006)(15):5744-58.

[0088] Interestingly, ALS type 20 is caused by a mutation in the hnRNPA1 gene. Krebs BB and De Mesquita JF, “Amyotrophic Lateral Sclerosis Type 20-In Silico Analysis and Molecular Dynamics Simulation of hnRNPA1.” PLoS One (2016 Jul 14;11(7):e0158939).

[0089] Antibodies against hnRNP A1 have been reported to contribute to neuronal loss in animal models of multiple sclerosis. See Libner CD et al., J Comp Neurol 2020 Jul;528(10):1704-1724. In fact, mislocalized hnRNP A1 levels were significantly higher in EAE mice compared to naive mice, and these models were reported to be directly correlated with neuronal death and clinical scores.

[0090] Post-transcriptional regulation of gene expression during various stimuli such as heat shock, oxidative stress, and viral infection is crucial for cell survival. SGs represent cytoplasmic regions where translationally stopped mRNA and numerous RNA-binding proteins form nuclei in response to stress, allowing cells to reprogram gene expression. SGs may also be sites where mRNA triage occurs, directing whether to degrade or translate the mRNA.

[0091] Example 4: In vitro antibody toxicity evaluation of TGM-010 This study focused on evaluating the potential neurotoxicity of the antibody TGM-010. Positive control antibodies were created by sequencing B cells derived from patients with autoimmune encephalitis and cloning anti-neuronal antibodies, such as "DS14".

[0092] Sy5y cells were incubated with antibodies at 37°C for 3 hours. For the last 30 minutes of incubation, the cells were incubated with 6 μM caspase-3 / 7 green detection reagent (INVITROGEN C10423, a fluorescence-generating substrate for cell lysis detection). The cells were then fixed with 4% (v / v) paraformaldehyde (PFA) and stained for immunocytochemistry using the following antibodies: mouse anti-G3BP1 (1:100 BD BIOSCIENCES 611126), goat anti-human conjugate ALEXAFLUOR 568 (1:1000 THERMO FISHER A-21090), and goat anti-mouse conjugate ALEXAFLUOR 647 (1:500 ABCAM ab150115). Coverslips were placed on the cells, and the cells were visualized using a spinning disk confocal microscope.

[0093] As shown in Figure 10, TGM-010 did not induce clear neuronal apoptosis, similar to the vehicle control, compared to DS14, a positive control that induced a statistically significant degree of apoptosis (approximately 35%).

[0094] These data, in particular, demonstrated that TGM-010, a neuron-targeting antibody, does not exhibit clear neurotoxicity in vitro.

[0095] Example 5: TGM-010 hnRNP binding domain test and epitope mapping To evaluate the binding site of TGM-010, enzyme-linked immunosorbent assay (ELISA) was performed on an hnRNP peptide array. The peptide array was generated using 24-mer with 8-amino acid length (8-mer) overlaps. Selected hnRNP family protein members, namely hnRNP A2B1, hnRNP A1, and hnRNP AB, were tested using a standard ELISA protocol and compared with TGM-010 against a non-specific control antibody (e.g., JRR7).

[0096] NUNC-IMMUNO MAXISORP 96-well plates (SIGMA ALDRICH) were incubated overnight at 4°C in 0.1 M sodium carbonate buffer (pH 9.5) with 5 μg / ml hnRNP peptide. The coated plates were searched with 20 μg / mL TGM-010 or JRR67 in assay diluent. After several washes, the plates were subsequently incubated with goat anti-human IgG secondary antibody (CHEMICON) conjugated with horseradish peroxidase. Colorimetric readings were obtained using an ELISA microplate reader (λ=450). Positive antibody reactivity was defined as a value greater than the mean value of the control plus two standard errors.

[0097] As shown in Figures 11A and 11B, the ELISA signals indicated that peptides 3-8 and 3-11 (SEQ ID NOs. 16 and 19, Table 1) bound to the TGM-010 antibody by more than two standard errors compared to the control antibody. These peptides are located within the RRM2 domain of hnRNP A1. Furthermore, peptides 6-11 and 6-14 (SEQ ID NOs. 20 and 23, Table 1) of hnRNP A / B bound to the TGM-010 antibody by more than two standard errors compared to the control antibody. These peptides are also located within the RRM2 domain of hnRNP A / B. These data, in particular, demonstrate the antibody's specificity within the RRM2 domain of selected hnRNP proteins exhibiting homology within this region.

[0098] These data demonstrate, in particular, that TGM-010 binds to the conserved RRM2 region of the hnRNP protein, which is associated with various neurological and neurodegenerative states characterized by neuronal loss.

[0099] Example 6: Translocation of TGM-010 into living cells In summary, the internal distribution capacity of TGM-010 was evaluated in vitro using the Sy5y human neuroblastoma cell line. Sy5y cells were cultured to confluence, incubated with either a nonspecific negative control antibody or TGM-010, then fixed, stained, and imaged using a fluorescence confocal microscope at various time points. Staining was performed using an anti-tubulin monoclonal antibody (e.g., Tub) to visualize the neuronal cell body, DAPI blue staining to visualize the nucleus, and control or TGM-010 antibody staining (e.g., rhAb).

[0100] As shown in Figures 12A and 12B, statistically significant internal migration of TGM-010 into Sy5y neuroblastoma cells was observed, as evidenced by the fluorescence intensity compared to the control antibody at 2, 6, and 24 hours post-exposure (scale bar = 20 μm). Furthermore, as shown in Figure 12B, the dynamics of internal migration indicate that TGM-010 rapidly entered the cytoplasm 60 minutes after exposure, while the control antibody clearly did not enter the cells.

[0101] A mouse cortical neuron model was also used to evaluate and confirm the in vitro internal transfer of TGM-010, which was performed substantially as described above for Sy5y cells. As shown in Figures 12C and 12D, statistically significant internal transfer of TGM-010 into mouse cortical neurons was observed at 30 minutes, 2 hours, and 6 hours post-exposure compared to nonspecific control antibodies (scale bar = 20 μm).

[0102] These data, in particular, showed that TGM-010, compared to nonspecific antibodies, increased in total volume and speed, and effectively internalized into neurons (across species). These results partially reflect the conserved nature of the RRM2 epitope across species.

[0103] Example 7: Evaluation of TGM-010 Fc domain binding Next, the effect of the presence of the Fc domain on TGM-010 binding was evaluated. Full-length IgG TGM-010 was pepsin-cleaved to remove the Fc domain, and the resulting F(ab')2 product was isolated, for example, as shown in Figure 13A. The effect of the Fc domain on TGM-010 binding was evaluated using the Sy5y human neuroblastoma cell line. Sy5y cells were cultured to confluence and incubated with either a nonspecific negative control antibody, full-length IgG TGM-010, or F(ab')2 TGM-010, then fixed, stained, and imaged using a fluorescence confocal microscope at various time points. Staining was performed with DAPI blue to visualize the nuclei, and antibody staining (e.g., rhAb) was performed with green, so that the color of the neurons would shift to cyan due to fluorescence overlap caused by internal migration of antibody or antigen-binding fragments.

[0104] As shown in Figure 13B, the full-length nonspecific control antibody (JRR67) did not show clear staining. In contrast, as shown in Figure 13C, both full-length IgG TGM-010 and antigen-binding fragment F(ab')2 TGM-010 resulted in significant neuronal connectivity, as is evident from the color shift of fluorescence intensity.

[0105] Example 8: In vitro neuroprotective effect of TGM-010 under stress conditions To evaluate the potential neuroprotective effects of TGM-010 in neurons under stress conditions, we employed a modified toxicity assay used in Example 4. Toxicity was compared to that of positive control anti-neuronal antibody DS14, which is an antibody cloned from B cells obtained from patients with autoimmune encephalitis.

[0106] Sy5y cells were incubated with antibody for 6 hours, followed by heat shock treatment at 43°C for 2 hours or maintenance at 37°C for 1.5 hours. Then, for the last 30 minutes of each incubation period, the cells were incubated with 6 μM caspase-3 / 7 green detection reagent (INVITROGEN C10423, a fluorescence-generating substrate for cell lysis detection). The cells were fixed with 4% (v / v) paraformaldehyde (PFA), then covered with a coverslip, and visualized by caspase-3 / 7 fluorescence using a spinning disk confocal microscope.

[0107] As shown in Figure 14, TGM-010 treatment provided a statistically significant degree of neuroprotection from heat-induced neuronal apoptosis compared to the vehicle alone and the neurotoxic antibody DS14. TGM-010 replicas showed approximately 60%+ non-apoptotic cells in culture and a reduction of about one-tenth in the presence of apoptotic neurons.

[0108] These data, in particular, demonstrated that TGM-010 has a clear neuroprotective effect in vitro against cellular stress (e.g., heat-induced stress).

[0109] Example 9: Co-localization of stress granule concentration with TGM-010 To further investigate the neuroprotective mechanisms of TGM-010, we examined the co-localization of antibodies with stress granule assembly factor 1 (G3BP1) in stress granules (SGs). G3BP1 is a known core protein of stress granules (SGs) and is used as a marker to assess SG enrichment, for example, as described in Fang et al., Neuron 2019.

[0110] Here, isolated mouse cortical neurons underwent heat-induced staghorn gland (SG) formation, were subsequently fixed, stained, and imaged using a fluorescence confocal microscope. Staining was performed using an anti-G3BP1 monoclonal antibody to visualize SGs, DAPI blue to visualize nuclei, MAP2 staining to visualize neuronal bodies, and an anti-human secondary antibody to visualize either a nonspecific control antibody or TGM-010 antibody.

[0111] As shown in Figure 15A, heat treatment at 45°C for 2 hours without antibody resulted in significant SG formation, and G3BP1 staining was observed in the granules under heat treatment conditions (e.g., indicated by white arrows). Next, a nonspecific negative control antibody (e.g., JRR67) was compared with TGM-010 to analyze the antibody's ability to co-localize with SG. As shown in Figure 15B, the control antibody did not show co-localization with SG, while qualitative and quantitative analyses, as shown in Figures 15C-15D, showed that TGM-010 treatment resulted in statistically significant co-localization with SG formation under heat stress conditions.

[0112] These data, in particular, showed that TGM-010 colocalizes with stress granules (SGs) in neurons under cellular stress.

[0113] Example 10: Neuroprotective effects of TGM-010 in an experimental autoimmune encephalitis (EAE) mouse model. Experimental autoimmune encephalitis (EAE) animals have conventionally been used to screen for neuroprotective agents. Myelin proteolipid protein residues 139-151 (PLP) along with 2 μg of complete Freund's adjuvant (CFA) have been used. 139~151EAE was induced in female SJL / j mice by immunization with 100 μg of ). The first “episode” or finding of disease severity due to demyelination typically occurs around day 10 and disappears around day 17 (e.g., shown as the peak of disease severity in Figures 16A-16B). Mice that had recovered from the first epidemic (n=28) were blinded and randomized, and administered either TGM-010 (5 μg) or a nonspecific control antibody by intraventricular (ICV) administration on day 15. Mice were scored for EAE for 20 days, or until the mouse had another epidemic and recovered. They were followed for 39 days and sacrificed.

[0114] As shown in Figure 16A, the mean EAE score was statistically significantly improved. The TGM-010 antibody showed a statistically significant reduction in disease severity compared to the control antibody immediately after administration, and the improvement lasted for at least approximately two weeks after injection. Subsequently, a blinded EAE in vivo study was performed in mice (n=26), and the results were confirmed by intraperitoneal (IP) systemic administration in six doses, for example, on days 19, 21, 23, 25, 27, and 29. As shown in Figure 16B, TGM-010 achieved a statistically significant reduction in disease severity compared to the control antibody even when administered systemically.

[0115] To confirm that TGM-010 can penetrate the intact blood-brain barrier and access its cellular targets, WT mice were intravenously injected with either TGM-010 or the control antibody JRR67, and their brains were collected 24 hours after injection. Serial fixed sections were then prepared from the brains of the treated mice. The administered antibody was detected in the serial brain sections by immunofluorescence staining using a goat anti-human AlexaFluor488 secondary antibody. Neurons were labeled with a mouse anti-NeuN primary antibody, followed by a goat anti-Ms AlexaFluor647 labeled secondary antibody. Images were acquired using a Zeiss Axioscope.A1 fluorescence microscope equipped with a 20x / 0.45NA lens.

[0116] As shown in Figure 17A, TGM-010 exhibits strong BBB permeability and neuronal labeling 24 hours after administration, while the control antibody does not. The punctate labeling observed in both slides is nonspecific background autofluorescence. Figure 17A quantifies TGM-010 staining in neurons compared to the control antibody JRR67, further demonstrating statistically significant levels of TGM-010 staining compared to JRR67.

[0117] Figure 17B shows labeling of anterior, middle, and distal brain sections of layer 1 of the mouse cerebral cortex 24 hours after drug administration, demonstrating strong staining of TGM-010 in all parts of the cortex. Figure 17B quantifies the signal intensity and shows a significant increase in TGM-010 staining at 24 hours compared to the control antibody and at 8 hours post-administration.

[0118] These data, in particular, demonstrated that TGM-010 exhibits neuroprotective effects in vivo and can cross the blood-brain barrier (BBB) ​​in vivo via various local and systemic administration methods.

[0119] Example 11: Internal transfer of TGM-010 into human cortical neurons derived from ALS. The internal distribution capacity of TGM-010 was evaluated in vitro using human cortical neurons derived from ALS. Human cortical neurons were cultured to confluence and incubated for 2 hours with either a nonspecific negative control antibody (JR667) or TGM-010, then fixed, stained, and imaged using a fluorescence confocal microscope at various time points. Anti-MAP2 monoclonal antibody was used to visualize neuronal boundaries, DAPI blue staining was used to visualize nuclei, and control or TGM-010 antibody staining (e.g., rhAb) was used.

[0120] As shown in Figure 18, statistically significant internal transfer of TGM-010 into human cortical neurons was observed at 0 and 30 minutes post-exposure, as evidenced by the fluorescence intensity compared to the vehicle. This was significantly increased compared to JRR67 at 30 minutes, indicating that TGM-010 rapidly penetrates the cytoplasm, whereas the control antibody clearly did not penetrate the cells.

[0121] These data, in particular, demonstrate that TGM-010, compared to nonspecific antibodies, appropriately internalizes into ALS-derived cortical neurons in increased total amounts and at a rapid rate.

[0122] All compositions and methods disclosed and claimed herein can be prepared and performed without undue experimentation in light of this disclosure. While the compositions and methods of this disclosure are described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations may be applied to the compositions and methods described herein, as well as to the steps or order of the steps, without departing from the concepts, spirit, and scope of this disclosure. More specifically, it will be apparent that certain chemically and physiologically related active substances may be substituted for the active substances described herein, insofar as the same or similar results are achieved. All such similar substitutions and modifications that are apparent to those skilled in the art are considered to fall within the spirit, scope, and concepts of this disclosure as defined by the appended claims.

[0123] TIFF2026522973000002.tif188165

Claims

1. A method for neuroprotection, comprising administering an effective amount of a substance that binds to cytoplasmic heteronuclear ribonucleoproteins (hnRNPs) in neurons and thereby inhibits neuronal cell death to a subject in need thereof.

2. The method according to claim 1, wherein the hnRNP includes hnRNP A1 or hnRNP A / B.

3. The method according to claim 1, wherein the hnRNP includes one or more of hnRNP A2 / B1, hnRNP C1 / C2, hnRNP A1, hnRNP A3, and hnRNP D0.

4. The method according to any one of claims 1 to 3, wherein the active substance binds to the RRM2 epitope in Table 1.

5. The method according to any one of claims 1 to 4, wherein the active substance is an antibody or an antigen-binding fragment or portion thereof.

6. The method according to claim 5, wherein the active substance is an IgG monoclonal antibody.

7. The method according to claim 5, wherein the active substance is scFv.

8. The active substance is F(ab') ２ The method according to claim 5.

9. The method according to any one of claims 1 to 8, wherein the active substance is a polypeptide expressed in neurons via delivered nucleic acids.

10. The method according to claim 9, wherein the nucleic acid is DNA or mRNA.

11. The method according to claim 9 or 10, wherein the nucleic acid is delivered by lipid nanoparticles or encoded in a viral vector.

12. The method according to any one of claims 1 to 11, wherein the active substance is a TGM-010 antibody or its antigen-binding fragment or portion, or a variant thereof.

13. The method according to claim 12, wherein the antibody or its antigen-binding fragment or portion comprises 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.

14. The method according to claim 13, wherein the antibody or its antigen-binding fragment or portion comprises a heavy chain variable domain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1, and a light chain variable domain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO:

5.

15. The method according to claim 13, wherein the antibody or its antigen-binding fragment or portion comprises a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 1 and a light chain variable domain having the amino acid sequence of SEQ ID NO:

5.

16. The method according to any one of claims 1 to 15, wherein the subject has a neurodegenerative disease or traumatic injury, or is at risk thereof.

17. The method according to 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's ataxia, Lewy body dementia, or spinal muscular atrophy.

18. The method according to any one of claims 1 to 15, wherein the subject is at risk of, has experienced, or has recovered from ischemic or hemorrhagic stroke, traumatic brain injury, or spinal cord injury.

19. The method according to any one of claims 1 to 18, wherein the active substance is administered systemically.

20. The method according to any one of claims 1 to 18, wherein the active substance is administered parenterally, and optionally by subcutaneous, intravenous, intraventricular, intraparenchymal, or intrathecal administration.

21. The method according to any one of claims 1 to 18, wherein the active substance is administered intranasally.

22. The method according to claim 20 or 21, wherein the active substance is administered once or multiple times, and optionally at a frequency of approximately daily to approximately monthly.

23. The method according to claim 22, wherein the active substance is administered during symptoms of neuronal damage or during an attack of a neurodegenerative disease, or during or after recovery from the damage or the attack.

24. A method for neuroprotection, comprising administering an effective amount of an antibody or its antigen-binding portion 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, to a subject in need thereof.

25. The method according to claim 24, wherein the antibody or its antigen-binding portion binds to one or more intracytoplasmic heteronuclear ribonucleoproteins (hnRNPs) in a neuron.

26. The method according to claim 25, wherein the hnRNP includes hnRNP A / B.

27. The method according to claim 25, wherein the hnRNP includes hnRNP A1.

28. The method according to any one of claims 24 to 27, wherein the antibody is an IgG antibody, and is optionally selected from IgG1, IgG2, IgG3, and IgG4.

29. The aforementioned antibody is scFv or F(ab') ２ The method according to any one of claims 24 to 28.

30. The method according to any one of claims 24 to 29, wherein the antibody or its antigen-binding fragment is expressed in a neuron via DNA or mRNA delivery.

31. The method according to claim 30, wherein the antibody or its antigen-binding fragment is delivered using lipid nanoparticles.

32. The method according to claim 30, wherein the antibody or its antigen-binding fragment is encoded in a viral vector.

33. The method according to any one of claims 24 to 32, wherein the antibody or antigen-binding fragment comprises a heavy chain variable domain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1, and a light chain variable domain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO:

5.

34. The method according to claim 33, wherein the antibody or antigen-binding fragment comprises a heavy chain variable domain having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 1, and a light chain variable domain having an amino acid sequence having at least 95% sequence identity with SEQ ID NO:

5.

35. The method according to claim 33, wherein the antibody or its antigen-binding fragment comprises a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 1 and a light chain variable domain having the amino acid sequence of SEQ ID NO:

5.

36. The method according to any one of claims 24 to 35, wherein the subject has or is at risk of having a neurodegenerative disease or acute or traumatic injury.

37. The method according to claim 36, wherein the neurodegenerative disease is multiple sclerosis (MS), clinical isolation 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's ataxia, Lewy body dementia, or spinal muscular atrophy.

38. The method according to claim 36, wherein the subject is at risk of, has experienced, or has recovered from ischemic or hemorrhagic stroke, traumatic brain injury, or spinal cord injury.

39. The method according to any one of claims 24 to 38, wherein the antibody or its antigen-binding fragment is administered parenterally.

40. The method according to claim 39, wherein the antibody or its antigen-binding fragment is administered by subcutaneous administration, intravenous administration, intraventricular administration, intraparenchymal administration, or intrathecal administration.

41. The method according to any one of claims 24 to 38, wherein the antibody or an antigen-binding fragment thereof, or DNA or mRNA encoding it, is administered intranasally.

42. The method according to claim 40 or 41, wherein the antibody or its antigen-binding fragment is administered at a frequency of approximately daily to approximately monthly.

43. The method according to any one of claims 34 to 42, wherein the antibody or its antigen-binding fragment is administered during symptoms of neuronal injury or during an attack of a 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5, and a pharmaceutically acceptable carrier, wherein the monoclonal antibody further comprises one or more modifications selected from a portion that enhances cell permeability, a modification that reduces Fc-mediated effector function, and an Fc modification that improves pharmacodynamic (PD) properties.

45. The pharmaceutical composition according to claim 44, wherein the monoclonal antibody comprises one or more modifications that increase the circulating half-life or serum half-life, modifications that increase blood-brain barrier (BBB) ​​permeability, modifications that increase neuronal uptake, and modifications that reduce off-target or nonspecific uptake.

47. A pharmaceutical composition comprising an antigen-binding fragment of an antibody, or a polynucleotide encoding the same, comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 1 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 5, and a pharmaceutically acceptable carrier.

48. The aforementioned fragment is scFv or F(ab') ２ The pharmaceutical composition according to claim 47.

49. The pharmaceutical composition according to claim 47 or 48, wherein the fragment further comprises a portion that enhances cell permeability.

50. The pharmaceutical composition according to claim 49, wherein the aforementioned portion comprises a cell membrane permeable peptide.

51. The pharmaceutical composition according to claim 47, comprising a polynucleotide encoding scFv, and optionally further comprising a peptide for enhancing cell permeability.

52. The pharmaceutical composition according to claim 51, wherein the polynucleotide is DNA or mRNA.

53. The pharmaceutical composition according to claim 51, wherein the polynucleotide is encapsulated by lipid nanoparticles.

54. The pharmaceutical composition according to claim 52, wherein the DNA or mRNA is encoded from or expressed by a viral vector.

55. A method for preparing a pharmaceutical composition to provide neuroprotection, To identify active agents that bind to one or more intracytoplasmic heteronuclear ribonucleoproteins (hnRNPs) or their RRM2 epitopes in stress granules, To determine whether the active substance reduces or inhibits apoptosis in neurons, and / or reduces or improves neurodegenerative diseases or neuronal loss in animal models, Formulating the aforementioned active substance for delivery to humans or animals. The method, including the method described above.

56. The method according to claim 55, wherein one or more hnRNPs include RNA recognition motif 2 (RRM2).

57. The method according to claim 55 or 56, wherein the hnRNP includes hnRNP A / B or hnRNP A1.

58. The method according to claim 55 or 56, wherein the one or more hnRNPs include hnRNP A2 / B1, hnRNP C1 / C2, hnRNP A1, hnRNP A3, and hnRNP D0.

59. The method according to any one of claims 55 to 58, wherein the active substance blocks the binding of TGM-010 to the hnRNP or RRM2 epitope when measured by an in vitro binding assay.

60. The effector substance is 1×10 －７ M or less, or 1×10 －８ M or less, 1×10 －９ M or less, 1×10 －１０ M or less, 1×10 －１１ M or less, or 1×10 －１２ The method according to any one of claims 55 to 59, which binds to the hnRNP or RRM2 epitope with an affinity of M or less.

61. The method according to claim 59 or 60, wherein the active substance is an antibody or an antigen-binding fragment thereof.

62. The method according to claim 59 or 60, wherein the active substance is a peptide, aptamer, adonectin, or DARPin.

63. The method according to claim 59 or 60, wherein the active substance is selected from single-domain antibodies, recombinant heavy chain-only antibodies (VHH), single-chain antibodies, single-chain variable fragments (scFv), shark heavy chain-only antibodies (VNAR), tetranectin, affibody, transbody, antikalin, affilin, microbody, phylomer, stradobody, fynomer, avimer, triomab, Fv, Fab, Fab', and F(ab')2.

64. The method according to claim 59 or 60, wherein the active substance is an antibody, and optionally a human antibody, and optionally a TGM-010 variant.

65. The method according to claim 64, wherein the active substance is a VH4 antibody that selectively binds to neurons, or selectively binds to neurons and astrocytes.

66. The TGM-010 variant is Introducing one to four amino acid substitutions into one or more of the TGM-010 CDRs (SEQ ID NOs: 2-5 or 6-8), and / or Introducing 1 to 6 amino acid substitutions, deletions, or insertions within the framework region of the TGM-010 VH (SEQ ID NO: 1) chain and / or VL (SEQ ID NO: 5) chain. The method according to claim 64, which is prepared by

67. The method according to claim 64, wherein the TGM-010 variant is prepared by modifying the Fc domain.

68. The method according to any one of claims 55 to 67, comprising screening candidate activators for binding to one or more peptides derived from the RRM2 domain epitope.

69. The method according to 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 according to claim 69, wherein the RRM2 domain epitope comprises the amino acid sequence shown in Table 1 or a portion thereof.

71. The method according to claim 69, wherein the RRM2 domain epitope has at least about 80%, at least about 90%, at least about 95%, or 100% amino acid sequence identity with respect to at least 20 amino acids, or at least 25 amino acids, or at least 40 amino acids, of the amino acid sequences listed in Table 1.

72. The method according to any one of claims 66 to 71, further comprising measuring the binding between a candidate binding agent and an RRM2 epitope by ELISA, surface plasmon resonance (SPR), and biolayer interferometry (BLI).

73. The method according to any one of claims 55 to 72, wherein inhibition of apoptosis in neurons is determined in vitro using a mouse or human nerve cell line.

74. The method according to any one of claims 55 to 73, wherein the inhibition of apoptosis in neurons is determined in vitro.

75. The method according to any one of claims 55 to 74, wherein a reduction or improvement of neurodegenerative disease or neuronal loss is determined in an animal model, and optionally the animal model is an EAE mouse model.

75. The method according to any one of claims 55 to 74, further comprising quantifying internal migration in neurons in vitro or using an in vivo animal model.

76. The method according to any one of claims 55 to 75, wherein the active substance is a polypeptide and is formulated to be expressed in neurons via gene or mRNA delivery.

77. The method according to any one of claims 55 to 75, comprising formulating the active substance for parenteral administration or intranasal administration.

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