ASIC1 channel antagonist antibody

Abstract

To provide compositions and methods of preventing or treating ischemic stroke.SOLUTION: The present technology relates to administering anti-ASIC1a antibodies in effective amounts to treat a subject suffering from, or predisposed to, ischemic stroke.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present technology relates generally to compositions and methods for preventing or treating diseases associated with ASIC1a activity and / or signaling. More particularly, the present technology provides for administering to a subject an effective amount of an anti-ASIC1a antibody to treat a subject suffering from or susceptible to ischemic stroke and related conditions caused by or associated with ASIC1a activity and / or signaling. [Background technology]

[0002] The following explanation is provided to aid the reader's understanding. None of the information provided or references cited are admitted to be prior art. Acid-sensing ion channels (ASICs) are gated by extracellular protons. ASICs are cation channels that are activated by extracellular acidosis. At least four genes have been identified that code for six ASIC subunits: ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3, and ASIC4, in which the designations "a" and "b" represent alternatively spliced ​​variants of the ASIC1 and ASIC2 genes, ACCN2 and ACCN1, respectively. Functional ASIC channels that are sensitive to blockade by amiloride are composed of three subunits assembled in either homomeric or heteromeric forms. ASIC1a is highly expressed in the brain and forms functional homomeric or heteromeric channels with other ASIC isoforms. With an activation threshold near pH 7.0, ASIC1a serves as the primary sensor of acidosis in the brain and is involved in normal as well as pathological conditions. Summary of the Invention [Problem to be solved by the invention]

[0003] The present invention provides compositions and methods for preventing or treating diseases associated with ASIC1a activity and / or signaling. [Means for solving the problem]

[0004] In one embodiment, the present technology provides a method for treating ischemic stroke in a subject in need thereof, comprising administering to a subject a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domains (V L administering to a subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof comprising: H is selected from the group consisting of SEQ ID NOs: 3, 13, 23, 33, 43, and 53 H - CDR1 sequence; V selected from the group consisting of SEQ ID NOs: 4, 14, 24, 34, 44, and 54 H - a CDR2 sequence; and a V selected from the group consisting of SEQ ID NOs: 5, 15, 25, 35, 45, and 55 H - comprising a CDR3 sequence; V L is selected from the group consisting of SEQ ID NOs: 8, 18, 28, 38, 48, and 58 L - CDR1 sequence; V selected from the group consisting of SEQ ID NOs: 9, 19, 29, 39, 49, and 59 L - a CDR2 sequence; and a V selected from the group consisting of SEQ ID NOs: 10, 20, 30, 40, 50, and 60 L -CDR3 sequence,

[0005] Additionally or alternatively, in some embodiments, V H comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 12, 22, 32, 42, and 52. Additionally or alternatively, in some embodiments, V Lcomprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 17, 27, 37, 47, and 57. In some embodiments, the antibody or antigen-binding fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and SEQ ID NO: 7 (ASC01); SEQ ID NOs: 12 and SEQ ID NO: 17 (ASC02); SEQ ID NOs: 22 and SEQ ID NO: 27 (ASC03); SEQ ID NOs: 32 and SEQ ID NO: 37 (ASC04); SEQ ID NOs: 42 and SEQ ID NO: 47 (ASC05); and SEQ ID NOs: 52 and SEQ ID NO: 57 (ASC06). H Amino acid sequence and V L Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment thereof is ASC06. Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment thereof further comprises an Fc domain of an isotype selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, and IgE. Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment thereof further comprises an Fc domain of an isotype selected from the group consisting of Fab, F(ab')2, Fab', scF V , and F V Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment thereof is a monoclonal antibody, a chimeric antibody, a humanized antibody, or a bispecific antibody.

[0006] Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:7 (ASC01); SEQ ID NO:12 and SEQ ID NO:17 (ASC02); SEQ ID NO:22 and SEQ ID NO:27 (ASC03); SEQ ID NO:32 and SEQ ID NO:37 (ASC04); SEQ ID NO:42 and SEQ ID NO:47 (ASC05); and SEQ ID NO:52 and SEQ ID NO:57 (ASC06), respectively. H Amino acid sequence and V L Contains the amino acid sequence. Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment thereof binds to ASIC1a protein. Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment thereof binds to cells expressing ASIC1a protein or fragment thereof. Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment thereof binds to the extracellular domain of ASIC1a protein or full-length ASIC1a protein. Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment thereof binds to an epitope that spans two ASIC1a subunits. Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment thereof inhibits proton-induced ASIC1a current.

[0007] In one embodiment, the present technology provides a method of alleviating one or more symptoms of ischemic stroke in a subject in need thereof, the method comprising administering to a subject a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domains (V L administering to a subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof comprising V H is selected from the group consisting of SEQ ID NOs: 3, 13, 23, 33, 43, and 53 H - CDR1 sequence; V selected from the group consisting of SEQ ID NOs: 4, 14, 24, 34, 44, and 54 H - a CDR2 sequence; and a V selected from the group consisting of SEQ ID NOs: 5, 15, 25, 35, 45, and 55 H - comprising a CDR3 sequence; L is selected from the group consisting of SEQ ID NOs: 8, 18, 28, 38, 48, and 58 L - CDR1 sequence; V selected from the group consisting of SEQ ID NOs: 9, 19, 29, 39, 49, and 59 L - a CDR2 sequence; and a V selected from the group consisting of SEQ ID NOs: 10, 20, 30, 40, 50, and 60 L -CDR3 sequence,

[0008] Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment thereof further comprises an Fc domain of an isotype selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, and IgE. Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment thereof further comprises an Fc domain of an isotype selected from the group consisting of Fab, F(ab')2, Fab', scF V , and F V Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment thereof is a monoclonal antibody, a chimeric antibody, a humanized antibody, or a bispecific antibody. Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment thereof binds to ASIC1a protein. Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment thereof binds to the extracellular domain of ASIC1a protein or full-length ASIC1a protein. Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment thereof binds to an epitope that spans two ASIC1a subunits. Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment thereof inhibits ASIC1a. Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment thereof inhibits ASIC1a current. Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment thereof inhibits proton-induced ASIC1a current.

[0009] Additionally or alternatively, in some embodiments, the one or more symptoms of ischemic stroke are sudden facial weakness, paralysis or numbness of the face, arms, or legs, drooping of one side of the face, confusion, difficulty speaking, impaired speech comprehension, impaired vision in one or both eyes, blurred vision, amaurosis, double vision, difficulty breathing, dizziness, difficulty walking, loss of balance, impaired coordination, unexplained falls, loss of consciousness, sudden or severe headache. Additionally or alternatively, in some embodiments, the one or more symptoms of ischemic stroke are sudden facial weakness, drooping of one side of the face, unilateral weakness of the upper limbs, or abnormal speech.

[0010] In one embodiment, the subject technology provides a method for treating ischemic stroke in a subject in need thereof, comprising: (a) administering to a subject a light chain immunoglobulin variable domain sequence (V) that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to any one of the light chain immunoglobulin variable domain sequences of SEQ ID NOs: 7, 17, 27, 37, 47, or 57; L and / or (b) a heavy chain immunoglobulin variable domain sequence (V) that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a heavy chain immunoglobulin variable domain sequence present in any one of SEQ ID NOs: 2, 12, 22, 32, 42, or 52. H (c) administering to a subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof comprising:

[0011] In one embodiment, the present technology provides a method for the production of a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domains (V L ), wherein V H is selected from the group consisting of SEQ ID NOs: 3, 13, 23, 33, and 43 H - CDR1 sequence; V selected from the group consisting of SEQ ID NOs: 4, 14, 24, 34, and 44 H - a CDR2 sequence; and a V selected from the group consisting of SEQ ID NOs: 5, 15, 25, 35, and 45H - comprising a CDR3 sequence; V L is selected from the group consisting of SEQ ID NOs: 8, 18, 28, 38, and 48 L - CDR1 sequence; V selected from the group consisting of SEQ ID NOs: 9, 19, 29, 39, and 49 L - a CDR2 sequence; and a V selected from the group consisting of SEQ ID NOs: 10, 20, 30, 40, and 50 L -CDR3 sequence. Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment thereof further comprises an Fc domain of an isotype selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, and IgE. Additionally or alternatively, in some embodiments, the antigen-binding fragment is a Fab, F(ab')2, Fab', scF V , and F V Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment thereof is a monoclonal antibody, a chimeric antibody, a humanized antibody, or a bispecific antibody. Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment thereof binds to ASIC1a protein. Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment thereof binds to the extracellular domain of ASIC1a protein or the full-length ASIC1a protein. Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment thereof binds to an epitope that spans two ASIC1a subunits. Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment thereof inhibits ASIC1a current. Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment thereof inhibits proton-induced ASIC1a current. In one aspect, the technology provides a composition comprising an antibody of any embodiment disclosed herein. Additionally or alternatively, in some embodiments, the nucleic acid sequence is selected from the group consisting of SEQ ID NO: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 51, and 56.

[0012] In one embodiment, the technology provides a host cell or vector expressing any of the nucleic acids disclosed herein. In one aspect, the technology provides a kit comprising the antibody or antigen-binding fragment thereof of any embodiment disclosed herein and instructions for use.Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment thereof of any embodiment disclosed herein is coupled to at least one detectable label selected from the group consisting of radioactive label, fluorescent label, and color label.Additionally or alternatively, in some embodiments, the kit further comprises a secondary antibody that specifically binds to the antibody or antigen-binding fragment thereof of any embodiment disclosed herein.

[0013] In one aspect, the technology provides a method for detecting ASIC1a protein in a biological sample, the method comprising the steps of contacting the biological sample with an antibody or antigen-binding fragment thereof of any embodiment disclosed herein, wherein the antibody or antigen-binding fragment thereof is conjugated to a detectable label; and detecting the presence and level of the detectable label in the biological sample. [Brief description of the drawings]

[0014] [Figure 1] Graph showing binding of purified scFv antibodies ASC01-ASC06 to Nanodiscs containing truncated hASIC1a (amino acids 13-464; hereafter "ΔhASIC1a") as measured by ELISA. Empty Nanodiscs were used as a negative control. [Diagram 2]Confocal microscopy images illustrating the interaction of six scFv antibodies with cells expressing hASIC1a on their surface. CHO-K1 cells were transiently transfected with a plasmid encoding hASIC1a-eYFP (green) and stained with ASC01-ASC06 (red), and nuclei were labeled with DAPI (blue), bar = 10 μm. [Figure 3A] Images showing CHO-K1 / hASIC1a cells stained with ASC06 scFv and ASC06-IgG1 antibody. CHO-K1 cells were transiently transfected with a plasmid encoding hASIC1a-eYFP (green) and stained with ASC06 scFv and ASC06-IgG1 antibody (red), nuclei were labeled with DAPI (blue), bar=10 μm. [Figure 3B] 1 is a graph showing the binding affinity of ASC06-IgG1 to ΔhASIC1a as determined using a Biacore SPR (surface plasmon resonance) system using the single cycle kinetics (SCK) model. A line graph depicting resonance units (RU, reflecting the change in analyte binding capacity of a surface) as a function of time. The binding affinity of ASC06-IgG1 to ΔhASIC1a was determined to be 7.9 nM. [Figure 4A] Confocal microscopy images illustrating the species specificity of ASC06-IgG1. CHO-K1 cells were transiently transfected with hASIC1a-eYFP, mASIC1a-eYFP, or rASIC1a-eYFP (green) plasmids and stained with ASC06-IgG1 (red). DAPI (blue) was used to stain the nuclei of the cells. Bars represent a 10 μm scale. [Figure 4B]Graph illustrating species specificity and ASIC1 subtype specificity of ASC06-IgG1 as determined by FACS sorting. CHO-K1 cells were transiently transfected with hASIC1a-eYFP, mASIC1a-eYFP, rASIC1a-eYFP, hASIC1b-eYFP, hASIC2a-eYFP, hASIC2a / 2b-eYFP, and hASIC3a-eYFP plasmids and stained with Alexa555-conjugated ASC06-IgG1. 10,000 cells were sorted. Alexa555 and eYFP double positive cells appearing in the upper right quadrant of the FACS profile indicate binding of ASC06-IgG1 to a given ASIC1 subtype or homologue. [Figure 5A] Representative current traces from a single hASIC1a stable CHO-K1 cell in the absence or presence of various doses of ASC06-IgG1 (100 nM, 300 nM). The grey lines above the current traces indicate the time when the extracellular pH value was decreased from pH 7.4 to pH 6.0 and recordings were made. The black lines above the current traces represent treatment with the indicated agents. Cells treated with pH 6 were used as a negative control and the venom peptide Psalmotoxin-1 (PcTx1) was used as a positive control. "Wash" represents the recovery of current after treatment with 300 nM ASC06-IgG1 followed by a 15 min injection of wash solution. [Figure 5B] FIG. 1 is a graph showing dose-dependent inhibition of acid-induced hASIC1a current by ASC06-IgG1. The apparent IC50 value is determined to be 85±6 nM (N=3-5). Data are presented as the mean±standard deviation for five replicates. [Figure 5C] Graph showing washout experiment illustrating hASIC1a current during washout period after treatment with ASC06-IgG1 or PcTx1. Line graph shows relative current as a function of washout time (min) (N=4). [Figure 6A]Graph showing inhibition of acid-induced ASIC1a-mediated calcium influx by ASC06-IgG1.Representative progress curves of acid-induced calcium influx in CHO-K1 cells stably overexpressing hASIC1a-mCherry (4C12) in the presence of various concentrations of ASC06-IgG1 (30-0.12 nM in 1:3 serial dilutions) are shown. [Figure 6B] FIG. 13 is a graph showing dose-dependent inhibition of acid-induced calcium influx by ASC06-IgG1 (N=6). [Figure 7A] FIG. 13 is a graph showing the effect of pH (pH=5.5, 6.0, and 7.4) on the viability of CHO-K1 cells in the presence or absence of membrane expression of hASIC1a-eYFP, as measured by cytoplasmic dehydrogenase activity (N=5-6). [Figure 7B] Graph showing the effect of pH (pH = 5.5, 6.0, and 7.4) on CHO-K1 cells with and without membrane expression of hASIC1a-eYFP. Bar graph shows released LDH content (N = 5-6). [Figure 7C] Graph showing dose-dependent protection from acidosis-induced cell death by ASC06-IgG1 as measured by viability of hASIC1a-eYFP stable cells in the presence of increasing concentrations of ASC06-IgG1. Data are shown as the mean ± standard deviation for four replicates (N=3-5). [Figure 7D] Graph showing dose-dependent protection from acidosis-induced cell death by ASC06-IgG1 as measured by LDH release by hASIC1a-eYFP stable cells in the presence of increasing concentrations of ASC06-IgG1. Data are shown as the mean ± standard deviation for 3-5 replicates (N=3-5). [Figure 8A] FIG. 1 is a schematic diagram depicting the experimental design for in vivo studies in a mouse middle cerebral artery occlusion (MCAO)-induced ischemic stroke model. [Figure 8B](Top panel) Images of brain cross sections illustrating the infarct area as detected by staining with the vital dye 2,3,5-triphenyltetrazolium hydrochloride (TTC) after treatment with PBS (sham control, N=6), isotype control (N=6), ASC06-IgG1 (N=6), and PcTx1 (N=6) in MCAO-induced ischemic stroke mouse models. (Bottom panel) Bar graph illustrating the volume of the infarct area in brains from mice treated as in (top panel). * indicates p-value<0.05 and ** indicates p-value<0.01 compared to the sham control group. NS=not significant. [Figure 9A] Negative staining electron microscopy images of the complex between the hASIC1a ectodomain (hASIC1a-ECD) and the ASC06-Fab complex. Figure 9A shows a partial area of ​​the raw image. Scale bar = 50 nm. [Figure 9B] Figure 9B shows a negative stain electron microscopy image of the complex between the hASIC1a ectodomain (hASIC1a-ECD) and the ASC06-Fab complex. Figure 9B shows a representative single particle selected from Figure 9A. Scale bar = 10 nm. [Figure 9C] Negative staining electron microscopy image of the hASIC1a ectodomain (hASIC1a-ECD) complex with ASC06-Fab complex. Figure 9C shows the corresponding representative class average based on single particles (N=5,064). Scale bar=10 nm. [Figure 9D] FIG. 1 shows the conformational epitope of ASC06-IgG1 as detected by molecular dynamics simulation. The first subunit is presented on the left side of the image and the second subunit is shown on the right side. The part of the first subunit containing the beta sheet near the bottom of the image is located behind the second subunit. The binding site of ASC06-IgG1, shown using dots (dark grey dots in the first subunit, light grey dots in the second subunit), is formed by two ASIC1a subunits. [Figure 9E]FIG. 1 shows the identification of amino acid residues in the hASIC1a protein that have a role in binding to ASC06-IgG1. CHO-K1 cells transiently transfected with the indicated alanine substitution mutant hASIC1a-eYFP proteins were assayed for binding to ASC06-IgG1 using a FACS-based assay. The top panel shows a negative control (NC) in which no first antibody but only the second antibody was included. The bottom panel shows an experiment in which ASC06-IgG1 was incubated with the cells. A positive cell is shown in which the cells express wild-type hASIC1a protein. Another negative control in which CHO-K1 cells do not express hASIC1a is also shown on the left. [Figure 10A] 1 is a graph showing dose-dependent binding of ASC06-IgG1 to the 6H7 cell line, a stable line expressing the hASIC1a-eYFP fusion protein. The data show an apparent binding affinity of 2.06±0.01 nM (n=3). [Figure 10B] (Top panel) Confocal microscopy images of ASC06-IgG1 stained primary neurons (green) from the cortex of mASIC1a KO mice lightly transfected with hASIC1a-mCherry (red). Differential interference contrast (DIC) views show brightfield images merged with fluorescent views of the neurons. (Bottom panel) Images show amplified views of neurites showing that ASC06-IgG1 binding occurs in postsynaptic dendrites. [Figure 11A] 1 is a graph showing the effect of ASC06-IgG1 on the steady-state desensitization (SSD) profile of hASIC1a (n=6 to 8). [Figure 11B] FIG. 1 is a graph showing the effect of ASC06-IgG1 on the proton activation profile of hASIC1a (n=5). [Figure 12A]Image showing SDS-PAGE analysis of the composition of ΔhASIC1a-Nanodiscs. ΔhASIC1a-Nanodiscs illustrated on the right using computational modeling, where the blue, yellow, and purple colored ribbons on top represent the predicted ΔhASIC1a trimeric configuration, the red ribbons on the sides of the disc represent MSP1, and the white globular clusters represent 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). Empty nanodiscs refer to nanodisc assemblies using acetylcholine in place of ΔhASIC1a. [Figure 12B] FIG. 1 shows the results of dynamic light scattering (DLS) measurements illustrating the size of Nanodisc assemblies. The inset image shows modeled particles labeled with calculated diameters. [Figure 12C] FIG. 1 is a graph showing the output of positive phagemids in "differential enrichment" panning. [Figure 12D] 1 is an image showing SDS-PAGE analysis of purified single chain (ASC06) and full length (ASC06-IgG1) antibodies. [Figure 13A] This is an image showing Western blot analysis of cell lysates from CHO-K1 cells ("-") or a CHO-K1 stable cell line expressing hASIC1a-eYFP ("+") using a commercial goat polyclonal anti-ASIC1a antibody or ASC06-IgG1. [Figure 13B] This is an image showing Western blots of cell lysates of CHO-K1 cells ("-") or a CHO-K1 stable cell line expressing hASIC1a-eYFP ("+") immunoprecipitated with ASC06-IgG1 and analyzed with a commercial anti-eYFP antibody. [Figure 14] Graph showing gel filtration profiles of hASIC1a-ECD and ASC06-Fab assembled complex. Gel filtration profiles of hASIC1a-ECD alone, ASC06-Fab alone, and the complex are shown. A Superdex 200 size exclusion chromatography column was used (24 mL volume). [Figure 15]1 is an SPR sensorgram of PcTx1-Fc. The binding affinity of PcTx1-Fc to ΔhASIC1a was determined to be 1.0 nM based on a single cycle kinetic method. [Figure 16A] 16A and 16B are graphs illustrating that ASC06-IgG1 does not interfere with the SSD or activation of ASIC1a. Figure 16A shows representative traces of ASIC1a currents evoked by pH 6.0 stimulation at various pH control values. The traces in the upper panel show the currents in the absence of ASC06-IgG1, and the traces in the lower panel show the currents in the presence of 300 nM ASC06-IgG1. [Figure 16B] Graphs illustrating that ASC06-IgG1 does not interfere with SSD or activation of ASIC1a. FIG. 16B shows representative current traces elicited by pH ranging from 6.8 to 6.0 with adjusted pH 7.4. Top, without ASC06-IgG1; bottom, with ASC06-IgG1 (300 nM). pH 7.2 and pH 7.0 activation traces are not shown as there were no currents elicited in these conditions. [Figure 17A] Graph showing maximum fluorescence intensity of calcium influx in the cytoplasm after acid induction in CHO-K1 cells or stable CHO-K1 cells overexpressing hASIC1a-mCherry (4C12). Where indicated, 4C12 cells were preincubated with amiloride, PcTx1, ASC06-IgG1, or isotype control antibody. [Figure 17B] Representative progress curves of acid-induced calcium influx in 4C12 in the presence of amiloride, PcTx1, ASC06-IgG1, or an isotype control antibody. An irrelevant antibody was chosen as an isotype control. [Figure 18] Figure 1 shows size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC) analysis of ASC06-IgG1. ASC06-IgG1 (0.5 mg / mL) was incubated with extracellular solutions of different pH for 6 hours at 37°C, followed by SEC-HPLC analysis. [Figure 19A] 1 is a table showing the nucleotide sequence of the VH domain of ASC01 (SEQ ID NO:1). [Figure 19B] 1 is a table showing the amino acid sequence of the VH domain of ASC01 (SEQ ID NO:2). VH CDR1 (SEQ ID NO:3), VH CDR2 (SEQ ID NO:4), and VH CDR3 (SEQ ID NO:5) are indicated by underlined bold font. [Figure 20A] 1 is a table showing the nucleotide sequence of the VL domain of ASC01 (SEQ ID NO:6). [Figure 20B] 1 is a table showing the amino acid sequence of the VL domain of ASC01 (SEQ ID NO:7). VL CDR1 (SEQ ID NO:8), VL CDR2 (SEQ ID NO:9), and VL CDR3 (SEQ ID NO:10) are indicated by underlined bold font. [Figure 21A] 1 is a table showing the nucleotide sequence of the VH domain of ASC02 (SEQ ID NO:11). [Figure 21B] 1 is a table showing the amino acid sequence of the VH domain of ASC02 (SEQ ID NO: 12). VH CDR1 (SEQ ID NO: 13), VH CDR2 (SEQ ID NO: 14), and VH CDR3 (SEQ ID NO: 15) are shown in bold underlined font. [Figure 22A] 1 is a table showing the nucleotide sequence of the VL domain of ASC02 (SEQ ID NO: 16). [Figure 22B] 1 is a table showing the amino acid sequence of the VL domain of ASC02 (SEQ ID NO: 17). VL CDR1 (SEQ ID NO: 18), VL CDR2 (SEQ ID NO: 19), and VL CDR3 (SEQ ID NO: 20) are shown in bold underlined font. [Figure 23A] 2 is a table showing the nucleotide sequence of the VH domain of ASC03 (SEQ ID NO:21). [Figure 23B] 2 is a table showing the amino acid sequence of the VH domain of ASC03 (SEQ ID NO:22). VH CDR1 (SEQ ID NO:23), VH CDR2 (SEQ ID NO:24), and VH CDR3 (SEQ ID NO:25) are shown in bold underlined font. [Figure 24A]2 is a table showing the nucleotide sequence of the VL domain of ASC03 (SEQ ID NO:26). [Figure 24B] 1 is a table showing the amino acid sequence of the VL domain of ASC03 (SEQ ID NO:27). VL CDR1 (SEQ ID NO:28), VL CDR2 (SEQ ID NO:29), and VL CDR3 (SEQ ID NO:30) are shown in bold underlined font. [Figure 25A] 1 is a table showing the nucleotide sequence of the VH domain of ASC04 (SEQ ID NO:31). [Figure 25B] 1 is a table showing the amino acid sequence of the VH domain of ASC04 (SEQ ID NO:32). VH CDR1 (SEQ ID NO:33), VH CDR2 (SEQ ID NO:34), and VH CDR3 (SEQ ID NO:35) are shown in bold underlined font. [Figure 26A] 1 is a table showing the nucleotide sequence of the VL domain of ASC04 (SEQ ID NO:36). [Figure 26B] 1 is a table showing the amino acid sequence of the VL domain of ASC04 (SEQ ID NO:37). VL CDR1 (SEQ ID NO:38), VL CDR2 (SEQ ID NO:39), and VL CDR3 (SEQ ID NO:40) are shown in bold underlined font. [Figure 27A] 1 is a table showing the nucleotide sequence of the VH domain of ASC05 (SEQ ID NO:41). [Figure 27B] 1 is a table showing the amino acid sequence of the VH domain of ASC05 (SEQ ID NO: 42). VH CDR1 (SEQ ID NO: 43), VH CDR2 (SEQ ID NO: 44), and VH CDR3 (SEQ ID NO: 45) are shown in bold underlined font. [Figure 28A] 1 is a table showing the nucleotide sequence of the VL domain of ASC05 (SEQ ID NO:46). [Figure 28B] 1 is a table showing the amino acid sequence of the VL domain of ASC05 (SEQ ID NO:47). VL CDR1 (SEQ ID NO:48), VL CDR2 (SEQ ID NO:49), and VL CDR3 (SEQ ID NO:50) are shown in bold underlined font. [Figure 29A]1 is a table showing the nucleotide sequence of the VH domain of ASC06 (SEQ ID NO:51). [Figure 29B] 1 is a table showing the amino acid sequence of the VH domain of ASC06 (SEQ ID NO:52). VH CDR1 (SEQ ID NO:53), VH CDR2 (SEQ ID NO:54), and VH CDR3 (SEQ ID NO:55) are shown in bold underlined font. [Figure 30A] 1 is a table showing the nucleotide sequence of the VL domain of ASC06 (SEQ ID NO:56). [Figure 30B] 1 is a table showing the amino acid sequence of the VL domain of ASC06 (SEQ ID NO:57). VL CDR1 (SEQ ID NO:58), VL CDR2 (SEQ ID NO:59), and VL CDR3 (SEQ ID NO:60) are indicated by underlined bold font. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Detailed Description It should be understood that certain aspects, modes, embodiments, variations, and characteristics of the present technology are described below at various levels of detail to provide a substantial understanding of the present technology. The present technology provides a method for treating ischemic stroke and / or related disorders. Although the exemplified antibodies targeting the ASIC1a protein described herein are scFv and IgG1 antibodies, the description is intended to broadly encompass any immunological binding agent, such as IgG, IgM, IgA, IgD, IgE, and genetically modified IgG, and fragments thereof, as well as polypeptides comprising antibody complementarity determining region (CDR) domains that retain the antigen-binding activity described herein.

[0016] definition Definitions of certain terms used herein are provided below. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to "a cell" includes a combination of two or more cells, etc. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry, and nucleic acid chemistry and hybridization described below are those well known and commonly employed in the art.

[0017] As used herein, the term "about" in reference to a number, unless otherwise stated or otherwise clear from the context, is generally taken to include numbers that fall within a range of 1%, 5%, or 10% in either direction of the number (greater or less than that) (except where such number would be less than 0% or greater than 100% of the possible value). As used herein, "administration" of an agent, drug or peptide to a subject includes any route of introducing or delivering a compound to a subject to perform its intended function. Administration can be by any suitable route, including oral, intranasal, parenteral (intravenous, intramuscular, intraperitoneal or subcutaneous), or topical. In some embodiments, the anti-ASIC1a antibody of the present technology is administered by intracranial (intraventricular) route, intrathecal route, or intraarterial route. Administration includes self-administration and administration by another person.

[0018] As used herein, the term "amino acid" is used to refer to any organic molecule that contains at least one amino group and at least one carboxyl group. Typically, at least one amino group is in the alpha position relative to the carboxyl group. The term "amino acid" includes naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are subsequently modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., α-carbons that are attached to hydrogen, carboxyl groups, amino groups, and R groups, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to chemical compounds that have structures that differ from the general chemical structure of amino acids, but function in a manner similar to naturally occurring amino acids. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0019] As used herein, the term "antibody" collectively refers to immunoglobulin or immunoglobulin-like molecules, including, by way of example and without limitation, IgA, IgD, IgE, IgG, and IgM, combinations thereof, and similar molecules produced during the immune response in any vertebrate, mammalian, e.g., human, goat, rabbit, and mouse, as well as non-mammalian species, such as shark immunoglobulin. As used herein, "antibody" (including intact immunoglobulins) and "antigen-binding fragment" refers to an antibody that specifically binds to a molecule of interest (or a group of closely similar molecules of interest) to the substantial exclusion of binding to other molecules (e.g., with a binding affinity at least 10 times greater than the binding affinity for other molecules in a biological sample). 3 M -1 Large, at least 10 4 M -1 Greater than or at least 10 5 M -1 (Antibodies and antibody fragments that have a large binding affinity for a molecule of interest). The term "antibody" also includes genetically engineered forms such as chimeric antibodies (e.g., humanized mouse antibodies), heteroconjugate antibodies (bispecific antibodies, etc.). Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology, 3 rd Ed., WH Freeman & Co., New York, 1997.

[0020] More specifically, an antibody refers to a polypeptide ligand that contains at least a light or heavy immunoglobulin chain variable region that specifically recognizes and binds to an epitope of an antigen. Antibodies are made up of a variable heavy chain (V H ) region and the variable light chain (V L The antibody is composed of heavy and light chains with variable regions called V H Area and V LThe regions are responsible for binding to the antigen recognized by the antibody. Typically, immunoglobulins have heavy (H) and light (L) chains interconnected by disulfide bonds. There are two types of light chains: lambda (λ) and kappa (κ). There are five main heavy chain classes (or isotypes) that determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE. Each heavy and light chain contains a constant region and a variable region (the regions are also known as "domains"). In combination, the heavy and light chain variable regions specifically bind to the antigen. The light and heavy chain variable regions contain a "framework" region separated by three hypervariable regions, also called "complementarity determining regions" or "CDRs". The extent of the framework regions and CDRs have been defined (see Kabat et al., Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991, hereby incorporated by reference). The Kabat database is currently maintained online. The sequences of framework regions of different light or heavy chains are relatively conserved within a species. The framework regions of antibodies, which are the combined framework regions of light and heavy chain components, mostly adopt a β-sheet conformation, and the CDRs form loops that connect to and in some cases form part of the β-sheet structure. Thus, the framework regions act to form a scaffold that provides for the positioning of the CDRs in the correct orientation by interchain non-covalent interactions.

[0021] The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are typically identified by the chain in which the particular CDR is located. Thus, V H CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, while V LCDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found. Antibodies that bind to the ASIC1a protein have a specific V H Area and V L The CDRs of the present invention are composed of a plurality of CDRs, each of which has a specific CDR sequence and therefore a specific CDR sequence. Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. Although it is the CDRs that vary from antibody to antibody, only a limited number of amino acid sites in the CDRs are directly involved in antigen binding. These sites in the CDRs are called specificity determining residues (SDRs). As used herein, the term "anti-ASIC1a antibody of the present technology" refers to antibodies (monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, multispecific antibodies, bispecific antibodies, etc.) as well as antibody fragments. An antibody or an antigen-binding fragment thereof specifically binds to an antigen.

[0022] As used herein, the term "antibody-related polypeptide" refers to antigen-binding antibody fragments, including single chain antibodies, which may contain a variable region alone or in combination with all or a portion of the following polypeptide elements: hinge region, CH1, CH2, and CH3 domains of an antibody molecule. Any combination of variable regions and hinge regions, CH1, CH2, and CH3 domains are also included in the technology. For example, Fab, Fab' and F(ab')2, Fd, single chain Fv (scFv), single chain antibodies, disulfide-linked Fv (sdFv), and VFv are also included in the technology. L or V H Antibody-related molecules, including but not limited to fragments containing any of the V domains, are useful in the present methods. Examples include: (i) L , V H , C L (ii) a bivalent fragment, F(ab')2, that contains two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a V H and an Fd fragment consisting of the CH1 domain; (iv) a V of a single arm of an antibody. L and V H(v) an Fv fragment consisting of a V domain; H (vi) isolated complementarity determining regions (CDRs). Thus, an "antibody fragment" or "antigen-binding fragment" can include a portion of a full-length antibody, typically the antigen-binding or variable region thereof. Examples of antibody fragments or antigen-binding fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; bispecific antibodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0023] As used herein, the term "conjugated" refers to the association of two molecules by any method known to those skilled in the art. Suitable types of association include chemical and physical bonds. Chemical bonds include, for example, covalent bonds and coordinate bonds. Physical bonds include, for example, hydrogen bonds, dipole interactions, van der Waals forces, electrostatic interactions, hydrophobic interactions, and aromatic stacking. As used herein, the term "diabody" refers to a small antibody fragment having two antigen-binding sites, the fragment comprising a light chain variable domain (V L ) connected to a heavy chain variable domain (V H ) in the same polypeptide chain (V H V L (By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain, creating two antigen-binding sites. Diabodies are more fully described, for example, in EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).

[0024] As used herein, the term "single chain antibody" or "single chain Fv (scFv)" refers to the two domains V of an Fv fragment. L and V H The term "single-chain antibody" refers to an antibody fusion molecule of the above-mentioned type. A single-chain antibody molecule may comprise a polymer having several individual molecules, such as a dimer, trimer, or other polymer. In addition, the two domains V of the Fv fragment may be combined to form a single-chain antibody fusion molecule. L and V H Although they are encoded by separate genes, they are L and V H The regions can be joined using recombinant methods by synthetic linkers that allow them to be made as a single protein chain that pairs to form monovalent molecules (known as single-chain Fv (scFv)). Bird et al. (1988) Science 242:423-426 and Huston et al. (1988) Proc. Natl. Acad Sci. USA 85:5879-5883. Such single-chain antibodies can be prepared by recombinant techniques or by enzymatic or chemical cleavage of intact antibodies. As used herein, "antigen" refers to a molecule that an antibody (or an antigen-binding fragment thereof) can selectively bind to. Target antigens can be proteins, carbohydrates, nucleic acids, lipids, haptens, or other naturally occurring or synthetic compounds. In some embodiments, target antigens can be polypeptides (e.g., ASIC1a polypeptides). Antigens can also be administered to animals to generate an immune response in the animal.

[0025] The term "antigen-binding fragment" refers to a fragment of the entire immunoglobulin structure that possesses a portion of the polypeptide involved in binding to an antigen. Examples of antigen-binding fragments useful in the present technology include, but are not limited to, scFv, (scFv)2, Fab, Fab', and F(ab')2. Any of the above-described antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for binding specificity and neutralizing activity in the same manner as are intact antibodies. By "binding affinity" is meant the strength of the total non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen or antigenic peptide). The affinity of a molecule X for its partner Y is generally measured by the dissociation constant (K D ) Affinity can be measured by standard methods known in the art, including those described herein. Low affinity complexes generally contain antibodies that tend to dissociate easily from the antigen, while high affinity complexes generally contain antibodies that tend to remain bound to the antigen for longer durations.

[0026] As used herein, the term "biological sample" refers to sample material derived from living cells. Biological samples can include tissues, cells, protein or membrane extracts of cells, and biological fluids (e.g., ascites or cerebrospinal fluid (CSF)) isolated from a subject, as well as tissues, cells, and fluids present within a subject. The biological samples of the present technology include, but are not limited to, samples taken from breast tissue, kidney tissue, cervix, endometrium, head and neck, gallbladder, parotid tissue, prostate, brain, pituitary gland, kidney tissue, muscle, esophagus, stomach, small intestine, colon, liver, spleen, pancreas, thyroid tissue, heart tissue, lung tissue, bladder, adipose tissue, lymph node tissue, uterus, ovarian tissue, adrenal tissue, testicular tissue, tonsils, thymus, blood, hair, cheek, skin, serum, plasma, CSF, semen, prostate fluid, seminal fluid, urine, feces, sweat, saliva, sputum, mucus, bone marrow, lymph, and tears. Biological samples can also be obtained from biopsies of internal organs or from cancer. Biological samples can be obtained from subjects for diagnosis or research, or from non-diseased individuals as controls or for basic research. Samples may be obtained by standard methods, including, for example, venipuncture and surgical biopsy. In certain embodiments, the biological sample is a skin tissue, hair, nail, sebaceous gland, or muscle biopsy sample.

[0027] As used herein, "control" refers to an alternative sample that is used in an experiment for comparison purposes.Control can be "positive" or "negative".For example, when the purpose of an experiment is to determine the correlation of the efficacy of a therapeutic agent for treating a specific type of disease, a positive control (a compound or composition that is known to exhibit the desired therapeutic effect) and a negative control (a subject or sample that does not receive therapy or receives a placebo) are typically employed. As used herein, the term "effective amount" refers to an amount sufficient to achieve a desired therapeutic and / or prophylactic effect, e.g., an amount that results in the prevention or reduction of a disease or condition described herein, or one or more signs or symptoms associated with a disease or condition described herein. In the context of therapeutic or prophylactic applications, the amount of the composition administered to a subject will vary depending on the composition, the extent, type, and severity of the disease, and individual characteristics such as general health, age, sex, weight, and tolerance to drugs. Those skilled in the art will be able to determine appropriate dosages depending on these and other factors. The composition may also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, the therapeutic composition may be administered to a subject having one or more signs or symptoms of a disease or condition described herein. As used herein, a "therapeutically effective amount" of a composition refers to a level of the composition at which the physiological effects of the disease or condition are ameliorated or eliminated. A therapeutically effective amount may be given in one or more administrations.

[0028] An "isolated" or "purified" polypeptide or peptide is substantially free of cellular material or other contaminating polypeptides from the cell or tissue source from which the agent is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized.For example, the isolated anti-ASIC1a antibody of the present technology will not contain materials that would interfere with the diagnostic or therapeutic use of the agent.Such interfering materials may include enzymes, hormones, and other proteinaceous and non-proteinaceous solutes.

[0029] As used herein, the term "epitope" refers to a protein determinant that can be specifically bound to an antibody. Epitopes are usually composed of chemically active surface groups of molecules such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former is lost in the presence of denaturing solvents, but the binding to the latter is not lost. In some embodiments, "epitope" is the region of the ASIC1a protein trimer that the anti-ASIC1a antibody of the present technology specifically binds to, including the extracellular domain of ASIC1a. In some embodiments, the epitope can span two ASIC1a subunits. In some embodiments, the epitope is a conformational epitope or a non-conformational epitope. To screen for anti-ASIC1a antibody that binds to epitopes, routine cross-blocking assays such as those described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988) can be carried out.This assay can be used to determine whether anti-ASIC1a antibody binds to the same site or epitope as the anti-ASIC1a antibody of the present technology.Alternatively or additionally, epitope mapping can be carried out by methods known in the art.For example, as shown in Figure 9E, antibody sequence can be mutated by alanine scanning, etc., to identify contact residues.In different methods, peptides corresponding to various regions of ASIC1a protein can be used in competitive assays with test antibody, or with test antibody and antibody with characterized or known epitopes.

[0030] As used herein, "expression" includes one or more of the following: transcription of a gene into precursor mRNA; splicing and other processing of precursor mRNA to produce mature mRNA; mRNA stability; translation of mature mRNA into protein (including codon usage and tRNA availability); and, if required for proper expression and function, glycosylation and / or other modifications of the translation product. As used herein, the term "gene" refers to a segment of DNA that contains all the information for the regulated biosynthesis of an RNA product, including the promoter, exons, introns, and other untranslated regions that control expression.

[0031] As used herein, the term "homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing positions in each sequence that can be aligned for comparison purposes. If a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matched or homologous positions shared by the sequences. A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) of "sequence identity" with another sequence, meaning that, when aligned, that percentage of bases (or amino acids) are the same when the two sequences are compared. This alignment and homology or sequence identity percentage can be determined using software programs known in the art. In some embodiments, default parameters are used for alignment. One alignment program is BLAST using default parameters. In particular, the programs are BLASTN and BLASTP using the following default parameters: Genetic code=standard; Filter=none; Strand=both; Cutoff=60; Expected=10; Matrix=BLOSUM62; Description=50 sequences; Filter=HIGH SCORE; Database=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at the National Center for Biotechnology Information. Biologically equivalent polynucleotides are those that have a specified percentage of homology and encode polypeptides that have the same or similar biological activity. Two sequences are considered "unrelated" or "non-homologous" if they share less than 40% identity with each other, or less than 25% identity.

[0032] As used herein, "humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins in which recipient hypervariable region residues are replaced by hypervariable region residues from a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, and capacity. In some embodiments, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance, such as binding affinity. Generally, a humanized antibody will have all or substantially all of its hypervariable loops corresponding to those of a non-human immunoglobulin, and all or substantially all of its FR regions of a human immunoglobulin consensus FR sequence, but the FR regions will include substantially all of at least one, and typically two, variable domains (e.g., Fab, Fab', F(ab')2, or Fv) that may contain one or more amino acid substitutions that improve binding affinity. The number of these amino acid substitutions in the FRs is typically no more than six in the H chain and no more than three in the L chain. The humanized antibody may also optionally include at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332: 323-327 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See, e.g., Ahmed & Cheung, FEBS Letters 588(2):288-297 (2014); Saxena & Wu, Frontiers in immunology 7: 580 (2016).

[0033] As used herein, the term "identical" or percent "identity", when used in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that are the same or have a specified percentage of identical amino acid residues or nucleotides (i.e., about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity over a specified region (e.g., a nucleotide sequence encoding an antibody described herein or an amino acid sequence of an antibody described herein)) when compared and aligned for maximum correspondence over a comparison window or specified region, as measured using the BLAST or BLAST 2.0 sequence comparison algorithm with default parameters described below or by manual alignment and visual inspection (e.g., the NCBI website). Such sequences are then said to be "substantially identical". The term can also refer to or be applied to the complement of a test sequence. The term also includes sequences that have deletions and / or additions, as well as those that have substitutions. In some embodiments, the identity exists over a region that is at least about 25 amino acids or nucleotides in length, or over a region that is 50-100 amino acids or nucleotides in length.

[0034] As used herein, the term "intact antibody" or "intact immunoglobulin" refers to an antibody having at least two heavy (H) chain polypeptides and two light (L) chain polypeptides interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable region (herein referred to as HCVR or V H The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (herein abbreviated as LCVR or V L The light chain constant region consists of one domain, C L It consists of: V H and V LThe regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). H and V L is composed of three CDRs and four FRs arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. As used herein, the terms "individual," "patient," or "subject" can be an individual organism, a vertebrate, a mammal, or a human. In some embodiments, the individual, patient, or subject is a human.

[0035] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. For example, a monoclonal antibody may be an antibody derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Monoclonal antibodies are highly specific, directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as obtained from a substantially homogeneous population of antibodies, and should not be taken as requiring the production of the antibody by any particular method. Monoclonal antibodies can be prepared using a wide variety of techniques known in the art, including, but not limited to, hybridoma, recombinant, and phage display techniques.For example, the monoclonal antibodies of interest for use in the present method can be produced by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or by recombinant DNA methods (see, for example, U.S. Patent No. 4,816,567)."Monoclonal antibodies" can also be isolated from phage antibody libraries, for example, by using the techniques described in Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991).

[0036] As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal compounds, isotonic and absorption delaying compounds, and the like, that are compatible with pharmaceutical administration. Pharmaceutically acceptable carriers and their formulation are known to those skilled in the art, and are described, for example, in Remington's Pharmaceutical Sciences (20 th edition, ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, PA. As used herein, "inhibition" or "inhibiting" a disorder or condition refers to a compound that, in a statistical sample, reduces the occurrence of a disorder or condition in a treated sample compared to an untreated control sample, or delays the onset of or reduces the severity of one or more symptoms of a disorder or condition compared to an untreated control sample.

[0037] As used herein, the terms "polypeptide", "peptide" and "protein" are used interchangeably herein to mean a polymer containing two or more amino acids joined together by peptide bonds or modified peptide bonds, i.e., peptide isosteres. Polypeptide refers to both short chains, commonly referred to as peptides, glycopeptides, or oligomers, and longer chains, commonly referred to as proteins. Polypeptides can contain amino acids other than the 20 gene-encoded amino acids. Polypeptides include amino acid sequences modified by natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art. As used herein, the term "separate" therapeutic use refers to the administration of at least two active ingredients by different routes, at the same or substantially the same time. As used herein, the term "sequential" therapeutic use refers to the administration of at least two active ingredients at different times, with the same or different administration routes.More specifically, sequential use refers to the full administration of one of the active ingredients before the administration of one or more other active ingredients begins.Thus, one of the active ingredients can be administered for several minutes, several hours, or several days before the administration of one or more other active ingredients.In this case, there is no simultaneous treatment.

[0038] As used herein, the term "concurrent" therapeutic use refers to the administration of at least two active ingredients by the same route and at the same or substantially the same time. As used herein, "specifically binds" refers to a molecule (e.g., an antibody or antigen-binding fragment thereof) that recognizes and binds to another molecule (e.g., an antigen) but does not substantially recognize or bind to other molecules. As used herein, the terms "specific binding to," "specifically binds to," or "is specific for" a particular molecule (e.g., a polypeptide, or an epitope on a polypeptide) refer to, for example, a specific binding activity of about 10% to the molecule to which it binds. -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 K of M D The term "specifically binds" can also refer to binding of a molecule (e.g., an antibody or antigen-binding fragment thereof) to a particular polypeptide (e.g., an ASIC1a polypeptide) or an epitope on a particular polypeptide without substantially binding to any other polypeptides or polypeptide epitopes.

[0039] As used herein, the terms "subject," "individual," or "patient" can be an individual organism, vertebrate, mammal, or human. As used herein, "treating," "treat," or "treatment" covers the treatment of a disease or disorder described herein in a subject, such as a human, and includes (i) inhibiting the disease or disorder, i.e., halting its progression; (ii) alleviating the disease or disorder, i.e., causing regression of the disorder; (iii) slowing the progression of the disorder; and / or (iv) inhibiting, alleviating, or slowing the progression of one or more symptoms of the disease or disorder. It should also be understood that the various modes of treatment of disorders described herein are intended to mean "substantial," including total treatment, but also including less than total treatment, where some biologically or medically relevant result is achieved. Treatment can be continuous long-term treatment for chronic diseases, or one or several doses for treatment of acute medical conditions.

[0040] Amino acid sequence modifications of the anti-ASIC1a antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of anti-ASIC1a antibodies are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of residues in the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions may be made to obtain the antibody of interest, so long as the obtained antibody possesses the desired properties. Modifications may also include changes in the glycosylation pattern of the protein. The most popular sites for substitutional mutagenesis include hypervariable regions, but FR changes are also contemplated. "Conservative substitutions" are shown in the table below.

[0041] TIFF2025063103000001.tif168160

[0042] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody. A convenient way to generate such substitutional variants involves affinity maturation using phage display. Specifically, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody variants so generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as disclosed herein. To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively or additionally, it may be beneficial to analyze a crystal structure of an antigen-antibody complex to identify contact points between the antibody and the antigen. Such contact and neighboring residues are candidates for substitution by the techniques detailed herein. Once such variants are generated, the panel of variants can be subjected to screening as described herein and antibodies with similar or superior properties in one or more relevant assays selected for further development.

[0043] Pathogenesis of ischemic stroke Stroke is caused by an interruption or reduction in the supply of oxygen-rich blood to parts of the brain. Without oxygen, brain cells begin to die within minutes. Stroke usually presents through symptoms such as sudden weakness; paralysis or numbness of the face, arm, or leg, especially on one side of the body; drooping of one side of the face; confusion; difficulty speaking or impaired language comprehension, such as slurred words; difficulty seeing in one or both eyes, such as blurred vision or amaurosis, or double vision in one or both eyes; difficulty breathing; dizziness; difficulty walking; loss of balance or coordination, for example causing an unexplained fall; loss of consciousness; and sudden and severe headache. The most common symptoms include sudden facial weakness (e.g., drooping of one side of the face), unilateral weakness of the upper limbs, and abnormal speech. These symptoms typically begin suddenly and last from a few seconds to a few minutes, without progressing further in most cases. Immediate emergency treatment is crucial to surviving a stroke with a minimal amount of damage to the brain and the ability to function.

[0044] Ischemic strokes, which account for about 87% of all strokes, occur when blood supply to parts of the brain is cut off due to blockage of a blood vessel by a blood clot or other particle. Fatty deposits called plaque can also cause blockages by building up in blood vessels. The blocked blood flow in an ischemic stroke can be caused by atherosclerosis, which over time causes the arteries to narrow. Ischemic strokes can be caused by blockages anywhere along the arteries that feed the brain. An ischemic stroke can be an embolic stroke, where a blood clot or plaque fragment forms elsewhere in the body (usually the heart) and travels to the brain. Once in the brain, the blood clot travels to a blood vessel that is small enough to block the blood clot from passing through. The blood clot lodges there, blocking the blood vessel and causing a stroke. Approximately 15% of embolic strokes occur in people with atrial fibrillation (Afib).

[0045] Ischemic stroke can be thrombotic stroke, which is caused by a clot forming inside one of the arteries that supply blood to the brain. This type of stroke is usually seen in people with high cholesterol levels and atherosclerosis. Thrombotic stroke can be large vessel thrombosis or small vessel disease. Large vessel thrombosis occurs in the larger arteries of the brain. In most cases, it is caused by long-term atherosclerosis in combination with rapid clot formation. High cholesterol is a common risk factor for this type of stroke. Small vessel disease or lacunar infarction is closely related to high blood pressure.

[0046] The anti-ASIC1a antibody of this technology The present technology describes the method and composition for making and using the anti-ASIC1a antibody of the present technology (for example, anti-ASIC1a antibody or its antigen-binding fragment).The anti-ASIC1a antibody of the present technology can be useful in the diagnosis or treatment of ischemic stroke.The anti-ASIC1a antibody of the present technology within the scope of the present technology includes, but is not limited to, for example, monoclonal, chimeric, humanized, bispecific antibody, and diabody, its homolog, derivative, or fragment, which specifically binds to target polypeptide.

[0047] The table below provides the complementarity determining region (CDR) sequences of the anti-ASIC1a antibodies of the present technology. TIFF2025063103000002.tif211153

[0048] Thus, the antibody or antigen-binding fragment thereof (the anti-ASIC1a antibody of the present technology) comprises a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domains (V L ), wherein V H is a complementarity determining region V H CDR1, V H CDR2, and V H CDR3; L is a complementarity determining region V L CDR1, VL CDR2, and V L Including CDR3, V H and V L may include.

[0049] In one embodiment, the present technology provides a method for the production of a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domains (V L ), wherein the V H is selected from the group consisting of SEQ ID NOs: 3, 13, 23, 33, 43, and 53 H - CDR1 sequence; V selected from the group consisting of SEQ ID NOs: 4, 14, 24, 34, 44, and 54 H - a CDR2 sequence; and a V selected from the group consisting of SEQ ID NOs: 5, 15, 25, 35, 45, and 55 H - comprising a CDR3 sequence; L is selected from the group consisting of SEQ ID NOs: 8, 18, 28, 38, 48, and 58 L - CDR1 sequence; V selected from the group consisting of SEQ ID NOs: 9, 19, 29, 39, 49, and 59 L - a CDR2 sequence; and a V selected from the group consisting of SEQ ID NOs: 10, 20, 30, 40, 50, and 60 L -CDR3 sequence,

[0050] Additionally or alternatively, in some embodiments, V H is selected from the group consisting of SEQ ID NOs: 3, 13, 23, 33, and 43 H - CDR1 sequence; V selected from the group consisting of SEQ ID NOs: 4, 14, 24, 34, and 44 H - a CDR2 sequence; and a V selected from the group consisting of SEQ ID NOs: 5, 15, 25, 35, and 45 H - comprising a CDR3 sequence; V L is selected from the group consisting of SEQ ID NOs: 8, 18, 28, 38, and 48 L- CDR1 sequence; V selected from the group consisting of SEQ ID NOs: 9, 19, 29, 39, and 49 L - a CDR2 sequence; and a V selected from the group consisting of SEQ ID NOs: 10, 20, 30, 40, and 50 L -CDR3 sequence. Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment thereof further comprises an Fc domain of an isotype selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, and IgE. Additionally or alternatively, in some embodiments, the antigen-binding fragment comprises an Fab, F(ab')2, Fab', scF V , and F V Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment thereof is a monoclonal antibody, a chimeric antibody, a humanized antibody, or a bispecific antibody. Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment thereof binds to ASIC1a protein. Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment thereof binds to the extracellular domain of ASIC1a. Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment thereof binds to an epitope that spans two ASIC1a subunits. Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment thereof inhibits ASIC1a. Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment thereof inhibits proton-induced ASIC1a current.

[0051] In one aspect, the technology provides a composition comprising an antibody of any embodiment disclosed herein. Additionally or alternatively, in some embodiments, the nucleic acid sequence is selected from the group consisting of SEQ ID NO: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 51, and 56. In one embodiment, the technology provides a host cell or vector expressing any of the nucleic acids disclosed herein. In one aspect, the technology provides a kit comprising the antibody or antigen-binding fragment thereof of any embodiment disclosed herein and instructions for use.Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment thereof of any embodiment disclosed herein is coupled to at least one detectable label selected from the group consisting of radioactive label, fluorescent label, and color label.Additionally or alternatively, in some embodiments, the kit further comprises a secondary antibody that specifically binds to the antibody or antigen-binding fragment thereof of any embodiment disclosed herein.

[0052] In one aspect, the technology provides a method for detecting ASIC1a protein in a biological sample, the method comprising: contacting the biological sample with an antibody or antigen-binding fragment thereof of any embodiment disclosed herein, conjugated to a detectable label; and detecting the presence and level of the detectable label in the biological sample. In some embodiments, V H CDR1 comprises the amino acid sequence set forth in SEQ ID NO:3, H CDR2 comprises the amino acid sequence set forth in SEQ ID NO:4, H CDR3 comprises the amino acid sequence set forth in SEQ ID NO:5; L CDR1 comprises the amino acid sequence set forth in SEQ ID NO:8, L CDR2 comprises the amino acid sequence set forth in SEQ ID NO:9, L CDR3 comprises the amino acid sequence set forth in SEQ ID NO:10. In some embodiments, V H CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 13, H CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 14, H CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 15; L CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 18, L CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 19, LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:20.

[0053] In some embodiments, V H CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 23, H CDR2 comprises the amino acid sequence set forth in SEQ ID NO:24, H CDR3 comprises the amino acid sequence set forth in SEQ ID NO:25; L CDR1 comprises the amino acid sequence set forth in SEQ ID NO:28, L CDR2 comprises the amino acid sequence set forth in SEQ ID NO:29, L CDR3 comprises the amino acid sequence set forth in SEQ ID NO:30. In some embodiments, V H CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 33, H CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 34, H CDR3 comprises the amino acid sequence set forth in SEQ ID NO:35; L CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 38, L CDR2 comprises the amino acid sequence set forth in SEQ ID NO:39, L CDR3 comprises the amino acid sequence set forth in SEQ ID NO:40.

[0054] In some embodiments, V H CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 43, H CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 44, H CDR3 comprises the amino acid sequence set forth in SEQ ID NO:45; L CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 48, L CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 49, L CDR3 comprises the amino acid sequence set forth in SEQ ID NO:50. In some embodiments, V H CDR1 comprises the amino acid sequence set forth in SEQ ID NO:53, H CDR2 comprises the amino acid sequence set forth in SEQ ID NO:54, H CDR3 comprises the amino acid sequence set forth in SEQ ID NO:55;L CDR1 comprises the amino acid sequence set forth in SEQ ID NO:58, L CDR2 comprises the amino acid sequence set forth in SEQ ID NO:59, L CDR3 comprises the amino acid sequence set forth in SEQ ID NO:60.

[0055] In some embodiments, V H In some embodiments, V comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 12, 22, 32, 42, and 52. L comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 17, 27, 37, 47, and 57. The antibody or its antigen-binding fragment (the anti-ASIC1a antibody of the present technology) can specifically bind to ASIC1a protein.In some embodiments, the anti-ASIC1a antibody of the present technology can bind to the extracellular domain of ASIC1a.In some embodiments, the anti-ASIC1a antibody of the present technology can bind to an epitope that spans two ASIC1a subunits.

[0056] In some embodiments, the anti-ASIC1a antibodies of the present technology inhibit the function of ASIC1a trimers. In some embodiments, the anti-ASIC1a antibodies of the present technology stabilize ASIC1a trimers. In some embodiments, the anti-ASIC1a antibodies of the present technology inhibit hetero-oligomerization (e.g., heterotrimerization) of ASIC1a with other ASIC1 isomers. In some embodiments, the antibody or antigen-binding fragment thereof is an antibody, scFv, (scFv)2, Fab, Fab', F(ab')2, or scFv-Fc antibody. In some embodiments, the antibody or antigen-binding fragment thereof is an scFv antibody. In some embodiments, the scFv antibody is ASC01, ASC02, ASC03, ASC04, ASC05, or ASC06.

[0057] Formulation For example, the anti-ASIC1a antibody of the present technology is formulated in a simple delivery vehicle.However, the anti-ASIC1a antibody of the present technology can be lyophilized or incorporated into a gel, cream, biomaterial, sustained release delivery vehicle. The anti-ASIC1a antibody of the present technology is generally combined with a pharma- ceutically acceptable carrier. The term "pharma- ceutically acceptable carrier" refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition and can be administered without undue toxicity. Suitable carriers can be large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, and amino acid copolymers. Such carriers are well known to those skilled in the art. Pharmaceutically acceptable carriers in therapeutic compositions can include liquids such as water, saline, glycerol, and ethanol. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, can also be present in such vehicles. Pharmaceutically acceptable salts, such as mineral acid salts, for example, hydrochloride, hydrobromide, phosphate, sulfate, and the like; and salts of organic acids, such as acetate, propionate, malonate, benzoate, and the like, can also be present in the pharmaceutical composition.

[0058] Mode of Administration and Effective Dosages Any method known to those skilled in the art can be employed to contact cells, organs or tissues with the immunoglobulin-related composition of the present technology.Suitable methods include in vitro, ex vivo, or in vivo methods.In vivo methods typically include administration of the anti-ASIC1a antibody of the present technology, such as those described above, to a mammal, suitably a human.When used in vivo for therapy, the anti-ASIC1a antibody of the present technology is administered to a subject in an effective amount (i.e., an amount that has a desired therapeutic effect).The dosage and dosing regimen will depend on the degree of symptoms in the subject, the characteristics of the specific anti-ASIC1a antibody of the present technology used, such as its therapeutic index, the subject, and the subject's medical history. Effective amount can be determined during preclinical and clinical trials by methods familiar to physicians and clinicians.An effective amount of the anti-ASIC1a antibody of the present technology useful in the method can be administered to a mammal in need thereof by any of several well-known methods for administering pharmaceutical compositions.The anti-ASIC1a antibody of the present technology can be administered systemically or locally.

[0059] The immunoglobulin-related compositions described herein can be incorporated into pharmaceutical compositions for administration alone or in combination to a subject for the treatment or prevention of diseases described herein. Such compositions typically include an active agent and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., that are compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the composition. A pharmaceutical composition is typically formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral (e.g., intravenous, intradermal, intraperitoneal, or subcutaneous), oral, inhalation, transdermal (topical), intraocular, iontophoretic, and transmucosal administration. The solutions or suspensions used for parenteral, intradermal, or subcutaneous application may contain the following components: sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates; and agents for adjusting tonicity such as sodium chloride or dextrose. pH may be adjusted using acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic. For the convenience of the patient or treating physician, dosage formulations can be provided in kits that contain all the necessary supplies (e.g., vials of drug, vials of diluent, syringes, and needles) for a course of treatment (e.g., 7 days of treatment).

[0060] In some embodiments, the immunoglobulin-related compositions described herein are administered by a parenteral route. In some embodiments, the antibodies or antigen-binding fragments thereof are administered by a topical route. Pharmaceutical compositions suitable for injectable use may include sterile aqueous solutions (where water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). In all cases, compositions for parenteral administration must be sterile and fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The anti-ASIC1a antibody composition of the present technology may contain a carrier, which may be a solvent or dispersion medium, for example, containing water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Prevention of microbial action may be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thiomerasol, and the like. Glutathione and other antioxidants may be included to prevent oxidation. In many cases, isotonic agents, such as sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride, are included in the composition. Prolonged absorption of an injectable composition may be achieved by including an agent that delays absorption, such as aluminum monostearate or gelatin, in the composition.

[0061] Sterile injectable solution can be prepared by incorporating the active compound in the required amount in suitable solvent with one or combination of the above-listed components as required, and then sterilizing by filtration.Generally, dispersion is prepared by incorporating active compound into a sterile vehicle that contains basic dispersion medium and other components required from above-listed.For the preparation of sterile powder for sterile injectable solution, the typical method of preparation includes vacuum drying and freeze-drying, which can produce the powder of active ingredient and any additional desired components from the solution that has been previously sterilized and filtered.

[0062] Oral compositions generally include an inert diluent or edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with an excipient and used in the form of tablets, troches, or capsules, such as gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, troches, etc. can contain any of the following ingredients, or compounds of a similar nature: binders such as microcrystalline cellulose, tragacanth, or gelatin; excipients such as starch or lactose, disintegrating agents such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate or Sterotes; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate, or orange flavoring. For administration by inhalation, the anti-ASIC1a antibodies of the present technology can be delivered in the form of an aerosol spray from a pressured container or dispenser containing a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Such methods include those described in U.S. Patent No. 6,468,798.

[0063] Systemic administration of the anti-ASIC1a antibody of the technology described herein can also be by transmucosal or transdermal means.For transmucosal or transdermal administration, a penetrant suitable for the barrier to be permeated is used in the formulation.Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.Transmucosal administration can be accomplished by using nasal spray.For transdermal administration, active compound is formulated into ointments, salves, gels, or creams as generally known in the art.In one embodiment, transdermal administration can be carried out by iontophoresis. The anti-ASIC1a antibody of the present technology can be formulated in a carrier system. The carrier can be a colloidal system. The colloidal system can be a liposome, a phospholipid bilayer vehicle. In one embodiment, the therapeutic anti-ASIC1a antibody of the present technology is encapsulated in liposomes while maintaining the integrity of the anti-ASIC1a antibody of the present technology. As those skilled in the art will understand, there are various methods for preparing liposomes. (See Lichtenberg et al., Methods Biochem. Anal., 33:337-462 (1988); Anselem et al., Liposome Technology, CRC Press (1993)). Liposome formulations can delay clearance and increase cellular uptake (See Reddy, Ann. Pharmacother., 34(7-8):915-923 (2000)). The active agent may also be carried within particles prepared from pharma- ceutically acceptable components, including, but not limited to, soluble, insoluble, permeable, non-permeable, biodegradable, or gastroretentive polymers or liposomes. Such particles include, but are not limited to, nanoparticles, biodegradable nanoparticles, microparticles, biodegradable microparticles, nanospheres, biodegradable nanospheres, microspheres, biodegradable microspheres, capsules, emulsions, liposomes, micelles, and viral vector systems.

[0064] The carrier can also be a polymer, for example a biodegradable biocompatible polymer matrix. In one embodiment, the anti-ASIC1a antibody of the present technology can be embedded in a polymer matrix while maintaining protein integrity. The polymer can be natural, such as a polypeptide, protein, or polysaccharide, or synthetic, such as poly-alpha-hydroxy acid. Examples include carriers made of, for example, collagen, fibronectin, elastin, cellulose acetate, cellulose nitrate, polysaccharides, fibrin, gelatin, and combinations thereof. In one embodiment, the polymer is polylactic acid (PLA) or copolylactic / glycolic acid (PGLA). The polymer matrix can be prepared and isolated in a variety of forms and sizes, including microspheres and nanospheres. The polymer formulation can lead to a long duration of therapeutic effect. (See Reddy, Ann. Pharmacother., 34(7-8):915-923 (2000)). Polymer formulations for human growth hormone (hGH) are used in clinical trials. (See Kozarich and Rich, Chemical Biology, 2:548-552 (1998)).

[0065] Examples of polymeric microsphere sustained release formulations are described in PCT Publication No. WO 99 / 15154 (Tracy et al.), U.S. Patent Nos. 5,674,534 and 5,716,644 (both to Zale et al.), PCT Publication No. WO 96 / 40073 (Zale et al.), and PCT Publication No. WO 00 / 38651 (Shah et al.). U.S. Patent Nos. 5,674,534 and 5,716,644, and PCT Publication No. WO 96 / 40073 describe polymer matrices containing particles of erythropoietin that are stabilized against aggregation with salts. In some embodiments, the anti-ASIC1a antibody of the present technology is prepared with a carrier that will protect the anti-ASIC1a antibody of the present technology from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, can be used. Such formulations can be prepared using known techniques. Materials can also be obtained commercially, for example, from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to specific cells using monoclonal antibodies against cell-specific antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0066] The anti-ASIC1a antibody of the present technology can also be formulated to enhance intracellular delivery.For example, liposome delivery systems are known in the art, see, for example, Chonn and Cullis, "Recent Advances in Liposome Drug Delivery Systems," Current Opinion in Biotechnology 6:698-708 (1995);Weiner, "Liposomes for Protein Delivery: Selecting Manufacturing and Development Processes," Immunomethods, 4(3):201-9 (1994);and Gregoriadis, "Engineering Liposomes for Drug Delivery: Progress and Problems," Trends Biotechnol., 13(12):527-37 (1995).Mizguchi et al., Cancer Lett., 100:63-69 (1996) describe the use of fusogenic liposomes to deliver proteins to cells both in vivo and in vitro. The dosage, toxicity, and therapeutic efficacy of the anti-ASIC1a antibody of the present technology can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine LD50 (the dose lethal to 50% of the population) and ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic effect and therapeutic effect is the therapeutic index, which can be expressed as the LD50 / ED50 ratio. In some embodiments, the anti-ASIC1a antibody of the present technology exhibits a high therapeutic index. Although the anti-ASIC1a antibody of the present technology that exhibits toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue to minimize potential damage to uninfected cells, thereby reducing side effects.

[0067] Data obtained from cell culture assays and animal tests can be used to formulate a range of dosages for use in humans. The dosage of such compounds is within a range of circulating concentrations that includes the ED50 with little or no toxicity. Dosages can vary within this range depending on the dosage form employed and the route of administration used. For any anti-ASIC1a antibody of the present technology used in the method, a therapeutically effective dose can be estimated initially from cell culture assays. Dosages are determined based on the IC determined in cell culture. 50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms). Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

[0068] Typically, an effective amount of the anti-ASIC1a antibody of the present technology sufficient to achieve a therapeutic or prophylactic effect ranges from about 0.000001 mg / kilogram body weight / day to about 10,000 mg / kilogram body weight / day. Suitably, the dosage range is from about 0.0001 mg / kilogram body weight / day to about 100 mg / kilogram body weight / day. For example, dosage can be 1 mg / kg body weight or 10 mg / kg body weight every day, every 2 days, or every 3 days, or can be in the range of 1 to 10 mg / kg every week, every 2 weeks, or every 3 weeks. In one embodiment, a single dosage of the anti-ASIC1a antibody of the present technology ranges from 0.001 to 10,000 micrograms / kg body weight. In one embodiment, the anti-ASIC1a antibody concentration of the present technology in the carrier ranges from 0.2 to 2000 micrograms per milliliter delivered. An exemplary treatment regime involves administration once a day or once a week. In therapeutic applications, relatively high dosages at relatively short intervals may be required until the progression of the disease is slowed or terminated and the subject shows partial or complete improvement of the symptoms of the disease. Thereafter, the patient may be administered a prophylactic regime.

[0069] In some embodiments, a therapeutically effective amount of an anti-ASIC1a antibody of the present technology is 10 -12 ~10 -6 Molar concentration, e.g., approximately 10 -7The concentration of the anti-ASIC1a antibody of the present technology in the target tissue, in molar terms, may be defined as the concentration. This concentration may be delivered by a systemic dose of 0.001 to 100 mg / kg, or an equivalent dose by body surface area. The dosing schedule will be optimized to maintain a therapeutic concentration in the target tissue. In some embodiments, dosing is administered by a daily or weekly administration, but may also include continuous administration (e.g., parenteral injection or transdermal application). In some embodiments, the dosage of the anti-ASIC1a antibody of the present technology is provided at a "low", "intermediate", or "high" dosage level. In one embodiment, the low dose is provided at about 0.0001 to about 0.5 mg / kg / hour, suitably about 0.001 to about 0.1 mg / kg / hour. In one embodiment, the intermediate dose is provided at about 0.01 to about 1.0 mg / kg / hour, suitably about 0.01 to about 0.5 mg / kg / hour. In one embodiment, the high dose is provided at about 0.5 to about 10 mg / kg / hour, suitably about 0.5 to about 2 mg / kg / hour.

[0070] For example, a therapeutically effective amount can partially or completely alleviate one or more symptoms of ischemic stroke, including sudden weakness; paralysis or numbness of face, arm or leg, especially on one side of the body; drooping of one side of face; confusion; difficulty in speaking or language comprehension, such as slurred words; difficulty in seeing in one or both eyes, such as blurred vision or amaurosis, or double vision in one or both eyes; difficulty in breathing; dizziness; difficulty in walking; loss of balance or coordination, such as causing an unexplained fall; loss of consciousness; and sudden and severe headache.A therapeutically effective amount can partially or completely alleviate one or more symptoms of ischemic stroke, including but not limited to sudden facial weakness of face (such as drooping on one side), unilateral weakness of upper limbs, and abnormal speech. Those skilled in the art will appreciate that certain factors, including but not limited to the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and other diseases present, may affect the dosage and timing required to effectively treat a subject. Moreover, treatment of a subject with a therapeutically effective amount of a therapeutic composition described herein may include a single treatment or a series of treatments.

[0071] The mammal treated according to the present method can be any mammal, including, for example, farm animals such as sheep, pigs, cows, and horses; pet animals such as dogs and cats; laboratory animals such as rats, mice, and rabbits. In some embodiments, the mammal is a human. Use of the anti-ASIC1a antibody of the present technology Overview. The anti-ASIC1a antibody of the present technology is useful in methods known in the art for localizing and / or quantifying ASIC1a protein or its variants (e.g., for measuring the level of ASIC1a protein in suitable physiological samples, for use in diagnostic methods, for use in imaging polypeptides, etc.). The anti-ASIC1a antibody of the present technology is useful for isolating ASIC1a protein by standard techniques such as affinity chromatography or immunoprecipitation. The anti-ASIC1a antibody of the present technology can facilitate the purification of natural immunoreactive ASIC1a protein from biological samples, such as mammalian serum or cells, as well as recombinantly produced immunoreactive ASIC1a protein expressed in host systems. In addition, the anti-ASIC1a antibody of the present technology can be used to detect immunoreactive ASIC1a protein or its fragments (e.g., in plasma, cell lysate, or cell supernatant) to evaluate the abundance and expression pattern of immunoreactive polypeptide. The anti-ASIC1a antibody of the present technology can be used diagnostically to monitor the immunoreactive ASIC1a protein level in tissue as part of clinical testing procedure, for example, to determine the efficacy of a given treatment regimen.As mentioned above, detection can be facilitated by coupling (i.e., physically linking) the anti-ASIC1a antibody of the present technology to a detectable substance.

[0072] Detection of ASIC1a protein.The exemplary method for detecting the presence or absence of immunoreactive ASIC1a protein in biological sample involves obtaining a biological sample from test subject, and contacting the biological sample with the anti-ASIC1a antibody of the present technology that can detect immunoreactive ASIC1a protein, thereby detecting the presence of immunoreactive ASIC1a protein in biological sample.Detection can be accomplished by the detectable label attached to the antibody. The term "labeled" with respect to the anti-ASIC1a antibody of the present technology is intended to include direct labeling of the antibody by coupling (i.e., physically linking) a detectable substance to the antibody, as well as indirect labeling of the antibody by reactivity with another compound that is directly labeled, such as a secondary antibody. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody, and end-labeling of a DNA probe with biotin so that it can be detected using fluorescently labeled streptavidin. In some embodiments, the anti-ASIC1a antibody of the technology disclosed herein is conjugated with one or more detectable labels.For such use, the anti-ASIC1a antibody of the technology disclosed herein can be detectably labeled by covalent or non-covalent attachment of chromogenic, enzymatic, radioisotopic, isotopic, fluorescent, toxin, chemiluminescent label, nuclear magnetic resonance contrast agent or other label.

[0073] Examples of suitable colorimetric labels include diaminobenzidine and 4-hydroxyazo-benzene-2-carboxylic acid. Examples of suitable enzyme labels include malate dehydrogenase, staphylococcal nuclease, Δ-5-steroid isomerase, yeast alcohol dehydrogenase, α-glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase. Examples of suitable radioisotope labels include: 3 H, 111 In, 125 I, 131 I, 32 P, 35 S, 14 C. 51 Cr, 57 To, 58 Co, 59 Fe, 75 Se, 152 EU,90 Y, 67 Cu, 217 Ci, 211 At, 212 Pb, 47 Sc, 109 Pd, etc. 111 In is, 125 I or 131 I is an exemplary isotope used for in vivo imaging, since it avoids the problem of dehalogenation by the liver of I-labeled ASIC1a protein-binding antibodies. In addition, this isotope has a more favorable gamma emission energy for imaging (Perkins et al, Eur. J. Nucl. Med. 70:296-301 (1985); Carasquillo et al., J. Nucl. Med. 25:281-287 (1987)). For example, 1-(P-isothiocyanatobenzyl)-DPTA coupled to a monoclonal antibody is used. 111 In exhibits little uptake in non-tumor tissues, especially the liver, enhancing the specificity of tumor localization (Esteban et al., J. Nucl. Med. 28:861-870 (1987)). Examples of suitable non-radioactive isotope labels include: 157 Gd, 55 Mn, 162 Dy, 52 Tr, and 56 Contains Fe.

[0074] Examples of suitable fluorescent labels include: 152 Examples of suitable toxin labels include Eu labels, fluorescein labels, isothiocyanate labels, rhodamine labels, phycoerythrin labels, phycocyanin labels, allophycocyanin labels, green fluorescent protein (GFP) labels, o-phthaldehyde labels, and fluorescamine labels. Examples of suitable toxin labels include diphtheria toxin, ricin, and cholera toxin. Examples of chemiluminescent labels include luminol labels, isoluminol labels, aromatic acridinium ester labels, imidazole labels, acridinium salt labels, oxalate ester labels, luciferin labels, luciferase labels, and aequorin labels. Examples of nuclear magnetic resonance imaging agents include heavy metal nuclei such as Gd, Mn, and iron.

[0075] The detection method of the present technology can be used to detect immunoreactive ASIC1a protein in biological samples in vitro and in vivo.The in vitro techniques for detecting immunoreactive ASIC1a protein include enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation, radioimmunoassay and immunofluorescence.In addition, the in vivo techniques for detecting immunoreactive ASIC1a protein include introducing the labeled anti-ASIC1a antibody of the present technology into a subject.For example, the anti-ASIC1a antibody of the present technology can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.In one embodiment, the biological sample contains ASIC1a protein molecules from a test subject. Immunological assays and imaging. Antibody-based techniques can be used to assay immunoreactive ASIC1a protein levels in biological samples (e.g., human plasma) using the anti-ASIC1a antibody of the present technology. For example, protein expression in tissues can be tested using classical immunohistological methods. Jalkanen, M. et al., J. Cell. Biol. 101: 976-985, 1985; Jalkanen, M. et al., J. Cell. Biol. 105: 3087-3096, 1987. Other antibody-based methods useful for detecting protein gene expression include immunological assays such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels such as glucose oxidase, and iodine ( 125 I, 121 I, 131 I), Carbon ( 14C), sulfur ( 35 S), tritium ( 3 H), Indium ( 112 In), and technetium ( 99 mTc) or other radioactive agents, as well as fluorescent labels, such as fluorescein, rhodamine, and green fluorescent protein (GFP), and biotin.

[0076] In addition to assaying the immunoreactive ASIC1a protein level in biological samples, the anti-ASIC1a antibody of the present technology can be used to image ASIC1a protein in vivo.Antibodies useful in this method include those that can be detected by X-ray radiography, NMR, or ESR.For X-ray radiography, suitable labels include radioisotopes such as barium or cesium that emit detectable radiation but are not overtly harmful to subjects.For NMR and ESR, suitable markers include those that have detectable characteristic spins, such as deuterium, which can be incorporated into the anti-ASIC1a antibody of the present technology by nutrient labeling to related scFv clones.

[0077] Radioisotopes (e.g. 131 I, 112 In, 99 An anti-ASIC1a antibody of the present technology that is labeled with a suitable detectable imaging moiety, such as mTc, a radiopaque substance, or a material detectable by nuclear magnetic resonance, is introduced into the subject (e.g., parenterally, subcutaneously, or intraperitoneally). It will be understood in the art that the size of the subject and the imaging system used will determine the amount of imaging moiety required to produce a diagnostic image. In the case of a radioisotope moiety, for a human subject, the amount of radioactivity injected will typically be in the range of about 5-20 millicuries. 99The labeled anti-ASIC1a antibody of the present technology will then accumulate at the location of cells that contain specific labeled polypeptide.For example, the labeled anti-ASIC1a antibody of the present technology will accumulate in the subject in the cells and tissues where ASIC1a protein is localized. Thus, the present technology provides a method for diagnosing a medical condition, involving: (a) assaying expression of immunoreactive ASIC1a protein by measuring binding of the anti-ASIC1a antibody of the present technology in an individual's cells or body fluids; and (b) comparing the amount of immunoreactive ASIC1a protein present in the sample with a standard reference, wherein an increase or decrease in immunoreactive ASIC1a protein levels compared to the standard is indicative of the medical condition.

[0078] Affinity purification. The anti-ASIC1a antibody of the present technology can be used to purify immunoreactive ASIC1a protein from a sample. In some embodiments, the antibody is immobilized on a solid support. Examples of such solid supports include plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, acrylic resins such as polyacrylamide and latex beads. The techniques for coupling antibodies to such solid supports are well known in the art (Weir et al., "Handbook of Experimental Immunology" 4th Ed., Blackwell Scientific Publications, Oxford, England, Chapter 10 (1986); Jacoby et al., Meth. Enzym. 34 Academic Press, NY (1974)). The simplest way to bind antigen to an antibody-support matrix is ​​to collect the beads in a column and pass the antigen solution through the column. The efficiency of this method depends on the contact time between the immobilized antibody and the antigen, which can be extended by using a low flow rate. The immobilized antibody captures the antigen as it flows past. Alternatively, the antigen can be contacted with the antibody-support matrix by mixing the antigen solution with the support (e.g., beads) and rotating or vibrating the slurry, allowing maximum contact between the antigen and the immobilized antibody. After the binding reaction is complete, the slurry is passed through the column for collection of the beads. The beads are washed using an appropriate washing buffer, and then the pure or substantially pure antigen is eluted.

[0079] The antibody or polypeptide of interest can be conjugated to a solid support such as beads.In addition, the first solid support such as beads can also be conjugated to a second solid support, which can be a second bead or other support, if desired, by any suitable means, including those disclosed herein for conjugating a polypeptide to a support.Therefore, any of the conjugation methods and means disclosed herein for conjugating a polypeptide to a solid support can also be applied to conjugating a first support to a second support, where the first and second solid supports can be the same or different.

[0080] Suitable linkers, which may be crosslinkers, for use in conjugating a polypeptide to a solid support include a variety of agents that can react with functional groups present on the surface of the support or with the polypeptide, or both. Reagents useful as crosslinkers include homobifunctional and, in particular, heterobifunctional reagents. Useful bifunctional crosslinkers include, but are not limited to, N-SIAB, dimaleimide, DTNB, N-SATA, N-SPDP, SMCC, and 6-HYNIC. Crosslinkers can be selected to provide a selectively cleavable bond between the polypeptide and the solid support. For example, photolabile crosslinkers such as 3-amino-(2-nitrophenyl)propionic acid can be employed as a means to cleave the polypeptide from the solid support. (Brown et al., Mol. Divers, pp, 4-12 (1995); Rothschild et al., Nucl. Acids Res., 24:351-66 (1996); and U.S. Patent No. 5,643,722). Other cross-linking reagents are known in the art (see, e.g., Wong (1991), supra; and Hermanson (1996), supra).

[0081] The antibody or polypeptide can be immobilized on a solid support such as a bead through a covalent amide bond formed between a carboxyl functionalized bead and the amino terminus of the polypeptide, or vice versa through a covalent amide bond formed between an amino functionalized bead and the carboxyl terminus of the polypeptide. In addition, a bifunctional trityl linker can be attached to the support through an amino or carboxyl group on the resin by an amino resin, for example, a 4-nitrophenyl active ester on a resin such as a Wang resin. Using the bifunctional trityl approach, the solid support may require treatment with a volatile acid such as formic acid or trifluoroacetic acid to ensure that the polypeptide can be cleaved and removed. In such cases, the polypeptide can be deposited as beadless spots on the bottom of the well of the solid support or on the flat surface of the solid support. After addition of a matrix solution, the polypeptide can be desorbed by MS.

[0082] Hydrophobic trityl linkers can also be used as acid-labile linkers by using volatile acid or a suitable matrix solution, such as a matrix solution containing 3-HPA, to cleave the amino-linked trityl group from the polypeptide. Acid lability can also be changed. For example, trityl, monomethoxytrityl, dimethoxytrityl, or trimethoxytrityl can be changed to a suitable p-substituted or more acid-labile tritylamine derivative of the polypeptide, i.e., trityl ether and tritylamine bonds can be made in the polypeptide. Thus, the polypeptide can be removed from the hydrophobic linker, for example, by disrupting the hydrophobic attraction, or by cleaving the trityl ether or tritylamine bond under acidic conditions, including typical MS conditions, where a matrix such as 3-HPA acts as an acid, if desired.

[0083] Orthogonal cleavable linkers can also be useful for binding a first solid support, such as beads, to a second solid support or for binding a polypeptide of interest to a solid support.Using such linkers, a first solid support, such as beads, can be selectively cleaved from a second solid support without cleaving the polypeptide from the support; the polypeptide can then be subsequently cleaved from the beads.For example, a disulfide linker that can be cleaved using a reducing agent such as DTT can be employed to bind beads to a second solid support, and an acid-cleavable bifunctional trityl group can be used to immobilize a polypeptide to the support.As desired, the link of the polypeptide to the solid support can be cleaved first, for example, leaving the link between the first and second supports intact.The trityl linker can provide covalent or hydrophobic conjugation, and regardless of the nature of the conjugation, the trityl group is easily cleaved in acidic conditions. For example, beads can be attached to a second support through a linking group that can be selected to have a length and chemical nature that facilitates high density binding of beads to a solid support or high density binding of polypeptides to beads. Such linking groups can have, for example, a "dendritic" structure, thereby providing multiple functional groups per attachment site on the solid support. Examples of such linking groups include polylysine, polyglutamic acid, penta-erythrole, and tris-hydroxy-aminomethane.

[0084] Association by non-covalent bonds. Through non-covalent interactions, antibodies or polypeptides can be conjugated to solid supports, or a first solid support can also be conjugated to a second solid support. For example, magnetic beads made of ferromagnetic materials that can be magnetized can be attracted to magnetic solid supports and released from the support by removing the magnetic field. Alternatively, solid supports can be provided with ionic or hydrophobic moieties, which can respectively allow interaction of the ionic or hydrophobic moieties with polypeptides, such as polypeptides containing attached trityl groups, or with a second solid support that has hydrophobic characteristics. A solid support can also be provided with a member of a specific binding pair and thus conjugated to a polypeptide or a second solid support containing a complementary binding moiety. For example, avidin- or streptavidin-coated beads can be bound to a polypeptide having a biotin moiety incorporated therein, or to a second solid support coated with biotin or a derivative of biotin such as iminobiotin. It should be recognized that any of the binding members disclosed herein or otherwise known in the art can be interchanged. Thus, biotin can be incorporated, for example, into either the polypeptide or the solid support, and conversely, avidin or other biotin-binding moieties can be incorporated into the support or the polypeptide, respectively. Other specific binding pairs contemplated for use herein include, but are not limited to, hormones and their receptors, enzymes and their substrates, nucleotide sequences and their complementary sequences, antibodies and the antigens with which they specifically interact, and other such pairs known to those skilled in the art.

[0085] A. Diagnostic Uses of the Anti-ASIC1a Antibodies of the Present Technology Overview. The anti-ASIC1a antibodies of the present technology are useful in diagnostic methods. Therefore, the present technology provides a method of using the antibodies in diagnosing ASIC1a protein activity in a subject. The anti-ASIC1a antibodies of the present technology can be selected so that they have any level of epitope binding specificity and very high binding affinity to ASIC1a protein. In general, the higher the binding affinity of the antibody, the more stringent washing conditions can be performed in an immunological assay to remove non-specifically bound materials without removing the target polypeptide. Thus, the anti-ASIC1a antibodies of the present technology useful in diagnostic assays are usually about 10 8 M -1 , 10 9 M -1 , 10 10 M -1 , 10 11 M -1 , or 10 12 M -1 Additionally, anti-ASIC1a antibodies of the present technology used as diagnostic reagents desirably have a kinetic on-rate sufficient to reach equilibrium under standard conditions within at least 12 hours, at least five (5) hours, or at least one (1) hour.

[0086] The anti-ASIC1a antibodies of the present technology can be used to detect immunoreactive ASIC1a protein in a variety of standard assay formats, including immunoprecipitation, Western blotting, ELISA, radioimmunoassay, and immunometric assays. Harlow & Lane, Antibodies, A Laboratory Manual (Cold Spring Harbor Publications, New York, 1988); see U.S. Patent Nos. 3,791,932; 3,839,153; 3,850,752; 3,879,262; 4,034,074, 3,791,932; 3,817,837; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; and 4,098,876. Biological sample can be obtained from any tissue or body fluid of subject.In certain embodiments, subject is in early stage of cancer.In one embodiment, early stage of cancer is determined by ASIC1a protein level or expression pattern in sample obtained from subject.In certain embodiments, sample is selected from the group consisting of urine, blood, serum, plasma, saliva, amniotic fluid, cerebrospinal fluid (CSF) and biopsied biological tissue.

[0087] Immunometric assay or sandwich assay is one format for the diagnostic method of the present technology.See US Patent No. 4,376,110, 4,486,530, 5,914,241 and 5,965,375.Such assay uses one kind of antibody, for example, the anti-ASIC1a antibody of the present technology or a group of anti-ASIC1a antibodies of the present technology immobilized on solid phase, and another anti-ASIC1a antibody of the present technology or a group of anti-ASIC1a antibodies of the present technology in solution.Typically, the anti-ASIC1a antibody of the present technology or a group of anti-ASIC1a antibodies of the present technology in solution is labeled.When an antibody group is used, the group can contain antibodies that bind to different epitope specificities in the target polypeptide.Therefore, the same group can be used for both solid phase and solution antibody.When the anti-ASIC1a antibody of the present technology is used, first and second ASIC1a protein monoclonal antibodies with different binding specificities are used for solid phase and liquid phase. The solid phase (also referred to as "capture") and solution (also referred to as "detection") antibodies may contact the target antigen in either order or simultaneously. If the solid phase antibody contacts first, the assay is referred to as a forward assay. Conversely, if the solution antibody contacts first, the assay is referred to as a backward assay. If the target contacts both antibodies simultaneously, the assay is referred to as a simultaneous assay. After contacting the ASIC1a protein with the anti-ASIC1a antibody of the present technology, the sample is incubated for a period that typically varies from about 10 minutes to about 24 hours (usually about 1 hour). A washing step is then performed to remove components of the sample that are not specifically bound to the anti-ASIC1a antibody of the present technology used as a diagnostic reagent. If the solid phase and solution antibodies are bound in separate steps, washing may be performed after either or both binding steps. After washing, binding is typically quantified by detecting a label linked to the solid phase through binding of the labeled solution antibody. Usually, for a given pair of antibodies or antibody populations and given reaction conditions, a calibration curve is made from samples containing known concentrations of target antigen.The concentration of immunoreactive ASIC1a protein in the sample being tested is then read by interpolation from the calibration curve (i.e., standard curve).The analyte can be measured from the amount of labeled solution antibody bound at equilibrium or by kinetic measurements of labeled solution antibody binding at a series of time points before equilibrium is reached. The slope of such a curve is a measure of the concentration of ASIC1a protein in the sample.

[0088] Suitable supports for use in the above method include, for example, nitrocellulose membrane, nylon membrane and derivatized nylon membrane, as well as particles, such as agarose, dextran-based gel, dipstick, particulate matter, microsphere, magnetic particle, test tube, microtiter well, SEPHADEX™ (Amersham Pharmacia Biotech, Piscataway NJ), etc. Immobilization can be by absorption or by covalent attachment.Optionally, the anti-ASIC1a antibody of the present technology can be conjugated to a linker molecule, such as biotin, for attachment to a surface-bound linker, such as avidin. In some embodiments, the present disclosure provides the anti-ASIC1a antibody of the present technology conjugated to a diagnostic agent.The diagnostic agent can include radioactive or non-radioactive labeling contrast agents (such as for magnetic resonance imaging, computed tomography, or ultrasound), and the radioactive label can be gamma, beta, alpha, Auger electron, or positron emitting isotopes.The diagnostic agent is a molecule that is conjugated to an antibody portion, i.e., an antibody or an antibody fragment or subfragment, and administered, and is useful in diagnosing or detecting disease by locating cells that contain antigens.

[0089] Useful diagnostic agents include, but are not limited to, radioisotopes, dyes (such as those with biotin-streptavidin complexes), contrast agents, fluorescent compounds or molecules, and enhancement agents for magnetic resonance imaging (MRI) (e.g., paramagnetic ions). U.S. Patent No. 6,331,175 describes MRI techniques and the preparation of antibodies conjugated to MRI enhancement agents, and is incorporated by reference in its entirety. In some embodiments, the diagnostic agent is selected from the group consisting of radioisotopes, enhancement agents for use in magnetic resonance imaging, and fluorescent compounds. To load an antibody component with a radiometal or paramagnetic ion, it may be necessary to react it with a reagent that has a long tail to which multiple chelating groups are attached for binding to the ion. Such tails can be polymers such as polylysine, polysaccharides, or other derivatized or derivatizable chains with pendant groups to which chelating groups can be attached, such as, for example, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrins, polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and similar groups known to be useful for this purpose. Chelates can be coupled to the antibodies of the present technology using standard chemistry. Chelates are usually linked to the antibody by a group that allows for the formation of bonds to the molecule with minimal loss of immunoreactivity and minimal aggregation and / or internal crosslinking. Other methods and reagents for conjugating chelates to antibodies are disclosed in U.S. Patent No. 4,824,659. Particularly useful metal-chelate combinations include 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs used with diagnostic isotopes for radioimaging. The same chelates when complexed with non-radioactive metals such as manganese, iron, and gadolinium are useful for MRI when used with the ASIC1a protein antibodies of the present technology.

[0090] B. Therapeutic Uses of Anti-ASIC1a Antibodies of the Present Technology Overview. In some embodiments, the anti-ASIC1a antibodies of the present technology are useful in the methods disclosed herein to provide therapies for preventing, ameliorating, or treating ischemic stroke and related conditions. In one embodiment, the present technology provides a method of treating ischemic stroke in a subject in need thereof, comprising administering to a subject a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domains (V L administering to a subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof comprising V H is selected from the group consisting of SEQ ID NOs: 3, 13, 23, 33, 43, and 53 H - CDR1 sequence; V selected from the group consisting of SEQ ID NOs: 4, 14, 24, 34, 44, and 54 H - a CDR2 sequence; and a V selected from the group consisting of SEQ ID NOs: 5, 15, 25, 35, 45, and 55 H - comprising a CDR3 sequence; L is selected from the group consisting of SEQ ID NOs: 8, 18, 28, 38, 48, and 58 L - CDR1 sequence; V selected from the group consisting of SEQ ID NOs: 9, 19, 29, 39, 49, and 59 L - a CDR2 sequence; and a V selected from the group consisting of SEQ ID NOs: 10, 20, 30, 40, 50, and 60 L -CDR3 sequence,

[0091] In some embodiments, V H V H CDR1, V H CDR2, and V H CDR3, V L V L CDR1, V L CDR2, and V L In some embodiments, the V H CDR1 comprises the amino acid sequence set forth in SEQ ID NO:3, H CDR2 comprises the amino acid sequence set forth in SEQ ID NO:4, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:5; L CDR1 comprises the amino acid sequence set forth in SEQ ID NO:8, L CDR2 comprises the amino acid sequence set forth in SEQ ID NO:9, L CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 10. H CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 13, H CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 14, H CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 15; L CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 18, L CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 19, L CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 20. H CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 23, H CDR2 comprises the amino acid sequence set forth in SEQ ID NO:24, H CDR3 comprises the amino acid sequence set forth in SEQ ID NO:25; L CDR1 comprises the amino acid sequence set forth in SEQ ID NO:28, L CDR2 comprises the amino acid sequence set forth in SEQ ID NO:29, L CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 30. H CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 33, H CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 34, H CDR3 comprises the amino acid sequence set forth in SEQ ID NO:35; L CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 38, L CDR2 comprises the amino acid sequence set forth in SEQ ID NO:39, L CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 40. H CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 43, H CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 44, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:45; L CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 48, L CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 49, L CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 50. H CDR1 comprises the amino acid sequence set forth in SEQ ID NO:53, H CDR2 comprises the amino acid sequence set forth in SEQ ID NO:54, H CDR3 comprises the amino acid sequence set forth in SEQ ID NO:55; L CDR1 comprises the amino acid sequence set forth in SEQ ID NO:58, L CDR2 comprises the amino acid sequence set forth in SEQ ID NO:59, L CDR3 comprises the amino acid sequence set forth in SEQ ID NO:60.

[0092] In some embodiments, V H In some embodiments, V comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 12, 22, 32, 42, and 52. L comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 17, 27, 37, 47, and 57.

[0093] In one embodiment, the present technology provides for alleviating one or more symptoms of ischemic stroke in a subject in need thereof, the method comprising administering to a subject a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domains (V L administering to a subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof comprising V H is selected from the group consisting of SEQ ID NOs: 3, 13, 23, 33, 43, and 53 H - CDR1 sequence; V selected from the group consisting of SEQ ID NOs: 4, 14, 24, 34, 44, and 54 H - a CDR2 sequence; and a V selected from the group consisting of SEQ ID NOs: 5, 15, 25, 35, 45, and 55 H - comprising a CDR3 sequence; V Lis selected from the group consisting of SEQ ID NOs: 8, 18, 28, 38, 48, and 58 L - CDR1 sequence; V selected from the group consisting of SEQ ID NOs: 9, 19, 29, 39, 49, and 59 L - a CDR2 sequence; and a V selected from the group consisting of SEQ ID NOs: 10, 20, 30, 40, 50, and 60 L -CDR3 sequence,

[0094] In some embodiments, V H V H CDR1, V H CDR2, and V H CDR3, V L V L CDR1, V L CDR2, and V L In some embodiments, the V H CDR1 comprises the amino acid sequence set forth in SEQ ID NO:3, H CDR2 comprises the amino acid sequence set forth in SEQ ID NO:4, H CDR3 comprises the amino acid sequence set forth in SEQ ID NO:5; L CDR1 comprises the amino acid sequence set forth in SEQ ID NO:8, L CDR2 comprises the amino acid sequence set forth in SEQ ID NO:9, L CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 10. H CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 13, H CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 14, H CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 15; L CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 18, L CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 19, L CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 20. H CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 23, H CDR2 comprises the amino acid sequence set forth in SEQ ID NO:24,H CDR3 comprises the amino acid sequence set forth in SEQ ID NO:25; L CDR1 comprises the amino acid sequence set forth in SEQ ID NO:28, L CDR2 comprises the amino acid sequence set forth in SEQ ID NO:29, L CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 30. H CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 33, H CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 34, H CDR3 comprises the amino acid sequence set forth in SEQ ID NO:35; L CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 38, L CDR2 comprises the amino acid sequence set forth in SEQ ID NO:39, L CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 40. H CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 43, H CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 44, H CDR3 comprises the amino acid sequence set forth in SEQ ID NO:45; L CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 48, L CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 49, L CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 50. H CDR1 comprises the amino acid sequence set forth in SEQ ID NO:53, H CDR2 comprises the amino acid sequence set forth in SEQ ID NO:54, H CDR3 comprises the amino acid sequence set forth in SEQ ID NO:55; L CDR1 comprises the amino acid sequence set forth in SEQ ID NO:58, L CDR2 comprises the amino acid sequence set forth in SEQ ID NO:59, L CDR3 comprises the amino acid sequence set forth in SEQ ID NO:60.

[0095] In one embodiment, the subject technology provides a method for treating ischemic stroke in a subject in need thereof, comprising administering to a subject a medicament comprising: (a) a light chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a light chain immunoglobulin variable domain sequence of any one of SEQ ID NOs: 7, 17, 27, 37, 47, or 57; and / or (b) a heavy chain immunoglobulin variable domain sequence (V H (c) administering to a subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof comprising:

[0096] In some embodiments, the antibody or its antigen-binding fragment binds to ASIC1a protein.In some embodiments, the antibody or its antigen-binding fragment binds to the extracellular domain of ASIC1a.In some embodiments, the antibody or its antigen-binding fragment binds to an epitope that spans two ASIC1a subunits.In some embodiments, the antibody or its antigen-binding fragment inhibits the function of ASIC1a trimer.

[0097] In some embodiments, one or more symptoms of ischemic stroke are sudden weakness; paralysis or numbness of face, arm or leg, especially on one side of the body; drooping of one side of face; confusion; difficulty in speaking or language comprehension, such as slurred words; difficulty in seeing in one or both eyes, such as blurred vision or amaurosis, or double vision in one or both eyes; difficulty in breathing; dizziness; difficulty in walking; loss of balance or coordination, for example causing unexplained falls; loss of consciousness; and sudden or severe headache.In some embodiments, one or more symptoms of ischemic stroke are selected from the group consisting of sudden facial weakness (such as drooping of one side of face), unilateral weakness of upper limbs, and abnormal speech. In some embodiments, the antibody or antigen-binding fragment thereof is an antibody, scFv, (scFv)2, Fab, Fab', F(ab')2, or scFv-Fc antibody. In some embodiments, the antibody or antigen-binding fragment thereof is an scFv antibody. In some embodiments, the scFv antibody is ASC01, ASC02, ASC03, ASC04, ASC05, or ASC06.

[0098] Thus, for example, one or more of the anti-ASIC1a antibodies of the present technology can be (1) co-formulated and administered or delivered alone or in a combination formulation with other active agents or the anti-ASIC1a antibodies of the present technology at the same time; (2) delivered alternately or in parallel as separate formulations; or (3) by any other combination therapy regimen known in the art.When delivered in alternating therapy, the method described herein can include sequentially administering or delivering the active ingredients, for example, in separate solutions, emulsions, suspensions, tablets, pills, or capsules, or by different injections in separate syringes.Generally, during alternating therapy, the effective dosage of each active ingredient is administered sequentially, i.e., consecutively, while in simultaneous therapy, the effective dosage of two or more active ingredients is administered together.Various sequences of intermittent combination therapy can also be used. Administering such combination of anti-ASIC1a antibody of the present technology and other active agents can produce synergistic biological effects when administered in therapeutically effective amounts to a subject suffering from a medical disease or condition and in need of treatment.The advantage of such an approach is that a lower dose of anti-ASIC1a antibody of the present technology and / or other active agents may be required to prevent, improve or treat subjects suffering from or susceptible to ischemic stroke in subjects.In addition, the use of lower dosages of anti-ASIC1a antibody of the present technology and / or other active agents can avoid potential side effects of treatment.

[0099] The anti-ASIC1a antibodies of the present technology described herein, such as ASC01, ASC02, ASC03, ASC04, ASC05, ASC06, are useful for preventing or treating disease. Specifically, the present disclosure provides both preventive and therapeutic methods of treating a subject suffering from or susceptible to ischemic stroke. Thus, the present method provides prevention and / or treatment to a subject suffering from or susceptible to ischemic stroke in a subject by administering to a subject in need thereof an effective amount of an anti-ASIC1a antibody of the present technology to the subject to restore the function of an ion channel protein trimer mutant. The present technology provides treatment of a subject suffering from or susceptible to ischemic stroke in a mammal through administration to a subject in need thereof of a therapeutically effective amount of an anti-ASIC1a antibody of the present technology disclosed herein, such as ASC01, ASC02, ASC03, ASC04, ASC05, ASC06.

[0100] Determining the biological effect of the anti-ASIC1a antibody of this technology In various embodiments, suitable in vitro or in vivo assays are carried out to determine the effect of specific therapy based on the anti-ASIC1a antibody of the present technology, and whether its administration is indicated for therapy. In various embodiments, in vitro assays can be carried out using CHO-K1 cells, such as the representative cell line CHO-K1 / hASIC1a (stable cell line expressing full-length hASIC1a) disclosed herein. These experiments can be used to determine whether a given anti-ASIC1a antibody of the present technology exerts the desired effect in inhibiting the activity of ASIC1a protein trimer. Compounds for use in therapy can be tested in suitable animal model systems, including but not limited to rats, mice, chickens, cows, monkeys, rabbits, etc., before testing in human subjects. Similarly, for in vivo testing, any of the animal model systems known in the art can be used before administration to human subjects. In some embodiments, ASIC1a activity is determined by assays well known in the art, including but not limited to electrophysiological assays such as patch clamp as disclosed herein.In some embodiments, ASIC1a activity is determined by assays that measure biological activity in animal models.In some embodiments, ASIC1a activity is determined by assays that measure the rescue of disease phenotypes in animal models, including but not limited to mouse middle cerebral artery occlusion (MCAO)-induced ischemic stroke model as disclosed herein.

[0101] Mode of Administration and Effective Dosage Any method known to those skilled in the art can be employed to contact cells, organs, or tissues with the immunoglobulin-related composition of the present technology.Suitable methods include in vitro, ex vivo, or in vivo methods.In vivo methods typically include the administration of immunoglobulin-related compositions such as those described above to a mammal, suitably a human.When used in vivo for therapy, the anti-ASIC1a antibody of the present technology is administered to a subject in an effective amount (i.e., an amount that has a desired therapeutic effect).The dosage and dosing regimen will depend on the degree of symptoms in the subject, the characteristics of the particular immunoglobulin used, such as its therapeutic index, the subject, and the subject's medical history. Effective amounts can be determined during preclinical and clinical trials by methods familiar to physicians and clinicians. An effective amount of immunoglobulin useful in the method can be administered to a mammal in need thereof by any of several well-known methods for administering pharmaceutical compounds. The immunoglobulin can be administered systemically or locally.

[0102] C.Kit The present technology provides a kit for detecting and / or treating ischemic stroke, comprising at least one immunoglobulin-related composition of the present technology (e.g., any antibody or antigen-binding fragment described herein) or a functional variant (e.g., substitution variant) thereof. Optionally, the above components of the kit of the present technology are packaged in a suitable container and labeled for the diagnosis and / or treatment of ischemic stroke. The above components can be stored in unit or multi-dose containers, such as sealed ampoules, vials, bottles, syringes, and test tubes, as aqueous, preferably sterile solutions, or as lyophilized, preferably sterile formulations for reconstitution. The kit can further comprise a second container holding a diluent suitable for diluting the pharmaceutical composition to a higher volume. Suitable diluents include, but are not limited to, pharma- ceutically acceptable excipients of the pharmaceutical composition and saline solution. In addition, the kit can comprise instructions for diluting the pharmaceutical composition and / or instructions for administering the pharmaceutical composition, whether diluted or not. The containers may be formed from a variety of materials, such as glass or plastic, and may have a sterile access port (for example, the container may be an intravenous solution bag or vial having a stopper that can be pierced by a hypodermic injection needle). The kit may further include more containers containing pharma- ceutically acceptable buffers, such as phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, culture media for one or more of the appropriate hosts. The kit may optionally include instructions typically included in commercial packaging of a therapeutic or diagnostic product, containing, for example, information on the indications, usage, dosage, manufacture, administration, contraindications, and / or warnings for the use of such a therapeutic or diagnostic product.

[0103] The kit is useful for detecting the presence of immunoreactive ASIC1a protein in biological samples, for example, any body fluid, including but not limited to serum, plasma, lymph, cyst fluid, urine, excreta, cerebrospinal fluid, ascites, or blood, and including biopsy samples of biological tissue.For example, the kit can include one or more humanized, chimeric, or bispecific anti-ASIC1a antibodies (or antigen-binding fragments thereof) of the present technology that can bind to ASIC1a protein in biological samples; a means for determining the amount of ASIC1a protein in samples; and a means for comparing the amount of immunoreactive ASIC1a protein in samples with a standard.One or more of the anti-ASIC1a antibodies can be labeled.The kit components (e.g., reagents) can be packaged in a suitable container.The kit can further include instructions for using the kit to detect immunoreactive ASIC1a protein. With respect to an antibody-based kit, the kit may include, for example, 1) a first antibody attached to a solid support that binds to the ASIC1a protein, such as a humanized or chimeric anti-ASIC1a antibody (or antigen-binding fragment thereof) of the present technology; and optionally, 2) a second, different antibody that binds to either the ASIC1a protein or the first antibody and is conjugated to a detectable label.

[0104] The kit may also include, for example, a buffer, a preservative, or a protein stabilizer. The kit may further include components necessary for detecting detectable labels, such as enzymes or substrates. The kit may also include a control sample or a series of control samples that can be assayed and compared with the test sample. Each component of the kit may be enclosed in an individual container, and all of the various containers may be in a single package, together with instructions for interpreting the results of the assay carried out using the kit. The kit of the present technology may include a written product on or in the kit container. The written product describes the method of using the reagent contained in the kit, for example, for detecting ASIC1a protein in vitro or in vivo, or for treating ischemic stroke in a subject in need thereof. In certain embodiments, the use of the reagent may be in accordance with the method of the present technology. EXAMPLES

[0105] The present technology is further illustrated by the following examples, which should not be taken as limiting in any way. For each of the examples below, any immunological binding agent and fragments thereof described herein may be used, such as IgG, IgM, IgA, IgD, IgE, and genetically modified IgG. By way of example, but not by way of limitation, the scFv or IgG1 antibody used in the examples below may be ASC01, ASC02, ASC03, ASC04, ASC05, or ASC06, etc. Example 1 material and method Reagents and Abbreviations. All reagents were purchased at the highest analytical grade from Sigma or as otherwise described. Recombinant protein symbols are used in a format where the initial letter of the species (e.g., human, mouse, rat, etc.) in italics is used as the first letter in the symbol followed by the gene name (e.g., hASIC1a represents hASIC1a protein). Antibodies were arbitrarily numbered according to panning results, such as ASC00. Any number with or without the "-IgG1" symbol represents the antibody in the form of scFv fused to Fc or full-length IgG1 (IgG1), respectively.

[0106] Cell culture. CHO-K1 cell lines (#CRL-9618; ATCC) were maintained in F-12k medium (#21127022; Gibco) containing 10% (vol / vol) FBS (#1600074; Gibco), while FreeStyle 293F (#R79007; ThermoFisher Scientific) cell lines were cultured in Freestyle 293 Expression medium (#12338026; ThermoFisher Scientific). Spodoptera frugiperda 9 (Sf9) cells (#12659017; ThermoFisher Scientific) were cultured at 27°C in ESF921 medium (#96-001-01; Expression Systems). E18-d-old C57 BL / 6 mice or ASIC1a - / -Primary cortical neurons dissected from mice were maintained in Neurobasal medium (#21103049; Gibco) supplemented with B27 (#17504044; Gibco) and Glutamx I (#35050061; Gibco). CHO-K1 / hASIC1a stable cell lines overexpressing full-length hASIC1a (1–528 amino acids) channels were generated as follows: cDNAs of full-length hASIC1a with C-terminal eYFP fusion or C-terminal mCherry fusion were constructed, cloned into pCDNA3.1-Neo expression vector, and transfected into CHO-K1 cells using Lipofectin 2000. CHO-K1 / hASIC1a stable cell lines were selected by the antibiotic geneticin (up to 1 mg / mL) and transfected with the fused fluorescent protein eYFP (λ ex = 488 nm; λ em = 528 nm) or mCherry fluorescence (λ ex = 587 nm; λ em The expression of the cells was verified using flow cytometry by NMR spectroscopy (NMR spectroscopy = 610 nm). More than 80% of positive cells survived when cultured for 30 days in the presence of 200 μg / mL Geneticin. Clonal stable lines were selected using a flow cytometry instrument (BD FACSAria III) based on expression of eYFP (6H7 cell line) and mCherry (4C12 cell line) and expanded using F-12K medium supplemented with 10% (vol / vol) FBS and 200 μg / mL Geneticin. Overexpression of hASIC1a was further confirmed using the hASIC1a-specific monoclonal antibody ASC06.

[0107] Purification of recombinant truncated hASIC1a. cDNA encoding truncated hASIC1a (ΔhASIC1a) fused to an N-terminal Flag tag was cloned into the pEG BacMam expression vector (courtesy of Eric Gouaux, Howard Hughes Medical Institute, Oregon Health and Science University, Portland, OR). The ΔhASIC1a protein was then heterologously expressed in HEK293F cells using the BacMam system. Goehring, et al., Nat Protoc 9:2574-2585 (2014). Briefly, the pEG BacMam-ΔhASIC1a construct was transformed into DH10Bac Escherichia coli cells (ΔhASIC1a bacmid). The resulting bacmid was then transfected into Sf9 cells using FuGENE transfection reagent (Promega) to obtain P3 baculovirus (amplified twice). HEK293F cells (2 × 10 per mL) 6 Cells (100 x 100 cells) were infected with baculovirus at a multiplicity of infection (MOI) of 1:25 (virus:cell) for 24 h at 37° C. Infected cells were incubated in the presence of 2 mM sodium butyrate for 48 h at 37° C. to boost expression of the target protein. Cells were harvested by centrifugation at 2,000×g for 1 min.

[0108] The following steps were performed on ice unless otherwise specified. The cell pellet from a 2 L culture was first resuspended in 100 mL of hypotonic buffer (10 mM Tris, pH 7.5, 10 mM MgCl2, 20 mM KCl) supplemented with a protease inhibitor mixture (1 tablet per 50 mL; Roche). The cells were ground using a Dounce homogenizer and centrifuged at 80,000 x g for 40 min. After repeating the process once, the resulting pellet was resuspended in 100 mL of hypertonic buffer (10 mM Tris, pH 7.5, 10 mM MgCl2, 20 mM KCl, 1 M NaCl), homogenized, and centrifuged. This procedure was also repeated once more. The resulting pellet was then mixed and treated with 2 mg / mL iodoacetamide in 50 mL hypotonic buffer containing protease inhibitors for 30 min. Recombinant ΔhASIC1a protein was extracted in cell lysis buffer (100 mM Tris, pH 7.5, 1.6 M NaCl, 1% n-dodecyl bD-maltopyranoside (DDM), 0.2% cholesteryl hemisuccinate (CHS)) under gentle agitation for 3 h at 4° C. After centrifugation at 15,000×g for 30 min, the supernatant was incubated overnight with anti-FLAG M2 magnetic beads (#M8823; Sigma) under gentle agitation. The magnetic beads were washed once each with Buffer A (50 mM Tris, pH 7.5, 800 mM NaCl, 10% glycerol, 0.1% DDM, 0.02% CHS, 10 mM MgCl2) and Buffer B (25 mM Tris, pH 7.5, 800 mM NaCl, 10% glycerol, 0.05% DDM, 0.01% CHS). ΔhASIC1a protein was eluted using Buffer B supplemented with 0.1 mg / mL Flag peptide, concentrated, and further purified using SEC on a Superdex 200 column (GE Heathcare) and concentrated to yield 60 μL of a 15 mg / mL stock solution. As shown in FIG. 12A, the resulting homogenous ΔhASIC1a was >90% pure as judged by SDS-PAGE analysis.

[0109] Nanodisc assembly of hASIC1a channels. To mimic the lipid bilayer environment of cell membranes, purified ΔhASIC1a protein was incorporated into lipid nanodiscs by mixing ΔhASIC1a protein with membrane scaffolding protein (MSP) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) in a molar ratio of 3:2:65. Associated detergent was removed by gentle agitation with Bio-Beads SM2 [Bio-Beads:DDM=10:1 (mass / mass); Bio-Rad] overnight. ΔhASIC1a-embedded nanodiscs were separated from unassembled nanodiscs using Flag peptide-specific magnetic beads. D'Antona et al., Biochemistry 53:127-134 (2014). The desired ΔhASIC1a Nanodisc assemblies were further purified and collected using a Superose 6 column (GE Heathcare) in a buffer containing 20 mM Tris, pH 7.5, and 150 mM NaCl. Empty Nanodiscs were packaged without the addition of ΔhASIC1a. The composition of assembled ΔhASIC1a Nanodiscs was analyzed by SDS-PAGE, EM, and dynamic light scattering (Figures 9A-C, 12A, 12B).

[0110] Differential enrichment-based screening of combinatorial antibody libraries. After three rounds of affinity enrichment against biotinylated ΔhASIC1a nanodiscs immobilized on streptavidin-coated magnetic beads (Pierce), hASIC1a-specific scFv antibodies were screened against a combinatorial human monoclonal scFv antibody phage library (10 11The phage antibody library panning was performed using a modified procedure. Xu, et al. Front Mol Neurosci 10: 298 (2017). Briefly, phagemids (displaying the antibody library) that bound to the antigen (ΔhASIC1a Nanodiscs) were enriched in each cycle and eluted with glycine-HCl (pH 2.2). XL1-Blue cells were used to express and amplify the output phagemids for the next round of panning. To minimize nonspecific enrichment, differential enrichment phage display selection was used, and nonspecific phagemids were pulled down using an excess amount of empty nanodiscs (2-fold more than the amount of ΔhASIC1a Nanodiscs) before panning against ΔhASIC1a Nanodiscs in the second and third cycles. After three iterations, 96 positive colonies were selected and analyzed by phage ELISA. Xu, et al. Front Mol Neurosci 10: 298 (2017). All of the positive clones were sequenced. Both DNA and protein sequences of the CDR3 domains were analyzed using the international ImMunoGeneTics information platform. After aligning the CDR3 sequences, a phylogenetic tree was constructed. Six unique scFv sequences were highly enriched.

[0111] Antibody expression and purification. Genes encoding candidate scFv sequences were cloned into a modified pFUSE expression vector (#pfuse-hg1fc2; Invivogen) to obtain scFv-Fc fusion proteins comprising the entire Fc domain of human IgG1. For full-length IgG1 antibodies, the heavy chain (V H domain) and light chain (V L The variable regions of the intact heavy chain domains (CH1, CH2, and CH3) and the intact light chain domains (C L) into a plasmid carrying the scFv-Fc vector. HEK293F cells transfected with scFv-Fc vector or co-transfected with equimolar heavy and light chain vectors were cultured for 4 days. After centrifugation, the scFv-Fc or full-length IgG1 antibody secreted into the medium was purified using a HiTrap Protein A HP column (#17-0403-03; GE Healthcare) with citrate elution buffer pH 3.4 in an AKTA purifier 100 (GE Healthcare). The purified antibody was then concentrated (15 mg / mL) and stored at -80°C in PBS buffer pH 7.4. As shown for example in Figure 12D, SDS-PAGE analysis showed that the purified single chain and full-length antibodies were at least 90% pure.

[0112] Negative staining electron microscopy (EM): (1) Sample preparation and data collection. An antigen-binding fragment of ASC06-IgG1 (ASC06-Fab) was prepared by digesting ASC06-IgG1 with papain, followed by protein A affinity purification and gel filtration. The ectodomain of hASIC1a with IL-2 and Flag tags fused to the N-terminus (hASIC1a-ECD) was expressed in HEK293F cells and purified by gel filtration using anti-Flag M2 magnetic beads and PBS buffer pH 7.4. ASC06-Fab and hASIC1a-ECD were incubated overnight at 4°C in a 3:1 molar ratio in PBS buffer pH 7.4 to form a tertiary complex. This complex was further isolated by gel filtration. As shown in Figure 14, the complex formed a unique peak that was distinguishable from both ASC06-Fab alone and hASIC1a-ECD alone. The peak fractions containing this complex were collected and subjected to negative staining EM. EM samples were prepared by diluting the purified complex of hASIC1a-ECD and ASC06-Fab to 0.01 mg / mL. A 4-μL sample droplet was then absorbed for 1 min onto a freshly glow-discharged copper grid covered with a thin and continuous carbon film (#BZ31024a; Beijing Zhongjingkeyi Technology). The grid was washed with two drops of deionized water, followed by negative staining with two drops of 0.75% (wt / vol) uranyl formate (#22450; Electron Microscopy Sciences) for 1 min, after which it was blotted with filter paper and then air-dried.

[0113] All micrographs were recorded using a Tecnai G2 Spirit transmission electron microscope (FEI) equipped with a LaB6 cathode operated at 120 kV and a 4,000 × 4,000 Eagle CCD camera with a calibrated magnification of 67,000, resulting in a pixel size of 1.74 Å. EM image processing and 3D reconstruction. The Scipion software suite was used for all image processing steps. de la Rosa-Trevin et al., J Struct Biol 157:38-46 (2016). All recorded micrographs were first decimated by a factor of 2 (resulting in a pixel size of 3.48 Å). A total of 5,064 particles were then manually selected from 100 micrographs using the EMAN2 Boxer function using a box size of 96 × 96 pixels. Tang et al., J Struct Biol. 157(1):38-46 (2007). The Xmipp CL2D function was used to produce a reference-free 2D class average analysis. de la Rosa-Trevin et al., J Struct Biol 184:321-328. (2013).

[0114] Molecular Dynamics Simulations. Models for hASIC1a and for ASC06 in Fab form were derived by homology by using the Swiss Model website. Biasini et al. Nucleic Acids Res 42:W252-W258 (2014). The template for ASIC1a protein was obtained from the structure of the truncated chicken protein [Protein Data Bank (PDB) ID code 4FZ0]. To compare the negative staining results with possible atomistic models, a series of different possible dockings of Fab (only residues 73-425 were kept) to the ECD of hASIC1a was first generated using the ClusPro 2.0 server and antibody docking mode. Comeau et al., Bioinformatics 28:2608-2614SR (2004); and Brenke et al., Bioinformatics 28(20):2608-14 (2012). Eight of the Fab dockings to the ECD of hASIC1a with geometries more compatible with those described by negative staining were then subselected and energy minimization was performed, followed by a 20-ns molecular dynamics simulation to loosen the interactions and stabilize the structure of the complex. After this procedure, only one model appeared to meet the overall geometry experimentally observed in the negative staining experiment. Simulations were performed using the Gromacs 4.6.7 package and the Amber14ffSB force field. Pronk et al., Bioinformatics 29: 845-854 (2013); and Lindorff-Larsen et al., Proteins 78: 1950-1958 (2010). All of the systems were doped with Cl at a concentration of approximately 0.15M. - and K. + All-atom TIP3P containing ions were solvated with water to mimic physiological ionic strength.

[0115] The temperature T and pressure P were kept constant at 300 K and 1 atm, respectively, using a Berendsen thermostat and a barostat. Berendsen et al., J Chem Phys 81:3684-3690 (1984). The fast Particle Mesh Ewald sum method was used for long-range electrostatic interactions with a cutoff of 1.0 nm for direct interactions. Darden et al., J Chem Phys 98:10089-10093 (1993). As shown in Figure 12A, starting from the crystal structure of MSP1 protein complexed with ASIC1a trimer (PDB ID code 2MSC) and packed with 91 DMPC molecules, we obtained a model of ASIC1a inserted into nanodiscs.

[0116] ELISA assay. Avidin (#21121; Pierce) was diluted in carbonate-bicarbonate buffer (#C3041; Sigma) to a final concentration of 2ng / μL. The resulting avidin solution was used to coat 96-well plates overnight at 4°C (25μL per well). The coated plates were washed once with phosphate buffered saline (PBST) containing 0.05% Tween-20 (150μL per well), followed by the addition of 25μL of biotinylated ΔhASIC1a nanodisc solution (2ng / μL) in each well and incubation for 1 hour at 37°C. PBST buffer alone and empty nanodisc solution (2ng / μL) were used as background and negative controls, respectively. After removal of the incubation solution, the resulting plate was rinsed once using PBST buffer and incubated with blocking solution (150 μL per well) containing 5% (volume / volume) milk in PBST for 1 h at 37° C. After blocking and PBST washing (1 time), 25 μL of scFv-Fc antibody solution (10 μg / mL in PBST containing 1% (volume / volume) milk) was added to each well and incubated for 1 h at 37° C. The resulting plate was rinsed 8 times using PBST and then subjected to HRP detection. A solution containing goat anti-human Fc HRP-conjugated secondary antibody (dilution factor 1:5,000; #A0170; Sigma) and anti-M13 HRP-conjugated secondary antibody (dilution factor 1:5,000; #27-9421-01; GE / Amersham / Whatman) was added to the plate (50 μL per well) and incubated for 1 h at 37° C. The plates were then washed eight times with PBST, followed by the addition of 50 μL of 2,2'-azinobis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt solution (#11684302001; Roche) to each well. After 20 min of incubation at room temperature, the absorbance change at 405 nm in each well was measured with a microplate reader (Enspire; PerkinElmer).

[0117] Affinity determination. Surface plasmon resonance (SPR) and flow cytometry were used to investigate the interaction between monoclonal antibodies and hASIC1a. SPR binding studies were performed on a Biacore T200 (GE Healthcare). Anti-Flag antibodies were amine-coupled to the surface of a biosensor chip CM5 series S (GE Healthcare). Flag-tagged recombinant ΔhASIC1a was then immobilized on the chip surface. Serial dilutions of antibody ASC06-IgG1 (1, 5, 10, 50, 100 nM) were applied in running buffer (pH 7.4) containing 25 mM HEPES, 500 mM NaCl, 0.05% EDTA, and 0.05% DDM. The binding / dissociation kinetics were measured as the change in SPR signal resonance units. The apparent binding affinity was calculated using the Biacore T200 evaluation software with a single cycle kinetics model.

[0118] For flow cytometry binding experiments, ASIC1a-eYFP-expressing stable cells (6H7) were harvested and resuspended in ice-cold FACS buffer (PBS, 0.05% BSA, 2 mM EDTA). The same number of 6H7 cells (50,000 cells per tube) were then incubated with various concentrations of ASC06-IgG1 for 20 minutes at 4°C, washed with 1 mL of ice-cold FACS buffer, centrifuged, and the pellet was resuspended in 100 μL of ice-cold FACS buffer containing Alexa555-conjugated secondary antibody recognizing human Fc (1:800 (volume / volume) dilution; Life Technology). After 15 minutes of incubation at 4°C, the cells were washed twice and resuspended in FACS buffer. The cells were sorted and analyzed on a flow cytometer (CytoFLEX S; Beckman Culter) to determine the relative binding levels of antibodies to the stable cell lines overexpressing hASIC1a. The mean fluorescence intensity of Alexa555 in eYFP-positive cells was recorded and analyzed to calculate the apparent binding affinity of the antibody.

[0119] Cell membrane colocalization studies using immunocytochemistry (ICC). Cells (CHO-K1) were plated at 2 × 10 per mL onto poly-D-lysine-coated glass-bottom 24-well plates. 5 The cells were seeded at a concentration of 1 x 10 cells. After 12 hours of culture, CHO-K1 cells were transfected with ASIC plasmids by Lipofectin 3000 reagent. 24 hours after transfection, cells were fixed in 4% paraformaldehyde and incubated in blocking solution (5% goat serum in PBS) for 30 minutes. The resulting cells were incubated with 2 μg / mL ASC06-IgG1 overnight at 4°C, followed by three PBS washes for 5 minutes each and incubation with Alexa555-conjugated goat anti-human secondary antibody (Life Technology). After three additional PBS washes for 5 minutes each, the nuclei of the cells were stained with DAPI (#10236276001; Roche). ICC analysis was performed at 25°C on a laser scanning confocal microscope (ZEISS LSM710) with a 63×NA1.4 objective. Images were collected with a 1,024 × 1,024 pixel EM-CCD camera. Data acquisition and analysis were performed using ZEN 2012 professional software.

[0120] Electrophysiological experiments. Cellular ASIC1a inward currents were recorded from single cells using standard whole-cell recording techniques with a patch clamp amplifier (Axon 200B; Axon Instruments). Membrane currents were sampled and analyzed using a personal computer with a Digidata 1320A interface and Clampex and Clampfit software (version 9.0.1; Axon Instruments). For a typical experiment, the extracellular fluid (ECF) used contained 150 mM NaCl, 5 mM KCl, 10 mM HEPES, 10 mM D-glucose, 2 mM CaCl2, and 1.0 mM MgCl2, with an osmolality in the range of 320-335 mOsm and pH 7.4 adjusted with 5 M NaOH. Patch electrodes (4–6 MΩ) were at 300 mOsm and contained 30 mM NaCl, 120 mM KCl, 0.5 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 2 mM MgATP, and 5 mM EGTA (pH 7.2 adjusted with 5 M KOH). ASIC1a-activated ECF was identical to normal ECF except that MES was used to replace HEPES to maintain the ECF at pH 6.0. Coverslips with individual CHO-ASIC1a stable cells (6H7) discretely distributed on their surface were placed in the recording chamber under constant perfusion (approximately 2–3 mL / min) produced by an ALA 8-channel perfusion system (ALA Scientific Instruments Inc.) mounted on an inverted microscope. After achieving break-in (whole-cell) positioning, cells were voltage-clamped at a holding potential of -70 mV. First, normal ECF (pH 7.4) was perfused into the patched cells, and then activated ECF at pH 6.0 was rapidly applied to elicit a maximum inward ASIC1a current as a control current. When testing the inhibitory effects of antibodies, ECF at pH 7.4 containing various concentrations of ASC06-IgG1 was perfused into the recording chamber for 10-15 min, from low to high concentrations. To measure inhibition of the current, activated ECF at pH 6.0 containing the corresponding concentration of the test antibody was applied to the same patched 6H7 cells to produce an inward current that could be normalized to the control current of the same cells.The small molecule inhibitor amiloride (#S1811; Selleck) and the venom peptide PcTx1 (#4435s; Peptide Institute) at concentrations of 30 μM and 100 nM, respectively, were used as positive controls for inhibition of ASIC1a currents. Data obtained from 4–5 cells were used for statistical calculation of the inhibitory effect.

[0121] For the measurement of steady state desensitization (SSD), the pH of the adjusted ECF was adjusted to 7.6, 7.4, 7.2, 7.0, and 6.8 (in HEPES buffer), while the activated ECF was maintained at pH 6.0 (in MES buffer). For the measurement of pH activation of hASIC1a in 6H7 stable cells, the adjusted ECF was fixed at pH 7.6 (in HEPES buffer), and the pH of the activated ECF was adjusted to 7.4, 7.2, 7.0, 6.8, 6.5, 6.3, and 6.0. hASIC1a was recorded when the stable cell line 6H7 was patched in the presence and absence of 300 nM ASC06-IgG1. The relative currents expressed as fractions of maximum amplitude (I / Imax) on the sensorgrams were calculated and plotted to construct the SSD and pH activation profiles. Each data point represents the average of at least five patched cells.

[0122] Calcium influx. The FLIPR Calcium 5 assay kit was used to determine acid-induced calcium influx in stable CHO-K1 cells (4C12) expressing hASIC1a-mCherry. Cells were seeded at a density of 10,000 cells per well in clear-bottom 96-well plates 24 h prior to the assay. Assay buffer and dye loading buffer (1.26 mM CaCl2, 0.5 mM MgCl2, 0.4 mM MgSO4, 5.33 mM KCl, 0.44 mM KH2PO4, 4.17 mM NaHCO3, 138 mM NaCl, 0.338 mM Na2HPO4, 20 mM HEPES, pH 7.4) were prepared according to the manufacturer's instructions. Probenecid was added to the dye loading buffer to a final concentration of 2.5 mM immediately before use. Fifty milliliters of assay buffer containing ASC06-IgG1 were incubated with 4C12 cells for 30 minutes at 37°C, and an equal volume of dye loading buffer was added. Amiloride and PcTx1 and isotype antibodies were used as positive and negative controls, respectively. Cells were incubated with dye loading buffer for 1 hour before reading on a FDSS / μCELL plate reader (Hamamatsu). 100 μL of pH 6.0 activation buffer (same composition as assay buffer; pH adjusted to 6.0) was added to the cells at a rate of 50 μL / s. Progress curves of calcium signals produced in each well were continuously recorded for 10 seconds before the addition of activation buffer (pre-read) and for 170 seconds after its addition to the FDSS / μCELL (excitation wavelength: 480 nm; emission wavelength: 540 nm) FLIPR reader. IC of ASC06-IgG1 in inhibiting ASIC1a-mediated currents 50 To determine the value, 1:3 serial dilutions of ASC06-IgG1 were applied to 4C12 cells with six replicates for each concentration.

[0123] Acidosis-induced cell death. hASIC1a-eYFP stable cells (6H7) and CHO-K1 cells were seeded in 96-well plates at a density of 10,000 cells per well one day before treatment. ECF solutions of pH 5.5, 6.0, and 7.4 were used to treat cells for 6 hours at 37°C. To test the rescue effect of ASC06-IgG1, the antibody was dissolved in acidic ECF (pH 5.5) to make final antibody concentrations of 0, 50, 100, 300, 500, and 1,000 nM. 6H7 cells were first pre-incubated with the antibody at the corresponding concentration in cell culture medium for 30 minutes at 37°C. After replacing the cell culture medium with the corresponding acidic ECF, the cells were incubated at 37°C for 6 hours, washed with PBS buffer (pH 7.4), and then cultured overnight in 1% FBS DMEM / F-12K medium. Supernatants from overnight cultures were collected for LDH release measurements using an LDH assay kit (#88953; Thermo Fisher Scientific). Cell viability was determined using a CCK-8 assay kit (#CK04; Dojindo).

[0124] Middle cerebral artery occlusion (MCAO) model. Animal care and experimental protocols were approved by the Animal Ethics Committee of Shanghai Jiao Tong University School of Medicine, Shanghai, China. Transient ischemic stroke in the left hemisphere was established in male mice (C57 BL / 6, 20-25 g) using 1 h of middle cerebral artery occlusion (MCAO) before reperfusion. Three hours after ischemia, a total of 4 μL of vehicle solution (PBS) in the absence or presence of neuroprotective agents (100 nM PcTx1 or 3.0 μg / μL endotoxin-free ASC06-IgG1 (20 μM)) was administered by intracerebroventricular (icv) injection into the contralateral side of the brain hemisphere. An irrelevant endotoxin-free antibody (3.0 μg / μL, 20 μM) was used as an isotype control. Cerebral blood flow was monitored by transcranical laser Doppler to ensure that ischemia was successfully induced. Mice were sacrificed 24 hours after icv injection. Mouse brains were rapidly dissected and sectioned transversely into serially spaced slices, followed by staining with 2% vital dye 2,3,5-triphenyltetrazolium hydrochloride (TTC). Infarct area was calculated by subtracting the normal area stained with TTC in the ischemic hemisphere from that in the nonischemic hemisphere. Infarct volume was calculated by summing the infarct areas of all sections and multiplying by slice thickness. Data analysis. Results were expressed as mean ± SD unless otherwise indicated. Data analysis was performed by one-way ANOVA using OriginPro 2017 statistical software. Significance was inferred at a P value < 0.05.

[0125] Example 2 Selection of functional antibodies against hASIC1a using Nanodiscs Truncated ASIC1a (amino acids 13-464; ΔhASIC1a) was overexpressed and purified to assemble a system displaying soluble hASIC1a. To mimic the native-like membrane environment and preserve the native extracellular conformation of hASIC1a, truncated human ASIC1a protein was assembled into Nanodiscs as described herein. Empty Nanodiscs were prepared using the same procedure, except for the addition of ΔhASIC1a. Nanodiscs loaded with ΔhASIC1a (ΔhASIC1a Nanodiscs) were purified using Superose 6 size-exclusion chromatography (SEC). The composition of ΔhASIC1a Nanodiscs was analyzed using SDS-PAGE. As shown in FIG. 12A, ΔhASIC1a protein and MSP1 protein were present in the Nanodiscs. The ΔhASIC1a protein in the Nanodiscs was more than 90% pure as judged by SDS-PAGE analysis. Densitometric analysis of the SDS / PAGE bands corresponding to membrane scaffold protein 1 (MSP1) and monomeric ΔhASIC1a revealed a molar ratio of MSP1 and ΔhASIC1a of approximately 2:3. ΔhASIC1a nanodiscs and empty nanodiscs were analyzed by dynamic light scattering. As shown in FIG. 12B, each of the nanodiscs (nanodsc) exhibited only one peak, indicating that the nanodiscs were homogeneous. The apparent diameter of assembled ΔhASIC1a nanodiscs was approximately 15.5 nm, while the empty nanodiscs had an apparent size of approximately 9.9 nm (FIG. 12B), consistent with the incorporation of ΔhASIC1a protein in ΔhASIC1a nanodiscs (FIG. 12B). As shown in FIG. 12B, a computational model of ΔhASIC1a nanodiscs calculated the distance from the extracellular domain (ECD) to the bottom edge of the nanodisc to be approximately 14.8 nm, which was the maximum distance calculated.

[0126] 10 11For panning of a human scFv combinatorial antibody library on phages containing members of the species, biotinylated nanodiscs were used in combination with streptavidin-coated magnetic beads. To minimize non-specific interactions between the phagemids and the nanodisc structure, a differential enrichment strategy using empty nanodiscs was used as described herein. After three rounds of panning, six species of enriched sequences were obtained, and the six antibodies were purified in scFv format and named ASC01-ASC06.

[0127] Figures 19A and 19B show the nucleotide and amino acid sequences of the heavy chain variable region of ASC01, respectively. Figures 20A and 20B show the nucleotide and amino acid sequences of the light chain variable region of ASC01, respectively. The nucleotide and amino acid sequences of the heavy chain variable region of ASC02 are shown in Figures 21A and 21B, respectively. The nucleotide and amino acid sequences of the light chain variable region of ASC02 are shown in Figures 22A and 22B, respectively. The nucleotide and amino acid sequences of the heavy chain variable region of ASC03 are shown in Figures 23A and 23B, respectively. The nucleotide and amino acid sequences of the light chain variable region of ASC03 are shown in Figures 24A and 24B, respectively. The nucleotide and amino acid sequences of the heavy chain variable region of ASC04 are shown in Figures 25A and 25B, respectively. The nucleotide and amino acid sequences of the light chain variable region of ASC04 are shown in Figures 26A and 26B, respectively. The nucleotide and amino acid sequences of the heavy chain variable region of ASC05 are shown in Figures 27A and 27B, respectively. The nucleotide and amino acid sequences of the light chain variable region of ASC05 are shown in Figures 28A and 28B, respectively. The nucleotide and amino acid sequences of the heavy chain variable region of ASC06 are shown in Figures 29A and 29B, respectively. The nucleotide and amino acid sequences of the light chain variable region of ASC06 are shown in Figures 30A and 30B, respectively.

[0128] Example 3 Binding ability to ΔhASIC1a-nanodiscs The binding ability of ASC01-ASC06 in scFv format to ΔhASIC1a-Nanodiscs compared to empty Nanodiscs was assessed using ELISA. As shown in Figure 1, the ELISA signal observed for ΔhASIC1a Nanodiscs was greater than that observed for empty Nanodiscs, indicating enrichment of scFv antibodies specific for the ΔhASIC1a component of the Nanodiscs. The affinity of the antibodies was then measured. The table below shows the binding affinity of ASC01-ASC06 (in scFv format) to ΔhASIC1a-Nanodiscs as measured using the Octet system. ASC04 and ASC06 showed the strongest affinity for ΔhASIC1a Nanodiscs.

[0129] TIFF2025063103000003.tif53162

[0130] Example 4 Binding of ASC01-ASC06 to ASIC1a protein expressed on the cell surface To understand whether ASC01–ASC06 specifically bind to ASIC1a protein expressed on the cell surface, the binding of the antibodies to CHO-K1 / hASIC1a cells was assessed using immunocytochemistry. CHO-K1 / hASIC1a cells overexpress full-length hASIC1a (1–528 amino acids) fused with eYFP. Immunocytochemistry (ICC) studies showed eYFP (green) expressed on the cell surface. As shown in Figure 2, ASC02, ASC03, ASC04, and ASC06 (red) were more localized to the plasma membrane of cells expressing hASIC1a-eYFP compared to ASC01 and ASC05. ASC06 showed more colocalization with hASIC1a-eYFP on the stained membrane compared to the other antibodies. ASC01–ASC05 may recognize specific ASIC1a conformational confirmation or subpopulations.

[0131] The ASC06 antibody was converted to a full-length IgG1 form (ASC06-IgG1) for further studies. The yields after purification of ASC06-IgG1 and ASC06 were comparable at approximately 78 mg / L. As shown in Figure 12D, SDS-PAGE analysis suggested that the single-chain (ASC06) and full-length (ASC06-IgG1) antibodies were greater than 90% pure and showed protein bands at the expected locations. Binding of ASC06-IgG1 to CHO-K1 / hASIC1a cells was assessed using immunocytochemistry in comparison to ASC06 (scFv format). As shown in Figure 3A, ASC06-IgG1, like ASC06, showed membrane co-localization with hASIC1a at the cell surface. The binding specificity of ASC06-IgG1 was confirmed in ASIC1a KO mice (ASIC1a - / - ) was also shown. As expected, primary neurons from KO mice showed no obvious binding to ASC06-IgG1 in integrated differential interference contrast (DIC) microscopy views. As shown in FIG. 10B, when neurons were transiently transfected with hASIC1a-mCherry, the expression of hASIC1a on the membrane of neurons was verified and visualized by ASC06-IgG1. Furthermore, the amplification view of the ASC06-IgG1 signal in neurites showed that the transiently expressed and ASC06-IgG1-labeled hASIC1a was mainly located in the postsynaptic dendrites of neurons (FIG. 10B).

[0132] Example 5 Binding affinity of ASC06-IgG1 to purified ΔhASIC1a protein The binding affinity of ASC06-IgG1 to the purified ΔhASIC1a protein was measured using surface plasmon resonance (SPR). The venom peptide PcTx1 fused to Fc (PcTx1-Fc) was used as a positive control for binding to the ΔhASIC1a protein. As shown in FIG. 15, PcTx1-Fc has an apparent K of 1.0 nM for binding to the ΔhASIC1a protein. d, consistent with reports in the literature. Hoagland et al., J Biol Chem 285:41852-41862 (2010). As shown in FIG. 3B, ASC06-IgG1 exhibited an apparent KD value of 7.9 nM for binding to purified ΔhASIC1a protein, as measured by SPR using a Biacore T200 instrument. As a positive control, venom peptide PcTx1 fused to Fc (PcTx1-Fc) was used.

[0133] Example 6 Binding affinity of ASC06-IgG1 to ASIC1a protein expressed on the cell surface The binding affinity of ASC06-IgG1 to the cell surface expressed full-length hASIC1a protein was determined using quantitative fluorescence-activated cell sorting (FACS). As shown in FIG. 10A, the apparent EC 50 The value (concentration providing 50% of maximal binding) was determined to be 2.06 ± 0.01 nM, which was consistent with the measured KD value for purified ΔhASIC1a (7.9 nM), indicating that truncated hASIC1a and full-length hASIC1a expressed on the cell surface share a similar extracellular conformation.

[0134] Example 7 Binding selectivity of ASC06-IgG1 To test the species and subtype selectivity of ASC06-IgG1, vectors encoding human ASIC1b-eYFP fusion, rat ASIC1a-eYFP fusion, mouse ASIC1a-eYFP fusion, hASIC2a-eYFP fusion, hASIC2b-eYFP fusion, and hASIC3a-eYFP fusion were constructed. CHO-K1 cells were transiently transfected with these plasmids. FACS binding assays and immunocytochemistry (ICC) were performed to determine whether ASC06-IgG1 could bind to ASIC homologs and isotypes expressed on the cell surface. As shown in Figure 4A, ICC staining demonstrated membrane colocalization of ASC06-IgG1 with hASIC1a-eYFP, rASIC1a-eYFP, and mASIC1a-eYFP, consistent with the high sequence homology between human and rodent ASIC1a. As shown in Figure 4B, the FACS results revealed that ASC06-IgG1 recognized only the human / mouse / rat homolog of ASIC1a, but not other ASIC1 subtypes, hASIC1b, hASIC2a, hASIC2a / 2b, or hASIC3a, demonstrating the subtype selectivity of the ASC06-IgG1 antibody.

[0135] Example 8 Efficacy of ASC06-IgG1 in cell culture: patch clamp experiments The function of ASC06-IgG1 was characterized using electrophysiological techniques. The effect of ASC06-IgG1 antibody on acid-induced hASIC1a-mediated currents in cells was analyzed. PcTx1 was used as a positive control. As shown in Figure 5A, whole-cell recording mode showed that decreasing the extracellular pH value (from pH 7.4 to pH 6.0) led to the formation of currents in hASIC1a-overexpressing stable cell lines. The magnitude of hASIC1a-mediated inward currents of stable cells was recorded and quantified in the absence and presence of ASC06-IgG1, with the venom peptide PcTx1 (100 nM) used as a positive control. As shown in Figures 5B-5C, ASC06-IgG1 induced an apparent IC of 85 ± 6 nM. 50PcTx1 exhibited sustained (30 min) and dose-dependent inhibition of up to 80% of the acid (pH 6.0)-induced current, with values ​​of 0.01 and 0.02. In contrast, the positive control PcTx1 exhibited almost complete inhibition of the current, as shown in Figures 5A & 5C. Unlike the sustained inhibition with the antibody after washing, the inhibition with PcTx1 could be quickly washed out within 5 min (Figure 5C).

[0136] To understand the mechanism by which ASC06-IgG1 blocks hASIC1a currents, we examined the SSD and pH activation of hASIC1a in the absence and presence of ASC06-IgG1. 50 (the drop in extracellular pH that can open 50% of ASIC1a channels) values ​​were measured and compared in the presence or absence of 300 nM ASC06-IgG1. As shown in Figures 11A-11B, SSD was induced when conditioned extracellular fluid (ECF) was applied at various pH values ​​and activated ECF at pH 6.0, while pH activation curves were constructed when activated ECF was applied at various pH values ​​and conditioned ECF at pH 7.6. The pH of SSD and pH activation 50 No significant difference in values ​​was observed for 300 nM ASC06-IgG1. In addition, ASC06-IgG1 did not interfere with either SSD or activation of ASIC1a, as shown by representative traces of both SSD and activation of ASIC1a currents treated with and without ASC06-IgG1, as shown in Figures 16A-16B. Therefore, ASC06-IgG1 appeared to block hASIC1a activation using a mechanism that is non-competitive with proton concentration.

[0137] Example 9 Efficacy of ASC06-IgG1 in cell culture: A FLIPR-based assay for inhibition of ASIC1a-mediated calcium influx by ASC06-IgG1 ASIC1a calcium influx was measured on a FLIPR instrument using a stable cell line 4C12 expressing a hASIC1a-mCherry fusion. The effect of ASC06-IgG1 antibody on acid-induced hASIC1a-mediated calcium influx in cells was analyzed using a FLIPR instrument by measuring the intracellular fluorescence signal of a calcium-sensitive dye (Calcium 5, Molecular Devices) using a stable cell line expressing a hASIC1a-mCherry fusion. In 4C12 cells, activation of homomeric hASIC1a at pH 6.0 in the tenth second of the recording appeared to induce a strong calcium influx, whereas no acid-induced calcium influx was observed for CHO-K1 cells without hASIC1a expression (FIG. 17A). As shown in FIG. 6A and 6B, activation of homomeric hASIC1a channels at pH 6.0 in stable cells in the tenth second of the recording appeared to induce a strong calcium influx. The hASIC1a-specific antibody, ASC06-IgG1, had an IC 50 In contrast, the nonselective small molecule ASIC blocker amiloride exhibited only 21% inhibition at 30 μM, as shown in FIG.

[0138] Example 10 pH stability of ASC06-IgG1 The resistance of ASC06-IgG1 to acidic conditions was tested. Concentrated ASC06-IgG1 solutions (3.3 μM) were incubated at various pH values ​​(7.4 to 5.0) at 37° C. for 6 hours and subjected to SEC-HPLC analysis. As shown in FIG. 18, no significant degradation or aggregation of ASC06-IgG1 was observed. Example 11 ASC06-IgG1 prevents acidosis-induced cell death in vitro Extracellular acidosis in stroke or ischemia-reperfusion injury is known to induce activation of ASIC1a channels, which leads to neuronal cell death in the central nervous system, possibly through a transient increase in intracellular calcium and associated cell signaling mediated by ASIC1a. The survival of hASIC1a-overexpressing stable cells was assessed by measuring the enzymatic activity of cytoplasmic dehydrogenases (CCK8 assay) and the concentration of lactate dehydrogenase (LDH) secreted into the cell medium, which reflect cell viability and cytotoxicity, respectively. As shown in Figures 7A and 7B, the ASIC1a stable cell line (right panel) exhibited a higher degree of pH sensitivity than the control CHO-K1 cells, especially at pH 5.5, suggesting that the observed enhancement of the response to acid is mediated through the hASIC1a channel on the membrane. Under the same experimental conditions and at pH 5.5, ASC06-IgG1 showed a significant dose-dependent protective effect (Figures 7C and 7D). In the presence of 1 μM ASC06-IgG1, approximately 45% of ASIC1a-expressing stable cells survived at pH 5.5, compared to a survival rate of approximately 5% in the absence of antibody. Consistent with the CCK8 results, the concentration of LDH released into the cell medium also decreased after addition of ASC06-IgG1 in a dose-dependent manner (Figure 7D). These results demonstrate that the anti-ASIC1a antibodies of the present technology are useful in methods for blocking acidosis-induced cell death.

[0139] Example 12 ASC06-IgG1 prevents acidosis-induced cell death in vivo To determine whether the protective effect of antibody ASC06-IgG1 in vitro could be extended to pathological conditions in vivo, a middle cerebral artery occlusion (MCAO) model was used to investigate the neuroprotective effect of the antibody. Ischemia was induced by MCAO to the left hemisphere of the mouse for 60 minutes before reperfusion. As shown in FIG. 8A, 3 hours after the induction of ischemia, a total of 4 μL of vehicle solution (PBS) containing 100 nM PcTx1 or 3.0 μg / L ASC06-IgG1 was intracerebroventricularly (icv) injected into the contralateral hemisphere of the mouse. An irrelevant antibody (isotype) with the same concentration as ASC06-IgG1 was administered as a negative control. 24 hours after injection, the infarct volumes of the cortex and striatum were calculated. As shown in FIG. 8B, ischemia induced significant infarction in the brain in the PBS group (approximately 42% volume when compared to the contralateral brain region). PcTx1 reduced the infarct volume to about 17% (Figure 8B). Isotype antibodies did not show any protective effect. On the other hand, similar to PcTx1, the group treated with ASC06-IgG1 showed a significantly reduced infarct volume (to about 23%), indicating a strong neuroprotective effect of the antibody against stroke (Figure 8B). These results demonstrate that the anti-ASIC1a antibodies of the present technology are useful in methods for preventing acidosis-induced cell death and for treating ischemic stroke.

[0140] Example 13 ASC06-IgG1 bound to a conformational epitope on ASIC1a As demonstrated herein, antibody ASC06-IgG1 was shown to be able to recognize and bind to purified ASIC1a protein and cell surface expressed ASIC1a protein. To characterize the interaction between ASC06-IgG1 and hASIC1a, 6H7 stable line expressing hASIC1a-eYFP fusion protein was used in Western blot analysis. As shown in Figure 13A, ASC06-IgG1 could not recognize hASIC1a in cell lysate of cells overexpressing ASIC1a in Western blot assay. In contrast, commercial goat polyclonal anti-ASIC1 antibody and anti-eYFP antibody could detect hASIC1a-eYFP protein in 6H7 lysate at molecular weight of around 100 kDa (Figure 13A and data not shown). This observation indicated that antibody recognizes ASIC1a in a conformation-dependent manner.

[0141] ASC06-IgG1 was used to pull down native hASIC1a-YFP in cell lysates, followed by anti-eYFP Western blot for detection of hASIC1a-YFP. As shown in Figure 13B, a clear band of approximately 100 kDa representing hASIC1a-eYFP was observed, indicating that ASC06-IgG1 recognizes hASIC1a in a conformation-dependent manner. To understand the nature of ASC06 binding to hASIC1a, complexes were formed by mixing hASIC1a-ECD and ASC06-Fab. The hASIC1a-ECD and ASC06-Fab complexes were resolved using gel filtration. As shown in Figure 14, hASIC1a-ECD existed as a homotrimer in solution and formed stable complexes with three ASC06-Fabs.

[0142] To further understand the nature of antibody binding, negative stain EM studies were performed using a complex of the ectodomain of hASIC1a (hASIC1a-ECD) and ASC06 in Fab format (ASC06-Fab). As shown in Figure 9A, the raw negative stain EM images showed that most of the particles showed a triangular arrangement for the formed complex; meanwhile, boomerang-shaped and other types of particles were also observed, which probably represent complexes with one or two ASC06-Fab missing. As shown in Figure 9A, the EM results showed that each ASC06-Fab bound to the hASIC1a-ECD-ASC06-Fab complex. As shown in Figure 9D, several amino acids of ASIC1a were predicted to be important for the binding of ASC06-IgG1 to hASIC1a based on a molecular dynamics simulation model. As shown in Figure 9C, the 2D average analysis showed a triangular complex with a clear domain division, indicating the interaction of ASC06-Fab with the trimeric conformation of hASIC1a-ECD. A combination of molecular docking and molecular dynamics simulations was used to obtain a model that roughly reproduced the apparent experimental geometry (Figure 9D). As shown in Figures 9A-9C, the epitope was located on the surface formed by the two hASIC1a-ECD subunits.

[0143] To confirm the role of these amino acids, alanine mutagenesis was performed to identify four amino acids that are very important for antibody binding. Plasmids encoding single amino acid mutations of hASIC1a-eYFP were constructed and transiently transfected into CHO-K1 cells. FACS binding assays were then performed to test whether ASC06-IgG1 still binds to the cell surface expressing mutated hASIC1a. As shown in Figure 9E, unlike WT hASIC1a that can bind to ASC06-IgG1, either hASIC1a Y360A mutation or hASIC1a N322A mutation reduces the binding ability of ASC06-IgG1; hASIC1a Y318A mutation abolishes the binding of ASC06-IgG1, as does hASIC1a F252A mutation.

[0144] Equivalent The present technology should not be limited in terms of the specific embodiments described in this application, which are intended as single illustrations of individual aspects of the technology. As would be apparent to one skilled in the art, many modifications and variations of the present technology can be made without departing from its spirit and scope. In addition to those enumerated herein, functionally equivalent methods and devices within the scope of the present technology would be apparent to one skilled in the art from the foregoing description. Such modifications and variations are intended to fall within the scope of the appended claims. The present technology should be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. It should be understood that the present technology is not limited to specific methods, reagents, compounds, compositions, or biological systems, which may of course vary. It should also be understood that the terminology used herein is for the purpose of describing only specific embodiments, and is not intended to be limiting.

[0145] Additionally, where features or aspects of the disclosure are described in terms of a Markush group, those of skill in the art will recognize that the disclosure is also thereby described in terms of any individual members or subgroups of members of the Markush group. As will be understood by those skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein encompass any and all possible subranges and subrange combinations thereof. Any recited range can be readily recognized as fully describing and enabling the same range to be divided into at least 2, 3, 4, 5, 10, etc. As a non-limiting example, each range discussed herein can be readily divided into a lower third, a middle third, and an upper third, etc. As will also be understood by those skilled in the art, all words such as "up to," "at least," "greater than," "less than," etc. refer to ranges that include the recited number and can then be divided into subranges as discussed above. Finally, as will be understood by those skilled in the art, ranges include each individual member. Thus, for example, a group having 1-3 cells refers to a group having 1, 2, or 3 cells. Similarly, a group having 1 to 5 cells refers to groups having 1, 2, 3, 4, or 5 cells, etc.

[0146] All patents, patent applications, provisional applications, and publications mentioned or cited in this specification are incorporated by reference in their entirety, including all figures and tables, to the extent they do not contradict the explicit teachings of this specification. Other embodiments are within the scope of the following claims.

Claims

1. A method for treating ischemic stroke in a subject in need thereof, comprising administering to a subject a heavy chain immunoglobulin variable domain (V H ) and a light chain immunoglobulin variable domain (V L administering to a subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof comprising The V H is selected from the group consisting of SEQ ID NOs: 3, 13, 23, 33, 43, and 53 H - CDR1 sequence; V selected from the group consisting of SEQ ID NOs: 4, 14, 24, 34, 44, and 54 H - CDR2 sequence; and a V selected from the group consisting of SEQ ID NOs: 5, 15, 25, 35, 45, and 55 H - comprises a CDR3 sequence; The V L is selected from the group consisting of SEQ ID NOs: 8, 18, 28, 38, 48, and 58 L - CDR1 sequence; V selected from the group consisting of SEQ ID NOs: 9, 19, 29, 39, 49, and 59 L - CDR2 sequence; and V selected from the group consisting of SEQ ID NOs: 10, 20, 30, 40, 50, and 60 L -CDR3 sequence.

2. 2. The method of claim 1, wherein the antibody or antigen-binding fragment thereof further comprises an Fc domain of an isotype selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, and IgE.

3. The antibody or antigen-binding fragment thereof may be Fab, F(ab') 2 , Fab', scF V , and F V The method of claim 1 , wherein the compound is selected from the group consisting of:

4. The method of any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody, a chimeric antibody, a humanized antibody, or a bispecific antibody.

5. The method of any one of claims 1 to 4, wherein the antibody or antigen-binding fragment thereof binds to the ASIC1a protein.

6. The method of any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof binds to the extracellular domain of ASIC1a.

7. The method of any one of claims 1 to 6, wherein the antibody or antigen-binding fragment thereof binds to an epitope spanning two ASIC1a subunits.

8. The method of any one of claims 1 to 7, wherein the antibody or antigen-binding fragment thereof inhibits proton-induced ASIC1a current.

9. V H The method of any one of claims 1 to 8, wherein said sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 12, 22, 32, 42, and 52.

10. V L The method of any one of claims 1 to 9, wherein said sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 17, 27, 37, 47, and 57.

11. The antibody or antigen-binding fragment thereof, SEQ ID NO:2 and SEQ ID NO:7 (ASC01); SEQ ID NO:12 and SEQ ID NO:17 (ASC02); SEQ ID NO:22 and SEQ ID NO:27 (ASC03); SEQ ID NO:32 and SEQ ID NO:37 (ASC04); SEQ ID NO:42 and SEQ ID NO:47 (ASC05); and SEQ ID NO:52 and SEQ ID NO:57 (ASC06) The method of any one of claims 1 to 10, comprising an HC amino acid sequence and an LC amino acid sequence selected from the group consisting of:

12. The method of any one of claims 1 to 11, wherein the antibody or antigen-binding fragment thereof is ASC06.

13. A method for alleviating one or more symptoms of ischemic stroke in a subject in need thereof, comprising administering to a subject a heavy chain immunoglobulin variable domain (V H ) and a light chain immunoglobulin variable domain (V L administering to a subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof comprising The V H is selected from the group consisting of SEQ ID NOs: 3, 13, 23, 33, 43, and 53 H - CDR1 sequence; V selected from the group consisting of SEQ ID NOs: 4, 14, 24, 34, 44, and 54 H - CDR2 sequence; and a V selected from the group consisting of SEQ ID NOs: 5, 15, 25, 35, 45, and 55 H - comprises a CDR3 sequence; The V L is selected from the group consisting of SEQ ID NOs: 8, 18, 28, 38, 48, and 58 L - CDR1 sequence; V selected from the group consisting of SEQ ID NOs: 9, 19, 29, 39, 49, and 59 L - CDR2 sequence; and V selected from the group consisting of SEQ ID NOs: 10, 20, 30, 40, 50, and 60 L -CDR3 sequence.

14. The antibody or antigen-binding fragment thereof, SEQ ID NO:2 and SEQ ID NO:7 (ASC01); SEQ ID NO:12 and SEQ ID NO:17 (ASC02); SEQ ID NO:22 and SEQ ID NO:27 (ASC03); SEQ ID NO:32 and SEQ ID NO:37 (ASC04); SEQ ID NO:42 and SEQ ID NO:47 (ASC05); and SEQ ID NO:52 and SEQ ID NO:57 (ASC06) The method of claim 13, comprising an HC amino acid sequence and an LC amino acid sequence selected from the group consisting of:

15. 15. The method of any one of claims 13 to 14, wherein the antibody or antigen-binding fragment thereof further comprises an Fc domain of an isotype selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, and IgE.

16. The antibody or antigen-binding fragment thereof may be Fab, F(ab') 2 , Fab', scF V , and F V The method according to any one of claims 13 to 15, wherein the compound is selected from the group consisting of:

17. The method of any one of claims 13 to 16, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody, a chimeric antibody, a humanized antibody, or a bispecific antibody.

18. The method of any one of claims 13 to 17, wherein the antibody or antigen-binding fragment thereof binds to the ASIC1a protein.

19. The method of any one of claims 13 to 18, wherein the antibody or antigen-binding fragment thereof binds to the extracellular domain of ASIC1a.

20. The method of any one of claims 13 to 19, wherein the antibody or antigen-binding fragment thereof binds to an epitope spanning two ASIC1a subunits.

21. The method of any one of claims 13 to 20, wherein the antibody or antigen-binding fragment thereof inhibits proton-induced ASIC1a current.

22. 22. The method of any one of claims 13 to 21, wherein the one or more symptoms of ischemic stroke are sudden weakness, paralysis or numbness in the face, arms, or legs, drooping of one side of the face, confusion, difficulty speaking, impaired speech comprehension, loss of vision in one or both eyes, blurred vision, amaurosis, double vision, difficulty breathing, dizziness, difficulty walking, loss of balance, loss of coordination, unexplained falls, loss of consciousness, sudden headache or severe headache.

23. 23. The method of any one of claims 13 to 22, wherein the one or more symptoms of ischemic stroke is sudden facial weakness, unilateral facial droop, unilateral upper limb weakness, or abnormal speech.

24. 1. A method for treating ischemic stroke in a subject in need thereof, comprising: (a) a light chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a light chain immunoglobulin variable domain sequence in any one of SEQ ID NOs: 7, 17, 27, 37, 47, or 57; and / or (b) a heavy chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a heavy chain immunoglobulin variable domain sequence present in any one of SEQ ID NOs: 2, 12, 22, 32, 42, or 52 (V H ) The method comprises administering to a subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof comprising:

25. Heavy chain immunoglobulin variable domain (V H ) and a light chain immunoglobulin variable domain (V L ) , The V H is selected from the group consisting of SEQ ID NOs: 3, 13, 23, 33, and 43 H - CDR1 sequence; V selected from the group consisting of SEQ ID NOs: 4, 14, 24, 34, and 44 H - CDR2 sequence; and V selected from the group consisting of SEQ ID NOs: 5, 15, 25, 35, and 45 H - comprises a CDR3 sequence; The V L is selected from the group consisting of SEQ ID NOs: 8, 18, 28, 38, and 48 L - CDR1 sequence; V selected from the group consisting of SEQ ID NOs: 9, 19, 29, 39, and 49 L - CDR2 sequence; and V selected from the group consisting of SEQ ID NOs: 10, 20, 30, 40, and 50 L - CDR3 sequences, An antibody or an antigen-binding fragment thereof.

26. 26. The antibody or antigen-binding fragment thereof of claim 25, further comprising an Fc domain of an isotype selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, and IgE.

27. The antigen-binding fragment is Fab, F(ab') 2 , Fab', scF V , and F V The antibody or antigen-binding fragment thereof according to any one of claims 25 to 26, selected from the group consisting of:

28. The antibody or antigen-binding fragment thereof according to any one of claims 25 to 27, which is a monoclonal antibody, a chimeric antibody, a humanized antibody, or a bispecific antibody.

29. An antibody or antigen-binding fragment thereof according to any one of claims 25 to 28, which binds to ASIC1a protein.

30. 30. The antibody or antigen-binding fragment thereof according to any one of claims 25 to 29, which binds to the extracellular domain of ASIC1a.

31. An antibody or antigen-binding fragment thereof according to any one of claims 25 to 30, which binds to an epitope spanning two ASIC1a subunits.

32. The antibody or antigen-binding fragment thereof according to any one of claims 25 to 31, which inhibits ASIC1a current.

33. A nucleic acid sequence encoding the antibody or antigen-binding fragment of any one of claims 25 to 32.

34. A nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 51, and 56.

35. A host cell or vector expressing a nucleic acid according to claim 33 or claim 34.

36. A kit comprising an antibody or antigen-binding fragment thereof according to any one of claims 25 to 32 and instructions for use.

37. 37. The kit of claim 36, wherein the antibody or antigen-binding fragment is coupled to at least one detectable label selected from the group consisting of a radioactive label, a fluorescent label, and a chromogenic label.

38. 38. The kit of claim 36 or 37, further comprising a secondary antibody that specifically binds to the antibody or antigen-binding fragment.

39. A method for detecting ASIC1a protein in a biological sample, comprising the steps of contacting the biological sample with an antibody or antigen-binding fragment thereof described in any one of claims 25 to 32, conjugated to a detectable label; and detecting the presence and level of the detectable label in the biological sample.

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