Polypeptides binding to MIRO1

Abstract

The present disclosure describes anti-Miro1 polypeptides or binding fragments thereof, and nucleic acids, compositions, methods, kits, and systems related to the polypeptides or binding fragments. In one aspect, the anti-Miro1 polypeptides or binding fragments thereof can be used as capture and detection reagents in a Miro1 ELISA assay. The Miro1 ELISA assay as described herein is capable of selective detection of Miro1 protein over Miro2 protein.

Description

Attorney Docket No.: 058367-508001WO POLYPEPTIDES BINDING TO MIRO1 CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No.63 / 559,012, filed February 28, 2024, which is incorporated herein in its entirety for all purposes. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted in XML format via Patent Center and is hereby incorporated by reference in its entirety. Said XML copy is named 2025-02-11 Sequence_Listing_ST26058367-508001WO.xml, created on February 11, 2025, and is 107,753 bytes in size. BACKGROUND

[0003] Neurons are metabolically active cells with high energy demands at locations distant from the cell body. As a result, these cells are particularly dependent on mitochondrial function, as reflected by the observation that diseases of mitochondrial dysfunction often have a neurodegenerative component. Recent discoveries have highlighted that neurons are reliant particularly on the dynamic properties of mitochondria. Mitochondria are dynamic organelles by several criteria. They engage in repeated cycles of fusion and fission, which serve to intermix the lipids and contents of a population of mitochondria. In addition, mitochondria are actively recruited to subcellular sites, such as the axonal and dendritic processes of neurons. Finally, the quality of a mitochondrial population is maintained through mitophagy, a form of autophagy in which defective mitochondria are selectively degraded. Defects in the key features of mitochondrial dynamics, such as mitochondrial fusion, fission, transport and mitophagy are associated with neurodegenerative disorder. Several major neurodegenerative disorders—including Parkinson’s, Alzheimer’s and Huntington’s disease—involve disruption of mitochondrial dynamics.

[0004] Mitochondrial movements are tightly controlled to maintain energy homeostasis and prevent oxidative stress. Mitochondrial motility ceases prior to the initiation of mitophagy, a crucial cellular mechanism by which depolarized mitochondria are degraded through autophagosomes and lysosomes. The arrest of motility may sequester damaged mitochondria, preventing them from moving and from reintroducing damage to other healthy mitochondria.

[0005] Miro is an outer mitochondrial membrane (OMM) protein that anchors the microtubule motors kinesin and dynein to mitochondria (Glater et al., “Axonal transport of mitochondria requires milton to recruit kinesin heavy chain and is light chain independent,” The Journal of cell biology, 2006,173:545–557; and Koutsopoulos et al., “Human Miltons associate with mitochondria and induce microtubule-dependent remodeling of mitochondrial networks,” Biochimica et biophysica acta, 2010,1803:564–574). This depolarization- triggered mitochondrial arrest is achieved by removal of Miro from the damaged mitochondrial surface (Wang et al., “PINK1 and Parkin target Miro for phosphorylation and degradation to arrest mitochondrial motility,” Cell, 2011,147:893–906). Miro is subsequently degraded by proteasomes (Wang et al., 2011). Evidence has shown that two PD-linked proteins, PINK1 (PTEN-induced putative kinase 1) and Parkin, act in concert to target Miro for degradation (Ashrafi et al., “Mitophagy of damaged mitochondria occurs locally in distal neuronal axons and requires PINK1 and Parkin,” The Journal of cell biology, 2014,206:655– 670; Liu et al., “Parkinson’s disease-associated kinase PINK1 regulates Miro protein level and axonal transport of mitochondria,” PLoS genetics, 2012,8:e1002537; and Wang et al., 2011). Mutations in PINK1 or Parkin are tied to rare forms of recessive early-onset PD.

[0006] Altered mitochondrial transport is one of the pathogenic changes in major adult- onset neurodegenerative diseases (Sheng ZH, Cai Q, “Mitochondrial transport in neurons: impact on synaptic homeostasis and neurodegeneration,” Nat Rev Neurosci, 2012 Jan 5;13(2):77-93). In mutant LRRK2 GS2019 cells, the mitochondrial outer membrane protein Miro is stabilized and remains on damaged mitochondria for longer than normal, prolonging active transport and inhibiting mitochondrial degradation (Hsieh et al., “Functional Impairment in Miro Degradation and Mitophagy Is a Shared Feature in Familial and Sporadic Parkinson's Disease,” Cell Stem Cell, 2016 Dec 1;19(6):709-724). Miro degradation and mitochondrial motility are also impaired in sporadic PD patients (Hsieh et al., 2016). Prolonged retention of Miro, and the downstream consequences that ensue, may constitute a central component of PD pathogenesis.

[0007] Two Miro GTPases, named as Miro1 and Miro2, are encoded by the RHOT1 gene located on chromosome 17 and RHOT2 gene located on chromosome 16, respectively. Miro1 and Miro2 are both ubiquitously expressed, consisting of 662 amino acid residues, and display a 60% peptide sequence homology. Miro GTPases are conserved in almost all eukaryotes containing mitochondria. Genetic screening suggests that Miro1 and Miro2 may be candidates for treating Parkinson’s disease. See, Anvret, et al. Open Neurology Journal2012, 6: 1-5. To date, it is unknown whether increased activity or levels of Miro1, Miro2, or a combination of Miro1 and Miro2, in a patient in comparison with a control individual gives rise to the altered mitochondrial transport leading to neurodegenerative disorders in humans.

[0008] The development of methods for the effective clinical detection of Miro1 for detecting and monitoring a disease or condition associated with its elevated levels requires selective and sensitive techniques for quantifying Miro1 levels, e.g., permitting measurement of Miro1 levels in a small amount of a blood sample from a human subject. Commercially available Miro1 antibodies fail to provide the required selectivity and / or sensitivity to achieve these objectives.

[0009] There is a need for novel methods to detect and quantify Miro1 level in a cell, for example, for diagnosing or treating neurodegenerative disease such as Parkinson’s disease. BRIEF SUMMARY

[0010] In some embodiments, a polypeptide or binding fragment thereof of the present disclosure binds specifically to Miro1, e.g., is an anti-Miro1 polypeptide or binding fragment thereof, which can be used in a biochemical assay, e.g., in an ELISA assay, to quantitate Miro1 levels in a biological sample. In some embodiments, the ELISA assay is selective for detection of Miro1 over Miro2.

[0011] In some embodiments, the present disclosure provides an anti-Miro1 polypeptide or binding fragment thereof that comprises (i) a VH chain comprising three CDRs; and (ii) a VL chain comprising three CDRs, wherein: VHCDR #1 is: SYAVR (SEQ ID NO: 21) or SNAIS (SEQ ID NO: 41); VH CDR #2 is: IISDSGTTYYANWAIG (SEQ ID NO: 22) or FIGYTGNTYYATWAKG (SEQ ID NO: 42); VHCDR #3 is: YDDYGDSILHI (SEQ ID NO: 23) or GSAWLDP (SEQ ID NO: 43); VL CDR #1 is: QASQNIDSNLV (SEQ ID NO: 24) or QSSQSVYTNTRLS (SEQ ID NO: 44); VLCDR #2 is: AASNLAS (SEQ ID NO: 25) or KASTLAS (SEQ ID NO: 45); and VL CDR #3 is: QCTYYSSSYVEV (SEQ ID NO: 26) or LGVYSGNIAV (SEQ ID NO: 46).

[0012] The present disclosure also describes herein an anti-Miro1 polypeptide or binding fragment thereof that comprises (i) a VH chain comprising three CDRs; and (ii) a VL chain comprising three CDRs; which is selective over Miro2.

[0013] Also provided herein is a composition comprising the anti-Miro1 polypeptide or binding fragment thereof, and an acceptable excipient.

[0014] Further provided is a nucleic acid comprising a nucleotide sequence encoding an anti-Miro1 polypeptide or binding fragment thereof as described herein.

[0015] The disclosure also provides a vector comprising the nucleic acid of the present disclosure. The present disclosure also describes host cells engineered to express the nucleic acid of the present disclosure. In some embodiments, the host cell comprises the vector of the present disclosure.

[0016] In some embodiments, a kit comprises the anti-Miro1 polypeptide or binding fragment thereof or the nucleic acid of the present disclosure, and instructions for use.

[0017] The present disclosure also provides a method of quantifying Miro1 in a sample from a subject comprising (a) contacting the sample with a carrier comprising a first anti- Miro1 polypeptide or binding fragment thereof; and (b) detecting binding between the carrier and the Miro1 with a second anti-Miro1 polypeptide or binding fragment thereof.

[0018] Further provided is a method of detecting a disease or disorder characterized by an elevated Miro1 in a subject, the method comprising: (a) quantifying Miro1 in a sample from the subject comprising: (i) contacting the sample with a carrier comprising a first anti-Miro1 polypeptide or binding fragment thereof; and (ii) determining binding between the carrier and the Miro1 with a second anti-Miro1 polypeptide or binding fragment thereof; (b) comparing the sample Miro1 level with a control Miro1 level; and (c) detecting the disease or disorder characterized by an elevated Miro1 if the sample Miro1 level is higher than the control Miro1 level.

[0019] Also provided herein is a method of treating a disease or disorder characterized by an elevated Miro1 in a subject, the method comprising: (a) quantifying Miro1 in a sample from the subject comprising: (i) contacting the sample with a carrier comprising a first anti- Miro1 polypeptide or binding fragment thereof; and (ii) determining binding between the carrier and the Miro1 with a second anti-Miro1 polypeptide or binding fragment thereof; (b) comparing the sample Miro1 level with a control Miro1 level; (c) detecting the disease or disorder characterized by an elevated Miro1 if the sample Miro1 level is higher than the control Miro1 level; and (d) administering a therapeutically effective amount of a Miro1- reducing agent to the subject.

[0020] In some embodiments, the method of the present disclosure is a method of treating a neurodegenerative disease in a subject, the method comprising: (a) quantifying Miro1 in asample from the subject comprising: (i) contacting the sample with a carrier comprising a first anti-Miro1 polypeptide or binding fragment thereof; and (ii) determining binding between the carrier and the Miro1 with a second anti-Miro1 polypeptide or binding fragment thereof; (b) comparing the sample Miro1 level with a control Miro1 level; (c) detecting the neurodegenerative disease if the sample Miro1 level is higher than the control Miro1 level; and (d) administering a therapeutically effective amount of a Miro1-reducing agent to the subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG.1 shows sensitivity of Miro1 ELISA using 77C5 capture / 40H2 detection antibodies against Miro1 (R2= 0.92) and Miro2 (line near baseline). The assay was developed using the MesoScale Discovery platform. The ELISA assay showed >100-fold selectivity for Miro1 vs. Miro2. Standard curves using recombinant Miro1 and Miro2 proteins are shown.

[0022] FIG.2 shows a selected capture and detection antibody pair that detected endogenous Miro1 protein levels in wildtype, but not in Miro1 knockout, HeLa cells. Intra- assay CV < 7.6%; inter-assay CV < 7%.

[0023] FIG.3 shows quantification of endogenous Miro1 protein levels in human peripheral blood mononuclear cells (PBMCs) using ELISA assay. Miro1 levels were reduced after challenge with a mitophagy-inducing mitochondrial stressor in healthy donor PBMCs.

[0024] FIG.4A-4B show one-dimensional fluorescence intensity histograms from flow cytometry experiments: FIG.4A shows x-axis = anti-Miro1 antibody 40H2-1. FIG.4B shows x-axis = anti-Miro1 antibody Abcam ab320019. DETAILED DESCRIPTIONI. GENERAL

[0025] The present disclosure generally relates to, inter alia, compositions, methods, kits, and systems for the diagnosis and / or treatment of various health conditions associated with elevated level and / or expression of Miro1 protein, e.g., Parkinson’s disease. In particular, some embodiments of the disclosure relate to anti-Miro1 polypeptides, e.g., isolated antibodies or binding fragments thereof, that bind specifically to an N-terminal, e.g., an N- terminal GTPase, or C-terminal, e.g., a C-terminal GTPase, region of human Miro1. Someembodiments of the disclosure relate to compositions and methods useful for producing such polypeptides and binding fragments, including recombinant nucleic acid molecules, recombinant cells, and transgenic animals that have been engineered to produce a polypeptide as disclosed herein or a binding fragment thereof. In some embodiments, the anti-Miro1 polypeptides are anti-Miro1 monoclonal antibodies that can be used as capture and detection reagents in a Miro1 ELISA assay. The Miro1 ELISA assay as described herein is capable of selective detection of Miro1 protein over Miro2 protein.

[0026] The Examples of the present disclosure describe illustrative embodiments of the invention. Anti-Miro1 monoclonal antibodies were generated in rabbits immunized using an N-terminal or C-terminal immunogen derived from human Miro1. See, Examples 1 and 2. Antibodies specific to the C-terminal region of Miro1, such as 77C5-1 and 72A12-3, and antibodies specific to the Miro1 N-terminus, such as 40H2-1, were identified and sequenced. See, Example 3. An ELISA assay matching a C-terminal capture antibody, such as 77C5-1 and 72A12-3, and an N-terminal detection antibody, such as 40H2-1, proved capable of detecting Miro1 selectively over Miro2. See, Example 4. Comparison of an ELISA comprising the anti-Miro1 antibodies of the present disclosure with a comparator ELISA with commercial anti-Miro1 antibodies showed that the currently disclosed antibodies afforded an ELISA with higher sensitivity to Miro1 in human PBMCs. See, Example 5. Comparison of an anti-Miro1 antibody of the present disclosure with a commercial anti-Miro1 antibody in a flow cytometry assay showed the Miro1 antibody of the present disclosure had higher sensitivity to Miro1 in human PBMCs. See, Example 6. The data suggest that the Miro1 antibody pairs of the present disclosure may be suitable in developing a high throughput clinical grade Miro1 biomarker assay, e.g., an ELISA or flow cytometry assay, that would require a relatively low number of cells and would have a lower patient burden, and that would provide a wider dynamic assay range to enable more precise quantification of different Miro1 protein levels.II. DEFINITIONS

[0027] Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this disclosure pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent asubstantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art.

[0028] The singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes one or more cells, including mixtures thereof. “A and / or B” is used herein to include all of the following alternatives: “A”, “B”, “A or B”, and “A and B”.III. ANTI-MIRO1 POLYPEPTIDES AND / OR BINDING FRAGMENTS

[0029] In some embodiments, the present disclosure relates to anti-Miro1 polypeptides or binding fragments thereof, e.g., polypeptides or binding fragments that specifically bind human Miro, such as human Miro1. In some embodiments, the human Miro1 has an amino acid sequence according to SEQ ID NO: 100.

[0030] The anti-Miro1 polypeptides and / or binding fragments and polynucleotides described herein are, in many embodiments, described by way of their respective polypeptide or polynucleotide sequences. Unless indicated otherwise, polypeptide sequences are provided in N→C orientation; polynucleotide sequences in 5'→3' orientation. For polypeptide sequences, the conventional three or one-letter abbreviations for the genetically encoded amino acids may be used.

[0031] In some embodiments, the anti-Miro1 polypeptide or binding fragment thereof is an antibody. The antibody may be polyclonal, monoclonal, genetically engineered, and / or otherwise modified in nature, including but not limited to chimeric antibodies, humanized antibodies, human antibodies, primatized antibodies, single chain antibodies, etc. In some embodiments, the anti-Miro1 polypeptide or binding fragment thereof is a polyclonal antibody. In some embodiments, the anti-Miro1 polypeptide or binding fragment thereof is a monoclonal antibody.

[0032] In some embodiments, the anti-Miro polypeptides are antibodies that are heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH)followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light-chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light-chain and heavy-chain variable domains.

[0033] In some embodiments, the anti-Miro1 antibodies comprise all or a portion of a constant region of an antibody. In some embodiments, the constant region is an isotype selected from: IgA (e.g., IgA1or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3or IgG4), and IgM.

[0034] The light constant region of an anti-Miro1 antibody may be a kappa (κ) light region or a lambda (λ) region. A λ light region can be any one of the known subtypes, e.g., λ1, λ2, λ3, or λ4. In some embodiments, the λ light region has a C-terminal residue truncation as compared to the corresponding wild type sequence. See, e.g., Shen et al., MAbs, 5(3): 418- 431 (May-June 2013). In some embodiments, the anti-Miro1 antibody comprises a kappa (κ) light region. In some embodiments, the anti-Miro1 antibody comprises a lambda (λ) light region.

[0035] The hypervariable region refers to the amino acid residues of an anti-Miro1 antibody or binding fragment that are responsible for antigen binding. The hypervariable region comprises amino acid residues from a complementarity-determining region or “CDR” (e.g., three sections in the light-chain variable domain and three sections in the heavy-chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5thEd. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and / or those residues from a hypervariable loop (e.g., three sections in the light-chain variable domain and three sections in the heavy-chain variable domain; Chothia and Lesk J. Mol. Biol.196:901-917 (1987)). Framework or FR residues are those variable domain residues other than the hypervariable region residues as herein defined.

[0036] Also disclosed herein are anti-Miro1 binding fragments that are capable of specifically binding human Miro1. Examples of binding fragments include by way of example and not limitation, Fab, Fab', F(ab')2, Fv fragments, single chain Fv fragments and single domain fragments.

[0037] A Fab fragment contains the constant and variable domains of the light chain and the first constant domain (CH1) and the variable domain of the heavy chain. Fab' fragmentsdiffer from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. F(ab') fragments are produced by cleavage of the disulfide bond at the hinge cysteines of the F(ab')2 pepsin digestion product. Additional chemical couplings of antibody fragments are known to those of ordinary skill in the art. Fab and F(ab')2 fragments lack the Fc fragment of an intact antibody, clear more rapidly from the circulation of animals, and may have less non- specific tissue binding than an intact antibody (see, e.g., Wahl et al., 1983, J. Nucl. Med. 24:316).

[0038] An Fv fragment is the minimum fragment of an antibody that contains a complete target recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in a tight, non-covalent association (VH-VL dimer). It is in this configuration that the three CDRs of each variable domain interact to define a target binding site on the surface of the VH-VL dimer. Often, the six CDRs confer target binding specificity to the antibody. However, in some instances even a single variable domain (or half of an Fv comprising only three CDRs specific for a target) can have the ability to recognize and bind target, although at a lower affinity than the entire binding site.

[0039] Single-chain Fv or "scFv" antibody binding fragments comprise the VHand VLdomains of an antibody, where these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VHand VLdomains which enables the scFv to form the desired structure for target binding.

[0040] Single domain fragments are composed of a single VH or VL domains which exhibit sufficient affinity to human Miro1, such as a camelized fragment (See, e.g., Riechmann, 1999, Journal of Immunological Methods 231:25-38). Single domain-based VHHs, also known as nanobodies, are included within the scope of single domain fragments as used herein. See, Jin, B.-K. et al. Int. J. Mol. Sci.2023 Mar; 24(6):5994. Miro1 nanobodies have been described in Fagbadebo, F. O. et al. Frontiers in Molecular Biosciences, March 2022, volume 9, Article 835302.

[0041] In some embodiments, the anti-Miro1 polypeptide or binding fragment thereof is generated using an immunogen derived from human Miro1 having the sequence: MKKDVRILLVGEPRVGKTSLIMSLVSEEFPEEVPPRAEEITIPADVTPERVPTHIVDYSEAE QSDEQLHQEISQANVICIVYAVNNKHSIDKVTSRWIPLINERTDKDSRLPLILVGNKSDLV EYSSMETILPIMNQYTEIETCVECSAKNLKNISELFYYAQKAVLHPTGPLYCPEEKEMKPACIKALTRIFKISDQDNDGTLNDAELNFFQRICFNTPLAPQALEDVKNVVRKHISDGVADSG LTLKGFLFLHTLFIQRGRHETTWTVLRRFGYDDDLDLTPEYLFPLLKIPPDCTTELNHHAY LFLQSTFDKHDLDRDCALSPDELKDLFKVFPYIPWGPDVNNTVCTNERGWITYQGFLSQ WTLTTYLDVQRCLEYLGYLGYSILTEQESQASAVTVTRDKKIDLQKKQTQRNVFRCNVI GVKNCGKSGVLQALLGRNLMRQKKIREDHKSYYAINTVYVYGQEKYLLLHDISESEFLT EAEIICDVVCLVYDVSNPKSFEYCARIFKQHFMDSRIPCLIVAAKSDLHEVKQEYSISPTDF CRKHKMPPPQAFTCNTADAPSKDIFVKLTTMAMYPHVTQADLKSSTFWLRASFGATVF AVLGFAMYKALLKQR (SEQ ID NO: 100).

[0042] In some embodiments, the anti-Miro1 polypeptide or binding fragment thereof is generated using an N-terminal Miro1 immunogen. In some embodiments, the N-terminal Miro1 immunogen comprises the N-terminal GTPase region of Miro1. In some embodiments, the anti-Miro1 polypeptide or binding fragment thereof is generated using an N-terminal Miro1 immunogen derived from human Miro1 amino acids 53-143 having the sequence: THIVDYSEAEQSDEQLHQEISQANVICIVYAVNNKHSIDKVTSRWIPLINERTDKDSRLPLI LVGNKSDLVEYSSMETILPIMNQYTEIE (SEQ ID NO: 101).

[0043] In some embodiments, the anti-Miro1 polypeptide or binding fragment thereof specifically binds to the N-terminal GTPase region of Miro1. In some embodiments, the anti- Miro1 polypeptide or binding fragment thereof specifically binds to SEQ ID NO: 101.

[0044] In some embodiments, the anti-Miro1 polypeptide or binding fragment thereof is generated using a C-terminal Miro1 immunogen. In some embodiments, the C-terminal Miro1 immunogen comprises the C-terminal GTPase region of Miro1. In some embodiments, the anti-Miro1 polypeptide or binding fragment thereof is generated using a C-terminal Miro1 immunogen was derived from human Miro1 amino acids 411-592 having the sequence: KKQTQRNVFRCNVIGVKNCGKSGVLQALLGRNLMRQKKIREDHKSYYAINTVYVYGQE KYLLLHDISESEFLTEAEIICDVVCLVYDVSNPKSFEYCARIFKQHFMDSRIPCLIVAAKSD LHEVKQEYSISPTDFCRKHKMPPPQAFTCNTADAPSKDIFVKLTTMAMYPHVTQADLKS STF (SEQ ID NO: 102).

[0045] In some embodiments, the anti-Miro1 polypeptide or binding fragment thereof specifically binds to the C-terminal GTPase region of Miro1. In some embodiments, the anti- Miro1 polypeptide or binding fragment thereof specifically binds to SEQ ID NO: 102.

[0046] In some embodiments, an anti-Miro1 polypeptide or binding fragment thereof that comprises (i) a VH chain comprising three CDRs VH CDR #1 (HCDR1), VH CDR #2 (HCDR2), and VHCDR #3 (HCDR3); and (ii) a VLchain comprising three CDRs VLCDR #1 (LCDR1), VL CDR #2 (LCDR2), and VL CDR #3 (LCDR3). In some embodiments, an anti-Miro1 polypeptide or binding fragment thereof that comprises (i) a VHchain comprising three CDRs; and (ii) a VL chain comprising three CDRs, wherein: VH CDR #1 is: SYAVR (SEQ ID NO: 21) or SNAIS (SEQ ID NO: 41); VHCDR #2 is: IISDSGTTYYANWAIG (SEQ ID NO: 22) or FIGYTGNTYYATWAKG (SEQ ID NO: 42); VH CDR #3 is: YDDYGDSILHI (SEQ ID NO: 23) or GSAWLDP (SEQ ID NO: 43); VLCDR #1 is: QASQNIDSNLV (SEQ ID NO: 24) or QSSQSVYTNTRLS (SEQ ID NO: 44); VL CDR #2 is: AASNLAS (SEQ ID NO: 25) or KASTLAS (SEQ ID NO: 45); and VLCDR #3 is: QCTYYSSSYVEV (SEQ ID NO: 26) or LGVYSGNIAV (SEQ ID NO: 46).

[0047] In some embodiments, the anti-Miro1 polypeptide or binding fragment thereof is an anti-Miro1 polypeptide or binding fragment thereof wherein (a) VHCDR #1 is SYAVR (SEQ ID NO: 21); VHCDR #2 is IISDSGTTYYANWAIG (SEQ ID NO: 22); VHCDR #3 is YDDYGDSILHI (SEQ ID NO: 23); VLCDR #1 is QASQNIDSNLV (SEQ ID NO: 24); VLCDR #2 is AASNLAS (SEQ ID NO: 25); and VLCDR #3 is QCTYYSSSYVEV (SEQ ID NO: 26); or(b) VHCDR #1 is SNAIS (SEQ ID NO: 41); VH CDR #2 is FIGYTGNTYYATWAKG (SEQ ID NO: 42); VHCDR #3 is GSAWLDP (SEQ ID NO: 43); VL CDR #1 is QSSQSVYTNTRLS (SEQ ID NO: 44); VLCDR #2 is KASTLAS (SEQ ID NO: 45); and VL CDR #3 is LGVYSGNIAV (SEQ ID NO: 46).

[0048] In some embodiments, the anti-Miro1 polypeptide or binding fragment thereof is an anti-Miro1 polypeptide or binding fragment thereof wherein VH CDR #1 is SYAVR (SEQ ID NO: 21); VHCDR #2 is IISDSGTTYYANWAIG (SEQ ID NO: 22); VH CDR #3 is YDDYGDSILHI (SEQ ID NO: 23); VLCDR #1 is QASQNIDSNLV (SEQ ID NO: 24); VL CDR #2 is AASNLAS (SEQ ID NO: 25); and VLCDR #3 is QCTYYSSSYVEV (SEQ ID NO: 26).

[0049] In some embodiments, the anti-Miro1 polypeptide or binding fragment thereof is an anti-Miro1 polypeptide or binding fragment thereof wherein VHCDR #1 is SNAIS (SEQ ID NO: 41); VH CDR #2 is FIGYTGNTYYATWAKG (SEQ ID NO: 42); VHCDR #3 is GSAWLDP (SEQ ID NO: 43); VL CDR #1 is QSSQSVYTNTRLS (SEQ ID NO: 44); VLCDR #2 is KASTLAS (SEQ ID NO: 45); and VL CDR #3 is LGVYSGNIAV (SEQ ID NO: 46).

[0050] In some embodiments, the anti-Miro1 polypeptide or binding fragment thereof is an anti-Miro1 polypeptide or binding fragment thereof that comprises (i) a VHchain comprising four framework regions HFWR1, HFWR2, HFWR3, and HFWR4; and (ii) a VL chain comprising four framework regions LFWR1, LFWR2, LFWR3, and LFWR4.

[0051] In some embodiments, the anti-Miro1 polypeptide or binding fragment thereof is an anti-Miro1 polypeptide or binding fragment thereof that comprises (i) a VH chain comprising four framework regions; and (ii) a VLchain comprising four framework regions, wherein: the first VHframework region (HFWR1) has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 1 or 10;the second VHframework region (HFWR2) has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 2 or 11; the third VHframework region (HFWR3) has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 3 or 12; the fourth VHframework region (HFWR4) has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 4 or 13; the first VLframework region (LFWR1) has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 5 or 14; the second VLframework region (LFWR2) has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 6 or 15; the third VLframework region (LFWR3) has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 7, 9, or 16; and the fourth VLframework region (LFWR4) has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 8.

[0052] In some embodiments, the anti-Miro1 polypeptide or binding fragment thereof is an anti-Miro1 polypeptide or binding fragment thereof that comprises (i) a VH chain comprising four framework regions; and (ii) a VLchain comprising four framework regions, wherein: the first VHframework region (HFWR1) corresponds in sequence to SEQ ID NO: 1 or 10; the second VH framework region (HFWR2) corresponds in sequence to SEQ ID NO: 2 or 11; the third VHframework region (HFWR3) corresponds in sequence to SEQ ID NO: 3 or 12; the fourth VH framework region (HFWR4) corresponds in sequence to SEQ ID NO: 4 or 13; the first VLframework region (LFWR1) corresponds in sequence to SEQ ID NO: 5 or 14; the second VL framework region (LFWR2) corresponds in sequence to SEQ ID NO: 6 or 15;the third VLframework region (LFWR3) corresponds in sequence to SEQ ID NO: 7, 9, or 16; and the fourth VLframework region (LFWR4) corresponds in sequence to SEQ ID NO: 8.

[0053] In some embodiments, the anti-Miro1 polypeptide or binding fragment thereof is an anti-Miro1 polypeptide or binding fragment thereof that comprises (i) a VH chain comprising four framework regions; and (ii) a VLchain comprising four framework regions, wherein: (a) the first VHframework region has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 1; the second VH framework region has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 2; the third VHframework region has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 3; the fourth VH framework region has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 4; the first VLframework region has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 5; the second VL framework region has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 6; the third VLframework region has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 7; and the fourth VLframework region has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 8; or(b) the first VHframework region has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 1; the second VH framework region has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 2; the third VHframework region has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 3; the fourth VH framework region has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 4; the first VLframework region has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 5; the second VL framework region has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 6; the third VLframework region has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 9; and the fourth VLframework region has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 8; or (c) the first VHframework region has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 10; the second VH framework region has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 11; the third VHframework region has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 12;the fourth VHframework region has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 13; the first VL framework region has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 14; the second VLframework region has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 15; the third VL framework region has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 16; and the fourth VLframework region has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 8.

[0054] In some embodiments, the anti-Miro1 polypeptide or binding fragment thereof is an anti-Miro1 polypeptide or binding fragment thereof that comprises (i) a VH chain comprising four framework regions; and (ii) a VLchain comprising four framework regions, wherein: (a) the first VHframework region corresponds in sequence to SEQ ID NO: 1; the second VH framework region corresponds in sequence to SEQ ID NO: 2; the third VHframework region corresponds in sequence to SEQ ID NO: 3; the fourth VH framework region corresponds in sequence to SEQ ID NO: 4; the first VLframework region corresponds in sequence to SEQ ID NO: 5; the second VLframework region corresponds in sequence to SEQ ID NO: 6; the third VLframework region corresponds in sequence to SEQ ID NO: 7; and the fourth VLframework region corresponds in sequence to SEQ ID NO: 8; or (b) the first VHframework region corresponds in sequence to SEQ ID NO: 1; the second VHframework region corresponds in sequence to SEQ ID NO: 2; the third VHframework region corresponds in sequence to SEQ ID NO: 3; the fourth VHframework region corresponds in sequence to SEQ ID NO: 4; the first VLframework region corresponds in sequence to SEQ ID NO: 5; the second VL framework region corresponds in sequence to SEQ ID NO: 6;the third VLframework region corresponds in sequence to SEQ ID NO: 9; and the fourth VL framework region corresponds in sequence to SEQ ID NO: 8; or (c) the first VH framework region corresponds in sequence to SEQ ID NO: 10; the second VHframework region corresponds in sequence to SEQ ID NO: 11; the third VH framework region corresponds in sequence to SEQ ID NO: 12; the fourth VHframework region corresponds in sequence to SEQ ID NO: 13; the first VL framework region corresponds in sequence to SEQ ID NO: 14; the second VLframework region corresponds in sequence to SEQ ID NO: 15; the third VL framework region corresponds in sequence to SEQ ID NO: 16; and the fourth VLframework region corresponds in sequence to SEQ ID NO: 8.

[0055] In some embodiments, the anti-Miro1 polypeptide or binding fragment thereof is an anti-Miro1 polypeptide or binding fragment thereof that comprises (i) a VH chain comprising four framework regions; and (ii) a VLchain comprising four framework regions, wherein: the first VHframework region has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 1; the second VH framework region has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 2; the third VHframework region has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 3; the fourth VHframework region has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 4; the first VLframework region has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 5; the second VLframework region has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 6;the third VLframework region has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 7; and the fourth VL framework region has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 8.

[0056] In some embodiments, the anti-Miro1 polypeptide or binding fragment thereof is an anti-Miro1 polypeptide or binding fragment thereof that comprises (i) a VHchain comprising four framework regions; and (ii) a VL chain comprising four framework regions, wherein: the first VH framework region corresponds in sequence to SEQ ID NO: 1; the second VHframework region corresponds in sequence to SEQ ID NO: 2; the third VH framework region corresponds in sequence to SEQ ID NO: 3; the fourth VHframework region corresponds in sequence to SEQ ID NO: 4; the first VL framework region corresponds in sequence to SEQ ID NO: 5; the second VLframework region corresponds in sequence to SEQ ID NO: 6; the third VL framework region corresponds in sequence to SEQ ID NO: 7; and the fourth VLframework region corresponds in sequence to SEQ ID NO: 8.

[0057] In some embodiments, the anti-Miro1 polypeptide or binding fragment thereof is an anti-Miro1 polypeptide or binding fragment thereof that comprises (i) a VH chain comprising four framework regions; and (ii) a VLchain comprising four framework regions, wherein: the first VHframework region has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 1; the second VH framework region has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 2; the third VHframework region has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 3; the fourth VH framework region has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 4;the first VLframework region has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 5; the second VL framework region has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 6; the third VLframework region has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 9; and the fourth VL framework region has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 8.

[0058] In some embodiments, the anti-Miro1 polypeptide or binding fragment thereof is an anti-Miro1 polypeptide or binding fragment thereof that comprises (i) a VHchain comprising four framework regions; and (ii) a VL chain comprising four framework regions, wherein: the first VH framework region corresponds in sequence to SEQ ID NO: 1; the second VHframework region corresponds in sequence to SEQ ID NO: 2; the third VH framework region corresponds in sequence to SEQ ID NO: 3; the fourth VHframework region corresponds in sequence to SEQ ID NO: 4; the first VL framework region corresponds in sequence to SEQ ID NO: 5; the second VLframework region corresponds in sequence to SEQ ID NO: 6; the third VL framework region corresponds in sequence to SEQ ID NO: 9; and the fourth VLframework region corresponds in sequence to SEQ ID NO: 8.

[0059] In some embodiments, the anti-Miro1 polypeptide or binding fragment thereof is an anti-Miro1 polypeptide or binding fragment thereof that comprises (i) a VH chain comprising four framework regions; and (ii) a VLchain comprising four framework regions, wherein: the first VHframework region has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 10; the second VH framework region has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 11;the third VHframework region has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 12; the fourth VH framework region at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 13; the first VLframework region has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 14; the second VL framework region has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 15; the third VLframework region has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 16; and the fourth VL framework region has at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 8.

[0060] In some embodiments, the anti-Miro1 polypeptide or binding fragment thereof is an anti-Miro1 polypeptide or binding fragment thereof that comprises (i) a VHchain comprising four framework regions; and (ii) a VL chain comprising four framework regions, wherein: the first VH framework region corresponds in sequence to SEQ ID NO: 10; the second VHframework region corresponds in sequence to SEQ ID NO: 11; the third VHframework region corresponds in sequence to SEQ ID NO: 12; the fourth VHframework region corresponds in sequence to SEQ ID NO: 13; the first VLframework region corresponds in sequence to SEQ ID NO: 14; the second VLframework region corresponds in sequence to SEQ ID NO: 15; the third VLframework region corresponds in sequence to SEQ ID NO: 16; and the fourth VLframework region corresponds in sequence to SEQ ID NO: 8.

[0061] In some embodiments, the anti-Miro1 polypeptide or binding fragment thereof is an anti-Miro1 polypeptide or binding fragment thereof having at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99%, sequence identity to a VHchain (HCVR) corresponding in sequence to SEQ ID NO: 27, 37, or 47, and a VL chain (LCVR) corresponding in sequence to SEQ ID NO: 28, 38, or 48.

[0062] In some embodiments, the anti-Miro1 polypeptide or binding fragment thereof is an anti-Miro1 polypeptide or binding fragment thereof having a VH chain (HCVR) corresponding in sequence to SEQ ID NO: 27, 37, or 47, and a VLchain (LCVR) corresponding in sequence to SEQ ID NO: 28, 38, or 48.

[0063] In some embodiments, the anti-Miro1 polypeptide or binding fragment thereof is an anti-Miro1 polypeptide or binding fragment thereof at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to a VHchain corresponding in sequence to SEQ ID NO: 27, and a VL chain corresponding in sequence to SEQ ID NO: 28. In some embodiments, the anti- Miro1 polypeptide or binding fragment thereof is an anti-Miro1 polypeptide or binding fragment thereof having an IgG heavy chain; and a kappa light chain.

[0064] In some embodiments, the anti-Miro1 polypeptide or binding fragment thereof is an anti-Miro1 polypeptide or binding fragment thereof having a VHchain corresponding in sequence to SEQ ID NO: 27, and a VL chain corresponding in sequence to SEQ ID NO: 28. In some embodiments, the anti-Miro1 polypeptide or binding fragment thereof is an anti- Miro1 polypeptide or binding fragment thereof having an IgG heavy chain; and a kappa light chain.

[0065] In some embodiments, the anti-Miro1 polypeptide or binding fragment thereof is an anti-Miro1 polypeptide or binding fragment thereof having at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to a VH chain corresponding in sequence to SEQ ID NO: 37, and a VLchain corresponding in sequence to SEQ ID NO: 38. In some embodiments, the anti-Miro1 polypeptide or binding fragment thereof is an anti-Miro1 polypeptide or binding fragment thereof having an IgG heavy chain; and a kappa light chain.

[0066] In some embodiments, the anti-Miro1 polypeptide or binding fragment thereof is an anti-Miro1 polypeptide or binding fragment thereof having a VH chain corresponding in sequence to SEQ ID NO: 37, and a VLchain corresponding in sequence to SEQ ID NO: 38. In some embodiments, the anti-Miro1 polypeptide or binding fragment thereof is an anti-Miro1 polypeptide or binding fragment thereof having an IgG heavy chain; and a kappa light chain.

[0067] In some embodiments, the anti-Miro1 polypeptide or binding fragment thereof is an anti-Miro1 polypeptide or binding fragment thereof having at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to a VHchain corresponding in sequence to SEQ ID NO: 47, and a VL chain corresponding in sequence to SEQ ID NO: 48. In some embodiments, the anti-Miro1 polypeptide or binding fragment thereof is an anti-Miro1 polypeptide or binding fragment thereof having an IgG heavy chain; and a kappa light chain.

[0068] In some embodiments, the anti-Miro1 polypeptide or binding fragment thereof is an anti-Miro1 polypeptide or binding fragment thereof having a VHchain corresponding in sequence to SEQ ID NO: 47, and a VL chain corresponding in sequence to SEQ ID NO: 48. In some embodiments, the anti-Miro1 polypeptide or binding fragment thereof is an anti- Miro1 polypeptide or binding fragment thereof having an IgG heavy chain; and a kappa light chain.

[0069] Illustrative anti-Miro1 polypeptides of the present disclosure are shown in Table 1. Table 1. Exemplary Polypeptides of the Disclosure. Antibody Polypeptide sequence identifiers (SEQ ID NOs)*HCVR HCVRHCDR1 HCDR2 HCDR3LCVR LCVRLCDR1 LCDR2 LCDR3light chain variable regions, respectively, without a signal peptide sequence. HCVR(2) and LCVR(2) correspond to the sequences of heavy chain variable region and light chain variable regions, respectively, including a signal peptide sequence.

[0070] In some embodiments, the anti-Miro1 polypeptide or binding fragment thereof is an anti-Miro1 polypeptide or binding fragment thereof having an IgG heavy chain; and / or a kappa light chain.

[0071] The present disclosure also includes variations of the anti-Miro1 polypeptides or binding fragments described herein. Variations in amino acid sequences of the anti-Miro1 polypeptides or binding fragments described herein may be naturally occurring, such assplicing variants or allelic variants. In addition or alternatively, variations in amino acid sequences of the anti-Miro1 polypeptides or binding fragments may be introduced by substitution, deletion or insertion of one or more codons into the nucleic acid sequences encoding the antibodies that results in a change in the amino acid sequences of the antibodies. Optionally, the variation may be resulted from substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids with any other amino acid in the antibodies. Amino acid substitutions in variants of anti-Miro1 polypeptides or binding fragments may be conservative or non-conservative. Those of skill in the art will understand that a “non-conservative substitution,” when used in reference to a polypeptide, refers to a substitution of an amino acid in a polypeptide with an amino acid with significantly differing side chain properties. Non-conservative substitutions may use amino acids between, rather than within, the defined groups and affects (a) the structure of the peptide backbone in the area of the substitution (e.g., serine for glycine), (b) the charge or hydrophobicity, or (c) the bulk of the side chain. A non-limiting exemplary non-conservative substitution can be an acidic amino acid substituted with a basic or aliphatic amino acid; an aromatic amino acid substituted with a small amino acid; and a hydrophilic amino acid substituted with a hydrophobic amino acid.

[0072] Conservatively modified variant anti-Miro1 polypeptides or binding fragments thereof are also contemplated as part of the present disclosure. As discussed above, a “conservatively modified variant” or a “conservative substitution” refers to a variant wherein there is one or more substitutions of amino acids in a polypeptide with other amino acids having similar characteristics (e.g., charge, side-chain size, hydrophobicity / hydrophilicity, backbone conformation and rigidity, etc.). Such changes can frequently be made without significantly disrupting the biological activity of the antibody or fragment. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity. In addition, substitutions of structurally or functionally similar amino acids are less likely to significantly disrupt biological activity.

[0073] Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartate andglutamate, and 7) sulfur-containing side chains: cysteine and methionine. Exemplary conservative amino acids substitution groups include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine- glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix as disclosed in Gonnet et al. (1992) Science 256: 144345.

[0074] Non-limiting exemplary embodiments of the anti-Miro1 polypeptides or binding fragments thereof of the disclosure can include one or more of the following features. In some embodiments, the anti-Miro1 polypeptides or binding fragments thereof of the disclosure can include a polypeptide including an amino acid sequence that is set forth herein except for one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) mutations such as, for example, missense mutations (e.g., conservative substitutions), non-sense mutations, deletions, or insertions. For example, the present disclosure includes polypeptides which include an immunoglobulin light chain variant comprising an LCVR amino acid sequence set forth in Table 1 and Sequence Listing but having one or more of such mutations and / or an immunoglobulin heavy chain variant comprising an HCVR amino acid sequence set forth in Table 1 and Sequence Listing but having one or more of such mutations. As described in greater detail below, in some embodiments, an anti-Miro1 polypeptide or binding fragment thereof of the disclosure can include an immunoglobulin light chain variant comprising LCDR1, LCDR2 and LCDR3 wherein one or more (e.g., 1 or 2 or 3) of such CDRs has one or more of such mutations (e.g., conservative substitutions) and / or an immunoglobulin heavy chain variant comprising HCDR1, HCDR2 and HCDR3 wherein one or more (e.g., 1 or 2 or 3) of such CDRs has one or more of such mutations (e.g., conservative substitutions). Such substitutions can be in a CDR, framework, and / or constant region of a polypeptide or binding fragment thereof.

[0075] Accordingly, in some embodiments, the anti-Miro1 polypeptides or binding fragments thereof of the disclosure can include one or more variant CDRs (e.g., any one or more of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and / or LCDR3) that are set forth herein with at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to, e.g., the heavy chain and light chain CDRs of Table 1 and Sequence Listing.

[0076] Illustrative anti-Miro1 polypeptides of the present disclosure are described in Table 2.Table 2. Exemplary Polypeptides of the Disclosure and Corresponding Framework Regions Antibody Polypeptide sequence identifiers (SEQ ID NOs)HFWR1 HFWR2 HFWR3 HFWR4 LFWR1 LFWR2 LFWR3 LFWR477 1 1 2 4 7disclosure includes a framework region having at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-16.

[0078] Affinity of anti-Miro1 polypeptides or binding fragments thereof for human Miro1 can be determined using techniques well known in the art or described herein, such as for example, but not by way of limitation, ELISA, FACS, isothermal titration calorimetry (ITC), surface plasmon resonance, or fluorescent polarization assay, including the methods described in Example 4.

[0079] In some embodiments, the methods disclosed herein include assessing the specific binding affinity (e.g., ability to bind, with varying degrees of specificity) of the anti-Miro1 polypeptide or binding fragment to the target antigen. In some embodiments, the methods disclosed herein optionally include identifying the anti-Miro1 polypeptide or binding fragment as having a binding specificity for a Miro1 protein if the anti-Miro1 polypeptide or binding fragment specifically binds to the Miro1 protein. Generally, binding affinity can be used as a measure of the strength of a non-covalent interaction between two molecules, e.g., an anti-Miro1 polypeptide or binding fragment thereof and an antigen (e.g., N-terminal region of a Miro1 protein, e.g., N-terminal GTPase region of a Miro1 protein). In some cases, binding affinity can be used to describe monovalent interactions (intrinsic activity). Binding affinity between two molecules can be quantified by determination of the equilibrium dissociation constant (KD). In turn, KD can be determined by measurement of the kinetics of complex formation and dissociation using, e.g., the surface plasmon resonance (SPR) method (Biacore, Carterra, ForteBio). The rate constants corresponding to the association and the dissociation of a monovalent complex are referred to as the association rate constants ka (orkon) and dissociation rate constant kd(or koff), respectively. KDis related to kaand kdthrough the equation KD = kd / ka. The value of the dissociation constant can be determined directly by various methods, and can be computed even for complex mixtures by methods such as those set forth in Caceci et al. (1984, Byte 9: 340-362). For example, the KD can be established using a double-filter nitrocellulose filter binding assay such as that disclosed by Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90: 5428- 5432).

[0080] Other assays to evaluate the binding ability (e.g., binding affinity and / or specificity) of the anti-Miro1 polypeptides or binding fragments of the present disclosure towards target antigens include, for example, ELISAs, Western blots, RIAs, and flow cytometry analysis. The binding kinetics and binding affinity of the polypeptide also can be assessed by standard assays known in the art, such as Surface Plasmon Resonance (SPR), e.g., by using a Biacore™ system, or KinExA. In some embodiments, the binding affinity of an antibody or an antigen-binding fragment for a target antigen (e.g., Miro1 protein antigen) can be calculated by the Scatchard method described by Frankel et al., Mol. Immunol, 16: 101-106, 1979. It will be understood that the binding affinity of an anti-Miro1 polypeptide or binding fragment for a target antigen is the strength of interaction between the anti-Miro1 polypeptide or binding fragment with the target antigen, whereas the binding specificity of anti-Miro1 polypeptide or binding fragment for a target antigen relates to the affinity to the target antigen relative to other antigens. It will also be understood that an anti-Miro1 polypeptide or binding fragment that “specifically binds” a target antigen (such as Miro1 protein) is an antigen- binding fragment that binds the target antigen but does not significantly bind non-target antigens. In some embodiments, the anti-Miro1 polypeptide or binding fragment “specifically binds” a target antigen if it does not significantly bind other antigens (e.g., non-target antigens) but binds the target antigen with high affinity, e.g., with an equilibrium dissociation constant (KD) of 100 nM or less, such as 60 nM or less, for example, 30 nM or less, such as, 15 nM or less, or 10 nM or less, or 5 nM or less, or 1 nM or less, or 500 pM or less, or 400 pM or less, or 300 pM or less, or 200 pM or less, or 100 pM or less. In some embodiments, the anti-Miro1 polypeptides or binding fragments of the disclosure that specifically bind a target antigen, such as a Miro1 protein (e.g., an N-terminal or C-terminal fragment of a Miro1 protein), have a binding affinity to the target antigen expressed as KD, of at least about 10−8M, as measured by real-time, label free bio-layer interferometry assay, for example, at 25° C. or 37°C, e.g., an Octet® HTX biosensor, or by surface plasmon resonance, e.g., BIACORE™, or by solution-affinity ELISA. In some embodiments, the anti-Miro1polypeptide or binding fragment has a binding affinity and / or binding specificity with an equilibrium dissociation constant (KD) value of less than 500 nM, for example, less than 400 nM, less than 300 nM, less than 200 nM, less than 150 nM, less than 120 nM, less than 100 nM, less than 90 nM, less than 80 nM, less than 70 nM, less than 60 nM, less than 50 nM, less than 40 nM, less than 20 nM, less than 15 nM, less than 10 nM, less than 5 nM, less than 5 nM, or less than 1 nM.

[0081] In some embodiments, the anti-Miro1 polypeptide or binding fragment thereof is an anti-Miro1 polypeptide or binding fragment thereof, which is selective over Miro2. In some embodiments, the anti-Miro1 polypeptide or binding fragment thereof is at least 3-fold, at least 10-fold, at least 100-fold, or at least 1000-fold selective over Miro2, or a selectivity ranging between any of the foregoing values.

[0082] In some embodiments, the anti-Miro1 polypeptide or binding fragment thereof is an anti-Miro1 polypeptide or binding fragment thereof that comprises (i) a VHchain comprising three CDRs; and (ii) a VL chain comprising three CDRs; which is selective over Miro2. In some embodiments, the anti-Miro1 polypeptide or binding fragment thereof is at least 3-fold, at least 10-fold, at least 100-fold, or at least 1000-fold selective over Miro2, or a selectivity ranging between any of the foregoing values.

[0083] Anti-Miro1 polypeptides or binding fragments thereof with affinity for human Miro1 and / or selectivity over Miro2 may be desirable for therapeutic and diagnostic uses. Accordingly, the present disclosure contemplates polypeptides or binding fragments having binding affinity to human Miro1. In specific embodiments, the anti-Miro1 polypeptides or binding fragments thereof bind human Miro1 with an affinity of at least about 1000 nM, but may exhibit higher affinity, for example, at least about 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 300 nM, 250 nM, 200 nM, 150 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.1 nM, 0.01 nM, or even higher. In some embodiments, the polypeptides or binding fragments thereof bind human Miro1 with an affinity in the range of about 1 pM to about 1000 nM, or an affinity ranging between any of the foregoing values.IV. NUCLEOTIDES ENCODING ANTI-MIRO1 POLYPEPTIDES OR BINDINGFRAGMENTS, EXPRESSION SYSTEMS, AND METHODS OF MAKING

[0084] The present disclosure encompasses nucleic acid molecules encoding immunoglobulin light and heavy chain genes for anti-Miro1 polypeptides or binding fragments thereof, vectors comprising such nucleic acids, and host cells capable of producing the anti-Miro1 polypeptides or binding fragments of the disclosure.

[0085] An anti-Miro1 polypeptide or binding fragment of the disclosure can be prepared by recombinant expression of immunoglobulin light and heavy chain genes in a host cell. To express an antibody recombinantly, a host cell is transfected with one or more recombinant expression vectors carrying DNA fragments encoding the immunoglobulin light and heavy chains of the antibody such that the light and heavy chains are expressed in the host cell and, optionally, secreted into the medium in which the host cells are cultured, from which medium the antibodies can be recovered. Standard recombinant DNA methodologies are used to obtain antibody heavy and light chain genes, incorporate these genes into recombinant expression vectors and introduce the vectors into host cells, such as those described in Molecular Cloning; A Laboratory Manual, Second Edition (Sambrook, Fritsch and Maniatis (eds), Cold Spring Harbor, N. Y., 1989), Current Protocols in Molecular Biology (Ausubel, F.M. et al., eds., Greene Publishing Associates, 1989) and in US 4,816,397.

[0086] To generate nucleic acids encoding such an anti-Miro1 polypeptide or binding fragment, DNA fragments encoding the light and heavy chain variable regions are first obtained. These DNAs can be obtained by amplification and modification of germline DNA or cDNA encoding light and heavy chain variable sequences, for example using the polymerase chain reaction (PCR). Germline DNA sequences for human heavy and light chain variable region genes are known in the art (See, e.g., the "VBASE" human germline sequence database; see also Kabat, E. A. et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No.91- 3242; Tomlinson et al., 1992, J. Mol. Biol.22T: 116-198; and Cox et al., 1994, Eur. J. Immunol.24:827-836).

[0087] Once DNA fragments encoding anti-Miro1 polypeptide or binding fragment-related VHand VLsegments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region genes to full-length antibody chain genes, to Fab fragment genes or to a scFv gene. In thesemanipulations, a VL- or VH-encoding DNA fragment is operatively linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operatively linked," as used in this context, is intended to mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in-frame.

[0088] The isolated DNA encoding the VHregion can be converted to a full-length heavy chain gene by operatively linking the VH-encoding DNA to another DNA molecule encoding heavy chain constant regions (CH1, CH2, CH3 and, optionally, CH4). The sequences of human heavy chain constant region genes are known in the art (See, e.g., Kabat, E.A., et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No.91-3242) and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG (e.g., IgG1, IgG2, IgG3, or IgG4), IgA, IgE, IgM or IgD constant region, but in certain embodiments is an IgG. For a Fab fragment heavy chain gene, the VH- encoding DNA can be operatively linked to another DNA molecule encoding only the heavy chain CH1 constant region.

[0089] The isolated DNA encoding the VLregion can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operatively linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL. The sequences of human light chain constant region genes are known in the art (See, e.g., Kabat, et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No.91-3242) and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region, but in certain embodiments is a kappa constant region. To create a scFv gene, the VH- and VL-encoding DNA fragments are operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4∼Ser)3(SEQ ID NO:90), such that the VHand VLsequences can be expressed as a contiguous single-chain protein, with the VL and VH regions joined by the flexible linker (See, e.g., Bird et al., 1988, Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., 1990, Nature 348:552-554).

[0090] To express the encoding anti-Miro1 polypeptide or binding fragment of the disclosure, DNAs encoding partial or full-length light and heavy chains, obtained asdescribed above, are inserted into expression vectors such that the genes are operatively linked to transcriptional and translational control sequences. As understood in the art, operatively linked is intended to mean that an antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors or, more typically, both genes are inserted into the same expression vector.

[0091] The genes for the anti-Miro1 polypeptide or binding fragment are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present). Prior to insertion of the anti-Miro1 polypeptide-related light or heavy chain sequences, the expression vector can already carry antibody constant region sequences. For example, one approach to converting the anti-Miro1 polypeptide-related VH and VLsequences to full-length antibody genes is to insert them into expression vectors already encoding heavy chain constant and light chain constant regions, respectively, such that the VHsegment is operatively linked to the CH segment(s) within the vector and the VLsegment is operatively linked to the CL segment within the vector. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0092] Accordingly, in some embodiments, provided herein is a nucleic acid molecule including a nucleotide sequence encoding an anti-Miro1 polypeptide or binding fragment thereof of the present disclosure.

[0093] In some embodiments, the nucleic acid comprising a nucleotide sequence encoding an anti-Miro1 polypeptide or binding fragment thereof has a sequence corresponding to: CAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACTCCCCTGACACTC ACCTGCACAGCTTCTGGATTCTCCCGCAGTAGCTATGCAGTGCGATGGGTCCGCCAG GCTCCAGGGAAGGGGCTGGAATGGATCGGAATCATTAGTGATAGTGGTACCACATACTACGCGAATTGGGCGATAGGCCGATTCACCATCTCCAAAACCTCGACCACGGTGGA TCTAAAAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGATA CGATGATTATGGTGATTCCATTTTACACATCTGGGGCCCAGGCACCCTGGTCACCGTC TCCTCA (SEQ ID NO: 29).

[0094] In some embodiments, the nucleic acid comprising a nucleotide sequence encoding an anti-Miro1 polypeptide or binding fragment thereof has a sequence corresponding to: GATGTTGTGATGACCCAGACTCCATCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTC ACCATCAAGTGCCAGGCCAGTCAGAATATTGATAGTAATTTAGTCTGGTATCAGCAG AAACCAGGGCAGCGTCCCAAGCTCCTGATCTCTGCTGCATCCAATCTGGCATCTGGG GTCCCATCGCGGTTCAGTGGCAGTGGATCTGGGTCAGAGTTCACTCTCACCATCAGC GACCTGGAGTGTGCCGATTCTGCCACTTACTACTGTCAATGTACTTATTATAGTAGTA GTTATGTTGAAGTTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA (SEQ ID NO: 30).

[0095] In some embodiments, the nucleic acid comprising a nucleotide sequence encoding an anti-Miro1 polypeptide or binding fragment thereof has a sequence corresponding to: CAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTC ACCTGCACAGCCTCTGGATTCTCCCGCAGTAGCTATGCAGTGCGATGGGTCCGCCAG GCTCCAGGGAAGGGGCTGGAATGGATCGGAATCATTAGTGATAGTGGTACCACATA CTACGCGAATTGGGCGATAGGCCGATTCACCATCTCCAAAACCTCGACCACGGTGGA TCTAAAAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGATA CGATGATTATGGTGATTCCATTTTACACATCTGGGGCCCAGGCACCCTGGTCACCGTC TCCTCA (SEQ ID NO: 39).

[0096] In some embodiments, the nucleic acid comprising a nucleotide sequence encoding an anti-Miro1 polypeptide or binding fragment thereof has a sequence corresponding to: GATGTTGTGATGACCCAGACTCCATCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTC ACCATCAAGTGCCAGGCCAGTCAGAATATTGATAGTAATTTAGTCTGGTATCAGCAG AAACCAGGGCAGCGTCCCAAGCTCCTGATCTCTGCTGCATCCAATCTGGCATCTGGG GTCCCATCGCGGTTCAGTGGCAGTGGATCTGGGTCAGAGTTCACTCTCACCATCAGC GACCTGGAGTGTGCCGATGCTGCCACTTACTACTGTCAATGTACTTATTATAGTAGTA GTTATGTTGAAGTTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA (SEQ ID NO: 40).

[0097] In some embodiments, the nucleic acid comprising a nucleotide sequence encoding an anti-Miro1 polypeptide or binding fragment thereof has a sequence corresponding to:CAGTCGGTGGGGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTC ACCTGCACAGTCTCTGGATTCTCCCTCAGTAGCAATGCAATAAGCTGGGTCCGCCAG GCTCCAGGGAAGGGGCTGGAATATATCGGATTCATTGGTTATACGGGTAACACATAC TACGCGACCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGACCACGGTGGAT CTGAAAATGACCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGAGGG TCTGCCTGGTTGGATCCCTGGGGCCAGGGCACCCTGGTCACCGTCTCTTCA (SEQ ID NO: 49).

[0098] In some embodiments, the nucleic acid comprising a nucleotide sequence encoding an anti-Miro1 polypeptide or binding fragment thereof has a sequence corresponding to: ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGT GCCACATTTGCTCAAGTGCTGACCCAGACTGCATCGCCCGTGTCTGCAGCTGTGGGA GGCACAGTTACCATCAAGTGCCAGTCCAGTCAGAGTGTTTATACTAACACCCGCTTA TCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACAAGGCATCC ACTCTGGCATCTGGGGTCCCATCGCGGTTCAGTGGCAGTGGATCTGGGACACAGTTC ACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACTTACTACTGTCTAGGC GTTTATAGTGGTAATATTGCTGTTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA (SEQ ID NO: 50).

[0099] In some embodiments, the nucleic acid comprising a nucleotide sequence encoding an anti-Miro1 polypeptide or binding fragment thereof has a sequence corresponding to any one of SEQ ID NO: 200, 202, 204, 206, 208, and 210.

[0100] In addition to the polypeptide chain genes, the recombinant expression vectors of the disclosure carry regulatory sequences that control the expression of the antibody chain genes in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA, 1990. It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. Suitable regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV) (such as the CMVpromoter / enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter / enhancer), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)) and polyoma. For further description of viral regulatory elements, and sequences thereof, see, e.g., U.S. Patent No. 5,168,062, U.S. Patent No.4,510,245, and U.S. Patent No.4,968,615.

[0101] In addition to the anti-Miro1 polypeptide chain genes and regulatory sequences, the recombinant expression vectors of the disclosure can carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (See, e.g., U.S. Patents Nos.4,399,216 , 4,634,665 and 5,179,017, all by Axel et al .). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Suitable selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in DHFR- host cells with methotrexate selection / amplification) and the neo gene (for G418 selection). For expression of the light and heavy chains, the expression vector(s) encoding the heavy and light chains is transfected into a host cell by standard techniques. The various forms of the term transfection are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, lipofection, calcium- phosphate precipitation, DEAE-dextran transfection and the like.

[0102] It is possible to express the anti-Miro1 polypeptide of the disclosure in either prokaryotic or eukaryotic host cells. In certain embodiments, expression of antibodies is performed in eukaryotic cells, e.g., mammalian host cells, of optimal secretion of a properly folded and immunologically active antibody. Exemplary mammalian host cells for expressing the recombinant antibodies of the disclosure include Chinese Hamster Ovary (CHO cells) (including DHFR- CHO cells, described in Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp, 1982, Mol. Biol.159:601-621), NSO myeloma cells, COS cells and SP2 cells. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods. Host cells can also be used to produce portions of intact antibodies, such as Fab fragments or scFv molecules. It isunderstood that variations on the above procedure are within the scope of the present disclosure. For example, it can be desirable to transfect a host cell with DNA encoding either the light chain or the heavy chain (but not both) of an anti-Miro1 polypeptide of this disclosure.

[0103] Recombinant DNA technology can also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that is not necessary for binding to human Miro1. The molecules expressed from such truncated DNA molecules are also encompassed by the antibodies of the disclosure.

[0104] For recombinant expression of an anti-Miro1 polypeptide or binding fragment thereof of the disclosure, the host cell can be co-transfected with two expression vectors of the disclosure, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors can contain identical selectable markers, or they can each contain a separate selectable marker. Alternatively, a single vector can be used which encodes both heavy and light chain polypeptides.

[0105] Once a nucleic acid encoding one or more portions of an anti-Miro1 polypeptide or binding fragment thereof, further alterations or mutations can be introduced into the coding sequence, for example to generate nucleic acids encoding antibodies with different CDR sequences, antibodies with reduced affinity to the Fc receptor, or antibodies of different subclasses.

[0106] The anti-Miro1 polypeptide or binding fragment thereof of the disclosure can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984, The Pierce Chemical Co., Rockford, Ill.). Variant antibodies can also be generated using a cell-free platform (See, e.g., Chu et al., Biochemia No.2, 2001 (Roche Molecular Biologicals) and Murray et al., 2013, Current Opinion in Chemical Biology, 17:420-426).

[0107] Once an anti-Miro1 polypeptide or binding fragment thereof of the disclosure has been produced by recombinant expression, it can be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Further, the anti-Miro1 polypeptide or binding fragment thereof of the present disclosure can be fused toheterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.

[0108] Once isolated, the anti-Miro1 polypeptide or binding fragment thereof can, if desired, be further purified, e.g., by high performance liquid chromatography (see, e.g., Fisher, Laboratory Techniques In Biochemistry And Molecular Biology, Work and Burdon, eds., Elsevier, 1980), or by gel filtration chromatography on a Superdex™ 75 column (Pharmacia Biotech AB, Uppsala, Sweden).

[0109] Additional examples of isolated anti-Miro1 polypeptides or binding fragments thereof include an anti-Miro1 polypeptide or binding fragment thereof derived from a rabbit. Examples of purification techniques suitable for the purification of the anti-Miro1 polypeptide or binding fragment thereof disclosed herein include affinity chromatography, anion exchange chromatography (AEX), cation exchange chromatography (CEX), hydroxyapatite chromatography, size exclusion chromatography (SEC), hydrophobic interaction chromatography (HIC), metal affinity chromatography, mixed mode chromatography (MMC), centrifugation, diafiltration, and ultrafiltration.

[0110] Generally, a polypeptide (e.g., anti-Miro1 polypeptide or binding fragment thereof of the present disclosure) is “substantially pure,” “substantially homogeneous,” or “substantially purified” when at least about 60 to 75% of a sample exhibits a single species of polypeptide. The polypeptide may be monomeric or multimeric. A substantially pure polypeptide generally includes about 50%, 60%, 70%, 80% or 90% W / W of a protein sample, more usually about 95%, 96%, 97%, 98%, or in some embodiments, over 99% pure. Protein purity or homogeneity may be indicated by a number of means available in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualizing a single polypeptide band upon staining the gel with a suitable stain available in the art. For certain purposes, higher resolution may be provided by using HPLC or other means available in the art for purification. Accordingly, in some embodiments, the anti-Miro1 polypeptide or binding fragment thereof of the disclosure has a purity of greater than 80% such as, for example, a purity of greater than 85%, 90%, 95%, 96%, 97%, 98%, or 99%.V. COMPOSITIONS

[0111] In some embodiments, the composition of the present disclosure is a composition comprising an anti-Miro1 polypeptide or binding fragment as described herein, and anacceptable excipient. The compositions may be formulated for specific uses, such as for veterinary uses or pharmaceutical uses in humans. The form of the composition (e.g., dry powder, liquid formulation, etc.) and the excipients, diluents and / or carriers used will depend upon the intended uses of the anti-Miro1 polypeptide or binding fragment and, for therapeutic uses, the mode of administration.

[0112] Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water / propylene glycol solutions.

[0113] Compositions may be prepared for storage as lyophilized formulations or aqueous solutions by mixing an anti-Miro1 polypeptide or binding fragment having the desired degree of purity with optional acceptable carriers, excipients or stabilizers typically employed in the art (all of which are referred to herein as “carriers”), i.e., buffering agents, stabilizing agents, preservatives, isotonifiers, non-ionic detergents, antioxidants, and other miscellaneous additives. See, Remington: The Science and Practice of Pharmacy, 21stedition (David Troy, ed.2005).

[0114] Buffering agents can help to maintain the pH in an appropriate range, e.g., approximating physiological conditions. They may be present at a wide variety of concentrations, but will typically be present in concentrations ranging from about 2 mM to about 50 mM. Suitable buffering agents for use with the present disclosure include both organic and inorganic acids and salts thereof such as citrate buffers (e.g., monosodium citrate-disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-monosodium citrate mixture, etc.), succinate buffers (e.g., succinic acid-monosodium succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid-disodium succinate mixture, etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture, etc.), phosphate buffers (e.g., phosphoric acid-monosodium phosphate mixture, phosphoric acid-disodium phosphate mixture, monosodium phosphate-disodium phosphate mixture, etc.), gluconate buffers (e.g., gluconic acid-sodium gluconate mixture, gluconic acid-sodium hydroxide mixture, gluconic acid- potassium gluconate mixture, etc.), oxalate buffer (e.g., oxalic acid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassium oxalate mixture, etc.), lactate buffers (e.g., lactic acid-sodium lactate mixture, lactic acid-sodium hydroxide mixture, lactic acid-potassium lactate mixture, etc.) and acetate buffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodium hydroxide mixture, etc.). Additionally, fumarate buffers,histidine buffers and trimethylamine salts such as 2-amino-2-hydroxymethyl-propane-1,3- diol (i.e., Tris, THAM, or tris(hydroxymethyl)aminomethane) can be used.

[0115] Isotonicifiers can be added to ensure isotonicity of liquid compositions of the present disclosure and include polyhydric sugar alcohols, for example trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol. Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which solubilizes the therapeutic agent or helps to prevent denaturation or adherence to the container wall. Typical stabilizers can be polyhydric sugar alcohols (enumerated above); amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinositol, galactitol, glycerol and the like, including cyclitols such as inositol; polyethylene glycol; amino acid polymers; sulfur containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol and sodium thiosulfate; low molecular weight polypeptides (e.g., peptides of 10 residues or fewer); hydrophilic polymers, such as polyvinylpyrrolidone monosaccharides, such as xylose, mannose, fructose, glucose; disaccharides such as lactose, maltose, sucrose and trehalose; and trisaccacharides such as raffinose; and polysaccharides such as dextran. Stabilizers may be present in amounts ranging from 0.5 to 10 weight % per weight of anti-Miro1 polypeptide or binding fragment.

[0116] Non-ionic surfactants or detergents (also known as wetting agents) may be added to help solubilize the anti-Miro1 polypeptide or binding fragment as well as to protect the polypeptide against agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stressed without causing denaturation of the protein. Suitable non- ionic surfactants include polysorbates (20, 80, etc.), poloxamers (184, 188 etc.), and pluronic polyols. Non-ionic surfactants may be present in a range of about 0.05 mg / mL to about 1.0 mg / mL.VI. METHODSA. Methods of Detection

[0117] An anti-Miro1 polypeptide or binding fragment thereof of the present disclosure can be used in a method of detecting, e.g., a method of quantifying, Miro1 as described herein. Insome embodiments, the anti-Miro1 polypeptide or binding fragment thereof is a monoclonal antibody. Without wishing to be bound by theory, the sensitivity of the anti-Miro1 polypeptides or binding fragments thereof of the present invention are believed to be superior to other available anti-Miro1 polypeptides. Accordingly, a method of detecting Miro1, e.g., by ELISA or flow cytometry, comprising an anti-Miro1 polypeptide or binding fragment thereof can be used with biological samples, such as urine, plasma, serum, or cerebrospinal fluid, that do not contain large numbers of cells. A method of detection of the present disclosure may be useful in quantifying Miro1 levels in cell populations, such as heart, liver, or kidney cells, where a biological sample may be more difficult to obtain.

[0118] In illustrative embodiments, the anti-Miro1 monoclonal antibodies 77C5-1, 72A12- 3, and 40H2-1 were capable of detecting Miro1 protein selectively over Miro2 in PBMCs. See, Example 4. In an ELISA format, the anti-Miro1 monoclonal antibodies of the present disclosure were more sensitive than commercial anti-Miro1 monoclonal antibodies. See, Example 5. Anti-Miro1 monoclonal antibody 40H2-1 was also capable of detecting Miro1 protein in human PBMCs in a flow cytometry assay format and afforded about two orders of magnitude higher signal for cells that exhibited Miro1 compared to the corresponding isotype control antibody, thereby distinguishing PBMCs with Miro1 and providing a wide dynamic assay range for detection of different Miro1 protein levels in PBMCs. In contrast, a commercial anti-Miro1 antibody afforded about an order of magnitude higher signal for cells that exhibited Miro1 compared to the isotype control antibody, and thus would not provide as wide of a dynamic assay range or distinguish PBMC Miro1 protein levels as precisely. See, Example 6 and FIGS.4A-4B.

[0119] Without wishing to be bound by theory, a Miro1 level in a cell from a subject having or suspected of having a disease or condition characterized by an elevated Miro1 level, e.g., Parkinson’s disease, is expected to exhibit delayed or retarded lowering, remain the same, or become even higher in response to a mitochondrial stressor in contrast to the reduction of a Miro1 level that can be observed in a healthy cell from a control subject. In some embodiments, a Miro1 level in a cell from a healthy subject, e.g., a peripheral cell, e.g., a fibroblast cell or PBMC, is lowered by from about 30% to about 80%, such as from about 40% to about 70% or from about 40% to about 60%, in the cell treated with a mitochondrial stressor, e.g., CCCP, or a combination of Antimycin A and Oligomycin, compared to Miro1 levels in the cell that was not treated with the mitochondrial stressor. By contrast, in some embodiments, a Miro1 level in a cell from a subject that has Parkinson’s disease is loweredby from about 0% to about 20% in the cell treated with a mitochondrial stressor compared to Miro1 levels in the cell not treated with the mitochondrial stressor. In some embodiments, a Miro1 level in a cell from a subject that has Parkinson’s disease is higher, e.g., from about 0% to about 100%, such as from about 20% to about 80%, in the cell treated with a mitochondrial stressor compared to Miro1 levels in the cell not treated with a mitochondrial stressor.

[0120] A “mitochondrial stressor” can be any number of agents that can induce mitochondrial stress, including mtDNA mutation, heat, ethidium bromide treatment, electron transport chain (ETC) protein mutation, ETC inhibition, or uncoupling of OXPHOS, and physiological stimuli-induced accumulation of ROS, which can disrupt membrane potential and / or increase the load of misfolded proteins, leading to dysregulation of mitochondrial homeostasis and function. In some embodiments, the mitochondrial stressor can include calcium dysregulation, ROS production, chemicals such as barbiturates, ginsenoside-Rh2, rotonene, Complex I inhibitors, Complex II inhibitors, Complex III inhibitors, Complex IV inhibitors, Complex V inhibitors, mycotoxins such as aurovertins A-E, leucinostatins A and B, venturicidin and ossamycin, efrapeptin, oligomycins A-D, vancomycin, antimycin, naturally occurring flavonoids, propranolol, local anesthetics, herbicide paraquat, pyrethroid, DDT, parathion, diethylstilbestrol, several cationic dyes and organotin compounds, uncouplers such as substituted phenols, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), carbonyl cyanide meta-chlorophenylhydrazone (CCCP), trifluoromethylbenzimidazoles, salicylanilides and carbonyl cyanide phenyl hydrazones, endogenous and exogenous free fatty acids (FFA) and fatty-acid like compounds such as perfluorodecanoic acid, sulfuramide and methyl-substituted hexadecanedioic acid, peptides such as adenine nucleotide translocase, ionophores such as gramicidins (gramicidin A, D and S), nigericin, valinomycin, cationic uncouplers such as cyanine dye tri-S-C4(5), Cu2`-(o- phenanthroline)2 complex, and pentamidine, membrane active peptides such as alamethicin, Mastoparan, alternative electron acceptors such as adriamycin, paraquat, and variously substituted naphthoquinones and nitrosoamines among others. In some embodiments, the mitochondrial stressor is CCCP. In some embodiments, the mitochondrial stressor is a combination of Antimycin A and Oligomycin.

[0121] Any method in the art for detecting the level and / or activity of Miro1 can be used with the anti-Miro1 polypeptide or binding fragment thereof described herein. For example, the level and / or the phosphorylation state of a Miro protein (Ser156, Thr298 or Thr299 ofMiro1 and Miro2, see, e.g., Wang et al. Cell 2011, 147(4): 893-906) may be detected, for example by immunoprecipitation with a mitochondrial transport protein-specific antibody followed by Western blotting with a phospho-specific or a general antibody, where an increase in phosphorylation of Miro proteins and / or a decrease of total Miro protein levels, or a decrease in phosphorylation of Khc following contact with the agent may indicate that the agent will treat Parkinson’s Disease. As another example, the level and / or the ubiquitination of a Miro1 protein may be detected, for example by immunoprecipitation with a mitochondrial transport protein-specific antibody followed by Western blotting with a ubiquitin-specific antibody, where an increase in ubiquitination following contact with the candidate agent indicates that the agent will treat Parkinson’s Disease. As another example, the ability of the target mitochondrial protein to transport mitochondria within a cell may be assessed by, for example, treating cultured cells (e.g., neurons) with the compound of the present disclosure and observing the transport of mitochondria in the cells as compared to cells not treating with the compound of the present disclosure, e.g., using live cell imaging techniques (see, e.g., Brickley and Stephenson J. Biol Chem 286(20): 18079-92 (2011); Misko et al. J Neurosci 30(19): 4232-40 (2010); Russo GJ et al. J. Neurosci 29(17):5443-55 (2009)). As another example, because the formation of a complex between Miro (e.g., Miro1 and Miro2), TRAK (e.g., TRAK1 and TRAK2), and Khc is essential for mitochondrial transport in neurons (see e.g., Brickley and Stephenson J. Biol Chem 286(20): 18079-92 (2011)), Miro1 function may be assessed by assessing the ability of Miro1, TRAK and Khc to form a complex in the presence of a Miro1-reducing agent. Such an assessment can be performed using any technique to determine protein-protein interaction including, but not limited to, co-immunoprecipitation and affinity purification techniques. In specific embodiments, the ability is assessed in a cell having a familial PD mutation, e.g., a PINK1 or LRRK2 mutation. Additional illustrative Miro1 assays are described in US publication nos. 2022 / 0334133 and 2022 / 0288071, and US application no.18 / 280,096, each of which is incorporated by reference in its entirety herein.

[0122] Affinity assays, which are often immunoassays, are an assay or analytic procedure that relies on the binding of the target molecule, i.e. Miro1, to receptors, antibodies or other macromolecules. A detection method is used to determine the presence and extent of the binding complexes that are formed. Many formats for such assays are known and used in the art, and are suitable for detection of Miro1 degradation following mitochondrial uncouplingor depolarization. In some embodiments, the assay format is suitable for high-throughput analysis.

[0123] Included in suitable assay formats are immunoassays that utilize antibodies specific for Miro1, including those described in the Examples. Additional suitable antibodies for this purpose are known and commercially available as polyclonal or monoclonal compositions, e.g., from Miro1 polyclonal from Proteintech (cat #11237-1-AP), LSBio, Abcam (ab154946); and the like.

[0124] Assays of interest include, for example, Western blots; immunohistochemistry; immunoprecipitation; etc., and particularly include immunoassays such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA); enzyme immunoassay (EIA). The detectable label or reporter molecule can be a radioisotope, such as3H,14C,32P,35S, or125I; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate, or rhodamine; or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Additional exemplary assays that can be used to detect or measure Miro1 in a sample include neutralization assays and fluorescence-activated cell sorting (FACS).

[0125] Enzyme-linked immunosorbent assays (ELISAs) can be used to qualitatively and / or quantitatively analyze the presence or concentration of a particular soluble antigen such as Miro1, in liquid samples, such as cell lysates. These assays generally make use of the ability of multiwell plates or others to bind antibodies which trap the cognate antigen. Usually a colorimetric endpoint that can be detected via absorbance wavelength and quantitated from a known standard curve of antigen or antibody dilutions is used. The detection antibody is often labelled with an enzyme such as horseradish peroxidase or alkaline phosphatase, or a fluorescent tag, or an electrochemiluminescent label or through an intermediary label such as biotin.

[0126] Common ELISA formats include the sandwich ELISA, so named because the analyte is “sandwiched” between two different antibodies. The capture substrate in this format is a capture antibody, often a monoclonal antibody, to increase the specificity of the assay and reduce background noise. The analyte is bound to the capture antibody, then detected by binding to a detection antibody. A variation of sandwich ELISA assay, called Single-Molecule Assay (Simoa), uses beads are coated with a capture antibody; each bead is bound to either one or zero target molecule, and individual beads are detected with another antibody (detection antibody) and a labeling enzyme.

[0127] Other ELISA formats include indirect ELISA, where the capture substrate is the specific antigen that is being tested and the detection step is mediated by a primary antibody and an enzyme-conjugated secondary antibody which is reactive against the primary antibody. Thus, the primary antibody that recognizes the antigen is not labeled. In a direct ELISA the capture substrate is the specific antigen that is being tested, and the enzyme that catalyzes the color-change reaction is conjugated to the antigen detector antibody.

[0128] Immuno-PCR (I-PCR) is a technique that combines the sensitivity of the nucleic acid amplification by PCR with the specificity of the antibody-based assays resulting in an increase of the detection sensitivity.

[0129] Mass spectrometry assays can qualitatively or quantitatively measure specific analytes in complex biological matrices (such as urine, blood or tissues).

[0130] One aspect of the present disclosure relates to methods for detecting the presence of Miro1, e.g., human Miro1, such as a Miro1 protein having a sequence of SEQ ID NO: 100, in a biological sample, the methods including contacting an anti-Miro1 polypeptide or binding fragment thereof as disclosed herein with a biological sample from a subject having or suspected of having a disease or condition characterized by an elevated Miro1 level. For example, the anti-Miro1 polypeptides or binding fragments thereof of the present disclosure can be used to detect and / or measure Miro1 in a sample, e.g., for diagnostic purposes.

[0131] In some embodiments, a subject is an animal, such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice and the like. In some embodiments, the subject is a human. In some embodiments, the subject is a patient.

[0132] In some embodiments, the methods include (i) contacting anti-Miro1 polypeptide or binding fragment thereof as disclosed herein with a biological sample from a subject having or suspected of having a disease or condition characterized by an elevated Miro1 level, (ii) detecting the formation of an antigen-polypeptide complex between the anti-Miro1 polypeptide or binding fragment and a Miro1 protein present in the biological sample. The formation of the antigen-polypeptide complex can be detected by one or more techniques known in the art, such as radioimmunoassay (RIA), enzyme linked immunosorbent assay (ELISA), immunofluorescence assay (IFA), dot blot or western blot. In some embodiments, the formation of the antigen-polypeptide complex can be detected by ELISA, dot blot orwestern blot. In some embodiments, the formation of the antigen-polypeptide complex can be detected by ELISA.

[0133] In some embodiments, the method of the present disclosure comprises a Miro1 ELISA. In some embodiments, the Miro1 ELISA comprises a first anti-Miro1 polypeptide or binding fragment thereof that is a capture antibody targeting a C-terminal region, e.g., the C-terminal GTPase region, of Miro1. In some embodiments, the Miro1 ELISA comprises asecond anti-Miro1 polypeptide or binding fragment thereof that is a detection antibody targeting an N-terminal region, e.g., the N-terminal GTPase region, of Miro1. In some embodiments, the Miro1 ELISA comprises a capture antibody that is 77C5-1 antibody or 72A12-3 antibody, and a detection antibody that is 40H2-1 antibody as described in Example 3 herein. In some embodiments, as illustrated in Example 4, the ELISA has over 100-fold selectivity for human Miro1 over Miro2 protein, and can measure wild type Miro1 levels as compared to those in Miro1 knockout cell lysates.

[0134] In some embodiments, the anti-Miro1 polypeptide or binding fragment thereof, e.g., antibodies of the present disclosure (e.g., of Example 3), can be used to detect and / or measure Miro1 in a sample. Exemplary assays for Miro1 include, but are not limited to, those described in Section VI(A) above. In some embodiments, a Miro1 assay can include contacting a biological sample with an anti-Miro1 polypeptide or binding fragment thereof (e.g., anti-Miro1 antibody) of the disclosure, wherein the anti-Miro1 polypeptide or binding fragment thereof is labeled with a detectable label or reporter molecule or used as a capture ligand to selectively isolate Miro1 from biological samples. The presence of an anti-Miro1 polypeptide or binding fragment thereof complexed with Miro1 indicates the presence of Miro1 in the sample. In some embodiments, an unlabeled anti-Miro1 antibody can be used in combination with a secondary antibody which can be detectably labeled. Thus, the present disclosure includes a method for detecting the presence of Miro1 in a sample comprising contacting the sample with an anti-Miro1 polypeptide or binding fragment thereof and detecting the presence of a Miro1 / anti-Miro1 polypeptide or binding fragment wherein the presence of the complex indicates the presence of Miro1.

[0135] In principle, there are no particular restrictions in regard to the types of biological samples suitable for use in the methods described herein. For example, samples that can be suitably used in Miro1 diagnostic assays according to the present disclosure include any tissue or fluid sample obtainable from a subject, which contains detectable quantities of eitherMiro1 protein, or fragments thereof, under normal or pathological conditions. In some embodiments, samples that can be suitably used in Miro1 diagnostic assays according to the present disclosure include any cell sample obtainable from a subject, which contains detectable quantities of either Miro1 protein, or fragments thereof, under normal or pathological conditions. In some embodiments, the biological sample includes sputum, bronchoalveolar lavage, pleural effusion, tissue, whole blood, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool, circulating tumor cells, circulating nucleic acids, bonemarrow, or any combination thereof. In some embodiments, the biological sample includescells or tissue. For example, the biological sample can be a tissue sample, such as a biopsy,core biopsy, needle aspirate, or fine needle aspirate. In some embodiments, the biological sample can be a fluid sample, such as a blood sample, urine sample, or saliva sample. In some embodiments, the biological sample can be a skin sample. In some embodiments, the biological sample can be a cheek swab. In some embodiments, the biological sample includes whole blood and blood components. In some embodiments, the biological sample includes peripheral blood mononuclear cells (PBMCs).

[0136] Any suitable cell can be used in a method of detecting Miro1 level described herein. Cultured cells may be derived from a subject (e.g., a patient), or control samples; and may be modified to generate genetically-modified cells, in vitro differentiated cells, cells exposed to a candidate therapeutic agent; and the like. In some embodiments, the cell is a skin cell. In some embodiments, the cell is a muscle cell. For example, the muscle cell can be a cardiac cell, that is, a cardiomyocyte. In some embodiments, the cell is a renal cell. In some embodiments, the cell is a liver cell. In some embodiments, the cell is a neuronal cell. The method can be performed in a cell in vitro, ex vivo, or in vivo. In some embodiments, the detecting Miro1 level is in vitro or ex vivo. In some embodiments, the detecting Miro1 level is in vivo.

[0137] Generally, levels of Miro1 protein in a particular sample obtained from a control subject (e.g., a healthy subject, e.g., a subject not having or suspected of having a disease or condition characterized by an elevated Miro1 level) are measured to initially establish a baseline, or standard, level of Miro1. This baseline level of Miro1 can then be compared against the levels of Miro1 measured in samples obtained from individuals suspected of having a Miro1-associated condition, or symptoms associated with such condition. For example, in some embodiments, a Miro1 level of a skin fibroblast from a subject is compared to a control Miro1 level of a control skin fibroblast from a control subject.

[0138] Any higher level of Miro1 compared to a control Miro1 level as determined in a method as described herein can lead to a determination of an “elevated Miro1 level”. In some embodiments, the level of Miro1 in a cell in a method as described herein is at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 200%, about 300%, about 400%, or at least about 500% higher than the control level of Miro1 in a control cell.

[0139] The anti-Miro1 polypeptide or binding fragment specific for Miro1 protein may contain no additional labels or moieties, or they may contain an N-terminal or C-terminal label or moiety. In some embodiments, the label or moiety is biotin. In a binding assay, the location of a label (if any) may determine the orientation of the peptide relative to the surface upon which the peptide is bound. For example, if a surface is coated with avidin, a peptide containing an N-terminal biotin will be oriented such that the C-terminal portion of the peptide will be distal to the surface. Sandwich ELISA

[0140] As understood in the art, an ELISA can be a sandwich ELISA comprising a capturereagent and reagent for a detection means, e.g., a detection reagent for the quantification of an analyte such as a protein. Accordingly, in some embodiments, a Miro1 ELISA is a sandwich ELISA comprising a first anti-Miro1 polypeptide or binding fragment that is a capture reagent and second anti-Miro1 polypeptide or binding fragment that is a detection means. In some embodiments, the Miro1 ELISA comprises a capture reagent specifically binding to the C-terminal GTPase region of human Miro1, and a detection means specifically binding to the N-terminal GTPase region of human Miro1. In some embodiments, the Miro1 ELISA comprises a capture reagent specifically binding to the C-terminal GTPase region of human Miro1 having a sequence according to SEQ ID NO: 102, and a detection means specifically binding to the N-terminal GTPase region of human Miro1 having a sequence according to SEQ ID NO: 101.

[0141] In some embodiments, the term “capture reagent” refers to a reagent capable of binding and capturing a target molecule in a sample such that under suitable condition, the capture reagent-target molecule complex can be separated from the rest of the sample. Typically, the capture reagent is immobilized or immobilizable. In a sandwich ELISA assay, the capture reagent can be a polypeptide, e.g., an antibody, e.g., an anti-Miro1 antibody, or a mixture of different antibodies against a target antigen.

[0142] In some embodiments, the term “detection means” refers to a moiety or technique used to detect the presence of the detection polypeptide, e.g., a detection antibody, in an ELISA as described herein and includes detection agents that amplify the immobilized label such as label captured onto a microtiter plate. In one embodiment, the detection means comprises a colorimetric detection agent such as avidin or streptavidin-HRP. Capture

[0143] In the capture step of the assay herein, the biological sample is contacted and incubated with the immobilized capture (or coat) reagent, which is an anti-Miro1 polypeptide or binding fragment, such as an anti-Miro1 antibody described herein. This antibody may be from any species, but in some embodiments, the anti-Miro1 monoclonal antibody is a rabbit monoclonal antibody, for example, derived from a New Zealand rabbit. Hence, in a specific embodiment, the immobilized monoclonal antibody is a rabbit monoclonal antibody. Immobilization conventionally is accomplished by insolubilizing the capture reagent either before the assay procedure, as by adsorption to a water-insoluble matrix or surface (U.S. Pat. No.3,720,760) or non-covalent or covalent coupling (for example, using glutaraldehyde or carbodiimide cross-linking, with or without prior activation of the support with, e.g., nitric acid and a reducing agent as described in U.S. Pat. No.3,645,852 or in Rotmans et al. J. Immunol. Methods 57:87-98 (1983)), or afterward, e.g., by immunoprecipitation.

[0144] In some embodiments, the Miro1 ELISA comprises a capture reagent specifically binding to the C-terminal GTPase region of human Miro1 having a sequence according to SEQ ID NO: 102. Illustrative capture reagents include 77C5-1 antibody and 72A12-3 antibody described in Example 3 below.

[0145] The solid phase used for immobilization may be any inert support or carrier that is essentially water insoluble and useful in immunometric assays, including supports in the form of, e.g., surfaces, particles, porous matrices, etc. Examples of commonly used supports include small sheets, Sephadex, polyvinyl chloride, plastic beads, and assay plates or test tubes manufactured from polyethylene, polypropylene, polystyrene, and the like including 96-well or greater microtiter plates, as well as particulate materials such as filter paper, agarose, cross-linked dextran, and other polysaccharides. Alternatively, reactive water- insoluble matrices such as cyanogen bromide-activated carbohydrates and the reactive substrates described in U.S. Pat. Nos.3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are suitably employed for capture reagent immobilization. In one embodimentthe immobilized capture reagent is coated on a microtiter plate, e.g., the solid phase used can be a multi-well microtiter plate that can be used to analyze several samples at one time, e.g., a microtest 96-well ELISA plate such as that sold as Nune Maxisorb or Immulon. In certain embodiments, the plate is a Meso Scale Discovery (MSD) 96-well standard ELISA plate.

[0146] In some embodiments, the solid phase is coated with the capture reagent as defined above, which may be linked by a non-covalent or covalent interaction or physical linkage as desired. Techniques for attachment include those described in U.S. Pat. No.4,376,110 and the references cited therein. If covalent, the plate or other solid phase is incubated with a cross-linking agent together with the capture reagent under conditions well known in the art, e.g., such as for 1 hour at room temperature.

[0147] Commonly used cross-linking agents for attaching the capture reagent to the solid phase substrate include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N- hydroxy-succinimide esters, for example, esters with 4-azido-salicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3′-dithiobis-(succinimidyl-propionate), and bifunctional maleimides such as bis-N-maleimido-1,8-octane. Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatable intermediates capable of forming cross-links in the presence of light.

[0148] If 96-well plates are utilized, they can be coated with the capture reagent (e.g., diluted in a buffer such as 0.05 M sodium carbonate by incubation for at least about 10 hours, e.g., overnight, at temperatures of about 4-20° C., or about 4-8° C., and at a pH of about 8-12, or about pH 9-10, or about pH 9.6). If shorter coating times are desired, one can coat, e.g., 96-well plates at room temperature for two hours. The plates may be stacked and coated long in advance of the assay itself, and then the assay can be carried out simultaneously on several samples in a manual, semi-automatic, or automatic fashion, such as by using robotics.

[0149] The coated plates can be treated with a blocking agent that binds non-specifically to and saturates the binding sites to prevent unwanted binding of the free ligand to the excess sites on the wells of the plate. Examples of appropriate blocking agents for this purpose include, e.g., gelatin, bovine serum albumin, egg albumin, casein, and non-fat milk. The blocking treatment typically takes place under conditions of ambient temperatures for about 1-4 hours, e.g., about 1 to 3 hours, or overnight at 0-4° C.

[0150] After coating and blocking, the Miro1 standard (purified Miro1 protein, e.g., having the sequence of SEQ ID NO: 100) or the biological sample to be analyzed, appropriatelydiluted, is added to the immobilized phase. The dilution rate can be about 1-15%, e.g., about 10%, by volume. Buffers that may be used for dilution for this purpose include (a) PBS containing 0.5% BSA, 0.05% TWEEN 20™ detergent (P20), 0.05% PROCLIN™ 300 antibiotic, 5 mM EDTA, 0.25% Chaps surfactant, 0.2% beta-gamma globulin, and 0.35M NaCl, pH 7.4; (b) PBS containing 0.5% bovine serum albumin, 0.05% polysorbate 20, 5 mM EDTA, 0.25% CHAPS, 0.2% bovine γ-globulins, and 0.35 M NaCl; pH 7.4 (c) PBS containing 0.5% BSA, 0.05% polysorbate 20 (P20), and 0.05% PROCLIN™ 300, pH 7; (d) PBS containing 0.5% BSA, 0.05% P20, 0.05% PROCLIN™ 300, 5 mM EDTA, and 0.35 M NaCl, pH 6.35; (e) PBS containing 0.5% BSA, 0.05% P20, 0.05% PROCLIN™ 300, 5 mM EDTA, 0.2% beta-gamma globulin, and 0.35 M NaCl, pH 7.4; and (f) PBS containing 0.5% BSA, 0.05% P20, 0.05% PROCLIN™ 300, 5 mM EDTA, 0.25% Chaps, and 0.35 M NaCl, pH 7.4. PROCLIN™ 300 acts as a preservative, and TWEEN 20™ acts as a detergent to eliminate non-specific binding.

[0151] While the concentration of the capture reagents will generally be determined by the concentration range of interest of the Miro1 taking any necessary dilution of the biological sample into account, the final concentration of the capture reagent will normally be determined empirically to maximize the sensitivity of the assay over the range of interest.

[0152] The conditions for incubation of sample and immobilized capture reagent are selected to maximize sensitivity of the assay and to minimize dissociation. In some embodiments, the incubation can be accomplished at constant temperatures, ranging from about 0° C to about 40° C, e.g., from about 20 to 25° C. The time for incubation depends primarily on the temperature, being generally no greater than about 10 hours to avoid an insensitive assay. In some embodiments, the incubation time is from about 0.5 to about 3 hours, such as from 1.5 to 3 hours at room temperature to maximize binding of free Miro1 to capture reagents. The duration of incubation may be longer if a protease inhibitor is added to prevent proteases in the biological fluid from degrading the Miro1.

[0153] In some embodiments, the pH of the incubation mixture can be in the range of about 4-9.5, e.g., in the range of about 6-9, e.g., about 7-8, e.g., the pH of the assay (ELISA) diluent can be pH 7.4. The pH of the incubation buffer is chosen to maintain a significant level of specific binding of the capture reagent to the Miro1 being captured. Various buffers may be employed to achieve and maintain the desired pH during this step, including borate, phosphate, carbonate, Tris-HCl or Tris-phosphate, acetate, barbital, and the like. Theparticular buffer employed is not believed to be critical to the invention, but in individual assays one buffer may be preferred over another. Detection

[0154] In some embodiments of the detection step, the immobilized capture reagent is contacted with an anti-Miro1 polypeptide or binding fragment, such as an anti-Miro1 antibody described herein acting as a detection antibody, at a temperature of, e.g., about 20- 40° C, such as about 20-25 °C, with the exact temperature and time for contacting the two being dependent primarily on the detection means employed. For example, when strepatavidin-peroxidase and 3,3′,5,5′-tetramethyl benzidine are used as the means for detection, e.g., in one embodiment, the contacting is carried out (e.g., about 1 hour or more) to amplify the signal to the maximum. In some embodiments, a molar excess of an antibody with respect to the maximum concentration of free Miro1 expected (as described above) is added to the plate after it is washed. The antibody may be directly or indirectly detectable. While the detection antibody may be a polyclonal or monoclonal antibody, e.g., in certain embodiments, it is a monoclonal antibody, in some embodiments, rabbit, and in some embodiments, derived from New Zealand rabbits. In some embodiments, the detection antibody can be directly detectable, and in one embodiment has a colorimetric label, and in another embodiment has a fluorometric label. In some embodiments, the detection antibody is biotinylated and the detection means is avidin or streptavidin-peroxidase and 3,3′,5,5′- tetramethyl benzidine. In some embodiments, the detection antibody is SULFO-TAGTMlabeled. The readout of the detection means can be fluorometric or colorimetric. The affinity of the detection antibody must be sufficiently high that small amounts of the free Miro1 can be detected, but not so high that it causes the Miro1 to be pulled from the capture reagents.

[0155] Subsequently, the level of free Miro1 that is now bound to the capture reagent can be measured using a detection means for the detection antibody. If the biological sample is from a subject having or suspected of having a disease or condition characterized by an elevated Miro1 level, the measuring step can comprise comparing the reaction that occurs as a result of the above steps with a standard curve to determine the level of Miro1 compared to control.

[0156] In some embodiments, the method has a greater selectivity for detecting Miro1 compared to Miro2. In some embodiments, the method comprises an anti-Miro1 polypeptide or binding fragment thereof of the present disclosure that is selective over Miro2. In someembodiments, the kit is at least 3-fold, at least 10-fold, at least 100-fold, or at least 1000-fold selective for Miro1 over Miro2, or a selectivity ranging between any of the foregoing values.

[0157] In some embodiments, the method has a high sensitivity for detecting, e.g., quantitating, Miro1 in a biological sample. In some embodiments, the method is capable of detecting and / or quantitating Miro1 in a smaller biological sample, e.g., containing fewer cells, compared to a literature method in the art. In some embodiments, the method is capable of quantitating Miro1 in a smaller biological sample, e.g., from about 10% to about 99%, e.g., at least 10%, such as at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, smaller than a biological sample required for quantitating Miro1 in a literature method. In some embodiments, the method is capable of quantitating Miro1 in fewer cells, e.g., from about 10% to about 99%, e.g., at least 10%, such as at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, fewer cells compared to the cells required for quantitating Miro1 in a literature method.

[0158] In some embodiments, the method of the present disclosure has a high sensitivity for detecting Miro1 in a biological sample, e.g., a sample that includes peripheral blood mononuclear cells (PBMCs). In some embodiments, the method is capable of detecting Miro1 in a biological sample containing less than about 1,000,000 PBMCs, e.g., less than about 500,000 PBMCs, less than about 400,000 PBMCs, less than about 300,000 PBMCs, less than about 200,000 PBMCs, or less than about 100,000 PBMCs. In some embodiments, the method is capable of detecting Miro1 in a biological sample containing from about 10,000 to about 1,000,000 PBMCs, e.g., from about 10,000 to about 500,000 PBMCs, from about 100,000 to about 500,000 PBMCs, from about 10,000 to about 400,000 PBMCs, from about 100,000 to about 400,000 PBMCs, from about 10,000 to about 300,000 PBMCs, from about 100,000 to about 300,000 PBMCs, from about 10,000 to about 200,000 PBMCs, or from about 100,000 to about 200,000 PBMCs. B. Kits for Methods of Detection

[0159] Kits that comprise an anti-Miro1 polypeptide or binding fragment thereof of the present disclosure, or pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing any of the above, for example, an immunoassay kit, such as an ELISA kit, are also included in the present disclosure. In some embodiments, a kit further includes instructions for use. In some embodiments, a kit includes an anti-Miro1 polypeptideor binding fragment of the disclosure, such as any one described in Section III above, and a label and / or instructions for use of the polypeptides described herein. In some embodiments, kits comprising an anti-Miro1 polypeptide or binding fragment of the present disclosure, or a pharmaceutically acceptable salt thereof, in combination with one or more (e.g., one, two, three, four, one or two, or one to three, or one to four) additional agents are provided.

[0160] In some embodiments, a kit can further include instructions for using the components of the kit to practice the method of the present disclosure. The instructions for practicing the methods are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kit as a package insert, in the labeling of the container of the kit or components thereof (e.g., associated with the packaging or subpackaging) etc. In some embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g., CD-ROM, diskette, flash drive, etc. In some embodiments, the instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, the means for obtaining the instructions is recorded on a suitable substrate.

[0161] In some embodiments, the kit comprises a capture antibody or binding fragment thereof and a detection antibody or binding fragment thereof. Accordingly, in some embodiments, the kit comprises a first anti-Miro1 polypeptide or binding fragment thereof and a second anti-Miro1 polypeptide or binding fragment thereof.

[0162] In some embodiments, the kit comprises the first anti-Miro1 polypeptide or binding fragment thereof that has a VHchain corresponding in sequence to SEQ ID NO: 27 or 37, and a VL chain corresponding in sequence to SEQ ID NO: 28 or 38; and the second anti-Miro1 polypeptide or binding fragment thereof that has a VHchain corresponding in sequence to SEQ ID NO: 47, and a VL chain corresponding in sequence to SEQ ID NO: 48.

[0163] In some embodiments, the kit comprises the first anti-Miro1 polypeptide or binding fragment thereof that has a VHchain corresponding in sequence to SEQ ID NO: 27, and a VLchain corresponding in sequence to SEQ ID NO: 28. In some embodiments, the kit comprises the first anti-Miro1 polypeptide or binding fragment thereof that has a VHchaincorresponding in sequence to SEQ ID NO: 37, and a VLchain corresponding in sequence to SEQ ID NO: 38.

[0164] Provided herein are also articles of manufacture that include an anti-Miro1 polypeptide or binding fragment of the present disclosure in a suitable container. The container may be a vial, jar, ampoule, preloaded syringe, and intravenous bag.

[0165] In some embodiments, the method of the present disclosure comprises quantifying Miro1 in a sample from a subject by (a) contacting the sample with a carrier comprising a first anti-Miro1 polypeptide or binding fragment thereof; and (b) detecting binding between the carrier and the Miro1 with a second anti-Miro1 polypeptide or binding fragment thereof. In some embodiments, the method comprises the first anti-Miro1 polypeptide or binding fragment thereof that specifically binds to the C-terminus of Miro1; and the second anti- Miro1 polypeptide or binding fragment thereof that specifically binds to the N-terminus of Miro1. In some embodiments, the method comprises the first anti-Miro1 polypeptide or binding fragment thereof that has a VH chain corresponding in sequence to SEQ ID NO: 27 or 37, and a VLchain corresponding in sequence to SEQ ID NO: 28 or 38; and the second anti- Miro1 polypeptide or binding fragment thereof that has a VH chain corresponding in sequence to SEQ ID NO: 47, and a VLchain corresponding in sequence to SEQ ID NO: 48. In some embodiments, the method comprises the first anti-Miro1 polypeptide or binding fragment thereof that has a VHchain corresponding in sequence to SEQ ID NO: 27, and a VLchain corresponding in sequence to SEQ ID NO: 28. In some embodiments, the method comprises the first anti-Miro1 polypeptide or binding fragment thereof that has a VHchain corresponding in sequence to SEQ ID NO: 37, and a VL chain corresponding in sequence to SEQ ID NO: 38.

[0166] In some embodiments, the kit, e.g., an ELISA kit, has a greater selectivity for detecting Miro1 compared to Miro2. In some embodiments, the kit comprises an anti-Miro1 polypeptide or binding fragment thereof of the present disclosure that is selective over Miro2. In some embodiments, the kit is at least 3-fold, at least 10-fold, at least 100-fold, or at least 1000-fold selective for Miro1 over Miro2, or a selectivity ranging between any of the foregoing values.

[0167] In some embodiments, the kit, e.g., an ELISA kit, has a high sensitivity for detecting, e.g., quantitating, Miro1 in a biological sample. In some embodiments, the kit is capable of detecting and / or quantitating Miro1 in a smaller biological sample, e.g., containingfewer cells, compared to a literature ELISA kit in the art. In some embodiments, the kit is capable of quantitating Miro1 in a smaller biological sample, e.g., from about 10% to about 99%, e.g., at least 10%, such as at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, smaller than a biological sample required for quantitating Miro1 in a literature ELISA kit. In some embodiments, the kit is capable of quantitating Miro1 in fewer cells, e.g., from about 10% to about 99%, e.g., at least 10%, such as at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, fewer cells compared to the cells required for quantitating Miro1 in a literature ELISA kit.

[0168] In some embodiments, the kit, e.g., an ELISA kit, has a high sensitivity for detecting Miro1 in a biological sample, e.g., a sample that includes peripheral blood mononuclear cells (PBMCs). In some embodiments, the kit is capable of detecting Miro1 in a biological sample containing less than about 1,000,000 PBMCs, e.g., less than about 500,000 PBMCs, less than about 400,000 PBMCs, less than about 300,000 PBMCs, less than about 200,000 PBMCs, or less than about 100,000 PBMCs. In some embodiments, the kit is capable of detecting Miro1 in a biological sample containing from about 10,000 to about 1,000,000 PBMCs, e.g., from about 10,000 to about 500,000 PBMCs, from about 100,000 to about 500,000 PBMCs, from about 10,000 to about 400,000 PBMCs, from about 100,000 to about 400,000 PBMCs, from about 10,000 to about 300,000 PBMCs, from about 100,000 to about 300,000 PBMCs, from about 10,000 to about 200,000 PBMCs, or from about 100,000 to about 200,000 PBMCs. C. Methods and / or Uses in Diseases Characterized by Elevated Miro1

[0169] A deficiency in the ability to degrade or clear Miro1 from cells is believed to correlate with the development of a disease or disorder characterized by elevated Miro1 level, for example, a neurodegenerative disease, such as Parkinson’s disease, before a subject displays an overt symptom of the disease or disorder, such as one or more of the symptoms described herein. Accordingly, in some embodiments, the subject is asymptomatic for a disease or disorder characterized by elevated Miro1 level. Miro1 may be used as a predictive biomarker for a neurodegenerative disorder in a subject at risk of developing such disorder, for example, as an initial step in treating the disease or disorder characterized by elevated Miro1 level before symptoms appear. The subject at risk can have familial history of developing a disease or disorder characterized by elevated Miro1 level, e.g., aneurodegenerative disease, can present a genetic marker associated with increased risk of developing a disease or disorder characterized by elevated Miro1 level, for example, LRRK2 G2019S mutation for Parkinson’s disease, or can have no known risk of developing a disease or disorder characterized by elevated Miro1 level.

[0170] A Miro1 level in cells derived from a subject having or suspected of having a disease or disorder characterized by elevated Miro1 level is expected to be higher than a control Miro1 level in healthy control cells due to mitophagy processes induced by mitochondrial stressors associated with the disease or disorder characterized by elevated Miro1 level. With biological sample cells derived from a subject, a Miro1 level that is similar or higher compared to a control Miro1 level in healthy control cells may indicate a disease or disorder characterized by elevated Miro1 level, e.g., a neurodegenerative disease such as Parkinson’s disease, that correlates with defective mitophagy processes.

[0171] The presently described assays and methods are believed to be useful in the detection and / or diagnosis of a disease or condition characterized by elevated Miro1 levels. Accordingly, in some embodiments, the method of the present disclosure is a method of detecting a disease or disorder characterized by an elevated Miro1 in a subject, the method comprising: (a) quantifying Miro1 in a sample from the subject comprising: (i) contacting the sample with a carrier comprising a first anti-Miro1 polypeptide or binding fragment thereof; and (ii) determining binding between the carrier and the Miro1 with a second anti-Miro1 polypeptide or binding fragment thereof; (b) comparing the sample Miro1 level with a control Miro1 level; and (c) detecting the disease or disorder characterized by an elevated Miro1 if the sample Miro1 level is higher than the control Miro1 level.

[0172] In some embodiments, the disease or disorder characterized by an elevated Miro1 is detected if the sample Miro1 level is at least 20%, e.g., at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, higher than the control Miro1 level.

[0173] In some embodiments, the method has a greater selectivity for detecting Miro1 compared to Miro2. In some embodiments, the method comprises an anti-Miro1 polypeptide or binding fragment thereof of the present disclosure that is selective over Miro2. In some embodiments, the method is at least 3-fold, at least 10-fold, at least 100-fold, or at least 1000- fold selective for Miro1 over Miro2, or a selectivity ranging between any of the foregoing values.

[0174] In some embodiments, the method has a high sensitivity for detecting, e.g., quantitating, Miro1 in a biological sample. In some embodiments, the method is capable of detecting and / or quantitating Miro1 in a smaller biological sample, e.g., containing fewer cells, compared to a literature method in the art. In some embodiments, the method is capable of quantitating Miro1 in a smaller biological sample, e.g., from about 10% to about 99%, e.g., at least 10%, such as at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, smaller than a biological sample required for quantitating Miro1 in a literature method. In some embodiments, the method is capable of quantitating Miro1 in fewer cells, e.g., from about 10% to about 99%, e.g., at least 10%, such as at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, fewer cells compared to the cells required for quantitating Miro1 in a literature method.

[0175] In some embodiments, the method of the present disclosure has a high sensitivity for detecting Miro1 in a biological sample, e.g., a sample that includes peripheral blood mononuclear cells (PBMCs). In some embodiments, the method is capable of detecting Miro1 in a biological sample containing less than about 1,000,000 PBMCs, e.g., less than about 500,000 PBMCs, less than about 400,000 PBMCs, less than about 300,000 PBMCs, less than about 200,000 PBMCs, or less than about 100,000 PBMCs. In some embodiments, the method is capable of detecting Miro1 in a biological sample containing from about 10,000 to about 1,000,000 PBMCs, e.g., from about 10,000 to about 500,000 PBMCs, from about 100,000 to about 500,000 PBMCs, from about 10,000 to about 400,000 PBMCs, from about 100,000 to about 400,000 PBMCs, from about 10,000 to about 300,000 PBMCs, from about 100,000 to about 300,000 PBMCs, from about 10,000 to about 200,000 PBMCs, or from about 100,000 to about 200,000 PBMCs.

[0176] In some embodiments, a method of the present disclosure comprises a kit, e.g., an ELISA kit, that has a high sensitivity for detecting Miro1 in a biological sample, e.g., a sample that includes peripheral blood mononuclear cells (PBMCs). In some embodiments, the kit is capable of detecting Miro1 in a biological sample containing less than about 1,000,000 PBMCs, e.g., less than about 500,000 PBMCs, less than about 400,000 PBMCs, less than about 300,000 PBMCs, less than about 200,000 PBMCs, or less than about 100,000 PBMCs. In some embodiments, the kit is capable of detecting Miro1 in a biological sample containing from about 10,000 to about 1,000,000 PBMCs, e.g., from about 10,000 to about 500,000 PBMCs, from about 100,000 to about 500,000 PBMCs, from about 10,000 to about400,000 PBMCs, from about 100,000 to about 400,000 PBMCs, from about 10,000 to about 300,000 PBMCs, from about 100,000 to about 300,000 PBMCs, from about 10,000 to about 200,000 PBMCs, or from about 100,000 to about 200,000 PBMCs. Methods of Identifying a Subject at Risk of Developing Disease

[0177] In some embodiments, provided is a method for identifying a subject at risk of developing a disease or disorder characterized by an elevated Miro1, the method comprising: a) detecting whether a Miro level is similar or higher in a biological sample obtained from the subject as compared to a control Miro1 level in a control biological sample; b) identifying the subject at risk of developing a disease or disorder characterized by an elevated Miro1 if the Miro1 level is similar or higher in the biological sample compared to the control Miro1 level in the control biological sample. In some embodiments, the level of Miro1 in a biological sample in a method as described herein is at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 200%, about 300%, about 400%, or at least about 500% higher than the control level of Miro1 in a control biological sample. In some embodiments, the subject at risk of developing a disease or disorder is asymptomatic. In some embodiments, the subject at risk of developing a disease or disorder has one or more symptoms associated with a disease or disorder characterized by an elevated Miro1.

[0178] A control Miro1 level can be obtained from a control cell. The control cell can be a cell that has been administered only cell media (that is, without a compound of the present disclosure or pharmaceutically acceptable salt thereof). Alternatively, a control cell can be a cell from or derived from a healthy subject that does not have or is not suspected of suffering from a disease or condition described herein, e.g., a neurodegenerative disorder such as Parkinson’s disease, that is administered the compound of the present disclosure or pharmaceutically acceptable salt thereof. A control cell can be obtained from or derived from a cell obtained from a healthy human volunteer or from a cell bank. For example, a control cell can be obtained from or derived from a cell obtained from American Type Culture Collection (ATCC), e.g., neural progenitor cells or neural tissue-derived cell lines such as astrocyctes.

[0179] In some embodiments, control values are measured from corresponding control samples from control, e.g., non-diseased, subjects. For example, in some embodiments, aMiro1 level of PBMCs from a subject, e.g., a patient, is compared to a control Miro1 level of control PBMCs from a control subject.

[0180] In some embodiments, the method has a greater selectivity for detecting Miro1 compared to Miro2. In some embodiments, the method comprises an anti-Miro1 polypeptide or binding fragment thereof of the present disclosure that is selective over Miro2. In some embodiments, the method is at least 3-fold, at least 10-fold, at least 100-fold, or at least 1000- fold selective for Miro1 over Miro2, or a selectivity ranging between any of the foregoing values.

[0181] In some embodiments, the method has a high sensitivity for detecting, e.g., quantitating, Miro1 in a biological sample. In some embodiments, the method is capable of detecting and / or quantitating Miro1 in a smaller biological sample, e.g., containing fewer cells, compared to a literature method in the art. In some embodiments, the method is capable of quantitating Miro1 in a smaller biological sample, e.g., from about 10% to about 99%, e.g., at least 10%, such as at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, smaller than a biological sample required for quantitating Miro1 in a literature method. In some embodiments, the method is capable of quantitating Miro1 in fewer cells, e.g., from about 10% to about 99%, e.g., at least 10%, such as at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, fewer cells compared to the cells required for quantitating Miro1 in a literature method.

[0182] In some embodiments, the method of the present disclosure has a high sensitivity for detecting Miro1 in a biological sample, e.g., a sample that includes peripheral blood mononuclear cells (PBMCs). In some embodiments, the method is capable of detecting Miro1 in a biological sample containing less than about 1,000,000 PBMCs, e.g., less than about 500,000 PBMCs, less than about 400,000 PBMCs, less than about 300,000 PBMCs, less than about 200,000 PBMCs, or less than about 100,000 PBMCs. In some embodiments, the method is capable of detecting Miro1 in a biological sample containing from about 10,000 to about 1,000,000 PBMCs, e.g., from about 10,000 to about 500,000 PBMCs, from about 100,000 to about 500,000 PBMCs, from about 10,000 to about 400,000 PBMCs, from about 100,000 to about 400,000 PBMCs, from about 10,000 to about 300,000 PBMCs, from about 100,000 to about 300,000 PBMCs, from about 10,000 to about 200,000 PBMCs, or from about 100,000 to about 200,000 PBMCs.

[0183] In some embodiments, a method of the present disclosure comprises a kit, e.g., an ELISA kit, that has a high sensitivity for detecting Miro1 in a biological sample, e.g., a sample that includes peripheral blood mononuclear cells (PBMCs). In some embodiments, the kit is capable of detecting Miro1 in a biological sample containing less than about 1,000,000 PBMCs, e.g., less than about 500,000 PBMCs, less than about 400,000 PBMCs, less than about 300,000 PBMCs, less than about 200,000 PBMCs, or less than about 100,000 PBMCs. In some embodiments, the kit is capable of detecting Miro1 in a biological sample containing from about 10,000 to about 1,000,000 PBMCs, e.g., from about 10,000 to about 500,000 PBMCs, from about 100,000 to about 500,000 PBMCs, from about 10,000 to about 400,000 PBMCs, from about 100,000 to about 400,000 PBMCs, from about 10,000 to about 300,000 PBMCs, from about 100,000 to about 300,000 PBMCs, from about 10,000 to about 200,000 PBMCs, or from about 100,000 to about 200,000 PBMCs.

[0184] In some embodiments, a disease or condition characterized by elevated Miro1 levels is associated with mitochondrial dysfunction.

[0185] Mitochondrial dysfunction contributes to various disease states. Some mitochondrial diseases are due to mutations or deletions in the mitochondrial genome. If a threshold proportion of mitochondria in the cell is defective, and if a threshold proportion of such cells within a tissue have defective mitochondria, symptoms of tissue or organ dysfunction can result. Practically any tissue can be affected, and a large variety of symptoms may be present, depending on the extent to which different tissues are involved. Some examples of mitochondrial diseases include Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, Friedreich's ataxia (FRDA), Leber's Hereditary Optic Neuropathy (LHON), mitochondrial myopathy, encephalopathy, lactacidosis, and stroke (MELAS), Myoclonus Epilepsy Associated with Ragged-Red Fibers (MERRF) syndrome, Maternally Inherited Diabetes and Deafness (MIDD), and respiratory chain disorders. Mitochondrial diseases can involve children who manifest the signs and symptoms of accelerated aging, including neurodegenerative diseases, stroke, blindness, hearing or balance impairment, diabetes, and heart failure.

[0186] Friedreich's ataxia is an autosomal recessive neurodegenerative and cardiodegenerative disorder caused by decreased levels of the protein Frataxin. The disease causes the progressive loss of voluntary motor coordination (ataxia) and cardiac complications. Symptoms typically begin in childhood, and the disease progressively worsensas the patient grows older; patients eventually become wheelchair-bound due to motor disabilities.

[0187] Leber's Hereditary Optic Neuropathy (LHON) is a disease characterized by blindness which occurs on average between 27 and 34 years of age. Other symptoms may also occur, such as cardiac abnormalities and neurological complications.

[0188] Mitochondrial myopathy, encephalopathy, lactacidosis, and stroke (MELAS) can manifest itself in infants, children, or young adults. Strokes, accompanied by vomiting and seizures, are one of the most serious symptoms; it is postulated that the metabolic impairment of mitochondria in certain areas of the brain is responsible for cell death and neurological lesions, rather than the impairment of blood flow as occurs in ischemic stroke.

[0189] Myoclonus Epilepsy Associated with Ragged-Red Fibers (MERRF) syndrome is one of a group of rare muscular disorders that are called mitochondrial encephalomyopathies. Mitochondrial encephalomyopathies are disorders in which a defect in the genetic material arises from a part of the cell structure that releases energy (mitochondria). This can cause a dysfunction of the brain and muscles (encephalomyopathies). The most characteristic symptom of MERRF syndrome is myoclonic seizures that are usually sudden, brief, jerking, spasms that can affect the limbs or the entire body, difficulty speaking (dysarthria), optic atrophy, short stature, hearing impairment, dementia, and involuntary jerking of the eyes (nystagmus) may also occur.

[0190] Leigh's disease is a rare inherited neurometabolic disorder characterized by degeneration of the central nervous system where the symptoms usually begin between the ages of 3 months to 2 years and progress rapidly. In most children, the first signs may be poor sucking ability and loss of head control and motor skills. These symptoms may be accompanied by loss of appetite, vomiting, irritability, continuous crying, and seizures. As the disorder progresses, symptoms may also include generalized weakness, lack of muscle tone, and episodes of lactic acidosis, which can lead to impairment of respiratory and kidney function. Heart problems may also occur.

[0191] Maternally Inherited Diabetes and Deafness (MIDD) is caused by a mutation in mitochondrial DNA (3243 tRNA). The diabetes is a non-insulin dependent type that usually presents before the age of 40 years; it is due to a defect in beta cell function with normal insulin sensitivity. The associated deafness is sensorineural and develops in most of the diabetic subjects. In keeping with other mitochondrial disorders, MIDD may have othermulti-organ features: for example, elevated serum lactate, neuromuscular and cardiac problems, pigmented retinopathy, and nephropathy with proteinuria.

[0192] Co-Enzyme Q10 Deficiency is a respiratory chain disorder, with syndromes such as myopathy with exercise intolerance and recurrent myoglobin in the urine manifested by ataxia, seizures or mental retardation and leading to renal failure, childhood-onset cerebellar ataxia and cerebellar atrophy; and infantile encephalomyopathy associated with nephrosis. Biochemical measurement of muscle homogenates of patients with CoQ10 deficiency showed severely decreased activities of respiratory chain complexes I and II+III, while complex IV (COX) was moderately decreased.

[0193] Complex I Deficiency or NADH dehydrogenase NADH-CoQ reductase deficiency is a respiratory chain disorder, with symptoms classified by three major forms: (1) fatal infantile multisystem disorder, characterized by developmental delay, muscle weakness, heart disease, congenital lactic acidosis, and respiratory failure; (2) myopathy beginning in childhood or in adult life, manifesting as exercise intolerance or weakness; and (3) mitochondrial encephalomyopathy (including MELAS), which may begin in childhood or adult life and consists of variable combinations of symptoms and signs, including ophthalmoplegia, seizures, dementia, ataxia, hearing impairment, pigmentary retinopathy, sensory neuropathy, and uncontrollable movements.

[0194] Complex II Deficiency or Succinate dehydrogenase deficiency is a respiratory chain disorder with symptoms including encephalomyopathy and various manifestations, including failure to thrive, developmental delay, hyoptonia, lethargy, respiratory failure, ataxia, myoclonus and lactic acidosis.

[0195] Complex III Deficiency or Ubiquinone-cytochrome C oxidoreductase deficiency is a respiratory chain disorder with symptoms categorized in four major forms: (1) fatal infantile encephalomyopathy, congenital lactic acidosis, hypotonia, dystrophic posturing, seizures, and coma; (2) encephalomyopathies of later onset (childhood to adult life): various combinations of weakness, short stature, ataxia, dementia, hearing impairment, sensory neuropathy, pigmentary retinopathy, and pyramidal signs; (3) myopathy, with exercise intolerance evolving into fixed weakness; and (4) infantile histiocytoid cardiomyopathy.

[0196] Complex IV Deficiency or Cytochrome C oxidase deficiency is a respiratory chain disorder with symptoms categorized in two major forms: (1) encephalomyopathy, which is typically normal for the first 6 to 12 months of life and then show developmental regression,ataxia, lactic acidosis, optic atrophy, ophthalmoplegia, nystagmus, dystonia, pyramidal signs, respiratory problems and frequent seizures; and (2) myopathy with two main variants: (a) fatal infantile myopathy—may begin soon after birth and accompanied by hypotonia, weakness, lactic acidosis, ragged-red fibers, respiratory failure, and kidney problems: and (b) benign infantile myopathy—may begin soon after birth and accompanied by hypotonia, weakness, lactic acidosis, ragged-red fibers, respiratory problems, but (if the child survives) followed by spontaneous improvement.

[0197] Complex V Deficiency or ATP synthase deficiency is a respiratory chain disorder including symptoms such as slow, progressive myopathy.

[0198] CPEO or Chronic Progressive External Ophthalmoplegia Syndrome is a respiratory chain disorder including symptoms such as visual myopathy, retinitis pigmentosa, or dysfunction of the central nervous system.

[0199] Kearns-Sayre Syndrome (KSS) is a mitochondrial disease characterized by a triad of features including: (1) typical onset in persons younger than age 20 years; (2) chronic, progressive, external ophthalmoplegia; and (3) pigmentary degeneration of the retina. In addition, KSS may include cardiac conduction defects, cerebellar ataxia, and raised cerebrospinal fluid (CSF) protein levels (e.g., >100 mg / dL). Additional features associated with KSS may include myopathy, dystonia, endocrine abnormalities (e.g., diabetes, growth retardation or short stature, and hypoparathyroidism), bilateral sensorineural deafness, dementia, cataracts, and proximal renal tubular acidosis.

[0200] In addition to congenital disorders involving inherited defective mitochondria, acquired mitochondrial dysfunction contributes to diseases, particularly neurodegenerative disorders such as Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), or Huntington's disease. The incidence of somatic mutations in mitochondrial DNA rises exponentially with age; diminished respiratory chain activity is found universally in aging people. Mitochondrial dysfunction is also implicated in excitoxic, neuronal injury, such as that associated with cerebral vascular accidents, seizures and ischemia.

[0201] Parkinson’s disease has been associated with mitochondrial dysfunction since 1983, when 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was found to cause parkinsonian-like symptoms in intravenous drug users. When MPTP penetrates the blood- brain barrier, the compound is bio-transformed into its toxic form 1-methyl-4- phenylpyridinium (MPP+) by glial monoamine oxidase (MAO). MPP+ specifically interfereswith the activity of respiratory chain (RC) complex I (NADH: Ubiquinone oxidoreductase) in dopaminergic (DA) neurons, causing selective neurodegeneration in both human and mousesubstantia nigra (SN). In post-mortem studies, varying degrees of Complex I and complex II(succinate dehydrogenase, SDH) deficiency have been found in individual SN neurons from PD patients (~60% Complex I and ~65% Complex II deficiency).

[0202] Mitochondria may mediate, drive, or contribute to a variety of Alzheimer’s disease (AD) pathologies. Amyloid-β (Aβ) may induce AD mitochondrial dysfunction. Alternatively, data indicate mitochondrial dysfunction exists independent of Aβ, potentially lies upstream of Aβ deposition, and suggest a primary mitochondrial cascade hypothesis that assumes mitochondrial pathology hierarchically supersedes Aβ pathology. Mitochondria, therefore, appear at least to mediate or possibly even initiate pathologic molecular cascades in AD.

[0203] Amyotrophic lateral sclerosis (ALS) is caused by selective degeneration of motor neurons in the brain and spinal cord, which may be mediated by mitochondrial dysfunction. Studies suggest that ferroptosis mediates selective motor neuron death in amyotrophic lateral sclerosis. See, Wang, T. et al. Cell Death Differ 29, 1187–1198 (2022). Methods of Aiding and / or Treatment

[0204] Anti-Miro1 polypeptides or binding fragments thereof of the disclosure or are also useful for methods of aiding the treatment of a disease, a disorder, and / or a health condition associated with a mitochondrial disease, such as Parkinson’s disease. As used herein, a “method of aiding” generally refers to methods of assisting in performing or practicing a method disclosed herein, for example, methods of assisting in (i) performing, (ii) practicing, and / or (iii) making a determination concerning the detection, classification, treatment regiment, or nature, of a mitochondrial disease (e.g., Parkinson’s disease), a disease, a disorder, and / or a health condition.

[0205] Accordingly, in some embodiments, a method of aiding in the treatment of a mitochondrial disease in a subject in need thereof comprises administering to the subject a therapeutically effective amount of a compound of the present disclosure or pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure. In some embodiments, the method of aiding in the treatment of a disease or disorder characterized by an elevated Miro1 in a subject comprises: (a) quantifying Miro1 in a sample from the subject comprising: (i) contacting the sample with a carrier comprising a first anti-Miro1 polypeptide or binding fragment thereof; and (ii) determining binding between the carrier and the Miro1with a second anti-Miro1 polypeptide or binding fragment thereof; (b) comparing the sample Miro1 level with a control Miro1 level; (c) detecting the disease or disorder characterized by an elevated Miro1 if the sample Miro1 level is higher than the control Miro1 level; and (d) administering a therapeutically effective amount of a Miro1-reducing agent to the subject.

[0206] Treatment as used herein refers to an approach for obtaining beneficial or desired results. For purposes of the present disclosure, beneficial or desired results include, but are not limited to, alleviation of a symptom and / or diminishment of the extent of a symptom and / or preventing a worsening of a symptom associated with a disease or condition. In some embodiments, “treatment” or “treating” includes one or more of the following: a) inhibiting the disease or condition (e.g., decreasing one or more symptoms resulting from the disease or condition, and / or diminishing the extent of the disease or condition); b) slowing or arresting the development of one or more symptoms associated with the disease or condition (e.g., stabilizing the disease or condition, delaying the worsening or progression of the disease or condition); and c) relieving the disease or condition, e.g., causing the regression of clinical symptoms, ameliorating the disease state, delaying the progression of the disease, increasing the quality of life, and / or prolonging survival.

[0207] Therapeutically effective amount as used herein refers to an amount that is effective to elicit the desired biological or medical response, including the amount of a compound that, when administered to a subject for treating a disease, is sufficient to effect such treatment for the disease. The effective amount can vary depending on the compound, the disease, and its severity and the age, weight, etc., of the subject to be treated. The effective amount can include a range of amounts. As is understood in the art, an effective amount may be in one or more doses, i.e., a single dose or multiple doses may be required to achieve the desired treatment endpoint. An effective amount may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable or beneficial result may be or is achieved. Suitable doses of any co-administered compounds may optionally be lowered due to the combined action (e.g., additive or synergistic effects) of the compounds.

[0208] Administering refers to oral administration, administration as a suppository, topical contact, parenteral, intravenous, intraperitoneal, intramuscular, intralesional, intranasal or subcutaneous administration, intrathecal administration, or the implantation of a slow-releasedevice e.g., a mini-osmotic pump, to the subject. The administration can be carried out according to a schedule specifying frequency of administration, dose for administration, and other factors.

[0209] In some embodiments, the present anti-Miro1 polypeptides or binding fragment thereof are useful in methods, compositions, kits, that can be used in the detection of a disease or disorder characterized by an elevated Miro1 in a subject before treatment with a Miro1-reducing agent. In some embodiments, the method of the present disclosure is a method of treating a disease or disorder characterized by an elevated Miro1 in a subject, the method comprising: (a) quantifying Miro1 in a sample from the subject comprising: (i) contacting the sample with a carrier comprising a first anti-Miro1 polypeptide or binding fragment thereof; and (ii) determining binding between the carrier and the Miro1 with a second anti-Miro1 polypeptide or binding fragment thereof; (b) comparing the sample Miro1 level with a control Miro1 level; (c) detecting the disease or disorder characterized by an elevated Miro1 if the sample Miro1 level is higher than the control Miro1 level; and (d) administering a therapeutically effective amount of a Miro1-reducing agent to the subject.

[0210] In some embodiments, the method of the present disclosure is a method of treating a neurodegenerative disease in a subject, the method comprising: (a) quantifying Miro1 in a sample from the subject comprising: (i) contacting the sample with a carrier comprising a first anti-Miro1 polypeptide or binding fragment thereof; and (ii) determining binding between the carrier and the Miro1 with a second anti-Miro1 polypeptide or binding fragment thereof; (b) comparing the sample Miro1 level with a control Miro1 level; (c) detecting the neurodegenerative disease if the sample Miro1 level is higher than the control Miro1 level; and (d) administering a therapeutically effective amount of a Miro1-reducing agent to the subject.

[0211] In some embodiments, the method has a greater selectivity for detecting Miro1 compared to Miro2. In some embodiments, the method comprises an anti-Miro1 polypeptide or binding fragment thereof of the present disclosure that is selective over Miro2. In some embodiments, the method is at least 3-fold, at least 10-fold, at least 100-fold, or at least 1000- fold selective for Miro1 over Miro2, or a selectivity ranging between any of the foregoing values.

[0212] In some embodiments, the method has a high sensitivity for detecting, e.g., quantitating, Miro1 in a biological sample. In some embodiments, the method is capable ofdetecting and / or quantitating Miro1 in a smaller biological sample, e.g., containing fewer cells, compared to a literature method in the art. In some embodiments, the method is capable of quantitating Miro1 in a smaller biological sample, e.g., from about 10% to about 99%, e.g., at least 10%, such as at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, smaller than a biological sample required for quantitating Miro1 in a literature method. In some embodiments, the method is capable of quantitating Miro1 in fewer cells, e.g., from about 10% to about 99%, e.g., at least 10%, such as at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, fewer cells compared to the cells required for quantitating Miro1 in a literature method.

[0213] In some embodiments, the method of the present disclosure has a high sensitivity for detecting Miro1 in a biological sample, e.g., a sample that includes peripheral blood mononuclear cells (PBMCs). In some embodiments, the method is capable of detecting Miro1 in a biological sample containing less than about 1,000,000 PBMCs, e.g., less than about 500,000 PBMCs, less than about 400,000 PBMCs, less than about 300,000 PBMCs, less than about 200,000 PBMCs, or less than about 100,000 PBMCs. In some embodiments, the method is capable of detecting Miro1 in a biological sample containing from about 10,000 to about 1,000,000 PBMCs, e.g., from about 10,000 to about 500,000 PBMCs, from about 100,000 to about 500,000 PBMCs, from about 10,000 to about 400,000 PBMCs, from about 100,000 to about 400,000 PBMCs, from about 10,000 to about 300,000 PBMCs, from about 100,000 to about 300,000 PBMCs, from about 10,000 to about 200,000 PBMCs, or from about 100,000 to about 200,000 PBMCs.

[0214] In some embodiments, a method of the present disclosure comprises a kit, e.g., an ELISA kit, that has a high sensitivity for detecting Miro1 in a biological sample, e.g., a sample that includes peripheral blood mononuclear cells (PBMCs). In some embodiments, the kit is capable of detecting Miro1 in a biological sample containing less than about 1,000,000 PBMCs, e.g., less than about 500,000 PBMCs, less than about 400,000 PBMCs, less than about 300,000 PBMCs, less than about 200,000 PBMCs, or less than about 100,000 PBMCs. In some embodiments, the kit is capable of detecting Miro1 in a biological sample containing from about 10,000 to about 1,000,000 PBMCs, e.g., from about 10,000 to about 500,000 PBMCs, from about 100,000 to about 500,000 PBMCs, from about 10,000 to about 400,000 PBMCs, from about 100,000 to about 400,000 PBMCs, from about 10,000 to about300,000 PBMCs, from about 100,000 to about 300,000 PBMCs, from about 10,000 to about 200,000 PBMCs, or from about 100,000 to about 200,000 PBMCs.

[0215] The neurodegenerative disease is any one recognized in the art. In some embodiments, the neurodegenerative disease is one described in the present section, e.g., Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, Friedreich's ataxia (FRDA), or Leber's Hereditary Optic Neuropathy (LHON). In some embodiments, the neurodegenerative disease is Parkinson’s disease.

[0216] A Miro1-reducing agent refers to any agent that decreases the level of a Miro1 nucleic acid, e.g., a Miro1 RNA or a Miro1 DNA, and / or a Miro1 protein in cells. In an exemplary embodiment, a Miro1-reducing agent may decrease at least one biological activity of a Miro1 protein in a cell with depolarized mitochondria. Exemplary biological activities of Miro1 include promoting mitochondrial transport, mitophagy, microtubule binding, mitochondrial fission and fusion among others. A Miro1-reducing agent can be, for example, a small molecule, a peptide, an aptamer, a protein or a functional fragment of a protein. A functional fragment of a protein, as used here, refers to all or part of the molecular elements of a protein which affect a specified function such as protein binding, signal transduction etc. In some embodiments, a Miro1-reducing agent is a compound, or pharmaceutically acceptable salt thereof, described in US publication no.20220334133; PCT publication no. WO 2022 / 216386; PCT application no. PCT / US23 / 73312; and US provisional application no. 63 / 609,598, which are each incorporated by reference herein in its entirety. For example, a Miro1-reducing agent can be Example 15 of PCT publication no. WO 2022 / 216386.VII. EXAMPLES

[0217] The following examples are provided to further aid in understanding the embodiments disclosed in the application, and presuppose an understanding of conventional methods well known to those persons having ordinary skill in the art to which the examples pertain. The materials and conditions described hereunder are intended to exemplify particular aspects of embodiments disclosed herein and should not be construed to limit the reasonable scope thereof. Example 1. Anti-Miro1 Monoclonal Antibody Production Materials

[0218] Monoclonal antibodies can be prepared by recovering spleen cells from immunized animals and immortalizing the cells in conventional fashion, e.g., by fusion with myeloma cells or by Epstein-Barr virus transformation, and screening for clones expressing the desired antibody. See, e.g., Kohler and Milstein Eur. J. Immunol.6:511 (1976).

[0219] Antigen Name: Miro1 Immunogen: Miro1 Host Strain: 4 New Zealand Rabbits

[0220] Indirect ELISA Coating Antigen: A: Miro2 protein B: Miro1 protein C: Miro2 Peptide D: Streptavidin + Miro1 Peptide-Biotin Coating Concentration: 1ug / ml, 100μl / well Coating Buffer: Phosphate Buffered Saline (PBS), pH 7.4 (“1X PBS”) Secondary Antibody: HRP Conjugated AffiniPure Donkey Anti-Rabbit IgG (H+L)

[0221] Immunogens: the immunogens were derived from human Miro1 (human mitochondrial Rho GTPase 1 variant 1): MKKDVRILLVGEPRVGKTSLIMSLVSEEFPEEVPPRAEEITIPADVTPERVPTHIVDYSEAE QSDEQLHQEISQANVICIVYAVNNKHSIDKVTSRWIPLINERTDKDSRLPLILVGNKSDLV EYSSMETILPIMNQYTEIETCVECSAKNLKNISELFYYAQKAVLHPTGPLYCPEEKEMKPA CIKALTRIFKISDQDNDGTLNDAELNFFQRICFNTPLAPQALEDVKNVVRKHISDGVADSG LTLKGFLFLHTLFIQRGRHETTWTVLRRFGYDDDLDLTPEYLFPLLKIPPDCTTELNHHAY LFLQSTFDKHDLDRDCALSPDELKDLFKVFPYIPWGPDVNNTVCTNERGWITYQGFLSQ WTLTTYLDVQRCLEYLGYLGYSILTEQESQASAVTVTRDKKIDLQKKQTQRNVFRCNVI GVKNCGKSGVLQALLGRNLMRQKKIREDHKSYYAINTVYVYGQEKYLLLHDISESEFLT EAEIICDVVCLVYDVSNPKSFEYCARIFKQHFMDSRIPCLIVAAKSDLHEVKQEYSISPTDF CRKHKMPPPQAFTCNTADAPSKDIFVKLTTMAMYPHVTQADLKSSTFWLRASFGATVF AVLGFAMYKALLKQR (SEQ ID NO: 100).

[0222] An N-terminal Miro1 immunogen was derived from amino acids 53-143 having the sequence: THIVDYSEAEQSDEQLHQEISQANVICIVYAVNNKHSIDKVTSRWIPLINERTDKDSRLPLI LVGNKSDLVEYSSMETILPIMNQYTEIE (SEQ ID NO: 101).

[0223] A C-terminal Miro1 immunogen was derived from amino acids 411-592 having the sequence: KKQTQRNVFRCNVIGVKNCGKSGVLQALLGRNLMRQKKIREDHKSYYAINTVYVYGQE KYLLLHDISESEFLTEAEIICDVVCLVYDVSNPKSFEYCARIFKQHFMDSRIPCLIVAAKSD LHEVKQEYSISPTDFCRKHKMPPPQAFTCNTADAPSKDIFVKLTTMAMYPHVTQADLKS STF (SEQ ID NO: 102). Methods Rabbit Antibody Generation

[0224] For rabbit monoclonal antibody production, 2-4 New Zealand white rabbits at 2.0- 2.5 kg were used for immunization with the designed immunogens. The immunogens were diluted with saline or sodium phosphate buffered saline (PBS) and mixed with Freund’s adjuvant and injected subcutaneously into four or six sites. One primary immunization and 2- 3 boost immunizations were given. Serum was collected after the second boost for analysis, and additional boost immunization was given if needed. Animals were exsanguinated at the end of the experiment. Indirect ELISA

[0225] The antigen was diluted in Coating Buffer and the ELISA plate was coated with 100 μL of the diluted antigen per well, with the concentration of coated antigen ranging from 1-10 μg / mL. The plate was covered with adhesive plastic and incubated at 37 °C for 1 hour or at 4 °C overnight. Buffer was removed and the plate washed 1X with 250 μL Washing Buffer (0.5 mL Tween 20 in 1000 mL PBS buffer). Blocking Buffer (100 mL Washing Buffer and 1 g bovine serum albumin) (150 μL) was added. The plate was covered with adhesive plastic and incubated at 37 °C for 1 hour or at 4 °C overnight. Buffer was removed and the plate washed 1X with 250 μL Washing Buffer.

[0226] The primary antibody or antiserum was diluted with Blocking Buffer and 100 μL of the diluted antibody was added per well. The plate was covered with adhesive plastic andincubated at 37 °C for 1 hour or at 4 °C overnight. Buffer was removed and the plate washed 1X with 250 μL Washing Buffer. The HRP-conjugated secondary antibody was diluted with Blocking Buffer and 100 μL of the diluted secondary antibody was added per well. The plate was covered with adhesive plastic and incubated at 37 °C for 1 hour or at 4 °C overnight. Buffer was removed and the plate washed 1X with 250 μL Washing Buffer.

[0227] TMB reagent (3, 3’, 5, 5’-tetramethylbenzidine, GenScript Cat. No. M00078) (100 μL) was added to each well, and the plate incubated at 25 °C. After about 15-20 minutes, Stop Solution (GenScript [83 mL 12 N HCl in 917 mL distilled water], 50 μL) was added to each well. The absorbance in each well was determined at 450 nm using a microplate reader. Example 2. Anti-Miro1 Antibody Cloning, Expression, and Purification Materials

[0228] Antigen Name: Miro1 Preservative: 0.02% ProClin 300 Buffer: PBS[1]Blocking Buffer as used in Example 1 Methods ELISA

[0229] Indirect ELISA was performed according to the protocol described in Example 1. Coating Antigen(s): A: Miro1 protein B: Miro2 proteinC: His tagged irrelevant proteinCoating Concentration: 1µg / ml, 100μl / well Coating Buffer: Phosphate Buffered Saline, pH 7.4 (“1X PBS”) Secondary Antibody: Anti-Rabbit IgG Fc Monoclonal Secondary Antibody (Min X Hu, Ms, Rt, Sh, Bv, Gt, Camel) (HRP conjugated) (GenScript, Cat. No. A01856). B cell cloning

[0230] The specific antigen was diluted with 1X PBS, filtered through 0.22 micron filter, added to a 10-mm dish to fix the antigen, sealed with plastic, and incubated at 4 °C overnight to coat. The dish was then washed with 1X PBS twice, and then Blocking Buffer was added at 37 °C.

[0231] PBMC separation: Histopaque-1077TM(5 mL / tube) was added to 15-mL tubes. Blood (5 mL) was mixed with 1X PBS (5 mL) and overlaid on top of the upper layer of the Histopaque-1077TM, and then centrifuged according to manufacturer’s specification at 400 X g for 35 minutes at room temperature. After centrifugation, the lymphocyte layer was moved to a new 50 mL centrifuge tube, 1X PBS was added, and the mixture centrifuged at 200 X g for 15 minutes at room temperature. The supernatant was discarded, red blood cell lysing buffer was added to resuspend the cells, and the mixture incubated at 37℃. Then an appropriate amount of 1X PBS was added to stop the reaction. The mixture was centrifuged at 200 X g for 15 minutes at room temperature, whereupon the supernatant was discarded. An appropriate amount of cell culture medium (CM) was added to fully suspend the PBMC, and cell number was counted.

[0232] The PBMCs obtained from the previous step were added to the dish coated with antigen, and plated in an incubator at 37 ℃ for enrichment culture. Unknotted cells were washed away. The enriched B cells were suspended in CM and mixed with feeder cells. The cell suspension was plated into 96-well cell culture plates and cultured at 37 ℃. After 6 days, the cultured supernatant was transferred to an ELISA plate for analysis. Recombinant Expression

[0233] Materials: ExpiFectamineTMCHO Transfection kit, GibcoTMExpiCHOTMExpression Medium, and OptiPROTMSFM (Thermo Fisher Scientific).

[0234] Method: CHO-S cells were cultured with GibcoTMExpiCHOTMExpression Medium (Fisher Scientific) and incubated at 36.5 °C, 8% CO2, 100 rpm in carbon dioxide oscillation incubator. Plasmids of the heavy and light chains were determined using micro- spectrophotometer. According to the transfection volume, an appropriate amount of the transfection mixture was prepared and added to the cell suspension to be transfected. The transfected cells were cultured in the incubator at 32 °C, 5% CO2, 120 rpm. Nutrient was added 24 hours after transfection and then again on day 5 after transfection. Cells were counted using a cell counter. The cell supernatant was collected at day 10-14.Antibody Purification

[0235] Protein A resin was completely suspended in a column. Binding / Wash Buffer (10x resin volume) was added on to the column to equilibrate the resin. The antibody sample was transferred to the column, and flow-through was collected. The column was connected to a nucleic acid protein detector and absorbance was measured at 280 nm. Binding / Wash Buffer was added onto the column to wash the resin until absorbance at 280 nm was stable. Binding / Wash Buffer was drained from the column, and the Elution Buffer was added to elute the antibody bound to the resin. Eluent containing the antibody was collected based on 280 nm absorbance. To the antibody eluent was added the Neutralization Buffer to neutralize pH. The antibody eluent was then transferred to a dialysis bag and dialyzed against PBS at 2- 8 °C overnight. ProClinTM300 Buffer (Sigma Aldrich) was added to the antibody solution with a final concentration of 0.02%, and the solution was stored at 2-8 °C.

[0236] Illustrative results for the above methods in the preparation of 40H2-1 antibody is shown in Table 3 and Table 4 below. Table 3. anti-Miro1 Polypeptide Concentration and Purity Clone ID ConcentrationVolume Quantity (mg / ml)[1][2] (ml)[2](mg)[2]Purity[3] 2H2O, NaH2PO4·2H2O,NaCl (PBS, pH 7.4). [2] Concentration is measured by NanoDrop Spectrophotometer at 280 nm absorbance. [3] Concentration, volume and quantity data are for specified single tube information. Table 4: ELISA results of the recombinant MonoRabTMantibody Concentration1,000.00 500.00 250.00 125.00 62.50 31.25 15.62l 003 56Concentration 7.81 3.90 1.95 / / / (ng / ml)[4]g

[0237] Storage condition: The antibody was stable at 2-8 C for up to one month. For long- term storage, the antibody was stored at -20°C or below, while avoiding repeated freeze-thaw cycles. Example 3. Sequencing of Anti-Miro1 Antibodies

[0238] Materials: B cells prepared by GenScript; RNA-easy Isolation Reagent (Vazyme, Cat. No.: R701-01-AA); PrimeScriptTM1st Strand cDNA Synthesis Kit (Takara, Cat. No.: 6110A).

[0239] Methods: Total RNA was isolated from the B cells following the technical manual of RNA-easy Isolation Reagent. Total RNA was then reverse-transcribed into cDNA using either isotype-specific anti-sense primers or universal primers following the technical manual of PrimeScriptTM1st Strand cDNA Synthesis Kit. Antibody fragments of heavy chain and light chain were amplified using specific primers according to the standard operating procedure (SOP) of GenScript. Amplified antibody fragments were cloned into a standard cloning vector separately. Colony PCR was performed to screen for clones with inserts of correct sizes. The consensus sequence was provided.

[0240] The following nucleotide and peptide sequences show signal sequences in italics and complementarity-determining regions (CDRs) in underline. Consistent with standard practice, the signal peptides were cleaved off during the expression process. 77C5-1 antibody sequences

[0241] Heavy chain: DNA sequence (405 bp): signal sequence in italics, nucleotides encoding CDRs underlined: Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGT CAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACTCCCCTGACA CTCACCTGCACAGCTTCTGGATTCTCCCGCAGTAGCTATGCAGTGCGATGGGTCC GCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAATCATTAGTGATAGTGGTA CCACATACTACGCGAATTGGGCGATAGGCCGATTCACCATCTCCAAAACCTCGAC CACGGTGGATCTAAAAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTT CTGTGCCAGATACGATGATTATGGTGATTCCATTTTACACATCTGGGGCCCAGGC ACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 200)

[0242] Heavy chain: Amino acid sequence (135 aa): signal sequence in italics, CDRs underlined: Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 METGLRWLLLVAVLKGVQCQSLEESGGRLVTPGTPLTLTCTASGFSRSSYAVRWVRQA PGKGLEWIGIISDSGTTYYANWAIGRFTISKTSTTVDLKITSPTTEDTATYFCARYDDY GDSILHIWGPGTLVTVSS (SEQ ID NO: 201)

[0243] Light chain: DNA sequence (396 bp): signal sequence in italics, nucleotides encoding CDRs underlined: Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGT GCCAGATGTGATGTTGTGATGACCCAGACTCCATCCTCCGTGTCTGCAGCTGTGGG AGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAATATTGATAGTAATTTAGTC TGGTATCAGCAGAAACCAGGGCAGCGTCCCAAGCTCCTGATCTCTGCTGCATCCA ATCTGGCATCTGGGGTCCCATCGCGGTTCAGTGGCAGTGGATCTGGGTCAGAGTT CACTCTCACCATCAGCGACCTGGAGTGTGCCGATTCTGCCACTTACTACTGTCAA TGTACTTATTATAGTAGTAGTTATGTTGAAGTTTTCGGCGGAGGGACCGAGGTGG TGGTCAAA (SEQ ID NO: 202)

[0244] Light chain: Amino acid sequence (132 aa): signal sequence in italics, CDRs underlined: Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4MDTRAPTQLLGLLLLWLPGARCDVVMTQTPSSVSAAVGGTVTIKCQASQNIDSNLVW YQQKPGQRPKLLISAASNLASGVPSRFSGSGSGSEFTLTISDLECADSATYYCQCTYY SSSYVEVFGGGTEVVVK (SEQ ID NO: 203) Table 5.77C5-1 Antibody Subtype and IMGT Analysis of V(D)J Junctions Seque Subty V-GENE Functio V- J- D- AA Junction nce pe and allele nality REGION GENE GENE Junction frame identity and and

[0245] Heavy chain: DNA sequence (405 bp): signal sequence in italics, nucleotides encoding CDRs underlined: Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGT CAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACA CTCACCTGCACAGCCTCTGGATTCTCCCGCAGTAGCTATGCAGTGCGATGGGTCC GCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAATCATTAGTGATAGTGGTA CCACATACTACGCGAATTGGGCGATAGGCCGATTCACCATCTCCAAAACCTCGAC CACGGTGGATCTAAAAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTT CTGTGCCAGATACGATGATTATGGTGATTCCATTTTACACATCTGGGGCCCAGGC ACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 204)

[0246] Heavy chain: Amino acid sequence (135 aa): signal sequence in italics, CDRs underlined: Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 METGLRWLLLVAVLKGVQCQSLEESGGRLVTPGTPLTLTCTASGFSRSSYAVRWVRQA PGKGLEWIGIISDSGTTYYANWAIGRFTISKTSTTVDLKITSPTTEDTATYFCARYDDY GDSILHIWGPGTLVTVSS (SEQ ID NO: 205)

[0247] Light chain: DNA sequence (396 bp): signal sequence in italics, nucleotides encoding CDRs underlined: Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGT GCCAGATGTGATGTTGTGATGACCCAGACTCCATCCTCCGTGTCTGCAGCTGTGGG AGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAATATTGATAGTAATTTAGTC TGGTATCAGCAGAAACCAGGGCAGCGTCCCAAGCTCCTGATCTCTGCTGCATCCA ATCTGGCATCTGGGGTCCCATCGCGGTTCAGTGGCAGTGGATCTGGGTCAGAGTT CACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTGCCACTTACTACTGTCAA TGTACTTATTATAGTAGTAGTTATGTTGAAGTTTTCGGCGGAGGGACCGAGGTGG TGGTCAAA (SEQ ID NO: 206)

[0248] Light chain: Amino acid sequence (132 aa): signal sequence in italics, CDRs underlined: Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 MDTRAPTQLLGLLLLWLPGARCDVVMTQTPSSVSAAVGGTVTIKCQASQNIDSNLVW YQQKPGQRPKLLISAASNLASGVPSRFSGSGSGSEFTLTISDLECADAATYYCQCTYY SSSYVEVFGGGTEVVVK (SEQ ID NO: 207) Table 6.72A12-3 Antibody Subtype and IMGT Analysis of V(D)J Junctions Seque Subty V-GENE Functio V- J- D- AA Junction nce and allele nality REGION GENE GENE Junction frame40H2-1 antibody sequences

[0249] Heavy chain: DNA sequence (393 bp): signal sequence in italics, nucleotides encoding CDRs underlined:Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGT CAGTCGGTGGGGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACA CTCACCTGCACAGTCTCTGGATTCTCCCTCAGTAGCAATGCAATAAGCTGGGTCC GCCAGGCTCCAGGGAAGGGGCTGGAATATATCGGATTCATTGGTTATACGGGTA ACACATACTACGCGACCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGA CCACGGTGGATCTGAAAATGACCAGTCCGACAACCGAGGACACGGCCACCTATT TCTGTGCCAGAGGGTCTGCCTGGTTGGATCCCTGGGGCCAGGGCACCCTGGTCAC CGTCTCTTCA (SEQ ID NO: 208)

[0250] Heavy chain: Amino acid sequence (131 aa): signal sequence in italics, CDRs underlined: Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 METGLRWLLLVAVLKGVQCQSVGESGGRLVTPGTPLTLTCTVSGFSLSSNAISWVRQA PGKGLEYIGFIGYTGNTYYATWAKGRFTISKTSTTVDLKMTSPTTEDTATYFCARGS AWLDPWGQGTLVTVSS (SEQ ID NO: 209)

[0251] Light chain: DNA sequence (396 bp): signal sequence in italics, nucleotides encoding CDRs underlined: Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGT GCCACATTTGCTCAAGTGCTGACCCAGACTGCATCGCCCGTGTCTGCAGCTGTGGG AGGCACAGTTACCATCAAGTGCCAGTCCAGTCAGAGTGTTTATACTAACACCCGC TTATCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACAAGG CATCCACTCTGGCATCTGGGGTCCCATCGCGGTTCAGTGGCAGTGGATCTGGGAC ACAGTTCACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACTTACTAC TGTCTAGGCGTTTATAGTGGTAATATTGCTGTTTTCGGCGGAGGGACCGAGGTGG TGGTCAAA (SEQ ID NO: 210)

[0252] Light chain: Amino acid sequence (132 aa): signal sequence in italics, CDRs underlined: Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4MDTRAPTQLLGLLLLWLPGATFAQVLTQTASPVSAAVGGTVTIKCQSSQSVYTNTRLS WYQQKPGQPPKLLIYKASTLASGVPSRFSGSGSGTQFTLTISGVQCDDAATYYCLGV YSGNIAVFGGGTEVVVK (SEQ ID NO: 211) Table 7.40H2-1 Antibody Subtype and IMGT Analysis of V(D)J Junctions Seque Subty V-GENE Functio V- J- D- AA Junction nce pe and allele nality REGION GENE GENE Junction frame identity and andExample 4. Miro1 ELISA Assay

[0253] The following protocol was adapted from literature procedures, for example,Kuster, D. W. D. et al. J. Vis. Exp. 2013; (78): 50786. The equipment and materials usedincluded a Meso Scale Discovery (MSD) standard plate (Meso Scale Diagnostics, Rockville, MD USA), MSD 4X reading buffer, MSD blocker A (bovine serum albumin, BSA) at 4 C, 1X PBS buffer (pH 7.4), and a plate shaker (Fisher).

[0254] The capture antibody (non-tagged) was the rabbit anti-Miro177C5-1 antibody described in Example 3. The detection antibody (SULFO-tagged) was the rabbit anti-Miro1 40H2-1 antibody described in Example 3.

[0255] Working solutions were prepared as follows: -blocker was a 3% blocker A solution prepared by dissolving 1.5 g blocker A in 50 mLPBS buffer; -diluent for antibodies: 1% blocker A solution prepared by diluting the above 3%blocker A solution with additional PBS buffer.30 mL of the 3% blocker A solution was further diluted by adding 60 mL PBS; -Wash buffer: PBS-T (0.05% Tween 20 in PBS); and- 2X reading buffer: MSD 4X reading buffer was diluted 1:1 with water.

[0256] The assay was performed at room temperature. The capture antibody was prepared by diluting in PBS 1:400 (250 ng / mL). The coating peptide (40 μL / well) was loaded in the assay plate. The diluent for antibodies was prepared. All of these reagents were incubated at 4 °C overnight.

[0257] After overnight incubation, the coated assay plate and blocker were allowed to warm to room temperature. The wells were washed with PBS-T (150 μL) three times. The sample and standard peptide (full length Miro1 from 0-1000 ng / mL 1:5 dilution, 8 concentration points (ab163047, Abcam, 250 ng / mL, 50 μL / well)) were loaded, and incubated for 2 hr with shaking at 700 rpm. The wells were washed with PBS-T (150 μL) three times. The detection antibody (1:500, 40 μL / well) was then loaded onto the plate, which was incubated for 2 hr with shaking at 700 rpm. The wells were washed with PBS-T (150 μL) three times. The 2X reading buffer (150 μL / well) was loaded. The plate was then read by determining absorbance in each well at 450 nm. Miro1 vs. Miro2 Peptide

[0258] Standard curves using recombinant Miro1 and Miro2 proteins were tested in the above ELISA protocol. An exemplary graph is shown in FIG.1. The ELISA assay demonstrated at least 100 times higher sensitivity in detecting Miro1 compared to Miro2. HeLa Cell Lysates

[0259] HeLa cell lysates were obtained from Abcam (Waltham, MA USA). Wild type and Miro1 knockout lysates (2 mg / mL) were each diluted 1:100 before use. The raw Miro1 readings of wild type (“WT”) cell lysate samples compared with Miro1 knockout (“KO”) lysates are shown in FIG.2. PBMC

[0260] A peripheral blood mononuclear cell (PBMC) sample was obtained from a healthy human subject, and divided into three groups: (1) vehicle control, (2) treated for 14 hr with 40 μM CCCP, and (3) treated for 14 hr with 20 μM Antimycin A and 20 μM Oligomycin. Plate loading was done at 1:1 dilution from 1.8x106cells, 8 sample dilution.

[0261] The goal of the PBMC study was to determine the number of cells required to see a reproducible Miro1 signal. FIG.3 shows PBMCs from healthy subjects that were treated with either a vehicle (DMSO) or a mitochondrial stressor (a combination of Antimycin A andOligomycin) to induce lowering of Miro1 levels. Different amounts of cell lysates, reflected in the corresponding PBMC cell counts shown on the x-axis, were tested to determine the minimal cell input material required to detect Miro1 lowering within the dynamic range of the ELISA. The results showed that a lowering of Miro1 levels was demonstrated with as few as 225,000 PBMCs per ELISA well. The data is shown as the ratio of Miro1 levels when comparing mitochondrial stressor normalized to DMSO at each input level. Example 5. Miro1 ELISA: Comparison with Commercial anti-Miro1 Antibodies

[0262] Illustrative anti-Miro1 antibodies of the present disclosure were compared with commercial anti-Miro1 antibodies, e.g., Abcam antibodies ab320018 and ab320019.

[0263] A 96-well MesoScale Discovery (MSD) ELISA plate was first coated with anti- Miro1 capture antibodies (77C5-1, 72A12-3, or Abcam ab320018) at a final concentration of 1.2 μg / mL and incubated overnight at 4 °C. The next day, the plate was washed with a standard MSD wash buffer to remove excess unbound antibody, and then the plate was blocked for 1 hour with standard MSD blocking buffer for 1 hour at room temperature. Some wells (for each antibody) then received a titration curve of recombinant human Miro1 peptide to generate a standard curve. Some wells (for each antibody) received lysates collected from either 500,000 or 250,000 naive human PBMCs. Incubations then proceeded for 2 hours at room temperature. The plate was washed with a standard MSD wash buffer to remove excess unbound protein. The wells then received the corresponding detection antibodies (40H2-1 to match with 77C5-1, 40H2-1 to match with 72A12-3, or Abcam ab320019 to match with Abcam ab320018) labeled with MSD SULFO-TAG and were incubated for 2 hours at room temperature. MSD read buffer was then added and the signals from the 96-well plate were measured using an MSD MESO QuickPlex SQ 120MM instrument. Standard curves of MSD raw signal vs. pg / well of recombinant human Miro1 peptide were then calculated and used to determine the amount of endogenous Miro1 protein detected in the PBMC lysates.

[0264] Based on the standard curve generated for each antibody pair, an interpolated value for Miro1 protein levels in naïve PBMCs was calculated using the detected signal for specific amounts of recombinant Miro1 protein (Table 8, Table 9, and Table 10.) The data suggests that the Miro1 antibody pairs of the present disclosure (77C5-1-40H2-1 and 77A12-3-40H2- 1) are more sensitive at detecting endogenous Miro1 protein levels in naïve human PBMCs than the Abcam Miro1 antibody pair. All antibody pairs tested were capable of detecting Miro1 protein when the lysate from 500,000 naïve PBMCs was loaded into a single well ofan ELISA assay plate. However, the Abcam antibody ELISA yielded a lower signal (Table 10). Only the Miro1 antibody pairs of the present disclosure were capable of detecting endogenous Miro1 protein when the lysate from 250,000 naïve PBMCs was loaded into a single well of an ELISA assay plate (Table 8 and Table 9).

[0265] This data suggests that the Miro1 antibody pairs of the present disclosure are more sensitive at detecting endogenous Miro1 protein levels in human PBMC lysates compared to the Abcam commercial Miro1 antibodies. A higher ELISA sensitivity may be critical for developing a high throughput clinical grade Miro1 ELISA biomarker assay that would require a relatively low number of cells and thus would have a lower patient burden. Table 8.77C5-1 Capture / 40H2-1 Detection Interpolated Miro1 Value (pg / well) Based on Standard Curve Number of Naïve PBMCs Miro1 (pg / well)Table 9.72A12-3 Capture / 40H2-1 Detection Interpolated Miro1 Value (pg / well) Based on Standard Curve Number of Naïve PBMCs Miro1 (pg / well)Table 10. Abcam ab320018 Capture / Abcam ab320019 Detection Interpolated Miro1 Value (pg / well) Based on Standard Curve Number of Naïve PBMCs Miro1 (pg / well)Standard curve R2= 0.9961 Example 6. Anti-Miro1 Antibodies in Flow Cytometry

[0266] Illustrative anti-Miro1 antibodies of the present disclosure were compared with commercial anti-Miro1 antibodies, e.g., Abcam antibodies ab320019, when used in flow cytometry.

[0267] Naïve PBMCs were fixed in 4% paraformaldehyde solution for 20 minutes at room temperature. This was then washed with PBS buffer. The resulting cell mixture was spun at 300g for 10 minutes at 4 °C and supernatant removed.

[0268] The cell suspension was blocked with blocking buffer (150 μL including 2.5 μL FC receptor blocker per 1 x 106cells) for 1 hr at room temperature. Blocking buffer was prepared by mixing 50 mL PBS and 0.25 g filtered bovine serum albumin with 5 mL normal goat serum. Triton X-100 detergent was added at 0.1% final concentration in the blocking buffer.

[0269] After blocking, anti-Miro1 primary antibody at 4, 2, 1, 0.5, or 0.25 μg / mL was added in blocking buffer for 30 minutes at room temperature with shaking. The resulting cell mixture was spun at 300 g for 10 minutes at 4 °C and supernatant removed. The mixture was then washed with PBS. Secondary antibody at 4, 2, 1, 0.5, or 0.25 μg / mL was then added and the mixture incubated for 20 minutes at room temperature. The cell mixture was then washed with PBS, and analyzed on the flow cytometer.

[0270] FIG.4A-4B show one-dimensional fluorescence intensity flow cytometry histograms as measured by flow cytometry using anti-Miro1 monoclonal antibody 40H2-1 (FIG.4A) and commercial anti-Miro1 antibody Abcam ab320019 (FIG.4B). FIG.4A showed that 40H2-1 could resolve populations of PBMCs that exhibited Miro1 with a peak signal about 100-fold higher than the corresponding isotype control antibody, in contrast to commercial ab320019 which achieved a weaker peak signal of about 10-fold compared with the isotype control antibody.

[0271] Although the foregoing invention has been described in some detail by way of illustration and Example for purposes of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference.Where a conflict exists between the instant application and a reference provided herein, the instant application shall dominate. Table 11. Sequences Name Sequence 77C5-1 VHQSLEESGGRLVTPGTPLTLTCTASGFSRS (SEQ ID NO: 1)Name Sequence 40H2-1 VLWYQQKPGQPPKLLIY (SEQ ID NO: 15) f k D H G G A C G T T G A G D H N C GName Sequence 72A12-3 TGCGATGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAA V TCATTAGTGATAGTGGTACCACATACTACGCGAATTGGGCGATAGGCC C G T T G A G Y W V C A T C T G G C C GName Sequence Miro1 MKKDVRILLVGEPRVGKTSLIMSLVSEEFPEEVPPRAEEITIPADVTPERVPT V i t 1 HIVDYSEAE SDE LH EIS ANVICIVYAVNNKHSIDKVTSRWIPLINERT L L R T F V H S I F G G C C S I ) G T C A C S S G G GName Sequence signal CGAATTGGGCGATAGGCCGATTCACCATCTCCAAAACCTCGACCAC T AT TAAAAAT A A T A AA A A A A C C S I ) G T C A C S S G T G C T C S K G C C A G T L

Claims

WHAT IS CLAIMED IS:

1. An anti-Miro1 polypeptide or binding fragment thereof that comprises (i) a VH chain comprising three CDRs; and (ii) a VL chain comprising three CDRs, wherein: VH CDR #1 is: SYAVR (SEQ ID NO: 21) or SNAIS (SEQ ID NO: 41); VH CDR #2 is: IISDSGTTYYANWAIG (SEQ ID NO: 22) or FIGYTGNTYYATWAKG (SEQ ID NO: 42); VH CDR #3 is: YDDYGDSILHI (SEQ ID NO: 23) or GSAWLDP (SEQ ID NO: 43); VL CDR #1 is: QASQNIDSNLV (SEQ ID NO: 24) or QSSQSVYTNTRLS (SEQ ID NO: 44); VL CDR #2 is: AASNLAS (SEQ ID NO: 25) or KASTLAS (SEQ ID NO: 45); and VL CDR #3 is: QCTYYSSSYVEV (SEQ ID NO: 26) or LGVYSGNIAV (SEQ ID NO: 46).

2. The anti-Miro1 polypeptide or binding fragment thereof of claim 1, wherein: (a) VHCDR #1 is SYAVR (SEQ ID NO: 21); VH CDR #2 is IISDSGTTYYANWAIG (SEQ ID NO: 22); VHCDR #3 is YDDYGDSILHI (SEQ ID NO: 23); VL CDR #1 is QASQNIDSNLV (SEQ ID NO: 24); VLCDR #2 is AASNLAS (SEQ ID NO: 25); and VL CDR #3 is QCTYYSSSYVEV (SEQ ID NO: 26); or (b) VH CDR #1 is SNAIS (SEQ ID NO: 41); VHCDR #2 is FIGYTGNTYYATWAKG (SEQ ID NO: 42);VHCDR #3 is GSAWLDP (SEQ ID NO: 43); VL CDR #1 is QSSQSVYTNTRLS (SEQ ID NO: 44); VLCDR #2 is KASTLAS (SEQ ID NO: 45); and VL CDR #3 is LGVYSGNIAV (SEQ ID NO: 46).

3. The anti-Miro1 polypeptide or binding fragment thereof of claim 1 or 2, that comprises (i) a VH chain comprising four framework regions; and (ii) a VL chain comprising four framework regions, wherein: the first VH framework region corresponds in sequence to SEQ ID NO: 1 or 10; the second VHframework region corresponds in sequence to SEQ ID NO: 2 or 11; the third VH framework region corresponds in sequence to SEQ ID NO: 3 or 12; the fourth VHframework region corresponds in sequence to SEQ ID NO: 4 or 13; the first VL framework region corresponds in sequence to SEQ ID NO: 5 or 14; the second VLframework region corresponds in sequence to SEQ ID NO: 6 or 15; the third VL framework region corresponds in sequence to SEQ ID NO: 7, 9, or 16; and the fourth VL framework region corresponds in sequence to SEQ ID NO:

8.

4. The anti-Miro1 polypeptide or binding fragment thereof of claim 1 or 2, that comprises (i) a VHchain comprising four framework regions; and (ii) a VLchain comprising four framework regions, wherein: (a) the first VHframework region corresponds in sequence to SEQ ID NO: 1; the second VH framework region corresponds in sequence to SEQ ID NO: 2; the third VHframework region corresponds in sequence to SEQ ID NO: 3; the fourth VH framework region corresponds in sequence to SEQ ID NO: 4; the first VLframework region corresponds in sequence to SEQ ID NO: 5; the second VL framework region corresponds in sequence to SEQ ID NO: 6; the third VLframework region corresponds in sequence to SEQ ID NO: 7; and the fourth VL framework region corresponds in sequence to SEQ ID NO: 8; or (b) the first VH framework region corresponds in sequence to SEQ ID NO: 1; the second VHframework region corresponds in sequence to SEQ ID NO: 2; the third VH framework region corresponds in sequence to SEQ ID NO: 3; the fourth VHframework region corresponds in sequence to SEQ ID NO: 4;the first VLframework region corresponds in sequence to SEQ ID NO: 5; the second VL framework region corresponds in sequence to SEQ ID NO: 6; the third VLframework region corresponds in sequence to SEQ ID NO: 9; and the fourth VL framework region corresponds in sequence to SEQ ID NO: 8; or (c) the first VH framework region corresponds in sequence to SEQ ID NO: 10; the second VHframework region corresponds in sequence to SEQ ID NO: 11; the third VH framework region corresponds in sequence to SEQ ID NO: 12; the fourth VHframework region corresponds in sequence to SEQ ID NO: 13; the first VL framework region corresponds in sequence to SEQ ID NO: 14; the second VLframework region corresponds in sequence to SEQ ID NO: 15; the third VL framework region corresponds in sequence to SEQ ID NO: 16; and the fourth VLframework region corresponds in sequence to SEQ ID NO:

8.

5. The anti-Miro1 polypeptide or binding fragment thereof of any one of claims 1 to 4, having a VHchain corresponding in sequence to SEQ ID NO: 27, 37, or 47, and a VL chain corresponding in sequence to SEQ ID NO: 28, 38, or 48.

6. The anti-Miro1 polypeptide or binding fragment thereof of claim 5, having a VHchain corresponding in sequence to SEQ ID NO: 27, and a VLchain corresponding in sequence to SEQ ID NO:

28.

7. The anti-Miro1 polypeptide or binding fragment thereof of claim 5, having a VH chain corresponding in sequence to SEQ ID NO: 37, and a VL chain corresponding in sequence to SEQ ID NO:

38.

8. The anti-Miro1 polypeptide or binding fragment thereof of claim 5, having a VH chain corresponding in sequence to SEQ ID NO: 47, and a VL chain corresponding in sequence to SEQ ID NO:

48.

9. The anti-Miro1 polypeptide or binding fragment thereof of any one of claims 1 to 8, which is a monoclonal antibody.

10. The anti-Miro1 polypeptide or binding fragment thereof of claims 1 to 8, which is a polyclonal antibody.

11. The anti-Miro1 polypeptide or binding fragment thereof of claim 9, having an IgG heavy chain; and / or a kappa light chain.

12. The anti-Miro1 polypeptide or binding fragment thereof of any one of claims 1 to 11, which is selective over Miro2.

13. The anti-Miro1 polypeptide or binding fragment thereof of claim 12, which is at least 3-fold, at least 10-fold, at least 100-fold, or at least 1000-fold selective over Miro2.

14. An anti-Miro1 polypeptide or binding fragment thereof that comprises (i) a VHchain comprising three CDRs; and (ii) a VLchain comprising three CDRs; which is at least 3-fold, at least 10-fold, at least 100-fold, or at least 1000-fold selective over Miro2.

15. A composition comprising the anti-Miro1 polypeptide or binding fragment thereof of any one of claims 1 to 14, and an acceptable excipient.

16. A nucleic acid comprising a nucleotide sequence encoding an anti- Miro1 polypeptide or binding fragment thereof of any one of claims 1 to 14.

17. A vector comprising the nucleic acid of claim 16.

18. A host cell engineered to express the nucleic acid of claim 16.

19. A host cell transformed with the vector of claim 17.

20. The host cell of claim 18 or 19 that is eukaryotic.

21. A kit comprising the anti-Miro1 polypeptide or binding fragment thereof of any one of claims 1 to 14 or the nucleic acid of claim 16, and instructions for use.

22. The kit of claim 21, comprising a first anti-Miro1 polypeptide or binding fragment thereof and a second anti-Miro1 polypeptide or binding fragment thereof.

23. The kit of claim 22, wherein:the first anti-Miro1 polypeptide or binding fragment thereof has a VHchain corresponding in sequence to SEQ ID NO: 27 or 37, and a VL chain corresponding in sequence to SEQ ID NO: 28 or 38; and the second anti-Miro1 polypeptide or binding fragment thereof has a VH chain corresponding in sequence to SEQ ID NO: 47, and a VLchain corresponding in sequence to SEQ ID NO:

48.

24. The kit of claim 23, wherein the first anti-Miro1 polypeptide or binding fragment thereof has a VH chain corresponding in sequence to SEQ ID NO: 27, and a VLchain corresponding in sequence to SEQ ID NO:

28.

25. The kit of claim 23, wherein the first anti-Miro1 polypeptide or binding fragment thereof has a VHchain corresponding in sequence to SEQ ID NO: 37, and a VL chain corresponding in sequence to SEQ ID NO:

38.

26. A method of quantifying Miro1 in a sample from a subject comprising: (a) contacting the sample with a carrier comprising a first anti-Miro1 polypeptide or binding fragment thereof; and (b) detecting binding between the carrier and the Miro1 with a second anti-Miro1 polypeptide or binding fragment thereof.

27. The method of claim 26, wherein the first anti-Miro1 polypeptide or binding fragment thereof specifically binds to the C-terminus of Miro1; and the second anti-Miro1 polypeptide or binding fragment thereof specifically binds to the N-terminus of Miro1.

28. The method of claim 26 or 27, wherein the first anti-Miro1 polypeptide or binding fragment thereof has a VH chain corresponding in sequence to SEQ ID NO: 27 or 37, and a VL chain corresponding in sequence to SEQ ID NO: 28 or 38; and the second anti-Miro1 polypeptide or binding fragment thereof has a VH chain corresponding in sequence to SEQ ID NO: 47, and a VL chain corresponding in sequence to SEQ ID NO: 48.

29. The method of claim 28, wherein the first anti-Miro1 polypeptide or binding fragment thereof has a VH chain corresponding in sequence to SEQ ID NO: 27, and a VL chain corresponding in sequence to SEQ ID NO:

28.

30. The method of claim 28, wherein the first anti-Miro1 polypeptide or binding fragment thereof has a VHchain corresponding in sequence to SEQ ID NO: 37, and a VL chain corresponding in sequence to SEQ ID NO:

38.

31. A method of detecting a disease or disorder characterized by an elevated Miro1 in a subject, the method comprising: (a) quantifying Miro1 in a sample from the subject comprising: (i) contacting the sample with a carrier comprising a first anti-Miro1 polypeptide or binding fragment thereof; and (ii) determining binding between the carrier and the Miro1 with a second anti- Miro1 polypeptide or binding fragment thereof; (b) comparing the sample Miro1 level with a control Miro1 level; and (c) detecting the disease or disorder characterized by an elevated Miro1 if the sample Miro1 level is higher than the control Miro1 level.

32. A method of treating a disease or disorder characterized by an elevated Miro1 in a subject, the method comprising: (a) quantifying Miro1 in a sample from the subject comprising: (i) contacting the sample with a carrier comprising a first anti-Miro1 polypeptide or binding fragment thereof; and (ii) determining binding between the carrier and the Miro1 with a second anti- Miro1 polypeptide or binding fragment thereof; (b) comparing the sample Miro1 level with a control Miro1 level; (c) detecting the disease or disorder characterized by an elevated Miro1 if the sample Miro1 level is higher than the control Miro1 level; and (d) administering a therapeutically effective amount of a Miro1-reducing agent to the subject.

33. A method of treating a neurodegenerative disease in a subject, the method comprising: (a) quantifying Miro1 in a sample from the subject comprising: (i) contacting the sample with a carrier comprising a first anti-Miro1 polypeptide or binding fragment thereof; and (ii) determining binding between the carrier and the Miro1 with a second anti- Miro1 polypeptide or binding fragment thereof; (b) comparing the sample Miro1 level with a control Miro1 level; (c) detecting the neurodegenerative disease if the sample Miro1 level is higher than the control Miro1 level; and (d) administering a therapeutically effective amount of a Miro1-reducing agent to the subject.

34. The method of claim 33, wherein the neurodegenerative disease is Parkinson’s disease.

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