Antibodies that bind to post-translationally modified c9ORF72 arginine-rich dipeptide repeat proteins and uses thereof

Abstract

Isolated monoclonal antibodies that bind to methylated dipeptide repeat proteins (DRPs), e.g., ADMe-GR and SDMe-GR, and related antibody-based compositions and molecules are disclosed. Also disclosed are diagnostic and therapeutic methods for using the antibodies.

Description

[0001] Attorney Docket No. ALSE-016PC

[0002] ANTIBODIES THAT BIND TO POST-TRANSLATIONALLY MODIFIED C9orf72 ARGININE-RICH DIPEPTIDE REPEAT PROTEINS AND USES THEREOF

[0003] Related Applications

[0004] This application is a U. S. Patent Application which claims priority to United States Provisional Application No. 63 / 740,196, filed on December 30, 2024, the contents of which are hereby incorporated by reference.

[0005] Background of the Invention

[0006] The most frequent known single genetic causes of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are intronic repeat expansion mutations (i.e., expansion of a hexanucleotide (GGGGCC) repeat) in the gene chromosome 9 open reading frame 72 C9orf72 gene). Both toxic gain-of-function and loss-of-function consequences are believed to result from a hexanucleotide repeat expansion mutation in C9orf72 (C9-HRE). Toxic gain-of-function from the C9-HRE manifests in the production of five unique dipeptide repeat proteins (DRPs). Specifically, RNA transcribed from a mutated C9ORF72 gene that contains expanded GGGGCC repeats is translated through a non-ATG initiated mechanism. This drives the formation and intracellular accumulation of dipeptide repeat proteins (DRPs). DRPs translated from all six reading frames in either the sense or antisense direction of the hexanucleotide repeat result in the expression of five DRPs: glycine-alanine (GA), glycinearginine (GR), proline-alanine (PA), proline-arginine (PR) and glycine-proline (GP).

[0007] DRPs polyGR and polyPR have been shown to bind nucleoli, impede RNA biogenesis, and are particularly detrimental to cellular viability via cellular dysfunction leading to cell death, especially in neuronal cells (Kwon, et al. Science, 545(6201), 1139— 1145). As a result, polyGR in cerebrospinal fluid (CSF) has been investigated as a sensitive fluid-based biomarker for C9orf72 -mediated ALS and FTD (C9-ALS / FTD) (Krishnan, et al. Nature Communications, 75(1), 2799). Recently, the methylation state of polyGR has been implicated in aspects of C9-ALS disease duration (Gittings, et al. Acta Neuropathologica, 139(2), 407-410), and serum asymmetric dimethylarginine (ADMA) has been correlated with C9-ALS progression and prognosis (Ikenaka, et al. European Journal of Neurology, 29(5), 1410-1416). However, the ability to detect methylated forms of polyGR in biomarker discovery has not been explored. Accordingly, reagents for detecting DRPs and inhibiting intronic repeat expansion mutations in the C9orf72 gene are needed and are desirable for a variety of therapeutic purposes.Attorney Docket No. ALSE-016PC

[0008] Summary of the Invention

[0009] The invention provides antibodies that bind to DRPs produced by repeat expansion mutation of the C9orf72 gene, e.g., methylated forms of polyGR, such as asymmetrically dimethylated polyGR (ADMe-GR) and symmetrically dimethylated polyGR (SDMe-GR) DRPs. The antibodies of the present invention have desirable therapeutic properties, such as the ability to detect methylated DRPs, e.g., ADMe-GR and SDMe-GR DRPs for diagnostic purposes. The antibodies also can be used to treat neurodegenerative diseases (e.g., ALS or FTD) based on their ability to inhibit the activity of methylated DRPs, e.g., ADMe-GR and SDMe-GR DRPs. DRP activity inhibited by the antibodies includes, for example, neuronal cell death.

[0010] Accordingly, in one aspect, the invention pertains to isolated antibodies, or antigen binding portions thereof, which bind to a methylated dipeptide repeat protein (DRP) produced by repeat expansion mutation of the C9orf72 gene, wherein the antibody, or antigen binding portion thereof, exhibits one or more of the following properties:

[0011] (a) inhibits neuronal cell death

[0012] (b) reduces the level of methylated DRP-associated toxicity in the cell expressing the DRP;

[0013] (c) reduces the level of methylated DRP-associated toxicity in cells exogenously exposed to the DRP;

[0014] (d) binds to a methylated DRP with a KD of approximately 10’8to 10-10M or less; and / or

[0015] (e) reduces circulating levels of methylated DRP in biological tissues or fluids.

[0016] In one embodiment, the DRP is an asymmetrically dimethylated (ADMe) dipeptide repeat protein or a symmetrically dimethylated (SDMe) dipeptide repeat protein e.g., wherein the ADMe or SDMe DRP comprises at least one poly-glycine-arginine dipeptide (GR) and / or poly-proline-arginine dipeptide (PR).

[0017] In another embodiment, the antibodies, or antigen binding portions thereof, comprise the CDR1, CDR2, and CDR3 amino acid sequences of the heavy and light chain variable region sequences respectively set forth in (a) SEQ ID NOs: 7 and 8 or (b) SEQ ID NOs: 21 and 22, e.g., the antibody (or binding portion) comprises:Attorney Docket No. ALSE-016PC

[0018] (a) heavy chain CDR1, CDR2, and CDR3 amino acid sequences respectively set forth in SEQ ID NOs: 1, 2, and 3, and / or light chain CDR1, CDR2, and CDR3 amino acid sequences respectively set forth in SEQ ID NOs: 4, 5, and 6; or

[0019] (b) heavy chain CDR1, CDR2, and CDR3 amino acid sequences respectively comprising SEQ ID NOs: 15, 16, and 17 and / or light chain CDR1, CDR2, and CDR3 amino acid sequences respectively comprising SEQ ID NOs: 18, 19, and 20.

[0020] In another embodiment, the antibody (or binding portion) comprises:

[0021] (a) heavy chain CDR1, CDR2, and CDR3 amino acid sequences respectively set forth in SEQ ID NOs: 1, 2, and 3 and light chain CDR1, CDR2, and CDR3 amino acid sequences respectively set forth in SEQ ID NOs: 4, 5, and 6: or

[0022] (b) heavy chain CDR1, CDR2, and CDR3 amino acid sequences respectively comprising SEQ ID NOs: 15, 16, and 17 and light chain CDR1, CDR2, and CDR3 amino acid sequences respectively comprising SEQ ID NOs: 18, 19, and 20.

[0023] In yet another embodiment, the antibody, or antigen binding portion thereof, comprises a heavy chain variable region which is at least 90% identical to the heavy chain amino acid sequence set forth in SEQ ID NO: 7 or 21 and / or a light chain variable region which is at least 90% identical to the light chain amino acid sequence set forth in SEQ ID NO: 8 or 22, e.g., the antibody (or binding portion) comprises heavy and light chain variable regions which are at least 90% identical to the heavy and light chain variable region amino acid sequences respectively set forth in:

[0024] (a) SEQ ID NOs: 7 and 8; or

[0025] (b) SEQ ID NOs: 21 and 22.

[0026] In another embodiment, the antibody, or antigen binding portion thereof, comprises a heavy chain which is at least 90% identical to the heavy chain amino acid sequence set forth in SEQ ID NO: 9 or 23 and / or a light chain which is at least 90% identical to the light chain amino acid sequence set forth in SEQ ID NO: 10 or 24, e.g., the antibody (or binding portion) comprises heavy and light chains which are at least 90% identical to the heavy and light chain amino acid sequences respectively set forth in:

[0027] (a) SEQ ID NOs: 9 and 10; or

[0028] (b) SEQ ID NOs: 23 and 24.

[0029] In another embodiment, the present invention includes isolated monoclonal antibodies, or antigen binding portions thereof, which bind to the same epitope as antibodies having the sequences set forth in Table 3.Attorney Docket No. ALSE-016PC

[0030] In another embodiment, antibodies (or binding portions thereof) of the present invention are conjugated to a detectable label, e.g., a radioactive label or a fluorescent label.

[0031] Isolated nucleic acid molecule encoding the heavy and / or light chain variable region of the antibodies of the present invention (or antigen binding portions thereof) also are provided, e.g., a nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO: 11, 13, 14, 15, 25, 26, 27, or 28, as well as expression vectors comprising such nucleic acid molecules and cells transformed an expression vector.

[0032] In another embodiment, compositions are provided which comprise the antibodies (or antigen binding portion thereof) or the nucleic acid molecules of the invention, along with one or more pharmaceutically acceptable excipient, e.g., a solvent, aqueous solvent, nonaqueous solvent, dispersion media, diluent, dispersion, suspension aid, surface active agent, isotonic agent, thickening or emulsifying agent, preservative, lipid, lipidoids liposome, lipid nanoparticle, core-shell nanoparticles, polymer, lipoplexes, peptide, protein, cell, hyaluronidase, or mixtures thereof.

[0033] In another embodiment, kits are provided which comprise the antibodies (or antigen binding portion thereof), the nucleic acid molecules, or compositions of the invention, along with instructions for use.

[0034] In another embodiment, the invention provides methods of detecting a methylated DRP produced by repeat expansion mutation of the C9orf72 gene in a cell, comprising contacting the cell (such as a neuronal cell, e.g., a sensory neuron, a motor neuron, or an interneuron) with the antibody (or antigen binding portion) and detecting the binding of the antibody (or binding portion), to the protein.

[0035] In yet another embodiment, the invention provides methods of diagnosing a disease associated with methylated DRPs produced by repeat expansion mutation of the C9orf72 gene in a subject, comprising administering the antibody (or antigen binding portion thereof) and detecting the binding of the antibody (or binding portion) to the protein.

[0036] In yet another embodiment, the invention provides methods of decreasing toxicity in a subject caused by methylated DRPs produced by a repeat expansion mutation of the C9orf72 gene in a subject, comprising administering an effective amount of an antibody (or antigen binding portion thereof), a nucleic acid molecule, or a composition of the present invention.

[0037] In one embodiment, the subject is diagnosed with a neurodegenerative disease, e.g., C9ORF72-linked Amyotrophic Lateral Sclerosis (ALS) or C9ORF72-linked frontotemporal dementia (FTD).Attorney Docket No. ALSE-016PC

[0038] In yet another embodiment, the invention provides methods of treating a subject diagnosed with a disease associated with the expression of DRPs produced by repeat expansion mutation of the C9orf72 gene, comprising administering an effective amount of an antibody (or antigen binding portion thereof), a nucleic acid molecule, or a composition of the invention.

[0039] In another embodiment, the invention provides methods of treating a subject diagnosed with a neurodegenerative disorder (e.g., ALS or FTD), comprising administering an effective amount of an antibody (or antigen binding portion thereof), a nucleic acid molecule, or a composition of the invention.

[0040] Brief Description of the Drawings

[0041] FIG.1 shows the ADMe-GR8 monoclonal antibody 9A6 plasmid sequence for the light chain.

[0042] FIG.2 shows the ADMe-GR8 monoclonal antibody 9A6 plasmid sequence for the heavy chain.

[0043] FIG.3 shows the SDMe-GR8 monoclonal antibody 7E2 plasmid sequence for the light chain.

[0044] FIG.4 shows the SDMe-GR8 monoclonal antibody 7E2 plasmid sequence for the heavy chain.

[0045] FIG.5 is a graph showing binding profiles of anti-ADMe-GRs monoclonal antibody at concentrations of 1 pg / mL to 1 ng / mL against peptide coats of either ADMe-GR8, SDMe-GR8, GR8, or ADMe-PR15. The antibody shows high specificity for ADMe-GRs peptide coat compared to related antigen peptide coats. Starting at a concentration of 62.5 ng / mL, the antibody only binds its intended target ADMe-GR8with a high absorbance signal.

[0046] FIG.6 is a bar graph showing quantification of mean ADMe-GR8antibody green (“GFP”) signal intensity within motor neuron nuclei which indicates significant increase in detection of ADMe-GR with 24h of treatment with 1 p M exogenous ADMe-GR15peptide. Motor neurons containing the C9-HRE exhibit significantly higher mean ADMe-GR staining intensity with ADMe-GR15treatment than C9 wild-type motor neurons without the mutation (Two-way ANOVA, ** = p<0.01, **** = p<0.0001).

[0047] FIG.7 is a graph showing binding profiles of anti-SDMe-GR8monoclonal antibody at concentrations of 1 pg / mL to 0.02 ng / mL against peptide coats of either ADMe-GR8, SDMe-GR8, GR8, PR15, or ADMe-PR15. The antibody shows high specificity for SDMe-GRsAttorney Docket No. ALSE-016PC

[0048] peptide coat compared to related antigen peptide coats. At all concentrations tested, the antibody only binds its intended target SDMe-GRs with a high absorbance signal.

[0049] FIG. 8 is a bar graph showing quantification of mean SDMe-GR8antibody green signal intensity within motor neuron nuclei which indicates significant increase in detection of SDMe-GR with 24h treatment with 1 µM exogenous SDMe-GR15peptide (Two-way ANOVA, **** = p < 0.0001). Motor neurons that contained the C9-HRE mutation (“29_C9_HRE”) exhibit non-significantly higher mean SDMe-GR staining intensity with SDMe-GR15treatment compared to motor neurons where the C9-HRE mutation was corrected using a CRISPR-Cas9 approach (“31_C9_HRE_Corrected”).

[0050] FIGs. 9A-9D are graphs showing show antibody binding profile data; (A) repeat binding profiles of custom anti-SDMe-GRs monoclonal antibody (i.e., “custom anti-SDMe-GRs” or “TDI anti-SDMe-GRs”) against peptide coats of either ADMe-GRs, SDMe-GRs, GRs, PRis, or ADMe-PRis; (B) binding profiles of publicly available anti-SDMe-RG monoclonal antibody mixture (i.e., “CST SDMe-RG monoclonal antibody mixture” or “CST multi-mAb”) against peptide coats of either ADMe-GRs, SDMe-GRs, GRs, PRi.s, or ADMe-PRis; and (C) binding profiles against intended antigen SDMe-GR8described in FIGs. 9A and 9B transposed to compare the custom mAb binding sensitivity directly to CST multi-mAb. FIG. 9D is a table showing EC50 calculation of binding to SDMe-GR8coat using non-linear regression model analysis of the custom mAb compared to the CST SDMe-RG multi-mAb.

[0051] FIG. 10 is a table showing SPR data for antibodies 9A6 and 7E2.

[0052] FIGs. 11A and 11B are sensorgrams showing SPR data for (A) ADMe-GR15 / anti-ADMe-GR and (B) SDMe-GR15 / anti-SDMe-GR.

[0053] FIGs. 12A-12D are graphs showing anti-ADMe-GR8and Anti-SDMe-GR8monoclonal antibodies significantly and dose-dependently reduce methylated polyGR-induced cytotoxicity; (A) anti-ADMe-GR8monoclonal antibody co-titrated on NSC-34 cells with ADMe-GR15challenge; (B) anti-ADMe-GR8monoclonal antibody in the absence of ADMe-GR15challenge; (C) anti-SDMe-GR8monoclonal antibody co-titrated on NSC-34 cells with SDMe-GR15challenge; and (D) anti-ADMe-GR8monoclonal antibody in the absence of SDMe-GR15challenge.

[0054] FIGs: 13A-13H are graphs showing anti-ADMe-GR8and Anti-SDMe-GR8monoclonal antibodies significantly and dose-dependently reduce methylated polyGR-induced dysmetabolism; (A) anti-ADMe-GR8monoclonal antibody co-titrated on NSC-34Attorney Docket No. ALSE-016PC

[0055] cells with ADMe-GR15challenge; (B) anti-ADMe-GR8monoclonal antibody (in the absence of ADMe-GR15challenge); (C) anti-SDMe-GR8monoclonal antibody co-titrated on NSC-34 cells with SDMe-GR15challenge; (D) anti-SDMe-GR8monoclonal antibody (in the absence of SDMe-GR15challenge); (E) anti-ADMe-GR8monoclonal antibody co-titrated on NSC-34 cells with ADMe-GR15challenge; (F) anti-ADMe-GR8monoclonal antibody (in the absence of ADMe-GR15challenge); (G) anti-SDMe-GR8monoclonal antibody co-titrated on NSC-34 cells with SDMe-GR15challenge; and (H) anti-SDMe-GR8monoclonal antibody (in the absence of SDMe-GR15challenge).

[0056] FIGs: 14A and 14B are graphs showing rabbit anti-ADMe-GR8monoclonal antibody dose-dependently captures ADMe-GR15analyte down to pg / mL range sensitivity, even in samples that have not been pre-concentrated; (A) custom MSD assay; and (B) detection sensitivity at low-end of tested curve.

[0057] FIGs: 15A-15D are graphs showing rabbit anti-ADMe-GR8monoclonal antibody detects elevated ADMe-GR8in spinal cord sections from two C9orf72-mediated ALS mouse models; (A) staining intensity in mouse spinal cord sections (cervical, thoracic, and sacral);

[0058] (B) cervical spinal cord; (C) thoracic spinal cord; and (D) sacral spinal cord.

[0059] FIG: 16 is a graph showing rabbit anti-ADMe-GR8monoclonal antibody detects elevated ADMe-GR8in C9orf72 ALS patient cerebrospinal fluid.

[0060] Detailed Description of the Invention

[0061] The following definitions are provided to facilitate understanding of the methods and compositions disclosed herein.

[0062] As used herein, the term “dipeptide repeat proteins” (DRPs) refers to peptides consisting of repeating units of two amino acids. Examples of DRPs include the DRPs which are formed when RNA transcribed from the mutated C9ORF72 gene (containing expanded GGGGCC repeats) is translated through a non- AUG initiated mechanism. DRPs translated from all six reading frames in either the sense or antisense direction of the hexanucleotide repeat result in the expression of five DRPs: glycine-alanine (polyGA), glycine-arginine (polyGR), proline-alanine (polyPA), proline-arginine (polyPR) and glycine-proline (polyGP; polyGP is generated from both the sense and antisense reading frames). Of these five DRPs, polyGR and polyPR are rich in the amino acid arginine (R). As a result, polyGR and polyPR have been demonstrated as potential substrates for protein methylating enzymes known as protein arginine methyltransferases (PRMTs).Attorney Docket No. ALSE-016PC

[0063] Like their unmethylated counterparts, methylated DRPs have been shown to be “toxic” to cells, e.g., by interfering with the normal function of genes (e.g., the C9orf72 gene), as well as interfering with cellular proteins, thus leading to cellular dysfunction, degeneration, and death. Accordingly, the terms “toxic” and “toxicity” are used interchangeably and refer to the degree to which a substance (e.g., a methylated DRP or a mixture of methylated DRPs) can damage a cell (e.g., a neuronal cell) and / or the organism comprising the cell, such as a human, animal, or bacterium. Such toxic effects include, e.g., loss of cell function and / or cell death.

[0064] The term “DRP activity” refers to activity associated with a methylated DRP or the effect a methylated DRP has on a cell, e.g., a neuronal cell. Examples of DRP activity include binding to nucleoli, impeding RNA biogenesis, cellular dysfunction and cell death.

[0065] The terms “ADMe-GR” and “SDMe-GR” refer to methylated forms of polyGR DRPs produced by a repeat expansion mutation of the C9orf72 gene, i.e., asymmetrically dimethylated polyGR (ADMe-GR) and symmetrically dimethylated polyGR (SDMe-GR) DRPs.

[0066] As used herein, the terms “inhibits” or “blocks” (e.g., referring to inhibition / blocking of DRP activity) are used interchangeably and encompass both partial and complete inhibition / blocking. The inhibition / blocking of DRP activity by the DRP antibodies of the invention (e.g., an ADMe-GR and / or SDMe-GR DRP antibody) reduces or alters the toxic effect of a methylated DRP (e.g., an ADMe-GR and / or SDMe-GR DRP) on a cell, such as a neuronal cell, compared to the methylated DRP activity without the presence of the DRP antibody. Such DRP activity includes, e.g., toxic gain-of-function and loss-of-function, and is intended to include any measurable decrease in such activity, e.g., inhibits activity associated with ADMe-GR and / or SDMe-GR by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0067] The term “antibody” as referred to herein includes whole antibodies. An “antibody” refers, in one preferred embodiment, to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The lightAttorney Docket No. ALSE-016PC

[0068] chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system e.g., effector cells) and the first component (Clq) of the classical complement system.

[0069] The term “antigen-binding portion” of an antibody (or simply “antibody portion”), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen {e.g., ADMe-GR or SDMe-GR). Such "fragments" are, for example between about 8 and about 1500 amino acids in length, suitably between about 8 and about 745 amino acids in length, suitably about 8 to about 300, for example about 8 to about 200 amino acids, or about 10 to about 50 or 100 amino acids in length. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term “antigenbinding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR) or (vii) a combination of two or more isolated CDRs which may optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. Antigen-Attorney Docket No. ALSE-016PC

[0070] binding portions can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.

[0071] A “bispecific” or “bifunctional antibody” is an artificial hybrid antibody having two different heavy / light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including transfecting cells (e.g., HEK cells) with selected heavy / light chain pairs and expressing the desired antibody, as well as fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol.

[0072] 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).

[0073] The term “monoclonal antibody,” as used herein, refers to an antibody which displays a single binding specificity and affinity for a particular epitope. Accordingly, the term “human monoclonal antibody” refers to an antibody which displays a single binding specificity and which has variable and optional constant regions derived from human germline immunoglobulin sequences. In one embodiment, human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

[0074] The term “recombinant antibody,” as used herein, includes all chimeric, humanized and human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom, (b) antibodies isolated from a host cell transformed to express the antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human or humanized antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences.

[0075] The term “humanized antibody” refers to antibodies having framework regions from human germline sequences and CDRs from a non-human species (e.g., mouse, rat, rabbit) and includes, for example, antibodies in which the human framework regions and / or the CDRs have undergone specific site directed mutagenesis to optimize binding.

[0076] The term “human antibody” includes antibodies having variable and constant regions (if present) of human germline immunoglobulin sequences. Human antibodies of the invention can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or byAttorney Docket No. ALSE-016PC

[0077] somatic mutation in vivo) (see, Lonberg, N. etal. (1994) Nature 368(6474): 856-859);

[0078] Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. Vol. 13: 65-93, and Harding, F. and Lonberg, N. (1995) Ann. N. Y. Acad. Sci 764:536-546). However, the term “human antibody” does not include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (j.e., humanized antibodies).

[0079] An “isolated antibody,” as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to human ADMe-GR or SDMe-GR is substantially free of antibodies that specifically bind antigens other than human ADMe-GR or SDMe-GR). An isolated antibody that specifically binds to an epitope of human ADMe-GR or SDMe-GR may, however, have cross-reactivity to other ADMe-GR or SDMe-GR dipeptide proteins from different species. However, the antibody preferably always binds to human ADMe-GR or SDMe-GR. In addition, an isolated antibody is typically substantially free of other cellular material and / or chemicals.

[0080] The term “epitope” or “antigenic determinant” refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatial conformation. Methods for determining what epitopes are bound by a given antibody (j.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, wherein overlapping or contiguous peptides from an ADMe-GR or SDMe-GR are tested for reactivity with the given antibody of the invention. Methods of determining spatial conformation of epitopes include techniques in the art and those described herein, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996)).

[0081] The term "epitope mapping" refers to the process of identification of the molecular determinants for antibody-antigen recognition.Attorney Docket No. ALSE-016PC

[0082] The term "binds to the same epitope" with reference to two or more antibodies means that the antibodies compete for binding to an antigen and bind to the same, overlapping or encompassing continuous or discontinuous segments of amino acids. Those of skill in the art understand that the phrase "binds to the same epitope" does not necessarily mean that the antibodies bind to exactly the same amino acids. The precise amino acids to which the antibodies bind can differ. For example, a first antibody can bind to a segment of amino acids that is completely encompassed by the segment of amino acids bound by a second antibody. In another example, a first antibody binds one or more segments of amino acids that significantly overlap the one or more segments bound by the second antibody. For the purposes herein, such antibodies are considered to "bind to the same epitope."

[0083] As used herein, the terms “specific binding,” “selective binding,” “selectively binds,” and “specifically binds,” refer to antibody binding to an epitope on a predetermined antigen as measured by its equilibrium dissociation constant (KD). The term “KD as used herein, is intended to refer to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is obtained from the ratio of KD to KA (z.e., KD / KA) and is expressed as a molar concentration (M). KD values for antibodies can be determined using methods well established in the art. A preferred method for determining the KD of an antibody is by using surface plasmon resonance, preferably using a biosensor system such as a Biacore® system. Typically, the antibodies of the invention bind to ADMe-GR or SDMe-GR with a dissociation equilibrium constant (KD) of approximately 10-8M or less, or 10’9M or less, or 10-10M or less, or even lower when determined by surface plasmon resonance (SPR) technology using recombinant human ADMe-GR or SDMe-GR as the analyte and the antibody as the ligand.

[0084] The phrases “an antibody recognizing an antigen” and “an antibody specific for an antigen” are used interchangeably herein with the term “an antibody which binds specifically to an antigen.”

[0085] The term “IC50” as used herein, refers to the concentration of an antibody or an antigen-binding portion thereof, which is needed, either in an in vitro or an in vivo assay, to inhibit a given biological response by half. That is, it is the half minimal (50%) inhibitory concentration (IC) of the antibody or antigen-binding portion thereof.

[0086] As used herein, “isotype” refers to the antibody class (e.g., IgM or IgGl) that is encoded by heavy chain constant region genes. In one embodiment, a human monoclonal antibody of the invention is of the IgGl isotype. In another embodiment, a humanAttorney Docket No. ALSE-016PC

[0087] monoclonal antibody of the invention is of the IgG2 isotype. In another embodiment, a human monoclonal antibody of the invention is of the IgG4 isotype.

[0088] The term “cross-reacts,” as used herein, refers to the ability of an antibody of the invention to bind to ADMe-GR or SDMe-GR from a different species. For example, an antibody of the present invention that binds human ADMe-GR or SDMe-GR may also bind another species of ADMe-GR or SDMe-GR, such as cynomolgus monkey or rabbit. As used herein, cross-reactivity is measured by detecting a specific reactivity with purified antigen in binding assays (e.g., SPR, ELISA) or binding to, or otherwise functionally interacting with, cells physiologically expressing ADMe-GR or SDMe-GR. Methods for determining crossreactivity include standard binding assays as described herein, for example, by Biacore™ surface plasmon resonance (SPR) analysis using a Biacore™ 2000 SPR instrument (Biacore AB, Uppsala, Sweden), or flow cytometric techniques.

[0089] As used herein, “glycosylation pattern’’ is defined as the pattern of carbohydrate units that are covalently attached to a protein, more specifically to an immunoglobulin protein. A glycosylation pattern of a heterologous antibody can be characterized as being substantially similar to glycosylation patterns which occur naturally on antibodies produced by the species of the nonhuman transgenic animal, when one of ordinary skill in the art would recognize the glycosylation pattern of the heterologous antibody as being more similar to said pattern of glycosylation in the species of the nonhuman transgenic animal than to the species from which the CH genes of the transgene were derived.

[0090] The term “naturally-occurring” as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring.

[0091] The term “rearranged” as used herein refers to a configuration of a heavy chain or light chain immunoglobulin locus wherein a V segment is positioned immediately adjacent to a D-J or J segment in a conformation encoding essentially a complete VH or VL domain, respectively. A rearranged immunoglobulin gene locus can be identified by comparison to germline DNA; a rearranged locus will have at least one recombined heptamer / nonamer homology element.Attorney Docket No. ALSE-016PC

[0092] The term “unrearranged” or “germline configuration” as used herein in reference to a V segment refers to the configuration wherein the V segment is not recombined so as to be immediately adjacent to a D or J segment.

[0093] The term “nucleic acid molecule,” as used herein, is intended to include DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or doublestranded, but preferably is double-stranded DNA.

[0094] The term “isolated nucleic acid molecule,” as used herein in reference to nucleic acids encoding antibodies or antibody portions (e.g., VH, VL, CDR3) that bind to ADMe-GR or SDMe-GR, is intended to refer to a nucleic acid molecule in which the nucleotide sequences encoding the antibody or antibody portion are free of other nucleotide sequences encoding antibodies or antibody portions that bind antigens other than ADMe-GR or SDMe-GR, which other sequences may naturally flank the nucleic acid in human genomic DNA.

[0095] The present invention also encompasses “conservative sequence modifications” of the sequences set forth in the sequences of Table 3, i.e., nucleotide and amino acid sequence modifications which do not abrogate the binding of the antibody encoded by the nucleotide sequence or containing the amino acid sequence, to the antigen. Such conservative sequence modifications include conservative nucleotide and amino acid substitutions, as well as nucleotide and amino acid additions and deletions. For example, modifications can be introduced into the sequences described herein by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in an antibody of the invention is preferably replaced with another amino acid residue from the same side chain family. Methods of identifying nucleotide and amino acid conservative substitutions which do not eliminate antigen binding are well-known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993):Attorney Docket No. ALSE-016PC

[0096] Kobayashi et al. Protein Eng. 12(10):879-884 (1999): and Burks et al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

[0097] Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a DRP antibody coding sequence, e.g., an ADMe-GR or SDMe-GR antibody coding sequence, such as by saturation mutagenesis, and the resulting modified ADMe-GR or SDMe-GR antibodies can be screened for binding activity.

[0098] The term “substantial homology” indicates that two nucleotide or amino acid sequences, when optimally aligned and compared, are identical, with appropriate nucleotide or amino acid insertions or deletions, in at least about 80% of the nucleotides or amino acids, usually at least about 90% to 95%, and more preferably at least about 98% to 99.5% of the nucleotides. Alternatively, substantial homology exists when the segments of two nucleotide sequences will hybridize under selective hybridization conditions, to the complement of the strand.

[0099] The per cent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = # of identical positions / total # of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of per cent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.

[0100] The per cent identity between two nucleotide or amino acid sequences can be determined using the GAP program in the GCG software package (available at www.gcg.com), using a NWSgapdna. CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. The per cent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CAB IOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the per cent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0101] The nucleic acid and protein sequences of the present invention can further be used as a “query sequence” to perform a search against public databases to, for example, identifyAttorney Docket No. ALSE-016PC

[0102] related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to the protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

[0103] The nucleic acids or proteins of the invention may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid or protein is “isolated" or “rendered substantially pure" when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and others well known in the art. See, F. Ausubel, et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

[0104] The nucleic acid compositions of the present invention, while often in a native sequence (except for modified restriction sites and the like), from either cDNA, genomic or mixtures thereof may be mutated, in accordance with standard techniques to provide gene sequences. For coding sequences, these mutations may affect amino acid sequence as desired. In particular, DNA sequences substantially homologous to or derived from native V, D, J, constant, switches and other such sequences described herein are contemplated (where "derived" indicates that a sequence is identical or modified from another sequence).

[0105] A nucleic acid is “operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. With respect to transcription regulatory sequences, operably linked means that the DNA sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame. For switch sequences, operably linked indicates that the sequences are capable of effecting switch recombination.Attorney Docket No. ALSE-016PC

[0106] The term “vector,” as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.

[0107] Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0108] The term “recombinant host cell” (or simply “host cell”), as used herein, is intended to refer to a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein.

[0109] As used herein, the term “linked” refers to the association of two or more molecules. The linkage can be covalent or non-covalent. The linkage also can be genetic (z.e., recombinantly fused). Such linkages can be achieved using a wide variety of art recognized techniques, such as chemical conjugation and recombinant protein production.

[0110] As used herein, “positron emission tomography” or “PET” refers to a non-invasive, nuclear medicine technique that produces a three-dimensional image of tracer location in the body. The method detects pairs of gamma rays emitted indirectly by a positron-emitting radionuclide (tracer), which is introduced into the body on a biologically active molecule. PET imaging tools have a wide variety of uses and aid in drag development both preclinicallyAttorney Docket No. ALSE-016PC

[0111] and clinically. Exemplary applications include direct visualization of in vivo saturation of targets; monitoring uptake in normal tissues to anticipate toxicity or patient to patient variation; quantifying diseased tissue; tumor metastasis; and monitoring drug efficacy over time, or resistance over time.

[0112] The terms “treat,” "treatment," and "treating" as used herein is intended to encompass slowing the progression, reversing, or otherwise ameliorating a neurological disorder such as a neurodegenerative and / or neuromuscular disorder.

[0113] The term “neurodegenerative disease,” as used herein, refers to a condition characterized by progressive dysfunction, degeneration and death of specific populations of neurons. Examples of neurodegenerative diseases include, e.g., ALS and FTD, which include “C9ORF72-linked ALS” and “C9ORF72-linked FTD” which can be inherited in an autosomal dominant manner, with age-dependent penetrance.

[0114] The term “effective dose” or “effective dosage” is defined as an amount sufficient to achieve or at least partially achieve the desired effect. The term “therapeutically effective dose” is defined as an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Amounts effective for this use will depend upon the severity of the disorder being treated and the general state of the patient’s own immune system.

[0115] In reference to the decrease in the toxicity of a cell, an effective amount, e.g., comprises an amount sufficient to restore the phenotype of at least one detectable cell viability parameter (e.g., rate of cell proliferation, percentage of cell death, etc.) to equal that of a healthy, untreated cell, where such a phenotype is not detectably aberrant. In reference to the treatment of a neurodegenerative disease (e.g., ALS or FTD), an effective amount is an amount sufficient to prevent or delay symptoms associated with the disease (e.g., muscle weakness and / or cognitive impairment). An effective amount can be administered in one or more administrations. In one example, an “effective amount” is the amount of a ADMe-GR or SDMe-GR antibody clinically proven to affect a significant improvement in the symptoms associated with the disease (e.g., ALS or FTD).

[0116] The term “neuronal cell” refers to a specialized, impulse-conducting cell that is the functional unit of the nervous system, consisting of the cell body and its processes, the axon and dendrites. Neuronal cells include sensory neurons, motor neurons, and interneurons.

[0117] The term “neural stem cell,” or "neural progenitor cell" or "neural precursor cell" refers to cells that can generate progeny that are either neuronal cells (such as neuronAttorney Docket No. ALSE-016PC

[0118] precursors or mature neurons) or glial cells (such as glial precursors, mature astrocytes, or mature oligodendrocytes). Typically, the cells express some of the phenotypic markers that are characteristic of the neural lineage.

[0119] Neurodegenerative diseases include, e.g., ALS (amyotrophic lateral sclerosis) and FTD (frontotemporal dementia). The terms “C9ORF72-linked ALS” and “C9ORF72-linked FTD” refer to forms of ALS and FTD, respectively, that afflict individuals who cany' expanded hexanucleotide (GGGGCC) repeat mutations, e.g., the C9ORF72 mutation discussed above. In the general population (unaffected by ALS or FTD) open frame region 72 of chromosome 9 will typically exhibit a tract of GGGGCC hexanucleotide repeats between 3 and 10 and almost always fewer than 20 repeats. Thus, an individual afflicted with ALS or FTD having greater than 20 hexanucleotide repeats, or greater than 30 hexanucleotide repeats, or more, in open frame region 72 of chromosome 9 may suffer from “C9ORF72-linked ALS” or “C9ORF72-linked FTD.”

[0120] The term “patient” includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment.

[0121] As used herein, the term "subject" is a human or other animal, e.g., a human having a neurological disorder. In some embodiments, the subjects are mammals. Examples of subjects can include, but are not limited to, humans, horses, monkeys, dogs, cats, mice, rats, cows, pigs, goats and sheep. In some embodiments, "subjects" are generally human patients diagnosed with ALS or FTD.

[0122] Various aspects of the invention are described in further detail in the following subsections.

[0123] I. Production of Antibodies to Methylated DRPs

[0124] The present invention encompasses antibodies, e.g., humanized antibodies, that bind methylated DRPs produced by repeat expansion mutation of the C9orf72 gene, e.g., methylated forms of polyGR e.g., human polyGR, such as asymmetrically dimethylated polyGR (ADMe-GR) and symmetrically dimethylated polyGR (SDMe-GR) DRPs.

[0125] Exemplary monoclonal antibodies include antibodies 9A6 and 7E2 which respectively bind ADMe-GR or SDMe-GR. The heavy chain variable region amino acid sequences of 9A6 and 7E2 are shown in SEQ ID NOs: 7 and 21, respectively, and the light chain variable region amino acid sequences of 9A6 and 7E2 are shown in SEQ ID NOs: 8 and 22, respectively. The heavy chain CDR1, 2 and 3 amino acid sequence of 9A6 are shown in SEQ ID NOs: 1,Attorney Docket No. ALSE-016PC

[0126] 2, and 3, respectively, and the light chain CDR1, 2 and 3 amino acid sequence of 9A6 are shown in SEQ ID NOs: 4, 5, and 6, respectively. The heavy chain CDR1, 2 and 3 amino acid sequence of 7E2 are shown in SEQ ID NOs: 15, 16, and 17, respectively, and the light chain CDR1, 2 and 3 amino acid sequence of 7E2 are shown in SEQ ID NOs: 18, 19, and 20, respectively.

[0127] Monoclonal antibodies of the invention can be produced using a variety of known techniques, such as the standard somatic cell hybridization technique described by Kohler and Milstein, Nature 256: 495 (1975). Other techniques for producing monoclonal antibodies also can be employed, e.g., viral or oncogenic transformation of B lymphocytes, phage display technique using libraries of human antibody genes and humanization techniques such as those known in the art.

[0128] Accordingly, in one embodiment, a dual-step approach starting with b-cell cloning from immunized host animal splenocytes and concluding with hyperexpression of monoclonal antibody via transfected plasmid DNA derived from host b-cells is used for producing an antibody that binds a methylated DRP, e.g., ADMe-GR or SDMe-GR. In this method, a rabbit, rat, mouse or other appropriate host animal can be immunized with a suitable antigen in order to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the antigen used for immunization (as described in Example 1). Sera from immunized host animals can be tested in a suitable assay to detect binding, such as an ELISA (as described in detail in the examples). Based upon antigen binding profiles, one or more host animals can be selected for spleen harvesting, from which splenocytes can be isolated.

[0129] Splenocytes from the selected host animals can then be co-incubated with a fluorescently labeled form of the antigen and sorted through fluorescence-activated cell sorting (FACS). During sorting, B-cells mounting the greatest reaction to the antigen can then be selected and plated for primary screening and culturing. Primary screening can include testing of cultured B-cell supernatant for ELISA-based detection of antibodies produced by B-cells against the intended antigen. Based on the results of primary screening, a set of B-cells can be chosen for cloning of heavy and light chain genes into a linear expression module (LEM). LEMs derived from chosen B-cells can be transfected into suitable host cells for gene expression, e.g., HEK cells (as described in the examples). The monoclonal antibodies secreted by the LEM-transfected subclones can be separated from the culture medium by conventional immunoglobulin purification procedures such as, forAttorney Docket No. ALSE-016PC

[0130] example, protein A or other affinity chromatography (as described in the examples), Sepharose, hydroxyapatite chromatography, gel electrophoresis, or dialysis.

[0131] A non-human monoclonal antibody, such as a rabbit or mouse antibody, can be humanized using methods known in the art. This approach is based on the principle that if a non-human and a human antibody have similarly structured CDRs, the human frameworks will also support the non-human CDRs, with good retention of affinity. Thus, in this method, the human framework sequences are chosen from the set of human germline genes based on the structural similarity of the human CDRs to those of the antibody to be humanized (same Chothia canonical structures). A phage display library of Fab variant sequences, containing deviating FR residues, is generated. After affinity-driven selections, individual clones are screened for binding and off-rate and the human identity and homology of the sequence is determined. Other approaches and methodologies for CDR-grafting and humanization that are well established in the art also can be used to generate humanized DRP antibody, e.g., a humanized ADMe-GR and SDMe-GR antibody.

[0132] In another embodiment, antibodies and antibody portions that bind a methylated DRP, e.g., ADMe-GR or SDMe-GR, can be isolated from antibody phage libraries generated using the techniques described in, for example, McCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991), Marks et al., J. Mol. Biol., 222:581-597 (1991) and Hoet et al (2005) Nature Biotechnology 23, 344-348; U. S. Patent Nos. 5,223,409:

[0133] 5,403,484; and 5,571,698 to Ladner et al.,' U. S. Patent Nos. 5,427,908 and 5,580,717 to Dower et al.,' U. S. Patent Nos. 5,969,108 and 6,172,197 to McCafferty et al.', and U. S. Patent Nos. 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and 6,593,081 to Griffiths et al.. Additionally, production of high affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology, 10:779-783 (1992)), as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al., Nuc. Acids. Res., 21:2265-2266 (1993)) may also be used.

[0134] In one embodiment, the antibody that binds a methylated DRP, e.g., ADMe-GR or SDMe-GR, is produced using the phage display technique described by Hoet et al., supra. This technique involves the generation of a human Fab library having a unique combination of immunoglobulin sequences isolated from human donors and having synthetic diversity in the heavy-chain CDRs. The library is then screened for Fabs that bind to the target.

[0135] In another embodiment, antibodies directed against a methylated DRP, e.g., ADMe-GR and SDMe-GR, are generated using methods known in the art which utilize transgenic orAttorney Docket No. ALSE-016PC

[0136] transchromosomal mice carrying parts of the human immune system rather than parts of the mouse immune system. (Taylor, L. et al. (1992) Nucleic Acids Research 20:6287-6295; PCT Publication WO 02 / 43478; U. S. Pat. NOs. 5,939,598; 6,075,181; 6,114,598; 6,150,584 and 6,162,963; Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97:722-727; Kuroiwaet al. (2002) Nature Biotechnology 20:889-894; WO 2009 / 15777; U. S. Pat. NOs. 5,476,996 and 5,698,767).

[0137] Antibodies of the invention also can be produced in a host cell transfectoma using, for example, a combination of recombinant DNA techniques and gene transfection methods as is well known in the art (Morrison, S. (1985) Science 229:1202). Exemplary embodiments for recombinant expression of DRP antibodies, e.g., ADMe-GR and SDMe-GR antibodies, are described further in examples. Furthermore, in one embodiment, the gene(s) of interest, e.g., antibody genes, can be ligated into an expression vector such as a eukaryotic expression plasmid such as used by GS gene expression system disclosed in WO 87 / 04462, WO 89 / 01036 and EP 338 841 or other expression systems well known in the art. The purified plasmid with the cloned antibody genes can be introduced in eukaryotic host cells such as CHO-cells or NSO-cells or alternatively other eukaryotic cells such as plant derived cells, fungi or yeast cells. The method used to introduce these genes could be methods described in the art such as electroporation, lipofectine, lipofectamine or other. After introducing these antibody genes in host cells, cells expressing the antibody can be identified and selected. The selected cells represent transfectomas that can then be amplified for their expression level and upscaled to produce antibodies. Recombinant antibodies can be isolated and purified from these culture supernatants and / or cells.

[0138] Alternatively, cloned antibody genes can be expressed in other systems such as E. coli, complete organisms, or synthetic expression systems.

[0139] Use of Partial Antibody Sequences to Express Intact Antibodies

[0140] In certain embodiments, a DRP antibody, e.g., an ADMe-GR or SDMe-GR antibody, of the invention comprises (a) a heavy chain CDR3 amino acid sequence shown in SEQ ID NO: 3 and a light chain CDR3 amino acid sequence shown in SEQ ID NO: 6 (i.e., antibody 9A6) or (b) a heavy chain CDR3 amino acid sequence shown in SEQ ID NO: 17 and a light chain CDR3 amino acid sequence shown in SEQ ID NO: 20 (i.e., antibody 7E2). The antibody can further comprise (a) a heavy chain CDR2 amino acid sequence shown in SEQ ID NO: 2 and a light chain CDR2 amino acid sequence shown in SEQ ID NO: 5 (i.e.,Attorney Docket No. ALSE-016PC

[0141] antibody 9A6) or (b) a heavy chain CDR2 amino acid sequence shown in SEQ ID NO: 16 and a light chain CDR2 amino acid sequence shown in SEQ ID NO: 18 (i.e., antibody 7E2). The antibody can still further comprise (a) a heavy chain CDR1 amino acid sequence shown in SEQ ID NO: 1 and a light chain CDR1 amino acid sequence shown in SEQ ID NO: 4 (i.e., antibody 9A6) or (b) a heavy chain CDR1 amino acid sequence shown in SEQ ID NO: 15 and a light chain CDR1 amino acid sequence shown in SEQ ID NO: 18 (i.e., antibody 7E2).

[0142] Antibodies interact with target antigens predominantly through amino acid residues located in the six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences within CDRs are more diverse between individual antibodies than sequences outside of CDRs. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies. This is achieved by constructing expression vectors that include CDR sequences from the specific naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann, L. etal., 1998, Nature 332P>23-y2T, Jones, P. et al., 1986, Nature 321:522-525; and Queen, C. et al., 1989, Proc. Natl. Acad. See. U. S. A. 86:10029-10033). Such framework sequences can be obtained from public DNA databases that include germline antibody gene sequences. These germline sequences will differ from mature antibody gene sequences because they will not include completely assembled variable genes, which are formed by V(D)J joining during B cell maturation. Germline gene sequences will also differ from the sequences of a high affinity secondary repertoire antibody at individual evenly across the variable region. For example, somatic mutations are relatively infrequent in the amino-terminal portion of framework region. For another example, somatic mutations are relatively infrequent in the amino terminal portion of framework region 1 and in the carboxy -terminal portion of framework region 4. Furthermore, many somatic mutations do not significantly alter the binding properties of the antibody. For this reason, it is not necessary to obtain the entire DNA sequence of a particular antibody in order to recreate an intact recombinant antibody having binding properties similar to those of the original antibody (see PCT / US99 / 05535). The use of partial heavy and light chain sequence spanning the CDR regions is typically sufficient for this purpose. The partial sequence is used to determine which germline variable and joining gene segments contribute to the recombined antibody variable genes. The germline sequence is then used to fill in missing portions of the variable regions. Heavy and light chain leader sequences are cleaved duringAttorney Docket No. ALSE-016PC

[0143] protein maturation and do not contribute to the properties of the final antibody. To add missing sequences, cloned cDNA sequences can be combined with synthetic oligonucleotides by ligation or PCR amplification. Alternatively, the entire variable region can be synthesized as a set of short, overlapping oligonucleotides and combined by PCR amplification to create an entirely synthetic variable region clone. This process has certain advantages such as elimination or inclusion of particular restriction sites, or optimization of particular codons.

[0144] The nucleotide sequences of heavy and light chain transcripts from B-cell clones are used to design an overlapping set of synthetic oligonucleotides to create synthetic V sequences with identical amino acid coding capacities as the natural sequences. The synthetic heavy and kappa chain sequences can differ from the natural sequences in three ways: strings of repeated nucleotide bases are interrupted to facilitate oligonucleotide synthesis and PCR amplification; optimal translation initiation sites are incorporated according to Kozak’s rules (Kozak, 1991, J. Biol. Chem. 266:19867-19870); and Hindlll sites are engineered upstream of the translation initiation sites.

[0145] For both the heavy and light chain variable regions, the optimized coding and corresponding non-coding strand sequences are broken down into 30 - 50 nucleotide lengths using the approximate midpoint of the corresponding non-coding oligonucleotide. Thus, for each chain, oligonucleotides can be assembled into overlapping double stranded sets that span segments of 150 - 400 nucleotides. The pools are then used as templates to produce PCR amplification products of 150 - 400 nucleotides. Typically, a single variable region oligonucleotide set will be broken down into two pools, which are separately amplified to generate two overlapping PCR products. These overlapping products are then combined by PCR amplification to form the complete variable region. It may also be desirable to include an overlapping fragment of the heavy or light chain constant region (including the BbsI site of the kappa light chain, or the Agel site if the gamma heavy chain) in the PCR amplification to generate fragments that can easily be cloned into the expression vector constructs.

[0146] The reconstructed heavy and light chain variable regions are then combined with other sequences, e.g., cloned promoter, leader sequence, translation initiation, constant region, 3’ untranslated, polyadenylation, and transcription termination, sequences, to form expression vector constructs. The heavy and light chain expression constructs can be combined into a single vector, co-transfected, serially transfected, or separately transfected into host cells which are then fused to form a host cell expressing both chains.Attorney Docket No. ALSE-016PC

[0147] Plasmids for use in construction of expression vectors have been constructed so that PCR amplified V heavy and V kappa light chain cDNA sequences could be used to reconstruct complete heavy and light chain minigenes. These plasmids can be used to express completely human IgGiK or IgG4K antibodies. Fully human and chimeric antibodies of the present invention also include IgG2, IgG3, IgE, IgA, IgM, and IgD antibodies. Similar plasmids can be constructed for expression of other heavy chain isotypes, or for expression of antibodies comprising lambda light chains.

[0148] Thus, in another aspect of the invention, structural features of DRP antibodies, e.g., ADMe-GR and SDMe-GR antibodies, of the invention are used to create structurally related antibodies that retain at least one functional property of the antibodies of the invention, such as, for example,

[0149] (a) binds the same epitope as a DRP antibody of the invention, such as 9A6 or 7E2; (b) inhibits neuronal cell death;

[0150] (c) reduces the level of methylated DRP-associated toxicity in the cell expressing the DRP;

[0151] (d) reduces circulating levels of methylated DRP in biological tissues or fluids;

[0152] (e) reduces the level of methylated DRP-associated toxicity in cells exogenously exposed to the DRP; and / or

[0153] (f) binds to a methylated DRP with a KD of approximately 10-8to IO-10M or less. In one embodiment, one or more CDR regions of antibodies of the invention can be combined recombinantly with known framework regions and CDRs to create additional, recombinantly engineered DRP antibodies, e.g., ADMe-GR or SDMe-GR antibodies, of the invention. The heavy and light chain variable framework regions can be derived from the same or different antibody sequences. The antibody sequences can be the sequences of naturally occurring antibodies or can be consensus sequences of several antibodies. See Kettleborough el al., Protein Engineering 4:773 (1991); Kolbinger el al., Prolein Engineering 6:971 (1993) and Carter et al., WO 92 / 22653.

[0154] Accordingly, in another embodiment, the invention provides a method for preparing an ADMe-GR or SDMe-GR antibody including: preparing an antibody including (1) heavy chain framework regions and heavy chain CDRs, where at least one of the heavy chain CDRs includes an amino acid sequence selected from the amino acid sequences of CDRs shown in SEQ ID NOs: 1, 2, 3, 15, 16, and 17; and (2) light chain framework regions and light chain CDRs, where at least one of the light chain CDRs includes an amino acid sequence selectedAttorney Docket No. ALSE-016PC

[0155] from the amino acid sequences of CDRs shown in SEQ ID NOs: 4, 5, 6, 18, 19; where the antibody retains the ability to bind to ADMe-GR or SDMe-GR. The ability of the antibody to bind the target can be determined using standard binding and / or functional assays, such as those set forth in the Examples. Preferably, the antibody exhibits at least one, or at least two or at least three or at least four or all five of the functional properties listed above as (a) through (c).

[0156] It is well known in the art that antibody heavy and light chain CDR3 domains play a particularly important role in the binding specificity / affinity of an antibody for an antigen (see, Hall et al., J. Imunol., 149:1605-1612 (1992); Polymenis et al., J. Immunol., 152:5318-5329 (1994); Jahn et al., Immunobiol., 193:400-419 (1995); Klimka et al., Brit. J. Cancer, 83:252-260 (2000); Beiboer et al., J. Mol. Biol, 296:833-849 (2000); Rader et al., Proc. Natl. Acad. Sci. USA, 95:8910-8915 (1998); Barbas et al., J. Am. Chem. Soc., 116:2161-2162 (1994); Ditzel et al., J. Immunol., 157:739-749 (1996)). Accordingly, the recombinant antibodies of the invention prepared as set forth above preferably comprise the heavy and / or light chain CDR3 of the antibody 9A6 or 7E2, as set forth in SEQ ID NOs: 3 and 17, respectively.

[0157] Moreover, in another embodiment, the invention further provides ADMe-GR or SDMe-GR antibodies comprising: (1) heavy chain framework regions, a heavy chain CDR1 region, a heavy chain CDR2 region, and a heavy chain CDR3 region, wherein the heavy chain CDR3 region comprises the sequence of SEQ ID NO: 3 or 17 and (2) light chain framework regions, a light chain CDR1 region, a light chain CDR2 region, and a light chain CDR3 region, wherein the light chain CDR3 region comprises the sequence of SEQ ID NO: 16 or 20, wherein the antibody binds ADMe-GR or SDMe-GR. The antibody may further include the heavy chain CDR2 and / or the light chain CDR2 of antibody 9A6, as respectively set forth in SEQ ID NOs: 2 and 5, or the heavy chain CDR2 and / or the light chain CDR2 of antibody 7E2, as respectively set forth in SEQ ID NOs: 16 and 18. The antibody may further comprise the heavy chain CDR1 and / or the light chain CDR1 of antibody 9A6, as respectively set forth in SEQ ID NOs: 1 and 4, or the heavy chain CDR1 and / or the light chain CDR1 of antibody 7E2, as respectively set forth in SEQ ID NOs: 15 and 18.

[0158] Generation of Antibodies Having Modified Sequences

[0159] Antibodies interact with target antigens predominantly through amino acid residues that are located in the six heavy and light chain complementarity determining regionsAttorney Docket No. ALSE-016PC

[0160] (CDRs). For this reason, the amino acid sequences within CDRs are more diverse between individual antibodies than sequences outside of CDRs. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies, by way of constructing expression vectors that include CDR sequences from the specific naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann, L. et al., 1998, Nature 332:323-327; Jones, P. et al,, 1986, Nature 321:522-525; and Queen, C. et al., 1989, Proc. Natl, Acad. See. U. S. A. 86: 10029-10033). Such framework sequences can be obtained from public DNA databases that include germline antibody gene sequences. These germline sequences will differ from mature antibody gene sequences because they will not include completely assembled variable genes, which are formed by V(D)J joining during B cell maturation. Germline gene sequences will also differ from the sequences of a high affinity secondary repertoire antibody at individual sites evenly across the variable region. For example, somatic mutations are relatively infrequent in the amino-terminal portion of a framework region. For example, somatic mutations are relatively infrequent in the amino terminal portion of framework region 1 and in the carboxy -terminal portion of framework region 4. Furthermore, many somatic mutations do not significantly alter the binding properties of the antibody. For this reason, it is not necessary to obtain the entire DNA sequence of a particular antibody in order to recreate an intact recombinant antibody having binding properties similar to those of the original antibody (see PCT / US99 / 05535 filed on March 12, 1999). A partial heavy and light chain sequence spanning the CDR regions is typically sufficient for this purpose. The partial sequence is used to determine which germline variable and joining gene segments contributed to the recombined antibody variable genes. The germline sequence is then used to fill in missing portions of the variable regions. Heavy and light chain leader sequences are cleaved during protein maturation and do not contribute to the properties of the final antibody. To add missing sequences, cloned cDNA sequences can be combined with synthetic oligonucleotides by ligation or PCR amplification. Alternatively, the entire variable region can be synthesized as a set of short, overlapping, oligonucleotides and combined by PCR amplification to create an entirely synthetic variable region clone. This process has certain advantages such as elimination or inclusion of particular restriction sites, or optimization of particular codons.Attorney Docket No. ALSE-016PC

[0161] The nucleotide sequences of heavy and light chain transcripts from isolated B-cells (from spleens harvested from chosen host animals) or a hybridoma are used to design an overlapping set of synthetic oligonucleotides to create synthetic V sequences with identical amino acid coding capacities as the natural sequences. The synthetic heavy and kappa chain sequences can differ from the natural sequences in three ways: strings of repeated nucleotide bases are interrupted to facilitate oligonucleotide synthesis and PCR amplification; optimal translation initiation sites are incorporated according to Kozak’s rules (Kozak, 1991, J. Biol. Chem. 266:19867-19870); and, Hindlll sites are engineered upstream of the translation initiation sites.

[0162] For both the heavy and light chain variable regions, the optimized coding and corresponding non-coding strand sequences are broken down into 30 - 50 nucleotide lengths using the approximate midpoint of the corresponding non-coding oligonucleotide. Thus, for each chain, the oligonucleotides can be assembled into overlapping double-stranded sets that span segments of 150 - 400 nucleotides. The pools are then used as templates to produce PCR amplification products of 150 - 400 nucleotides. Typically, a single variable region oligonucleotide set will be broken down into two pools which are separately amplified to generate two overlapping PCR products. These overlapping products are then combined by PCR amplification to form the complete variable region. It may also be desirable to include an overlapping fragment of the heavy or light chain constant region (including the BbsI site of the kappa light chain, or the Agel site if the gamma heavy chain) in the PCR amplification to generate fragments that can easily be cloned into the expression vector constructs.

[0163] The reconstructed heavy and light chain variable regions are then combined with other sequences, e.g., cloned promoter, leader sequence, translation initiation, constant region, 3’ untranslated, polyadenylation, and transcription termination, sequences to form expression vector constructs. The heavy and light chain expression constructs can be combined into a single vector, co-transfected, serially transfected, or separately transfected into host cells which are then fused to form a host cell expressing both chains.

[0164] Plasmids for use in construction of expression vectors were constructed so that PCR amplified V heavy and V kappa light chain cDNA sequences could be used to reconstruct complete heavy and light chain minigenes. These plasmids can be used to express completely human IgGiK or IgG4K antibodies. Fully human and chimeric antibodies of the present invention also include IgG2, IgG3, IgE, IgA, IgM, and IgD antibodies. SimilarAttorney Docket No. ALSE-016PC

[0165] plasmids can be constructed for expression of other heavy chain isotypes, or for expression of antibodies comprising lambda light chains.

[0166] Thus, in another aspect of the invention, structural features of DRP antibodies, e.g., ADMe-GR or SDMe-GR antibodies, of the invention are used to create structurally related antibodies that retain at least one functional property of the antibodies of the invention, such as, for example,

[0167] (a) binds the same epitope as a DRP antibody of the invention, such as 9A6 or 7E2; (b) inhibits neuronal cell death;

[0168] (c) reduces the level of methylated DRP-associated toxicity in the cell expressing the methylated DRP;

[0169] (d) reduces circulating levels of methylated DRP in biological tissues or fluids;

[0170] (e) reduces the level of methylated DRP-associated toxicity in cells exogenously exposed to the DRP; and / or

[0171] (f) binds to a methylated DRP with a KD of approximately 10’8to 10-10M or less. In one embodiment, one or more CDR regions of antibodies of the invention can be combined recombinantly with known framework regions and CDRs to create additional, recombinantly-engineered, ADMe-GR or SDMe-GR antibodies of the invention. The heavy and light chain variable framework regions can be derived from the same or different antibody sequences. The antibody sequences can be the sequences of naturally occurring antibodies or can be consensus sequences of several antibodies. See Kettleborough et al., Protein Engineering 4:773 (1991); Kolbinger et al., Protein Engineering 6:971 (1993) and Carter et al., WO 92 / 22653.

[0172] Accordingly, in another embodiment, the invention provides a method for preparing an ADMe-GR or SDMe-GR antibody including: preparing an antibody including (1) heavy chain framework regions and heavy chain CDRs, where at least one of the heavy chain CDRs includes an amino acid sequence selected from the amino acid sequences of CDRs shown in SEQ ID NOs: 1, 2, 3, 15, 16, or 17; and (2) light chain framework regions and light chain CDRs, where at least one of the light chain CDRs includes an amino acid sequence selected from the amino acid sequences of CDRs shown in SEQ ID NOs: 4, 5, 6, 18, 19, or 20; where the antibody retains the ability to bind to the target. The ability of the antibody to bind ADMe-GR or SDMe-GR can be determined using standard binding assays, such as those set forth in the Examples {e.g., an ELISA or a FLISA).Attorney Docket No. ALSE-016PC

[0173] It is well known in the art that antibody heavy and light chain CDR3 domains play a particularly important role in the binding specificity / affinity of an antibody for an antigen (see, Hall et al., J. Iniunol., 149:1605-1612 (1992); Polymenis et al., J. Immunol., 152:5318-5329 (1994); Jahn et al., Immunobiol., 193:400-419 (1995); Klimka et al., Brit. J. Cancer, 83:252-260 (2000); Beiboer et al., J. Mol. Biol, 296:833-849 (2000); Rader et al., Proc. Natl. Acad. Sci. USA, 95:8910-8915 (1998); Barbas et al., J. Am. Chem. Soc., 116:2161-2162 (1994); Ditzel etal., J. Immunol.,

[0174]

[0175] (1996)). Accordingly, the recombinant antibodies of the invention prepared as set forth above preferably comprise the heavy and / or light chain CDR3 amino acid sequences of antibodies 9A6 and 7E2. The antibodies further can comprise the CDR2 amino acid sequences of antibodies 9A6 and 7E2. The antibodies further can comprise the CDR1 sequences of antibodies 9A6 and 7E2. The antibodies can further comprise any combinations of the CDR amino acid sequences.

[0176] Accordingly, in another embodiment, the invention further provides ADMe-GR and SDMe-GR antibodies comprising: (1) heavy chain framework regions, a heavy chain CDR1 region, a heavy chain CDR2 region, and a heavy chain CDR3 region, wherein the heavy chain CDR3 region is selected from the CDR3 amino acid sequences of 9A6 or 7E2, and (2) light chain framework regions, a light chain CDR1 region, a light chain CDR2 region, and a light chain CDR3 region, wherein the light chain CDR3 region is selected from the CDR3 amino acid sequences of 9A6 or 7E2, wherein the antibody binds ADMe-GR or SDMe-GR. The antibody may further include the heavy chain CDR2 and / or the light chain CDR2 amino acid sequences of antibodies 9A6 or 7E2. The antibody may further comprise the heavy chain CDR1 and / or the light chain CDR1 amino acid sequence of antibodies 9A6 or 7E2.

[0177] In another embodiment, the CDR1, 2, and / or 3 regions of the engineered antibodies described above can comprise the exact amino acid sequence(s) as those of antibodies 9A6 or 7E2 disclosed herein. However, in other aspects of the invention, the antibodies comprise derivatives from the exact CDR sequences of 9A6 or 7E2 yet still retain the ability to bind ADMe-GR and SDMe-GR effectively. Such sequence modifications may include one or more amino acid additions, deletions, or substitutions, e.g., conservative sequence modifications as described above. Sequence modifications may also be based on the consensus sequences described above for the particular CDR1, CDR2, and CDR3 sequences of antibodies 9A6 or 7E2.

[0178] Accordingly, in another embodiment, the engineered antibody may be composed of one or more CDRs that are, for example, 90%, 95%, 98% or 99.5% identical to one or moreAttorney Docket No. ALSE-016PC

[0179] CDR amino acid sequences of antibodies 9A6 or 7E2. Ranges intermediate to the aboverecited values, e.g., CDRs that are 90-95%, 95-98%, or 98-100% identical identity to one or more of the above sequences are also intended to be encompassed by the present invention.

[0180] In another embodiment, one or more residues of a CDR may be altered to modify binding to achieve a more favored on-rate of binding, a more favored off-rate of binding, or both, such that an idealized binding constant is achieved. Using this strategy, an antibody having ultra-high binding affinity of, for example, IO10M1or more, can be achieved.

[0181] Affinity maturation techniques, well known in the art and those described herein, can be used to alter the CDR region(s) followed by screening of the resultant binding molecules for the desired change in binding. Accordingly, as CDR(s) are altered, changes in binding affinity as well as immunogenicity can be monitored and scored such that an antibody optimized for the best combined binding and low immunogenicity are achieved.

[0182] In addition to or instead of modifications within the CDRs, modifications can also be made within one or more of the framework regions, FR1, FR2, FR3 and FR4, of the heavy and / or the light chain variable regions of an antibody, so long as these modifications do not eliminate the binding affinity of the antibody. For example, one or more non-germline amino acid residues in the framework regions of the heavy and / or the light chain variable region of an antibody of the invention, is substituted with a germline amino acid residue, i.e., the corresponding amino acid residue in the human germline sequence for the heavy or the light chain variable region, which the antibody has significant sequence identity with. For example, an antibody chain can be aligned to a germline antibody chain which it shares significant sequence identity with, and the amino acid residues which do not match between antibody framework sequence and the genuline chain framework can be substituted with corresponding residues from the germline sequence. When an amino acid differs between an antibody variable framework region and an equivalent human germline sequence variable framework region, the antibody framework amino acid should usually be substituted by the equivalent human germline sequence amino acid if it is reasonably expected that the amino acid falls within one of the following categories:

[0183] (1) an amino acid residue which noncovalently binds antigen directly, (2) an amino acid residue which is adjacent to a CDR region, (3) an amino acid residue which otherwise interacts with a CDR region e.g., is within about 3-6 A of a CDR region as determined by computer modeling), or (4) an amino acid reside which participates in the VL-VH interface.Attorney Docket No. ALSE-016PC

[0184] Residues which “noncovalently bind antigen directly” include amino acids in positions in framework regions which have a good probability of directly interacting with amino acids on the antigen according to established chemical forces, for example, by hydrogen bonding, Van der Waals forces, hydrophobic interactions, and the like.

[0185] Accordingly, in one embodiment, an amino acid residue in the framework region of an antibody of the invention is substituted with the corresponding gemiline amino acid residue which noncovalently binds the antigen directly.

[0186] Residues which are “adjacent to a CDR region” include amino acid residues in positions immediately adjacent to one or more of the CDRs in the primary sequence of the antibody, for example, in positions immediately adjacent to a CDR as defined by Kabat, or a CDR as defined by Chothia (see e.g., Chothia and Lesk J. Mol. Biol. 196:901 (1987)).

[0187] Accordingly, in one embodiment, an amino acid residue within the framework region of an antibody of the invention is substituted with a corresponding gemiline amino acid residue which is adjacent to a CDR region.

[0188] Residues that “otherwise interact with a CDR region” include those that are determined by secondary structural analysis to be in a spatial orientation sufficient to affect a CDR region. Such amino acids will generally have a side chain atom within about 3 angstrom units (A) of some atom in the CDRs, and must contain an atom that could interact with the CDR atoms according to established chemical forces such as those listed above. Accordingly, in one embodiment, an amino acid residue within the framework region of an antibody of the invention is substituted with the corresponding germline amino acid residue which otherwise interacts with a CDR region.

[0189] The amino acids at several positions in the framework are known to be important for determining CDR confirmation (e.g., capable of interacting with the CDRs) in many antibodies (Chothia and Lesk, supra, Chothia et al., supra and Tramontane et al., J. Mol. Biol. 215: 175 (1990), all of which are incorporated herein by reference). These authors identified conserved framework residues important for CDR conformation by analysis of the structures of several known antibodies. The antibodies analyzed fell into a limited number of structural or “canonical” classes based on the conformation of the CDRs. Conserved framework residues within members of a canonical class are referred to as “canonical” residues. Canonical residues include residues 2, 25, 29, 30, 33, 48, 64, 71, 90, 94 and 95 of the light chain and residues 24, 26, 29, 34, 54, 55, 71 and 94 of the heavy chain. Additional residues (e.g., CDR structure-determining residues) can be identified according to theAttorney Docket No. ALSE-016PC

[0190] methodology of Martin and Thorton (1996) J. Mol. Biol. 263:800. Notably, the amino acids at positions 2, 48, 64 and 71 of the light chain and 26-30, 71 and 94 of the heavy chain (numbering according to Kabat) are known to be capable of interacting with the CDRs in many antibodies. The amino acids at positions 35 in the light chain and 93 and 103 in the heavy chain are also likely to interact with the CDRs. Additional residues which may effect conformation of the CDRs can be identified according to the methodology of Foote and Winter (1992) J. Mol. Biol. 224:487. Such residues are termed “vernier” residues and are those residues in the framework region closely underlying (z. e., forming a “platform” under) the CDRs.

[0191] Residues which “participate in the VL-VH interface” or “packing residues” include those residues at the interface between VL and VH as defined, for example, by Novotny and Haber, Proc. Natl. Acad. Sci. USA, 82:4592-66 (1985) or Chothia el al, supra.

[0192] Occasionally, there is some ambiguity about whether a particular amino acid falls within one or more of the above-mentioned categories. In such instances, alternative variant antibodies are produced, one of which has that particular substitution, the other of which does not. Alternative variant antibodies so produced can be tested in any of the assays described herein for the desired activity, and the preferred antibody selected.

[0193] Additional candidates for substitution within the framework region are amino acids that are unusual or “rare” for an antibody at that position. These amino acids can be substituted with amino acids from the equivalent position of the human germline sequence or from the equivalent positions of more typical antibodies. For example, substitution may be desirable when the amino acid in a framework region of the antibody is rare for that position, and the corresponding amino acid in the germline sequence is common for that position in immunoglobulin sequences; or, when the amino acid in the antibody is rare for that position, and the corresponding amino acid in the germline sequence is also rare relative to other sequences. It is contemplated that by replacing an unusual amino acid with an amino acid from the gemiline sequence that happens to be typical for antibodies, the antibody may be made less immunogenic. Substitution may also be desirable, for example, in cases of unpaired cysteine residues or putative N-linked glycosylation sites.

[0194] The term “rare”, as used herein, indicates an amino acid occurring at that position in less than about 20%, preferably less than about 10%, more preferably less than about 5%, even more preferably less than about 3%, even more preferably less than about 2% and even more preferably less than about 1% of sequences in a representative sample of sequences, andAttorney Docket No. ALSE-016PC

[0195] the term “common”, as used herein, indicates an amino acid occurring in more than about 25% but usually more than about 50% of sequences in a representative sample. For example, all light and heavy chain variable region sequences are respectively grouped into “subgroups” of sequences that are especially homologous to each other and have the same amino acids at certain critical positions (Kabat et al., supra). When deciding whether an amino acid in an antibody sequence is “rare” or “common” among sequences, it will often be preferable to consider only those sequences in the same subgroup as the antibody sequence.

[0196] In general, the framework regions of antibodies are usually substantially identical, and more usually, identical to the framework regions of the human germline sequences from which they were derived. Of course, many of the amino acids in the framework region make little or no direct contribution to the specificity or affinity of an antibody. Thus, many individual conservative substitutions of framework residues can be tolerated without appreciable change of the specificity or affinity of the resulting immunoglobulin. Thus, in one embodiment, the variable framework region of the antibody shares at least 85% sequence identity to a human germline variable framework region sequence or consensus of such sequences. In another embodiment, the variable framework region of the antibody shares at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a human germline variable framework region sequence or consensus of such sequences.

[0197] In addition to simply binding a methylated DRP (such as ADMe-GR or SDMe-GR) an antibody may be selected for its retention of other functional properties of antibodies of the invention, such as, for example:

[0198] (a) inhibits neuronal cell death;

[0199] (b) reduces the level of methylated DRP-associated toxicity in the cell expressing the methylated DRP;

[0200] (c) reduces circulating levels of methylated DRP in biological tissues or fluids;

[0201] (d) reduces the level of methylated DRP-associated toxicity in cells exogenously exposed to the DRP; and / or

[0202] (e) binds to a methylated DRP with a KD of approximately 10’8to 10-10M or less.

[0203] Characterization of Monoclonal Antibodies to DRPs

[0204] Monoclonal antibodies of the invention can be characterized for binding to a methylated DRP, e.g., ADMe-GR or SDMe-GR, using a variety of known techniques.

[0205] Generally, the antibodies are initially characterized by ELISA (as described in the examples).Attorney Docket No. ALSE-016PC

[0206] Preferably, spleens from rabbits which develop the highest titers are harvested, from which splenocytes were collected. Using cell-sorting (FACs) on splenocytes co-incubated with fluorescently-labeled antigen, reactive B-cells are separated out from pooled splenocytes. These B-cells are cultured and cloned to determine which B-cell clones are the highest monoclonal antibody expressors in the population (via ELISA testing of B-cell supernatants). From high-expressor B-cell clones, heavy and light chain plasmid linear expression modules (LEMs) are generated. These LEMs are then transfected into a suitable cell line for protein overexpression (HEK cells) and HEK supernatants were collected and purified using a protein-A approach for final mAb product.

[0207] To determine if the selected antibodies bind to unique epitopes, each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, IL). Biotinylated MAb binding can be detected with a streptavidin labeled probe. To determine the isotype of purified antibodies, isotype ELIS As can be performed using art recognized techniques. For example, wells of microtiter plates can be coated with 10 pg / ml of anti- Ig overnight at 4°C. After blocking with 5% BSA, the plates are reacted with 10 pg / ml of monoclonal antibodies or purified isotype controls, at ambient temperature for two hours. The wells can then be reacted with either IgGl or other isotype specific conjugated probes. Plates are developed and analyzed as described above.

[0208] To test the binding of monoclonal antibodies to live cells expressing a methylated DRP, e.g., ADMe-GR or SDMe-GR, flow cytometry can be used, e.g., by growing the cell lines mixed with various concentrations of monoclonal antibodies in PBS containing 0.1% BSA at 4°C for 1 hour. After washing, the cells can be reacted with a labeled antibody under the same conditions as the primary antibody staining. The samples can be analyzed by FACScan instrument using light and side scatter properties to gate on single cells and binding of the labeled antibodies is determined. An alternative assay using fluorescence microscopy may be used (in addition to or instead of) the flow cytometry assay. Cells can be stained exactly as described above and examined by fluorescence microscopy. This method allows visualization of individual cells, but may have diminished sensitivity depending on the density of the antigen.

[0209] DRP antibodies, e.g., ADMe-GR or SDMe-GR antibodies, can be further tested for reactivity, i.e., inhibition of DRPs by other methods. For example, an exogenous DRP challenge assay wherein either ADMe-GRis or SDMe-GR is exogenous peptides are dosed into cells culture media, followed by administration of either custom antibody. Binding ofAttorney Docket No. ALSE-016PC

[0210] the antibody to exogenous peptide, thus preventing either cellular entry or DRP toxicity upon cellular entry, indicates reactivity.

[0211] Methods for analyzing binding affinity, cross-reactivity, and binding kinetics of various DRP antibodies, e.g., ADMe-GR or SDMe-GR antibodies, include standard assays known in the art, for example, Biacore™ surface plasmon resonance (SPR) analysis using a Biacore™ 2000 SPR instrument (Biacore AB, Uppsala, Sweden), or bio-layer interferometry (BLI) using an Octet™ QKe instrament as described in the examples.

[0212] Antibodies which bind to the same epitope as that of antibodies 9A6 and 7E2 (as determined by a given epitope mapping technique) also are provided herein. As described herein, techniques for determining antibodies that bind to the "same epitope on ADMe-GR or SDMe-GR" with the antibodies described herein include, for example, epitope mapping methods, such as, x-ray analyses of crystals of antigen: antibody complexes which provides atomic resolution of the epitope. Other methods monitor the binding of the antibody to antigen fragments or mutated variations of the antigen where loss of binding due to a modification of an amino acid residue within the antigen sequence is often considered an indication of an epitope component. In addition, computational combinatorial methods for epitope mapping can also be used. Methods may also rely on the ability of an antibody of interest to affinity isolate specific short peptides (either in native three-dimensional form or in denatured form) from combinatorial phage display peptide libraries. The peptides are then regarded as leads for the definition of the epitope corresponding to the antibody used to screen the peptide library. For epitope mapping, computational algorithms have also been developed which have been shown to map conformational discontinuous epitopes.

[0213] II. Uses and Methods of the Invention

[0214] As further described herein, the antibodies of the present invention can be used for detecting the presence or absence of a methylated DRP, e.g., an ADMe-GR or SDMe-GR DRP, in a subject or biological sample. The antibodies of the present invention also can be used to treat diseases associated with expression of methylated DRPs, such as neurodegenerative diseases (e.g., ALS or FTD).

[0215] Diagnostic / Prognostic Uses and In vivo imaging with DRP Antibodies

[0216] The DRP antibodies (e.g., ADMe-GR and SDMe-GR antibodies) described herein are useful in a variety of diagnostic and prognostic applications, e.g., as an agent for measuringAttorney Docket No. ALSE-016PC

[0217] the amount of methylated DRPs or the amount of activity associated with methylated DRPs in a sample from a subject (e.g., a subject diagnosed with a neurodegenerative disease, such as ALS or FTD). Methods of diagnosing or monitoring the progress of a disease associated with the expression or activity of methylated DRPs in a subject, comprise

[0218] (a) administering to the subject the antibody, or antigen binding portion thereof, described herein at a first time point and measuring the level of methylated DRPs (or the activity of methylated DRPs) in a sample from the subject (e.g., a biological fluid or tissue from the subject;

[0219] (b) administering to the subject the antibody, or antigen binding portion thereof, at one or more subsequent time points and measuring the level of methylated DRPs (or the activity of methylated DRPs) in a sample from the subject; wherein the level of methylated DRPs at each time point is indicative of the progress of the disease and the efficacy of the treatment administered to the subject.

[0220] The DRP antibodies (e.g., ADMe-GR and SDMe-GR antibodies) described herein also are useful in imaging applications, i.e., as an imaging agent. In certain embodiments, a DRP antibody is labelled with a moiety that is detectable in vivo and is used as an in vivo imaging agent, e.g., in a method for detecting expression or amount of methylated DRPs in a subject comprising administering to the subject a DRP antibody (e.g., a ADMe-GR or SDMe-GR antibody) linked to a detectable label and, after the appropriate time, detecting the label in the subject.

[0221] As an imaging agent, a DRP antibody may be used to diagnose a disorder or disease associated with increased levels or toxicity of methylated DRPs, for example, a neurodegenerative disease, such as ALS or FTD. In a similar manner, a DRP antibody can be used to monitor DRP levels in a subject, e.g., a subject that is being treated to reduce DRP levels. The DRP antibodies may be used with or without modification and may be labeled by covalent or non-covalent attachment of a detectable moiety.

[0222] Detectable moieties that may be used include fluorescent labeling reagents, i.e., chemicals that provide fluorophores. In fluorescence labeling technology, commonly used reagents include rhodamines and fluoresceins. Other fluorescent labeling reagents include polycyclic aromatic compounds, aromatic heterocyclic compounds, and some chelates of rare earth elements.

[0223] Other detectable moieties that may be used include radioactive agents, such as: radioactive heavy metals such as iron chelates, radioactive chelates of gadolinium orAttorney Docket No. ALSE-016PC

[0224] manganese, positron emitters of oxygen, nitrogen, iron, carbon, or gallium,1SF,60Cu,61Cu,62Cu,64Cu,l24I,86Y,s9Zr,6CGa,67Ga,68Ga,44Sc,47Sc,1JC.lllln,I14l,,In.!l4In,1251,1241,13!I.l23I,131I,123I,32C1,33C1,34C1.74Br,75Br,76Br,77Br,78Br,89Zr,186Re,1S8Re,S6Y,90X177Lu, " Tc,212Bi,213Bi,212Pb,225Ac, or153Sm.

[0225] In certain embodiments, the radioactive agent is conjugated to the DRP antibody at one or more amino acid residues. In certain embodiments, one or more, such as two or more, three or more, four or more, or a greater number of radionuclides can be present in the labelled probe. In certain embodiments, the radionuclide is attached directly to the DRP antibody by a chelating agent (e.g., see U. S. Patent 8,808,665). In certain embodiments, the radionuclide is present in a prosthetic group conjugated to the DRP antibody by a bifunctional chelator or conjugating (BFC) moiety. In certain embodiments, the radionuclide chelating agent and / or conjugating moiety is DFO, DOTA and its derivatives (CB-DO2A, 3p-C-DEPA, TCMC, Oxo-DO3A), DBCO, TE2A, CB-TE2A, CB-TE1A1P, CB-TE2P, MM-TE2A, DM-TE2A, diamsar and derivatives, NODASA, NOD AGA, NOTA, NETA, TACN-TM, DTPA, 1B4M-DTPA, CHX-A"-DTPA, TRAP (PRP9), NOPO, AAZTA and derivatives (DATA), FFdedpa, Hioctapa, FEazapa, Hsdecapa, FEphospa, HBED, SHBED, BPCA, CP256, PCTA, HEHA, PEPA, EDTA, TETA, and TRITA based chelating agents, and close analogs and derivatives thereof.

[0226] In certain embodiments, the radionuclide chelating or conjugating (BFC) moiety is maleamide-NODAGA or maleamide-DBCO, which can be attached covalently to a polypeptide via cysteine residues near the C-terminus of the polypeptide. In certain embodiments, a DRP antibody is modified at its C-terminus by the addition of a cysteine. For example, PxCy may be linked C-terminal to the amino acid residues NYRT, wherein P is proline, C is cysteine, and x and y are integers that are at least 1. Maleimide-NODAGA or maleimide-DBCO can be reacted with the cysteine, to yield DRP antibody-NODAGA or DRP antibody-DBCO, respectively.

[0227] In certain embodiments, the radionuclide chelating agent is DFO, which can be attached, e.g., at random surface lysines.

[0228] In certain embodiments, the chelator for64Cu is DOTA, NOTA, EDTA, Df, DTPA, or TETA. Suitable combinations of chelating agents and radionuclides are extensively reviewed in Price et al., Chem Soc Rev 2014;43:260-90.Attorney Docket No. ALSE-016PC

[0229] In certain embodiments, a DRP antibody is labelled with the PET tracer18E A DRP antibody may be labelled with a prosthetic group, such as [lsF]-3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-fluoropyridine ([18F]-FFPEGA).

[0230] In certain embodiments, a DRP antibody is labelled with64Cu.64Cu may be linked to DRP antibody with a chelating agent, such as NOD AGA.

[0231] Other art-recognized methods for labelling polypeptides with radionuclides such as64Cu and18F for synthesizing the DRP antibody imaging agents described herein may also be used. See, e.g., US2014 / 0271467; Gill et al., Nature Protocols 2011;6:1718-25; Berndt et al. Nuclear Medicine and Biology 2007;34:5-15, Inkster et al., Bioorganic & Medicinal Chemistry Letters 2013;23:3920-6, the contents of which are herein incorporated by reference in their entirety.

[0232] Administration and Imaging

[0233] In certain embodiments, the labelled DRP antibody can be used to image cells expressing a methylated DRP, e.g., neuronal cells. For example, the labelled DRP antibody is administered to a subject in an amount sufficient to uptake the labelled DRP antibody into the tissue of interest (e.g., neural tissue). Die subject is then imaged using an imaging system (such as PET) for an amount of time appropriate for the particular radionuclide being used. The labelled DRP antibody-bound DRP-expressing cells or tissues are then detected by the imaging system.

[0234] PET imaging with a DRP antibody imaging agent may be used to qualitatively or quantitatively detect a methylated DRP. A DRP antibody imaging agent may be used as a biomarker, and the presence or absence of a positive signal in a subject may be indicative that, e.g., the subject would be responsive to a given therapy or that the subject is responding or not to a therapy.

[0235] In certain embodiments, the progression or regression of disease (e.g., a neurodegenerative disease) can be imaged as a function of time or treatment. For instance, the level of toxicity caused by methylated DRPs can be monitored in a subject undergoing therapy and the extent of regression of the level of toxicity can be monitored in real-time based on detection of the labeled DRP antibody.

[0236] The amount effective to result in uptake of the imaging agent (e.g.,i8F-DRP antibody imaging agent,64Cu- DRP antibody imaging agent) into the cells or tissue of interest (e.g., neuronal cells) may depend upon a variety of factors, including for example, the age, bodyAttorney Docket No. ALSE-016PC

[0237] weight, general health, sex, and diet of the host; the time of administration; the route of administration; the rate of excretion of the specific probe employed; the duration of the treatment; the existence of other drugs used in combination or coincidental with the specific composition employed; and other factors.

[0238] Also disclosed herein are methods of diagnosing the presence of a disease associated with methylated DRPs in a subject, the method comprising

[0239] (a) administering to a subject in need thereof a DRP antibody (e.g., a ADMe-GR or SDMe-GR antibody) imaging agent described herein; and

[0240] (b) obtaining a radio-image of at least a portion of the subject to detect the presence or absence of the imaging agent, wherein the presence and location of the imaging agent above background is indicative of the presence of a disease associated with methylated DRPs, e.g., a neurodegenerative disease, such as ALS or FTD.

[0241] Also disclosed herein are methods of monitoring the progress of a disease associated with the expression of methylated DRPs (e.g., a neurodegenerative disease, such as ALS or FTD) in the cells of a subject, the method comprising

[0242] (a) administering to a subject in need thereof a DRP antibody imaging agent described herein at a first time point and obtaining an image of at least a portion of the subject to determine the amount of methylated DRPs expressed by the cells;

[0243] (b) administering to the subject the imaging agent at one or more subsequent time points and obtaining an image of at least a portion of the subject at each time point; wherein the amount of the methylated DRPs at each time point is indicative of the progress of the disease.

[0244] In certain embodiments, the labelled DRP antibody can be used to monitor disease progression in a subject (e.g., humans or animal subjects diagnosed with a neurodegenerative disease, such as ALS or FTD) receiving one or more drug therapies (e.g., one or more drug therapies to treat ALS) to assess the efficacy of the therapy. For example, the method comprises

[0245] (a) administering to the subject diagnosed with ALS a DRP antibody imaging agent described herein at a first time point and obtaining an image of at least a portion of the subject to determine the amount of methylated DRPs expressed by the cells;

[0246] (b) administering to the subject the imaging agent at one or more subsequent time points and obtaining an image of at least a portion of the subject at each time point;Attorney Docket No. ALSE-016PC

[0247] wherein the amount of the methylated DRPs at each time point is indicative of the progress of the disease and the efficacy of the therapy.

[0248] PET Imaging

[0249] Typically, for PET imaging purposes it is desirable to provide the recipient with a dosage of DRP antibody imaging agent that is in the range of from about 1 mg to 200 mg as a single intravenous infusion, although a lower or higher dosage also may be administered as circumstances dictate. In certain embodiments, administration occurs in an amount of radiolabeled DRP antibody of between about 0.005 pg / kg of body weight to about 50 pg / kg of body weight per day, usually between 0.02pg / kg of body weight to about 3 pg / kg of body weight. The mass associated with a PET tracer is in the form of the natural isotope (e.g.,19F for an18F PET tracer). A particular analytical dosage for the instant composition includes from about 0.5 pg to about 100 pg of a radiolabeled protein. The dosage will usually be from about Ipg to about 50pg of a radiolabeled protein.

[0250] Dosage regimens are adjusted to provide the optimum detectable amount for obtaining a clear image of the tissue or cells which uptake the radiolabeled DRP antibody. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to which the radiolabeled DRP antibody is to be administered. The specification for the dosage unit forms described herein are dictated by and directly dependent on (a) the unique characteristics of the targeting portion of the radiolabeled DRP antibody; (b) the tissue or cells to be targeted; (c) the limitations inherent in the imaging technology used.

[0251] Actual dosage levels of the active ingredients in the pharmaceutical compositions described herein may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired uptake of the radiolabeled DRP antibody in the cells or tissues of a particular patient, composition, and mode of administration, without being toxic to the patient. It will be understood, however, that the total daily usage of the radiolabeled DRP antibody of the present disclosure will be decided by the attending physician or other attending professional within the scope of sound medical judgment. The specific effective dose level for any particular subject will depend upon a variety of factors, including for example, the activity of the specific composition employed: the specific composition employed; the age, body weight, general health, sex, and diet of the host; the time ofAttorney Docket No. ALSE-016PC

[0252] administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; other drugs, compounds and / or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0253] In vitro imaging with DRP Antibodies

[0254] In addition to detecting methylated DRPs in vivo, DRP antibody imaging agents, such as those described herein, may be used for detecting a target molecule in a sample. A method may comprise contacting the sample with a DRP antibody (e.g., an ADME-GR or SDMe-GR antibody) described herein, wherein said contacting is carried out under conditions that allow a complex to form between the antibody and the target; and detecting said complex, thereby detecting said target in said sample. Detection may be carried out using any art-recognized technique, such as, e.g., radiography, immunological assay, fluorescence detection, mass spectroscopy, or surface plasmon resonance. The sample may be from a human or other mammal. For diagnostic purposes, appropriate agents are detectable labels that include radioisotopes, for whole body imaging, and radioisotopes, enzymes, fluorescent labels and other suitable antibody tags for sample testing.

[0255] The detectable labels can be any of the various types used currently in the field of in vitro diagnostics, including particulate labels including metal sols such as colloidal gold, isotopes such as I125or Tc99presented for instance with a peptidic chelating agent of the N2S2, N3S or N4 type, chromophores including fluorescent markers, biotin, luminescent markers, phosphorescent markers and the like, as well as enzyme labels that convert a given substrate to a detectable marker, and polynucleotide tags that are revealed following amplification such as by polymerase chain reaction. A biotinylated antibody would then be detectable by avidin or streptavidin binding. Suitable enzyme labels include horseradish peroxidase, alkaline phosphatase and the like. For instance, the label can be the enzyme alkaline phosphatase, detected by measuring the presence or formation of chemiluminescence following conversion of 1,2 dioxetane substrates such as adamantyl methoxy phosphoryloxy phenyl dioxetane (AMPPD), disodium 3-(4-(methoxyspiro { 1,2-dioxetane-3,2'-(5'-chloro)tricyclo { 3.3.1.1 3, 7} decan} -4-yl) phenyl phosphate (CSPD), as well as CDP and CDP-star® or other luminescent substrates well-known to those in the art, for example the chelates of suitable lanthanides such as Terbium(III) and Europium(III). Other labels include those set forthAttorney Docket No. ALSE-016PC

[0256] above in the imaging section. The detection means is determined by the chosen label.

[0257] Appearance of the label or its reaction products can be achieved using the naked eye, in the case where the label is particulate and accumulates at appropriate levels, or using instruments such as a spectrophotometer, a luminometer, a fluorimeter, and the like, all in accordance with standard practice.

[0258] In certain embodiments, conjugation methods result in linkages which are substantially (or nearly) non-immunogenic, e.g., peptide- (i.e. amide-), sulfide-, (sterically hindered), disulfide-, hydrazone-, and ether linkages. These linkages are nearly non-immunogenic and show reasonable stability within serum (see e.g. Senter, P. D., Curr. Opin. Chem. Biol. 13 (2009) 235-244; WO 2009 / 059278; WO 95 / 17886).

[0259] Depending on the biochemical nature of the moiety and the DRP antibody, different conjugation strategies can be employed. In a case where the moiety is a naturally occurring or recombinant polypeptide of between 50 to 500 amino acids, there are standard procedures in textbooks describing the chemistry for synthesis of protein conjugates, which can be easily followed by the skilled artisan (see e.g. Hackenberger, C. P. R., and Schwarzer, D., Angew. Chem. Int. Ed. Engl. 47 (2008) 10030-10074). In one embodiment, the reaction of a maleinimido moiety with a cysteine residue within the DRP antibody or the moiety is used. Alternatively, coupling to the C-terminal end of the DRP antibody is performed. C-terminal modification of a protein can be performed as described in, e.g., Sunbul, M. and Yin, J., Org. Biomol. Chem. 7 (2009) 3361 -3371 ). When the moiety is a peptide or polypeptide, the DRP antibody and moiety can be fused by standard genetic fusion, optionally with a linker disclosed herein.

[0260] Therapeutic Methods

[0261] The DRP antibodies (e.g., ADMe-GR or SDMe-GR antibodies) of the invention also are capable of functionally inhibiting the effects of intronic repeat expansion mutations in the C9orf72 gene, both in vitro and in vivo. Specifically, by binding to the methylated DRPs generated by the intronic repeat expansion mutations in the C9orf72 gene (such as ADMe-GR and SDMe-GR DRPs) such that the toxic activity associated with the methylated DRPs is inhibited. Accordingly, in another aspect, the invention pertains to a method of inhibiting DRP activity in a subject, the method comprising administering to the subject the antibody of the invention in an amount effective to inhibit DRP activity in the subject. In another embodiment, the invention provides a method of treating, preventing, or reducing symptomsAttorney Docket No. ALSE-016PC

[0262] of a disorder mediated by undesired activity of methylated DRP activity in a subject, the method comprising administering to the subject an effective amount of an antibody of the invention. Examples of such disorders include neurodegenerative disorders, such as Amyotrophic Lateral Sclerosis (ALS, sometimes called Lou Gehrig's disease), Frontotemporal Dementia (FTD), Charcot-Marie-Tooth disease (Hereditary Motor and Sensory Neuropathy, HMSN) and Huntington Disease (HD).

[0263] In another embodiment, the present invention provides a composition, e.g., a composition containing one or a combination of antibodies of the present invention (i.e., a DRP antibody, such as an ADMe-GR or SDMe-GR antibody), formulated together with a carrier e.g., a pharmaceutically acceptable carrier) for use in a therapeutic method (e.g., the treatment of a neurodegenerative disease). Pharmaceutical compositions of the invention also can be administered in combination therapy, i.e., combined with other agents. For example, the combination therapy can include a composition of the present invention with at least one or more additional therapeutic agents, such as a therapeutic agent for treating a neurodegenerative disease.

[0264] As used herein, the terms “carrier” and “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., antibody, bispecific and multispecific molecule, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0265] Examples of adjuvants which may be used with the antibodies and constructs of the present invention include: Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N. J.); AS-2 (SmithKline Beecham, Philadelphia, Pa.); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc: an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatised polysaccharides; polyphosphazenes; biodegradable microspheres; cytokines, such as GM-CSF, interleukin-2, -7, -12, and other like factors; 3D-MPL; CpG oligonucleotide; and monophosphoryl lipid A, for example 3-de-O-acylated monophosphoryl lipid A.Attorney Docket No. ALSE-016PC

[0266] A “pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl -substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N, N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.

[0267] A composition of the present invention can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and / or mode of administration will vary depending upon the desired results. The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, poly anhydrides, polyglycolic acid, collagen, poly orthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0268] To administer a compound of the invention by certain routes of administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation. For example, the compound may be administered to a subject in an appropriate carrier, for example, liposomes, or a diluent. Acceptable diluents include saline and aqueous buffer solutions. Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes (Strejan et al. (1984) J. Neuroimmunol. 7:27).

[0269] Carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof inAttorney Docket No. ALSE-016PC

[0270] the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0271] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[0272] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0273] In certain embodiments, the antibodies of the invention can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the invention cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g., U. S. Patents 4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery see, e.g., V. V. Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folate or biotin (see, e.g., U. S. Patent 5,416,016 to Low et al.) mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140; M.Attorney Docket No. ALSE-016PC

[0274] Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233: 134), different species of which may comprise the formulations of the inventions, as well as components of the invented molecules; pl 20 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion; I. I. Fidler (1994) Immunomethods 4:273. In one embodiment of the invention, the therapeutic compounds of the invention are formulated in liposomes; in a more preferred embodiment, the liposomes include a targeting moiety. The composition must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0275] Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. For example, the antibodies of the invention may be administered once or twice weekly by subcutaneous or intramuscular injection or once or twice monthly by subcutaneous or intramuscular injection.

[0276] It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

[0277] Examples of pharmaceutically-acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.Attorney Docket No. ALSE-016PC

[0278] For the therapeutic compositions, formulations of the present invention include those suitable for intravenous, intraperitoneal, oral, nasal, topical (including buccal and sublingual), rectal, vaginal and / or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 0.001 per cent to about ninety per cent of active ingredient, preferably from about 0.005 per cent to about 70 per cent, most preferably from about 0.01 per cent to about 30 per cent.

[0279] The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

[0280] Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0281] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.Attorney Docket No. ALSE-016PC

[0282] When the compounds of the present invention are administered as pharmaceuticals, to humans and animals, they can be given alone or as a pharmaceutical composition containing, for example, 0.001 to 90% (more preferably, 0.005 to 70%, such as 0.01 to 30%) of active ingredient in combination with a pharmaceutically acceptable carrier.

[0283] Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrated form, and / or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.

[0284] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable daily dose of a composition of the invention will be that amount of the compound which is the lowest dose effective in producing a therapeutic effect. Such an effective dose will generally depend upon the factors described above. It is preferred that administration be intravenous, intramuscular, intraperitoneal, or subcutaneous, preferably administered proximal to the site of the target. If desired, the effective daily dose of a therapeutic composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition).Attorney Docket No. ALSE-016PC

[0285] Therapeutic compositions can be administered with medical devices known in the art. For example, in a preferred embodiment, a therapeutic composition of the invention can be administered with a needleless hypodermic injection device, such as the devices disclosed in U. S. Patent Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556. Examples of well-known implants and modules useful in the present invention include: U. S. Patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U. S. Patent No. 4,486,194, which discloses a therapeutic device for administering medicants through the skin; U. S. Patent No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U. S. Patent No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U. S. Patent No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and U. S. Patent No. 4,475,196, which discloses an osmotic drug delivery system. Many other such implants, delivery systems, and modules are known to those skilled in the art.

[0286] The composition must be sterile and fluid to the extent that the composition is deliverable by syringe. In addition to water, the carrier can be an isotonic buffered saline solution, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by use of coating such as lecithin, by maintenance of required particle size in the case of dispersion and by use of surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition. Long-term absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.

[0287] When the active compound is suitably protected, as described above, the compound may be orally administered, for example, with an inert diluent or an assimilable edible carrier.

[0288] The present invention is further illustrated by the following examples, which should not be construed as further limiting. The contents of Sequence Listing, figures and all references, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.Attorney Docket No. ALSE-016PC

[0289] Examples

[0290] Two monoclonal antibodies that bind methylated DRPs, i.e., ADMe-GR (antibody 9A6) and SDMe-GR (antibody 7E2) were generated, and the four corresponding plasmid sequences were obtained (see FIGs. 1-4) according to the protocols described in Examples 1 and 2.

[0291] Example 1: Generation of anti-ADMe-GRs rabbit monoclonal antibody 9A6

[0292] Study 1 A: Rabbit immunization sequence and candidate selection for monoclonal antibody

[0293] Three rabbits, herein referred to as AL139, AL140, and AL141, underwent the following immunization process with a KLH-conjugated form of ADMe-GRs. As shown in the ELISA results, T50% titers were included for both the intended antigen, ADMe-GRs, and related counter-antigens, GRs and SDMe-GRs. Sequences for all mentioned peptides are included in Table 1. Additional ELISAs on the same sera samples were performed to confirm titer profiles (data not shown). Also, AL139 was immunized first as a “test” animal, with AL140 and AL141 immunized later once successful immunization was achieved with AL139. Accordingly, the dates below vary between AL139 and AL140-141 throughout.

[0294] • Day 0: Rabbits were bled before immunization (“pre-bleed”), and subcutaneously immunized with 250 pg of KLH-ADMe-GRs.

[0295] o AL139: 11 / 4 / 22

[0296] o AL140-141: 12 / 6 / 22

[0297] • Day 14: Rabbits were subcutaneously boosted with 125 pg of KLH-ADMe-GRs. o AL139: 11 / 18 / 22

[0298] o AL140-141: 12 / 20 / 22

[0299] • Day 24: Rabbits were bled (“production bleed 1”), serum was collected, and an ELISA was run to measure antibody titers.

[0300] o AL139: 11 / 28 / 22

[0301] □ AL139 Production Bleed 1 ELISA run 11 / 30 / 22

[0302] • T50% Titer vs. ADMe-GRs: 170,000

[0303] • T50% Titer vs. GRs: 32,600

[0304] • T50% Titer vs. SDMe-GRs: 30,700

[0305] o AL140-141: 12 / 30 / 22

[0306] □ AL140 Production Bleed 1 ELISA run 1 / 5 / 23

[0307] • T50% Titer vs. ADMe-GRs: 271,000

[0308] • T50% Titer vs. GR8: 20,700

[0309] • T50% Titer vs. SDMe-GRs: 33,900

[0310] □ AL141 Production Bleed 1 ELISA run 1 / 5 / 23

[0311] • T50% Titer vs. ADMe-GRs: 400,000

[0312] • T50% Titer vs. GRS: 11,600

[0313] • T50% Titer vs. SDMe-GRs: 17,000Attorney Docket No. ALSE-016PC

[0314] • Day 35: Rabbits were subcutaneously boosted with 125 pg of KLH-ADMe-GRs. o AL139: 12 / 9 / 22

[0315] o AL140-141: 1 / 10 / 23

[0316] • Day 45: Rabbits were bled (“production bleed 2”), serum was collected, and an ELISA was run to measure antibody titers.

[0317] o AL139: 12 / 19 / 22

[0318] □ AL139 Production Bleed 2 ELISA run 12 / 22 / 22

[0319] • T50% Titer vs. ADMe-GR8: 140,000

[0320] • T50% Titer vs. GR8: 20,700

[0321] • T50% Titer vs. SDMe-GRs: 33,900

[0322] o AL140-141: 1 / 20 / 23

[0323] □ AL140 Production Bleed 2 ELISA run 1 / 25 / 23

[0324] • T50% Titer vs. ADMe-GRs: 410,000

[0325] • T50% Titer vs. GR8: 75,500

[0326] • T50% Titer vs. SDMe-GRs: 128,000

[0327] □ AL141 Production Bleed 2 ELISA run 1 / 25 / 23

[0328] • T50% Titer vs. ADMe-GR8: 286,000

[0329] • T50% Titer vs. GR8: 6,470

[0330] • T50% Titer vs. SDMe-GRs: 39,300

[0331] • Day 56: Rabbits were subcutaneously boosted with 125 pg of KLH-ADMe-GR8. o AL139: 12 / 30 / 22

[0332] o AL140-141: 1 / 31 / 23

[0333] • Day 66: Rabbits were bled (“production bleed 3”), serum was collected, and an ELISA was run to measure antibody titers.

[0334] o AL139: 1 / 9 / 23

[0335] □ AL139 Production Bleed 3 ELISA run 1 / 11 / 23

[0336] • T50% Titer vs. ADMe-GRs: 74,200

[0337] • T50% Titer vs. GR8: 10,900

[0338] • T50% Titer vs. SDMe-GR8: 25,400

[0339] o AL140-141: 2 / 10 / 23

[0340] □ AL140 Production Bleed 3 ELISA run 2 / 15 / 23

[0341] • T50% Titer vs. ADMe-GRs: 186,000

[0342] • T50% Titer vs. GRs: 64,500

[0343] • T50% Titer vs. SDMe-GRs: 89,100

[0344] □ AL141 Production Bleed 3 ELISA run 2 / 15 / 23

[0345] • T50% Titer vs. ADMe-GRs: 218,000

[0346] • T50% Titer vs. GR8: 6,550

[0347] • T50% Titer vs. SDMe-GR8: 73,800

[0348] • Days 184 and 216: Rabbits were subcutaneously boosted with 125 pg of KLH- ADMe-GRs.

[0349] o Day 216: AL139: 6 / 8 / 23

[0350] o Day 184: AL140-141: 6 / 8 / 23

[0351] • Days 192 and 224: Rabbits were bled (“production bleed 4’’), serum was collected, and an ELISA was run to measure antibody titers.

[0352] o Day 224: AL139: 6 / 16 / 23Attorney Docket No. ALSE-016PC

[0353] □ AL139 Production Bleed 4 ELISA run 6 / 20 / 23

[0354] • T50% Titer vs. ADMe-GRs: 25,400

[0355] • T50% Titer vs. GRs: not tested by Labcorp

[0356] • T50% Titer vs. SDMe-GRs: 40,700

[0357] o Day 192: AL140-141: 6 / 16 / 23

[0358] □ AL140 Production Bleed 4 ELISA run 6 / 20 / 23

[0359] • T50% Titer vs. ADMe-GRs: 45,400

[0360] • T50% Titer vs. GRs: not tested by Labcorp

[0361] • T50% Titer vs. SDMe-GRs: 18,000

[0362] □ AL141 Production Bleed 4 ELISA run 6 / 20 / 23

[0363] • T50% Titer vs. ADMe-GRs: 127,000

[0364] • T50% Titer vs. GRs: not tested by Labcorp

[0365] • T50% Titer vs. SDMe-GRs: 66,900

[0366] • Days 255 and 287: Rabbits were intravenously boosted with (not specified) pg of KLH-ADMe-GRs.

[0367] o Day 287: AL139: 8 / 18 / 23

[0368] o Day 255: AL140-141: 8 / 18 / 23

[0369] • Days 259 and 291: Rabbits were terminated, followed by spleen harvests on AL141 (chosen candidate) and AL139 (backup candidate) and terminal bleed (all animals).

[0370] o Day 287: AL 139: 8 / 22 / 23

[0371] o Day 259: AL140-141: 8 / 22 / 23

[0372] • End of Study 1A: Rabbit AL141’s spleen was chosen to proceed to monoclonal antibody preparation in Study IB. A polyclonal antibody from this animal was also isolated and purified from reagents collected during Study 1A.

[0373] Study IB: B-Cell isolation, cloning, and monoclonal anti -ADMe-GRs antibody production from chosen candidate AL141

[0374] The monoclonal antibody production process was split into three main phases. Phase I included the collection and isolation of antigen-reactive b-cells from the spleen of animal AL141. Phase II included cloning of the b-cells isolated in phase I, selection of clones with high binding to the intended antigen, and generation of heavy and light-chain gene linear expression modules (LEMs) for top clone candidates. HEK cells were then transfected with resulting LEMs. Phase III included selection of one high-expressing LEM-transfected HEK clone, small-scale antibody expression and purification followed by validation testing, and final large-scale antibody expression and purification. The final monoclonal antibody reagent was produced by the conclusion of Phase III.

[0375] • Phase la: Splenocytes were isolated from spleen harvested from animal AL141.

[0376] • Phase lb: Splenocytes were incubated with fluorescent DRP antigen and sorted using flow cytometry to select for b-cells with high antigen affinity.

[0377] • Phase Ila: Sorted b-cells were plated into 384-well plates overnight. The following day, b-cell supernatants were collected and tested by ELISA. Ten b-cell clones were chosen to move forward based on having their supernatant samples having the highestAttorney Docket No. ALSE-016PC

[0378] antibody binding to the desired antigen and lowest antibody binding to relevant counter- antigens.

[0379] • Phase lib: Heavy and light-chain genes from top b-cell clones were cloned into linear expression module (LEM) plasmids. These LEM plasmids were transfected into HEK cells. Supernatants from the transfected HEK cells, referred to as “LEM- supernatants,” were collected and tested by ELISA. Based on results, one LEM- transfected HEK clone was selected to move forward to final phase.

[0380] • Phase Illa: Selected HEK clone candidate was used for small-scale antibody expression. The resulting antibody was purified using a Protein A purification approach, then validated by ELISA and SDS-PAGE.

[0381] • Phase Illb: Antibody expression was repeated at a larger scale and resulting antibody was purified using a Protein A purification approach.

[0382] • End of Study IB: Samples of two plasmids: one encoding the heavy-chain gene sequence and one encoding the light -chain gene sequence for the final validated monoclonal antibody were collected. The sequences for both plasmids were determined. Final purified samples of monoclonal anti-ADMe-GRs antibody from phases Illa and Illb are being stored in-house.

[0383] Example 2: Generation of anti-SDMe-GRs rabbit monoclonal antibody 7E2

[0384] Study 2A: Rabbit immunization sequence and candidate selection for monoclonal development

[0385] Three rabbits, herein referred to as AL142, AL143, and AL144, underwent the following immunization process with a KLH-conjugated form of SDMe-GRs. As shown in the ELISA results, T50% titers are included for both the intended antigen, SDMe-GRs, and related counter-antigens, GRs and ADMe-GRs. Sequences for all mentioned peptides are included in Table 1. Additional testing on the same sera samples to confirm titer profiles was performed (data not shown). Also, in contrast to Study 1 A, no “test animal” was designated. Therefore, each step was performed on the same date for all three animals.

[0386] • Day 0: Rabbits were bled before immunization (“pre-bleed”), and subcutaneously immunized with 250 pg of KLH-SDMe-GRs.

[0387] o AL142-144: 7 / 21 / 23

[0388] • Day 21: Rabbits were subcutaneously boosted with 125 pg of KLH-SDMe-GRs. o AL142-144: 8 / 11 / 23

[0389] • Day 31: Rabbits were bled (“production bleed 1”), serum was collected, and an ELISA was run to measure antibody titers.

[0390] o AL142-144: 8 / 21 / 23

[0391] □ AL142 Production Bleed 1 ELISA run 8 / 24 / 23

[0392] • T50% Titer vs. SDMe-GRs: 375,000

[0393] □ AL142 Production Bleed 1 ELISA follow-up run 12 / 12 / 23

[0394] • T50% Titer vs. ADMe-GRs: 32,500

[0395] • T50% Titer vs. GRs: 9,120

[0396] □ AL143 Production Bleed 1 ELISA run 8 / 24 / 23Attorney Docket No. ALSE-016PC

[0397] • T50% Titer vs. SDMe-GR8: 364,000

[0398] □ AL143 Production Bleed 1 ELISA follow-up run 12 / 12 / 23 • T50% Titer vs. ADMe-GR8: 56,700

[0399] • T50% Titer vs. GR8: < 1,000

[0400] □ AL144 Production Bleed 1 ELISA run 8 / 24 / 23

[0401] • T50% Titer vs. SDMe-GR8: 740,000

[0402] □ AL144 Production Bleed 1 follow-up run 12 / 12 / 23

[0403] • T50% Titer vs. ADMe-GR8: 100,000

[0404] • T50% Titer vs. GR8: 54,600

[0405] • Day 42: Rabbits were subcutaneously boosted with 125 pg of KLH-SDMe-GR8.

[0406] • Day 52: Rabbits were bled (“production bleed 2”), serum was collected, and an ELISA was run to measure antibody titers.

[0407] o AL142-144: 9 / 11 / 23

[0408] □ AL142 Production Bleed 2 ELISA run 9 / 12 / 23

[0409] • T50% Titer vs. SDMe-GR8: 411,000

[0410] □ AL142 Production Bleed 2 ELISA follow-up run 12 / 13 / 23 • T50% Titer vs. ADMe-GRs: 65,100

[0411] • T50% Titer vs. GR8: 13,600

[0412] □ AL143 Production Bleed 2 ELISA run 9 / 12 / 23

[0413] • T50% Titer vs. SDMe-GR8: 387,000

[0414] □ AL143 Production Bleed 2 ELISA follow-up run 12 / 12 / 23 • T50% Titer vs. ADMe-GR8: 88,200

[0415] • T50% Titer vs. GR8: 567

[0416] □ AL144 Production Bleed 2 ELISA run 9 / 12 / 23

[0417] • T50% Titer vs. SDMe-GR8: 699,000

[0418] □ AL144 Production Bleed 2 follow-up run 12 / 12 / 23

[0419] • T50% Titer vs. ADMe-GR8: 176,000

[0420] • T50% Titer vs. GR8: 95,400

[0421] • Day 63: Rabbits were subcutaneously boosted with 125 pg of KLH-SDMe-GR8.

[0422] • Day 73: Rabbits were bled (“production bleed 3”), serum was collected, and an ELISA was run to measure antibody titers.

[0423] o AL 142- 144: 10 / 2 / 23

[0424] □ AL142 Production Bleed 3 ELISA run 10 / 4 / 23

[0425] • T50% Titer vs. SDMe-GR8: 173,000

[0426] □ AL142 Production Bleed 3 ELISA follow-up ran 12 / 13 / 23 • T50% Titer vs. ADMe-GR8: 31,800

[0427] • T50% Titer vs. GR8: 9,390

[0428] □ AL143 Production Bleed 3 ELISA run 10 / 4 / 23

[0429] • T50% Titer vs. SDMe-GR8: 120,000

[0430] □ AL143 Production Bleed 3 ELISA follow-up ran 12 / 13 / 23 • T50% Titer vs. ADMe-GR8: 67,200

[0431] • T50% Titer vs. GR8: 1,150

[0432] • Day 231? (not specified): Rabbits were subcutaneously boosted with 125 pg of KLH-SDMe-GRs.Attorney Docket No. ALSE-016PC

[0433] • Day 241: Rabbits were bled (“production bleed 4”), serum was collected, and an ELISA was run to measure antibody titers.

[0434] o AL142- 144: 3 / 18 / 24

[0435] □ AL142 Production Bleed 4 ELISA run 3 / 19 / 24

[0436] • T50% Titer vs. SDMe-GRs: 25,300

[0437] • T50% Titer vs. ADMe-GRs: 12,000

[0438] • T50% Titer vs. GRs: 4,650

[0439] □ AL143 Production Bleed 4 ELISA run 3 / 19 / 24

[0440] • T50% Titer vs. SDMe-GRs: 31,600

[0441] • T50% Titer vs. ADMe-GRs: 14,900

[0442] • T50% Titer vs. GR8: 9,930

[0443] □ AL144 Production Bleed 4 ELISA run 3 / 19 / 24

[0444] • T50% Titer vs. SDMe-GRs: 120,000

[0445] • T50% Titer vs. ADMe-GRs: 19,500

[0446] • T50% Titer vs. GRS: 12,100

[0447] • Day 269: Rabbits were terminated, followed by spleen harvests on AL144 (chosen candidate) and AL142 (backup candidate) and terminal bleed (all animals).

[0448] o AL142- 144: 4 / 15 / 24

[0449] • End of Study 2A: Rabbit AL144’s spleen was chosen to proceed to monoclonal antibody preparation in Study 2B. A polyclonal antibody from this animal was also isolated and purified from reagents collected during Study 2A.

[0450] Study 2B: B-Cell isolation, cloning, and monoclonal anti-SDMe-GRs antibody production from chosen candidate AL 144

[0451] The monoclonal antibody production process was split into three main phases. Phase I included the collection and isolation of antigen-reactive b-cells from the spleen of animal AL144. Phase II included cloning of the b-cells isolated in phase I, selection of clones with high binding to the intended antigen, and generation of heavy and light-chain gene linear expression modules (LEMs) for top clone candidates. HEK cells were then transfected with resulting LEMs. Phase III included selection of one top LEM-transfected HEK clone, small-scale antibody expression and purification followed by validation testing, and final large-scale antibody expression and purification. The final monoclonal antibody reagent was produced by the conclusion of Phase III.

[0452] • Phase la: Splenocytes were isolated from spleen harvested from animal AL144.

[0453] • Phase lb: Splenocytes were incubated with fluorescent DRP antigen and sorted using flow cytometry to select for b-cells with high antigen affinity.

[0454] • Phase Ila: Sorted b-cells were plated into 384-well plates overnight. The following day, b-cell supernatants were collected and tested by ELISA. Ten b-cell clones were chosen to move forward based on having the highest antibody binding to the desired antigen and lowest antibody binding to relevant counter-antigens.Attorney Docket No. ALSE-016PC

[0455] • Phase lib: Heavy and light-chain genes from top b-cell clones were cloned into linear expression module (LEM) plasmids. These LEM plasmids were transfected into HEK cells. Supernatants from the transfected HEK cells, referred to as “LEM- supernatants," were collected and tested by ELISA. Based on results, one LEM- transfected HEK clone was selected to move forward to final phase.

[0456] • Phase Illa: Selected HEK clone candidate was used for small-scale antibody expression. The resulting antibody was purified using a Protein A purification approach, then validated by ELISA and SDS-PAGE.

[0457] • Phase Illb: Antibody expression was repeated at a larger scale and resulting antibody was purified using a Protein A purification approach.

[0458] End of Study 2B: Samples of two plasmids: one encoding the heavy-chain gene sequence and one encoding the light-chain gene sequence for the final validated monoclonal antibody were collected. The sequences for both plasmids were determined. Final purified samples of monoclonal anti-SDMe-GRs antibody from phases Illa and Illb are being stored in-house. Table 1: List of peptides used in Examples 1 and 2 and related assays

[0459] Peptide Use in Study Peptide Sequence

[0460] Name

[0461] KLH- Immunizations of KLH-Cys-K(PEG4)-G-R(Me2)asym-G- Conjugated animals AL139, R(Me2)asym-G-R(Me2)asym-G-R(Me2)asym-G- ADMe-GRs AL 140, and AL141 R(Me2)asym-G-R(Me2)asym-G-R(Me2)asym-G- R(Me2)asym-OH

[0462] ADMe-GRs ELISA detection of H2N-G-R(Me2)asym-G-R(Me2)asym-G- antibody binding R(Me2)asym-G-R(Me2)asym-G-R(Me2)asym-G- R(Me2)asym-G-R(Me2)asym-G-R(Me2)asym-OH KLH- Immunizations of KLH-Cys-K(PEG4)-G-R(Me2)sym-G-R(Me2)sym- Conjugated animals AL 142, G-R-(Me2)sym-G-R(Me2)sym-G-R(Me2)sym-G- SDMe-GRs AL143, AL144 R(Me2) sy m-G-R(Me2) sym-G-R(Me2) sy m-OH SDMe-GRs ELISA detection of H2N-G-R(Me2)sym-G-R(Me2)sym-G-R-(Me2)sym- antibody binding G-R(Me2)sym-G-R(Me2)sym-G-R(Me2)sym-G- R(Me2)sym-G-R(Me2)sym-OH

[0463] GRs ELISA detection of H2N-G-R-G-R-G-R-G-R-G-R-G-R-G-R-G-R-OH antibody binding to

[0464] counter-antigen

[0465] PRs ELISA detection of H2N-P-R-P-R-P-R-P-R-P-R-P-R-P-R-P-R-OH antibody binding to

[0466] counter-antigen

[0467] ADMe-PRis ELISA detection of H2N-P-R(Me2)asym-P-R(Me2)asym-P- antibody binding to R(Me2)asym-P-R(Me2)asym-P-R(Me2)asym-P- counter-antigen R(Me2)asym-P-R(Me2)asym-P-R(Me2)asym-P-

[0468]

[0469] R(Me2)asym-P-R(Me2)asym-P-R(Me2)asym-P-Attorney Docket No. ALSE-016PC

[0470] R(Me2)asym-P-R(Me2)asym-P-R(Me2)asym-P- R(Me2)asym-P-R-OH

[0471] ADMe-GRis ELISA detection of H2N-G-R(Me2)asym-G-R(Me2)asym-G- antibody binding R(Me2)asym-G-R(Me2)asym-G-R(Me2)asym-G- R(Me2)asym-G-R(Me2)asym-G-R(Me2)asym G- R(Me2)asym-G-R(Me2)asym-G-R(Me2)asym-G- R(Me2)asym-G-R(Me2)asym-G-R(Me2)asym-G- R(Me2)asym-OH

[0472] SDMe-GRis ELISA detection of H2N-G-R(Me2)sym-G-R(Me2)sym-G-R-(Me2)sym- antibody binding G-R(Me2)sym-G-R(Me2)sym-G-R(Me2)sym-G- R(Me2)sym-G-R(Me2)sym-G-R(Me2)sym-G- R(Me2)sym-G-R-(Me2)sym-G-R(Me2)sym-G- R(Me2) sy m-G-R(Me2) sym-G-R(Me2) sy m-OH

[0473]

[0474] Example 3: ELISA for Detection of Anti-ADMe-GRs Antibody Binding Profiles To confirm high binding specificity for ADMe-GRs antibody (described in Example 1 above) over related counter-antigens the following assay was performed. Custom-synthesized peptides ADMe-GR8, SDMe-GR8, GR8, or ADMe-PR15were coated onto 96-well plates. Following a standard ELISA format, the ADMe-GRs antibody (FIG. 5) was titrated onto each peptide coat and binding activity was assessed. Specifically, ADMe-GRs (Vivitide®), SDMe-GRs (Anaspec®), GRs (Vivitide), or ADMe-PRis (Anaspec) were dissolved in DMSO and aliquoted for storage at -20°C to avoid freeze-thaw cycles. Each peptide was diluted in IX PBS to 1 pg / mL and used to coat half of a clear, half-volume, high-binding 96-well plate. Plates were sealed and left at 4°C overnight to coat. Peptide coat was shaken out and plates were washed two times with wash buffer (IX PBS, 0.1% Tween20) and two times with IX PBS using a wash bottle. Plates were blotted dry on paper towels before adding block buffer (IX PBS, 0.1% BSA) and incubating for Ih at room temperature with low-speed shaking. Block buffer was next shaken out and plates were washed two times with IX PBS. Plates were blotted dry on paper towels before anti-ADMe-GRs monoclonal antibody was diluted first 1:100 and next 1:29.3 in assay buffer (IX PBS, 0.1% BSA, 0.1% Tween20) prior to being diluted serially 1:2 from 1 pg / ml to 1 ng / mL onto each peptide coat. One column of each half-plate received assay buffer only to serve as a secondary antibody background control. Primary antibody was incubated on plates for Ih at room temperature with low-speed shaking. Primary antibody was next shaken out and plates were washed three times with wash buffer and three times with IX PBS. Plates were blotted dry before HRP anti-rabbit secondary antibody (1:10,000 in assay buffer) was applied and incubated for Ih at room temperature covered from light with low-speed shaking. Secondary antibody was nextAttorney Docket No. ALSE-016PC

[0475] shaken out and plates were washed three times with wash buffer and three times with IX PBS. Plates were blotted dry before TMB substrate solution was applied and incubated for 5 minutes at room temperature. The reaction was stopped with the addition of 2M H2SO4 and plates were read for absorbance at 450 nm.

[0476] As shown in FIG. 5, ADMe-GR antibody demonstrates high specificity for ADMe-GRs peptide coat compared to related antigen peptide coats. Starting at a concentration of 62.5 ng / mL, the antibody only binds its intended target ADMe-GR8with a high absorbance signal.

[0477] Example 4: Immunocytochemistry for Anti- ADMe-GRs Monoclonal Antibody Staining Profile

[0478] Induced human pluripotent stem cells (iPSCs) were differentiated into motor neurons using a modified dual-SMAD inhibition protocol (see Du et al. Nature Communications, 6(1), 6626. doi.org / 10.1038 / ncomms7626) from individuals with or without the C9orf72 hexanucleotide repeat expansion mutation (C9-HRE). Day 19 motor neurons were plated in 96-well plates. At day 16 post-plating, motor neurons were treated with 1 pM ADMe-GRs peptide for 24 hours prior to fixation and immunocytochemical imaging. Specifically, motor neurons cultured in a 96-well plate were fixed using 4% paraformaldehyde (PF A). After fixation, cells were rinsed with PBS three times before permeabilization with 0.5% Triton X-100 in Tris-buffered saline with Tween 20 (TBS-T) for 10 minutes, followed by blocking using 10% normal donkey serum for 1 hour at room temperature. The neurons were then incubated overnight at 4°C with a solution containing the ADMe-GRs monoclonal antibody (1:7,500). The following day, the primary antibody solution was rinsed off five times using IX TBST, and neurons were subsequently incubated with secondary antibody solution for 1 hour at room temperature followed by five more IX TBST rinses. Nuclei were counterstained with 4’ 6-daimidino-2-phenylindole (DAPI). Imaging was performed with a 20x confocal objective using Cytation 10 (BioTek) microscope, where DAPI, GFP, and TRITC channels corresponded to nuclear staining, ADMe-GRs monoclonal antibody staining, and Tuj-1 neuronal marker staining, respectively. Mean nuclear anti-ADMe-GRs staining intensity was quantified using Signal Image Artist software (Revity Signals).

[0479] Representative images of day 16 post-plating iPSC-derived motor neurons were obtained. Motor neurons with either wild type C9orf72 or hexanucleotide repeat-expanded C9orf72 were treated with either DMSO vehicle or 1 pM ADMe-GR15peptide for 24h (dataAttorney Docket No. ALSE-016PC

[0480] not provided). As shown in FIG. 6, quantification of mean ADMe-GR8antibody green (“GFP”) signal intensity within motor neuron nuclei indicates significant increase in detection of ADMe-GR after 24h of treatment with 1 pM exogenous ADMe-GR15peptide. Motor neurons containing the C9-HRE exhibit significantly higher mean ADMe-GR staining intensity with ADMe-GRis treatment than C9 wild-type motor neurons without the mutation (Two-way ANOVA, ** = p<0.01, **** = p<0.0001).

[0481] Example 5: ELISA for Detection of Anti-SDMe-GR8 Antibody Binding Profiles To confirm high binding specificity for SDMe-GRs antibody (described in Example 2 above) over related counter-antigens the following assay was performed. ADMe-GRs (Vivitide), SDMe-GRs (Anaspec), GRs (Vivitide), PRis (GenicBio Ltd.) or ADMe-PRis (Anaspec) were dissolved in DMSO and aliquoted for storage at -20°C to avoid freeze-thaw cycles. Each peptide was diluted in IX PBS to 1 pg / mL and used to coat half of a clear, halfvolume, high-binding 96-well plate. Plates were sealed and left at 4°C overnight to coat. Peptide coat was shaken out and plates were washed two times with wash buffer (IX PBS, 0.05% Tween20) and two times with IX PBS using a wash bottle. Plates were blotted dry on paper towels before adding block buffer (IX PBS, 0.1% BSA) and incubating for Ih at room temperature with low-speed shaking. Block buffer was next shaken out and plates were washed two times with IX PBS. Plates were blotted dry on paper towels before anti-SDMe-GRs monoclonal antibody was diluted first 1400 and next 1:28.8 in assay buffer (IX PBS, 0.1% BSA, 0.1% Tween20) before being diluted serially 1:3 from 1 pg / ml to 0.02 ng / mL onto each peptide coat. One column of each half-plate received assay buffer only to serve as a secondary antibody background control. Primary antibody was incubated on plates for Ih at room temperature with low-speed shaking. Primary antibody was next shaken out and plates were washed three times with wash buffer and three times with IX PBS. Plates were blotted dry before HRP anti-rabbit secondary antibody (1:10,000 in assay buffer) was applied and incubated for Ih at room temperature covered from light with low-speed shaking.

[0482] Secondary antibody was next shaken out and plates were washed three times with wash buffer and three times with IX PBS. Plates were blotted dry before TMB substrate solution was applied and incubated for 5 minutes at room temperature. The reaction was stopped with the addition of 2M H2SO4 and plates were read for absorbance at 450 nm.

[0483] As shown in FIG. 7, SDMe-GRs antibody (at concentrations of 1 pg / mL to 0.02 ng / mL against peptide coats of either ADMe-GRs, SDMe-GRs, GRs, PR 15, or ADMe-PRis)Attorney Docket No. ALSE-016PC

[0484] shows high specificity for SDMe-GR8peptide coat compared to related antigen peptide coats. At all concentrations tested, the antibody only binds its intended target SDMe-GR8with a high absorbance signal.

[0485] Example 6: Immunocytochemistry for Anti-SDMe-GRs Monoclonal Antibody Staining Profile

[0486] Induced human pluripotent stem cells (iPSCs) were differentiated into motor neurons using a modified dual-SMAD inhibition protocol (see Du et al. Nature Communications, 6(1), 6626. doi.org / 10.1038 / ncomms7626) from individuals with the C9orf72 hexanucleotide repeat expansion mutation (C9-HRE). In a subset of these motor neurons, the C9-HRE was corrected to wild type using a CRISPR-Cas9 approach (31_C9_HRE_Corrected). Day 19 motor neurons were thawed and plated in 96-well plates. At day 4 post-plating, motor neurons were treated with 1 u M SDMe-GRs peptide for 24 hours prior to fixation and immunocytochemical imaging. Specifically, motor neurons were plated at 10,000 cells per well (31,250 cells / cm2) in a 96-well plate. Four days post-plating, motor neurons were treated with 1 pM SDMe-GRis and incubated for 24h at 37°C, 5% CO2 prior to fixation. Motor neurons were fixed using 4% paraformaldehyde (PF A). After fixation, cells were rinsed with PBS three times before permeabilization with 0.5% Triton X-100 in Tris-buffered saline with Tween20 (TBS-T) for 10 minutes. After permeabilization, a blocking step was performed using 10% normal donkey serum for 1 hour at room temperature. The neurons were then incubated overnight at 4°C with a primary antibody solution containing the SDMe-GRs monoclonal antibody (1:10,000). The following day, the primary antibody solution was rinsed off five times using IX TBS-T, and neurons were subsequently incubated with secondary antibody solution for 1 hour at room temperature followed by five more IX TBS-T rinses. Nuclei were counterstained with 4’ 6-daimidino-2-phenylindole (DAPI). Imaging was performed with a 20X confocal objective using a Cytation 10 (Biotek) microscope, where DAPI, GFP, and Texas Red channels corresponded to nuclear staining, SDMe-GRs monoclonal antibody staining, and Tuj-1 neuronal marker staining, respectively. DAPI and GFP channel images were taken using a confocal filter and Texas Red channel images were taken using widefield. Mean nuclear anti-SDMe-GRs staining intensity was quantified using Signal Image Artist software (Revity Signals).

[0487] Representative images of day 4 post-plating iPSC-derived motor neurons were obtained. Motor neurons with either a corrected C9orf72 mutationAttorney Docket No. ALSE-016PC

[0488] (“31_C9_HRE_Corrected”) or hexanucleotide repeat-expanded C9orf72 (“29_C9_HRE”) were treated with either DMSO vehicle or 1 pM SDMe-GRis peptide for 24h followed by imaging detection of fluorescent antibody signal (data not provided). As shown in FIG. 8, quantification of mean SDMe-GRis antibody green (“GFP”) signal intensity within motor neuron nuclei indicates significant increase in detection of SDMe-GR after 24h of treatment with 1 pM exogenous SDMe-GRis peptide. (Two-way ANOVA, ** = p<0.01, **** = p<0.0001). Motor neurons with the C9-HRE exhibit non-significantly higher mean SDMe-GR staining intensity with SDMe-GRis treatment than motor neurons in which the C9-HRE mutation has been corrected.

[0489] Example 7: ELISA for Comparison of Anti-SDMe-GRs Binding Potency and Specificity Custom-synthesized peptides, ADMe-GRs, SDMe-GRs, GRs, ADMe-PRis, or PRis, were coated onto 96-well plates. Following a standard ELISA format, a symmetric di-Methyl arginine motif (sdme-RG) multi-monoclonal antibody mixture (“multi-mAb” obtained from Cell Signaling Technologies® Cat.#12333T) which recognizes endogenous levels of proteins that are symmetrically dimethylated on arginine residues and the anti-SDMe-GRs monoclonal antibody described above in Example 2 (i.e., the “custom antibody”) were titrated onto each peptide coat to assess binding activity. The goal of the assay was to compare binding specificity and binding potency between the two.

[0490] ADMe-GRs (Vivitide), SDMe-GRs (Anaspec), GRs (Vivitide), or ADMe-PRis (Anaspec) and PRis (GenicBio Ltd.®) were dissolved in DMSO and aliquoted for storage at -20°C to avoid freeze-thaw cycles. Each peptide was diluted in IX PBS to 1 pg / mL and used to coat half of a clear, half- volume, high-binding 96-well plate. Plates were sealed and left at 4°C overnight to coat. Peptide coat was shaken out and plates were washed two times with wash buffer (IX PBS, 0.1% Tween20) and two times with IX PBS using a wash bottle. Plates were blotted dry on paper towels before adding block buffer (IX PBS, 0.1% BSA) and incubating for Ih at room temperature with low-speed shaking. Block buffer was next shaken out and plates were washed two times with IX PBS. Plates were blotted dry on paper towels before custom anti-SDMe-GRs monoclonal antibody was diluted first 1: 100 and next 1:28.8 in assay buffer (IX PBS, 0.1% BSA, 0.1% Tween20) before being diluted serially 1:3 from 1 pg / ml to 0.02 ng / mL onto each peptide coat in duplicate. At the same time anti-SDMe-GRs custom antibody was applied, an anti-SDMe-RG multi-mAb mix (Cell Signaling Technology Cat.#12333T) was diluted first 1: 100 and next 1:9.17 in assay buffer beforeAttorney Docket No. ALSE-016PC

[0491] being diluted serially 1:3 from 1 pg / ml to 0.02 ng / mL onto each peptide coat in duplicate. One column of each half-plate received assay buffer only to serve as a secondary antibody background control. Primary antibody was incubated on plates for Ih at room temperature with low-speed shaking. Primary antibody was next shaken out and plates were washed three times with wash buffer and three times with IX PBS. Plates were blotted dry before HRP anti-rabbit secondary antibody (1:10,000 in assay buffer) was applied and incubated for Ih at room temperature covered from light with low-speed shaking. Secondary antibody was next shaken out and plates were washed three times with wash buffer and three times with IX PBS. Plates were blotted dry before TMB substrate solution was applied and incubated for 5 minutes at room temperature. The reaction was stopped with the addition of 2M H2SO4 and plates were read for absorbance at 450 nm.

[0492] As shown in FIG. 9, custom anti-SDMe-GRs antibody binds SDMe-GRs more potently than the known mixture of monoclonal antibodies raised against SDMe-RG.

[0493] Specifically, FIG. 9A shows repeat binding profiles of custom anti-SDMe-GRs monoclonal antibody at concentrations of 1 pg / mL to 0.02 ng / mL against peptide coats of either ADMe-GRs, SDMe-GRs, GRs, PR15, or ADMe-PRis. As shown, the custom antibody shows high specificity for SDMe-GR8peptide coat compared to related antigen peptide coats. At all concentrations tested, the antibody only binds its intended target SDMe-GR8with a high absorbance signal. FIG. 9B shows the binding profiles of publicly available anti-SDMe-RG monoclonal antibody mixture (Cell Signaling Technology Cat.#12333T) at concentrations of 1 pg / mL to 0.02 ng / mL against peptide coats of either ADMe-GRs, SDMe-GRs, GRs, PR15, or ADMe-PRis. The monoclonal antibody mixture (multi -mAb) shows high specificity for SDMe-GR8peptide coat compared to related antigen peptide coats. At all concentrations tested, the antibody only binds its intended target SDMe-GR8with a high absorbance signal. FIG. 9C shows the binding profiles against intended antigen SDMe-GRs described in FIGs.

[0494] 9A and 9B transposed to compare the custom mAb binding sensitivity directly to CST multi-mAb. The custom mAb binds SDMe-GRs coat more potently than CST multi-mAb reagent. FIG. 9C shows an EC50 calculation of binding to SDMe-GR8coat using non-linear regression model analysis of the custom mAb compared to the SDMe-RG multi-mAb. As shown the custom mAb binds intended antigen 3.4 times more potently than the multi-mAb, with an estimated EC50 of 24.21 ng / ml (custom antibody) compared to 83.29 ng / ml (multi-mAb).Attorney Docket No. ALSE-016PC

[0495] Example 8: Surface Plasmon Resonance (SPR) for Detection of Antibody Binding Kinetics

[0496] Antibody binding was measured using standard SPR protocol and recombinant human ADMe-GR or SDMe-GR as the analyte and the custom antibodies (described above, see Examples 1 and 2) as the ligand. The senor chip, Series S CM5 Sensor chip (Cytiva®), was used. Running buffer included 10 mM HEPES pH 7.4, 150 mM NaCl, 0.05% Tween-20 (HBS-P+) with 1% DMSO and an assay temperature of 25°C.

[0497] Sensor chip preparation included the following:

[0498] (a) Instrument: Biacore 8K+

[0499] (b) Running buffer: HBS-P+

[0500] (c) Sensor chip: Series S CM5

[0501] (d) Goat Anti-Rabbit IgG, Fc Fragment Specific (GAR-Fc) was immobilized to the sensor chip to a level of -6400-8000 RU

[0502] TABLE 2

[0503] Channel Flow Cell Target Immobilization Level (RU)

[0504] 2 1 GAR-Fc 7571.6

[0505] 3 1 GAR-Fc 7679.5

[0506] 2 2 GAR-Fc 6369.9

[0507] 3 2 GAR-Fc 7899.3

[0508]

[0509] Analyte testing included the following:

[0510] (a) Running buffer: HBS-P+, 1% DMSO

[0511] (b) Method: Single-cycle kinetics (SCK)

[0512] (c) Highest concentration: see table

[0513] (d) Dilution scheme: 3-fold, 5 point

[0514] (e) The data were reference subtracted and buffer blank subtracted (double referenced) and solvent corrected

[0515] (f) A 1:1 kinetic binding model was used to fit the data

[0516] Each antibody was tested against its intended antigen only (ADMe-GRs mAb vs. ADMe-GR15; SDMe-GRs mAb vs. SDMe-GRis). Data shown in FIG. 10 and FIGs. 11 AAttorney Docket No. ALSE-016PC

[0517] and 1 IB. It was also determined that each antibody is bivalent.

[0518] Example 9: Cell-Based Lactate Dehydrogenase (LDH) Assay for Detection of Anti-GRs Antibody Abrogation of Methylated PolyGR-Induced Cytotoxicity

[0519] Anti-ADMe-GRs or anti-SDMe-GRs monoclonal antibodies (described above, see Examples 1 and 2) were titrated onto murine spinal cord motor neuron hybrid cells (NSC-34) challenged with either ADMe- or SDMe-polyGR DRPs. Using a 24-hour DRP challenge paradigm, monoclonal antibodies and methylated DRPs were co-titrated onto NSC-34 cells and incubated for one day before cytotoxicity endpoint measurement. Lactate dehydrogenase (LDH) release was used as a measurable cytotoxic endpoint, reflecting leakage of LDH enzyme into culture media following cellular damage. Specifically, NSC-34 cells were plated in proliferation medium (DMEM High-Glucose, 10% FBS, 1% Penicillin-Streptomycin, 1% L-Glutamine) at 3.77xl04cells / well in optically clear 96-well plates and incubated overnight at 37°C, 5% CO2. Next, one plate of cells received a titration of anti-ADMe-GRs monoclonal antibody (333 ng / ml serial threefold to 0.02 ng / ml) diluted in proliferation medium. Immediately after antibody application, ADMe-GR15peptide (Anaspec, 3 pM) diluted in proliferation medium was added to each well that received antibody. Assay controls were also included: 2x untreated (one becomes lysed control), lx DMSO vehicle control, 2x MS023 inhibitor (one receives DRP challenge, neither should exhibit rescue against pre-methylated polyGR peptides), and 2x media only without cells (one becomes LDH spike control). All treatment groups were applied in triplicate wells. A second plate was prepared in the same manner as above using anti-SDMe-GRs monoclonal antibody for treatment and SDMe-GRis peptide for challenge in experimental wells. On a third plate, half of the cells received a titration of anti-ADMe-GRs monoclonal antibody (333 ng / ml serial threefold to 1 ng / ml) and half of the cells received a titration of anti-SDMe-GRs monoclonal antibody (333 ng / ml serial threefold to 1 ng / ml). Immediately after dosing, each plate was returned to 37°C overnight. On the day of testing, reagents from Abcam’s LDH-Cytotoxicity Assay Kit II (ab65393) were thawed to room temperature and prepared according to manufacturer instructions. To begin endpoint testing, 10 pL / well cell lysis solution was added to lysed control wells in each plate and incubated 5 minutes. Next, plates were spun for 10 minutes at 600xg in a plate centrifuge (Beckman Coulter Allegra X-22R). New optically clear 96-well transfer plates were prepared, and after testing plates spun, 10 pL of supernatant from each testing plate well was transferred to corresponding transfer plateAttorney Docket No. ALSE-016PC

[0520] wells. Three wells in each transfer plate were spotted with 5 p L of LDH positive control solution for LDH spike controls. Using freshly prepared LDH reaction mixture containing LDH assay buffer and substrate, 100 L / well LDH reaction mixture was added to each well before plates were incubated 30 minutes at room temperature. After 30 minutes, reactions were stopped with 10 pL stop solution and plates were read at absorbance 450 nm using an absorbance microplate reader (Molecular Devices SpectraMax M5). To calculate % LDH release from absorbance measurements, the following equation was used per manufacturer’s instructions: % LDH release = ((A450 test sample - A450 untreated control) / (A450 lysed control - A450 untreated control))* 100%.

[0521] As shown in FIG. 12, rabbit Anti-ADMe-GRs and Anti-SDMe-GRs monoclonal antibodies significantly and dose-dependently reduce methylated polyGR-induced cytotoxicity. Specifically in FIG. 12A, when co-titrated on NSC-34 cells with ADMe-GRis challenge (3 pM), custom anti-ADMe-GRs monoclonal antibody significantly reduces % LDH release at 1-333 ng / ml concentrations (One-way ANOVA: ****, p < 0.0001; ***, p < 0.001; **, p < 0.01; *, p < 0.05). In FIG. 12B, in the absence of ADMe-GRis challenge, custom anti-ADMe-GRs monoclonal antibody is well-tolerated by NSC-34 cells when applied at 1-333 ng / ml concentrations. In FIG. 12C, when co-titrated on NSC-34 cells with SDMe-GRis challenge (3 pM), custom anti-SDMe-GRs monoclonal antibody significantly reduces % LDH release at 0.5-333 ng / ml concentrations (One-way ANOVA: ****, p < 0.0001; **, p < 0.01). Lastly, in FIG. 12D, in the absence of SDMe-GRis challenge, custom anti-SDMe-GRs monoclonal antibody is well-tolerated by NSC-34 cells when applied at 1-333 ng / ml concentrations.

[0522] Example 10: Cell-Based Stable Tetrazolium Salt (WST-1) Assay for Detection of Anti-GRs Antibody Abrogation of Methylated PolyGR-induced Dysmetabolism

[0523] Anti-ADMe-GRs or anti-SDMe-GRs monoclonal antibodies were titrated onto murine spinal cord motor neuron hybrid cells (NSC-34) challenged with either ADMe- or SDMe-polyGR DRPs. Using a 24-hour DRP challenge paradigm, monoclonal antibodies and methylated DRPs were co-titrated onto NSC-34 cells and incubated for one day before metabolic endpoint measurement. WST-1 salt cleavage to colored formazan product via the succinate-tetrazolium reductase system within the mitochondrial respiratory chain was used as a measurable metabolic endpoint, reflecting activity of a pathway exclusive to metabolically intact cells.Attorney Docket No. ALSE-016PC

[0524] NSC-34 cells were plated in proliferation medium (DMEM High-Glucose, 10% FBS, 1% Penicillin-Streptomycin, 1% L-Glutamine) at 3.77xl04cells / well in optically clear 96-well plates and incubated overnight at 37°C, 5% CO2. Next, one plate of cells received a titration of anti-ADMe-GRs monoclonal antibody (333 ng / ml serial threefold to 0.02 ng / ml) diluted in proliferation medium. Immediately after antibody application, ADMe-GR15peptide (Anaspec, 3 pM) diluted in proliferation medium was added to each well that received antibody. Assay controls were also included: 2x untreated (one later receives WST-1, one does not and is used forbackground absorbance subtraction), lx DMSO vehicle control, 2x MS023 inhibitor (one receives DRP challenge, neither should exhibit rescue against pre-methylated polyGR peptides), and 2x media only without cells (one later receives WST-1, one does not). All treatment groups were applied in triplicate wells. A second plate was prepared in the same manner as above using anti-SDMe-GRs monoclonal antibody for treatment and SDMe-GRis peptide for challenge in experimental wells. On a third plate, half of the cells received a titration of anti-ADMe-GRs monoclonal antibody (333 ng / ml serial threefold to 1 ng / ml) and half of the cells received a titration of anti-SDMe-GRs monoclonal antibody (333 ng / ml serial threefold to 1 ng / ml). Immediately after dosing, each plate was returned to 37°C overnight. On the day of testing, aliquots of cell proliferation reagent WST-1 (Millipore-Sigma) were thawed to room temperature, and later gently warmed at 37 °C immediately prior to reagent addition. A solution of DPBS-Glucose (4.5 g / L) was also warmed to 37°C prior to testing. To begin endpoint testing, medium in wells of each plate was gently aspirated and replaced with 200 pL / well warmed DPBS-glucose solution. Next, 20 pL / well warmed WST-1 solution was added to each well before plates were immediately returned to 37°C. Every 15 minutes for a total testing duration of Ih, each plate was read at absorbance 450 nm using an absorbance microplate reader (Molecular Devices SpectraMax M5). To calculate final WST-1 absorbance values, background subtraction was performed on each plate by subtracting the average absorbance of cells that did not receive WST-1 reagent from absorbance values in every experimental well.

[0525] As shown in FIG. 13, rabbit anti-ADMe-GRs and anti-SDMe-GRs monoclonal antibodies significantly and dose-dependently reduce methylated polyGR-induced dysmetabolism. Specifically, in FIG. 13 A when co-titrated on NSC-34 cells with ADMe-GR15 challenge (3 pM), custom anti-ADMe-GRs monoclonal antibody significantly increases WST-1 conversion at 1-333 ng / ml concentrations (One-way ANOVA: ****, p < 0.0001: **, p < 0.01; *, p < 0.05). In FIG. 13B, in the absence of ADMe-GRis challenge, custom anti-Attorney Docket No. ALSE-016PC

[0526] ADMe-GRs monoclonal antibody is well-tolerated by NSC-34 cells when applied at 1-333 ng / ml concentrations. In FIG. 13C, when co-titrated on NSC-34 cells with SDMe-GRi5 challenge (3 pM), custom anti-SDMe-GRs monoclonal antibody significantly increases WST-1 conversion at 12-333 ng / ml concentrations (One-way ANOVA: ****, p < 0.0001; **, p < 0.01). In FIG. 13D, in the absence of SDMe-GRi5 challenge, custom anti-SDMe-GRs monoclonal antibody is well-tolerated by NSC-34 cells when applied at 1-333 ng / ml concentrations. In Fig. 13E, when co-titrated on NSC-34 cells with ADMe-GRis challenge (3 pM), custom anti-ADMe-GRs monoclonal antibody increases metabolic rate over time as indicated by slopes of best-fit lines of WST-1 conversion over time. In Fig. 13F, in the absence of ADMe-GRis challenge, custom anti-ADMe-GRs monoclonal antibody does not change metabolic rate over time as indicated by slopes of best-fit lines of WST-1 conversion. In Fig. 13G, when co-titrated on NSC-34 cells with SDMe-GRis challenge (3 pM), custom anti-SDMe-GRs monoclonal antibody increases metabolic rate over time as indicated by slopes of best-fit lines of WST-1 conversion over time. In Fig. 13H, in the absence of SDMe-GRis challenge, custom anti-SDMe-GRs monoclonal antibody does not change metabolic rate overtime as indicated by slopes of best-fit lines of WST-1 conversion.

[0527] Example 11: Custom Meso-Scale Discovery (MSD) Assay for Low-Level Detection of ADMe-GR8 in Biological Samples

[0528] Rabbit anti-ADMe-GRs monoclonal antibody was conjugated to biotin using Thermo Scientific EZ-Link Sulfo-NHS-LC-Biotin (Cat no. 21335). Biotin conjugation efficiency was verified using Thermo Scientific Pierce Biotin Quantitation Kit (Cat no. 28005). Conjugated antibody concentration was verified using Thermo Scientific Pierce Dilution-Free Rapid-Gold BCA Assay (Cat no. A55860). A polyclonal anti-ADMe-GRs antibody raised and purified from the same rabbit was conjugated to MSD GOLD SULFO-TAG NHS-Ester using the tag’s corresponding conjugation kit (Cat no. R31 AA-1). Sulfo-tag conjugation efficiency was verified using 455 nm spectrophotometry readings on a Nanodrop One-C device, and conjugated antibody concentration was verified using the same BCA assay as above.

[0529] Biotinylated rabbit anti-ADMe-GRs monoclonal capture antibody was coated on an MSD 1-Spot Streptavidin QuickPlex plate (Cat no. L55SA-2) at a concentration of 2 pg / ml with a volume of 25 pL / well overnight, sealed at 4°C. The following day, biotinylated rabbit anti-ADMe-GRs monoclonal antibody coat was removed and washed three times with IX Tris-Buffered Saline, 0.05% Tween-20 (IX TBST 0.05%), 200 pL / well per wash. Plate wasAttorney Docket No. ALSE-016PC

[0530] gently blotted dry on paper towels before MSD Blocker A solution (prepared using MSD Blocker A Kit, Cat no. R93AA-2) was applied 150 pL / well, plate was sealed and incubated Ih at room temperature with vigorous shaking (speed 7 on a microplate orbital shaker). After Ih, blocking solution was removed and washed once with IX TBS, 200 pL / well per wash, before blotting dry and adding in duplicate three titration curves of exogenous ADMe-GRis (Anaspec) diluted in MSD assay buffer (Diluent 100, Cat no. R50AA-2). Curve 1 titrated ADMe-GRis from 1 pg / mL serial 1:3 down to 1 ng / mL, curve 2 titrated ADMe-GRis from 100 ng / mL serial 1:3 down to 130 pg / mL, and curve 3 titrated ADMe-GRn from 10 ng / mL serial 1:3 down to 13 pg / mL, all in duplicate with 50 pL sample loaded per well. Control wells containing MSD assay buffer only were included in each titration curve to reflect background assay signal. Following sample addition, plate was sealed and incubated at 4°C overnight with low-speed (1.5x) orbital shaking. The following day, samples were shaken out and plate was washed three times with IX TBST 0.05%, 200 pL / well per wash. Plate was gently blotted dry on paper towels before sulfo-tagged rabbit polyclonal anti-ADMe-GRs detector antibody was added at 0.5 pg / mL, 25 pL / well and plate was sealed before vigorous shaking (speed 7 on a microplate orbital shaker) for Ih at room temperature in the dark. After Ih detector antibody was shaken out and plate was washed three more times with IX TBST 0.05%, 200 pL / well per wash. Plate was thoroughly blotted dry on paper towels before addition of MSD Read Buffer A, 150 pL / well, and immediate read of ECL signal on MSD MESO QuickPlex SQ 120 Reader. Before plotting, ECL signal values were background-subtracted based on signals in assay buffer-only wells.

[0531] As shown in FIG. 14, when biotinylated for use as a detector antibody in a custom MSD assay, rabbit anti-ADMe-GR8monoclonal antibody dose-dependently captures ADMe-GR15analyte down to pg / mL range sensitivity, even in samples that have not been preconcentrated. Specifically, in Fig. 14B, rabbit anti-ADMe-GRs monoclonal antibody is able to capture and significantly detect analyte levels as low as 41 pg / mL (One-way ANOVA: **, p = 0.0017), when used in combination with a sulfo-tagged rabbit anti-ADMe-GRs polyclonal antibody detector.

[0532] Example 12: Staining of Rabbit Anti-ADMe-GRs Monoclonal Antibody to Detect ADMe-GRs in Spinal Cord Sections from Two C9orf72-Mediated ALS Mouse Models One wild-type (C57BL / 6J, Jackson Laboratories Strain no. 000664), one “C9-112” C9orf72 model (C57BL-6J TgC9orf72_3-l 12, Jackson Laboratories Strain no. 023099), andAttorney Docket No. ALSE-016PC

[0533] one “C9-500” C9orf72 model (FVBNK-500J C9orf72_500, Jackson Laboratories Strain no.

[0534] 029099) mice were harvested to collect whole spinal columns. Mice were aged out to four months of age prior to tissue collection, to allow for possible C9orf72 dipeptide repeat protein accumulation in each tested C9 mouse model. Mice were saline-perfused at time of spinal column collection, and spinal columns were incubated in 4% PFA for 24 hours at 4°C immediately following harvest. After 24 hours, spinal columns were washed 2x with saline, and transferred to 70% ethanol at 4°C. Spinal columns in 70% ethanol were shipped overnight to Applied Pathology Systems (“APS”; Shrewsbury, MA, USA) for processing. One sample of rabbit anti-ADMe-GR8 monoclonal antibody was shipped on dry ice overnight to APS for validation staining on provided mouse tissues.

[0535] Upon arrival, fixed, whole mouse spinal columns were processed to gently remove the muscles attached to the spinal column and split the column into 4 different regions: cervical, thoracic, lumbar, and sacral (4 samples per mouse, 12 samples total). Histology ink was used to mark the starting point of the spinal column for each designated region. The samples were moved to a cassette and put into 20% EDTA, an immunohistochemistry (“IHC”)-friendly decalcifier, until the decalcification process was completed. The decalcification process was considered “complete” once samples became soft and flexible. Each sample was then dissected into two or three pieces starting from the second vertebrae from each region (e.g. “C2,” “T2,” “L2,” “S2”), and the area of interest was inked using histology ink before being placed back into the cassette for processing. The samples were processed in a Leica ASP3005 processer overnight using a standard protocol of incubation in the following solutions: 70% alcohol for 1 hour and 30 minutes, 95% alcohol for 45 minutes, 95% alcohol for 45 minutes, 100% alcohol for 15 minutes, 100% alcohol for 30 minutes, 100% alcohol for 45 minutes, xylene for 45 minutes, xylene for 45 minutes, xylene for 45 minutes, paraffin for 30 minutes at 60°C with pressure / vacuum, paraffin for 45 minutes at 60°C with pressure / vacuum, and paraffin for 45 minutes at 60°C with pressure / vacuum. Samples were embedded in paraffin vertically on the inked marked side facing down in the mold. Paraffin-embedded tissue blocks were trimmed carefully until the full shape of the spinal cord was visible. Then, paraffin-embedded tissue blocks were sectioned on a 5 pm thickness and mounted carefully onto slides. Slides were air-dried for 30 minutes- 1 hour before backing to remove water drops from each section and improve tissue attachment. Slides were then baked at 60°C for 1 hour before staining protocol began.Attorney Docket No. ALSE-016PC

[0536] Before staining, slides were deparaffinized and tissue sections were rehydrated using a xylene and graded alcohol series. Following rehydration, slides were washed for 2 minutes in distilled water and 2 minutes in tris-buffered saline with tween-20 (TBST). Heat-induced epitope retrieval (HIER) was performed by placing slides in a pressure cooker in IX citrate buffer (pH 6.0). Following HIER, slides were cooled down for 30 minutes at room temperature. After cooling, slides were rinsed in TBST for 2 minutes. Tissue sections were circled on each slide using a hydrophobic barrier pen. Slides were then blocked with BLOXALL (Vector, SP-6000-100) at room temperature for 10 minutes before another 2-minute TBST rinse. Slides were blocked a second time with 2.5% normal horse serum at room temperature for 20 minutes. Blocking reagent was drained off before applying working primary antibody solutions to the slides and incubating at room temperature for 1 hour. Working primary antibody solution contained rabbit monoclonal anti-GRs antibody at a 1:50 dilution. After incubating the primary antibody solution for Ih, slides were washed twice in TBST for 2 minutes each. Washed slides were then incubated with goat anti-rabbit IgG amplifier antibody (Vector, 30130) at room temperature for 15 minutes. Slides were then washed twice in TBST for 2 minutes each. Next, slides were incubated with ImmPRESS-HRP Horse Anti-Goat IgG Polymer Reagent (Vector, 30036) at room temperature for 30 minutes. Slides were then washed in TBST and then in DI water for 2 minutes per wash. Washed slides were then counterstained with Gill II stain for 15 seconds, followed by dehydration in an auto-stainer set to program 10. Dehydrated slides were cover slipped before whole slide-scan imaging using PhenoImager (Akoya Biosciences).

[0537] As shown in FIG. 15, when used as a primary antibody stain on wild type and C9orf72-mediated ALS mouse model spinal cord tissues, staining intensity in C9orf72-mediated ALS mice trends higher in both ALS mouse models when compared to wild-type controls. Specifically, in FIG. 15 A, significant differences are detectable between wild type and both C9-ALS mouse models when comparing technical replicate staining across two separate staining runs per region (2 cervical, 2 thoracic, or 2 sacral), per mouse model (Oneway ANOVA, Dunnett’s Multiple Comparisons Test vs. wild-type) for demonstrative purposes. As shown in FIG. 15B-D, when these data are separated out by spinal cord region, consistent trends comparing wild type controls to C9-ALS models are observed across repeat staining in cervical, thoracic, and sacral spinal regions. Image quantification was performed using Fiji / Image-J software, in which stained and unstained areas in color-deconvoluted images — where DAB color channel (anti-ADMe-GRs staining) had entire 10X zoom slideAttorney Docket No. ALSE-016PC

[0538] field quantified and compared to background staining in each image — were quantified before calculations of optical density (OD) and DAB mean were obtained for graphing. Values were graphed and analyzed using GraphPad Prism 10.6.0 software.

[0539] Example 13: Using Custom Meso-Scale Discovery (MSD) Assay for ADMe-GRs Detection in Human Cerebrospinal Fluid Samples

[0540] Healthy cerebrospinal fluid samples were provided by the National Disease Research Interchange (NDRI), and C9orf72-mediated ALS patient cerebrospinal fluid samples were provided by the Center for Disease Control (CDC)’s ALS Registry. In this example, the custom MSD assay developed in Example 14 was used to test neat (non-concentrated) samples of cerebrospinal fluid in three healthy individuals and three C9orf72-ALS patients.

[0541] Biotinylated rabbit anti-ADMe-GRs monoclonal capture antibody was coated on an MSD 1-Spot Streptavidin QuickPlex plate (Cat no. L55SA-2) at a concentration of 2 pg / ml with a volume of 25 pL / well overnight, sealed at 4°C. The following day, biotinylated rabbit anti-ADMe-GRs monoclonal antibody coat was removed and washed three times with IX Tris-Buffered Saline, 0.05% Tween-20 (IX TBST 0.05%), 200 pL / well per wash. Plate was gently blotted dry on paper towels before MSD Blocker A solution (prepared using MSD Blocker A Kit, Cat no. R93AA-2) was applied 150 pL / well, plate was sealed and incubated Ih at room temperature with vigorous shaking (speed 7 on a microplate orbital shaker). After Ih, blocking solution was removed and washed once with IX TBS, 200 pL / well per wash, before blotting dry and adding in duplicate the following standard curve in quadruplicate: ADMe-GRis in MSD Diluent 100 assay buffer from 100 ng / mL down serial 1:5 to 6.4 pg / mL (48 uL / well). Control wells containing MSD assay buffer-only were included in the curve to reflect background assay signal at 0 pg / mL. At the same time as standard curve addition, healthy and ALS CSF samples were loaded neat, in duplicate wells (48 uL / well). Following sample addition, plate was sealed and incubated at 4°C overnight with low-speed (1.5x) orbital shaking. The following day, samples were shaken out and plate was washed three times with IX TBST 0.05%, 200 pL / well per wash. Plate was gently blotted dry on paper towels before sulfo-tagged rabbit polyclonal anti-ADMe-GRs detector antibody was added at 0.5 pg / mL, 25 pL / well and plate was sealed before vigorous shaking (speed 7 on a microplate orbital shaker) for Ih at room temperature in the dark. After Ih detector antibody was shaken out and plate was washed three more times with IX TBST 0.05%, 200 pL / well per wash. Plate was thoroughly blotted dry on paper towels before addition of MSD Read Buffer A, 150Attorney Docket No. ALSE-016PC

[0542] pL / well, and immediate read of ECL signal on MSD MESO QuickPlex SQ 120 Reader. Before plotting, ECL signal values were background-subtracted based on signals in assay buffer-only wells. ADMe-GR levels in healthy and patient CSF were interpolated using a non-linear 4PL regression model of the assay’s standard curve (R-squared of fit = 0.9906) in GraphPad Prism 10.6.0 software. Only two out of three ALS patient samples were included in FIG. 16, due to Prism being unable to accurately estimate the third patient’s pg / mL ADMe-GR value from the standard curve.

[0543] As shown in FIG. 16, when biotinylated for use as a detector antibody in a custom MSD assay, rabbit anti-ADMe-GRs monoclonal antibody captures ADMe-GRis analyte in non-concentrated cerebrospinal fluid samples. Specifically, the custom MSD assay is able to distinguish C9orf72-ALS patients positive and negative for elevated levels of ADMe-GR in cerebrospinal fluid from one another, and from healthy control samples (One-Way ANOVA, Dunnett’s Multiple Comparisons Test, All compared to C9 Patient 1).

[0544] Equivalents

[0545] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.Attorney Docket No. ALSE-016PC

[0546] Table 3: SUMMARY OF SEQUENCE LISTING

[0547] SEQ ID NO: DESCRIPTION

[0548] ADMe-GR Antibody 9A6

[0549] 1 Heavy Chain CDR1 Amino Acid Sequence

[0550] SYFYMC

[0551] 2 Heavy Chain CDR2 Amino Acid Sequence

[0552] CIWTSSGTTYYANWARG

[0553] 3 Heavy Chain CDR3 Amino Acid Sequence

[0554] DGSADYGTYYGMDL

[0555] 4 Light Chain CDR1 Amino Acid Sequence

[0556] QASEDISSWLS

[0557] 5 Light Chain CDR2 Amino Acid Sequence

[0558] DASTLAS

[0559] 6 Light Chain CDR3 Amino Acid Sequence

[0560] QQDYNSINVDNS

[0561] 7 Heavy Chain Variable Region Amino Acid Sequence QSLEESGGDLVKPGASLTLTCTASGIDFSSYFYMCWVRQAPGKGLE WIGCIWTSSGTTYYANWARGRFTISKSPSTTVTLQMTSLTAADTAIY FC ARDGS ADYGTYYGMDLWGPGTLVTVS S

[0562] 8 Light Chain Variable Region Amino Acid Sequence DMTQTPASVEVAVGGTVTIKCQASEDISSWLSWYQQKSGQRPKLLI YDASTLASGVSSRFKGSGSGTQFTLTISGVECADAATYYCQQDYNSI NVDNSFGGGTEVVVK

[0563] 9 Heavy Chain Amino Acid Sequence METGLRWLLEVAVLKGVOCOSLEESGGDLVKPGASLTLTCTASGID FSSYFYMCWVROAPGKGLEWIGCIWTSSGTTYYANWARGRFTISKS PSTTVTLOMTSLTAADTAIYFCARDGSADYGTYYGMDLWGPGTLVT VSSGOPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSG TLTNGVRTFPS VRQS SGLYSLS S VVS VTS S SQPVTCNVAHPATNTKV DKTVAPSTCSKPMCPPPELPGGPSVFIFPPKPKDTLMISRTPEVTCVVV DVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQ DWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREEL SSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYF LYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK

[0564]

[0565] Attorney Docket No. ALSE-016PC

[0566] SEQ ID NO: DESCRIPTION

[0567] 10 Light Chain Amino Acid Sequence MDTRAPTOLLCTLLLLWLPCTARCAYDMTOTPASVEVAVGGTVTIKCO ASEDISSWLSWYOOKSGORPKLLIYDASTLASGVSSRFKGSGSGTOF TLTISGVECADAATYYCOODYNSINVDNSFGGGTEVVVKGDPVAPTV LIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKT PQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGD

[0568] C

[0569] 11 Heavy Chain Open Reading Frame Nucleotide Sequence GAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAA GGTGTCCAGTGTCAGTCGTTGGAGGAGTCCGGGGGAGACCTGGTC AAGCCTGGGGCATCCCTGACACTCACCTGCACAGCCTCTGGAATC GACTTCAGTAGCTACTTCTACATGTGTTGGGTCCGCCAGGCTCCAG GGAAGGGGCTGGAGTGGATCGGATGCATTTGGACTAGTAGTGGTA CCACTTACTACGCGAACTGGGCGAGAGGCCGATTCACCATCTCCA AAAGCCCGTCGACCACGGTGACTCTGCAAATGACCAGTCTGACAG CCGCGGACACGGCCATCTATTTCTGTGCGAGAGATGGCAGTGCTG ATTATGGGACCTACTACGGCATGGACCTCTGGGGCCCAGGGACCCT CGTCACCGTCTCTTCAGGGCAACCTAAGGCTCCATCAGTCTTCCCA CTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTG GGCTGCCTGGTCAAAGGCTACCTCCCGGAGCCAGTGACCGTGACC TGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCC GTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGC GTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCA GCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGC AGCAAGCCCATGTGCCCACCCCCTGAACTCCCGGGGGGACCGTCT GTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCAC GCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATG ACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGC GCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACG ATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTG AGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCC GGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCT GGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGA GCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACC CTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAG GACAACTACAAGACCACGCCGACCGTGCTGGACAGCGACGGCTC CTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCA GCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCA CAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATA

[0570] G

[0571] 12 Light Chain Open Reading Frame Nucleotide Sequence

[0572]

[0573] Attorney Docket No. ALSE-016PC

[0574] SEQ ID NO: DESCRIPTION GACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTC TGGCTCCCAGGTGCCAGATGTGCCTATGATATGACCCAGACTCCA GCCTCTGTGGAGGTAGCTGTGGGAGGCACAGTCACCATCAAGTGC CAGGCCAGTGAGGATATTAGTAGTTGGTTATCCTGGTATCAGCAG AAATCAGGGCAGCGTCCCAAGCTCCTGATCTATGATGCATCCACT CTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGATCTGGG ACACAGTTCACTCTCACCATCAGCGGCGTGGAGTGTGCCGATGCT GCCACTTACTACTGTCAACAGGATTATAATAGCATTAATGTTGAT AATAGTTTCGGCGGAGGGACCGAGGTGGTGGTCAAAGGTGATCC AGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTG GCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTT CCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAAC AACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATT GTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGT ACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACG ACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAG

[0575] 13 Heavy Chain Full Plasmid Nucleotide Sequence GTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGC GTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGG TCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTAC GGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATT GACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACT TTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACT TGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGA CGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATG ACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCAT CGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGT GGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTG ACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTC CAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTA GGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGA ACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCC ATAGAAGACACCGGGACCGATCCAGCCTCCGGACTCTAGAATTCA CCATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCA AAGGTGTCCAGTGTCAGTCGTTGGAGGAGTCCGGGGGAGACCTG GTCAAGCCTGGGGCATCCCTGACACTCACCTGCACAGCCTCTGGA ATCGACTTCAGTAGCTACTTCTACATGTGTTGGGTCCGCCAGGCTC CAGGGAAGGGGCTGGAGTGGATCGGATGCATTTGGACTAGTAGTG GTACCACTTACTACGCGAACTGGGCGAGAGGCCGATTCACCATCT CCAAAAGCCCGTCGACCACGGTGACTCTGCAAATGACCAGTCTGA CAGCCGCGGACACGGCCATCTATTTCTGTGCGAGAGATGGCAGTG CTGATTATGGGACCTACTACGGCATGGACCTCTGGGGCCCAGGGA CCCTCGTCACCGTCTCTTCAGGGCAACCTAAGGCTCCATCAGTCTT

[0576]

[0577] CCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACAttorney Docket No. ALSE-016PC

[0578] SEQ ID NO: DESCRIPTION CCTGGGCTGCCTGGTCAAAGGCTACCTCCCGGAGCCAGTGACCGT GACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCC GTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGT GAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCC ACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCG ACATGCAGCAAGCCCATGTGCCCACCCCCTGAACTCCCGGGGGGA CCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGA TCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCC AGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGC AGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAAC AGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGAC TGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGC ACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCA GCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGA GCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTT CTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGG CAGAGGACAACTACAAGACCACGCCGACCGTGCTGGACAGCGAC GGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAG TGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCC TTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGT AAATAGGGATCCAGCTTAAGGGTTCGATCCCTACCGGTTAGTAATG AGTTTGATATCTCGACAATCAACCTCTGGATTACAAAATTTGTGAA AGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGG ATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGC TTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGA GGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGT GTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTG TCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACG GCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCT CGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTG ACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGC GCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGG ACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCG TCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCC TCCCCGCCTGGAAACGGGGGAGGCTAACTGAAACACGGAAGGAG ACAATACCGGAAGGAACCCGCGCTATGACGGCAATAAAAAGACAG AATAAAACGCACGGGTGTTGGGTCGTTTGTTCATAAACGCGGGGT TCGGTCCCAGGGCTGGCACTCTGTCGATACCCCACCGAGACCCCA TTGGGGCCAATACGCCCGCGTTTCTTCCTTTTCCCCACCCCACCCC CCAAGTTCGGGTGAAGGCCCAGGGCTCGCAGCCAACGTCGGGGC GGCAGGCCCTGCCATAGCAGATCTGCGCAGCTGGGGCTCTAGGGG GTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGT GGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGC GCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCG

[0579]

[0580] GCTTTCCCCGTCAAGCTCTAAATCGGGGCATCCCTTTAGGGTTCCGAttorney Docket No. ALSE-016PC

[0581] SEQ ID NO: DESCRIPTION ATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGT GATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCC CTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCA AACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTAT AAGGGATTTTGGGGATTTCGGCCTATTGGTTAAAAAATGAGCTGAT TTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGT TAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGC AAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCC CAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATT AGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTTATATCTTTCTCAGTCTTCACTAGGCTATGCACGGGCCCCCGATATGCGTCCCCGGCCCCTCCCTAGTCGCGTCCTTGGAACGTCATAATTTTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAA AAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAAGAG ACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCAC GCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACT GGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGC TGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGT CCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGT GGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTG TCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCG GGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTAT CCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGC TACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGC ACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGA CGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCT CAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGG CGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCT GGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAG GACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCG AATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGA TTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGA GCGGGACTCTGGGGTTCGCGAAATGACCGACCAAGCGACGCCCA ACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAG GTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTC CAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGT TTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAAT TTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTC CAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACCTCTA GCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAA TTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATA AAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTA ATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGT GCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTT

[0582]

[0583] TGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGAttorney Docket No. ALSE-016PC

[0584] SEQ ID NO: DESCRIPTION CTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGC GGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAA CATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGG CCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCA TCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAG GACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCG CTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTT CTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGT ATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCA CGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTAT CGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCA GCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGT GCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGA AGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCG GAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTG GTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAA AAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGA CGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAG ATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAA GTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGT TACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATT TCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACG ATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGC GAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGC CAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCG CCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAG TTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGC ATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCG GTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAA AAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAA GTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAAT TCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGA GTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGT TGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCA GAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAA AACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACC CACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGC GTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAA GGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTT TTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGG ATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGC GCACATTTCCCCGAAAAGTGCCACCTGACGTCGACGGATCGGGAG ATCTCCCGATCCCCTATGGTCGACTCTCAGTACAATCTGCTCTGATG

[0585]

[0586] CCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTAttorney Docket No. ALSE-016PC

[0587] SEQ ID NO: DESCRIPTION CGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGG CTTGACCGACAATTGCATGAAGAATCTGCTTAGG

[0588] 14 Light Chain Full Plasmid Nucleotide Sequence GTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACG CGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACG GGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAA CTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGC CCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATA GGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACT GCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCC CCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCC CAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTAC GTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTAC ATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGT CTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAAT CAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACG CAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAG AGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACG CTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCG GACTCTAGGAATTCACCATGGACACGAGGGCCCCCACTCAGCTGC TGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCAGATGTGCCTATG ATATGACCCAGACTCCAGCCTCTGTGGAGGTAGCTGTGGGAGGCA CAGTCACCATCAAGTGCCAGGCCAGTGAGGATATTAGTAGTTGGT TATCCTGGTATCAGCAGAAATCAGGGCAGCGTCCCAAGCTCCTGA TCTATGATGCATCCACTCTGGCATCTGGGGTCTCATCGCGGTTCA AAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGGCG TGGAGTGTGCCGATGCTGCCACTTACTACTGTCAACAGGATTATA ATAGCATTAATGTTGATAATAGTTTCGGCGGAGGGACCGAGGTGG TGGTCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACC AGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGT GGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGA TGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGC AGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACAC TGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAG GTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGT GACTGTTAGGGATCCAGCTTAAGGGTTCGATCCCTACCGGTTAGT AATGAGTTTGATATCTCGACAATCAACCTCTGGATTACAAAATTT GTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCT ATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCC CGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGT CTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGG TGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTG CCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCC

[0589]

[0590] TATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGAttorney Docket No. ALSE-016PC

[0591] SEQ ID NO: DESCRIPTION GACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTC GGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCAC CTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTC AATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGG CCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCC TTTGGGCCGCCTCCCCGCCTGGAAACGGGGGAGGCTAACTGAAA CACGGAAGGAGACAATACCGGAAGGAACCCGCGCTATGACGGCA ATAAAAAGACAGAATAAAACGCACGGGTGTTGGGTCGTTTGTTC ATAAACGCGGGGTTCGGTCCCAGGGCTGGCACTCTGTCGATACCC CACCGAGACCCCATTGGGGCCAATACGCCCGCGTTTCTTCCTTTTC CCCACCCCACCCCCCAAGTTCGGGTGAAGGCCCAGGGCTCGCAGC CAACGTCGGGGCGGCAGGCCCTGCCATAGCAGATCTGCGCAGCT GGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAA GCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTG CCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTC GCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGCATC CCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAA AAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGA TAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATA GTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGG TCTATTCTTTTGATTTATAAGGGATTTTGGGGATTTCGGCCTATTG GTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAATT CTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCC CCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGC AACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTA TGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCC TAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTC TCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAG GCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTT TTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATA TCCATTTTCGGATCTGATCAAGAGACAGGATGAGGATCGTTTCGC ATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGG GTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGG CTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCC GGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACT GCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCG TTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGG ACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCAT CTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAA TGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACC ACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAA GCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGG GCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCC CGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCC

[0592]

[0593] GAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGAttorney Docket No. ALSE-016PC

[0594] SEQ ID NO: DESCRIPTION TGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGC TACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCG CTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATC GCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGG GTTCGCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACG AGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGG AATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGA TCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCT TATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAAT AAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCA TCAATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAG CTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTAT CCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTG TAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGC GTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCA GCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGC GTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTC GGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGT AATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACA TGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCC GCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCAT CACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGG ACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCG CTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTT CTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGG TATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTG CACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAAC TATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTG GCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGG CGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACAC TAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTAC CTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCAC CGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCG CAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGG GTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGT CATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTA AAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTG GTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGC GATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTG TAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCT GCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCA GCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCC TGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAA GCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTT

[0595]

[0596] GCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGAttorney Docket No. ALSE-016PC

[0597] SEQ ID NO: DESCRIPTION GCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGA TCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCG ATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTT ATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGA TGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAA TAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGG GATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATT GGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTG TTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCT TCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACA GGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGA AATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCAT TTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATT TAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAA AGTGCCACCTGACGTCGACGGATCGGGAGATCTCCCGATCCCCTA TGGTCGACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGC CAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGC GCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAA TTGCATGAAGAATCTGCTTAGG

[0598]

[0599] SDMe-GR Antibody 7E2

[0600] 15 Heavy Chain CDR1 Amino Acid Sequence

[0601] SYYYMC

[0602] 16 Heavy Chain CDR2 Amino Acid Sequence

[0603] CIYTGSSGSTYYASWAKG

[0604] 17 Heavy Chain CDR3 Amino Acid Sequence

[0605] DLPAGYAGYGYFNF

[0606] 18 Light Chain CDR1 Amino Acid Sequence

[0607] RASEDIESYLA

[0608] 19 Light Chain CDR2 Amino Acid Sequence

[0609] DSSGLAS

[0610] 20 Light Chain CDR3 Amino Acid Sequence

[0611] QVGDYTTSDNV

[0612] 21 Heavy Chain Variable Region Amino Acid Sequence

[0613]

[0614] Attorney Docket No. ALSE-016PC

[0615] OSLEESGGDLVKPGASLTLTCTASGIDFSSYYYMCWVROAPGKGLE WIACIYTGSSGSTYYASWAKGRFTISKTSSTTVTLOMTSLTAADTAT YFC ARDLPAGYAGYGYFNFWGPGTLVTVS S

[0616] Light Chain Variable Region Amino Acid Sequence AIKMTOTPSSVSAAVGGTVTINCRASEDIESYLAWYOOKPGOPPKLLT YDSSGLASGVPSRFKGSGSGKOFTLTISGVOCDDAATYYCOVGDYT TSDNVFGGGTEVVVK

[0617] Heavy Chain Amino Acid Sequence METGLRWLLLVAVLKGVOCOSLEESGGDLVKPGASLTLTCTASGID FSSYYYMCWVROAPGKGLEWIACIYTGSSGSTYYASWAKGRFTISK TSSTTVTLOMTSLTAADTATYFCARDLPAGYAGYGYFNFWGPGTLV TVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNS GTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTK VDKTVAPSTCSKPMCPPPELPGGPSVFIFPPKPKDTLMISRTPEVTCVV VDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAH QDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREE LSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSY FLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK

[0618] Light Chain Amino Acid Sequence

[0619] MDTR APTQLT. GT J J J. WI. PG ATFAIKMTOTPS S VS A AVGGTVTINCR A SEDIESYLAWYOOKPGOPPKLLIYDSSGLASGVPSRFKGSGSGKOFTL TISGVOCDDAATYYCOVGDYTTSDNVFGGGTEVVVKGDPVAPTVLI FPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTP QNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC

[0620] Heavy Chain Open Reading Frame Nucleotide Sequence ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAA GGTGTCCAGTGTCAGTCGTTGGAGGAGTCCGGGGGAGACCTGGTC AAGCCTGGGGCATCCCTGACACTCACCTGCACAGCCTCTGGAATC GACTTCAGTTCCTACTACTACATGTGCTGGGTCCGCCAGGCTCCAG GAAAGGGGCTGGAGTGGATCGCATGCATTTATACTGGTAGTAGTGG TAGCACTTACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTC CAAAACCTCGTCGACCACGGTGACTCTGCAAATGACCAGTCTGAC AGCCGCGGACACGGCCACCTATTTCTGTGCGAGAGATCTCCCTGC TGGTTATGCTGGTTATGGTTACTTTAACTTTTGGGGCCCAGGCACC CTGGTCACCGTCTCCTCAGGGCAACCTAAGGCTCCATCAGTCTTCC CACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCC TGGGCTGCCTGGTCAAAGGCTACCTCCCGGAGCCAGTGACCGTGA CCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGT CCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGA

[0621]

[0622] GCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCAttorney Docket No. ALSE-016PC

[0623] CAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACAT GCAGCAAGCCCATGTGCCCACCCCCTGAACTCCCGGGGGGACCGT CTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTC ACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGG ATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGG TGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGC ACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGG CTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACT CCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCC CCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCT GAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTA CCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAG AGGACAACTACAAGACCACGCCGACCGTGCTGGACAGCGACGGC TCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGG CAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTG CACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAA TAG

[0624] Light Chain Open Reading Frame Nucleotide Sequence ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTC TGGCTCCCAGGTGCCACATTTGCCATCAAAATGACCCAGACTCCAT CCTCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAATTGCC GGGCCAGTGAGGACATTGAAAGCTATTTAGCCTGGTATCAGCAGA AACCAGGGCAGCCTCCCAAGCTCCTGATCTATGATTCATCCGGTCT GGCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGAA ACAGTTCACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGC CACTTACTACTGTCAAGTTGGTGATTATACTACTAGTGATAATGTTT TCGGCGGAGGGACCGAGGTGGTGGTCAAAGGTGATCCAGTTGCA CCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTG GAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGT CACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCAT CGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAA CCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCA CAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCG TCCAGAGCTTCAATAGGGGTGACTGTTAG

[0625] Heavy Chain Full Plasmid Nucleotide Sequence GTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGC GTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGG TCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTAC GGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATT GACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACT TTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACT TGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGA CGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATG

[0626]

[0627] ACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATAttorney Docket No. ALSE-016PC

[0628] CGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGT GGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTG ACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTC CAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTA GGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGA ACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCC ATAGAAGACACCGGGACCGATCCAGCCTCCGGACTCTAGAATTCA CCATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCA AAGGTGTCCAGTGTCAGTCGTTGGAGGAGTCCGGGGGAGACCTG GTCAAGCCTGGGGCATCCCTGACACTCACCTGCACAGCCTCTGGA ATCGACTTCAGTTCCTACTACTACATGTGCTGGGTCCGCCAGGCTC CAGGAAAGGGGCTGGAGTGGATCGCATGCATTTATACTGGTAGTAG TGGTAGCACTTACTACGCGAGCTGGGCGAAAGGCCGATTCACCAT CTCCAAAACCTCGTCGACCACGGTGACTCTGCAAATGACCAGTCT GACAGCCGCGGACACGGCCACCTATTTCTGTGCGAGAGATCTCCC TGCTGGTTATGCTGGTTATGGTTACTTTAACTTTTGGGGCCCAGGC ACCCTGGTCACCGTCTCCTCAGGGCAACCTAAGGCTCCATCAGTC TTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTG ACCCTGGGCTGCCTGGTCAAAGGCTACCTCCCGGAGCCAGTGACC GTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTC CCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTG GTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCC CACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTC GACATGCAGCAAGCCCATGTGCCCACCCCCTGAACTCCCGGGGGG ACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATG ATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGC CAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAG CAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAA CAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGA CTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGG CACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGC AGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGG AGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCT TCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGG CAGAGGACAACTACAAGACCACGCCGACCGTGCTGGACAGCGAC GGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAG TGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCC TTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGT AAATAGGGATCCAGCTTAAGGGTTCGATCCCTACCGGTTAGTAATG AGTTTGATATCTCGACAATCAACCTCTGGATTACAAAATTTGTGAA AGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGG ATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGC TTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGA GGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGT GTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTG

[0629]

[0630] TCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGAttorney Docket No. ALSE-016PC

[0631] GCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCT CGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTG ACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGC GCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGG ACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCG TCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCC TCCCCGCCTGGAAACGGGGGAGGCTAACTGAAACACGGAAGGAG ACAATACCGGAAGGAACCCGCGCTATGACGGCAATAAAAAGACAG AATAAAACGCACGGGTGTTGGGTCGTTTGTTCATAAACGCGGGGT TCGGTCCCAGGGCTGGCACTCTGTCGATACCCCACCGAGACCCCA TTGGGGCCAATACGCCCGCGTTTCTTCCTTTTCCCCACCCCACCCC CCAAGTTCGGGTGAAGGCCCAGGGCTCGCAGCCAACGTCGGGGC GGCAGGCCCTGCCATAGCAGATCTGCGCAGCTGGGGCTCTAGGGG GTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGT GGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGC GCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCG GCTTTCCCCGTCAAGCTCTAAATCGGGGCATCCCTTTAGGGTTCCG ATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGT GATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCC CTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCA AACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTAT AAGGGATTTTGGGGATTTCGGCCTATTGGTTAAAAAATGAGCTGAT TTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGT TAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGC AAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCC CAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATT AGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCT AACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAA AAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAAGAG ACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCAC GCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACT GGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGC TGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGT CCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGT GGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTG TCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCG GGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTAT CCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGC TACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGC ACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGA CGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCT CAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGG CGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCT

[0632]

[0633] GGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGAttorney Docket No. ALSE-016PC

[0634] GACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCG AATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGA TTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGA GCGGGACTCTGGGGTTCGCGAAATGACCGACCAAGCGACGCCCA ACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAG GTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTC CAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGT TTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAAT TTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTC CAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACCTCTA GCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAA TTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATA AAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTA ATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGT GCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTT TGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCG CTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGC GGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAA CATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGG CCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCA TCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAG GACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCG CTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTT CTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGT ATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCA CGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTAT CGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCA GCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGT GCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGA AGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCG GAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTG GTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAA AAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGA CGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAG ATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAA GTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGT TACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATT TCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACG ATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGC GAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGC CAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCG CCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAG TTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGC ATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCG GTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAA

[0635]

[0636] AAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAAttorney Docket No. ALSE-016PC

[0637] GTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAAT TCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGA GTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGT TGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCA GAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAA AACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACC CACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGC GTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAA GGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTT TTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGG ATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGC GCACATTTCCCCGAAAAGTGCCACCTGACGTCGACGGATCGGGAG ATCTCCCGATCCCCTATGGTCGACTCTCAGTACAATCTGCTCTGATG CCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGT CGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGG CTTGACCGACAATTGCATGAAGAATCTGCTTAGG

[0638] Light Chain Full Plasmid Nucleotide Sequence GTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACG CGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACG GGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAA CTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGC CCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATA GGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACT GCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCC CCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCC CAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTAC GTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTAC ATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGT CTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAAT CAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACG CAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAG AGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACG CTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCG GACTCTAGGAATTCACCATGGACACGAGGGCCCCCACTCAGCTGC TGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCCATCA AAATGACCCAGACTCCATCCTCCGTGTCTGCAGCTGTGGGAGGCA CAGTCACCATCAATTGCCGGGCCAGTGAGGACATTGAAAGCTATT TAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGA TCTATGATTCATCCGGTCTGGCATCTGGGGTCCCATCGCGGTTCA AAGGCAGTGGATCTGGGAAACAGTTCACTCTCACCATCAGCGGC GTGCAGTGTGACGATGCTGCCACTTACTACTGTCAAGTTGGTGAT TATACTACTAGTGATAATGTTTTCGGCGGAGGGACCGAGGTGGTG GTCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCA GCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTG

[0639]

[0640] GCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATAttorney Docket No. ALSE-016PC

[0641] GGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCA GAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACT GACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGG TGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTG ACTGTTAGGGATCCAGCTTAAGGGTTCGATCCCTACCGGTTAGTA ATGAGTTTGATATCTCGACAATCAACCTCTGGATTACAAAATTTG TGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTA TGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCC GTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCT CTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTG TGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCC ACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTA TTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGA CAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGG GGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCT GGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCA ATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGC CTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCT TTGGGCCGCCTCCCCGCCTGGAAACGGGGGAGGCTAACTGAAAC ACGGAAGGAGACAATACCGGAAGGAACCCGCGCTATGACGGCAA TAAAAAGACAGAATAAAACGCACGGGTGTTGGGTCGTTTGTTCAT AAACGCGGGGTTCGGTCCCAGGGCTGGCACTCTGTCGATACCCCA CCGAGACCCCATTGGGGCCAATACGCCCGCGTTTCTTCCTTTTCCC CACCCCACCCCCCAAGTTCGGGTGAAGGCCCAGGGCTCGCAGCC AACGTCGGGGCGGCAGGCCCTGCCATAGCAGATCTGCGCAGCTG GGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAG CGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGC CAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTC GCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGCATC CCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAA AAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGA TAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATA GTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGG TCTATTCTTTTGATTTATAAGGGATTTTGGGGATTTCGGCCTATTG GTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAATT CTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCC CCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGC AACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTA TGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCC TAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTC TCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAG GCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTT TTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATA TCCATTTTCGGATCTGATCAAGAGACAGGATGAGGATCGTTTCGC ATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGG

[0642]

[0643] GTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGAttorney Docket No. ALSE-016PC

[0644] CTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCC GGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACT GCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCG TTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGG ACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCAT CTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAA TGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACC ACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAA GCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGG GCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCC CGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCC GAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTG TGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGC TACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCG CTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATC GCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGG GTTCGCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACG AGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGG AATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGA TCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCT TATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAAT AAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCA TCAATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAG CTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTAT CCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTG TAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGC GTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCA GCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGC GTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTC GGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGT AATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACA TGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCC GCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCAT CACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGG ACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCG CTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTT CTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGG TATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTG CACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAAC TATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTG GCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGG CGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACAC TAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTAC CTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCAC CGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCG

[0645]

[0646] CAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGAttorney Docket No. ALSE-016PC

[0647] GTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGT CATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTA AAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTG GTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGC GATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTG TAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCT GCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCA GCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCC TGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAA GCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTT GCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATG GCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGA TCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCG ATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTT ATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGA TGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAA TAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGG GATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATT GGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTG TTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCT TCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACA GGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGA AATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCAT TTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATT TAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAA AGTGCCACCTGACGTCGACGGATCGGGAGATCTCCCGATCCCCTA TGGTCGACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGC CAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGC GCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAA TTGCATGAAGAATCTGCTTAGG

[0648]

Claims

Attorney Docket No. ALSE-016PCCLAIMS1. An isolated antibody, or antigen binding portion thereof, which binds to a methylated dipeptide repeat protein (DRP) produced by repeat expansion mutation of the C9orf72 gene, wherein the antibody, or antigen binding portion thereof, exhibits one or more of the following properties:(a) inhibits neuronal cell death(b) reduces the level of methylated DRP-associated toxicity in the cell expressing the DRP;(c) reduces circulating levels of methylated DRP in biological tissues or fluids(d) reduces the level of methylated DRP-associated toxicity in cells exogenously exposed to the methylated DRP; and / or(e) binds to a methylated DRP with a KD of approximately ICT8to 10-10M or less.

2. The antibody, or antigen binding portion thereof, of claim 1, wherein the DRP is an asymmetrically dimethylated (ADMe) dipeptide protein or a symmetrically dimethylated (SDMe).

3. The antibody, or antigen binding portion thereof, of claim 1 or 2, wherein the ADMe or SDMe DRP comprises at least one poly-glycine-arginine dipeptide (GR) and / or poly-proline-arginine dipeptide (PR).

4. The antibody, or antigen binding portion thereof, of any one of the preceding claims, wherein the ADMe or SDMe DRP is poly-GR or poly-PR.

5. The antibody, or antigen binding portion thereof, of any one of the preceding claims, comprising the CDR1, CDR2, and CDR3 amino acid sequences of the heavy and light chain variable region sequences respectively set forth in:(a) SEQ ID NOs: 7 and 8; or(b) SEQ ID NOs: 21 and 22.Attorney Docket No. ALSE-016PC6. The antibody, or antigen binding portion thereof, of any one of the preceding claims, comprising:(a) heavy chain CDR1, CDR2, and CDR3 amino acid sequences respectively set forth in SEQ ID NOs: 1, 2, and 3, and / or light chain CDR1, CDR2, and CDR3 amino acid sequences respectively set forth in SEQ ID NOs: 4, 5, and 6; or(b) heavy chain CDR1, CDR2. and CDR3 amino acid sequences respectively comprising SEQ ID NOs: 15, 16, and 17 and / or light chain CDR1, CDR2, and CDR3 amino acid sequences respectively comprising SEQ ID NOs: 18, 19, and 20.

7. The antibody, or antigen binding portion thereof, of any one of the preceding claims, comprising:(a) heavy chain CDR1, CDR2, and CDR3 amino acid sequences respectively set forth in SEQ ID NOs: 1, 2, and 3 and light chain CDR1, CDR2, and CDR3 amino acid sequences respectively set forth in SEQ ID NOs: 4, 5, and 6; or(b) heavy chain CDR1, CDR2, and CDR3 amino acid sequences respectively comprising SEQ ID NOs: 15, 16, and 17 and light chain CDR1, CDR2, and CDR3 amino acid sequences respectively comprising SEQ ID NOs: 18, 19, and 20.

8. An isolated antibody, or antigen binding portion thereof, which binds to a methylated dipeptide repeat protein (DRP) produced by repeat expansion mutation of the C9orf72 gene, wherein the antibody, or antigen binding portion thereof, comprises a heavy chain variable region which is at least 90% identical to the heavy chain amino acid sequence set forth in SEQ ID NO: 7 or 21 and / or a light chain variable region which is at least 90% identical to the light chain amino acid sequence set forth in SEQ ID NO: 8 or 22.

9. An isolated antibody, or antigen binding portion thereof, which binds to a methylated dipeptide repeat protein (DRP) produced by repeat expansion mutation of the C9orf72 gene, wherein the antibody, or antigen binding portion thereof, comprises heavy and light chain variable regions which are at least 90% identical to the heavy and light chain variable region amino acid sequences respectively set forth in:(a) SEQ ID NOs: 7 and 8; orAttorney Docket No. ALSE-016PC(b) SEQ ID NOs: 21 and 22.

10. An isolated antibody, or antigen binding portion thereof, which binds to a methylated dipeptide repeat protein (DRP) produced by repeat expansion mutation of the C9orf72 gene, wherein the antibody, or antigen binding portion thereof, comprises a heavy chain which is at least 90% identical to the heavy chain amino acid sequence set forth in SEQ ID NO: 9 or 23 and / or a light chain which is at least 90% identical to the light chain amino acid sequence set forth in SEQ ID NO: 10 or 24.

11. An isolated antibody, or antigen binding portion thereof, which binds to a methylated dipeptide repeat protein (DRP) produced by repeat expansion mutation of the C9orf72 gene, wherein the antibody, or antigen binding portion thereof, comprises heavy and light chains which are at least 90% identical to the heavy and light chain amino acid sequences respectively set forth in:(a) SEQ ID NOs: 9 and 10: or(b) SEQ ID NOs: 23 and 24.

12. An isolated monoclonal antibody, or antigen binding portion thereof, which binds to the same epitope as the antibody of any one of the preceding claims.

13. The antibody, or antigen binding portion thereof, of any one of the preceding claims, conjugated to a detectable label.

14. The antibody, or antigen binding portion thereof, of claim 13, wherein the detectable label is a radioactive label or a fluorescent label.

15. An isolated nucleic acid molecule encoding the heavy and / or light chain variable region of the antibody, or antigen binding portion thereof, of any one of claims 1 to 14.

16. The nucleic acid molecule of claim 15, comprising the nucleotide sequence set forth in SEQ ID NO: 11, 13, 25. or 26.Attorney Docket No. ALSE-016PC17. The nucleic acid molecule of claim 15 or 16, comprising the nucleotide sequence set forth in SEQ ID NO: 14, 15, 27, or 28.

18. An expression vector comprising the nucleic acid molecule of any one of claims 15 to 17.

19. A cell transformed with an expression vector of claim 18.

20. A composition comprising the antibody, or antigen binding portion thereof, of any one of claims 1 to 14 or the nucleic acid molecule of any one of claims 15 to 17, and one or more pharmaceutically acceptable excipient.

21. The composition of claim 20, wherein the one or more excipient is selected from a solvent, aqueous solvent, non-aqueous solvent, dispersion media, diluent, dispersion, suspension aid, surface active agent, isotonic agent, thickening or emulsifying agent, preservative, lipid, lipidoids liposome, lipid nanoparticle, core-shell nanoparticles, polymer, lipoplexes, peptide, protein, cell, hyaluronidase, or mixtures thereof.

22. A kit comprising the antibody, or antigen binding portion thereof, the nucleic acid molecule, or composition of any one of claims 1 to 17 or 20 to 21, and instructions for use.

23. A method of detecting a methylated DRP produced by repeat expansion mutation of the C9orf72 gene in a cell, comprising contacting the cell with the antibody, or antigen binding portion thereof, of any one of claims 1 to 14 and detecting the binding of the antibody, or antigen binding portion thereof, to the protein.

24. The method of claim 23, wherein the cell is a neuronal cell.

25. The method of claim 24, wherein the cell is a sensory neuron, a motor neuron, or an interneuron.Attorney Docket No. ALSE-016PC26. A method of diagnosing a disease associated with methylated DRPs produced by repeat expansion mutation of the C9orf72 gene in a subject, comprising administering the antibody, or antigen binding portion thereof, of any one of claims 1 to 14 and detecting the binding of the antibody, or antigen binding portion thereof, to the protein.

27. A method of monitoring the progress of a disease associated with methylated DRPs produced by repeat expansion mutation of the C9orf72 gene in a subject, comprising(a) administering to the subject the antibody, or antigen binding portion thereof, of any one of claims 1 to 14 at a first time point and obtaining an image of at least a portion of the subject to determine the amount of methylated DRPs expressed by the cells;(b) administering to the subject the antibody, or antigen binding portion thereof, at one or more subsequent time points and obtaining an image of at least a portion of the subject at each time point; wherein the amount of the methylated DRPs at each time point is indicative of the progress of the disease and the efficacy of the treatment.

28. A method of monitoring the progress of a disease associated with methylated DRPs produced by repeat expansion mutation of the C9orf72 gene in a subject, comprising(a) administering to the subject the antibody, or antigen binding portion thereof, of any one of claims 1 to 14 at a first time point and measuring the level of methylated DRPs or the activity associated with methylated DRPs in a sample from the subject;(b) administering to the subject the antibody, or antigen binding portion thereof, at one or more subsequent time points and measuring the level of methylated DRPs or the activity associated with methylated DRPs in a sample from the subject; wherein the level of methylated DRPs at each time point is indicative of the progress of the disease and the efficacy of the treatment.

29. The method of claim 28, wherein the sample is a biological fluid or tissue from the subject.

30. A method of decreasing toxicity in a subject caused by methylated DRPs produced by repeat expansion mutation of the C9orf72 gene in a subject, comprising administering an effective amount of the antibody, or antigen binding portion thereof, of any one of claims 1 toAttorney Docket No. ALSE-016PC14, the nucleic acid molecule of any one of claims 15 to 17, or the composition of claim 20 or 21.

31. The method of any one of 26 to 30, wherein the subject is diagnosed with a neurodegenerative disease.

32. The method of claim 31, wherein the subject is diagnosed with C9ORF72-linked Amyotrophic Lateral Sclerosis (ALS) or C9ORF72-linked frontotemporal dementia (FTD).

33. A method of treating a subject diagnosed with a disease associated with the expression of DRPs produced by repeat expansion mutation of the C9orf72 gene, comprising administering an effective amount of the antibody, or antigen binding portion thereof, of any one of claims 1 to 14, the nucleic acid molecule of any one of claims 15 to 17, or the composition of claim 20 or 21.

34. A method of treating a subject diagnosed with a neurodegenerative disorder, comprising administering an effective amount of the antibody, or antigen binding portion thereof, of any one of claims 1 to 14, the nucleic acid molecule of any one of claims 15 to 17, or the composition of claim 20 or 21.

35. The method of claim 32, wherein the disorder is ALS or FTD.

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