WO2025193741A1

WO2025193741A1 - 3e10 antibody nucleic acid conjugates

Info

Publication number: WO2025193741A1
Application number: PCT/US2025/019428
Authority: WO
Inventors: Robert Shaffer
Original assignee: Vrata Therapeutics Inc
Current assignee: Vrata Therapeutics Inc
Priority date: 2024-03-12
Filing date: 2025-03-11
Publication date: 2025-09-18
Anticipated expiration: 2026-09-12

Abstract

Antibody-drug conjugate (ADC) comprised of a humanized 3E10 monoclonal antibody (mAb) or derivative thereof conjugated to a nucleic acid payload through a heterobifunctional linker are provided. Methods of using the ADC for delivering nucleic acid pay loads to a variety of tissue are also provided.

Description

3E10 ANTIBODY NUCLEIC ACID CONJUGATES

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application number 63/564,461, filed March 12, 2024; and U.S. provisional application number 63/660,390, filed June 14, 2024, the contents of each of which are incorporated by reference herein in its entirety.

SEQUENCE LISTING

The content of the electronic sequence listing (V038770000WO00-SEQ-EMB.xml; Size: 77,859 bytes; and Date of Creation: March 11, 2025) is herein incorporated by reference in its entirety.

BACKGROUND OF INVENTION

The ability to deliver drugs to target tissue remains a significant obstacle to effective systemic drug delivery. Targeted antibodies have emerged as an effective tool in some instances for targeting agents to specific tissues. However, once the agent is delivered to a tissue, the drug must be taken up by the key cells and delivered to the proper intracellular compartment in order to generate a therapeutic response. These challenges continue to impede effective delivery of many therapeutic agents.

SUMMARY

In some aspects, the techniques described herein relate to an antibody-drug conjugate (ADC), including a humanized 3E10 monoclonal antibody (mAb) or derivative thereof conjugated to a nucleic acid payload through a non-cleavable heterobifunctional linker, wherein the humanized 3E10 mAb is conjugated to the linker through a chemical reaction of an N-terminal primary amine or the primary amine of a solvent-exposed lysine of the mAb and an N-hydroxysuccinimide functional group.

In some embodiments, the techniques described herein relate to a nucleic acid payload is conjugated to the linker through a click chemistry reaction, wherein an azide functional group is reacted with a bicyclo [6.1.0] nonyne functional group.

In some embodiments, the techniques described herein relate to a linker comprising an N-hydroxysuccinimide functional group, a bicyclo [6.1.0] nonyne functional group, and a polyethylene glycol (PEG)-based space or 1 or more repeating PEG units. In some embodiments, the techniques described herein relate to a conjugate comprising a humanized 3E10 monoclonal antibody (mAb) or derivative thereof conjugated to a nucleic acid pay load through cleavable linker, wherein the humanized 3E10 mAb is conjugated to the linker through a chemical reaction of an N-terminal primary amine or the primary amine of a solvent-exposed lysine of the mAb and an N-hydroxysuccinimide functional group and wherein the cleavable linker is cleavable by enzyme or reduction.

In some embodiments, the techniques described herein relate to a linker comprising an N-hydroxysuccinimide functional group, a bicyclo[6.1.0]nonyne functional group, and a poly-ethylene glycol (PEG)-based spacer of 1 or more repeating PEG units, and an internal reducible disulfide bond.

In some embodiments, the techniques described herein relate to a linker comprising an N-hydroxysuccinimide functional group, a bicyclo[6.1.0]nonyne functional group, and a poly-ethylene glycol (PEG)-based spacer of 1 or more repeating PEG units, and an internal valine-citrulline enzymatic cleavage site.

In some embodiments, the techniques described herein relate to a nucleic acid payload is a phosphorodiamidate morpholino oligomer, an antisense oligonucleotide, a short interfering RNA (siRNA), or a splice-switching oligonucleotide.

In some embodiments, the techniques described herein relate to a humanized 3E10 monoclonal antibody (mAb) or derivative thereof comprises a sequence having at least 80% sequence identity to a sequence selected from any one of SEQ ID NO. 1-6, 9-10, 20-21, 32- 38, 40-43, 49, 55-60, or 65-66.

In some embodiments, the techniques described herein relate to a humanized 3E10 mAb or derivative thereof comprises a sequence having at least 95% sequence identity to a sequence selected from any one of SEQ ID NO. 1-6, 9-10, 20-21, 32-38, 40-43, 49, 55-60, or 65-66.

In some embodiments, the techniques described herein relate to a humanized 3E10 mAb or derivative thereof comprises a sequence selected from any one of SEQ ID NO. 1-6, 9-10, 20-21, 32-38, 40-43, 49, 55-60, or 65-66.

In some embodiments, the techniques described herein relate to a humanized 3E10 mAb or derivative thereof that is an antigen-binding fragment of the humanized 3E10 mAb, optionally wherein the antigen-binding fragment is an scFv, a Fab, a single-chain antibody, or a nanobody.

In some embodiments, the techniques described herein relate to a nucleic acid payload that comprises up to 6 oligonucleotides linked to each humanized 3E10 mAb.

In some embodiments, the techniques described herein relate to a nucleic acid payload that acts on a nucleus-localized substrate of a cell.

In some embodiments, the techniques described herein relate to a nucleic acid payload that acts on a cytoplasm-localized substrate of a cell.

In some embodiments, the techniques described herein relate to a nucleus-localized substrate that is mRNA or pre-mRNA and the nucleic acid payload degrades the mRNA in the cell nucleus.

In some embodiments, the techniques described herein relate to a nucleus-localized substrate that is pre-mRNA and the nucleic acid payload modulates splicing of the pre- mRNA in the cell nucleus.

In some embodiments, the techniques described herein relate to a nucleic acid payload that is complementary to a nucleic acid of a cardiac cell, a skeletal muscle cell, a liver cell, a lung cell, and/or a kidney cell.

In some embodiments, the techniques described herein relate to a humanized 3E10 mAb that is a full-length antibody, which is optionally an IgG molecule.

In some aspects, the techniques described herein relate to an antibody-drug conjugate (ADC), comprising a humanized 3E10 monoclonal antibody (mAb) conjugated to a nucleic acid pay load, wherein the humanized 3E10 mAb comprises an amino acid sequence having at least 98% sequence identity to a sequence of SEQ ID NO. 65 or 66.

In some embodiments, the techniques described herein relate to a humanized 3E10 mAb that comprises an amino acid sequence of SEQ ID NO. 65 or 66.

In some embodiments, the techniques described herein relate to a humanized 3E10 mAb that comprises a heavy chain of SEQ ID NO. 65 and a light chain of SEQ ID NO. 66.

In some embodiments, the techniques described herein relate to a humanized 3E10 mAb that is conjugated to the nucleic acid payload by a cleavable or non-cleavable heterobifunctional linker.

In some embodiments, the techniques described herein relate to a nucleic acid payload that is a phosphorodiamidate morpholino oligomer, an antisense oligonucleotide, a short interfering RNA (siRNA), or a splice-switching oligonucleotide. In some embodiments, the techniques described herein relate to a humanized 3E10 mAb that is an IgG molecule.

In some aspects, the techniques described herein relate to an antibody-drug conjugate (ADC), comprising a humanized 3E10 monoclonal antibody (mAb) or derivative thereof conjugated to a nucleic acid payload through a cleavable or non-cleavable heterobifunctional linker, wherein the nucleic acid payload comprises at least 4-9, 4-8, 4-7, 4-6, 4-5, 5-9, 5-8, 5- 6, 5-7, 5-6, 6-7, 6-8, 6-9, 7-8, 7-9, or 8-9 oligonucleotides linked to each humanized 3E10 mAb.

In some aspects, the techniques described herein relate to an antibody-drug conjugate (ADC), comprising a humanized 3E10 antigen binding fragment conjugated to a nucleic acid payload through a cleavable or non-cleavable heterobifunctional linker, wherein the antigenbinding fragment is an scFv, a Fab, a single-chain antibody, or a nanobody.

In some embodiments, the techniques described herein relate to a nucleic acid payload that is conjugated to the linker through a click chemistry reaction, wherein an azide functional group is reacted with a bicyclo [6.1.0] nonyne functional group.

In some embodiments, the techniques described herein relate to a linker that comprises an N-hydroxysuccinimide functional group, a bicyclo [6.1.0] nonyne functional group, and a poly-ethylene glycol (PEG)-based spacer of 1 or more repeating PEG units.

In some embodiments, the techniques described herein relate to a nucleic acid payload that is a phosphorodiamidate morpholino oligomer, an antisense oligonucleotide, a short interfering RNA (siRNA), or a splice-switching oligonucleotide.

In some embodiments, the techniques described herein relate to a humanized 3E10 mAb or derivative thereof that is an antigen-binding fragment of the humanized 3E10 mAb, optionally wherein the antigen-binding fragment is an scFv, a Fab, a single-chain antibody, or a nanobody or wherein the humanized 3E10 mAb comprises an amino acid sequence having at least 98% sequence identity to a sequence of SEQ ID NO. 65 or 66. In some embodiments, the techniques described herein relate to a nucleic acid payload that comprises up to 6 oligonucleotides linked to each humanized 3E10 mAb.

In some aspects, the techniques described herein relate to a method for delivering a nucleic acid payload to a tissue, comprising: administering an antibody-drug conjugate (ADC), comprising a humanized 3E10 monoclonal antibody (mAb) or derivative thereof conjugated to a nucleic acid payload through a heterobifunctional linker to a subject to deliver the nucleic acid payload to a tissue, wherein the tissue is selected from cardiac, lung, kidney, liver, and skeletal muscle.

In some embodiments, the techniques described herein relate to a method of delivering any ADC provided herein.

In some embodiments, the techniques described herein relate to intravenous administration or subcutaneous administration.

In some embodiments, the techniques described herein relate to delivering any ADC provided herein to lung tissue or cardiac tissue. In some embodiments, where any ADC provided herein is delivered to cardiac tissue, the nucleic acid payload is a splice switching oligonucleotide.

Each of the limitations of the disclosure can encompass various embodiments of the disclosure. It is, therefore, anticipated that each of the limitations of the disclosure involving any one element or combinations of elements can be included in each aspect of the disclosure. This disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The disclosure is capable of other embodiments and of being practiced or of being carried out in various ways.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in various figures is represented by a like numeral. For purposes of clarity, not every component may be labeled in every drawing. In the drawings:

FIG. 1 is a schematic showing an exemplary reaction linking an antibody to a payload such as an oligonucleotide.

FIGs. 2A-2B show chromatograms depicting reaction products of an antibody-linker reacted with a 5’-BCN-oliognucleotide (2A) and reaction product arising from humanized 3E10 mAb reduced with DTT or TCEP and then reacted with a linker that contains a free SH group (2B).

FIG. 3 show results of an assay to demonstrate delivery of a phosphorodiamidate morpholino oligomer (PMO) sequence targeting mouse DMD intron/exon 23 boundary to inhibit retention of exon 23 during splicing, in the form of an antibody-oligonucleotide conjugate. The data is presented in bar graph form in FIG. 3, wherein the VEEmAb-PMO results are shown as the last column.

FIG. 4 shows results of an assay to demonstrate delivery of an antisense oligonucleotide (ASO) sequence targeting GYSI, in the form of an antibody-oligonucleotide conjugate. Untreated is shown as the first column at each time point. VEEmAb-ASO results are shown as the last column at each time point.

FIG. 5 shows results in the form of a bar graph of an in vivo assay to demonstrate systemic delivery of a humanized 3E10 monoclonal antibody (mAb) to the nuclei of cells of cardiac tissue. Cardiac tissue was stained following administration of the antibody to mice by intravenous injection. The data was compared with untreated control cardiac tissue and nucleus/VEEmAb co-staining was quantified to assess the extent of co-localization.

FIG. 6 shows results in the form of a bar graph of an in vivo assay to demonstrate systemic delivery of a humanized 3E10 mAb to the nuclei of cells of skeletal muscle tissue. Bicep muscle tissue was stained following administration of the antibody to mice by intravenous injection. The data was compared with untreated control muscle tissue and nucleus/VEEmAb co-staining was quantified to assess the extent of co-localization.

FIG. 7 shows results of an in vivo assay in a non-human primate to demonstrate systemic delivery of a humanized 3E10 mAb to multiple tissues, including brain, bicep, heart, quadricep, liver, spleen, lung, tongue, and gastric.

FIGs. 8 show results of an in vivo assay in mouse cardiac tissue to demonstrate the efficacy of humanized 3E10 mAb conjugated to a morpholino (“VEEmAb AOC”) to skip DMD exon 23 in Mdx mice (X-linked muscular dystrophy). “Untreated” are wild-type C57 untreated mice. “PMO” is phosphorodiamidate morpholino oligomers delivered to Mdx mice, without an antibody. VEEmAb AOC was administered at 10 mg/kg or 30 mg/kg.

FIG. 9 shows results of an in vitro assay in mouse muscle progenitor cells to demonstrate the efficacy of humanized 3E10 mAb conjugated to siRNA (“AOC”) to knockdown target mRNA in mouse cells. “Untreated” are untreated C2C12 mouse cells, “Positive control” are C2C12 mouse cells treated with 500 nM control siRNA, and “AOC Day 3” and “AOC Day 5” are C2C12 mouse cells treated with 500 nM siRNA- AOC 3 days and 5 days after treatment, respectively.

FIG. 10 shows results of an in vitro assay in HL-1 mouse cardiomyocytes to show the efficacy of labeled humanized 3E10 antibody conjugated to antisense oligonucleotide (“AF488-AOC”) to knockdown target mRNA. The data demonstrate that AF488-AOC was detectable and produced significant knockdown of target mRNA, relative to control. Untreated HL-1 mouse cells; unconjugated VEEmAb (“VEEmAb alone” -humanized 3E10 antibody without AOC); and AOC alone (“AOC” -oligonucleotide not linked to humanized 3E10 antibody) were used as controls.

FIG. 11 shows results of an in vitro assay in mouse muscle progenitor cells to demonstrate the efficacy of cleavable humanized 3E10 antibody (VEEmAb )- antisense oligonucleotide (ASO) conjugate (AOC) to knockdown target mRNA. The data depict target mRNA expression as fold change relative to untreated and are normalized to GAPDH. “Untreated” depict results from C2C12 mouse cells which have not been treated by VEEmAb or ASO; “Cleavable linker” depict results from C2C12 mouse cells treated with 300 nM VEEmAb linked to ASO by a cleavable linker; and “Non-cleavable linker” depict results from C2C12 mouse cells treated with 300 nM VEEmAb non-cleavable linker conjugated to antisense oligonucleotide.

FIG. 12 shows results of an assay to demonstrate delivery of a PMO sequence targeting mouse DMD intron/exon 23 boundary to inhibit retention of exon 23 during splicing, in the form of an antibody fragment-oligonucleotide conjugate. The data in the form of a picture of a gel is shown and demonstrate that 3E10 antibody fragments (VEEmAb Fab) conjugated to PMO effectively induces exon skipping in differentiated C2C12 mouse muscle progenitor cells.

FIG. 13 shows the ability to achieve a high drug antibody ratio (DAR) and related stability using humanized 3E10 antibody - PMO antibody oligonucleotide conjugates (VEEmAb-PMO AOC).

FIGs. 14A-14C show results of in situ detection of PMO in mouse heart tissue treated with humanized 3E10 - PMO antibody oligonucleotide conjugates (VEEmAb AOCs). FIGs. 14A and 14B show detection of PMO in MDX mice treated with VEEmAb AOCs. FIG. 14C is a control and shows detection in MDX mice that are not treated with VEEmAb AOCs.

DETAILED DESCRIPTION

Described herein are compositions (including pharmaceutical compositions) and methods for the design, preparation, manufacture and/or formulation of humanized 3E10 monoclonal antibodies and antigen binding fragments thereof linked to nucleic acids. Also provided are systems, processes, devices and kits for the selection, design and/or utilization of the antibody nucleic acid conjugates described herein. The present disclosure is based, at least in part, on the development of antibody nucleic acid conjugates, which possess unexpected superior features compared with nucleic acids delivered by other methods. For instance, the antibody nucleic acid conjugates disclosed herein may possess superior/unexpected features, for example, (a) delivering the nucleic acids to the nucleus of cells such that the nucleic acid is able to exert a physiological effect on substrate nucleic acids within the nucleus, (b) the resulting physiological effect is accelerated relative to nucleic acid delivery alone; and (c) achieves an extensive biodistribution to a variety of tissues following systemic administration, including to cardiac, liver, kidney, lung, and skeletal muscle.

Accordingly, provided herein are conjugates comprised of antibodies linked to nucleic acid payloads via heterobifunctional linkers, and uses thereof for therapeutic, research, and diagnostic purposes. Also provided herein are kits for therapeutic and/or diagnostic use of the antibodies, as well as methods for producing the conjugates.

The compositions disclosed herein have unique properties which enable the delivery of functional nucleic acids to a variety of tissues in vivo, such that therapeutic targets may be modified and diagnostic targets labeled. The compositions, which are equivalently referred to herein as antibody drug conjugates (ADC) or antibody-oligonucleotide conjugates (AOC) are comprised of a humanized 3E10 antibody or antigen binding fragments thereof linked to a nucleic acid cargo through a linker, preferably a heterobifunctional linker. The humanized 3E10 antibody or antigen binding fragment thereof is also equivalently referred to herein as humanized 3E10 antibody, hu3E10 antibody or 3E10Ab. In some embodiments the humanized 3E10 antibody is a humanized 3E10 monoclonal antibody (also sometimes referred to as 3E10mAb or VEEmAb) or an antibody fragment such as a Fab or scFv.

Once delivered to the tissue, the humanized 3E10 antibody of the ADC is capable of being internalized through equilibrative nucleoside transporter 2 (ENT2) such that the nucleic acid is delivered into the cell. Internalization of the nucleic acid in this manner has the benefit of being independent of endocytosis. Functioning through this mechanism the ADCs have been demonstrated to successfully be delivered in vivo to target tissues such that the nucleic acid moiety may achieve a therapeutic or diagnostic function and/or such that the target cells can be labeled, for instance for diagnostic purposes. As shown in the Examples, the ADCs when delivered to cells or tissues result in nucleic acid knock down, splice switching, etc.

The ADC disclosed have a unique biodistribution. It is disclosed herein that high levels of the ADC accumulate in tissues such as the heart and lung. This contrasts with prior art 3E10 antibodies, which have been identified as accumulating in and having high levels present in the liver of mice (see Rackear et al., Oncotarget, 2024, Vol. 15, pp: 699-713). The data presented in the Examples, such as in Figure 13 demonstrate detection of VEEmAb- PMO in mouse heart tissue by in situ hybridization.

Quite surprisingly it was also discovered that the ADC disclosed herein are capable of being conjugated to multiple nucleic acids to achieve drug-antibody ratios (DARs) greater than 2, and in some cases as high as 6 and above, for instance 9. This finding is surprising, at least in part, because the production of antibody conjugates with charged RNA species is challenging due to the charged species interfering with the antibodies and thus limiting the amount of RNA that can be attached to the antibodies. The ADC disclosed herein were able to overcome these limitations.

The surprising aspects and unique properties of compositions provided herein may be due, at least in part, to the specific heterobifunctional linkers that link a humanized 3E10 antibody or antigen binding fragments thereof to a nucleic acid cargo. Disclosed herein are heterobifunctional linkers that may be either non-cleavable or cleavable and which provide effective production of fully functional ADC. Due to the charge difference between highly positively charged antibodies and negatively charged nucleic acids, identifying linkers which are capable of retaining function of the antibody and nucleic acid, while also being produced effectively is unpredictable. Further, unexpectedly, it was discovered that the order of reactions for forming such a linker between a humanized 3E10 antibody or antigen binding fragment thereof linked to a nucleic acid cargo may be important to the success of producing such a molecule. In some embodiments the ADC production is significantly more effective when the linker is linked to the antibody first and then the linker- antibody is linked to the nucleic acid.

In some embodiments the surprising aspects and unique properties of compositions provided herein may be due, at least in part, to a humanized antibody of the disclosure comprising a heavy chain comprising: SEQ ID NO: 67 and a light chain comprising SEQ ID NO: 68 or fragments or variants thereof.

Antibodies

The term “antibody” (interchangeably used in plural form) refers to an immunoglobulin capable of specific binding to a target, such as a polypeptide, carbohydrate, nucleic acid, etc., through at least one antigen recognition site located in the variable region of the immunoglobulin molecule.

In some embodiments the antibody of the antibody conjugate is a humanized monoclonal antibody. In some embodiments the antibody is a humanized 3E10 monoclonal antibody. As used herein, the term “antibody” and/or “antibody or derivatives thereof’ encompasses not only intact (i.e., full-length) polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof. For instance, an antibody or derivative thereof includes but is not limited to Fab, Fab', F(ab')2, Fv), single chain (scFv), mutants thereof, fusion proteins comprising an antibody portion, humanized antibodies, chimeric antibodies, diabodies, nanobodies, linear antibodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies) and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.

An antibody includes an antibody of any class, such as IgD, IgE, IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant domain of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

In some embodiments, the antibody used in the methods described herein is a humanized antibody. Humanized antibodies refer to forms of non-human (e.g. murine) antibodies that are specific chimeric immunoglobulins, immunoglobulin chains, or antigenbinding fragments thereof that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, the humanized antibody may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, six) which are altered with respect to the original antibody, which are also termed one or more CDRs “derived from” one or more CDRs from the original antibody. Humanized antibodies may also involve affinity maturation.

The disclosure provides antibodies and antigen-binding fragments. In certain embodiments, these antibodies and antigen-binding fragments are humanized (e.g., comprise at least a humanized VH or a humanized VL). In certain embodiments, these antibodies and antigen-binding fragments bind DNA and/or transit cellular membranes, such as via an ENT transporter (e.g., an ENT2 and/or ENT3 transporter). In certain embodiments, antibodies and antigen-binding fragments of the disclosure bind DNA (e.g., single stranded DNA or double stranded DNA) with a KD of less than 100 nM, less than 75 nM, less than 50 nM, or less than 30 nM, as measured by SPR or QCM using currently standard protocols. In certain embodiments, antibodies and antigen-binding fragments of the disclosure bind DNA with a KD of less 20 nM, less than 10 nM, or less than 1 nM, as measured by SPR or QCM using currently standard protocols. In certain embodiments, the antibodies or antigen binding fragments of the disclosure compete for binding to DNA with 3E10, as produced by the hybridoma having ATCC accession No. PTA-2439 and as disclosed in U.S. Pat. No. 7,189,396. In some embodiments, the antibodies and antigen-binding fragments of the disclosure have properties consistent with the 3E10 parent antibody. In certain embodiments, antibodies and antigen binding fragments of the disclosure comprise Kabat CDRs that differ in comparison to murine 3E10 or variants thereof (e.g., comprise one or more changes in the CDRs, relative to murine 3E10, such as differing at one or more of VH CDR2, VL CDR1 and/or VL CDR2). In some embodiments, the antibody is a humanized 3E10 antibody or antigen binding fragment thereof as disclosed in U.S. Patent No. 10,221,250, which is hereby incorporated by reference for the disclosure of antibodies and antigen binding fragments thereof.

In some embodiments, the antibody or antigen-binding fragment comprises a humanized antibody or antigen-binding fragment, wherein the humanized antibody or antigen-binding fragment comprises a light chain variable (VL) domain and a heavy chain variable (VH) domain; wherein the VH domain is humanized and comprises: a VH CDR1 having the amino acid sequence of SEQ ID NO: 1; a VH CDR2 having the amino acid sequence of SEQ ID NO: 2; and a VH CDR3 having the amino acid sequence of SEQ ID NO: 3; and the VL is humanized and comprises: a VL CDR1 having the amino acid sequence of SEQ ID NO: 4; a VL CDR2 having the amino acid sequence of YAS; and a VL CDR3 having the amino acid sequence of SEQ ID NO: 6; which CDRs are in accordance with the IMGT system.

In some embodiments, a humanized antibody or antigen binding fragment of the disclosure comprises a VH domain and a VL domain comprising: a VH CDR1 having the amino acid sequence of SEQ ID NO: 32; a VH CDR2 having the amino acid sequence of SEQ ID NO: 33; and a VH CDR3 having the amino acid sequence of SEQ ID NO: 34, which CDRs are according to Kabat; and a VL CDR1 having the amino acid sequence of SEQ ID NO: 4; a VL CDR2 having the amino acid sequence of YAS; and a VL CDR3 having the amino acid sequence of SEQ ID NO: 6, which CDRs are according to the IMGT system.

In some embodiments, a humanized antibody or antigen binding fragment of the disclosure comprises a VH domain and a VL domain comprising: a VH CDR1 having the amino acid sequence of SEQ ID NO: 1; a VH CDR2 having the amino acid sequence of SEQ ID NO: 2; and a VH CDR3 having the amino acid sequence of SEQ ID NO: 3, which CDRs are according to the IMGT system, and a VL CDR1 having the amino acid sequence of SEQ ID NO: 35; a VL CDR2 having the amino acid sequence of SEQ ID NO: 36; and a VL CDR3 having the amino acid sequence of SEQ ID NO: 37, which CDRs are according to Kabat.

In some embodiments, an asparagine is mutated to another amino acid residue in the VH or VL domains in order to reduce N-linked glycosylation of the humanized antibody or antibody fragment.

In some embodiments, a humanized antibody or antigen binding fragment of the disclosure comprises a VH domain and a VL domain comprising: a VH CDR1 having the amino acid sequence of any one of SEQ ID NO: 1; or 32 a VH CDR2 having the amino acid sequence of any one of SEQ ID NO: 2; 33, or 49 and a VH CDR3 having the amino acid sequence of any one of SEQ ID NO: 3; or 34 and the VL is humanized and comprises: a VL CDR1 having the amino acid sequence of any one of SEQ ID NO: 4; 35, or 50 a VL CDR2 having the amino acid sequence of any one of YAS; 36, or 51 and a VL CDR3 having the amino acid sequence of any one of SEQ ID NO: 6; or 37 which CDRs are in accordance with the IMGT system.

In some embodiments, a humanized antibody or antigen binding fragment of the disclosure comprises a light chain variable domain (hVL2) comprising: SEQ ID NO. 8.

In some embodiments, a humanized antibody or antigen binding fragment of the disclosure comprises a heavy chain variable domain (hVH2) comprising: SEQ ID NO. 10 or 39.

In some embodiments, a humanized antibody or antigen binding fragment of the disclosure comprises a light chain variable domain (hVLl) comprising: SEQ ID NO. 40.

In some embodiments, a humanized antibody or antigen binding fragment of the disclosure comprises a heavy chain variable domain (hVHl) comprising: SEQ ID NO. 38.

In some embodiments, a humanized antibody or antigen binding fragment of the disclosure comprises a light chain comprising: SEQ ID NO. 41.

In some embodiments, a humanized antibody or antigen binding fragment of the disclosure comprises a heavy chain comprising: SEQ ID NO. 42.

In some embodiments, a humanized antibody or antigen binding fragment of the disclosure comprises: SEQ ID NO. 43.

In some embodiments, a humanized antibody of the disclosure comprises a heavy chain having at least 80% sequence identity to a sequence comprising: SEQ ID NO: 65. In some embodiments, a humanized antibody of the disclosure comprises a heavy chain having at least 81% sequence identity to a sequence comprising: SEQ ID NO: 65. In some embodiments, a humanized antibody of the disclosure comprises a heavy chain having at least 82% sequence identity to a sequence comprising: SEQ ID NO: 65. In some embodiments, a humanized antibody of the disclosure comprises a heavy chain having at least 83% sequence identity to a sequence comprising: SEQ ID NO: 65. In some embodiments, a humanized antibody of the disclosure comprises a heavy chain having at least 84% sequence identity to a sequence comprising: SEQ ID NO: 65. In some embodiments, a humanized antibody of the disclosure comprises a heavy chain having at least 85% sequence identity to a sequence comprising: SEQ ID NO: 65. In some embodiments, a humanized antibody of the disclosure comprises a heavy chain having at least 86% sequence identity to a sequence comprising: SEQ ID NO: 65. In some embodiments, a humanized antibody of the disclosure comprises a heavy chain having at least 87% sequence identity to a sequence comprising: SEQ ID NO: 65. In some embodiments, a humanized antibody of the disclosure comprises a heavy chain having at least 88% sequence identity to a sequence comprising: SEQ ID NO: 65. In some embodiments, a humanized antibody of the disclosure comprises a heavy chain having at least 89% sequence identity to a sequence comprising: SEQ ID NO: 65. In some embodiments, a humanized antibody of the disclosure comprises a heavy chain having at least 90% sequence identity to a sequence comprising: SEQ ID NO: 65. In some embodiments, a humanized antibody of the disclosure comprises a heavy chain having at least 91% sequence identity to a sequence comprising: SEQ ID NO: 65. In some embodiments, a humanized antibody of the disclosure comprises a heavy chain having at least 92% sequence identity to a sequence comprising: SEQ ID NO: 65. In some embodiments, a humanized antibody of the disclosure comprises a heavy chain having at least 93% sequence identity to a sequence comprising: SEQ ID NO: 65. In some embodiments, a humanized antibody of the disclosure comprises a heavy chain having at least 94% sequence identity to a sequence comprising: SEQ ID NO: 65. In some embodiments, a humanized antibody of the disclosure comprises a heavy chain having at least 95% sequence identity to a sequence comprising: SEQ ID NO: 65. In some embodiments, a humanized antibody of the disclosure comprises a heavy chain having at least 96% sequence identity to a sequence comprising: SEQ ID NO: 65. In some embodiments, a humanized antibody of the disclosure comprises a heavy chain having at least 97% sequence identity to a sequence comprising: SEQ ID NO: 65. In some embodiments, a humanized antibody of the disclosure comprises a heavy chain having at least 98% sequence identity to a sequence comprising: SEQ ID NO: 65. In some embodiments, a humanized antibody of the disclosure comprises a heavy chain having at least 99% sequence identity to a sequence comprising: SEQ ID NO: 65. In some embodiments, a humanized antibody of the disclosure comprises a heavy chain having 100% sequence identity to a sequence comprising: SEQ ID NO: 65. In some embodiments the percent sequence identity referred to herein refers to the percent sequence identity in regions of the heavy chain outside of the CDRs. In some embodiments the percent sequence identity referred to herein refers to the percent sequence identity in regions of the heavy chain in the CDRs. In some embodiments the percent sequence identity referred to herein refers to the percent sequence identity over the full length of the heavy chain, both outside of the CDRs and in the CDRs.

In some embodiments, a humanized antibody of the disclosure comprises a light chain having at least 80% sequence identity to a sequence comprising: SEQ ID NO: 66. In some embodiments, a humanized antibody of the disclosure comprises a light chain having at least 81% sequence identity to a sequence comprising: SEQ ID NO: 66. In some embodiments, a humanized antibody of the disclosure comprises a light chain having at least 82% sequence identity to a sequence comprising: SEQ ID NO: 66. In some embodiments, a humanized antibody of the disclosure comprises a light chain having at least 83% sequence identity to a sequence comprising: SEQ ID NO: 66. In some embodiments, a humanized antibody of the disclosure comprises a light chain having at least 84% sequence identity to a sequence comprising: SEQ ID NO: 66. In some embodiments, a humanized antibody of the disclosure comprises a light chain having at least 85% sequence identity to a sequence comprising: SEQ ID NO: 66. In some embodiments, a humanized antibody of the disclosure comprises a light chain having at least 86% sequence identity to a sequence comprising: SEQ ID NO: 66. In some embodiments, a humanized antibody of the disclosure comprises a light chain having at least 87% sequence identity to a sequence comprising: SEQ ID NO: 66. In some embodiments, a humanized antibody of the disclosure comprises a light chain having at least 88% sequence identity to a sequence comprising: SEQ ID NO: 66. In some embodiments, a humanized antibody of the disclosure comprises a light chain having at least 89% sequence identity to a sequence comprising: SEQ ID NO: 66. In some embodiments, a humanized antibody of the disclosure comprises a light chain having at least 90% sequence identity to a sequence comprising: SEQ ID NO: 66. In some embodiments, a humanized antibody of the disclosure comprises a light chain having at least 91% sequence identity to a sequence comprising: SEQ ID NO: 66. In some embodiments, a humanized antibody of the disclosure comprises a light chain having at least 92% sequence identity to a sequence comprising: SEQ ID NO: 66. In some embodiments, a humanized antibody of the disclosure comprises a light chain having at least 93% sequence identity to a sequence comprising: SEQ ID NO: 66. In some embodiments, a humanized antibody of the disclosure comprises a light chain having at least 94% sequence identity to a sequence comprising: SEQ ID NO: 66. In some embodiments, a humanized antibody of the disclosure comprises a light chain having at least 95% sequence identity to a sequence comprising: SEQ ID NO: 66. In some embodiments, a humanized antibody of the disclosure comprises a light chain having at least 96% sequence identity to a sequence comprising: SEQ ID NO: 66. In some embodiments, a humanized antibody of the disclosure comprises a light chain having at least 97% sequence identity to a sequence comprising: SEQ ID NO: 66. In some embodiments, a humanized antibody of the disclosure comprises a light chain having at least 98% sequence identity to a sequence comprising: SEQ ID NO: 66. In some embodiments, a humanized antibody of the disclosure comprises a light chain having at least 99% sequence identity to a sequence comprising: SEQ ID NO: 66. In some embodiments, a humanized antibody of the disclosure comprises a light chain having 100% sequence identity to a sequence comprising: SEQ ID NO: 66. In some embodiments the percent sequence identity referred to herein refers to the percent sequence identity in regions of the light chain outside of the CDRs. In some embodiments the percent sequence identity referred to herein refers to the percent sequence identity in regions of the light chain in the CDRs. In some embodiments the percent sequence identity referred to herein refers to the percent sequence identity over the full length of the light chain, both outside of the CDRs and in the CDRs.

In some embodiments a humanized antibody of the disclosure comprises a heavy chain comprising: SEQ ID NO: 65 and a light chain comprising SEQ ID NO: 66. In some embodiments a humanized antibody of the disclosure comprises a substituted heavy chain comprising: SEQ ID NO: 65 and/or a substituted light chain comprising SEQ ID NO: 66, wherein the heavy chain has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids different from SEQ ID NO: 65 and/or wherein the light chain has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids different from SEQ ID NO: 66. In some embodiments, the substituted heavy chain comprises a CDR1 having a sequence of SEQ ID NO: 1 or 31, a CDR2 having a sequence of SEQ ID NO: 2, 33, or 49, and/or a CDR3 having a sequence of SEQ ID NO: 3 or 34. In some embodiments, the substituted light chain comprises a CDR1 having a sequence of SEQ ID NO: 4, 35 or 50, a CDR2 having a sequence of SEQ ID NO: 36 or 51 or a sequence of YAS, and/or a CDR3 having a sequence of SEQ ID NO: 6 or 37. In some embodiments, an asparagine is mutated to another amino acid residue in the VH or VL domains in order to reduce N-linked glycosylation of the humanized antibody or antibody fragment.

A typical antibody molecule comprises a heavy chain variable region (VH) and a light chain variable region (VL), which are usually involved in antigen binding. The VH and VL regions can be further subdivided into regions of hypervariability, also known as “complementarity determining regions” (“CDR”), interspersed with regions that are more conserved, which are known as “framework regions” (“FR”). Each VH and VL is typically 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. Thus, an antibody variable region consists of a “framework” region interrupted by three “antigen binding sites”. The antigen binding sites are defined using various terms: (i) Complementarity Determining Regions (CDRs), three in the VH (HCDR1, HCDR2, HCDR3), and three in the VL (LCDR1, LCDR2, LCDR3), are based on sequence variability; (ii) “Hypervariable regions,” “HVR,” or “HV,” three in the VH (Hl, H2, H3) and three in the VL (LI, L2, L3), refer to the regions of an antibody variable domains which are hypervariable in structure as defined by Chothia and Lesk. “Framework” or “framework sequences” are the remaining sequences of a variable region other than those defined to be antigen binding sites. Because the antigen binding sites can be defined by various terms as described above, the exact amino acid sequence of a framework depends on how the antigen-binding site was defined.

The antibodies described herein can be murine, rat, human, primate, porcine, or any other origin (including chimeric or humanized antibodies). Such antibodies are non-naturally occurring, i.e., would not be produced in an animal without human act (e.g., immunizing such an animal with a desired antigen or fragment thereof). In preferred embodiments the antibodies are humanized monoclonal antibodies. A “monoclonal antibody” refers to a homogenous antibody population.

In another example, the antibody described herein is a chimeric antibody, which can include a heavy constant region and a light constant region from a human antibody. Chimeric antibodies refer to antibodies having a variable region or part of variable region from a first species and a constant region from a second species. Typically, in these chimeric antibodies, the variable region of both light and heavy chains mimics the variable regions of antibodies derived from one species of mammals (e.g., a non-human mammal such as mouse, rabbit, and rat), while the constant portions are homologous to the sequences in antibodies derived from another mammal such as human. In some embodiments, amino acid modifications can be made in the variable region and/or the constant region.

Antibody heavy and light chain constant regions are well known in the art, e.g., those provided in the IMGT database (imgt.org) or at vbase2.org/vbstat.php., both of which are incorporated by reference herein.

In some embodiments the antibody is an antigen binding fragment. As used herein, the term “antigen binding fragment” refers to an antibody fragment such as, for example, a diabody, a Fab, a Fab', a F(ab')2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv) 2, a bispecific dsFv (dsFv-dsFv'), a disulfide stabilized diabody (ds diabody), a singlechain antibody molecule (scFv), a single domain antibody (sdab) an scFv dimer (bivalent diabody), a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a camelized single domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not comprise a complete antibody structure. An antigen-binding fragment is capable of binding to the same antigen to which the parent antibody or a parent antibody fragment binds.

According to particular embodiments, the antigen-binding fragment comprises a light chain variable region, a light chain constant region, and an Fd segment (i.e., portion of the heavy chain which is included in the Fab fragment). According to other particular embodiments, the antigen-binding fragment comprises Fab and F(ab'). A humanized antigen binding fragment is an antigen binding fragment that includes one or more of the human CDRs, optionally, the CDRs disclosed herein.

In yet another example, the antibody described herein can be a single-domain antibody, which interacts with the target antigen via only one single variable domain such as a single heavy chain domain (as opposed to traditional antibodies, which interact with the target antigen via heavy chain and light chain variable domains). A single-domain antibody can be a heavy-chain antibody (VHH) which contains only an antibody heavy chain and is devoid of light chain. In additional to a variable region (for example, a VH), a single-domain antibody may further comprise a constant region, for example, CHi, CH2, CH3, CFU, or a combination thereof. In some embodiments the antibodies are humanized single domain antibodies.

In some embodiments, the antibodies and antigen binding fragments thereof comprise a fragment crystallizable (Fc) region. The Fc region is the tail region of an antibody and antigen binding fragments thereof which contains constant domains (e.g., CH2 and CH3); the other region of the antibodies and antigen binding fragments thereof being the Fab region which contains a variable domain (e.g., VH) and a constant domain (e.g., CHi), the former of which defines binding specificity.

As described herein, antibodies can comprise a VH domain. In some embodiments, the VH domain further comprises one or more constant domains (e.g., CH2 and/or CH3) of an Fc region and/or one or more constant domains (e.g., CHi) of a Fab region. In some embodiments, each of the one or more constant domains (e.g., CHi, CH2, and/or CH3) can comprise or consist of portions of a constant domain. For example, in some embodiments, the constant domain comprises 99% or less, 98% or less, 97% or less, 96% or less, 95% or less, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less of the corresponding full sequence.

In some embodiments, a humanized 3E10 Fab of the disclosure comprises a heavy chain having at least 80% sequence identity to a sequence comprising: SEQ ID NO: 67. In some embodiments, a humanized 3E10 Fab of the disclosure comprises a heavy chain having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or has 100% sequence identity to a sequence comprising: SEQ ID NO: 67. In some embodiments the percent sequence identity referred to herein refers to the percent sequence identity in regions of the heavy chain outside of the CDRs. In some embodiments the percent sequence identity referred to herein refers to the percent sequence identity in regions of the heavy chain in the CDRs. In some embodiments the percent sequence identity referred to herein refers to the percent sequence identity over the full length of the heavy chain, both outside of the CDRs and in the CDRs.

In some embodiments, a humanized 3E10 Fab of the disclosure comprises a light chain having at least 80% sequence identity to a sequence comprising: SEQ ID NO: 68. In some embodiments, a humanized 3E10 Fab of the disclosure comprises a light chain having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or has 100% sequence identity to a sequence comprising: SEQ ID NO: 68. In some embodiments the percent sequence identity referred to herein refers to the percent sequence identity in regions of the light chain outside of the CDRs. In some embodiments the percent sequence identity referred to herein refers to the percent sequence identity in regions of the light chain in the CDRs. In some embodiments the percent sequence identity referred to herein refers to the percent sequence identity over the full length of the light chain, both outside of the CDRs and in the CDRs.

In some embodiments a humanized 3E10 Fab of the disclosure comprises a heavy chain comprising: SEQ ID NO: 67 and a light chain comprising SEQ ID NO: 68. In some embodiments a humanized 3E10 Fab of the disclosure comprises a substituted heavy chain comprising: SEQ ID NO: 67 and/or a substituted light chain comprising SEQ ID NO: 68, wherein the heavy chain has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids different from SEQ ID NO: 67 and/or wherein the light chain has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids different from SEQ ID NO: 68. In some embodiments, the substituted heavy chain comprises a CDR1 having a sequence of SEQ ID NO: 1 or 31, a CDR2 having a sequence of SEQ ID NO: 2, 33, or 49, and/or a CDR3 having a sequence of SEQ ID NO: 3 or 34. In some embodiments, the substituted light chain comprises a CDR1 having a sequence of SEQ ID NO: 4, 35 or 50, a CDR2 having a sequence of SEQ ID NO: 36 or 51 or a sequence of YAS, and/or a CDR3 having a sequence of SEQ ID NO: 6 or 37.

Alternatively, an antibody described herein may comprise up to 5 (e.g., 4, 3, 2, or 1) amino acid residue variations in one or more of the CDR regions of one of the antibodies exemplified herein and binds the same epitope of antigen with substantially similar affinity (e.g., having a KD value in the same order). In one example, the amino acid residue variations are conservative amino acid residue substitutions. As used herein, a “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

In some embodiments the antibody is conjugated to a nucleic acid through a heterobifunctional linker. Such compounds are referred to herein, interchangeably, as “conjugates”, “antibody conjugates”, “antibody drug conjugates (ADC)”, “antibody nucleic acid (or oligonucleotide) conjugates” or “antibody -linker-nucleic acid (or oligonucleotide) conjugates”. Each of these conjugates is comprised of a humanized 3E10 monoclonal antibody or derivative thereof, a heterobifunctional linker and a payload (preferably a nucleic acid pay load) for targeted drug delivery. Any of the antibodies or portions thereof described herein conjugated via linker with any nucleic acid payload with any feasible technology/chemistry at any feasible site of antibody molecule is a conjugate of the disclosure.

In further embodiments, the antibodies may include modifications to improve properties of the antibody, for example, stability, oxidation, isomerization and deamidation. In some embodiments, the antibody disclosed herein may comprise heavy chain CDRs that collectively are at least 80% (e.g., 85%, 90%, 95%, or 98%) identical to the heavy chain CDRs of a reference antibody such as humanized 3E10 monoclonal antibody. Alternatively or in addition, the antibody may comprise light chain CDRs that collectively are at least 80% (e.g., 85%, 90%, 95%, or 98%) identical to the light chain CDRs of the reference antibody. In some embodiments, the antibody may comprise a heavy chain variable region that is at least 80% (e.g., 85%, 90%, 95%, or 98%) identical to the heavy chain variable region of a reference antibody such as humanized 3E10 monoclonal antibody and/or a light chain variable region that is at least 80% (e.g., 85%, 90%, 95%, or 98%) identical to the light chain variable region of the reference antibody.

The “percent identity” of two amino acid sequences may be determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990. 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 interest. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

The present disclosure also provides germlined variants of any of the exemplary humanized 3E10 monoclonal antibodies disclosed herein. A germlined variant contains one or more mutations in the framework regions as relative to its parent antibody towards the corresponding germline sequence. To make a germline variant, the heavy or light chain variable region sequence of the parent antibody or a portion thereof (e.g., a framework sequence) can be used as a query against an antibody germline sequence database (e.g., bioinfo.org.uk/abs/, vbase2.org, or imgt.org) to identify the corresponding germline sequence used by the parent antibody and amino acid residue variations in one or more of the framework regions between the germline sequence and the parent antibody. One or more amino acid substitutions can then be introduced into the parent antibody based on the germline sequence to produce a germlined variant.

The antibodies are referred to as synthetic or isolated. The phrase “isolated antibody or antibody fragment” refers to an antibody or antibody fragment that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody specifically binding a target antigen is substantially free of antibodies that specifically do not bind the target antigen). Moreover, an isolated antibody or antibody fragment can be substantially free of other cellular material and/or chemicals. Isolated antibodies according to embodiment of the invention can be synthetic. A synthetic antibody is an antibody that is not naturally occurring. The antibodies, while derived from human immunoglobulin sequences, can be generated using systems such as phage display incorporating synthetic CDRs and/or synthetic frameworks, or can be subjected to in vitro mutagenesis to improve antibody properties, resulting in antibodies that do not naturally exist within the human antibody germline repertoire in vivo.

Linkers

In some embodiments, the invention relates to an antibody conjugated to an active moiety such as a nucleic acid payload through a linker. The conjugates described herein may include a heterobifunctional linker.

As used herein the term “conjugated” refers to the joining or connection of two or more objects together. When referring to chemical or biological compounds, conjugated can refer to a covalent connection between the two or more chemical or biological compounds. By way of a non-limiting example, an antibody can be conjugated with a compound such as a nucleic acid to form an antibody conjugated nucleic acid. In certain embodiments the pharmaceutically active moiety can be linked directly to the antibody or in other embodiments it may be covalently coupled with the antibody through a linker. The linker can be modified chemically to allow for the conjugation of the antibody to the pharmaceutically active moiety.

In some embodiments the antibody-oligonucleotide conjugate includes a heterobifunctional non-cleavable linker. A heterobifunctional linker is a molecule containing two different functional groups at each end, enabling the selective conjugation of two different biomolecules or chemical entities. These linkers serve as intermediates for connecting various molecules in bioconjugation. The two functional groups typically have orthogonal reactivities, meaning they can react selectively with distinct reactive groups under specific conditions. This ideally allows for the conjugation of antibodies with nucleic acid pay loads.

A non-cleavable heterobifunctional linker contains two different functional groups at each end and also contain bonds that can not be easily broken under physiological conditions (non-cleavable). These linkers are designed to provide stable connections between biomolecules or chemical entities without undergoing spontaneous cleavage. Non-cleavable linkers are particularly useful when the goal is to maintain the integrity of the conjugate over an extended period or under various physiological conditions. Thus, a non-cleavable heterobifunctional linker, as used herein comprises two different functional groups, one at each end of a core region, wherein the two different functional groups form a non-cleavable bond with the linked compound (antibody or nucleic acid pay load). Examples of functional groups of non-cleavable heterobifunctional linkers include: NHS ester (NHS (N- hydroxysuccinimide) group reacts specifically with primary amines (e.g., lysine residues on proteins) to form stable amide bonds), Maleimide (maleimide group reacts with thiol (-SH) groups to form stable thioether linkages), Azide group ((-N3) reacts via copper-free or copper-catalyzed azide-alkyne cycloaddition (CuAAC) with alkyne groups (-C=CH) to form stable 1,2,3-triazole linkages), Alkyne group ((-C=CH) reacts via copper- free or CuAAC with azide groups to form stable 1,2,3-triazole linkages). In some embodiments a non-cleavable heterobifunctional linker comprises an NHS ester functional group that covalently attaches to primary amines on antibodies (e.g., lysine residues), a spacer core of repeating units such as PEG1, PEG2, PEG3, PEG4, PEG5, PEG6[...] PEG20, and an azide functional group that covalently attaches to nucleic acids via click chemistry. In some embodiments, a non- cleavable heterobifunctional linker comprises a maleimide group that covalently attaches to thiols on antibodies (e.g., cysteine residues), a spacer core of repeating units such as PEG1, PEG2, PEG3, PEG4, PEG5, PEG6[...] PEG20 and an azide functional group that covalently attaches to nucleic acids via click chemistry. In some embodiments, a non-cleavable heterobifunctional linker comprises a succinimidyl ester group that covalently attaches to a primary amine on antibodies (e.g., lysine residues), a spacer core of repeating units such as PEG1, PEG2, PEG3, PEG4, PEG5, PEG6[...] PEG20 and a maleimide group that covalently attaches to thiols on nucleic acids.

In some embodiments the linker is a cleavable heterobifunctional linker. Cleavable heterobifunctional linkers are designed to connect two different entities with reversible bonds that can be selectively cleaved under specific conditions, such as changes in pH or other conditions, enzymatic activity, or the presence of certain reagents. These linkers allow for controlled release of the connected molecules, making them particularly useful in drug delivery systems where controlled release is desired. Thus, a cleavable heterobifunctional linker, as used herein comprises two different functional groups for covalent conjugation at each end of a spacer core region, with an additional functional group in the core region that enables release of the linked compound (antibody or nucleic acid pay load). Some examples of spacer core region functional groups (optionally referred to herein as a cleavage sensitive moieties) of cleavable heterobifunctional linkers include enzyme or protease sensitive linkers, linkers cleavable by reduction, esterase-cleavable linkers, and pho tocleav able linkers. An enzyme cleavable linker can be used to connect the antibody to the nucleic acid payload with a bond that can be selectively cleaved by specific enzymes. Similarly protease-cleavable linkers are designed to be cleaved by proteolytic enzymes, such as serine proteases, cysteine proteases, metalloproteases, and aspartic proteases, which selectively hydrolyze peptide bonds. An exemplary linker may be a reducible 2,2'-dithiodipyridine disulfide bond or a PEG-based spacer of 1 or more repeating PEG units, and a valine-citrulline enzymatic cleavage site. An advantage of including a cleavage sensitive moiety in a linker is that the moiety may be designed for selective release at a desired site. For instance a valine-citrulline enzymatic cleavage site is stable in human plasma and will remained intact until it is delivered to the cells. Another exemplary linker comprises azidoethyl- SS -propionic NHS ester: . In some embodiments a cleavable heterobifunctional linker comprises an azidoethyl- SS -propionic NHS ester -core- azidoethyl- SS -propionic NHS ester.

The linker also includes a core that connects the two different functional groups. In some embodiments the core can be, for example, a chemical linker core, a polyethylene glycol (PEG) linker core, a polypropylene glycol (PPG) linker core, a hybrid linker core consisting of PEG and a chemical linker core. The size of the core can vary. For instance, a core comprised of PEG linkers can comprise, for example, 1-24 PEG units. In some embodiments the PEG linker core comprises 1-10, 1-20, 2-10, 3-10, 4-10, 5-10, 6-10, 7-10, 8- 10, 9-10, 3-15, 4-15, 5-15, 6-15, 7-15, 8-15, 9-15, 10-15, 11-15, 8-12, Or 9-11 PEG units. In some embodiments the PEG linker core comprises 10 PEG units.

The linkers can be conjugated to the antibody and nucleic acid payloads through a variety of reactions. In some embodiments a random amine coupling is used to attach the linker to the antibody. For instance, primary amines of the antibody can be reacted with N- hydroxysuccinimide functional groups of the linker. A schematic of the reaction is shown in FIG. 1. In the Figure, random amine coupling is used to attach the linker to the antibody. Primary amines of the antibody were reacted with N-hydroxysuccinimide functional groups of the linker. In a separate reaction the Ab-linker conjugate was further conjugated to the nucleic acid payload via click chemistry. For instance, azide functional groups of the oligonucleotide are reacted with bicyclo [6.1.0] nonyne functional groups of the linker.

In some embodiments the linker can be attached to the nucleic acid payload or antibody using click chemistry. Click chemistry involves a set of highly selective, and modular reactions that are used to rapidly construct molecular entities. One of the most prominent reactions in click chemistry is the copper-catalyzed azide-alkyne cycloaddition (CuAAC), also known as the Huisgen cycloaddition.

For example an antibody may be modified with functional groups that can participate in click chemistry. For example, the antibody can be modified to introduce azide or alkyne groups. These modifications can be achieved through various methods, including chemical synthesis or enzymatic approaches. The linker to be conjugated with the antibody is also modified to contain the complementary functional group. Under optimized conditions, the azide and alkyne groups react selectively to form a stable triazole linkage, resulting in the conjugation to the antibody. Similarly, click chemistry can be used to conjugate nucleic acids. An azide or an alkyne group can be included in a nucleic acid during solid-phase synthesis. These modified nucleotides can then undergo CuAAC with the linker bearing the complementary reactive group.

The conjugate may also include a spacer unit. A spacer links the antibody to the drug, with an optional linker and stretcher. Spacer units typically are of two general types: self- immolative and non self-immolative. A non self-immolative spacer unit is one in which part or all of the spacer unit remains bound to the drug after enzymatic cleavage of the antibodydrug conjugate. Examples of a non self-immolative spacer unit include, but are not limited to a (glycine-glycine) spacer unit and a glycine spacer unit. To release the drug, an independent hydrolysis reaction may take place within the target cell to cleave the glycine-drug unit bond. In some embodiments, a non self-immolative the spacer is Gly. Alternatively, a conjugate contains a self-immolative spacer that can release the drug without the need for a separate hydrolysis step. In these embodiments, the spacer may be substituted and unsubstituted 4- aminobutyric acid amides, appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems and 2- aminophenylpropionic acid amides. Thus, the conjugate has as its most basic structure an Ab-Nucleic acid. Optionally the structure is Ab-linker-Nucleic acid; Ab-linker- spacer-Nucleic acid; or Ab-linker-spacer-stretcher-Nucleic acid.

Payload

In some embodiments the antibody is conjugated to a payload. An exemplary payload for use in the conjugates of the invention is a nucleic acid payload. A nucleic acid or oligonucleotide payload may be a therapeutic nucleic acid that acts on a target nucleic acid substrate. An example of a nucleic acid payload is an inhibitory oligonucleotide such as an antisense DNA oligonucleotide, an antisense RNA-DNA-RNA (gapmer) oligonucleotide, a short interfering RNA (siRNA), an aptamer, a short hairpin RNA (shRNA), a DNAzyme, or an aptazyme. Inhibitory oligonucleotides may interact with a target nucleic acid in a nucleus, disrupting the ability of the target nucleic acid to be translated into a protein. Inhibitory oligonucleotides may also interact with a target nucleic acid in a cytoplasm. Interacting with a target nucleic acid in a nucleus or in a cytoplasm may, in some embodiments, promote degradation of the target nucleic acid, inhibit the ability of a target nucleic acid to be translated into another molecule (e.g., protein, pre-mRNA, etc.), inhibit the ability of a target nucleic acid to be bound by a protein (e.g., ribosome), inhibit the ability of a target nucleic acid to bind to another nucleic acid, or some combination thereof.

An antisense nucleic acid for instance, in some embodiments, may have a length of about 3 to about 30 nucleotides and may contain one or more modifications to improve characteristics such as stability in the serum, in a cell or in a place where the compound is likely to be delivered.

In some embodiments the nucleic acid payload is a splice switching oligonucleotide. Splice- switching or splice modulating oligonucleotides direct pre-mRNA splicing by binding sequence elements and blocking access to the transcript by the spliceosome and other splicing factors. They can be applied to (1) restore correct splicing of an aberrantly spliced transcript, (2) produce a novel splice variant that is not normally expressed, or (3) manipulate alternative splicing from one splice variant to another. Through the latter mechanism, splice- switching oligonucleotides may therefore downregulate a deleterious transcript while simultaneously upregulating expression of a preferred transcript. Notably, their activity is enhanced with increased target gene expression because this enables increased production of the preferred splice variant. This is in contrast to traditional anti-sense approaches and small-interfering RNA, which exhibit decreased potency with increased target gene expression.

In some embodiments the nucleic acid payload is a DNA, RNA or RNA:DNA hybrid. Any one strand may include a mixture of DNA and RNA, as well as modified forms that cannot readily be classified as either DNA or RNA. For example, in some cases, the nucleic acid is single stranded, and is a hybrid of RNA and DNA nucleobases. Likewise, a double stranded compound may be DNA:DNA, DNA:RNA or RNA:RNA, and any one strand may also include a mixture of DNA and RNA, as well as modified forms that cannot readily be classified as either DNA or RNA. For example, in some cases, the nucleic acid is a duplex with one, or the other, or both strands made of RNA and DNA nucleobases.

In some embodiments, the nucleic acid payload is an RNA. An RNA payload as provided herein may be any RNA payload known in the art. Non-limiting examples of RNA payloads include: messenger RNAs (mRNAs), short interfering RNAs (siRNAs), microRNAs (miRNAs), antisense oligonucleotides (ASOs,) that bind to a target nucleic acid, single guide RNAs (sgRNAs), and non-coding RNAs (ncRNAs). In some embodiments, the nucleic acid pay load is an RNA that is antisense oligonucleotides (ASOs). In some embodiments the nucleic acid payload is designed to induce editing of the target mRNA, pre-mRNA, or DNA via endogenous or exogenous nucleic acid editing machinery. For example, in some cases, the nucleic acid recruits and directs ADAR1 to edit mRNA or IncRNA. For example, in other cases, the nucleic acid is a guide RNA and recruits and directs a Cas protein to a specific site in the genome.

The nucleic acid or oligonucleotide may include any of a variety of modifications, including one or modifications to the backbone (the sugar-phosphate portion in a natural nucleic acid, including internucleotide linkages) or the base portion (the purine or pyrimidine portion of a natural nucleic acid). For example, in some cases one or more of the nucleobases used to fabricate the nucleic acid are modified with certain chemical moieties such as, for example, phosphorthioate, morpholino, 2'-F, and 2'-0Me. In some embodiments, the nucleic acid is modified with a fluorophore, or other imaging agent (e.g., gadolinium, radionuclide).

Catalytic or enzymatic nucleic acids may, in some cases, be ribozymes or DNA enzymes and may also contain modified forms. In certain embodiments, compounds described herein comprise or consist of an oligonucleotide comprising a region that is complementary to a target nucleic acid. In certain embodiments, the target nucleic acid is an endogenous RNA molecule. In certain embodiments, the target nucleic acid encodes a protein. In certain such embodiments, the target nucleic acid is selected from an mRNA and a pre-mRNA, including intronic, exonic and untranslated regions. In certain embodiments, the target RNA is an mRNA. In certain embodiments, the target nucleic acid is a pre-mRNA. In certain such embodiments, the target region is entirely within an intron. In certain embodiments, the target region spans an intron/exon junction. In certain embodiments, the target region is at least 50% within an intron.

Compositions and Methods of Use

In some embodiments, the humanized antibody promotes delivery of the nucleic acid payload into a cell (i.e., penetrate desired cell; transport across a cellular membrane; deliver across cellular membranes to, the nucleus). The humanized antibody and antigen binding fragments promote entry into cells via an ENT transporter, such as an ENT2 transporter and/or an ENT3 transporter. ENT2 is expressed preferentially in certain cell types, including muscle (skeletal and cardiac), neuronal, liver and/or kidney cells. Accordingly, conjugates (e.g., conjugates in which a humanized antibody or antigen binding fragment of the disclosure is conjugated to a nucleic acid payload) are delivered into cells of particular tissues, including skeletal muscle, cardiac muscle, lung, liver, and kidney. It has been further discovered herein that the antibody conjugates effectively deliver the nucleic acid payload to the nucleus of these cells where it can act directly on nucleic acid target substrates.

Thus, in some embodiments, the conjugates are useful for modulating nucleic acid expression in a variety of tissues.

In some embodiments, the antibody conjugates provided herein effectively deliver the nucleic acid payload to cardiac tissue. Cardiac tissue may contain any cardiac cells. Nonlimiting examples of cardiac tissue cells include: cardiac muscle cells, endothelial cells, mesothelial cells, fibroblasts, pericytes, adipocytes, neurons, lymphoid cells, myeloid cells, and erythrocytes. In some embodiments, the antibody conjugates provided herein effectively deliver the nucleic acid pay load to cardiac muscle cells.

Effectively deliver the nucleic acid payload means that antibody conjugates provided herein increase the delivery of nucleic acid payload by 5%-100% or more compared with a control. A control may be an antibody conjugate without a payload, another means for delivering a nucleic acid payload known in the art, or a combination thereof. Means for delivering a nucleic acid payload known in the art include, but are not limited to, viruses (e.g., adeno-associated viruses, lentiviruses, retroviruses), particles (e.g., nanoparticles, lipidoids, dendrimers), and targeting conjugates (e.g., GalNAc). Cardiac muscle is a smooth, involuntary muscle tissue that makes up the majority of the heart wall. Cardiac muscle is composed of cardiac muscle cells (cardiomyocytes) joined by intercalated discs and encased by collagen fibers and other substances that form the extracellular matrix. Cardiac muscle cells may be atrial cardiomyocytes that are located in the right and left atrium, ventricular cardiomyocytes that are located in the right and left ventricle, or some combination thereof.

Cardiac muscle cells contract in concert to pump blood through the heart to the rest of the body. Several barriers exist for delivering of therapeutic materials (e.g., antibody conjugates with nucleic acid payload) to cardiac muscle including, but not limited to: anatomical challenges of access to the cardiac muscle cells, mechanical force of blood flow, barrier of endothelial cells, and the immune response. The present application therefore advances the art by providing an antibody conjugate that efficiently overcomes these barriers and delivers nucleic acid pay load to cardiac muscle.

In some embodiments, antibody conjugates provided herein increase the delivery of nucleic acid payload by 5% - 200%, 15% - 190%, 25% - 180%, 35% - 170%, 45% - 160%, 55% - 150%, 65% - 140%, 75% - 130%, 85% - 120%, 95% - 110% compared with a control. In some embodiments, antibody conjugates provided herein increase the delivery of nucleic acid payload by at least or equal to 5%, 10%, 15%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, or 200% or more compared with a control.

Antibody conjugates, pharmaceutical compositions, compositions, or a combination thereof as described herein may be used in a method to modify nucleic acids (e.g., DNA, RNA, genes) in a cell. A nucleic acid may be RNA (e.g., messenger RNA (mRNA), pre- mRNA, ribosomal RNA (rRNA), transfer RNA (tRNA), DNA (e.g., nuclear DNA (gene), chromosomal DNA, extra-chromosomal DNA, mitochondrial DNA (mtDNA) or some combination thereof. Modifying nucleic acids may be modifying nucleic acid splicing, deleting nucleic acid sequences, adding nucleic acid sequences, mutating nucleic acid sequences, altering expression of nucleic acid sequences using, for instance, siRNA, shRNA or antisense oligonucleotides, or any combination thereof.

In some embodiments, antibody conjugates, pharmaceutical compositions, compositions, or a combination thereof are used in a method to modify nucleic acid splicing in a cell. In some embodiments, RNA is spliced in a cell, and methods provided herein provide antibody conjugates, pharmaceutical compositions, compositions, and combinations thereof that modify RNA splicing in a cell. RNA splicing refers to the processing of a pre- mRNA transcript into a mature mRNA transcript. Pre-mRNA is made up of, at least in part, exons and introns. Exons are nucleic acid sequences that encode amino acid sequences and introns are nucleic acid sequences that do not encode amino acid sequences. Introns are intervening sequences between exons and need to be spliced out (removed from) pre-mRNA. During pre-mRNA processing, introns are spliced out of (removed from) pre-mRNA and exons are spliced together and joined once the intervening intron sequences are removed.

Modifying nucleic acid (e.g., RNA) splicing may be increasing nucleic acid splicing, decreasing RNA splicing, or splicing out an exon that would typically be left in mature mRNA. Splicing out an exon that would typically be left in mature mRNA may be beneficial, for example, for removing a mutation from an mRNA. A mutation is a change in the nucleic acid (e.g., mRNA) sequence relative to a corresponding wild-type nucleic sequence. Removing a mutation from an mRNA may be useful if the mutation is associated with a disease or disorder. Associated with a disease or disorder means that the mutation is, at least partially, causative of the disease or disorder or exacerbates the symptoms of the disease or disorder (relative to a subject that does not have the mutation). In some embodiments, an exon containing a mutation is removed from an mRNA and an exon that does not contain a mutation is inserted into the mRNA by splicing. A disease or disorder associated with a mutation that is removed by splicing may be any disease or disorder associated with splicing. Associated with splicing means that the nucleic acid has a mutation associated with a disease or disorder that may be removed (or inserted) by splicing.

Modifying nucleic acid expression may be achieved by interfering RNA including but not limited to siRNA, aiRNA, miRNA, Dicer- substrate ds RNA, shRNA, and mixtures thereof. RNA interference (RNAi) is an evolutionarily conserved process in which recognition of double-stranded RNA (dsRNA) ultimately leads to posttranscriptional suppression of gene expression. RNAi induces specific degradation of mRNA through complementary base pairing between the dsRNA and the target mRNA. RNAi is typically mediated by short dsRNAs such as small interfering RNA (siRNA) duplexes of 21-23 nucleotides in length or by longer Dicer-substrate dsRNAs of 25-30 nucleotides in length. Unlike siRNAs, Dicer- substrate dsRNAs are cleaved by Dicer endonuclease, a member of the RNase III family, to produce smaller functional 21-mer siRNA duplexes. The 21-mer siRNA (whether synthesized or processed by Dicer) recruits the RNA-induced silencing complex (RISC) and enables effective gene silencing via sequence- specific cleavage of the target sequence. RNAi is useful for downregulating or silencing the transcription and translation of a gene of interest. In particular, for the treatment of cardiovascular disorders, RNAi may be used to modulate (e.g., reduce) the expression of certain genes in cardiac tissue.

The pharmaceutical compositions to be used in the present methods can comprise pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations used, and may comprise buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrans; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). In some examples, the pharmaceutical composition described herein comprises liposomes containing the antibodies which can be prepared by methods known in the art.

The pharmaceutical compositions to be used for in vivo administration may be sterile. This is readily accomplished by, for example, filtration through sterile filtration membranes. Therapeutic antibody compositions are generally placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

The pharmaceutical compositions described herein can be in unit dosage forms such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories, for oral, parenteral or rectal administration, or administration by inhalation or insufflation. For preparing solid compositions such as tablets, the principal active ingredient can be mixed with a pharmaceutical carrier, e.g., conventional tableting ingredients such as com starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g., water, to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention, or a non-toxic pharmaceutically acceptable salt thereof. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid preformulation composition is then subdivided into unit dosage forms of the type described above containing from 0.1 to about 500 mg of the active ingredient of the present invention. The tablets or pills of the novel composition can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer that serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, acetyl alcohol and cellulose acetate.

Suitable surface-active agents include, in particular, non-ionic agents, such as polyoxyethylenesorbitans (e.g., Tween™ 20, 40, 60, 80 or 85) and other sorbitans (e.g., Span™ 20, 40, 60, 80 or 85). Compositions with a surface-active agent will conveniently comprise between 0.05 and 5% surface-active agent, and can be between 0.1 and 2.5%. It will be appreciated that other ingredients may be added, for example mannitol or other pharmaceutically acceptable vehicles, if necessary.

Suitable emulsions may be prepared using commercially available fat emulsions, such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™ and Lipiphysan™. The active ingredient may be either dissolved in a pre-mixed emulsion composition or alternatively it may be dissolved in an oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil) and an emulsion formed upon mixing with a phospholipid (e.g., egg phospholipids, soybean phospholipids or soybean lecithin) and water. It will be appreciated that other ingredients may be added, for example glycerol or glucose, to adjust the tonicity of the emulsion. Suitable emulsions will typically contain up to 20% oil, for example, between 5 and 20%. The fat emulsion can comprise fat droplets between 0.1 and 1.0 .im, particularly 0.1 and 0.5 .im, and have a pH in the range of 5.5 to 8.0. The emulsion compositions can be those prepared by mixing an antibody with Intralipid™ or the components thereof (soybean oil, egg phospholipids, glycerol and water).

Pharmaceutical compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as set out above. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect.

Compositions in preferably sterile pharmaceutically acceptable solvents may be nebulized by use of gases. Nebulized solutions may be breathed directly from the nebulizing device or the nebulizing device may be attached to a face mask, tent or intermittent positive pressure breathing machine. Solution, suspension or powder compositions may be administered, preferably orally or nasally, from devices which deliver the formulation in an appropriate manner.

To practice the method disclosed herein, an effective amount of the pharmaceutical composition described herein can be administered to a subject (e.g., a human) in need of the treatment via a suitable route, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra- articular, intrasynovial, intrathecal, oral, inhalation or topical routes. Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers are useful for administration. Liquid formulations can be directly nebulized and lyophilized powder can be nebulized after reconstitution. Alternatively, the antibodies as described herein can be aerosolized using a fluorocarbon formulation and a metered dose inhaler, or inhaled as a lyophilized and milled powder.

The subject to be treated by the methods described herein can be a mammal, more preferably a human. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice and rats. A human subject who needs the treatment may be a human patient having, at risk for, or suspected of having a disease or disorder. A subject having a target disease or disorder can be identified by routine medical examination, e.g., laboratory tests, organ functional tests, CT scans, or ultrasounds. A subject suspected of having any of such target disease/disorder might show one or more symptoms of the disease/disorder. A subject at risk for the disease/disorder can be a subject having one or more of the risk factors for that disease/disorder. As used herein, “an effective amount” refers to the amount of each active agent required to confer therapeutic effect on the subject, either alone or in combination with one or more other active agents.

Generally, for administration of any of the antibodies described herein, an initial candidate dosage can be about 2 mg/kg. For the purpose of the present disclosure, a typical daily, weekly, every two weeks, or every three weeks dosage might range from about any of 0.1 pg/kg to 3 pg/kg to 30 pg/kg to 100 pg/kg to 300 pg/kg to 0.6 mg/kg, 1 mg/kg, 3 mg/kg, to 10 mg/kg, to 30 mg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days, weeks, months, or longer, depending on the condition, the treatment is sustained until a desired suppression of symptoms occurs or until sufficient therapeutic levels are achieved to alleviate a target disease or disorder, or a symptom thereof, and may continue thereafter for symptom maintenance. An exemplary dosing regimen comprises administering an initial dose of about 3 mg/kg every 3 weeks, followed by a maintenance dose of about 1 mg/kg of the antibody once in 6 weeks, or followed by a maintenance dose of about 1 mg/kg every 3 weeks. However, other dosage regimens may be useful, depending on the pattern of pharmacokinetic decay that the practitioner wishes to achieve. In some embodiments, dosing ranging from about 3 pg/mg to about 3 mg/kg (such as about 3 pg/mg, about 10 pg/mg, about 30 pg/mg, about 100 pg/mg, about 300 pg/mg, about 1 mg/kg, and about 3 mg/kg) may be used. In some embodiments, dosing frequency is once every week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, or every 10 weeks; or once every month, every 2 months, or every 3 months, or longer. The progress of this therapy is easily monitored by conventional techniques and assays. The dosing regimen (including the antibody used) can vary over time. In some embodiments the dose of the conjugate delivered to the subject is calculated based on the amount of antibody per dose. For instance, in some embodiments the dose may be any one of the following ranges in milligram per kilogram (mg/kg) or any integer dose therebetween: 10-500, 100- 500, 150-500; 200- 500, 250-500, 300-500, 350-500, 400- 500, 450-500, 100-1,000, 100-1,000, 100- 1,000, 150-1,000; 200- 1,000, 250-1,000, 300-1,000, 350-1,000, 400- 1,000, 450-1,000, 500-1,000, 600-1,000, 700-1,000, 800-1,000, or 900-1,000 mg/kg.

In some embodiments the dose of the conjugate delivered to the subject is calculated based on the amount of nucleic acid per dose. For instance, in some embodiments the dose may be any one of the following ranges in milligram per kilogram (mg/kg) or any integer dose therebetween: 1-500, 10- 30, 10-45 mg/kg; 10- 20 mg/kg, 20- 30 mg/kg or 30- 40 mg/kg.

In embodiments, the dosage of the nucleic acid in the conjugate is provided in an amount of about 0.1 pg up to l,000pg such as, but not limited to, greater than 0.1 pg and less than 0.5pg, less than l.Opg, less than 5pg, less than lOpg, less than 15pg, less than 20pg, less than 25 pg, less than 30pg, less than 35pg, less than 40pg, less than 50pg, less than 55pg, less than 60pg, less than 65pg, less than 70pg, less than 75pg, less than 80pg, less than 85pg, less than 90pg, less than 95pg, less than lOOpg, less than 125 pg, less than 150pg, less than 175pg, less than 200pg, less than 225 pg, less than 250pg, less than 275pg, less than 300pg, less than 325 pg, less than 350pg, less than 375pg, less than 400pg, less than 425 pg, less than 450pg, less than 475pg, less than 500pg, less than 525 pg, less than 550pg, less than 575pg, less than 600pg, less than 625pg, less than 650pg, less than 675pg, less than 700pg, less than 725pg, less than 750pg, less than 775pg, less than 800pg, less than 825 pg, less than 850pg, less than 875pg, less than 900pg, less than 925 pg, less than 950pg, or less than 975pg.

In embodiments, the dosage of the nucleic acid in the conjugate is provided in an amount of about lOpg up to l,000pg such as, but not limited to, greater than lOpg and less than 15pg, less than 20pg, less than 25 pg, less than 30pg, less than 35pg, less than 40pg, less than 50pg, less than 55pg, less than 60pg, less than 65pg, less than 70pg, less than 75pg, less than 80pg, less than 85pg, less than 90pg, less than 95pg, less than lOOpg, less than 125pg, less than 150pg, less than 175pg, less than 200pg, less than 225 pg, less than 250pg, less than 275pg, less than 300pg, less than 325 pg, less than 350pg, less than 375pg, less than 400pg, less than 425 pg, less than 450pg, less than 475pg, less than 500pg, less than 525 pg, less than 550pg, less than 575pg, less than 600pg, less than 625 pg, less than 650pg, less than 675pg, less than 700pg, less than 725pg, less than 750pg, less than 775pg, less than 800pg, less than 825pg, less than 850pg, less than 875pg, less than 900pg, less than 925 pg, less than 950pg, or less than 975pg.

In embodiments, the dosage of the nucleic acid in the conjugate is provided in an amount of about O.lmg up to 500mg such as, but not limited to, greater than O.lmg and less than 0.5mg, less than l.Omg, less than 5mg, less than lOmg, less than 15mg, less than 20mg, less than 25mg, less than 30mg, less than 35mg, less than 40mg, less than 50mg, less than 55mg, less than 60mg, less than 65mg, less than 70mg, less than 75mg, less than 80mg, less than 85mg, less than 90mg, less than 95mg, less than lOOmg, less than 125mg, less than 150mg, less than 175mg, less than 200mg, less than 225mg, less than 250mg, less than 275mg, less than 300mg, less than 325mg, less than 350mg, less than 375mg, less than 400mg, less than 425mg, less than 450mg, less than 475mg, or less than 500mg.

In embodiments, the dosage of the nucleic acid in the conjugate is provided in an amount of about lOmg up to 500mg such as, but not limited to, greater than lOmg and less than 15mg, less than 20mg, less than 22mg, less than 24mg, less than 25mg, less than 28mg, less than 30mg, less than 32mg, less than 35mg, less than 40mg, less than 50mg, less than 55mg, less than 60mg, less than 65mg, less than 70mg, less than 75mg, less than 80mg, less than 85mg, less than 90mg, less than 95mg, less than lOOmg, less than 125mg, less than 150mg, less than 175mg, less than 200mg, less than 225mg, less than 250mg, less than 275mg, less than 300mg, less than 325mg, less than 350mg, less than 375mg, less than 400mg, less than 425mg, less than 450mg, less than 475mg, or less than 500mg.

In each instance where a dosage range is recited as greater than or less than a recited value that range also includes the recited value. Thus the term less than includes less than or equal to and the term greater than includes greater than or equal to.

As used herein, the term “treating” refers to the application or administration of a composition including one or more active agents to a subject, who has a target disease or disorder, a symptom of the disease/disorder, or a predisposition toward the disease/disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disorder, the symptom of the disease, or the predisposition toward the disease or disorder. Alleviating a target disease/disorder includes delaying the development or progression of the disease, or reducing disease severity.

Alleviating the disease does not necessarily require curative results. As used therein, “delaying” the development of a target disease or disorder means to defer, hinder, slow, retard, stabilize, and/or postpone progression of the disease. This delay can be of varying lengths of time, depending on the history of the disease and/or individuals being treated. A method that “delays” or alleviates the development of a disease, or delays the onset of the disease, is a method that reduces probability of developing one or more symptoms of the disease in a given time frame and/or reduces extent of the symptoms in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a number of subjects sufficient to give a statistically significant result.

“Development” or “progression” of a disease means initial manifestations and/or ensuing progression of the disease. Development of the disease can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that may be undetectable. For purpose of this disclosure, development or progression refers to the biological course of the symptoms. “Development” includes occurrence, recurrence, and onset. As used herein “onset” or “occurrence” of a target disease or disorder includes initial onset and/or recurrence.

In some embodiments, the conjugates described herein are administered to a subject in need of the treatment at an amount sufficient to inhibit the activity of the target nucleic acid (substrate nucleic acid) by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater) in vivo. In other embodiments, the conjugate is administered in an amount effective in reducing the activity level of a target nucleic acid (substrate nucleic acid) by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater). In other embodiments, the conjugate is administered in an amount effective in modulating the splice pattern of a target nucleic acid (substrate nucleic acid) by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater).

Conventional methods, known to those of ordinary skill in the art of medicine, can be used to administer the pharmaceutical composition to the subject, depending upon the type of disease to be treated or the site of the disease. This composition can also be administered via other conventional routes, e.g., administered parenterally, topically, orally, by inhalation spray, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term “parenteral” as used herein includes subcutaneous, intracutaneous, intravenous, intraperitoneal, intratumor, intramuscular, intraarticular, intraarterial, intrasynovial, intrastemal, intrathecal, intralesional, and intracranial injection or infusion techniques. In addition, it can be administered to the subject via injectable depot routes of administration such as using 1-, 3-, or 6-month depot injectable or biodegradable materials and methods. In some examples, the pharmaceutical composition is administered intraocularly or intravitreally. Injectable compositions may contain various carriers such as vegetable oils, dimethylactamide, dimethyformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, and the like). For intravenous injection, water soluble antibodies can be administered by the drip method, whereby a pharmaceutical formulation containing the antibody and a physiologically acceptable excipient is infused. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer’s solution or other suitable excipients. Intramuscular preparations, e.g., a sterile formulation of a suitable soluble salt form of the antibody, can be dissolved and administered in a pharmaceutical excipient such as Water-for- Injection, 0.9% saline, or 5% glucose solution.

In some embodiments the antibody conjugate as described herein may be conjugated with a detectable label e.g., an imaging agent such as a contrast agent). In some embodiments, the detectable label is an agent suitable for imaging compounds or cells in vivo, which can be a radioactive molecule, a radiopharmaceutical, or an iron oxide particle. Radioactive molecules suitable for in vivo imaging include, but are not limited to, ¹²²I, ¹²³I,¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁸F, ⁷⁵Br, ⁷⁶Br, ⁷⁶Br, ⁷⁷Br, ²¹¹At, ²²⁵ Ac, ¹⁷⁷Lu, ¹⁵³Sm, ¹⁸⁶Re, ¹⁸⁸Re, ⁶⁷Cu, ²¹³Bi, ²¹²Bi, ²¹²Pb, and ⁶⁷Ga. Exemplary radiopharmaceuticals suitable for in vivo imaging include ^{n i}In Oxy quinoline, ¹³¹1 Sodium iodide, "^mTc Mebrofenin, and "^mTc Red Blood Cells, ¹²³I Sodium iodide, "^mTc Exametazime, "^mTc Macroaggregate Albumin, "^mTc Medronate, "^mTc Mertiatide, "^mTc Oxidronate, "^mTc Pentetate, "^mTc Pertechnetate, "^mTc Sestamibi, "^mTc Sulfur Colloid, "^mTc Tetrofosmin, Thallium-201, and Xenon- 133. The label can also be a dye, e.g., a fluorophore.

EXAMPLES

Example 1 - Synthesis of humanized 3E10 mAh conjugated to a heterobifunctional non- cleavable linker and an oligonucleotide payload

An exemplary antibody-oligonucleotide conjugate was synthesized from a humanized 3E10 mAb (i.e., heavy chain sequence of SEQ ID NO: 65 and light chain sequence of SEQ ID NO: 66) and an oligonucleotide payload through a heterobifunctional non-cleavable linker. A schematic of the reaction scheme is shown in FIG. 1. Random amine coupling was used to attach the linker to the mAb. Primary amines of the antibody were reacted with N- hydroxysuccinimide functional groups of the linker. Humanized 3E10 mAb or derivative was reacted with the linker at 1:5, 1: 10, 1:20, or 1:40 mAbdinker ratios for 8, 16, or 24 hours at 22, 25, or 37°C and passed through a desalting column in PBS, pH 7.4. Unless otherwise specified the linker comprised 10 PEG units.

In a separate reaction the Ab-linker conjugate was further conjugated to the nucleic acid payload via click chemistry. For instance, azide functional groups of the oligonucleotide were reacted with bicyclo [6.1.0] nonyne functional groups of the linker. The linker was reacted with the 5’-BCN-oligonucleotide at 1:5, 1:10, 1:20, or 1:40 nucleic aciddinker ratios for 8, 16, or 24 hours at 22, 25, or 37°C. Then, the full conjugate was passed through a desalting column in PBS, pH 7.4. Chromatograms demonstrating that AOC is made following this synthesis scheme, by incorporating either of these two linker types were generated.

Although the linker- Ab conjugate was formed first in the exemplary reaction, the oligonucleotide: linker reaction may be performed first, followed by the antibody: linker reaction. Additionally, other chemical reactions may be used to prepare the antibody-linker oligonucleotide conjugate. For instance, chemical modifications to the oligonucleotide may influence actual conditions used in the reaction. For example, inclusion of ENA nucleotides in the oligonucleotide benefits from reactions at higher temperatures in order to improve yield.

Example 2 - Synthesis of humanized 3E10 mAh or derivative conjugated to a heterobifunctional linker cleavable by enzyme or reduction and an oligonucleotide payload

An exemplary antibody-oligonucleotide conjugate (AOC) was synthesized from a humanized 3E10 mAb and an oligonucleotide pay load through a heterobifunctional cleavable linker. Random amine coupling was used to attach the linker to the mAb. Primary amines of the antibody were reacted with N-hydroxy succinimide functional groups of the linker. Humanized 3E10 mAb was reacted with the linker at 1:5, 1:10, 1:20, or 1:40 mAbdinker ratios for 8, 16, or 24 hours at 22, 25, or 37°C and passed through a desalting column in PBS, pH 7.4. Einkers may include, for instance, a PEG-based spacer of 1 or more repeating PEG units, and a reducible 2,2 '-dithiodipyridine disulfide bond or a PEG-based spacer of 1 or more repeating PEG units, and a valine-citrulline enzymatic cleavage site. An exemplary linker used in the example is azidoethyl- SS -propionic NHS ester: The antibody-linker conjugate was then reacted with the 5’-BCN-oligonuclotide at similar ratios for similar reaction times and temperatures. Then, the full conjugate was passed through a desalting column in PBS, pH 7.4. FIG. 2A shows a chromatogram depicting reaction products of an antibody-linker reacted with a 5’-BCN-oliognucleotide.

In another example, humanized 3E10 mAb was reduced with DTT or TCEP for 10, 60, or 150 minutes at 22 or 37°C and passed through a desalting column. A linker that contains a free SH group, from 2,2'-dithiodipyridine for example, was then reacted with the reduced mAb for 3 hours at 22°C before being passed through a desalting column in PBS, at pH 7.4. The data depicted in chromatograph is shown in FIG. 2B.

Example 3 - Use of a humanized 3E10 mAb conjugated to a phosphorodiamidate morpholino oligomer

The humanized 3E10 mAb linker conjugates were used to deliver phosphorodiamidate morpholino oligomers (PMOs) to cells to examine cell uptake and delivery to intracellular compartment. The PMO nucleic acid sequence was designed to target the mouse DMD intron/exon 23 boundary to inhibit retention of exon 23 during splicing. mAb-PMO conjugation was performed as described in Example 1. Terminally-differentiated C2C12 mouse muscle cells were treated with PMO alone (10 pM), positive control (10 pM PMO transfected using endoporter reagent), or 6 pM mAb-PMO conjugate (VEEmAb AOC; concentration is of PMO in the conjugate). Following 48 hours, the cells were lysed and cDNA was generated from cellular mRNA via reverse transcriptase PCR. cDNA was then amplified by a second round of PCR with primers designed to flank exon 23. A third round of PCR was performed as a nested PCR to further amplify the PCR product. The nested PCR product was resolved via agarose gel electrophoresis (FIGs. 3A). % exon skipping was determined by first quantifying intensity of the top band (includes exon 23) and bottom band (exon 23 was skipped), then taking the ratio of bottom band intensity to total intensity. The data is shown in bar graph format in FIG. 3B. The results demonstrate that mAb AOC at 6 pM displays robust exon skipping activity at 48 hours that is approximately 6-fold greater than positive control -10 pM unconjugated PMO. (Endop. = endoporter reagent).

Example 4 - Use of a humanized 3E10 mAb conjugated to an antisense oligonucleotide

An assay was performed to demonstrate delivery of an antisense oligonucleotide (ASO) sequence targeting GYSI, in the form of an antibody-oligonucleotide conjugate. In this example, the ASO sequence is a gapmer design that recruits RNAse H to degrade GYSI mRNA. mAb-ASO conjugation was performed as described in Example 1. C2C12 mouse muscle progenitor cells were treated with ASO alone (10 pM), positive control (10 pM ASO transfected using Lipofectamine reagent), or 6 pM mAb-ASO conjugate (mAb AOC; concentration is of ASO). Following 24 or 48 hours as indicated, cells were lysed and prepared for QPCR in situ using a Cells-to-Ct kit (Thermo Fisher). QPCR was performed on the product and GYSI Ct values were obtained and normalized to a housekeeping gene (GAPDH). % normalized expression was calculated by normalizing ACt values to the highest value for each timepoint, set to 100%.

The results are shown in FIG. 4. mAb AOC at 6 pM potently knocked down expression of GYSI at 24 hours, with strong knockdown continuing through 48 hours posttreatment. This knockdown is remarkably fast relative to endocytosed, Eipofectamine transfected positive control ASO, which does not reach >50% knockdown until after the experiment period.

Example 5 - In vivo systemic delivery of a humanized 3E10 mAb to the local cardiac and skeletal muscle tissue

An in vivo assay was performed to demonstrate systemic delivery of a humanized 3E10 mAb to the nuclei of cells of cardiac tissue. Wild-type C57 mice were treated with a single intravenous injection of 100 mg/kg mAb on day 0. Euthanasia and tissue collection occurred on day 3. Cardiac tissue was perfused and fixed in formalin and embedded in paraffin per standard procedures. Sections were mounted on slides and stained via immunohistochemistry using a mouse serum pre-adsorbed goat anti-human IgG primary antibody and a rabbit anti-goat secondary antibody. Images were obtained via microscopy and % positive nuclei were determined as the average of two independent manual analyses. Black arrows denote exemplary positive staining.

The data is shown in FIG. 5. Cardiac tissue was stained following administration of the antibody to mice by intravenous injection. The results are compared to the untreated control cardiac tissue. The results for the % of nuclei positive for mAb co-staining are shown in bar graph form in FIG. 5.

An in vivo assay was also performed to demonstrate systemic delivery of a humanized 3E10 mAb to the nuclei of cells of skeletal muscle tissue. Wild-type C57 mice were treated with a single intravenous injection of 100 mg/kg mAb on day 0. Euthanasia and tissue collection occurred on day 3. Tissues were fixed in formalin and embedded in paraffin per standard procedures. Sections were mounted on slides and stained via immunohistochemistry using a mouse serum pre-adsorbed goat anti-human IgG primary antibody and a rabbit anti-goat secondary antibody. Images were obtained via microscopy and % positive nuclei were determined as the average of two independent manual analyses. Black arrows denote exemplary positive staining.

The results are shown in FIG. 6. Bicep muscle tissue was stained following administration of the antibody to mice by intravenous injection. The results are compared to untreated control muscle tissue. FIG. 6 shows the results for the % of nuclei positive for mAb co-staining in bar graph form.

Example 6 - In vivo systemic delivery in a non-human primate of a humanized 3E10 mAb to multiple tissues

An in vivo assay was performed in a non-human primates to demonstrate systemic delivery of a humanized 3E10 mAb to multiple tissues, including brain, bicep, heart, quadricep, liver, spleen, lung, tongue, and kidney. Wild-type Chinese cynomolgus macaques were treated with a single intravenous injection of 50 mg/kg VEEmAb on day 0. Euthanasia and tissue collection occurred on day 3. Tissues were perfused as relevant and flash frozen. Sections were taken, homogenized, and processed for western blot analysis using an NHP serum pre-adsorbed goat anti-human IgG primary antibody and rabbit anti-goat HRP secondary antibody. All tissues were analyzed at the same concentration. Peak area was quantified using image! and plotted. The data is shown in FIG. 7.

Example 7 - In vivo efficacy of a humanized 3E10 mAb to murine cardiac muscle tissue An in vivo assay was performed in Mdx mice to demonstrate the efficacy of a humanized 3E10 mAb to skip DMD exon 23 in cardiac muscle tissue. Mdx mice were treated on day 0 with a single intravenous injection of 10 or 30 mg/kg VEEmAb conjugated to a morpholino designed to skip murine DMD exon 23 (sequence: GGCCAAACCTCGGCTTACCTGAAAT (SEQ ID NO: 64)). Dose refers to payload. Wildtype C57 mice were untreated (“Unt”). Euthanasia and tissue collection occurred on day 7. Cardiac tissue was perfused and stored in RNAlater. Tissue was then homogenized and lysed, and mRNA was extracted. Total RNA was converted to cDNA and the relevant section of DMD was amplified using nested PCR. Final PCR product was analyzed by agarose gel. FIG. 8. Wild-type DMD PCR amplicon expected size is 633 bp (top arrow), and exon 23- skipping DMD expected size is 420 bp (bottom arrow). Results confirmed by Sanger sequencing.

Example 8 - Use of a humanized 3E10 mAh conjugated to a small interfering RNA (siRNA) to deliver functional siRNA into cells

Humanized 3E10 mAb linker conjugates were used to deliver small interfering RNAs (siRNAs) to cells to examine cell uptake and delivery to an intracellular compartment. The siRNA nucleic acid sequence was designed to target a gene associated with cardiomyopathy. mAb-siRNA conjugation was performed as described in Example 1. Terminally- differentiated C2C12 mouse muscle cells were untreated, positive control (500 nM siRNA transfected using endoporter reagent), or 500 nM mAb-siRNA conjugate (VEEmAb AOC; concentration is of siRNA in the conjugate). Following 72 and 120 hours, the cells were lysed and cDNA was generated from cellular mRNA via reverse transcriptase PCR. The data is shown in bar graph format in FIG. 9. The results demonstrate that mAb AOC at 500 nM displays reduced target mRNA expression.

Example 9 - Use of a humanized 3E10 mAb conjugated to an siRNA and a detectable label

Humanized 3E10 mAb linker conjugates were used to deliver small interfering RNAs (siRNAs) and detectable labels to cells to examine cell uptake and delivery to intracellular compartment. The siRNA nucleic acid sequence was designed to target a gene associated with cardiomyopathy. mAb-siRNA conjugation was performed as described in Example 1, and the mAb-siRNA was labeled with Alexa Fluor 488 (AF488) via random amine coupling (VEEmAb ASO-AOC). HL-1 mouse cardiomyocytes were untreated, treated with VEEmAb alone, treated with siRNAs alone (“AOC”) or treated with 300 nM VEEmAb-AF488-AOC (“AF488-labeled AOC”). Following 96 hours, the cells were lysed and cDNA was generated from cellular mRNA via reverse transcriptase PCR. The data is shown in bar graph format in FIG. 10. The results demonstrate that VEEmAb-AF488-AOC is effective at reducing target mRNA expression in cardiomyocytes. It was further demonstrated that VEEmAb-AF488- AOC co-localizes with nucleic acids in the nuclei of cardiomyocytes.

Example 10 - Use of a humanized 3E10 mAb conjugated to an antisense oligonucleotide (ASO) with a cleavable linker was successful in delivering functional oligonucleotide into cells. Humanized 3E10 mAb linker conjugates were used to deliver antisense oligonucleotides (ASOs) to cells to examine the delivery and efficacy of the ASOs. The ASO nucleic acid sequence was designed to a gene associated with cardiomyopathy. mAb-ASO conjugation was performed as described in Example 1. C2C12 mouse muscle progenitor cells were untreated, treated with 300 nM cleavable VEEmAb-ASO linker (“Cleavable linker”) or 300 nM non-cleavable VEEmAb-ASO linker (“Non-cleavable linker”). Following 48 hours, the cells were lysed and cDNA was generated from cellular mRNA via reverse transcriptase PCR. The data is shown in bar graph format in FIG. 11. The results demonstrate that VEEmAb-AOC with a cleavable linker is active in reducing target mRNA expression.

Example 11 - Use of a 3E10 Fab conjugated to an oligonucleotide was successful in delivering functional oligonucleotide into cells.

A humanized 3E10 Fab was conjugated to a phosphorodiamidate morpholino oligomer (PMO). The 3E10 Fab was produced by digesting a mAb using LysC. The digestion produced a Fab comprising a heavy chain of SEQ ID NO: 67 and a light chain of SEQ ID NO: 68. The Fab-PMO conjugate was delivered to cells to examine cell uptake. The PMO nucleic acid sequence was designed to target the mouse DMD intron/exon 23 boundary to inhibit retention of exon 23 during splicing. Fab-PMO conjugation was performed as described in Example 1. Terminally-differentiated C2C12 mouse muscle cells were untreated or treated with 0.94 pM, or 4.7 pM FAb-PMO conjugate (VEE-Fab AOC; concentration is of PMO in the conjugate). Following 48 hours, the cells were lysed and cDNA was generated from cellular mRNA via reverse transcriptase PCR. cDNA was then amplified by a second round of PCR with primers designed to flank exon 23. A third round of PCR was performed as a nested PCR to further amplify the PCR product. The nested PCR product was resolved via agarose gel electrophoresis (FIG. 12). % exon skipping was determined by first quantifying intensity of the top band (includes exon 23) and bottom band (exon 23 was skipped), then taking the ratio of bottom band intensity to total intensity. The results demonstrate that Fab AOC displays robust exon skipping activity at 4.7 pM that is greater than untreated cells and cells treated with 0.94 pM Fab-PMO conjugate.

Example 12 - VEEmAb was successfully linked to oligonucleotides at high drug antibody ratio (DAR).

The number of oligonucleotides linked to humanized 3E10 mAb (VEEmAb) and the stability of the resultant AOCs was examined. mAb-PMO conjugation was performed as described in Example 1. Humanized VEEmAb PMO antisense oligonucleotide conjugates (VEEmAb-PMO AOCs) were produced with varied linker and PMO ratios, while holding PMO or linker constant as relevant. AOCs were resolved under non-reducing conditions and visualized using silver stain. A drug antibody ratio (DAR) for PMO to VEEmAb linker of up to 6 was stable as shown in FIG. 13. Further studies demonstrated that higher numbers of oligonucleotides, including up to 9 could be linked (data not shown).

Example 13 - VEEmAb PMO conjugates are delivered to heart tissue

Humanized 3E10 mAb (VEEmAb) linker conjugates were used to deliver PMOs to heart tissue. MDX mice were treated with VEEmAb PMOs (VEEmAb AOCs) by intravenous administration. Heart tissue samples were collected on day 7 and were analyzed by in situ hybridization (ISH). The results in FIGs. 14A-14B show increased hybridization of the detection probe (Probe2-ISH) in treated heart tissue versus untreated heart tissue (14C). These results demonstrate that VEEmAb-AOCs are successfully delivered to heart tissue in vivo.

SEQUENCES:

SEQ. Construct Sequence

ID NO

1 heavy chain variable (V_H) GFTFSNYG domain CDR1 of exemplary 3E10 molecule, in accordance with CDRs as defined by the IMGT system

2 heavy chain variable (V_H) ISSGSSTI domain CDR2 of exemplary 3E10 molecule, in accordance with CDRs as defined by the IMGT system

3 heavy chain variable (V_H) ARRGLLLDY domain CDR3 of exemplary 3E10 molecule, in accordance with CDRs as defined by the IMGT system

4 light chain variable (V_L) KSVSTSSYSY domain CDR1 of exemplary 3E10 molecule, in accordance with CDRs as defined by the IMGT system light chain variable (V_L) YAS domain CDR2 of exemplary 3E10 molecule, in accordance with CDRs as defined by the IMGT system light chain variable (V_L) domain QHSREFPWT CDR3 of exemplary 3E10 molecule, in accordance with CDRs as defined by the IMGT system amino acid sequence of murine D I VLTQS PAS LAVS LGQR ATISC R AS KSVSTSSYSYM H WYQQ 3E10 light chain variable KPGQPPKLLIKYASYLESGVPARFSGSGSGTDFHLNIHPVEEE domain (V_L) DAATYYCQHSREFPWTFGGGTKLELK used as parent VL amino acid sequence of DIQMTQSPSSLSASVGDRVTISCRAS KSVSTSSYSYM HWYQQ humanized 3E10 light chain KPEKAPKLLIKYASYLQSGVPSRFSGSGSGTDFTLTISSLQPEDV variable domain (hVL2) ATYYCQHSREFPWTFGAGTKLELK amino acid sequence of murine EVQLVESGGGLVKPGGSRKLSCAASGFTFSNYGMHWVRQA 3E10 heavy chain variable PEKGLEWVAYISSGSSTIYYADTVKGRFTISRDNAKNTLFLQM domain (V_H) used as parent VH TSLRSEDTAMYYCARRGLLLDYWGQGTTLTVSS amino acid sequence of EVQLQESGGGVVQPGGSLRLSCAASGFTFSNYGMHWIRQA humanized 3E10 heavy chain PGKGLEWVSYISSGSSTIYYADSVKGRFTISRDNSKNTLYLQM variable domain (hVH3) NSLRSEDTAVYYCARRGLLLDYWGQGTLVTVSS amino acid sequence of the ASASTSKYNSHSLENESIKRTSRDGVNRDLTEAVPRLPGETLIT human MTM1 protein DKEVIYICPFNGPIKGRVYITNYRLYLRSLETDSSLILDVPLGVIS (NP_000243.1) RIEKMGGATSRGENSYGLDITCKDMRNLRFALKQEGHSRRD MFEILTRYAFPLAHSLPLFAFLNEEKFNVDGWTVYNPVEEYRR QGLPNHHWRITFINKCYELCDTYPALLVVPYRASDDDLRRVA TFRSRNRIPVLSWIHPENKTVIVRCSQPLVGMSGKRNKDDEK YLDVIRETNKQISKLTIYDARPSVNAVANKATGGGYESDDAY HNAELFFLDIHNIHVMRESLKKVKDIVYPNVEESHWLSSLEST HWLEHIKLVLTGAIQVADKVSSGKSSVLVHCSDGWDRTAQL TSLAMLMLDSFYRSIEGFEILVQKEWISFGHKFASRIGHGDKN HTD AD RS P I F LQF I DCVWQM S KQF PTAF E F N EQF LI 11 LD H LYS CRFGTFLFNCESARERQKVTERTVSLWSLINSNKEKFKNPFYT

KEINRVLYPVASMRHLELWVNYYIRWNPRIKQQQPNPVEQR YMELLALRDEYIKRLEELQLANSAKLSDPPTSPSSPSQMMPH VQTHF The amino acid sequence of AVSVTPIRDTKWLTLEVCREFQRGTCSRPDTECKFAHPSKSC the human MBNLl protein, QVENGRVIACFDSLKGRCSRENCKYLHPPPHLKTQLEINGRN isoform a (GenBank Accession N LIQQKN MAM LAQQM QLAN AM M PG APLQPVPM FSVAP No. NP_066368.2) SLATNASAAAFNPYLGPVSPSLVPAEILPTAPMLVTGNPGVP VPAAAAAAAQKLMRTDRLEVCREYQRGNCNRGENDCRFAH PADSTMIDTNDNTVTVCMDYIKGRCSREKCKYFHPPAHLQA KIKAAQYQVNQAAAAQAAATAAAMGIPQAVLPPLPKRPALE KTNGATAVFNTGIFQYQQALANMQLQQHTAFLPPGSILCMT PATSVVPMVHGATPATVSAATTSATSVPFAATATANQIPIISA EHLTSHKYVTQM The amino acid sequence of MAVSVTPIRDTKWLTLEVCREFQRGTCSRPDTECKFAHPSKS the human MBNLl protein, CQVENGRVIACFDSLKGRCSRENCKYLHPPPHLKTQLEINGR isoform b (GenBank Accession N N LIQQKN MAM LAQQM QLAN AM M PG APLQPVPM FSVA No. NP_997175.1) PSLATNASAAAFNPYLGPVSPSLVPAEILPTAPMLVTGNPGV PVPAAAAAAAQKLMRTDRLEVCREYQRGNCNRGENDCRFA HPADSTMIDTNDNTVTVCMDYIKGRCSREKCKYFHPPAHLQ AKIKAAQYQVNQAAAAQAAATAAAMGIPQAVLPPLPKRPA LEKTNGATAVFNTGIFQYQQALANMQLQQHTAFLPPVPMV HGATPATVSAATTSATSVPFAATATANQIPIISAEHLTSHKYVT QM The amino acid sequence of MAVSVTPIRDTKWLTLEVCREFQRGTCSRPDTECKFAHPSKS the human M BNLl protein, CQVENGRVIACFDSLKGRCSRENCKYLHPPPHLKTQLEINGR isoform c (GenBank Accession N N LIQQKN MAM LAQQMQLAN AM M PG APLQPVPM FSVA No. NP_997176.1) PSLATNASAAAFNPYLGPVSPSLVPAEILPTAPM LVTGNPGV

PVPAAAAAAAQKLM RTDRLEVCREYQRGNCNRGENDCRFA HPADSTMIDTNDNTVTVCMDYIKGRCSREKCKYFHPPAHLQ AKIKAAQYQVNQAAAAQAAATAAAMTQSAVKSLKRPLEAT FDLGIPQAVLPPLPKRPALEKTNGATAVFNTGIFQYQQALAN MQLQQHTAFLPPVPMVHGATPATVSAATTSATSVPFAATA TANQIPIISAEHLTSHKYVTQM The amino acid sequence of MAVSVTPIRDTKWLTLEVCREFQRGTCSRPDTECKFAHPSKS the human M BNLl protein, CQVENGRVIACFDSLKGRCSRENCKYLHPPPHLKTQLEINGR isoform d (GenBank Accession NNLIQQKNMAM LAQQMQLANAMMPGAPLQPVVCREYQ No. NP_997177.1) RGNCNRGENDCRFAHPADSTM IDTNDNTVTVCMDYIKGRC SREKCKYFHPPAHLQAKIKAAQYQVNQAAAAQAAATAAAM GIPQAVLPPLPKRPALEKTNGATAVFNTGIFQYQQALANMQ LQQHTAFLPPVPMVHGATPATVSAATTSATSVPFAATATAN QIPIISAEHLTSHKYVTQM The amino acid sequence of MAVSVTPIRDTKWLTLEVCREFQRGTCSRPDTECKFAHPSKS the human M BNLl protein, CQVENGRVIACFDSLKGRCSRENCKYLHPPPHLKTQLEINGR isoform e (GenBank Accession NNLIQQKNMAM LAQQMQLANAMMPGAPLQPVVCREYQ No. NP_997178.1) RGNCNRGENDCRFAHPADSTM IDTNDNTVTVCMDYIKGRC SREKCKYFHPPAHLQAKIKAAQYQVNQAAAAQAAATAAAM GIPQAVLPPLPKRPALEKTNGATAVFNTGIFQYQQALANMQ LQQHTAFLPPGSILCMTPATSVVPMVHGATPATVSAATTSA TSVPFAATATANQIPIISAEHLTSHKYVTQM The amino acid sequence of MAVSVTPIRDTKWLTLEVCREFQRGTCSRPDTECKFAHPSKS the human M BNLl protein, CQVENGRVIACFDSLKGRCSRENCKYLHPPPHLKTQLEINGR isoform f (GenBank Accession N N LIQQKN MAM LAQQMQLAN AM M PG APLQPVPM FSVA No. NP_997179.1) PSLATNASAAAFNPYLGPVSPSLVPAEILPTAPM LVTGNPGV PVPAAAAAAAQKLM RTDRLEVCREYQRGNCNRGENDCRFA HPADSTMIDTNDNTVTVCMDYIKGRCSREKCKYFHPPAHLQ AKIKAAQYQVNQAAAAQAAATAAAMFPWCTVLRQPLCPQ QQHLPQVFPSLQQPQPTSPILDASTLLGATSCPAAAGKM IPII

SAEHLTSHKYVTQM The amino acid sequence of MAVSVTPIRDTKWLTLEVCREFQRGTCSRPDTECKFAHPSKS the human M BNLl protein, CQVENGRVIACFDSLKGRCSRENCKYLHPPPHLKTQLEINGR isoform g (GenBank Accession N N LIQQKN MAM LAQQMQLAN AM M PG APLQPVPM FSVA No. NP_997180.1) PSLATNASAAAFNPYLGPVSPSLVPAEILPTAPM LVTGNPGV PVPAAAAAAAQKLM RTDRLEVCREYQRGNCNRGENDCRFA HPADSTMIDTNDNTVTVCMDYIKGRCSREKCKYFHPPAHLQ AKIKAAQYQVNQAAAAQAAATAAAMGIPQAVLPPLPKRPA LEKTNGATAVFNTGIFQYQQALANMQLQQHTAFLPPGSILC

MTPATSVDTHNICRTSD The amino acid sequence of MGHSKQIRILLLNEMEKLEKTLFRLEQGYELQFRLGPTLQGKA the human AGL protein, VTVYTNYPFPGETFNREKFRSLDWENPTEREDDSDKYCKLNL isoform 1 (GenBank Accession QQSGSFQYYFLQGNEKSGGGYIVVDPILRVGADNHVLPLDC No. NP_000019.2) VTLQTFLAKCLGPFDEWESRLRVAKESGYNMIHFTPLQTLGL SRSCYSLANQLELNPDFSRPNRKYTWNDVGQLVEKLKKEWN VICITDVVYNHTAANSKWIQEHPECAYNLVNSPHLKPAWVL DRALWRFSCDVAEGKYKEKGIPALIENDHHMNSIRKIIWEDIF PKLKLWEFFQVDVNKAVEQFRRLLTQENRRVTKSDPNQHLTI IQDPEYRRFGCTVDMNIALTTFIPHDKGPAAIEECCNWFHKR MEELNSEKHRLINYHQEQAVNCLLGNVFYERLAGHGPKLGP VTRKHPLVTRYFTFPFEEIDFSMEESMIHLPNKACFLMAHNG WVMGDDPLRNFAEPGSEVYLRRELICWGDSVKLRYGNKPE DCPYLWAHMKKYTEITATYFQGVRLDNCHSTPLHVAEYMLD AARNLQPNLYVVAELFTGSEDLDNVFVTRLGISSLIREAMSAY NSHEEGRLVYRYGGEPVGSFVQPCLRPLMPAIAHALFMDITH DNECPIVHRSAYDALPSTTIVSMACCASGSTRGYDELVPHQIS WSEERFYTKWNPEALPSNTGEVNFQSGIIAARCAISKLHQEL GAKGFIQVYVDQVDEDIVAVTRHSPSIHQSVVAVSRTAFRNP KTSFYSKEVPQMCIPGKIEEVVLEARTIERNTKPYRKDENSING TPDITVEIREHIQLNESKIVKQAGVATKGPNEYIQEIEFENLSPG SVIIFRVSLDPHAQVAVGILRNHLTQFSPHFKSGSLAVDNADP ILKIPFASLASRLTLAELNQILYRCESEEKEDGGGCYDIPNWSAL KYAGLQGLMSVLAEIRPKNDLGHPFCNNLRSGDWMIDYVS NRLISRSGTIAEVGKWLQAMFFYLKQIPRYLIPCYFDAILIGAYT TLLDTAWKQMSSFVQNGSTFVKHLSLGSVQLCGVGKFPSLPI LSPALMDVPYRLNEITKEKEQCCVSLAAGLPHFSSGIFRCWGR DTFIALRGILLITGRYVEARNIILAFAGTLRHGLIPNLLGEGIYAR YNCRDAVWWWLQCIQDYCKMVPNGLDILKCPVSRMYPTD DSAPLPAGTLDQPLFEVIQEAMQKHMQGIQFRERNAGPQID RN M KD EG F N ITAG VD E ETG FVYGG N R F N CGTWM D KM G ES DRARNRGIPATPRDGSAVEIVGLSKSAVRWLLELSKKNIFPYH EVTVKRHGKAIKVSYDEWNRKIQDNFEKLFHVSEDPSDLNEK HPNLVHKRGIYKDSYGASSPWCDYQLRPNFTIAMVVAPELFT TEKAWKALEIAEKKLLGPLGMKTLDPDDMVYCGIYDNALDN

DNYNLAKGFNYHQGPEWLWPIGYFLRAKLYFSRLMGPETTA KTIVLVKNVLSRHYVHLERSPWKGLPELTNENAQYCPFSCET QAWSIATILETLYDL The amino acid sequence of M S LLTCAFYLG YE LQF R LG PTLQG KAVTVYTN YP F PG ETF N R E the human AGL protein, KFRSLDWENPTEREDDSDKYCKLNLQQSGSFQYYFLQGNEK isoform 2 (GenBank Accession SGGGYIVVDPILRVGADNHVLPLDCVTLQ.TFLAKCLGPFDEW No. NM_000645.2) ESRLRVAKESGYNMIHFTPLQ.TLGLSRSCYSLANQLELNPDFS RPNRKYTWNDVGQLVEKLKKEWNVICITDVVYNHTAANSK WIQEHPECAYNLVNSPHLKPAWVLDRALWRFSCDVAEGKY KEKGIPALIENDHHMNSIRKIIWEDIFPKLKLWEFFQVDVNKA VEQFRRLLTQENRRVTKSDPNQHLTIIQDPEYRRFGCTVDMN IALTTFIPHDKGPAAIEECCNWFHKRMEELNSEKHRLINYHQE QAVNCLLGNVFYERLAGHGPKLGPVTRKHPLVTRYFTFPFEEI DFSMEESMIHLPNKACFLMAHNGWVMGDDPLRNFAEPGS EVYLRRELICWGDSVKLRYGNKPEDCPYLWAHMKKYTEITAT YFQGVRLDNCHSTPLHVAEYMLDAARNLQPNLYVVAELFTG SEDLDNVFVTRLGISSLIREAMSAYNSHEEGRLVYRYGGEPVG SFVQPCLRPLMPAIAHALFMDITHDNECPIVHRSAYDALPSTT IVSMACCASGSTRGYDELVPHQISVVSEERFYTKWNPEALPS NTGEVNFQSGIIAARCAISKLHQELGAKGFIQVYVDQVDEDIV AVTRHSPSIHQSVVAVSRTAFRNPKTSFYSKEVPQMCIPGKIE EVVLEARTIERNTKPYRKDENSINGTPDITVEIREHIQLNESKIV KQAG VATKG P N EYI QE I E F E N LS PGSVI I F RVS LD P H AQVAVG I LRNHLTQFSPHFKSGSLAVDNADPILKIPFASLASRLTLAELNQ ILYRCESEEKEDGGGCYDIPNWSALKYAGLQGLMSVLAEIRPK NDLGHPFCNNLRSGDWMIDYVSNRLISRSGTIAEVGKWLQA MFFYLKQIPRYLIPCYFDAILIGAYTTLLDTAWKQMSSFVQNG STFVKHLSLGSVQLCGVGKFPSLPILSPALMDVPYRLNEITKEK EQCCVSLAAGLPHFSSGIFRCWGRDTFIALRGILLITGRYVEAR NIILAFAGTLRHGLIPNLLGEGIYARYNCRDAVWWWLQCIQD YCKMVPNGLDILKCPVSRMYPTDDSAPLPAGTLDQPLFEVIQ EAMQKHMQGIQFRERNAGPQIDRNMKDEGFNITAGVDEE TGFVYGGNRFNCGTWMDKMGESDRARNRGIPATPRDGSA VEIVGLSKSAVRWLLELSKKNIFPYHEVTVKRHGKAIKVSYDE WNRKIQDNFEKLFHVSEDPSDLNEKHPNLVHKRGIYKDSYGA SSPWCDYQLRPNFTIAMVVAPELFTTEKAWKALEIAEKKLLG PLGMKTLDPDDMVYCGIYDNALDNDNYNLAKGFNYHQGPE WLWPIGYFLRAKLYFSRLMGPETTAKTIVLVKNVLSRHYVHLE RSPWKGLPELTNENAQYCPFSCETQAWSIATILETLYDL The amino acid sequence of MAPILSINLFIGYELQFRLGPTLQGKAVTVYTNYPFPGETFNRE the human AGL protein, KFRSLDWENPTEREDDSDKYCKLNLQQSGSFQYYFLQGNEK isoform 3 (GenBank Accession SGGGYIVVDPILRVGADNHVLPLDCVTLQ.TFLAKCLGPFDEW No. NM_000646.2) ESRLRVAKESGYNMIHFTPLQ.TLGLSRSCYSLANQLELNPDFS RPNRKYTWNDVGQLVEKLKKEWNVICITDVVYNHTAANSK WIQEHPECAYNLVNSPHLKPAWVLDRALWRFSCDVAEGKY KEKGIPALIENDHHMNSIRKIIWEDIFPKLKLWEFFQVDVNKA VEQFRRLLTQENRRVTKSDPNQHLTIIQDPEYRRFGCTVDMN IALTTFIPHDKGPAAIEECCNWFHKRMEELNSEKHRLINYHQE QAVNCLLGNVFYERLAGHGPKLGPVTRKHPLVTRYFTFPFEEI DFSMEESMIHLPNKACFLMAHNGWVMGDDPLRNFAEPGS EVYLRRELICWGDSVKLRYGNKPEDCPYLWAHMKKYTEITAT YFQGVRLDNCHSTPLHVAEYMLDAARNLQPNLYVVAELFTG SEDLDNVFVTRLGISSLIREAMSAYNSHEEGRLVYRYGGEPVG SFVQPCLRPLMPAIAHALFMDITHDNECPIVHRSAYDALPSTT IVSMACCASGSTRGYDELVPHQISVVSEERFYTKWNPEALPS NTGEVNFQSGIIAARCAISKLHQELGAKGFIQVYVDQVDEDIV AVTRHSPSIHQSVVAVSRTAFRNPKTSFYSKEVPQMCIPGKIE EVVLEARTIERNTKPYRKDENSINGTPDITVEIREHIQLNESKIV KQAG VATKG P N EYI QE I E F E N LS PGSVI I F RVS LD P H AQVAVG I LRNHLTQFSPHFKSGSLAVDNADPILKIPFASLASRLTLAELNQ ILYRCESEEKEDGGGCYDIPNWSALKYAGLQGLMSVLAEIRPK NDLGHPFCNNLRSGDWMIDYVSNRLISRSGTIAEVGKWLQA MFFYLKQIPRYLIPCYFDAILIGAYTTLLDTAWKQMSSFVQNG STFVKHLSLGSVQLCGVGKFPSLPILSPALMDVPYRLNEITKEK EQCCVSLAAGLPHFSSGIFRCWGRDTFIALRGILLITGRYVEAR NIILAFAGTLRHGLIPNLLGEGIYARYNCRDAVWWWLQCIQD YCKMVPNGLDILKCPVSRMYPTDDSAPLPAGTLDQPLFEVIQ EAMQKHMQGIQFRERNAGPQIDRNMKDEGFNITAGVDEE TGFVYGGNRFNCGTWMDKMGESDRARNRGIPATPRDGSA VEIVGLSKSAVRWLLELSKKNIFPYHEVTVKRHGKAIKVSYDE

WNRKIQDNFEKLFHVSEDPSDLNEKHPNLVHKRGIYKDSYGA SSPWCDYQLRPNFTIAMVVAPELFTTEKAWKALEIAEKKLLG PLGMKTLDPDDMVYCGIYDNALDNDNYNLAKGFNYHQGPE WLWPIGYFLRAKLYFSRLMGPETTAKTIVLVKNVLSRHYVHLE RSPWKGLPELTNENAQYCPFSCETQAWSIATILETLYDL full-length, immature GAA MGVRHPPCSHRLLAVCALVSLATAALLGHILLHDFLLVPREL amino acid sequence (952 SGSSPVLEETHPAH amino acids; signal sequence QQGASRPGPRDAQAHPGRPRAVPTQCDVPPNSRFDCAPDK indicated in bold/underline) AITQEQCEARGCCYIPAKQGLQGAQMGQPWCFFPPSYPSYK LE N LSSS E M G YTATLTRTTPTF F P KD I LTLR LDVM M ETE N R LH FTIKDPANRRYEVPLETPHVHSRAPSPLYSVEFSEEPFGVIVRR QLDGRVLLNTTVAPLFFADQFLQLSTSLPSQYITGLAEHLSPL M LSTSWTRITLWNRDLAPTPGANLYGSH PFYLALEDGGSAH GVFLLNSNAM DVVLQPSPALSWRSTGGILDVYI FLGPEPKSV VQQYLDVVGYPFM PPYWGLGFHLCRWGYSSTAITRQVVEN MTRAHFPLDVQWNDLDYM DSRRDFTFN KDGFRDFPAMV QELHQGGRRYM M IVDPAISSSGPAGSYRPYDEGLRRGVFIT NETGQPLIGKVWPGSTAFPDFTN PTALA WWEDMVAEFHD QVPFDGMWI DM N EPSN FIRGSEDGCPNN ELENPPYVPGVV GGTLQAATICASSHQFLSTHYNLHN LYGLTEAIASH RALVKAR GTRPFVISRSTFAGHGRYAGHWTGDVWSSWEQLASSVPEI L QFN LLGVPLVGADVCGFLGNTSEELCVRWTQLGAFYPFM RN HNSLLSLPQEPYSFSEPAQQAM RKALTLRYALLPHLYTLFHQA HVAGETVARPLFLEFPKDSSTWTVDHQLLWGEALLITPVLQA GKAEVTGYFPLGTWYDLQTVPVEALGSLPPPPAAPREPAI HS EGQWVTLPAPLDTINVHLRAGYI IPLQGPGLTTTESRQQPMA LAVALTKGGEARGELFWDDGESLEVLERGAYTQVI FLARNNT IVNELVRVTSEGAGLQLQKVTVLGVATAPQQVLSNGVPVSN FTYS P DTKVLD I CVS LLM G EQF LVS WC full-length, immature GAA MGVRHPPCSHRLLAVCALVSLATAALLGHILLHDFLLVPREL amino acid sequence (957 SGSSPVLEETHPAH amino acids; signal sequence QQGASRPGPRDAQAHPGRPRAVPTQCDVPPNSRFDCAPDK indicated in bold/underline) AITQEQCEARGCCYIPAKQGLQGAQMGQPWCFFPPSYPSYK (GenBank Accession No. LE N LSSS E M G YTATLTRTTPTF F P KD I LTLR LDVM M ETE N R LH EAW89583.1) FTIKDPANRRYEVPLETPHVHSRAPSPLYSVEFSEEPFGVIVRR QLDGRVLLNTTVAPLFFADQFLQLSTSLPSQYITGLAEHLSPL M LSTSWTRITLWNRDLAPTPGANLYGSH PFYLALEDGGSAH GVFLLNSNAM DVVLQPSPALSWRSTGGILDVYI FLGPEPKSV VQQYLDVVGYPFM PPYWGLGFHLCRWGYSSTAITRQVVEN MTRAHFPLDVQWNDLDYM DSRRDFTFN KDGFRDFPAMV QELHQGGRRYM M IVDPAISSSGPAGSYRPYDEGLRRGVFIT NETGQPLIGKVWPGSTAFPDFTN PTALAWWEDMVAEFHD QVPFDGMWI DM N EPSN FIRGSEDGCPNN ELENPPYVPGVV GGTLQAATICASSHQFLSTHYNLHN LYGLTEAIASH RALVKAR GTRPFVISRSTFAGHGRYAGHWTGDVWSSWEQLASSVPEI L QFN LLGVPLVGADVCGFLGNTSEELCVRWTQLGAFYPFM RN HNSLLSLPQEPYSFSEPAQQAM RKALTLRYALLPHLYTLFHQA HVAGETVARPLFLEFPKDSSTWTVDHQLLWGEALLITPVLQA GKAEVTGYFPLGTWYDLQTVPIEALGSLPPPPAAPREPAIHSE GQWVTLPAPLDTINVHLRAGYII PLQGPGLTTTESRQQPMAL AVALTKGGEARGELFWDDGESLEVLERGAYTQVI FLARNNTI VN ELVRVTSEGAGLQLQKVTVLGVATAPQQVLSNGVPVSN F TYSPDTKARGPRVLDICVSLLMGEQFLVSWC exemplary mature GAA amino GQPWCFFPPSYPSYKLENLSSSEMGYTATLTRTTPTFFPKDILT acid sequence (corresponding LRLDVM M ETENRLHFTIKDPANRRYEVPLETPHVHSRAPSPL to residues 123-782 of SEQ ID YSVEFSEEPFGVIVRRQLDGRVLLNTTVAPLFFADQFLQLSTSL NO: 22; one embodiment of a PSQYITGLAEHLSPLM LSTSWTRITLWN RDLAPTPGANLYGS mature GAA polypeptide) HPFYLALEDGGSAHGVFLLNSNAM DVVLQPSPALSWRSTGG I LDVYI FLG PE PKSVVQQYLDVVGYPFM PPYWG LG FH LCRW GYSSTAITRQVVENMTRAH FPLDVQWNDLDYM DSRRDFTF NKDGFRDFPAMVQELHQGGRRYMMIVDPAISSSGPAGSYR PYDEGLRRGVFITNETGQPLIGKVWPGSTAFPDFTNPTALAW WEDMVAEFHDQVPFDGMWIDMNEPSNFIRGSEDGCPNN ELENPPYVPGVVGGTLQAATICASSHQFLSTHYNLHNLYGLTE AIASHRALVKARGTRPFVISRSTFAGHGRYAGHWTGDVWSS WEQLASSVPEILQFNLLGVPLVGADVCGFLGNTSEELCVRWT QLGAFYPFMRNHNSLLSLPQEPYSFSEPAQQAMRKALTLRY ALLPHLYTLFHQAHVAGETVARPLFLEFPKDSSTWTVDHQLL WGEALLITPVLQAGKAEVTGYFPLGTWYDLQTVPVEA exemplary mature GAA amino GANLYGSHPFYLALEDGGSAHGVFLLNSNAMDVVLQPSPAL acid sequence (corresponding SWRSTGGILDVYIFLGPEPKSVVQQYLDVVGYPFMPPYWGL to residues 288-782 of SEQ ID GFHLCRWGYSSTAITRQVVENMTRAHFPLDVQWNDLDYM NO: 22; one embodiment of a DSRRDFTFNKDGFRDFPAMVQELHQGGRRYMMIVDPAISS mature GAA polypeptide) SGPAGSYRPYDEGLRRGVFITNETGQPLIGKVWPGSTAFPDF TNPTALAWWEDMVAEFHDQVPFDGMWIDMNEPSNFIRG SEDGCPNNELENPPYVPGVVGGTLQAATICASSHQFLSTHYN LHNLYGLTEAIASHRALVKARGTRPFVISRSTFAGHGRYAGH WTGDVWSSWEQLASSVPEILQFNLLGVPLVGADVCGFLGN TS E E LC V R WTQLG AFYP F MRNHNSLLSLPQEPYSFSE P AQQA MRKALTLRYALLPHLYTLFHQAHVAGETVARPLFLEFPKDSST WTVDHQLLWGEALLITPVLQAGKAEVTGYFPLGTWYDLQTV PVEA Exemplary GAA polypeptide SRPGPRDAQAHPGRPRAVPTQCDVPPNSRFDCAPDKAITQE comprising mature GAA QCEARGCCYIPAKQGLQGAQMGQPWCFFPPSYPSYKLENLS (residues 61-952; one SSEMGYTATLTRTTPTFFPKDILTLRLDVMMETENRLHFTIKD embodiment of a GAA PANRRYEVPLETPHVHSRAPSPLYSVEFSEEPFGVIVRRQLDG polypeptide) RVLLNTTVAPLFFADQFLQLSTSLPSQYITGLAEHLSPLMLSTS WTRITLWNRDLAPTPGANLYGSHPFYLALEDGGSAHGVFLL NSNAMDVVLQPSPALSWRSTGGILDVYIFLGPEPKSVVQQYL DVVGYPFMPPYWGLGFHLCRWGYSSTAITRQVVENMTRAH FPLDVQWNDLDYMDSRRDFTFNKDGFRDFPAMVQELHQG GRRYMMIVDPAISSSGPAGSYRPYDEGLRRGVFITNETGQPLI GKVWPGSTAFPDFTNPTALAWWEDMVAEFHDQVPFDGM WIDMNEPSNFIRGSEDGCPNNELENPPYVPGVVGGTLQAAT ICASSHQFLSTHYNLHNLYGLTEAIASHRALVKARGTRPFVISR STFAGHGRYAGHWTGDVWSSWEQLASSVPEILQFNLLGVPL VG ADVCG F LG NTS E E LCVR WTQLG AFYP F M R N H N 8 LLS LPQ EPYSFSEPAQQAMRKALTLRYALLPHLYTLFHQAHVAGETVA RPLFLEFPKDSSTWTVDHQLLWGEALLITPVLQAGKAEVTGY FPLGTWYDLQTVPVEALGSLPPPPAAPREPAIHSEGQWVTLP APLDTINVHLRAGYIIPLQGPGLTTTESRQQPMALAVALTKG GEARGELFWDDGESLEVLERGAYTQVIFLARNNTIVNELVRV TS EG AG LQLQKVTVLG VATAPQQVLS N G VPVS N FTYS PDTK VLDICVSLLMGEQFLVSWC

Exemplary GAA polypeptide DAQAHPGRPRAVPTQCDVPPNSRFDCAPDKAITQEQCEAR comprising mature GAA GCCYIPAKQGLQGAQMGQPWCFFPPSYPSYKLENLSSSEMG (residues 67-952; one YTATLTRTTPTFFPKDILTLRLDVMMETENRLHFTIKDPANRR embodiment of a GAA YEVPLETPHVHSRAPSPLYSVEFSEEPFGVIVRRQLDGRVLLNT polypeptide TVAPLFFADQFLQLSTSLPSQYITGLAEHLSPLMLSTSWTRITL WNRDLAPTPGANLYGSHPFYLALEDGGSAHGVFLLNSNAM DVVLQPSPALSWRSTGGILDVYIFLGPEPKSVVQQYLDVVGY PFMPPYWGLGFHLCRWGYSSTAITRQVVENMTRAHFPLDV QWNDLDYMDSRRDFTFNKDGFRDFPAMVQELHQGGRRY MMIVDPAISSSGPAGSYRPYDEGLRRGVFITNETGQPLIGKV

WPGSTAFPDFTNPTALAWWEDMVAEFHDQVPFDGMWID

MNEPSNFIRGSEDGCPNNELENPPYVPGVVGGTLQAATICA

SSHQFLSTHYNLHNLYGLTEAIASHRALVKARGTRPFVISRSTF

AGHGRYAGHWTGDVWSSWEQLASSVPEILQFNLLGVPLVG

ADVCGFLGNTSEELCVRWTQLGAFYPFMRNHNSLLSLPQEP

YSFSEPAQQAMRKALTLRYALLPHLYTLFHQAHVAGETVARP

LFLEFPKDSSTWTVDHQLLWGEALLITPVLQAGKAEVTGYFP

LGTWYDLQTVPVEALGSLPPPPAAPREPAIHSEGQWVTLPA

PLDTINVHLRAGYIIPLQGPGLTTTESRQQPIVIALAVALTKGGE

ARGELFWDDGESLEVLERGAYTQVIFLARNNTIVNELVRVTSE

GAGLQLQKVTVLGVATAPQQVLSNGVPVSNFTYSPDTKVLDI

CVSLLMGEQFLVSWC

AGIH" AGIH

"SAGIH" SAGIH linker sequence "GS3" GGGGSGGGGSGGGGS linker sequence "GSTS" GSTSGSGKSSEGKG heavy chain variable domain NYGM H CDR1 of VH (as that VH is defined with reference to SEQ ID NO: 9), in accordance with CDRs as defined by Kabat heavy chain variable domain YISSGSSTIYYADTVKG CDR2 of VH (as that VH is defined with reference to SEQ ID NO: 9), in accordance with CDRs as defined by Kabat heavy chain variable domain RGLLLDY CDR3 of VH (as that VH is defined with reference to SEQ ID NO: 9), in accordance with CDRs as defined by Kabat light chain variable domain RASKSVSTSSYSYM H CDR1 of VL (as that VL is defined with reference to SEQ ID NO: 7), in accordance with CDRs as defined by Kabat light chain variable domain YASYLES CDR2 of VL (as that VL is defined with reference to SEQ ID NO: 7), in accordance with CDRs as defined by Kabat light chain variable domain QHSREFPWT CDR3 of VL (as that VL is defined with reference to SEQ ID NO: 7), in accordance with CDRs as defined by Kabat amino acid sequence of EVQLVQSGGGLIQPGGSLRLSCAASGFTFSNYGIVI HWVRQA humanized 3E10 heavy chain PGKGLEWVSYISSGSSTIYYADSVKGRFTISRDNSKNTLYLQM (hVHl) NSLRAEDTAVYYCARRGLLLDYWGQGTTVTVSS amino acid sequence of EVQLVESGGGLIQPGGSLRLSCAASGFTFSNYGMHWVRQAP humanized 3E10 heavy chain GKGLEWVSYISSGSSTIYYADSVKGRFTISRDNSKNTLYLQMT (hVH2) SLRAEDTAVYYCARRGLLLDYWGQGTTLTVSS amino acid sequence of D IQMTQSPSS LS ASVG D RVTITC R AS KSVSTSSYSYLAWYQQ humanized 3E10 light chain KPEKAPKLLIKYASYLQSGVPSRFSGSGSGTDFTLTISSLQPEDF (hVLl) ATYYCQHSREFPWTFGAGTKLELK amino acid sequence of D I VLTQS PAS LAVS PGQR ATITCR AS KSVSTSSYSYM H WYQQ humanized 3E10 light chain KPGQPPKLLIYYASYLESGVPARFSGSGSGTDFTLTINPVEAND TANYYCQHSREFPWTFGQGTKVEIK amino acid sequence of EVQLVESGGGLVQPGGSLRLSCSASGFTFSNYGMHWVRQA humanized 3E10 heavy chain PGKGLEYVSYISSGSSTIYYADTVKGRFTISRDNSKNTLYLQMS SLRAEDTAVYYCVKRGLLLDYWGQGTLVTVSS

Humanized Fv3E10 D I VLTQS PAS LAVS PG R ATITCR AS KSVSTSSYSYM H WYQQ KPGQPPKLLIYYASYLESGVPARFSGSGSGTDFTLTINPVEAND TANYYCQHSREFPWTFGQGTKVEIKGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCSASGFTFSNYGMHWVRQA PGKGLEYVSYISSGSSTIYYADTVKGRFTISRDNSKNTLYLQMS SLRAEDTAVYYCVKRGLLLDYWGQGTLVTVSS

Exemplary GAA polypeptide AHPGRPRAVPTQCDVPPNSRFDCAPDKAITQEQCEARGCCY comprising mature GAA IPAKQGLQGAQMGQPWCFFPPSYPSYKLENLSSSEMGYTAT (residues 70-952; one LTRTTPTFFPKDILTLRLDVMM ETENRLHFTIKDPANRRYEVP embodiment of a GAA LETPHVHSRAPSPLYSVEFSEEPFGVIVRRQLDGRVLLNTTVA polypeptide) PLFFADQFLQLSTSLPSQYITGLAEHLSPLMLSTSWTRITLWN RDLAPTPGANLYGSHPFYLALEDGGSAHGVFLLNSNAMDVV LQPSPALSWRSTGGILDVYIFLGPEPKSVVQQYLDVVGYPFM PPYWGLGFHLCRWGYSSTAITRQVVENMTRAHFPLDVQW NDLDYMDSRRDFTFNKDGFRDFPAMVQELHQGGRRYMMI VDPAISSSGPAGSYRPYDEGLRRGVFITNETGQPLIGKVWPG STAFPDFTNPTALAWWEDMVAEFHDQVPFDGMWIDM NE PSNFIRGSEDGCPNNELENPPYVPGVVGGTLQAATICASSHQ FLSTHYNLHNLYGLTEAIASHRALVKARGTRPFVISRSTFAGH G RYAG H WTG DVWSS WEQLASSVP E I LQF N LLG VP LVG ADV CGFLGNTSEELCVRWTQLGAFYPFMRNHNSLLSLPQEPYSFS EPAQQAM RKALTLRYALLPHLYTLFHQAHVAGETVARPLFLE FPKDSSTWTVDHQLLWGEALLITPVLQAGKAEVTGYFPLGT WYDLQTVPVEALGSLPPPPAAPREPAIHSEGQWVTLPAPLD TINVHLRAGYIIPLQGPGLTTTESRQQPMALAVALTKGGEAR GELFWDDGESLEVLERGAYTQVIFLARNNTIVNELVRVTSEG

AGLQLQKVTVLGVATAPQQVLSNGVPVSNFTYSPDTKVLDIC VSLLMGEQFLVSWC linker sequence GGSGGGSGGGSGG full linker region (residues 57- HILLHDFLLVPRELSGSSPVLEETHPAH 78 of GAA) His Tag HHHHHH

Exemplary c-myc Tag EQKLISEEDL heavy chain variable domain YISSGSSTIYYADSVKG CDR2 of certain antibodies of the disclosure, in accordance with CDRs as defined by Kabat light chain variable domain RASKSVSTSSYSYLA CDR1 of certain antibodies of the disclosure, in accordance with CDRs as defined by Kabat light chain variable domain YASYLQS CDR2 of certain antibodies of the disclosure, in accordance with CDRs as defined by Kabat bovine GAA precursor protein MMRWPPCSRPLLGVCTLLSLALLGHILLHDLEVVPRELRGFS (GenBank Accession No. QDEIHQACQPGASSPECRGSPRAAPTQCDLPPNSRFDCAPD NP_776338.1) KGITPQQCEARGCCYMPAEWPPDAQMGQPWCFFPPSYPS YRLENLTTTETGYTATLTRAVPTFFPKDIMTLRLDMLM ETESR LHFTIKDPANRRYEVPLETPRVYSQAPFTLYSVEFSEEPFGVVV

RRKLDGRVLLNTTVAPLFFADQFLQLSTSLPSQHITGLAEHLG SLMLSTNWTKITLWNRDIAPEPNVNLYGSHPFYLVLEDGGLA HGVFLLNSNAMDVVLQPSPALSWRSTGGILDVYIFLGPEPKS

WQQYLDVVGYPFMPPYWGLGFHLCRWGYSTSAITRQVVE

NMTRAYFPLDVQWNDLDYMDARRDFTFNKDHFGDFPAM VQELHQGGRRYIMIVDPAISSSGPAGTYRPYDEGLRRGVFIT NETGQPLIGQVWPGLTAFPDFTNPETLDWWQDMVTEFHA QVPFDGMWIDMNEPSNFVRGSVDGCPDNSLENPPYLPGV VGGTLRAATICASSHQFLSTHYDLHNLYGLTEALASHRALVKA

RGM RPFVISRSTFAGHGRYSGHWTGDVWSNWEQLSYSVPE ILLFNLLGVPLVGADICGFLGNTSEELCVRWTQLGAFYPFMR NHNALNSQPQEPYRFSETAQQAMRKAFTLRYVLLPYLYTLFH RAHVRGETVARPLFLEFPEDPSTWTVDRQLLWGEALLITPVL EAEKVEVTGYFPQGTWYDLQTVPMEAFGSLPPPAPLTSVIHS

KGQWVTLSAPLDTI N VH LRAG H 11 PM QG PALTTTESRKQH M ALAVALTASGEAQGELFWDDGESLGVLDGGDYTQLIFLAKN NTFVNKLVHVSSEGASLQLRNVTVLGVATAPQQVLCNSVPV SNFTFSPDTETLAIPVSLTMGEQFVISWS Nucleotide sequence encoding GACATTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATC murine 3E10 light chain TCTGGGGCAGAGGGCCACCATCTCCTGCAGGGCCAGCAA Genbank accession number AAGTGTCAGTACATCTAGCTATAGTTACATGCACTGGTACC L34051 AACAGAAACCAGGACAGCCACCCAAACTCCTCATCAAGTA TGCATCCTACCTAGAATCTGGGGTTCCTGCCAGGTTCAGT GGCAGTGGGTCTGGGACAGACTTTCACCTCAACATCCATC CTGTGGAGGAGGAGGATGCTGCAACATATTACTGTCAGCA CAGTAGGGAGTTTCCGTGGACGTTCGGTGGAGGCACCAA

GCTGGAGTTGAAA Nucleotide sequence encoding GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAG murine heavy chain sequence CCTGGAGGGTCCCGGAAACTCTCCTGTGCAGCCTCTGGAT (Genbank accession number TCACTTTCAGTAACTATGGAATGCACTGGGTCCGTCAGGCT L16982) CCAGAGAAGGGGCTGGAGTGGGTTGCATACATTAGTAGT GGCAGTAGTACCATCTACTATGCAGACACAGTGAAGGGCC

GATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTTC CTGCAAATGACCAGTCTAAGGTCTGAGGACACAGCCATGT ATTACTGTGCAAGGCGGGGGTTACTACTTGACTACTGGGG CCAAGGCACCACTCTCACAGTCTCCTCA The amino acid sequence of MALNVAPVRDTKWLTLEVCRQFQRGTCSRSDEECKFAHPPK the human M BNL2 protein, SCQVENGRVIACFDSLKGRCSRENCKYLHPPTHLKTQLEINGR isoform 1 (GenBank Accession NNLIQQKTAAAMLAQQMQFM FPGTPLHPVPTFPVGPAIGT No. NP_659002.1) NTAISFAPYLAPVTPGVGLVPTEILPTTPVIVPGSPPVTVPGST ATQKLLRTDKLEVCREFQRGNCARGETDCRFAHPADSTM ID

TSDNTVTVCMDYIKGRCMREKCKYFHPPAHLQAKIKAAQHQ ANQAAVAAQAAAAAATVMAFPPGALHPLPKRQALEKSNGT SAVFNPSVLHYQQALTSAQLQQHAAFIPTGSVLCMTPATSIV PM M HSATSATVSAATTPATSVPFAATATANQII LK The amino acid sequence of MALNVAPVRDTKWLTLEVCRQFQRGTCSRSDEECKFAHPPK the human M BNL2 protein, SCQVENGRVIACFDSLKGRCSRENCKYLHPPTHLKTQLEINGR isoform 3 (GenBank Accession NNLIQQKTAAAMLAQQMQFM FPGTPLHPVPTFPVGPAIGT No. NP_997187.1) NTAISFAPYLAPVTPGVGLVPTEILPTTPVIVPGSPPVTVPGST ATQKLLRTDKLEVCREFQRGNCARGETDCRFAHPADSTM ID TSDNTVTVCMDYIKGRCMREKCKYFHPPAHLQAKIKAAQHQ ANQAAVAAQAAAAAATVMAFPPGALHPLPKRQALEKSNGT SAVFNPSVLHYQQALTSAQLQQHAAFIPTDNSEIISRNGMEC QESALRITKHCYCTYYPVSSSIELPQTA

The amino acid sequence of MTAVNVALIRDTKWLTLEVCREFQRGTCSRADADCKFAHPP the human M BNL3 protein, RVCHVENGRVVACFDSLKGRCTRENCKYLHPPPHLKTQLEIN isoform G (GenBank Accession GRNNLIQQKTAAAMFAQQMQLM LQNAQMSSLGSFPMTP No. NP_060858.2) SIPANPPMAFNPYIPHPGMGLVPAELVPNTPVLIPGNPPLAM PGAVGPKLM RSDKLEVCREFQRGNCTRGENDCRYAHPTDA SM IEASDNTVTICMDYIKGRCSREKCKYFHPPAHLQARLKAA HHQM NHSAASAMALQPGTLQLIPKRSALEKPNGATPVFNP TVFHCQQALTNLQLPQPAFIPAGPILCMAPASNIVPMMHGA TPTTVSAATTPATSVPFAAPTTGNQLKF

The amino acid sequence of MTAVNVALIRDTKWLTLEVCREFQRGTCSRADADCKFAHPP the human M BNL3 protein, RVCHVENGRVVACFDSLKGRCTRENCKYLHPPPHLKTQLEIN isoform R (GenBank Accession GRNNLIQQKTAAAMFAQQMQLM LQNAQMSSLGSFPMTP No. NP_597846.1) SIPANPPMAFNPYIPHPGMGLVPAELVPNTPVLIPGNPPLAM PGAVGPKLM RSDKLEVCREFQRGNCTRGENDCRYAHPTDA SM IEASDNTVTICMDYIKGRCSREKCKYFHPPAHLQARLKAA HHQM NHSAASAMALTNLQLPQPAFIPAGPILCMAPASNIVP M M H G ATPTTVS AATTP ATSVP F AAPTTG N QI PQLS I D E LNSS MFVSQM

Amino acids 1-116 of the MAVSVTPIRDTKWLTLEVCREFQRGTCSRPDTECKFAHPSKS human MBNL1 protein, CQVENGRVIACFDSLKGRCSRENCKYLHPPPHLKTQLEINGR isoform a (GenBank Accession N N LIQQKN MAM LAQQM LAN AM M PG APLQPVP No. NP_066368.2) or isoform b human kappa light chain RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC

Exemplary signal sequence MDMRVPAQLLGLLLLWLRGARC Exemplary signal sequence MEFGLSWLFLVAILKGVQC Exemplary blunt end DNA GGGTGAACCTGCAGGTGGGCAAAGATGTCC substrate

Morpholino to skip murine GGCCAAACCTCGGCTTACCTGAAAT DMD exon 23 in Mdx mice Hu3E10 H3 N279Q MEFGLSWLFLVAILKGVQCEVQLQESGGGVVQPGGSLRLSC aglycosylated huIgGl heavy AASGFTFSNYGMHWIRQAPGKGLEWVSYISSGSSTIYYADSV chain KGRFTISRDNSKNTLYLQM NSLRSEDTAVYYCARRGLLLDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF

A native human signal PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL sequence compatible with the GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL human acceptor heavy chain GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW HV3 germline gene framework YVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGK in hu3E10 is bolded and EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN underlined, with signalP QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS predicting appropriate signal FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS cleavage PG The human IgGl constant domain represents the Glml7,l allotype commonly expressed among caucasoid, negroid, and mongoloid populations (Glml7 Lys214 in Kabat numbering is italicized, versus Arg214 in Glm3 allotype; and Glml Asp356 and Leu358 is bolded, versus Glu356 and Met358 in nGlml allotype).

Hu3E10 L2 kappa light chain M DM RVP AQLLG LLLLWLRG ARC D IQMTQSPSS LS ASVG D R

VTISCRASKSVSTSSYSYM HWYQQKPEKAPKLLIKYASYLQSG

A native human signal VPSRFSGSGSGTDFTLTISSLQPEDVATYYCQHSREFPWTFGA sequence compatible with the GTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA human acceptor kappa light KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY chain KV1 germline gene EKHKVYACEVTHQGLSSPVTKSFNRGEC framework in hu3E10 is bolded, with signalP predicting appropriate signal cleavage.

The human kappa constant domain above represents the Km3 allotype (Km3 Alal53 and Vall91 in Kabat numbering are italicized, versus Kml Vall53 and Leul91 or Kml, 2 Alal53 and Leul91 Hu3E10 Fab heavy chain MEFGLSWLFLVAILKGVQCEVQLQESGGGVVQPGGSLRLSC

AASGFTFSNYGMHWIRQAPGKGLEWVSYISSGSSTIYYADSV

A native human KGRFTISRDNSKNTLYLQM NSLRSEDTAVYYCARRGLLLDYW immunoglobulin signal GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF sequence is bolded PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL

GTQTYICNVNHKPSNTKVDKKVEPKSCDK

Human IgGl CHI and truncated hinge constant domains of Glml7 allotype Hu3E10 Fab light chain M DM RVP AQLLG LLLLWLRG ARC D IQMTQSPSS LS ASVG D R

VTISCRASKSVSTSSYSYM HWYQQKPEKAPKLLIKYASYLQSG

A native human VPSRFSGSGSGTDFTLTISSLQPEDVATYYCQHSREFPWTFGA immunoglobulin signal GTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA sequence is bolded KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY

E KH KVYAC EVTH QG LSS PVTKS F N RG EC

Human kappa constant domain (Km3 allotype)

Embodiments The disclosure provides at least the following embodiments:

Embodiment 1. An antibody-drug conjugate (ADC), comprising a humanized 3E10 monoclonal antibody (mAb) or derivative thereof conjugated to a nucleic acid payload through a heterobifunctional linker.

Embodiment 2. The ADC of Embodiment 1, wherein the linker is a non- cleavable linker.

Embodiment 3. The ADC of Embodiment 2, wherein the humanized 3E10 mAb is conjugated to the linker through a chemical reaction of a primary amine or the primary amine of a solvent-exposed lysine of the mAb and an N-hydroxysuccinimide functional group.

Embodiment 4. The ADC of any one of Embodiments 2-3, wherein the nucleic acid payload is conjugated to the linker through a click chemistry reaction, wherein an azide functional group is reacted with a bicyclo [6.1.0] nonyne functional group.

Embodiment 5. The ADC of any one of Embodiments 2-4, wherein the linker comprises an N-hydroxysuccinimide functional group, a bicyclo [6.1.0] nonyne functional group, and a poly-ethylene glycol (PEG)-based spacer of 1 or more repeating PEG units.

Embodiment 6. The ADC of Embodiment 1, wherein the linker is a cleavable linker.

Embodiment 7. The ADC of Embodiment 6, wherein the humanized 3E10 mAb is conjugated to the linker through a chemical reaction of an N-terminal primary amine or the primary amine of a solvent-exposed lysine of the mAb and an N-hydroxysuccinimide functional group.

Embodiment 8. The ADC of any one of Embodiments 6-7, wherein the nucleic acid payload is conjugated to the linker through a click chemistry reaction, wherein an azide functional group is reacted with a bicyclo [6.1.0] nonyne functional group. Embodiment 9. The ADC of any one of Embodiments 6-8, wherein the cleavable linker is cleavable by enzyme or reduction.

Embodiment 10. The ADC of any one of Embodiments 6-8, wherein the linker comprises an N-hydroxysuccinimide functional group, a bicyclo [6.1.0] nonyne functional group, and a poly-ethylene glycol (PEG)-based spacer of 1 or more repeating PEG units, and an internal reducible disulfide bond.

Embodiment 11. The ADC of any one of Embodiments 6-8, wherein the linker comprises an N-hydroxysuccinimide functional group, a bicyclo [6.1.0] nonyne functional group, and a poly-ethylene glycol (PEG)-based spacer of 1 or more repeating PEG units, and an internal valine-citrulline enzymatic cleavage site.

Embodiment 12. The ADC of any one of Embodiments 1-11, wherein the nucleic acid payload is a phosphorodiamidate morpholino oligomer.

Embodiment 13. The ADC of any one of Embodiments 1-11, wherein the nucleic acid payload is an antisense oligonucleotide.

Embodiment 14. The ADC of any one of Embodiments 1-11, wherein the nucleic acid pay load is a short interfering RNA (siRNA).

Embodiment 15. The ADC of any one of Embodiments 1-11, wherein the nucleic acid payload acts on a nucleus-localized substrate of a cell.

Embodiment 16. The ADC of any one of Embodiments 1-11, wherein the nucleic caid payload acts on a cytoplasm-localized substrate of a cell.

Embodiment 17. The ADC of Embodiment 15, wherein the substrate is mRNA and the nucleic acid payload degrades the mRNA in the cell nucleus.

Embodiment 18. The ADC of Embodiment 15, wherein the substrate is pre- mRNA and the nucleic acid pay load modulates splicing of the pre-mRNA in the cell nucleus. Embodiment 19. The ADC of any one of Embodiments 15-18, wherein the cell is a cardiac tissue cell, and optionally wherein the nucleic acid payload is a splice switching oligonucleotide.

Embodiment 20. The ADC of any one of Embodiments 15-18, wherein the cell is a skeletal muscle tissue cell.

Embodiment 21. The ADC of any one of Embodiments 15-18, wherein the cell is a liver tissue cell.

Embodiment 22. The ADC of any one of Embodiments 15-18, wherein the cell is a lung tissue cell.

Embodiment 23. The ADC of any one of Embodiments 15-18, wherein the cell is a kidney tissue cell.

Embodiment 24. The ADC of any one of Embodiments 1-23, wherein the humanized 3E10 mAb is a full-length antibody.

Embodiment 25. The ADC of Embodiment 24, wherein the full-length antibody is an IgG molecule.

Embodiment 26. The ADC of any one of Embodiments 1-23, wherein the humanized 3E10 mAb is a derivative of a full-length antibody.

Embodiment 27. The ADC of Embodiment 24, wherein the derivative is an antigen-binding fragment of the humanized 3E10 mAb.

Embodiment 28. The ADC of Embodiment 27, wherein the antigen-binding fragment is an scFv or a bi-, tri-, tetra-, penta- or hexa-valent scFv tandem repeat (TaFv).

Embodiment 29. The ADC of Embodiment 27, wherein the antigen-binding fragment is Fab, Fab', F(ab')2, or Fv. Embodiment 30. The ADC of Embodiment 27, wherein the antigen-binding fragment is a single-chain antibody or a nanobody.

Embodiment 31. The ADC of any one of Embodiments 1-29, wherein the ADC is conjugated to a detectable label.

Embodiment 32. A method for delivering a nucleic acid payload to a tissue, comprising: administering an antibody-drug conjugate (ADC), comprising a humanized 3E10 monoclonal antibody (mAb) or derivative thereof conjugated to a nucleic acid payload through a heterobifunctional linker to a subject to deliver the nucleic acid payload to a tissue, wherein the tissue is selected from cardiac, lung, kidney, liver, and skeletal muscle.

Embodiment 33. The method of Embodiment 32, wherein the administration is intravenous administration.

Embodiment 34. The method of Embodiment 32, wherein the administration is subcutaneous administration.

Embodiment 35. The method of any one of Embodiments 32-34, wherein the tissue is lung tissue.

Embodiment 36. The method of any one of Embodiments 32-35, wherein the 3E10 antibody is a humanized 3E10 mAb.

Embodiment 37. The method of any one of Embodiments 32-36, wherein the pay load is a nucleic acid payload.

Embodiment 38. The method of any one of Embodiments 32-37, wherein the ADC is an ADC of any one of Embodiments 1-31.

Embodiment 39. The method of Embodiment 37, wherein the tissue is a cardiac tissue, and optionally wherein the nucleic acid payload is a splice switching oligonucleotide. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.

While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document. The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be appreciated that embodiments described in this document using an open-ended transitional phrase (e.g., “comprising”) are also contemplated, in alternative embodiments, as “consisting of’ and “consisting essentially of’ the feature described by the open-ended transitional phrase. For example, if the disclosure describes “a composition comprising A and B”, the disclosure also contemplates the alternative embodiments “a composition consisting of A and B” and “a composition consisting essentially of A and B”.

Unless otherwise stated, any numerical values, such as a concentration or a concentration range described herein, are to be understood as being modified in all instances by the term “about.” Thus, a numerical value typically includes ±10% of the recited value. For example, a concentration of 1 mg/mE includes 0.9 mg/mE to 1.1 mg/mL. Likewise, a concentration range of 1% to 10% (w/v) includes 0.9% (w/v) to 11% (w/v). As used herein, the use of a numerical range expressly includes all possible subranges, all individual numerical values within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise.

Unless otherwise indicated, the term “at least” preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the invention. It should also be understood that the terms “about,” “approximately,” “generally,” “substantially” and like terms, used herein when referring to a dimension or characteristic of a component of the preferred invention, indicate that the described dimension/characteristic is not a strict boundary or parameter and does not exclude minor variations therefrom that are functionally the same or similar, as would be understood by one having ordinary skill in the art. At a minimum, such references that include a numerical parameter would include variations that, using mathematical and industrial principles accepted in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.), would not vary the least significant digit.

Claims

1. An antibody-drug conjugate (ADC), comprising a humanized 3E10 monoclonal antibody (mAb) or derivative thereof conjugated to a nucleic acid payload through a non-cleavable heterobifunctional linker, wherein the humanized 3E10 mAb is conjugated to the linker through a chemical reaction of an N-terminal primary amine or the primary amine of a solvent-exposed lysine of the mAb and an N-hydroxysuccinimide functional group.

2. The ADC of claim 1, wherein the nucleic acid pay load is conjugated to the linker through a click chemistry reaction, wherein an azide functional group is reacted with a bicyclo [6.1.0] nonyne functional group.

3. The ADC of claim 1 or claim 2, wherein the linker comprises an N- hydroxysuccinimide functional group, a bicyclo[6.1.0]nonyne functional group, and a polyethylene glycol (PEG)-based spacer of 1 or more repeating PEG units.

4. An antibody-drug conjugate (ADC), comprising a humanized 3E10 monoclonal antibody (mAb) or derivative thereof conjugated to a nucleic acid payload through cleavable linker, wherein the humanized 3E10 mAb is conjugated to the linker through a chemical reaction of an N-terminal primary amine or the primary amine of a solvent-exposed lysine of the mAb and an N-hydroxysuccinimide functional group and wherein the cleavable linker is cleavable by enzyme or reduction.

5. The ADC of claim 4, wherein the nucleic acid pay load is conjugated to the linker through a click chemistry reaction, wherein an azide functional group is reacted with a bicyclo [6.1.0] nonyne functional group.

6. The ADC of claim 4 or claim 5, wherein the linker comprises an N- hydroxysuccinimide functional group, a bicyclo[6.1.0]nonyne functional group, and a polyethylene glycol (PEG)-based spacer of 1 or more repeating PEG units, and an internal reducible disulfide bond.

7. The ADC of claim 4 or claim 5, wherein the linker comprises an N- hydroxysuccinimide functional group, a bicyclo[6.1.0]nonyne functional group, and a poly- ethylene glycol (PEG)-based spacer of 1 or more repeating PEG units, and an internal valinecitrulline enzymatic cleavage site.

8. The ADC of any one of claims 1-7, wherein the nucleic acid pay load is a phosphorodiamidate morpholino oligomer, an antisense oligonucleotide, a short interfering RNA (siRNA), or a splice- switching oligonucleotide.

9. The ADC of any one of claims 1-8, wherein the humanized 3E10 monoclonal antibody (mAb) or derivative thereof comprises a sequence having at least 80% sequence identity to a sequence selected from any one of SEQ ID NO. 1-6, 9-10, 20-21, 32-38, 40-43, 49, 55-60, or 65-66.

10. The ADC of any one of claims 1-8, wherein the humanized 3E10 mAb or derivative thereof comprises a sequence having at least 95% sequence identity to a sequence selected from any one of SEQ ID NO. 1-6, 9-10, 20-21, 32-38, 40-43, 49, 55-60, or 65-66.

11. The ADC of any one of claims 1-8, wherein the humanized 3E10 mAb or derivative thereof comprises a sequence selected from any one of SEQ ID NO. 1-6, 9-10, 20- 21, 32-38, 40-43, 49, 55-60, or 65-66.

12. The ADC of any one of claims 1-8, wherein the humanized 3E10 mAb or derivative thereof is an antigen-binding fragment of the humanized 3E10 mAb, optionally wherein the antigen-binding fragment is an scFv, a Fab, a single-chain antibody, or a nanobody.

13. The ADC of any one of claims 1-12, wherein the nucleic acid pay load comprises up to 6 oligonucleotides linked to each humanized 3E10 mAb.

14. The ADC of any one of claims 1-13, wherein the nucleic acid payload acts on a nucleus-localized substrate of a cell.

15. The ADC of any one of claims 1-13, wherein the nucleic acid pay load acts on a cytoplasm-localized substrate of a cell.

16. The ADC of claim 14, wherein the nucleus-localized substrate is mRNA or pre-mRNA and the nucleic acid pay load degrades the mRNA in the cell nucleus.

17. The ADC of claim 14, wherein the nucleus-localized substrate is pre-mRNA and the nucleic acid payload modulates splicing of the pre-mRNA in the cell nucleus.

18. The ADC of any one of claims 1-17, wherein the nucleic acid pay load is complementary to a nucleic acid of a cardiac cell, a skeletal muscle cell, a liver cell, a lung cell, and/or a kidney cell.

19. The ADC of any one of claims 1-11 or 14-17, wherein the humanized 3E10 mAb is a full-length antibody, which is optionally an IgG molecule.

20. An antibody-drug conjugate (ADC), comprising a humanized 3E10 monoclonal antibody (mAb) conjugated to a nucleic acid payload, wherein the humanized 3E10 mAb comprises an amino acid sequence having at least 98% sequence identity to a sequence of SEQ ID NO. 65 or 66.

21. The ADC of claim 20, wherein the humanized 3E10 mAb comprises an amino acid sequence of SEQ ID NO. 65 or 66.

22. The ADC of claim 20, wherein the humanized 3E10 mAb comprises a heavy chain of SEQ ID NO. 65 and a light chain of SEQ ID NO. 66.

23. The ADC of any one of claims 20-22, wherein the humanized 3E10 mAb is conjugated to the nucleic acid payload by a cleavable or non-cleavable heterobifunctional linker.

24. The ADC of any one of claims 20-23, wherein the nucleic acid payload is a phosphorodiamidate morpholino oligomer, an antisense oligonucleotide, a short interfering RNA (siRNA), or a splice- switching oligonucleotide.

25. The ADC of any one of claims 20-24, wherein the humanized 3E10 mAb is an IgG molecule.

26. An antibody-drug conjugate (ADC), comprising a humanized 3E10 monoclonal antibody (mAb) or derivative thereof conjugated to a nucleic acid payload through a cleavable or non-cleavable heterobifunctional linker, wherein the nucleic acid pay load comprises at least 4-9 oligonucleotides linked to each humanized 3E10 mAb.

27. An antibody-drug conjugate (ADC), comprising a humanized 3E10 antigen binding fragment conjugated to a nucleic acid payload through a cleavable or non-cleavable heterobifunctional linker, wherein the antigen-binding fragment is an scFv, a Fab, a singlechain antibody, or a nanobody.

28. The ADC of claim 26 or 27, wherein the nucleic acid payload is conjugated to the linker through a click chemistry reaction, wherein an azide functional group is reacted with a bicyclo [6.1.0] nonyne functional group.

29. The ADC of any one of claims 26-28, wherein the linker comprises an N- hydroxysuccinimide functional group, a bicyclo[6.1.0]nonyne functional group, and a polyethylene glycol (PEG)-based spacer of 1 or more repeating PEG units.

30. The ADC of any one of claims 26-29, wherein the nucleic acid payload is a phosphorodiamidate morpholino oligomer, an antisense oligonucleotide, a short interfering RNA (siRNA), or a splice- switching oligonucleotide.

31. The ADC of claim 26, wherein the humanized 3E10 mAb or derivative thereof is an antigen-binding fragment of the humanized 3E10 mAb, optionally wherein the antigenbinding fragment is an scFv, a Fab, a single-chain antibody, or a nanobody or wherein the humanized 3E10 mAb comprises an amino acid sequence having at least 98% sequence identity to a sequence of SEQ ID NO. 65 or 66.

32. The ADC of claim 27, wherein the nucleic acid payload comprises up to 6 oligonucleotides linked to each humanized 3E10 mAb.

33. The ADC of any one of claims 26-32, wherein the nucleic acid pay load is complementary to a nucleic acid of a cardiac cell, a skeletal muscle cell, a liver cell, a lung cell, and/or a kidney cell.

34. A method for delivering a nucleic acid payload to a tissue, comprising: administering an antibody-drug conjugate (ADC), comprising a humanized 3E10 monoclonal antibody (mAb) or derivative thereof conjugated to a nucleic acid payload through a heterobifunctional linker to a subject to deliver the nucleic acid payload to a tissue, wherein the tissue is selected from cardiac, lung, kidney, liver, and skeletal muscle.

35. The method of claim 34, wherein the ADC is an ADC of any one of claims 1- 33.

36. The method of claim 34 or 35, wherein the administration is intravenous administration or subcutaneous administration.

37. The method of any one of claims 34-36, wherein the tissue is lung tissue.

38. The method of any one of claims 34-36, wherein the tissue is a cardiac tissue, and optionally wherein the nucleic acid payload is a splice switching oligonucleotide.