Humanized 3E10 antibody, variants thereof, and antigen-binding fragments thereof

Abstract

The present disclosure provides humanized 3E10 antibodies and antigen-binding fragments thereof. Also disclosed are compositions and methods of using said humanized 3E10 antibodies and antigen-binding fragments thereof to deliver cargo. TIFF2025507840000048.tif212164

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 316,338, filed March 3, 2022, the disclosure of which is incorporated by reference in its entirety herein.

[0002] Technical Field The present disclosure relates to humanized 3E10 antibodies and antigen-binding fragments thereof, for example, for delivering therapeutic cargo to cells.

[0003] Brief Description of the Sequence Listing This submission is being submitted as an XML file via the USPTO Electronic Patent Filing System in accordance with 37 CFR §§ 1.831-1.835, with the attachment of a "Sequence Listing XML" containing SEQ ID NOs:1-161, 169 KB, created on March 1, 2023. 37 CFR § 1.835(a)(1). [Background technology]

[0004] background Among polynucleotide-based cancer therapies, many different strategies have been deployed. For example, immunostimulatory polynucleotides, such as pattern recognition receptors, have been used to stimulate proinflammatory cytokine mediators in various cancer immunotherapies. For example, gene regulatory polynucleotides, such as siRNA, miRNA, and ASO, have been used to silence target genes and control signaling pathways involved in cancer progression. Polynucleotides encoding therapeutic proteins, such as mRNA or plasmids encoding antigens or cancer immunotherapy proteins, have been used in therapy. Functional nucleic acids, such as aptamers, have been used as well as antibody-based cancer therapies, for example by binding to and blocking tumor targets such as PD-1. Gene editing polynucleotides have also been used to silence the expression of cancer mediators. For a review of these various polynucleotide-based cancer therapies, see, for example, Zhou S.et at., Medicine in Drug Discovery, 2020.6:100023 (Non-Patent Document 1) and Hager et al., Cells.2020.9(9):2061 (Non-Patent Document 2). The contents of that document are incorporated herein by reference.

[0005] Although these polynucleotide-based therapies have shown some success in preclinical trials, they have been disappointing in clinical trials when evaluated for therapeutic efficacy. See, for example, Lopes et al., Cancer DNA vaccine: Current preclinical and clinical developments and future perspectives. J. Exp. Clin. Cancer Res. 2019. 38, 146 (Non-Patent Document 3); Dome et al., Therapeutic Cancer Vaccination with ex vivo RNA-Transfected Dendritic Cells-An Update. Pharmaceutics. 2020. 12, 92 (Non-Patent Document 4), the contents of which are incorporated herein by reference in their entirety. One obstacle is that nucleic acids cannot easily cross cell membranes. Furthermore, nucleic acids are easily degraded by extracellular nucleases present in skin, tissues, and blood. Kowalski PS et al., Mol Ther., 27(4): 710-28 (2019) (Non-Patent Document 5). The contents of that document are incorporated herein by reference in their entirety.

[0006] The mouse anti-DNA antibody 3E10 is known to penetrate cells and at least partially localize in the nucleus of cells. See, for example, Weisbart RH et al. 1998. J.Autoimmun.;11:539-546 (Non-Patent Document 6), the contents of which are incorporated herein by reference in their entirety. Thus, it has been suggested that 3E10 and its derivatives may function as targeting agents for delivering therapeutic agents in vivo. However, mouse antibodies are immunogenic when administered to humans, especially in chronic administration. Several techniques are known in the art for reducing the immunogenicity of non-human antibodies by humanization. However, not all humanized antibodies retain the advantageous characteristics of their parent non-human antibodies. [Prior art documents]

Non-Patent Literature

[0007]

Non-Patent Literature 1

Non-Patent Literature 2

Non-Patent Literature 3

Non-Patent Literature 4

Non-Patent Literature 5

Non-Patent Literature 6

Summary of the Invention

[0008] Summary In view of the above background, there is a need in the art for humanized 3E10 antibodies and antigen-binding fragments thereof. Such humanized 3E10 antibodies would facilitate improved methods for delivering therapeutic cargos, such as therapeutic polynucleotides, polypeptides, and chemical agents, to target cells. Polynucleotide-based therapeutics offer a promising avenue for the treatment of diseases due to their versatility in encoding any polypeptide, availability of highly reproducible manufacturing methods, ability to make simple and precise adjustments to polynucleotide sequences, low cost, and ability to specifically target and / or edit any gene sequence. However, there are many challenges in delivering polynucleotide therapeutics to specific tissues in vivo, including rapid degradation of exogenous nucleic acids in the body and immunogenicity caused by common delivery vehicles, such as liposomes and viral vectors. See, for example, Zhou et al., Medicine in Drug Discovery, 6 (2020) 100023 and Dahlman et al., Nature Nanotechnol. 9 (8): 648-655 (2014), the contents of which are incorporated herein by reference in their entirety.

[0009] Advantageously, the present disclosure provides humanized 3E10 antibodies and antigen-binding fragments thereof that retain core 3E10 properties such as ENT2-based cell penetrating activity and nucleic acid binding activity. Accordingly, as described herein, the present disclosure provides humanized 3E10 antibodies and antigen-binding fragments thereof, pharmaceutical compositions, and methods for using same to treat various medical disorders.

[0010] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a light chain variable domain (3E10-VL) comprising an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of 3E10-VL-h1 (SEQ ID NO: 85), 3E10-VL-h2 (SEQ ID NO: 86), 3E10-VL-h3 (SEQ ID NO: 87), 3E10-VL-h4 (SEQ ID NO: 88), 3E10-VL-h5 (SEQ ID NO: 89), and 3E10-VL-h6 (SEQ ID NO: 90).

[0011] In some embodiments, the light chain variable domain (3E10-VL) comprises one or more amino acid residues selected from proline (Pro) at position 15, threonine (Thr) at position 22, tyrosine (Tyr) at position 49, Thr at position 74, asparagine (Asn) at position 76, alanine (Ala) at position 80, Asn at position 81, Thr at position 83, Asn at position 85, and valine (Val) at position 104, numbered by Kabat numbering. In some embodiments, 3E10-VL comprises a set of CDRs having a total of six or fewer amino acid substitutions compared to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NO: 9), 3E10-VL-CDR2 (SEQ ID NO: 10), 3E10-VL-CDR3 (SEQ ID NO: 11).

[0012] Similarly, in some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a heavy chain variable domain (3E10-VH (SEQ ID NO: 2)) comprising an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of 3E10-VH-h1 (SEQ ID NO: 64), 3E10-VH-h2 (SEQ ID NO: 65), 3E10-VH-h3 (SEQ ID NO: 66), 3E10-VH-h4 (SEQ ID NO: 67), 3E10-VH-h5 (SEQ ID NO: 68), 3E10-VH-h6 (SEQ ID NO: 69), and 3E10-VH-h7 (SEQ ID NO: 70).

[0013] In some embodiments, the heavy chain variable domain (3E10-VH) comprises one or more amino acid residues selected from glutamine (Gln) at position 13, leucine (Leu) at position 18, arginine (Arg) at position 19, glycine (Gly) at position 42, serine (Ser) at position 49, Ser at position 77, tyrosine (Tyr) at position 79, Asn at position 82, Ala at position 84, Val at position 89, leucine (Leu) at position 108, Val at position 109, and Ser at position 113, numbered by the Kabat numbering system. In some embodiments, 3E10-VH comprises a set of CDRs having a total of six or fewer amino acid substitutions compared to the set of CDRs having the amino acid sequences of 3E10-VH-CDR1_D31N (sequence number 15), 3E10-VH-CDR2 (sequence number 4), and 3E10-VH-CDR3 (sequence number 5).

[0014] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a heavy chain CDR1 having an aspartic acid at position 31 as numbered by Kabat numbering.

[0015] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a lysine at position 72 of 3E10-VL numbered by Kabat numbering, which supports nucleic acid binding affinity. Mutation of this amino acid residue to tyrosine abolishes high affinity binding to DNA substrates. Additionally, arginine and lysine at positions 37 and 38 in 3E10-VH are believed to be beneficial for nucleic acid binding. Mutation of these residues to leucine and arginine, respectively, reduces the ability of the antibody to bind to DNA substrates with high affinity.

[0016] In some embodiments, the disclosure provides compositions and methods for improving delivery of nucleic acids to cells without relying on a separate delivery vehicle, such as, for example, liposomes, viral vectors, etc. In some embodiments, the disclosure provides compositions and methods for improving delivery of nucleic acids to cells. In some embodiments, the composition includes (i) a humanized 3E10 antibody or antigen-binding fragment thereof having nucleic acid binding activity, and (ii) a nucleic acid cargo, such as a therapeutic polynucleotide, a nucleic acid encoding a polypeptide, a functional nucleic acid, a nucleic acid encoding a functional nucleic acid, or a combination thereof. In some embodiments, elements (i) and (ii) are non-covalently linked to form a complex. In some embodiments, elements (i) and (ii) are covalently linked. Nucleic acids in various embodiments include DNA (single-stranded or double-stranded), RNA, PNA, and other modified nucleic acids.

[0017] In some embodiments, the disclosure provides a humanized 3E10 antibody or antigen-binding fragment thereof covalently linked to a therapeutic agent. In some embodiments, the therapeutic agent is a therapeutic nucleic acid. In some embodiments, the therapeutic agent is a chemical agent, and in some embodiments, the therapeutic agent is a therapeutic protein or polypeptide. In some embodiments, these conjugates are used in methods for delivering a therapeutic agent to a cell without the need for a separate delivery vehicle, such as, for example, a liposome, viral vector, etc. In some embodiments, the conjugate is a fusion protein in which the 3E10 antibody or antigen-binding fragment thereof polypeptide and the therapeutic polypeptide are encoded and translated from the same open reading frame.

[0018] Also provided is a method of delivering cargo, such as a nucleic acid, a chemical agent, or a therapeutic protein or polypeptide, to a cell by contacting the cell with the complexes and an effective amount of the complexes described herein. The contacting can be in vitro, ex vivo, or in vivo. In some embodiments, an effective amount of ex vivo treated cells is administered to a subject in need thereof, for example, in an amount effective to treat one or more symptoms of a disease or disorder.

[0019] In some embodiments, the contacting is performed in vivo after administration to a subject in need thereof. The subject may have a disease or disorder, such as a genetic disorder or cancer. The composition may be administered to the subject, for example, by injection or infusion, in an amount effective to reduce one or more symptoms of the disease or disorder in the subject.

[0020] Applications of the compositions and methods are also provided, including, but not limited to, gene therapy and T cell or CAR T cell manufacturing, formation, and / or treatment. [Brief description of the drawings]

[0021] [Figure 1] FIG. 1 (SEQ ID NOs: 1-12) shows the amino acid sequence of the parent 3E10 monoclonal antibody. [Figure 2A] Figures 2A (SEQ ID NOs: 13-25), 2B, and 2C show the amino acid sequences of the D31N variant (Figure 2A (SEQ ID NOs: 13-25)), other CDR variants (Figure 2B), and additional contemplated CDR variants (Figure 2C) of the 3E10 monoclonal antibody according to some embodiments of the present disclosure. [Figure 2B] See legend to Figure 2A. [Figure 2C] See legend to Figure 2A. [Diagram 3] 1 shows examples of charge-conserving CDR variants of the 3E10 monoclonal antibody according to various embodiments of the disclosure. [Figure 4] 1 shows examples of CDR variants comprising combinations of amino acid substitutions, charge-conservative amino acid substitutions, and rationally-designed amino acid substitutions of the 3E10 monoclonal antibody, according to various embodiments of the disclosure. [Diagram 5] 1 shows the amino acid sequence of a humanized 3E10 variable heavy chain (3E10-VH) domain according to various embodiments of the present disclosure. [Figure 6] 1 shows the amino acid sequence of the mature humanized 3E10 heavy chain lacking the signal peptide (3E10-HC) according to various embodiments of the present disclosure. [Figure 7]1 shows the amino acid sequence of humanized 3E10 heavy chain (3E10-HC) according to various embodiments of the present disclosure. [Figure 8] 1 shows the amino acid sequence of a humanized 3E10 variable light chain (3E10-VL) domain according to various embodiments of the present disclosure. [Figure 9] 1 shows the amino acid sequence of the mature humanized 3E10 light chain lacking the signal peptide (3E10-LC) according to various embodiments of the present disclosure. [Figure 10] 1 shows the amino acid sequence of humanized 3E10 light chain (3E10-LC) according to various embodiments of the present disclosure. [Figure 11A] Figures 11A and 11B show electrostatic surface potential renderings of a molecular model of the 3E10-scFv construct, revealing a putative nucleic acid binding pocket (NAB1). In addition, Figure 11A shows the predicted structural and electrostatic potential changes induced by an amino acid substitution at residue HC CDR1 residue 31. Figure 11B is a representation of the molecular modeling (Pymol) of 3E10-scFv with the NAB1 amino acid residues highlighted with punctate dots. [Figure 11B] See legend to Figure 11A. [Figure 12-1] 12A, 12B, 12C, 12D, and 12E summarize the results of the nucleic acid binding characterization of various humanized 3E10 constructs described in Example 1, according to some implementations of the present disclosure. [Figure 12-2] See description of Figure 12-1. [Figure 12-3] See description of Figure 12-1. [Figure 13] FIG. 1 shows a histogram summarizing the 4-day time course of type 1 IFN responses in THP-1 monocytes PBS (control), 3p-hpRNA RIG-I agonist alone (1 ug / well), increasing amounts of humanized 3E10 antibody alone, and humanized 3E10 antibody / 3p-hpRNA (1 ug 3p-hpRNA / well) as described in Example 3. [Figure 14]FIG. 1 shows a histogram summarizing the 4-day time course of type 1 IFN responses in THP-1 monocytes PBS (control), 3p-hpRNA RIG-I agonist alone (1 ug / well), increasing amounts of humanized 3E10 antibody alone, and humanized 3E10 antibody / 3p-hpRNA (1 ug 3p-hpRNA / well) as described in Example 3. [Figure 15] FIG. 1 shows a histogram summarizing the 4-day time course of type 1 IFN responses in THP-1 monocytes PBS (control), 3p-hpRNA RIG-I agonist alone (1 ug / well), increasing amounts of humanized 3E10 antibody alone, and humanized 3E10 antibody / 3p-hpRNA (1 ug 3p-hpRNA / well) as described in Example 4. [Figure 16] FIG. 1 shows a histogram summarizing the 4-day time course of type 1 IFN responses in THP-1 monocytes PBS (control), 3p-hpRNA RIG-I agonist alone (1 ug / well), increasing amounts of humanized 3E10 antibody alone, and non-humanized 3E10 antibody / 3p-hpRNA (1 ug 3p-hpRNA / well) as described in Example 3. [Figure 17] 1 shows histograms summarizing humanized 3E10 antibody uptake (+ / -) dipyridamole in tumor, liver, kidney, spleen, quadriceps, and gastrocnemius in tumor-bearing mice (CT-26 colorectal cancer model). [Figure 18] FIG. 1 shows an experimental scheme for measuring the biodistribution of 3E10-D31N IgG4 variant in a pancreatic ductal adenocarcinoma (PDAC) mouse model. [Figure 19-1] 19A, 19B, 19C, 19D, and 19E show binding kinetics and affinity measurements of 3E10-D31N and the 3E10-D31N IgG4 Fc variant, as described in Example 8. [Figure 19-2] See description of Figure 19-1. [Figure 20]Figure 20A shows chimeric 3E10-D31N delivery of GFP mRNA in an MDA-MB-231 mouse model. Figure 20B shows a comparative study testing chimeric 3E10-D31N delivery of GFP mRNA and humanized 3E10 antibody construct (V66) delivery of GFP mRNA in a KPC syngeneic tumor model. [Figure 21] Tumor and normal tissue expression for targeted functional delivery of GFP mRNA payload is shown. [Figure 22] 1 shows the comparison of chimeric 3E10 D31N and humanized 3E10 antibody (V66) in the B16 tumor model measuring tumor volume days after implantation of antibody:3p-hpRNA complexes. [Figure 23A] Figures 23A, 23B, 23C, 23D, 23E, 23F, 23G, 23H, 23I, 23J, 23K, 23L, 23M, 23N, 23O, 23P, and 23Q summarize the serum and tissue pharmacokinetic profiles of the humanized 3E10(D31N) antibody V66, as indicated in each figure, from a single-dose, dose-escalation, pharmacokinetic study conducted in C57Bl / 6 mice, as described in Example 12. [Figure 23B] See legend to Figure 23A. [Figure 23C] See legend to Figure 23A. [Figure 23D] See legend to Figure 23A. [Figure 23E] See legend to Figure 23A. [Figure 23F] See legend to Figure 23A. [Figure 23G] See legend to Figure 23A. [Figure 23H] See legend to Figure 23A. [Figure 23I] See legend to Figure 23A. [Figure 23J] See legend to Figure 23A. [Figure 23K] See legend to Figure 23A. [Figure 23L] See legend to Figure 23A. [Figure 23M] See legend to Figure 23A. [Figure 23N] See legend to Figure 23A. [Figure 23O] See legend to Figure 23A. [Figure 23P] See legend to Figure 23A. [Figure 23Q] See legend to Figure 23A. [Figure 24A] 24A, 24B, 24C, and 24D provide a summary analysis and statistics for the serum and tissue pharmacokinetic studies of humanized 3E10(D31) antibody V66, as described in Example 12. [Figure 24B] See legend to Figure 24A. [Figure 24C] See legend to Figure 24A. [Figure 24D] See legend to Figure 24A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] Detailed Description In various aspects and embodiments, the present disclosure provides humanized 3E10 antibodies and antigen-binding fragments thereof, and methods for using same to deliver cargo, such as, for example, polynucleotides, polypeptides, or chemical agents, to cells. The humanized 3E10 antibodies and antigen-binding fragments thereof described herein can penetrate cells and aid in the delivery of cargo, such as, for example, polynucleotides, polypeptides, or chemical agents, across the cell membrane into the cytoplasm and / or nucleus without the need for a separate delivery vehicle. Advantageously, the 3E10 antibodies and antigen-binding fragments described herein are humanized and therefore are believed to be less immunogenic when administered to humans compared to the murine and chimeric 3E10 parent antibodies. Furthermore, the humanized 3E10 antibodies and antigen-binding fragments described herein retain important 3E10 properties, such as sequence-nonspecific nucleic acid binding and antigen-mediated cell penetration. Furthermore, as reported in the Examples, various combinations of humanized 3E10 variable heavy and light chain domains have different nucleic acid binding affinities, allowing for better control of nucleic acid binding and release in vivo.

[0023] definition The terms used in this disclosure are merely for describing certain embodiments and are not intended to limit the present invention. When used in the detailed description and the appended claims, the singular forms "a", "an" and "the" are intended to include the plural forms unless the context clearly indicates otherwise. It will also be understood that the term "and / or" as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items. It will be further understood that, unless the context otherwise requires, when used herein, the terms "includes", "comprising", or any variation thereof, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. Additionally, to the extent the terms "including," "includes," "having," "has," "with," or variations thereof, are used in any of the detailed description and / or claims, such terms are intended to be inclusive in a similar manner as the term "comprising."

[0024] Recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated herein as if it were individually recited herein.

[0025] Use of the term "about" is intended to describe a value that is either above or below the stated value within approximately ±10%.

[0026] As used herein, the term "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items.

[0027] Unless the context requires otherwise, as used herein, the terms "includes," "comprising," or any variation thereof, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. Furthermore, to the extent the terms "including," "includes," "having," "has," "with," or variants thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in a similar manner as the term "comprising."

[0028] Recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated herein as if it were individually recited herein.

[0029] As used herein, the term "subject" means any individual who is the target of administration. A subject can be a vertebrate, e.g., a mammal. Thus, a subject can be a human. The term does not denote a particular age or sex.

[0030] As used herein, the term "pharmaceutical effective amount" means that the amount of the composition used is sufficient to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration requires only reduction or alteration, not necessarily elimination. The exact dosage will vary depending on a variety of factors, including subject-dependent variables (e.g., age, immune health, etc.), the disease or disorder being treated, and the route of administration and pharmacokinetics of the agent being administered.

[0031] As used herein, the term "carrier" or "excipient" refers to an organic or inorganic, natural or synthetic inactive ingredient in a formulation with which one or more active ingredients are combined. The carrier or excipient will necessarily be selected to minimize degradation of the active ingredient or to minimize adverse side effects in the subject, as is well known to those of skill in the art.

[0032] As used herein, the term "treat" refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. The term includes active treatment, i.e., treatment specifically aimed at improving the disease, pathological condition, or disorder, and also includes causal treatment, i.e., treatment aimed at eliminating the cause of the associated disease, pathological condition, or disorder. In addition, the term includes palliative treatment, i.e., treatment designed for the relief of symptoms rather than a cure of the disease, pathological condition, or disorder, preventive treatment, i.e., treatment aimed at minimizing or partially or completely preventing the occurrence of the associated disease, pathological condition, or disorder, and supportive treatment, i.e., treatment used to complement another specific therapy aimed at ameliorating the associated disease, pathological condition, or disorder. As used herein, the term "inhibit" or "reduce" means to decrease an activity, response, condition, disease, or other biological parameter. This can include, but is not limited to, the complete disappearance of an activity, response, condition, or disease. It may also include, for example, a statistically significant decrease in an activity, response, condition, or disease as compared to native or control levels.

[0033] In this disclosure, the term "CNS cancer" or "cancer of the central nervous system" refers to an abnormal proliferation of cells derived from any tissue of the central nervous system, including the brain, spinal cord, meninges, or hematopoietic tissue of the main CNS of a subject. Non-limiting examples of CNS cancers include neuroepithelial cancers (such as, for example, gliomas, mature neuronal carcinomas, primitive neuroectodermal tumors, and undifferentiated brain tumors), meningeal cancers, and primary central nervous system hematopoietic cancers.

[0034] Antibodies and their variants "Antigen binding domain" or "ABD" herein refers to a set of six complementarity determining regions (CDRs) that, when present as part of a polypeptide sequence, specifically binds to a target antigen as discussed herein. As known in the art, these CDRs generally exist as a first set of variable heavy chain CDRs (vhCDRs or VHCDRs) and a second set of variable light chain CDRs (vlCDRs or VLCDRs), each of which includes three CDRs, vhCDR1, vhCDR2, vhCDR3 for the heavy chain, and vlCDR1, vlCDR2, and vlCDR3 for the light chain. The CDRs are present in the variable heavy chain domain and the variable light chain domain, respectively, and together form the Fv region. Various CDR numbering schemes are known, as outlined in the table below and the related discussion. Thus, in some cases, the six CDRs of the antigen binding domain are contributed by the variable heavy chain domain and the variable light chain domain. In the "Fab" format, the set of six CDRs is contributed by two different polypeptide sequences, namely a variable heavy domain (vh or VH; containing vhCDR1, vhCDR2, and vhCDR3) and a variable light domain (vl or VL; containing vlCDR1, vlCDR2, and vlCDR3), with the C-terminus of the vh domain attached to the N-terminus of the CH1 domain of the heavy chain and the C-terminus of the vl domain attached to the N-terminus of the constant light domain (thus forming the light chain). In the scFv format, the vh and vl domains are generally covalently linked into a single polypeptide sequence by the use of a linker ("scFv linker") as outlined herein, which can be either vh-linker-vl or vl-linker-vh (starting from the N-terminus), with optional domain linkers on each side, depending on the format used. Generally, the C-terminus of the scFv domain is attached to the N-terminus of the hinge in the second monomer.

[0035] As will be appreciated by those skilled in the art, the exact numbering and arrangement of CDRs may vary among various numbering systems. However, it should be understood that the disclosure of a variable heavy chain sequence and / or a variable light chain sequence includes the disclosure of the associated (unique) CDRs. Thus, the disclosure of each variable heavy chain region is the disclosure of the vhCDRs (e.g., vhCDR1, vhCDR2, and vhCDR3), and the disclosure of each variable light chain region is the disclosure of the vlCDRs (e.g., vlCDR1, vlCDR2, and vlCDR3). A useful comparison for CDR numbering is as follows, see Lafranc et al., Dev. Comp. Immunol. 2003. 27(1):55-77.

[0036] Comparison of CDR numbering TIFF2025507840000002.tif75159

[0037] For all amino acid positions discussed in this disclosure related to antibodies, the numbering of amino acid positions is according to the EU index. EU index or EU index as in Kabat or EU numbering scheme refers to EU antibody numbering. Kabat et al. collected a large number of primary sequences of heavy and light chain variable regions. Based on the degree of sequence conservation, Kabat et al. classified each primary sequence into CDR and framework and created a list thereof. SEQUENCES OF IMMUNOLOGICAL INTEREST , 5th edition, NIH publication, No. 91-3242, EA Kabat et al.; Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, the contents of which are incorporated herein by reference in their entireties. Modifications can be additions, deletions, or substitutions.

[0038] As used herein, "antibody variant" or "variant antibody" refers to an antibody that differs from a parent antibody by at least one amino acid modification, and as used herein, "IgG variant" or "variant IgG" refers to an antibody that differs from a parent IgG (again, often a human IgG sequence) by at least one amino acid modification, and as used herein, "immunoglobulin variant" or "variant immunoglobulin" refers to an immunoglobulin sequence that differs from a parent immunoglobulin sequence by at least one amino acid modification. As used herein, "Fc variant" or "variant Fc" refers to a protein that comprises an amino acid modification in the Fc domain compared to the Fc domain of human IgG1, IgG2, IgG3, or IgG4, as further described herein.

[0039] In some embodiments, the parent polypeptide, e.g., the Fc parent polypeptide, is a human wild-type sequence, such as a heavy chain constant domain or Fc region from IgG1, IgG2, IgG3, or IgG4, although human sequences with variants can also function as "parent polypeptides", e.g., the IgG1 / 2 hybrids of US Publication No. 2006 / 0134105. The protein variant sequences herein preferably have at least about 75% identity to the parent protein sequence, or at least about 80% identity to the parent protein sequence, and most preferably at least about 90% identity, more preferably at least about 95%, or at least about 98%, or at least about 99% sequence identity.

[0040] In some embodiments, a protein variant sequence herein has at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a parent protein sequence.

[0041] As used herein, "isotype" means any of the subclasses of immunoglobulins, which are defined by the chemical and antigenic properties of their constant regions. It is understood that therapeutic antibodies may also include hybrids of isotypes and / or subclasses.

[0042] As used herein, "Fab" or "Fab region" refers to a polypeptide that comprises the VH, CH1, VL, and CL immunoglobulin domains, generally on two different polypeptide chains (e.g., VH-CH1 on one chain and VL-CL on the other chain). Fab may refer to this region alone or in the context of an antibody of the disclosure. In the context of a Fab, the Fab comprises the Fv region in addition to the CH1 and CL domains.

[0043] As used herein, "Fv" or "Fv fragment" or "Fv region" refers to a polypeptide comprising the VL and VH domains of the ABD. The Fv region can be formatted as both a Fab (which, as discussed above, are generally two different polypeptides (also including the constant regions outlined above)) and an scFv, where the vl and vh domains combine to form an scFv (generally with a linker as discussed herein).

[0044] As used herein, "single chain Fv" or "scFv" refers to a variable heavy domain covalently linked to a variable light domain, typically using an scFv linker as discussed herein to form an scFv domain or scFv domains. The scFv domains can be in either orientation (vh-linker-vl or vl-linker-vh) from N-terminus to C-terminus. In this disclosure, the order of the vh and vl domains is indicated in the names, e.g., from N-terminus to C-terminus, H.X_L.Y is vh-linker-vl, and L.Y_H.X is vl-linker-vh.

[0045] As used herein, "Fc" or "Fc region" or "Fc domain" refers to a polypeptide comprising the CH2-CH3 domain of an IgG molecule, optionally including the hinge. In the EU numbering of human IgG1, the CH2-CH3 domain comprises amino acids 231-447, and the hinge is 216-230. Thus, the definition of "Fc domain" includes both amino acids 231-447 (CH2-CH3) or 216-447 (hinge-CH2-CH3), or fragments thereof. An "Fc fragment" in this context may comprise a smaller number of amino acids from either or both the N-terminus and C-terminus, but still retains the ability to form a dimer with another Fc domain or Fc fragment, which can generally be detected using standard size-based methods (e.g., non-denaturing chromatography, size exclusion chromatography, etc.). Human IgG Fc domains are particularly useful in the present disclosure and can be Fc domains from human IgG1, IgG2, or IgG4.

[0046] As used herein, a "variant Fc domain" comprises an amino acid modification compared to the parent Fc domain. Thus, a "variant human IgG1 Fc domain" comprises an amino acid modification (generally an amino acid substitution, but in the case of an ablation variant, including an amino acid deletion) compared to the human IgG1 Fc domain. Generally, the variant Fc domain has at least about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, or about 99% identity (using the identity algorithm discussed below, one embodiment using the BLAST algorithm known in the art using default parameters) with the corresponding parent human IgG Fc domain. Alternatively, the variant Fc domain can have 1 to about 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) amino acid modifications compared to the parent Fc domain. In addition, as discussed herein, the variant Fc domains herein retain the ability to form dimers with another Fc domain, as measured using known techniques described herein, such as non-denaturing gel electrophoresis.

[0047] As used herein, a "heavy chain constant region" refers to the CH1-hinge-CH2-CH3 portion of an antibody (or fragment thereof) excluding the variable heavy domain, which in the EU numbering of human IgG1 is amino acids 118 to 447. As used herein, a "heavy chain constant region fragment" refers to a heavy chain constant region that includes fewer amino acids from either or both of the N-terminus and C-terminus, but still retains the ability to form a dimer with another heavy chain constant region.

[0048] As used herein, a "variable region" or "variable domain" refers to a region of an immunoglobulin that includes one or more Ig domains substantially encoded by any of the Vκ, Vλ, and / or VH genes that constitute the kappa, lambda, and heavy chain immunoglobulin loci, respectively, and includes CDRs that confer antigen specificity. Thus, a "variable heavy chain domain" pairs with a "variable light chain domain" to form an antigen-binding domain ("ABD"). In addition, each variable domain includes three hypervariable regions ("complementarity determining regions", "CDRs") (vhCDR1, vhCDR2, and vhCDR3 for the variable heavy chain domain, vlCDR1, vlCDR2, and vlCDR3 for the variable light chain domain) and four framework (FR) regions arranged from amino-terminus to carboxy-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0049] The term "full length heavy chain" as used herein refers to the entire heavy chain of an antibody, including the signal peptide (S) and having the structure S-VH-CH1-hinge-CH2-CH3.

[0050] The term "mature heavy chain" as used herein refers to the portion of an antibody heavy chain having the structure VH-CH1-hinge-CH2-CH3, excluding the signal peptide.

[0051] The term "full length light chain" as used herein refers to the entire light chain of an antibody, including the signal peptide (S) and having the structure S-VL-CL.

[0052] The term "mature light chain" as used herein refers to the portion of the light chain of an antibody having the structure S-VL-CL, excluding the signal peptide.

[0053] As used herein, the term "IgG subclass modification" or "isotype modification" refers to an amino acid modification that converts one amino acid of one IgG isotype to the corresponding amino acid of a different aligned IgG isotype. For example, an F296Y substitution in IgG2 is considered an IgG subclass modification because IgG1 contains a tyrosine and IgG2 contains a phenylalanine at EU position 296.

[0054] The term "non-naturally occurring modification" as used herein refers to a non-isotypic amino acid modification. For example, the substitution 434S in IgG1, IgG2, IgG3, or IgG4 (or hybrids thereof) is considered a non-naturally occurring modification, since none of the human IgGs contain a serine at position 434.

[0055] The antibodies and antigen-binding fragments thereof of the present disclosure are engineered to have various properties as described herein and are generally recombinant antibodies that are isolated prior to use. As used herein, the term "isolated" when used to describe various polypeptides described herein means that the polypeptide has been identified, separated and / or recovered from the cell or cell culture in which it is expressed. Typically, an isolated polypeptide is prepared by at least one purification step. An "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities. "Recombinant" means that the antibodies are produced in an exogenous host cell using recombinant nucleic acid techniques and can also be isolated.

[0056] As used herein, "3E10 antibody" refers to an antibody that has a set of heavy chain CDRs (VH CDR1, VH CDR2, and VH CDR3) that comprise amino acid sequences that vary by no more than two amino acids each from SEQ ID NOs: 58, 59, and 60, respectively, as identified by the Kabat system, and a set of light chain CDRs (VL CDR1, VL CDR2, and VL CRD3) that comprise amino acid sequences that vary by no more than two amino acids each from SEQ ID NOs: 61, 62, and 63, respectively, and that binds nucleic acid. As described herein, the 3E10 antigen is a polynucleotide.

[0057] The term "cell-permeable" as used herein refers to an antibody or antigen-binding fragment thereof that is capable of penetrating a cell, e.g., a mammalian cell, without the aid of an exogenous delivery vehicle, such as a liposome, or a conjugated cell-permeable peptide. With respect to the 3E10 antibody and antigen-binding fragment thereof, the cell-permeable antibody or antigen-binding fragment thereof can penetrate a cell expressing the ENT2 receptor on its cell surface, for example, in the presence of a nucleic acid non-covalently bound and / or conjugated to the 3E10 antibody or antigen-binding fragment thereof, resulting in internalization of the 3E10 antibody and antigen-binding fragment thereof. In some embodiments, the cell-permeable 3E10 antibody or antigen-binding fragment thereof is conjugated to a functional molecule, e.g., a chemical agent, a polynucleotide, or a polypeptide.

[0058] As used herein, "variant protein" or "protein variant" or "variant" refers to a protein that differs from that of a parent protein by at least one amino acid modification. A protein variant has at least one amino acid modification compared to the parent protein, but not so many that the variant protein does not align with the parent protein using an alignment program such as those described below. Generally, a variant protein (such as a variant Fc domain as outlined herein) will generally align with the parent protein at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, at least 100%, at least 102%, at least 104%, at least 106%, at least 108%, at least 109%, at least 110%, at least 112%, at least 114%, at least 116%, at least 118%, at least 118%, at least 119%, at least 120%, at least 122%, at least 124%, at least 126%, at least 128%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least 137%, at least 138%, at least 139%, at least 140%, at least 141%, at least 142%, at least 143%, at least 144%, at least 145%, at least 146%, at least 147%, at least 14 or at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical. Amino acid sequence modifications to the 3E10 antibody or antigen-binding fragment thereof described herein may produce proteins and / or polypeptides referred to as variant 3E10 antibodies or antigen-binding fragments thereof, but such variants are still included within the classification of the 3E10 antibody or antigen-binding fragment thereof so long as they maintain the CDR sequence and cell-permeability activity requirements of the 3E10 antibody or antigen-binding fragment thereof.

[0059] Sequence identity between two similar sequences (e.g., antibody variable domains) can be determined by the methods described in Smith, TF & Waterman, MS (1981) "Comparison Of Biosequences," Adv. Appl. Math. 2:482 [local homology algorithm], Needleman, SB & Wunsch, CD. (1970) "A General Method Applicable To The Search For Similarities In The Amino Acid Sequence Of Two Proteins," J. Mol. Biol., 48:443 [homology alignment algorithm], Pearson, WR & Lipman, DJ (1988) "Improved Tools For Biological Sequence Comparison," Proc. Natl. Acad. Sci. (USA) 85:2444 [search for similarity method], or Altschul, SF et al, (1990) "Basic Local Alignment Search Tool," J. Mol. Biol.215:403-10, for the "BLAST" algorithm, see the URL website blast.ncbi.nlm.nih.gov / Blast.cgi. When using any of the above algorithms, default parameters (window length, gap penalty, etc.) are used. Unless otherwise specified, sequence identity is determined using the BLAST algorithm with default parameters.

[0060] 3E10 ANTIBODIES, VARIANTS, AND FRAGMENTS THEREOF In embodiments, the present disclosure relates to humanized 3E10 antibodies and antigen-binding fragments thereof, and their use to deliver therapeutic agents for the treatment of various diseases. As discussed herein, the humanized 3E10 antibodies that find use in the methods and compositions of the present disclosure can incorporate a range of amino acid substitutions in any portion of the antibody. Furthermore, the antigen-binding fragments of the humanized 3E10 antibodies can take many forms, including, but not limited to, the exemplary forms described herein.

[0061] The amino acid sequences of the 3E10 monoclonal antibody and its binding fragments are known in the art. Examples of 3E10 heavy and light chain sequences are provided below and shown in various figures. When present, single underlines indicate CDR regions identified by the Kabat system, italics indicate variable domains, and double underlines indicate signal peptides. The mouse version of the 3E10 antibody is described in Zack, et al., Immunology and Cell Biology, 72:513-520 (1994). The heavy and light chains of mouse 3E10, as well as the variable regions and CDRs, are shown in Figure 1 (SEQ ID NOs: 1-12).

[0062] Amino acid variants of the 3E10 antibody are also known in the art, as described, for example, in Zack, et al., J. Immunol., 157(5):2082-8 (1996). For example, amino acid position 31 in CDR1 of the heavy chain variable region of 3E10 affects the antibody's ability to bind nucleic acid and penetrate the nucleus. Substitution of the "wild-type" (e.g., compared to the original mouse antibody) aspartic acid with asparagine (D31N mutation) improves the antibody's nucleic acid binding and nuclear penetration compared to the wild-type mouse antibody. See, for example, Zack, et al., Immunology and Cell Biology, 72:513-520 (1994); Weisbart, et al., J. Autoimmun., 11, 539-546 (1998); and Weisbart, Int. J. Oncol., 25, 1867-1873 (2004). The sequence of murine 3E10 with a D31N substitution is shown in Figure 2. Thus, in some embodiments, the humanized 3E10 antibodies and binding fragments thereof disclosed herein comprise a D31N substitution. In other embodiments, the humanized 3E10 antibodies and binding fragments thereof disclosed herein have other amino acid substitutions at position 31. For example, as modeled in Figure 11A, some embodiments of the present disclosure incorporate a D31R or D31K substitution.

[0063] Other 3E10 light chain sequences are known in the art, e.g., Zack, et al., J. Immunol., 15;154(4):1987-94 (1995), GenBank: L16981.1--mouse Ig rearranged light chain gene, partial cds, GenBank: AAA65681.1--immunoglobulin light chain, partial [Mus musculus]).

[0064] Conventional antibody structural units typically include a tetramer. Each tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one "light chain" (typically having a molecular weight of about 25 kDa) and one "heavy chain" (typically having a molecular weight of about 50-70 kDa). Human light chains are classified as kappa light chains and lambda light chains. The present disclosure is generally directed to antibodies based on the IgG class, which has several subclasses, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. In general, IgG1, IgG2, and IgG4 are more frequently used than IgG3. It should be noted that IgG1 has different allotypes with polymorphisms at 356 (D or E) and 358 (L or M).

[0065] The light chain generally comprises two domains: a variable light domain (which comprises the light chain CDRs and which together with the variable heavy domain form the Fv region) and a constant light domain (often referred to as CL or Cκ).The heavy chain comprises a variable heavy domain and a constant domain, comprising a CH1-optional hinge-Fc domain, which comprises CH2-CH3.

[0066] The hypervariable region of an antibody generally consists of amino acid residues from about 24-34 (LCDR1, "L" refers to light chain), 50-56 (LCDR2), and 89-97 (LCDR3) in the light chain variable region, and from about 31-35B (HCDR1, "H" refers to heavy chain), 50-65 (HCDR2), and 95-102 (HCDR3) in the heavy chain variable region (Kabat et al., 2003). SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)), and / or residues forming the hypervariable loops (e.g., residues 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3) in the light chain variable region, and 26-32 (HCDR1), 53-55 (HCDR2), and 96-101 (HCDR3) in the heavy chain variable region, Chothia and Lesk (1987) J. Mol. Biol. 1987. 196:901-917). Particular CDRs useful in the compositions and methods described herein are described below.

[0067] As will be appreciated by those skilled in the art, the exact numbering and arrangement of the CDRs may vary among various numbering systems. However, it should be understood that the disclosure of a variable heavy chain sequence and / or a variable light chain sequence includes the disclosure of the associated (unique) CDRs. Thus, the disclosure of each variable heavy chain region is the disclosure of the vhCDRs (e.g., vhCDR1, vhCDR2, and vhCDR3), and the disclosure of each variable light chain region is the disclosure of the vlCDRs (e.g., vlCDR1, vlCDR2, and vlCDR3). A useful comparison for CDR numbering is provided in Lafranc et al., Dev. Comp. Immunol. 2003. 27(1):55-77(2003).

[0068] Throughout this disclosure, the Kabat numbering system is generally used when referring to residues in the variable domain (approximately residues 1-107 for light chain variable regions and residues 1-113 for heavy chain variable regions), and the EU numbering system is generally used when referring to the Fc region (e.g., Kabat et al., supra (1991)).

[0069] The present disclosure provides a number of different CDR sets. In this case, the "complete CDR set" includes three variable light chain and three variable heavy chain CDRs, e.g., vlCDR1, vlCDR2, vlCDR3, vhCDR1, vhCDR2, and vhCDR3. These may be part of a larger variable light chain domain or variable heavy chain domain, respectfully. In addition, as more fully outlined herein, the variable heavy chain domain and the variable light chain domain may be on separate polypeptide chains, when heavy and light chains are used (e.g., when Fabs are used), or on a single polypeptide chain, in the case of scFv sequences. The CDRs contribute to the formation of the antigen-binding site of the antibody, more specifically, the epitope-binding site. "Epitope" refers to a determinant that interacts with a specific antigen-binding site within the variable region of an antibody molecule, known as the paratope.

[0070] An epitope is a grouping of molecules, such as nucleic acids, amino acids, or sugar side chains, that usually have specific structural characteristics as well as specific charge characteristics. A single antigen may have more than one epitope. The antibodies described herein bind to nucleic acid epitopes in a partially sequence-independent manner. That is, while the antibodies described herein bind to some polynucleotide structures and sequences with higher affinity than other nucleic acid structures and sequences, they have some general affinity for polynucleotides.

[0071] The "Fc domain" of a heavy chain comprises the -CH2-CH3 domain and optionally the hinge domain (-H-CH2-CH3). In the case of IgG, the Fc domain comprises the immunoglobulin domains CH2 and CH3 (Cγ2 and Cγ3) and the lower hinge region between CH1 (Cγ1) and CH2 (Cγ2). Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined to include residues C226 or P230 at its carboxyl terminus, numbering according to the EU index as per Kabat et al. Thus, the "CH" domains in the context of IgG are as follows: "CH1" refers to positions 118-215 according to the EU index as per Kabat et al. "Hinge" refers to positions 216-230 according to the EU index as per Kabat et al. "CH2" refers to positions 231-340 according to the EU index as in Kabat et al., and "CH3" refers to positions 341-447 according to the EU index as in Kabat et al. Thus, an "Fc domain" includes the -CH2-CH3 domain, and optionally a hinge domain (hinge-CH2-CH3). In embodiments herein, when an scFv is attached to an Fc domain, it is generally the C-terminus of the scFv construct that is attached to all or part of the hinge of the Fc domain, e.g., it is generally attached to the sequence EPKS, which is the beginning of the hinge. In some embodiments, as described more fully below, amino acid modifications are made to the Fc region, e.g., to alter binding to one or more FcγR or FcRn receptors, and to allow for the formation and purification of heterodimers, as outlined herein.

[0072] Another part of the heavy chain is the hinge region. By "hinge" or "hinge region" or "antibody hinge region" or "hinge domain" herein is meant a flexible polypeptide comprising amino acids between the first and second constant domains of an antibody. Structurally, the IgG CH1 domain ends at EU position 215 and the IgG CH2 domain starts at residue EU position 231. Thus, for IgG, the antibody hinge is defined herein to comprise positions 216 (E216 in IgG1) to 230 (p230 in IgG1), the numbering being according to the EU index as in Kabat et al. In some cases, "hinge fragments" are used that comprise fewer amino acids at either or both the N-terminus and C-terminus of the hinge domain.

[0073] An scFv comprises a variable heavy chain, an scFv linker, and a variable light chain domain. In many of the constructs and sequences outlined herein, the C-terminus of the variable heavy chain is attached to the N-terminus of the scFv linker, which is attached to the N-terminus of the variable light chain (N-vh-linker-vl-C), although this can be switched (N-vl-linker-vh-C).

[0074] Thus, the present disclosure relates to various antibody domains. As described herein and known in the art, the heterodimeric antibodies described in certain embodiments of the present disclosure contain different domains in the heavy and light chains, which may also overlap. These domains include, but are not limited to, Fc domain, CH1 domain, CH2 domain, CH3 domain, hinge domain, heavy chain constant domain (CH1-hinge-Fc domain or CH1-hinge-CH2-CH3), variable heavy chain domain, variable light chain domain, light chain constant domain, Fab domain, and scFv domain.

[0075] In certain embodiments, an antibody of the present disclosure comprises a heavy chain variable region derived from a particular germline heavy chain immunoglobulin gene and / or a light chain variable region derived from a particular germline light chain immunoglobulin gene. For example, such an antibody may comprise or consist of a human antibody comprising a heavy chain variable region or a light chain variable region that is "the product of" or "derived from" a particular germline sequence, e.g., the germline sequence of the 3E10 antibody. A human antibody that is "the product of" or "derived from" a human germline immunoglobulin sequence can be identified as such (using the methods outlined herein) by comparing the amino acid sequence of the human antibody to the amino acid sequence of the human germline immunoglobulin and selecting the human germline immunoglobulin sequence that is closest (i.e., the highest % identity) to the sequence of the human antibody. A human antibody that is "the product of" or "derived from" a particular human germline immunoglobulin sequence may contain amino acid differences compared to the germline sequence, for example, due to the deliberate introduction of naturally occurring somatic mutations or site-specific mutations. However, a humanized antibody typically has an amino acid sequence that is at least 90% identical to an amino acid sequence encoded by a human germline immunoglobulin gene and contains amino acid residues that identify the antibody as derived from a human sequence as compared to germline immunoglobulin amino acid sequences of other species (e.g., mouse germline sequences). In certain cases, a humanized antibody may have an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99%, or even at least 96%, 97%, 98%, or 99% identical to an amino acid sequence encoded by a germline immunoglobulin gene. Typically, a humanized antibody derived from a particular human germline sequence will display no more than 10-20 amino acid differences from the amino acid sequence encoded by a human germline immunoglobulin gene. In certain cases, a humanized antibody may display no more than 5, or no more than 4, no more than 3, no more than 2, or no more than 1 amino acid difference from the amino acid sequence encoded by a germline immunoglobulin gene.

[0076] In one embodiment, the parent antibody is affinity matured as known in the art. Structure-based methods can be used for humanization and affinity maturation, for example, as described in U.S. patent application Ser. No. 11 / 004,590, the contents of which are incorporated herein by reference in their entirety. Selection-based methods can be used to humanize and / or affinity mature antibody variable regions, including, but not limited to, those described in Wu et al., 1999, J. Mol. Biol. 294:151-162; Baca et al., 1997, J. Biol. Chem. 272(16):10678-10684; Rosok et al., 1996, J. Biol. Chem. 271(37):22611-22618; Rader et al., 1998, Proc. Natl. Acad. Sci. USA 95:8910-8915; Krauss et al., 2003, Protein Engineering 16(10):753-759, all of which are incorporated herein by reference. Other humanization methods may involve grafting only a portion of the CDRs, including, but not limited to, those described in U.S. Application 09 / 810,510; Tan et al., 2002, J. Immunol. 169:1119-1125; and De Pascalis et al., 2002, J. Immunol. 169:3076-3084, the contents of which are incorporated herein by reference in their entireties.

[0077] In some aspects, the disclosure relates to the use of antigen binding domains (ABDs) that bind nucleic acids, specifically ABDs derived from the 3E10 antibody that bind therapeutic polynucleotides for use in diseases such as cancer. The amino acid sequences of the heavy and light chains of the parent 3E10 antibody are shown in Figure 1 (SEQ ID NOs: 1-12). Thus, in some embodiments, the compositions described herein comprise the 3E10 antibody or an antigen-binding fragment thereof.

[0078] As used herein, "antigen-binding fragments" of humanized 3E10 antibodies include, but are not limited to, fragments, variants, and fusion proteins such as scFv, di-scFv, tr-scFv, and other single-chain variable fragments that have nucleic acid binding properties.

[0079] The humanized 3E10 antibody or antigen-binding fragment thereof can be transported into the cytoplasm and / or nucleus of a cell without the aid of a carrier or conjugate. For example, monoclonal antibody 3E10 and its active fragments that are transported in vivo to the nucleus of a mammalian cell without cytotoxic effects are disclosed in U.S. Patent Nos. 4,812,397 and 7,189,396 to Richard Weisbart.

[0080] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof binds to and / or inhibits Rad51. See, e.g., Turchick, et al., Nucleic Acids Res., 45(20):11782-11799 (2017), WO2020 / 047344, and WO2020 / 047353, each of which is specifically incorporated by reference in its entirety.

[0081] The humanized 3E10 antibody or antigen-binding fragment thereof that can be used in the compositions and methods includes any class of whole immunoglobulin (i.e., intact antibody), fragments thereof, and synthetic proteins that contain at least the nucleic acid binding and ENT2-mediated cellular internalization characteristic of the 3E10 antibody. Antigen-binding activity is typically concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in both the light and heavy chain variable domains. The more highly conserved parts of the variable domains are called frameworks (FRs). Natural heavy and light chain variable domains each contain four FR regions and predominantly adopt a beta-sheet configuration connected by three CDRs that form loops to connect and sometimes form part of the beta-sheet structure. The CDRs in each chain are held together in close proximity to the CDRs from the other chain by the FR regions and contribute to the formation of the antigen-binding site of the antibody.

[0082] Humanized 3E10 antibody and antigen-binding fragment thereof Generally, a humanized antibody is the result of a process in which the sequence of a parent antibody from a non-human species is modified to increase the overall similarity of the parent antibody to a human antibody while retaining the antigen-binding activity of the parent antibody. Generally, the process involves identifying a human antibody, sometimes called a scaffold antibody, and then either (i) replacing amino acids in the parent (non-human) antibody with equivalent amino acids from the scaffold (human) antibody (e.g., framework amino acids that have little or no effect on antigen binding), or (ii) replacing amino acids in the scaffold (human) antibody with equivalent amino acids from the parent (non-human) antibody (e.g., CDR and other amino acids that have significant effect on antigen binding). Various methods for humanization are known in the art, including framework homology-based humanization, germline humanization, complementarity determining region (CDR) homology-based humanization, and specificity determining residue (SDR) grafting. For a review of these methods, see, e.g., Safdari Y. et al., Biotechnology and Genetic Engineering Reviews, 29:2, 175-86 (2013).

[0083] As described in the Examples, seven humanized 3E10 variable light domains and six humanized 3E10 variable heavy domains were generated, the sequences of which are shown in Figure 5 (heavy chain variable region), Figure 6 (heavy chain without signal sequence), Figure 7 (heavy chain with signal peptide), Figure 8 (light chain variable region), Figure 9 (light chain without signal sequence), and Figure 10 (light chain with signal peptide). These variable light and variable heavy domains can be combined in any of 42 possible combinations (each of the seven variable light domains with each of the variable heavy domains) to form humanized 3E10 antibodies and nucleic acid binding fragments thereof (e.g., scFv). As described in the Examples, 22 antibodies incorporating different combinations of these humanized VL and VH sequences were generated, all of which bound to nucleic acid. Furthermore, when human leukemia cells were exposed to complexes formed between these antibodies and RIG-I agonist polynucleotides, all of the complexes were capable of generating a type I IFN response, suggesting that all of the antibodies tested were able to deliver functional polynucleotides into cells and affect RIG-I-mediated responses.

[0084] Thus, in some embodiments, the disclosure provides humanized antibodies and antigen-binding fragments thereof that incorporate any combination of humanized VL and VH sequences shown in Figures 5-10, as well as any combination of VL and VH sequences that have sequence identity thereto, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the VH or VL sequences shown in Figures 5-10.

[0085] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a light chain variable domain (3E10VL-h1, 3E10VL-h2, 3E10VL-h3, 3E10VL-h4, 3E10VL-h5, 3E10VL-h6, 3E10VL-h7, 3E10VL-h8, 3E10VL-h9, 3E10VL-h10, 3E10VL-h11, 3E10VL-h12, 3E10VL-h13, 3E10VL-h14, 3E10VL-h15, 3E10VL-h16, 3E10VL-h17, 3E10VL-h18, 3E10VL-h19, 3E10VL-h20, 3E10VL-h21, 3E10VL-h22, 3E10VL-h23, 3E10VL-h24, 3E10VL-h25, 3E10VL-h26, 3E10VL-h27, 3E10VL-h28, 3E10VL-h29, 3E10VL-h30, 3E10VL-h31, 3E10VL-h32, 3E10VL-h33, 3E10VL-h34, 3E10VL-h35, 3E10VL-h36, 3E10VL-h37, 3E10VL-h38, 3E10VL-h39, 3E10VL-h40, 3E10VL-h41, and a heavy chain variable domain (3E10-VH) comprising an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of 3E10-VH-h1 (SEQ ID NO: 64), 3E10-VH-h2 (SEQ ID NO: 65), 3E10-VH-h3 (SEQ ID NO: 66), 3E10-VH-h4 (SEQ ID NO: 67), 3E10-VH-h5 (SEQ ID NO: 68), 3E10-VH-h6 (SEQ ID NO: 69), and 3E10-VH-h7 (SEQ ID NO: 70).

[0086] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a light chain variable domain (3E10VL-h1, 3E10VL-h2, 3E10VL-h3, 3E10VL-h4, 3E10VL-h5, 3E10VL-h6, 3E10VL-h7, 3E10VL-h8, 3E10VL-h9, 3E10VL-h10, 3E10VL-h11, 3E10VL-h12, 3E10VL-h13, 3E10VL-h14, 3E10VL-h15, 3E10VL-h16, 3E10VL-h17, 3E10VL-h18, 3E10VL-h19, 3E10VL-h20, 3E10VL-h21, 3E10VL-h22, 3E10VL-h23, 3E10VL-h24, 3E10VL-h25, 3E10VL-h26, 3E10VL-h27, 3E10VL-h28, 3E10VL-h29, 3E10VL-h30, 3E10VL-h31, 3E10VL-h32, 3E10VL-h33, 3E10VL-h34, 3E10VL-h35, 3E10VL-h36, 3E10VL-h37, 3E10VL-h38, 3E10VL-h39, 3E10VL-h40, 3E10VL-h41, and a heavy chain variable domain (3E10-VH) comprising an amino acid sequence that is at least 97% identical to an amino acid sequence selected from the group consisting of 3E10-VH-h1 (SEQ ID NO: 64), 3E10-VH-h2 (SEQ ID NO: 65), 3E10-VH-h3 (SEQ ID NO: 66), 3E10-VH-h4 (SEQ ID NO: 67), 3E10-VH-h5 (SEQ ID NO: 68), 3E10-VH-h6 (SEQ ID NO: 69), and 3E10-VH-h7 (SEQ ID NO: 70).

[0087] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a light chain variable domain (3E10VL-h1, 3E10VL-h2, 3E10VL-h3, 3E10VL-h4, 3E10VL-h5, 3E10VL-h6, 3E10VL-h7, 3E10VL-h8, 3E10VL-h9, 3E10VL-h10, 3E10VL-h11, 3E10VL-h12, 3E10VL-h13, 3E10VL-h14, 3E10VL-h15, 3E10VL-h16, 3E10VL-h17, 3E10VL-h18, 3E10VL-h19, 3E10VL-h20, 3E10VL-h21, 3E10VL-h22, 3E10VL-h23, 3E10VL-h24, 3E10VL-h25, 3E10VL-h26, 3E10VL-h27, 3E10VL-h28, 3E10VL-h29, 3E10VL-h30, 3E10VL-h31, 3E10VL-h32, 3E10VL-h33, 3E10VL-h34, 3E10VL-h35, 3E10VL-h36, 3E10VL-h37, 3E10VL-h38, 3E10VL-h39, 3E10VL-h40, 3E10VL-h41, and a heavy chain variable domain (3E10-VH) comprising an amino acid sequence at least 98% identical to an amino acid sequence selected from the group consisting of 3E10-VH-h1 (SEQ ID NO: 64), 3E10-VH-h2 (SEQ ID NO: 65), 3E10-VH-h3 (SEQ ID NO: 66), 3E10-VH-h4 (SEQ ID NO: 67), 3E10-VH-h5 (SEQ ID NO: 68), 3E10-VH-h6 (SEQ ID NO: 69), and 3E10-VH-h7 (SEQ ID NO: 70).

[0088] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a light chain variable domain (3E10VL-h1, 3E10VL-h2, 3E10VL-h3, 3E10VL-h4, 3E10VL-h5, 3E10VL-h6, 3E10VL-h7, 3E10VL-h8, 3E10VL-h9, 3E10VL-h10, 3E10VL-h11, 3E10VL-h12, 3E10VL-h13, 3E10VL-h14, 3E10VL-h15, 3E10VL-h16, 3E10VL-h17, 3E10VL-h18, 3E10VL-h19, 3E10VL-h20, 3E10VL-h21, 3E10VL-h22, 3E10VL-h23, 3E10VL-h24, 3E10VL-h25, 3E10VL-h26, 3E10VL-h27, 3E10VL-h28, 3E10VL-h29, 3E10VL-h30, 3E10VL-h31, 3E10VL-h32, 3E10VL-h33, 3E10VL-h34, 3E10VL-h35, 3E10VL-h36, 3E10VL-h37, 3E10VL-h38, 3E10VL-h39, 3E10VL-h40, 3E10VL-h41, and a heavy chain variable domain (3E10-VH) comprising an amino acid sequence that is at least 99% identical to an amino acid sequence selected from the group consisting of 3E10-VH-h1 (SEQ ID NO: 64), 3E10-VH-h2 (SEQ ID NO: 65), 3E10-VH-h3 (SEQ ID NO: 66), 3E10-VH-h4 (SEQ ID NO: 67), 3E10-VH-h5 (SEQ ID NO: 68), 3E10-VH-h6 (SEQ ID NO: 69), and 3E10-VH-h7 (SEQ ID NO: 70).

[0089] In some embodiments, the sequence of 3E10-VL is at least 97% identical to 3E10-VL-h1 (SEQ ID NO: 85). In some embodiments, the sequence of 3E10-VL is at least 98% identical to 3E10-VL-h1 (SEQ ID NO: 85). In some embodiments, the sequence of 3E10-VL is at least 99% identical to 3E10-VL-h1 (SEQ ID NO: 85). In some embodiments, the sequence of 3E10-VL is 3E10-VL-h1 (SEQ ID NO: 85).

[0090] In some embodiments, the sequence of 3E10-VL is at least 97% identical to 3E10-VL-h2 (SEQ ID NO: 86). In some embodiments, the sequence of 3E10-VL is at least 98% identical to 3E10-VL-h2 (SEQ ID NO: 86). In some embodiments, the sequence of 3E10-VL is at least 99% identical to 3E10-VL-h2 (SEQ ID NO: 86). In some embodiments, the sequence of 3E10-VL is 3E10-VL-h2 (SEQ ID NO: 86).

[0091] In some embodiments, the sequence of 3E10-VL is at least 97% identical to 3E10-VL-h3 (SEQ ID NO: 87). In some embodiments, the sequence of 3E10-VL is at least 98% identical to 3E10-VL-h3 (SEQ ID NO: 87). In some embodiments, the sequence of 3E10-VL is at least 99% identical to 3E10-VL-h3 (SEQ ID NO: 87). In some embodiments, the sequence of 3E10-VL is 3E10-VL-h3 (SEQ ID NO: 87).

[0092] In some embodiments, the sequence of 3E10-VL is at least 97% identical to 3E10-VL-h4 (SEQ ID NO: 88). In some embodiments, the sequence of 3E10-VL is at least 98% identical to 3E10-VL-h4 (SEQ ID NO: 88). In some embodiments, the sequence of 3E10-VL is at least 99% identical to 3E10-VL-h4 (SEQ ID NO: 88). In some embodiments, the sequence of 3E10-VL is 3E10-VL-h4 (SEQ ID NO: 88).

[0093] In some embodiments, the sequence of 3E10-VL is at least 97% identical to 3E10-VL-h5 (SEQ ID NO: 89). In some embodiments, the sequence of 3E10-VL is at least 98% identical to 3E10-VL-h5 (SEQ ID NO: 89). In some embodiments, the sequence of 3E10-VL is at least 99% identical to 3E10-VL-h5 (SEQ ID NO: 89). In some embodiments, the sequence of 3E10-VL is 3E10-VL-h5 (SEQ ID NO: 89).

[0094] In some embodiments, the sequence of 3E10-VL is at least 97% identical to 3E10-VL-h6 (SEQ ID NO: 90). In some embodiments, the sequence of 3E10-VL is at least 98% identical to 3E10-VL-h6 (SEQ ID NO: 90). In some embodiments, the sequence of 3E10-VL is at least 99% identical to 3E10-VL-h6 (SEQ ID NO: 90). In some embodiments, the sequence of 3E10-VL is 3E10-VL-h6 (SEQ ID NO: 90).

[0095] In some embodiments, the sequence of 3E10-VH is at least 95% identical to 3E10-VH-h1 (SEQ ID NO: 64). In some embodiments, the sequence of 3E10-VH is at least 96% identical to 3E10-VH-h1 (SEQ ID NO: 64). In some embodiments, the sequence of 3E10-VH is at least 97% identical to 3E10-VH-h1 (SEQ ID NO: 64). In some embodiments, the sequence of 3E10-VH is at least 98% identical to 3E10-VH-h1 (SEQ ID NO: 64). In some embodiments, the sequence of 3E10-VH is at least 99% identical to 3E10-VH-h1 (SEQ ID NO: 64). In some embodiments, the sequence of 3E10-VH is 3E10-VH-h1 (SEQ ID NO: 64).

[0096] In some embodiments, the sequence of 3E10-VH is at least 95% identical to 3E10-VH-h2 (SEQ ID NO: 65). In some embodiments, the sequence of 3E10-VH is at least 96% identical to 3E10-VH-h2 (SEQ ID NO: 65). In some embodiments, the sequence of 3E10-VH is at least 97% identical to 3E10-VH-h2 (SEQ ID NO: 65). In some embodiments, the sequence of 3E10-VH is at least 98% identical to 3E10-VH-h2 (SEQ ID NO: 65). In some embodiments, the sequence of 3E10-VH is at least 99% identical to 3E10-VH-h2 (SEQ ID NO: 65). In some embodiments, the sequence of 3E10-VH is 3E10-VH-h2 (SEQ ID NO: 65).

[0097] In some embodiments, the sequence of 3E10-VH is at least 95% identical to 3E10-VH-h3 (SEQ ID NO: 66). In some embodiments, the sequence of 3E10-VH is at least 96% identical to 3E10-VH-h3 (SEQ ID NO: 66). In some embodiments, the sequence of 3E10-VH is at least 97% identical to 3E10-VH-h3 (SEQ ID NO: 66). In some embodiments, the sequence of 3E10-VH is at least 98% identical to 3E10-VH-h3 (SEQ ID NO: 66). In some embodiments, the sequence of 3E10-VH is at least 99% identical to 3E10-VH-h3 (SEQ ID NO: 66). In some embodiments, the sequence of 3E10-VH is 3E10-VH-h3 (SEQ ID NO: 66).

[0098] In some embodiments, the sequence of 3E10-VH is at least 95% identical to 3E10-VH-h4 (SEQ ID NO: 67). In some embodiments, the sequence of 3E10-VH is at least 96% identical to 3E10-VH-h4 (SEQ ID NO: 67). In some embodiments, the sequence of 3E10-VH is at least 97% identical to 3E10-VH-h4 (SEQ ID NO: 67). In some embodiments, the sequence of 3E10-VH is at least 98% identical to 3E10-VH-h4 (SEQ ID NO: 67). In some embodiments, the sequence of 3E10-VH is at least 99% identical to 3E10-VH-h4 (SEQ ID NO: 67). In some embodiments, the sequence of 3E10-VH is 3E10-VH-h4 (SEQ ID NO: 67).

[0099] In some embodiments, the sequence of 3E10-VH is at least 95% identical to 3E10-VH-h5 (SEQ ID NO: 68). In some embodiments, the sequence of 3E10-VH is at least 96% identical to 3E10-VH-h5 (SEQ ID NO: 68). In some embodiments, the sequence of 3E10-VH is at least 97% identical to 3E10-VH-h5 (SEQ ID NO: 68). In some embodiments, the sequence of 3E10-VH is at least 98% identical to 3E10-VH-h5 (SEQ ID NO: 68). In some embodiments, the sequence of 3E10-VH is at least 99% identical to 3E10-VH-h5 (SEQ ID NO: 68). In some embodiments, the sequence of 3E10-VH is 3E10-VH-h5 (SEQ ID NO: 68).

[0100] In some embodiments, the sequence of 3E10-VH is at least 95% identical to 3E10-VH-h6 (SEQ ID NO: 69). In some embodiments, the sequence of 3E10-VH is at least 96% identical to 3E10-VH-h6 (SEQ ID NO: 69). In some embodiments, the sequence of 3E10-VH is at least 97% identical to 3E10-VH-h6 (SEQ ID NO: 69). In some embodiments, the sequence of 3E10-VH is at least 98% identical to 3E10-VH-h6 (SEQ ID NO: 69). In some embodiments, the sequence of 3E10-VH is at least 99% identical to 3E10-VH-h6 (SEQ ID NO: 69). In some embodiments, the sequence of 3E10-VH is 3E10-VH-h6 (SEQ ID NO: 69).

[0101] In some embodiments, the sequence of 3E10-VH is at least 95% identical to 3E10-VH-h7 (SEQ ID NO: 70). In some embodiments, the sequence of 3E10-VH is at least 96% identical to 3E10-VH-h7 (SEQ ID NO: 70). In some embodiments, the sequence of 3E10-VH is at least 97% identical to 3E10-VH-h7 (SEQ ID NO: 70). In some embodiments, the sequence of 3E10-VH is at least 98% identical to 3E10-VH-h7 (SEQ ID NO: 70). In some embodiments, the sequence of 3E10-VH is at least 99% identical to 3E10-VH-h7 (SEQ ID NO: 70). In some embodiments, the sequence of 3E10-VH is 3E10-VH-h7 (SEQ ID NO: 70).

[0102] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein has an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of 3E10-LC-h1m (SEQ ID NO: 91), 3E10-LC-h1m (SEQ ID NO: 91), 3E10-LC-h2m (SEQ ID NO: 92), 3E10-LC-h3m (SEQ ID NO: 93), 3E10-LC-h4m (SEQ ID NO: 94), 3E10-LC-h5m (SEQ ID NO: 95), and 3E10-LC-h6m (SEQ ID NO: 96). and a heavy chain (3E10-HC) comprising an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of 3E10-HC-h1m (SEQ ID NO: 71), 3E10-HC-h2m (SEQ ID NO: 72), 3E10-HC-h3m (SEQ ID NO: 73), 3E10-HC-h4m (SEQ ID NO: 74), 3E10-HC-h5m (SEQ ID NO: 75), 3E10-HC-h6m (SEQ ID NO: 76), and 3E10-HC-h7m (SEQ ID NO: 77).

[0103] In some embodiments, the sequence of 3E10-LC is at least 95% identical to 3E10-LC-h1m (SEQ ID NO: 91). In some embodiments, the sequence of 3E10-LC is at least 96% identical to 3E10-LC-h1m (SEQ ID NO: 91). In some embodiments, the sequence of 3E10-LC is at least 97% identical to 3E10-LC-h1m (SEQ ID NO: 91). In some embodiments, the sequence of 3E10-LC is at least 98% identical to 3E10-LC-h1m (SEQ ID NO: 91). In some embodiments, the sequence of 3E10-LC is at least 99% identical to 3E10-LC-h1m (SEQ ID NO: 91). In some embodiments, the sequence of 3E10-LC is 3E10-LC-h1m (SEQ ID NO: 91).

[0104] In some embodiments, the sequence of 3E10-LC is at least 95% identical to 3E10-LC-h2m (SEQ ID NO: 92). In some embodiments, the sequence of 3E10-LC is at least 96% identical to 3E10-LC-h2m (SEQ ID NO: 92). In some embodiments, the sequence of 3E10-LC is at least 97% identical to 3E10-LC-h2m (SEQ ID NO: 92). In some embodiments, the sequence of 3E10-LC is at least 98% identical to 3E10-LC-h2m (SEQ ID NO: 92). In some embodiments, the sequence of 3E10-LC is at least 99% identical to 3E10-LC-h2m (SEQ ID NO: 92). In some embodiments, the sequence of 3E10-LC is 3E10-LC-h2m (SEQ ID NO: 92).

[0105] In some embodiments, the sequence of 3E10-LC is at least 95% identical to 3E10-LC-h3m (SEQ ID NO: 93). In some embodiments, the sequence of 3E10-LC is at least 96% identical to 3E10-LC-h3m (SEQ ID NO: 93). In some embodiments, the sequence of 3E10-LC (SEQ ID NO: 7) is at least 97% identical to 3E10-LC-h3m (SEQ ID NO: 93). In some embodiments, the sequence of 3E10-LC is at least 98% identical to 3E10-LC-h3m (SEQ ID NO: 93). In some embodiments, the sequence of 3E10-LC is at least 99% identical to 3E10-LC-h3m (SEQ ID NO: 93). In some embodiments, the sequence of 3E10-LC is 3E10-LC-h3m (SEQ ID NO: 93).

[0106] In some embodiments, the sequence of 3E10-LC is at least 95% identical to 3E10-LC-h4m (SEQ ID NO: 94). In some embodiments, the sequence of 3E10-LC is at least 96% identical to 3E10-LC-h4m (SEQ ID NO: 94). In some embodiments, the sequence of 3E10-LC is at least 97% identical to 3E10-LC-h4m (SEQ ID NO: 94). In some embodiments, the sequence of 3E10-LC (SEQ ID NO: 7) is at least 98% identical to 3E10-LC-h4m (SEQ ID NO: 94). In some embodiments, the sequence of 3E10-LC is at least 99% identical to 3E10-LC-h4m (SEQ ID NO: 94). In some embodiments, the sequence of 3E10-LC is 3E10-LC-h4m (SEQ ID NO: 94).

[0107] In some embodiments, the sequence of 3E10-LC is at least 95% identical to 3E10-LC-h5m (SEQ ID NO: 95). In some embodiments, the sequence of 3E10-LC is at least 96% identical to 3E10-LC-h5m (SEQ ID NO: 95). In some embodiments, the sequence of 3E10-LC is at least 97% identical to 3E10-LC-h5m (SEQ ID NO: 95). In some embodiments, the sequence of 3E10-LC is at least 98% identical to 3E10-LC-h5m (SEQ ID NO: 95). In some embodiments, the sequence of 3E10-LC is at least 99% identical to 3E10-LC-h5m (SEQ ID NO: 95). In some embodiments, the sequence of 3E10-LC is 3E10-LC-h5m (SEQ ID NO: 95).

[0108] In some embodiments, the sequence of 3E10-LC is at least 95% identical to 3E10-LC-h6m (SEQ ID NO: 96). In some embodiments, the sequence of 3E10-LC is at least 96% identical to 3E10-LC-h6m (SEQ ID NO: 96). In some embodiments, the sequence of 3E10-LC is at least 97% identical to 3E10-LC-h6m (SEQ ID NO: 96). In some embodiments, the sequence of 3E10-LC is at least 98% identical to 3E10-LC-h6m (SEQ ID NO: 96). In some embodiments, the sequence of 3E10-LC is at least 99% identical to 3E10-LC-h6m (SEQ ID NO: 96). In some embodiments, the sequence of 3E10-LC is 3E10-LC-h6m (SEQ ID NO: 96).

[0109] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a light chain that comprises an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of 3E10-LC-h1m (SEQ ID NO: 97), 3E10-LC-h2 (SEQ ID NO: 98), 3E10-LC-h3 (SEQ ID NO: 99), 3E10-LC-h4 (SEQ ID NO: 100), 3E10-LC-h5 (SEQ ID NO: 101), and 3E10-LC-h6 (SEQ ID NO: 102). (3E10-LC) and a heavy chain (3E10-HC) comprising an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of 3E10-HC-h1 (SEQ ID NO:78), 3E10-HC-h2 (SEQ ID NO:79), 3E10-HC-h3 (SEQ ID NO:80), 3E10-HC-h4 (SEQ ID NO:81), 3E10-HC-h5 (SEQ ID NO:82), 3E10-HC-h67 (SEQ ID NO:83), and 3E10-HC-h7 (SEQ ID NO:84).

[0110] In some embodiments, the sequence of 3E10-LC is at least 95% identical to 3E10-LC-h1m (SEQ ID NO: 97). In some embodiments, the sequence of 3E10-LC is at least 96% identical to 3E10-LC-h1m (SEQ ID NO: 97). In some embodiments, the sequence of 3E10-LC is at least 97% identical to 3E10-LC-h1m (SEQ ID NO: 97). In some embodiments, the sequence of 3E10-LC is at least 98% identical to 3E10-LC-h1m (SEQ ID NO: 97). In some embodiments, the sequence of 3E10-LC is at least 99% identical to 3E10-LC-h1m (SEQ ID NO: 97). In some embodiments, the sequence of 3E10-LC is 3E10-LC-h1m (SEQ ID NO: 97).

[0111] In some embodiments, the sequence of 3E10-LC is at least 95% identical to 3E10-LC-h2 (SEQ ID NO: 98). In some embodiments, the sequence of 3E10-LC is at least 96% identical to 3E10-LC-h2 (SEQ ID NO: 98). In some embodiments, the sequence of 3E10-LC is at least 97% identical to 3E10-LC-h2 (SEQ ID NO: 98). In some embodiments, the sequence of 3E10-LC is at least 98% identical to 3E10-LC-h2 (SEQ ID NO: 98). In some embodiments, the sequence of 3E10-LC is at least 99% identical to 3E10-LC-h2 (SEQ ID NO: 98). In some embodiments, the sequence of 3E10-LC is 3E10-LC-h2 (SEQ ID NO: 98).

[0112] In some embodiments, the sequence of 3E10-LC is at least 95% identical to 3E10-LC-h3 (SEQ ID NO: 99). In some embodiments, the sequence of 3E10-LC is at least 96% identical to 3E10-LC-h3 (SEQ ID NO: 99). In some embodiments, the sequence of 3E10-LC is at least 97% identical to 3E10-LC-h3 (SEQ ID NO: 99). In some embodiments, the sequence of 3E10-LC is at least 98% identical to 3E10-LC-h3 (SEQ ID NO: 99). In some embodiments, the sequence of 3E10-LC is at least 99% identical to 3E10-LC-h3 (SEQ ID NO: 99). In some embodiments, the sequence of 3E10-LC is 3E10-LC-h3 (SEQ ID NO: 99).

[0113] In some embodiments, the sequence of 3E10-LC is at least 95% identical to 3E10-LC-h4 (SEQ ID NO: 100). In some embodiments, the sequence of 3E10-LC is at least 96% identical to 3E10-LC-h4 (SEQ ID NO: 100). In some embodiments, the sequence of 3E10-LC is at least 97% identical to 3E10-LC-h4 (SEQ ID NO: 100). In some embodiments, the sequence of 3E10-LC is at least 98% identical to 3E10-LC-h4 (SEQ ID NO: 100). In some embodiments, the sequence of 3E10-LC is at least 99% identical to 3E10-LC-h4 (SEQ ID NO: 100). In some embodiments, the sequence of 3E10-LC is 3E10-LC-h4 (SEQ ID NO: 100).

[0114] In some embodiments, the sequence of 3E10-LC is at least 95% identical to 3E10-LC-h5 (SEQ ID NO: 101). In some embodiments, the sequence of 3E10-LC is at least 96% identical to 3E10-LC-h5 (SEQ ID NO: 101). In some embodiments, the sequence of 3E10-LC is at least 97% identical to 3E10-LC-h5 (SEQ ID NO: 101). In some embodiments, the sequence of 3E10-LC is at least 98% identical to 3E10-LC-h5 (SEQ ID NO: 101). In some embodiments, the sequence of 3E10-LC is at least 99% identical to 3E10-LC-h5 (SEQ ID NO: 101). In some embodiments, the sequence of 3E10-LC is 3E10-LC-h5 (SEQ ID NO: 101).

[0115] In some embodiments, the sequence of 3E10-LC is at least 95% identical to 3E10-LC-h6 (SEQ ID NO: 102). In some embodiments, the sequence of 3E10-LC is at least 96% identical to 3E10-LC-h6 (SEQ ID NO: 102). In some embodiments, the sequence of 3E10-LC is at least 97% identical to 3E10-LC-h6 (SEQ ID NO: 102). In some embodiments, the sequence of 3E10-LC is at least 98% identical to 3E10-LC-h6 (SEQ ID NO: 102). In some embodiments, the sequence of 3E10-LC is at least 99% identical to 3E10-LC-h6 (SEQ ID NO: 102). In some embodiments, the sequence of 3E10-LC is 3E10-LC-h6 (SEQ ID NO: 102).

[0116] In some embodiments, the sequence of 3E10-HC is at least 95% identical to 3E10-HC-h1m (SEQ ID NO: 71). In some embodiments, the sequence of 3E10-HC is at least 96% identical to 3E10-HC-h1m (SEQ ID NO: 71). In some embodiments, the sequence of 3E10-HC is at least 97% identical to 3E10-HC-h1m (SEQ ID NO: 71). In some embodiments, the sequence of 3E10-HC is at least 98% identical to 3E10-HC-h1m (SEQ ID NO: 71). In some embodiments, the sequence of 3E10-HC is at least 99% identical to 3E10-HC-h1m (SEQ ID NO: 71). In some embodiments, the sequence of 3E10-HC is 3E10-HC-h1m (SEQ ID NO: 71).

[0117] In some embodiments, the sequence of 3E10-HC is at least 95% identical to 3E10-HC-h2m (SEQ ID NO: 72). In some embodiments, the sequence of 3E10-HC is at least 96% identical to 3E10-HC-h2m (SEQ ID NO: 72). In some embodiments, the sequence of 3E10-HC is at least 97% identical to 3E10-HC-h2m (SEQ ID NO: 72). In some embodiments, the sequence of 3E10-HC is at least 98% identical to 3E10-HC-h2m (SEQ ID NO: 72). In some embodiments, the sequence of 3E10-HC is at least 99% identical to 3E10-HC-h2m (SEQ ID NO: 72). In some embodiments, the sequence of 3E10-HC is 3E10-HC-h2m (SEQ ID NO: 72).

[0118] In some embodiments, the sequence of 3E10-HC is at least 95% identical to 3E10-HC-h3m (SEQ ID NO: 73). In some embodiments, the sequence of 3E10-HC is at least 96% identical to 3E10-HC-h3m (SEQ ID NO: 73). In some embodiments, the sequence of 3E10-HC is at least 97% identical to 3E10-HC-h3m (SEQ ID NO: 73). In some embodiments, the sequence of 3E10-HC is at least 98% identical to 3E10-HC-h3m (SEQ ID NO: 73). In some embodiments, the sequence of 3E10-HC is at least 99% identical to 3E10-HC-h3m (SEQ ID NO: 73). In some embodiments, the sequence of 3E10-HC is 3E10-HC-h3m (SEQ ID NO: 73).

[0119] In some embodiments, the sequence of 3E10-HC is at least 95% identical to 3E10-HC-h4m (SEQ ID NO: 74). In some embodiments, the sequence of 3E10-HC is at least 96% identical to 3E10-HC-h4m (SEQ ID NO: 74). In some embodiments, the sequence of 3E10-HC is at least 97% identical to 3E10-HC-h4m (SEQ ID NO: 74). In some embodiments, the sequence of 3E10-HC is at least 98% identical to 3E10-HC-h4m (SEQ ID NO: 74). In some embodiments, the sequence of 3E10-HC is at least 99% identical to 3E10-HC-h4m (SEQ ID NO: 74). In some embodiments, the sequence of 3E10-HC is 3E10-HC-h4m (SEQ ID NO: 74).

[0120] In some embodiments, the sequence of 3E10-HC is at least 95% identical to 3E10-HC-h5m (SEQ ID NO: 75). In some embodiments, the sequence of 3E10-HC is at least 96% identical to 3E10-HC-h5m (SEQ ID NO: 75). In some embodiments, the sequence of 3E10-HC is at least 97% identical to 3E10-HC-h5m (SEQ ID NO: 75). In some embodiments, the sequence of 3E10-HC is at least 98% identical to 3E10-HC-h5m (SEQ ID NO: 75). In some embodiments, the sequence of 3E10-HC is at least 99% identical to 3E10-HC-h5m (SEQ ID NO: 75). In some embodiments, the sequence of 3E10-HC is 3E10-HC-h5m (SEQ ID NO: 75).

[0121] In some embodiments, the sequence of 3E10-HC is at least 95% identical to 3E10-HC-h6m (SEQ ID NO: 76). In some embodiments, the sequence of 3E10-HC is at least 96% identical to 3E10-HC-h6m (SEQ ID NO: 76). In some embodiments, the sequence of 3E10-HC is at least 97% identical to 3E10-HC-h6m (SEQ ID NO: 76). In some embodiments, the sequence of 3E10-HC is at least 98% identical to 3E10-HC-h6m (SEQ ID NO: 76). In some embodiments, the sequence of 3E10-HC is at least 99% identical to 3E10-HC-h6m (SEQ ID NO: 76). In some embodiments, the sequence of 3E10-HC is 3E10-HC-h6m (SEQ ID NO: 76).

[0122] In some embodiments, the sequence of 3E10-HC is at least 95% identical to 3E10-HC-h7m (SEQ ID NO: 77). In some embodiments, the sequence of 3E10-HC is at least 96% identical to 3E10-HC-h7m (SEQ ID NO: 77). In some embodiments, the sequence of 3E10-HC is at least 97% identical to 3E10-HC-h7m (SEQ ID NO: 77). In some embodiments, the sequence of 3E10-HC is at least 98% identical to 3E10-HC-h7m (SEQ ID NO: 77). In some embodiments, the sequence of 3E10-HC is at least 99% identical to 3E10-HC-h7m (SEQ ID NO: 77). In some embodiments, the sequence of 3E10-HC is 3E10-HC-h7m (SEQ ID NO: 77).

[0123] In some embodiments, the sequence of 3E10-HC is at least 95% identical to 3E10-HC-h1 (SEQ ID NO: 78). In some embodiments, the sequence of 3E10-HC is at least 96% identical to 3E10-HC-h1 (SEQ ID NO: 78). In some embodiments, the sequence of 3E10-HC is at least 97% identical to 3E10-HC-h1 (SEQ ID NO: 78). In some embodiments, the sequence of 3E10-HC is at least 98% identical to 3E10-HC-h1 (SEQ ID NO: 78). In some embodiments, the sequence of 3E10-HC is at least 99% identical to 3E10-HC-h1 (SEQ ID NO: 78). In some embodiments, the sequence of 3E10-HC is 3E10-HC-h1 (SEQ ID NO: 78).

[0124] In some embodiments, the sequence of 3E10-HC is at least 95% identical to 3E10-HC-h2 (SEQ ID NO: 79). In some embodiments, the sequence of 3E10-HC is at least 96% identical to 3E10-HC-h2 (SEQ ID NO: 79). In some embodiments, the sequence of 3E10-HC is at least 97% identical to 3E10-HC-h2 (SEQ ID NO: 79). In some embodiments, the sequence of 3E10-HC is at least 98% identical to 3E10-HC-h2 (SEQ ID NO: 79). In some embodiments, the sequence of 3E10-HC is at least 99% identical to 3E10-HC-h2 (SEQ ID NO: 79). In some embodiments, the sequence of 3E10-HC is 3E10-HC-h2 (SEQ ID NO: 79).

[0125] In some embodiments, the sequence of 3E10-HC is at least 95% identical to 3E10-HC-h3 (SEQ ID NO: 80). In some embodiments, the sequence of 3E10-HC is at least 96% identical to 3E10-HC-h3 (SEQ ID NO: 80). In some embodiments, the sequence of 3E10-HC is at least 97% identical to 3E10-HC-h3 (SEQ ID NO: 80). In some embodiments, the sequence of 3E10-HC is at least 98% identical to 3E10-HC-h3 (SEQ ID NO: 80). In some embodiments, the sequence of 3E10-HC is at least 99% identical to 3E10-HC-h3 (SEQ ID NO: 80). In some embodiments, the sequence of 3E10-HC is 3E10-HC-h3 (SEQ ID NO: 80).

[0126] In some embodiments, the sequence of 3E10-HC is at least 95% identical to 3E10-HC-h4 (SEQ ID NO: 81). In some embodiments, the sequence of 3E10-HC is at least 96% identical to 3E10-HC-h4 (SEQ ID NO: 81). In some embodiments, the sequence of 3E10-HC is at least 97% identical to 3E10-HC-h4 (SEQ ID NO: 81). In some embodiments, the sequence of 3E10-HC is at least 98% identical to 3E10-HC-h4 (SEQ ID NO: 81). In some embodiments, the sequence of 3E10-HC is at least 99% identical to 3E10-HC-h4 (SEQ ID NO: 81). In some embodiments, the sequence of 3E10-HC is 3E10-HC-h4 (SEQ ID NO: 81).

[0127] In some embodiments, the sequence of 3E10-HC is at least 95% identical to 3E10-HC-h5 (SEQ ID NO: 82). In some embodiments, the sequence of 3E10-HC is at least 96% identical to 3E10-HC-h5 (SEQ ID NO: 82). In some embodiments, the sequence of 3E10-HC is at least 97% identical to 3E10-HC-h5 (SEQ ID NO: 82). In some embodiments, the sequence of 3E10-HC is at least 98% identical to 3E10-HC-h5 (SEQ ID NO: 82). In some embodiments, the sequence of 3E10-HC is at least 99% identical to 3E10-HC-h5 (SEQ ID NO: 82). In some embodiments, the sequence of 3E10-HC is 3E10-HC-h5 (SEQ ID NO: 82).

[0128] In some embodiments, the sequence of 3E10-HC is at least 95% identical to 3E10-HC-h67 (SEQ ID NO: 83). In some embodiments, the sequence of 3E10-HC is at least 96% identical to 3E10-HC-h67 (SEQ ID NO: 83). In some embodiments, the sequence of 3E10-HC is at least 97% identical to 3E10-HC-h67 (SEQ ID NO: 83). In some embodiments, the sequence of 3E10-HC is at least 98% identical to 3E10-HC-h67 (SEQ ID NO: 83). In some embodiments, the sequence of 3E10-HC is at least 99% identical to 3E10-HC-h67 (SEQ ID NO: 83). In some embodiments, the sequence of 3E10-HC is 3E10-HC-h67 (SEQ ID NO: 83).

[0129] In some embodiments, the sequence of 3E10-HC is at least 95% identical to 3E10-HC-h7 (SEQ ID NO: 84). In some embodiments, the sequence of 3E10-HC is at least 96% identical to 3E10-HC-h7 (SEQ ID NO: 84). In some embodiments, the sequence of 3E10-HC is at least 97% identical to 3E10-HC-h7 (SEQ ID NO: 84). In some embodiments, the sequence of 3E10-HC is at least 98% identical to 3E10-HC-h7 (SEQ ID NO: 84). In some embodiments, the sequence of 3E10-HC is at least 99% identical to 3E10-HC-h7 (SEQ ID NO: 84). In some embodiments, the sequence of 3E10-HC is 3E10-HC-h7 (SEQ ID NO: 84).

[0130] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein is selected from the group consisting of 3E10-VH-h1 (SEQ ID NO: 64) and 3E10-VL-h1 (SEQ ID NO: 85), 3E10-VH-h1 (SEQ ID NO: 64) and 3E10-VL-h2 (SEQ ID NO: 86), 3E10-VH-h1 (SEQ ID NO: 64) and 3E10-VL-h3 (SEQ ID NO: 87), 3E10-VH-h1 (SEQ ID NO: 64) and 3E10-VL-h4 (SEQ ID NO: 88), 3E10-VH-h2 (SEQ ID NO: 65 ...3 (SEQ ID NO: 87), 2 (SEQ ID NO: 65) and 3E10-VL-h2 (SEQ ID NO: 86), 3E10-VH-h2 (SEQ ID NO: 65) and 3E10-VL-h3 (SEQ ID NO: 87), 3E10-VH-h2 (SEQ ID NO: 65) and 3E10-VL-h4 (SEQ ID NO: 88), 3E10-VH-h3 (SEQ ID NO: 66) and 3E10-VL-h1 (SEQ ID NO: 85), 3E10-VH-h3 (SEQ ID NO: 66) and 3E10-VL-h2 (SEQ ID NO: 86), 3E10-VH-h3 (SEQ ID NO: 66) and 3E10-VL-h3 (SEQ ID NO: 87), 3E10-VH-h3 (SEQ ID NO: 66) and 3E10-VL-h4 (SEQ ID NO: 88), 3E10-VH-h4 (SEQ ID NO: 67) and 3E10-VL-h1 (SEQ ID NO: 85), 3E10-VH-h4 (SEQ ID NO: 67) and 3E10-VL-h2 (SEQ ID NO: 86), 3E10-VH-h4 (SEQ ID NO: 67) and 3E10-VL-h3 (SEQ ID NO: 87), 3E10-VH-h4 (SEQ ID NO: 67) and 3E10-VL-h4 (SEQ ID NO: 88), 3E10-VH-h5 (SEQ ID NO: 68) and 3E10-VL-h5 (SEQ ID NO: 89), 3E10-VH-h5 (SEQ ID NO: 68) and 3E10-VL-h6 ( and a combination of heavy chain variable domains (VH) and light chain variable domains (VL) comprising amino acid sequences having at least 97% sequence identity to a pair of VL and VH selected from: 3E10-VH-h6 (SEQ ID NO: 69) and 3E10-VL-h5 (SEQ ID NO: 89), 3E10-VH-h6 (SEQ ID NO: 69) and 3E10-VL-h6 (SEQ ID NO: 90), 3E10-VH-h7 (SEQ ID NO: 70) and 3E10-VL-h5 (SEQ ID NO: 89), and 3E10-VH-h7 (SEQ ID NO: 70) and 3E10-VL-h6 (SEQ ID NO: 90).

[0131] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein is selected from the group consisting of 3E10-VH-h1 (SEQ ID NO: 64) and 3E10-VL-h1 (SEQ ID NO: 85), 3E10-VH-h1 (SEQ ID NO: 64) and 3E10-VL-h2 (SEQ ID NO: 86), 3E10-VH-h1 (SEQ ID NO: 64) and 3E10-VL-h3 (SEQ ID NO: 87), 3E10-VH-h1 (SEQ ID NO: 64) and 3E10-VL-h4 (SEQ ID NO: 88), 3E10-VH-h2 (SEQ ID NO: 65 ...3 (SEQ ID NO: 87), 2 (SEQ ID NO: 65) and 3E10-VL-h2 (SEQ ID NO: 86), 3E10-VH-h2 (SEQ ID NO: 65) and 3E10-VL-h3 (SEQ ID NO: 87), 3E10-VH-h2 (SEQ ID NO: 65) and 3E10-VL-h4 (SEQ ID NO: 88), 3E10-VH-h3 (SEQ ID NO: 66) and 3E10-VL-h1 (SEQ ID NO: 85), 3E10-VH-h3 (SEQ ID NO: 66) and 3E10-VL-h2 (SEQ ID NO: 86), 3E10-VH-h3 (SEQ ID NO: 66) and 3E10-VL-h3 (SEQ ID NO: 87), 3E10-VH-h3 (SEQ ID NO: 66) and 3E10-VL-h4 (SEQ ID NO: 88), 3E10-VH-h4 (SEQ ID NO: 67) and 3E10-VL-h1 (SEQ ID NO: 85), 3E10-VH-h4 (SEQ ID NO: 67) and 3E10-VL-h2 (SEQ ID NO: 86), 3E10-VH-h4 (SEQ ID NO: 67) and 3E10-VL-h3 (SEQ ID NO: 87), 3E10-VH-h4 (SEQ ID NO: 67) and 3E10-VL-h4 (SEQ ID NO: 88), 3E10-VH-h5 (SEQ ID NO: 68) and 3E10-VL-h5 (SEQ ID NO: 89), 3E10-VH-h5 (SEQ ID NO: 68) and 3E10-VL-h6 ( and a combination of heavy chain variable domains (VH) and light chain variable domains (VL) comprising amino acid sequences having at least 98% sequence identity to a pair of VL and VH selected from: 3E10-VH-h6 (SEQ ID NO: 69) and 3E10-VL-h5 (SEQ ID NO: 89), 3E10-VH-h6 (SEQ ID NO: 69) and 3E10-VL-h6 (SEQ ID NO: 90), 3E10-VH-h7 (SEQ ID NO: 70) and 3E10-VL-h5 (SEQ ID NO: 89), and 3E10-VH-h7 (SEQ ID NO: 70) and 3E10-VL-h6 (SEQ ID NO: 90).

[0132] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein is selected from the group consisting of 3E10-VH-h1 (SEQ ID NO: 64) and 3E10-VL-h1 (SEQ ID NO: 85), 3E10-VH-h1 (SEQ ID NO: 64) and 3E10-VL-h2 (SEQ ID NO: 86), 3E10-VH-h1 (SEQ ID NO: 64) and 3E10-VL-h3 (SEQ ID NO: 87), 3E10-VH-h1 (SEQ ID NO: 64) and 3E10-VL-h4 (SEQ ID NO: 88), 3E10-VH-h2 (SEQ ID NO: 65 ...3 (SEQ ID NO: 87), 2 (SEQ ID NO: 65) and 3E10-VL-h2 (SEQ ID NO: 86), 3E10-VH-h2 (SEQ ID NO: 65) and 3E10-VL-h3 (SEQ ID NO: 87), 3E10-VH-h2 (SEQ ID NO: 65) and 3E10-VL-h4 (SEQ ID NO: 88), 3E10-VH-h3 (SEQ ID NO: 66) and 3E10-VL-h1 (SEQ ID NO: 85), 3E10-VH-h3 (SEQ ID NO: 66) and 3E10-VL-h2 (SEQ ID NO: 86), 3E10-VH-h3 (SEQ ID NO: 66) and 3E10-VL-h3 (SEQ ID NO: 87), 3E10-VH-h3 (SEQ ID NO: 66) and 3E10-VL-h4 (SEQ ID NO: 88), 3E10-VH-h4 (SEQ ID NO: 67) and 3E10-VL-h1 (SEQ ID NO: 85), 3E10-VH-h4 (SEQ ID NO: 67) and 3E10-VL-h2 (SEQ ID NO: 86), 3E10-VH-h4 (SEQ ID NO: 67) and 3E10-VL-h3 (SEQ ID NO: 87), 3E10-VH-h4 (SEQ ID NO: 67) and 3E10-VL-h4 (SEQ ID NO: 88), 3E10-VH-h5 (SEQ ID NO: 68) and 3E10-VL-h5 (SEQ ID NO: 89), 3E10-VH-h5 (SEQ ID NO: 68) and 3E10-VL-h6 ( and 3E10-VH-h7 (SEQ ID NO: 70) and 3E10-VL-h6 (SEQ ID NO: 90).

[0133] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein is selected from the group consisting of 3E10-VH-h1 (SEQ ID NO: 64) and 3E10-VL-h1 (SEQ ID NO: 85), 3E10-VH-h1 (SEQ ID NO: 64) and 3E10-VL-h2 (SEQ ID NO: 86), 3E10-VH-h1 (SEQ ID NO: 64) and 3E10-VL-h3 (SEQ ID NO: 87), 3E10-VH-h1 (SEQ ID NO: 64) and 3E10-VL-h4 (SEQ ID NO: 88), 3E10-VH-h2 (SEQ ID NO: 65) and 3E10-VL-h1 (SEQ ID NO: 85), 3E 10-VH-h2 (SEQ ID NO: 65) and 3E10-VL-h2 (SEQ ID NO: 86), 3E10-VH-h2 (SEQ ID NO: 65) and 3E10-VL-h3 (SEQ ID NO: 87), 3E10-VH-h2 (SEQ ID NO: 65) and 3E10-VL-h4 (SEQ ID NO: 88), 3E10-VH-h3 (SEQ ID NO: 66) and 3E10-VL-h1 (SEQ ID NO: 85), 3E10-VH-h3 (SEQ ID NO: 66) and 3E10-VL-h2 (SEQ ID NO: 86), 3E10-VH-h3 (SEQ ID NO: 66) and 3E10-VL-h3 (SEQ ID NO: 87), 3E10-V H-h3 (SEQ ID NO: 66) and 3E10-VL-h4 (SEQ ID NO: 88), 3E10-VH-h4 (SEQ ID NO: 67) and 3E10-VL-h1 (SEQ ID NO: 85), 3E10-VH-h4 (SEQ ID NO: 67) and 3E10-VL-h2 (SEQ ID NO: 86), 3E10-VH-h4 (SEQ ID NO: 67) and 3E10-VL-h3 (SEQ ID NO: 87), 3E10-VH-h4 (SEQ ID NO: 67) and 3E10-VL-h4 (SEQ ID NO: 88), 3E10-VH-h5 (SEQ ID NO: 68) and 3E10-VL-h5 (SEQ ID NO: 89), 3E10-VH-h5 (SEQ ID NO: 68) and 3E10-VL-h6 (SEQ ID NO: 90), 3E10-VH-h6 (SEQ ID NO: 69) and 3E10-VL-h5 (SEQ ID NO: 89), 3E10-VH-h6 (SEQ ID NO: 69) and 3E10-VL-h6 (SEQ ID NO: 90), 3E10-VH-h7 (SEQ ID NO: 70) and 3E10-VL-h5 (SEQ ID NO: 89), and 3E10-VH-h7 (SEQ ID NO: 70) and 3E10-VL-h6 (SEQ ID NO: 90).

[0134] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a heavy chain variable domain (VH) comprising an amino acid sequence having at least 97% sequence identity to 3E10-VH-h6 (SEQ ID NO: 69) and a light chain variable domain (VL) comprising an amino acid sequence having at least 97% sequence identity to 3E10-VL-h6 (SEQ ID NO: 90). In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a heavy chain variable domain (VH) comprising an amino acid sequence having at least 98% sequence identity to 3E10-VH-h6 (SEQ ID NO: 69) and a light chain variable domain (VL) comprising an amino acid sequence having at least 98% sequence identity to 3E10-VL-h6 (SEQ ID NO: 90). In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a heavy chain variable domain (VH) comprising an amino acid sequence having at least 99% sequence identity to 3E10-VH-h6 (SEQ ID NO: 69) and a light chain variable domain (VL) comprising an amino acid sequence having at least 99% sequence identity to 3E10-VL-h6 (SEQ ID NO: 90). In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a heavy chain variable domain (VH) comprising the amino acid sequence of 3E10-VH-h6 (SEQ ID NO: 69) and a light chain variable domain (VL) comprising the amino acid sequence of 3E10-VL-h6 (SEQ ID NO: 90).

[0135] In some embodiments, the humanized 3E10 antibodies or antigen-binding fragments thereof described herein have CDR sequences corresponding to the parent 3E10 antibody shown in FIG. 1 (SEQ ID NOs: 1-12), which optionally contains a D31N amino acid substitution in VH CDR1 as shown in FIG. Thus, in some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) complementarity determining region (CDR) 1 comprising the amino acid sequence of 3E10-VL-CDR1 (SEQ ID NO: 9), a VL CDR2 comprising the amino acid sequence of 3E10-VL-CDR2 (SEQ ID NO: 10), a VL CDR3 comprising the amino acid sequence of 3E10-VL-CDR3 (SEQ ID NO: 11), a heavy chain variable region (VH) CDR1 comprising the amino acid sequence of 3E10-VH-CDR1a (SEQ ID NO: 16), a VH CDR2 comprising the amino acid sequence of 3E10-VH-CDR2 (SEQ ID NO: 4), and a VH CDR3 comprising the amino acid sequence of 3E10-VH-CDR3 (SEQ ID NO: 5).

[0136] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises the CDR sequences from a variant humanized 3E10 antibody comprising a D31N amino acid substitution in VH CDR1 as shown in FIG.

[0137] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a set of complementarity determining regions (CDRs) having a total of no more than seven amino acid substitutions compared to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NO: 9), 3E10-VL-CDR2 (SEQ ID NO: 10), 3E10-VL-CDR3 (SEQ ID NO: 11), 3E10-VH-CDR1_D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NO: 4), and 3E10-VH-CDR3 (SEQ ID NO: 5).

[0138] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a set of complementarity determining regions (CDRs) having a total of 10 or fewer amino acid substitutions compared to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NO: 9), 3E10-VL-CDR2 (SEQ ID NO: 10), 3E10-VL-CDR3 (SEQ ID NO: 11), 3E10-VH-CDR1_D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NO: 4), and 3E10-VH-CDR3 (SEQ ID NO: 5).

[0139] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a set of complementarity determining regions (CDRs) having a total of nine or fewer amino acid substitutions compared to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NO: 9), 3E10-VL-CDR2 (SEQ ID NO: 10), 3E10-VL-CDR3 (SEQ ID NO: 11), 3E10-VH-CDR1_D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NO: 4), and 3E10-VH-CDR3 (SEQ ID NO: 5).

[0140] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a set of complementarity determining regions (CDRs) having a total of eight or fewer amino acid substitutions compared to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NO: 9), 3E10-VL-CDR2 (SEQ ID NO: 10), 3E10-VL-CDR3 (SEQ ID NO: 11), 3E10-VH-CDR1_D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NO: 4), and 3E10-VH-CDR3 (SEQ ID NO: 5).

[0141] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a set of complementarity determining regions (CDRs) having a total of no more than seven amino acid substitutions compared to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NO: 9), 3E10-VL-CDR2 (SEQ ID NO: 10), 3E10-VL-CDR3 (SEQ ID NO: 11), 3E10-VH-CDR1_D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NO: 4), and 3E10-VH-CDR3 (SEQ ID NO: 5).

[0142] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a set of complementarity determining regions (CDRs) having a total of six or fewer amino acid substitutions compared to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NO: 9), 3E10-VL-CDR2 (SEQ ID NO: 10), 3E10-VL-CDR3 (SEQ ID NO: 11), 3E10-VH-CDR1_D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NO: 4), and 3E10-VH-CDR3 (SEQ ID NO: 5).

[0143] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a set of complementarity determining regions (CDRs) having a total of five or fewer amino acid substitutions compared to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NO: 9), 3E10-VL-CDR2 (SEQ ID NO: 10), 3E10-VL-CDR3 (SEQ ID NO: 11), 3E10-VH-CDR1_D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NO: 4), and 3E10-VH-CDR3 (SEQ ID NO: 5).

[0144] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a set of complementarity determining regions (CDRs) having a total of no more than four amino acid substitutions compared to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NO: 9), 3E10-VL-CDR2 (SEQ ID NO: 10), 3E10-VL-CDR3 (SEQ ID NO: 11), 3E10-VH-CDR1_D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NO: 4), and 3E10-VH-CDR3 (SEQ ID NO: 5).

[0145] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a set of complementarity determining regions (CDRs) having a total of no more than three amino acid substitutions compared to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NO: 9), 3E10-VL-CDR2 (SEQ ID NO: 10), 3E10-VL-CDR3 (SEQ ID NO: 11), 3E10-VH-CDR1_D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NO: 4), and 3E10-VH-CDR3 (SEQ ID NO: 5).

[0146] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a set of complementarity determining regions (CDRs) having a total of no more than two amino acid substitutions compared to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NO: 9), 3E10-VL-CDR2 (SEQ ID NO: 10), 3E10-VL-CDR3 (SEQ ID NO: 11), 3E10-VH-CDR1_D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NO: 4), and 3E10-VH-CDR3 (SEQ ID NO: 5).

[0147] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a set of complementarity determining regions (CDRs) having a total of no more than one amino acid substitution compared to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NO: 9), 3E10-VL-CDR2 (SEQ ID NO: 10), 3E10-VL-CDR3 (SEQ ID NO: 11), 3E10-VH-CDR1_D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NO: 4), and 3E10-VH-CDR3 (SEQ ID NO: 5).

[0148] Thus, in some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) complementarity determining region (CDR) 1 comprising the amino acid sequence of 3E10-VL-CDR1 (SEQ ID NO: 9), a VL CDR2 comprising the amino acid sequence of 3E10-VL-CDR2 (SEQ ID NO: 10), a VL CDR3 comprising the amino acid sequence of 3E10-VL-CDR3 (SEQ ID NO: 11), a heavy chain variable region (VH) CDR1 comprising the amino acid sequence of 3E10-VH-CDR1_D31N (SEQ ID NO: 15), a VH CDR2 comprising the amino acid sequence of 3E10-VH-CDR2 (SEQ ID NO: 4), and a VH CDR3 comprising the amino acid sequence of 3E10-VH-CDR3 (SEQ ID NO: 5).

[0149] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a set of complementarity determining regions (CDRs) having a total of no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions selected from, but not limited to, a G to S substitution at position 5 of VH CDR2, a T to S substitution at position 14 of VH CDR2, an S to T substitution at position 5 of VL CDR1, an M to L substitution at position 14 of VL CDR1, an H to A substitution at position 15 of VL CDR1, and an E to Q substitution at position 6 of VL CDR2, compared to the CDR sequences of the 3E10-D31N variant (shown in FIG. 2 ).

[0150] Thus, in some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof comprises a VH CDR2 comprising the amino acid sequence of 3E10-VH-CDR2.1 (SEQ ID NO:26) or 3E10-VH-CDR2.2 (SEQ ID NO:27). In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDR1-3 and VH CDR1 and 3 in accordance with the parent 3E10 antibody (shown in FIG. 1 (SEQ ID NOs:1-12)). In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDR1-3 and VH CDR1 and 3 in accordance with the 3E10-D31N variant (shown in FIG. 2A (SEQ ID NOs:13-25)).

[0151] Similarly, in some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof comprises a VL CDR1 comprising the amino acid sequence of 3E10-VL-CDR1.1 (SEQ ID NO:28) or 3E10-VL-CDR1.1 (SEQ ID NO:29). In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDR2 and 3, and VH CDR1-3 in accordance with the parent 3E10 antibody (shown in FIG. 1 (SEQ ID NOs:1-12)). In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDR2 and 3, and VH CDR1-3 in accordance with the 3E10-D31N variant (shown in FIG. 2A (SEQ ID NOs:13-25)).

[0152] Similarly, in some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof comprises a VL CDR2 comprising the amino acid sequence of 3E10-VL-CDR2.1 (SEQ ID NO:30). In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDR1 and 3, and VH CDR1-3 in accordance with the parent 3E10 antibody (shown in FIG. 1 (SEQ ID NOs:1-12)). In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDR1 and 3, and VH CDR1-3 in accordance with the 3E10-D31N variant (shown in FIG. 2A (SEQ ID NOs:13-25)).

[0153] While some of the amino acid substitutions mentioned above, e.g., S to T at position 5 of VL CDR1, are fairly conservative substitutions, others, e.g., M to L at position 14 of VL CDR1, H to A at position 15 of VL CDR1, and E to Q at position 6 of VL CDR2, are substitutions of amino acids with very different properties, which suggests, without being bound by theory, that at least these positions within the 3E10 CDR framework are tolerant to other amino acid substitutions.

[0154] Thus, in some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof comprises a VH CDR2 comprising the amino acid sequence of 3E10-VH-CDR2.3 (SEQ ID NO:31). In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDR1-3, and VH CDR1 and 3, in accordance with the parent 3E10 antibody (shown in FIG. 1 (SEQ ID NOs:1-12)). In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDR1-3, and VH CDR1 and 3, in accordance with the 3E10-D31N variant (shown in FIG. 2A (SEQ ID NOs:13-25)).

[0155] Similarly, in some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof comprises a VL CDR1 comprising the amino acid sequence of 3E10-VL-CDR1.3 (SEQ ID NO:32). In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDR2 and 3, and VH CDR1-3 in accordance with the parent 3E10 antibody (shown in FIG. 1 (SEQ ID NOs:1-12)). In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDR2 and 3, and VH CDR1-3 in accordance with the 3E10-D31N variant (shown in FIG. 2A (SEQ ID NOs:13-25)).

[0156] Similarly, in some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof comprises a VL CDR2 comprising the amino acid sequence of 3E10-VL-CDR2.2 (SEQ ID NO:33). In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDR1 and 3, and VH CDR1-3 in accordance with the parent 3E10 antibody (shown in FIG. 1 (SEQ ID NOs:1-12)). In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDR1 and 3, and VH CDR1-3 in accordance with the 3E10-D31N variant (shown in FIG. 2A (SEQ ID NOs:13-25)).

[0157] Thus, in some embodiments, a humanized 3E10 antibody or antigen-binding fragment thereof comprises a VH CDR1 comprising the amino acid sequence of 3E10-VH-CDR1.c1 (SEQ ID NO: 34), 3E10-VH-CDR1.c2 (SEQ ID NO: 35), 3E10-VH-CDR1.c3 (SEQ ID NO: 36), 3E10-VH-CDR1.c4 (SEQ ID NO: 37), or 3E10-VH-CDR1.c5 (SEQ ID NO: 38). In some embodiments, a humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDR1-3, and VH CDR2 and 3 in accordance with the parent 3E10 antibody (shown in Figure 1 (SEQ ID NOs: 1-12)).

[0158] Similarly, in some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof comprises a VH CDR2 comprising the amino acid sequence of 3E10-VH-CDR2.c1 (SEQ ID NO:39), 3E10-VH-CDR2.c2 (SEQ ID NO:40), or 3E10-VH-CDR2.c3 (SEQ ID NO:41). In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDR1-3 and VH CDR1 and 3 in accordance with the parent 3E10 antibody (shown in FIG. 1 (SEQ ID NOs:1-12)). In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDR1-3 and VH CDR1 and 3 in accordance with the 3E10-D31N variant (shown in FIG. 2A (SEQ ID NOs:13-25)).

[0159] Similarly, in some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof comprises a VH CDR3 comprising the amino acid sequence of 3E10-VH-CDR3.c1 (SEQ ID NO:42), 3E10-VH-CDR3.c2 (SEQ ID NO:43), or 3E10-VH-CDR3.c3 (SEQ ID NO:44). In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDR1-3 and VH CDR1 and 2 in accordance with the parent 3E10 antibody (shown in FIG. 1 (SEQ ID NOs:1-12)). In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDR1-3 and VH CDR1 and 2 in accordance with the 3E10-D31N variant (shown in FIG. 2A (SEQ ID NOs:13-25)).

[0160] Similarly, in some embodiments, a humanized 3E10 antibody or antigen-binding fragment thereof comprises a VL CDR1 comprising the amino acid sequence of 3E10-VL-CDR1.c1 (SEQ ID NO:45), 3E10-VL-CDR1.c2 (SEQ ID NO:46), 3E10-VL-CDR1.c3 (SEQ ID NO:47), 3E10-VL-CDR1.c4 (SEQ ID NO:48), 3E10-VL-CDR1.c5 (SEQ ID NO:49), or 3E10-VL-CDR1.c6 (SEQ ID NO:50). In some embodiments, a humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDR2 and 3, and VH CDR1-3 in accordance with the parent 3E10 antibody (shown in Figure 1 (SEQ ID NOs:1-12)). In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDRs 2 and 3, and VH CDRs 1-3 according to the 3E10-D31N variant (shown in FIG. 2A (SEQ ID NOs: 13-25)).

[0161] Similarly, in some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof comprises a VL CDR2 comprising the amino acid sequence of 3E10-VL-CDR2.c1 (SEQ ID NO:51). In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDR1 and 3, and VH CDR1-3 in accordance with the parent 3E10 antibody (shown in FIG. 1 (SEQ ID NOs:1-12)). In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDR1 and 3, and VH CDR1-3 in accordance with the 3E10-D31N variant (shown in FIG. 2A (SEQ ID NOs:13-25)).

[0162] Similarly, in some embodiments, a humanized 3E10 antibody or antigen-binding fragment thereof comprises a VL CDR3 comprising the amino acid sequence of 3E10-VL-CDR3.c1 (SEQ ID NO:52), 3E10-VL-CDR3.c2 (SEQ ID NO:53), 3E10-VL-CDR3.c3 (SEQ ID NO:54), 3E10-VL-CDR3.c4 (SEQ ID NO:55), 3E10-VL-CDR3.c5 (SEQ ID NO:56), or 3E10-VL-CDR3.c6 (SEQ ID NO:57). In some embodiments, a humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDR1 and 2, and VH CDR1-3 in accordance with the parent 3E10 antibody (shown in Figure 1 (SEQ ID NOs:1-12)). In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDR1 and 2, and VH CDR1-3 according to the 3E10-D31N variant (shown in FIG. 2A (SEQ ID NOs: 13-25)).

[0163] It is also contemplated that the humanized 3E10 antibodies or antigen-binding fragments thereof described herein contain no more than 7, 6, 5, 4, 3, 2, or 1 of the CDR amino acid substitutions described above. Further examples of 3E10 variant CDR sequences described herein are shown in FIG.

[0164] Thus, in some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof comprises a VH CDR1 comprising the amino acid sequence of 3E10-VH-CDR1m (SEQ ID NO: 58). In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDR1-3, and VH CDR2 and 3 according to the parent 3E10 antibody (shown in Figure 1 (SEQ ID NOs: 1-12)).

[0165] Similarly, in some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof comprises a VH CDR2 comprising the amino acid sequence of 3E10-VH-CDR2m (SEQ ID NO:59). In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDR1-3, and VH CDR1 and 3, in accordance with the parent 3E10 antibody (shown in FIG. 1 (SEQ ID NOs:1-12)). In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDR1-3, and VH CDR1 and 3, in accordance with the 3E10-D31N variant (shown in FIG. 2A (SEQ ID NOs:13-25)).

[0166] Similarly, in some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof comprises a VH CDR3 comprising the amino acid sequence of 3E10-VH-CDR3m (SEQ ID NO: 60). In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDR1-3 and VH CDR1 and 2 in accordance with the parent 3E10 antibody (shown in FIG. 1 (SEQ ID NOs: 1-12)). In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDR1-3 and VH CDR1 and 2 in accordance with the 3E10-D31N variant (shown in FIG. 2A (SEQ ID NOs: 13-25)).

[0167] Similarly, in some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof comprises a VL CDR1 comprising the amino acid sequence of 3E10-VL-CDR1m (SEQ ID NO:61). In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDR2 and 3, and VH CDR1-3 in accordance with the parent 3E10 antibody (shown in FIG. 1 (SEQ ID NOs:1-12)). In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDR2 and 3, and VH CDR1-3 in accordance with the 3E10-D31N variant (shown in FIG. 2A (SEQ ID NOs:13-25)).

[0168] Similarly, in some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof comprises a VL CDR2 comprising the amino acid sequence of 3E10-VL-CDR2m (SEQ ID NO: 62). In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDR1 and 3, and VH CDR1-3 in accordance with the parent 3E10 antibody (shown in FIG. 1 (SEQ ID NOs: 1-12)). In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDR1 and 3, and VH CDR1-3 in accordance with the 3E10-D31N variant (shown in FIG. 2A (SEQ ID NOs: 13-25)).

[0169] Similarly, in some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof comprises a VL CDR3 comprising the amino acid sequence of 3E10-VL-CDR3m (SEQ ID NO: 63). In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDR1 and 2, and VH CDR1-3 in accordance with the parent 3E10 antibody (shown in FIG. 1 (SEQ ID NOs: 1-12)). In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDR1 and 2, and VH CDR1-3 in accordance with the 3E10-D31N variant (shown in FIG. 2A (SEQ ID NOs: 13-25)).

[0170] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a light chain variable domain (3E10-VL) comprising an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of 3E10-VL-h1 (SEQ ID NO: 85), 3E10-VL-h2 (SEQ ID NO: 86), 3E10-VL-h3 (SEQ ID NO: 87), 3E10-VL-h4 (SEQ ID NO: 88), 3E10-VL-h5 (SEQ ID NO: 89), and 3E10-VL-h6 (SEQ ID NO: 90), wherein the light chain variable domain (3E10-VL) comprises proline (Pro) at position 15, threonine (Thr) at position 22, tyrosine (Tyr) at position 49, Thr at position 74, asparagine (Asn) at position 76, according to the Kabat numbering of 3E10-VL. (Asn), alanine (Ala) at position 80, Asn at position 81, Thr at position 83, Asn at position 85, and valine (Val) at position 104, and a set of 3E10-VL (SEQ ID NO: 8) CDRs having a total of six or fewer amino acid substitutions compared to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NO: 9), 3E10-VL-CDR2 (SEQ ID NO: 10), and 3E10-VL-CDR3 (SEQ ID NO: 11), and the antibody comprises a set of 3E10-VL CDRs having a total of six or fewer amino acid substitutions compared to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NO: 9), 3E10-VL-CDR2 (SEQ ID NO: 10), and 3E10-VL-CDR3 (SEQ ID NO: 11).

[0171] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof comprises a set of 3E10-VL CDRs that comprise no more than 5 amino acid substitutions compared to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NO: 9), 3E10-VL-CDR2 (SEQ ID NO: 10), 3E10-VL-CDR3 (SEQ ID NO: 11). In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof comprises a set of 3E10-VL CDRs that comprise no more than 4 amino acid substitutions compared to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NO: 9), 3E10-VL-CDR2 (SEQ ID NO: 10), 3E10-VL-CDR3 (SEQ ID NO: 11). In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof comprises a set of 3E10-VL CDRs that comprise no more than three amino acid substitutions compared to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NO: 9), 3E10-VL-CDR2 (SEQ ID NO: 10), 3E10-VL-CDR3 (SEQ ID NO: 11). In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof comprises a set of 3E10-VL CDRs that comprise no more than two amino acid substitutions compared to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NO: 9), 3E10-VL-CDR2 (SEQ ID NO: 10), 3E10-VL-CDR3 (SEQ ID NO: 11). In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof comprises a set of 3E10-VL CDRs that comprise no more than one amino acid substitution compared to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NO: 9), 3E10-VL-CDR2 (SEQ ID NO: 10), 3E10-VL-CDR3 (SEQ ID NO: 11). In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof comprises a set of 3E10-VL CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NO: 9), 3E10-VL-CDR2 (SEQ ID NO: 10), 3E10-VL-CDR3 (SEQ ID NO: 11).

[0172] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof has a lysine (Lys) residue at position 49 of 3E10-VL according to the Kabat numbering. In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof has a glutamic acid (Glu) residue at position 81 of 3E10-VL according to the Kabat numbering. In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof has a proline (Pro) residue at position 15 of 3E10-VL according to the Kabat numbering. In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof has a valine (Val) residue at position 104 of 3E10-VL according to the Kabat numbering.

[0173] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a heavy chain variable domain (3E10-VH) comprising an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of 3E10-VH-h1 (SEQ ID NO: 64), 3E10-VH-h2 (SEQ ID NO: 65), 3E10-VH-h3 (SEQ ID NO: 66), 3E10-VH-h4 (SEQ ID NO: 67), 3E10-VH-h5 (SEQ ID NO: 68), 3E10-VH-h6 (SEQ ID NO: 69), and 3E10-VH-h7 (SEQ ID NO: 70), wherein the heavy chain variable domain (3E10-VH) comprises a glutamate at position 13 according to the Kabat numbering of 3E10-VH. and one or more amino acid residues selected from among Amino Acid Sequence Listing (Asp) at position 1, Amino Acid Sequence Listing (Ala) at position 2, Amino Acid Sequence Listing (Gln) at position 3, Amino Acid Sequence Listing (Leu) at position 4, Amino Acid Sequence Listing (Arg) at position 5, Amino Acid Sequence Listing (Ala) at position 6, Amino Acid Sequence Listing (Ala) at position 7, Amino Acid Sequence Listing (Ala) at position 8, Amino Acid Sequence Listing (Ala) at position 9, Amino Acid Sequence Listing (Ala) at position 10, Amino Acid Sequence Listing (Ala) at position 11, Amino Acid Sequence Listing (Ala) at position 12, Amino Acid Sequence Listing (Ala) at position 13, Amino Acid Sequence Listing (Ala) at position 14, Amino Acid Sequence Listing (Ala) at position 15, Amino Acid Sequence Listing (Ala) at position 16, Amino Acid Sequence Listing (Ala) at position 17, Amino Acid Sequence Listing (Ala) at position 18, Amino Acid Sequence Listing (Arg) at position 19, Amino Acid Sequence Listing (Gln) at position 20, Amino Acid Sequence Listing (Leu) at position 21, Amino Acid Sequence Listing (Arg) at position 22, Amino Acid Sequence Listing (Gly) at position 42, Amino Acid Sequence Listing (Ser) at position 49, Amino Acid Sequence Listing (Ser) at position 77, Amino Acid Sequence Listing (Tyr) at position 79, Amino Acid Sequence Listing (Asn) at position 82, Amino Acid Sequence Listing (Ala) at position 84, Amino Acid Sequence Listing (Val) at position 89, Amino Acid Sequence Listing (Leu) at position 108, Amino Acid Sequence Listing (Val) at position 109, and Amino Acid Sequence Listing (S

[0174] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof comprises a set of 3E10-VH CDRs that comprise no more than 5 amino acid substitutions compared to the set of CDRs having the amino acid sequences of 3E10-VH-CDR1_D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NO: 4), and 3E10-VH-CDR3 (SEQ ID NO: 5). In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof comprises a set of 3E10-VH CDRs that comprise no more than 4 amino acid substitutions compared to the set of CDRs having the amino acid sequences of 3E10-VH-CDR1_D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NO: 4), and 3E10-VH-CDR3 (SEQ ID NO: 5). In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof comprises a set of 3E10-VH CDRs that comprise no more than three amino acid substitutions compared to the set of CDRs having the amino acid sequences of 3E10-VH-CDR1_D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NO: 4), and 3E10-VH-CDR3 (SEQ ID NO: 5). In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof comprises a set of 3E10-VH CDRs that comprise no more than two amino acid substitutions compared to the set of CDRs having the amino acid sequences of 3E10-VH-CDR1_D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NO: 4), and 3E10-VH-CDR3 (SEQ ID NO: 5). In some embodiments, a humanized 3E10 antibody or antigen-binding fragment thereof comprises a set of 3E10-VH CDRs that comprise no more than one amino acid substitution compared to the set of CDRs having the amino acid sequences of 3E10-VH-CDR1_D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NO: 4), and 3E10-VH-CDR3 (SEQ ID NO: 5). Similarly, in some embodiments, a humanized 3E10 antibody or antigen-binding fragment thereof comprises a set of 3E10-VH CDRs having the amino acid sequences of 3E10-VH-CDR1_D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NO: 4), and 3E10-VH-CDR3 (SEQ ID NO: 5).

[0175] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof has an arginine (Arg) residue at position 18 of 3E10-VH according to the Kabat numbering. In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof has a (Lys) residue at position 19 of 3E10-VH (SEQ ID NO:2) according to the Kabat numbering. In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof has an alanine (Ala) residue at position 49 of 3E10-VH according to the Kabat numbering. In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof has a glutamine (Gln) residue at position 13 of 3E10-VH (SEQ ID NO:2) according to the Kabat numbering. In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof has a leucine (Leu) residue at position 108 of 3E10-VH according to the Kabat numbering. In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof has a Val residue at position 109 of 3E10-VH according to the Kabat numbering. In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof has a serine (Ser) residue at position 113 of 3E10-VH according to the Kabat numbering.

[0176] In some embodiments, a humanized 3E10 antibody or antigen-binding fragment thereof of the present disclosure has a nucleic acid binding affinity for a polynucleotide, e.g., DNA, RNA, PNA, morpholino, etc., that is similar to the affinity that a reference 3E10 antibody or antigen-binding fragment thereof, such as the 3E10 monoclonal antibody produced by hybridoma ATCC No. PTA 2439, or its D31N variant, has for the same polynucleotide. Thus, in some embodiments, a humanized 3E10 antibody or antigen-binding fragment thereof has an affinity for a polynucleotide that is 0.05-50 times stronger than the affinity that a reference 3E10 antibody has for the same polynucleotide.

[0177] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof of the present disclosure has a nucleic acid binding affinity for a polynucleotide, e.g., DNA, RNA, PNA, morpholino, etc., that is less than the affinity that a reference 3E10 antibody or antigen-binding fragment thereof, e.g., the 3E10 monoclonal antibody produced by ATCC No. PTA 2439 hybridoma or its D31N variant, has for the same polynucleotide. Thus, in some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof has an affinity for a polynucleotide that is 0.00001-fold to less than 1-fold stronger than the affinity that a reference 3E10 antibody has for the same polynucleotide. In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof has an affinity for a polynucleotide that is 0.00001-fold to 0.001-fold stronger than the affinity that a reference 3E10 antibody has for the same polynucleotide. In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof has an affinity for a polynucleotide that is 0.0001-0.01 times stronger than the affinity that a reference 3E10 antibody has for the same polynucleotide. In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof has an affinity for a polynucleotide that is 0.001-0.1 times stronger than the affinity that a reference 3E10 antibody has for the same polynucleotide. In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof has an affinity for a polynucleotide that is 0.01-1 times stronger than the affinity that a reference 3E10 antibody has for the same polynucleotide.

[0178] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof of the present disclosure has a nucleic acid binding affinity for a polynucleotide, e.g., DNA, RNA, PNA, morpholino, etc., that is greater than the affinity that a reference 3E10 antibody or antigen-binding fragment thereof, e.g., the 3E10 monoclonal antibody produced by ATCC No. PTA 2439 hybridoma or its D31N variant, has for the same polynucleotide. Thus, in some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof has an affinity for a polynucleotide that is greater than 1-fold to 10,000-fold stronger than the affinity that a reference 3E10 antibody has for the same polynucleotide. In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof has an affinity for a polynucleotide that is 100-fold to 10,000-fold stronger than the affinity that a reference 3E10 antibody has for the same polynucleotide. In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof has an affinity for a polynucleotide that is 10-1000 times stronger than the affinity that a reference 3E10 antibody has for the same polynucleotide. In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof has an affinity for a polynucleotide that is more than 1-100 times stronger than the affinity that a reference 3E10 antibody has for the same polynucleotide.

[0179] Binding affinity can be determined by the association (Ka) and dissociation (Kd) rates. The equilibrium affinity constant KD is the ratio of Ka / Kd. A humanized 3E10 antibody or antigen-binding fragment thereof having the same binding affinity as another 3E10 antibody means that the dissociation constant (Kd) of each antibody is within about 1-10 fold (1-10 fold greater affinity or 1-10 fold less affinity, or any number or range or value within such range). An example of an affinity for a target antigen (DNA (e.g., single-stranded and / or double-stranded DNA)) is one with a dissociation constant (Kd) of 5×10 -2 Less than M, 10 -2 Less than M, 5×10 -3 Less than M, 10 -3 Less than M, 5×10 -4 Less than M, 10 -4 Less than M, 5×10 -5Less than M, 10 -5 Less than M, 5×10 -8 Less than M, 10 -8 Less than M, 5×10 -7 Less than M, 10 -7 Less than M, 5×10 -8 Less than M, 10 -8 Less than M, 5×10 -9 Less than M, 10 -9 Less than M, 5×10 -10 Less than M, 10 -10 Less than M, 5×10 -11 Less than M, 10 -11 Less than M, 5×10 -12 Less than M, 10 -12 Less than M, 5×10 -13 Less than M, 10 -13 Less than M, 5×10 -14 Less than M, 10 -14 Less than M, 5×10 -15 Less than M or 10 -15 In some embodiments, the binding affinity (Kd) for the target is less than 10 -7 Less than M, 5×10 -8 Less than M, 10 -8 Less than M, 5×10 -9 Less than M, 10 -9 Less than M, 5×10 -10 Less than M, 10 -10 Less than M, 5×10 -11 Less than M, 10 -11 Less than M, 5×10 -12 Less than M or 10 -12 Less than M.

[0180] In some embodiments, binding affinity is assessed by determining the solution concentration of humanized 3E10 antibody or antigen-binding fragment thereof required to achieve half-maximal binding of a polynucleotide coated on a solid surface (EC50). In some embodiments, the nucleic acid is a poly-dT oligonucleotide. In some embodiments, binding of an oligonucleotide, e.g., a poly-dT oligonucleotide, is detected using an ELISA assay with a secondary anti-human immunoglobulin antibody.

[0181] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein has a weak binding affinity to poly-dT oligonucleotides. In some embodiments, the weak binding affinity is an EC50 value at least 100 times greater than the EC50 value of a reference 3E10 antibody, for example, a chimeric 3E10 antibody having a D31N amino acid substitution. In some embodiments, the weak binding affinity is an EC50 value that is 100 times greater to 10,000 times greater than the EC50 value of a reference 3E10 antibody, for example, a chimeric 3E10 antibody having a D31N amino acid substitution. In some embodiments, the weak binding affinity is an EC50 value that is 100 times greater to 5000 times greater than the EC50 value of a reference 3E10 antibody, for example, a chimeric 3E10 antibody having a D31N amino acid substitution. In some embodiments, a weak binding affinity is an EC50 value that is 100-fold greater to 1000-fold greater than the EC50 value of a reference 3E10 antibody, e.g., a chimeric 3E10 antibody with a D31N amino acid substitution. In some embodiments, a weak binding affinity is an EC50 value that is 100-fold greater to 500-fold greater than the EC50 value of a reference 3E10 antibody, e.g., a chimeric 3E10 antibody with a D31N amino acid substitution.

[0182] In some embodiments, a humanized 3E10 antibody or antigen-binding fragment thereof described herein has a moderate binding affinity for a poly-dT oligonucleotide, in some embodiments, a moderate binding affinity is an EC50 value that is 25-100 times greater than the EC50 value of a reference 3E10 antibody, e.g., a chimeric 3E10 antibody having a D31N amino acid substitution.

[0183] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein has a moderate binding affinity to poly-dT oligonucleotides. In some embodiments, the moderate binding affinity is an EC50 value that is not more than 25 times the EC50 value of a reference 3E10 antibody, e.g., a chimeric 3E10 antibody with a D31N amino acid substitution. In some embodiments, the moderate binding affinity is an EC50 value that is not more than 20 times the EC50 value of a reference 3E10 antibody, e.g., a chimeric 3E10 antibody with a D31N amino acid substitution. In some embodiments, the moderate binding affinity is an EC50 value that is not more than 15 times the EC50 value of a reference 3E10 antibody, e.g., a chimeric 3E10 antibody with a D31N amino acid substitution. In some embodiments, the moderate binding affinity is an EC50 value that is not more than 10 times the EC50 value of a reference 3E10 antibody, e.g., a chimeric 3E10 antibody with a D31N amino acid substitution. In some embodiments, a moderate binding affinity is an EC50 value that is no more than 5-fold the EC50 value of a reference 3E10 antibody, for example, a chimeric 3E10 antibody having a D31N amino acid substitution.

[0184] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein has weak binding affinity for a 3p-hpRNA RIG-I agonist having the nucleotide sequence 5'-pppGGAGCAAAAGCAGGGUGACAAAGACAUAAUGGAUCCAAACACUGUGUCAAGCUUUCAGGUAGAUUGCUUUCUUUGGCAUGUCCGCAAAC-3' (SEQ ID NO: 103).

[0185] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein has a moderate binding affinity for a 3p-hpRNA RIG-I agonist having the nucleotide sequence 5'-pppGGAGCAAAAGCAGGGUGACAAAGACAUAAUGGAUCCAAACACUGUGUCAAGCUUUCAGGUAGAUUGCUUUCUUUGGCAUGUCCGCAAAC-3' (SEQ ID NO: 103).

[0186] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein has strong binding affinity for a 3p-hpRNA RIG-I agonist having the nucleotide sequence 5'-pppGGAGCAAAAGCAGGGUGACAAAGACAUAAUGGAUCCAAACACUGUGUCAAGCUUUCAGGUAGAUUGCUUUCUUUGGCAUGUCCGCAAAC-3' (SEQ ID NO: 103).

[0187] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a fragment crystallizable (Fc) region. In some embodiments, the Fc region is human IgG1 Fc, human IgG2a Fc, human IgG2b Fc, human IgG3 Fc, and human IgG4 Fc.

[0188] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein has a human IgG1 Fc domain. In some embodiments, the human IgG1 domain is ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK The present invention includes an amino acid sequence having a high sequence identity, for example, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, or 100% identity, to TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 115).

[0189] In some embodiments, the IgG1 Fc domain sequence comprises one or more naturally occurring amino acid variants found in the human population, e.g., IgG1 Fc allotypes. Non-limiting examples of IgG1 Fc allotype amino acid variants include G1m(z,a), G1m(f), and G1m(f,a). The G1m(f) allele is found only in Caucasians, and the G1m(f,a) allele is common in Asian populations, although other variants G1m(z,a,x) and G1m(z,a,v) have also been described (see Vitarsson et al. Front. Immunol., October 2014, Vol. 5, Article 520, the disclosure of which is incorporated herein by reference in its entirety).

[0190] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof has an IgG1 domain containing one or more engineered amino acid substitutions, for example, to reduce Fc effector function, improve half-life in vivo, and / or otherwise change the properties of the antibody in vivo. Several approved antibodies have shown strong in vitro CDC activity, such as the anti-CD20 mAbs rituximab and ofatumumab, and there are many ways in which researchers have used Fc engineering to enhance complement-based effector functions. Idusogie et al. demonstrated that K326W / E333S enhances C1q binding and CDC activity compared to IgG1, suggesting that these two residues play a structural role in the interaction between C1q and IgG. Moore et al. demonstrated that the Fc mutation S267E / H268F / S324T enhances C1q binding 47-fold and CDC activity 6.9-fold over IgG1 (see Tringling JLet al. J.Immunol. 2006 177:362-371; Idusogie EEet al. J.Immunol. 2001 166:2571-2575; Moore GLett al. MAbs.2010 2:181-189; and Wang et al. Protein & Cell,Volume 9, Issue 1,2018,63-73, the disclosures of which are incorporated herein by reference in their entireties.

[0191] Thus, in some embodiments, the IgG1 Fc domain comprises an amino acid substitution at a position selected from L234A according to the EU index in the Kabat numbering scheme. In some embodiments, the IgG1 Fc domain comprises an amino acid substitution selected from L235A. In some embodiments, the IgG1 Fc domain comprises a combination of amino acid substitutions selected from L234A / L235A. In some embodiments, the IgG1 Fc domain comprises a combination of amino acid substitutions selected from N297D. In some embodiments, the IgG1 Fc domain comprises a combination of amino acid substitutions selected from L234A / L235A / N297D.

[0192] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein has a human IgG1 Fc domain that comprises the L234A / L235A amino acid substitutions. In some embodiments, the IgG1 Fc domain comprises residues 234A / 235A, The present invention includes amino acid sequences having a high degree of sequence identity to TIFF2025507840000003.tif39158, for example, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, or 100% identity.

[0193] In embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein has a human IgG1 Fc domain that comprises a N297D amino acid substitution. In some embodiments, the IgG1 Fc domain comprises residue 297D, The present invention includes amino acid sequences having a high degree of sequence identity to TIFF2025507840000004.tif39158, for example, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, or 100% identity.

[0194] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein has a human IgG1 Fc domain comprising the L234A / L235A / N297D amino acid substitutions. Amino acid substitutions. In some embodiments, the IgG1 Fc domain comprises residues 234A / 235A / 297D, The present invention includes amino acid sequences having a high degree of sequence identity to TIFF2025507840000005.tif39158, for example, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, or 100% identity.

[0195] In some embodiments, the IgG1 constant heavy chain region 1 comprises an amino acid sequence that has high sequence identity, e.g., at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, or 100% identity, to ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV (SEQ ID NO: 154).

[0196] In some embodiments, the IgG1 hinge region comprises an amino acid sequence that comprises high sequence identity to EPKSCDKTHTCP (SEQ ID NO: 155), e.g., at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, or 100% identity.

[0197] In some embodiments, the IgG1 L2345A / L235A constant heavy chain region 2 is The present invention includes an amino acid sequence having a high degree of sequence identity to TIFF2025507840000006.tif18160, for example, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, or 100% identity.

[0198] In some embodiments, the IgG1 constant heavy chain region 3 comprises an amino acid sequence that comprises high sequence identity, e.g., at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, or 100% identity, to GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 157).

[0199] In some embodiments, the IgG1 N297D constant heavy chain region 2 is The present invention includes an amino acid sequence having a high degree of sequence identity to TIFF2025507840000007.tif18159, for example, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, or 100% identity.

[0200] In some embodiments, the IgG1 L2345A / L235A / N297D constant heavy chain region 2 is The present invention includes an amino acid sequence having a high degree of sequence identity to TIFF2025507840000008.tif18160, for example, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, or 100% identity.

[0201] In some embodiments, the unmodified constant heavy chain region 2 comprises an amino acid sequence that comprises high sequence identity, e.g., at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, or 100% identity, to PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO: 160).

[0202] In some embodiments, the full-length light chain sequence comprises an amino acid sequence that comprises high sequence identity, e.g., at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, or 100% identity, to DIQMTQSPSSLSASLGDRATITCRASKTVSTSSYSYMHWYQQKPGQPPKLLIKYASYLESGVPSRFSGSGSGTDFTLTISSLQPEDAATYYCQHSREFPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 161).

[0203] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein has a human IgG4 Fc domain. In some embodiments, the human IgG4 domain is ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT The present invention includes an amino acid sequence having a high sequence identity, for example, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, or 100% identity, to ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 104).

[0204] In some embodiments, the IgG4 Fc domain sequence comprises one or more naturally occurring amino acid variants found in the human population, e.g., IgG4 Fc allotypes. Non-limiting examples of IgG4 Fc allotype amino acid variants include nG4m(a) and nG4m(b) (see Vidarsson et al. Front. Immunol., October 2014, Vol. 5, Article 520, the disclosure of which is incorporated herein by reference in its entirety).

[0205] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof has an IgG4 domain that contains one or more engineered amino acid substitutions, for example, to reduce Fc effector function, improve half-life in vivo, and / or otherwise change the properties of the antibody in vivo. Indeed, the majority of IgG4-based therapeutic antibodies approved for marketing or in late-stage clinical trials contain at least one such amino acid substitution. For a review of amino acid substitutions in such antibodies, see, for example, Dumet C. et al., MABS, 11(8):1341-50 (2019), the disclosure of which is incorporated herein by reference in its entirety. Other IgG4 Fc mutations have been suggested to reduce immune effector function while retaining other Fc properties. For example, Tam SH et al., Antibodies, 6(12):1-34 (2017), the disclosure of which is incorporated herein by reference in its entirety, reports the characterization of two IgG4 Fc variant designs, huIgG4σ1 and huIgG4σ2. The huIgG4σ1 construct contains S228P, F234A, L235A, G237A, and P238S amino acid substitutions, while the huIgG4σ2 construct contains G236>del in addition to S228P, F234A, L235A, G237A, and P238S amino acid substitutions. Other IgG4 Fc mutations are described in Liu R. et al., Antibodies, 9(64):1-34 (2020), the disclosure of which is incorporated herein by reference in its entirety. These mutations include M252Y, S254T, T256E, H433K, and N434F.

[0206] Still other IgG4 Fc amino acid substitutions that can be incorporated into the IgG4 Fc domain of the humanized 3E10 antibody or antigen-binding fragment thereof have been proposed in the art, including but not limited to WO1989 / 007142, US5,885,573, WO1994 / 029351, US6,407,214, US2006 / 0024298, US7,863,419, US2007 / 0041972, US8,961,967, US9,187,552, US S8,969,526, US9,359,437, WO2017 / 079369, US7,371,826, US7,083,784, WO2004 / 035752, US9, 200,079, US11,046,784, US8,802,820, US2020 / 0255502, US2010 / 0098730, USRE45992, US2010 / 0204454, US8,637,641, US2014 / 0302028, GB201302878, US2015 / 0065690, US2014 / 0294812, US2020 / 0071423, US11,319,383, US2018 / 0037634, KR101792191, US2019 / 0010243, US8,911,7 No. 2013 / 0100140, US9,085,625, US10,562,966, US2017 / 029521, US11,254,753, and WO2018 / 119380, the disclosures of which are incorporated by reference in their entireties for all purposes.

[0207] Thus, in some embodiments, the IgG4 Fc domain comprises an amino acid substitution at a position selected from 196, 228, 234, 234, 235, 235, 236, 237, 238, 252, 254, 256, 265, 296, 233, 310, 331, 356, 409, 428, 433, 434, 435, 445, 446, and K447, according to the EU index in the Kabat numbering scheme. In some embodiments, the IgG4 Fc domain comprises amino acid substitutions selected from K196Q, S228P, F234A, F234V, L235A, L235E, G236>del, G237A, P238S, M252Y, S254T, T256E, D265A, F296Y, E233P, T307Q, H310Q, P331S, E356K, R409K, M428L, H433K, N434A, N434F, N434S, H435R, L445P, G446>del, and K447>del. The Fc domains are S228P / L234A / L235A, L234F / L235E / P331S, M252Y / S254T / T256E, M252Y / S254T / T256E / H433K / N434F, S228P / F234A / L235A / H310Q, S228P / F234A / L235A / M25 Includes a combination of amino acid substitutions selected from 2Y / S254T / T256E, S228P / F234A / L235A / T307Q / N434A, S228P / F234A / L235A / G237A / P238S, and S228P / F234A / L235A / G236>del / G237A / P238S.

[0208] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein has a human IgG4 Fc domain that comprises S228P / F234A / L235A amino acid substitutions. In some embodiments, the IgG4 Fc domain comprises residues 228P / 234A / 235A, The present invention includes amino acid sequences having a high degree of sequence identity to TIFF2025507840000009.tif40159, for example, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, or 100% identity.

[0209] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein has a human IgG4 Fc domain that comprises S228P / F234A / L235A / T307Q / N434A amino acid substitutions. The present invention relates to a method for the preparation of a nucleic acid sequence comprising the steps of: (a) administering a nucleic acid sequence comprising the amino acid sequence 228P / 234A / 235A / 307Q / 434A; and (b) administering a nucleic acid sequence comprising the amino acid sequence 228P / 234A / 235A / 307Q / 434A, the amino acid sequence having a high sequence identity to TIFF2025507840000010.tif39159, e.g., at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, or 100% identity.

[0210] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein has a human IgG4 Fc domain that comprises S228P / F234A / L235A / M252Y / S254T / T256E amino acid substitutions. The present invention relates to a method for the preparation of a nucleic acid sequence comprising the steps of: (a) administering a nucleic acid sequence comprising the amino acid sequence 228P / 234A / 235A / 252Y / 254T / 256E to a mammalian animal having a human subject; and (b) administering a human subject a human subject having ...

[0211] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein has a human IgG4 Fc domain that comprises S228P / F234A / L235A / H310Q amino acid substitutions. The present invention relates to a method for the preparation of a nucleic acid sequence comprising the steps of: (a) administering a nucleic acid sequence comprising the amino acid sequence 228P / 234A / 235A / 310Q of a nucleic acid sequence having a high degree of sequence identity to TIFF2025507840000012.tif39159, e.g., at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, or 100% identity.

[0212] In some embodiments, the disclosure provides humanized 3E10 antibodies and antigen-binding fragments thereof comprising heavy chain constant domain (CH)1.

[0213] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof comprises an Fc region selected from human γ1 CH1, human γ2 CH1, human γ3 CH1, and human γ4 CH1.

[0214] In some embodiments, the disclosure provides a humanized 3E10 antibody or antigen-binding fragment thereof comprising a light chain constant domain (CL).

[0215] In another embodiment, the humanized 3E10 antibody or antigen-binding fragment thereof comprises an Fc region selected from the group consisting of human λCL and human κCL.

[0216] In various embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof is covalently linked to the therapeutic moiety.

[0217] In some embodiments, the therapeutic moiety is a therapeutic polypeptide, a cytotoxic moiety, a chemotherapeutic moiety, or a detectable moiety.

[0218] Cell penetration and nuclear localization The disclosed compositions and methods typically employ antibodies that maintain the ability to penetrate the cell and, optionally, the nucleus.

[0219] The mechanisms of cell internalization by autoantibodies are diverse: some are taken up into cells through electrostatic interactions or FcR-mediated endocytosis, while others use mechanisms based on association with cell surface myosin or calreticulin followed by endocytosis (Ying-Chyi et al., Eur. J. Immunol. 38, 3178-3190 (2008); Yanase et al., J. Clin. Invest. 100, 25-31 (1997)). 3E10 penetrates cells via an Fc-independent mechanism (as evidenced by the ability of an Fc-less 3E10 fragment to penetrate cells), but requires the presence of the nucleoside transporter ENT2 (Weisbart et al., Scientific Reports volume 5, Article number: 12022 (2015); Zack et al., J Immunol 157, 2082-2088 (1996); Hansen et al., J Biol Chem 282, 20790-20793 (2007)). Thus, in some embodiments, the antibodies used in the compositions and methods of the present disclosure penetrate cells via an Fc-independent mechanism, but include the presence of the nucleoside transporter ENT2.

[0220] Mutations in 3E10 that interfere with its ability to bind to nucleic acids may render the antibody unable to penetrate the nucleus. Thus, typically, the disclosed variants and humanized forms of the antibody maintain the ability to bind to nucleic acids. Also, it has been previously shown that 3E10 scFv can penetrate into biological cells and nucleic acids in an ENT2-dependent manner, with the efficiency of uptake being impaired in ENT2-deficient cells (Hansen, et al., J. Biol. Chem. 282, 20790-20793 (2007)). Thus, in some embodiments, the disclosed variants and humanized forms of the antibody maintain the ability to penetrate into the cell nucleus in an ENT-dependent manner, preferably in an ENT2-dependent manner.

[0221] As discussed in US2021 / 0054102 and US2021 / 0137960, some humanized 3E10 variants have been found to penetrate the cell nucleus more efficiently than the original murine 3E10(D31N) di-scFv, while others have been found to have lost the ability to penetrate the nucleus. In particular, variants 10 and 13 penetrated the nucleus very well compared to the murine antibody.

[0222] A potential bipartite nuclear localization signal (NLS) in the humanized 3E10 VL has been identified, with the following sequence: It may contain part or all of TIFF2025507840000013.tif19128.

[0223] Exemplary consensus NLSs are: TIFF2025507840000014.tif4128 (wherein (X) = any residue, but preferentially a basic residue (R or K)) (SEQ ID NO:112), or a variant thereof having at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 percent sequence identity to SEQ ID NO:112.

[0224] Thus, in some embodiments, particularly where import into the nucleus is important, the disclosed antibodies can comprise the sequence of any one of SEQ ID NOs: 109-112, or fragments and variants thereof (e.g., at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% amino acid sequence identity to any one of SEQ ID NOs: 109-112) that are capable of translocating to the nucleus of a cell.

[0225] The presence of the NLS indicates that the humanized 3E10 antibody or antigen-binding fragment thereof can cross the nuclear membrane via an import pathway into the nucleus. In some embodiments, the NLS improves import by interacting with one or more members of the import pathway. Thus, in some embodiments, the NLS can bind to importin β, importin β / importin α heterodimer, or a combination thereof.

[0226] Nucleic acid binding In some embodiments, the disclosed compositions and methods typically employ a humanized 3E10 antibody or antigen-binding fragment thereof that retains the ability to bind to nucleic acid, such as DNA, RNA, or a combination thereof.

[0227] The following examples illustrate molecular modeling of wild-type 3E10 and additional 3E10 variants. Molecular modeling (Pymol) of 3E10 revealed a putative nucleic acid binding pocket (NAB1) (see, e.g., Figures 11A and 11B), shown in the underlined sequence below. WT heavy chain scFv sequence TIFF2025507840000015.tif18152 Light chain scFv sequence TIFF2025507840000016.tif17152

[0228] In some embodiments, the disclosed humanized 3E10 antibodies comprise some or all of the underlined NAB1 sequences. In some embodiments, the antibodies comprise variant sequences that have altered ability to bind nucleic acids. In some embodiments, the mutations (e.g., substitutions, insertions, and / or deletions) in NAB1 improve the binding of the antibody to nucleic acids, such as DNA, RNA, or combinations thereof. In some embodiments, the mutations are conservative substitutions. In some embodiments, these mutations increase the cationic charge of the NAB1 pocket.

[0229] As discussed and exemplified herein, mutation of residue 31 of CDR1 from aspartic acid to asparagine increased the cationic charge of this residue and enhanced nucleic acid binding and delivery in vivo (3E10-D31N).

[0230] Additional exemplary variants include mutation of the aspartic acid at residue 31 of CDR1 to arginine (3E10-D31R), which modeling indicates an expansion of the cationic charge, or to lysine (3E10-D31K), which modeling indicates a change in charge orientation. Thus, in some embodiments, a 3E10 binding protein includes a D31R or D31K substitution.

[0231] Additional exemplary variants include mutation of arginine (R) 96 to asparagine (N) and / or serine (S) 30 to aspartic acid (D), alone or in combination with D31N, D31R, or D31K.

[0232] All sequences disclosed herein that have residues corresponding to 3E10 D31 or N31 are expressly disclosed with the D31R or D31K or N31R or N31K substitution.

[0233] Molecular modeling (Pymol) of 3E10 revealed a putative nucleic acid binding pocket (NAB1) (Figures 11A-11B). Mutation of residue 31 of CDR1 from aspartic acid to asparagine increased the cationic charge of this residue and enhanced nucleic acid binding and delivery in vivo (3E10-D31N).

[0234] Mutation of residue 31 of CDR1 from aspartic acid to arginine (3E10-D31R) further extended the cationic charge, while mutation to lysine (3E10-D31K) altered the charge orientation (FIG. 11A).

[0235] NAB1 amino acids predicted from molecular modeling are underlined in the heavy and light chain sequences above. Figure 11B shows molecular modeling (Pymol) of 3E10-scFv with NAB1 amino acid residues illustrated with punctuated dots.

[0236] All of the sequences disclosed herein that have a residue corresponding to R96 are explicitly disclosed with the R96N substitution.

[0237] All sequences disclosed herein that have a residue corresponding to S30 are explicitly disclosed by S30D.

[0238] Any of the substitutions can be included in any combination. Thus, sequences with two or three substitutions at any combination of residues 31, 30, and 96 are explicitly provided.

[0239] In certain embodiments, the sequence has 31N, 31K, or 31R, alone or in combination with 30D, without the R96N substitution. Thus, in some embodiments, the residue corresponding to 96 is not N, and in more specific embodiments remains R.

[0240] Fragments, variants, and fusion proteins The anti-nucleic acid antibody can be comprised of an antibody fragment or fusion protein that contains an amino acid sequence of a variable heavy chain and / or a variable light chain that is at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence of the variable heavy chain and / or variable light chain of a humanized 3E10 form thereof (e.g., any of SEQ ID NOs: 64-102).

[0241] The anti-nucleic acid antibody can be comprised of an antibody fragment or fusion protein that contains one or more CDRs that are at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence of the CDRs of a humanized 3E10 form or variant thereof (e.g., any of the CDRs of SEQ ID NOs: 3-5, 9-11, 15-18, 22-24, and 26-63). Determination of the percent identity of two amino acid sequences can be determined by BLAST protein comparison. In some embodiments, the antibody contains one, two, three, four, five, or all six of the 3E10 CDRs described herein.

[0242] Preferably, the antibody comprises one each of the heavy chain CDR1, CDR2, and CDR3 in combination with one each of the light chain CDR1, CDR2, and CDR3.

[0243] The predicted complementarity determining regions (CDRs) of the light chain variable sequence for 3E10 are provided above. See also GenBank: AAA65681.1-immunoglobulin light chain, partial [Mus musculus] and GenBank: L34051.1-mouse Ig rearranged kappa chain mRNA V region. The predicted complementarity determining regions (CDRs) of the heavy chain variable sequence for 3E10 are provided above. See also, e.g., Zack, et al., Immunology and Cell Biology, 72:513-520 (1994), GenBank Accession No. AAA65679.1. Zach, et al., J. Immunol. 154(4), 1987-1994 (1995) and GenBank: L16982.1-mouse Ig rearranged H chain gene, partial cds.

[0244] Also included are fragments of antibodies that have nucleic acid delivery activity, which fragments include insertions, deletions, substitutions, or other selected modifications of specific regions or specific amino acid residues, whether or not attached to other sequences, provided that the activity of the fragment is not significantly altered or impaired compared to the unmodified antibody or antibody fragment.

[0245] The technique can also be adapted for the production of single chain antibodies specific for the nucleic acids of the present disclosure. Methods for the production of single chain antibodies are well known to those skilled in the art. Single chain antibodies can be made by fusing together the variable domains of heavy and light chains using a short peptide linker, thereby reconstituting an antigen binding site on a single molecule. Single chain antibody variable fragments (scFv) have been developed in which the C-terminus of one variable domain is tethered to the N-terminus of the other variable domain via a peptide or linker of 15-25 amino acids without significantly disrupting antigen binding or specificity of binding. The linker is chosen to allow the heavy and light chains to bind together in their proper conformational orientation.

[0246] Anti-nucleic acid antibodies can be modified to improve their nucleic acid delivery capabilities. For example, in some embodiments, the cell-permeable anti-nucleic acid antibody is conjugated to another antibody specific for a therapeutic target in the cytoplasm and / or nucleus of the target cell. For example, the cell-permeable anti-nucleic acid antibody can be a fusion protein comprising 3E10 Fv and a single-chain variable fragment of a monoclonal antibody that specifically binds to the therapeutic target. In other embodiments, the cell-permeable anti-nucleic acid antibody is a bispecific antibody having a first heavy chain and a first light chain from 3E10, and a second heavy chain and a second light chain from a monoclonal antibody that specifically binds to the therapeutic target.

[0247] Bispecific antibodies and other binding proteins having a first heavy chain and a first light chain from 3E10 and a second heavy chain and a second light chain from a monoclonal antibody that specifically binds to a target are discussed in Weisbart, et al., Mol. Cancer Ther., 11(10):2169-73 (2012), and Weisbart, et al., Int. J. Oncology, 25:1113-8 (2004), and U.S. Patent Application No. 2013 / 0266570, which are specifically incorporated by reference in their entirety. In some embodiments, the target is specific for a target cell type, tissue, organ, etc. In this way, the second heavy chain and the second light chain can serve as a targeting moiety that targets the complex to a target cell type, tissue, organ. In some embodiments, the second heavy chain and the second light chain are specific for a hematopoietic stem cell, CD34 + In some embodiments, the second heavy chain and the second light chain target a thymus, spleen, or cancer cell, for example, by targeting a receptor or ligand expressed on the preferred cell type.

[0248] In some embodiments, the antibodies can be specifically targeted to target T cells in vivo, e.g., antigen-specific T cells, CAR T cells, immune cells, or for in vivo production of T cell markers such as CD3, CD7, or CD8. For example, anti-CD8 antibodies and anti-CD3 Fab fragments have both been used to target T cells in vivo (Pfeiffer, et al., EMBO Mol Med., 10(11)(2018).pii:e9158.doi:10.15252 / emmm.201809158., Smith, et al., Nat Nanotechnol., 12(8):813-820(2017).doi:10.1038 / nnano.2017.57). Thus, in some embodiments, the 3E10 antibody or antigen-binding fragment or fusion protein is a bispecific antibody, portions of which can specifically bind to CD3, CD7, CD8, or another immune cell (e.g., T cell) marker, or a marker for a particular tissue, such as the thymus, spleen, or liver.

[0249] Bivalent single chain variable fragments (di-scFv) can be engineered by linking two scFvs. This can be done by producing a single peptide chain with two VH and two VL regions, resulting in tandem scFvs. ScFvs can also be designed with a linker peptide that is too short (about 5 amino acids) to fold the two variable regions together, causing the scFvs to dimerize. This type is known as a diabody. Diabodies have been shown to have dissociation constants up to 40 times lower than the corresponding scFvs, meaning that they have much higher affinity for their targets. Even shorter linkers (one or two amino acids) lead to the formation of trimers (triabodies or tribodies). Tetrabodies have also been produced. They show even higher affinity for their targets than diabodies. In some embodiments, the anti-nucleic acid antibody may comprise two or more linked single chain variable fragments of 3E10 (e.g., 3E10 di-scFv, 3E10 tri-scFv), or conservative variants thereof. In some embodiments, the anti-nucleic acid antibody is a diabody or triabody (e.g., 3E10 diabody, 3E10 triabody). Sequences for single and two or more linked single chain variable fragments of 3E10 are provided in US2019 / 0247515 and US2017 / 0291961.

[0250] The function of an antibody can be enhanced by coupling the antibody or a fragment thereof to a therapeutic agent, which can be achieved by creating an immunoconjugate or a fusion protein, or by linking the antibody or fragment to a nucleic acid, such as DNA or RNA (e.g., siRNA), that comprises the antibody or antibody fragment and the therapeutic agent.

[0251] In some embodiments, the cell-permeable antibody is modified to change its half-life. In some embodiments, it is desirable to increase the half-life of the antibody so that the antibody is present in the circulation or at the site of treatment for a longer period of time. For example, it may be desirable to maintain the titer of the antibody in the circulation or at the site of treatment for an extended period of time. In other embodiments, the half-life of the anti-nucleic acid antibody is decreased to reduce potential side effects. Antibody fragments, such as 3E10Fv, may have a shorter half-life than full-sized antibodies. Other methods of altering half-life are known and can be used in the methods described. For example, the antibody can be engineered with an Fc variant that extends the half-life, for example, using Xtend™ antibody half-life extension technology (Xencor, Monrovia, Calif.).

[0252] Linker The term "linker" as used herein includes, but is not limited to, peptide linkers. Peptide linkers can be of any size, provided they do not interfere with epitope binding by the variable region. In some embodiments, the linker comprises one or more glycine and / or serine amino acid residues. Monovalent single chain antibody variable fragments (scFv) in which the C-terminus of one variable domain is tethered to the N-terminus of the other variable domain, typically via a 15-25 amino acid peptide or linker. The linker is chosen to allow the heavy and light chains to bind together in their proper conformational orientation. Linkers in diabodies, triabodies, etc. typically comprise shorter linkers than those of monovalent scFvs, as discussed above. Bivalent, trivalent, and other multivalent scFvs typically comprise three or more linkers. The linkers can be the same or different in length and / or amino acid composition. Thus, the number of linkers, the composition of the linker, and the length of the linker can be determined based on the desired value of the scFv, as known in the art. Linkers can enable or drive the formation of bivalent, trivalent, and other multivalent scFvs.

[0253] For example, the linker can include 4 to 8 amino acids. In certain embodiments, the linker includes the amino acid sequence GQSSRSS (SEQ ID NO: 119). In another embodiment, the linker includes 15 to 20 amino acids, for example, 18 amino acids. In certain embodiments, the linker includes the amino acid sequence GQSSRSSSGGGSSGGGGS (SEQ ID NO: 120). Other flexible linkers include, but are not limited to, the amino acid sequences Gly-Ser, Gly-Ser-Gly-Ser (SEQ ID NO: 121), Ala-Ser, Gly-Gly-Gly-Ser (SEQ ID NO: 122), (Gly4-Ser)2 (SEQ ID NO: 123) and (Gly4-Ser)4 (SEQ ID NO: 124), and (Gly-Gly-Gly-Gly-Ser)3 (SEQ ID NO: 125).

[0254] Other exemplary linkers include, for example, RADAAPGGGGSGGGGSGGGGS (SEQ ID NO: 126) and ASTKGPSVFPLAPLESSGS (SEQ ID NO: 127).

[0255] Nucleic Acid Cargo When used in some embodiments of the methods and compositions provided herein, the humanized 3E10 antibody or antigen-binding fragment thereof is complexed with a nucleic acid cargo (polynucleotide). In some embodiments, the polynucleotide is non-covalently attached to the humanized 3E10 antibody or antigen-binding fragment thereof. In some embodiments, the polynucleotide is covalently attached to the humanized 3E10 antibody or antigen-binding fragment thereof.

[0256] The nucleic acid cargo can be single-stranded or double-stranded. The nucleic acid cargo can be or include DNA, RNA, a nucleic acid analog, or a combination thereof. As discussed in more detail below, the nucleic acid analog can be modified at the base moiety, sugar moiety, or phosphate backbone. Such modifications can, for example, improve the stability, hybridization, or solubility of the nucleic acid.

[0257] Nucleic acid cargoes are typically functional in the sense that they are or encode agents that are biologically active once delivered into a cell. Exemplary cargoes are discussed in more detail below, but include, for example, mRNA or DNA encoding a polypeptide of interest, including expression constructs and vectors, inhibitory nucleic acids, such as siRNAs, or nucleic acids encoding inhibitory nucleic acids, including, for example, expression constructs and vectors.

[0258] The disclosed compositions can include a plurality of single nucleic acid cargo molecules, hi some embodiments, the compositions include a plurality (e.g., 2, 3, 4, 5, 6, 7, 8, 9 10, or more) of different nucleic acid molecules.

[0259] In some embodiments, the cargo molecule is about 0.001, about 0.01, about 1, 10's 100's, 1,000's, 10,000's, and / or 100,000's kilobases in length.

[0260] In some embodiments, for example, the cargo is between 0.001 kb and 100 kb, or between 0.001 kb and 50 kb, or between 0.001 kb and 25 kb, or between 0.001 kb and 12.5 kb, or between 0.001 kb and 10 kb, or between 0.001 kb and 8 kb, or between 0.001 kb and 5 kb, or between 0.001 kb and 2.5 kb, or between 0.001 kb and 1 kb, or between 0.01 kb and 100 kb, or between 0.01 kb and 50 kb, or between 0.01 kb and 25 kb, or between 0.01 kb and 12.5 kb, or between 0.01 kb and 10 kb, or between 0.01 kb and 8 kb, or between 0.01 kb and 5 kb, or between 0.01 kb and 2.5 kb, or between 0.01 kb and 1 kb, or between 0.1 kb and 100 kb, or between 0.1 kb and 50 kb, or between 0.1 kb and 25 kb, or between 0.1 kb and 12.5 kb, or between 0.1 kb and 10 kb, or between 0.1 kb and 8 kb, or between 0.1 kb and 5 kb, or between 0.1 kb and 2.5 kb, or between 0.1 kb and 1 kb, or between 1 kb and 100 kb, or between 1 kb and 50 kb, or between 1 kb and 25 kb, or between 1 kb and 12.5 kb, or between 1 kb and 10 kb, or between 1 kb and 8 kb, or between 1 kb and 5 kb, or between 1 kb and 2.5 kb (each inclusive).

[0261] In some embodiments, for example, the cargo is between about 0.001 kb and about 100 kb, or between about 0.001 kb and about 50 kb, or between about 0.001 kb and about 25 kb, or between about 0.001 kb and about 12.5 kb, or between about 0.001 kb and about 10 kb, or between about 0.001 kb and about 8 kb, or between about 0.001 kb and about 5 kb, or about 0.001 kb. b to about 2.5 kb, or between about 0.001 kb to about 1 kb, or between about 0.01 kb to about 100 kb, or between about 0.01 kb to about 50 kb, or between about 0.01 kb to about 25 kb, or between about 0.01 kb to about 12.5 kb, or between about 0.01 kb to about 10 kb, or between about 0.01 kb to about 8 kb, or between about 0.01 kb to about 5 kb, or about 0. between about 0.01 kb and about 2.5 kb, or between about 0.01 kb and about 1 kb, or between about 0.1 kb and about 100 kb, or between about 0.1 kb and about 50 kb, or between about 0.1 kb and about 25 kb, or between about 0.1 kb and about 12.5 kb, or between about 0.1 kb and about 10 kb, or between about 0.1 kb and about 8 kb, or between about 0.1 kb and about 5 kb, or between about 0.1 kb and about 2.5 kb, or between about 0.1 kb and about 1 kb, or between about 1 kb and about 100 kb, or between about 1 kb and about 50 kb, or between about 1 kb and about 25 kb, or between about 1 kb and about 12.5 kb, or between about 1 kb and about 10 kb, or between about 1 kb and about 8 kb, or between about 1 kb and about 5 kb, or between about 1 kb and about 2.5 kb (each inclusive).

[0262] In some embodiments, for example, the cargo is between 0.2kb and 10kb, or between 0.2kb and 5kb, or between 0.2kb and 2.5kb, or between 0.2kb and 1kb, or between 0.2kb and 0.5kb, or between 0.2kb and 0.25kb, or between 0.5kb and 10kb, or between 0.5kb and 5kb, or between 1kb and 5kb, or between 1kb and 3kb, or between 2kb and 10kb, or between 3kb and 5kb.

[0263] In some embodiments, for example, the cargo is between about 0.2 kb and about 10 kb, or between about 0.2 kb and about 5 kb, or between about 0.2 kb and about 2.5 kb, or between about 0.2 kb and about 1 kb, or between about 0.2 kb and about 0.5 kb, or between about 0.2 kb and about 0.25 kb, or between about 0.5 kb and about 10 kb, or between about 0.5 kb and about 5 kb, or between about 1 kb and about 5 kb, or between about 1 kb and about 3 kb, or between about 2 kb and about 10 kb, or between about 3 kb and about 5 kb.

[0264] It will be appreciated that for certain applications, the nucleic acid cargo may be one or more individual lengths, for example, within one of the ranges (inclusive) above, although specific values ​​for each are expressly disclosed. For example, the size may be as small as a single nucleotide or nucleic acid base. In an exemplary application, the cargo is a cyclic dinucleotide such as cGAMP, a STING agonist. In other embodiments, the cargo is a short oligomer. For example, oligomers as short as 8mers can be used for antisense or splice switching. Slightly longer ones (e.g., 18-20mers) can be used for gene editing.

[0265] Immunostimulatory Oligonucleotides Polynucleotide agonists of macromolecular stimulators of the innate immune system, particularly pattern recognition receptors (PRRs), are highly promising for the treatment of cancer. Pattern recognition receptors (PRRs) recognize pathogen-associated molecular patterns as well as endogenous damage-associated molecular patterns. Upon ligand binding, a signaling cascade occurs within the cell to activate effector molecules, resulting in the recruitment and activation of antitumor immune cells and the release of inflammatory cytokines. Thus, PRR agonists have been successfully used as immunotherapies for the treatment of a wide range of cancers. For a review of PRRs and the use of PRR agonists in cancer immunotherapy, see, for example, Bai L., et al., “Promising targets based on pattern recognition receptors for cancer immunotherapy,” Pharmacological Research, 159 (2020) 105017. The contents of this document are incorporated herein by reference in their entirety.

[0266] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof is complexed with a polynucleotide immunostimulator, such as a polynucleotide capable of stimulating a pattern recognition receptor (PRR). In some embodiments, the polynucleotide immunostimulator is non-covalently bound to the humanized 3E10 antibody or antigen-binding fragment thereof. In some embodiments, the polynucleotide immunostimulator is covalently bound to the humanized 3E10 antibody or antigen-binding fragment thereof.

[0267] Pattern Recognition Receptor (PRR) Agonists In one aspect, the present disclosure relates to compositions and methods for treating cancer in a subject by administering to the subject a therapeutically effective amount of a composition comprising a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof, and (ii) a polynucleotide ligand capable of stimulating a pattern recognition receptor (PRR), as described herein. As recognized in the art, stimulation of the innate immune system, for example, via activation of pattern recognition receptors, is a promising therapeutic approach, particularly for cancer treatment. Generally, PRRs stimulate the innate immune system after recognition of pathogen-associated patterns (PAMPs) and / or damage-associated patterns (DAMPs). Traditionally, PRRs are grouped into five categories: Toll-like receptors (TLRs), C-type lectin receptors (CLRs), RIG-I-like receptors (RLRs), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), and cytoplasmic DNA sensors (CDSs). The methods and compositions described herein act through any of these classes of PRRs that recognize and are activated by polynucleotide antigens.

[0268] RIG-I-like receptors (RLRs) are a family of RNA helicases that function as cytoplasmic sensors of pathogen-associated molecular patterns (PAMPs) in viral RNA. Thus, in another aspect, compositions and methods are provided for treating cancer by administering to a subject a therapeutically effective amount of a composition comprising a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof, and (ii) a polynucleotide ligand capable of stimulating a RIG-I-like receptor (RLR). Identified RLRs include RIG-I (retinoic acid-inducible gene I), MDA5 (melanoma differentiation associated factor 5), and LGP2 (laboratory of genetics and physiology 2). Thus, in some embodiments, the present disclosure provides a composition formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof, and (ii) a polynucleotide ligand capable of stimulating RIG-I. Methods are also provided for treating cancer by administering such a composition to a subject in need of cancer treatment. In some embodiments, the complex comprises a covalent conjugate between the humanized 3E10 antibody, or antigen-binding fragment thereof, and the polynucleotide ligand.

[0269] Exemplary RIG-I ligands include, but are not limited to, 5'ppp-dsRNA, which is a specific agonist of RIG-I, 3p-hpRNA, which is a specific agonist of RIG-I, poly(I:C) / LyoVec complexes recognized by RIG-I and / or MDA-5 depending on the size of poly(I:C), and poly(dA:dT) / LyoVec complexes recognized indirectly by RIG-I. In some embodiments, the 3p-hpRNA is a 5' triphosphate hairpin RNA generated by in vitro transcription of a sequence derived from influenza A (H1N1). In some embodiments, the 3p-hpRNA is an RNA oligonucleotide containing an uncapped 5' triphosphate end and a double-stranded fragment. In some embodiments, the 3p-hpRNA is about 50 bp, about 55 bp, about 60 bp, about 65 bp, about 70 bp, about 75 bp, about 80 bp, about 85 bp, about 90 bp, about 100 bp, or more. In some embodiments, the 3p-hpRNA is 89 bp in length.

[0270] In some embodiments, the polynucleotide capable of stimulating RIG-I is non-covalently linked to the humanized 3E10 antibody or antigen-binding fragment thereof. In some embodiments, the polynucleotide capable of stimulating RIG-I is covalently linked to the humanized 3E10 antibody or antigen-binding fragment thereof.

[0271] Thus, in some embodiments, compositions and methods are provided for treating cancer by administering to a subject a therapeutically effective amount of a composition comprising a complex formed between (i) a humanized 3E10 antibody or antigen-binding fragment thereof and (ii) an RNA molecule that is at least partially double-stranded and capable of stimulating RIG-I. In some embodiments, the at least partially double-stranded RNA molecule comprises two separate RNA strands that anneal to form the double-stranded portion of the molecule. In other embodiments, the at least partially double-stranded RNA molecule is a single RNA strand that has self-complementarity such that under physiological conditions it anneals to itself to form the double-stranded portion of the molecule, e.g., forming one or more hairpin structures.

[0272] Similarly, in some embodiments, compositions and methods are provided for treating cancer by administering to a subject a therapeutically effective amount of a composition comprising a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof, and (ii) a polynucleotide that is at least partially double-stranded, contains at least one 5' triphosphate moiety, and is capable of stimulating RIG-I. In some embodiments, the at least partially double-stranded RNA molecule comprises two separate RNA strands that anneal to form the double-stranded portion of the molecule. In other embodiments, the at least partially double-stranded RNA molecule is a single RNA strand that has self-complementarity such that it anneals to itself under physiological conditions to form the double-stranded portion of the molecule, for example, forming one or more hairpin structures. Examples of polynucleotide RIG-I agonists are provided in the literature. Generally, any one of these polynucleotide RIG-I agonists will find use in the methods and compositions described herein.

[0273] In some embodiments, the RIG-I agonist is a single-stranded negative-sense RNA virus having the sequence 5'-pppGGAGCAAAAGCAGGGUGACAAAGACAUAAUGGAUCCAAACACUGUGUCAAGCUUUCAGGUAGAUUGCUUUCUUUGGCAUGUCCGCAAAC-3' (SEQ ID NO: 103) derived from influenza A (H1N1) virus, or a 5' triphosphate hairpin RNA (3p-hpRNA) generated by in vitro transcription of a highly conserved nucleotide sequence thereto. See, for example, Rehwinkel J. et al., Cell, 140:397-408 (2010) and Liu G. et al., J Virol. 89(11):6067-79 (2015), the contents of which are incorporated herein by reference in their entirety for all purposes. Thus, in some embodiments, compositions and methods are provided for treating cancer by administering to a subject a therapeutically effective amount of a composition comprising a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof and (ii) a polynucleotide having the sequence of 3p-hpRNA (sequence number 103), which is capable of stimulating RIG-I.

[0274] In some embodiments, the RIG-I agonist has a sequence that is at least 80% identical to the sequence of 3p-hpRNA (SEQ ID NO: 103). In some embodiments, the RIG-I agonist has a sequence that is at least 85% identical to the sequence of 3p-hpRNA (SEQ ID NO: 103). In some embodiments, the RIG-I agonist has a sequence that is at least 90% identical to the sequence of 3p-hpRNA (SEQ ID NO: 103). In some embodiments, the RIG-I agonist has a sequence that is at least 95% identical to the sequence of 3p-hpRNA (SEQ ID NO: 103). In some embodiments, the RIG-I agonist has a sequence that is at least 96% identical to the sequence of 3p-hpRNA (SEQ ID NO: 103). In some embodiments, the RIG-I agonist has a sequence that is at least 97% identical to the sequence of 3p-hpRNA (SEQ ID NO: 103). In some embodiments, the RIG-I agonist has a sequence that is at least 98% identical to the sequence of 3p-hpRNA (SEQ ID NO: 103). In some embodiments, the RIG-I agonist has a sequence that is at least 99% identical to the sequence of 3p-hpRNA (SEQ ID NO: 103).Thus, in some embodiments, compositions and methods are provided for treating cancer by administering to a subject a therapeutically effective amount of a composition comprising a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof and (ii) a polynucleotide capable of stimulating RIG-I and having a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the nucleotide sequence of 3p-hpRNA (SEQ ID NO: 103).

[0275] Other RIG-I agonists that find use in the methods and compositions disclosed herein are known in the art. For example, useful RIG-I agonists that can be conjugated to the humanized 3E10 antibody and antigen-binding fragments thereof disclosed herein include those described in WO2023 / 278897, US2010 / 0178272, US9,738,680, US2011 / 0184045, US10,059,943, US2018 / 0195063, US11,382,966, US11,542,505, WO2020 / 260547, and the like. , US2021 / 0260093, US9,226,959, US2014 / 0286998, US9,861,574, US9,775,894, US2021 / 0046168, US2019 / 0076463, US11,499,157, US10,907,161, and US2022 / 0333113, the disclosures of which are incorporated by reference in their entireties herein.

[0276] In some embodiments, compositions and methods are provided for treating cancer by administering to a subject a therapeutically effective amount of a composition comprising a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof and (ii) a polynucleotide ligand capable of stimulating a Toll-like receptor (TLR). At least thirteen Toll-like receptors have been identified, including TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and TLR13. Each of these Toll-like receptors has an affinity for a different antigen. According to various embodiments of the present disclosure, the methods and compositions described herein include the use of polynucleotide agonists of TLRs. For example, each of TLR3, TLR7, TLR8, and TLR9 has an affinity for, and is activated by, various polynucleotides. Thus, in some embodiments, the agonists used in the methods and compositions described herein are capable of stimulating TLR3, TLR7, TLR8, or TLR9. In some embodiments, the complex is a non-covalent complex. In some embodiments, the complex comprises a covalent conjugate between the humanized 3E10 antibody, or antigen-binding fragment thereof, and the polynucleotide ligand.

[0277] For example, unmethylated CpG sites can be detected by TLR9 on plasmacytoid dendritic cells and B cells in humans (Zaida, et al., Infection and Immunity, 76(5):2123-2129, (2008)). Thus, the sequence of the oligonucleotide can contain one or more unmethylated cytosine-guanine (CG or CpG, used interchangeably) dinucleotide motifs. "p" refers to the phosphodiester backbone of DNA, however, in some embodiments, oligonucleotides containing CG can have a modified backbone, e.g., a phosphorothioate (PS) backbone.

[0278] In some embodiments, the oligonucleotide can contain two or more CG dinucleotides, either consecutively or separated by intervening nucleotides. The CpG motif can be internal to the oligonucleotide sequence. Numerous nucleotide sequences stimulate TLR9 with variations in the number and position of CG dinucleotides and the exact base sequence adjacent to the CG dimer.

[0279] Typically, CG ODNs are classified based on their sequence, secondary structure, and effect on human peripheral blood mononuclear cells (PBMCs). The five classes are class A (D type), class B (K type), class C, class P, and class S (Vollmer, J & Krieg, AM, Advanced Drug Delivery Reviews 61(3):195-204(2009), incorporated herein by reference). CG ODNs can stimulate the production of type I interferons (e.g., IFNα) and induce the maturation of dendritic cells (DCs). Some classes of ODNs are also potent activators of natural killer (NK) cells through indirect cytokine signaling. Some classes are potent stimulators of human B cell and monocyte maturation (Weiner, GL, PNAS USA 94(20):10833-7 (1997); Dalpke, AH, Immunology 106(1):102-12 (2002); Hartmann, G, J. of Immun. 164(3):1617-2 (2000), each of which is incorporated herein by reference).

[0280] In some embodiments, the polynucleotide immunostimulatory agent is a stimulator of cyclic-GMP-AMP synthase (cGAS) or interferon genes ( ST emulator of I Interferon G ene)(STING).

[0281] Polynucleotides encoding effector polypeptides In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof disclosed herein is covalently or non-covalently complexed with a therapeutic polynucleotide encoding a protein or peptide, e.g., an effector polypeptide, for cancer therapy. In some embodiments, the therapeutic polynucleotide is non-covalently bound to the humanized 3E10 antibody or antigen-binding fragment thereof. In some embodiments, the therapeutic polynucleotide is covalently bound to the humanized 3E10 antibody or antigen-binding fragment thereof.

[0282] In some embodiments, the polynucleotide is conjugated to or further encodes one or more of an RNAi agent, an siRNA, an shRNA, an miRNA, an miRNA binding site, an antisense RNA, a ribozyme, a catalytic DNA, a tRNA, an RNA that induces triple helix formation, an aptamer, or a vector, and the like.

[0283] In other embodiments, the polynucleotides may be designed to encode one or more effector polypeptides or fragments thereof against cancer tissue. Such effector polypeptides may include, but are not limited to, an entire polypeptide, multiple polypeptides, or fragments of a polypeptide, which may be independently encoded by one or more regions or portions of the effector polynucleotide, or the entirety thereof.

[0284] Effector polypeptide refers to any polypeptide selected to be encoded within or whose function is affected by the polynucleotides of the present disclosure. Effector polypeptides can modulate the activation of the immune system and can directly or indirectly provide a therapeutic effect on cancer, for example, by slowing the progression of cancer, inducing cell death in cancer cells, inducing senescence in cancer cells, etc. For example, in some embodiments, effector polypeptides stimulate immune cells to upregulate the production of cytokines that target cancer cells and cause cell death. In other embodiments, effector polypeptides stimulate tumor cells to express or upregulate the expression of tumor antigens that are markers for immune cells to identify tumor cells. For reviews of effector polypeptides, see, for example, Esensten et al., “CD28 costimulation: from mechanism to therapy,” Immunity Review, 44, (2016) 973; Immunity, 2016, 44, 973-988; Smolle et al., Noncoding RNAs and immune checkpoints, FEBS Journal, 2017, 284, 1952-1966; Chen et al., Anti-PD-1-PD-L1 therapy of human cancer past, present, and future, Journal of Clinical Investigation, 2015, Volume 125, 9, 3384-3391; Rowshanravan et al., CTLA-4 a moving target in immunotherapy, Blood, 2018, Volume 131, 1, 58-67; and Dougal et al., TIGIT and CD96 New checkpoint receptor targets for cancer immunotherapy, Immunological Reviews, 2017, 276, 112-120, the contents of each of which are incorporated herein by reference in their entirety for all purposes.

[0285] Accordingly, in one aspect, the disclosure relates to compositions and methods for treating cancer in a subject by administering to the subject a therapeutically effective amount of a composition comprising a complex formed between (i) a humanized 3E10 antibody, or an antigen-binding fragment thereof, and (ii) a therapeutic polynucleotide encoding a protein or peptide, e.g., an effector polypeptide, for treating cancer, as described herein.

[0286] Tumor antigens In some embodiments, the present disclosure provides compositions and methods for treating cancer in a subject by administering to the subject a therapeutically effective amount of a composition comprising a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof, and (ii) a therapeutic polynucleotide encoding a tumor-associated antigen, as described herein. In some embodiments, the therapeutic polynucleotide is non-covalently bound to the humanized 3E10 antibody or an antigen-binding fragment thereof. In some embodiments, the therapeutic polynucleotide is covalently bound to the humanized 3E10 antibody or an antigen-binding fragment thereof.

[0287] Tumor antigens are peptides that are almost exclusively presented on the cell surface of cancerous cells, which distinguish them from non-cancerous cells that do not present tumor antigens. When cancer cells die, these tumor antigens are released into the tumor microenvironment and can be recognized by the immune system as foreign peptides, altered self-peptides, or self-peptides. When a sufficient amount of tumor antigens are released, the immune system can generate anti-tumor immunity by generating an immune response against the tumor antigen. Specifically, the immune system targets and destroys cancerous cells that present the tumor antigen used to generate the immune response.

[0288] Several types of antigens have been experimentally exploited to generate anti-tumor immunity. Specifically, tumor antigens are exogenously administered to patients with cancers that present the tumor antigen on their cell surface, either as peptides or as nucleic acids encoding the antigen. A sufficient amount of antigen is then presented to the immune system to generate an immune response against the tumor antigen, resulting in anti-tumor immunity. Examples of types of tumor antigens include tumor viral protein antigens, neoantigens, and antigens derived from cancer germline genes. In many cases, tumor antigens, or polynucleotides encoding tumor antigens, are co-administered with adjuvants that activate dendritic cells, or with dendritic cells themselves, to promote the generation of anti-tumor immunity. For general reviews, see, for example, Haen et al., “Towards new horizons Characterization, classification and implications of the tumor antigenic repertoire,” Nature Reviews-Clinical Oncology, 2020, Volume 17, 595-610; Saxena M. et al., Nat. Rev. Cancer 21, 360-378 (2021). The contents of the literature are incorporated herein by reference in their entirety for all purposes.

[0289] In some embodiments, the present disclosure relates to compositions and methods for treating cancer in a subject by administering to the subject a therapeutically effective amount of a composition comprising a complex formed between (i) a humanized 3E10 antibody, or an antigen-binding fragment thereof, and (ii) a therapeutic polynucleotide encoding a tumor virus protein antigen, as described herein.

[0290] At least six tumor and cancer-causing viruses are known to date, including hepatitis B virus, hepatitis C virus, Epstein-Barr virus (EBV or HHV-4), human papillomavirus, human T-lymphotropic virus type 1, and Kaposi's sarcoma-associated herpesvirus (KSHV or HHV-8), but the pathogenic mechanisms have not yet been fully elucidated.

[0291] Tumor virus protein antigens are antigens that are presented on the cell surface of cancer and are derived from oncogenic viruses associated with cancer.For example, the majority of cervical cancers are related, even if they are not caused by HPV infection.Therefore, the antigens that are presented on the cell surface of cervical cancer cells and derived from HPV represent tumor virus protein antigens.Non-limiting examples of tumor virus protein antigens and examples of cancers that these antigens are associated with are shown in Table 1 below.

[0292] Table 1. Examples of tumor virus proteins and associated cancer types TIFF2025507840000017.tif98165

[0293] Thus, in some embodiments, the present disclosure relates to compositions and methods for treating cancer in a subject by administering to the subject a therapeutically effective amount of a composition comprising a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof, and (ii) a therapeutic polynucleotide encoding a tumor viral protein antigen derived from a viral protein listed in Table 1, as described herein. In some embodiments, the therapeutically effective amount of the composition is co-administered with an adjuvant. In some embodiments, the therapeutically effective amount of the composition is co-administered with dendritic cells.

[0294] In some embodiments, the present disclosure relates to compositions and methods for treating cancer in a subject by administering to the subject a therapeutically effective amount of a composition comprising a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof, and (ii) a therapeutic polynucleotide encoding a neoantigen, as described herein. Neoantigens are peptides that are presented on the surface of cancer cells and have an amino acid sequence that is new to the cancer tissue. That is, neoantigens have an amino acid sequence that is not present in the germline (wild type) human genome. Neoantigens are generated by genomic mutations during or after the development of cancer, and in this manner are specific to an individual patient.

[0295] Deep sequencing techniques can be used to identify mutations present in the exome of individual tumors and predict neo-antigens. This is done by identifying neo-antigens that can be recognized by T cells. For example, in some embodiments, tumor material is analyzed for non-synonymous somatic mutations. RNA sequencing data is used to focus on mutations in expressed genes. Peptide stretches containing any of the identified non-synonymous mutations are generated in silico and either unfiltered, filtered using predictive algorithms, or used to identify MHC-associated neo-antigens in mass spectrometry data generated from patient cancer tissue. Modeling of the effect of mutations on the resulting peptide-MHC complexes may be used as an additional filter to identify particularly promising neo-antigens. The resulting epitope set can also be used to identify physiologically occurring neo-antigen-specific T cell responses by MHC multimer-based screening. See, for example, Science, 03 Apr 2015: Vol. 348, Issue 6230, pp. 69-74. However, other techniques, including exome analysis and proteomic analysis, can also be used to identify novel genomic or peptide sequences, respectively, that correspond to neoantigens. Non-limiting examples of neoantigens identified from individual cancers using transcriptome, exome, and proteomic analysis are described, for example, in Haen et al., Towards new horizons Characterization, classification and implications of the tumor antigenic repertoire, Nature Reviews-Clinical Oncology, 2020, Volume 17, 595-610.

[0296] Thus, in some embodiments, the present disclosure provides a method for treating cancer in a subject by first identifying a neo-antigen in the subject's cancer, and second, administering to the subject a therapeutically effective amount of a composition comprising a complex formed between (i) a humanized 3E10 antibody, or an antigen-binding fragment thereof, and (ii) a therapeutic polynucleotide encoding the identified neo-antigen, as described herein.

[0297] In some embodiments, the present disclosure relates to compositions and methods for treating cancer in a subject by administering to the subject a therapeutically effective amount of a composition comprising a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof, and (ii) a therapeutic polynucleotide encoding an antigen derived from a cancer germline gene, as described herein. Cancer germline antigens are a type of immunogenic tumor antigen that are encoded by genes expressed in gamete cells of the testis and / or ovary, and in human cancers. Examples of cancer germline antigens include, but are not limited to, antigens derived from synovial sarcoma X-2 (SSX-2), New York-esophageal squamous cell carcinoma-1 (NY-ESO-1), melanoma associated antigen 1 (MAGA1), and melanoma associated antigen 3 (MAGA3), each of which are overexpressed in different human cancers, such as melanoma and lung cancer.

[0298] Thus, in some embodiments, the present disclosure relates to compositions and methods for treating cancer in a subject by administering to the subject a therapeutically effective amount of a composition comprising a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof and (ii) a therapeutic polynucleotide encoding an antigen derived from an SSX-2, NY-ESO-1, MAGA1, or MAGA3 protein, as described herein. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is lung cancer.

[0299] In some embodiments, the present disclosure relates to compositions and methods for treating cancer in a subject by administering to the subject a therapeutically effective amount of a composition comprising a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof and (ii) a therapeutic polynucleotide encoding a tumor-associated antigen (TAA), as described herein. In some embodiments, the therapeutically effective amount of the composition is co-administered with an adjuvant. In some embodiments, the therapeutically effective amount of the composition is co-administered with dendritic cells.

[0300] Tumor-associated antigens are peptides derived from wild-type protein sequences or glycoproteins synthesized by tumor cells. TAA proteins can be present in any intracellular compartment of tumor cells, for example, they can be membrane-bound, cytoplasmic, nuclear-localized, or even secreted by tumor cells. TAAs are primarily generated by gene amplification or post-translational modification, making the underlying proteins differentially expressed in cancer cells compared to non-cancerous cells, allowing preferential recognition of tumor cells by specific T cells or immunoglobulins. Non-limiting examples of tumor-associated antigens that have been identified are shown in Tables 2-3.

[0301] Table 2. Examples of tumor-associated antigens (TAA) and associated cancers TIFF2025507840000018.tif41133

[0302] Table 3: Examples of tumor-associated antigens (TAA) and related references, all of which are incorporated herein by reference for purposes of identifying the referenced tumor markers. TIFF2025507840000019.tif239170TIFF2025507840000020.tif255168TIFF2025507840000021.tif104170

[0303] Exemplary TAAs include, for example, membrane-bound complement-regulating glycoproteins: CD46, CD55, and CD59, which have been found to be expressed on most tumor cells in vivo and in vitro. Human mucins, such as MUC1, are known tumor markers, as are gp100, tyrosinase, and MAGE, which are found in melanoma, see Tables 2-3. The wild-type Wilms tumor gene WT1 is expressed at high levels in most acute myeloid leukemias, acute lymphocytic leukemias, and chronic myeloid leukemias, as well as in various types of solid tumors, including lung cancer.

[0304] Acute lymphocytic leukemia is characterized by TAA HLA-Dr, CD1, CD2, CD5, CD7, CD19, and CD20. Acute myeloid leukemia is characterized by TAA HLA-Dr, CD7, CD13, CD14, CD15, CD33, and CD34. Breast cancer is characterized by the markers EGFR, HER2, MUC1, Tag-72. Various cancers are characterized by MUC1, TAG-72, and CEA. Chronic lymphocytic leukemia is characterized by CD3, CD19, CD20, CD21, CD25, and HLA-DR. Hairy cell leukemia is characterized by CD19, CD20, CD21, CD25. Hodgkin's disease is characterized by the Leu-Ml marker. Various melanomas are characterized by the HMB45 marker. Non-Hodgkin's lymphomas are characterized by the markers CD20, CD19, and la, and various prostate cancers are characterized by the markers PSMA and SE10.

[0305] Thus, in some embodiments, the present disclosure relates to compositions and methods for treating cancer in a subject by administering to the subject a therapeutically effective amount of a composition comprising a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof, and (ii) a therapeutic polynucleotide encoding a tumor-associated antigen derived from a protein listed in Tables 2-3, as described herein. In some embodiments, the therapeutically effective amount of the composition is co-administered with an adjuvant. In some embodiments, the therapeutically effective amount of the composition is co-administered with dendritic cells.

[0306] In some embodiments, the present disclosure relates to compositions and methods for treating cancer in a subject by administering to the subject a therapeutically effective amount of a composition comprising a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof, and (ii) a therapeutic polynucleotide encoding a tumor-associated antigen derived from a protein listed in Table 2, as described herein, wherein the cancer is a cancer associated with the respective tumor-associated antigen in Table 2. In some embodiments, a therapeutically effective amount of the composition is co-administered with an adjuvant. In some embodiments, a therapeutically effective amount of the composition is co-administered with dendritic cells.

[0307] In some embodiments, the tumor antigen is a tumor viral protein antigen, a neoantigen, or an antigen derived from a cancer germline gene.

[0308] In some embodiments, the tumor virus protein antigen is folate receptor, HER2, papillomavirus oncoprotein E6 and papillomavirus oncoprotein E7, carcinoembryonic antigen (CEA), mucin 1, EGFR, squamous cell carcinoma antigen recognized by T cells 3 (SART3), beta-human chorionic gonadotropin (beta-hCG), Wilms' Tumor antigen 1 (WT1), survivin, MAGE3, p53, ring finger protein 43 and translocase of the outer mitochondrial membrane 34 (TOMM34), prostate specific antigen (PSA)-TRICOM, or KRAS.

[0309] In some embodiments, the tumor associated antigen (TAA) is 5-alpha reductase, a-fetoprotein, AM-1, APC, APRIL, BAGE, β-catenin, Bcl2, bcr-abl (b3a2), CA-125, CASP-8 / FLICE, cathepsin, CD19, CD20, CD21, CD23, CD22, CD38, CD33, CD35, CD44, CD45, CD46, CD5, CD52, CD55, CD59 (791Tgp72), CDC2 7, CDK4, CEA, c-myc, Cox-2, DCC, DcR3, E6 / E7, EGFR, EMBP, Ena78, FGF8b and FGF8a, FLK-1 / KDR, folate receptor, G250, GAGE ​​family, gastrin-17, gastrin-releasing hormone (bombesin), GD2 / GD3 / GM2, GnRH, GnTV, gp100 / Pmell7, gp-100-in4, gpis, gp75 / TRP-1, hCG, heparanase, Her2 / neu Her3, HMTV, Hsp70, hTERT (telomerase), IGFR1, IL-13R, iNOS, Ki 67, KIAA0205, K-ras, H-ras, N-ras, KSA (CO17-1A), LDLR-FUT, MAGE family (MAGE1, MAGE3, etc.), mammaglobin, MAP17, Melan-A / MART-1, mesothelin, MIC A / B, MT-MMPs such as MMP2, MMP3, MMP7, MMP9, Moxl, mucins such as MUC-1, MUC-2, MUC-3, and MUC-4, MUM-1, NY-ESO-1, osteonectin, p15, P170 / MDR1, p53, p97 / melanotransferrin, PAI-1, PDGF, plasminogen (uPA), PRAME, probasin, progenipoietin, PSA, PSM, RAGE-1, Rb, RCAS1, SART-1, SSX gene family, STAT3 (mucin-associated), TAG-72, TGF-ct, TGF-β, thymosin β15, IFN-ct, TPA, TPI, TRP-2, tyrosinase, VEGF, ZAG, pl6INK4, or glutathione S-transferase.

[0310] In some embodiments, the neoantigen is BRCA1, BRCA2 BRAF, KRAS, EGFR, IDH1, PIK3CA, ROS1, HLA, JAK1, JAK2, PARK2, ATM, p53, TP53, erbb2 interacting protein (ERBB2IP), beta-2-microglobulin (β2m), cyclin-dependent kinase inhibitor 2A (CDKN2A), alternate reading frame (ARF), or cyclin-dependent kinase 4 (CDK4).

[0311] In some embodiments, the cancer germline gene is MAGEA1, MAGEA2, MAGEA3, MAGEA4, MAGEA5, MAGEA6, MAGEA8, MAGEA9, MAGEA10, MAGEA11, MAGEA12, BAGE, BAGE2, BAGE3, BAGE4, ​​BAGE5, MAGEB1, MAGEB2, MAGEB5, MAGEB6, MAGEB3, MAGEB4, GAGE1, GAGE2A, GAGE3, GAGE4, ​​GAGE5, GAGE6, GAGE7, GAGE8, SSX1, SSX2, SSX2b, SSX3, SSX4 , CTAG1B, LAGE-1b, CTAG2, MAGEC1, MAGEC3, SYCP1, BRDT, MAGEC2, SPANXA1, SPANXB1, SPANXC, SPANXD, SPANXN1, SPANXN2, SPANXN3, SPANXN4, SPANXN5, XAGE1D, XAGE1C, XAGE1B, XAGE1, XAGE2, XAGE3, XAGE-3b, XAGE-4 / RP11-167P23.2, XAGE5, DDX43, SAGE1, ADAM2, PAGE5, CT16.2, PAGE1, PAGE2, PAGE2B, PAGE3, PAGE4, ​​LIPI, VENTXP1, IL13RA2, TSP50, CTAGE1, CTAGE-2, CTAGE5, SPA17, ACRBP, CSAG1, CSAG2, DSCR8, MMA1b, DDX53, CTCFL, LUZP4, CASC5, TFD P3, JARID1B, LDHC, MORC1, DKKL1, SPO11, CRISP2, FMR1NB, FTHL17, NXF2, TAF7L, TDRD1, TDRD6, TDRD4, TEX15, FATE1, TPTE, CT45A1, CT45A2, CT45A3, CT 45A4, CT45A5, CT45A6, HORMAD1, HORMAD2, CT47A1, CT47A2, CT47A3, CT47A4, CT47A5, CT47A6, CT47A7, CT47A8, CT47A9, CT47A10, CT47A11, CT47B1, SLC O6A1, TAG, LEMD1, HSPB9, CCDC110, ZNF165, SPACA3, CXorf48, THEG, ACTL8, NLRP4, COX6B2, LOC348120, CCDC33, LOC196993, PASD1, LOC647107, TULP2,<h2 style=";text-align:left;direction:ltr">CT66 / AA884595、PRSS54、RBM46、CT69 / BC040308、CT70 / BI818097、SPINLW1、TSSK6、ADAM29、CCDC36、LO C440934、SYCE1、CPXCR1、TSPY3、TSGA10、HIWI、MIWI、PIWI、PIWIL2、ARMC3、AKAP3、Cxorf61、PBK、C21or f99、OIP5、CEP290、CABYR、SPAG9、MPHOSPH1、ROPN1、PLAC1、CALR3、PRM1、PRM2、CAGE1、TTK、LY6K、IMP-3 、AKAP4、DPPA2、KIAA0100、DCAF12、SEMG1、POTED、POTEE、POTEA、POTEG、POTEB、POTEC、POTEH、GOLGAGL2 FA, CDCA1, PEPP2, OTOA, CCDC62, GPATCH2, CEP55, FAM46D, TEX14, CTNNA2, FAM133A, LOC130576, ANKRD45, ELOVL4, IGSf11, TMEFF1, TMEFF2, ARX, SPEF2, GPAT2, TMEM108, NOL4, PTP N20A、SPAG4、MAEL、RQCD1、PRAME、TEX101、SPATA19、ODF1、ODF2、ODF3、ODF4、ATAD2、ZNF64 5、MCAK、SPAG1、SPAG6、SPAG8、SPAG17、FBXO39、RGS22、サイクリンA1、C15orf60、CCDC83、TEKT5 、NR6A1、TMPRSS12、TPPP2、PRSS55、DMRT1、EDAG、NDR、DNAJB8、CSAG3B、CTAG1A、GAGE12B、G AGE12C、GAGE12D、GAGE12E、GAGE12F、GAGE12G、GAGE12H、GAGE12I、GAGE12J、GAGE13、LOC7 28137、MAGEA2B、MAGEA9B / LOC728269、NXF2B、SPANXA2、SPANXB2、SPANXE、SSX4B、SSX5、SS X6.<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0312] <h2 style=";text-align:left;direction:ltr"> CD28 is a member of a subfamily of costimulatory molecules characterized by an extracellular variable immunoglobulin-like domain. Human CD28 is composed of four exons encoding a 220 amino acid protein and is expressed on the cell surface as a glycosylated disulfide-linked homodimer of 44 kDa. Members of the CD28 family share many common features, such as paired V-set immunoglobulin superfamily (IgSF) domains linked to a single transmembrane domain, and a cytoplasmic domain containing important signaling motifs. (Esensten et al., Immunity Review (2016)). CD28 has been reported to control T cell activation through interactions with signaling motifs. For example, tyrosine phosphorylation of CD28 plays a role in early signaling events characterized by CD28 costimulation and the resulting control of T cell activation. Thus, in some embodiments, the compositions for treating cancer provided herein comprise a complex formed between (i) a humanized 3E10 antibody, or an antigen-binding fragment thereof, and (ii) a polynucleotide encoding a signaling motif of a costimulatory molecule having paired V-set immunoglobulin superfamily (IgSF) domains linked to a single transmembrane domain and a cytoplasmic domain.

[0313] Proinflammatory cytokines Proinflammatory cytokines limit tumor cell proliferation directly by direct antiproliferative or proapoptotic activity, or indirectly by stimulating the cytotoxic activity of immune cells against tumor cells. Proinflammatory cytokines are secreted by Th1 cells, CD4+ cells, macrophages, and dendritic cells. They are characterized by the production of several interleukins (ILs), IL-1, IL-2, IL-12, IL-17, IL-18, IFN-γ, and TNF-α. The main proinflammatory cytokines are IL-1, IL-6, and TNF-α. These cytokines signal through type I cytokine receptors (CCR1), which are structurally distinct from other cytokine receptor types. They are essential for orchestrating cell-mediated immune responses and play a key role in regulating the immune system. Proinflammatory cytokines generally control immune cell proliferation, cell activation, differentiation, and homing to the site of infection with the aim of combating and eliminating intracellular pathogens, including viruses.

[0314] IL-1 is subdivided into IL-1α and IL-1β. IL-1β is a potent pro-inflammatory cytokine, induced mainly by lymphocytes, macrophages, and monocytes in response to microbial molecules. During viral infection, pattern recognition receptors (PPRs) and toll-like receptors (TLRs) are expressed, which then lead to enhanced expression of IL-1β. IL-1β stimulates CD4+ cells and differentiates them into Th17 cells. In addition to the stimulatory effects of the IL-1 family, there are also members (IL-1RA and IL-1R2) that can inhibit or suppress IL-1 cytokine expression. IL-1RA is secreted by neutrophils, macrophages, monocytes, and hepatocytes with the aim of reducing inflammation. However, in order to efficiently inhibit or suppress the expression of IL-1β, the expression of IL-RA needs to be expressed up to 1,000-fold.

[0315] IL-2 is approved for the treatment of advanced renal cell carcinoma (RCC) and metastatic melanoma, and IFN-α is approved for the treatment of hairy cell leukemia, follicular non-Hodgkin's lymphoma, melanoma, and AIDS-related Kaposi's sarcoma (Berraondo et al., Cytokines in clinical cancer immunotherapy. British Journal of Cancer, 2019, 120, 6-15; Fyfe et al., Results of treatment of 255 patients with metastatic renal cell carcinoma who received high-dose recombinant interleukin-2 therapy. J. Clin. Oncol., 1995, 13, 688-696; Atkins et al., High-dose recombinant interleukin 2 therapy for patients with metastatic melanoma: analysis of 270 patients treated between 1985 and 1993.J.Clin.Oncol.,1999,17,2105-2116, Golomb et al.,α-2 interferon therapy of hairy-cell leukemia: a multicenter study of 64 patients.J.Clin.Oncol.,1986,4,900-905, Solal-Celigny et al.,Recombinant interferon alfa-2b combined with a regimen containing doxorubicin in patients with advanced follicular lymphoma. Groupe d'Etude des Lymphomes de l'Adulte. New Engl. J. Med., 1993, 329, 1608-1614, Kirkwood et al.,Interferon alfa-2b adjuvant therapy of high-risk resected cutaneous melanoma:the Eastern Cooperative Oncology Group Trial EST 1684.J.Clin.Oncol.,1996,14,7-17,Groopman et al.,Recombinant alpha-2 interferon therapy for Kaposi's sarcoma associated with the acquired immunodeficiency syndrome.Ann.Intern.Med.,1984,100,671-676). .

[0316] IL-6 is a pleiotropic cytokine that not only affects the immune system, but also acts in other biological systems and many physiological events, such as controlling cell growth, as well as gene activation, proliferation, survival, and differentiation. IL-6 is produced by a variety of cell types, including monocytes, fibroblasts, and endothelial cells. Upon stimulation, IL-6 is secreted by many additional cell types, including macrophages, T cells, B cells, mast cells, glial cells, eosinophils, keratinocytes, and granulocytes. IL-6 stimulates the production of acute phase proteins in several types of white blood cells and the liver. It is particularly important in differentiating B cells into antibody-forming cells (plasma cells). Binding of IL-6 to its receptor initiates cellular events, including activation of JAK (Janus kinase) kinases and activation of Ras-mediated signaling.

[0317] Like other Th1 proinflammatory cytokines, TNF-α plays an important role in structuring the inflammatory response both locally and in the circulation. TNF-α induces the expression of vascular endothelial cells and enhances leukocyte adhesion molecules that stimulate immune cell infiltration. It plays a key role in the early response to viral infections by enhancing lymphocyte infiltration to the site of infection.

[0318] Thus, in some embodiments, compositions for treating cancer described herein comprise a humanized 3E10 antibody, or an antigen-binding fragment thereof, and a polynucleotide encoding a pro-inflammatory cytokine.

[0319] In some embodiments, the disclosure provides a composition comprising a complex formed between (i) a humanized 3E10 antibody or antigen-binding fragment thereof and (ii) a therapeutic polynucleotide encoding a pro-inflammatory cytokine, as described herein, and a method for treating cancer by administering such a composition to a subject in need of such treatment. In some embodiments, the therapeutic polynucleotide is non-covalently bound to the humanized 3E10 antibody or antigen-binding fragment thereof. In some embodiments, the therapeutic polynucleotide is covalently bound to the humanized 3E10 antibody or antigen-binding fragment thereof.

[0320] In some embodiments, the cytokine is IL-1, IL-6, IL-8, IL-12, IFN-gamma, IL-18, IL-15, IL-2, TNF-alpha, IL-10, TGF-b, CSF-1, CCL2, CCL3, CCL5, or VEGF.

[0321] Gene Regulatory Polynucleotides In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein is used to deliver a gene-regulating polynucleotide that reduces or silences the expression of a gene product that promotes cancer growth and / or progression, for example, by targeting the gene or its transcript. Non-limiting examples of gene-regulating polynucleotides include siRNA, miRNA, saRNA, antagomir, antisense oligonucleotides, and decoy oligonucleotides. In some embodiments, the gene-regulating polynucleotide is a non-replicating, modified or unmodified mRNA. In some embodiments, the gene-regulating polynucleotide is a self-amplifying mRNA. In some embodiments, the gene-regulating polynucleotide is a plasmid that encodes a protein or peptide. In some embodiments, the gene-regulating polynucleotide is an expression-regulating polynucleotide. For a review of the various types of gene-regulating polynucleotides that have been investigated for their therapeutic potential, see, for example, Roberts TC, Langer R, Wood MJA, “Advances in oligonucleotide drug delivery,” Nat. Rev. Drug Discov., 19(10):673-94 (2020). The contents of that document are incorporated herein by reference.

[0322] Thus, in one aspect, the present disclosure relates to compositions and methods for treating cancer in a subject by administering to the subject a therapeutically effective amount of a composition comprising a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof and (ii) a gene regulatory polynucleotide, as described herein. In some embodiments, the therapeutic polynucleotide is non-covalently bound to the humanized 3E10 antibody or an antigen-binding fragment thereof. In some embodiments, the therapeutic polynucleotide is covalently bound to the humanized 3E10 antibody or an antigen-binding fragment thereof. In some embodiments, the gene regulatory polynucleotide is non-covalently bound to the humanized 3E10 antibody or an antigen-binding fragment thereof. In some embodiments, the gene regulatory polynucleotide is covalently bound to the humanized 3E10 antibody or an antigen-binding fragment thereof.

[0323] siRNA The present disclosure relates to compositions and methods for treating cancer, including a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof, and (ii) an siRNA, as described herein. Small interfering RNA (siRNA), also known as short interfering RNA or silencing RNA, is a type of non-coding RNA molecule, typically 20-27 base pairs in length, double-stranded RNA, that operates within the RNA interference (RNAi) pathway. For example, gene regulatory nucleic acid agents such as siRNA can control post-transcriptional gene expression, silence target genes, and even control intracellular signaling pathways involved in cancer progression (Zhou et al., Delivery of nucleic acid therapeutics for cancer immunotherapy, Medicine in Drug Discovery, March 24, 2020; Dahlman et al., In vivo endothelial siRNA delivery using polymeric nanoparticles with low molecular weight, Nature Nanotechnol. 2014; 9(8):648-655). In some embodiments, the siRNA is non-covalently linked to the humanized 3E10 antibody or antigen-binding fragment thereof. In some embodiments, the siRNA is covalently linked to the humanized 3E10 antibody or antigen-binding fragment thereof.

[0324] Thus, siRNA can be used to regulate the expression of, for example, immune checkpoint molecules described herein by controlling post-translational gene expression and / or silencing the corresponding gene. Similarly, siRNA can be used to indirectly regulate the activity of immune checkpoint molecules by regulating the expression of agonists or inhibitors of immune checkpoint molecules. Thus, in some embodiments, the compositions for treating cancer described herein include a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof and (ii) an siRNA that targets an mRNA transcript from a gene encoding an immune checkpoint molecule, as described herein.

[0325] PD-1 and certain of its homologues, such as PD-L1, suppress T cell responses, particularly in the tumor microenvironment. Thus, inhibitors of PD-1 and / or PD-L1 may improve the effectiveness of T cells in attacking and killing tumor cells. Suppression of PD-1 and / or PD-L1 activity can be achieved, for example, by inhibiting the production of PD-1 and / or PD-L1 in cells. Thus, in some embodiments, the compositions for cancer provided herein include a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof and (ii) an siRNA targeting an mRNA transcript of PD-1 or PD-L1.

[0326] It is known that suppression of the activity of cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) leads to rapid infiltration of T cells. Therefore, an inhibitor of CTLA-4 may lead to promotion of T cell response. Thus, in some embodiments, the composition for treating cancer provided herein comprises a complex formed between (i) humanized 3E10 antibody or its antigen-binding fragment and (ii) siRNA targeting the mRNA transcript of CTLA-4.

[0327] siRNA can also be used to silence genes that control tumor growth or angiogenesis. For example, siRNA has been used to target vascular endothelial growth factor (VEGF) and kinesin spindle protein (KSP) (liver metastases from solid tumors, such as colon cancer). Other gene targets that can be silenced using siRNA include, but are not limited to, genes encoding protein kinase N3 (PKN3) (e.g., metastatic pancreatic cancer), M2 subunit of ribonucleotide reductase (RRM2) (e.g., solid tumors), Myc oncoprotein (e.g., hepatocellular carcinoma), ephrin type-A receptor 2 (EphA2) (e.g., advanced cancer), and KRAS G12D mutation (e.g., advanced pancreatic cancer). See, e.g., International Journal of Nanomedicine 2019:14 3111-3128. Thus, in some embodiments, the compositions for treating cancer provided herein comprise a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof and (ii) an siRNA targeting an mRNA transcript of VEGF, KSP, PKN3, RRM2, EphA2, ERBB2 / HER2, SOCS1, PLK1, or KRAS.

[0328] Other examples of siRNAs that find use in the methods and compositions described herein include, but are not limited to, siRNAs that target mRNA transcripts from the gene encoding CD25 (the IL-2 receptor), which downregulate IL-2 signaling in CD8+ T cells.

[0329] Other non-limiting examples of siRNAs and associated cancer types that have been studied are provided in Table 4 below (see, e.g., Int. J. Mol. Sci. 22 (2021) 3295).

[0330] Table 4. Examples of siRNAs and associated cancer types TIFF2025507840000022.tif88164

[0331] Accordingly, in some embodiments, the present disclosure relates to compositions and methods for treating cancer in a subject by administering to the subject a therapeutically effective amount of a composition comprising a complex formed between (i) a humanized 3E10 antibody, or an antigen-binding fragment thereof, and (ii) an siRNA listed in Table 4, as described herein.

[0332] In some embodiments, the present disclosure relates to compositions and methods for treating cancer in a subject by administering to the subject a therapeutically effective amount of a composition comprising a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof and (ii) each of the siRNAs listed in Table 4, as described herein, wherein the cancer is a cancer associated with each of the siRNAs in Table 4.

[0333] In some embodiments, the disclosure relates to compositions and methods for treating cancer in a subject by administering to the subject a therapeutically effective amount of a composition comprising a complex formed between (i) a humanized 3E10 antibody, or an antigen-binding fragment thereof, and (ii) an siRNA targeting a transcript from a gene listed in Table 4, as described herein.

[0334] In some embodiments, the disclosure relates to compositions and methods for treating cancer in a subject by administering to the subject a therapeutically effective amount of a composition comprising a complex formed between (i) a humanized 3E10 antibody, or an antigen-binding fragment thereof, and (ii) an siRNA targeting a transcript from each of the genes listed in Table 4, as described herein, wherein the cancer is a cancer associated with each of the genes in Table 4.

[0335] miRNA In some embodiments, the compositions and methods for treating cancer described herein include a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof and (ii) a miRNA, as described herein. MicroRNA (miRNA) is a type of non-coding RNA that plays an important role in controlling gene expression. miRNA is an endogenous small non-coding RNA with a length of about 18-24 nt, and can control the expression of target genes by a mechanism similar to that of siRNA (Zhou et al., Delivery of nucleic acid therapeutics for cancer immunotherapy, Medicine in Drug Discovery, March 24, 2020; Xiao et al., MicroRNA control in the immune system; basic principles, Cell, 2009; 136(1):26-36). One of the main challenges of miRNA delivery is to deliver it into tumor tissue while efficiently penetrating deep tissues. Furthermore, the complexity of the tumor microenvironment also hampers efficient intracellular delivery of miRNA to target tumor cells (Rupaimoole et al., MiRNA deregulation in cancer cells and the tumor microenvironment. Cancer Discov. 2016;6(3):235-46). In some embodiments, the miRNA is non-covalently attached to the humanized 3E10 antibody or antigen-binding fragment thereof. In some embodiments, the miRNA is covalently attached to the humanized 3E10 antibody or antigen-binding fragment thereof.

[0336] However, expression of miRNAs specific to individual tumors is beneficial, and miRNAs are involved in early control of immune responses. One way to treat cancer is to regulate the expression of immune checkpoint molecules, such as those described herein, by regulating the levels of miRNAs. Examples of miRNAs that control immune checkpoint-related processes include, but are not limited to, miR-15a, -15b, -16, -195, -424, -497, -503, which control the expression of PD-L1 and CD80. Another example of a miRNA with tumor suppressive function is miR-28, which inhibits the expression of TIM3, BTLA, and PD-1 in T cells by binding to their respective 3'UTRs. Yet another example of a miRNA is miR-138, which inhibits the expression of PD-1 and CTLA-4 on the surface of both effector and regulatory T cells. The miR-34 family includes miR-34a, -34b, and -34c, which inhibit the expression of PD-L1.

[0337] Expression of miR-138-5p is known to inhibit CRC cell proliferation, prevent the G1 to S phase transition of the cell cycle, and directly inhibit PD-L1 expression.

[0338] Other miRNAs, such as miR-20b, -21, and -130b, that are overexpressed in certain types of cancer cells may be effective in indirectly mitigating T cell activation through PTEN expression in the tumor microenvironment.

[0339] Thus, in some embodiments, the compositions and methods for treating cancer described herein comprise a complex formed between (i) a humanized 3E10 antibody, or an antigen-binding fragment thereof, and (ii) an miRNA that directly or indirectly regulates the expression of an immune checkpoint molecule, as described herein.

[0340] In some embodiments, the miRNA is miR-15a, miR-15b, miR-16, miR-20b, miR-21, miR-28, miR-34a, miR-34b, miR-34c, miR-125b, miR-130b, miR-138, miR-138-5p, miR-155, miR-195, miR-197, miR-200, miR-210, miR-221, miR-222, miR-424, miR-497, miR-503, or miR-513.

[0341] In some embodiments, the compositions for treating cancer described herein comprise a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof, and (ii) a miRNA mimic molecule that directly or indirectly regulates expression of an immune checkpoint molecule, as described herein. The miRNA may be a double-stranded synthetic RNA that is identical in sequence to and therefore mimics an endogenous miRNA.

[0342] In some embodiments, the compositions for treating cancer described herein comprise a complex formed between (i) a humanized 3E10 antibody, or an antigen-binding fragment thereof, and (ii) an miRNA expression vector encoding an miRNA that directly or indirectly regulates the expression of an immune checkpoint molecule, as described herein.

[0343] In some embodiments, the compositions and methods for treating cancer described herein include a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof, and (ii) an LNA-modified antisense oligodeoxyribonucleotide (ASO) targeting an miRNA that directly or indirectly regulates the expression of an immune checkpoint molecule, as described herein. LNA is a bicyclic RNA analog in which the ribose is locked in a C3'-endo conformation by the introduction of a 2'-O,4'-C methylene bridge.

[0344] In some embodiments, the compositions and methods for treating cancer described herein comprise a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof, and (ii) an antagomir that targets a miRNA that directly or indirectly regulates the expression of an immune checkpoint molecule, as described herein. The antagomir can be a single-stranded 23-nucleotide RNA molecule that is complementary to the target miRNA and is partially modified with a phosphorothioate backbone in addition to 2'-O-methoxyethyl. This is known to increase the stability of the miRNA by protecting it from degradation.

[0345] In some embodiments, the compositions and methods for treating cancer described herein comprise a complex formed between (i) a humanized 3E10 antibody, or an antigen-binding fragment thereof, and (ii) an antisense oligodeoxyribonucleotide (ASO) targeting an miRNA that directly or indirectly regulates the expression of an immune checkpoint molecule, as described herein.

[0346] In some embodiments, the compositions and methods for treating cancer described herein comprise a complex formed between (i) a humanized 3E10 antibody, or an antigen-binding fragment thereof, and (ii) an miRNA sponge that directly or indirectly regulates expression of an immune checkpoint molecule, as described herein. The miRNA sponge can be an RNA that contains multiple tandem binding sites for a miRNA of interest transcribed from an expression vector.

[0347] Similarly, miRNAs that regulate the expression of proteins associated with tumor growth or angiogenesis can also be delivered by complexing with the humanized 3E10 antibody or antigen-binding fragment thereof, as described herein. Non-limiting examples of miRNAs and the cancers with which they are associated are shown in Table 5.

[0348] Table 5: Examples of miRNAs being studied for cancer treatment (see, for example, Journal of the International Federation of Clinical Chemistry and Laboratory Medicine (2019) Vol. 30, No. 2, pp. 114-127) TIFF2025507840000023.tif52170

[0349] Accordingly, in some embodiments, the present disclosure relates to compositions and methods for treating cancer in a subject by administering to the subject a therapeutically effective amount of a composition comprising a complex formed between (i) a humanized 3E10 antibody, or an antigen-binding fragment thereof, and (ii) an miRNA listed in Table 5, as described herein.

[0350] In some embodiments, the present disclosure relates to compositions and methods for treating cancer in a subject by administering to the subject a therapeutically effective amount of a composition comprising a complex formed between (i) a humanized 3E10 antibody, or an antigen-binding fragment thereof, and (ii) each miRNA listed in Table 5, as described herein, wherein the cancer is a cancer associated with each miRNA in Table 5.

[0351] saRNA In some embodiments, the compositions and methods for treating cancer described herein include a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof and (ii) a saRNA, as described herein. Small activating RNA (saRNA) is a type of non-coding dsRNA that is approximately 21 nt long and has 2 nt overhangs at both ends (Zhou et al., Medicine in Drug Discovery, March 24, 2020; Kwok et al., Ther. Deliv. 2019; 10(3): 151-64). It shares a similar structure to siRNA but has an opposite mechanism of gene regulation. The saRNA in the cytoplasm is specifically loaded into the AGO2 protein, and this RNA-AGO2 complex is transported to the nucleus to induce target gene promoters for gene activation (Li et al., Proc. Natl. Acad. Sci. USA. 2006; 103(46): 17337-42). The saRNA-AGO2 complex in the nucleus has been reported to recruit proteins essential for transcription initiation, such as RNA helicase A, RNA polymerase-associated protein CTR9 homolog (CTR9), and RNA polymerase II-associated factor 1 homolog (PAF1) (Portnoy et al., Cell Res. 2016;26(3):320-35). Due to their ability to upregulate genes, saRNAs show potential for applications such as cancer immunotherapy. Thus, in some embodiments, the compositions for treating cancer described herein comprise a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof, and (ii) a saRNA that induces activation of a gene encoding an immune checkpoint molecule, as described herein. In some embodiments, the saRNA is non-covalently bound to the humanized 3E10 antibody or an antigen-binding fragment thereof. In some embodiments, the saRNA is covalently bound to the humanized 3E10 antibody or an antigen-binding fragment thereof.

[0352] In one example, saRNA can upregulate the transcription factor CCATT / enhancer binding protein alpha (CEBPA), which leads to an increase in functional C / EBP proteins and albumin, inhibiting liver cancer growth in a rat model. Other non-limiting examples of saRNAs that have been investigated for the treatment of cancer are listed in Table 6.

[0353] Table 6. saRNAs being investigated for cancer treatment TIFF2025507840000024.tif20170

[0354] Antagomir In some embodiments, the compositions and methods for treating cancer described herein include a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof, and (ii) an antagomir, as described herein. An antagomir is a small synthetic RNA that is complementary to a specific miRNA target and inhibits Ago2 cleavage by either mispairing at the Ago2 cleavage site or by certain base modifications. An antagomir sequesters specific endogenous microRNAs that are in competition with cellular target mRNAs, inducing miRNA repression to prevent degradation of the mRNA target by RISC. Thus, an antagomir can be used in treatments where loss of function of an miRNA is beneficial. In some embodiments, an antagomir is non-covalently attached to a humanized 3E10 antibody or an antigen-binding fragment thereof. In some embodiments, an antagomir is covalently attached to a humanized 3E10 antibody or an antigen-binding fragment thereof.

[0355] One example of an antagomir is anti-miR21. Studies have shown that silencing miR21 with anti-miR21 affects survival, apoptosis, and cell cycle in colon cancer cells (Song et al., "The anti-miR21 antagomir, a therapeutic tool for colorectal cancer, has a potential synergistic effect by perturbing an angiogenesis-associated miR30," Front. Genet., January 2014).

[0356] Another example of antagomir is antagomir-221. Studies have shown that antagomir-221 can reduce cell proliferation by suppressing the function of miR-221, which plays an important role in HCC, and miR-221 inhibits tumor-suppressive target proteins, such as P27KIP1, P57KIP2, and phosphatase and tensin homolog (PTEN). Similarly, some studies have shown that antagomir-21 reversed epithelial-mesenchymal transition (EMT) through inactivation of AKT serine / threonine kinase 1 (AKT) and ERK1 / 2 pathways by targeting PTEN. This effect of antagomir-21 may be used to target mechanisms responsible for the malignant tendency of breast cancer. See, for example, Atri, et al., AGO-Driven Non-Coding RNAs (2019).

[0357] AntagomiRs targeting miR-155 are in Phase I clinical trials (NCT02580552) and Phase II clinical trials (NCT03713320). miR-155 controls the differentiation and proliferation of blood and lymphatic cells and is a suitable target for treating certain types of lymphoma and leukemia. See, for example, “RNA-Based Therapeutics: From Antisense Oligonucleotides to miRNAs,” Cells 9 (2020), 137.

[0358] Thus, in some embodiments, the compositions for treating cancer described herein include a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof, and (ii) an antagomir that targets a microRNA that regulates the translation of tumor-associated mRNA, as described herein. Non-limiting examples of antagomir include antagomir-221, antagomir-21, and antagomir-155.

[0359] Antisense Oligonucleotides (ASOs) In some embodiments, the compositions for the treatment of cancer described herein comprise a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof and (ii) an antisense oligonucleotide, as described herein. Antisense oligonucleotides (ASOs) are small, chemically modified, synthetic nucleotide chains that have the potential to target any gene product of interest. Typically, ASOs are single-stranded sequences complementary to the sequence of messenger RNA (mRNA) transcribed from a target gene in cells (Rinaldi et al., "Antisense oligonucleotides: the next frontier for treatment of neurological disorders," Nat. Rev. Neurol. 2018; 14(1): 9-21; Bennett, Therapeutic Antisense Oligonucleotides Are Coming of Age. Ann. Rev. Med. 2019; 70: 307-321). ASOs target the corresponding mRNA and degrade the target complex by mechanisms such as endogenous cellular RNase H. In some embodiments, the antisense oligonucleotide is non-covalently linked to the humanized 3E10 antibody or antigen-binding fragment thereof. In some embodiments, the antisense oligonucleotide is covalently linked to the humanized 3E10 antibody or antigen-binding fragment thereof.

[0360] One example of an ASO used in cancer therapy is an ASO targeting CD39 mRNA, which improves CD8+ T cell proliferation and improves antitumor immune responses. Zhou,et al. Medicine in Drug Discovery,6(2020)100023.

[0361] Other non-limiting examples of ASOs and the cancers they are associated with are shown in Table 7.

[0362] (Table 7) Examples of ASOs being investigated for the treatment of cancer (see, for example, Int. J. Mol. Sci 22 (2021) 3295) TIFF2025507840000025.tif216154TIFF2025507840000026.tif60166

[0363] Accordingly, in some embodiments, the present disclosure relates to compositions and methods for treating cancer in a subject by administering to the subject a therapeutically effective amount of a composition comprising a complex formed between (i) a humanized 3E10 antibody, or an antigen-binding fragment thereof, and (ii) an antisense oligonucleotide listed in Table 7, as described herein.

[0364] In some embodiments, the present disclosure relates to compositions and methods for treating cancer in a subject by administering to the subject a therapeutically effective amount of a composition comprising a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof and (ii) each of the antisense oligonucleotides listed in Table 7, as described herein, wherein the cancer is a cancer associated with each of the antisense oligonucleotides in Table 7.

[0365] In some embodiments, the disclosure relates to compositions and methods for treating cancer in a subject by administering to the subject a therapeutically effective amount of a composition comprising a complex formed between (i) a humanized 3E10 antibody, or an antigen-binding fragment thereof, and (ii) an antisense oligonucleotide targeted to a transcript from a gene listed in Table 7, as described herein.

[0366] In some embodiments, the disclosure relates to compositions and methods for treating cancer in a subject by administering to the subject a therapeutically effective amount of a composition comprising a complex formed between (i) a humanized 3E10 antibody, or an antigen-binding fragment thereof, and (ii) an antisense oligonucleotide targeting a transcript from each of the genes listed in Table 7, as described herein, wherein the cancer is a cancer associated with each of the genes in Table 7.

[0367] Decoy oligonucleotide In some embodiments, the present disclosure relates to compositions and methods for treating cancer in a subject by administering to the subject a therapeutically effective amount of a composition comprising a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof and (ii) a decoy oligonucleotide or a polynucleotide encoding the decoy oligonucleotide. Transfection of cis-element double-stranded oligonucleotides, called decoy oligodeoxynucleotides, has been reported to be a powerful tool that provides a new class of anti-gene strategy in gene therapy (Crinelli et al., Design and characterization of decoy oligonucleotides containing locked nucleic acids. Nucleic Acid Res. 2002;30(11):2435-2443). One such example is the STAT3 decoy oligonucleotide, a 15-mer double-stranded oligonucleotide that corresponds closely to the signal transducer and activator of transcription 3 (STAT3) response element in the c-fos promoter and has potential anti-tumor activity. STAT3 decoy oligonucleotides specifically bind to activated STAT3 and block STAT3 binding to DNA sequences on various STAT3-responsive promoters. This results in inhibition of STAT3-mediated transcription and potentially tumor cell proliferation. STAT3 is constitutively activated in various cancers, including squamous cell carcinoma of the head and neck, leading to loss of cell proliferation control and neoplastic transformation. In some embodiments, the decoy oligonucleotide or polynucleotide encoding the decoy oligonucleotide is non-covalently linked to the humanized 3E10 antibody or antigen-binding fragment thereof. In some embodiments, the decoy oligonucleotide or polynucleotide encoding the decoy oligonucleotide is covalently linked to the humanized 3E10 antibody or antigen-binding fragment thereof.

[0368] Examples of polynucleotides encoding complexes capable of genome editing are described in the following sections.

[0369] Zinc Finger Nucleases In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein is complexed with a polynucleotide encoding a zinc finger nuclease. The zinc finger nuclease is a genome editing nuclease. The zinc finger nuclease is an artificial restriction enzyme generated by fusing a zinc finger DNA-binding domain to a DNA cleavage domain. The binding specificity of the designed zinc finger domain directs the zinc finger nuclease to a specific genome site.

[0370] In some embodiments, the present disclosure relates to compositions and methods for performing gene editing in a subject in need of gene editing by administering to the subject a therapeutically effective amount of a composition comprising a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof and (ii) a polynucleotide encoding a zinc finger nuclease. In some embodiments, the polynucleotide encoding the zinc finger nuclease is non-covalently linked to the humanized 3E10 antibody or an antigen-binding fragment thereof. In some embodiments, the polynucleotide encoding the zinc finger nuclease is covalently linked to the humanized 3E10 antibody or an antigen-binding fragment thereof.

[0371] With success in multiple organisms including Drosophila, zebrafish, and rats, ZFN technology has also been successfully used in human cell cultures such as K562 cells, T cells, and hPSCs. It was first demonstrated that ZFN-driven target gene addition to the endogenous CCR5 locus in hESCs generates green fluorescent protein (GFP) reporter cell lines that stably express GFP for at least 2 months in both undifferentiated and differentiated states. It was reported that the eGFP gene introduced into the OCT4 (also known as POU5F1) locus faithfully reflects its transcriptional state, resulting in an endogenous pluripotent reporter cell line. ZFN-driven gene addition has also been successfully used to introduce fluorescent reporter and drug resistance genes into ubiquitous loci such as AAVS1 for constitutive or inducible expression, or into lineage-specific loci such as PITX3 to monitor pluripotency and track cell differentiation. In all of these studies, specific and stable gene addition was achieved with high efficiency without loss of pluripotency, and more importantly, the integrated genes retained high expression levels even as the stem cells differentiated.

[0372] Despite the success of targeted genome editing in hPSCs, ZFN technology has some limitations. First, two ZFNs and homologous DNA need to be co-delivered into hPSCs, which requires an efficient delivery method, typically using a viral vector. However, viral vectors can randomly integrate viral sequences into the target genome, potentially disrupting important genes in hPSCs. To circumvent viral vectors and introduce ZFNs and DNA into the cells of interest, transfection and electroporation methods, or even direct protein delivery methods, have been used. Another obstacle to successfully applying ZFN technology is that designing ZFNs is always difficult and time-consuming, because the incomplete modular nature of tandem zinc fingers means that assembled ZFNs do not necessarily have high affinity for the target sequence, which is a complex of the 3-bp binding sequences of each individual zinc finger. Moreover, the incomplete modular structure of zinc finger assembly and non-specific site binding of the FokI cleavage domain also increase the risk of off-target activity and cytotoxicity. To address this issue, structure- and selection-based approaches, including oligomerized pool engineering and directed evolution, were applied to generate improved ZFNs with optimized DNA-binding specificity and reduced cytotoxicity.

[0373] Transcription activator-like effector nucleases (TALENs) In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein is complexed with a polynucleotide encoding a transcription activator-like effector nuclease (TALEN). TALENs are artificial endonucleases (e.g., restriction enzymes) that are generated by fusion of a transcription activator-like effector (TALE) DNA binding domain and a DNA cleavage domain. TALENs can be engineered to bind to any DNA sequence of interest. In some embodiments, the present disclosure relates to compositions and methods for performing gene editing in a subject by administering to the subject a therapeutically effective amount of a composition comprising a complex formed between (i) a humanized 3E10 antibody or antigen-binding fragment thereof and (ii) a polynucleotide encoding a TALEN. In some embodiments, the polynucleotide encoding the TALEN is non-covalently linked to the humanized 3E10 antibody or antigen-binding fragment thereof. In some embodiments, the polynucleotide encoding the TALEN is covalently linked to the humanized 3E10 antibody or antigen-binding fragment thereof.

[0374] In one embodiment, an engineered TALEN is composed of, from N-terminus to C-terminus, a first spacer, a TALE DNA binding domain, a second spacer, and a FokI nuclease catalytic domain fused to the C-terminus. Since the DNA cleavage domain cleaves the DNA strand, the fusion with the TALE DNA binding domain can be specific to the DNA sequence of interest for genome editing by inducing double-strand breaks. TALENs can function alone, in pairs, or in multiple pairs. For example, the TALE DNA binding domains can bind to targets positioned opposite each other across the spacer where the FokI domains together generate DNA breaks. In one aspect, a TALE DNA binding domain can be designed for use with the disclosed TALENs. A single TALEN (also referred to herein as a monomeric TALEN or TALEN monomer) is composed of a TALE DNA binding domain and a FokI nuclease catalytic domain fused to the C-terminus. TALENs can be engineered for use in TALEN pairs (also referred to herein as pairs of TALENs or TALEN pairs) designed to bind to target nucleotide sequences arranged from N-terminus to C-terminus on opposite strands of DNA. The TALENs of a TALEN pair can have the same sequence or can be different in sequence.

[0375] CRISPR / Cas system In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein is complexed with a polynucleotide encoding Cas. In some embodiments, the present disclosure relates to compositions and methods for performing gene editing in a subject by administering to the subject a therapeutically effective amount of a composition comprising a complex formed between (i) a humanized 3E10 antibody or antigen-binding fragment thereof and (ii) a polynucleotide encoding a Cas endonuclease. In some embodiments, the polynucleotide encoding the Cas endonuclease is non-covalently bound to the humanized 3E10 antibody or antigen-binding fragment thereof. In some embodiments, the polynucleotide encoding the Cas endonuclease is covalently conjugated to the humanized 3E10 antibody or antigen-binding fragment thereof.

[0376] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein is complexed with a CRISPR / Cas guide polynucleotide, e.g., a single guide RNA (sgRNA). In some embodiments, the present disclosure relates to compositions and methods for performing gene editing in a subject by administering to the subject a therapeutically effective amount of a composition comprising a complex formed between (i) a humanized 3E10 antibody or antigen-binding fragment thereof and (ii) a CRISPR / Cas guide polynucleotide. In some embodiments, the CRISPR / Cas guide polynucleotide is non-covalently linked to the humanized 3E10 antibody or antigen-binding fragment thereof. In some embodiments, the CRISPR / Cas guide polynucleotide is covalently linked to the humanized 3E10 antibody or antigen-binding fragment thereof.

[0377] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-associated (Cas) endonucleases were originally discovered as an adaptive immune system evolved by bacteria and archaea to protect against invading viruses and plasmids. Naturally occurring CRISPR / Cas systems in bacteria consist of one or more Cas genes and one or more CRISPR arrays consisting of short palindromic repeats of base sequences separated by genomic targeting sequences (called spacers) acquired from previously encountered viruses and plasmids. (Wiedenheft, B., et.al. Nature. 2012; 482: 331, Bhaya, D., et.al., Annu. Rev. Genet. 2011; 45: 231, and Terms, MPet. al., Curr. Opin. Microbiol. 2011; 14: 321). Bacteria and archaea harboring one or more CRISPR loci respond to viral or plasmid attack by integrating short pieces of foreign sequences (protospacers) into the host chromosome at the proximal end of the CRISPR array. Transcription of the CRISPR locus generates a library of CRISPR-derived RNAs (crRNAs) that contain sequences complementary to previously encountered invading nucleic acids (Haurwitz, RE, et.al., Science. 2012:329;1355; Gesner, EM, et.al. Nat. Struct. Mol. Biol. 2001:18;688; Jinek, M., et.al., Science. 2012:337;816-21). Target recognition by the crRNA occurs via complementary base pairing with the target DNA, directing cleavage of the foreign sequence by the Cas protein. (Jinek et.al.2012 “A Programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.”Science.2012:337;816-821).

[0378] There are at least six major CRISPR system types (Type I, Type II, Type III, Type IV, Type V, and Type VI) and at least 16 distinct subtypes. See, e.g., Makarova, K.S., et al., Nat. Rev. Microbiol. 2015. Nat. Rev. Microbiol.13,722-736, and Liu, Z., Dong, H., Cui, Y. et al., Application of different type of CRISPR / Cas-based systems in bacteria. Microb Cell Fact 19,172(2020). CRISPR systems are also classified based on their effector proteins. Class 1 systems have a multi-subunit crRNA effector complex, while in class 2 systems, all functions of the effector complex are carried out by a single protein (e.g., Cas9 or Cpf1). In some embodiments, the present disclosure teaches using type II and / or type V single subunit effector systems. Thus, in some embodiments, the present disclosure teaches using Class 2 CRISPR systems.

[0379] CRISPR / Cas9 In some embodiments, the present disclosure provides a gene editing method using a type II CRISPR system. In some embodiments, the type II CRISPR system uses the Cas9 enzyme. The type II system relies on i) a single endonuclease protein, ii) a transactivating crRNA (tracrRNA), and iii) a crRNA in which an approximately 20 nucleotide (nt) portion of the 5' end of the crRNA is complementary to the target nucleic acid. The region of the CRISPR crRNA strand that is complementary to its target DNA protospacer is referred to herein as the "guide sequence."

[0380] In some embodiments, the tracrRNA and crRNA components of the Type II system may be replaced by a single guide RNA (sgRNA). The sgRNA may, for example, comprise a nucleotide sequence that includes at least a 12-20 nucleotide sequence that is complementary to a target DNA sequence (guide sequence) and may include a common scaffold RNA sequence at its 3' end. As used herein, "common scaffold RNA" refers to any RNA sequence that mimics the tracrRNA sequence or functions as a tracrRNA.

[0381] The Cas9 endonuclease generates blunt-ended DNA breaks and is recruited to the target DNA by a combination of crRNA and tracrRNA oligos, which tether the endonuclease via complementary hybridization of the RNA CRISPR complex.

[0382] In some embodiments, DNA recognition by the crRNA / endonuclease complex requires additional complementary base pairing with a protospacer adjacent motif (PAM) (e.g., 5'-NGG-3') located in the 3' portion of the target DNA downstream of the target protospacer. (Jinek, M., et.al., Science. 2012:337; 816-821). In some embodiments, the PAM motif recognized by Cas9 varies for different Cas9 proteins.

[0383] Aptamers In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein is complexed with a polynucleotide encoding an aptamer. Nucleic acid aptamers are single-stranded (ss) oligonucleotide molecules (DNA or RNA) that fold into distinct secondary or tertiary structures, thereby obtaining high affinity and specific binding capacity for the corresponding target (Zhu et al., Nucleic Acid Aptamer-Mediated Drug Delivery for Targeted Cancer Therapy, ChemMedChem 2015,10,39-45). Aptamers are selected from random libraries of 1013-1016 ssDNA or ssRNA molecules via an in vitro technique known as SELEX (systematic evolution of ligands by exponential enrichment) (Ellington et al., Nature. 1990, 346, 818-822; Tuerk et al., Science. 1990, 249, 505-510).

[0384] After an aptamer sequence is identified by SELEX, modified nucleotides may be inserted into the sequence, for example to improve stability and / or resistance to nuclease degradation and / or to increase the efficiency of the aptamer. For example, the aptamer APTA-12 contains gemcitabine residues, which are 2',2'-difluoro analogs of 2'deoxycytidine. See, for example, Park JY et al., Mol. Ther. Nucleic Acids 2018,12,543-553.

[0385] Generally, aptamers are used in cancer therapy to either directly inhibit the activity of a target molecule (where the aptamer acts as a functional therapeutic molecule) or to target therapeutic molecules, such as chemotherapeutic agents or other anti-cancer agents, to cancer tissue. In some embodiments, the aptamers used in the methods and compositions described herein directly inhibit the activity of a target molecule rather than targeting cancer tissue. This is because the humanized 3E10 antibody or antigen-binding fragment thereof conjugated to an aptamer, as described herein, already targets various cancer tissues. Generally, therapeutic aptamers used in cancer therapy act as antagonists of oncoproteins or one of their ligands by binding to them, blocking protein-protein interactions or blocking receptor-ligand interactions that promote the development and / or progression of cancer. For reviews of the use of aptamers for the treatment of cancer, see, e.g., Han et al., Application and development of aptamer in cancer-from clinical diagnosis to cancer therapy, Journal of Cancer, 2020, 11, 6902-6915; Zhu et al., “Nucleic Acid Aptamer-Mediated Drug Delivery for Targeted Cancer Therapy”, ChemMedChem 2015, 10, 39-45; and Subjakova et al., 'Polymer Nanoparticles and Nanomotors Modified by DNA RNA Aptamers and Antibodies in Targeted Therapy of Cancer”, Polymers, 2021, 13, 341; Morita Y et al., Cancers (Basel), 2018; 10(3):80, the disclosures of which are incorporated herein by reference.

[0386] In some embodiments, the present disclosure relates to compositions and methods for treating a subject in need of treatment (e.g., cancer) by administering to the subject a therapeutically effective amount of a composition comprising a complex formed between (i) a humanized 3E10 antibody or antigen-binding fragment thereof and (ii) an aptamer, as described herein. In some embodiments, the aptamer is non-covalently bound to the humanized 3E10 antibody or antigen-binding fragment thereof. In some embodiments, the aptamer is covalently bound to the humanized 3E10 antibody or antigen-binding fragment thereof.

[0387] In some embodiments, the aptamer is a PSMA aptamer, a HER2 aptamer, a MUC1 aptamer, a CD117 aptamer, a PTK7 aptamer, a CTLA-4 aptamer, a TLS11a aptamer, a PD-1 aptamer, a PD-1 aptamer, a Macugen aptamer, AS1411, Sgc8, TD05, ARC1779, a-Thrombin (TBA), Macugen, E10030, AS1411, ARC1779, NU172, NOX In some embodiments, the aptamer targets an immune checkpoint control protein. In some embodiments, the immune checkpoint control protein is B7-H3, B7-H4, BTLA, CD160, CTLA4, KIR, LAG3, PD-1, PD-L1, PD-L2, TIM3, or TIGIT.

[0388] Non-limiting examples of aptamers being investigated for the treatment of cancer are provided in Table 8 below.

[0389] Table 8. Examples of aptamers being studied for cancer treatment TIFF2025507840000027.tif103161TIFF2025507840000028.tif208161TIFF202 5507840000029.tif205161TIFF2025507840000030.tif199161TIFF20255078400 00031.tif219161TIFF2025507840000032.tif208161TIFF2025507840000033.t if221161TIFF2025507840000034.tif212161TIFF2025507840000035.tif163161

[0390] Thus, in some embodiments, the present disclosure relates to compositions and methods for treating cancer in a subject by administering to the subject a therapeutically effective amount of a composition comprising a complex formed between (i) a humanized 3E10 antibody or antigen-binding fragment thereof, and (ii) an aptamer selected from the aptamers listed in Table 8, as described herein. In some embodiments, the aptamer is non-covalently bound to the humanized 3E10 antibody or antigen-binding fragment thereof. In some embodiments, the aptamer is covalently bound to the humanized 3E10 antibody or antigen-binding fragment thereof.

[0391] In some embodiments, the present disclosure provides compositions and methods for treating cancer in a subject by administering to the subject a therapeutically effective amount of a composition comprising a complex formed between (i) a humanized 3E10 antibody or antigen-binding fragment thereof and (ii) an aptamer that specifically binds to a respective molecular target selected from the molecular targets listed in Table 8, as described herein, wherein the cancer is a cancer associated with a molecular target in Table 8. In some embodiments, the aptamer is non-covalently bound to the humanized 3E10 antibody or antigen-binding fragment thereof. In some embodiments, the aptamer is covalently bound to the humanized 3E10 antibody or antigen-binding fragment thereof.

[0392] Ribozymes In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein is complexed with a polynucleotide encoding a ribozyme. Ribozymes are catalytically active RNA molecules. Ribozymes occur naturally in a variety of sizes and shapes. Ribozymes catalyze the cleavage and ligation of specific phosphodiester bonds. The formation of peptide bonds during protein synthesis in ribosomes is catalyzed by ribosomal RNA. The biological functions of ribozymes are diverse and play a central role in transfer RNA maturation, intron splicing, replication of RNA viruses or viroids, control of messenger RNA stability, and protein synthesis (Westhof et al. in Encyclopedia of Virology (Third Edition), 2008). In some embodiments, the ribozyme targets human telomerase reverse transcriptase (hTERT) RNA.

[0393] Thus, in some embodiments, the present disclosure provides compositions and methods for treating a disorder (e.g., cancer) by administering to a subject in need of treatment a therapeutically effective amount of a composition comprising a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof and (i) a ribozyme or a polynucleotide encoding the ribozyme. In some embodiments, the ribozyme or polynucleotide encoding the ribozyme is non-covalently attached to the humanized 3E10 antibody or an antigen-binding fragment thereof. In some embodiments, the ribozyme or polynucleotide encoding the ribozyme is covalently attached to the humanized 3E10 antibody or an antigen-binding fragment thereof.

[0394] Treatment method In some embodiments, the disclosure provides compositions and methods for treating a disorder by administering to a subject in need of such treatment a therapeutically effective amount of a composition comprising a complex formed between (i) a humanized 3E10 antibody or antigen-binding fragment thereof, and (i) a therapeutic agent. In some embodiments, the therapeutic agent is covalently attached to the humanized 3E10 antibody or antigen-binding fragment thereof.

[0395] In some embodiments, the therapeutic agent is non-covalently bound to the humanized 3E10 antibody or antigen-binding fragment thereof. In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof is associated with, e.g., conjugated to, a delivery vehicle for the therapeutic agent. In some embodiments, the delivery vehicle is a liposome, lipid nanoparticle, nanoparticle, microparticle, bead system, micelle, biomimetic exosome, or dendrimer. For a review of drug delivery systems, see, e.g., Tiwari G. et al., International Journal of Pharmaceutical Investigation, 2(1):2-11 (2012), the contents of which are incorporated herein by reference in their entirety. For a review of strategies for functionalizing nanoparticle drug delivery systems, see, e.g., Seidu TA et al., Pharmaceutics, 14(5):1113 (2022), the contents of which are incorporated herein by reference in their entirety.

[0396] In some embodiments, the therapeutic agent is a DNA damage inducing agent, a DNA repair inhibitor, an immunomodulatory molecule, an alkylating agent, a microtubule inhibitor, an immune checkpoint inhibitor, an angiogenesis inhibitor, an adoptive cellular therapy, or a topoisomerase inhibitor. In some embodiments, the therapeutic agent is an anti-tumor drug. In some embodiments, the therapeutic agent is a maytansinoid, a benzodiazepine, an auristatin, tecan, a taxoid, CC-1065, (4S)-4,11-diethyl-4,9-dihydroxy-1,4-dihydro-3H,14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14-dione (SN38), exatecan, monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), pyrrolobenzodiazepine (PBD), a proteolysis-inducing chimeric molecule (PROTAC), deruxtecan (Dxd), calicheamicin, duocarmycin, a stimulator of interferon genes (STING) agonist, PNU-159682, NMS249, IMGN Camp 1, duocarmycin hydroxybenzamide azaindole (DUBA), or a prodrug thereof. In some embodiments, the therapeutic agent is a maytansinoid. In some embodiments, the therapeutic agent is N(2')-deacetyl-N(2')-(3-mercapto-1-oxopropyl)-maytansine (DM1). In some embodiments, the therapeutic agent is N2'-deacetyl-N2'-(4-mercapto-4-methyl-1-oxopentyl)maytansine (DM4). In some embodiments, the therapeutic agent is (4S)-4,11-diethyl-4,9-dihydroxy-1,4-dihydro-3H,14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14-dione (SN38). In some embodiments, the therapeutic agent is PNU-159682. In some embodiments, the therapeutic agent is PNU-159682. In some embodiments, the therapeutic agent is NMS249.

[0397] In some embodiments, the therapeutic agent is a therapeutic polynucleotide, e.g., as described herein. In some embodiments, the therapeutic polynucleotide is non-covalently attached to the humanized 3E10 antibody or antigen-binding fragment thereof. In some embodiments, the therapeutic polynucleotide is covalently attached to the humanized 3E10 antibody or antigen-binding fragment thereof. In some embodiments, the therapeutic polynucleotide is a non-replicating, non-modified mRNA. In some embodiments, the therapeutic polynucleotide is a non-replicating, modified mRNA. In some embodiments, the therapeutic polynucleotide is a self-amplifying mRNA. In some embodiments, the therapeutic polynucleotide is a plasmid encoding a protein or peptide. In some embodiments, the therapeutic polynucleotide is a gene regulatory polynucleotide.

[0398] In some embodiments, the disorder is cancer. In some embodiments, the cancer is carcinoma, sarcoma, blastoma, papilloma, or adenoma. In some embodiments, the cancer is metastatic cancer. In some embodiments, the cancer is bladder cancer, blood cancer, brain cancer, breast cancer, bone cancer, cervical cancer, colorectal cancer, endocrine cancer, esophageal cancer, gastric cancer, head and neck cancer, hepatobiliary cancer, leukemia, lung cancer, lymphoma, melanoma, myeloma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, thyroid cancer, or uterine cancer.

[0399] In some embodiments, the present invention is directed to cancer types, blastomas, and sarcomas, as well as certain leukemias or lymphoid malignancies, benign and malignant tumors, such as malignant tumors, e.g., sarcomas, carcinomas, and melanomas; blood cancers of the blood or bone marrow; hematological (or hematologic) cancers, such as acute leukemias (acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloid leukemia, and myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythrocytic leukemias), chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (low-grade and high-grade), multiple myeloma, Waldenstrom's hypergammaglobulinemia, heavy chain disease, myelodysplastic syndromes. Solid tumors such as sarcomas and carcinomas, e.g. fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, other sarcomas, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid malignancies, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma Methods are provided for treating a subject suffering from a tumor, including a tumor of the cervix, a pheochromocytoma, a sebaceous carcinoma, a papillary carcinoma, a papillary adenocarcinoma, a medullary carcinoma, a bronchial carcinoma, a renal cell carcinoma, a hepatoma, a cholangiocarcinoma, a choriocarcinoma, a Wilms' tumor, a cervical carcinoma, a testicular tumor, a seminoma, a bladder cancer, a melanoma, as well as a CNS tumor (e.g., a glioma (e.g., brain stem glioma and mixed glioma), a glioblastoma (also known as glioblastoma multiforme), an astrocytoma, a CNS lymphoma, a germ cell tumor, a medulloblastoma, a schwannoma, a craniopharyngioma, an ependymoma, a pinealoma, a hemangioblastoma, an acoustic neuroma, an oligodendroglioma, a meningioma, a neuroblastoma, a retinoblastoma, or a brain metastasis.

[0400] In some embodiments, the disclosure provides a method for treating cancer of the central nervous system by parenterally administering to a subject a therapeutically effective amount of a composition comprising a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof and (ii) a therapeutic substance, as described herein.

[0401] In some embodiments, the cancer is skin cancer. In some embodiments, the skin cancer is basal cell carcinoma, squamous cell carcinoma, or melanoma. In one embodiment, the cancer is melanoma.

[0402] In one aspect, the disclosure provides a method for treating a skeletal muscle disease in a subject, comprising administering to the subject a therapeutically effective amount of a composition comprising a non-covalent complex of (i) a humanized 3E10 antibody or an antigen-binding fragment thereof and (i) an mRNA encoding a protein mutated in a genetic skeletal muscle disease. In some embodiments, the mRNA is non-covalently linked to the humanized 3E10 antibody or an antigen-binding fragment thereof. In some embodiments, the mRNA is covalently linked to the humanized 3E10 antibody or an antigen-binding fragment thereof. In some embodiments, the mRNA encodes dystrophin (DMD) or a fragment thereof, such as a mini-dystrophin or a micro-dystrophin construct.

[0403] Stoichiometry of Therapeutic Polynucleotides In some embodiments, for example, when the therapeutic polynucleotide is non-covalently bound to the humanized 3E10 antibody or antigen-binding fragment thereof, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of at least 2:1. The use of a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide in the compositions described herein protects the therapeutic polynucleotide from degradation. For example, the parent 3E10 antibody and the 3E10(D31N) variant antibody protect mRNA from RNAseA-mediated RNA degradation at molar ratios of 2:1 and 20:1, but the protection obtained at the molar ratio of 20:1 is greater than that obtained at 2:1.

[0404] In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of at least 2:1. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of at least 2.5:1. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of at least 5:1. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of at least 7.5:1. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of at least 10:1. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of at least 15:1. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of at least 20:1. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of at least 25:1. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of at least 30:1. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of at least 40:1.

[0405] In some embodiments, the compositions described herein comprise at least 3:1, at least 4:1, at least 5:1, at least 6:1, at least 7:1, at least 8:1, at least 9:1, at least 10:1, at least 11:1, at least 12:1, at least 13:1, at least 14:1, at least 15:1, at least 16:1, at least 17:1, at least 18:1, at least 19:1, at least 20:1, at least 21:1, at least 22:1, at least 23:1, at least 24:1, at least 25:1 , at least 26:1, at least 27:1, at least 28:1, at least 29:1, at least 30:1, at least 31:1, at least 32:1, at least 33:1, at least 34:1, at least 35:1, at least 36:1, at least 37:1, at least 38:1, at least 39:1, at least 40:1, at least 41:1, at least 42:1, at least 43:1, at least 44:1, at least 45:1, or more, molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide.

[0406] In some embodiments, the pharmaceutical compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1 or more.

[0407] In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 50:1 or less. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 40:1 or less. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 30:1 or less. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 25:1 or less. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 20:1 or less. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 15:1 or less. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody, or antigen-binding fragment thereof, to therapeutic polynucleotide of 10:1 or less.

[0408] In some embodiments, the compositions described herein comprise at least one of: 50:1 or less, 49:1 or less, 48:1 or less, 47:1 or less, 46:1 or less, 45:1 or less, 44:1 or less, 43:1 or less, 42:1 or less, 41:1 or less, 40:1 or less, 39:1 or less, 38:1 or less, 37:1 or less, 36:1 or less, 35:1 or less, 34:1 or less, 33:1 or less, 32:1 or less, 31:1 or less, 30:1 or less, 29:1 or less, 28:1 or less, 27:1 or less, 26:1 or less, 28:1 or less, 27 ... The humanized 3E10 antibody or antigen-binding fragment thereof has a molar ratio of therapeutic polynucleotide of 0.1:1 or less, 25:1 or less, 24:1 or less, 23:1 or less, 22:1 or less, 21:1 or less, 20:1 or less, 19:1 or less, 18:1 or less, 17:1 or less, 16:1 or less, 15:1 or less, 14:1 or less, 13:1 or less, 12:1 or less, 11:1 or less, 10:1 or less, 9:1 or less, 8:1 or less, 7:1 or less, 6:1 or less, 5:1 or less, or less.

[0409] In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 2:1 to 50:1. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 2:1 to 40:1. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 2:1 to 30:1. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 2:1 to 25:1. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 2:1 to 20:1. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 2:1 to 15:1. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 2:1 to 10:1. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 2:1 to 7.5:1. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 2:1 to 5:1. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 2:1 to 5:1. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 2:1 to 3:1.

[0410] In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 5:1 to 50:1. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 5:1 to 40:1. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 5:1 to 30:1. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 5:1 to 25:1. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 5:1 to 20:1. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 5:1 to 15:1. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of between 5:1 and 10:1. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of between 5:1 and 7.5:1.

[0411] In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 10:1 to 50:1. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 10:1 to 40:1. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 10:1 to 30:1. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 10:1 to 25:1. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 10:1 to 20:1. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody, or antigen-binding fragment thereof, to therapeutic polynucleotide of 10:1 to 15:1.

[0412] In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 15:1 to 50:1. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 15:1 to 40:1. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 15:1 to 30:1. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 15:1 to 25:1. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 15:1 to 20:1.

[0413] In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 20:1 to 50:1. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 20:1 to 40:1. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 20:1 to 30:1. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 20:1 to 25:1.

[0414] In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 25:1 to 50:1. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 25:1 to 40:1. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 25:1 to 30:1. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 30:1 to 50:1. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 30:1 to 40:1. In still other embodiments, other ranges within the range of 2:1 to 50:1 are also contemplated.

[0415] In some embodiments, the compositions described herein are 2:1 to 50:1, 2:1 to 40:1, 2:1 to 30:1, 2:1 to 25:1, 2:1 to 20:1, 2:1 to 15:1, 2:1 to 10:1, 2:1 to 7.5:1, 2:1 to 5:1, 5:1 to 50:1, 5:1 to 40:1, 5:1 to 30:1, 5:1 to 25:1, 5:1 to 20:1, 5:1 to 15:1, 5:1 to 10:1, 5:1 to 7.5:1, 10:1 to 50:1, 10:1 to 40:1, 10:1 to 30:1, 10:1 to 2 The molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide is 5:1, 10:1 to 20:1, 10:1 to 15:1, 15:1 to 50:1, 15:1 to 40:1, 15:1 to 30:1, 15:1 to 25:1, 15:1 to 20:1, 20:1 to 50:1, 20:1 to 40:1, 20:1 to 30:1, 20:1 to 25:1, 25:1 to 50:1, 25:1 to 40:1, 25:1 to 30:1, 30: to 50:1, 30:1 to 40:1, or 40:1 to 50:1. In yet other embodiments, other ranges within the range of 2:1 to 50:1 are also contemplated.

[0416] In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 1:1 to 50:1. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 1:1 to 30:1. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 1:1 to 20:1. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 1:1 to 10:1. In some embodiments, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen-binding fragment thereof to therapeutic polynucleotide of 1:1 to 5:1.

[0417] In some embodiments, the molar ratio is related to the size of the nucleic acid (i.e., therapeutic polynucleotide), e.g., long polynucleotides are complexed at high molar ratios and short polynucleotides are complexed at low molar ratios.

[0418] In some embodiments, the size of the therapeutic polynucleotide is about 10bp, 15bp, 20bp, 25bp, 30bp, 35bp, 40bp, 45bp, 50bp, 55bp, 60bp, 65bp, 70bp, 75bp, 80bp, 85bp, 90bp, 95bp, 100bp, 105bp, 110bp, 115bp, 120bp, 125bp, 130bp, 135bp, 140bp, p, 145bp, 150bp, 155bp, 160bp, 165bp, 170bp, 175bp, 180bp, 185bp, 190bp, 195bp, 200bp, 205bp, 210bp , 215bp, 220bp, 225bp, 230bp, 235bp, 240bp, 245bp, 250bp, 255bp, 260bp, 265bp, 270bp, 275bp, 280bp, 285bp, 290bp, 295bp, 300bp, 305bp, 310bp, 315bp, 320bp, 325bp, 330bp, 335bp, 340bp, 345bp, 350bp, 3 55bp, 360bp, 365bp, 370bp, 375bp, 380bp, 385bp, 390bp, 395bp, 400bp, 405bp, 410bp, 415bp, 420bp, 42 5bp, 430bp, 435bp, 440bp, 445bp, 450bp, 455bp, 460bp, 465bp, 470bp, 475bp, 480bp, 485bp, 490bp, 495bp, 500bp, 505bp, 510bp, 515bp, 520bp, 525bp, 530bp, 535bp, 540bp, 545bp, 550bp or more, and any range in between.

[0419] In some embodiments, the molar ratio disclosed herein is related to the size of the therapeutic polynucleotide disclosed herein.For example, long polynucleotides are complexed at high molar ratios, and short polynucleotides are complexed at low molar ratios.In some embodiments, any molar ratio disclosed herein can be combined with any size of therapeutic polynucleotide. Non-limiting examples include molar ratios of 3E10 antibody to therapeutic polynucleotide of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, 100:1, 105:1, 110:1, 115:1, 120:1, 125:1, 130:1, 135:1, 140:1, 145:1, 150:1, 160:1, 170:1, 180:1, 190:1, 200:1, 210:1, 220:1, 230:1, 240:1, 250:1, 260:1, 270:1, 280:1, 290:1, 300:1, 310:1, 320:1, 330:1, 340:1, 350:1, 360:1, 370:1, 380:1, 390:1, 40 ... 1, 140:1, 145:1, 150:1, 155:1, 160:1, 165:1, 170:1, 175:1, 180:1, 185:1, 190:1, 195:1, 200:1, 205:1, 210:1, 215:1, 220:1, 225:1, 230:1, 235:1, 240:1, 245:1, 250:1 or more, and any range therebetween, and the therapeutic polynucleotide has a size of about 10 bp, 15 bp, 20 bp, 25 bp, 30 bp, 35 bp, 40 bp, 45 bp, 50 bp, 55 bp, 60 bp, 65 bp, 70 bp, 75 bp, 80 bp, 85 bp, 90 bp, 95 bp, 100 bp, 105 bp, 110 bp, 120 bp, 130 bp, 140 bp, 150 bp, 160 bp, 170 bp, 185 bp, 190 bp, 195 bp, 200 bp, 205 bp, 210 bp, 215 bp, 220 bp, 225 bp, 230 bp, 235 bp, 240 bp, 245 bp, 250 bp, or more, and any range therebetween. 0bp, 45bp, 50bp, 55bp, 60bp, 65bp, 70bp, 75bp, 80bp, 85bp, 90bp, 95bp, 100bp, 105bp, 110bp, 115bp, 120bp, 125bp, 130bp, 13 5bp, 140bp, 145bp, 150bp, 155bp, 160bp, 165bp, 170bp, 175bp, 180bp, 185bp, 190bp, 195bp, 200bp, 205bp, 210bp, 215bp, 220 bp, 225bp, 230bp, 235bp, 240bp, 245bp, 250bp, 255bp, 260bp, 265bp, 270bp, 275bp, 280bp, 285bp, 290bp, 295bp, 300bp, 305b p, 310bp, 315bp, 320bp, 325bp, 330bp, 335bp, 340bp, 345bp, 350bp, 355bp, 360bp, 365bp, 370bp, 375bp, 380bp, 385bp, 390bp,Examples of such compositions include those having a length of 395bp, 400bp, 405bp, 410bp, 415bp, 420bp, 425bp, 430bp, 435bp, 440bp, 445bp, 450bp, 455bp, 460bp, 465bp, 470bp, 475bp, 480bp, 485bp, 490bp, 495bp, 500bp, 505bp, 510bp, 515bp, 520bp, 525bp, 530bp, 535bp, 540bp, 545bp, 550bp or more, and any range therebetween.

[0420] All methods described herein can be performed in any suitable order unless otherwise indicated or clearly contradicted by context. Any and all examples provided herein, or the use of exemplary language (e.g., "for example," "etc.") are intended merely to more clearly describe the embodiments and do not limit the scope of the embodiments unless otherwise specified. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. EXAMPLES

[0421] Example 1: Binding kinetics and affinity measurements of humanized 3E10 antibody Briefly, seven humanized versions of the 3E10 variable heavy domain (VH-h1 through VH-h7) and six humanized versions of the 3E10 variable light domain (VL-h1 through VL-h6) were constructed. Figures 5-10 provide the amino acid sequences of the humanized VH domain, the mature portion of the complete HC incorporating the humanized VH, the full-length heavy chain incorporating the humanized VH, the humanized VL domain, the mature portion of the complete LC incorporating the humanized VL, and the full-length heavy chain incorporating the humanized VL, respectively.

[0422] Of the 42 possible humanized 3E10 antibodies that can be made from these humanized VH and VL domains, 22 constructs were generated, as outlined in Table 1. In Table 1, the variant number refers to a particular combination of humanized VH and VL. For example, variant 12 refers to a humanized 3E10 antibody that includes humanized VH-h1 and humanized VL-h2, and variant 41 refers to a humanized 3E10 antibody that includes humanized VH-h4 and humanized VL-h1.

[0423] Table 1. Humanized 3E10 antibody constructs TIFF2025507840000036.tif191128TIFF2025507840000037.tif215128TIFF2025507840000038.tif191128

[0424] The nucleic acid affinity of 22 humanized 3E10 antibodies was investigated using a solid-phase ELISA poly-dT binding assay. Briefly, microtiter plates were coated with 100 μl of μg / ml streptavidin in PBS per well by incubating overnight at 4° C. After removing the coating solution, the wells were washed twice with 0.05% PBST. The wells were then blocked by incubating with 1% bovine serum albumin (BSA) in PBS for 1 h at 37° C. After removing the blocking solution, the wells were washed once with 0.05% PBST. The wells were then coated with poly-dT by incubating with 100 μl of 0.1 μM biotin-labeled poly-dT in ELISA buffer for 40 min at room temperature. After removing the coating solution, the wells were washed three times with 0.05% PBST. A three-fold dilution series with antibody concentrations from 100 μg / ml to 1.69 ng / ml was established for each of the 22 humanized antibodies mentioned above. 100 μl of each dilution was incubated in separate wells at 4 °C for 4 h. After removing the dilution solution, the wells were washed again three times with 0.05% PBST. Antibodies bound to poly-dT were detected by placing 100 μl of a 1:10,000 diluted horseradish peroxidase (HRP)-conjugated secondary mouse anti-human IgG Fc antibody in each well and incubating for 1.5 h at room temperature. After removing the secondary antibody solution, the wells were washed again three times with 0.05% PBST. Secondary antibodies were detected by incubating 3,3',5,5'-tetramethylbenzidine (TMB) in each well for 10 min.

[0425] Binding curves for the various binding assays are shown in Figures 12A, 12B, 12C, 12D, and 12E. EC50 values, representing the concentration of antibody at which half-maximal binding of poly-dT was achieved, were calculated from the curves and then normalized to the EC50 value determined for each experiment using the chimeric 3E10 D31N variant with mouse VH and VL sequences.

[0426] The results indicate that lysine at position 72 of the 3E10 light chain contributes significantly to the nucleic acid binding activity of 3E10. Mutation of this residue to tyrosine in the VL-h1 variant significantly reduces the affinity of the antibody for DNA binding. Similarly, arginine and lysine at positions 37 and 38 of the variable heavy chain appear to contribute to the nucleic acid binding activity of 3E10. Mutation of these residues to leucine and arginine, respectively, reduces the affinity of the antibody for DNA binding.

[0427] Example 2: Humanized 3E10 antibody-mediated delivery of RIG-I ligand induces type I IFN responses in THP-1 monocytes (low affinity candidate) We investigated whether humanized 3E10 antibody-mediated delivery of RIG-I stimulatory ligand to monocytic cells derived from acute monocytic leukemia effectively induces type I IFN response, a hallmark of immunotherapy. Briefly, THP-1 monocytes were seeded in wells and incubated at 20,000 cells / well in DMEM supplemented with 20% FBS and 1% P / S. The cells were then treated with PBS (control), 3p-hpRNA RIG-I agonist alone (1ug / well), increasing amounts of humanized 3E10 antibody alone, and humanized 3E10 antibody / 3p-hpRNA complex (1ug 3p-hpRNA / well), as shown in Figure 13. Sample media was collected at the indicated time points and measured for luciferase activity (a reporter for type I IFN). IFN response was monitored for 4 consecutive days at 24, 48, 72, and 96 hours, as shown in Figure 13.

[0428] As shown in FIG. 13, exposure of THP-1 monocytes to 3p-hpRNA RIG-I with 3E10 variants 11, 21, 31, or 41 resulted in a mean peak increase in type 1 IFN response (approximately 135-fold) 24 hours after treatment, which steadily decreased at 48, 72, and 96 hours compared to control samples (untreated, 3p-hpRNA alone, and 3E10 variants 11, 21, 31, or 41 alone).

[0429] Example 3: Humanized 3E10 antibody-mediated delivery of RIG-I ligand induces type I IFN responses in THP-1 monocytes (medium affinity candidate) We investigated whether humanized 3E10 antibody-mediated delivery of RIG-I stimulatory ligand to monocytic cells derived from acute monocytic leukemia effectively induces type I IFN response, a hallmark of immunotherapy. Briefly, THP-1 monocytes were seeded in wells and incubated at 20,000 cells / well in DMEM supplemented with 20% FBS and 1% P / S. The cells were then treated with PBS (control), 3p-hpRNA RIG-I agonist alone (1ug / well), increasing amounts of humanized 3E10 antibody alone, and humanized 3E10 antibody / 3p-hpRNA complex (1ug 3p-hpRNA / well), as shown in Figure 14. Sample media was sampled at the indicated time points and measured for luciferase activity (a reporter for type I IFN). IFN response was monitored for 4 consecutive days at 24, 48, 72, and 96 hours, as shown in Figure 14.

[0430] As shown in Figure 14, exposure of THP-1 monocytes to 3p-hpRNA RIG-I with 3E10 variants 22, 12 or 13 resulted in a mean peak increase in type 1 IFN response (approximately 132-fold) 72 hours after treatment. At 96 hours, the IFN response begins to decline. The data suggest a mechanism for controlled dissociation of the payload, i.e., 3p-hpRNA, from the humanized 3E10 antibody over time when compared to control samples (untreated, 3p-hpRNA alone, and 3E10 variants 22, 12, or 13 alone).

[0431] The experiment was repeated using non-humanized 3E10 WT and 3E10 D31N instead of the humanized antibodies, and the results are shown in Figure 16.

[0432] Example 4: Humanized 3E10 antibody-mediated delivery of RIG-I ligand induces type I IFN responses in THP-1 monocytes (high affinity candidate) We investigated whether humanized 3E10 antibody-mediated delivery of RIG-...

Claims

1. A humanized 3E10 antibody or antigen-binding fragment thereof, comprising a light chain variable domain (3E10-VL) and a heavy chain variable domain (3E10-VH), the 3E10-VL comprises an amino acid sequence at least 97% identical to an amino acid sequence selected from the group consisting of 3E10-VL-h1 (SEQ ID NO: 85), 3E10-VL-h2 (SEQ ID NO: 86), 3E10-VL-h3 (SEQ ID NO: 87), 3E10-VL-h4 (SEQ ID NO: 88), 3E10-VL-h5 (SEQ ID NO: 89), and 3E10-VL-h6 (SEQ ID NO: 90); the 3E10-VH comprises an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of 3E10-VH-h1 (SEQ ID NO: 64), 3E10-VH-h2 (SEQ ID NO: 65), 3E10-VH-h3 (SEQ ID NO: 66), 3E10-VH-h4 (SEQ ID NO: 67), 3E10-VH-h5 (SEQ ID NO: 68), 3E10-VH-h6 (SEQ ID NO: 69), and 3E10-VH-h7 (SEQ ID NO: 70); The humanized 3E10 antibody or an antigen-binding fragment thereof.

2. the humanized 3E10 antibody or antigen-binding fragment thereof comprises a light chain (3E10-LC) and a heavy chain (3E10-HC); the 3E10-LC comprises an amino acid sequence at least 97% identical to an amino acid sequence selected from the group consisting of 3E10-LC-h1m (SEQ ID NO:91), 3E10-LC-h2m (SEQ ID NO:92), 3E10-LC-h3m (SEQ ID NO:93), 3E10-LC-h4m (SEQ ID NO:94), 3E10-LC-h5m (SEQ ID NO:95), and 3E10-LC-h6m (SEQ ID NO:96); the 3E10-HC comprises an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of 3E10-HC-h1m (SEQ ID NO:71), 3E10-HC-h2m (SEQ ID NO:72), 3E10-HC-h3m (SEQ ID NO:73), 3E10-HC-h4m (SEQ ID NO:74), 3E10-HC-h5m (SEQ ID NO:75), 3E10-HC-h6m (SEQ ID NO:76), and 3E10-HC-h7m (SEQ ID NO:77); The humanized 3E10 antibody or antigen-binding fragment thereof according to claim 1.

3. the humanized 3E10 antibody or antigen-binding fragment thereof comprises a light chain (3E10-LC) and a heavy chain (3E10-HC); the 3E10-LC comprises an amino acid sequence at least 97% identical to an amino acid sequence selected from the group consisting of 3E10-LC-h1m (SEQ ID NO:97), 3E10-LC-h2 (SEQ ID NO:98), 3E10-LC-h3 (SEQ ID NO:99), 3E10-LC-h4 (SEQ ID NO:100), 3E10-LC-h5 (SEQ ID NO:101), and 3E10-LC-h6 (SEQ ID NO:102); the 3E10-HC comprises an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of 3E10-HC-h1 (SEQ ID NO:78), 3E10-HC-h2 (SEQ ID NO:79), 3E10-HC-h3 (SEQ ID NO:80), 3E10-HC-h4 (SEQ ID NO:81), 3E10-HC-h5 (SEQ ID NO:82), 3E10-HC-h6 (SEQ ID NO:83), and 3E10-HC-h7 (SEQ ID NO:84); The humanized 3E10 antibody or antigen-binding fragment thereof according to claim 1.

4. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1 to 3, wherein the 3E10-VL comprises an amino acid sequence that is at least 97%, 98%, 99%, or 100% identical to 3E10-VL-h1 (sequence number 85).

5. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1 to 3, wherein the 3E10-VL comprises an amino acid sequence that is at least 97%, 98%, 99%, or 100% identical to 3E10-VL-h2 (sequence number 86).

6. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1 to 3, wherein the 3E10-VL comprises an amino acid sequence that is at least 97%, 98%, 99%, or 100% identical to 3E10-VL-h3 (sequence number 87).

7. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1 to 3, wherein the 3E10-VL comprises an amino acid sequence that is at least 97%, 98%, 99%, or 100% identical to 3E10-VL-h4 (sequence number 88).

8. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1 to 3, wherein the 3E10-VL comprises an amino acid sequence that is at least 97%, 98%, 99%, or 100% identical to 3E10-VL-h5 (sequence number 89).

9. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1 to 3, wherein the 3E10-VL comprises an amino acid sequence that is at least 97%, 98%, 99%, or 100% identical to 3E10-VL-h6 (sequence number 90).

10. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1 to 3, wherein the 3E10-VH comprises an amino acid sequence that is at least 96%, 97%, 98%, 99%, or 100% identical to 3E10-VH-h1 (sequence number 64).

11. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1 to 3, wherein the 3E10-VH comprises an amino acid sequence that is at least 96%, 97%, 98%, 99%, or 100% identical to 3E10-VH-h2 (sequence number 65).

12. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1 to 3, wherein the 3E10-VH comprises an amino acid sequence that is at least 96%, 97%, 98%, 99%, or 100% identical to 3E10-VH-h3 (sequence number 66).

13. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1 to 3, wherein the 3E10-VH comprises an amino acid sequence that is at least 96%, 97%, 98%, 99%, or 100% identical to 3E10-VH-h4 (sequence number 67).

14. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1 to 3, wherein the 3E10-VH comprises an amino acid sequence that is at least 96%, 97%, 98%, 99%, or 100% identical to 3E10-VH-h5 (sequence number 68).

15. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1 to 3, wherein the 3E10-VH comprises an amino acid sequence that is at least 96%, 97%, 98%, 99%, or 100% identical to 3E10-VH-h6 (sequence number 69).

16. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1 to 3, wherein the 3E10-VH comprises an amino acid sequence that is at least 96%, 97%, 98%, 99%, or 100% identical to 3E10-VH-h7 (SEQ ID NO: 70).

17. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1 to 3, wherein the 3E10-VH comprises an amino acid sequence at least 98%, 99%, or 100% identical to 3E10-VH-h6 (SEQ ID NO: 69), and the 3E10-VL comprises an amino acid sequence at least 97%, 98%, 99%, or 100% identical to 3E10-VL-h6 (SEQ ID NO: 90).

18. The humanized 3E10 antibody or its antigen-binding fragment according to claim 17, wherein the 3E10-VH comprises the amino acid sequence of 3E10-VH-h6 (sequence number 69), and the 3E10-VL comprises the amino acid sequence of 3E10-VL-h6 (sequence number 90).

19. The humanized 3E10 antibody or antigen-binding fragment thereof of claim 17, wherein the 3E10-HC comprises the amino acid sequence of 3E10-HC-h6m (sequence number 76) and the 3E10-LC comprises the amino acid sequence of 3E10-LC-h6m (sequence number 96).

20. The humanized 3E10 antibody or antigen-binding fragment thereof of claim 17, wherein the 3E10-HC comprises the amino acid sequence of 3E10-HC-h67 (SEQ ID NO: 83) and the 3E10-LC comprises the amino acid sequence of 3E10-LC-h6 (SEQ ID NO: 102).

21. The humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, comprising a set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NO: 9), 3E10-VL-CDR2 (SEQ ID NO: 10), 3E10-VL-CDR3 (SEQ ID NO: 11), 3E10-VH-CDR1_D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NO: 4), and 3E10-VH-CDR3 (SEQ ID NO: 5).

22. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1 to 3, further comprising a fragment crystallizable (Fc) region.

23. A composition comprising a covalent or non-covalent complex of (i) the humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1 to 3 and (ii) a polynucleotide.

24. A method for treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1 to 3.

25. A polynucleotide encoding the humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

Images