Humanized 3E10 antibodies, variants and antigen binding fragments thereof

Abstract

The present disclosure provides humanized 3E10 antibodies and antigen binding fragments thereof. Compositions and methods for delivering cargo using the humanized 3E10 antibodies and antigen-binding fragments thereof are also disclosed.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 316,338, filed on March 3, 2022, the disclosure of which is incorporated herein in its entirety. Technical Field

[0003] The present disclosure relates to humanized 3E10 antibodies and antigen-binding fragments thereof, for example, for use in delivering therapeutic cargo into cells.

[0004] Brief Description of Sequence Listing

[0005] This submission is accompanied by a "Sequence Listing XML" (169 KB) containing SEQ ID NOs: 1-161, created on March 1, 2023, which was submitted as an XML file through the USPTO Patent Electronic Submission System in accordance with 37 CFR §§ 1.831 to 1.835. 37 CFR § 1.835(a)(1). Background Art

[0006] In cancer therapy based on polynucleotides, many different strategies have been developed. For example, in various cancer immunotherapies, immunostimulatory polynucleotides such as pattern recognition receptors have been used to excite mediators of proinflammatory cytokines. Gene regulatory polynucleotides (e.g., siRNA, miRNA, ASO, etc.) have been used to silence targeted genes, thereby regulating the signal transduction pathways involved in cancer progression. Polynucleotides encoding therapeutic proteins, such as mRNA or plasmids encoding antigens or cancer immunotherapy proteins, have also been used in treatment. Functional nucleic acids such as aptamers have been similarly used in antibody-based cancer therapies, such as by binding and blocking key tumor targets, such as PD-1. Gene editing polynucleotides have also been used to silence the expression of cancer mediators. For reviewing these various cancer therapies based on polynucleotides. See, for example, Zhou S. et al., Medicine in Drug Discovery, 2020.6: 100023 and Hager et al., Cell. 2020.9 (9): 2061, the contents of which are incorporated herein by reference in their entirety.

[0007] Although these polynucleotide-based therapies have shown some success in preclinical studies, when evaluating the therapeutic effect, the polynucleotide-based therapies have not met expectations in clinical trials. See, for example, Lopes et al., Cancer DNA vaccines: Current preclinical and clinical developments and future perspectives. "Experimental and Clinical Cancer Research (J.Exp.Clin.Cancer Res.)" 2019.38,146; Dome et al., Therapeutic Cancer Vaccination with ex vivoRNA-Transfected Dendritic Cells-An Update. "Pharmaceutics". 2020.12,92, the contents of which are incorporated herein by reference in their entirety. One obstacle is that nucleic acids are not easy to cross cell membranes. In addition, 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), the contents of which are incorporated herein by reference in their entirety.

[0008] The murine anti-DNA antibody 3E10 is known to penetrate cells and localize at least partially to the nucleus of cells. See, e.g., Weisbart RH et al., 1998. J. Autoimmun.; 11:539–546., the contents of which are incorporated herein by reference in their entirety. Thus, it has been shown that 3E10 and its derivatives can act as targeting agents for in vivo delivery of therapeutic agents. However, when administered to humans, especially during chronic administration, murine antibodies are immunogenic. Several techniques for reducing the immunogenicity of non-human antibodies by humanization are known in the art. However, not all humanized antibodies retain the favorable characteristics of their parent non-human antibodies. Summary of the invention

[0009] 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 will facilitate improved methods for delivering therapeutic cargoes such as therapeutic polynucleotides, polypeptides, and chemical agents to targeted cells. Polynucleotide-based therapies, for example, provide a promising approach for targeted diseases because of their versatility in encoding any polypeptide, the availability of highly reproducible manufacturing methods, the ability to simply and precisely adjust polynucleotide sequences, their inexpensive nature, their ability to specifically target and / or edit any gene sequence, etc. However, the delivery of polynucleotide therapeutics to specific tissues in vivo presents many challenges, including rapid degradation of exogenous nucleic acids in vivo 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.

[0010] 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. Thus, as described herein, the present disclosure provides humanized 3E10 antibodies and antigen-binding fragments thereof, pharmaceutical compositions, and methods for using the humanized 3E10 antibodies and antigen-binding fragments thereof, the pharmaceutical compositions to treat various medical conditions.

[0011] In some embodiments, a humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a light chain variable domain (3E10-VL) comprising an amino acid sequence 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).

[0012] In some embodiments, the light chain variable domain (3E10-VL) comprises one or more amino acid residues numbered according to Kabat numbering selected from the group consisting of: 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. In some embodiments, the 3E10-VL comprises a set of CDRs having no more than 6 amino acid substitutions in common relative to a set of CDRs having the following amino acid sequences: 3E10-VL-CDR1 (SEQ ID NO: 9), 3E10-VL-CDR2 (SEQ ID NO: 10), 3E10-VL-CDR3 (SEQ ID NO: 11),.

[0013] 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 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).

[0014] In some embodiments, the heavy chain variable domain (3E10-VH) comprises one or more amino acid residues numbered according to Kabat numbering selected from the following: 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. In some embodiments, the 3E10-VH comprises a set of CDRs having no more than 6 amino acid substitutions in common relative to a set of CDRs having the following amino acid sequences: 3E10-VH-CDR1_D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NO: 4), and 3E10-VH-CDR3 (SEQ ID NO: 5).

[0015] In some embodiments, a humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a heavy chain CDR1 having an aspartic acid at position 31 numbered according to Kabat numbering.

[0016] In some embodiments, the humanized 3E10 antibodies or antigen-binding fragments thereof described herein include a lysine numbered according to Kabat numbering at position 72 in 3E10-VL (which supports nucleic acid binding affinity). Mutation of this amino acid residue to tyrosine abolishes high affinity binding to DNA substrates. In addition, arginine and lysine at positions 37 and 38, respectively, in 3E10-VH appear 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.

[0017] In some embodiments, the present disclosure provides compositions and methods for improving nucleic acid delivery into cells, for example, the compositions and methods do not rely on a separate delivery vehicle (such as liposomes, viral vectors, etc.). In some embodiments, the present disclosure provides compositions and methods for improving nucleic acid delivery into cells. In some embodiments, the composition comprises (i) a humanized 3E10 antibody or its antigen-binding fragment with 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) non-covalently associate to form a complex. In some embodiments, elements (i) and (ii) covalently associate. In various embodiments, nucleic acids include DNA (single-stranded or double-stranded), RNA, PNA or other modified nucleic acids.

[0018] In some embodiments, the present disclosure provides a humanized 3E10 antibody or antigen-binding fragment thereof covalently conjugated 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 therapeutic agents to cells, for example, without the need for a separate delivery vehicle such as liposomes, viral vectors, etc. In some embodiments, the conjugate is a fusion protein in which the polypeptide of the 3E10 antibody or antigen-binding fragment thereof and the therapeutic polypeptide are encoded and translated from the same open reading frame.

[0019] Also provided are methods of delivering cargo to cells (e.g., nucleic acids, chemicals, or therapeutic proteins or polypeptides) by contacting the cells with an effective amount of the complexes and complexes described herein. The contacting can occur in vitro, ex vivo, or in vivo. In some embodiments, an effective amount of cells treated ex vivo are administered to a subject in need thereof, for example, in an effective amount for treating one or more symptoms of a disease or disorder.

[0020] In some embodiments, the contacting occurs in vivo following administration to a subject in need thereof. The subject may suffer from a disease or condition, such as a genetic disorder or cancer. An effective amount of the composition may be administered to the subject, for example by injection or infusion, to reduce one or more symptoms of the disease or condition in the subject.

[0021] Applications of the compositions and methods are also provided and include, but are not limited to, gene therapy and T cell or CAR T cell manufacturing, formation and / or therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 (SEQ ID NO: 1-12) show the amino acid sequences of the parent 3E10 monoclonal antibodies.

[0023] Figure 2A (SEQ ID NO: 13-25), 2B and 2C show the D31N variant of the 3E10 monoclonal antibody according to some embodiments of the present disclosure ( Figure 2A (SEQ ID NO: 13-25)), other CDR variants ( Figure 2B ) and additionally contemplated CDR variants ( Figure 2C )'s amino acid sequence.

[0024] Figure 3 Exemplary charge-conservative CDR variants of the 3E10 monoclonal antibody according to various embodiments of the present disclosure are shown.

[0025] Figure 4 Exemplary CDR variants containing 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 present disclosure are shown.

[0026] Figure 5 Shown are the amino acid sequences of humanized 3E10 variable heavy (3E10-VH) domains according to various embodiments of the present disclosure.

[0027] Figure 6 The amino acid sequence of the mature humanized 3E10 heavy chain (3E10-HC) lacking the signal peptide according to various embodiments of the present disclosure is shown.

[0028] Figure 7 The amino acid sequence of the humanized 3E10 heavy chain (3E10-HC) according to various embodiments of the present disclosure is shown.

[0029] Figure 8 Shown is the amino acid sequence of the humanized 3E10 light (3E10-VL) domain according to various embodiments of the present disclosure.

[0030] Fig. 9 The amino acid sequence of the mature humanized 3E10 light chain (3E10-LC) lacking the signal peptide according to various embodiments of the present disclosure is shown.

[0031] Fig.10 The amino acid sequence of the humanized 3E10 light chain (3E10-LC) according to various embodiments of the present disclosure is shown.

[0032] Fig.11A and 11B Electrostatic surface potential rendering of the molecular model of the 3E10-scFv construct is shown, revealing the putative nucleic acid binding pocket (NAB1). Fig.11A Also shown are the predicted structural and electrostatic potential changes induced by the amino acid substitution at residue 31 of HC CDR1. Fig. 11Bis an illustration of the molecular modeling of 3E10-scFv (Pymol), in which the NAB1 amino acid residues are highlighted by dots.

[0033] Fig. 12A , 12B , 12C, 12D and 12E collectively show the results of nucleic acid binding characterization of various humanized 3E10 constructs as described in Example 1 according to some embodiments of the present disclosure.

[0034] Fig.13 Shown are histograms showing a 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 indicated in Example 3.

[0035] Fig.14 Shown are histograms showing a 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 indicated in Example 3.

[0036] Fig.15 Shown are histograms showing a 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 indicated in Example 4.

[0037] Fig.16 Shown are histograms showing a 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 indicated in Example 3.

[0038] Fig.17 Shown are histograms collectively showing humanized 3E10 antibody uptake (+ / -) dipyridamole in tumor, liver, kidney, spleen, quadriceps, and gastrocnemius muscles of tumor-bearing mice (CT-26 colorectal cancer model).

[0039] Fig.18 Demonstrated is the experimental protocol used to measure the biodistribution of the 3E10-D31N IgG4 variant in a murine model of pancreatic ductal adenocarcinoma (PDAC).

[0040] Fig.19A , 19B , 19C, 19D and 19E show the binding kinetics and affinity measurements of 3E10-D31N and 3E10-D31N IgG4 Fc variants, as indicated in Example 8.

[0041] Fig. 20A Chimeric 3E10-D31N delivery of GFP mRNA in the MDA-MB-231 murine model was demonstrated.

[0042] Fig. 20B Comparative studies testing chimeric 3E10-D31N delivery of GFP mRNA and humanized 3E10 antibody construct (V66) delivery of GFP mRNA in a KPC syngeneic tumor model are presented.

[0043] Fig.21 Tumor and normal tissue expression of targeted functional delivery of GFP mRNA payload was demonstrated.

[0044] Fig. 22 Comparability of chimeric 3E10 D31N and humanized 3E10 antibody (V66) in the B16 tumor model measuring tumor volume several days after implantation of antibody:3p-hpRNA complexes was demonstrated.

[0045] Fig.23A , 23B , 23C, 23D, 23E, 23F, 23G, 23H, 23I, 23J, 23K, 23L, 23M, 23N, 23O, 23P and 23Q collectively demonstrate the serum and tissue pharmacokinetic profiles of the humanized 3E10 (D31N) antibody V66 from a single-dose, dose-escalation, pharmacokinetic study in C57Bl / 6 mice, as described in Example 12.

[0046] Fig.24A , 24B , 24C and 24D collectively provide summary analysis and statistics of serum and tissue pharmacokinetic studies of humanized 3E10(D31) antibody V66, as described in Example 12. DETAILED DESCRIPTION

[0047] In various aspects and embodiments, the present disclosure provides humanized 3E10 antibodies and antigen-binding fragments thereof, and methods for delivering cargo (e.g., polynucleotides, polypeptides, or chemicals) into cells using the humanized 3E10 antibodies and antigen-binding fragments thereof. The humanized 3E10 antibodies and antigen-binding fragments thereof described herein can penetrate cells, thereby facilitating the delivery of cargo (e.g., polynucleotides, polypeptides, or chemicals) across the plasma membrane into the cytoplasm and / or nucleus without the need for a separate delivery vehicle. Advantageously, because the 3E10 antibodies and antigen-binding fragments described herein have been humanized, they are less immunogenic when administered to humans than murine and chimeric 3E10 parent antibodies. Further, the humanized 3E10 antibodies and antigen-binding fragments described herein retain key 3E10 properties such as sequence nonspecific nucleic acid binding and antigen-mediated cell penetration. In addition, as reported in the Examples, various combinations of humanized 3E10 variable heavy domains and variable light domains have different nucleic acid binding affinities, thereby allowing for better control of nucleic acid binding and in vivo release.

[0048] definition

[0049] The terms used in the present disclosure are only used for the purpose of describing specific embodiments and are not intended to limit the present invention. As used in the specification of the present invention and the appended claims, the singular forms "a / an" and "the" are intended to include plural forms as well, unless the context clearly indicates. It should also be understood that, as used herein, the term "and / or" refers to and covers any and all possible combinations of one or more items in the relevant enumeration items. Unless the context otherwise requires, it should be further understood that the term "comprising", "including" or any variant thereof specifies the existence of stated features, integers, steps, operations, elements and / or components when used in this specification, but does not exclude the existence or addition of one or more other features, integers, steps, operations, elements, components and / or their groups. In addition, in the case of using the term "including / include", "having / has / with" or its variant in the specific embodiments and / or claims, such terms are intended to be inclusive in a manner similar to the term "including".

[0050] Recitation of ranges of values ​​herein are 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 into the specification as if it were individually recited herein.

[0051] Use of the term "about" is intended to describe values ​​that are within a range of approximately + / - 10% above or below the stated value.

[0052] As used herein, the term "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0053] Unless the context requires otherwise, the terms "comprises," "includes," or any variations 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. In addition, where the terms "including / include," "having / has / with," or variations thereof are used in the detailed description and / or claims, such terms are intended to be inclusive in a manner similar to the term "comprising."

[0054] Recitation of ranges of values ​​herein are 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 into the specification as if it were individually recited herein.

[0055] As used herein, the term "subject" means any individual that is a target for administration. The subject may be a vertebrate, such as a mammal. Thus, the subject may be a human. The term does not indicate a specific age or sex.

[0056] As used herein, the term "pharmaceutically effective amount" means that the amount of the composition used is sufficient to improve one or more causes or symptoms of a disease or condition. Such improvements only require reduction or modification, and do not necessarily require elimination. The exact dosage will vary according to a variety of factors, such as subject-dependent variables (e.g., age, immune system health, etc.), the disease or condition being treated, and the route of administration and the pharmacokinetics of the administered agent.

[0057] As used herein, the term "carrier" or "excipient" refers to an organic or inorganic component, natural or synthetic, inactive ingredient in a formulation, with which one or more active ingredients are combined. As is well known to those skilled in the art, a carrier or excipient will naturally be selected to minimize degradation of the active ingredient or to minimize adverse side effects in a subject.

[0058] As used herein, the term "treatment" refers to the medical management of a patient, wherein the disease, pathological condition or illness is cured, improved, stabilized or prevented. This term includes active treatment, i.e., treatment specifically for the improvement of a disease, pathological condition or illness, and also includes etiological treatment, i.e., treatment for the removal of the cause of the disease, pathological condition or illness. In addition, this term includes palliative treatment, i.e., treatment designed to relieve symptoms rather than cure a disease, pathological condition or illness; preventive treatment, i.e., treatment intended to minimize or partially or completely inhibit the development of a disease, pathological condition or illness; and supportive treatment, i.e., treatment for another specific therapy for supplementing the improvement of a disease, pathological condition or illness. As used herein, the term "inhibit" or "reduce" means to reduce activity, response, condition, disease or other biological parameters. This may include, but is not limited to, complete ablation of activity, response, condition or disease. This may also include, for example, statistically significant reduction in activity, response, condition or disease compared to natural or control levels.

[0059] In the present disclosure, the term "CNS cancer" or "cancer of the central nervous system" refers to an abnormal growth of cells from any tissue of the central nervous system, including the brain, spinal cord, meninges, or hematopoietic tissue of the primary CNS of a subject. Non-limiting examples of CNS cancers include neuroepithelial cancers (such as gliomas, mature neuronal cancers, primitive neuroectodermal tumors, and primitive brain cancers), meningeal cancers, and primary CNS hematopoietic cancers.

[0060] Antibodies and their variants

[0061] As used herein, "antigen binding domain" or "ABD" means a set of six complementary determining regions (CDRs) that specifically bind to a target antigen as discussed herein when present as part of a polypeptide sequence or sequence. As known in the art, these CDRs are typically present as a first set of variable heavy CDRs (vhCDR or VHCDR) and a second set of variable light CDRs (vlCDR or VLCDR), each of which includes three CDRs: vhCDR1, vhCDR2, vhCDR3 of the heavy chain and light vlCDR1, vlCDR2 and vlCDR3. The CDRs are present in the variable heavy domain and the variable light domain, respectively, and together form the Fv region. Various CDR numbering schemes are known, as outlined in the following table and related discussions. Therefore, in some cases, the six CDRs of the antigen binding domain are derived from the variable heavy domain and the variable light domain. In the "Fab" format, a set of 6 CDRs are derived from two different polypeptide sequences, 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), wherein the C-terminus of the vh domain is connected to the N-terminus of the CH1 domain of the heavy chain and the C-terminus of the vl domain is connected to the N-terminus of the constant light domain (and thus forms a light chain). In the scFv format, the vh domain and the vl domain are usually covalently linked to a single polypeptide sequence by using a linker as outlined herein ("scFv linker"), which can be (starting from the N-terminus) vh-linker-vl or vl-linker-vh (including optional domain linkers on each side, depending on the format used). Typically, the C-terminus of the scFv domain is connected to the N-terminus of the hinge in the second monomer.

[0062] As will be appreciated by those skilled in the art, the precise numbering and placement of CDRs may be different in different numbering systems. However, it should be understood that the disclosure of variable heavy and / or variable light sequences includes the disclosure of related (intrinsic) CDRs. Therefore, the disclosure of each variable heavy region is the disclosure of vhCDRs (e.g., vhCDR1, vhCDR2, and vhCDR3), and the disclosure of each variable light region is the disclosure of vlCDRs (e.g., vlCDR1, vlCDR2, and vlCDR3). Useful comparisons of CDR numbering are as follows, see Lafranc et al., Development and Comparative Immunology (Dev. Comp. Immunol.) 2003.27 (1): 55-77.

[0063] Comparison of CDR numbers

[0064]

[0065] For all amino acid positions related to antibodies discussed in this disclosure, the amino acid position numbering is according to the EU index. The EU index or EU index as in Kabat or EU numbering scheme refers to the EU antibody numbering. Kabat et al. collected a number of primary sequences of the variable regions of heavy and light chains. Based on the degree of conservation of the sequence, they classified each primary sequence into CDR and framework and made a list thereof. See Sequences of Immunological Interest ( SEQUENCES OF IMMUNOLOGICAL INTEREST )》, 5th edition, NIH publication number 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 entirety. Modifications may be additions, deletions or substitutions.

[0066] As used herein, the term "antibody variant" or "variant antibody" refers to an antibody that differs from a parent antibody due to at least one amino acid modification, as used herein, "IgG variant" or "variant IgG" means an antibody that differs from a parent IgG (again, in many cases, from a human IgG sequence) due to at least one amino acid modification, and as used herein, "immunoglobulin variant" or "variant immunoglobulin" means an immunoglobulin sequence that differs from a parent immunoglobulin sequence due to at least one amino acid modification. As used herein, "Fc variant" or "variant Fc" means a protein comprising amino acid modifications in an Fc domain as compared to the Fc domain of human IgG1, IgG2, IgG3 or IgG4, as further described herein.

[0067] In some embodiments, the parent polypeptide (e.g., Fc parent polypeptide) is a human wild-type sequence, such as a heavy constant domain or Fc region from IgG1, IgG2, IgG3 or IgG4, but a human sequence with a variant may also serve as a "parent polypeptide", for example, the IgG1 / 2 hybrid of U.S. Publication 2006 / 0134105 may be employed, the U.S. Publication being incorporated herein by reference in its entirety. The protein variant sequence herein will 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.

[0068] In some embodiments, the protein variant sequences herein have 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 the parent protein sequence.

[0069] As used herein, "isotype" refers to any subclass of immunoglobulins defined by the chemical and antigenic properties of their constant regions. It should be understood that therapeutic antibodies may also include hybrids of isotypes and / or subclasses.

[0070] As used herein, "Fab" or "Fab region" refers to a polypeptide that includes the VH, CH1, VL, and CL immunoglobulin domains, typically on two different polypeptide chains (e.g., VH-CH1 on one chain and VL-CL on another chain). Fab can refer to this region in isolation or in the context of an antibody of the present disclosure. In the context of Fab, in addition to the CH1 and CL domains, Fab also includes an Fv region.

[0071] As used herein, "Fv" or "Fv fragment" or "Fv region" refers to a polypeptide comprising the VL domain and the VH domain of the ABD. The Fv region can be formatted as both a Fab (as discussed above, typically two different polypeptides that also contain a constant region as outlined above) and a scFv, in which the vl and vh domains are combined (typically with a linker as discussed herein) to form an scFv.

[0072] As used herein, "single-chain Fv" or "scFv" refers to a variable heavy domain that is covalently linked to a variable light domain using an scFv linker as discussed herein to form an scFv or scFv domain. The scFv domain can be in either orientation (vh-linker-vl or vl-linker-vh) from the N-terminus to the C-terminus. In the present disclosure, specifically outlined in the figures, the order of the vh and vl domains is indicated by name, such as H.X_L.Y, which is vh-linker-vl from the N-terminus to the C-terminus, and L.Y_H.X is vl-linker-vh.

[0073] As used herein, "Fc" or "Fc region" or "Fc domain" refers to a polypeptide comprising the CH2-CH3 domain of an IgG molecule and, in some cases, a hinge. When human IgG1 is EU numbered, the CH2-CH3 domain includes amino acids 231 to 447, and the hinge is 216 to 230. Therefore, the definition of "Fc domain" includes amino acids 231-447 (CH2-CH3) or 216-447 (hinge-CH2-CH3) or fragments thereof. In this context, an "Fc fragment" may contain fewer amino acids from either or both of the N-terminus and the C-terminus but still remain able to form a dimer with another Fc domain or Fc fragment, as can be detected using standard methods generally based on size (e.g., non-denaturing chromatography, size exclusion chromatography, etc.). Human IgG Fc domains are particularly useful in the present disclosure, and may be Fc domains from human IgG1, IgG2, or IgG4.

[0074] As used herein, a "variant Fc domain" contains amino acid modifications as compared to a parent Fc domain. Thus, a "variant human IgG1 Fc domain" is a variant Fc domain that contains amino acid modifications (typically amino acid substitutions, but in the case of ablation variants, amino acid deletions) as compared to a human IgG1 Fc domain. Typically, a variant Fc domain has at least about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, or about 99% identity to the corresponding parent human IgG Fc domain (using an identity algorithm discussed below, one embodiment of which utilizes a BLAST algorithm as known in the art, using default parameters). Alternatively, the variant Fc domain may 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. Additionally, as discussed herein, the variant Fc domains herein still retain the ability to form a dimer with another Fc domain as measured using known techniques described herein, such as non-denaturing gel electrophoresis.

[0075] As used herein, the term "heavy chain constant region" refers to the CH1-hinge-CH2-CH3 portion of an antibody (or a fragment thereof), excluding the variable heavy domain; in the EU numbering of human IgG1, this is amino acids 118-447. A "heavy chain constant region fragment" herein means a heavy chain constant region that contains fewer amino acids from either or both of the N-terminus and the C-terminus but still retains the ability to form a dimer with another heavy chain constant region.

[0076] As used herein, the term "variable region" or "variable domain" refers to a region of an immunoglobulin that includes one or more Ig domains that are substantially encoded by any Vκ, Vλ and / or VH genes that constitute κ, λ and heavy chain immunoglobulin gene loci, respectively, and contains CDRs that confer antigen specificity. Therefore, a "variable heavy domain" is paired with a "variable light 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 of variable heavy domains and vlCDR1, vlCDR2 and vlCDR3 of variable light domains) and four framework regions (FRs) arranged in the following order from the amino terminus to the carboxyl terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0077] As used herein, the term "full-length heavy chain" refers to the entire heavy chain of an antibody having the structure S-VH-CH1-hinge-CH2-CH3, including the signal peptide (S).

[0078] As used herein, the term "mature heavy chain" refers to a portion of an antibody's heavy chain having the structure VH-CH1-hinge-CH2-CH3, excluding the signal peptide.

[0079] As used herein, the term "full-length light chain" refers to the entire light chain of an antibody having the structure S-VL-CL, including the signal peptide (S).

[0080] As used herein, the term "mature light chain" refers to a portion of the light chain of an antibody having the structure S-VL-CL, excluding the signal peptide.

[0081] 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 in a different aligned IgG isotype. For example, because IgG1 includes tyrosine and IgG2 includes phenylalanine at EU position 296, a F296Y substitution of IgG2 is considered an IgG subclass modification.

[0082] As used herein, the term "non-naturally occurring modification" refers to an amino acid modification that is not an isotype. For example, because human IgG does not include a serine at position 434, the substitution 434S of IgG1, IgG2, IgG3 or IgG4 (or a hybrid thereof) is considered a non-naturally occurring modification.

[0083] Antibodies and antigen-binding fragments thereof disclosed herein are recombinant antibodies that have been engineered to have various properties described herein and are typically separated prior to use. As used herein, the term "isolated" when used to describe various polypeptides described herein refers to a polypeptide that has been identified and separated and / or recovered from a cell or cell culture expressing the polypeptide. Typically, an isolated polypeptide will be prepared by at least one purification step. An "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigenic specificities. "Recombinant" means that antibodies are produced using recombinant nucleic acid technology in exogenous host cells, and antibodies may also be isolated.

[0084] As used herein, "3E10 antibody" refers to an antibody having a set of heavy chain CDRs (VHCDR1, VH CDR2, and VH CDR3) identified according to the Kabat system (including amino acid sequences that differ from SEQ ID NOs: 58, 59, and 60, respectively, by no more than two amino acids), a set of light chain CDRs (VL CDR1, VL CDR2, and VL CRD3) (including amino acid sequences that differ from SEQ ID NOs: 61, 62, and 63, respectively, by no more than two amino acids), and binding to nucleic acids. As described herein, the 3E10 antigen is a polynucleotide.

[0085] As used herein, the term "cell penetrating" refers to an antibody or antigen-binding fragment thereof that can penetrate cells (e.g., mammalian cells) without the aid of an exogenous transport vehicle (e.g., liposomes or conjugated cell-penetrating peptides). With respect to the 3E10 antibody and its antigen-binding fragment, the cell-penetrating antibody or its antigen-binding fragment can penetrate cells expressing the ENT2 receptor on its cell surface in the presence of nucleic acids, e.g., non-covalently bind and / or conjugate with the 3E10 antibody or its antigen-binding fragment, thereby resulting in internalization of the 3E10 antibody and its antigen-binding fragment. In some embodiments, the cell-penetrating 3E10 antibody or its antigen-binding fragment is conjugated to a functional molecule such as a chemical agent, a polynucleotide, or a polypeptide.

[0086] As used herein, "variant protein" or "protein variant" or "variant" means a protein that is different from a parent protein due to at least one amino acid modification. Compared to the parent protein, the protein variant has at least one amino acid modification, but not so much that the variant protein will not be compared with the parent protein using the alignment program described below. Typically, using the alignment program described below, such as BLAST, the variant protein (variant Fc domains summarized herein, etc.) is 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% identical to the parent protein. Although amino acid sequence modifications to the 3E10 antibody or antigen-binding fragment thereof as described herein can produce proteins and / or polypeptides referred to as variant 3E10 antibodies or antigen-binding fragments thereof, as long as the variant maintains the CDR sequences and cell penetrating activity requirements of the 3E10 antibody or antigen-binding fragment thereof, such variants still fall within the classification of the 3E10 antibody or antigen-binding fragment thereof.

[0087] Sequence identity between two similar sequences (e.g., antibody variable domains) can be measured by algorithms such as Smith, TF and Waterman, MS (1981) "Comparison Of Biosequences," Adv. Appl. Math. 2:482 [local homology algorithm]; Needleman, SB and 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 [homologous alignment algorithm], Pearson, WR and Lipman, DJ (1988) "Improved Tools For Biological Sequence Comparison." or Altschul, SF et al., (1990) "Basic Local Alignment Search Tool", Biochem. J "Journal of Molecular Biology" 215:403-10, "BLAST" algorithm, see the webpage at URL blast.ncbi.nlm.nih.gov / Blast.cgi. When using any of the above algorithms, the default parameters (for window length, gap penalty, etc.) are used. Unless otherwise stated, the BLAST algorithm is used to determine sequence identity using the default parameters.

[0088] 3E10 Antibody, Variants and Fragments Thereof

[0089] In embodiments, the present disclosure relates to humanized 3E10 antibodies and antigen-binding fragments thereof, and their use for delivering therapeutic agents to treat various diseases. As discussed herein, humanized 3E10 antibodies useful in the methods and compositions of the present disclosure can incorporate an array of amino acid substitutions in any portion of the antibody. Further, antigen-binding fragments of humanized 3E10 antibodies can take many forms, including but not limited to the example formats described herein.

[0090] The amino acid sequences of the 3E10 monoclonal antibody and its binding fragments are known in the art. Example sequences of the 3E10 heavy and light chains are provided below and are shown in the various figures. Where present, single underlining indicates CDR regions identified according to the Kabat system, italics indicate variable domains, and double underlining indicates signal peptides. The murine 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 murine 3E10 and the variable regions and CDRs are described in Figure 1 (SEQ ID NO: 1-12).

[0091] Amino acid variants of the 3E10 antibody are also known in the art, for example, as described 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 nucleic acid binding and the ability of the antibody to penetrate nuclear. Relative to the 'wild-type' murine antibody, substitution of the 'wild-type' (e.g., relative to the original murine antibody) aspartic acid with asparagine ('D31N' mutation) improves nucleic acid binding and nuclear penetration of the 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, other amino acids are substituted at position 31 in the humanized 3E10 antibodies and binding fragments thereof disclosed herein. For example, Fig.11A As shown in the modeling in , D31R or D31K substitutions are incorporated into some embodiments of the present disclosure.

[0092] Other 3E10 light chain sequences are known in the art. See, e.g., Zack et al., Journal of Immunology, 15; 154(4): 1987-94 (1995); GenBank: L16981.1 - Mouse Ig rearranged L chain gene, partial cds; GenBank: AAA65681.1 - Immunoglobulin light chain, partial [Mus musculus])

[0093] Traditional antibody structural units typically include tetramers. Each tetramer is typically composed of two identical pairs of polypeptide chains, each pair having a "light" chain (typically, a molecular weight of about 25 kDa) and a "heavy" chain (typically, a molecular weight of about 50-70 kDa). Human light chains are classified as κ and λ light chains. The present disclosure relates to antibodies typically based on the IgG class, which has several subclasses, including but not limited to IgG1, IgG2, IgG3 and IgG4. Typically, IgG1, IgG2 and IgG4 are used more frequently than IgG3. It should be noted that IgG1 has different allotypes with polymorphisms at 356 (D or E) and 358 (L or M).

[0094] The light chain generally includes two domains: a variable light domain (containing the light chain CDRs and forming the Fv region together with the variable heavy domain), and a constant light chain region (usually referred to as CL or Ck). The heavy chain includes a variable heavy domain and a constant domain comprising a CH1-optional hinge-Fc domain including CH2-CH3.

[0095] The hypervariable region of an antibody generally encompasses amino acid residues around about 24-34 (LCDR1; "L" for light chain), 50-56 (LCDR2), and 89-97 (LCDR3) from the light chain variable region and about 31-35B (HCDR1; "H" for heavy chain), 50-65 (HCDR2), and 95-102 (HCDR3) from the heavy chain variable region; Kabat et al., Sequences of Proteins of Immunological Interest ( SEQUENCESOFPROTEINS OF IMMUNOLOGICALINTEREST ), 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991) and / or those residues that form a hypervariable loop (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, J. Mol. Biol. 1987. 196: 901-917. Specific CDRs that can be used in the compositions and methods described herein are described below.

[0096] As will be appreciated by those skilled in the art, the precise numbering and placement of CDRs may be different in different numbering systems. However, it should be understood that the disclosure of variable heavy and / or variable light sequences includes the disclosure of related (intrinsic) CDRs. Therefore, the disclosure of each variable heavy region is the disclosure of vhCDRs (e.g., vhCDR1, vhCDR2, and vhCDR3), and the disclosure of each variable light region is the disclosure of vlCDRs (e.g., vlCDR1, vlCDR2, and vlCDR3). Useful comparisons of CDR numbering are described in Lafranc et al., Development and Comparative Immunology 2003.27 (1): 55-77 (2003).

[0097] Throughout this disclosure, the Kabat numbering system is generally used when referring to residues in the variable domain (roughly, residues 1-107 in the light chain variable region and residues 1-113 in the heavy chain variable region), and the EU numbering system is used for the Fc region (eg, Kabat et al., supra (1991)).

[0098] The present disclosure provides a large number of different CDR combinations. In this case, a "complete CDR set" includes three variable light and three variable heavy CDRs, such as vlCDR1, vlCDR2, vlCDR3, vhCDR1, vhCDR2 and vhCDR3. These can be a part of a larger variable light domain or a variable heavy domain, respectively. In addition, as more fully outlined herein, when using heavy and light chains (for example, when using Fab), variable heavy domains and variable light domains can be on separate polypeptide chains, or on a single polypeptide chain in the case of scFv sequences. CDR contributes to the formation of antigen binding or more specifically the epitope binding site of an antibody. "Epitope" refers to the determinant cluster that interacts with the specific antigen binding site in the variable region of an antibody molecule known as a paratope.

[0099] Epi-position is the group (grouping) of such molecules as nucleic acid, amino acid or sugar side chain and usually has concrete structural characteristics and concrete charge characteristics.Single antigen can have more than one epi-position.Antibody described herein is combined with nucleic acid epi-position in a partial sequence independent manner.That is to say, although antibody described herein is combined with some polynucleotide structures and sequences with avidity greater than other nucleic acid structures and sequences, it has some general affinities to polynucleotide.

[0100] The "Fc domain" of the heavy chain comprises a -CH2-CH3 domain and optionally a hinge domain (-H-CH2-CH3). For IgG, the Fc domain includes the immunoglobulin domains CH2 and CH3 (Cy2 and Cy3) and the lower hinge region between CH1 (Cy1) and CH2 (Cy2). Although the boundaries of the Fc region can vary, the human IgG heavy chain Fc region is generally defined as comprising residues C226 or P230 at its carboxyl terminus, wherein numbering is performed according to the EU index as in Kabat. Therefore, in the context of IgG, the "CH" domain is as follows: "CH1" refers to positions 118-215 according to the EU index as in Kabat. "Hinge" refers to positions 216-230 according to the EU index as in Kabat. "CH2" refers to positions 231-340 according to the EU index as in Kabat, and "CH3" refers to positions 341-447 according to the EU index as in Kabat. Thus, an "Fc domain" comprises a -CH2-CH3 domain and optionally a hinge domain (hinge-CH2-CH3). In the embodiments herein, when a scFv is linked to an Fc domain, typically the C-terminus of the scFv construct is linked to all or part of the hinge of the Fc domain; for example, it is typically linked to the sequence EPKS that is the beginning of the hinge. In some embodiments, as described more fully below, the Fc region is amino acid modified, for example to alter binding to one or more FcyR receptors or FcRn receptors, and heterodimer formation and purification are achieved as outlined herein.

[0101] The other part of heavy chain is hinge region. "Hinge" or "hinge region" or "antibody hinge region" or "hinge domain" herein means a flexible polypeptide comprising the amino acid between the first constant domain and the second constant domain of an antibody. Structurally, the IgG CH1 domain ends at EU position 215, and the IgG CH2 domain begins at residue EU position 231. Therefore, for IgG, antibody hinge is defined herein as comprising position 216 (E216 in IgG1) to 230 (p230 in IgG1), wherein numbering is performed according to the EU index as in Kabat. In some cases, "hinge fragment" is used, which contains less amino acid at either or both of the N-terminal and C-terminal ends of the hinge domain.

[0102] scFv includes variable heavy chain, scFv linker and variable light domain.In most constructs and sequences summarized herein, the C-terminus of the variable heavy chain is connected to the N-terminus of the scFv linker, and the C-terminus of the scFv linker is connected to the N-terminus of the variable light chain (N-vh-linker-vl-C), but can be switched (N-vl-linker-vh-C).

[0103] Therefore, the present disclosure relates to different antibody domains. As described herein and known in the art, the heterodimeric antibodies described in certain embodiments of the present disclosure include different domains in the heavy chain and light chain, and the domains may also overlap. These domains include but are not limited to Fc domains, CH1 domains, CH2 domains, CH3 domains, hinge domains, heavy constant domains (CH1-hinge-Fc domains or CH1-hinge-CH2-CH3), variable heavy domains, variable light domains, light constant domains, Fab domains, and scFv domains.

[0104] In certain embodiments, the antibodies of the present disclosure include a heavy chain variable region from a specific germline heavy chain immunoglobulin gene and / or a light chain variable region from a specific germline light chain immunoglobulin gene. For example, such antibodies may include or be composed of a human antibody, the human antibody including a heavy chain variable region or a light chain variable region as a "product" or "derived from" a specific germline sequence, such as a 3E10 antibody. Human antibodies that are "products" or "derived from" human germline immunoglobulin sequences can be identified as such: by comparing the amino acid sequence of the human antibody with the amino acid sequence of the human germline immunoglobulin, and selecting the human germline immunoglobulin sequence that is closest (i.e., the greatest % identity) to the sequence of the human antibody in terms of sequence (using the methods outlined herein). Human antibodies that are "products" or "derived from" specific human germline immunoglobulin sequences may contain amino acid differences compared to the germline sequences due to, for example, naturally occurring somatic mutations or intentionally introduced site-directed mutations. However, humanized antibodies are usually at least 90% identical in amino acid sequence to the amino acid sequence encoded by human germline immunoglobulin genes, and when compared with the germline immunoglobulin amino acid sequences of other species (e.g., murine germline sequences), contain amino acid residues that identify the antibody as being derived from human sequences. In some cases, humanized antibodies can be at least 95%, 96%, 97%, 98% or 99% identical in amino acid sequence to the amino acid sequence encoded by germline immunoglobulin genes, or even at least 96%, 97%, 98% or 99%. Typically, humanized antibodies derived from specific human germline sequences will show no more than 10-20 amino acid differences with the amino acid sequence encoded by human germline immunoglobulin genes. In some cases, humanized antibodies can show no more than 5, or even no more than 4, 3, 2 or 1 amino acid differences with the amino acid sequence encoded by germline immunoglobulin genes.

[0105] In one embodiment, the parent antibody has been affinity matured, as known in the art. Humanization and affinity maturation can be performed using a structure-based approach, such as described in U.S. Application 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 the methods described in the following references: Wu et al., 1999, Journal of Mol. Biol. 294: 151-162; Baca et al., 1997, Journal of Biol. Chem. 272(16): 10678-10684; Rosok et al., 1996, Journal of 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 the transplantation of only a portion of the CDRs, including but not limited to the methods described in: U.S. Application No. 09 / 810,510; Tan et al., 2002, Journal of Immunology 169: 1119-1125; De Pascalis et al., 2002, Journal of Immunology 169: 3076-3084, the contents of which are incorporated herein by reference in their entirety.

[0106] In some embodiments, the present disclosure relates to the use of antigen binding domains (ABDs) that bind to nucleic acids, and in particular to therapeutic polynucleotides derived from the 3E10 antibody for treating a disease (e.g., cancer). The amino acid sequences of the heavy and light chains of the parent 3E10 antibody are Figure 1 (SEQ ID NO: 1-12). Thus, in some embodiments, the compositions described herein comprise the 3E10 antibody or an antigen-binding fragment thereof.

[0107] As used herein, "antigen-binding fragments" of the humanized 3E10 antibody include but are not limited to fragments, variants and fusion proteins with nucleic acid binding properties, such as scFv, di-scFv, tri-scFv and other single-chain variable fragments.

[0108] The humanized 3E10 antibody or its antigen-binding fragment can be transported into the cytoplasm and / or nucleus of the cell without the aid of a carrier or conjugate. For example, U.S. Pat. Nos. 4,812,397 and 7,189,396 to Richard Weisbart disclose monoclonal antibody 3E10 and its active fragments that are transported into the nucleus of mammalian cells in vivo without cytotoxic effects.

[0109] 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), WO 2020 / 047344, and WO 2020 / 047353, each of which is incorporated herein by reference in its entirety.

[0110] Humanized 3E10 antibodies and antigen-binding fragments thereof that can be used in the compositions and methods include any class of intact immunoglobulins (i.e., intact antibodies), fragments thereof, and synthetic proteins that contain at least nucleic acid binding and ENT2-mediated cellular internalization as hallmarks of the 3E10 antibody. Antigen binding activity is typically concentrated in three segments of both the light chain variable domain and the heavy chain variable domain, known as the complementarity determining regions (CDRs) or hypervariable regions. The more highly conserved portions of the variable domains are known as the framework (FR). The variable domains of native heavy and light chains each include: four FR regions that primarily adopt a β-sheet configuration connected by three CDRs, which form loops connecting the β-sheet structure and in some cases form a portion of the β-sheet structure. The CDRs in each chain are held together closely by the FR regions and contribute to the formation of the antigen binding site of the antibody together with the CDRs from the other chain.

[0111] Humanized 3E10 antibody and antigen-binding fragments thereof

[0112] Generally, humanized antibodies are sequences of parent antibodies from non-human species that are modified to increase the overall similarity of parent antibodies to human antibodies, while retaining the result of the method for the antigen-binding activity of parent antibodies. Generally, the method is related to identifying human antibodies, sometimes referred to as scaffold antibodies, and then (i) replaces the amino acid in the parent (non-human) antibody with the equivalent amino acid from the scaffold (human) antibody, for example, the framework amino acid that has little effect on antigen binding, or (ii) replaces the amino acid in the scaffold (human) antibody with the equivalent amino acid from the parent (non-human) antibody, for example, the CDR and other amino acids that have a significant effect on antigen binding. Various methods for humanization are known in the art, including humanization based on framework homology, germline humanization, humanization based on complementary determining region (CDR) homology and specificity determining residue (SDR) grafting. For a review of these methods, see, for example, Safdari Y. et al., Biotechnology and Genetic Engineering Reviews, 29: 2, 175-86 (2013).

[0113] 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 Figure 5 10 (heavy chain variable region), 6 (heavy chain without signal sequence), 7 (heavy chain with signal peptide), 8 (light chain variable region), 9 (light chain without signal sequence) and 10 (light chain with signal peptide). These variable light domains and variable heavy domains can be combined in any of the 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 prepared, all of which were bound to nucleic acids. Further, when human leukemia cells were exposed to complexes formed between these antibodies and RIG-I agonist polynucleotides, all of the complexes were able to produce a type I IFN response, indicating that all of the antibodies tested were able to deliver functional polynucleotides into cells and affect RIG-I mediated responses.

[0114] Therefore, in some embodiments, the present disclosure provides humanized antibodies and antigen-binding fragments thereof, which incorporate Figure 5-10 Any combination of the humanized VL and VH sequences shown in , and VL and VH sequences having sequence identity thereto, e.g., with Figure 5-10 The VH or VL sequences shown in are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical.

[0115] In some embodiments, a humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a light chain variable domain (3E10-VL) comprising 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), and a heavy chain variable domain (3E10-VH) comprising 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: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). 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).

[0116] In some embodiments, a humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a light chain variable domain (3E10-VL) comprising 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), and a heavy chain variable domain (3E10-VH) comprising an amino acid sequence 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: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). 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).

[0117] In some embodiments, a humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a light chain variable domain (3E10-VL) comprising an amino acid sequence at least 98% 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), 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: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). 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).

[0118] In some embodiments, a humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a light chain variable domain (3E10-VL) comprising an amino acid sequence at least 99% 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), and a heavy chain variable domain (3E10-VH) comprising an amino acid sequence 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: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). 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).

[0119] 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).

[0120] 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).

[0121] 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).

[0122] 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).

[0123] 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).

[0124] 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).

[0125] 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).

[0126] 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).

[0127] 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).

[0128] 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).

[0129] 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).

[0130] 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).

[0131] 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).

[0132] In some embodiments, the humanized 3E10 antibodies or antigen-binding fragments thereof described herein comprise a light chain (3E10-LC) comprising an amino acid sequence 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 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: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). 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).

[0133] 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).

[0134] 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).

[0135] 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).

[0136] 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).

[0137] 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).

[0138] 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).

[0139] In some embodiments, a humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a light chain (3E10-LC) comprising an amino acid sequence 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), 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:99), 3E10-LC-h4 (SEQ ID NO:100), 3E10-LC-h5 (SEQ ID NO:101), and 3E10-LC-h6 (SEQ ID NO:102). 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).

[0140] 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).

[0141] 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).

[0142] 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).

[0143] 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).

[0144] 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).

[0145] 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).

[0146] 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).

[0147] 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).

[0148] 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).

[0149] 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).

[0150] 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).

[0151] 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).

[0152] 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)

[0153] In some embodiments, the sequence of 3E10-HC is at least 95% identical to 3E10-HC-hl (SEQ ID NO: 78). In some embodiments, the sequence of 3E10-HC is at least 96% identical to 3E10-HC-hl (SEQ ID NO: 78). In some embodiments, the sequence of 3E10-HC is at least 97% identical to 3E10-HC-hl (SEQ ID NO: 78). In some embodiments, the sequence of 3E10-HC is at least 98% identical to 3E10-HC-hl (SEQ ID NO: 78). In some embodiments, the sequence of 3E10-HC is at least 99% identical to 3E10-HC-hl (SEQ ID NO: 78). In some embodiments, the sequence of 3E10-HC is 3E10-HC-hl (SEQ ID NO: 78).

[0154] 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).

[0155] 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).

[0156] 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).

[0157] 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).

[0158] 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).

[0159] 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).

[0160] In some embodiments, a humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a combination of a heavy chain variable domain (VH) and a light chain variable domain (VL) comprising an amino acid sequence having at least 97% sequence identity to a pair of VL and VH 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:89), NO:85), 3E10-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-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:67) 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 IDNO: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).

[0161] In some embodiments, a humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a combination of a heavy chain variable domain (VH) and a light chain variable domain (VL) comprising an amino acid sequence having at least 98% sequence identity to a pair of VL and VH 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:89), NO:85), 3E10-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-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 (SEQ IDNO: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).

[0162] In some embodiments, a humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a combination of a heavy chain variable domain (VH) and a light chain variable domain (VL) comprising an amino acid sequence having at least 99% sequence identity to a pair of VL and VH 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:89), NO:85), 3E10-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-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:67) 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 IDNO: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).

[0163] In some embodiments, the humanized 3E10 antibodies or antigen-binding fragments thereof described herein include a combination of a heavy chain variable domain (VH) and a light chain variable domain (VL) comprising an amino acid sequence 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), 3E10-VH-h2 (SEQ ID NO:65) and 3E10-VL-h3 (SEQ ID NO:87), 3E10-VH-h1 (SEQ ID NO:64) and 3E10-VL-h4 (SEQ ID NO:88), 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 (SEQID 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 (SEQID 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 (SEQID 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).

[0164] In some embodiments, the humanized 3E10 antibodies or antigen-binding fragments thereof described herein include a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH includes an amino acid sequence having at least 97% sequence identity with 3E10-VH-h6 (SEQ ID NO: 69), and the VL includes an amino acid sequence having at least 97% sequence identity with 3E10-VL-h6 (SEQ ID NO: 90). In some embodiments, the humanized 3E10 antibodies or antigen-binding fragments thereof described herein include a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH includes an amino acid sequence having at least 98% sequence identity with 3E10-VH-h6 (SEQ ID NO: 69), and the VL includes an amino acid sequence having at least 98% sequence identity with 3E10-VL-h6 (SEQ ID NO: 90). In some embodiments, the humanized 3E10 antibodies or antigen-binding fragments thereof described herein include a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH includes an amino acid sequence having at least 99% sequence identity with 3E10-VH-h6 (SEQ ID NO: 69), and the VL includes an amino acid sequence having at least 99% sequence identity with 3E10-VL-h6 (SEQ ID NO: 90). In some embodiments, the humanized 3E10 antibodies or antigen-binding fragments thereof described herein include a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH includes an amino acid sequence of 3E10-VH-h6 (SEQ ID NO: 69), and the VL includes an amino acid sequence of 3E10-VL-h6 (SEQ ID NO: 90).

[0165] In some embodiments, the humanized 3E10 antibodies or antigen-binding fragments thereof described herein have CDR sequences corresponding to the parent 3E10 antibody, such as Figure 1as shown in (SEQ ID NO: 1-12), optionally comprising a D31N amino acid substitution in VH CDR1, as shown in FIG2 . Thus, in some embodiments, a humanized 3E10 antibody or antigen-binding fragment thereof comprises a light chain variable domain (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 domain (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).

[0166] In some embodiments, a humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a CDR sequence from a variant humanized 3E10 antibody comprising a D31N amino acid substitution in VHCDR1, as shown in FIG. 2 .

[0167] In some embodiments, a humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a set of complementary determining regions (CDRs) having no more than seven amino acid substitutions in common relative to a set of CDRs having the following amino acid sequences: 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).

[0168] In some embodiments, a humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a set of complementary determining regions (CDRs) having no more than ten amino acid substitutions in common relative to a set of CDRs having the following amino acid sequences: 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).

[0169] In some embodiments, a humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a set of complementary determining regions (CDRs) having no more than nine amino acid substitutions in common relative to a set of CDRs having the following amino acid sequences: 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).

[0170] In some embodiments, a humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a set of complementary determining regions (CDRs) having no more than eight amino acid substitutions in common relative to a set of CDRs having the following amino acid sequences: 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).

[0171] In some embodiments, a humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a set of complementary determining regions (CDRs) having no more than seven amino acid substitutions in common relative to a set of CDRs having the following amino acid sequences: 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).

[0172] In some embodiments, a humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a set of complementary determining regions (CDRs) having no more than six amino acid substitutions in common relative to a set of CDRs having the following amino acid sequences: 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).

[0173] In some embodiments, a humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a set of complementary determining regions (CDRs) having no more than five amino acid substitutions in common relative to a set of CDRs having the following amino acid sequences: 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).

[0174] In some embodiments, a humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a set of complementary determining regions (CDRs) having no more than four amino acid substitutions in common relative to a set of CDRs having the following amino acid sequences: 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).

[0175] In some embodiments, a humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a set of complementary determining regions (CDRs) having no more than three amino acid substitutions in common relative to a set of CDRs having the following amino acid sequences: 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).

[0176] In some embodiments, a humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a set of complementary determining regions (CDRs) having no more than two amino acid substitutions in common relative to a set of CDRs having the following amino acid sequences: 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).

[0177] In some embodiments, a humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a set of complementary determining regions (CDRs) having no more than one amino acid substitution in common relative to a set of CDRs having the following amino acid sequences: 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).

[0178] Thus, in some embodiments, a humanized 3E10 antibody or antigen-binding fragment thereof comprises a light chain variable domain (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 domain (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).

[0179] Thus, in some embodiments, the humanized 3E10 antibodies or antigen-binding fragments thereof described herein comprise a set of complementarity determining regions (CDRs) having in common no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution selected from the group consisting of 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, relative to the CDR sequences of the 3E10-D31N variant (shown in FIG. 2 ).

[0180] 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 CDR 1 and 3 according to the parent 3E10 antibody (e.g., Figure 1 In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDRs 1-3 and VH CDRs 1 and 3 according to the 3E10-D31N variant (such as Figure 2A As shown (SEQ ID NO: 13-25)).

[0181] 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 CDRs 2 and 3 and VH CDRs 1-3 (e.g., Figure 1 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 (e.g. Figure 2A As shown (SEQ ID NO: 13-25)).

[0182] 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 CDRs 1 and 3 and VH CDRs 1-3 (e.g., Figure 1 In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDRs 1 and 3 and VH CDRs 1-3 according to the 3E10-D31N variant (such as Figure 2A As shown (SEQ ID NO: 13-25)).

[0183] While some of the amino acid substitutions described above are fairly conservative substitutions, e.g., the S to T substitution at position 5 of VL CDR1—other substitutions are of amino acids with very different properties, e.g., the M to L substitution at position 14 of VL CDR1, the H to A substitution at position 15 of VL CDR1, and the E to Q substitution at position 6 of VL CDR2. This suggests, without being bound by theory, that at least these positions within the 3E10 CDR framework are tolerant to other amino acid substitutions.

[0184] 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 CDRs 1-3 and VH CDRs 1 and 3 according to the parent 3E10 antibody (e.g. Figure 1 In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDRs 1-3 and VH CDRs 1 and 3 according to the 3E10-D31N variant (such as Figure 2A As shown (SEQ ID NO: 13-25)).

[0185] 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 CDRs 2 and 3 and VH CDRs 1-3 (e.g., Figure 1 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 (such as Figure 2A As shown (SEQ ID NO: 13-25)).

[0186] 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 CDRs 1 and 3 and VH CDRs 1-3 (e.g., Figure 1In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDRs 1 and 3 and VH CDRs 1-3 according to the 3E10-D31N variant (such as Figure 2A As shown (SEQ ID NO: 13-25)).

[0187] Thus, in some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof comprises a VH CDR1 comprising the following amino acid sequence: 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, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDRs 1-3 and VH CDRs 2 and 3 (e.g., Figure 1 As shown (SEQ ID NO: 1-12)).

[0188] Similarly, in some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof comprises a VH CDR2 comprising the following amino acid sequence: 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 CDRs 1-3 and VH CDRs 1 and 3 according to the parent 3E10 antibody (e.g., Figure 1 In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDRs 1-3 and VH CDRs 1 and 3 according to the 3E10-D31N variant (such as Figure 2A As shown (SEQ ID NO: 13-25)).

[0189] Similarly, in some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof comprises a VH CDR3 comprising the following amino acid sequence: 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 CDRs 1-3 and VH CDRs 1 and 2 according to the parent 3E10 antibody (e.g., Figure 1 In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDRs 1-3 and VH CDRs 1 and 2 according to the 3E10-D31N variant (e.g. Figure 2A As shown (SEQ ID NO: 13-25)).

[0190] Similarly, in some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof comprises a VL CDR1 comprising the following amino acid sequence: 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, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDRs 2 and 3 and VH CDRs 1-3 (e.g., Figure 1 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 (e.g. Figure 2A As shown (SEQ ID NO: 13-25)).

[0191] 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 CDRs 1 and 3 and VH CDRs 1-3 (e.g., Figure 1In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDRs 1 and 3 and VH CDRs 1-3 according to the 3E10-D31N variant (e.g. Figure 2A As shown (SEQ ID NO: 13-25)).

[0192] Similarly, in some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof comprises a VL CDR3 comprising the following amino acid sequence: 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, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDRs 1 and 2 and VH CDRs 1-3 (e.g., Figure 1 In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDRs 1 and 2 and VH CDRs 1-3 according to the 3E10-D31N variant (such as Figure 2A As shown (SEQ ID NO: 13-25)).

[0193] It is also envisioned that the humanized 3E10 antibodies or antigen-binding fragments thereof as described herein comprise no more than 7, 6, 5, 4, 3, 2, or 1 CDR amino acid substitutions described above. Figure 4 Additional examples of 3E10 variant CDR sequences described herein are shown in .

[0194] 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 CDRs 1-3 and VH CDRs 2 and 3 according to the parent 3E10 antibody (e.g., Figure 1 As shown (SEQ ID NO: 1-12)).

[0195] 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 CDRs 1-3 and VH CDRs 1 and 3 according to the parent 3E10 antibody (e.g. Figure 1 In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDRs 1-3 and VH CDRs 1 and 3 according to the 3E10-D31N variant (such as Figure 2A As shown (SEQ ID NO: 13-25)).

[0196] 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 CDRs 1-3 and VH CDRs 1 and 2 according to the parent 3E10 antibody (e.g. Figure 1 In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDRs 1-3 and VH CDRs 1 and 2 according to the 3E10-D31N variant (such as Figure 2A As shown (SEQ ID NO: 13-25)).

[0197] 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 CDRs 2 and 3 and VH CDRs 1-3 (e.g., Figure 1 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 (such as Figure 2A As shown (SEQ ID NO: 13-25)).

[0198] 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 CDRs 1 and 3 and VH CDRs 1-3 (e.g., Figure 1 In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDRs 1 and 3 and VH CDRs 1-3 according to the 3E10-D31N variant (such as Figure 2A As shown (SEQ ID NO: 13-25)).

[0199] 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 CDRs 1 and 2 and VH CDRs 1-3 (e.g., Figure 1 In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises VL CDRs 1 and 2 and VH CDRs 1-3 according to the 3E10-D31N variant (such as Figure 2A As shown (SEQ ID NO: 13-25)).

[0200] In some embodiments, a humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a light chain variable domain (3E10-VL) comprising an amino acid sequence 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:81). NO:90), wherein the light chain variable domain (3E10-VL) comprises, according to Kabat numbering, one or more amino acid residues of the 3E10-VL selected from the group consisting of: 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, and a set of 3E10-VL (SEQ ID NO:8) CDRs having no more than 6 amino acid substitutions in common relative to a set of CDRs having the following amino acid sequences: 3E10-VL-CDR1 (SEQ ID NO:9), 3E10-VL-CDR2 (SEQ ID NO:10), 3E10-VL-CDR3 (SEQ ID NO:11), NO:11), and wherein the antibody comprises a set of 3E10-VL CDRs having no more than 6 amino acid substitutions in common relative to a set of CDRs having the following amino acid sequences: 3E10-VL-CDR1 (SEQ ID NO:9), 3E10-VL-CDR2 (SEQ ID NO:10), 3E10-VL-CDR3 (SEQ ID NO:11).

[0201] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof comprises a set of 3E10-VL CDRs comprising no more than 5 amino acid substitutions relative to a set of CDRs having the following amino acid sequences: 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 comprising no more than 4 amino acid substitutions relative to a set of CDRs having the following amino acid sequences: 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 comprising no more than 3 amino acid substitutions relative to a set of CDRs having the following amino acid sequences: 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 comprising no more than 2 amino acid substitutions relative to a set of CDRs having the following amino acid sequences: 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 comprising no more than 1 amino acid substitution relative to a set of CDRs having the following amino acid sequences: 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 following amino acid sequences: 3E10-VL-CDR1 (SEQ ID NO:9), 3E10-VL-CDR2 (SEQ ID NO:10), 3E10-VL-CDR3 (SEQ ID NO:11).

[0202] 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 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 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 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 Kabat numbering.

[0203] In some embodiments, a humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a heavy chain variable domain (3E10-VH) comprising an amino acid sequence 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). NO:70), wherein the heavy chain variable domain (3E10-VH) comprises one or more amino acid residues of the 3E10-VH selected from the group consisting of 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, according to Kabat numbering, and wherein the antibody comprises a set of 3E10-VH CDRs having no more than 6 amino acid substitutions in common relative to a set of CDRs having the following amino acid sequence: 3E10-VH-CDR1_D31N (SEQ ID NO:15), 3E10-VH-CDR2 (SEQ ID NO:4) and 3E10-VH-CDR3 (SEQ ID NO:5).

[0204] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof comprises a set of 3E10-VH CDRs comprising no more than 5 amino acid substitutions relative to a set of CDRs having the following amino acid sequences: 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 comprising no more than 4 amino acid substitutions relative to a set of CDRs having the following amino acid sequences: 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 comprising no more than 3 amino acid substitutions relative to a set of CDRs having the following amino acid sequences: 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 comprising no more than 2 amino acid substitutions relative to a set of CDRs having the following amino acid sequences: 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 comprising no more than 1 amino acid substitution relative to a set of CDRs having the following amino acid sequences: 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 having the following amino acid sequences: 3E10-VH-CDR1_D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NO: 4), and 3E10-VH-CDR3 (SEQ ID NO: 5).

[0205] 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 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 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 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 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 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 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 Kabat numbering.

[0206] In some embodiments, the humanized 3E10 antibodies or antigen-binding fragments thereof of the present disclosure have a nucleic acid binding affinity for a polynucleotide (e.g., DNA, RNA, PNA, morpholino, etc.) that is similar to the affinity of a reference 3E10 antibody or antigen-binding fragment thereof (e.g., 3E10 monoclonal antibody or D31N variant thereof produced by hybridoma No. ATCC PTA2439) for the same polynucleotide. Thus, in some embodiments, the affinity of a humanized 3E10 antibody or antigen-binding fragment thereof for a polynucleotide is 0.05 to 50 times greater than the affinity of a reference 3E10 antibody for the same polynucleotide.

[0207] In some embodiments, the humanized 3E10 antibodies or antigen-binding fragments thereof of the present disclosure have a nucleic acid binding affinity for a polynucleotide (e.g., DNA, RNA, PNA, morpholino, etc.) that is less than the affinity of a reference 3E10 antibody or antigen-binding fragment thereof (e.g., 3E10 monoclonal antibody or D31N variant thereof produced by hybridoma ATCC No. PTA 2439) for the same polynucleotide. Thus, in some embodiments, the affinity of a humanized 3E10 antibody or antigen-binding fragment thereof for a polynucleotide is from 0.00001 to less than 1 times the affinity of a reference 3E10 antibody for the same polynucleotide. In some embodiments, the affinity of a humanized 3E10 antibody or antigen-binding fragment thereof for a polynucleotide is from 0.00001 to 0.001 times the affinity of a reference 3E10 antibody for the same polynucleotide. In some embodiments, the affinity of the humanized 3E10 antibody or antigen-binding fragment thereof for the polynucleotide is 0.0001 to 0.01 times the affinity of the reference 3E10 antibody for the same polynucleotide. In some embodiments, the affinity of the humanized 3E10 antibody or antigen-binding fragment thereof for the polynucleotide is 0.001 to 0.1 times the affinity of the reference 3E10 antibody for the same polynucleotide. In some embodiments, the affinity of the humanized 3E10 antibody or antigen-binding fragment thereof for the polynucleotide is 0.01 to less than 1 times the affinity of the reference 3E10 antibody for the same polynucleotide.

[0208] In some embodiments, the nucleic acid binding affinity of the humanized 3E10 antibody or antigen-binding fragment thereof of the present disclosure for a polynucleotide (e.g., DNA, RNA, PNA, morpholino, etc.) is greater than the affinity of a reference 3E10 antibody or antigen-binding fragment thereof (e.g., 3E10 monoclonal antibody or D31N variant thereof produced by hybridoma No. ATCC PTA2439) for the same polynucleotide. Thus, in some embodiments, the affinity of the humanized 3E10 antibody or antigen-binding fragment thereof for the polynucleotide is greater than 1 to 10,000 times the affinity of the reference 3E10 antibody for the same polynucleotide. In some embodiments, the affinity of the humanized 3E10 antibody or antigen-binding fragment thereof for the polynucleotide is 100 to 10,000 times the affinity of the reference 3E10 antibody for the same polynucleotide. In some embodiments, the affinity of the humanized 3E10 antibody or antigen-binding fragment thereof for the polynucleotide is 10 to 1000 times the affinity of the reference 3E10 antibody for the same polynucleotide. In some embodiments, the affinity of the humanized 3E10 antibody or antigen-binding fragment thereof for a polynucleotide is greater than 1 to 100 times greater than the affinity of the reference 3E10 antibody for the same polynucleotide.

[0209] Binding affinity can be determined by 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 to 10 times (1-10 times higher affinity or 1-10 times lower affinity, or any number or range or value within such ranges). The dissociation constant (Kd) of an exemplary affinity for a target antigen (DNA (e.g., single-stranded and / or double-stranded DNA)) is less than 5×10 -2 M, less than 10 -2 M, less than 5×10 -3 M, less than 10 -3 M, less than 5×10 -4 M, less than 10 -4 M, less than 5×10 -5 M, less than 10 -5 M, less than 5×10 -8 M, less than 10 -8 M, less than 5×10 -7 M, less than 10 -7 M, less than 5×10 -8 M, less than 10 -8 M, less than 5×10 -9 M, less than 10 -9 M, less than 5×10 -10 M, less than 10 -10 M, less than 5×10 -11 M, less than 10 -11 M, less than 5×10 -12 M, less than 10 -12 M, less than 5×10 -13 M, less than 10 -13 M, less than 5×10 -14 M, less than 10 -14 M, less than 5×10 -15 M or less than 10 -15 M. In some embodiments, the binding affinity (Kd) of the target is less than 10 -7 M, less than 5×10 -8 M, less than 10 -8 M, less than 5×10 -9 M, less than 10 -9 M, less than 5×10 -10 M, less than 10 -10 M, less than 5×10 -11 M, less than 10 -11 M, less than 5×10 -12 M or less than 10 -12 M.

[0210] In some embodiments, binding affinity is assessed by determining the solution concentration of the humanized 3E10 antibody or antigen-binding fragment thereof required to achieve half-maximal binding (EC50) of the polynucleotide coated on the solid surface. 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.

[0211] In some embodiments, the humanized 3E10 antibodies or antigen-binding fragments thereof described herein have weak binding affinity for poly-dT oligonucleotides. In some embodiments, the weak binding affinity is an EC50 value that is at least 100 times greater than the EC50 value of a reference 3E10 antibody (e.g., a chimeric 3E10 antibody having a D31N amino acid substitution). In some embodiments, the weak binding affinity is an EC50 value that is 100 to 10,000 times greater than the EC50 value of a reference 3E10 antibody (e.g., a chimeric 3E10 antibody having a D31N amino acid substitution). In some embodiments, the weak binding affinity is an EC50 value that is 100 to 5000 times greater than the EC50 value of a reference 3E10 antibody (e.g., a chimeric 3E10 antibody having a D31N amino acid substitution). In some embodiments, the weak binding affinity is an EC50 value that is 100-fold to 1000-fold greater than the EC50 value of a reference 3E10 antibody (e.g., a chimeric 3E10 antibody having a D31N amino acid substitution). In some embodiments, the weak binding affinity is an EC50 value that is 100-fold to 500-fold greater than the EC50 value of a reference 3E10 antibody (e.g., a chimeric 3E10 antibody having a D31N amino acid substitution).

[0212] In some embodiments, the humanized 3E10 antibodies or antigen-binding fragments thereof described herein have a moderate binding affinity for a poly-dT oligonucleotide. In some embodiments, the moderate binding affinity is an EC50 value that is 25 to 100 times greater than the EC50 value of a reference 3E10 antibody (e.g., a chimeric 3E10 antibody having a D31N amino acid substitution).

[0213] In some embodiments, the humanized 3E10 antibodies or antigen-binding fragments thereof described herein have a moderate binding affinity for poly-dT oligonucleotides. In some embodiments, the moderate binding affinity is an EC50 value that is no more than 25 times greater than the EC50 value of a reference 3E10 antibody (e.g., a chimeric 3E10 antibody having a D31N amino acid substitution). In some embodiments, the moderate binding affinity is an EC50 value that is no more than 20 times greater than the EC50 value of a reference 3E10 antibody (e.g., a chimeric 3E10 antibody having a D31N amino acid substitution). In some embodiments, the moderate binding affinity is an EC50 value that is no more than 15 times greater than the EC50 value of a reference 3E10 antibody (e.g., a chimeric 3E10 antibody having a D31N amino acid substitution). In some embodiments, the moderate binding affinity is an EC50 value that is no more than 10 times greater than the EC50 value of a reference 3E10 antibody (e.g., a chimeric 3E10 antibody having a D31N amino acid substitution). In some embodiments, the moderate binding affinity is an EC50 value that is no more than 5-fold greater than the EC50 value of a reference 3E10 antibody (eg, a chimeric 3E10 antibody having a D31N amino acid substitution).

[0214] In some embodiments, the humanized 3E10 antibodies or antigen-binding fragments thereof described herein have weak binding affinity for the 3p-hpRNA RIG-I agonist having the nucleotide sequence 5'-pppGGAGCAAAAGCAGGGUGACAAAGACAUAAUGGAUCC AAACACUGUGUCAAGCUUUCAGGUAGAUUGCUUUCUUUGGCAUGUCCGCAAAC-3' (SEQ ID NO: 103).

[0215] In some embodiments, the humanized 3E10 antibodies or antigen-binding fragments thereof described herein have moderate binding affinity for the 3p-hpRNA RIG-I agonist having the nucleotide sequence 5'-pppGGAGCAAAAGCAGGGUGACAAAGACAUAAUGGAUCCAAACACUGUGUCAAGCUUUCAGGUAGAUUGCUUUCUUUGGCAUGUCCGCAAAC-3' (SEQ ID NO: 103).

[0216] In some embodiments, the humanized 3E10 antibodies or antigen-binding fragments thereof described herein have a strong binding affinity for the 3p-hpRNA RIG-I agonist having the nucleotide sequence 5'-pppGGAGCAAAAGCAGGGUGACAAAGACAUAAUGGAUCCAAACACUGUGUCAAGCUUUCAGGUAGAUUGCUUUCUUUGGCAUGUCCGCAAAC-3' (SEQ ID NO: 103).

[0217] In some embodiments, the humanized 3E10 antibodies or antigen-binding fragments thereof described herein include 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.

[0218] In some embodiments, the humanized 3E10 antibodies or antigen-binding fragments thereof described herein have a human IgG1 Fc domain. In some embodiments, the human IgG1 domain comprises an amino acid sequence that is similar to ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:115) has 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.

[0219] In some embodiments, the IgG1 Fc domain sequence includes one or more natural amino acid variants found in the human population, for example, it is an IgG1 Fc allotype. 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 only found in Caucasians, while the G1m (f, a) allele is common in Asian populations, but other variants G1m (z, a, x) and G1m (z, a, v) are also described (see, Vidarsson et al., Frontiers in Immunology (Front. Immunol.), October 2014, Vol. 5, Chapter 520, the disclosure of which is incorporated herein by reference in its entirety).

[0220] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof has an IgG1 domain containing one or more engineered amino acid substitutions, e.g., to reduce Fc effector function, improve in vivo half-life, and / or otherwise alter the in vivo properties of the antibody. Several approved antibodies have demonstrated potent in vitro CDC activity, such as the anti-CD20 mAbs rituximab and ofatumumab, and there are a variety of approaches that allow researchers to use Fc engineering to enhance complement-based effector function. Idusogie et al. demonstrated that K326W / E333S enhanced C1q binding and CDC activity relative to IgG1, leading the authors to suggest 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 enhanced C1q binding 47-fold and CDC activity 6.9-fold relative to IgG1 (see, Teeling JL et al., Journal of Immunology 2006 177:362–371, Idusogie EE et al., Journal of Immunology 2001 166:2571–2575, Moore GL et al., Monoclonal Antibodies (MAbs.) 2010 2:181–189, and Wang et al., Protein & Cell, Vol. 9, No. 1, 2018, 63–73, the disclosures of which are incorporated herein by reference in their entirety).

[0221] Thus, in some embodiments, the IgG1 Fc domain comprises an amino acid substitution at a position selected from L234A according to the EU index as 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.

[0222] In some embodiments, the humanized 3E10 antibodies or antigen-binding fragments thereof described herein have a human IgG1 Fc domain comprising L234A / L235A amino acid substitutions. In some embodiments, the IgG1 Fc domain comprises an amino acid sequence comprising residues 234A / 235A and conjugated to ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 116) 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.

[0223] In embodiments, the humanized 3E10 antibodies or antigen-binding fragments thereof described herein have a human IgG1 Fc domain comprising an N297D amino acid substitution. In some embodiments, the IgG1 Fc domain comprises an amino acid sequence comprising residue 297D and conjugated to ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY D STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 117) 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.

[0224] In some embodiments, the humanized 3E10 antibodies or antigen-binding fragments thereof described herein have a human IgG1 Fc domain comprising L234A / L235A / N297D amino acid substitutions. In some embodiments, the IgG1 Fc domain comprises an amino acid sequence comprising residues 234A / 235A / 297D and conjugated to ASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY DSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 118) 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.

[0225] In some embodiments, the IgG1 constant heavy region 1 comprises an amino acid sequence having 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 ASTKGPSVFPLAPSSK STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTQTYICNVNHKPSNTKVDKKV (SEQ ID NO: 154).

[0226] In some embodiments, the IgG1 hinge region comprises an amino acid sequence having 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.

[0227] In some embodiments, IgG1L2345A / L235A constant heavy region 2 comprises a AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO: 156) with high sequence identity, e.g., an amino acid sequence with 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.

[0228] In some embodiments, the IgG1 constant heavy region 3 comprises an amino acid sequence having 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 GQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 157).

[0229] In some embodiments, IgG1N297D constant heavy region 2 comprises a polypeptide comprising PCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQY D STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO: 158) has high sequence identity, for example, an amino acid sequence with 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.

[0230] In some embodiments, IgG1L2345A / L235A / N297D constant heavy region 2 comprises a AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQY D STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK(IgG1 L2345A / L235A / N297D (SEQ ID NO: 159) has high sequence identity, for example, an amino acid sequence of 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.

[0231] In some embodiments, the unmodified constant heavy region 2 comprises an amino acid sequence having 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 PCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO: 160).

[0232] In some embodiments, the full length light chain sequence comprises an amino acid sequence having 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 DIQMTQSPSSLSASLGD RATITCRASKTVSTSSYSYMHWYQQKPGQPPKLLIKYASYLESGVPSRFSGSGSGTDFTLTISSLQPEDAATYYCQHSREFPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 161).

[0233] In some embodiments, the humanized 3E10 antibodies or antigen-binding fragments thereof described herein have a human IgG4 Fc domain. In some embodiments, the human IgG4 domain comprises an amino acid sequence that is the same as ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:104) has 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.

[0234] In some embodiments, the IgG4 Fc domain sequence includes one or more natural amino acid variants found in the human population, e.g., it is an IgG4 Fc allotype. Non-limiting examples of IgG4 Fc allotype amino acid variants include nG4m (a) and nG4m (b) (see, Vidarsson et al., Frontiers in Immunology, October 2014, Vol. 5, Chapter 520, the disclosure of which is incorporated herein by reference in its entirety).

[0235] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof has an IgG4 domain containing one or more engineered amino acid substitutions, for example, to reduce Fc effector function, improve in vivo half-life, and / or otherwise alter the properties of the antibody in vivo. In fact, most 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., Monoclonal Antibodies, 11(8):1341-50(2019), the disclosure of which is incorporated herein by reference in its entirety. Other IgG4 Fc mutations have been suggested for reducing immune effector function while retaining other Fc features. For example, Tam SH et al., Antibodies, 6(12):1-34(2017) (the disclosure of which is incorporated herein by reference in its entirety) reported 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 hereby incorporated by reference in its entirety. These mutations include M252Y, S254T, T256E, H433K, and N434F.

[0236] Still other IgG4 Fc amino acid substitutions that can be incorporated into the IgG4 Fc domain of a humanized 3E10 antibody or antigen-binding fragment thereof have been proposed in the art, including but not limited to WO 1989 / 007142, US 5,885,573, WO 1994 / 029351, US 6,407,214, US2006 / 0024298, US 7,863,419, US2007 / 0041972, US8,961,967, US 9,187,552, US 8,969,526, US 9,359,437, WO 2017 / 079369, US 7,371,826, US 7,083,784, WO 2004 / 035752, US 9,200,079, US11,046,784, US 8,802,820, US2020 / 0255502, US2010 / 0098730, US RE45992, US2010 / 0204454, US8,637,641, US2014 / 0302028, GB 201302878、US2015 / 0065690、US2014 / 0294812、US2020 / 0071423、US11,319,383、US2018 / 0037634、KR101792191、US2019 / 0010243、US 8,911,726、US2010 / 0267934、US 9,688,762, US2012 / 0100140, US 9,085,625, US10,562,966, US2017 / 029521, US11,254,753 and WO 2018 / 119380, the disclosures of which are hereby incorporated by reference in their entirety for all purposes.

[0237] Thus, in some embodiments, the IgG4 Fc domain comprises an amino acid substitution at a position selected from the group consisting of: 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 as in the Kabat numbering scheme. In some embodiments, the IgG4 Fc domain comprises an amino acid substitution selected from the group consisting of 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 domain comprises a combination of amino acid substitutions selected from the group consisting of S228P / L234A / L235A, L234F / L235E / P331S, M252Y / S254T / T256E, M252Y / S254T / T256E / H433K / N434F, S228P / F234A / L235A / H310Q, S22 8P / F234A / L235A / M252Y / S254T / T256E, S228P / F234A / L235A / T307Q / N434A, S22 8P / F234A / L235A / G237A / P238S and S228P / F234A / L235A / G236>del / G237A / P238S.

[0238] In some embodiments, the humanized 3E10 antibodies or antigen-binding fragments thereof described herein have a human IgG4 Fc domain comprising S228P / F234A / L235A amino acid substitutions. In some embodiments, the IgG4 Fc domain comprises an amino acid sequence comprising residues 228P / 234A / 235A and having

[0239] Having 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.

[0240] In some embodiments, the humanized 3E10 antibodies or antigen-binding fragments thereof described herein have a human IgG4 Fc domain comprising S228P / F234A / L235A / T307Q / N434A amino acid substitutions. In some embodiments, the IgG4 Fc domain comprises an amino acid sequence comprising residues 228P / 234A / 235A / 307Q / 434A and

[0241] Having 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.

[0242] In some embodiments, the humanized 3E10 antibodies or antigen-binding fragments thereof described herein have a human IgG4 Fc domain comprising S228P / F234A / L235A / M252Y / S254T / T256E amino acid substitutions. In some embodiments, the IgG4 Fc domain comprises an amino acid sequence comprising residues 228P / 234A / 235A / 252Y / 254T / 256E and

[0243]

[0244] Having 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.

[0245] In some embodiments, the humanized 3E10 antibodies or antigen-binding fragments thereof described herein have a human IgG4 Fc domain comprising S228P / F234A / L235A / H310Q amino acid substitutions. In some embodiments, the IgG4 Fc domain comprises an amino acid sequence comprising residues 228P / 234A / 235A / 310Q and

[0246] Having 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.

[0247] In some embodiments, the present disclosure provides humanized 3E10 antibodies and antigen-binding fragments thereof comprising a heavy chain constant domain (CH) 1.

[0248] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof comprises an Fc region selected from the group consisting of human γ1 CH1, human γ2 CH1, human γ3 CH1, and human γ4 CH1.

[0249] In some embodiments, the present disclosure provides a humanized 3E10 antibody or antigen-binding fragment thereof comprising a light chain constant domain (CL).

[0250] In another aspect, the humanized 3E10 antibody or antigen-binding fragment thereof comprises an Fc region selected from the group consisting of human λCL and human κCL.

[0251] In various embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof is covalently linked to a therapeutic moiety.

[0252] In some embodiments, the therapeutic moiety is a therapeutic polypeptide, a cytotoxic moiety, a chemotherapeutic moiety, or a detectable moiety.

[0253] Cell penetration and nuclear localization

[0254] The disclosed compositions and methods generally utilize antibodies that maintain the ability to penetrate cells, and optionally utilize nuclei.

[0255] The mechanisms of cellular internalization of autoantibodies are diverse. Some enter cells through electrostatic interactions or FcR-mediated endocytosis, while others utilize 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 in an Fc-independent mechanism (as demonstrated by the ability of 3E10 fragments lacking Fc to penetrate cells), but involves the presence of the nucleoside transporter ENT2 (Weisbart et al., Scientific Reports Vol. 5, Article No.: 12022 (2015), Zack et al., Journal of Immunology 157, 2082-2088 (1996), Hansen et al., Journal of Biological Chemistry 282, 20790-20793 (2007)). Thus, in some embodiments, the antibodies utilized in the disclosed compositions and methods are antibodies that penetrate cells in an Fc-independent mechanism but involve the presence of the nucleoside transporter ENT2.

[0256] Mutations in 3E10 that interfere with its ability to bind nucleic acids may render the antibody incapable of nuclear penetration. Thus, generally, variants and humanized forms of the disclosed antibodies maintain the ability to bind nucleic acids. Additionally, it has been previously shown that the 3E10 scFv is able to penetrate into living cells and nuclei in an ENT2-dependent manner, with impaired uptake efficiency in ENT2-deficient cells (Hansen et al., Journal of Biol. Chem. 282, 20790-20793 (2007)). Thus, in some embodiments, humanized forms and variants of the disclosed antibodies maintain the ability to penetrate into the cell nucleus in an ENT-dependent, preferably ENT2-dependent manner.

[0257] As discussed in US2021 / 0054102 and US2021 / 0137960, some humanized 3E10 variants were found to penetrate the nucleus more effectively than the original murine 3E10 (D31N) discFv, while others were found to have lost the ability to penetrate the nucleus. Specifically, variants 10 and 13 penetrated the nucleus well compared to the murine antibody.

[0258] Potential dual nuclear localization signals (NLS) in humanized 3E10VL have been identified and may include part or all of the following sequences:

[0259] RASK S VSTSSYSYMHWYQQKPGQPPKLLIKY(SEQ ID NO:109);

[0260] RASK T VSTSSYSYMHWYQQKPGQPPKLLIKY (SEQ ID NO: 110); or

[0261] RVTITCRASKSVSTSSYSYMH WYQQKPGKAPKL (SEQ ID NO: 111).

[0262] An example consensus sequence NLS may be or include ( X )RASKTVSTSSYSYMHWYQQKP GQPPKLL( X )KY (wherein (X) = any residue, but preferably a basic residue (R or K) having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 112) (SEQ ID NO: 112) or a variant thereof.

[0263] Thus, in some embodiments, particularly where nuclear import is important, the disclosed antibodies can comprise the sequence of any one of SEQ ID NOs: 109-112, or fragments and variants thereof that can translocate into the nucleus (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).

[0264] The presence of an NLS indicates that the humanized 3E10 antibody or antigen-binding fragment thereof can cross the nuclear envelope via the nuclear import pathway. 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 heterodimers, or a combination thereof.

[0265] Nucleic acid binding

[0266] In some embodiments, the disclosed compositions and methods utilize humanized 3E10 antibodies and antigen-binding fragments thereof that maintain the ability to bind nucleic acids, such as DNA, RNA, or a combination thereof.

[0267] The following examples show molecular modeling of the wild-type 3E10 sequence and additional 3E10 variants. Molecular modeling of 3E10 (Pymol) revealed a putative nucleic acid binding pocket (NAB1) (see, e.g., Fig.11A and 11B ), and underline the following sequence.

[0268] WT heavy chain scFv sequence

[0269] E V LVESGGG VKV R C AASGFTFSD GMHWQ AP E KG L E WVAYI

[0270] SSGSSTI YYA DTVKGRFT I S RDNAKNT LFL QMTSLRSEDT AM YYCARRG LLL QDJ LTVS (SEQ ID NO: 113)

[0271] Light chain scFv sequence

[0272] D IVLT QSPASL AVSLGQRATI SC RASKSVS TSS SYMH W Y Q QKPGQPPKL L

[0273] I K YASYLESG VPARFSGSGS GTDFTLNIHP VEEEDA ATYY QUR EFPW TFGGG TKLEIKRADAAPGGGG SGGGGSGGGGS(SEQ ID NO:114)

[0274] 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 having altered abilities to bind to nucleic acids. In some embodiments, mutations (e.g., substitutions, insertions and / or deletions) in NAB1 improve binding of the antibody to nucleic acids such as DNA, RNA, or a combination thereof. In some embodiments, the mutations are conservative substitutions. In some embodiments, the mutations increase the cationic charge of the NAB1 pocket.

[0275] As discussed and exemplified herein, an aspartic acid to asparagine mutation at residue 31 of CDR1 increases the cationic charge of this residue and enhances nucleic acid binding and delivery in vivo (3E10-D31N).

[0276] Additional example variants include a mutation of aspartic acid to arginine (3E10-D31R) at residue 31 of CDR1, which modeling indicates an amplification of the cationic charge, or lysine (3E10-D31K), which modeling indicates a change in charge orientation. Thus, in some embodiments, the 3E10 binding protein includes a D31R or D31K substitution.

[0277] Additional exemplary variants include a mutation of arginine (R) 96 to asparagine (N), and / or a mutation of serine (S) 30 to aspartic acid (D), alone or in combination with D31N, D31R or D31K.

[0278] All sequences disclosed herein having residues corresponding to 3E10D31 or N31 are specifically disclosed as having D31R or D31K or N31R or N31K substitutions.

[0279] Molecular modeling of 3E10 (Pymol) revealed a putative nucleic acid binding pocket (NAB1) ( Figures 11A-11B ). An aspartic acid to asparagine mutation at residue 31 of CDR1 increases the cationic charge of this residue and enhances nucleic acid binding and delivery in vivo (3E10-D31N).

[0280] Mutation of aspartate to arginine (3E10-D31R) at residue 31 of CDR1 further amplifies the cationic charge, whereas mutation to lysine (3E10-D31K) changes the charge orientation ( Fig.11A ).

[0281] The NAB1 amino acids predicted from molecular modeling have been underlined in the heavy and light chain sequences above. Fig. 11B is a diagram showing molecular modeling of 3E10-scFv (Pymol) with NAB1 amino acid residues displayed as dots.

[0282] All sequences disclosed herein having a residue corresponding to R96 are explicitly disclosed as having an R96N substitution.

[0283] All sequences disclosed herein having a residue corresponding to S30 are explicitly disclosed as having S30D.

[0284] Any of the substitutions may be included in any combination. Sequences having two or three substitutions at any combination of residues 31, 30 and 96 are specifically provided.

[0285] In specific embodiments, the sequence has 31N, 31K or 31R alone or in combination with 30D, and does not have an R96N substitution. Thus, in some embodiments, the residue corresponding to 96 is not N, and in more specific embodiments remains R.

[0286] Fragments, variants and fusion proteins

[0287] The anti-nucleic acid antibody can be composed of an antibody fragment or a fusion protein comprising 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 light chain of its humanized 3E10 form (e.g., any one of SEQ ID NOs: 64-102).

[0288] The anti-nucleic acid antibody can be composed of an antibody fragment or fusion protein comprising 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 a CDR of humanized 3E10 or a variant thereof (e.g., a CDR of any one of SEQ ID NOs: 3-5, 9-11, 15-18, 22-24, and 26-63). The percent identity of two amino acid sequences can be determined by BLAST protein comparison. In some embodiments, the antibody comprises one, two, three, four, five, or all six of the 3E10 CDRs described herein.

[0289] Preferably, the antibody comprises one of each of the heavy chain CDR1, CDR2 and CDR3 in combination with one of each of the light chain CDR1, CDR2 and CDR3.

[0290] The predicted complementary determining regions (CDRs) of the light chain variable sequence of 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 complementary determining regions (CDRs) of the heavy chain variable sequence of 3E10 are provided above. See, for example, Zack et al., Immunology and Cell Biology, 72:513-520 (1994), GenBank accession number AAA65679.1. Zach et al., Journal of Immunology 154 (4), 1987-1994 (1995) and GenBank: L16982.1 - Mouse Ig rearranged H chain gene, partial cds.

[0291] Also included are antibody fragments having nucleic acid delivery activity. Whether or not attached to other sequences, the fragments include insertions, deletions, substitutions or other selected modifications of specific regions or specific amino acid residues, provided that the activity of the fragment is not significantly altered or impaired compared to the unmodified antibody or antibody fragment.

[0292] Can also adopt the technology for producing the single-chain antibody that nucleic acid of the present disclosure has specificity.The method for producing single-chain antibody is well-known to those skilled in the art.Can be by using short peptide linker, the variable domain of heavy chain and light chain is fused together to create single-chain antibody, thus reconstruct antigen binding site on single molecule.Under the situation that antigen binding or binding specificity are not significantly destroyed, single-chain antibody variable fragment (scFv) has been developed, in which the C-terminal of one variable domain is connected with the N-terminal bolt of another variable domain by having 15 to 25 amino acid whose peptide or linker.Select linker to allow heavy chain and light chain to be combined together with its suitable conformation towards.

[0293] Anti-nucleic acid antibodies can be modified to improve their nucleic acid delivery potential. For example, in some embodiments, a cell penetrating anti-nucleic acid antibody is conjugated to another antibody that is specific for a therapeutic target in the cytoplasm and / or nucleus of a target cell. For example, the cell penetrating anti-nucleic acid antibody can be a fusion protein containing a 3E10 Fv and a single chain variable fragment of a monoclonal antibody that specifically binds to a therapeutic target. In other embodiments, the cell penetrating 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 a therapeutic target.

[0294] Bispecific antibodies and other binding proteins have 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, as discussed in Weisbart et al., Mol. Cancer Ther., 11(10):2169-73 (2012), and Weisbart et al., Int. Oncol., 25:1113-8 (2004), and U.S. Patent Application No. 2013 / 0266570, which are specifically incorporated herein by reference in their entirety. In some embodiments, the target is specific for a target cell type, tissue, organ, etc. Thus, the second heavy chain and the second light chain can serve as a targeting portion of the targeting complex to a target cell type, tissue, organ. In some embodiments, the second heavy chain and the second light chain target, hematopoietic stem cell, CD34 + Cells, T cells or any other cell type, for example by targeting a receptor or ligand expressed on the cell type. In some embodiments, the second heavy chain and the second light chain target the thymus, spleen or cancer cells.

[0295] In some embodiments, particularly for targeting T cells in vivo, such as those for generating antigen-specific T cells, CAR T cells in vivo, immune cells or T cell markers such as CD3, CD7 or CD8 can be targeted. For example, both anti-CD8 antibodies and anti-CD3 Fab fragments have 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., Nature Nanotechnology, 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 portion that can specifically bind to CD3, CD7, CD8 or another immune cell (e.g., T cell) marker, or a marker for a specific tissue, such as the thymus, spleen or liver.

[0296] A bivalent single-chain variable fragment (two scFv) can be engineered by connecting two scFvs. This can be accomplished by producing a single peptide chain with two VH regions and two VL regions, thereby producing a series of scFvs. ScFv can also be designed to have a linker peptide that is too short for the two variable regions to fold together (about five amino acids), thereby forcing the scFv to dimerize. This type is called a bifunctional antibody. It has been shown that the dissociation constant of a bifunctional antibody is at least 1 / 40 of the dissociation constant of the corresponding scFv, which means that the bifunctional antibody has a higher affinity for its target. Still shorter linkers (one or two amino acids) lead to the formation of trimers (three functional antibodies or three bodies). Four antibodies have also been produced. Its affinity for the target is much higher than that of a bifunctional antibody. In some embodiments, the anti-nucleic acid antibody can contain two or more connected single-chain variable fragments of 3E10 (e.g., 3E10 two scFv, 3E10 three scFv) or conservative variants thereof. In some embodiments, the anti-nucleic acid antibody is a bifunctional antibody or a trifunctional antibody (e.g., 3E10 bifunctional antibody, 3E10 trifunctional antibody). Sequences of single and two or more linked single-chain variable fragments of 3E10 are provided in US2019 / 0247515 and US2017 / 0291961.

[0297] The function of the antibody can be enhanced by coupling the antibody or its fragment to a therapeutic agent. Such coupling of an antibody or fragment to a therapeutic agent can be achieved by preparing an immunoconjugate or by preparing a fusion protein or by connecting the antibody or fragment to a nucleic acid such as DNA or RNA (e.g., siRNA) including the antibody or antibody fragment and the therapeutic agent.

[0298] In some embodiments, the cell penetrating 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 exists in the circulation or at the treatment site 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 to be treated for an extended period of time. In other embodiments, the half-life of the anti-nucleic acid antibody is reduced to reduce potential side effects. Antibody fragments, such as 3E10Fv, may have a shorter half-life than full-size antibodies. Other methods of changing half-life are known and can be used in the described methods. For example, an antibody can be engineered to have an Fc variant with an extended half-life, such as using Xtend TM Antibody Half-Life Extension Technology (Xencor, Monrovia, CA).

[0299] Connectors

[0300] As used herein, the term "joint" includes but is not limited to peptide joints. The peptide joint can be of any size, as long as it does not interfere with the combination of the variable region and the epitope. In certain embodiments, the joint comprises one or more glycine and / or serine amino acid residues. Monovalent single-chain antibody variable fragment (scFv), wherein the C-terminus of one variable domain is usually bolted to the N-terminus of another variable domain by 15 to 25 amino acid peptides or joints. The joint is selected to allow the heavy chain and light chain to be combined together with its suitable conformation orientation. As discussed above, the joint in bifunctional antibodies, trifunctional antibodies, etc. usually comprises a joint shorter than the joint of the monovalent scFv. Divalent, trivalent and other multivalent scFvs usually comprise three or more joints. The length and / or amino acid composition of the joint can be the same or different. Therefore, as known in the art, the number of joints, the composition of the joint and the length of the joint can be determined based on the desired valence of the scFv. The joint can allow or drive the formation of divalent, trivalent and other multivalent scFvs.

[0301] For example, the linker can comprise 4-8 amino acids. In a specific embodiment, the linker comprises the amino acid sequence GQSSRSS (SEQ ID NO: 119). In another embodiment, the linker comprises 15-20 amino acids, such as 18 amino acids. In a specific embodiment, the linker comprises 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), (Gly 4 -Ser) 2(SEQ ID NO: 123) and (Gly 4 -Ser) 4 (SEQ ID NO:124) and (Gly-Gly-Gly-Gly-Ser) 3 (SEQ ID NO: 125).

[0302] Other exemplary linkers include, e.g., RADAAPGGGGSGGGGSGGGGS (SEQ ID NO: 126) and ASTKGPSVFPLAPLESSGS (SEQ ID NO: 127).

[0303] Nucleic acid cargo

[0304] As 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 bound to the humanized 3E10 antibody or antigen-binding fragment thereof. In some embodiments, the polynucleotide is covalently conjugated to the humanized 3E10 antibody or antigen-binding fragment thereof.

[0305] The nucleic acid cargo can be single-stranded or double-stranded. The nucleic acid cargo can be or comprise DNA, RNA, nucleic acid analogs or a combination thereof. As discussed in more detail below, nucleic acid analogs can be modified at the base moiety, sugar moiety or phosphate backbone. Such modifications can improve, for example, the stability, hybridization or solubility of the nucleic acid.

[0306] Nucleic acid cargo is generally of functional significance, i.e., is or encodes an agent that is 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, for example, expression constructs and vectors, inhibitory nucleic acids such as siRNA, or nucleic acids encoding inhibitory nucleic acids, including, for example, expression constructs and vectors.

[0307] The disclosed compositions can include multiple individual nucleic acid cargo molecules. In some embodiments, the composition includes multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) different nucleic acid molecules.

[0308] In some embodiments, the cargo molecule is about 0.001, about 0.01, about 1, 10, 100, 1,000, 10,000, and / or 100,000 kilobases in length.

[0309] 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 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 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 12.5 ... between 0.01 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 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 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 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 12. ... .1kb and 2.5kb, or between 0.1kb and 1kb, or between 1kb and 100kb, or between 1kb and 50kb, or between 1kb and 25kb, or between 1kb and 12.5kb, or between 1kb and 10kb, or between 1kb and 8kb, or between 1kb and 5kb, or between 1kb and 2.5kb, each including the end value.

[0310] 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 between about 0.001 kb and about 2 0.5 kb, or between about 0.001 kb and about 1 kb, or between about 0.01 kb and about 100 kb, or between about 0.01 kb and about 50 kb, or between about 0.01 kb and about 25 kb, or between about 0.01 kb and about 12.5 kb, or between about 0.01 kb and about 10 kb, or between about 0.01 kb and about 8 kb, or between about 0.01 kb and about 5 kb, or between about 0.0 1 kb to 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.

[0311] In some embodiments, for example, the cargo is between 0.2 kb and 10 kb, or between 0.2 kb and 5 kb, or between 0.2 kb and 2.5 kb, or between 0.2 kb and 1 kb, or between 0.2 kb and 0.5 kb, or between 0.2 kb and 0.25 kb, or between 0.5 kb and 10 kb, or between 0.5 kb and 5 kb, or between 1 kb and 5 kb, or between 1 kb and 3 kb, or between 2 kb and 10 kb, or between 3 kb and 5 kb.

[0312] 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.

[0313] It should be understood that for specific applications, the nucleic acid cargo can be, for example, one or more discrete lengths falling within the aforementioned ranges (including one of the aforementioned ranges), with the specific value of each discrete length explicitly disclosed. For example, the size can be as small as a single nucleotide or a nucleobase. In an example application, the cargo is a cyclic dinucleotide, like cGAMP, which is a STING agonist. In other embodiments, the cargo is a short oligomer. For example, oligomers as short as 8-mers can be used for antisense or splicing switches. Slightly longer oligomers (e.g., 18-mer to 20-mer) can be used for gene editing.

[0314] Immunostimulatory oligonucleotides

[0315] Macromolecular stimulators of the innate immune system, especially polynucleotide agonists of pattern recognition receptors (PRRs), have brought great hope for the treatment of cancer. Pattern recognition receptors (PRRs) recognize pathogen-associated and endogenous damage-associated molecular patterns. Once ligand binding occurs, signal transduction cascades develop within the cell to activate effector molecules, leading to the recruitment and activation of anti-tumor immune cells and the release of inflammatory cytokines. Therefore, PRR agonists have been successfully used as immunotherapy for the treatment of a variety of cancers. For a review of the use of PRRs and PRR agonists in cancer immunotherapy, see, for example, Bai L. et al., "Promising targetsbased on pattern recognition receptors for cancer immunotherapy", "Pharmacological Research", 159 (2020) 105017, the contents of which are incorporated herein by reference in their entirety.

[0316] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof is complexed with a polynucleotide immunostimulator (e.g., 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 conjugated to the humanized 3E10 antibody or antigen-binding fragment thereof.

[0317] Pattern recognition receptor (PRR) agonists

[0318] In one aspect, as described herein, 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 recognized in the art, stimulation of the innate immune system, for example by activating pattern recognition receptors, represents a promising therapeutic approach specifically for treating cancer. Typically, PRRs stimulate the innate immune system after recognizing pathogen-associated patterns (PAMPs) and / or damage-associated patterns (DAMPs). Typically, 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 by PRRs of any of these categories that recognize and are activated by polynucleotide antigens.

[0319] RIG-I-like receptors (RLRs) are a family of RNA helicases that are used as cytoplasmic sensors of pathogen-associated molecular patterns (PAMPs) within viral RNA. Therefore, on the other hand, compositions and methods for treating cancer by administering a therapeutically effective amount of a composition to a subject are provided, the composition comprising a complex formed between (i) a humanized 3E10 antibody or its antigen-binding fragment and (ii) a polynucleotide ligand capable of stimulating a RIG-I-like receptor (RLR). Identified RLRs include RIG-I (retinoic acid-induced gene 1), MDA5 (melanoma differentiation-related factor 5), and LGP2 (Genetics and Physiology Laboratory 2). Therefore, in some embodiments, the present disclosure provides a composition that is formed between (i) a humanized 3E10 antibody or its antigen-binding fragment and (ii) a polynucleotide ligand capable of stimulating RIG-I. A method for treating cancer by administering such a composition to a subject in need is also provided. In some embodiments, the complex includes a covalent conjugate between a humanized 3E10 antibody or its antigen-binding fragment and a polynucleotide ligand.

[0320] Example RIG-I ligands include but are not limited to 5'ppp-dsRNA, a specific agonist of RIG-I; 3p-hpRNA, 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); Poly(dA:dT) / LyoVec complexes indirectly recognized by RIG-I. In some embodiments, 3p-hpRNA is a 5' triphosphate hairpin RNA produced by in vitro transcription of the sequence of influenza A (H1N1). In some embodiments, 3p-hpRNA is an RNA oligonucleotide containing an uncapped 5' triphosphate end and a double-stranded fragment. In some embodiments, 3p-hpRNA is about 50bp, about 55bp, about 60bp, about 65bp, about 70bp, about 75bp, about 80bp, about 85bp, about 90bp, about 100bp or more. In some embodiments, the length of 3p-hpRNA is 89bp.

[0321] In some embodiments, the polynucleotide capable of stimulating RIG-I is non-covalently bound to the humanized 3E10 antibody or antigen-binding fragment thereof. In some embodiments, the polynucleotide capable of stimulating RIG-I is covalently conjugated to the humanized 3E10 antibody or antigen-binding fragment thereof.

[0322] Thus, in some embodiments, compositions and methods 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) an RNA molecule that is at least partially double-stranded and capable of stimulating RIG-I are provided. 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 having self-complementarity such that under physiological conditions, it anneals to itself to form the double-stranded portion of the molecule, e.g., thereby forming one or more hairpin structures.

[0323] Similarly, in some embodiments, compositions and methods 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 chains 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 chain with self-complementarity such that under physiological conditions, it anneals to form the double-stranded portion of the molecule, for example, thereby forming one or more hairpin structures. Examples of polynucleotide RIG-I agonists are provided in the literature. Typically, any of these polynucleotide RIG-I agonists can be used in the methods and compositions described herein.

[0324] In some embodiments, the RIG-I agonist is a 5' triphosphate hairpin RNA produced by in vitro transcription of a sequence from influenza A (H1N1) virus, a single-stranded negative sense RNA virus (3p-hpRNA) having the sequence 5'-pppGGAGCAAAAGCAGGGUGACAAAGACAUAAUG GAUCCAAACACUGUGUCAAGCUUUCAGGUAGAUUGCUUUCUUUGGC AUGUCCGCAAAC-3' (SEQ ID NO: 103), or a highly conserved nucleotide sequence thereof. See, e.g., 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 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 having the sequence of 3p-hpRNA (SEQ ID NO: 103) are provided.

[0325] 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 (SEQID 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 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 that has 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%, or at least 99% identical to the nucleotide sequence of 3p-hpRNA (SEQ ID NO: 103).

[0326] Other RIG-I agonists that can be used in the methods and compositions disclosed herein are known in the art. For example, useful RIG-I agonists that can be complexed with the humanized 3E10 antibodies and antigen-binding fragments thereof disclosed herein are described in WO 2023 / 278897, US20100178272, US 9,738,680, US2011 / 0184045, US10,059,943, US2018 / 0195063, US11,382,966, US11,542,505, WO 2020 / 260547, US2021 / 0260093, US9,226,959, US2014 / 0286998, US 9,861,574, US 9,775,894, US2021 / 0046168, US2019 / 0076463, US11,499,157, US10,907,161 and US2022 / 0333113, the disclosures of which are hereby incorporated by reference in their entirety.

[0327] In some embodiments, compositions and methods 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 13 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 affinity for a different antigen. According to various embodiments of the present disclosure, the methods and compositions described herein include polynucleotide agonists using TLRs. For example, each of TLR3, TLR7, TLR8, and TLR9 has affinity for various polynucleotides and is activated by various polynucleotides. Therefore, in some embodiments, the agonists used in the methods and compositions described herein can stimulate 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 a humanized 3E10 antibody or antigen-binding fragment thereof and a polynucleotide ligand.

[0328] For example, unmethylated CpG sites can be detected by TLR9 on human plasmacytoid dendritic cells and B cells (Zaida et al., Infection and Immunity, 76(5):2123-2129, (2008)). Therefore, 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, the oligonucleotide containing CG can have a modified backbone, such as a phosphorothioate (PS) backbone.

[0329] In some embodiments, the oligonucleotide may contain more than one CG dinucleotide arranged in series or separated by intermediate nucleotides. The CpG motif may be internal to the oligonucleotide sequence. Many nucleotide sequences stimulate TLR9 in which the number and position of CG dinucleotides and the precise base sequence of the flanking CG dimers vary.

[0330] Generally, CG ODN is classified based on its sequence, secondary structure and effect on human peripheral blood mononuclear cells (PBMC). The five categories are category A (type D), category B (type K), category C, category P and category S (Vollmer, J and Krieg, AM, Advanced Drug Delivery Reviews 61 (3): 195-204 (2009), which is incorporated herein by reference). CG ODN can stimulate the production of type I interferons (e.g., IFNα) and induce the maturation of dendritic cells (DC). Some categories of ODN are also strong activators of natural killer (NK) cells through indirect cytokine signaling. Some classes are potent stimulators of human B cell and monocyte maturation (Weiner, GL, Proc. Natl. Acad. Sci. USA 94(20):10833-7 (1997); Dalpke, AH, Immunology 106(1):102-12 (2002); Hartmann, G, J. Immunol. 164(3):1617-2 (2000), each of which is incorporated herein by reference).

[0331] In some embodiments, the polynucleotide immunostimulator is cyclic GMP-AMP synthase (cGAS) or stimulator of interferon genes (STING).

[0332] Polynucleotide encoding effector polypeptide

[0333] 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 conjugated to the humanized 3E10 antibody or antigen-binding fragment thereof.

[0334] In some embodiments, the polynucleotide is conjugated to or further encodes one or more of an RNAi agent, siRNA, shRNA, miRNA, miRNA binding site, antisense RNA, ribozyme, catalytic DNA, tRNA, RNA inducing triple helix formation, aptamer, or vector, or the like.

[0335] In other embodiments, polynucleotides can be designed to encode one or more effector polypeptides or fragments thereof directed against cancer tissues. Such effector polypeptides can include, but are not limited to, whole polypeptides, multiple polypeptides, or polypeptide fragments that can be independently encoded by one or more regions or portions or the entirety of an effector polynucleotide.

[0336] Effector polypeptides refer to any polypeptides selected to be encoded within the polynucleotides of the present disclosure or whose functions are affected by the polynucleotides. Effector polypeptides can directly or indirectly modulate the activity of the immune system and affect cancer treatment, such as by slowing cancer progression, inducing cell death of cancer cells, inducing senescence of cancer cells, etc. For example, in some embodiments, effector polypeptides stimulate immune cells to upregulate the production of cytokines, so that cancer cells are targeted, thereby causing cell death. In other embodiments, effector polypeptides express or stimulate tumor cells to upregulate the expression of tumor antigens, which are markers for immune cells to identify tumor cells. For reviews of effector peptides, see, e.g., 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 Future), Journal of Clinical Investigation, 2015, Vol. 125, 9, 3384-3391; Rowshanravan et al., CTLA-4a moving target in immunotherapy, Blood, 2018, Vol. 131, 1, 58-67; and Dougall 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.

[0337] 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 antigen-binding fragment thereof and (ii) a therapeutic polynucleotide encoding a protein or peptide (e.g., an effector polypeptide), for use in cancer therapy, as described herein.

[0338] Tumor Antigens

[0339] In some embodiments, as described herein, 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. 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 conjugated to the humanized 3E10 antibody or an antigen-binding fragment thereof.

[0340] Tumor antigens are peptides presented almost exclusively on the cell surface of cancer cells, thereby distinguishing cancer cells from non-cancerous cells that do not display 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 is released, the immune system can produce anti-tumor immunity by generating an immune response against tumor antigens. Specifically, the immune system targets and destroys cancer cells that display tumor antigens for generating an immune response.

[0341] Several classes of antigens have been experimentally developed to generate anti-tumor immunity. Specifically, tumor antigens are exogenously administered as peptides or nucleic acids encoding antigens to patients with cancer that displays tumor antigens on their cell surfaces. In turn, the immune system is presented with sufficient amounts of antigens to generate an immune response against the tumor antigens, thereby generating anti-tumor immunity. Examples of tumor antigen classes include tumor virus protein antigens, new antigens, and antigens derived from cancer germline genes. Typically, tumor antigens or polynucleotides encoding tumor antigens are co-administered with adjuvants that activate dendritic cells or dendritic cells themselves to promote the generation of anti-tumor immunity. For reviews, see, for example, Haen et al., "Towards new horizons Characterization, classification and implications of the tumor antigenic repertoire", Nature Reviews - Clinical Oncology, 2020, Vol. 17, 595-610; Saxena M. et al., Nature Reviews: Cancer (Nat. Rev. Cancer) 21, 360-378 (2021), all of which are incorporated herein by reference.

[0342] In some embodiments, as described herein, 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.

[0343] To date, at least six oncoviruses (cancer-causing viruses) are known, 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 are not yet fully understood.

[0344] Tumor virus protein antigens are antigens derived from cancer-associated oncogenic viruses that are presented on the cell surface of cancer. For example, the vast majority of cervical cancers are associated with HPV infection, even if they are not caused by the infection. Therefore, HPV-derived antigens presented on the cell surface of cervical cancer cells represent tumor virus protein antigens. Non-limiting examples of tumor virus protein antigens, as well as examples of cancers associated with these antigens, are presented in Table 1 below.

[0345] Table 1. Examples of tumor virus proteins and associated cancer types.

[0346]

[0347]

[0348] Thus, in some embodiments, as described herein, 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) a therapeutic polynucleotide encoding a tumor virus protein antigen derived from a viral protein listed in Table 1. 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 a dendritic cell.

[0349] In some embodiments, as described herein, 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. Neoantigens are peptides presented on the surface of cancer cells, having an amino acid sequence that is novel to cancer tissue. That is, the neoantigen has an amino acid sequence that is not present in the germline (wild-type) human genome. Neoantigens are generated by genomic mutations during or after cancer development, in this way, specific to individual patients.

[0350] Deep sequencing technology can be used to identify mutations present in the exome of an individual tumor to predict new antigens. This is achieved by identifying new antigens that can be recognized by T cells. For example, in some embodiments, non-synonymous somatic mutations of tumor materials are analyzed. RNA sequencing data is used to focus on mutations of expressed genes. Peptides containing any identified non-synonymous mutations are generated on a computer and are not filtered, filtered using a prediction algorithm, or used to identify MHC-related new antigens in mass spectrometry data generated by the patient's cancer tissue. Modeling of the effects of mutations on the resulting peptide-MHC complex can be used as an additional filter to identify particularly promising new antigens. Through screening based on MHC polymers, the resulting epitope group can also be used to identify physiologically occurring new antigen-specific T cell responses. (See, for example, Science, April 3, 2015: Vol. 348, No. 6230, pp. 69-74). However, other techniques including exome analysis and proteome analysis can also be used to identify new genomes or new peptide sequences corresponding to new antigens, respectively. Non-limiting examples of neoantigens identified from individual cancers using transcriptome, exome, and proteome analyses are described, for example, in Haen et al., Towards new heights: characterization, classification, and significance of the tumor antigen repertoire, Nature Reviews Clinical Oncology, 2020, Vol. 17, 595-610.

[0351] Thus, in some embodiments, as described herein, the present disclosure provides a method for treating a cancer in a subject by first identifying a neoantigen in the subject's cancer and secondly 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 therapeutic polynucleotide encoding the identified neoantigen.

[0352] In some embodiments, as described herein, 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. Cancer germline antigens are a class of immunogenic tumor antigens encoded by genes expressed in gametogenic 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 is overexpressed in different human cancers such as melanoma and lung cancer.

[0353] Thus, in some embodiments, as described herein, 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) a therapeutic polynucleotide encoding an antigen derived from an SSX-2, NY-ESO-1, MAGA1 or MAGA3 protein. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is lung cancer.

[0354] In some embodiments, as described herein, 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). 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 a dendritic cell.

[0355] Tumor associated antigens are peptides derived from wild-type protein sequences or glycoproteins synthesized by tumor cells. TAA proteins can reside in any subcellular compartment of tumor cells; for example, the protein can be membrane-bound, cytoplasmic, nuclear localized, or even secreted by tumor cells. TAA is mainly produced by gene amplification or post-translational modification, which results in differential expression of potential proteins in cancer cells relative to non-cancerous cells and allows specific T cells or immunoglobulins to preferentially recognize tumor cells. Non-limiting examples of tumor associated antigens that have been identified are presented in Tables 2-3.

[0356] Table 2. Examples of tumor-associated antigens (TAAs) and associated cancers.

[0357]

[0358] Table 3. Examples of tumor associated antigens (TAAs) and related references, all of which are incorporated herein by reference for the purpose of identifying the tumor markers referenced.

[0359]

[0360]

[0361]

[0362]

[0363] Illustrative TAAs include, for example, membrane-bound complement regulatory glycoproteins: CD46, CD55, and CD59, which have been found to be expressed on most tumor cells in vivo and in vitro. Human mucins (e.g., MUC1) are known tumor markers, such as gp100, tyrosinase, and MAGE found in melanoma, see Table 2-3. Wild-type Wilms' tumor gene WT1 is expressed at high levels not only in most acute myeloid leukemias, acute lymphocytic leukemias, and chronic myeloid leukemias, but also in various types of solid tumors including lung cancer.

[0364] Acute lymphocytic leukemia is characterized by TAA HLA-Dr, CD1, CD2, CD5, CD7, CD 19 and CD20. Acute myeloid leukemia is characterized by TAA HLA-Dr, CD7, CD13, CD14, CD15, CD33 and CD34. Breast cancer is characterized by markers EGFR, HER2, MUC1, Tag-72. Various carcinomas are characterized by markers MUC1, TAG-72 and CEA. Chronic lymphocytic leukemia is characterized by markers CD3, CD19, CD20, CD21, CD25 and HLA-DR. Hairy cell leukemia is characterized by markers CD19, CD20, CD21, CD25. Hodgkin's disease is characterized by Leu-M1 markers. Various melanomas are characterized by HMB 45 markers. Non-Hodgkin's lymphomas are characterized by the CD20, CD19 and La markers, and various prostate cancers are characterized by the PSMA and SE10 markers.

[0365] Thus, in some embodiments, as described herein, 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) a therapeutic polynucleotide encoding a tumor-associated antigen derived from a protein listed in Tables 2-3. 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 a dendritic cell.

[0366] In some embodiments, as described herein, 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) a therapeutic polynucleotide encoding a corresponding tumor-associated antigen derived from a protein listed in Table 2, wherein the cancer is a cancer associated with a corresponding tumor-associated antigen in Table 2. 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 a dendritic cell.

[0367] In some embodiments, tumor antigens are tumor virus protein antigens, neoantigens, and antigens derived from cancer germline genes.

[0368] 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 3 recognized by T cells (SART3), β-human chorionic gonadotropin (β-hCG), Wilms tumor antigen 1 (WT1), survivin, MAGE3, p53, RING finger protein 43 and mitochondrial outer membrane translocase 34 (TOMM34), prostate specific antigen (PSA)-TRICOM or KRAS.

[0369] In some embodiments, the tumor-associated antigen (TAA) is 5-alpha reductase, alpha-fetoprotein, AM-1, APC, APRIL, BAGE, beta-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), CD C27, 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, heparinase, 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 et al.), Mammaglobin, MAP 17, Melan-A / MART-1, Mesothelin, MIC A / B, MT-MMP, such as MMP2, MMP3, MMP7, MMP9, Mox1, 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 loading, STAT3 (mucin associated), TAG-72, TGF-ct, TGF-β, thymosin β15, IFN-ct, TPA, TPI, TRP-2, tyrosinase, VEGF, ZAG, p16INK4 or glutathione S-transferase.

[0370] In some embodiments, the neoantigen is BRCA1, BRCA2BRAF, 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), alternative reading frame (ARF), or cyclin-dependent kinase 4 (CDK4).

[0371] 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, L AGE-1b, CTAG2, MAGEC1, MAGEC3, SYCP1, BRDT, MAGEC2, SPANXA1, SPANXB1, SPANXC, SPANXD, SPANXN1, SPANXN2, SPANXN3, SPANXN4, SPANXN5, XAGE1D, XAG E1C, XAGE1B, XAGE1, XAGE2, XAGE3, XAGE-3b, XAGE-4 / RP11-167P23.2, , LIPI, VENTXP1, IL13RA2, TSP50, CTAGE1, CTAGE-2, CTAGE5, SPA17, ACRBP, CSAG1, CSAG2, DSCR8, MMA1b, DDX53, CTCFL, LUZP4, CASC5, TFDP3, JARID1B, LDHC, MORC1, DKKL1, SPO11, CRISP2, FMR1NB, FTHL17, NXF2, TAF7L, TDRD1, TDRD6, TDRD4, TEX15, FATE1, TPTE, CT45A1, CT45A2, CT45A3, CT45A4, CT45A5, CT45A6, HORMAD1, HORMAD2, CT47A1, CT47A2, CT47A3, CT47A4, CT47A5, CT47A6, CT47A7, CT47A8, CT47A9, CT47A10, CT47A11, CT47B1, SLCO6A1, TAG, LEMD 1. HSPB9, CCDC110, ZNF165, SPACA3, CXorf48, THEG, ACTL8, NLRP4, COX6B2, LOC348120, CCDC33, LOC196993, PASD1, LOC647107, TULP2, CT66 / AA884595,PRSS54, RBM46, CT69 / BC040308, CT70 / BI818097, SPINLW1, TSSK6, ADAM29, CCDC36, LOC440934, SYCE1, CPXCR1, TSPY3, TSGA10, HIWI, MIWI, PIWI, PIWIL2, ARMC3, AKAP3, Cxorf61, PBK, C21orf99, 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, PTPN20A, SPAG4, MAEL, RQCD1, PRAME, TEX101, SPATA19, ODF1, ODF2, ODF3, ODF4, ATAD2, ZNF645, MCAK, SPAG1, SPAG6, SPAG8, SPAG17, FBXO39, RGS22, Cyclin A1, C15orf60, CCDC83, TEKT5, NR6A1, TMPRSS12, TPPP2, PRSS55, DMRT1, EDAG, NDR, DNAJB8, CSAG3B, CTAG1A, GAGE12B, GAGE12C, GAGE12D, GAGE12E, GAGE12F, GAGE12G, GAGE12H, GAGE12I, GAGE12J, GAGE13, LOC728137, MAGEA2B, MAGEA9B / LOC728269, NXF2B, SPANXA2, SPANXB2, SPANXE, SSX4B, SSX5, SSX6, SSX7, SSX9, TSPY1D, TSPY1E, TSPY1F, TSPY1G, TSPY1H, TSPY1I, TSPY2 or XAGE1E.,

[0372] CD28 is a member of the costimulatory molecule subfamily characterized by an extracellular variable immunoglobulin-like domain. Human CD28 is composed of four exons encoding a protein with 220 amino acids, which is expressed as a glycosylated disulfide-bonded homodimer of 44kDa on the cell surface. The members of the CD28 family have many common features, such as paired V sets of immunoglobulin superfamily (IgSF) domains connected with a single transmembrane domain and a cytoplasmic domain containing a key signal transduction motif. (Esensten et al., "Immune Review" (2016)). It is reported that CD28 regulates T cell activation by interacting with a signal transduction motif. For example, the tyrosine phosphorylation of CD28 plays a role in characterizing CD28 costimulation and subsequently regulating the early signal transduction events of T cell activation. Thus, in some embodiments, provided herein is a composition for treating cancer, the composition comprising 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 co-stimulatory molecule having a pair of V-set immunoglobulin superfamily (IgSF) domains connected to a single transmembrane domain and a cytoplasmic domain.

[0373] Pro-inflammatory cytokines

[0374] Proinflammatory cytokines indirectly limit tumor cell growth by direct anti-proliferation or pro-apoptotic activity, or by stimulating the cytotoxic activity of immune cells to tumor cells. Proinflammatory cytokines are secreted from Th1 cells, CD4+ cells, macrophages and dendritic cells. It is characterized by producing some interleukins (IL), IL-1, IL-2, IL-12, IL-17, IL-18, IFN-γ and TNF-α. Key proinflammatory cytokines are IL-1, IL-6 and TNF-α. These cytokines send signals through type I cytokine receptors (CCR1) that are different from other cytokine receptor types in structure. It is essential for coordinating cell-mediated immune responses and plays a key role in regulating the immune system. Proinflammatory cytokines usually regulate the growth, cell activation, differentiation and homing of immune cells to the site of infection, with the purpose of controlling and eradicating intracellular pathogens (including viruses).

[0375] IL-1 is subdivided into IL-1α and IL-1β. IL-1β is an effective pro-inflammatory cytokine mainly induced by lymphocytes, macrophages and monocytes in response to microbial molecules. During viral infection, pattern recognition receptors (PPR) and toll-like receptors (TLR) are expressed, which in turn lead to enhanced expression of IL-1β. IL-1β stimulates CD4+ cells and will differentiate towards Th17 cells. In addition to the stimulatory effects of the IL-1 family, there are also membranes (IL-1RA and IL-1R2) that can inhibit (inhibit / suppress) the expression of IL-1 cytokines. IL-1RA is secreted by neutrophils, macrophages, monocytes and hepatocytes, aiming to reduce inflammation. However, the expression of IL-RA needs to be expressed up to 1,000 times in order to effectively inhibit (inhibit / suppress) the expression of IL-1β.

[0376] 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. 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. 64 patients). Journal of Clinical Oncology, 1986, 4, 900-905; Solal-Celigny et al., Recombinant interferon alfa-2b combined with a regimen containing doxorubicin in patients with advanced follicular lymphoma. Study of the Adult Lymphoma Study Group (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. Journal of Clinical Oncology, 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).

[0377] IL-6 is a pleiotropic cytokine that not only affects the immune system, but also acts on other biological systems and many physiological events (such as regulating cell growth and gene activation, proliferation, survival and differentiation). IL-6 is produced by a variety of cell types including monocytes, fibroblasts and endothelial cells. When stimulated, IL-6 is secreted by many other 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 leukocytes and liver. It is particularly important in inducing B cell differentiation into antibody-forming cells (plasma cells). The combination of IL-6 and its receptor triggers cell events, which include the activation of JAK (Janus kinase) kinases and the activation of Ras-mediated signal transduction.

[0378] Like other Th1 pro-inflammatory cytokines, TNF-α has an important role in inflammatory responses, both locally and in the circulation. TNF-α triggers the expression of 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.

[0379] Thus, in some embodiments, the compositions and methods described herein for treating cancer comprise a humanized 3E10 antibody or antigen-binding fragment thereof and a polynucleotide encoding a pro-inflammatory cytokine.

[0380] In some embodiments, the present disclosure provides compositions and methods for treating cancer by administering such compositions to a subject in need thereof, the compositions comprising a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof and (ii) a therapeutic polynucleotide encoding a proinflammatory cytokine, 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 conjugated to the humanized 3E10 antibody or an antigen-binding fragment thereof.

[0381] In some embodiments, the cytokine is IL-1, IL-6, IL-8, IL-12, IFN-, IL-18, IL-15, IL-2, TNF-, IL-10, TGF-b, CSF-1, CCL2, CCL3, CCL5 or VEGF.

[0382] Gene regulatory polynucleotides

[0383] In some embodiments, the humanized 3E10 antibody or its antigen-binding fragment as described herein is used to deliver a gene regulatory polynucleotide, which, for example, reduces or silences the expression of a gene product that promotes cancer growth and / or progression by targeting a gene or its transcript. Non-limiting examples of gene regulatory polynucleotides include siRNA, miRNA, saRNA, antagomir, antisense oligonucleotides, and decoy oligonucleotides. In some embodiments, the gene regulatory polynucleotide is a non-replicating modified or unmodified mRNA. In some embodiments, the gene regulatory polynucleotide is a self-amplifying mRNA. In some embodiments, the gene regulatory polynucleotide is a plasmid encoding a protein or peptide. In some embodiments, the gene regulatory polynucleotide is an expression regulatory polynucleotide. For a review of the therapeutic capabilities of various types of gene regulatory polynucleotides that have been studied, 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 which are incorporated herein by reference.

[0384] Thus, in one aspect, as described herein, 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. 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 conjugated 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 conjugated to the humanized 3E10 antibody or an antigen-binding fragment thereof.

[0385] siRNA

[0386] As described herein, the present disclosure relates to compositions and methods for treating cancer and comprises a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof and (ii) siRNA. Small interfering RNA (siRNA), also known as short interfering RNA or silencing RNA, is a class of double-stranded RNA non-coding RNA molecules that are typically 20-27 base pairs in length and operate within the RNA interference (RNAi) pathway. Gene regulatory nucleic acid drugs (such as siRNA) can regulate post-transcriptional gene expression and silence targeted genes, further regulating 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 bound to the humanized 3E10 antibody or its antigen-binding fragment. In some embodiments, the siRNA is covalently conjugated to the humanized 3E10 antibody or its antigen-binding fragment.

[0387] Thus, siRNA can be used to regulate the expression of immune checkpoint molecules (such as the immune checkpoint molecules described herein) by regulating post-translational gene expression and / or silencing corresponding genes. 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, as described herein, the composition for treating cancer described herein comprises a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof and (ii) a siRNA targeting an mRNA transcript from a gene encoding an immune checkpoint molecule.

[0388] PD-1 and some of its homologues (e.g., PD-L1) inhibit T cell responses, particularly in tumor microenvironments. Therefore, inhibitors of PD-1 and / or PD-L1 can improve the efficacy of T cells attacking and killing tumor cells. The inhibition of PD-1 and / or PD-L1 activity can be achieved, for example, by inhibiting the production of intracellular PD-1 and / or PD-L1. Therefore, in some embodiments, the composition for cancer provided herein comprises 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.

[0389] It is known that inhibition of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) activity leads to rapid infiltration of T cells. Therefore, inhibitors of CTLA-4 may lead to promotion of T cell responses. Therefore, in some embodiments, the composition for treating cancer provided herein comprises a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof and (ii) an siRNA targeting an mRNA transcript of CTLA-4.

[0390] siRNA can similarly be used to silence genes that regulate tumor growth or angiogenesis. For example, siRNA has been used to target vascular endothelial growth factor (VEGF) and kinesin spindle protein (KSP) (for solid tumors, such as liver metastasis of colon cancer). Other gene targets that can be silenced using siRNA include but are not limited to encoding protein kinase N3 (PKN3) (for example, for metastatic pancreatic cancer), the M2 subunit of ribonucleotide reductase (RRM2) (for example, for solid tumors), Myc oncoprotein (for example, for hepatocellular carcinoma), ephrin type A receptor 2 (EphA2) (for example, for advanced cancer) and KRAS G12D mutation (for example, for advanced pancreatic cancer) genes. See, for example, International Journal of Nanomedicine 2019: 143111–3128. Thus, in some embodiments, the compositions provided herein for treating cancer 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.

[0391] 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 (IL-2 receptor) to downregulate IL-2 signaling in CD8+ T cells.

[0392] Table 4 below provides other non-limiting examples of siRNAs and associated cancer types under study (see, e.g., Int. J. Mol. Sci. 22 (2021) 3295).

[0393] Table 4. Example siRNAs and associated cancer types.

[0394]

[0395] 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) an siRNA listed in Table 4, as described herein.

[0396] In some embodiments, as described herein, 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 corresponding siRNA listed in Table 4, wherein the cancer is a cancer associated with the corresponding siRNA in Table 4.

[0397] In some embodiments, as described herein, 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.

[0398] In some embodiments, as described herein, 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 targeted to a transcript from a corresponding gene listed in Table 4, wherein the cancer is a cancer associated with a corresponding gene in Table 4.

[0399] miRNA

[0400] In some embodiments, as described herein, 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. MicroRNA (miRNA) is a class of non-coding RNAs that play an important role in regulating gene expression. MiRNA is an endogenous non-coding small RNA of about 18-24 nt in length that can regulate target gene expression by a mechanism similar to 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). A major challenge in miRNA delivery is to efficiently deliver it to tumor tissue with deep tissue penetration. In addition, the complexity of the tumor microenvironment also prevents 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 bound to the humanized 3E10 antibody or its antigen-binding fragment. In some embodiments, the miRNA is covalently conjugated to the humanized 3E10 antibody or its antigen-binding fragment.

[0401] However, advantageously, the expression of miRNA is specific to different tumors, and miRNA is related to the early regulation of immune response. A method for treating cancer is to regulate the expression of immune checkpoint molecules (such as immune checkpoint molecules described herein) by regulating the level of miRNA. Examples of miRNAs regulating immune checkpoint-related processes include but are not limited to miR-15a, -15b, -16, -195, -424, -497, -503, which regulate the expression of PD-L1 and CD80. Another example of miRNA with tumor suppressor function is miR-28, which suppresses their expression by binding to the corresponding 3'UTR of TIM3, BTLA and PD-1 in T cells. Another example of miRNA is miR-138, which suppresses the expression of PD-1 and CTLA-4 on the surface of both effector T cells and regulatory T cells. The miR-34 family includes miR-34a, -34b and -34c, which suppress the expression of PD-L1.

[0402] It is known that the expression of miR-138-5p impedes the proliferation of CRC cells, blocks their transition from the G1 to S phase of the cell cycle, and directly inhibits the expression of PD-L1.

[0403] Other miRNAs that are overexpressed in certain types of cancer cells (e.g., miR-20b, -21, and -130b) can effectively mitigate T cell activation in the tumor microenvironment indirectly through the expression of PTEN.

[0404] Thus, as described herein, the compositions and methods described herein for treating cancer comprise a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof and (ii) a miRNA that directly or indirectly regulates the expression of an immune checkpoint molecule.

[0405] In some embodiments, the miRNA ismiR-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.

[0406] In some embodiments, as described herein, 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) a miRNA mimic molecule that directly or indirectly regulates the expression of an immune checkpoint molecule. Due to the same sequence, the miRNA can be a double-stranded synthetic RNA that mimics endogenous miRNA.

[0407] In some embodiments, as described herein, the compositions and methods described herein for treating cancer comprise a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof and (ii) a miRNA expression vector encoding a miRNA that directly or indirectly regulates the expression of an immune checkpoint molecule.

[0408] In some embodiments, as described herein, the compositions and methods described herein for treating cancer comprise a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof and (ii) an LNA-modified antisense oligodeoxyribonucleotide (ASO) targeting a miRNA that directly or indirectly regulates the expression of an immune checkpoint molecule. 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.

[0409] In some embodiments, as described herein, the compositions and methods described herein for treating cancer include a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof and (ii) an antagoumiao targeting a miRNA that directly or indirectly regulates the expression of an immune checkpoint molecule. Antagoumiao can be a single-stranded 23-nucleotide RNA molecule that is complementary to the targeted miRNA and is modified with a partial thiophosphate backbone in addition to a 2'-O-methoxyethyl. This is known to increase the stability of miRNA by protecting it from degradation.

[0410] In some embodiments, as described herein, the compositions and methods described herein for treating cancer comprise a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof and (ii) an antisense oligodeoxyribonucleotide (ASO) targeting a miRNA that directly or indirectly regulates the expression of an immune checkpoint molecule.

[0411] In some embodiments, as described herein, the compositions and methods described herein for treating cancer comprise a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof and (ii) a miRNA sponge that directly or indirectly regulates the expression of an immune checkpoint molecule. The miRNA sponge can be an RNA containing multiple tandem binding sites for a miRNA of interest transcribed from an expression vector.

[0412] Similarly, miRNAs that modulate the expression of proteins associated with tumor growth or angiogenesis can also be delivered by complexing with a humanized 3E10 antibody or antigen-binding fragment thereof, as described herein. Table 5 gives non-limiting examples of miRNAs and cancers associated therewith.

[0413] Table 5. Example miRNAs being studied for the treatment of cancer (see, e.g., Journal of the International Federation of Clinical Chemistry and Laboratory Medicine (2019) Vol. 30, No. 2, pp. 114-127).

[0414] miRNA cancer miR-122 HCV miR-155 Lymphoma and leukemia miR-16 Mesothelioma miR-34 Renal cell carcinoma, acral melanoma, hepatocellular carcinoma MRX34 Liver cancer, lung cancer, lymphoma, melanoma, multiple myeloma, renal cell carcinoma INT-1B3 Solid Tumors TargomiR Malignant pleural mesothelioma, non-small cell lung cancer Cobomarsen(MRG-106) Cutaneous T-cell lymphoma, colorectal cancer

[0415] Thus, in some embodiments, as described herein, 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 miRNA listed in Table 5.

[0416] In some embodiments, as described herein, 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 corresponding miRNA listed in Table 5, wherein the cancer is a cancer associated with the corresponding miRNA in Table 5.

[0417] saRNA

[0418] In some embodiments, as described herein, 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) saRNA. Small activating RNA (saRNA) is a class of non-coding dsRNAs with a length of about 21 nt and 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). Although it shares a similar structure with siRNA, it has an opposite gene regulation mechanism. The saRNA in the cytoplasm is specifically loaded into the AGO2 protein, and this RNA-AGO2 complex is transported to the nucleus to induce the targeted gene promoter for gene activation (Li et al., Proceedings of the National Academy of Sciences of the United States of America 2006; 103(46): 17337–42). It is reported that the saRNA-AGO2 complex in the nucleus recruits essential proteins 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 its ability to upregulate genes, saRNA shows potential for application as cancer immunotherapy. Therefore, in some embodiments, as described herein, the composition for treating cancer described herein comprises 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. In some embodiments, saRNA is non-covalently bound to a humanized 3E10 antibody or an antigen-binding fragment thereof. In some embodiments, saRNA is covalently conjugated to a humanized 3E10 antibody or an antigen-binding fragment thereof.

[0419] In one example, saRNA can upregulate the transcription factor CCATT / enhancer binding protein alpha (CEBPA), which leads to an increase in functional C / EBP protein and albumin, and inhibits the growth of liver cancer in a rat model. Table 6 lists other non-limiting examples of saRNAs being studied for the treatment of cancer.

[0420] Table 6. saRNAs being studied for cancer treatment

[0421] saRNA cancer C / EBPa-saRNA Hepatocellular carcinoma, pancreatic ductal adenocarcinoma dsP21-322 Bladder cancer

[0422] Anta is wonderful

[0423] In some embodiments, as described herein, the compositions and methods described herein for treating cancer comprise a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof and (ii) Antagoumiao. Antagoumiao is a small synthetic RNA that is complementary to a specific miRNA target and has a mismatch or certain base modification at the cleavage site of Ago2 to inhibit Ago2 cleavage. Antagoumiao is an isolated specific endogenous microRNA that competes with cellular target mRNA, induces miRNA repression and prevents mRNA target degradation by RISC. Therefore, Antagoumiao can be used for treatments that are beneficial to miRNA function loss. In some embodiments, Antagoumiao is non-covalently bound to a humanized 3E10 antibody or an antigen-binding fragment thereof. In some embodiments, Antagoumiao is covalently conjugated to a humanized 3E10 antibody or an antigen-binding fragment thereof.

[0424] An example of an antagomir is anti-miR21. Studies have shown that silencing miR21 by using anti-miR21 affects the viability, apoptosis and cell cycle of colon cancer cells (Song et al., "The anti-miR21 antagomir, atherapeutic tool for colorectal cancer, has a potential synergistic effect by perturbing an angiogenesis-associated miR30", Front. Genet, January 2014).

[0425] Another example of Anta Gou Miao is Anta Gou Miao-221. Studies have shown that Anta Gou Miao-221 can reduce cell proliferation by inhibiting the function of miR-221, which plays an important role in HCC because it inhibits tumor suppressor target proteins such as P27KIP1, P57KIP2, and phosphatase and tension protein homolog (PTEN). Similarly, several studies have shown that Anta Gou Miao-21 reverses epithelial-mesenchymal transition (EMT) by targeting PTEN to inactivate AKT serine / threonine kinase 1 (AKT) and ERK1 / 2 pathways. This effect of Anta Gou Miao-21 can potentially be used to target the etiological mechanism of malignant tendency of breast cancer. See, for example, Atri et al., AGO-Driven Non-Coding RNAs (2019).

[0426] Antagomir, which targets miR-155, is in phase 1 (NCT02580552) and phase 2 clinical trials (NCT03713320). MiR-155 regulates the differentiation and proliferation of blood and lymphocytes and is a suitable target for treating certain types of lymphoma and leukemia. See, for example, “RNA-Based Therapeutics: From Antisense Oligonucleotides to miRNAs” Cell 9 (2020), 137.

[0427] Thus, in some embodiments, as described herein, the compositions described herein for treating cancer comprise a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof and (ii) an antagonist targeting a microRNA that regulates the translation of tumor-associated mRNA. Non-limiting examples of antagonists include antagonists-221, antagonists-21, and antagonists-155.

[0428] Antisense Oligonucleotides (ASOs)

[0429] In some embodiments, as described herein, the composition for treating cancer described herein comprises a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof and (ii) an antisense oligonucleotide. Antisense oligonucleotides (ASOs) are short synthetic chemically modified nucleotide chains with the potential to target any gene product of interest. Typically, ASOs are single-stranded sequences complementary to the messenger RNA (mRNA) sequence transcribed from a target gene in a cell (Rinaldi et al., "Antisense oligonucleotides: thenext 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). The ASO targets the corresponding mRNA to degrade the targeting complex by mechanisms such as endogenous cellular RNase H. In some embodiments, the antisense oligonucleotide is non-covalently bound to the humanized 3E10 antibody or antigen-binding fragment thereof. In some embodiments, the antisense oligonucleotide is covalently conjugated to the humanized 3E10 antibody or antigen-binding fragment thereof.

[0430] An example of an ASO for cancer therapy is an ASO that targets CD39 mRNA to improve CD8+ T cell proliferation, thereby improving anti-tumor immune responses. Zhou et al., Medicine in Drug Discovery, 6:(2020)100023.

[0431] Table 7 gives other non-limiting examples of ASOs and cancers associated therewith.

[0432] Table 7. Example ASOs being studied for the treatment of cancer (see, e.g., Int J Molecular Sci 22(2021)3295).

[0433]

[0434]

[0435]

[0436] Thus, in some embodiments, as described herein, 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.

[0437] In some embodiments, as described herein, 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 corresponding antisense oligonucleotide listed in Table 7, wherein the cancer is a cancer associated with the corresponding antisense oligonucleotide in Table 7.

[0438] In some embodiments, as described herein, 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.

[0439] In some embodiments, as described herein, 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 corresponding gene listed in Table 7, wherein the cancer is a cancer associated with a corresponding gene in Table 7.

[0440] Decoy Oligonucleotide

[0441] 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 a decoy oligonucleotide. It has been reported that transfection of cis-element double-stranded oligonucleotides (referred to as decoy oligodeoxynucleotides) is a powerful tool that provides a new class of antigenic strategies for gene therapy (Crinelli et al., Design and characterization of decoy oligonucleotides containing lockednucleic acids. Nucleic Acids Research 2002; 30(11): 2435-2443). One such example is a STAT3 decoy oligonucleotide, which is a double-stranded 15-mer oligonucleotide that closely corresponds to the signal transducer and activator of transcription 3 (STAT3) response element within the c-fos promoter and has potential anti-tumor activity. STAT3 decoy oligonucleotides specifically bind to activated STAT3 and block the binding of STAT3 to DNA sequences on a variety of STAT3-responsive promoters, thereby inhibiting STAT3-mediated transcription and potentially inhibiting tumor cell proliferation. STAT3 is constitutively activated in a variety of cancers including head and neck squamous cell carcinoma, leading to uncontrolled cell growth and neoplastic transformation. In some embodiments, the decoy oligonucleotide or a polynucleotide encoding the decoy oligonucleotide is non-covalently bound to a humanized 3E10 antibody or an antigen-binding fragment thereof. In some embodiments, the decoy oligonucleotide or a polynucleotide encoding the decoy oligonucleotide is covalently conjugated to a humanized 3E10 antibody or an antigen-binding fragment thereof.

[0442] Examples of polynucleotides encoding complexes that can perform genome editing are described in the following sections.

[0443] Zinc finger nuclease

[0444] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein is complexed with a polynucleotide encoding a zinc finger nuclease. Zinc finger nucleases are genome editing nucleases. Zinc finger nucleases are artificial restriction enzymes produced by fusing zinc finger DNA binding domains to DNA cleavage domains. The binding specificity of the designed zinc finger domains guides the zinc finger nuclease to specific genomic sites.

[0445] In some embodiments, the present disclosure relates to compositions and methods for performing gene editing in a subject in need thereof by administering to the subject a therapeutically effective amount of the composition, the 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 bound to the humanized 3E10 antibody or an antigen-binding fragment thereof. In some embodiments, the polynucleotide encoding the zinc finger nuclease is covalently conjugated to the humanized 3E10 antibody or an antigen-binding fragment thereof.

[0446] ZFN technology has been successful in a variety of organisms including fruit flies, zebrafish and rats, and has also been successfully used in human cell culture, such as K562 cells, T cells and hPSC. First, it was demonstrated that the targeted gene driven by ZFN was added to the endogenous CCR5 locus in hESC to generate a green fluorescent protein (GFP) reporter cell line that stably expresses GFP in both undifferentiated and differentiated states for at least 2 months. It is reported that the eGFP gene introduced into the OCT4 (also known as POU5F1) locus truly reflects its transcriptional state, thereby generating an endogenous pluripotency reporter cell line. ZFN-driven gene addition has also been successfully used to introduce fluorescent reporter genes and drug resistance genes into ubiquitous loci (such as AAVS1) for constitutive or inducible expression or differentiated lineage-specific loci (such as PITX3) to monitor pluripotency and track cell differentiation. In all these studies, specific and stable gene addition is achieved with high efficiency without losing pluripotency, and more importantly, the integrated gene maintains high expression levels when stem cells differentiate.

[0447] Although ZFN technology has been successful in target genome editing in hPSC, it still has several limitations. First, two ZFNs and homologous DNA must be delivered to hPSC together, which requires an efficient delivery method, which usually uses a viral vector. However, viral vectors can randomly integrate viral sequences into the target genome, thereby destroying key genes in hPSC. Transfection and electroporation methods or even direct protein delivery methods have been used to avoid viral vectors to introduce ZFN and DNA into the cells being focused on. Another obstacle to the successful application of ZFN technology is that due to the imperfect modular nature of tandem zinc fingers, the design of ZFN is always difficult and time-consuming, wherein the assembled ZFN does not necessarily have a high affinity for the targeted sequence, and the target sequence is a complex of the 3-bp binding sequence of each individual zinc finger. In addition, the imperfect modular structure of zinc finger assembly and the 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 oligomerization pool engineering and directed evolution, have been applied to generate improved ZFNs with optimized DNA-binding specificity and reduced cytotoxicity.

[0448] Transcription activator-like effector nuclease (TALEN)

[0449] In some embodiments, the humanized 3E10 antibody or its antigen-binding fragment described herein is compounded with a polynucleotide encoding a transcription activator-like effector nuclease (TALEN). TALEN is an artificial endonuclease (e.g., a restriction enzyme) and is produced by the fusion of a transcription activator-like effector (TALE) DNA binding domain with a DNA cleavage domain. TALEN can be engineered to bind to any DNA sequence of interest. In some embodiments, the disclosure relates to compositions, and methods for performing gene editing in a subject by administering a therapeutically effective amount of a composition to a subject, the composition comprising a complex formed between (i) a humanized 3E10 antibody or its antigen-binding fragment and (ii) a polynucleotide encoding TALEN. In some embodiments, the polynucleotide encoding TALEN is non-covalently bound to a humanized 3E10 antibody or its antigen-binding fragment. In some embodiments, the polynucleotide encoding TALEN is covalently conjugated to a humanized 3E10 antibody or its antigen-binding fragment.

[0450] In one embodiment, the engineered TALEN includes a first spacer, a TALE DNA binding domain, a second spacer, and a FokI nuclease catalytic domain fused to the C-terminus from the N-terminus to the C-terminus. The DNA cleavage domain cuts the DNA chain, and therefore the fusion with the TALE DNA binding domain may have specificity for editing the DNA sequence of interest of the genome by inducing double-strand breaks. TALEN can work individually, in pairs, or in pairs. For example, the TALE DNA binding domain can bind to targets positioned relative to each other across spacers, wherein the FokI domains gather together to produce breaks in DNA. On the one hand, the TALE DNA binding domain can be designed to be used in the disclosed TALEN. A single TALEN (also referred to as a monomer TALEN or TALEN monomer herein) includes a TALE DNA binding domain and a FokI nuclease catalytic domain fused to the C-terminus. TALEN can be engineered for use in TALEN pairs (or also referred to as a pair of TALEN or TALEN pairs herein), and the TALEN pairs are designed to bind to a target nucleotide sequence configured from the N-terminus to the C-terminus on the relative strand of DNA. The TALENs in a TALEN pair can have the same sequence or can differ in sequence.

[0451] CRISPR / Cas system

[0452] In some embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof described herein is complexed with a polynucleotide encoding a Cas endonuclease. In some embodiments, the present disclosure relates to compositions, and methods for performing gene editing in a subject by administering a therapeutically effective amount of the composition to the subject, the 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.

[0453] 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 the composition, the 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 bound to the humanized 3E10 antibody or antigen-binding fragment thereof. In some embodiments, the CRISPR / Cas guide polynucleotide is covalently conjugated to the humanized 3E10 antibody or antigen-binding fragment thereof.

[0454] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-associated (Cas) endonucleases were originally discovered as adaptive immune systems evolved by bacteria and archaea to prevent viral and plasmid invasion. The CRISPR / Cas system naturally occurring in bacteria consists of one or more Cas genes and one or more CRISPR arrays, which consist of short palindromic repeats of base sequences separated by genome targeting sequences acquired from previously encountered viruses and plasmids (called spacers). (Wiedenheft, B. et al., Nature. 2012; 482: 331; Bhaya, D. et al., Annals of Medicine 2011; 45: 231; and Terms, MP et al., Current Perspectives in Microbiology (Curr. Opin. Microbiol.) 2011; 14: 321). Bacteria and archaea with one or more CRISPR loci respond to viral or plasmid challenges by integrating short fragments of foreign sequences (protospacers) into the host chromosome at the proximal end of the CRISPR array. Transcription of the CRISPR loci produces a library of CRISPR-derived RNA (crRNA) containing 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 crRNA occurs through complementary base pairing with target DNA, which guides the cutting of foreign sequences by Cas proteins. (Jinek et al., 2012 “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Science 2012: 337; 816-821).

[0455] 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 different subtypes. See, for example, Makarova, KS et al., "Natural Microbiology Review (Nat. Rev. Microbiol.)" 2015. "Natural Microbiology Review" 13,722-736, and Liu, Z., Dong, H., Cui, Y. et al., Application of different types of CRISPR / Cas systems in bacteria. "Microb Cell Fact" 19,172 (2020). CRISPR systems are also classified based on their effector proteins. Class 1 systems have multi-subunit crRNA-effect complexes, while in Class 2 systems, all functions of the effector complex are performed by a single protein (e.g., Cas9 or Cpf1). In some embodiments, the present disclosure teaches the use of type II and / or type V single-subunit effector systems. Thus, in some embodiments, the present disclosure teaches the use of a Class 2 CRISPR system.

[0456] CRISPR / Cas9

[0457] In some embodiments, the present disclosure provides a method for gene editing using a type II CRISPR system. In some embodiments, a type II CRISPR system uses a Cas9 enzyme. The type II system relies on i) a single endonuclease protein, ii) a trans-activating crRNA (tracrRNA) and iii) a crRNA, wherein a portion of about 20 nucleotides (nt) at the 5' end of the crRNA is complementary to the target nucleic acid. The region of the CRISPR crRNA chain that is complementary to its target DNA protospacer is hereby referred to as a "guide sequence".

[0458] In some embodiments, the tracrRNA and crRNA components of the type II system can be replaced by a single guide RNA (sgRNA). The sgRNA can include, for example, a nucleotide sequence including at least 12-20 nucleotide sequences complementary to the target DNA sequence (guide sequence) and can include a common scaffold RNA sequence at its 3' end. As used herein, "common scaffold RNA" refers to any RNA sequence that simulates a tracrRNA sequence or any RNA sequence used as a tracrRNA.

[0459] The Cas9 endonuclease generates blunt-ended DNA breaks and is recruited to the target DNA by a combination of crRNA and tracrRNA oligonucleotides, which tether the endonuclease through complementary hybridization of the RNA CRISPR complex.

[0460] 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 with different Cas9 proteins.

[0461] Aptamer

[0462] In some embodiments, the humanized 3E10 antibody or its antigen-binding fragment described herein is complexed with an aptamer or a polynucleotide encoding an aptamer. Nucleic acid aptamers are single-stranded (ss) oligonucleotide molecules (DNA or RNA) folded into different secondary structures or tertiary structures, which give them high affinity and specific binding ability to their corresponding targets (Zhu et al., Nucleic Acid Aptamer-Mediated Drug Delivery for Targeted Cancer Therapy, Chem Med Chem 2015, 10, 39-45). Aptamers are selected from a random library of 1013-1016 ssDNA or ssRNA molecules by an in vitro technique called SELEX (systematic evolution of ligands by exponential enrichment) (Ellington et al., Nature 1990, 346, 818-822; Tuerk et al., Science 1990, 249, 505-510).

[0463] After the aptamer sequence is identified by SELEX, modified nucleotides can be incorporated into the sequence, for example, to improve stability and / or resistance to nuclease degradation and / or increase the efficiency of the aptamer. For example, the aptamer APTA-12 comprises a gemcitabine residue, which is a 2',2'-difluoro analog of 2'deoxycytidine. See, for example, Park JY et al., Mol. Ther. Nucleic Acids 2018, 12, 543-553.

[0464] In general, aptamers can be used in cancer therapy to directly inhibit the activity of a target molecule (where the aptamer acts as a functional therapeutic molecule), or to target a therapeutic molecule (e.g., a chemotherapeutic agent or other anticancer agent) to a 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 a cancer tissue. This is because the humanized 3E10 antibody or antigen-binding fragment thereof complexed with the aptamer is already targeted to various cancer tissues, as described herein. In general, therapeutic aptamers used in cancer therapy act as antagonists of an oncoprotein or one of its ligands by binding to the oncoprotein or one of its ligands, thereby blocking protein-protein or receptor-ligand interactions that promote cancer development and / or progression. For reviews on the use of aptamers to treat cancer, see, for example, 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, Chem. Biochem., 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 et al., Y et al., Cancer (Basel), 2018;10(3):80, the disclosure of which is incorporated herein by reference.

[0465] In some embodiments, as described herein, the present disclosure relates to compositions, and methods for treating a subject in need thereof (e.g., cancer) by administering to the subject a therapeutically effective amount of the composition, the composition comprising a complex formed between (i) a humanized 3E10 antibody or antigen-binding fragment thereof and (ii) an aptamer. 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 conjugated to the humanized 3E10 antibody or antigen-binding fragment thereof.

[0466] 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, a aptamer), AS1411, Sgc8, TD05, ARC1779, α-thrombin (TBA), makugen, E10030, AS1411, ARC1779, NU172, NOX-A12, NOX-E36, NOX-H94, ARC1905, REG1, ARC19499, AS1411, AS1411, EpCAM, A10-3-J1, Sgc8c, TSA14, 5TR1, Endo28, EGFR, A10, Sgc8c, AS1411, NOX-A12, KH1C12, K19, TD05, AS1411, HB5, HeA2_3, H2, S6, SYL3C, APTA-12, M17, S-1, SL2B, CAA01, CA50 A02, CA72-4 A01, APT-43, TA6, CA125.1, Apt928, R13, HF3-58 or HA5-68. In some embodiments, the aptamer is an immune checkpoint regulatory protein. In some embodiments, the immune checkpoint regulatory protein is B7-H3, B7-H4, BTLA, CD160, CTLA4, KIR, LAG3, PD-1, PD-L1, PD-L2, TIM3 or TIGIT.

[0467] Non-limiting examples of aptamers that have been investigated for the treatment of cancer are presented in Table 8 below.

[0468] Table 8. Example aptamers investigated for the treatment of cancer.

[0469]

[0470]

[0471]

[0472]

[0473]

[0474] Thus, in some embodiments, as described herein, 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 those listed 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 conjugated to the humanized 3E10 antibody or antigen-binding fragment thereof.

[0475] In some embodiments, as described herein, 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) an aptamer that specifically binds to a corresponding molecular target selected from those listed in Table 8, wherein the cancer is a cancer associated with a molecular target within Table 8. In some embodiments, the aptamer is non-covalently bound to the humanized 3E10 antibody or an antigen-binding fragment thereof. In some embodiments, the aptamer is covalently conjugated to the humanized 3E10 antibody or an antigen-binding fragment thereof.

[0476] Ribozyme

[0477] In some embodiments, the humanized 3E10 antibodies and antigen-binding fragments thereof described herein are complexed with ribozymes or polynucleotides encoding ribozymes. Ribozymes are RNA molecules with catalytic activity. Ribozymes exist naturally in various sizes and shapes. The ribozymes catalyze the cleavage and ligation of specific phosphodiester bonds. Peptide bond formation during protein synthesis on the ribosome is catalyzed by ribosomal RNA. The biological functions of ribozymes are diverse, and the ribozymes play an important role in transfer RNA maturation, intron splicing, RNA virus or viroid replication, messenger RNA stability regulation and protein synthesis (Westhof et al. Encyclopedia of Virology ( Encyclopedia of Virology ) (3rd edition), 2008). In some embodiments, the ribozyme targets human telomerase reverse transcriptase (hTERT) RNA.

[0478] Thus, in some embodiments, the present disclosure provides compositions, and methods for treating a condition (such as cancer) by administering a therapeutically effective amount of a composition to a subject in need thereof, the composition comprising a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof and (ii) a ribozyme or a polynucleotide encoding a ribozyme. In some embodiments, the ribozyme or the polynucleotide encoding the ribozyme is non-covalently bound to the humanized 3E10 antibody or an antigen-binding fragment thereof. In some embodiments, the ribozyme or the polynucleotide encoding the ribozyme is covalently conjugated to the humanized 3E10 antibody or an antigen-binding fragment thereof.

[0479] Treatment

[0480] In some embodiments, the present disclosure provides compositions and methods for treating a condition by administering a therapeutically effective amount of a composition to a subject in need thereof, the composition comprising a complex formed between (i) a humanized 3E10 antibody or an antigen-binding fragment thereof and (ii) a therapeutic agent. In some embodiments, the therapeutic agent is covalently conjugated to the humanized 3E10 antibody or an antigen-binding fragment thereof.

[0481] In some embodiments, the therapeutic agent is non-covalently bound to the humanized 3E10 antibody or its antigen-binding fragment. In some embodiments, the humanized 3E10 antibody or its antigen-binding fragment is associated with a delivery vehicle of the therapeutic agent, such as conjugated. In some embodiments, the delivery vehicle is a liposome, a lipid nanoparticle, a nanoparticle, a microparticle, a beaded system, a micelle, a biomimetic exosome, or a 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.

[0482] In some embodiments, the therapeutic agent is a DNA damage inducer, a DNA repair inhibitor, an immunomodulatory molecule, an alkylating agent, a microtubule inhibitor, an immune checkpoint inhibitor, an angiogenesis inhibitor, an adoptive cell therapy, and 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, a 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, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), pyrrolobenzodiazepine (PBD), proteolysis targeting chimera (PROTAC), deruxtecan (Dxd), calicheamicin, duocarmycin, interferon gene stimulator (STING) agonist, PNU-159682, NMS249, IMGN Camp1, duocarmycin hydroxybenzamide azaindole (DUBA) or its prodrug. In some embodiments, the therapeutic agent is a maytansinoid alkaloid. In some embodiments, the therapeutic agent is N (2 ') -deacetyl-N (2 ') -(3-mercapto-1-oxopropyl) -maytansinoid (DM1). In some embodiments, the therapeutic agent is N2 '-deacetyl-N2 '-(4-mercapto-4-methyl-1-oxopentyl) maytansinoid (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.

[0483] In some embodiments, the therapeutic agent is a therapeutic polynucleotide, e.g., as described herein. In some embodiments, the therapeutic polynucleotide is non-covalently bound to a humanized 3E10 antibody or an antigen-binding fragment thereof. In some embodiments, the therapeutic polynucleotide is covalently linked to a humanized 3E10 antibody or an antigen-binding fragment thereof. In some embodiments, the therapeutic polynucleotide is a non-replicating, unmodified 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, wherein the therapeutic polynucleotide is a gene regulatory polynucleotide.

[0484] In some embodiments, the condition is cancer. In some embodiments, the cancer is a carcinoma, a sarcoma, a blastoma, a papilloma, or an adenoma. In some embodiments, the cancer is a 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.

[0485] In some embodiments, methods are provided for treating a subject having: carcinomas, blastomas and sarcomas and certain leukemias or lymphoid malignancies, benign and malignant tumors and malignancies (e.g., sarcomas, carcinomas, and melanomas); hematological cancers of the blood or bone marrow; hematological (or blood-borne) cancers, including acute leukemias (such as acute lymphocytic leukemia, acute granulocytic leukemia, acute myeloid leukemia, and myeloblastic leukemia, promyelocytic, myelomonocytic, monocytic, and erythroleukemias); chronic leukemias (such as chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia; solid tumors (such as sarcomas and carcinomas), including fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovial tumors, mesothelioma, Ewing's tumors 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, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumors, seminoma, bladder cancer, melanoma, and CNS tumors such as gliomas (such as brain stem gliomas and mixed gliomas), glioblastomas (also called glioblastoma multiforme), astrocytomas, CNS lymphomas, germ cell tumors, medulloblastomas, Schwannoma craniopharyogioma), ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, or brain metastases.

[0486] In some embodiments, the present disclosure provides methods for treating cancer of the central nervous system by peripherally 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 agent, as described herein.

[0487] 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.

[0488] In one aspect, the present disclosure provides a method for treating a skeletal muscle disease in a subject, the method 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 hereditary skeletal muscle disease. In some embodiments, the mRNA is non-covalently bound to the humanized 3E10 antibody or an antigen-binding fragment thereof. In some embodiments, the mRNA is covalently conjugated to the humanized 3E10 antibody or an antigen-binding fragment thereof. In some embodiments, the mRNA encodes a dystrophin (DMD) or a fragment thereof, such as a micro-dystrophin or a micro-dystrophin construct.

[0489] Stoichiometry of therapeutic polynucleotides

[0490] In some embodiments, for example, where the therapeutic polynucleotide is non-covalently bound to the humanized 3E10 antibody or antigen-binding fragment thereof, the molar ratio of the humanized 3E10 antibody or antigen-binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is at least 2: 1. The molar ratio of the humanized 3E10 antibody or antigen-binding fragment thereof to the therapeutic polynucleotide used in the compositions described herein protects the therapeutic polynucleotide from degradation. For example, the parent 3E10 and 3E10(D31N) variant antibodies protect mRNA from RNase A-mediated RNA degradation at molar ratios of 2: 1 and 20: 1, but the protection provided by the molar ratio of 20: 1 exceeds the protection provided by 2: 1.

[0491] In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is at least 2: 1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is at least 2.5: 1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is at least 5: 1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is at least 7.5: 1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is at least 10: 1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is at least 15: 1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is at least 20: 1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is at least 25: 1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is at least 30: 1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is at least 40: 1.

[0492] In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen-binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is 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 greater.

[0493] In some embodiments, the molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide of the compositions described herein is 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 greater.

[0494] In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein does not exceed 50:1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein does not exceed 40:1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein does not exceed 30:1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein does not exceed 25:1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein does not exceed 20:1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein does not exceed 15:1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein does not exceed 10:1.

[0495] In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen-binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is no more than 50:1, no more than 49:1, no more than 48:1, no more than 47:1, no more than 46:1, no more than 45:1, no more than 44:1, no more than 43:1, no more than 42:1, no more than 41:1, no more than 40:1, no more than 39:1, no more than 38:1, no more than 37:1, no more than 36:1, no more than 35:1, no more than 34:1, no more than 33:1, no more than 32:1, no more than 31:1, No more than 30:1, no more than 29:1, no more than 28:1, no more than 27:1, no more than 26:1, no more than 25:1, no more than 24:1, no more than 23:1, no more than 22:1, no more than 21:1, no more than 20:1, no more than 19:1, no more than 18:1, no more than 17:1, no more than 16:1, no more than 15:1, no more than 14:1, no more than 13:1, no more than 12:1, no more than 11:1, no more than 10:1, no more than 9:1, no more than 8:1, no more than 7:1, no more than 6:1, no more than 5:1 or less.

[0496] In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is 2:1 to 50:1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is 2:1 to 40:1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is 2:1 to 30:1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is 2:1 to 25:1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is 2:1 to 20:1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is 2:1 to 15:1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is 2:1 to 10:1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is 2: 1 to 7.5: 1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is 2: 1 to 5: 1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is 2: 1 to 5: 1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is 2: 1 to 3: 1.

[0497] In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is 5:1 to 50:1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is 5:1 to 40:1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is 5:1 to 30:1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is 5:1 to 25:1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is 5:1 to 20:1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is 5:1 to 15:1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is 5:1 to 10:1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen-binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is 5:1 to 7.5:1.

[0498] In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is 10:1 to 50:1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is 10:1 to 40:1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is 10:1 to 30:1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is 10:1 to 25:1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is 10:1 to 20:1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is 10:1 to 15:1.

[0499] In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is 15: 1 to 50: 1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is 15: 1 to 40: 1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is 15: 1 to 30: 1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is 15: 1 to 25: 1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is 15: 1 to 20: 1.

[0500] In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is 20: 1 to 50: 1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is 20: 1 to 40: 1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is 20: 1 to 30: 1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is 20: 1 to 25: 1.

[0501] In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof of the compositions described herein to the therapeutic polynucleotide is 25: 1 to 50: 1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof of the compositions described herein to the therapeutic polynucleotide is 25: 1 to 40: 1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof of the compositions described herein to the therapeutic polynucleotide is 25: 1 to 30: 1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof of the compositions described herein to the therapeutic polynucleotide is 30: 1 to 50: 1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof of the compositions described herein to the therapeutic polynucleotide is 30: 1 to 40: 1. In yet other embodiments, other ranges falling within the range of 2: 1 to 50: 1 are contemplated.

[0502] In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen-binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is 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 5:1. 1, 10:1 to 30:1, 10:1 to 25:1, 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, 30:1 to 50:1, 30:1 to 40:1, or 40:1 to 50: 1. In still other embodiments, other ranges falling within the range of 2:1 to 50:1 are contemplated.

[0503] In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is 1:1 to 50:1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is 1:1 to 30:1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is 1:1 to 20:1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is 1:1 to 10:1. In some embodiments, the molar ratio of the humanized 3E10 antibody or antigen binding fragment thereof to the therapeutic polynucleotide of the compositions described herein is 1:1 to 5:1.

[0504] In some embodiments, the molar ratio is related to the size of the nucleic acid (ie, therapeutic polynucleotide). For example, longer polynucleotides are complexed at higher molar ratios, and shorter polynucleotides are complexed at lower molar ratios.

[0505] In some embodiments, the size of the therapeutic polynucleotide is 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, 115 bp, 120 bp, 125 bp, 130 bp, 135 bp, 140 bp, 145 bp, 150bp, 155bp, 160bp, 165bp, 170bp, 175bp, 180bp, 185bp, 190bp, 195bp, 200bp, 205bp, 210bp, 21 5bp, 220bp, 225bp, 230bp, 235bp, 240bp, 245bp, 250bp, 255bp, 260bp, 265bp, 270bp, 275bp, 280bp, 28 5bp, 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, 4 25bp, 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.

[0506] In some embodiments, the molar ratios disclosed herein are related to the size of the therapeutic polynucleotide as disclosed herein. For example, longer polynucleotides are complexed at higher molar ratios, and shorter polynucleotides are complexed at lower molar ratios. In some embodiments, any molar ratio disclosed herein can be combined with any size of the therapeutic polynucleotide. Non-limiting examples include compositions having a molar ratio 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, 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 ranges therebetween,wherein 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, 145bp, 15 0bp, 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, 355 bp, 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 therebetween. ,

[0507] Unless otherwise specified or clearly contradicted by the context, all methods described herein may be performed in any suitable order. Unless otherwise required, the use of any and all examples or example language (e.g., "such as") provided herein is intended only to better illustrate the embodiments and does not constitute a limitation on the scope of the embodiments. Any language in this specification should not be interpreted as indicating that any unclaimed element is necessary for practicing the present invention.

[0508] Examples

[0509] Example 1: Binding kinetics and affinity measurements of humanized 3E10 antibody

[0510] Briefly, seven humanized versions of the 3E10 variable heavy domain (VH-h1 to VH-h7) and six humanized versions of the 3E10 variable light domain (VL-h1-VL-h6) were constructed. Figure 5-10 The amino acid sequences of the humanized VH domain, the mature portion of the full HC incorporated into the humanized VH, the full length heavy chain incorporated into the humanized VH, the humanized VL domain, the mature portion of the full LC incorporated into the humanized VL, and the full length heavy chain incorporated into the humanized VL are provided respectively.

[0511] Of the 42 possible humanized 3E10 antibodies that could be generated from these humanized VH and VL domains, twenty-two constructs were generated, as summarized in Table 1. In Table 1, the variant number refers to a specific combination of humanized VH and VL. For example, variant 12 refers to a humanized 3E10 antibody comprising humanized VH-h1 and humanized VL-h2, and variant 41 refers to a humanized 3E10 antibody comprising humanized VH-h4 and humanized VL-h1.

[0512] Table 1 .Humanized 3E10 antibody construct

[0513]

[0514]

[0515] The nucleic acid affinity of 22 humanized 3E10 antibodies was studied using a solid phase ELISA poly-dT binding assay. Briefly, microtiter plates were coated by incubating 100 μl of PBS / well (μg / ml) containing streptavidin at 4°C overnight. After removing the coating solution, the wells were washed twice with 0.05% PBST. The wells were then blocked by incubating for 1 hour at 37°C with PBS containing 1% bovine serum albumin (BSA). After removing the blocking solution, the wells were washed once with 0.05% PBST. The wells were then coated with poly-dT by incubating for 40 minutes at room temperature with 100 μl of ELISA buffer containing 0.1 μM biotin-labeled poly-dT. After removing the coating solution, the wells were washed three times with 0.05% PBST. For each of the 22 humanized antibodies described above, a 3-fold dilution series of antibodies was established from 100 μg / ml to 1.69 ng / ml. 100 μl of each dilution was incubated in a separate well for four hours at 4°C. After removing the dilution solution, the wells were washed three times with 0.05% PBST again. Antibodies bound to poly-dT were detected by incubating 100 μl of a 1:10,000 dilution of a secondary mouse anti-human IgG Fc antibody conjugated with horseradish peroxidase (HRP) in each well for 1.5 hours at room temperature. After removing the secondary antibody solution, the wells were washed three times with 0.05% PBST again. Secondary antibodies were detected by incubating 3,3',5,5'-tetramethylbenzidine (TMB) for 10 minutes in each well.

[0516] Binding curves for various binding assays are shown in Fig. 12A , 12B , 12C, 12D and 12E. EC50 values ​​representing the antibody concentration that achieves half-maximal binding of poly-dT were calculated from the curves and then normalized for the EC50 values ​​determined for each experiment using the chimeric 3E10 D31N variant with murine VH and VL sequences.

[0517] The results indicate that the lysine at position 72 in 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 antibody's affinity for DNA binding. Similarly, the arginine and lysine at positions 37 and 38, respectively, in the variable heavy chain also appear to contribute to the nucleic acid binding activity of 3E10. Mutation of these residues to leucine and arginine, respectively, reduces the antibody's affinity for DNA binding.

[0518] Example 2: Humanized 3E10 antibody-mediated delivery of RIG-I ligands induces type I IFN responses in THP-1 monocytes (low affinity candidate)

[0519] It was investigated whether humanized 3E10 antibody-mediated delivery of RIG-I stimulating ligands to monocytes derived from acute monocytic leukemia effectively induces type I IFN responses characteristic of immunotherapy. Briefly, THP-1 monocytes were seeded into wells and incubated at 20,000 cells / well in DMEM supplemented with 20% FBS and 1% P / S. Cells were then treated with 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 in Fig.13 As indicated in. Sample culture medium was sampled at the indicated time points and luciferase activity (reporter gene for type I IFN) was measured. Fig.13 IFN responses were monitored for four consecutive days, 24 hours, 48 ​​hours, 72 hours and 96 hours as indicated.

[0520] like Fig.13 As shown, exposure of THP-1 monocytes with 3p-hpRNA RIG-I with 3E10 variants 11, 21, 31 or 41 resulted in an average peak increase in type 1 IFN response (approximately 135-fold) 24 hours after treatment, and a steady decrease at 48 hours, 72 hours and 96 hours when compared to control samples (untreated, 3p-hpRNA alone and 3E10 variants 11, 21, 31 or 41 alone).

[0521] Example 3: Humanized 3E10 antibody-mediated delivery of RIG-I ligands induces type I IFN responses in THP-1 monocytes (intermediate affinity candidate)

[0522] It was investigated whether humanized 3E10 antibody-mediated delivery of RIG-I stimulating ligands to monocytes derived from acute monocytic leukemia effectively induces type I IFN responses characteristic of immunotherapy. Briefly, THP-1 monocytes were seeded into wells and incubated at 20,000 cells / well in DMEM supplemented with 20% FBS and 1% P / S. Cells were then treated with 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 in Fig.14 As indicated in. Sample culture medium was sampled at the indicated time points and luciferase activity (reporter gene for type I IFN) was measured. Fig.14 IFN responses were monitored for four consecutive days, 24 hours, 48 ​​hours, 72 hours and 96 hours as indicated.

[0523] like Fig.14As shown, exposure of THP-1 monocytes with 3p-hpRNARIG-I with 3E10 variants 22, 12 or 13 resulted in an average peak increase (about 132-fold) in type 1 IFN responses 72 hours after treatment. At 96 hours, the IFN response began to decrease. The data indicate a mechanism for the 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).

[0524] The experiment was repeated using nonhumanized 3E10WT and 3E10 D31N instead of the humanized antibody. Fig.16 Shown in.

[0525] Example 4: Humanized 3E10 antibody-mediated delivery of RIG-I ligands induces type I IFN responses in THP-1 monocytes (high affinity candidate)

[0526] It was investigated whether humanized 3E10 antibody-mediated delivery of RIG-I stimulating ligands to monocytes derived from acute monocytic leukemia effectively induces type I IFN responses characteristic of immunotherapy. Briefly, THP-1 monocytes were seeded into wells and incubated at 20,000 cells / well in DMEM supplemented with 20% FBS and 1% P / S. Cells were then treated with 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 in Fig.15 As indicated in...

Claims

1. A humanized 3E10 antibody or an antigen-binding fragment thereof, comprising a light chain variable domain (3E10-VL) and a heavy chain variable domain (3E10-VH), wherein: The 3E10-VL comprises an amino acid sequence that is 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), and The 3E10-VH comprises 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).

2. The humanized 3E10 antibody or antigen-binding fragment thereof according to claim 1, wherein the humanized 3E10 antibody or antigen-binding fragment thereof comprises a light chain (3E10-LC) and a heavy chain (3E10-HC), wherein: The 3E10-LC comprises an amino acid sequence that is 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), and The 3E10-HC comprises 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).

3. The humanized 3E10 antibody or antigen-binding fragment thereof according to claim 1, wherein the humanized 3E10 antibody or antigen-binding fragment thereof comprises a light chain (3E10-LC) and a heavy chain (3E10-HC), wherein: The 3E10-LC comprises an amino acid sequence that is 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), and The 3E10-HC comprises an amino acid sequence that is 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).

4. The humanized 3E10 antibody or antigen-binding fragment thereof according to 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 (SEQ ID NO: 85).

5. The humanized 3E10 antibody or antigen-binding fragment thereof according to 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 (SEQ ID NO: 86).

6. The humanized 3E10 antibody or antigen-binding fragment thereof according to 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 (SEQ ID NO: 87).

7. The humanized 3E10 antibody or antigen-binding fragment thereof according to 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 (SEQ ID NO: 88).

8. The humanized 3E10 antibody or antigen-binding fragment thereof according to 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 (SEQ ID NO: 89).

9. The humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the 3E10-VL (SEQ ID NO: 8) comprises an amino acid sequence that is at least 97%, 98%, 99% or 100% identical to 3E10-VL-h6 (SEQ ID NO: 90).

10. The humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the 3E10-VH comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to 3E10-VH-h1 (SEQ ID NO: 64).

11. The humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the 3E10-VH comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to 3E10-VH-h2 (SEQ ID NO: 65).

12. The humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the 3E10-VH comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to 3E10-VH-h3 (SEQ ID NO: 66).

13. The humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the 3E10-VH comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to 3E10-VH-h4 (SEQ ID NO: 67).

14. The humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the 3E10-VH comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to 3E10-VH-h5 (SEQ ID NO: 68).

15. The humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the 3E10-VH comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to 3E10-VH-h6 (SEQ ID NO: 69).

16. The humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the 3E10-VH comprises an amino acid sequence that is at least 95%, 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 according to any one of claims 1 to 3, wherein the 3E10-VH comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to 3E10-VH-h6 (SEQ ID NO: 69), and the 3E10-VL comprises an amino acid sequence that is at least 97%, 98%, 99% or 100% identical to 3E10-VL-h6 (SEQ ID NO: 90).

18. The humanized 3E10 antibody or antigen-binding fragment thereof according to claim 17, wherein the 3E10-VH comprises the amino acid sequence of 3E10-VH-h6 (SEQ ID NO: 69), and the 3E10-VL comprises the amino acid sequence of 3E10-VL-h6 (SEQ ID NO: 90).

19. The humanized 3E10 antibody or antigen-binding fragment thereof according to claim 17, wherein the 3E10-HC comprises the amino acid sequence of 3E10-HC-h6m (SEQ ID NO: 76), and the 3E10-LC comprises the amino acid sequence of 3E10-LC-h6m (SEQ ID NO: 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 20, comprising a set of complementarity determining regions (CDRs) having no more than 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions in common relative to a set of CDRs having the following amino acid sequences: 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 according to any one of claims 1 to 20, comprising a set of CDRs having the following amino acid sequences: 3E10-VL-CDR1 (SEQ ID NO: 9), 3E10-VL-CDR2 (SEQ ID NO: 10), 3E10-VL-CDR3 (SEQ ID NO: 11), 3E10-VH-CDR1_D3 1N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NO: 4) and 3E10-VH-CDR3 (SEQ ID NO: 5).

23. A humanized 3E10 antibody or antigen-binding fragment thereof, comprising 3E10-VL and 3E10-VH, wherein the 3E10-VL comprises: 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), According to Kabat numbering, one or more of the 3E10-VL is selected from the following amino acid residues: 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, and a set of 3E10-VL CDRs having no more than 6 amino acid substitutions in common relative to a set of CDRs having the following amino acid sequences: 3E10-VL-CDR1 (SEQ ID NO: 9), 3E10-VL-CDR2 (SEQ ID NO: 10), 3E10-VL-CDR3 (SEQ ID NO: 11); and Wherein the 3E10-VH comprises: 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), According to Kabat numbering, one or more of the 3E10-VH is selected from the following amino acid residues: 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, A1a at position 84, Va1 at position 89, leucine (Leu) at position 108, Val at position 109 and Ser at position 113; and A set of 3E10-VH CDRs having no more than 6 amino acid substitutions in common relative to a set of CDRs having the following amino acid sequences: 3E10-VH-CDR1_D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NO: 4), and 3E10-VH-CDR3 (SEQ ID NO: 5).

24. The humanized 3E10 antibody or antigen-binding fragment thereof of claim 23, wherein the 3E10-VL comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to 3E10-VL-h1 (SEQ ID NO: 85).

25. The humanized 3E10 antibody or antigen-binding fragment thereof of claim 23, wherein the 3E10-VL comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to 3E10-VL-h2 (SEQ ID NO: 86).

26. The humanized 3E10 antibody or antigen-binding fragment thereof of claim 23, wherein the 3E10-VL comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to 3E10-VL-h3 (SEQ ID NO: 87).

27. The humanized 3E10 antibody or antigen-binding fragment thereof of claim 23, wherein the 3E10-VL comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to 3E10-VL-h4 (SEQ ID NO: 88).

28. The humanized 3E10 antibody or antigen-binding fragment thereof of claim 23, wherein the 3E10-VL comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to 3E10-VL-h5 (SEQ ID NO: 89).

29. The humanized 3E10 antibody or antigen-binding fragment thereof of claim 23, wherein the 3E10-VL (SEQ ID NO: 8) comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to 3E10-VL-h6 (SEQ ID NO: 90).

30. The humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 23 to 29, wherein the 3E10-VH comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to 3E10-VH-h1 (SEQ ID NO: 64).

31. The humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 23 to 29, wherein the 3E10-VH comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to 3E10-VH-h2 (SEQ ID NO: 65).

32. The humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 23 to 29, wherein the 3E10-VH comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to 3E10-VH-h3 (SEQ ID NO: 66).

33. The humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 23 to 29, wherein the 3E10-VH comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to 3E10-VH-h4 (SEQ ID NO: 67).

34. The humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 23 to 29, wherein the 3E10-VH comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to 3E10-VH-h5 (SEQ ID NO: 68).

35. The humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 23 to 29, wherein the 3E10-VH comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to 3E10-VH-h6 (SEQ ID NO: 69).

36. The humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 23 to 29, wherein the 3E10-VH comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to 3E10-VH-h7 (SEQ ID NO: 70).

37. The humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 23 to 36, wherein the set of 3E10-VL CDRs have no more than 5, 4, 3, 2 or 1 amino acid substitutions in common relative to a set of CDRs having the following amino acid sequences: 3E10-VL-CDR1 (SEQ ID NO: 9), 3E10-VL-CDR2 (SEQ ID NO: 10), 3E10-VL-CDR3 (SEQ ID NO: 11).

38. The humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 23 to 36, wherein the set of 3E10-VL CDRs has the following amino acid sequence: 3E10-VL-CDR1 (SEQ ID NO: 9), 3E10-VL-CDR2 (SEQ ID NO: 10), 3E10-VL-CDR3 (SEQ ID NO: 11).

39. The humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 23 to 38, wherein the set of 3E10-VH CDRs have no more than 5, 4, 3, 2 or 1 amino acid substitutions in common relative to a set of CDRs having the following amino acid sequences: 3E10-VH-CDR1_D3 1N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NO: 4) and 3E10-VH-CDR3 (SEQ ID NO: 5).

40. The humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 23 to 38, wherein the set of VH CDRs has the following amino acid sequence: 3E10-VH-CDR1_D3 1N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NO: 4) and 3E10-VH-CDR3 (SEQ ID NO: 5).

41. The humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 1 to 40, comprising a lysine (Lys) residue at position 49 of the 3E10-VL according to Kabat numbering.

42. The humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 1 to 41, comprising a glutamic acid (Glu) residue at position 81 of the 3E10-VL according to Kabat numbering.

43. The humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 1 to 42, comprising an arginine (Arg) residue at position 18 of the 3E10-VH according to Kabat numbering.

44. The humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 1 to 43, comprising a Lys residue at position 19 of the 3E10-VH according to Kabat numbering.

45. The humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 1 to 44, comprising an alanine (Ala) residue at position 49 of the 3E10-VH according to Kabat numbering.

46. ​​The humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 1 to 45, comprising a proline (Pro) residue at position 15 of the 3E10-VL according to Kabat numbering.

47. The humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 1 to 46, comprising a valine (Val) residue at position 104 of the 3E10-VL according to Kabat numbering.

48. The humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 1 to 47, comprising a glutamine (Gln) residue at position 13 of the 3E10-VH according to Kabat numbering.

49. The humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 1 to 48, comprising a leucine (Leu) residue at position 108 of the 3E10-VH according to Kabat numbering.

50. The humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 1 to 49, comprising a Val residue at position 109 of the 3E10-VH according to Kabat numbering.

51. The humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 1 to 50, comprising a serine (Ser) residue at position 113 of the 3E10-VH according to Kabat numbering.

52. The humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 1 to 51, wherein the humanized 3E10 antibody or antigen-binding fragment thereof has a weak binding affinity to the 3p-hpRNA RIG-I agonist having the nucleotide sequence 5' pppGGAGCAAAAGCAGGGUGACAAAGACAUAAUGGAUCCAAACACUGUGUCAAGCUUUCAGGUAGAUUGCUUUCUUUGGCAUGUCCGCAAAC-3' (SEQ ID NO: 103).

53. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1 to 51, wherein the humanized 3E10 antibody or antigen-binding fragment thereof has an intermediate binding affinity for a 3p-hpRNA RIG-I agonist having the nucleotide sequence 5' pppGGAGCAAAAGCAGGGUGACAAAGACAUAAUGGAUCCAAACACUGUGUCAAGCUUUCAGGUAGAUUGCUUUCUUUGGCAUGUCCGCAAAC-3' (SEQ ID NO: 103).

54. The humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 1 to 51, wherein the humanized 3E10 antibody or antigen-binding fragment thereof has a strong binding affinity to the 3p-hpRNA RIG-I agonist having the nucleotide sequence 5' pppGGAGCAAAAGCAGGGUGACAAAGACAUAAUGGAUCCAAACACUGUGUCAAGCUUUCAGGUAGAUUGCUUUCUUUGGCAUGUCCGCAAAC-3' (SEQ ID NO: 103).

55. The humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 1 to 54, wherein the humanized 3E10 antibody or antigen-binding fragment thereof further comprises a fragment crystallizable (Fc) region.

56. The humanized 3E10 antibody or antigen-binding fragment thereof according to claim 55, wherein the Fc region is selected from the group consisting of a human IgG1 Fc region, a human IgG2a Fc region, a human IgG2b Fc region, a human IgG3 Fc region, and a human IgG4 Fc region.

57. The humanized 3E10 antibody or antigen-binding fragment thereof of claim 56, wherein the human Fc region is an IgG1 Fc region.

58. The humanized 3E10 antibody or antigen-binding fragment thereof of claim 56, wherein the human Fc region is an IgG4 Fc region.

59. The humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 56 to 58, wherein the human Fc region comprises an alanine at position 234 and an alanine at position 235 according to Kabat numbering.

60. The humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 56 to 58, wherein the human Fc region comprises an alanine at position 234 and a glutamic acid at position 235 according to Kabat numbering.

61. The humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 56 to 60, wherein the human Fc region comprises aspartic acid at position 297 according to Kabat numbering.

62. The humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 56 to 61, wherein the human Fc region comprises a proline at position 228 according to Kabat numbering.

63. The humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 56 to 62, wherein the human Fc region comprises a glutamine at position 307 and an alanine at position 434 according to Kabat numbering.

64. The humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 56 to 63, wherein the human Fc region comprises a methionine at position 252, a threonine at position 254, and a glutamic acid at position 256 according to Kabat numbering.

65. The humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 56 to 64, wherein the human Fc region comprises a glutamine at position 310 according to Kabat numbering.

66. The humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 1 to 65, wherein the humanized 3E10 antibody or antigen-binding fragment thereof further comprises a heavy chain constant domain (CH1). The humanized 3E10 antibody or antigen-binding fragment thereof according to claim 66 , wherein the CH1 is selected from the group consisting of human γ1 CH1, human γ2 CH1, human γ3 CH1 and human γ4 CH1.

68. The humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 1 to 67, wherein the humanized 3E10 antibody or antigen-binding fragment thereof further comprises a light chain constant domain (CL).

69. The humanized 3E10 antibody or antigen-binding fragment thereof according to claim 68, wherein the CL is selected from the group consisting of human λ CL and human κ CL.

70. The humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 1 to 54, wherein the humanized 3E10 antibody or antigen-binding fragment thereof is a single-chain Fv (scFv) or an antigen-binding fragment (Fab).

71. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1 to 70, wherein the humanized 3E10 antibody or antigen-binding fragment thereof is covalently linked to a therapeutic moiety.

72. The humanized 3E10 antibody or antigen-binding fragment thereof of claim 71, wherein the therapeutic moiety is a therapeutic polynucleotide.

73. The humanized 3E10 antibody or antigen-binding fragment thereof of claim 71, wherein the therapeutic moiety is a therapeutic polypeptide.

74. The humanized 3E10 antibody or antigen-binding fragment thereof of claim 71, wherein the therapeutic moiety is a cytotoxic moiety.

75. The humanized 3E10 antibody or antigen-binding fragment thereof of claim 71, wherein the therapeutic moiety is a chemotherapeutic moiety.

76. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1 to 70, wherein the humanized 3E10 antibody or antigen-binding fragment thereof is covalently linked to a detectable moiety.

77. A humanized 3E10 antibody or antigen-binding fragment thereof, comprising a combination of 3E10-VL and 3E10-VH selected from the group consisting of: 3E10-VL-h1 (SEQ ID NO: 85) and 3E10-VH-h1 (SEQ ID NO: 64), 3E10-VL-h1 (SEQ ID NO: 85) and 3E10-VH-h2 (SEQ ID NO: 65), 3E10-VL-h1 (SEQ ID NO: 85) and 3E10-VH-h3 (SEQ ID NO: 66), 3E10-VL-h1 (SEQ ID NO: 85) and 3E10-VH-h4 (SEQ ID NO: 67), 3E10-VL-h2 (SEQ ID NO: 86) and 3E10-VH-h1 (SEQ ID NO: 64), 3E10-VL-h2 (SEQ ID NO: 86) and 3E10-VH-h2 (SEQ ID NO: 65), 3E10-VL-h2 (SEQ ID NO: 86) and 3E10-VH-h3 (SEQ ID NO: 66), 3E10-VL-h2 (SEQ ID NO: 86) and 3E10-VH-h4 (SEQ ID NO: 67), 3E10-VL-h3 (SEQ ID NO: 87) and 3E10-VH-h1 (SEQ ID NO: 64), 3E10-VL-h3 (SEQ ID NO: 87) and 3E10-VH-h2 (SEQ ID NO: 65), 3E10-VL-h3 (SEQ ID NO: 87) and 3E10-VH-h3 (SEQ ID NO: 66), 3E10-VL-h3 (SEQ ID NO: 87) and 3E10-VH-h4 (SEQ ID NO: 67), 3E10-VL-h4 (SEQ ID NO: 88) and 3E10-VH-h1 (SEQ ID NO: 64), 3E10-VL-h4 (SEQ ID NO: 88) and 3E10-VH-h2 (SEQ ID NO: 65), 3E10-VL-h4 (SEQ ID NO: 88) and 3E10-VH-h3 (SEQ ID NO: 66), 3E10-VL-h4 (SEQ ID NO: 88) and 3E10-VH-h4 (SEQ ID NO: 67), 3E10-VL-h5 (SEQ ID NO: 89) and 3E10-VH-h5 (SEQ ID NO: 68), 3E10-VL-h5 (SEQ ID NO: 89) and 3E10-VH-h6 (SEQ ID NO: 69), 3E10-VL-h6 (SEQ ID NO: 90) and 3E10-VH-h5 (SEQ ID NO: 68), 3E10-VL-h6 (SEQ ID NO: 90) and 3E10-VH-h6 (SEQ ID NO: 69), 3E10-VL-h7 (SEQ ID NO: 91) and 3E10-VH-h5 (SEQ ID NO: 68), and 3E10-VL-h7 (SEQ ID NO:91) and 3E10-VH-h6 (SEQ ID NO:69).

78. A composition comprising a non-covalent complex of: (i) the humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 1 to 77, and (ii) a polynucleotide.

79. The composition of claim 78, wherein the molar ratio of the humanized 3E10 antibody or antigen-binding fragment thereof (i) to the polynucleotide (ii) in the composition is at least 2:1, at least 5:1, at least 20:1, at least 50:1 or at least 100:

1.

80. The composition of claim 78 or 79, wherein the molar ratio of the humanized 3E10 antibody or antigen-binding fragment thereof (i) to the polynucleotide (ii) in the composition is no more than 200:1 or no more than 100:

1.

81. The composition of claim 78, wherein the molar ratio of the humanized 3E10 antibody or antigen-binding fragment thereof (i) to the polynucleotide (ii) in the composition is 2:1 to 50:1, and wherein the length of the polynucleotide does not exceed 2000 nucleotides.

82. The composition of claim 78, wherein the molar ratio of the humanized 3E10 antibody or antigen-binding fragment thereof (i) to the polynucleotide (ii) in the composition is 2:1 to 30:1, and wherein the length of the polynucleotide does not exceed 1000 nucleotides.

83. The composition of claim 78, wherein the molar ratio of the humanized 3E10 antibody or antigen-binding fragment thereof (i) to the polynucleotide (ii) in the composition is 20:1 to 200:1, and wherein the polynucleotide is at least 2000 nucleotides in length.

84. A composition comprising a covalent complex of: (i) the humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 1 to 77, and (ii) a polynucleotide, a polypeptide or a chemical agent.

85. The composition of any one of claims 78 to 84, wherein the polynucleotide is a therapeutic polynucleotide.

86. The composition of claim 85, wherein the therapeutic polynucleotide is a polynucleotide immunostimulatory agent.

87. The composition of claim 86, wherein the polynucleotide immunostimulatory agent is a polynucleotide ligand capable of stimulating a pattern recognition receptor (PRR).

88. The composition of claim 87, wherein the PRR is retinoic acid inducible gene 1 (RIG-I).

89. The composition of claim 87, wherein the polynucleotide ligand comprises a 5' triphosphate and double-stranded RNA.

90. The composition of claim 87, wherein the polynucleotide ligand comprises the nucleic acid sequence 5' pppGGAGCAAAAGCAGGGUGACAAAGACAUAAUGGAUCCAAACACUGUGUCAAGCUUUCAGGUAGAUUGCUUUCUUUGGCAUGUCCGCAAAC-3' (SEQ ID NO: 103).

91. The composition of claim 87, wherein the polynucleotide ligand comprises a 3' phosphate and a hairpin RNA.

92. The composition of claim 87, wherein the PRR is a Toll-like receptor (TLR).

93. The composition of claim 92, wherein the TLR is TLR3, TLR7, TLR8 or TLR9.

94. The composition of claim 87, wherein the PRR is melanoma differentiation-associated protein 5 (MDA5).

95. The composition of claim 86, wherein the polynucleotide immunostimulator is a polynucleotide ligand capable of stimulating cyclic GMP-AMP synthase (cGAS).

96. The composition of claim 86, wherein the polynucleotide immunostimulator is a polynucleotide ligand capable of stimulating stimulator of interferon genes (STING).

97. The composition of claim 85, wherein the therapeutic polynucleotide is a DNA or mRNA encoding a protein or peptide for cancer therapy.

98. The composition of claim 97, wherein the protein or peptide for cancer therapy is a tumor antigen.

99. The composition of claim 98, wherein the tumor antigen is selected from the group consisting of a tumor-associated antigen, a tumor virus protein antigen, a neoantigen, and an antigen derived from a cancer germline gene.

100. The composition of claim 99, wherein the tumor antigen is derived from a protein selected from the group consisting of folate receptor, HER2, papillomavirus oncoprotein E6 and papillomavirus oncoprotein E7 carcinoembryonic antigen (CEA), mucin 1, EGFR, squamous cell carcinoma antigen 3 recognized by T cells (SART3), β-human chorionic gonadotropin (β-hCG), Wilms' Tumor antigen 1 (WT1), survivin, MAGE3, p53, RING finger protein 43 and mitochondrial outer membrane translocase 34 (TOMM34), prostate specific antigen (PSA)-TRICOM and KRAS.

101. The composition of claim 99, wherein the tumor antigen is a neoantigen derived from a mutant protein selected from the group consisting of 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), and cyclin-dependent kinase 4 (CDK4).

102. The composition of claim 99, wherein the tumor antigen is an antigen derived from a cancer germline gene selected from the group consisting of: 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, SPAN XN3, SPANXN4, SPANXN5, XAGE1D, XAGE1C, XAGE1B, XAGE1, XAGE2, XAGE3, XAGE-3b, XAGE-4 / RP11-167P23.2, PAGE1, PAGE2, PAGE2B, PAGE3, PAGE4, ​​LIPI, VENTXP1, IL13RA2, TSP50, CTAGE1, CTAGE-2, CTAGE5, SPA17, ACRBP, CSAG1, CSAG2, DSCR8, MMA1b, DDX53, CT CFL, LUZP4, CASC5, TFDP3, JARID1B, LDHC, MORC1, DKKL1, SPO11, CRISP2, FMR1NB, FTHL17, NXF2, TAF7L, TDRD1, TDRD6, TDRD4, TEX15, FATE1, TPTE, CT45A 1. CT45A2, CT45A3, CT45A4, CT45A5, CT45A6, HORMAD1, HORMAD2, CT47A1, CT47A2, CT47A3, CT47A4, CT47A5, CT47A6, CT47A7, CT47A8, CT47A9, CT47A10, C T47A11, CT47B1, SLCO6A1, TAG, LEMD1, HSPB9, CCDC110, ZNF165, SPACA3, CXorf48, THEG, ACTL8, NLRP4, COX6B2, LOC348120, CCDC33, LOC196993, PASD1,LOC647107, TULP2, CT66 / AA884595, PRSS54, RBM46, CT69 / BC040308, CT70 / BI818097, SPINLW1, TSSK6, ADAM 29. CCDC36, LOC440934, SYCE1, CPXCR1, TSPY3, TSGA10, HIWI, MIWI, PIWI, PIWIL2, ARMC3, AKAP3, Cxorf61, P.S BK, C21orf99, OIP5, CEP290, CABYR, SPAG9, MPHOSPH1, ROPN1, PLAC1, CALR3, PRM1, PRM2, CAGE1, TTK, LY6K, I MP-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, LOC1 30576. ANKRD45, ELOVL4, IGSF11, TMEFF1, TMEFF2, ARX, SPEF2, GPAT2, TMEM108, NOL4, PT PN20A, SPAG4, MAEL, RQCD1, PRAME, TEX101, SPATA19, ODF1, ODF2, ODF3, ODF4, ATAD2, ZNF 645. MCAK, SPAG1, SPAG6, SPAG8, SPAG17, FBXO39, RGS22, A1, C15orf60, CCDC83, TE KT5, NR6A1, TMPRSS12, TPPP2, PRSS55, DMRT1, EDAG, NDR, DNAJB8, CSAG3B, CTAG1A, GAGE1 2B、GAGE12C、GAGE12D、GAGE12E、GAGE12F、GAGE12G、GAGE12H、GAGE12I、GAGE12J、GAGE13、 LOC728137, MAGEA2B, MAGEA9B / LOC728269, NXF2B, SPANXA2, SPANXB2, SPANXE, SSX4B, SS X5, SSX6, SSX7, SSX9, TSPY1D, TSPY1E, TSPY1F, TSPY1G, TSPY1H, TSPY1I, TSPY2, and XAGE1E.

103. The composition of claim 97, wherein the protein or peptide for cancer therapy is a cytokine.

104. The composition of claim 103, wherein the cytokine is selected from IL-1, IL-6, IL-8, IL-12, IFN-γ, IL-18, IL-15, IL-2, TNF-α, IL-10, TGF-β, CSF-1, CCL2, CCL3, CCL5 and VEGF.

105. The composition of any one of claims 97 to 104, wherein the therapeutic polynucleotide is a non-replicating modified or unmodified mRNA.

106. The composition of any one of claims 97 to 104, wherein the therapeutic polynucleotide is a self-amplifying mRNA.

107. The composition of any one of claims 97 to 104, wherein the therapeutic polynucleotide is a plasmid encoding the protein or peptide.

108. The composition of claim 85, wherein the therapeutic polynucleotide is an expression regulating polynucleotide.

109. The composition of claim 108, wherein the expression regulating polynucleotide is siRNA.

110. The composition of claim 109, wherein the siRNA targets an mRNA transcript from a gene selected from the group consisting of KRAS, ERBB2 / HER2, VEGF, SOCS1, PLK1, and BCL2.

111. The composition of claim 108, wherein the expression regulating polynucleotide is a miRNA.

112. The composition of claim 111, wherein the miRNA is selected from 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, and miR-513.

113. The composition of claim 108, wherein the expression modulating polynucleotide is a small activating RNA (saRNA).

114. The composition of claim 113, wherein the saRNA targets the promoter region of the CEBPA gene.

115. The composition of claim 108, wherein the expression regulating polynucleotide is antagomir.

116. The composition of claim 108, wherein the expression regulating polynucleotide is an antisense oligonucleotide.

117. The composition of claim 108, wherein the expression regulating polynucleotide is a decoy oligonucleotide.

118. The composition of claim 85, wherein the therapeutic polynucleotide encodes a genome editing effector.

119. The composition of claim 85, wherein the therapeutic polynucleotide encodes a zinc finger nuclease.

120. The composition of claim 85, wherein the therapeutic polynucleotide encodes a transcription activator-like effector nuclease (TALEN).

121. The composition of claim 85, wherein the therapeutic polynucleotide encodes a CRISPR system comprising a Cas protein and a guide RNA.

122. The composition of claim 85, wherein the therapeutic polynucleotide is an effector polynucleotide.

123. The composition of claim 122, wherein the effector polynucleotide is an aptamer.

124. The composition of claim 123, wherein the aptamer is selected from the group consisting of 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, a aptamer), AS1411, Sgc8, TD05, ARC1779, α-thrombin (TBA), makugen, E10030, AS1411, ARC1779, NU172, NOX-A12, NOX-E36, NOX-H94, ARC1905, REG1, ARC19499, AS1411, AS1411, EpCAM, A10-3-J1, Sgc8c, TSA14, 5TR1, Endo28, EGFR, A10, Sgc8c, AS1411, NOX-A12, KH1C12, K19, TD05, AS1411, HB5, HeA23, H2, S6, SYL3C, APTA-12, M17, S-1, SL2B, CAA01, CA50A02, CA72-4 A01, APT-43, TA6, CA125.1, Apt928, R13, HF3-58 and HA5-68.

125. The composition of claim 122, wherein the effector polynucleotide is a ribozyme.

126. The composition of claim 125, wherein the ribozyme targets human telomerase reverse transcriptase (hTERT) RNA.

127. The composition of claim 84, wherein the chemical agent is selected from the group consisting of: DNA damage inducers, DNA repair inhibitors, immunomodulatory molecules, alkylating agents, microtubule inhibitors, immune checkpoint inhibitors, angiogenesis inhibitors, adoptive cell therapy, and topoisomerase inhibitors.

128. The composition of claim 84, wherein the chemical agent is an anti-tumor drug.

129. The composition of claim 84, wherein the chemical agent is selected from the group consisting of maytansinoids, benzodiazepines, auristatins, tecans, taxoids, CC-1065, (4S)-4,11-diethyl-4,9-dihydroxy-1,4-dihydro-3H,14H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,1 4-dione (SN38), exatecan, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), pyrrolobenzodiazepine (PBD), proteolysis targeting chimera (PROTAC), deruxtecan (Dxd), calicheamicin, duocarmycin, interferon gene stimulator (STING) agonist, PNU-159682, NMS249, IMGNCamp 1, duocarmycin hydroxybenzamide azaindole (DUBA) and its prodrugs.

130. The composition of claim 84, wherein the chemical agent is a maytansinoid.

131. The composition of claim 84, wherein the chemical agent is N(2')-deacetyl-N(2')-(3-mercapto-1-oxopropyl)-maytansinoid (DM1).

132. The composition of claim 84, wherein the chemical agent is N2'-deacetyl-N2'-(4-mercapto-4-methyl-1-oxopentyl) maytansinoid (DM4).

133. A composition according to claim 84, wherein the chemical 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).

134. The composition of claim 84, wherein the chemical agent is PNU- 159682. In some embodiments, the therapeutic agent is PNU-159682.

135. The composition of claim 84, wherein the chemical agent is NMS249.

136. A method for treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 1 to 77.

137. A method for treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition according to any one of claims 78 to 135.

138. The method of claim 136 or 137, wherein the cancer is a carcinoma, sarcoma, blastoma, papilloma, or adenoma.

139. The method of claim 136 or 137, wherein the cancer is a metastatic cancer.

140. The method of any one of claims 136 to 139, wherein the cancer is selected from the group consisting of 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, and uterine cancer.

141. The method of claim 140, wherein the cancer is a skin cancer selected from the group consisting of basal cell carcinoma, squamous cell carcinoma, and melanoma.

142. The method of claim 141, wherein the cancer is melanoma.

143. The method of any one of claims 136 to 139, wherein the cancer is a cancer of the central nervous system.

144. The method of claim 143, wherein the cancer is a neuroepithelial brain or spinal cord tumor selected from the group consisting of medulloblastoma, astrocytic tumor, oligodendroglial tumor, oligoastrocytic tumor, ependymal tumor, choroid plexus tumor, neuronal or mixed neuronal-glial tumor, pineal region tumor, embryonal tumor, or an otherwise unclassified neuroepithelial tumor.

145. The method of any one of claims 136 to 144, wherein the administration is performed by parenteral administration.

146. The method of claim 145, wherein the parenteral administration is intramuscular, intravenous, or subcutaneous.

147. The method of claim 136, wherein the cancer is melanoma and the administering is by parenteral administration.

148. A method for treating a skeletal muscle disease in a subject in need thereof, the method comprising: administering to the subject a therapeutically effective amount of a composition comprising a complex of: (i) a humanized 3E10 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 77, and (ii) an mRNA encoding a protein mutated in an inherited skeletal muscle disease.

149. The method of claim 148, wherein the mRNA encodes dystrophin (DMD).

150. The method of claim 148, wherein the administering is performed by intravenous or subcutaneous administration.

151. The method of any one of claims 148 to 150, wherein the mRNA encoding the protein that is mutated in an inherited skeletal muscle disease is covalently conjugated to the humanized 3E10 antibody or antigen-binding fragment thereof.

152. The method of any one of claims 148 to 150, wherein the mRNA encoding the protein that is mutated in an inherited skeletal muscle disease is non-covalently bound to the humanized 3E10 antibody or antigen-binding fragment thereof.

153. The method of claim 152, wherein the molar ratio of the humanized 3E10 antibody or antigen-binding fragment thereof (i) to the mRNA encoding the protein mutated in a hereditary skeletal muscle disease in the composition is at least 2:1, at least 5:1, at least 20:1, at least 50:1 or at least 100:

1.

154. The method of claim 152 or 153, wherein the molar ratio of the humanized 3E10 antibody or antigen-binding fragment thereof (i) to the mRNA encoding the protein mutated in an inherited skeletal muscle disease in the composition is no more than 200:1 or no more than 100:

1.

155. The method of claim 152, wherein the molar ratio of the humanized 3E10 antibody or antigen-binding fragment thereof (i) to the mRNA encoding the protein mutated in an inherited skeletal muscle disease in the composition is 2:1 to 50:1, and wherein the length of the polynucleotide does not exceed 2000 nucleotides.

156. The method of claim 152, wherein the molar ratio of the humanized 3E10 antibody or antigen-binding fragment thereof (i) to the mRNA encoding the protein mutated in an inherited skeletal muscle disease in the composition is 2:1 to 30:1, and wherein the polynucleotide is no more than 1000 nucleotides in length.

157. The method of claim 152, wherein the molar ratio of the humanized 3E10 antibody or antigen-binding fragment thereof (i) to the mRNA encoding the protein mutated in an inherited skeletal muscle disease in the composition is 20:1 to 200:1, and wherein the polynucleotide is at least 2000 nucleotides in length.

158. A polynucleotide encoding the humanized 3E10 antibody or antigen-binding fragment thereof according to any one of claims 1 to 77.

159. A host cell carrying the polynucleotide composition according to claim 158.

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