Humanized 3e10 antibodies and antigen binding fragments optimized for RAD51 binding

Abstract

The disclosure provides humanized 3E10 antibodies and antigen binding fragments thereof that have been optimized for RAD51 binding. Compositions and methods of using the humanized 3E10 antibodies and antigen binding fragments thereof are also disclosed.

Description

HUMANIZED 3E10 ANTIBODIES AND ANTIGEN BINDING FRAGMENTS OPTIMIZED FOR RAD51 BINDINGCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. Provisional Application No. 63 / 683,631, filed August 15, 2024, is hereby incorporated by reference herein in its entirety.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The content of the electronically submitted sequence listing (Name: is 2681_158PC01_SequenceListing_ST26.xml; Size: 70,427 bytes; and Date of Creation: August 15, 2025) is herein incorporated by reference in its entirety.TECHNICAL FIELD

[0003] The present disclosure relates to humanized 3E10 antibodies and antigen binding fragments thereof that have been optimized for RAD51 binding.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0004] This invention was made with government support under grant numbers R35 CA197574 and R21 CA278934, awarded by National Institutes of Health. The Government has certain rights in the invention.BACKGROUND

[0005] The murine anti-DNA antibody 3E10 is known to penetrate cells and at least partially localizes to the nucleus of the cell. See, for example, Weisbart R.H.et al. 1998. J. Autoimmun.; 11 :539-546., the contents of which is incorporated by reference herein in its entirety. Cell penetration of the 3E10 antibody is dependent on the presence of extracellular DNA as well as the ability to bind DNA, which are important properties for tumor localization as extracellular DNA tends to accumulate in necrotic tumors and is correlated with poor patient survival. The 3E10 antibody is an ideal molecular deliveryvehicle due to its efficiency in penetrating into living cells with specific nuclear localization, absence of toxicity, and successful delivery of therapeutic cargo proteins in vitro and in vivo. Previously reports demonstrated that 3E10 binds to the DNA repair protein RAD51, inhibiting homology-directed repair (HDR), and is consequently synthetically lethal to cells deficient in DNA repair, including BRCA2-deficient and PTEN-deficient cells. Previous studies of humanized version of full-length 3E10, which were engineered to avoid human anti-mouse antibody responses, have been limited in scope however, evaluating only synthetic lethality as opposed to the important nucleic acid binding, nucleic acid delivery, and direct RAD51 binding properties. Accordingly, there is a need in the art for humanized 3E10 antibodies and antigen binding fragments thereof that have been optimized for direct RAD51 binding.SUMMARY

[0006] 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, while demonstrating superior RAD51 binding. Accordingly, as described herein, the present disclosure provides humanized 3E10 antibodies and antigen binding fragments thereof, pharmaceutical compositions, and methods for treating various medical disorders using the same.

[0007] In some aspects, provided herein are humanized 3E10 antibodies or antigenbinding fragments thereof comprising a light chain variable region (VL) and a heavy chain variable region (VH), wherein: the VL comprises a complementary determining region (CDR) 2 comprising the amino acid sequence XYASYLES (SEQ ID NO: 1), wherein X = D, Y, E, F, or W and corresponds to the amino acid residue at position 53 of the 3E10 light chain according to Kabat numbering; and the VH comprises a complementary determining region (CDR) 1 comprising the amino acid sequence XYGMH (SEQ ID NO: 2), wherein X = D, Y, E, F, or W and corresponds to the amino acid residue at position 31 of the 3E10 heavy chain according to Kabat numbering. In some aspects, the VL CDR2 comprises Y, F, or W at the amino acid residue corresponding to position 53 of the 3E10 light chain. In some aspects, the VL CDR2 comprises D or E at the amino acid residue corresponding to position 53 of the 3E10 light chain. In some aspects, the VH CDR1 comprises D or E at the amino acid residuecorresponding to position 31 of the 3E10 heavy chain. In some aspects, the VH CDR1 comprises Y, F, or W at the amino acid residue corresponding to position 31 of the 3E10 heavy chain. In some aspects, the VL CDR2 comprises the amino acid sequence YYASYLES (SEQ ID NO: 3). In some aspects, the VL CDR2 comprises the amino acid sequence FYASYLES (SEQ ID NO: 4). In some aspects, the VL CDR2 comprises the amino acid sequence WYASYLES (SEQ ID NO: 5). In some aspects, the VL CDR2 comprises the amino acid sequence DYASYLES (SEQ ID NO: 6). In some aspects, the VL CDR2 comprises the amino acid sequence EYASYLES (SEQ ID NO: 7). In some aspects, the VH CDR1 comprises the amino acid sequence DYGMH (SEQ ID NO: 8). In some aspects, the VH CDR1 comprises the amino acid sequence YYGMH (SEQ ID NO: 9). In some aspects, the VH CDR1 comprises the amino acid sequence FYGMH (SEQ ID NO: 10). In some aspects, the VH CDR1 comprises the amino acid sequence WYGMH (SEQ ID NO: 11). In some aspects, the VH CDR1 comprises the amino acid sequence EYGMH (SEQ ID NO: 12).

[0008] In some aspects, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 13; a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 14; a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15; and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16.

[0009] In some aspects, provided herein are humanized 3E10 antibodies or antigenbinding fragments thereof comprising a light chain variable domain (3E10-VL) and a heavy chain variable domain (3E10-VH), wherein the 3E10-VL comprises the amino acid sequence set forth in SEQ ID NO: 17; and the 3E10-VH comprises the amino acid sequence set forth in SEQ ID NO: 23.

[0010] In some aspects, provided herein are humanized 3E10 antibodies or antigenbinding fragments thereof comprising a light chain variable domain (3E10-VL) and a heavy chain variable domain (3E10-VH), wherein the 3E10-VL comprises the amino acid sequence set forth in SEQ ID NO: 18; and the 3E10-VH comprises the amino acid sequence set forth in SEQ ID NO: 24.

[0011] In some aspects, provided herein are humanized 3E10 antibodies or antigenbinding fragments thereof comprising a light chain variable domain (3E10-VL) and a heavy chain variable domain (3E10-VH), wherein the 3E10-VL comprises am amino acidsequence set forth in SEQ ID NOs: 19-22; and the 3E10-VH comprises the amino acid sequence set forth in SEQ ID NO: 25-28.

[0012] In some aspects, provided herein are humanized 3E10 antibodies or antigenbinding fragments 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 91% identical to an amino acid sequence of 3E10-VL-hl (SEQ ID NO: 18); and the 3E10-VH comprises an amino acid sequence that is at least 93% identical to an amino acid sequence of 3E10-VH-WT (SEQ ID NO: 24).

[0013] In some aspects, the antibody or antigen-binding fragment thereof comprises one or more amino acid residues selected from proline (P) at position 15, threonine (T) at position 22, tyrosine (Y) at position 49, T at position 74, asparagine (N) at position 76, alanine (A) at position 80, N at position 81, T at position 83, N at position 85, and valine (V) at position 104, of the 3E10-VL according to Kabat numbering. In some aspects, the antibody or antigen-binding fragment thereof comprises one or more amino acid residues selected from glutamine (Q) at position 13, leucine (L) at position 18, arginine (R) at position 19, glycine (G) at position 42, serine (S) at position 49, S at position 77, tyrosine (Y) at position 79, N at position 82, A at position 84, valine (V) at position 89, L at position 108, V at position 109, and S at position 113, of the 3E10-VH according to Kabat numbering. In some aspects, the humanized 3E10 antibody or antigen-binding fragment thereof comprises a lysine (Lys) residue at position 49 of the 3E10-VL according to Kabat numbering. In some aspects, the humanized 3E10 antibody or antigenbinding fragment thereof comprises a glutamic acid (Glu) residue at position 81 of the 3E10-VL according to Kabat numbering. In some aspects, the humanized 3E10 antibody or antigen-binding fragment thereof comprises an arginine (Arg) residue at position 18 of the 3E10-VH according to Kabat numbering. In some aspects, the humanized 3E10 antibody or antigen-binding fragment thereof comprises a Lys residue at position 19 of the 3E10-VH according to Kabat numbering. In some aspects, the humanized 3E10 antibody or antigen-binding fragment thereof comprises an alanine (Ala) residue at position 49 of the 3E10-VH according to Kabat numbering.

[0014] In some aspects, the humanized 3E10 antibody or antigen-binding fragment thereof comprises a proline (Pro) residue at position 15 of the 3E10-VL according to Kabat numbering. In some aspects, the humanized 3E10 antibody or antigen-binding fragment thereof comprises a valine (Vai) residue at position 104, of the 3E10-VLaccording to Kabat numbering. In some aspects, the humanized 3E10 antibody or antigenbinding fragment thereof comprises a glutamine (Gin) residue at position 13, of the 3E10- VH according to Kabat numbering. In some aspects, the humanized 3E10 antibody or antigen-binding fragment thereof comprises a leucine (Leu) residue at position 108, of the 3E10-VH according to Kabat numbering. In some aspects, the humanized 3E10 antibody or antigen-binding fragment comprises a Vai residue at position 109, of the 3E10-VH according to Kabat numbering. In some aspects, the humanized 3E10 antibody or antigenbinding fragment comprises a serine (Ser) residue at position 113, of the 3E10-VH according to Kabat numbering.

[0015] In some aspects, the humanized 3E10 antibody or antigen-binding fragment thereof includes the 3E10-VL comprises the amino acid sequence set forth in SEQ ID NO: 18; and the 3E10-VH comprises the amino acid sequence set forth in SEQ ID NO: 24. In some aspects, the amino acid residue at position 53 of the 3E10 light chain according to Kabat numbering does not comprise a K. In some aspects, the amino acid residue at position 31 of the 3E10 heavy chain according to Kabat numbering does not comprise a N.

[0016] In some aspects, provided herein are humanized 3E10 antibodies or antigenbinding fragment thereof comprises a light chain (LC) and a heavy chain (HC), wherein the 3E10 LC comprises an amino acid sequence that is at least 91% identical to an amino acid sequence of 3E10-LC (SEQ ID NO: 29); and the 3E10 HC comprises an amino acid sequence that is at least 93% identical to an amino acid sequence of 3E10-HC (SEQ ID NO: 30). In some aspects, the 3E10 LC comprises the amino acid sequence set forth in SEQ ID NO: 29; and the 3E10 HC comprises the amino acid sequence set forth in SEQ ID NO: 30.

[0017] In some aspects, the humanized 3E10 antibody or antigen-binding fragment thereof has a strong binding affinity for RAD51. In some aspects, the humanized 3E10 antibody or antigen-binding fragment thereof has a stronger binding affinity for RAD51 compared to a humanized 3E10 antibody or antigen-binding fragment thereof comprising a VL amino acid sequence set forth in SEQ ID NOs: 31. In some aspects, the humanized 3E10 antibody or antigen-binding fragment thereof has a stronger binding affinity for RAD51 compared to a humanized 3E10 antibody or antigen-binding fragment thereof comprising a VH amino acid sequence set forth in SEQ ID NO: 32. In some aspects, the humanized 3E10 antibody or antigen-binding fragment thereof has a stronger bindingaffinity for RAD51 compared to a humanized 3E10 antibody or antigen-binding fragment thereof comprising (i) a VL amino acid sequence set forth in SEQ ID NOs: 31, and / or (ii) a VH amino acid sequence set forth in SEQ ID NO: 32.

[0018] In some aspects, the humanized 3E10 antibody or antigen-binding fragment thereof binds to and inhibits RAD51.

[0019] In some aspects, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises a fragment crystallizable (Fc) region. In some aspects, the human Fc region is an IgGl Fc region. In some aspects, the human Fc region comprises an alanine at position 234 and an alanine at position 235, according to Kabat numbering. In some aspects, the human Fc region comprises an alanine at position 234 and a glutamic acid at position 235, according to Kabat numbering. In some aspects, the human Fc region comprises an aspartic acid at position 297, according to Kabat numbering. In some aspects, the human Fc region comprises a proline at position 228, according to Kabat numbering. In some aspects, the human Fc region comprises a glutamine at position 307 and an alanine at position 434, according to Kabat numbering. In some aspects, 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. In some aspects, the human Fc region comprises a glutamine at position 310, according to Kabat numbering.

[0020] In some aspects, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises a heavy chain constant domain (CHI). In some aspects, the CHI is selected from the group consisting of a human yl CHI, a human y2 CHI, a human y3 CHI, and a human y4 CHI.

[0021] In some aspects, the humanized 3E10 antibody or antigen-binding fragment thereof further comprises a light chain constant domain (CL). In some aspects, the CL is selected from the group consisting of a human Z. CL and a human K CL.

[0022] In some aspects, the humanized 3E10 antibody or antigen-binding fragment thereof is a single-chain Fv (scFv) or an antigen-binding fragment (Fab).

[0023] In some aspects, the humanized 3E10 antibody or antigen-binding fragment thereof is non-covalently associated with a therapeutic moiety. In some aspects, the therapeutic moiety is a therapeutic polypeptide. In some aspects, the therapeutic moiety is a cytotoxic moiety. In some aspects, the therapeutic moiety is a chemotherapeutic moiety. In some aspects, the humanized 3E10 antibody or antigen-binding fragment thereof is covalently linked to a detectable moiety.

[0024] In some aspects, provided herein are compositions comprising a non-covalent complex of: (i) a humanized 3E10 antibody or antigen-binding fragment thereof described herein, and (ii) a polynucleotide. In some aspects, the molar ratio of (i) the humanized 3E10 antibody or antigen-binding fragment thereof to (ii) the polynucleotide in the composition is at least about 2: 1, at least about 5: 1, at least about 20: 1, at least about 50: 1, or at least about 100: 1. In some aspects, the molar ratio of (i) the humanized 3E10 antibody or antigen-binding fragment thereof to (ii) the polynucleotide in the composition is no more than about 2: 1, no more than about 5:1, no more than about 20: 1, no more than about 100: 1, or no more than about 200: 1.

[0025] In some aspects, the polynucleotide is about 15-4000 nucleotides in length, about 15-100 nucleotides in length, about 100-500 nucleotides in length, about 15-40 nucleotides in length, about 1000-4000 nucleotides in length or about 300-900 nucleotides in length. In some aspects, the polynucleotide is at least about 15 nucleotides in length, at least about 25 nucleotides in length, at least about 35 nucleotides in length, at least about 100 nucleotides in length, at least about 300 nucleotides in length, at least about 500 nucleotides in length, at least about 900 nucleotides in length, at least about 1000 nucleotides in length, at least about 2000 nucleotides in length, or at least about 3000 nucleotides in length. In some aspects, the polynucleotide is no more than about 25 nucleotides in length, no more about 35 nucleotides in length, no more than about 100 nucleotides in length, no more than about 300 nucleotides in length, no more than about 500 nucleotides in length, no more than about 900 nucleotides in length, no more than about 1000 nucleotides in length, no more than about 2000 nucleotides in length, no more than about 3000 nucleotides in length, no more than about 4000 nucleotides in length.

[0026] In some aspects, the molar ratio of the humanized 3E10 antibody or antigenbinding fragment thereof (i) to the polynucleotide (ii) in the composition is from 2: 1 to 50: 1, and wherein the polynucleotide is no more than 2000 nucleotides in length. In some aspects, the molar ratio of the humanized 3E10 antibody or antigen-binding fragment thereof (i) to the polynucleotide (ii) in the composition is from 2: 1 to 30: 1, and wherein the polynucleotide is no more than 1000 nucleotides in length. In some aspects, the molar ratio of the humanized 3E10 antibody or antigen-binding fragment thereof (i) to the polynucleotide (ii) in the composition is from 20: 1 to 200: 1, and wherein the polynucleotide is at least 2000 nucleotides in length.

[0027] In some aspects, the polynucleotide is released from the non-covalent complex in less time than a corresponding non-covalent complex comprising a 3E10 antibody or antigen-binding fragment thereof comprising (i) a VL amino acid sequence set forth in SEQ ID NO: 3 land / or (ii) a VH amino acid sequence set forth in SEQ ID NO: 32.

[0028] In some aspects, the non-covalent complex has a larger equilibrium dissociation constant (KD) nanomolar value than a corresponding non-covalent complex comprising a 3E10 antibody or antigen-binding fragment thereof comprising (i) a VL amino acid sequence set forth in SEQ ID NO: 31, and / or (ii) a VH amino acid sequence set forth in SEQ ID NO: 32.

[0029] In some aspects, the non-covalent complex has a KD value of about 10'8to 10'7M. In some aspects, the non-covalent complex has a KD value of about 2 x 10'8to 3 x 10'8M.

[0030] In some aspects, the non-covalent complex has a faster off-rate (Kais) than a corresponding non-covalent complex comprising a 3E10 antibody or antigen-binding fragment thereof comprising (i) a VL amino acid sequence set forth in SEQ ID NO: 31, and / or (ii) a VH amino acid sequence set forth in SEQ ID NO: 32. In some aspects, the non-covalent complex has a Kais value of about 7 x 10'3to 4 x 10'2S'1. In some aspects, the non-covalent complex has a Kais value of about 1 x 10'2to 2 x 10'2S'1.

[0031] In some aspects, provided herein are compositions comprising a covalent complex of: (i) a humanized 3E10 antibody or antigen-binding fragment thereof described herein, and (ii) a polynucleotide, polypeptide, or a chemical agent. In some aspects, the polynucleotide is a therapeutic polynucleotide. In some aspects, the therapeutic polynucleotide is a polynucleotide immunostimulant. In some aspects, the polynucleotide immunostimulant is a polynucleotide ligand capable of stimulating a pattern recognition receptor (PRR). In some aspects, the PRR is retinoic acid-inducible gene I (RIG-I). In some aspects, the polynucleotide ligand comprises a 5’ triphosphate and double-stranded RNA. In some aspects, the polynucleotide ligand comprises the nucleic acid sequence 5 ’ -pppGGAGC AAAAGC AGGGUGAC AAAGAC AUAAUGGAUCC AAAC ACUGUG UCAAGCUUUCAGGUAGAUUGCUUUCUUUGGCAUGUCCGCAAAC- 3’ (SEQ ID NO: 51). In some aspects, the polynucleotide ligand comprises a 3’ phosphate and hairpin RNA. In some aspects, the PRR is a Toll-like receptor (TLR). In some aspects, the TLR is TLR3, TLR7, TLR8, or TLR9. In some aspects, the PRR is melanoma differentiation- associated protein 5 (MDA5). In some aspects, the polynucleotide immunostimulant is a polynucleotide ligand capable of stimulating cyclic-GMP-AMP-synthase (cGAS). Insome aspects, the polynucleotide immunostimulant is a polynucleotide ligand capable of stimulating Stimulator of interferon genes (STING).

[0032] In some aspects, the therapeutic polynucleotide is DNA or mRNA that encodes a protein or peptide for cancer therapy. In some aspects, the protein or peptide for cancer therapy is a tumor antigen. In some aspects, the tumor antigen is selected from a tumor associated antigen, an oncoviral protein antigen, a neoantigen, and an antigen derived from a cancer-germline gene. In some aspects, the tumor antigen is derived from a protein selected from folate receptor, HER2, papillomavirus oncoprotein E6 and papillomavirus oncoprotein E7 carcinoembryonic antigen (CEA), mucin 1, EGFR, squamous cell carcinoma antigen recognized by T cells 3 (SART3), beta-human chorionic gonadotropin (beta-hCG), Wilms’ Tumor antigen 1 (WT1), Survivin, MAGE3, p53, ring finger protein 43 and translocase of the outer mitochondrial membrane 34 (TOMM34), prostate-specific antigen (PSA)-TRICOM, and KRAS. In some aspects, the tumor antigen is a neoantigen derived from a mutant protein selected from BRCA1, BRCA2 BRAF, KRAS, EGFR, IDH1, PIK3CA, ROS1, HLA, JAK1, JAK2, PARK2, ATM, p53, TP53, erbb2 interacting protein (ERBB2IP), Beta-2-Microglobulin (P2m), cyclin-dependent kinase inhibitor 2A (CDKN2A), alternate reading frame (ARF), and cyclin-dependent kinase 4 (CDK4). In some aspects, the tumor antigen is an antigen derived from a cancer-germline gene selected from 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-lb, CTAG2, MAGECI, MAGEC3, SYCP1, BRDT, MAGEC2, SPANXA1, SPANXB1, SPANXC, SPANXD, SPANXN1, SPANXN2, SPANXN3, SPANXN4, SPANXN5, XAGE1D, XAGE1C, XAGE1B, XAGE1, XAGE2, XAGE3, XAGE-3b, XAGE-4 / RP11-167P23.2, XAGE5, DDX43, SAGE1, ADAM2, PAGE5, CT16.2, PAGE1, PAGE2, PAGE2B, PAGE3, PAGE4, LIPI, VENTXP1, IL13RA2, TSP50, CTAGE1, CTAGE-2, CTAGE5, SPA17, ACRBP, CSAG1, CSAG2, DSCR8, MMAlb, 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, H0RMAD1, H0RMAD2, CT47A1, CT47A2, CT47A3, CT47A4, CT47A5, CT47A6, CT47A7, CT47A8, CT47A9, CT47A10,CT47A11, CT47B1, SLC06A1, 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, ADAM29, CCDC36, LOC440934, SYCE1, CPXCR1, TSPY3, TSGA10, HIWI, MIWI, PIWI, PIWIL2, ARMC3, AKAP3, Cxorf61, PBK, C21orf99, OIP5, CEP290, CAB YR, 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 Al, 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, and XAGE1E.

[0033] In some aspects, the protein or peptide for cancer therapy is a cytokine. In some aspects, the cytokine is selected from IL-1, IL-6, IL-8, IL-12, IFN-y, IL-18, IL-15, IL-2, TNF-a, IL-10, TGF-P, CSF-1, CCL2, CCL3, CCL5, and VEGF.

[0034] In some aspects, the therapeutic polynucleotide is a non-replicating modified or unmodified mRNA. In some aspects, the therapeutic polynucleotide is a self-amplifying mRNA.

[0035] In some aspects, the therapeutic polynucleotide is a plasmid encoding the protein or peptide. In some aspects, the therapeutic polynucleotide is an expression-regulating polynucleotide.

[0036] In some aspects, the expression-regulating polynucleotide is an siRNA. In some aspects, the siRNA targets an mRNA transcript from a gene selected from KRAS, ERBB2 / HER2, VEGF, S0CS1, PLK1, and BCL2. In some aspects, the expressionregulating polynucleotide is an miRNA. In some aspects, 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. In some aspects, the expression-regulating polynucleotide is a small-activating RNA (saRNA). In some aspects, the saRNA targets the promoter region of the CEBPA gene. In some aspects, the expression-regulating polynucleotide is an antagomir. In some aspects, the expression-regulating polynucleotide is an antisense oligonucleotide. In some aspects, the expression-regulating polynucleotide is a decoy oligonucleotide.

[0037] In some aspects, the therapeutic polynucleotide encodes a genome editing effector. In some aspects, the therapeutic polynucleotide encodes a zinc-finger nuclease. In some aspects, the therapeutic polynucleotide encodes a transcription activator-like effector nuclease (TALEN). In some aspects, the therapeutic polynucleotide encodes a CRISPR system comprising a Cas protein and a guide RNA.

[0038] In some aspects, the therapeutic polynucleotide is an effector polynucleotide. In some aspects, the effector polynucleotide is an aptamer. In some aspects, the aptamer is selected from a PSMA aptamer, a HER2 aptamer, a MUC1 aptamer, a CD117 aptamer, a PTK7 aptamer, CTLA-4 aptamer, TLS1 la aptamer, PD-1 aptamer, a PD-1 aptamer, a Macugen aptamer, AS1411, Sgc8, TD05, ARC1779, a- Thrombin (TBA), Macugen, 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-l, SL2B, CAA01, CA50 A02, CA72-4 A01, APT-43, TA6, CA125.1, Apt928, R13, HF3-58, and HA5-68. In some aspects, the effector polynucleotide is a ribozyme. In some aspects, the ribozyme targets human telomerase reverse transcriptase (hTERT) RNA.

[0039] In some aspects, the chemical agent is selected from the group consisting of a DNA damage inducing agent, a DNA repair inhibitor, an immune modulatory molecule, an alkylating agent, a microtubule inhibitor, an immune checkpoint inhibitor, an angiogenesis inhibitor, adoptive cell therapy, and a topoisomerase inhibitor. In some aspects, the chemical agent is an anti-tumor drug. In some aspects, the chemical agent is selected from the group consisting of a maytansinoid, a benzodiazepine, an auristatin, a tecan, a taxoid, CC-1065, (4S)-4,l l-Diethyl-4,9-dihydroxy-l,4-dihydro-3H,14H- pyrano[3',4':6,7]indolizino[l,2-b]quinoline-3, 14-dione (SN38), exatecan, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), a pyrrolobenzodiazepine(PBD), PROteolysis TArgeting Chimera (PROTAC), deruxtecan (Dxd), a calicheamicin, a duocarmycin, a stimulator of interferon genes (STING) agonist, PNU- 159682, NMS249, IMGN Camp 1, duocarmycin hydroxybenzamide azaindole (DUB A), and a prodrug thereof. In some aspects, the chemical agent is a maytansinoid. In some aspects, the chemical agent is N(2')-deacetyl-N(2')-(3-mercapto-l-oxopropyl)-maytansine (DM1). In some aspects, the chemical agent is N2’-deacetyl-N2’-(4-mercapto-4-methyl-l- oxopentyl) maytansine (DM4). In some aspects, the chemical agent is (4S)-4,11-Diethyl- 4, 9-dihydroxy-l,4-dihydro-3H,14H-pyrano[3',4':6,7]indolizino[l,2-b]quinoline-3, 14- dione (SN38). In some aspects, the chemical agent is PNU- 159682. In some embodiments, the therapeutic agent is PNU- 159682. In some aspects, the chemical agent is NMS249.

[0040] In some aspects, provided herein are methods for treating a cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a humanized 3E10 antibody or antigen-binding fragment thereof described herein. In some aspects, the method comprising administering to the subject a therapeutically effective amount of a composition described herein.

[0041] In some aspects, the cancer is a carcinoma, a sarcoma, a blastoma, a papilloma, or an adenoma. In some aspects, the cancer is metastatic cancer. In some aspects, 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, renal cancer, thyroid cancer, and uterine cancer. In some aspects, the cancer is a skin cancer selected from the group consisting of basal cell carcinoma, squamous cell carcinoma, and melanoma. In some aspects, the cancer is melanoma. In some aspects, the cancer is a cancer of the central nervous system. In some aspects, the cancer is a neuroepithelial brain or spinal tumor selected from the group consisting of a medulloblastoma, an astrocytic tumor, an oligodendroglial tumor, an oligoastrocytic tumor, an ependymal tumor, a choroid plexus tumor, a neuronal or mixed neuronal-glial tumor, a tumor of the pineal region, an embryonal tumor, or an otherwise uncategorized neuroepithelial tumor. In some aspects, the cancer comprises one or more mutations in one or more DNA damage and response genes. In some aspects, the cancer comprises a mutation in PTEN, FANCA, FANCB, FANCC, FANCD1 / BRCA2, FANCD2, FANCE, FANCF, FANCG,FANCI, FANCJ / BRIP1, FANCL, FANCM, FANCN / PALB2, FANC0 / RAD51C, FANCP / SLX4, FANCQ / ERCC4, FANCR / RAD51, FANCS / BRCA1, FANCT / UBE2T, FANCU / XRCC2, FANCV / REV7, FANCW / RFWD3, FANCY / FAP100, or any combination thereof.

[0042] In some aspects, the humanized 3E10 antibody or antigen-binding fragment thereof is synthetically lethal to the cancer.

[0043] In some aspects, the administering is by parenteral administration. In some aspects, the parenteral administration is intramuscular administration, intravenous administration, or subcutaneous administration.

[0044] In some aspects, the cancer is melanoma and the administering is by parenteral administration.

[0045] In some aspects, provided herein are polynucleotides encoding the humanized 3E10 antibody or antigen-binding fragment thereof described herein. In some aspects, provided herein are host cells harboring the polynucleotide composition described herein. In some aspects, provided herein are methods of making a humanized 3E10 antibody or antigen-binding fragment thereof comprising culturing the host cell described herein under suitable conditions.BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 illustrates in silico electrostatic modeling of V66, V13, and V31 antibodies using IgFold.

[0047] Figures 2A-2B illustrate variable heavy chain (A) and variable light chain (B) sequence alignments, with deviations from WT sequence.

[0048] Figures 3A-3B illustrate representative western blot images (A) and quantification (B) showing antibody RAD51 binding affinity as assessed following coimmunoprecipitation.

[0049] Figures 4A-4B illustrate a schematic (A) and quantification (B) of homology- directed repair (HDR) in U2OS DR-GFP cells containing an inducible GFP reporter.

[0050] Figure 5 illustrates quantification of survival assays in isogenic cell line VC8 ±BRCA2.

[0051] Figure 6 illustrates quantification of survival assays in isogenic cell line U251 ±PTEN.

[0052] Figure 7 illustrates an in silico interaction model of RAD51 (8PBD) and WT 3E10 generated using HADDOCK.

[0053] Figure 8 illustrates an in silico electrostatic modeling of RAD51 (8PBD) with important interaction residues labeled.DETAILED DESCRIPTION

[0054] In various aspects, the present disclosure provides humanized 3E10 antibodies and binding fragments thereof, as well as methods for delivering cargo, e.g., polynucleotides, polypeptides, or chemical agents, into cells using the same.Definitions

[0055] The terminology used in the present disclosure is for the purpose of describing particular aspects only and is not intended to be limiting of the disclosure. As used in the description of the disclosure and the attached claims, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will also be understood that the term “and / or” as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items. Unless the context requires otherwise, it will be further understood that the terms “includes,” “comprising,” or any variation thereof, when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. Furthermore, to the extent that the terms “including,” “includes,” “having,” “has,” “with,” or variants thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

[0056] 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.

[0057] Use of the term “about” is intended to describe values either above or below the stated value in a range of approx. + / - 10%.

[0058] The term “and / or” as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0059] Unless the context requires otherwise, the terms “includes,” “comprising,” 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. Furthermore, to the extent that the terms “including,” “includes,” “having,” “has,” “with,” or variants thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

[0060] 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.

[0061] As used herein, the term “subject” means any individual who is the target of administration. The subject can be a vertebrate, for example, a mammal. Thus, the subject can be a human. The term does not denote a particular age or sex.

[0062] As used herein, the term “pharmaceutically effective amount” means that the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination. The precise dosage will vary according to a variety of factors such as subject-dependent variables (e.g., age, immune system health, etc.), the disease or disorder being treated, as well as the route of administration and the pharmacokinetics of the agent being administered.

[0063] As used herein, the term “carrier” or “excipient” refers to an organic or inorganic ingredient, natural or synthetic inactive ingredient in a formulation, with which one or more active ingredients are combined. The carrier or excipient would naturally be selected to minimize degradation of the active ingredient or to minimize adverse side effects in the subject, as would be well known to one of skill in the art.

[0064] As used herein, the term “treat” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than thecuring of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. As used herein, the term “inhibit” or “reduce” means to decrease an activity, response, condition, disease, or other biological parameter. This can include, but is not limited to, the complete ablation of the activity, response, condition, or disease. This may also include, for example, a statistically significant reduction in the activity, response, condition, or disease as compared to the native or control level.Antibodies and variants thereof

[0065] As used herein, an “antigen binding domain” or “ABD” refers to a set of six Complementary Determining Regions (CDRs) that, when present as part of a polypeptide sequence or sequences, specifically binds a target antigen as discussed herein. As is known in the art, these CDRs are generally present as a first set of variable heavy CDRs (vhCDRs or VHCDRs) and a second set of variable light CDRs (vlCDRs or VLCDRs), each comprising three CDRs: vhCDRl, vhCDR2, vhCDR3 for the heavy chain and vlCDRl, vlCDR2 and vlCDR3 for the light. The CDRs are present in the variable heavy and variable light domains, respectively, and together form an Fv region. Various CDR numbering schemes are known, as outlined in the table and related discussion below. Thus, in some cases, the six CDRs of the antigen binding domain are contributed by a variable heavy and a variable light domain. In a “Fab” format, the set of 6 CDRs are contributed by two different polypeptide sequences, the variable heavy domain (vh or VH; containing the vhCDRl, vhCDR2 and vhCDR3) and the variable light domain (vl or VL; containing the vlCDRl, vlCDR2 and vlCDR3), with the C-terminus of the vh domain being attached to the N-terminus of the CHI domain of the heavy chain and the C-terminus of the vl domain being attached to the N-terminus of the constant light domain (and thus forming the light chain). In a scFv format, the vh and vl domains are covalently attached, generally through the use of a linker (a “scFv linker”) as outlined herein, into a single polypeptide sequence, which can be either (starting from the N- terminus) vh-linker-vl or vl-linker-vh, including optional domain linkers on each side,depending on the format used. In general, the C-terminus of the scFv domain is attached to the N-terminus of the hinge in the second monomer.

[0066] As will be appreciated by those in the art, the exact numbering and placement of the CDRs can be different among different numbering systems. However, it should be understood that the disclosure of a variable heavy and / or variable light sequence includes the disclosure of the associated (inherent) CDRs. Accordingly, the disclosure of each variable heavy region is a disclosure of the vhCDRs (e.g., vhCDRl, vhCDR2 and vhCDR3) and the disclosure of each variable light region is a disclosure of the vlCDRs (e.g., vlCDRl, vlCDR2 and vlCDR3). CDRs can be defined by several different, but overlapping, conventions. For example, by Kabat etal., U.S. Dept, of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983) and by Chothia et al., J. Mol. Biol. 196:901-17 (1987), which are incorporated herein by reference. The Kabat and Chothia definitions include overlapping or subsets of amino acids when compared against each other. Other overlapping CDR definitions can be found, e.g., in Al-Lazikani B. et al., J. Mol. Biol. 273:927-48 (1997); MacCallum et al., J. Mol. Biol. 262:732-45 (1996); Abhinandan and Martin, Mol. Immunol. 45:3832-39 (2008); Lefranc et al., Dev. Comp. Immunol. 27:55-77 (2003); and Honegger and Pliickthun, J. Mol. Biol. 309:657- 70 (2001), which are incorporated herein by reference. Antibody variable domains can also be analyzed, e.g., using the IMGT information system (imgt dot cines dot fr / ) (IMGT® / V-Quest) to identify variable region segments, including CDRs. (See, e.g., Brochet et al., NucL Acids Res. 36:W503-08 (2008). Application of any particular definition (or other definitions known to those of ordinary skill in the art) to refer to a CDR of an antibody or variant thereof is intended to be within the scope of the term as defined and used herein, unless otherwise indicated. CDRs defined by any of these conventions can be easily determined for any given VH or VL amino acid sequence using readily available software applications, such as abYsis (www.abysis.org). While nonlimiting, the CDR sequences provided for the antibodies described herein have been defined according to the Kabat scheme.

[0067] The phrases “amino acid position numbering as in Kabat,” “Kabat position,” and grammatical variants thereof refer to the numbering system used for heavy chain variable domains or light chain variable domains of the compilation of antibodies in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linearamino acid sequence can contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain. For example, a heavy chain variable domain can include a single amino acid insert (residue 52a according to Kabat) after residue 52 of CDR2 and inserted residues (e.g., residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FW residue 82. See Table 1.Table 1.Loop Kab&t Ab Chothia

[0068] The Kabat numbering of residues can be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence. Chothia refers instead to the location of the structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35 A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular’s AbM antibody modeling software.

[0069] For all amino acid positions discussed in the disclosure relating to antibodies, 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 numbering of the EU antibody. Kabat et al. collected numerous primary sequences of the variable regions of heavy chains and light chains. Based on the degree of conservation of the sequences, they classified individual primary sequences into the CDR and the framework and made a list thereof. See, SEQUENCES OF IMMUNOLOGICAL INTEREST, 5th edition, NIH publication,No. 91-3242, E.A. Kabat et al.; Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, the contents of which are incorporated by reference herein in their entirety. The modification can be an addition, deletion, or substitution.

[0070] As used herein, the terms "antibody variant" or "variant antibody" refer to an antibody that differs from a parent antibody by virtue of at least one amino acid modification, "IgG variant" or "variant IgG" as used herein is meant an antibody that differs from a parent IgG (again, in many cases, from a human IgG sequence) by virtue of at least one amino acid modification, and "immunoglobulin variant" or "variant immunoglobulin" as used herein is meant an immunoglobulin sequence that differs from that of a parent immunoglobulin sequence by virtue of at least one amino acid modification. "Fc variant" or "variant Fc" as used herein is meant a protein comprising an amino acid modification in an Fc domain as compared to an Fc domain of human IgGl, IgG2, IgG3, or IgG4, as further described herein.

[0071] In some aspects, a parent polypeptide, e.g., an Fc parent polypeptide, is a human wild type sequence, such as the heavy constant domain or Fc region from IgGl, IgG2, IgG3 or IgG4, although human sequences with variants can also serve as “parent polypeptides,” for example the IgGl / 2 hybrid of US Publication 2006 / 0134105, the contents of which are incorporated by reference herein in their entirety, can be employed. The protein variant sequence herein will preferably possess at least about 75% identity with a parent protein sequence, or at least about 80% identity with a 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. In some aspects, the protein variant sequence herein has at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with a parent protein sequence.

[0072] As used herein, an “isotype” refers to any of the subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions. It should be understood that therapeutic antibodies can also comprise hybrids of isotypes and / or subclasses.

[0073] As used herein, a "Fab" or "Fab region" refers to a polypeptide that comprises the VH, CHI, VL, and CL immunoglobulin domains, generally on two different polypeptidechains (e.g., VH-CH1 on one chain and VL-CL on the other). Fab may refer to this region in isolation, or this region in the context of an antibody of the disclosure. In the context of a Fab, the Fab comprises an Fv region in addition to the CHI and CL domains.

[0074] As used herein, an "Fv" or "Fv fragment" or "Fv region" refers to a polypeptide that comprises the VL and VH domains of an ABD. Fv regions can be formatted as both Fabs (as discussed above, generally two different polypeptides that also include the constant regions as outlined above) and scFvs, where the vl and vh domains are combined (generally with a linker as discussed herein) to form an scFv.

[0075] As used herein, a “single chain Fv” or “scFv” refers to a variable heavy domain covalently attached to a variable light domain, generally using a scFv linker as discussed herein, to form a scFv or scFv domain. A scFv domain can be in either orientation from N- to C-terminus (vh-linker-vl or vl-linker-vh). In the present disclosure, particularly outlined in the figures, the order of the vh and vl domain is indicated in the name, e.g., H.X L.Y which, from N- to C-terminal, is vh-linker-vl, and L.Y H.X is vl-linker-vh.

[0076] As used herein, an “Fc” or “Fc region” or “Fc domain” refers to a polypeptide comprising the CH2-CH3 domains of an IgG molecule, and in some cases, inclusive of the hinge. In EU numbering for human IgGl, the CH2-CH3 domain comprises amino acids 231 to 447, and the hinge is 216 to 230. Thus, the definition of “Fc domain” includes both amino acids 231-447 (CH2-CH3) or 216-447 (hinge-CH2-CH3), or fragments thereof. An “Fc fragment” in this context may contain fewer amino acids from either or both of the N- and C-termini but still retains the ability 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 of particular use in the present disclosure, and can be the Fc domain from human IgGl, IgG2 or IgG4.

[0077] As used herein, a “variant Fc domain” contains amino acid modifications as compared to a parental Fc domain. Thus, a “variant human IgGl Fc domain” is one that contains amino acid modifications (generally amino acid substitutions, although in the case of ablation variants, amino acid deletions are included) as compared to the human IgGl Fc domain. In general, variant Fc domains have at least about 80, about 85, about 90, about 95, about 97, about 98 or about 99 percent identity to the corresponding parental human IgG Fc domain (using the identity algorithms discussed below, with one embodiment utilizing the BLAST algorithm as is known in the art, using defaultparameters). Alternatively, the variant Fc domains can have from 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 as compared to the parental 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 as described herein, such as non-denaturing gel electrophoresis.

[0078] As used herein, the term “heavy chain constant region” refers to the CHl-hinge- CH2-CH3 portion of an antibody (or fragments thereof), excluding the variable heavy domain; in EU numbering of human IgGl this is amino acids 118-447 By “heavy chain constant region fragment” herein is meant a heavy chain constant region that contains fewer amino acids from either or both of the N- and C-termini but still retains the ability to form a dimer with another heavy chain constant region.

[0079] As used herein, the terms "variable region" or “variable domain” refer to the region of an immunoglobulin that comprises one or more Ig domains substantially encoded by any of the VK, V , and / or VH genes that make up the kappa, lambda, and heavy chain immunoglobulin genetic loci respectively, and contains the CDRs that confer antigen specificity. Thus, a “variable heavy domain” pairs with a “variable light domain” to form an antigen binding domain (“ABD”). In addition, each variable domain comprises three hypervariable regions (“complementary determining regions,” “CDRs”) (vhCDRl, vhCDR2 and vhCDR3 for the variable heavy domain and vlCDRl, vlCDR2 and vlCDR3 for the variable light domain) and four framework (FR) regions, arranged from aminoterminus to carboxy -terminus in the following order: FR1-CDR1-FR2-CDR2-FR3- CDR3-FR4.

[0080] As used herein, the term “full-length heavy chain” refers to the entire heavy chain of an antibody, inclusive of the signal peptide (S), having the structure S-VH-CHl-hinge- CH2-CH3.

[0081] As used herein, the term “mature heavy chain” refers to the portion of the heavy chain of the antibody that excludes the signal peptide, having the structure VH-CH1- hinge-CH2-CH3.

[0082] As used herein, the term “full-length light chain” refers to the entire light chain of an antibody, inclusive of the signal peptide (S), having the structure S-VL-CL.

[0083] As used herein, the term “mature light chain” refers to the portion of the light chain of the antibody that excludes the signal peptide, having the structure S-VL-CL.

[0084] As used herein, the terms "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 IgGl comprises a tyrosine and IgG2 a phenylalanine at EU position 296, a F296Y substitution in IgG2 is considered an IgG subclass modification.

[0085] As used herein, the term "non-naturally occurring modification" refers to an amino acid modification that is not isotypic. For example, because none of the human IgGs comprise a serine at position 434, the substitution 434S in IgGl, IgG2, IgG3, or IgG4 (or hybrids thereof) is considered a non-naturally occurring modification.

[0086] The antibodies and antigen-binding fragments thereof of the disclosure are recombinant antibodies that have been engineered to have the various properties described herein and are generally isolated prior to use. As used herein, the term “isolated,” when used to describe the various polypeptides described herein, refers to a polypeptide that has been identified and separated and / or recovered from a cell or cell culture from which it was expressed. Ordinarily, an isolated polypeptide will be prepared by at least one purification step. An “isolated antibody,” refers to an antibody which is substantially free of other antibodies having different antigenic specificities. “Recombinant” means the antibodies are generated using recombinant nucleic acid techniques in exogenous host cells, and they can be isolated as well.

[0087] As used herein, the term “cell-penetrating” refers to an antibody or antigen binding fragment thereof that can penetrate a cell, e.g., a mammalian cell, without the aid of an exogeneous transport vehicle, such as a liposome, or a conjugated cell-penetrating peptide. With respect to 3E10 antibodies and antigen binding fragments thereof, the cellpenetrating antibody or antigen binding fragment thereof can penetrate a cell expressing an ENT2 receptor on its cell surface in the presence of nucleic acids, e.g., non-covalently bound or conjugated to the 3E10 antibody or antigen binding fragment thereof. In some aspects, the cell-penetrating 3E10 antibody or antigen binding fragment thereof is conjugated to a functional molecule, e.g., a chemical agent, polynucleotide, or polypeptide.

[0088] By "variant protein" or "protein variant" or "variant" as used herein is meant a protein that differs from that of a parent protein by virtue of at least one amino acid modification. The protein variant has at least one amino acid modification compared to the parent protein, yet not so many that the variant protein will not align with the parentalprotein using an alignment program such as that described below. In general, variant proteins (such as variant Fc domains, etc., outlined herein, are generally 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, using the alignment programs described below, such as BLAST. Although amino acid sequence modifications to a 3E10 antibody or antigen binding fragment thereof as described herein may produce a protein and / or polypeptide that is referred to as a variant 3E10 antibody or antigen binding fragment thereof, such variants still fall within the classification of a 3E10 antibody or antigen binding fragment thereof as long as they maintain the CDR sequence and cell penetrating activity requirements of a 3E10 antibody or antigen binding fragment thereof.

[0089] Sequence identity between two similar sequences (e.g., antibody variable domains) can be measured by algorithms such as that of Smith, T.F. & Waterman, M.S. (1981) "Comparison Of Biosequences," Adv. Appl. Math. 2:482 [local homology algorithm]; Needleman, S.B. & Wunsch, CD. (1970) "A General Method Applicable To The Search For Similarities In The Amino Acid Sequence Of Two Proteins," J. Mol. Biol., 48:443 [homology alignment algorithm], Pearson, W.R. & Lipman, D.J. (1988) "Improved Tools For Biological Sequence Comparison," Proc. Natl. Acad. Sci. (U.S.A.) 85:2444 [search for similarity method]; or Altschul, S.F. et al, (1990) "Basic Local Alignment Search Tool," J. Mol. Biol. 215:403-10 , the “BLAST” algorithm, see the webpage located at URL blast.ncbi.nlm.nih.gov / Blast.cgi. When using any of the aforementioned algorithms, the default parameters (for Window length, gap penalty, etc.) are used. Unless specifically stated otherwise, sequence identity is determined using the BLAST algorithm, using default parameters3E10 Antibodies, Variants, and Fragments Thereof

[0090] In aspects, the present disclosure relates to humanized 3E10 antibodies and fragments thereof and use of the same for delivering therapeutic agents for the treatment of various diseases. As is discussed herein, humanized 3E10 antibodies that find use in the methods and compositions of the disclosure can incorporate an array of amino acid substitutions in any portion of the antibody.

[0091] Amino acid sequences of 3E10 monoclonal antibodies and binding fragments thereof are known in the art. Example sequences of 3E10 heavy and light chains are provided herein. 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 the murine 3E10, as well as the variable regions and CDRs are provided in the sequence list herein. 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, influences nucleic acid binding and the antibody’s ability to penetrate nuclei. Substitution of the ‘wild type’ (e.g., relative to the original murine antibody) aspartic acid by asparagine (the ‘D3 IN’ mutation) improves nucleic acid binding and nuclei penetration of the antibody, relative to the ‘wild type’ murine 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, Ini. J. Oncol., 25, 1867-1873 (2004).

[0092] Other 3E10 light chain sequences are known in the art. See, for example, Zack, et al., J. Immunol., 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 comprise a tetramer. Each tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one “light” (typically having a molecular weight of about 25 kDa) and one “heavy” chain (typically having a molecular weight of about 50-70 kDa). Human light chains are classified as kappa and lambda light chains. The present disclosure is directed to antibodies that generally are based on the IgG class, which has several subclasses, including, but not limited to IgGl, IgG2, IgG3, and IgG4. In general, IgGl, IgG2 and IgG4 are used more frequently than IgG3. It should be noted that IgGl has different allotypes with polymorphisms at 356 (D or E) and 358 (L or M).

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

[0095] The hypervariable region of an antibody generally encompasses amino acid residues from about amino acid residues 24-34 (LCDR1; “L” denotes light chain), 50-56 (LCDR2) and 89-97 (LCDR3) in the light chain variable region and around about 31-35B (HCDR1; “H” denotes heavy chain), 50-65 (HCDR2), and 95-102 (HCDR3) in the heavy chain variable region; Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) and / or those residues forming 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 useful for the compositions and methods described herein are described below.

[0096] As will be appreciated by those in the art, the exact numbering and placement of the CDRs can be different among different numbering systems. However, it should be understood that the disclosure of a variable heavy and / or variable light sequence includes the disclosure of the associated (inherent) CDRs. Accordingly, the disclosure of each variable heavy region is a disclosure of the vhCDRs (e.g., vhCDRl, vhCDR2 and vhCDR3) and the disclosure of each variable light region is a disclosure of the vlCDRs (e.g., vlCDRl, vlCDR2 and vlCDR3). A useful comparison of CDR numbering is described in Lafranc et al., Dev. Comp. Immunol. 2003. 27(l):55-77 (2003).

[0097] Throughout the present disclosure, the Kabat numbering system is generally used when referring to a residue in the variable domain (approximately, residues 1-107 of the light chain variable region and residues 1-113 of the heavy chain variable region) and the EU numbering system for Fc regions (e.g., Kabat et al., supra (1991)).

[0098] The present disclosure provides CDR sets. In this case, a “full CDR set” comprises the three variable light and three variable heavy CDRs, e.g., a vlCDRl, vlCDR2, vlCDR3, vhCDRl, vhCDR2 and vhCDR3. These can be part of a larger variable light or variable heavy domain, respectfully. In addition, as more fully outlined herein, the variable heavy and variable light domains can be on separate polypeptide chains, when a heavy and light chain is used (for example when Fabs are used), or on a single polypeptide chain in the case of scFv sequences. The CDRs contribute to the formation of the antigen-binding, or more specifically, epitope binding site of antibodies. “Epitope” refers to a determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope.

[0099] Epitopes are groupings of molecules such as nucleic acids, amino acids, or sugar side chains and usually have specific structural characteristics, as well as specific charge characteristics. A single antigen may have more than one epitope. The antibodies described herein bind to nucleic acid epitopes in a partially sequence-independent manner. That is, while the antibodies described herein bind to some polynucleotide structures and sequences with greater affinity than other nucleic acid structures and sequences, they have some general affinity for polynucleotides.

[0100] The “Fc domain” of the heavy chain includes the -CH2-CH3 domain, and optionally a hinge domain (-H-CH2-CH3). For IgG, the Fc domain comprises immunoglobulin domains CH2 and CH3 (Cy2 and Cy3) and the lower hinge region between CHI (Cyl) and CH2 (Cy2). Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined to include residues C226 or P230 to its carboxyl-terminus, wherein the numbering is according to the EU index as in Kabat. Accordingly, “CH” domains in the context of IgG are as follows: “CHI” 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, the “Fc domain” includes the -CH2-CH3 domain, and optionally a hinge domain (hinge-CH2-CH3). In the aspects herein, when a scFv is attached to an Fc domain, it is generally the C-terminus of the scFv construct that is attached to all or part of the hinge of the Fc domain; for example, it is generally attached to the sequence EPKS which is the beginning of the hinge. In some aspects, as is more fully described below, amino acid modifications are made to the Fc region, for example to alter binding to one or more FcyR receptors or to the FcRn receptor, and to enable heterodimer formation and purification, as outlined herein.

[0101] Another part of the heavy chain is the hinge region. By “hinge” or “hinge region” or “antibody hinge region” or “hinge domain” herein is meant the flexible polypeptide comprising the amino acids between the first and second constant domains of an antibody. Structurally, the IgG CHI domain ends at EU position 215, and the IgG CH2 domain begins at residue EU position 231. Thus, for IgG the antibody hinge is herein defined to include positions 216 (E216 in IgGl) to 230 (p230 in IgGl), wherein the numbering is according to the EU index as in Kabat. In some cases, a “hinge fragment” isused, which contains fewer amino acids at either or both of the N- and C-termini of the hinge domain.

[0102] An scFv comprises a variable heavy chain, an scFv linker, and a variable light domain. In most of the constructs and sequences outlined herein, the C-terminus of the variable heavy chain is attached to the N-terminus of the scFv linker, the C-terminus of which is attached to the N-terminus of a variable light chain (N-vh-linker-vl-C) although that can be switched (N-vl- linker-vh-C).

[0103] Thus, the present disclosure relates to different antibody domains. As described herein and known in the art, the heterodimeric antibodies described in certain aspects of the disclosure comprise different domains within the heavy and light chains, which can be overlapping as well. These domains include, but are not limited to, the Fc domain, the CHI domain, the CH2 domain, the CH3 domain, the hinge domain, the heavy constant domain (CHI - hinge-Fc domain or CHl-hinge-CH2-CH3), the variable heavy domain, the variable light domain, the light constant domain, Fab domains and scFv domains.

[0104] In certain aspects, the antibodies of the disclosure comprise a heavy chain variable region from a particular germline heavy chain immunoglobulin gene and / or a light chain variable region from a particular germline light chain immunoglobulin gene. For example, such antibodies may comprise or consist of a human antibody comprising heavy or light chain variable regions that are "the product of or "derived from" a particular germline sequence, e.g., that of the 3E10 antibody. A human antibody that is "the product of or "derived from" a human germline immunoglobulin sequence can be identified as such by comparing the amino acid sequence of the human antibody to the amino acid sequences of human germline immunoglobulins and selecting the human germline immunoglobulin sequence that is closest in sequence (i.e., greatest % identity) to the sequence of the human antibody (using the methods outlined herein). A human antibody that is "the product of or "derived from" a particular human germline immunoglobulin sequence may contain amino acid differences as compared to the germline sequence, due to, for example, naturally occurring somatic mutations or intentional introduction of site- directed mutation. However, a humanized antibody typically is at least 90% identical in amino acids sequence to an amino acid sequence encoded by a human germline immunoglobulin gene and contains amino acid residues that identify the antibody as being derived from human sequences when compared to the germline immunoglobulin amino acid sequences of other species (e.g., murine germline sequences). In certain cases,a humanized antibody may be at least 95, 96, 97, 98 or 99%, or even at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, a humanized antibody derived from a particular human germline sequence will display no more than 10-20 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene. In certain cases, the humanized antibody may display no more than 5, or even no more than 4, 3, 2, or 1 amino acid difference from the amino acid sequence encoded by the germline immunoglobulin gene.

[0105] In some aspects, the present disclosure relates to the use of antigen binding domains (ABDs) that bind to nucleic acids, and specifically that bind to therapeutic polynucleotides used to a disease, e.g., cancer, derived from a 3E10 antibody.

[0106] A humanized 3E10 antibody or antigen binding fragment thereof is capable of being transported into the cytoplasm and / or nucleus of the cells without the aid of a carrier or conjugate. For example, the monoclonal antibody 3E10 and active fragments thereof that are transported in vivo to the nucleus of mammalian cells without cytotoxic effect are disclosed in U.S. Patent Nos. 4,812,397 and 7,189,396 to Richard Weisbart.

[0107] Humanized 3E10 antibodies and binding fragments thereof that can be used in the compositions and methods include whole immunoglobulin (i.e., an intact antibody) of any class, fragments thereof, and synthetic proteins containing at least the nucleic acid and ENT2 binding activities that are the hallmark of a 3E10 antibody.Humanized 3E10 antibodies and Antigen Fragments Thereof

[0108] The humanized antibodies described herein are useful as a single agent or for combined treatment of diseases, e.g., cancers. Abnormalities in DNA repair and genomic instability, including HDR deficiency, represent a key hallmark of cancer and constitute one of the most common drivers of tumorigenesis. RAD51, a key factor in homology- directed repair (HDR), has long been considered an attractive target for cancer therapy. See, e.g., Turchick, et al., Nucleic Acids Res., 45(20): 11782-11799 (2017), WO 2020 / 047344, and WO 2020 / 047353, which is specifically incorporated by reference herein, in its entirety. Thus, understanding the determinants of the 3E10-RAD51 interaction is imperative for the advancement of a 3E10 antibody for the treatment of patients with DNA-repair deficient tumors. As described in the Examples, humanized variants of the full-length, cell-penetrating, and nucleic-acid binding 3E10 antibody werecompared based on their nucleic acid binding affinities. As described therein, an example human variant designated as a RAD51 “superbinder” with optimized HDR-deficient tumor synthetic lethality was generated, with additional variants described herein.

[0109] Generally, a humanized antibody is the result of a process in which the sequence of a parental antibody from a non-human species is modified to increase the overall similarity of the parental antibody to human antibodies, while retaining antigen binding activity of the parental antibody. Generally, the process involves identifying a human antibody, sometimes referred to as a scaffold antibody, and then either (i) replacing amino acids in the parent (non-human) antibody with equivalent amino acids from the scaffold (human) antibody, e.g., framework amino acids having little to no effect on antigen binding or (ii) replacing amino acids in the scaffold (human) antibody with equivalent amino acids from the parent (non-human) antibody, e.g., CDRs and other amino acids with significant effects on antigen binding. Various methods for humanization are known in the art, including framework-homology-based humanization, germline humanization, complementary determining regions (CDR)-homology-based humanization, and specificity determining residues (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).

[0110] Accordingly, in some aspects the disclosure provides a humanized 3E10 antibody or antigen-binding fragment thereof comprising a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises a complementary determining region (CDR) 2 comprising the amino acid sequence XYASYLES (SEQ ID NO: 1), wherein X = D, Y, E, F, or W and corresponds to the amino acid residue at position 53 of the 3E10 light chain according to Kabat numbering; and the VH comprises a complementary determining region (CDR) 1 comprising the amino acid sequence XYGMH (SEQ ID NO: 2), wherein X = D, Y, E, F, or W and corresponds to the amino acid residue at position 31 of the 3E10 heavy chain according to Kabat numbering.[OHl] In some aspects, the humanized 3E10 antibody or antigen-binding fragment thereof described herein includes the VL CDR2 comprises Y, F, or W at the amino acid residue corresponding to position 53 of the 3E10 light chain. In some aspects, the VL CDR2 comprises the amino acid sequence YYASYLES (SEQ ID NO: 3). In some aspects, the VL CDR2 comprises the amino acid sequence FYASYLES (SEQ ID NO: 4).In some aspects, the VL CDR2 comprises the amino acid sequence WYASYLES (SEQ ID NO: 5).

[0112] In some aspects, the humanized 3E10 antibody or antigen-binding fragment thereof described herein includes the VL CDR2 comprises D or E at the amino acid residue corresponding to position 53 of the 3E10 light chain. In some aspects, the VL CDR2 comprises the amino acid sequence DYASYLES (SEQ ID NO: 6). In some aspects, the VL CDR2 comprises the amino acid sequence EYASYLES (SEQ ID NO: 7).

[0113] In some aspects, the humanized 3E10 antibody or antigen-binding fragment thereof described herein includes the VH CDR1 comprises Y, F, or W at the amino acid residue corresponding to position 31 of the 3E10 heavy chain. In some aspects, the VH CDR1 comprises the amino acid sequence YYGMH (SEQ ID NO: 9). In some aspects, the VH CDR1 comprises the amino acid sequence FYGMH (SEQ ID NO: 10). In some aspects, the VH CDR1 comprises the amino acid sequence WYGMH (SEQ ID NO: 11).

[0114] In some aspects, the humanized 3E10 antibody or antigen-binding fragment thereof described herein includes the VH CDR1 comprises D or E at the amino acid residue corresponding to position 31 of the 3E10 heavy chain. In some aspects, the VH CDR1 comprises the amino acid sequence DYGMH (SEQ ID NO: 8). In some aspects, the VH CDR1 comprises the amino acid sequence EYGMH (SEQ ID NO: 12).

[0115] In some aspects, the humanized 3E10 antibody or antigen-binding fragment thereof described herein further comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 13; a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 14; a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15; and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16.

[0116] In some aspects, the disclosure provides a humanized 3E10 antibody or antigenbinding fragment thereof comprising a light chain variable domain (3E10-VL) and a heavy chain variable domain (3E10-VH), wherein the 3E10-VL comprises the amino acid sequence set forth in SEQ ID NO: 18; and the 3E10-VH comprises the amino acid sequence set forth in SEQ ID NO: 24.

[0117] In some aspects, the disclosure provides a humanized 3E10 antibody or antigenbinding fragment thereof comprising a light chain variable domain (3E10-VL) and a heavy chain variable domain (3E10-VH), wherein the 3E10-VL comprises am amino acid sequence set forth in SEQ ID NOs: 17; and the 3E10-VH comprises the amino acidsequence set forth in SEQ ID NO: 23. In some aspects, the 3E10-VL comprises am amino acid sequence set forth in SEQ ID NO: 18. In some aspects, the 3E10-VL comprises am amino acid sequence set forth in SEQ ID NO: 19. In some aspects, the 3E10-VL comprises an amino acid sequence set forth in SEQ ID NO: 20. In some aspects, the 3E10-VL comprises am amino acid sequence set forth in SEQ ID NO: 21. In some aspects, the 3E10-VL comprises an amino acid sequence set forth in SEQ ID NO: 22. In some aspects, the 3E10-VH comprises the amino acid sequence set forth in SEQ ID NO: 24. In some aspects, the 3E10-VH comprises the amino acid sequence set forth in SEQ ID NO: 25. In some aspects, the 3E10-VH comprises the amino acid sequence set forth in SEQ ID NO: 26. In some aspects, the 3E10-VH comprises the amino acid sequence set forth in SEQ ID NO: 27. In some aspects, the 3E10-VH comprises the amino acid sequence set forth in SEQ ID NO: 28. In some aspects, wherein the 3E10-VL comprises am amino acid sequence set forth in SEQ ID NO: 18; and the 3E10-VH comprises the amino acid sequence set forth in SEQ ID NO: 24.

[0118] In some aspects, the disclosure provides a humanized 3E10 antibody or antigenbinding 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 91% identical to an amino acid sequence of 3E10-VL-hl (SEQ ID NO: 18); and the 3E10-VH comprises an amino acid sequence that is at least 93% identical to an amino acid sequence of 3E10-VH-WT (SEQ ID NO: 24). In some aspects, the 3E10-VL does not comprise the amino acid sequence of 3E10-VL-h6 (SEQ ID NO: 31); and the 3E10-VH does not comprise the amino acid sequence of 3E10-VH-h6 (SEQ ID NO: 32).

[0119] In some aspects, the 3E10-VL comprises an amino acid sequence that is at least 92% identical to an amino acid sequence of 3E10-VL-hl (SEQ ID NO: 18). In some aspects, the 3E10-VL comprises an amino acid sequence that is at least 93% identical to an amino acid sequence of 3E10-VL-hl (SEQ ID NO: 18). In some aspects, the 3E10-VL comprises an amino acid sequence that is at least 94% identical to an amino acid sequence of 3E10-VL-hl (SEQ ID NO: 18). In some aspects, the 3E10-VL comprises an amino acid sequence that is at least 95% identical to an amino acid sequence of 3E10-VL- hl (SEQ ID NO: 18). In some aspects, the 3E10-VL comprises an amino acid sequence that is at least 96% identical to an amino acid sequence of 3E10-VL-hl (SEQ ID NO: 18). In some aspects, the 3E10-VL comprises an amino acid sequence that is at least 97%identical to an amino acid sequence of 3E10-VL-hl (SEQ ID NO: 18). In some aspects, the 3E10-VL comprises an amino acid sequence that is at least 98% identical to an amino acid sequence of 3E10-VL-hl (SEQ ID NO: 18). In some aspects, the 3E10-VL comprises an amino acid sequence that is at least 99% identical to an amino acid sequence of 3E10-VL-hl (SEQ ID NO: 18). In some aspects, the 3E10-VL comprises the amino acid sequence set forth in 3E10-VL-hl (SEQ ID NO: 18). In some aspects, the 3E10-VL does not comprise the amino acid sequence of 3E10-VL-h6 (SEQ ID NO: 31).

[0120] In some aspects, the 3E10-VH comprises an amino acid sequence that is at least 93% identical to an amino acid sequence of 3E10-VH-WT (SEQ ID NO: 24). In some aspects, the 3E10-VH comprises an amino acid sequence that is at least 94% identical to an amino acid sequence of 3E10-VH-WT (SEQ ID NO: 24). In some aspects, the 3E10- VH comprises an amino acid sequence that is at least 95% identical to an amino acid sequence of 3E10-VH-WT (SEQ ID NO: 24). In some aspects, the 3E10-VH comprises an amino acid sequence that is at least 96% identical to an amino acid sequence of 3E10- VH-WT (SEQ ID NO: 24). In some aspects, the 3E10-VH comprises an amino acid sequence that is at least 97% identical to an amino acid sequence of 3E10-VH-WT (SEQ ID NO: 24). In some aspects, the 3E10-VH comprises an amino acid sequence that is at least 98% identical to an amino acid sequence of 3E10-VH-WT (SEQ ID NO: 24). In some aspects, the 3E10-VH comprises an amino acid sequence that is at least 99% identical to an amino acid sequence of 3E10-VH-WT (SEQ ID NO: 24). In some aspects, the 3E10-VH comprises the amino acid sequence set forth in 3E10-VH-WT (SEQ ID NO: 24). In some aspects, the 3E10-VH does not comprise the amino acid sequence of 3E10- VH-h6 (SEQ ID NO: 32).

[0121] In some aspects, a humanized 3E10 antibody or antigen binding fragment thereof described herein comprises one or more amino acid residues selected from proline (Pro) at position 15, threonine (Thr) at position 22, tyrosine (Tyr) at position 49, Thr at position 74, asparagine (Asn) at position 76, alanine (Ala) at position 80, Asn at position 81, Thr at position 83, Asn at position 85, and valine (Vai) at position 104, of the 3E10-VL according to Kabat numbering. In some aspects, the humanized 3E10 antibody or antigenbinding fragment thereof described herein comprises a lysine (Lys) residue at position 49 of the 3E10-VL according to Kabat numbering. In some aspects, the humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a glutamic acid (Glu) residue at position 81 of the 3E10-VL according to Kabat numbering.

[0122] In some aspects, the humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a proline (Pro) residue at position 15 of the 3E10-VL according to Kabat numbering. In some aspects, the humanized 3E10 antibody or antigenbinding fragment thereof described herein comprises a valine (Vai) residue at position 104, of the 3E10-VL according to Kabat numbering.

[0123] In some aspects, a humanized 3E10 antibody or antigen binding fragment thereof described herein comprises one or more amino acid residues selected from glutamine (Gin) 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, Vai at position 89, leucine (Leu) at position 108, Vai at position 109, and Ser at position 113, of the 3E10-VH according to Kabat numbering. In some aspects, the humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises an arginine (Arg) residue at position 18 of the 3E10-VH according to Kabat numbering. In some aspects, the humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a Lys residue at position 19 of the 3E10-VH according to Kabat numbering. In some aspects, the humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises an alanine (Ala) residue at position 49 of the 3E10-VH according to Kabat numbering.

[0124] In some aspects, the humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a glutamine (Gin) residue at position 13, of the 3E10- VH according to Kabat numbering. In some aspects, the humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a leucine (Leu) residue at position 108, of the 3E10-VH according to Kabat numbering. In some aspects, the humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a Vai residue at position 109, of the 3E10-VH according to Kabat numbering. In some aspects, the humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises a serine (Ser) residue at position 113, of the 3E10-VH according to Kabat numbering.

[0125] In some aspects, the humanized 3E10 antibody or antigen-binding fragment thereof described herein comprises the 3E10-VL comprising the amino acid sequence set forth in SEQ ID NO: 18; and the 3E10-VH comprising the amino acid sequence set forth in SEQ ID NO: 24.

[0126] In some aspects, the amino acid residue at position 53 of the 3E10 light chain according to Kabat numbering does not comprise a K. In some aspects, the amino acid residue at position 31 of the 3E10 heavy chain according to Kabat numbering does not comprise a N.

[0127] In some aspects, the disclosure provides a humanized 3E10 antibody or antigenbinding fragment thereof comprising a light chain (LC) and a heavy chain (HC), wherein the 3E10 LC comprises an amino acid sequence that is at least 91% identical to an amino acid sequence of 3E10-LC (SEQ ID NO: 29); and the 3E10 HC comprises an amino acid sequence that is at least 93% identical to an amino acid sequence of 3E10-HC (SEQ ID NO: 30). In some aspects, the 3E10 LC comprises the amino acid sequence set forth in SEQ ID NO: 29; and the 3E10 HC comprises the amino acid sequence set forth in SEQ ID NO: 30.

[0128] In some aspects, the 3E10 LC comprises an amino acid sequence that is at least 92% identical to an amino acid sequence of 3E10-LC (SEQ ID NO: 29). In some aspects, the 3E10 LC comprises an amino acid sequence that is at least 93% identical to an amino acid sequence of 3E10-LC (SEQ ID NO: 29). In some aspects, the 3E10 LC comprises an amino acid sequence that is at least 94% identical to an amino acid sequence of 3E10-LC (SEQ ID NO: 29). In some aspects, the 3E10 LC comprises an amino acid sequence that is at least 95% identical to an amino acid sequence of 3E10-LC (SEQ ID NO: 29). In some aspects, the 3E10 LC comprises an amino acid sequence that is at least 96% identical to an amino acid sequence of 3E10-LC (SEQ ID NO: 29). In some aspects, the 3E10 LC comprises an amino acid sequence that is at least 97% identical to an amino acid sequence of 3E10-LC (SEQ ID NO: 29). In some aspects, the 3E10 LC comprises an amino acid sequence that is at least 98% identical to an amino acid sequence of 3E10-LC (SEQ ID NO: 29). In some aspects, the 3E10 LC comprises an amino acid sequence that is at least 99% identical to an amino acid sequence of 3E10-LC (SEQ ID NO: 29). In some aspects, the 3E10 LC comprises the amino acid sequence set forth in SEQ ID NO: 29.

[0129] In some aspects, the 3E10 HC comprises an amino acid sequence that is at least 94% identical to an amino acid sequence of 3E10-HC (SEQ ID NO: 30). In some aspects, the 3E10 HC comprises an amino acid sequence that is at least 95% identical to an amino acid sequence of 3E10-HC (SEQ ID NO: 30). In some aspects, the 3E10 HC comprises an amino acid sequence that is at least 96% identical to an amino acid sequence of 3E10-HC (SEQ ID NO: 30). In some aspects, the 3E10 HC comprises an amino acid sequence that is at least 97% identical to an amino acid sequence of 3E10-HC (SEQ ID NO: 30). In some aspects, the 3E10 HC comprises an amino acid sequence that is at least 98% identical to an amino acid sequence of 3E10-HC (SEQ ID NO: 30). In some aspects, the 3E10 HC comprises an amino acid sequence that is at least 99% identical to an amino acid sequence of 3E10-HC (SEQ ID NO: 30). In some aspects, the 3E10 HC comprises the amino acid sequence set forth in SEQ ID NO: 30.

[0130] In some aspects, a 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), which is specifically incorporated by reference herein, in its entirety.

[0131] In some aspects, a humanized 3E10 antibody or antigen binding fragment thereof of the present disclosure has a strong binding affinity for RAD51, as compared to 3E10 antibodies known in the art. In some aspects, the humanized 3E10 antibody or antigenbinding fragment thereof has a stronger binding affinity for RAD51 compared to a humanized 3E10 antibody or antigen-binding fragment thereof comprising a VL amino acid sequence set forth in SEQ ID NOs: 18. In some aspects, the humanized 3E10 antibody or antigen-binding fragment thereof has a stronger binding affinity for RAD51 compared to a humanized 3E10 antibody or antigen-binding fragment thereof comprising a VH amino acid sequence set forth in SEQ ID NO: 24. In some aspects, the humanized 3E10 antibody or antigen-binding fragment thereof has a stronger binding affinity for RAD51 compared to a humanized 3E10 antibody or antigen-binding fragment thereof comprising (i) a VL amino acid sequence set forth in SEQ ID NO: 18, and / or (ii) a VH amino acid sequence set forth in SEQ ID NO: 24.

[0132] In some aspects, a humanized 3E10 antibody or antigen binding fragment thereof of the present disclosure has a nucleic acid binding affinity for a polynucleotide, e.g., a DNA, RNA, PNA, morpholino, etc., that is similar to the affinity of a reference 3E10 antibody or antigen binding fragment thereof, for example, the 3E10 monoclonal antibody produced by ATCC No. PTA 2439 hybridoma or a D3 IN variant thereof, has for the same polynucleotide. Accordingly, in some aspects, the humanized 3E10 antibody or antigen binding fragment thereof, has an affinity for a polynucleotide that is from 0.05- fold to 50-fold as strong as the affinity the reference 3E10 antibody has for the same polynucleotide.

[0133] In some aspects, a humanized 3E10 antibody or antigen binding fragment thereof, of the present disclosure has a nucleic acid binding affinity for a polynucleotide, e.g., a DNA, RNA, PNA, morpholino, etc., that is less than the affinity of a reference 3E10 antibody or antigen binding fragment thereof, for example, the 3E10 monoclonal antibody produced by ATCC No. PTA 2439 hybridoma or a D3 IN variant thereof, has for the same polynucleotide. Accordingly, in some aspects, the humanized 3E10 antibody or antigen binding fragment thereof, has an affinity for a polynucleotide that is from 0.00001-fold to less than 1-fold as strong as the affinity the reference 3E10 antibody has for the same polynucleotide. In some aspects, the humanized 3E10 antibody or antigen binding fragment thereof, has an affinity for a polynucleotide that is from 0.00001-fold to 0.001-fold as strong as the affinity the reference 3E10 antibody has for the same polynucleotide. In some aspects, the humanized 3E10 antibody or antigen binding fragment thereof, has an affinity for a polynucleotide that is from 0.0001-fold to 0.01-fold as strong as the affinity the reference 3E10 antibody has for the same polynucleotide. In some aspects, the humanized 3E10 antibody or antigen binding fragment thereof, has an affinity for a polynucleotide that is from 0.001-fold to 0.1-fold as strong as the affinity the reference 3E10 antibody has for the same polynucleotide. In some aspects, the humanized 3E10 antibody or antigen binding fragment thereof, has an affinity for a polynucleotide that is from 0.01-fold to less than 1-fold as strong as the affinity the reference 3E10 antibody has for the same polynucleotide.

[0134] In some aspects, a humanized 3E10 antibody or antigen binding fragment thereof, of the present disclosure has a nucleic acid binding affinity for a polynucleotide, e.g., a DNA, RNA, PNA, morpholino, etc., that is greater than the affinity of a reference 3E10 antibody or antigen binding fragment thereof, for example, the 3E10 monoclonal antibody produced by ATCC No. PTA 2439 hybridoma or a D3 IN variant thereof, has for the same polynucleotide. Accordingly, in some aspects, the humanized 3E10 antibody or antigen binding fragment thereof, has an affinity for a polynucleotide that is from greater than 1-fold to 10,000-fold as strong as the affinity the reference 3E10 antibody has for the same polynucleotide. In some aspects, the humanized 3E10 antibody or antigen binding fragment thereof, has an affinity for a polynucleotide that is from 100- fold to 10,000-fold as strong as the affinity the reference 3E10 antibody has for the same polynucleotide. In some aspects, the humanized 3E10 antibody or antigen binding fragment thereof, has an affinity for a polynucleotide that is from 10-fold to 1000-fold asstrong as the affinity the reference 3E10 antibody has for the same polynucleotide. In some aspects, the humanized 3E10 antibody or antigen binding fragment thereof, has an affinity for a polynucleotide that is from greater than 1-fold to 100-fold as strong as the affinity the reference 3E10 antibody has for the same polynucleotide.

[0135] Binding affinity may be determined by association (Ka) and dissociation (Kd) rate. 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) for each antibody is within about 1 to 10-fold (1-10 fold greater affinity or 1-10 fold less affinity, or any numerical value or range or value within such ranges). Example affinities for a target antigen (DNA (e.g., single-stranded and / or double-stranded DNA)) have a dissociation constant (Kd) less than 5* 102M, less than 102M, less than 5* I O3M, less than I O3M, less than 5 / 104M, less than 104M, less than 5 / 105M, less than I O5M, less than 5 / 108M, less than 108M, less than 5^ 107M, less than 107M, less than 5 x 108M, less than 108M, less than 5 / 109M, less than 109M, less than 5x l0'10M, less than 1010M, less than 5 x 101 1M, less than 101 1M, less than 5 x 1012M, less than 1012M, less than 5 x 1013M, less than 1013M, less than 5x l0'14M, less than 1014M, less than 5 / 1015M, or less than 1015M. In some aspects, the binding affinity (Kd) for a target is less than 107M, less than 5 x 108M, less than 108M, less than 5 x 109M, less than 109M, less than 5 x 1010M, less than 1010M, less than 5 x 101 1M, less than 101 1M, less than 5 x 1012M, or less than 1012M.

[0136] In some aspects, binding affinity is evaluated by determining a solution concentration of the humanized 3E10 antibody or antigen binding fragment thereof needed to achieve half maximum binding (EC50) of a polynucleotide coated on a solid surface. In some aspects, the nucleic acid is a poly-dT oligonucleotide. In some aspects, binding of the oligonucleotide, e.g., a poly-dT oligonucleotide, is detected using an ELISA assay with a secondary anti-human immunoglobulin antibody.

[0137] In some aspects, a humanized 3E10 antibody or antigen binding fragment thereof described herein has a weak binding affinity for a poly-dT oligonucleotide. In some aspects, 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 with a D31N amino acid substitution. In some aspects, the weak binding affinity is an EC50 value that is from 100 times greater to 10,000 times greater than the EC50 value of areference 3E10 antibody, e.g., a chimeric 3E10 antibody with a D31N amino acid substitution. In some aspects, the weak binding affinity is an EC50 value that is from 100 times greater to 5000 times greater than the EC50 value of a reference 3E10 antibody, e.g., a chimeric 3E10 antibody with a D31N amino acid substitution. In some aspects, the weak binding affinity is an EC50 value that is from 100 times greater to 1000 times greater than the EC50 value of a reference 3E10 antibody, e.g., a chimeric 3E10 antibody with a D3 IN amino acid substitution. In some aspects, the weak binding affinity is an EC50 value that is from 100 times greater to 500 times greater than the EC50 value of a reference 3E10 antibody, e.g., a chimeric 3E10 antibody with a D31N amino acid substitution.

[0138] In some aspects, a humanized 3E10 antibody or antigen binding fragment thereof described herein has an intermediate binding affinity for a poly-dT oligonucleotide. In some embodimentsaspects, the intermediate binding affinity is an EC50 value that is from 25 times to 100 times greater than the EC50 value of a reference 3E10 antibody, e.g., a chimeric 3E10 antibody with a D31N amino acid substitution.

[0139] In some aspects, a humanized 3E10 antibody or antigen binding fragment thereof described herein has an intermediate binding affinity for a poly-dT oligonucleotide. In some aspects, the intermediate 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 with a D31N amino acid substitution. In some aspects, the intermediate 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 with a D31N amino acid substitution. In some aspects, the intermediate 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 with a D31N amino acid substitution. In some aspects, the intermediate 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 with a D31N amino acid substitution. In some aspects, the intermediate binding affinity is an EC50 value that is no more than 5 times greater than the EC50 value of a reference 3E10 antibody, e.g., a chimeric 3E10 antibody with a D31N amino acid substitution.

[0140] In some aspects, a humanized 3E10 antibody or antigen binding fragment thereof described herein has a weak binding affinity for a 3p-hpRNA RIG-I agonist having the nucleotide sequence 5’-pppGGAGCAAAAGCAGGGUGACAAAGACAUAAUGGAUCCAAACACUGUGUCAAGCUUUCA GGUAGAUUGCUUUCUUUGGCAUGUCCGCAAAC- 3’ (SEQ ID NO: 51).

[0141] In some aspects, a humanized 3E10 antibody or antigen binding fragment thereof described herein has an intermediate binding affinity for the 3p-hpRNA RIG-I agonist having the nucleotide sequence 5’- pppGGAGCAAAAGCAGGGUGACAAAGACAUAAUGGAUCCAAACACUGUGUCAAGCUUUCA GGUAGAUUGCUUUCUUUGGCAUGUCCGCAAAC- 3’ (SEQ ID NO: 51).

[0142] In some aspects, a humanized 3E10 antibody or antigen binding fragment thereof described herein has a strong binding affinity for the 3p-hpRNA RIG-I agonist having the nucleotide sequence 5’- pppGGAGCAAAAGCAGGGUGACAAAGACAUAAUGGAUCCAAACACUGUGUCAAGCUUUCA GGUAGAUUGCUUUCUUUGGCAUGUCCGCAAAC- 3’ (SEQ ID NO: 51).

[0143] In some aspects, a humanized 3E10 antibody or antigen binding fragment thereof described herein comprises a fragment crystallizable (Fc) region. In some aspects, the Fc region is a human IgGl Fc, a human IgG2a Fc, a human IgG2b Fc, a human IgG3 Fc, and a human IgG4 Fc.

[0144] In some aspects, a humanized 3E10 antibody or antigen binding fragment thereof described herein has a human IgGl Fc domain. In some aspects, the human IgGl domain 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 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLJQSS GLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 33).

[0145] In some aspects, the IgGl Fc domain sequence comprises one or more natural amino acid variants found in the human population, e.g., it is an IgGl Fc allotype. Nonlimiting examples of IgGl Fc allotype amino acid variants include Glm (z,a), Glm (f), and Glm (f,a). The Glm (f) allele is only found in Caucasians, whereas the Glm (f,a) allele is common in Asian populations but other variants, Glm (z,a,x) and Glm (z,a,v),have also been described (See, Vidarsson et al. Front. Immunol., October 2014, Vol. 5, Article 520, the disclosures of which is incorporated herein by reference in its entirety.

[0146] In some aspects, a humanized 3E10 antibody or antigen-binding fragment thereof has an IgGl domain containing one or more engineered amino acid substitutions, e.g., to reduce Fc effector function, improve half-life in vivo, and / or otherwise alter the properties of the antibody in vivo.

[0147] Accordingly, in some aspects, the IgGl 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 aspects, the IgGl Fc domain comprises an amino acid substitution selected from from L235A. In some aspects, the IgGl Fc domain comprises a combination of amino acid substitutions selected from L234A / L235A. In some embodiments, the IgGl Fc domain comprises a combination of amino acid substitutions selected from N297D. In some embodiments, the IgGl Fc domain comprises a combination of amino acid substitutions selected from L234A / L235A / N297D.

[0148] In some aspects, a humanized 3E10 antibody or antigen binding fragment thereof described herein has a human IgGl Fc domain comprising L234A / L235A amino acid substitutions. In some aspects, the IgGl Fc domain comprises an amino acid se quence comprising residues 234A / 235A and 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 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGG PSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 34).

[0149] In aspects, a humanized 3E10 antibody or antigen binding fragment thereof described herein has a human IgGl Fc domain comprising an N297D amino acid substitution. In some aspects, the IgGl Fc domain comprises an amino acid sequence comprising residue 297D and 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 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYD STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 35).

[0150] In some aspects, a humanized 3E10 antibody or antigen binding fragment thereof described herein has a human IgGl Fc domain comprising L234A / L235A / N297D amino acid substitutions. In some aspects, the IgGl Fc domain comprises an amino acid sequence comprising residues 234A / 235A / 297D and 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 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYD STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 36).

[0151] In some aspects, a humanized 3E10 antibody or antigen binding fragment thereof described herein has a human IgG4 Fc domain. In some aspects, the human IgG4 domain 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 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSV FLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY RWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 38).

[0152] In some aspects, the human Fc region comprises an alanine at position 234 and an alanine at position 235, according to Kabat numbering. In some aspects, the human Fc region comprises an alanine at position 234 and a glutamic acid at position 235, according to Kabat numbering. In some aspects, the human Fc region comprises an aspartic acid at position 297, according to Kabat numbering. In some aspects, the human Fc regioncomprises a proline at position 228, according to Kabat numbering. In some aspects, the human Fc region comprises a glutamine at position 307 and an alanine at position 434, according to Kabat numbering. In some aspects, 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. In some aspects, the human Fc region comprises a glutamine at position 310, according to Kabat numbering.

[0153] In some aspects, the IgG4 Fc domain sequence comprises one or more natural amino acid variants found in the human population, e.g., it is an IgG4 Fc allotype. Nonlimiting examples of IgG4 Fc allotype amino acid variants include nG4m (a) and nG4m (b) (See, Vidarsson et al. Front. Immunol., October 2014, Vol. 5, Article 520, the disclosures of which is incorporated herein by reference in its entirety.

[0154] In some aspects, a humanized 3E10 antibody or antigen-binding fragment thereof has an IgG4 domain containing one or more engineered amino acid substitutions, e.g., to reduce Fc effector function, improve half-life in vivo, and / or otherwise alter the properties of the antibody in vivo. In fact, the majority of IgG4-based therapeutic antibodies approved for marketing or in late-stage clinical trials, contain at least one such amino acid substitution. For review of the amino acid substitutions in such antibodies see, for example, Dumet C. et al., MABS, 11(8): 1341-50 (2019), the disclosure of which is incorporated herein by reference in its entirety. Other IgG4 Fc mutations have been suggested for reducing immune effector functions while retaining other Fc characteristics. For example, Tam S.H. et al., Antibodies, 6(12): 1-34 (2017), the disclosure of which is incorporated by reference herein in its entirety, reports on the characterization of two IgG4 Fc variant designs, huIgG4o l and huIgG4o2. The huIgG4o l construct includes S228P, F234A, L235A, G237A, and P238S amino acid substitutions while the hu!gG4c2 construct includes a G236> del, in addition to the 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 herein, in its entirety. These mutations include M252Y, S254T, T256E, H433K, and N434F.

[0155] Yet other IgG4 Fc amino acid substitutions that may be integrated into an IgG4 Fc domain of a humanized 3E10 antibody or antigen-binding fragment thereof are suggested in the art including, without limitation, WO 1989 / 007142, US 5,885,573, WO 1994 / 029351, US 6,407,214, US 2006 / 0024298, US 7,863,419, US 2007 / 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, US 11,046,784, US 8,802,820, US 2020 / 0255502, US 2010 / 0098730, US RE45992, US 2010 / 0204454, US 8,637,641, US 2014 / 0302028, GB 201302878, US 2015 / 0065690, US 2014 / 0294812, US 2020 / 0071423, US 11,319,383, US 2018 / 0037634, KR 101792191, US 2019 / 0010243, US 8,911,726, US 2010 / 0267934, US 9,688,762, US 2012 / 0100140, US 9,085,625, US 10,562,966, US 2017 / 029521, US 11,254,753, and WO 2018 / 119380, the disclosures of which are hereby incorporated by reference herein, in their entireties, for all purposes.

[0156] Accordingly, in some aspects, the IgG4 Fc domain comprises an amino acid substitution at a position selected from 196, 228, 234, 234, 235, 235, 236, 237, 238, 252, 254, 256, 265, 296, 233, 310, 331, 356, 409, 428, 433, 434, 435, 445, 446, and K447, according to the EU index as in the Kabat numbering scheme. In some aspects, the IgG4 Fc domain comprises an amino acid substitution selected from K196Q, S228P, F234A, F234V, L235A, L235E, G236> del, G237A, P238S, M252Y, S254T, T256E, D265A, F296Y, E233P, T307Q, H310Q, P331S, E356K, R409K, M428L, H433K, N434A, N434F, N434S, H435R, L445P, G446> del, and K447> del. In some aspects, the IgG4 Fc domain comprises a combination of amino acid substitutions selected from S228P / L234A / L235A, L234F / L235E / P331S, M252Y / S254T / T256E, M252Y / S254T / T256E / H433K / N434F, S228P / F234A / L235A / H310Q, S228P / F234A / L235A / M252Y / S254T / T256E, S228P / F234A / L235A / T307Q / N434A, S228P / F234A / L235A / G237A / P238S, and S228P / F234A / L235A / G236> del / G237A / P238S.

[0157] In some aspects, a humanized 3E10 antibody or antigen binding fragment thereof described herein has a human IgG4 Fc domain comprising S228P / F234A / L235A amino acid substitutions. In some aspects, the IgGl Fc domain comprises an amino acid sequence comprising residues 228P / 234A / 235A and 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 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSV FLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY RWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 37).

[0158] In some aspects, a humanized 3E10 antibody or antigen binding fragment thereof described herein has a human IgG4 Fc domain comprising S228P / F234A / L235A / T307Q / N434A amino acid substitutions. In some aspects, the IgGl Fc domain comprises an amino acid sequence comprising residues 228P / 234A / 235A / 307Q / 434A 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 ASTKGPSVFPLAPCSRSTSESTAALJGCLVKDYFPEPVTVSWNSGALJTSGVHTFPAVLJQSS GLYSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSV FLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY RWSVLQVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHAHYTQKSLSLSLGK (SEQ ID NO: 39).

[0159] In some aspects, a humanized 3E10 antibody or antigen binding fragment thereof described herein has a human IgG4 Fc domain comprising S228P / F234A / L235A / M252Y / S254T / T256E amino acid substitutions. In some aspects, the IgGl Fc domain comprises an amino acid sequence comprising residues 228P / 234A / 235A / 252Y / 254T / 256E 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 ASTKGPSVFPLAPCSRSTSESTAALJGCLVKDYFPEPVTVSWNSGALJTSGVHTFPAVLJQSS GLYSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSV FLFPPKPKDTLYITREPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY RWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 40).

[0160] In some aspects, a humanized 3E10 antibody or antigen binding fragment thereof described herein has a human IgG4 Fc domain comprising S228P / F234A / L235A / H310Q amino acid substitutions. In some aspects, the IgGl Fc domain comprises an amino acid sequence comprising residues 228P / 234A / 235A / 310Q 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 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSV FLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY RWSVLTVLQQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 41).

[0161] In some aspects, the present disclosure provides humanized 3E10 antibodies or antigen-binding fragments thereof comprising a heavy chain constant domain (CHI).

[0162] In some aspects, the humanized 3E10 antibody or antigen binding fragment thereof comprises an Fc region selected from a human yl CHI, a human y2 CHI, a human y3 CHI, and a human y4 CHI.

[0163] In some aspects, the present disclosure provides a humanized 3E10 antibody or antigen binding fragment thereof comprising a light chain constant domain (CL).

[0164] In another aspect, the humanized 3E10 antibody or antigen binding fragment thereof comprises an Fc region selected from the group consisting of a human Z. CL and a human K CL.

[0165] In some aspects, the humanized 3E10 antibody or antigen-binding fragment thereof is a single-chain Fv (scFv) or an antigen-binding fragment (Fab).

[0166] In various aspects, the humanized 3E10 antibody or antigen binding fragment thereof is covalently linked to a therapeutic moiety. In various aspects, the humanized 3E10 antibody or antigen binding fragment thereof is non-covalently linked to a therapeutic moiety. In some aspects, the therapeutic moiety is a therapeutic polypeptide.

[0167] In some aspects, the therapeutic moiety is a therapeutic polypeptide, a cytotoxic moiety, a chemotherapeutic moiety, or a moiety that is detectable.Complexes comprising Therapeutic Polynucleotides

[0168] In some aspects, the disclosure provides a composition comprising a covalent complex of (i) a humanized 3E10 antibody or antigen-binding fragment thereof described herein, and (ii) a polynucleotide, polypeptide, or a chemical agent. In some aspects, the polynucleotide is a therapeutic polynucleotide.

[0169] In some aspects, the disclosure provides a composition comprising a non-covalent complex of (i) a humanized 3E10 antibody or antigen-binding fragment thereof described herein, and (ii) a polynucleotide.

[0170] In some aspects, e.g., where a therapeutic polynucleotide is non-covalently bound to a humanized 3E10 antibody or antigen binding fragment thereof, the compositions described herein have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide of at least 2:1. The use of molar ratios of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide in the compositions described herein protects the therapeutic polynucleotide from degradation. For instance, the parental 3E10 and 3E10 (D31N) variant antibodies protect mRNA from RNAseA-mediated RNA degradation at molar ratios of 2: 1 and 20: 1, the protection afforded by the 20: 1 molar ratio exceeds the protection afforded at 2:1.

[0171] In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is at least 2: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is at least 2.5:1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is at least 5: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is at least 7.5: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is at least 10: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is at least 15 : 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is at least 20: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is at least 25: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is at least 30: 1. In some aspects, the compositions described here have a molar ratio ofhumanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is at least 40:1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is at least 50:1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is at least 75:1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is at least 100: 1.

[0172] In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that 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, at least 46:1, at least 47:1, at least 48:1, at least 49:1, at least 50:1, at least 55:1, at least 60:1, at least 65:1, at least 70:1, at least 75:1, at least 80:1, at least 85:1, at least 90:1, at least 95:1, at least 100:1, or greater.

[0173] In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that 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.

[0174] In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is no more than 200:1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is no more than 150:1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is no more than 100:1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is no more than 50: 1. In someaspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is no more than 40: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is no more than 30: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is no more than 25: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is no more than 20: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is no more than 15: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is no more than 10: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is no more than 5: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is no more than 2: 1.

[0175] In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is no more than 200: 1, no more than 150:1, no more than 100: 1, 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, no more than 2: 1, or less.

[0176] In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 2: 1 to 200: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 2: 1 to 150: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 2: 1 to 100: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 2: 1 to 50: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 2: 1 to 40: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 2: 1 to 30: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 2: 1 to 25: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 2: 1 to 20: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 2: 1 to 15: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 2: 1 to 10: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 2: 1 to 7.5: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 2: 1 to 5 : 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 2:1 to 5: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 2: 1 to 3: 1.

[0177] In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 5: 1 to 200: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 5: 1 to 150: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 5: 1 to 100: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 5 : 1 to 50: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 5: 1 to 40: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 5: 1 to 30: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 5: 1 to 25: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 5 : 1 to 20: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 5: 1 to 15: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 5: 1 to 10: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 5: 1 to 7.5:1.

[0178] In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 10:1 to 200: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 10: 1 to 150: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 10: 1 to 100: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigenbinding fragment thereof to therapeutic polynucleotide that is of from 10: 1 to 50: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 10: 1 to 40: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 10: 1 to 30: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 10: 1 to 25: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 10: 1 to 20: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 10: 1 to 15: 1.

[0179] In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 15:1 to 200: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 15: 1 to 150: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 15: 1 to 100: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 15 : 1 to 50: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 15: 1 to 40: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 15: 1 to 30: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 15: 1 to 25: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 15 : 1 to 20: 1.

[0180] In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is offrom 20:1 to 200: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 20: 1 to 150: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 20: 1 to 100: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 20: 1 to 50: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 20: 1 to 40: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 20: 1 to 30: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 20: 1 to 25 : 1.

[0181] In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 25:1 to 200: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 25: 1 to 150: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 25: 1 to 100: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 25 : 1 to 50: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 25: 1 to 40: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 25: 1 to 30: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 30: 1 to 50: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 30: 1 to 40: 1. In yet other aspects, other ranges falling with the range of 2: 1 to 50: 1 are contemplated.

[0182] In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 2:1 to 200:1, 2:1 to 150:1, 2:1 to 100:1, 2:1 to 50:1, from 2:1 to 40:1, from 2:1 to 30:1, from 2:1 to 25:1, from 2:1 to 20:1, from 2:1 to 15:1, from 2:1 to 10:1, from 2:1 to 7.5:1, from 2:1 to 5:1, from 5:1 to 50:1, from 5:1 to 40:1, from 5:1 to 30:1, from 5:1 to 25:1, from 5:1 to 20:1, from 5:1 to 15:1, from 5:1 to 10:1, from 5:1 to 7.5:1, from 10:1 to 50:1, from 10:1 to 40:1, from 10:1 to 30:1, from 10:1 to 25:1, from 10:1 to 20:1, from 10:1 to 15:1, from 15:1 to 50:1, from 15:1 to 40:1, from 15:1 to 30:1, from 15:1 to 25:1, from 15:1 to 20:1, from 20:1 to 50:1, from 20:1 to 40:1, from 20:1 to 30:1, from 20:1 to 25:1, from 25:1 to 50:1, from 25:1 to 40:1, from 25:1 to 30:1, from 30:1 to 50:1, from 30: 1 to 40: 1, or from 40: 1 to 50: 1. In yet other aspects, other ranges falling with the range of from 2:1 to 50:1 are contemplated.

[0183] In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 1 : 1 to 50: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 1:1 to 30:1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 1 : 1 to 20: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 1 : 1 to 10: 1. In some aspects, the compositions described here have a molar ratio of humanized 3E10 antibody or antigen binding fragment thereof to therapeutic polynucleotide that is of from 1:1 to 5:1.

[0184] In some aspects, the molar ratio is related to the size of the nucleic acid (i.e., the therapeutic polynucleotide). For instance, longer polynucleotides are complexed at higher molar ratios and shorter polynucleotides are complexed at lower molar ratios.

[0185] In some aspects, the polynucleotide is about 15-4000 nucleotides in length, about 15-100 nucleotides in length, about 100-500 nucleotides in length, about 15-40 nucleotides in length, about 1000-4000 nucleotides in length or about 300-900 nucleotides in length. In some aspects, the polynucleotide is at least about 15 nucleotides in length, at least about 25 nucleotides in length, at least about 35 nucleotides in length, at least about 100 nucleotides in length, at least about 300 nucleotides in length, at leastabout 500 nucleotides in length, at least about 900 nucleotides in length, at least about 1000 nucleotides in length, at least about 2000 nucleotides in length, or at least about 3000 nucleotides in length. In some aspects, the polynucleotide is no more than about 25 nucleotides in length, no more about 35 nucleotides in length, no more than about 100 nucleotides in length, no more than about 300 nucleotides in length, no more than about 500 nucleotides in length, no more than about 900 nucleotides in length, no more than about 1000 nucleotides in length, no more than about 2000 nucleotides in length, no more than about 3000 nucleotides in length, no more than about 4000 nucleotides in length.

[0186] In some aspects, the size of the therapeutic polynucleotide is about lObp, 15bp, 20bp, 25bp, 30bp, 35bp, 40bp, 45bp, 50bp, 55bp, 60bp, 65bp, 70bp, 75bp, 80bp, 85bp, 90bp, 95bp, lOObp, 105bp, HObp, 115bp, 120bp, 125bp, 130bp, 135bp, 140bp, 145bp, 150bp, 155bp, 160bp, 165bp, 170bp, 175bp, 180bp, 185bp, 190bp, 195bp, 200bp, 205bp, 210bp, 215bp, 220bp, 225bp, 230bp, 235bp, 240bp, 245bp, 250bp, 255bp, 260bp, 265bp, 270bp, 275bp, 280bp, 285bp, 290bp, 295bp, 300bp, 305bp, 310bp, 315bp, 320bp, 325bp, 330bp, 335bp, 340bp, 345bp, 350bp, 355bp, 360bp, 365bp, 370bp, 375bp, 380bp, 385bp, 390bp, 395bp, 400bp, 405bp, 410bp, 415bp, 420bp, 425bp, 430bp, 435bp, 440bp, 445bp, 450bp, 455bp, 460bp, 465bp, 470bp, 475bp, 480bp, 485bp, 490bp, 495bp, 500bp, 505bp, 510bp, 515bp, 520bp, 525bp, 530bp, 535bp, 540bp, 545bp, 550bp, or more and any range in between.

[0187] In some aspects, the molar ratios disclosed herein are related to the size of the therapeutic polynucleotide as disclosed herein. For instance, longer polynucleotides are complexed at higher molar ratios and shorter polynucleotides are complexed at lower molar ratios. In some aspects, any molar ratio disclosed herein can be combined with any size of the therapeutic polynucleotide. Nonlimiting examples, include the composition that has a molar ratio of 3E10 antibody to therapeutic polynucleotide that is 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 greater and any ranges in between, wherein the size therapeutic polynucleotide is about lObp, 15bp, 20bp, 25bp, 30bp, 35bp, 40bp, 45bp, 50bp, 55bp, 60bp, 65bp, 70bp, 75bp, 80bp, 85bp, 90bp, 95bp, lOObp, 105bp, HObp, 115bp, 120bp, 125bp, 130bp, 135bp, 140bp, 145bp, 150bp, 155bp, 160bp, 165bp, 170bp, 175bp, 180bp, 185bp, 190bp, 195bp, 200bp, 205bp,210bp, 215bp, 220bp, 225bp, 230bp, 235bp, 240bp, 245bp, 250bp, 255bp, 260bp, 265bp, 270bp, 275bp, 280bp, 285bp, 290bp, 295bp, 300bp, 305bp, 310bp, 315bp, 320bp, 325bp, 330bp, 335bp, 340bp, 345bp, 350bp, 355bp, 360bp, 365bp, 370bp, 375bp, 380bp, 385bp, 390bp, 395bp, 400bp, 405bp, 410bp, 415bp, 420bp, 425bp, 430bp, 435bp, 440bp, 445bp, 450bp, 455bp, 460bp, 465bp, 470bp, 475bp, 480bp, 485bp, 490bp, 495bp, 500bp, 505bp, 510bp, 515bp, 520bp, 525bp, 530bp, 535bp, 540bp, 545bp, 550bp, or more and any ranges in between.

[0188] In some aspects, the polynucleotide is released from the non-covalent complex in less time than a corresponding non-covalent complex comprising a 3E10 antibody or antigen-binding fragment thereof comprising (i) a VL amino acid sequence set forth in SEQ ID NO: 18, and / or (ii) a VH amino acid sequence set forth in SEQ ID NO: 24.

[0189] In some emdodiments, the non-covalent complex has a larger equilibrium dissociation constant (KD) nanomolar value than a corresponding non-covalent complex comprising a 3E10 antibody or antigen-binding fragment thereof comprising (i) a VL amino acid sequence set forth in SEQ ID NO: 18, and / or (ii) a VH amino acid sequence set forth in SEQ ID NO: 24. In some aspects, the non-covalent complex has a KD value of about 10'8to 10'7M. In some aspects, the non-covalent complex has a KD value of about 2 x 10'8to 3 x 10'8M. In some aspects, the Kd value is less than 107M, less than 5* 108M, less than 108M, less than 5 * 109M, less than 109M, less than 5 * 1010M, less than 1010M, less than 5 * 101 1M, less than 101 1M, less than 5 * 1012M, or less than 1012M.

[0190] In some aspects, the non-covalent complex has a faster off-rate (Kais) than a corresponding non-covalent complex comprising a 3E10 antibody or antigen-binding fragment thereof comprising (i) a VL amino acid sequence set forth in SEQ ID NO: 18, and / or (ii) a VH amino acid sequence set forth in SEQ ID NO: 24. In some aspects, the non-covalent complex has a Kais value of about 7 x 10'3to 4 x 10'2S'1. In some aspects, the non-covalent complex has a Kais value of about 1 x 10'2to 2 x 10'2S'1.Cell Penetration and Nuclear Localization

[0191] 3E10 penetrates cells in an Fc-independent mechanism (as evidenced by the ability of 3E10 fragments lacking an Fc to penetrate cells) but involves presence of the nucleoside transporter ENT2 (Weisbart et al., Scientific Reports volume 5, Article number: 12022 (2015), Zack et al., J Immunol 157, 2082-2088 (1996), Hansen et al., JBiol Chem 282, 20790-20793 (2007)). Thus, in some aspects, the antibodies utilized in the disclosed compositions and methods are ones that penetrate cells in an Fc-independent mechanism but involves presence of the nucleoside transporter ENT2.

[0192] Mutations in 3E10 that interfere with its ability to bind nucleic acids may render the antibody incapable of nuclear penetration. Thus, typically the disclosed variants and humanized forms of the antibody maintain the ability to bind nucleic acids. In addition, 3E10 scFv has previously been shown capable of penetrating into living cells and nuclei in an ENT2-dependent manner, with efficiency of uptake impaired in ENT2-deficient cells (Hansen, et al., J. Biol. Chem. 282, 20790-20793 (2007)). Thus, in some aspects, the disclosed humanized forms and variants of the antibody maintain the ability to penetrate into cell nuclei in an ENT-dependent, preferably ENT2-dependent manner.

[0193] As discussed in US 2021 / 0054102 and US 2021 / 0137960, some humanized 3E10 variants were found to penetrate cell nuclei more efficiently than the original murine 3E10 (D3 IN) di-scFv, while others were found to have lost the ability to penetrate nuclei.Nucleic Acid Binding

[0194] In some aspects, 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.

[0195] The Examples below illustrate molecular modeling of wild type 3E10 sequences and additional 3E10 variants. Molecular modeling of 3E10 (Pymol) revealed a putative Nucleic Acid Binding pocket (NAB1).

[0196] In some aspects, the antibodies include a variant sequence that has an altered ability to bind nucleic acids. In some aspects, the mutations (e.g., substitutions, insertions, and / or deletions) in the NAB1 improve binding of the antibody to nucleic acids such as DNA, RNA, or a combination thereof. In some aspects, the mutations are conservative substitutions. In some aspects, the mutations increase the cationic charge of the NAB1 pocket.

[0197] For example, mutation of aspartic acid at residue 31 of CDR1 to asparagine increased the cationic charge of this residue and enhanced nucleic acid binding and delivery in vivo (3E10-D31N). In some aspects, the 3E10 antibody disclosed herein does not include a D3 IN substitution.

[0198] Additional example variants include mutation of aspartic acid at residue 31 of CDR1 to Y, E, F, or W. Thus, in some aspects, the 3E10 binding protein includes a D31 Y, D3 IF, D31W, or D3 IE substitution.

[0199] All of the sequences disclosed herein having the residue corresponding to 3E10 D31, are expressly disclosed with a D31 Y, D3 IF, D31W, or D3 IE substitution.Fragments, Variants, and Fusion Proteins

[0200] Also included are fragments of antibodies which have nucleic acid delivery activity. The fragments, whether attached to other sequences or not, include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the fragment is not significantly altered or impaired compared to the nonmodified antibody or antibody fragment.

[0201] Techniques can also be adapted for the production of single-chain antibodies specific for nucleic acids of the present disclosure. Methods for the production of singlechain antibodies are well known to those of skill in the art. A single chain antibody can be created by fusing together the variable domains of the heavy and light chains using a short peptide linker, thereby reconstituting an antigen binding site on a single molecule. Single-chain antibody variable fragments (scFvs) in which the C-terminus of one variable domain is tethered to the N-terminus of the other variable domain via a 15 to 25 amino acid peptide or linker have been developed without significantly disrupting antigen binding or specificity of the binding. The linker is chosen to permit the heavy chain and light chain to bind together in their proper conformational orientation.

[0202] The 3E10 antibody or antigen-binding fragment thereof can be modified to improve their nucleic acid delivery potential. For example, in some aspects, the cellpenetrating anti-nucleic acid antibody is conjugated to another antibody specific for a therapeutic target in the cytoplasm and / or nucleus of a target cell. For example, the cellpenetrating anti-nucleic acid antibody can be a fusion protein containing 3E10 Fv and a single chain variable fragment of a monoclonal antibody that specifically binds the therapeutic target. In other aspects, 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 a therapeutic target.

[0203] Bispecific antibodies and other binding proteins having a first heavy chain and a first light chain from 3E10 and a second heavy chain and a second light chain from a monoclonal antibody that specifically binds a target are discussed in Weisbart, et al., Mol. Cancer Ther., 11(10):2169-73 (2012), and Weisbart, et al., Ini. J. Oncology, 25: 1113-8 (2004), and U.S. Patent Application No. 2013 / 0266570, which are specifically incorporated by reference herein in their entireties. In some aspects, the target is specific for a target cell-type, tissue, organ etc. Thus, the second heavy chain and second light chain can serve as a targeting moiety that targets the complex to the target cell-type, tissue, or organ. In some aspects, the second heavy chain and second light chain target, hematopoietic stem cells, CD34+cells, T cells or any another cell type, e.g., by targeting a receptor or ligand expressed on the cell type. In some aspects, the second heavy chain and second light chain target the thymus, spleen, or cancer cells.

[0204] In some aspects, particularly those for targeting T cells in vivo, for example, for in vivo production of antigen-specific T cells, CAR T cells, immune cell, or T cell markers such as CD3, CD7, or CD8 can be targeted. For example, anti-CD8 antibodies and anti- CD3 Fab fragments have both been used to target T cells in vivo (Pfeiffer, et al., EMBO Mol Med., 10(11) (2018). pii: e9158. doi: 10.15252 / emmm.201809158., Smith, et al., Nanotechnol., 12(8):813-820 (2017). doi: 10.1038 / nnano.2017.57). Thus, in some aspects, the 3E10 antibody or antigen binding fragment or fusion protein is a bispecific antibody part of which can bind specifically 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.

[0205] Divalent single-chain variable fragments (di-scFvs) can be engineered by linking two scFvs. This can be done by producing a single peptide chain with two VH and two VL regions, yielding tandem scFvs. ScFvs can also be designed with linker peptides that are too short for the two variable regions to fold together (about five amino acids), forcing scFvs to dimerize. This type is known as diabodies. Diabodies have been shown to have dissociation constants up to 40-fold lower than corresponding scFvs, meaning that they have a much higher affinity to their target. Still shorter linkers (one or two amino acids) lead to the formation of trimers (triabodies or tribodies). Tetrabodies have also been produced. They exhibit an even higher affinity to their targets than diabodies. In some aspects, the anti-nucleic acid antibody may contain two or more linked single chain variable fragments of 3E10 (e.g., 3E10 di-scFv, 3E10 tri-scFv), or conservative variants thereof. In some aspects, the anti-nucleic acid antibody is a diabody or triabody (e.g.,3E10 diabody, 3E10 triabody). Sequences for single and two or more linked single chain variable fragments of 3E10 are provided in US 2019 / 0247515 and US 2017 / 0291961.

[0206] The function of the antibody may be enhanced by coupling the antibody or a fragment thereof with a therapeutic agent. Such coupling of the antibody or fragment with the therapeutic agent can be achieved by making an immunoconjugate or by making a fusion protein, or by linking the antibody or fragment to a nucleic acid such as DNA or RNA (e.g., siRNA), comprising the antibody or antibody fragment and the therapeutic agent.

[0207] In some aspects, the cell-penetrating antibody is modified to alter its half-life. In some aspects, it is desirable to increase the half-life of the antibody so that it is present in the circulation or at the site of treatment for longer periods of time. For example, it may be desirable to maintain titers of the antibody in the circulation or in the location to be treated for extended periods of time. In other aspects, the half-life of the anti-nucleic acid antibody is decreased to reduce potential side effects. Antibody fragments, such as 3E10Fv may have a shorter half-life than full size antibodies. Other methods of altering half-life are known and can be used in the described methods. For example, antibodies can be engineered with Fc variants that extend half-life, e.g., using Xtend™ antibody half-life prolongation technology (Xencor, Monrovia, CA).Linkers

[0208] The term “linker” as used herein includes, without limitation, peptide linkers. The peptide linker can be any size provided it does not interfere with the binding of the epitope by the variable regions. In some aspects, the linker includes one or more glycine and / or serine amino acid residues. Monovalent single-chain antibody variable fragments (scFvs) in which the C-terminus of one variable domain are typically tethered to the N- terminus of the other variable domain via a 15 to 25 amino acid peptide or linker. The linker is chosen to permit the heavy chain and light chain to bind together in their proper conformational orientation. Linkers in diabodies, triabodies, etc., typically include a shorter linker than that of a monovalent scFv as discussed above. Di-, tri-, and other multivalent scFvs typically include three or more linkers. The linkers can be the same, or different, in length and / or amino acid composition. Therefore, the number of linkers, composition of the linker(s), and length of the linker(s) can be determined based on thedesired valency of the scFv as is known in the art. The linker(s) can allow for or drive formation of a di-, tri-, and other multivalent scFv.

[0209] For example, a linker can include 4-8 amino acids. In a particular embodiment, a linker includes the amino acid sequence GQSSRSS (SEQ ID NO: 42). In another embodiment, a linker includes 15-20 amino acids, for example, 18 amino acids. In a particular embodiment, the linker includes the amino acid sequence GQSSRSSSGGGSSGGGGS (SEQ ID NO: 43). Other flexible linkers include, but are not limited to, the amino acid sequences Gly-Ser (SEQ ID NO: 54), Gly-Ser-Gly-Ser (SEQ ID NO: 44), Ala-Ser (SEQ ID NO: 55), Gly-Gly-Gly-Ser (SEQ ID NO: 45), (Gly4-Ser)2(SEQ ID NO: 46) and (Gly4-Ser)4(SEQ ID NO: 47), and (Gly-Gly-Gly-Gly-Ser)3(SEQ ID NO: 48).

[0210] Other example linkers include, for example, RADAAPGGGGSGGGGSGGGGS (SEQ ID NO: 49) and ASTKGPSVFPLAPLESSGS (SEQ ID NO: 50).Nucleic Acid Cargo

[0211] As used in some aspects of the methods and composition provided herein, the humanized 3E10 antibody or antigen binding fragment thereof is complexed with a nucleic acid cargo (a polynucleotide). In some aspects, the polynucleotide is non- covalently bound to the humanized 3E10 antibody or antigen binding fragment thereof. In some aspects, the polynucleotide is covalently conjugated to the humanized 3E10 antibody or antigen binding fragment thereof.

[0212] The nucleic acid cargo can be single stranded or double stranded. The nucleic acid cargo can be or include 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 modification can improve, for example, stability, hybridization, or solubility of the nucleic acid.

[0213] The nucleic acid cargo is typically functional in the sense that it is or encodes an agent that is biologically active once delivered into cells. Example cargo is discussed in more detail below, but includes, for example, mRNA or DNA encoding polypeptides of interest including, for example expression constructs and vectors, inhibitory nucleic acids such as siRNA, or nucleic acid encoding the inhibitory nucleic acid including, for example expression constructs and vectors.

[0214] The disclosed compositions can include a plurality of a single nucleic acid cargo molecule. In some aspects, the compositions include a plurality of a multiplicity (e.g., 2, 3, 4, 5, 6, 7, 8, 9 10, or more) of different nucleic acid molecules.

[0215] In some aspects, the cargo molecules are about 0.001, about 0.01, about 1, 10’s, 100’s, 1,000’s, 10,000’s, and / or 100,000’s of kilobases in length.

[0216] In some aspects, e.g., 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 0.001 kb and 5 kb, or between 0.001 kb and 2.5 kb, or between 0.001 kb and 1 kb, or between 0.01 kb and 100 kb, or between 0.01 kb and 50 kb, or between 0.01 kb and 25 kb, or between 0.01 kb and12.5 kb, or between 0.01 kb and 10 kb, or between 0.01 kb and 8 kb, or 0.01 kb and 5 kb, or between 0.01 kb and 2.5 kb, or between 0.01 kb and 1 kb, or between 0.1 kb and 100 kb, or between 0.1 kb and 50 kb, or between 0.1 kb and 25 kb, or between 0.1 kb and 12.5 kb, or between 0.1 kb and 10 kb, or between 0.1 kb and 8 kb, or 0.1 kb and 5 kb, or between 0.1 kb and 2.5 kb, or between 0.1 kb and 1 kb, or between 1 kb and 100 kb, or between 1 kb and 50 kb, or between 1 kb and 25 kb, or between 1 kb and 12.5 kb, or between 1 kb and 10 kb, or between 1 kb and 8 kb, or 1 kb and 5 kb, or between 1 kb and2.5 kb, each inclusive.

[0217] In some aspects, e.g., 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 about 0.001 kb and about 5 kb, or between about 0.001 kb and about 2.5 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 about 0.01 kb and about 5 kb, or between about 0.01 kb and about 2.5 kb, or between about 0.01 kb and about 1 kb, or between about 0.1 kb and about 100 kb, or between about 0.1 kb and about 50 kb, or between about 0.1 kb and about 25 kb, or between about 0.1 kb and about 12.5 kb, or between about 0.1 kb and about 10 kb, or between about 0.1 kb and about 8 kb, or between about 0.1 kb and about 5 kb, or between about 0.1 kb and about 2.5 kb, or between about 0.1 kb and about 1 kb, or between about 1 kb and about 100 kb, or between about 1 kb and about 50 kb, or between about 1 kb and about 25 kb, or betweenabout 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.

[0218] In some aspects, e.g., 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.

[0219] In some aspects, e.g., 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.

[0220] It will be appreciated that for specific application the nucleic acid cargo may be one or more discrete lengths that, for example, falls within one of the foregoing ranges (inclusive), the specific values for each are expressly disclosed. For example, the size can be as small as a single nucleotide or nucleobase. In an example application the cargo is a cyclic dinucleotide like cGAMP, which is a STING agonist. In other aspects, the cargo is a short oligomer. For example, oligomers as short as 8-mers can be used for anti-sense or splice switching. Slightly longer ones (e.g., 18 to 20 mers) can be used for gene editing.

[0221] Suitable and exemplary nucleic cargo, e.g., polynucleotides, for example, mRNA or DNA encoding polypeptides of interest including, for example expression constructs and vectors, inhibitory nucleic acids such as siRNA, or nucleic acid encoding the inhibitory nucleic acid including, for example expression constructs and vectors, that are suitable for use in the methods and compositions provided herein are explicitly provided in US 20230303719, the content of which is incorporated herein by reference in its entirety.Polynucleotide Immunostimulant

[0222] In some aspects, a humanized 3E10 antibody or antigen binding fragment thereof is complexed with a polynucleotide immunostimulant, e.g., a polynucleotide capable ofstimulating a pattern recognition receptor (PRR). In some aspects, the polynucleotide immunostimulant is non-covalently bound to the humanized 3E10 antibody or antigen binding fragment thereof. In some aspects, the polynucleotide immunostimulant is covalently conjugated to the humanized 3E10 antibody or antigen binding fragment thereof. Suitable polynucleotide immunostimulants for use in the methods and composition provided herein are explicitly provided in US 20230303719, the content of which is incorporated herein by reference in its entirety.Pattern Recognition Receptor (PRR) Agonists

[0223] In one aspect, the present disclosure relates to compositions and methods for treating cancer in subject by administering to the subject a therapeutically effective amount of a composition including a complex formed between (i) a humanized 3E10 antibody or antigen binding fragment thereof, and (ii) a polynucleotide ligand capable of stimulating a pattern recognition receptor (PRR). Polynucleotide ligands capable of stimulating a PRR that are suitable for use in the methods and compositions provided herein are explicitly provided in US 20230303719, the content of which is incorporated herein by reference in its entirety.

[0224] In some aspects, compositions and methods are provided for treating a cancer by administering to the subject a therapeutically effective amount of a composition including a complex formed between (i) a humanized 3E10 antibody or antigen binding fragment thereof, and (ii) an RNA molecule that is at least partially double-stranded and is capable of stimulating RIG-I. In some aspects, the at least partially double-stranded RNA molecule comprises two separate RNA strands that anneal to form a double-stranded portion of the molecule. In other aspects, the at least partially double-stranded RNA molecule is a single RNA strand with self-complementarity, such that under physiological conditions it anneals to itself to form a double-stranded portion of the molecule, e.g., thereby forming one or more hairpin structures. Suitable RNA molecules that capable of stimulating RIG-I for use in the methods and compositions provided herein are explicitly provided in US 20230303719, the content of which is incorporated herein by reference in its entirety. Additional examples of polynucleotide RIG-I agonists are provided in the literature. Generally, any one of these polynucleotide RIG-I agonists finds use in the methods and compositions described herein.

[0225] In some aspects, the RIG-I agonist is a 5’ triphosphate hairpin RNA that was generated by in vitro transcription of a sequence from the influenza A (H1N1) virus, a single-stranded negative-sense RNA virus (3p-hpRNA) having the sequence 5’- pppGGAGCAAAAGCAGGGUGACAAAGACAUAAUGGAUCCAAACACUGUGUC AAGCUUUCAGGUAGAUUGCUUUCUUUGGCAUGUCCGCAAAC- 3’ (SEQ ID NO: 51), or a highly conserved nucleotide sequence thereto. See, for example, Rehwinkel J. et al., Cell, 140:397-408 (2010) and Liu G. et al., J Virol. 89(11):6067-79 (2015), the contents of which are incorporated herein by reference, in their entireties, for all purposes. Accordingly, in some aspects, compositions and methods are provided for treating a cancer by administering to the subject a therapeutically effective amount of a composition including a complex formed between (i) a humanized 3E10 antibody or antigen binding fragment thereof, and (ii) a polynucleotide having the sequence of 3p-hpRNA (SEQ ID NO: 51) that is capable of stimulating RIG-I.

[0226] Other RIG-I agonists that find use in the methods and compositions disclosed herein are known in the art. For example, useful RIG-I agonists that can be complexed with the humanized 3E10 antibodies and antigen-binding fragments thereof disclosed herein are described in WO 2023 / 278897, WO 2008 / 017473, WO 2009 / 141146, WO 2010 / 002851, WO 2014 / 049079, WO 2017 / 001702, WO 2018 / 172546, WO 2019 / 204179, WO 2020 / 260547, WO 2019 / 246450, WO 2009 / 095226, WO 2013 / 064584, WO 2014 / 169049, US 9,775,894, WO 2019 / 152884, WO 2017 / 173427, WO 2019 / 143297, WO 2019 / 204743, WO 2021 / 076713, the disclosures of which are hereby incorporated herein the reference, in their entireties.

[0227] In some aspects, compositions and methods are provided for treating a cancer by administering to the subject a therapeutically effective amount of a composition including a complex formed between (i) a humanized 3E10 antibody or 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. In accordance with various aspects of the present disclosure, the methods and compositions described herein include use a polynucleotide agonist of a TLR. For example, each of TLR3, TLR7, TLR8, and TLR9 have affinity for, and are activated by, various polynucleotides. Accordingly, in some aspects, the agonists used in the methods and compositions described herein arecapable of stimulating TLR3, TLR7, TLR8, or TLR9. In some aspects, the complex is a non-covalent complex. In some aspects, the complex comprises a covalent conjugate between the humanized 3E10 antibody or antigen binding fragment thereof and the polynucleotide ligand. Polynucleotide ligands capable of stimulating a TLR that are suitable for use in the methods and compositions provided herein are explicitly provided in US 20230303719, the content of which is incorporated herein by reference in its entirety.

[0228] In some aspects, the polynucleotide immunostimulant is cyclic-GMP-AMP synthase (cGAS) or STimulator of Interferon Genes (STING).Polynucleotides Encoding Effector Polypeptides

[0229] In some aspects, a humanized 3E10 antibody or antigen binding fragment thereof disclosed herein is complexed, covalently or non-covalently, with a therapeutic polynucleotide that encodes a protein or peptide, e.g., an effector polypeptide, for cancer therapy. In some aspects, the therapeutic polynucleotide is non-covalently bound to the humanized 3E10 antibody or antigen binding fragment thereof. In some aspects, the therapeutic polynucleotide is covalently conjugated to the humanized 3E10 antibody or antigen binding fragment thereof.

[0230] In some aspects, the polynucleotide is conjugated to or further encodes one or more of RNAi agents, siRNAs, shRNAs, miRNAs, miRNA binding sites, antisense RNAs, ribozymes, catalytic DNA, tRNA, RNAs that induce triple helix formation, aptamers or vectors, and the like.

[0231] In other aspects, the polynucleotide(s) may be designed to encode one or more effector polypeptide(s) or fragments thereof to a cancerous tissue. Such effector polypeptide(s) may include, but is not limited to, whole polypeptides, a plurality of polypeptides, or fragments of polypeptides, which independently may be encoded by one or more regions or parts or the whole of an effector polynucleotide. Effector polypeptides that are suitable for use in the methods and compositions provided herein are explicitly provided in US 20230303719, the content of which is incorporated herein by reference in its entirety.Tumor Antigens

[0232] In some aspects, the present disclosure provides compositions, as well as methods for treating a cancer in a subject by administering to the subject a therapeutically effective amount of the compositions, including a complex formed between (i) a humanized 3E10 antibody or antigen binding fragment thereof, and (ii) a therapeutic polynucleotide encoding a tumor associated antigen, as described herein. In some aspects, the therapeutic polynucleotide is non-covalently bound to the humanized 3E10 antibody or antigen binding fragment thereof. In some aspects, the therapeutic polynucleotide is covalently conjugated to the humanized 3E10 antibody or antigen binding fragment thereof.

[0233] Tumor antigens are peptides that are presented almost exclusively on the cell surface of cancerous cells, thereby distinguishing cancerous cells from non-cancerous cells that do not display the tumor antigen. When cancerous 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. Upon release of a sufficient amount of a tumor antigen, the immune system can generate antitumor immunity by generating an immune response to the tumor antigen. Specifically, the immune system targets and destroys cancerous cells displaying the tumor antigen that was used to generate the immune response.

[0234] Several classes of antigens have been exploited experimentally to generate antitumor immunity. Specifically, tumor antigens are exogenously administered, as either a peptide or a nucleic acid encoding the antigen, to a patient with a cancer displaying the tumor antigen on its cell surface. In turn, the immune system is presented with sufficient amounts of the antigen to generate an immune response against the tumor antigen, resulting in antitumor immunity. Examples of classes of tumor antigens include oncoviral protein antigens, neoantigens, and antigens derived from a cancer-germline gene. Often, the tumor antigen, or polynucleotide encoding the tumor antigen, is co-administered with an adjuvant that activated dendritic cells, or with dendritic cells themselves, to promote generation of the antitumor immunity. For review, see, for example, Haen et al., “Towards new horizons Characterization, classification and implications of the tumor antigenic repertoire,” Nature Review s-Clinical Oncology, 2020, Volume 17, 595-610; Saxena M. et al., Nat. Rev. Cancer 21, 360-378 (2021), all the contents of which are incorporated herein by reference.

[0235] In some aspects, the present disclosure relates to compositions and methods for treating a cancer in a subject by administering to the subject a therapeutically effective amount of a composition including a complex formed between (i) a humanized 3E10 antibody or antigen binding fragment thereof, and (ii) a therapeutic polynucleotide encoding an oncoviral protein antigen, as described herein. Therapeutic polynucleotides encoding an oncoviral protein antigen that are suitable for use in the methods and compositions provided herein are discussed at length in US 20230303719, the content of which is incorporated herein by reference in its entirety.

[0236] In some aspects, the tumor antigen is an oncoviral protein antigen, a neoantigen, or an antigen derived from a cancer-germline gene.

[0237] In some aspects, the oncoviral protein antigen is a folate receptor, HER2, papillomavirus oncoprotein E6 and papillomavirus oncoprotein E7 carcinoembryonic antigen (CEA), mucin 1, EGFR, squamous cell carcinoma antigen recognized by T cells 3 (SART3), beta-human chorionic gonadotropin (beta-hCG), Wilms’ Tumor antigen 1 (WT1), Surviving, MAGE3, p53, ring finger protein 43 and translocase of the outer mitochondrial membrane 34 (TOMM34), prostate-specific antigen (PSA)-TRICOM, or KRAS.

[0238] In some aspects, the neoantigen is BRCA1, BRCA2 BRAF, KRAS, EGFR, IDH1, PIK3CA, ROS1, HLA, JAK1, JAK2, PARK2, ATM, p53, TP53, erbb2 interacting protein (ERBB2IP), Beta-2-Microglobulin (P2m), cyclin-dependent kinase inhibitor 2A (CDKN2A), alternate reading frame (ARF), or cyclin-dependent kinase 4 (CDK4).

[0239] In some aspects, the cancer germline gene is MAGEA1, MAGEA2, MAGEA3, MAGEA4, MAGEA5, MAGEA6, MAGEA8, MAGEA9, MAGEA10, MAGEA11, MAGEA12, BAGE, BAGE2, BAGE3, BAGE4, BAGE5, MAGEB1, MAGEB2, MAGEB5, MAGEB6, MAGEB3, MAGEB4, GAGE1, GAGE2A, GAGE3, GAGE4, GAGE5, GAGE6, GAGE7, GAGE8, SSX1, SSX2, SSX2b, SSX3, SSX4, CTAG1B, LAGE-lb, CTAG2, MAGECI, MAGEC3, SYCP1, BRDT, MAGEC2, SPANXA1, SPANXB1, SPANXC, SPANXD, SPANXN1, SPANXN2, SPANXN3, SPANXN4, SPANXN5, XAGE1D, XAGE1C, XAGE1B, XAGE1, XAGE2, XAGE3, XAGE-3b, XAGE-4 / RP11-167P23.2, XAGE5, DDX43, SAGE1, ADAM2, PAGE5, CT16.2, PAGE1, PAGE2, PAGE2B, PAGE3, PAGE4, LIPI, VENTXP1, IL13RA2, TSP50, CTAGE1, CTAGE-2, CTAGE5, SPA17, ACRBP, CSAG1, CSAG2, DSCR8, MMAlb, DDX53, CTCFL, LUZP4, CASC5, TFDP3, JARID1B, LDHC, MORC1, DKKL1,SP011, CRISP2, FMR1NB, FTHL17, NXF2, TAF7L, TDRD1, TDRD6, TDRD4, TEX15, FATE1, TPTE, CT45A1, CT45A2, CT45A3, CT45A4, CT45A5, CT45A6, H0RMAD1, H0RMAD2, CT47A1, CT47A2, CT47A3, CT47A4, CT47A5, CT47A6, CT47A7, CT47A8, CT47A9, CT47A10, CT47A11, 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, ADAM29, CCDC36, LOC440934, SYCE1, CPXCR1, TSPY3, TSGA10, HIWI, MIWI, PIWI, PIWIL2, ARMC3, AKAP3, Cxorf61, PBK, C21orf99, OIP5, CEP290, CAB YR, 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, OTO A, CCDC62, GPATCH2, CEP55, FAM46D, TEX14, CTNNA2, FAM133A, LOC130576, ANKRD45, ELOVL4, IGSF11, TMEFF1, TMEFF2, ARX, SPEF2, GPAT2, TMEM108, NOL4, PTPN20A, SPAG4, MAEL, RQCD1, PRAME, TEX101, SPATAI 9, ODF1, ODF2, ODF3, ODF4, ATAD2, ZNF645, MCAK, SPAG1, SPAG6, SPAG8, SPAG17, FBXO39, RGS22, cyclin Al, 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 XAGElE.Proinflammatory Cytokines

[0240] Proinflammatory cytokines limit tumor cell growth by a direct anti-proliferative or pro-apoptotic activity, or indirectly by stimulating the cytotoxic activity of immune cells against tumor cells. The pro-inflammatory cytokines are secreted from Thl cells, CD4+ cells, macrophages, and dendritic cells. They are characterized by production of several Interleukins (IL), IL-1, IL-2, IL- 12, IL- 17, IL- 18, IFN-y, and TNF-a. The key pro- inflammatory cytokines are IL-1, IL-6, and TNF-a. These cytokines signal via type I cytokine receptors (CCR1) that are structurally divergent from other cytokine receptor types. They are crucial for coordinating cell mediated immune response and play a critical role in modulating the immune system. Pro-inflammatory cytokines generally regulategrowth, cell activation, differentiation, and homing of the immune cells to the sites of infection with the aim to control and eradicate the intracellular pathogens, including viruses. Accordingly, in some aspects, the compositions and methods for treating cancer described herein includes a humanized 3E10 antibody or antigen binding fragment thereof and a polynucleotide that encodes a proinflammatory cytokine.

[0241] In some aspects, the present disclosure provides compositions, and methods for treating cancer by administering such compositions to a subject in need thereof, including a complex formed between a (i) a humanized 3E10 antibody or antigen binding fragment thereof, and (ii) a therapeutic polynucleotide encoding a proinflammatory cytokine, as described herein. In some aspects, the therapeutic polynucleotide is non-covalently bound to the humanized 3E10 antibody or antigen binding fragment thereof. In some aspects, the therapeutic polynucleotide is covalently conjugated to the humanized 3E10 antibody or antigen binding fragment thereof.

[0242] In some aspects, the cytokine is IL-1, IL-6, IL-8, IL-12, IFN-y, IL-18, IL-15, IL-2, TNF-a, IL-10, TGF-b, CSF-1, CCL2, CCL3, CCL5, or VEGF. Therapeutic polynucleotides encoding a proinflammatory cytokine that are suitable for use in the methods and compositions provided herein are discussed at length in US 20230303719, the content of which is incorporated herein by reference in its entirety.Gene Regulating Polynucleotides

[0243] In some aspects, a humanized 3E10 antibody or antigen binding fragment thereof, as described herein, is used to deliver a gene-regulating polynucleotide that reduces or silences expression a gene product that promotes cancer growth and / or progression, e.g., by targeting the gene or a transcript thereof. Non-limiting examples of gene-regulating polynucleotides include siRNA, miRNA, saRNA, antagomirs, antisense oligonucleotides, and decoy oligonucleotides. In some aspects, the gene-regulating polynucleotide is a nonreplicating modified or unmodified mRNA. In some aspects, the gene-regulating polynucleotide is a self-amplifying mRNA. In some aspects, the gene-regulating polynucleotide is a plasmid encoding a protein or peptide. In some aspects, the generegulating polynucleotide is an expression-regulating polynucleotide. For a review of the various types of gene-regulating polynucleotides that have been researched for therapeutic capability see, for example, Roberts TC, Langer R, Wood MJA, “Advances in oligonucleotide drug delivery,” Nat. Rev. Drug Discov., 19(10): 673 -94 (2020), thecontent of which is incorporated herein by reference. Gene-regulating polynucleotides that are suitable for use in the methods and compositions provided herein are discussed at length in US 20230303719, the content of which is incorporated herein by reference in its entirety. siRNA

[0244] The present disclosure relates to compositions and methods for treating a cancer and includes a complex formed between (i) a humanized 3E10 antibody or antigen binding fragment thereof, and (ii) an siRNA, as described herein. Small interfering RNA (siRNA), also known as short interfering RNA or silencing RNA, is a class of doublestranded RNA non-coding RNA molecules, typically 20-27 base pairs in length, and operating within the RNA interference (RNAi) pathway. Gene-regulating nucleic acid drugs such as siRNA can regulate post-transcriptional gene expression, and silence targeted genes, further regulating intracellular signaling pathway 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 aspects, the siRNA is non-covalently bound to the humanized 3E10 antibody or antigen binding fragment thereof. In some aspects, the siRNA is covalently conjugated to the humanized 3E10 antibody or antigen binding fragment thereof.

[0245] siRNAs can, thus, be used for modulating the expression of immune checkpoint molecules, such as those described herein, by regulating the post-translational gene expression and / or silencing corresponding genes. Similarly, siRNAs can be used for indirectly regulating the activity of immune checkpoint molecules by modulating the expression of agonists or inhibitors of immune checkpoint molecules. Accordingly, in some aspects, the composition for treatment of cancer described herein includes a complex formed between (i) a humanized 3E10 antibody or antigen binding fragment thereof, and (ii) an siRNA targeting an mRNA transcript from a gene encoding an immune checkpoint molecule, as described herein. Accordingly, in some aspects, the compositions for treating a cancer provided herein include a complex formed between (i) a humanized 3E10 antibody or antigen binding fragment thereof, and (ii) siRNA targeting an mRNA transcript for VEGF, KSP, PKN3, RRM2, EphA2, ERBB2 / HER2, SOCS1,PLK1, or KRAS. siRNAs that are suitable for use in the methods and compositions provided herein are discussed at length in US 20230303719, the content of which is incorporated herein by reference in its entirety. miRNA

[0246] In some aspects, the compositions and methods for treating a cancer described herein includes a complex formed between (i) a humanized 3E10 antibody or antigen binding fragment thereof, and (ii) an miRNA, as described herein. MicroRNAs (miRNAs) are a class of non-coding RNAs that play important roles in regulating gene expression. miRNA is endogenous small non-coding 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(l):26-36). One main challenge of miRNA delivery is to deliver them into tumor tissue with deep tissue penetration efficiently. Moreover, the complexation of tumor microenvironment also prevents miRNA from efficient intracellular delivery into target tumor cells (Rupaimoole et al., MiRNA deregulation in cancer cells and the tumor microenvironment. Cancer Discov . 2016;6(3):235-46). In some aspects, the miRNA is non-covalently bound to the humanized 3E10 antibody or antigen binding fragment thereof. In some aspects, the miRNA is covalently conjugated to the humanized 3E10 antibody or antigen binding fragment thereof.

[0247] Accordingly, the compositions and methods for treating a cancer described herein includes a complex formed between (i) a humanized 3E10 antibody or antigen binding fragment thereof (ii) an miRNA which directly or indirectly modulates the expression of immune checkpoint molecules, as described herein. In some aspects, the miRNA is miR- 15a, miR-15b, miR-16, miR-20b, miR-21, miR-28, miR-34a, miR-34b, miR-34c, miR- 125b, miR-130b, miR-138, miR-138-5p, miR-155, miR-195, miR-197, miR-200, miR- 210, miR-221, miR-222, miR-424, miR-497, miR-503, or miR-513. miRNAs that are suitable for use in the methods and compositions provided herein are discussed at length in US 20230303719, the content of which is incorporated herein by reference in its entirety.saRNA

[0248] In some aspects, the compositions and methods for treating a cancer described herein includes a complex formed between (i) a humanized 3E10 antibody or antigen binding fragment thereof, and (ii) an saRNA, as described herein. Small-activating RNA (saRNA) is a class of noncoding dsRNA about 21 nt in length with 2 nt overhangs at both end (Zhou et al., Medicine in Drug Discovery, March 24, 2020; Kwok et al., Ther. Deliv. 2019; 10(3): 151-64). Although it shares similar structure with siRNA, it has the opposite mechanism of gene regulation. An saRNA in the cytoplasm is specifically loaded to an AGO2 protein and this RNA-AGO2 complex is transported to the nucleus to induce targeted gene promoters for gene activation (Li et al., Proc. Natl. Acad. Sci. USA. 2006; 103(46): 17337-42). It has been reported that saRNA-AGO2 complex in the nucleus recruits essential protein 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 of gene upregulation, saRNA shows the potential for applications such as cancer immunotherapy. In one example, an saRNA can upregulate the transcription factor CCATT / enhancer binding protein alpha (CEBPA) which leads to an increase in functional CZEBP protein and albumin and inhibits growth of liver cancer in a rat model. Accordingly, in some aspects, the composition for treatment of cancer described herein includes a complex formed between (i) a humanized 3E10 antibody or antigen binding fragment thereof, and (ii) an saRNA inducing activation of a gene encoding an immune checkpoint molecule, as described herein. In some aspects, the saRNA is non-covalently bound to the humanized 3E10 antibody or antigen binding fragment thereof. In some aspects, the saRNA is covalently conjugated to the humanized 3E10 antibody or antigen binding fragment thereof. saRNAs that are suitable for use in the methods and compositions provided herein are discussed at length in US 20230303719, the content of which is incorporated herein by reference in its entirety.Antagomirs

[0249] In some aspects, the compositions and methods for treating a cancer described herein includes a complex formed between (i) a humanized 3E10 antibody or antigen binding fragment thereof, and (ii) an antagomir, as described herein. An antagomir is a small synthetic RNA that is complementary to the specific miRNA target with eithermispairing at the cleavage site of Ago2 or some sort of base modification to inhibit Ago2 cleavage. Antagomirs are sequestered specific endogenous microRNA in competition with cellular target mRNAs, inducing miRNA repression and preventing mRNA target degradation via RISC. Thus, antagomirs can be used in treatments where miRNA loss of function is advantageous. In some aspects, the antagomir is non-covalently bound to the humanized 3E10 antibody or antigen binding fragment thereof. In some aspects, the antagomir is covalently conjugated to the humanized 3E10 antibody or antigen binding fragment thereof. Antagomirs that are suitable for use in the methods and compositions provided herein are discussed at length in US 20230303719, the content of which is incorporated herein by reference in its entirety.Antisense Oligonucleotides (ASOs)

[0250] In some aspects, the composition for treatment of cancer described herein includes a complex formed between (i) a humanized 3E10 antibody or antigen binding fragment thereof, and (ii) an antisense oligonucleotide, as described herein. Antisense oligonucleotides (ASOs) are short, synthetic, chemically modified chains of nucleotides that have the potential to target any gene product of interest. Typically, an ASO is a single-stranded sequence complementary to the sequence of the target gene’s transcribed messenger RNA (mRNA) within a cell (Rinaldi et al., “Antisense oligonucleotides: the next frontier for treatment of neurological disorders,” Nat. Rev. Neurol. 2018; 14(l):9-21 ; Bennett, Therapeutic Antisense Oligonucleotides Are Coming of Age. Ann. Rev. Med. 2019; 70:307-321). An ASO targets the corresponding mRNA to degrade the targeted complex by mechanisms such as endogenous cellular RNase H. In some aspects, the antisense oligonucleotide is non-covalently bound to the humanized 3E10 antibody or antigen binding fragment thereof. In some aspects, the antisense oligonucleotide is covalently conjugated to the humanized 3E10 antibody or antigen binding fragment thereof.

[0251] Non-limiting Examples of ASOs and cancers they are associated with are discussed at length in US 20230303719, the content of which is incorporated herein by reference in its entirety.Decoy Oligonucleotides

[0252] In some aspects, the present disclosure relates to compositions, as well as methods for treating a cancer in a subject by administering to the subject a therapeutically effective amount of a composition, including a complex formed between (i) a humanized 3E10 antibody or antigen binding fragment thereof, and (ii) a decoy oligonucleotide or a polynucleotide that encodes a decoy oligonucleotide. Transfection of cis-element doublestranded oligonucleotides, referred to as decoy oligodeoxynucleotides, has been reported to be a powerful tool that provides a new class of antigene strategies for gene therapy (Crinelli et al., Design and characterization of decoy oligonucleotides containing locked nucleic acids. Nucleic Acid Res. 2002; 30(11): 2435-2443). One such example is STAT3 decoy oligonucleotide, which is a double-stranded 15-mer oligonucleotide, corresponding closely to the signal transducer and activator of transcription 3 (STAT3) response element within the c-fos promoter, with potential antineoplastic activity. STAT3 decoy oligonucleotide binds specifically to activated STAT3 and blocks binding of STAT3 to DNA sequences on a variety of STAT3 -responsive promoters, which results in the inhibition of STAT3 -mediated transcription and, potentially, the inhibition of tumor cell proliferation. STAT3 is constitutively activated in a variety of cancers including squamous cell carcinoma of the head and neck, contributing to the loss of cell growth control and neoplastic transformation. In some aspects, the decoy oligonucleotide or polynucleotide that encodes the decoy oligonucleotide is non-covalently bound to the humanized 3E10 antibody or antigen binding fragment thereof. In some aspects, the decoy oligonucleotide or polynucleotide that encodes the decoy oligonucleotide is covalently conjugated to the humanized 3E10 antibody or antigen binding fragment thereof. Decoy oligonucleotides that are suitable for use in the methods and compositions provided herein are discussed at length in US 20230303719, the content of which is incorporated herein by reference in its entirety.Polynucleotides that Encode Complexes that Can Perform Genome Editing

[0253] In some aspects, a humanized 3E10 antibody or antigen binding fragment thereof described herein can be complexed to a polynucleotide that encodes complexes that can perform genome editing.

[0254] In some aspects, a humanized 3E10 antibody or antigen binding fragment thereof described herein is complexed to a polynucleotide that encodes a zinc-finger nuclease.Zinc-finger nucleases are genome editing nucleases. They are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. The binding specificity of the designed zinc-finger domain directs the zinc-finger nuclease to a specific genomic site.

[0255] In some aspects, a humanized 3E10 antibody or antigen binding fragment thereof described herein is complexed to a polynucleotide that encodes a Transcription Activatorlike Effector Nucleases (TALEN). TALENs are artificial endonucleases (e.g., restriction enzymes) and are produced by the fusion of a transcription activator-like effector (TALE) DNA binding domain with a DNA cleavage domain. TALENs can be engineered to bind any DNA sequence of interest.

[0256] In some aspects, a humanized 3E10 antibody or antigen binding fragment thereof described herein is complexed to a polynucleotide that encodes Cas endonuclease. In some aspects, a humanized 3E10 antibody or antigen binding fragment thereof described herein is complexed to a CRISPR / Cas guide polynucleotide, e.g., a single guide RNA (sgRNA).

[0257] In some aspects, a humanized 3E10 antibody or antigen binding fragment thereof described herein is complexed with an aptamer or a polynucleotide encoding an aptamer. In some aspects, the aptamer is a PSMA aptamer, a HER2 aptamer, a MUC1 aptamer, a CD 117 aptamer, a PTK7 aptamer, CTLA-4 aptamer, TLSl la aptamer, PD-1 aptamer, a PD-1 aptamer, a Macugen aptamer, AS1411, Sgc8, TD05, ARC1779, a-Thrombin (TBA), Macugen, E10030, AS1411, ARC1779, NU172, N0X-A12, NOX-E36, NOX-H94, ARC1905, REG1, ARC19499, AS1411, AS1411, EpCAM, A10-3-J1, Sgc8c, TSA14, 5TR1, Endo28, EGFR, A10, Sgc8c, AS1411, N0X-A12, KH1C12, K19, TD05, AS1411, HB5, HeA2_3, H2, S6, SYL3C, APTA-12, M17, S-l, SL2B, CAA01, CA50 A02, CA72- 4 A01, APT-43, TA6, CA125.1, Apt928, R13, HF3-58, or HA5-68. In some aspects, the aptamer targets an immune checkpoint regulatory protein. In some aspects, the immune checkpoint regulatory protein is B7-H3, B7-H4, BTLA, CD160, CTLA4, KIR, LAG3, PD-1, PD-L1, PD-L2, TIM3, or TIGIT.

[0258] In some aspects, humanized 3E10 antibodies and antigen-binding fragments thereof described herein are complexed with a ribozyme or a polynucleotide that encodes a ribozyme. In some aspects, the ribozyme targets human telomerase reverse transcriptase (hTERT) RNA.

[0259] Further examples of polynucleotides that encode complexes that can perform genome editing are described in US 20230303719, the content of which is incorporated herein by reference in its entirety.Methods of Treatment

[0260] In some aspects, the present disclosure provides compositions, as well as methods for treating a disorder by administering to a subject in need thereof a therapeutically effective amount of a composition, including a complex formed between (i) a humanized 3E10 antibody or antigen binding fragment thereof, and (i) a therapeutic agent. In some aspects, the therapeutic agent is covalently conjugated to the humanized 3E10 antibody or antigen fragment thereof.

[0261] In some aspects, the therapeutic agent is non-covalently associated to the humanized 3E10 antibody or antigen fragment thereof. In some aspects, the humanized 3E10 antibody or antigen fragment thereof is associated, e.g., conjugated, with a delivery vehicle for the therapeutic agent. In some aspects, 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, for example, Tiwari G. et al., International Journal of Pharmaceutical Investigation, 2(1):2-11 (2012), the content of which is incorporated herein by reference in its entirety. For a review of strategies for functionalizing nanoparticulate drug delivery systems see, for example, Seidu TA et al., Pharmaceutics, 14(5): 1113 (2022), the content of which is incorporated herein by reference in its entirety.

[0262] In some aspects, the therapeutic agent is a DNA damage inducing agent, a DNA repair inhibitor, an immune modulatory molecule, an alkylating agent, a microtubule inhibitor, an immune checkpoint inhibitor, an angiogenesis inhibitor, adoptive cell therapy, or a topoisomerase inhibitor. In some embodiment, the therapeutic agent is an anti-tumor drug. In some aspects, the therapeutic agent is a maytansinoid, a benzodiazepine, an auristatin, a tecan, a taxoid, CC-1065, (4S)-4, 11 -Diethyl-4, 9- dihydroxy-l,4-dihydro-3H,14H-pyrano[3',4':6,7]indolizino[l,2-b]quinoline-3, 14-dione (SN38), exatecan, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), a pyrrolobenzodiazepine (PBD), PROteolysis TArgeting Chimera (PROTAC), deruxtecan (Dxd), a calicheamicin, a duocarmycin, a stimulator of interferon genes (STING) agonist, PNU- 159682, NMS249, IMGN Camp 1, duocarmycin hydroxybenzamide azaindole(DUBA), or a prodrug thereof. In some aspects, the therapeutic agent is a maytansinoid. In some aspects, the therapeutic agent is N(2')-deacetyl-N(2')-(3-mercapto-l-oxopropyl)- maytansine (DM1). In some aspects, the therapeutic agent is N2’-deacetyl-N2’-(4- mercapto-4-methyl-l -oxopentyl) maytansine (DM4). In some aspects, the therapeutic agent is (4S)-4,l l-Diethyl-4,9-dihydroxy-l,4-dihydro-3H,14H- pyrano[3',4':6,7]indolizino[l,2-b]quinoline-3, 14-dione (SN38). In some aspects, the therapeutic agent is PNU-159682. In some aspects, the therapeutic agent is PNU-159682. In some aspects, the therapeutic agent is NMS249.

[0263] In some aspects, the therapeutic agent is a therapeutic polynucleotide, e.g., as described herein. In some aspects, the therapeutic polynucleotide is non-covalently bound to the humanized 3E10 antibody or antigen binding fragment thereof. In some aspects, the therapeutic polynucleotide is covalently attached to the humanized 3E10 antibody or antigen binding fragment thereof. In some aspects, the therapeutic polynucleotide is nonreplicating unmodified mRNA. In some aspects, the therapeutic polynucleotide is nonreplicating modified mRNA. In some aspects, the therapeutic polynucleotide is a selfamplifying mRNA. In some aspects, the therapeutic polynucleotide is a plasmid encoding the protein or peptide. In some aspects, wherein the therapeutic polynucleotide is a generegulating polynucleotide

[0264] In some aspects, methods are provided for treating a cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a humanized 3E10 antibody or antigen-binding fragment thereof described herein. In some aspects, the disclosure provides methods for treating a cancer in a subject in need thereof, the method comprising to the subject a therapeutically effective amount of a composition described herein. In some aspects, the cancer is a carcinoma, a sarcoma, a blastoma, a papilloma, or an adenoma. In some aspects, the cancer is a metastatic cancer. In some aspects, 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, renal cancer, thyroid cancer, or uterine cancer.

[0265] In some aspects, methods are provided for treating a subject suffering from a carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignancies e.g., sarcomas, carcinomas, andmelanomas; hematologic cancers of the blood or bone marrow; hematological (or hematogenous) cancers, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, nonHodgkin'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, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors (such as a glioma (such as brainstem glioma and mixed gliomas), glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNS lymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, or brain metastases.

[0266] In some aspects, the disclosure provides methods for treating a cancer of the central nervous system by parenterally administering to the periphery of the subject a therapeutically effective amount of a composition including a complex formed between (i) a humanized 3E10 antibody or antigen binding fragment thereof and (ii) a therapeutic agent, as described herein.

[0267] In some aspects, the cancer is a neuroepithelial brain or spinal tumor selected from the group consisting of a medulloblastoma, an astrocytic tumor, an oligodendroglial tumor, an oligoastrocytic tumor, an ependymal tumor, a choroid plexus tumor, a neuronal or mixed neuronal-glial tumor, a tumor of the pineal region, an embryonal tumor, or an otherwise uncategorized neuroepithelial tumor.

[0268] In some aspects, the cancer is a skin cancer. In some aspects, the skin cancer is basal cell carcinoma, squamous cell carcinoma, or melanoma. In one embodiment, the cancer is melanoma.

[0269] In some aspects, the cancer comprises one or more mutations in one or more DNA damage and response genes. In some aspects, the cancer comprises a mutation in PTEN, FANCA, FANCB, FANCC, FANCD1 / BRCA2, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ / BRIP1, FANCL, FANCM, FANCN / PALB2, FANCO / RAD51C, FANCP / SLX4, FANCQ / ERCC4, FANCR / RAD51, FANCS / BRCA1, FANCT / UBE2T, FANCU / XRCC2, FANCV / REV7, FANCW / RFWD3, FANCY / FAP100, or any combination thereof.

[0270] In some aspects, the humanized 3E10 antibody or antigen-binding fragment thereof is synthetically lethal to the cancer.

[0271] In various aspects, the humanized 3E10 antibody or antigen-binding fragment thereof is administered by parenteral, local, or systemic administration. In some aspects, the humanized 3E10 antibody or antigen-binding fragment thereof is administered by parenteral administration. In some aspects, the parenteral administration is intramuscular administration, intravenous administration, or subcutaneous administration.Methods of Making

[0272] In some aspects, the present disclosure provides compositions, methods for treating a disorder, as well as methods of making a humanized 3E10 antibody or antigenbinding fragment thereof comprising culturing a host cell described herein under suitable conditions. In some aspects, the present disclosure provides a polynucleotide encoding the humanized 3E10 antibody or antigen-binding fragment thereof described herein. In some aspects, the present disclosure provides a host cell harboring the polynucleotide composition described herein.

[0273] All methods described herein can be performed in any suitable order unless otherwise indicated or otherwise clearly contradicted by context. The use of any and all examples, or example language (e.g., “such as”) provided herein, is intended merely to better illuminate the aspects and does not pose a limitation on the scope of the aspects unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.EXAMPLESExample 1: Nucleic acid affinity measurements

[0274] In previous studies, 3E10 was found to bind RAD51 and be synthetically lethal in PTEN-deficient cells. It is known that a substitution of asparagine in place of aspartic acid at position 31 in heavy chain CDR1 leads to higher affinity DNA binding and more robust cellular penetration. Using this information, humanized next generation 3E10 antibodies with tunable nucleic acid binding and release, cellular uptake, and RAD51 binding for various clinical applications were engineered. To explore which if any of these residues are critical for nucleic acid ligand biding, IgFold, an open-source deep learning tool, was used to predict the structures of 3E10 variant scFvs and model their surface electrostatic potentials. In accordance with previous work, a cationic pocket in a region of the antibody flanked by N31 and K53 was found in antibodies V66, V13, and V31 (FIG. 1). The hypothesis that this was the 3E10 nucleic acid binding pocket was strengthened by the fact that mutations in each of these residues (i.e. D31 in WT, K53 Y in V31) were associated with greatly altered nucleic acid affinity. It was found that, in general, as antibody variant affinity decreases, this binding pocket becomes increasingly anionic. Given the anionic nature of nucleic acids, this again reinforced the hypothesis that 3E10’s relevant ligand binding region was identified. More specifically, the modeling predicted that the global landscape of amino acid changes in V31 causes changes in the 3D structure of light chain CDR2, thereby exposing an anionic pocket adjacent to K53Y and orienting it directly within the nucleic acid binding site.Example 2: Superior binding to RAD51 by low nucleic acid affinity variants and synthetic lethality to BRCA2- and PTEN-deficient cells

[0275] Hypothesizing that point mutational differences are responsible for the drastic changes in nucleic acid affinities, the heavy and light chain variable regions of WT and D31N chimeric 3E10 and V66, V13, and V31 were aligned (FIGs. 2A-2B). Previous work showed that V31 antibody binds nucleic acids with low affinity. In addition to the previously established critical nucleic acid binding residue at heavy chain position 31, it was also noted that additional residues might affect nucleic acid affinity, including light chain residues K53 and E85, both of which were mutated in the lowest affinity V31variant. Previous reports have demonstrated that chimeric 3E10 binds to the N-terminal domain of RAD51 and, via the inhibition of the homology-directed repair (HDR) pathway, is synthetically lethal to BRCA2-deficient and PTEN-deficient cells in vitro. Abnormalities in DNA repair and genomic instability, including HDR deficiency, represent a key hallmark of cancer and constitute one of the most common drivers of tumorigenesis; thus, understanding the determinants of the 3E10-RAD51 interaction is imperative for the advancement of the mAb toward the most effective therapy for the large cohort of patients with DNA-repair deficient tumors. Investigators thus wanted to assess RAD51 binding and HDR inhibition in the humanized 3E10 variants. Since prior work showed that the lower affinity chimeric WT 3E10 has a higher affinity interaction with purified RAD51, it was hypothesized that, like cellular penetration and tumor localization, nucleic acid affinity could be predictive of RAD51 binding efficiency. To test this, RAD51 binding was first assayed by treating cells in culture with humanized antibodies and performing co-immunoprecipitation (co-IP) and western blot analysis of RAD51 abundance. Using WT and D31N antibodies as positive controls, out of the three humanized variants tested, only the low affinity antibody, V31, demonstrates RAD51 binding (FIGs. 3A-3B).

[0276] To test the biological relevance of this observation, the effects of the humanized 3E10 antibodies on HDR efficiency were assessed using the cell-based DR-GFP reporter assay. Briefly, cells were first treated with 3E10 antibodies, followed by transfection of an I-A'ccI plasmid which creates a site-specific double-stranded break (DSB). In the case of proficient HDR, the DSB is repaired and GFP expression is activated. Using this assay, cell treated with WT and V31 antibodies show a significant decrease in the percentage of HDR-proficient cells, in keeping with the conclusions of our co-IP data (FIGs. 4A-4B). A moderate decrease in HDR following D3 IN treatment was also observed, which is also in accordance with previous findings.

[0277] It was then investigated whether the previously observed synthetic lethality in BRCA2- and PTEN-deficient cells with the humanized 3E10 variants could be recapitulated. Isogenic VC8 Chinese hamster cells (either BRCA2-expressing or BRCA2- null) were first treated with chimeric and humanized 3E10 variants, and survival was assessed using the CellTiter-Glo ATP -based viability assay. In accordance with the RAD51 co-IP and HDR inhibition assays, some 3E10 variants were synthetically lethal in BRCA2-null but not BRCA2-proficient VC8 cells. Treatment with WT 3E10 lead to thelargest decrease in BRCA2-null cell viability, followed by V31, and then D31N (FIG. 5). No cell death was observed following treatment with V66 and V13, which also did not show evidence of RAD51 binding or HDR inhibition. This same assay was then repeated with the three identified RAD51-binding variants in a second isogenic cell line, U251 glioblastoma cells that are either PTEN-expressing or PTEN-null. Again, the largest decrease in PTEN-null cell viability occured following treatment with WT 3E10, followed by V31 and then D3 IN (FIG. 6).

[0278] The robust data showing that V31, a variant with low nucleic acid affinity, exhibited RAD51 binding, HDR inhibition and synthetic lethality with BRCA2 and PTEN deficient cells led to the hypothesis that 3E10 RAD51 binding can also be related to differential nucleic acid affinity, and also to the upstream driver of this affinity, electrostatics. A model of the predicted protein-protein interaction (PPI) was needed. AlphaFold Multimer was used to predict de novo this interaction, but due to the highly unstructured nature of the RAD51 N-terminal domain, investigators were unable to generate models of sufficient confidence through this method. Instead, a single monomer was used from the crystal structure of RAD51 oligomers (8PBD) to avoid any erroneously predicted interaction sites stemming from disordered regions. The PPI- prediction software HADDOCK was used to dock WT 3E10 and a RAD51 monomer. One heavy chain and one light chain residue in 3E10 were each predicted to interact with one of two residues on RAD51 critical for its homo-oligomerization. 3E10 D31 in CDR1 was predicted to interact with alanine in RAD51 at position 89 (FIG. 7). Additionally, 3E10 light chain residue K53 was predicted to interact with RAD51 phenylalanine at position 86. The interaction at these two specific amino acids in RAD51 suggests a potential explanation for the observed 3E10-mediated inhibition of HDR.

[0279] The identification of these amino acids is consistent with the findings concerning the differences in RAD51 binding between the chimeric and human 3E10 variants which were assayed. Only the WT variant contained an aspartic acid at heavy chain residue 31, while only V31 contained a tyrosine at light chain position 53. This finding supports a model in which the D31N mutation ablates 3E10 RAD51 binding while an additional mutation K53 Y can rescue the interaction and overcome the negative effects of D3 IN. Specifically, tyrosine is significantly more hydrophobic than lysine, which can cause a more favorable interaction with the hydrophobic phenylalanine in RAD51. Aspartic acid is more anionic than asparagine, which is likely to be impactful in the 3E10-RAD51interaction based on electrostatic modeling. In fact, the same 3E10 residues which are imperative for this PPI also determine the nucleic acid binding pocket electrostatic charge, and thus nucleic acid affinity. Interestingly, electrostatic modeling of RAD51 revealed that the predicted binding area in the N-terminal domain is slightly cationic (FIG. 8). This evidence not only supports the hypothesis and corroborates the observations that low affinity 3E10 variants have the highest affinity for RAD51, but also provides a potential structural explanation for this phenomenon.

[0280] Taken together, the data presented in this example demonstrates that a 3E10 variant WT variant containing an aspartic acid at heavy chain residue 31 and a tyrosine at light chain position 53 can be engineered as a RAD51 “superbinder” with optimized HDR-deficient tumor synthetic lethality.Example 3: RAD51 Binding Experiments

[0281] A variant antibody containing an aspartic acid at heavy chain residue 31 and a tyrosine at light chain position 53 is generated and its affinity for RAD51 is investigated.

[0282] To investigate the direct interaction between the superbinder antibody and RAD51, cross-link and immunoprecipitation (CLIP) will be used. Briefly, MCF7 cells will be transfected with a plasmid containing human RAD51 under a cytomegalovirus (CMV) promoter using a lipid reagent. The next day, cells will be treated with 500 nM purified superbinder antibody for 6 hours. Cells will be UV crosslinked prior to cellular lysate harvesting. Then, 500 pg of cell lysates will be mixed with protein A / G magnetic beads over-end for 2 hours. Following low-pH elution, eluates will be run on gradient SDS-PAGE gels and western blot analysis will be performed to analyze the abundance of RAD51 in each sample. Using WT and variant antibodies as positive controls, the “superbinder” antibody succesfully demonstrates RAD51 binding.

[0283] To investigate the synthetic lethality of the superbinder antibody in the context of HR-deficient genetic backgrounds, an ATP -based cell viability assay will be used. This assay will be conducted in two HR-deficient matched pair cell lines: VC8 cells ± BRCA2, and U251 cells ± PTEN. Briefly, cells will be seeded at 500 cells per well in a 96-well plate and treated with purified superbinder mAb (500 nM in VC8 and 1 pM in U251). Five days after treatment, viability will be assessed using the CellTiter-Glo Luminescent Cell Viability Assay (Promega) according to the manufacturer’s protocol. Luminescence will be read on a Synergy Hl Multi-Mode Microplate Reader (Biotek). Compared to WTand variant antibodies controls, the superbinder antibody demonstrates successful decrease in BRCA2-null cell viability and / or PTEN-null cell viability.

[0284] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosure belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[0285] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific aspects of the disclosure described herein. Such equivalents are intended to be encompassed by the following claims.SEQUENCES

Claims

WHAT IS CLAIMED IS:

1. A humanized 3E10 antibody or antigen-binding fragment thereof comprising a light chain variable region (VL) and a heavy chain variable region (VH), wherein: a. the VL comprises a complementary determining region (CDR) 2 comprising the amino acid sequence XYASYLES (SEQ ID NO: 1), wherein X = D, Y, E, F, or W and corresponds to the amino acid residue at position 53 of the 3E10 light chain according to Kabat numbering; and b. the VH comprises a complementary determining region (CDR) 1 comprising the amino acid sequence XYGMH (SEQ ID NO: 2), wherein X = D, Y, E, F, or W and corresponds to the amino acid residue at position 31 of the 3E10 heavy chain according to Kabat numbering.

2. The humanized 3E10 antibody or antigen-binding fragment thereof of claim 1, wherein the VL CDR2 comprises Y, F, or W at the amino acid residue corresponding to position 53 of the 3E10 light chain.

3. The humanized 3E10 antibody or antigen-binding fragment thereof of claim 1, wherein the VL CDR2 comprises D or E at the amino acid residue corresponding to position 53 of the 3E10 light chain.

4. The humanized 3E10 antibody or antigen-binding fragment thereof of claim 1, wherein the VH CDR1 comprises D or E at the amino acid residue corresponding to position 31 of the 3E10 heavy chain.

5. The humanized 3E10 antibody or antigen-binding fragment thereof of claim 1, wherein the VH CDR1 comprises Y, F, or W at the amino acid residue corresponding to position 31 of the 3E10 heavy chain.

6. The humanized 3E10 antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the VL CDR2 comprises the amino acid sequence YYASYLES (SEQ ID NO: 3).

7. The humanized 3E10 antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the VL CDR2 comprises the amino acid sequence FYASYLES (SEQ ID NO: 4).

8. The humanized 3E10 antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the VL CDR2 comprises the amino acid sequence WYASYLES (SEQ ID NO: 5).

9. The humanized 3E10 antibody or antigen-binding fragment thereof of claim 1 or 3, wherein the VL CDR2 comprises the amino acid sequence DYASYLES (SEQ ID NO: 6).

10. The humanized 3E10 antibody or antigen-binding fragment thereof of claim 1 or 3, wherein the VL CDR2 comprises the amino acid sequence EYASYLES (SEQ ID NO: 7).

11. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1-4, wherein the VH CDR1 comprises the amino acid sequence DYGMH (SEQ ID NO: 8).

12. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1-3 or 5, wherein the VH CDR1 comprises the amino acid sequence YYGMH (SEQ ID NO: 9).

13. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1-3 or 5, wherein the VH CDR1 comprises the amino acid sequence FYGMH (SEQ ID NO: 10).

14. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1-3 or 5, wherein the VH CDR1 comprises the amino acid sequence WYGMH (SEQ ID NO: 11).

15. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1-4, wherein the VH CDR1 comprises the amino acid sequence EYGMH (SEQ ID NO:

16. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1-15, further comprising: a. a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 13; b. a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 14; c. a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15; and d. a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16.

17. A humanized 3E10 antibody or antigen-binding fragment thereof comprising a light chain variable domain (3E10-VL) and a heavy chain variable domain (3E10-VH), wherein: a. the 3E10-VL comprises the amino acid sequence set forth in SEQ ID NO: 18; and b. the 3E10-VH comprises the amino acid sequence set forth in SEQ ID NO: 24.

18. A humanized 3E10 antibody or antigen-binding fragment thereof comprising a light chain variable domain (3E10-VL) and a heavy chain variable domain (3E10-VH), wherein: a. the 3E10-VL comprises an amino acid sequence set forth in SEQ ID NOs: 19- 22; and b. the 3E10-VH comprises the amino acid sequence set forth in SEQ ID NO: 25- 28.

19. A humanized 3E10 antibody or antigen-binding fragment thereof comprising a light chain variable domain (3E10-VL) and a heavy chain variable domain (3E10-VH), wherein: a. the 3E10-VL comprises an amino acid sequence that is at least 91% identical to an amino acid sequence of 3E10-VL-hl (SEQ ID NO: 18); and b. the 3E10-VH comprises an amino acid sequence that is at least 93% identical to an amino acid sequence of 3E10-VH-WT (SEQ ID NO: 24).

20. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1-19, wherein the antibody or antigen-binding fragment thereof comprises one or more amino acid residues selected from proline (P) at position 15, threonine (T) at position 22, tyrosine (Y) at position 49, T at position 74, asparagine (N) at position 76, alanine (A) atposition 80, N at position 81, T at position 83, N at position 85, and valine (V) at position 104, of the 3E10-VL according to Kabat numbering.

21. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1-20, wherein the antibody or antigen-binding fragment thereof comprises one or more amino acid residues selected from glutamine (Q) at position 13, leucine (L) at position 18, arginine (R) at position 19, glycine (G) at position 42, serine (S) at position 49, S at position 77, tyrosine (Y) at position 79, N at position 82, A at position 84, valine (V) at position 89, L at position 108, V at position 109, and S at position 113, of the 3E10-VH according to Kabat numbering.

22. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1-21, comprising a lysine (Lys) residue at position 49 of the 3E10-VL according to Kabat numbering.

23. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1-22, comprising a glutamic acid (Glu) residue at position 81 of the 3E10-VL according to Kabat numbering.

24. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1-23, comprising an arginine (Arg) residue at position 18 of the 3E10-VH according to Kabat numbering.

25. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1-24, comprising a Lys residue at position 19 of the 3E10-VH according to Kabat numbering.

26. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1-25, comprising an alanine (Ala) residue at position 49 of the 3E10-VH according to Kabat numbering.

27. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1-26, comprising a proline (Pro) residue at position 15 of the 3E10-VL according to Kabat numbering.

28. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1-27, comprising a valine (Vai) residue at position 104, of the 3E10-VL according to Kabat numbering.

29. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1-28, comprising a glutamine (Gin) residue at position 13, of the 3E10-VH according to Kabat numbering.

30. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1-29, comprising a leucine (Leu) residue at position 108, of the 3E10-VH according to Kabat numbering.

31. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1-30, comprising a Vai residue at position 109, of the 3E10-VH according to Kabat numbering.

32. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1-31, comprising a serine (Ser) residue at position 113, of the 3E10-VH according to Kabat numbering.

33. The humanized 3E10 antibody or antigen-binding fragment thereof of claim 19, wherein: a. the 3E10-VL comprises the amino acid sequence set forth in SEQ ID NO: 18; and b. the 3E10-VH comprises the amino acid sequence set forth in SEQ ID NO: 24.

34. The humanized 3E10 antibody or antigen-binding fragment thereof of claim 19, wherein the amino acid residue at position 53 of the 3E10 light chain according to Kabat numbering does not comprise a K.

35. The humanized 3E10 antibody or antigen-binding fragment thereof of claim 19, wherein the amino acid residue at position 31 of the 3E10 heavy chain according to Kabat numbering does not comprise a N.

36. A humanized 3E10 antibody or antigen-binding fragment thereof comprising a light chain (LC) and a heavy chain (HC), wherein: a. the 3E10 LC comprises an amino acid sequence that is at least 93% identical to an amino acid sequence of 3E10-LC (SEQ ID NO: 29); and b. the 3E10 HC comprises an amino acid sequence that is at least 91% identical to an amino acid sequence of 3E10-HC (SEQ ID NO: 30).

37. The humanized 3E10 antibody or antigen-binding fragment thereof of claim 36, wherein: a. the 3E10 LC comprises the amino acid sequence set forth in SEQ ID NO: 29; and b. the 3E10 HC comprises the amino acid sequence set forth in SEQ ID NO: 30.

38. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1-37, wherein the humanized 3E10 antibody or antigen-binding fragment thereof has a strong binding affinity for RAD51.

39. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1-38, wherein the humanized 3E10 antibody or antigen-binding fragment thereof has a stronger binding affinity for RAD51 compared to a humanized 3E10 antibody or antigenbinding fragment thereof comprising a VL amino acid sequence set forth in SEQ ID NO:31.

40. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1-39, wherein the humanized 3E10 antibody or antigen-binding fragment thereof has a stronger binding affinity for RAD51 compared to a humanized 3E10 antibody or antigenbinding fragment thereof comprising a VH amino acid sequence set forth in SEQ ID NO:32.

41. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1-40, wherein the humanized 3E10 antibody or antigen-binding fragment thereof has a stronger binding affinity for RAD51 compared to a humanized 3E10 antibody or antigenbinding fragment thereof comprising (i) a VL amino acid sequence set forth in SEQ ID NO: 31, and / or (ii) a VH amino acid sequence set forth in SEQ ID NO: 32.

42. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1-41, wherein the humanized 3E10 antibody or antigen-binding fragment thereof binds to and inhibits RAD 51.

43. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1-42, wherein the humanized 3E10 antibody or antigen-binding fragment thereof has a weak binding affinity for a 3p-hpRNA RIG-I agonist having the nucleotide sequence5 ’ -pppGGAGC AAAAGC AGGGUGAC AAAGAC AUAAUGGAUCC AAAC ACUGUG UCAAGCUUUCAGGUAGAUUGCUUUCUUUGGCAUGUCCGCAAAC- 3’ (SEQ ID NO: 51).

44. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1-43, wherein the humanized 3E10 antibody or antigen-binding fragment thereof further comprises a fragment crystallizable (Fc) region.

45. The humanized 3E10 antibody or antigen-binding fragment thereof of claim 44, wherein the human Fc region is an IgGl Fc region.

46. The humanized 3E10 antibody or antigen-binding fragment thereof of claim 44 or 45, wherein the human Fc region comprises an alanine at position 234 and an alanine at position 235, according to Kabat numbering.

47. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 44-46, wherein the human Fc region comprises an alanine at position 234 and a glutamic acid at position 235, according to Kabat numbering.

48. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 44-47, wherein the human Fc region comprises an aspartic acid at position 297, according to Kabat numbering.

49. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 44-48, wherein the human Fc region comprises a proline at position 228, according to Kabat numbering.

50. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 44-49, wherein the human Fc region comprises a glutamine at position 307 and an alanine at position 434, according to Kabat numbering.

51. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 44-50, 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.

52. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 44-51, wherein the human Fc region comprises a glutamine at position 310, according to Kabat numbering.

53. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 44-52, wherein the humanized 3E10 antibody or antigen-binding fragment thereof further comprises a heavy chain constant domain (CHI).

54. The humanized 3E10 antibody or antigen-binding fragment thereof of claim 53, wherein the CHI is selected from the group consisting of a human yl CHI, a human y2 CHI, a human y3 CHI, and a human y4 CHI.

55. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1-54, wherein the humanized 3E10 antibody or antigen-binding fragment thereof further comprises a light chain constant domain (CL).

56. The humanized 3E10 antibody or antigen-binding fragment thereof of claim 55, wherein the CL is selected from the group consisting of a human X CL and a human K CL.

57. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1-56, wherein the humanized 3E10 antibody or antigen-binding fragment thereof is a single-chain Fv (scFv) or an antigen-binding fragment (Fab).

58. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1-57, wherein the humanized 3E10 antibody or antigen-binding fragment thereof is non- covalently associated with a therapeutic moiety.

59. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1-58, wherein the humanized 3E10 antibody or antigen-binding fragment thereof is covalently linked to a therapeutic moiety.

60. Same as belowThe humanized 3E10 antibody or antigen-binding fragment thereof of claim 58 or 59, wherein the therapeutic moiety is a therapeutic polypeptide.

61. The humanized 3E10 antibody or antigen-binding fragment thereof of claim 60, wherein the therapeutic moiety is a cytotoxic moiety.

62. The humanized 3E10 antibody or antigen-binding fragment thereof of claim 60, wherein the therapeutic moiety is a chemotherapeutic moiety.

63. The humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1-62, wherein the humanized 3E10 antibody or antigen-binding fragment thereof is covalently linked to a detectable moiety.

64. A composition comprising a non-covalent complex of: (i) a humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1-63, and (ii) a polynucleotide.

65. The composition of claim 64, wherein the molar ratio of (i) the humanized 3E10 antibody or antigen-binding fragment thereof to (ii) the polynucleotide in the composition is at least about 2:1, at least about 5: 1, at least about 20: 1, at least about 50: 1, or at least about 100: 1.

66. The composition of claim 64 or 65, wherein the molar ratio of (i) the humanized 3E10 antibody or antigen-binding fragment thereof to (ii) the polynucleotide in the composition is no more than about 2: 1, no more than about 5: 1, no more than about 20: 1, no more than about 100: 1, or no more than about 200: 1.

67. The composition of any one of claims 64-66, wherein the polynucleotide is about 15-4000 nucleotides in length, about 15-100 nucleotides in length, about 100-500 nucleotides in length, about 15-40 nucleotides in length, about 1000-4000 nucleotides in length or about 300-900 nucleotides in length.

68. The composition of any one of claims 64-67, wherein the polynucleotide is at least about 15 nucleotides in length, at least about 25 nucleotides in length, at least about 35 nucleotides in length, at least about 100 nucleotides in length, at least about 300 nucleotides in length, at least about 500 nucleotides in length, at least about 900 nucleotides in length, at least about 1000 nucleotides in length, at least about 2000 nucleotides in length, or at least about 3000 nucleotides in length.

69. The composition of any one of claims 64-68, wherein the polynucleotide is no more than about 25 nucleotides in length, no more about 35 nucleotides in length, no more than about 100 nucleotides in length, no more than about 300 nucleotides in length, no more than about 500 nucleotides in length, no more than about 900 nucleotides in length, no more than about 1000 nucleotides in length, no more than about 2000 nucleotides in length, no more than about 3000 nucleotides in length, no more than about 4000 nucleotides in length.

70. The composition of claim 69, wherein the molar ratio of the humanized 3E10 antibody or antigen-binding fragment thereof (i) to the polynucleotide (ii) in the composition is from 2: 1 to 50: 1, and wherein the polynucleotide is no more than 2000 nucleotides in length.

71. The composition of claim 69, wherein the molar ratio of the humanized 3E10 antibody or antigen-binding fragment thereof (i) to the polynucleotide (ii) in the composition is from 2: 1 to 30: 1, and wherein the polynucleotide is no more than 1000 nucleotides in length.

72. The composition of claim 69, wherein the molar ratio of the humanized 3E10 antibody or antigen-binding fragment thereof (i) to the polynucleotide (ii) in the composition is from 20: 1 to 200: 1, and wherein the polynucleotide is at least 2000 nucleotides in length.

73. The composition of any one of claims 64-72, wherein the polynucleotide is released from the non-covalent complex in less time than a corresponding non-covalent complex comprising a 3E10 antibody or antigen-binding fragment thereof comprising (i) a VL amino acid sequence set forth in SEQ ID NO: 31, and / or (ii) a VH amino acid sequence set forth in SEQ ID NO: 32.

74. The composition of any one of claims 64-73, wherein the non-covalent complex has a larger equilibrium dissociation constant (KD) nanomolar value than a corresponding non- covalent complex comprising a 3E10 antibody or antigen-binding fragment thereof comprising (i) a VL amino acid sequence set forth in SEQ ID NO: 31, and / or (ii) a VH amino acid sequence set forth in SEQ ID NO: 32.

75. The composition of claim 74, wherein the non-covalent complex has a KD value of about IO’10to IO’7M.

76. The composition of claim 74, wherein the non-covalent complex has a KD value of about 2 x 10'8to 3 x IO’8M.

77. The composition of any one of claims 64-76, wherein the non-covalent complex has a faster off-rate (Kdis) than a corresponding non-covalent complex comprising a 3E10antibody or antigen-binding fragment thereof comprising (i) a VL amino acid sequence set forth in SEQ ID NO: 31, and / or (ii) a VH amino acid sequence set forth in SEQ ID NO: 32.

78. The composition of claim 77, wherein the non-covalent complex has a Kdis value of about 7 x 10'3to 4 x IO’2S’1.

79. The composition of claim 77, wherein the non-covalent complex has a Kdis value of about 1 x IO’2to 2 x IO’2S’1.

80. A composition comprising a covalent complex of: (i) a humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1-81, and (ii) a polynucleotide, polypeptide, or a chemical agent.

81. The composition of any one of claims 64-80, wherein the polynucleotide is a therapeutic polynucleotide.

82. The composition of claim 81, wherein the therapeutic polynucleotide is a polynucleotide immunostimulant.

83. The composition of claim 82, wherein the polynucleotide immunostimulant is a polynucleotide ligand capable of stimulating a pattern recognition receptor (PRR).

84. The composition of claim 83, wherein the PRR is retinoic acid-inducible gene I (RIG-I).

85. The composition of claim 83, wherein the polynucleotide ligand comprises a 5’ triphosphate and double-stranded RNA.

86. The composition of claim 83, wherein the polynucleotide ligand comprises the nucleic acid sequence5 ’ -pppGGAGC AAAAGC AGGGUGAC AAAGAC AUAAUGGAUCC AAAC ACUGUGUCAAGCUUUCAGGUAGAUUGCUUUCUUUGGCAUGUCCGCAAAC- 3’ (SEQ ID NO: 51).

87. The composition of claim 83, wherein the polynucleotide ligand comprises a 3’ phosphate and hairpin RNA.

88. The composition of claim 83, wherein the PRR is a Toll-like receptor (TLR).

89. The composition of claim 88, wherein the TLR is TLR3, TLR7, TLR8, or TLR9.

90. The composition of claim 83, wherein the PRR is melanoma differentiation-associated protein 5 (MDA5).

91. The composition of claim 82, wherein the polynucleotide immunostimulant is a polynucleotide ligand capable of stimulating cyclic-GMP-AMP-synthase (cGAS).

92. The composition of claim 82, wherein the polynucleotide immunostimulant is a polynucleotide ligand capable of stimulating Stimulator of interferon genes (STING).

93. The composition of claim 81, wherein the therapeutic polynucleotide is DNA or mRNA that encodes a protein or peptide for cancer therapy.

94. The composition of claim 93, wherein the protein or peptide for cancer therapy is a tumor antigen.

95. The composition of claim 94, wherein the tumor antigen is selected from a tumor associated antigen, an oncoviral protein antigen, a neoantigen, and an antigen derived from a cancer-germline gene.

96. The composition of claim 95, wherein the tumor antigen is derived from a protein selected from folate receptor, HER2, papillomavirus oncoprotein E6 and papillomavirusoncoprotein E7 carcinoembryonic antigen (CEA), mucin 1, EGFR, squamous cell carcinoma antigen recognized by T cells 3 (SART3), beta-human chorionic gonadotropin (beta-hCG), Wilms’ Tumor antigen 1 (WT1), Survivin, MAGE3, p53, ring finger protein 43 and translocase of the outer mitochondrial membrane 34 (TOMM34), prostate-specific antigen (PSA)-TRICOM, and KRAS.

97. The composition of claim 95, wherein the tumor antigen is a neoantigen derived from a mutant protein selected from BRCA1, BRCA2 BRAF, KRAS, EGFR, IDH1, PIK3CA, ROS1, HLA, JAK1, JAK2, PARK2, ATM, p53, TP53, erbb2 interacting protein (ERBB2IP), Beta-2-Microglobulin (P2m), cyclin-dependent kinase inhibitor 2A (CDKN2A), alternate reading frame (ARF), and cyclin-dependent kinase 4 (CDK4).

98. The composition of claim 95, wherein the tumor antigen is an antigen derived from a cancer-germline gene selected from 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-lb, CTAG2, MAGECI, MAGEC3, SYCP1, BRDT, MAGEC2, SPANXA1, SPANXB1, SPANXC, SPANXD, SPANXN1, SPANXN2, SPANXN3, SPANXN4, SPANXN5, XAGE1D, XAGE1C, XAGE1B, XAGE1, XAGE2, XAGE3, XAGE-3b, XAGE-4 / RP11- 167P23.2, XAGE5, DDX43, SAGE1, ADAM2, PAGE5, CT16.2, PAGE1, PAGE2, PAGE2B, PAGE3, PAGE4, LIPI, VENTXP1, IL13RA2, TSP50, CTAGE1, CTAGE-2, CTAGE5, SPA17, ACRBP, CSAG1, CSAG2, DSCR8, MMAlb, DDX53, CTCFL, LUZP4, CASC5, TFDP3, JARID1B, LDHC, M0RC1, DKKL1, SPO11, CRISP2, FMR1NB, FTHL17, NXF2, TAF7L, TDRD1, TDRD6, TDRD4, TEX15, FATE1, TPTE, CT45A1, CT45A2, CT45A3, CT45A4, CT45A5, CT45A6, H0RMAD1, H0RMAD2, CT47A1, CT47A2, CT47A3, CT47A4, CT47A5, CT47A6, CT47A7, CT47A8, CT47A9, CT47A10, CT47A11, 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, ADAM29, CCDC36, LOC440934, SYCE1, CPXCR1, TSPY3, TSGA10, HIWI, MIWI, PIWI, PIWIL2,ARMC3, AKAP3, Cxorf61, PBK, C21orf99, 0IP5, CEP290, CAB YR, SPAG9, MPH0SPH1, 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, OTO A, CCDC62, GPATCH2, CEP55, FAM46D, TEX14, CTNNA2, FAM133A, LOC130576, ANKRD45, ELOVL4, IGSF11, TMEFF1, TMEFF2, ARX, SPEF2, GPAT2, TMEM108, NOL4, PTPN20A, SPAG4, MAEL, RQCD1, PRAME, TEX101, SPATAI 9, ODF1, ODF2, ODF3, ODF4, ATAD2, ZNF645, MCAK, SPAG1, SPAG6, SPAG8, SPAG17, FBXO39, RGS22, cyclin Al, 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, and XAGE1E.

99. The composition of claim 93, wherein the protein or peptide for cancer therapy is a cytokine.

100. The composition of claim 99, wherein the cytokine is selected from IL-1, IL-6, IL-8, IL- 12, IFN-y, IL-18, IL-15, IL-2, TNF-a, IL-10, TGF-P, CSF-1, CCL2, CCL3, CCL5, and VEGF.

101. The composition of any one of claims 93-100, wherein the therapeutic polynucleotide is a non-replicating modified or unmodified mRNA.

102. The composition of any one of claims 93-100, wherein the therapeutic polynucleotide is a self-amplifying mRNA.

103. The composition of any one of claims 93-100, wherein the therapeutic polynucleotide is a plasmid encoding the protein or peptide.

104. The composition of claim 81, wherein the therapeutic polynucleotide is an expressionregulating polynucleotide.

105. The composition of claim 104, wherein the expression-regulating polynucleotide is an siRNA106. The composition of claim 105, wherein the siRNA targets an mRNA transcript from a gene selected from KRAS, ERBB2 / HER2, VEGF, SOCS1, PLK1, and BCL2.

107. The composition of claim 108, wherein the expression-regulating polynucleotide is an miRNA.

108. The composition of claim 107, 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.

109. The composition of claim 108, wherein the expression-regulating polynucleotide is a small-activating RNA (saRNA).

110. The composition of claim 109, wherein the saRNA targets the promoter region of the CEBPA gene.

111. The composition of claim 108, wherein the expression-regulating polynucleotide is an antagomir.

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

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

114. The composition of claim 81, wherein the therapeutic polynucleotide encodes a genome editing effector.

115. The composition of claim 81, wherein the therapeutic polynucleotide encodes a zinc- finger nuclease.

116. The composition of claim 81, wherein the therapeutic polynucleotide encodes a transcription activator-like effector nuclease (TALEN).

117. The composition of claim 81, wherein the therapeutic polynucleotide encodes a CRISPR system comprising a Cas protein and a guide RNA.

118. The composition of claim 81, wherein the therapeutic polynucleotide is an effector polynucleotide.

119. The composition of claim 118, wherein the effector polynucleotide is an aptamer.

120. The composition of claim 119, wherein the aptamer is selected from a PSMA aptamer, a HER2 aptamer, a MUC1 aptamer, a CD117 aptamer, a PTK7 aptamer, CTLA-4 aptamer, TLSl la aptamer, PD-1 aptamer, a PD-1 aptamer, a Macugen aptamer, AS 1411, Sgc8, TD05, ARC1779, a-Thrombin (TBA), Macugen, E10030, AS1411, ARC1779, NU172, N0X-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, and HA5-68.

121. The composition of claim 118, wherein the effector polynucleotide is a ribozyme.

122. The composition of claim 121, wherein the ribozyme targets human telomerase reverse transcriptase (hTERT) RNA.

123. The composition of claim 80, wherein the chemical agent is selected from the group consisting of a DNA damage inducing agent, a DNA repair inhibitor, an immune modulatory molecule, an alkylating agent, a microtubule inhibitor, an immune checkpoint inhibitor, an angiogenesis inhibitor, adoptive cell therapy, and a topoisomerase inhibitor.

124. The composition of claim 80, wherein the chemical agent is an anti-tumor drug.

125. The composition of claim 80, wherein the chemical agent is selected from the group consisting of a maytansinoid, a benzodiazepine, an auristatin, a tecan, a taxoid, CC-1065, (4S)-4,11 -Diethyl-4, 9-dihydroxy-l,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), a pyrrolobenzodiazepine (PBD), PROteolysis TArgeting Chimera (PROTAC), deruxtecan (Dxd), a calicheamicin, a duocarmycin, a stimulator of interferon genes (STING) agonist, PNU-159682, NMS249, IMGN Camp 1, duocarmycin hydroxybenzamide azaindole (DUB A), and a prodrug thereof.

126. The composition of claim 80, wherein the chemical agent is a maytansinoid.

127. The composition of claim 80, wherein the chemical agent is N(2')-deacetyl-N(2')-(3- mercapto-l-oxopropyl)-maytansine (DM1).

128. The composition of claim 80, wherein the chemical agent is N2’-deacetyl-N2’-(4- m ercapto-4-m ethyl- 1 -oxopentyl) maytansine (DM4).

129. The composition of claim 80, wherein the chemical agent is (4S)-4,11 -Diethyl-4, 9- dihydroxy-l,4-dihydro-3H,14H-pyrano[3',4':6,7]indolizino[l,2-b]quinoline-3, 14-dione (SN38).

130. The composition of claim 80, wherein the chemical agent is PNU-159682.

131. The composition of claim 80, wherein the chemical agent is NMS249.

132. A method for treating a cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1-63.

133. A method for treating a cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition of any one of claims 64-131.

134. The method of claim 132 or 133, wherein the cancer is a carcinoma, a sarcoma, a blastoma, a papilloma, or an adenoma.

135. The method of claim 132 or 133, wherein the cancer is metastatic cancer.

136. The method of any one of claims 132-135, 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, renal cancer, thyroid cancer, and uterine cancer.

137. The method of claim 136, wherein the cancer is a skin cancer selected from the group consisting of basal cell carcinoma, squamous cell carcinoma, and melanoma.

138. The method of claim 137, wherein the cancer is melanoma.

139. The method of any one of claims of any one of claims 132-135, wherein the cancer is a cancer of the central nervous system.

140. The method of claim 139, wherein the cancer is a neuroepithelial brain or spinal tumor selected from the group consisting of a medulloblastoma, an astrocytic tumor, an oligodendroglial tumor, an oligoastrocytic tumor, an ependymal tumor, a choroid plexustumor, a neuronal or mixed neuronal-glial tumor, a tumor of the pineal region, an embryonal tumor, or an otherwise uncategorized neuroepithelial tumor.

141. The method of any one of claims 132-140, wherein the cancer comprises one or more mutations in one or more DNA damage and response genes.

142. The method of claim 141, wherein the cancer comprises a mutation in PTEN, FANCA, FANCB, FANCC, FANCD1 / BRCA2, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ / BRIP1, FANCL, FANCM, FANCN / PALB2, FANCO / RAD51C, FANCP / SLX4, FANCQ / ERCC4, FANCR / RAD51, FANCS / BRCA1, FANCT / UBE2T, FANCU / XRCC2, FANCV / REV7, FANCW / RFWD3, FANCY / FAP100, or any combination thereof.

143. The method of claim any one of claims 132-142, wherein the humanized 3E10 antibody or antigen-binding fragment thereof is synthetically lethal to the cancer.

144. The method of any one of claims 132-143, wherein the administering is by parenteral administration.

145. The method of claim 144, wherein the parenteral administration is intramuscular administration, intravenous administration, or subcutaneous administration.

146. The method of claim 132, wherein the cancer is melanoma and the administering is by parenteral administration.

147. A polynucleotide encoding the humanized 3E10 antibody or antigen-binding fragment thereof of any one of claims 1-64.

148. A host cell harboring the polynucleotide composition of claim 147.

149. A method of making a humanized 3E10 antibody or antigen-binding fragment thereof comprising culturing the host cell of claim 148 under suitable conditions.

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