Structurally defined siRNA-dual variable domain immunoglobulin conjugates

Abstract

Dual variable domain immunoglobulin double-stranded RNA conjugates advantageous for the inhibition of target gene expression and compositions suitable for therapeutic use are provided. [Solution] Dual variable domain immunoglobulins comprise a first variable domain that binds to a binding target and a second variable domain that contains a reactive residue, and a linker is covalently conjugated to the reactive residue. A dsRNA is linked to the linker and has the ability to inhibit target gene expression by RNA interference. The disclosure also provides pharmaceutical compositions containing these conjugates and methods of inhibiting target gene expression by administering these conjugates, for example, to treat various disease states.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62 / 864,755, filed June 21, 2019, the contents of which are incorporated herein by reference in their entirety.

[0002] FIELD OF THE INVENTION The present invention relates to dual variable domain immunoglobulin siRNA conjugates that are advantageous for inhibiting target gene expression, and compositions suitable for therapeutic use. In addition, the present invention provides methods for inhibiting target gene expression by administering these conjugates, for example, to treat various diseases. [Background technology]

[0003] background RNA interference, or "RNAi," was first coined by Fire and colleagues to describe the observation that double-stranded RNAi (dsRNA) can block gene expression (Fire et al. (1998) Nature 391, 806-811; Elbashir et al. (2001) Genes Dev. 15, 188-200). Short dsRNAs direct gene-specific posttranscriptional silencing in many organisms, including vertebrates, and have become a new tool for studying gene function. RNAi is mediated by the RNA-induced silencing complex (RISC), a sequence-specific multicomponent nuclease that destroys messenger RNAs homologous to the silencing trigger. RISC is known to contain short RNAs (approximately 22 nucleotides) derived from double-stranded RNA triggers, but the protein components of this activity remained unknown.

[0004] The discovery of RNA-mediated post-transcriptional gene silencing in 1998 was crucial in biological research. This process, known as RNA interference (RNAi), enabled the specific knockdown of any gene and has become a commonly used technique in all biological research. From a therapeutic perspective, RNAi has the advantage of being able to target any RNA-based disease-related factor, many of which are considered "undruggable" by small molecules. Furthermore, sequence-specific target recognition reduces the concern of off-target toxicity. Several RNAi-based strategies utilize different types of RNA for efficient knockdown. Short interfering RNA (siRNA) is one type of RNA that is perfectly complementary to the target sequence on a transcript and is introduced into target cells as a duplex. After entering the cell, the siRNA is loaded into the RNA-induced silencing complex (RISC). During this loading process, the passenger (sense) strand is removed, and the guide (antisense) strand remains in the RISC and binds to its complementary site on the target mRNA. The bound mRNA is then cleaved by the nuclease activity of RISC and then further degraded by cellular nucleases. 1 .

[0005] Although siRNA-mediated gene silencing is highly efficient, several challenges must be overcome to enable their use as therapeutics: 1) the size and large negative charge of siRNA hinder passive uptake into cells, 2) unmodified siRNA is rapidly degraded by nucleases and exhibits a short half-life in biological matrices, and 3) siRNA is potentially immunogenic. For certain tissues, such as the eye and lung, local administration of siRNA by intravitreal injection and inhalation, respectively, can overcome some of these challenges. For siRNA delivery to the liver, significant advances over the past few years have led to several clinically validated delivery technologies that have been shown to be safe and effective in humans. One system involves multicomponent lipid nanoparticles (LNPs), in which siRNA is encapsulated for most of its journey. 2,3The LNPs are designed to release their respective siRNA payloads into the cytoplasm of hepatocytes, where they can engage the RISC machinery. 4エラー!ブックマークが定義されていない,エラー!ブックマークが定義されていない Another well-established and clinically proven approach utilizes multivalent N-acetylgalactosamine (GalNAc) ligands covalently conjugated to siRNA. These ligands are designed to bind with high affinity and specificity to the asialoglycoprotein receptor (ASGPR), a cell surface receptor expressed on hepatocytes. 4 In addition to the use of GalNAc-targeting ligands, the development of advanced siRNA chemistry was critical to the success of this approach. 5 These approaches to deliver siRNA to the liver have transformed the field and, in 2018, led to the first FDA-approved RNAi-based therapy (patisiran; Onpattro; Alnylam Pharmaceuticals) for the treatment of hereditary transthyretin amyloidosis with polyneuropathy (hATTR).

[0006] Although there have been some advances in liver-directed RNAi-based therapeutics, the ability to target other tissues is highly desirable. Monoclonal antibodies (mAbs) are particularly suitable as delivery vehicles due to their high specificity for antigens expressed on target tissues and their long circulating half-lives. 5 These properties contribute to the fact that mAbs are a remarkably successful therapeutic class, with over 60 antibody-based FDA-approved therapeutics to date. 6 Furthermore, mAbs have already been validated as delivery vehicles for generating antibody-drug conjugates (ADCs), which involve the conjugation of highly potent small molecules to deliver them to target cancer cells. 7 Therefore, the creation of antibody-RNA conjugates (ARCs) is a promising strategy for delivering siRNA to target cells. Although several methods for preparing ARCs exist, these strategies use nonspecific conjugation. For example,8~14 Site-specific methods that result in mixtures or require multiple steps and introduction of mutations 15~17 Please refer to. Summary of the Invention

[0007] overview The present disclosure provides dual variable domain (DVD) immunoglobulin conjugates, and uses thereof. Generally, the conjugates comprise a DVD immunoglobulin molecule having a first variable domain and a second variable domain, and a dsRNA molecule covalently conjugated to the second variable domain via a linker. Methods for making and using the conjugates to inhibit target gene expression and therapeutic uses are also provided.

[0008] Generally, the conjugates of the invention comprise a dual variable domain immunoglobulin molecule (Ig) or antigen-binding fragment thereof and a double-stranded RNA (dsRNA) molecule linked together via a linker. Typically, the dual variable domain immunoglobulin molecule comprises (i) a first variable domain that binds to a binding target and (ii) a second variable domain that comprises a reactive residue, to which the linker is covalently conjugated.

[0009] The conjugates disclosed herein have the formula: Ig-(LR) nwherein Ig is a dual variable domain immunoglobulin molecule or immunoglobulin fragment (antigen-binding fragment) thereof, the dual variable domain immunoglobulin molecule comprising a first variable domain that binds to a binding target and a second variable domain comprising a reactive residue, L is a linker covalently conjugated to the reactive residue of the second variable domain of the Ig, R is a double-stranded RNA molecule, and n is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. In certain aspects, the reactive residue allows for stoichiometric attachment of L and includes, but is not limited to, natural and unnatural amino acids containing SH, NH, OH, SeH, N, alkyne, alkene, strained alkyne, strained alkene, C=O, and activated CH as reactive functional groups. In some embodiments of the various aspects disclosed herein, the reactive residue of the second variable domain is lysine or arginine.

[0010] The double-stranded RNA molecule is conjugated to a linker. The double-stranded RNA molecule comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity to a target sequence to mediate RNA interference, and the dsRNA has the ability to inhibit expression of a target gene. In some embodiments of the various aspects disclosed herein, the dsRNA molecule has at least one, for example, two, three, four, five, six, seven, eight, or nine, or all ten of the following characteristics: (i) a melting temperature (T) of about 40°C to about 80°C; m), (ii) the antisense strand contains two, three, four, five, or six 2'-fluoro modifications; (iii) the antisense strand contains one, two, three, or four phosphorothioate internucleotide linkages; (iv) the sense strand is conjugated with a linker; (v) the sense strand contains two, three, four, or five 2'-fluoro modifications; (vi) the sense strand contains one, two, three, or four phosphorothioate internucleotide linkages; (vii) the dsRNA contains at least four 2'-fluoro modifications; (viii) the dsRNA contains a duplex region 12 to 40 nucleotide pairs in length; (ix) the dsRNA has a blunt end at the 5' end of the antisense strand; and (x) the dsRNA has an overhang at the 3' end of the antisense strand.

[0011] In another aspect, the present disclosure provides a method for inhibiting expression of a target gene sequence. Generally, the method comprises administering to a cell a conjugate described herein in an amount sufficient to inhibit expression of the target gene. The cell can be in vitro or in vivo.

[0012] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising a conjugate described herein.

[0013] In yet another aspect, the present disclosure provides methods for treating a subject with a conjugate described herein. Generally, the treatment methods include administering a therapeutically effective amount of a conjugate described herein to a subject in need thereof. [The present invention 1001] (a) (i) a first variable domain that binds to a binding target; (ii) a second variable domain containing a reactive residue; a dual variable domain immunoglobulin molecule (Ig) or an antigen-binding fragment thereof, comprising: (b) a linker (L) covalently conjugated to a reactive residue of the second variable domain of an Ig; and (c) a double-stranded RNA (dsRNA) molecule conjugated to a linker, wherein the dsRNA has the ability to inhibit expression of a target gene, the dsRNA comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, the antisense strand having sufficient complementarity to a target sequence to mediate RNA interference, and the dsRNA is (i) a melting temperature (T m ), (ii) the antisense strand contains two, three, four, five, or six 2'-fluoro modifications; (iii) the antisense strand contains one, two, three, or four phosphorothioate internucleotide linkages; (iv) the sense strand is conjugated with a linker; (v) the sense strand contains two, three, four, or five 2'-fluoro modifications; (vi) the sense strand contains 1, 2, 3, or 4 phosphorothioate internucleotide linkages; (vii) the dsRNA contains at least four 2'-fluoro modifications; (viii) the dsRNA contains a double-stranded region of 12 to 40 nucleotide pairs in length; (ix) the dsRNA has a blunt end at the 5' end of the antisense strand, and (x) dsRNA has an overhang at the 3' end of the antisense strand and a dsRNA molecule further comprising at least one of the following characteristics: , a conjugate comprising: [The present invention 1002] dsRNA has a melting temperature (T m 1001. A conjugate of the present invention having [The present invention 1003] 1001. The conjugate of the present invention, wherein the dsRNA has a melting temperature of at least 60°C. [The present invention 1004] 1001. The conjugate of claim 10, wherein the dsRNA comprises at least four 2'-fluoro modifications. [The present invention 1005] 1001. The conjugate of the invention, wherein the dsRNA comprises a duplex region of 12 to 40 nucleotide base pairs in length. [The present invention 1006] 1005. The conjugate of the invention, wherein the dsRNA comprises a duplex region of 18 to 25 nucleotide base pairs in length. [The present invention 1007] 1001. The conjugate of the present invention, wherein the dsRNA comprises a blunt end at the 5' end of the antisense strand. [The present invention 1008] 1001. The conjugate of the present invention, wherein the dsRNA comprises an overhang at the 3' end of the antisense strand. [The present invention 1009] 1008. The conjugate of the invention, wherein the dsRNA comprises an overhang of at least 2 nucleotides at the 3' end of the antisense strand. [The present invention 1010] 1001. The conjugate of claim 10, wherein the sense strand is covalently conjugated to a linker. [The present invention 1011] The conjugate of the present invention 1010, wherein the 5' end of the sense strand is covalently conjugated to a linker. [The present invention 1012] The conjugate of the present invention 1010, wherein the 3' end of the sense strand is covalently conjugated to a linker. [The present invention 1013] 1001. The conjugate of the present invention, wherein the sense strand is 19 to 25 nucleotides in length. [The present invention 1014] 1013. A conjugate of the invention, wherein the sense strand is 21 nucleotides in length. [The present invention 1015] 1001. A conjugate of the present invention, wherein the sense strand comprises 2, 3, 4 or 5 2'-fluoro modifications. [The present invention 1016] A conjugate of the invention 1015, wherein the sense strand comprises three or four 2'-fluoro modifications. [The present invention 1017] 1001. A conjugate of the present invention, wherein the sense strand comprises 2'-fluoro modifications at the 7th, 10th and 11th positions counting from the 5' end. [The present invention 1018] 1001. A conjugate of the present invention, wherein the sense strand comprises 2'-fluoro modifications at the 7th, 9th, 10th and 11th positions counting from the 5' end. [The present invention 1019] 1001. The conjugate of claim 10, wherein the sense strand comprises 0, 1, 2, 3, or 4 phosphorothioate internucleotide linkages. [The present invention 1020] 1001. A conjugate of the present invention, wherein the sense strand comprises phosphorothioate internucleotide linkages between the first and second nucleotides and between the second and third nucleotides counting from the 5' end. [The present invention 1021] 1001. The conjugate of the present invention, wherein the antisense strand is 19 to 25 nucleotides in length. [The present invention 1022] A conjugate of the present invention 1021, wherein the antisense is 23 nucleotides in length. [The present invention 1023] 1001. A conjugate of the present invention, wherein the antisense comprises 2, 3, 4, 5 or 6 2'-fluoro modifications. [The present invention 1024] 1001. A conjugate of the present invention, wherein the antisense comprises 2'-fluoro modifications at positions 2, 14 and 16 counting from the 5' end. [The present invention 1025] 1001. A conjugate of the present invention, wherein the antisense comprises 2'-fluoro modifications at the 2nd, 6th, 9th, 14th and 16th positions counting from the 5' end. [The present invention 1026] 1001. A conjugate of the present invention, wherein the antisense comprises 2'-fluoro modifications at the 2nd, 6th, 8th, 9th, 14th and 16th positions counting from the 5' end. [The present invention 1027] 1001. A conjugate of the invention, wherein the antisense comprises 1, 2, 3 or 4 phosphorothioate internucleotide linkages. [The present invention 1028] 1001. A conjugate of the present invention, wherein the antisense comprises phosphorothioate internucleotide linkages between the 21st and 22nd nucleotides and between the 22nd and 23rd nucleotides counting from the 5' end. [The present invention 1029] A conjugate of the present invention 1001, wherein the antisense comprises phosphorothioate internucleotide linkages between the first and second nucleotides, between the second and third nucleotides, between the 21st and 22nd nucleotides, and between the 22nd and 23rd nucleotides counting from the 5' end. [The present invention 1030] 1001. The conjugate of the present invention, wherein the antisense strand comprises at least one duplex thermodestabilizing modification within the first 9 nucleotide positions of the 5' region. [The present invention 1031] A conjugate of the invention 1030, wherein the thermodestabilizing modification is at the 4th, 5th, 6th, 7th, 8th or 9th position counting from the 5' end of the antisense strand. [The present invention 1032] A conjugate of the present invention 1031, wherein the thermolabile modification is at the 7th position counting from the 5' end of the antisense strand. [The present invention 1033] 1001. A conjugate of the present invention, wherein the antisense comprises a 5'-vinylphosphonate nucleotide at the 5' end. [The present invention 1034] 1001. The conjugate of the present invention, wherein the dsRNA comprises at least one 2'-OMe modification. [This invention 1035] 1001. The conjugate of the present invention, wherein the sense strand comprises at least one 2'-OMe modification. [The present invention 1036] 1001. A conjugate of the present invention, wherein the antisense strand comprises at least one 2'-OMe modification. [This invention 1037] 1001. The conjugate of the present invention, wherein the dsRNA comprises at least one locked nucleic acid (LNA) modification. [The present invention 1038] 1001. A conjugate of the present invention, wherein the reactive residue is lysine. [This invention 1039] 1001. A conjugate of the present invention, wherein the first variable domain of the Ig is positioned closer to the N-terminus than the second variable domain. [The present invention 1040] 1001. A conjugate of the present invention, wherein Ig is a bispecific immunoglobulin molecule. [The present invention 1041] 1001. A conjugate according to the present invention, wherein the antigen-binding fragment comprises the first and second variable domains of an Ig and is selected from Fab, Fab', F(ab')2, Fv or scFv. [The present invention 1042] 1001. The conjugate of the present invention, wherein the antigen-binding fragment comprises a Fab. [This invention 1043] 1001. A conjugate of the present invention, wherein the Ig comprises a chimeric immunoglobulin sequence. [This invention 1044] 1001. A conjugate of the present invention, wherein Ig comprises a humanized immunoglobulin sequence. [This invention 1045] 1001. A conjugate of the present invention, wherein Ig comprises a human immunoglobulin sequence. [The present invention 1046] The conjugate of the present invention 1001, wherein the binding target is a tumor cell surface antigen. [This invention 1047] Any of the aforementioned conjugates of the invention, wherein the first variable domain binds to CD138, B-cell maturation antigen (BCMA), SLAMF7, HER2, FOLR1 or CD79b. [This invention 1048] 1001. A conjugate according to the present invention, wherein the linker L is a reversible linker. [This invention 1049] 1001. A conjugate according to the present invention, wherein the linker L is a non-reversible linker. [The present invention 1050] 1001. A conjugate according to the present invention, wherein the linker L is a cleavable linker. [This invention 1051] 1001. A conjugate according to the present invention, wherein the linker L is a non-cleavable linker. [This invention 1052] 1001. A conjugate according to the present invention, wherein the linker L is a branched linker. [This invention 1053] 1001. A conjugate according to the present invention, wherein the linker L is a linear linker. [This invention 1054] 1001. The conjugate of claim 10, wherein the Ig further comprises a ligand. [This invention 1055] A conjugate of the present invention 1054, wherein the ligand is an endosomolytic ligand. [This invention 1056] A conjugate of the present invention 1054, wherein the ligand is linked to the light chain. [This invention 1057] 1001. The conjugate of the present invention, wherein the second variable domain of the Ig comprises an amino acid sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and any combination thereof. [This invention 1058] 1001. A conjugate of the present invention, wherein the Ig comprises a peptide linker between the first and second variable domains. [This invention 1059] The peptide linker is 1055. A conjugate of the present invention comprising an amino acid sequence selected from the group consisting of TIFF2026041866000002.tif25147 and any combination thereof. [The present invention 1060] 1001. The conjugate of the present invention, wherein the Ig comprises an amino acid sequence selected from the group consisting of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and any combination thereof. [This invention 1062] A pharmaceutical composition comprising a conjugate of the present invention 1001, alone or in combination with a pharmaceutically acceptable carrier or excipient. [This invention 1063] A gene silencing kit comprising the conjugate of the present invention. [This invention 1064] A method for silencing a target gene in a cell, comprising the step of introducing a conjugate of the present invention into the cell. [This invention 1065] Use of the conjugate of the present invention in the preparation of a medicament. [Brief explanation of the drawings]

[0014] [Figure 1A] Schematic diagram showing a comparison of h38C2 IgG1 and anti-multiple myeloma (MM) DVD-IgG1. DVD-IgG1 consists of an anti-CD138, anti-BCMA, or anti-SLAMF7 variable domain (blue), h38C2 (green) with a reactive lysine (Lys; K, yellow circle), and a constant domain (gray). The two variable domains were joined together using a fully human spacer sequence (ASTKGP, red line). [Figure 1B]Coomassie stained SDS-PAGE confirming the purity of all MM-targeted DVD-IgG1s under non-reducing conditions (expected approx. 200 kDa) and reducing conditions (heavy chain approx. 63 kDa, light chain approx. 36 KDa). Molecular weights from a prestained protein ladder are shown on the left. [Figure 1C] Flow cytometry analysis showing specific binding of DVD-IgG1 to three MM cell lines (U-266, NCI-H929, and RPMI-8226). h38C2 IgG1 (black) was used as a negative control. [Figure 2-1] The structures of siRNA compounds are shown. 3'-end (4, SEQ ID NOs: 40 and 54) and 5'-end (5, SEQ ID NOs: 39 and 54) β-lactam hapten (blue) functionalized siRNA targeting CTNNB1 for DVD-IgG1 attachment. Control 3'-end β-lactam hapten functionalized siRNA (6, SEQ ID NOs: 41 and 55) targeting human transthyretin (TTR Error! Bookmark not defined), an irrelevant target in this study. Control siRNA targeting CTNNB1 lacking the β-lactam hapten moiety for DVD-IgG1 conjugation (7, SEQ ID NOs: 37 and 54). Black circles in the siRNA indicate 2'-OMe-modified nucleosides, green circles represent 2'-F-modified nucleosides, and blue circles represent 2'-NMA 5-Me-U nucleosides containing a 5'-vinylphosphonate (VP) moiety. Yellow bars represent phosphorothioate (PS) linkages for exonuclease protection. The combination of 2'-OMe-modified nucleosides and 2'-F-modified nucleosides with PS linkages follows the previously reported "enhanced stabilization chemistry" (ESC) template. Error! Bookmark not defined. [Figure 2-2] This is a continuation of Figure 2-1. [Figure 3A]Schematic diagram of ARC assembly. ARCs (8–13) were assembled by incubating DVD-IgG1 (1–3) with 10 equivalents (eq) of β-lactam siRNA (4 and 5) for 2 h at room temperature (rt). Attachment of the siRNA (red and black helices) occurs at the reactive Lys (K, yellow circle) of h38C2, located in the inner heavy chain variable domain of the DVD, forming a stable amide bond. [Figure 3B] This is a line graph showing the catalytic retroaldol activity of the reactive Lys of h38C2. Activity was measured using methodol as a substrate, which is converted to a fluorescent aldehyde and detected. Signals are reported in relative fluorescence units (RFU; mean ± SD of triplicate determinations). Assembled ARCs (8–13) are catalytically inactive due to amide formation at the reactive Lys, indicating complete conjugation. Unconjugated DVD-IgG1 (1–3) was used as a positive control, and trastuzumab IgG1 (black) was used as a negative control. [Figure 4] Figure 4A is a bar graph showing CTNNB1 mRNA knockdown in NCI-H929 cells after treatment with ARCs targeting SLAMF7 (8 and 11), BCMA (9 and 12), or CD138 (10 and 13) at 90 nM (antibody concentration) at 37°C for 72 hours. Unconjugated DVD-IgG1 (1-3) (black) and transfected free siRNA (Figure 2, 7) (white) were used as negative and positive controls, respectively. Figure 4B is a bar graph showing the dose response of BCMA-targeting ARCs (9 and 12) in NCI-H929 cells. BCMA ARC (14) conjugated to siRNA targeting human TTR (Figure 2, 6) was used as a negative control. Error bars in (A) and (B) correspond to biological duplicates. When comparing each group with the untreated group, significance was determined using a Student's t-test. [Figure 5]Figure 1 is a bar graph showing CTNNB1 mRNA knockdown in NCI-H929 cells after treatment with unpurified or purified BCMA-targeted ARCs (9 and 12) at 90 nM (antibody concentration) at 37°C for 72 hours. Unconjugated anti-BCMA DVD-IgG1 (2), anti-BCMA ARC conjugated to siRNA targeting human TTR (14), and anti-HER2 ARC conjugated to siRNA targeting CTNNB1 (15 and 16) were used as negative controls. Transfected free siRNA (Figure 2, 7) was used as a positive control. Error bars correspond to biological triplicates. [Figure 6] This is a gel photograph showing CTNNB1 protein knockdown in NCI-H929 cells after treatment with BCMA-targeting ARC (9 and 12, lanes 3 and 4) at 90 nM (antibody concentration) for 72 hours at 37°C. Untreated cells (lane 1), unconjugated anti-BCMA DVD-IgG1 (2, lane 2), anti-BCMA ARC targeting TTR (14, lane 5), and anti-HER2 ARC targeting CTNNB1 (15 and 16, lanes 6 and 7) were used as negative controls. Transfected free siRNA (7, lane 8) was used as a positive control. The Western blot shown is a representative example from three biological replicates. [Figure 7] 1 is a schematic diagram showing the synthesis of exemplary sense strand 19 (SEQ ID NO:39) containing a β-lactam moiety by reaction of bis-β-lactam derivative 15 with the corresponding single-stranded siRNA 16 (SEQ ID NO:36) containing an amino functionality added to the 5′ end. Compounds 20 and 21, containing a β-lactam moiety at the 3′ end of the strand, were prepared in a similar manner from the corresponding precursor strands 17 and 18. [Figure 8A]Figures 8A and 8B are HPLC chromatograms of 19 (SEQ ID NO: 18) without (Figure 8A) and with (Figure 8B) butylamine pretreatment. Complete cleavage of the product by the HPLC eluent was observed. After partial pretreatment with excess butylamine, the product was converted to a butylamine adduct prior to chromatography. [Figure 8B] See legend to Figure 8A. [Figure 9] Figure 3 shows the catalytic retroaldol activity of the reactive Lys of h38C2. Activity was measured using methodol as a substrate, which is converted to a fluorescent aldehyde and detected. Signals are reported in relative fluorescence units (RFU; mean ± SD of triplicate determinations). Anti-BCMA DVD-IgG1 (2) was conjugated to siRNA targeting human TTR (Figures 2, 6) using the conditions shown in Figure 3A to generate the antibody-RNA conjugate (14). This ARC is catalytically inactive due to amide formation at the reactive Lys, indicating complete conjugation. Unconjugated anti-BCMA DVD-IgG1 (2) was used as a positive control, and trastuzumab IgG1 (black) was used as a negative control. [Figure 10A] Schematic showing the optimized assembly of anti-BCMA ARCs 9 and 12. ARCs were assembled by incubating anti-BCMA DVD-IgG1 (2) with two equivalents (eq) of β-lactam siRNA (4 and 5, Figure 2) for 4 hours at room temperature (rt). Attachment of the siRNA (red and black helices) occurs at the reactive Lys (K, yellow circle) of h38C2, located in the inner heavy chain variable domain of DVD-IgG1, forming a stable amide bond. [Figure 10B]Figure 10A shows the catalytic retroaldol activity of the reactive Lys of h38C2 in anti-BCMA ARCs 9 and 12. Activity was measured using methodol as a substrate, which was converted to a fluorescent aldehyde and detected. Signals are reported in relative fluorescence units (RFU; mean ± SD of triplicate determinations). Assembled ARCs (9 and 12) are catalytically inactive due to amide formation at the reactive Lys, indicating complete conjugation. Unconjugated anti-BCMA DVD-IgG1 (2) was used as a positive control, and trastuzumab IgG1 (black) was used as a negative control. [Figure 11] Figure 11A is a size exclusion chromatograph showing purified anti-BCMA DVD-IgG1 2 (Figure 11A) and the following ARCs: anti-BCMA ARC 9 (Figure 11B), anti-BCMA ARC 12 (Figure 11C) and anti-BCMA ARC 14 (Figure 11D). The main peak is shown in mL. [Figure 12] Figure 12A shows the assembly of anti-HER2 control ARCs and their size-exclusion chromatograms. Figure 12A shows the catalytic retroaldol activity of the reactive Lys of h38C2. Activity was measured using methodol as a substrate after conjugation of anti-HER2 DVD-IgG1 with β-lactam siRNAs (4 and 5), as shown in Figure 3. Signals are reported in relative fluorescence units (RFU; mean ± SD of triplicate determinations). Assembled ARCs (15 and 16) are catalytically inactive due to amide formation at the reactive Lys, indicating complete conjugation. Unconjugated anti-HER2 DVD (blue) was used as a positive control, and trastuzumab IgG1 (black) was used as a negative control. (B) Anti-HER2 DVD. (C) Anti-HER2 ARC (15). (D) Anti-HER2 ARC (16). 12B-12D are size-exclusion chromatographs showing purified anti-HER2 DVD (FIG. 12B), anti-HER2 ARC 15 (FIG. 12C), and anti-HER2 ARC 16 (FIG. 12D). The major peaks are shown in mL. [Figure 13]Bar graphs showing β-catenin knockdown in U266 cells (FIG. 13A) and RPMI-8226 cells (FIG. 13B) after treatment with SLAMF7-, BCMA-, or CD138-targeted ARCs (8-13) at 90 nM for 72 hours at 37°C. Unconjugated DVDs (1-3) (black) and transfected free siRNA (6) (gray) were used as controls. Student's t-test was used to determine significance when comparing each group to the untreated group. Error bars correspond to biological triplicates. [Figure 14] Figure 14 shows the toxicity of ARCs (1, 8, 11, 2, 9, 12, 3, 10, 13, and 25 from top to bottom; CD138 ADC is 28) after 72 hours of incubation at 37°C with MM cell lines U266 (Figure 14A), NCI-H929 (Figure 14B), and RPMI-8226 (Figure 14C) (mean ± SD of triplicate experiments). Unconjugated DVD was used as a negative control, and anti-CD138 DVD conjugated to cytotoxic MMAF (22, black) was used as a positive control. [Figure 15A] Figure 15 shows surface plasmon resonance (SPR) binding analysis of an exemplary anti-BSMA Fab-DVD conjugated with siRNA_4 (Figure 15A) and siRNA_5 (Figure 15B), as well as without siRNA conjugation (Figure 15C). The calculated equilibrium dissociation constant (Kd) was identical before and after conjugation, indicating that conjugation with siRNA does not affect binding of the outer variable domain to BCMA. [Figure 15B] See legend to Figure 15A. [Figure 15C] See legend to Figure 15A. [Figure 16] Schematic representation of the DVD-IgG1 ARC construct (1:2 conjugation). [Figure 17] Schematic representation of an exemplary DVD-IgG1 ARC / EEP construct (1:2 conjugation) with a fusogenic peptide. [Figure 18]1 shows size exclusion chromatography and flow cytometry analysis of an exemplary DVD-IgG1 ARC (BCMA / CTNNB 1-A). [Figure 19] 1 shows the Fab format, binding and surface plasmon resonance analysis of an exemplary DVD-IgG1 ARC (BCMA / CTNNB 1-A). [Figure 20] 20 shows knockdown of CTNNB1 mRNA by the exemplary DVD-IgG1 ARC (BCMA / CTNNB 1-A). The sequences in Figure 20 are SEQ ID NO: 39 (top) and SEQ ID NO: 54 (bottom). [Figure 21] 1 shows knockdown of CTNNB1 protein by the exemplary DVD-IgG1 ARC (BCMA / CTNNB 1-A). [Figure 22] Figure 1 shows the pharmacokinetics of exemplary DVD-IgG1 ARC (BCMA / CTNNB 1-A). The sequences are SEQ ID NO: 51 (top) and SEQ ID NO: 54 (bottom). [Figure 23] Illustrated diagram showing the effect of interferon regulatory factor 4 (IRF4) in multiple myeloma. Source: Shaffer et al. Nature (2008), 454(7201):226-31 and Shaffer et al. Clin Cancer Res (2009), 15(9):2954-2961. [Figure 24] 1 shows knockdown of IRF4 mRNA by the exemplary DVD-IgG1 ARC (BCMA / CTNNB 1-A). The sequences are SEQ ID NO:52 (top) and SEQ ID NO:53 (bottom). [Figure 25] 1 shows the cytotoxicity of exemplary ARC (BCMA / IRF4 and SLAMF7 / IRF4 (4-A=1:2) conjugates) with and without endosomal escape peptides. DETAILED DESCRIPTION OF THE INVENTION

[0015] Detailed Description Although the preparation of structurally defined conjugates of dual variable domain (DVD) antibodies containing unique reactive lysine residues that are site-specifically conjugated to small molecule cargoes such as cytotoxic agents using hapten-like β-lactam moieties as attachment anchors has been described in the art (Nanna et al., Nat. Commun. 2017 and International Patent Publication No. WO 2017 / 049139), the practical preparation of the corresponding DVD-siRNA conjugates for therapeutic use has been largely unknown and has presented a significant challenge. For example, when conjugated to a DVD, large, positively charged siRNA cargo molecules may impair binding affinity and cellular internalization; when conjugated to a DVD, siRNA molecules may lose gene silencing activity; when conjugated to a DVD, nucleolytic degradation of siRNA may be enhanced; the chemistry for conjugating drugs to DVDs (e.g., unstable β-lactams) is not compatible with currently established solid-phase synthesis of siRNA; the reactivity of β-lactam moieties attached to siRNA may not be high enough to support efficient conjugation to the uniquely reactive Lys residue in the hydrophobic pocket of the DVD using mild, neutral conditions so as not to compromise the integrity of the DVD and siRNA; and purification of DVD-siRNA conjugates is not trivial. Furthermore, the attachment point on the siRNA may contain the activity of the siRNA.

[0016] The present inventors have now surprisingly and unexpectedly discovered that conjugation of two siRNA molecules to a DVD does not impair the binding affinity or internalization of the DVD-Ig / dsRNA conjugates described herein. Furthermore, the present inventors have also surprisingly and unexpectedly discovered that DVD conjugation at the 3' or 5' position of the sense strand of the siRNA does not impair gene silencing activity. Furthermore, the novel and unobvious non-cleavable and cleavable bis-β-lactam linkers described herein can be used for post-synthesis conjugation to alkylamino group-containing siRNAs after solid-phase synthesis. Additionally, siRNAs containing 3-aryl-β-lactam moieties can be efficiently and quantitatively conjugated to the corresponding DVDs using mild, neutral conditions in aqueous buffer (phosphate-buffered saline, pH 7.4) without impairing the integrity of either the siRNA or the DVD. It was also surprising and unexpected that DVD-siRNA conjugates can be efficiently purified using size-exclusion chromatography.

[0017] In one aspect, the present invention provides a conjugate comprising: (i) a dual variable domain immunoglobulin molecule (Ig) or antigen-binding fragment thereof, comprising a first variable domain that binds to a binding target and a second variable domain comprising a reactive residue; (ii) a linker covalently conjugated to the reactive residue of the second variable domain of the Ig; and (iii) a double-stranded RNA (dsRNA) molecule conjugated to the linker, wherein the dsRNA is capable of inhibiting expression of a target gene, the dsRNA comprising a sense strand and an antisense strand, each strand having from 14 to 40 nucleotides, and the antisense strand has sufficient complementarity to mediate RNA interference with the target sequence.

[0018] dsRNA molecule Aspects of the present invention include double-stranded RNA molecules. Generally, dsRNA molecules include a sense strand (also referred to as a passenger strand) and an antisense strand (also referred to as a guide strand). Each strand of a dsRNA molecule can independently range from 12 to 40 nucleotides in length. For example, each strand can independently be 14 to 40 nucleotides, 17 to 37 nucleotides, 25 to 37 nucleotides, 27 to 30 nucleotides, 17 to 23 nucleotides, 17 to 21 nucleotides, 17 to 19 nucleotides, 19 to 25 nucleotides, 19 to 23 nucleotides, 19 to 21 nucleotides, 21 to 25 nucleotides, or 21 to 23 nucleotides in length. Without limitation, the sense and antisense strands can be equal or unequal in length. In some embodiments, the antisense strand is longer than the sense strand, e.g., by 1, 2, 3, 4, or 5 nucleotides.

[0019] In some embodiments, the antisense strand is 18-35 nucleotides in length. In some embodiments, the antisense strand is 21-25, 19-25, 19-21, or 21-23 nucleotides in length. In some specific embodiments, the antisense strand is 23 nucleotides in length.

[0020] Like the antisense strand, the sense strand can be 18-35 nucleotides in length in some embodiments. In some embodiments, the sense strand is 21-25, 19-25, 19-21, or 21-23 nucleotides in length. In some specific embodiments, the antisense strand is 21 nucleotides in length.

[0021] In some specific embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

[0022] A double-stranded RNA molecule has a double-stranded or duplex region. Generally, the duplex region (double-stranded region) is 12 to 40 nucleotide base pairs in length. For example, a dsRNA has a duplex region of 12 to 25 nucleotide base pairs in length. In some embodiments, a dsRNA has a duplex region of 18, 19, 20, 21, 22, 22, 23, 24, or 25 nucleotide base pairs in length. In some specific embodiments, a dsRNA has a duplex region of 19, 20, 21, or 22 nucleotide base pairs in length.

[0023] The dsRNA molecule can include thermostabilizing modifications, for example, the dsRNA molecule can include at least 4, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more thermostabilizing modifications.

[0024] Without being limited thereto, all of the thermostabilizing modifications can be present in one strand. In some embodiments, both the sense strand and the antisense strand contain at least one, for example, two, three, or four or more thermostabilizing modifications. The thermostabilizing modification can be present on any nucleotide of the sense strand or the antisense strand. For example, the thermostabilizing modification can be present on every nucleotide of the sense strand and / or the antisense strand, or each thermostabilizing modification can be present in an alternating pattern on the sense strand or the antisense strand, or both the sense strand and the antisense strand contain the thermostabilizing modification in an alternating pattern. The alternating pattern of the thermostabilizing modification on the sense strand can be the same or different from that of the antisense strand, and the alternating pattern of the thermostabilizing modification on the sense strand can be shifted relative to the alternating pattern of the thermostabilizing modification on the antisense strand.

[0025] The antisense strand of a dsRNA molecule can contain at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more thermostabilizing modifications. In some embodiments, the antisense strand contains 2, 3, 4, 5, or 6 thermostabilizing modifications. Without limitation, the thermostabilizing modifications in the antisense strand can be located at any position. In some embodiments, the antisense strand contains at least three thermostabilizing modifications. For example, the antisense strand contains thermostabilizing modifications at least at the second, 14th, and 16th positions from the 5' end. In other embodiments, the antisense strand contains at least four thermostabilizing modifications. For example, the antisense strand contains thermostabilizing modifications at least at the second, 6th, 14th, and 16th positions from the 5' end. In some further embodiments, the antisense strand contains at least five thermostabilizing modifications. For example, the antisense strand contains thermostabilizing modifications at least at the second, 6th, 9th, 14th, and 16th positions from the 5' end. In still some further embodiments, the antisense strand contains at least six thermostabilizing modifications. For example, the antisense strand contains thermostabilizing modifications at least at positions 2, 6, 8, 9, 14, and 16 from the 5' end.

[0026] The sense strand of a dsRNA molecule can contain at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more thermostabilizing modifications. In some embodiments, the sense strand contains 2, 3, 4, or 5 thermostabilizing modifications. For example, the sense strand contains 3 or 4 thermostabilizing modifications. Without limitation, the thermostabilizing modifications in the sense strand can be located at any position. In some embodiments, the sense strand contains at least three thermostabilizing modifications. For example, the sense strand contains thermostabilizing modifications at least at the 7th, 10th, and 11th positions from the 5' end. In other embodiments, the sense strand contains at least four thermostabilizing modifications. For example, the sense strand contains thermostabilizing modifications at least at the 7th, 9th, 10th, and 11th positions from the 5' end.

[0027] In some embodiments, the sense strand comprises thermostabilizing modifications at positions opposite or complementary to positions 11, 12, and 15 of the antisense strand, counting from the 5' end of the antisense strand. In other embodiments, the sense strand comprises thermostabilizing modifications at positions opposite or complementary to positions 11, 12, 13, and 15 of the antisense strand, counting from the 5' end of the antisense strand. In some embodiments, the sense strand comprises blocks of two, three, or four thermostabilizing modifications.

[0028] In some embodiments, the sense strand comprises thermostabilizing modifications at least at positions 7, 9, and 11 from the 5' end, and the antisense strand comprises thermostabilizing modifications at least at positions 2, 14, and 16 from the 5' end. In some other embodiments, the sense strand comprises thermostabilizing modifications at least at positions 7, 9, and 11 from the 5' end, and the antisense strand comprises thermostabilizing modifications at least at positions 2, 6, 9, 14, and 16 from the 5' end. In yet some other embodiments, the sense strand comprises thermostabilizing modifications at least at positions 7, 9, and 11 from the 5' end, and the antisense strand comprises thermostabilizing modifications at least at positions 2, 6, 8, 9, 14, and 16 from the 5' end.

[0029] In some embodiments, the sense strand comprises a thermostabilizing modification at least at positions 7, 9, 10, and 11 from the 5' end, and the antisense strand comprises a thermostabilizing modification at least at positions 2, 14, and 16 from the 5' end. In some other embodiments, the sense strand comprises a thermostabilizing modification at least at positions 7, 9, 10, and 11 from the 5' end, and the antisense strand comprises a thermostabilizing modification at least at positions 2, 6, 9, 14, and 16 from the 5' end. In yet some other embodiments, the sense strand comprises a thermostabilizing modification at least at positions 7, 9, 10, and 11 from the 5' end, and the antisense strand comprises a thermostabilizing modification at least at positions 2, 6, 8, 9, 14, and 16 from the 5' end.

[0030] In some embodiments, the sense strand does not contain a thermostabilizing modification at a position opposite or complementary to a thermodestabilizing modification of the duplex in the antisense strand.

[0031] Exemplary thermostabilizing modifications include, but are not limited to, 2'-fluoro modifications and locked nucleic acids (LNAs).

[0032] dsRNA molecules can contain 2'-fluoro nucleotides, i.e., 2'-fluoro modifications. For example, dsRNA molecules can contain at least four, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more 2'-fluoro nucleotides. Without being limited thereto, all 2'-fluoro nucleotides can be present in one strand. In some embodiments, both the sense strand and the antisense strand contain at least two 2'-fluoro nucleotides. 2'-fluoro modifications can be present on any nucleotide of the sense strand or antisense strand. For example, 2'-fluoro modifications can be present on every nucleotide of the sense strand and / or antisense strand, or each 2'-fluoro modification can be present in an alternating pattern on the sense strand or antisense strand, or both the sense strand and the antisense strand contain 2'-fluoro modifications in an alternating pattern. The alternating pattern of 2'-fluoro modifications on the sense strand can be the same as or different from the antisense strand, and the alternating pattern of 2'-fluoro modifications on the sense strand can be shifted relative to the alternating pattern of 2'-fluoro modifications on the antisense strand.

[0033] The antisense strand of a dsRNA molecule can contain at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) 2'-fluoro nucleotides. In some embodiments, the antisense strand contains 2, 3, 4, 5, or 6 2'-fluoro nucleotides. Without limitation, the 2'-fluoro modification in the antisense strand can be present at any position. In some embodiments, the antisense strand contains at least three 2'-fluoro nucleotides. For example, the antisense strand contains 2'-fluoro nucleotides at least at the second, 14th, and 16th positions from the 5' end. In other embodiments, the antisense strand contains at least four 2'-fluoro nucleotides. For example, the antisense strand contains 2'-fluoro nucleotides at least at the second, 6th, 14th, and 16th positions from the 5' end. In some further embodiments, the antisense strand contains at least five 2'-fluoro nucleotides. For example, the antisense strand contains 2'-fluoro nucleotides at least at the second, 6th, 9th, 14th, and 16th positions from the 5' end. In some further embodiments, the antisense strand comprises at least six 2'-fluoro nucleotides, for example, the antisense strand comprises at least 2'-fluoro nucleotides at the 2nd, 6th, 8th, 9th, 14th, and 16th positions from the 5' end.

[0034] The sense strand of a dsRNA molecule can contain at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) 2'-fluoro nucleotides. In some embodiments, the sense strand contains two, three, four, or five 2'-fluoro nucleotides. For example, the sense strand contains three or four 2'-fluoro nucleotides. Without being limited thereto, the 2'-fluoro modification in the sense strand can be present at any position. In some embodiments, the sense strand contains at least three 2'-fluoro nucleotides. For example, the sense strand contains 2'-fluoro nucleotides at least at the 7th, 10th, and 11th positions from the 5' end. In other embodiments, the sense strand contains at least four 2'-fluoro nucleotides. For example, the sense strand contains 2'-fluoro nucleotides at least at the 7th, 9th, 10th, and 11th positions from the 5' end.

[0035] In some embodiments, the sense strand comprises 2'-fluoro nucleotides at positions opposite or complementary to the 11th, 12th, and 15th positions of the antisense strand, counting from the 5' end of the antisense strand. In other embodiments, the sense strand comprises 2'-fluoro nucleotides at positions opposite or complementary to the 11th, 12th, 13th, and 15th positions of the antisense strand, counting from the 5' end of the antisense strand. In some embodiments, the sense strand comprises blocks of two, three, or four 2'-fluoro nucleotides.

[0036] In some embodiments, the sense strand contains 2'-fluoro nucleotides at least at the 7th, 9th, and 11th positions from the 5' end, and the antisense strand contains 2'-fluoro nucleotides at least at the 2nd, 14th, and 16th positions from the 5' end. In other embodiments, the sense strand contains 2'-fluoro nucleotides at least at the 7th, 9th, and 11th positions from the 5' end, and the antisense strand contains 2'-fluoro nucleotides at least at the 2nd, 6th, 9th, 14th, and 16th positions from the 5' end. In still other embodiments, the sense strand contains 2'-fluoro nucleotides at least at the 7th, 9th, and 11th positions from the 5' end, and the antisense strand contains 2'-fluoro nucleotides at least at the 2nd, 6th, 8th, 9th, 14th, and 16th positions from the 5' end.

[0037] In some embodiments, the sense strand contains 2'-fluoro nucleotides at least at the 7th, 9th, 10th, and 11th positions from the 5' end, and the antisense strand contains 2'-fluoro nucleotides at least at the 2nd, 14th, and 16th positions from the 5' end. In other embodiments, the sense strand contains 2'-fluoro nucleotides at least at the 7th, 9th, 10th, and 11th positions from the 5' end, and the antisense strand contains 2'-fluoro nucleotides at least at the 2nd, 6th, 9th, 14th, and 16th positions from the 5' end. In still other embodiments, the sense strand contains 2'-fluoro nucleotides at least at the 7th, 9th, 10th, and 11th positions from the 5' end, and the antisense strand contains 2'-fluoro nucleotides at least at the 2nd, 6th, 8th, 9th, 14th, and 16th positions from the 5' end.

[0038] In some embodiments, the antisense strand does not contain 2'-fluoro nucleotides at positions 3-9 counting from the 5' end.

[0039] A dsRNA molecule can contain at least one, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more 2'-OMe nucleotides. Without being limited thereto, all 2'-OMe nucleotides can be present in one strand. In some embodiments, both the sense strand and the antisense strand contain at least one 2'-OMe nucleotide. The 2'-OMe modification can be present on any nucleotide of the sense strand or the antisense strand. For example, the 2'-OMe modification can be present on every nucleotide of the sense strand and / or the antisense strand, or each thermostabilizing modification can be present in an alternating pattern on the sense strand or the antisense strand, or both the sense strand and the antisense strand contain the 2'-OMe modification in an alternating pattern. The alternating pattern of thermostabilizing modifications on the sense strand can be the same as or different from the antisense strand, and the alternating pattern of thermostabilizing modifications on the sense strand can be shifted relative to the alternating pattern of 2'-OMe modifications on the antisense strand.

[0040] The antisense strand of dsRNA molecule can comprise at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 or more 2'-OMe modification.Without being limited, the thermostabilizing modification in antisense strand can be present at any position.In some embodiments, antisense strand comprises at least 3 thermostabilizing modifications.

[0041] For example, the antisense strand does not contain 2'-OMe modifications at least at positions 2, 14, and 16 from the 5' end. In some other embodiments, the antisense strand does not contain 2'-OMe modifications at least at positions 2, 6, 14, and 16 from the 5' end. In some further embodiments, the antisense strand does not contain 2'-OMe modifications at least at positions 2, 6, 9, 14, and 16 from the 5' end. In still some further embodiments, the antisense strand does not contain 2'-OMe modifications at least at positions 2, 6, 8, 9, 14, and 16 from the 5' end.

[0042] The sense strand of dsRNA molecule can comprise at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 or more 2'-OMe modifications.Without being limited, the 2'-OMe modifications in the sense strand can be present at any position.In some embodiments, the sense strand does not comprise 2'-OMe modifications at least at the 7th, 10th and 11th positions from the 5' end.In other embodiments, the sense strand does not comprise 2'-OMe modifications at least at the 7th, 9th, 10th and 11th positions from the 5' end.

[0043] The dsRNA molecule can contain locked nucleic acid (LNA). For example, the dsRNA molecule can contain at least one, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more LNA modifications. Without limitation, all LNA nucleotides can be present in one strand. In some embodiments, both the sense strand and the antisense strand contain at least an LNA modification. The LNA modification can be present on any nucleotide of the sense strand or the antisense strand. For example, the LNA modification can be present on every nucleotide of the sense strand and / or the antisense strand, or each LNA modification can be present in an alternating pattern on the sense strand or the antisense strand, or both the sense strand and the antisense strand contain the LNA modification in an alternating pattern. The alternating pattern of LNA modifications on the sense strand may be the same as or different from the antisense strand, and the alternating pattern of LNA modifications on the sense strand may be shifted relative to the alternating pattern of 2'-fluoro modifications on the antisense strand.

[0044] The antisense strand of the dsRNA molecule can contain at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more LNA modifications. Without being limited thereto, the LNA modifications in the antisense strand can be present in any position.

[0045] The sense strand of dsRNA molecule can comprise at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more LNA modifications.Without being limited, the LNA modifications in sense strand can be present at any position.In some embodiments, the sense strand comprises at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more LNA modifications, and the antisense strand does not comprise 2'-fluoro nucleotides at the 3rd to 9th positions counting from the 5' end.

[0046] The dsRNA molecule can contain bridged nucleic acid (BNA). For example, the dsRNA molecule can contain at least one, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more BNA modifications. Without limitation, all BNA nucleotides can be present in one strand. In some embodiments, both the sense strand and the antisense strand contain at least a BNA modification. The BNA modification can be present on any nucleotide of the sense strand or the antisense strand. For example, the BNA modification can be present on every nucleotide of the sense strand and / or the antisense strand, or each BNA modification can be present in an alternating pattern on the sense strand or the antisense strand, or both the sense strand and the antisense strand contain the BNA modification in an alternating pattern. The alternating pattern of BNA modifications on the sense strand can be the same as or different from the antisense strand, and the alternating pattern of BNA modifications on the sense strand can be shifted relative to the alternating pattern of 2'-fluoro modifications on the antisense strand.

[0047] The antisense strand of a dsRNA molecule can contain at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more BNA modifications. Without being limited thereto, the BNA modifications in the antisense strand can be present in any position.

[0048] The sense strand of dsRNA molecule can comprise at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more BNA modifications.Without being limited, the BNA modifications in sense strand can be present at any position.In some embodiments, sense strand comprises at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more BNA modifications, and antisense strand does not comprise 2'-fluoro nucleotide at the 3rd to 9th positions counting from 5' end.

[0049] dsRNA molecules can contain cyclohexene nucleic acid (CeNA). For example, dsRNA molecules can contain at least one CeNA modification, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more. Without being limited thereto, all CeNA nucleotides can be present in one strand. In some embodiments, both the sense strand and the antisense strand contain at least CeNA modification. CeNA modification can be present on any nucleotide of the sense strand or antisense strand. For example, CeNA modification can be present on every nucleotide of the sense strand and / or antisense strand, or each CeNA modification can be present in an alternating pattern on the sense strand or antisense strand, or both the sense strand and the antisense strand contain CeNA modification in an alternating pattern. The alternating pattern of CeNA modifications on the sense strand can be the same as or different from the antisense strand, and the alternating pattern of CeNA modifications on the sense strand can be shifted relative to the alternating pattern of 2'-fluoro modifications on the antisense strand.

[0050] The antisense strand of a dsRNA molecule can contain at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more CeNA modifications. Without being limited thereto, the CeNA modifications in the antisense strand can be present in any position.

[0051] The sense strand of dsRNA molecule can comprise at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more CeNA modifications.Not limited to, the CeNA modifications in sense strand can be present at any position.In some embodiments, the sense strand comprises at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more CeNA modifications, and the antisense strand does not comprise 2'-fluoro nucleotides at the 3rd to 9th positions counting from the 5' end.

[0052] In some embodiments, the dsRNA molecule comprises one or more overhang regions (i.e., single-stranded regions) and / or capping groups at the 3' or 5' end or both ends of the strand. Without limitation, the overhangs can be 1-10 nucleotides, 1-6 nucleotides, 1-5 nucleotides, 1-4 nucleotides, 1-3 nucleotides, 2-6 nucleotides, 2-5 nucleotides, 2-4 nucleotides, 2-3 nucleotides, or 1-2 nucleotides in length. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being offset from each other. The overhangs can be mismatched with the targeted sequence, complementary to the targeted sequence, or other sequences. The first and second strands can be joined, for example, by additional bases to form a hairpin, or can be joined by other non-basic linkers. Without limitation, the overhangs can be present at the 3' end of the sense strand, the antisense strand, or both strands.

[0053] In some embodiments, dsRNA molecule comprises a single overhang.For example, dsRNA molecule has a single overhang, and this overhang is at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides long.In some embodiments, overhang is present at the 3' end of antisense strand.In some particular embodiments, dsRNA comprises a 2-nucleotide overhang at the 3' end of antisense strand.

[0054] dsRNA can also have blunt ends. For example, one end of dsRNA is blunt, and the other end has an overhang. Without being limited thereto, the blunt end can be located at the 5' end of the antisense strand (or the 3' end of the sense strand), or vice versa. Generally, the antisense strand of dsRNA has a nucleotide overhang at its 3' end, and the 5' end is blunt. Without being bound by theory, the asymmetric blunt end at the 5' end of the antisense strand and the 3' end overhang of the antisense strand are advantageous for the loading process of the guide strand into RISC. In some embodiments, dsRNA has a 2-nucleotide overhang at the 3' end of the antisense strand, and a blunt end at the 5' end of the antisense strand.

[0055] In some other embodiments, the dsRNA molecule has two blunt ends, ie, at both ends of the dsRNA.

[0056] The nucleotides in the overhang region of dsRNA molecule can each independently be modified nucleotides or unmodified nucleotides, for example, but not limited to, 2'-sugar modified nucleotides, such as 2'-fluoro, 2'-O-methyl, thymidine (T), 2'-O-methoxyethyl-5-methyluridine, 2'-O-methoxyethyl adenosine, 2'-O-methoxyethyl-5-methylcytidine, GNA, SNA, hGNA, hhGNA, mGNA, TNA, h'GNA, and any combination thereof.For example, TT (or UU) can be the overhang sequence at either end of either strand.The 5'-overhang or 3'-overhang of the sense strand, antisense strand, or both strands of dsRNA molecule can be phosphorylated.In some embodiments, the overhang region contains two nucleotides with phosphorothioate internucleotide linkage between the two nucleotides, and the two nucleotides in the overhang region can be the same or different.

[0057] A dsRNA molecule can comprise at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more phosphorothioate internucleotide linkages or methylphosphonate internucleotide linkages.The phosphorothioate internucleotide linkage modification or methylphosphonate internucleotide linkage modification can be present at any position of the strand, on any nucleotide of the sense strand or antisense strand, or both.For example, the internucleotide linkage modification can be present at every nucleotide on the sense strand and / or antisense strand, or each internucleotide linkage modification can be present in an alternating pattern on the sense strand or antisense strand, or both the sense strand and the antisense strand contain internucleotide linkage modifications in an alternating pattern.The alternating pattern of the internucleotide linkage modification on the sense strand can be the same or different from that of the antisense strand, and the alternating pattern of the internucleotide linkage modification on the sense strand can be shifted relative to the alternating pattern of the internucleotide linkage modification on the antisense strand.

[0058] In some embodiments, dsRNA molecules comprise phosphorothioate internucleotide linkage modification or methylphosphonate internucleotide linkage modification in overhang region.For example, overhang region comprises two nucleotides with phosphorothioate internucleotide linkage or methylphosphonate internucleotide linkage between two nucleotides.Internucleotide linkage modification can be used to connect overhang nucleotide with the terminal paired nucleotide in double-stranded region.For example, at least 2, 3 or 4 or all overhang nucleotides can be linked with phosphorothioate internucleotide linkage or methylphosphonate internucleotide linkage, and optionally there can be an additional phosphorothioate internucleotide linkage or methylphosphonate internucleotide linkage that connects the overhang nucleotide and the paired nucleotide adjacent to the overhang nucleotide.For example, there can be at least two phosphorothioate internucleotide linkages between the terminal three nucleotides, and two of these three nucleotides can be overhang nucleotides, and the third can be the paired nucleotide adjacent to the overhang nucleotide. Preferably, these terminal three nucleotides may be at the 3' end of the antisense strand.

[0059] In some embodiments, the sense strand of the dsRNA molecule comprises 1 to 10 blocks of 2 to 10 phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position in the oligonucleotide sequence, and the sense strand is paired with an antisense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate internucleotide linkages.

[0060] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of two phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising either a phosphorothioate or methylphosphonate internucleotide linkage or a phosphate internucleotide linkage.

[0061] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of three phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising either a phosphorothioate or methylphosphonate internucleotide linkage or a phosphate internucleotide linkage.

[0062] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of four phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising either a phosphorothioate or methylphosphonate or phosphate internucleotide linkage.

[0063] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of five phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising either a phosphorothioate or methylphosphonate internucleotide linkage or a phosphate internucleotide linkage.

[0064] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of six phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising either a phosphorothioate or methylphosphonate or phosphate internucleotide linkage.

[0065] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of seven phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, or 8 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising either a phosphorothioate or methylphosphonate internucleotide linkage or a phosphate internucleotide linkage.

[0066] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of eight phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, or 6 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising either a phosphorothioate or methylphosphonate or phosphate internucleotide linkage.

[0067] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of nine phosphorothioate or methylphosphonate internucleotide linkages separated by one, two, three, or four phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising either a phosphorothioate or methylphosphonate or phosphate internucleotide linkage.

[0068] In some embodiments, the dsRNA molecule comprises one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within one to ten of the terminal positions of the sense and / or antisense strands. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides at one or both ends of the sense and / or antisense strands can be linked by phosphorothioate or methylphosphonate internucleotide linkages.

[0069] In some embodiments, the dsRNA molecule comprises one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within 1 to 10 internal regions of the duplex of each of the sense and / or antisense strands. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in positions 8 to 16 of the duplex region, counting from the 5' end of the sense strand, can be linked by phosphorothioate-methylphosphonate internucleotide linkages, and the dsRNA molecule can optionally further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within 1 to 10 of the terminal positions.

[0070] In some embodiments, the dsRNA molecule contains 1 to 5 phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) of the sense strand and 1 to 5 phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 18-23 of the antisense strand, and 1 to 5 phosphorothioate or methylphosphonate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) of the antisense strand and 1 to 5 within positions 18-23 of the antisense strand.

[0071] In some embodiments, the dsRNA molecule contains one phosphorothioate internucleotide linkage modification within positions 1-5 (counting from the 5' end) of the sense strand and one phosphorothioate or methylphosphonate internucleotide linkage modification within positions 18-23 of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 (counting from the 5' end) of the antisense strand and two phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 18-23 of the antisense strand.

[0072] In some embodiments, the dsRNA molecule comprises two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within positions 18-23 of the sense strand, and one phosphorothioate internucleotide linkage modification within positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand.

[0073] In some embodiments, the dsRNA molecule comprises two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand.

[0074] In some embodiments, the dsRNA molecule comprises two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand.

[0075] In some embodiments, the dsRNA molecule comprises one phosphorothioate internucleotide linkage modification within positions 1-5 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within positions 18-23 of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand.

[0076] In some embodiments, the dsRNA molecule contains one phosphorothioate internucleotide linkage modification within positions 1-5 (counting from the 5' end) and one within positions 18-23 of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand.

[0077] In some embodiments, the dsRNA molecule contains one phosphorothioate internucleotide linkage modification within positions 1-5 (counting from the 5' end) of the sense strand, two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) of the antisense strand, and one phosphorothioate internucleotide linkage modification within positions 18-23.

[0078] In some embodiments, the dsRNA molecule contains two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) of the sense strand, one phosphorothioate internucleotide linkage modification at positions 1 and 2 (counting from the 5' end) of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18-23.

[0079] In some embodiments, the dsRNA molecule contains two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) and one within positions 18-23 of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand.

[0080] In some embodiments, the dsRNA molecule comprises two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within positions 18-23 of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand.

[0081] In some embodiments, the dsRNA molecule comprises two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within positions 18-23 of the sense strand, and one phosphorothioate internucleotide linkage modification within positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand.

[0082] In some embodiments, the dsRNA molecule contains two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications at positions 20 and 21 of the sense strand, and one phosphorothioate internucleotide linkage modification at position 1 and one at position 21 (counting from the 5' end) of the antisense strand.

[0083] In some embodiments, the dsRNA molecule comprises one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 20 and 21 (counting from the 5' end) of the antisense strand.

[0084] In some embodiments, the dsRNA molecule comprises two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications at positions 21 and 22 of the sense strand, and one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 of the antisense strand (counting from the 5' end).

[0085] In some embodiments, the dsRNA molecule comprises one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 21 and 22 (counting from the 5' end) of the antisense strand.

[0086] In some embodiments, the dsRNA molecule comprises two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications at positions 22 and 23 of the sense strand, and one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 of the antisense strand (counting from the 5' end).

[0087] In some embodiments, the dsRNA molecule comprises one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 23 and 23 (counting from the 5' end) of the antisense strand.

[0088] In some exemplary dsRNA molecules, the sense strand can contain 0, 1, 2, 3, or 4 phosphorothioate internucleotide linkages. For example, the sense strand contains a phosphorothioate internucleotide linkage between the first and second nucleotide and between the second and third nucleotide.

[0089] In some exemplary dsRNA molecules, antisense strand can comprise 1, 2, 3 or 4 phosphorothioate internucleotide linkages.For example, sense strand comprises phosphorothioate internucleotide linkages between the 21st and 22nd nucleotide and between the 22nd and 23rd nucleotide.In another example, antisense strand comprises phosphorothioate internucleotide linkages between the 1st and 2nd nucleotide, between the 2nd and 3rd nucleotide, between the 21st and 22nd nucleotide, and between the 22nd and 23rd nucleotide.

[0090] In some embodiments, the sense strand comprises phosphorothioate internucleotide linkages between the first and second nucleotides and between the second and third nucleotides, and the antisense strand comprises phosphorothioate internucleotide linkages between the 21st and 22nd nucleotides and between the 22nd and 23rd nucleotides. For example, the sense strand comprises phosphorothioate internucleotide linkages between the first and second nucleotides and between the second and third nucleotides, and the antisense strand comprises phosphorothioate internucleotide linkages between the first and second nucleotides, between the second and third nucleotides, between the 21st and 22nd nucleotides, and between the 22nd and 23rd nucleotides.

[0091] 5'-modified In some embodiments, the dsRNA molecule can be 5' phosphorylated or contain a phosphoryl analog at the 5' end. Exemplary 5'-phosphate modifications include those compatible with RISC-mediated gene silencing. Suitable modifications include 5'-monophosphate ((HO)2(O)PO-5'); 5'-diphosphate ((HO)2(O)POP(HO)(O)-O-5'); 5'-triphosphate ((HO)2(O)PO-(HO)(O)POP(HO)(O)-O-5'); 5'-guanosine cap (7-methylated or unmethylated) (7m-GO-5'-(HO)(O)PO-(HO)(O)POP(HO)( 5'-adenosine cap (Appp) and any modified or unmodified nucleotide cap structure (NO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5'); 5'-monothiophosphate (phosphorothioate; (HO)2(S)PO-5'); 5'-monodithiophosphate (phosphorodithioate; (HO)(HS)(S) PO-5'), 5'-phosphorothiolate ((HO)2(O)PS-5'); any further combination of oxygen / sulfur substituted monophosphate, diphosphate and triphosphate (e.g., 5'-alpha-thiotriphosphate, 5'-gamma-thiotriphosphate, etc.), 5'-phosphoramidate ((HO)2(O)P-NH-5', (HO)(NH2)(O)PO-5'), 5'-alkylphosphonate (R = alkyl = methyl, ethyl, isopropyl, propyl, etc., e.g., RP(OH)(O)-O-5'-, 5'-alkenylphosphonate (i.e., vinyl, substituted vinyl), (OH)2(O)P-5'-CH2-), 5'-alkyl ether phosphonate (R = alkyl ether = methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g., RP(OH)(O)-O-5'-). Modifications can be placed in the antisense strand of the dsRNA molecule. For example, the antisense strand can include a 5'-vinylphosphonate nucleotide at the 5' end.

[0092] In some embodiments, the antisense strand comprises 5'-E-vinyl phosphate. In some embodiments, the antisense strand has 5'-E-vinyl phosphate and a nucleoside at position N-1 that reduces or inhibits the activity of siRNA compared to the siRNA with the same antisense strand sequence but not modified at N-1 position, and a nucleoside at position N-1 that reduces or inhibits the activity of siRNA compared to the siRNA with the same antisense strand sequence but not modified at N-1 position.

[0093] In some embodiments, the sense strand comprises a 5'-morpholino, 5'-dimethylamino, 5'-deoxy, inverted abasic, or inverted abasic locked nucleic acid modification at the 5' end.

[0094] The linker between the Ig and the dsRNA molecule can be attached to the sense strand, the antisense strand, or both strands.Furthermore, the linker can be conjugated at the 3' end, the 5' end, or both ends of the strand.For example, the linker can be conjugated to the sense strand.In some embodiments, the linker is conjugated to the 3' end of the sense strand.In other embodiments, the linker is conjugated to the 3' end of the sense strand.

[0095] Generally, dsRNA has a melting temperature within the range of about 40°C to about 80°C. For example, the dsRNA has a melting temperature with a lower limit of about 40°C, 45°C, 50°C, 55°C, 60°C, or 65°C and an upper limit of about 70°C, 75°C, or 80°C. In some embodiments, the dsRNA has a melting temperature within the range of about 55°C to about 70°C or within the range of about 60°C to about 75°C. In some embodiments, the dsRNA has a melting temperature within the range of about 57°C to about 67°C. In some specific embodiments, the dsRNA has a melting temperature within the range of about 60°C to about 67°C. In some further embodiments, the dsRNA has a melting temperature within the range of about 62°C to about 66°C.

[0096] Without wishing to be bound by theory, dsRNA molecules that have a melting temperature of at least 60° C. are more effective in vivo and in vitro. Thus, in some embodiments, the dsRNA has a melting temperature of at least 60° C.

[0097] Without being bound by theory, for dsRNA molecules to be more effective in vivo, antisense strand must have some metabolic stability.In other words, for dsRNA molecules to be more effective in vivo, after administration, after a certain period, some amount of antisense strand may need to exist in vivo.Therefore, in some embodiments, after 5 days of in vivo administration, at least 40%, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80% of the antisense strand of dsRNA exists in vivo, for example, in mouse liver.In some embodiments, after 6 days of in vivo administration, at least 40%, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80% of the antisense strand of dsRNA exists in vivo, for example, in mouse liver. In some embodiments, after 7 days of in vivo administration, at least 40% of the antisense strand of dsRNA exists in vivo, for example, in mouse liver, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80%.In some embodiments, after 8 days of in vivo administration, at least 40% of the antisense strand of dsRNA exists in vivo, for example, in mouse liver, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80%.In some embodiments, after 9 days of in vivo administration, at least 40% of the antisense strand of dsRNA exists in vivo, for example, in mouse liver, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80%.In some embodiments, after 10 days of in vivo administration, at least 40%, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80% of the antisense strand of dsRNA exists in vivo, for example, in mouse liver.In some embodiments, after 11 days of in vivo administration, at least 40%, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80% of the antisense strand of dsRNA exists in vivo, for example, in mouse liver.In some embodiments, after 12 days of in vivo administration, at least 40%, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80% of the antisense strand of dsRNA exists in vivo, for example, in mouse liver. In some embodiments, after 13 days of in vivo administration, at least 40% of the antisense strand of dsRNA exists in vivo, for example, in mouse liver, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80%.In some embodiments, after 14 days of in vivo administration, at least 40% of the antisense strand of dsRNA exists in vivo, for example, in mouse liver, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80%.In some embodiments, after 15 days of in vivo administration, at least 40% of the antisense strand of dsRNA exists in vivo, for example, in mouse liver, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80%.

[0098] Without being bound by theory, thermodestabilizing modifications in the seed region of the antisense strand (i.e., the second to ninth nucleotides from the 5' end of the antisense strand) can reduce or inhibit off-target gene silencing.Thus, in some embodiments, the antisense strand comprises at least one (e.g., one, two, three, four, five or more) thermodestabilizing modifications of the duplex within the first nine nucleotide positions of the 5' region of the antisense strand.The term "thermodestabilizing modifications" encompasses modifications that result in a dsRNA with a reduced overall melting temperature (Tm) (preferably 1, 2, 3 or 4 degrees lower than the Tm of a dsRNA that does not have such modifications).

[0099] In some embodiments, the thermally destabilizing modification is located at position 2, 3, 4, 5, 6, 7, 8, or 9 from the 5' end of the antisense strand, or preferably at position 4, 5, 6, 7, or 8. In some embodiments, the thermally destabilizing modification is located at position 2, 3, 4, 5, or 9 from the 5' end of the antisense strand. In some other embodiments, the thermally destabilizing modification is located at position 6, 7, or 8 from the 5' end of the antisense strand. In some specific embodiments, the thermally destabilizing modification is located at position 7 from the 5' end of the antisense strand.

[0100] Thermally destabilizing modifications can include, but are not limited to, abasic modifications; mismatches with opposing nucleotides in opposing strands; and sugar modifications such as 2'-deoxy modifications or acyclic nucleotides, e.g., unlocked nucleic acid (UNA) or glycol nucleic acid (GNA).

[0101] Exemplary abasic modifications include, but are not limited to, the following modifications: TIFF2026041866000003.tif67140, in which R is H, Me, Et, or OMe, R' is H, Me, Et, or OMe, R'' is H, Me, Et, or OMe, and * represents either R, S, or racemic.

[0102] Exemplary, but non-limiting, destabilizing sugar modifications include the following sugar modifications: TIFF2026041866000004.tif50148, where B is a modified or unmodified nucleobase.

[0103] Additional sugar modifications include, but are not limited to, the following sugar modifications: TIFF2026041866000005.tif100168, where B is a modified or unmodified nucleobase.

[0104] In some embodiments, the thermodestabilizing modification is TIFF2026041866000006.tif71128, wherein B is a modified or unmodified nucleobase, and the asterisk on each structure represents R, S, or racemic.

[0105] The term "acyclic nucleotide" refers to any nucleotide having an acyclic ribose sugar, for example, one in which any of the bonds between the ribose carbons (e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', or C1'-O4') is absent and / or at least one of the ribose carbons or oxygens (e.g., C1', C2', C3', C4', or O4'), independently or in combination, is missing from the nucleotide. In some embodiments, an acyclic nucleotide is TIFF2026041866000007.tif35150, where B is a modified or unmodified nucleobase, and R 1 and R 2are independently H, halogen, OR3, or alkyl, and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar. The term "UNA" refers to an unlocked acyclic nucleic acid in which one of the sugar bonds has been removed to form an unlocked "sugar" residue. In one example, a UNA also encompasses a monomer in which the C1'-C4' bond (i.e., the carbon-oxygen-carbon covalent bond between the C1' and C4' carbons) has been removed. In another example, the C2'-C3' bond (i.e., the carbon-carbon covalent bond between the C2' and C3' carbons) of the sugar has been removed (see Mikhailov et al., Tetrahedron Letters, 26(17):2059 (1985) and Fluiter et al., Mol. Biosyst., 10:1039 (2009), which are incorporated herein by reference in their entireties). Acyclic derivatives increase backbone flexibility without affecting Watson-Crick pairing. Acyclic nucleotides can be linked by 2'-5' or 3'-5' linkages.

[0106] The term "GNA" refers to glycol nucleic acid, a polymer that is similar to DNA or RNA but differs in that its "backbone" composition consists of repeating glycerol units linked by phosphodiester bonds: Points to TIFF2026041866000008.tif57128.

[0107] The thermally destabilizing modification of the duplex can be a mismatch (i.e., non-complementary base pair) between the thermally destabilizing nucleotide in the dsRNA duplex and the opposite nucleotide in the opposite strand.Exemplary mismatch base pairs include G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, U:T, or combinations thereof.Other mismatch base pairings known in the art can also be applied to the present invention.Mismatches can occur between nucleotides that are either natural or modified nucleotides.That is, mismatch base pairing can occur between the nucleobases of each nucleotide, regardless of the modification on the ribose sugar of the nucleotide.In certain embodiments, the dsRNA molecule comprises at least one nucleobase that is a 2'-deoxynucleobase in mismatch pairing, for example, the 2'-deoxynucleobase is in the sense strand.

[0108] In some embodiments, the thermodestabilizing modification in the seed region of the antisense strand comprises a nucleotide that has impaired WCH binding to a complementary base on the target mRNA. Examples of nucleotides that have impaired WCH binding to a complementary base on the target mRNA include: TIFF2026041866000009.tif72135.

[0109] Further examples of abasic nucleotide modifications, acyclic nucleotide modifications (including UNA and GNA), and mismatch modifications are detailed in WO 2011 / 133876, which is incorporated herein by reference in its entirety.

[0110] Thermodestabilizing modifications can also include universal nucleobases that have reduced or eliminated hydrogen bonding ability.

[0111] In some embodiments, thermal destabilizing modification comprises the nucleotide with non-standard base, for example but not limited to, the nucleobase modification that the ability to form hydrogen bond with the base in the opposite strand is impaired or completely lost.These nucleobase modifications have been evaluated for destabilizing the central region of dsRNA duplex, as described in WO 2010 / 0011895, and this document is incorporated herein by reference in its entirety.The example of such nucleobase modification is: The file is TIFF2026041866000010.tif63157.

[0112] In some embodiments, the thermodestabilizing modification is one or more □-nucleotides complementary to a base on the target mRNA, e.g., TIFF2026041866000011.tif17146, where R is H, OH, OCH3, F, NH2, NHMe, NMe2 or O-alkyl.

[0113] Exemplary phosphate modifications known to decrease the thermal stability of dsRNA duplexes relative to natural phosphodiester linkages include: Examples of modifications include, but are not limited to, TIFF2026041866000012.tif31140.

[0114] The alkyl R group can be a C1-C6 alkyl. Specific alkyl R groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, pentyl, and hexyl.

[0115] In some embodiments, the destabilizing modification is Selected from TIFF2026041866000013.tif85128.

[0116] In some embodiments, the antisense strand comprises at least one stabilizing modification adjacent to the destabilizing modification.For example, the stabilizing modification can be the nucleotide at the 5' or 3' end of the destabilizing modification, i.e., the nucleotide at the -1 or +1 position from the position of the destabilizing modification.In some embodiments, the antisense strand comprises the stabilizing modification at the 5' end and 3' end of the destabilizing modification, i.e., the nucleotide at the -1 position and +1 position from the position of the destabilizing modification.

[0117] In some embodiments, the antisense strand comprises at least two stabilizing modifications 3' to the destabilizing modification, i.e., at least two stabilizing modifications at positions +1 and +2 from the position of the destabilizing modification.

[0118] In some embodiments, the sense strand does not contain a thermostabilizing modification at a position opposite or complementary to a thermodestabilizing modification of the duplex in the antisense strand.

[0119] In some embodiments, antisense strand comprises at least one 2'-fluoro nucleotide adjacent to destabilizing modification.For example, 2'-fluoro nucleotide can be the nucleotide at the 5' end or 3' end of destabilizing modification, that is, the nucleotide at the -1 or +1 position from the position of destabilizing modification.In some embodiments, antisense strand comprises 2'-fluoro nucleotide at each of the 5' end and 3' end of destabilizing modification, that is, the -1 position and +1 position from the position of destabilizing modification.

[0120] In some embodiments, the antisense strand comprises at least two 2'-fluoro nucleotides at the 3' end of the destabilizing modification, i.e., at least two 2'-fluoro nucleotides at positions +1 and +2 from the position of the destabilizing modification.

[0121] In some embodiments, the sense strand does not contain 2'-fluoro nucleotides at positions opposite or complementary to the thermally destabilizing modifications of the duplex in the antisense strand.

[0122] In some embodiments, all nucleotides in the sense strand and / or antisense strand of dsRNA molecule can be modified.Each nucleotide can be modified with the same or different modifications, and these modifications can include one or more modifications of one or both of the non-linked phosphate oxygens and / or one or more of the linked phosphate oxygens;Modification of the ribose sugar component, for example, modification of the 2' hydroxyl on the ribose sugar;Fully replacing phosphate moiety with "dephospho" linker;Modification or replacement of natural base;And replacement or modification of ribose-phosphate backbone.

[0123] Because nucleic acids are polymers of monomers, many modifications occur at positions that are repeated within the nucleic acid, such as modifications of bases, phosphate moieties, or non-linked Os in phosphate moieties. In some cases, modifications will occur at all of the target positions in the nucleic acid, but in many cases, this will not be the case. For example, modifications can occur only at the 3'- or 5'-terminal positions, or in terminal regions, such as at terminal nucleotide positions, or in the last 2, 3, 4, 5, or 10 nucleotides of the chain. Modifications can occur in double-stranded regions, single-stranded regions, or both. Modifications can occur only in double-stranded regions of RNA, or only in single-stranded regions of RNA. For example, phosphorothioate modifications of non-linked O positions can occur only at one or both ends, or only in terminal regions, such as at terminal nucleotide positions, or in the last 2, 3, 4, 5, or 10 nucleotides of the chain, or in double-stranded and single-stranded regions, especially at the ends. One or more 5'-ends can be phosphorylated.

[0124] For example, it may be possible to enhance stability, include specific bases in the overhang, or include modified nucleotides or nucleotide substitutes in the single-stranded overhang, e.g., the 5' overhang or the 3' overhang, or both. For example, it may be desirable to include purine nucleotides in the overhang. In some embodiments, all or a portion of the bases in the 3' or 5' overhang may be modified, e.g., with the modifications described herein. Modifications can include, for example, the use of modifications at the 2' position of the ribose sugar, such as modifications known in the art, e.g., the use of deoxyribonucleotides, 2'-deoxy-2'-fluoro (2'-F) or 2'-O-methyl modifications in place of the ribose sugar of the nucleobase, and modifications in the phosphate group, e.g., phosphorothioate modifications. The overhang need not be homologous to the target sequence.

[0125] In some embodiments, each residue of sense strand and antisense strand is independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy or 2'-fluoro.Strands can contain two or more modifications.In some embodiments, each residue of sense strand and antisense strand is independently modified with 2'-O-methyl or 2'-fluoro.It should be understood that these modifications are in addition to at least one thermostabilizing modification of the double strand that exists in antisense strand.

[0126] Typically, there are at least two different modifications on the sense strand and the antisense strand. These two modifications can be 2'-deoxy, 2'-O-methyl, or 2'-fluoro modifications, acyclic nucleotides, etc. In some embodiments, the sense strand and the antisense strand each contain two nucleotides with different modifications selected from 2'-O-methyl or 2'-deoxy. In some embodiments, each residue of the sense strand and the antisense strand is independently modified with a 2'-O-methyl nucleotide, a 2'-deoxy nucleotide, a 2'-deoxy-2'-fluoro nucleotide, a 2'-ON-methylacetamide (2'-O-NMA) nucleotide, a 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) nucleotide, a 2'-O-aminopropyl (2'-O-AP) nucleotide, or a 2'-ara-F nucleotide. Again, it should be understood that these modifications are in addition to at least one thermostabilizing modification of the duplex present in the antisense strand.

[0127] In some embodiments, the dsRNA molecule contains an alternating pattern of modifications, particularly in the B1, B2, B3, B1', B2', B3', and B4' regions. As used herein, the term "alternating motif" or "alternating pattern" refers to a motif having one or more modifications, each occurring on alternating nucleotides of a single strand. Alternating nucleotides can refer to patterns such as every other nucleotide or every third nucleotide. For example, if A, B, and C each represent a type of modification to a nucleotide, the alternating motif can be "ABABABABABAB...," "AABBAABBAABB...," "AABAABAABAAB...," "AAABAAABAAAB...," "AAABBBAAABBB...," or "ABCABCABCABC...," etc.

[0128] The types of modifications within the alternating motif can be the same or different. For example, if A, B, C, and D each represent one type of modification on a nucleotide, the alternation pattern, i.e., the modifications at every other nucleotide, can be the same, but each of the sense or antisense strands can be selected from several possible modifications within the alternating motif, such as "ABABAB...", "ACACAC...", "BDBDBD..." or "CDCDCD...".

[0129] In some embodiments, the dsRNA molecule comprises a modification pattern for the alternating motif on the sense strand that is shifted relative to the modification pattern for the alternating motif on the antisense strand. The shift can be such that the modified group of nucleotides in the sense strand corresponds to a different modified group of nucleotides in the antisense strand, or vice versa. For example, when the sense strand is paired with the antisense strand in a dsRNA duplex, the alternating motif in the sense strand can begin with "ABABAB" from 5' to 3' of the strand, and the alternating motif in the antisense strand can begin with "BABABA" from 3' to 5' of the strand within the duplex region. As another example, the alternating motif in the sense strand can begin with "AABBAABB" from 5' to 3' of the strand, and the alternating motif in the antisense strand can begin with "BBAABBAA" from 3' to 5' of the strand within the duplex region, such that a complete or partial shift in the modification pattern occurs between the sense strand and the antisense strand.

[0130] In some embodiments, the dsRNA molecule comprises mismatches with the target, mismatches within the duplex, or a combination thereof. Mismatches can be present in the overhang region or the duplex region. Base pairs can be ranked based on their tendency to promote dissociation or melting (e.g., with respect to the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pair on an individual pair basis, but analysis such as next neighbor or similarity analysis can also be used). In terms of promoting dissociation, A:U is more preferable than G:C, G:U is more preferable than G:C, and I:C is more preferable than G:C (I=inosine). Mismatches, such as non-canonical pairings, i.e., pairings other than canonical pairings (as described in other sections of this specification), are more preferable than canonical (A:T, A:U, G:C) pairings, and pairings containing universal bases are more preferable than canonical pairings.

[0131] In some embodiments, in a dsRNA molecule, at least one of the first 1, 2, 3, 4, or 5 base pairs from the 5' end of the antisense strand within the duplex region can be independently selected from the group of non-canonical pairings, i.e., pairings other than canonical pairings, including A:U, G:U, I:C, and mismatched pairs, e.g., universal bases, to promote dissociation of the antisense strand at the 5' end of the duplex.

[0132] In some embodiments, the first nucleotide from the 5' end of the antisense strand in the double-stranded region is selected from the group consisting of A, dA, dU, U and dT. Alternatively, at least one of the first 1, 2 or 3 base pairs from the 5' end of the antisense strand in the double-stranded region is an AU base pair. For example, the first base pair from the 5' end of the antisense strand in the double-stranded region is an AU base pair.

[0133] Without wishing to be bound by theory, the introduction of 4'- and / or 5'-modified nucleotides at the 3'-end of a dinucleotide phosphodiester (PO), phosphorothioate (PS) and / or phosphorodithioate (PS2) linkage at any position in a single- or double-stranded oligonucleotide produces a steric effect at the internucleotide linkage, thereby protecting or stabilizing it against nucleases.

[0134] In some embodiments, 5'-modified nucleosides are introduced at the 3'-end of dinucleotides at any position in dsRNA molecules.For example, 5'-alkylated nucleosides can be introduced at the 3'-end of dinucleotides at any position in dsRNA.The alkyl group at the 5'-position of ribose sugar can be racemic or chirally pure R or S isomer.An exemplary 5'-alkylated nucleoside is 5'-methyl nucleoside.5'-methyl can be racemic or chirally pure R or S isomer.

[0135] In some embodiments, 4'-modified nucleosides are introduced at the 3'-end of dinucleotides at any position in dsRNA. For example, 4'-alkylated nucleosides can be introduced at the 3'-end of dinucleotides at any position in dsRNA. The alkyl group at the 4'-position of the ribose sugar can be racemic or chirally pure R or S isomer. An exemplary 4'-alkylated nucleoside is 4'-methyl nucleoside. The 4'-methyl can be racemic or chirally pure R or S isomer. Alternatively, 4'-O-alkylated nucleosides can be introduced at the 3'-end of dinucleotides at any position in single-stranded or double-stranded siRNA. The 4'-O-alkyl of the ribose sugar can be racemic or chirally pure R or S isomer. An exemplary 4'-O-alkylated nucleoside is 4'-O-methyl nucleoside. The 4'-O-methyl can be racemic or chirally pure R or S isomer.

[0136] In some embodiments, 5'-alkylated nucleosides are introduced at any position on the sense strand or antisense strand of dsRNA, and such modifications maintain or improve the efficacy of dsRNA. 5'-alkyl can be racemic or chirally pure R or S isomer. An exemplary 5'-alkylated nucleoside is 5'-methyl nucleoside. 5'-methyl can be racemic or chirally pure R or S isomer.

[0137] In some embodiments, 4'-alkylated nucleosides are introduced into any position on the sense strand or antisense strand of dsRNA, and such modifications maintain or improve the efficacy of dsRNA. 4'-alkyl can be racemic or chirally pure R or S isomer. An exemplary 4'-alkylated nucleoside is 4'-methyl nucleoside. 4'-methyl can be racemic or chirally pure R or S isomer.

[0138] In some embodiments, 4'-O-alkylated nucleosides are introduced at any position on the sense strand or antisense strand of dsRNA, and such modifications maintain or improve the efficacy of dsRNA. 5'-Alkyl can be racemic or chirally pure R or S isomer. An exemplary 4'-O-alkylated nucleoside is 4'-O-methyl nucleoside. 4'-O-methyl can be racemic or chirally pure R or S isomer.

[0139] In some embodiments, dsRNA molecules can comprise 2'-5' linkages (with 2'-H, 2'-OH and 2'-OMe, and with P=O or P=S).For example, 2'-5' linkage modifications can be used to improve nuclease resistance, or to inhibit the binding of sense strand to antisense strand, or can be used at the 5' end of sense strand to avoid sense strand activation by RISC.In some embodiments, sense strands comprise 2'-5' linkages at N-1 and N-2 positions counting from the 5' end.

[0140] In some embodiments, dsRNA molecules can contain L-sugars (e.g., L-ribose, L-arabinose with 2'-H, 2'-OH, and 2'-OMe). For example, these L-sugar modifications can be used to enhance nuclease resistance, inhibit binding of the sense strand to the antisense strand, or can be used at the 5' end of the sense strand to prevent sense strand activation by RISC. In some embodiments, the sense strand contains an L-sugar nucleotide at the 5' end.

[0141] Exemplary dsRNA Embodiments In some embodiments, the dsRNA molecule comprises: (i) a sense strand having a length of 21 nucleotides, a linker attached to the 3' end, 2'-fluoro modifications at positions 7, 9, 10, and 11 (counting from the 5' end), and phosphorothioate internucleotide linkages between the first and second nucleotides and between the second and third nucleotides (counting from the 5' end); and (ii) an antisense strand having a length of 23 nucleotides, 2'-fluoro modifications at positions 2, 6, 8, 9, 14, and 16 (counting from the 5' end), and phosphorothioate internucleotide linkages between the first and second nucleotides, between the second and third nucleotides, between the 21st and 22nd nucleotides, and between the 22nd and 23rd nucleotides (counting from the 5' end), and optionally a duplex heat destabilizing modification at position 7 (counting from the 5' end), wherein the dsRNA molecule has a 2-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0142] In yet another embodiment, the dsRNA molecule comprises (i) a sense strand having a length of 21 nucleotides and a linker attached to its 3'-end, and 2'-fluoro modifications at positions 7, 9, 10, and 11 (counting from the 5'-end), and phosphorothioate internucleotide linkages between the first and second nucleotides and between the second and third nucleotides (counting from the 5'-end); and (ii) an antisense strand having a length of 23 nucleotides and 2'-fluoro modifications at positions 2, 6, 9, 14, and 16 (counting from the 5'-end), and phosphorothioate internucleotide linkages between the first and second nucleotides, the second and third nucleotides, the 21st and 22nd nucleotides, and the 22nd and 23rd nucleotides (counting from the 5'-end), and optionally a duplex heat-destabilizing modification at position 7 (counting from the 5'-end), wherein the dsRNA molecule has a two-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

[0143] In some embodiments, the dsRNA molecule comprises: (i) a sense strand having a length of 21 nucleotides, a linker attached to the 3' end, and 2'-fluoro modifications at positions 7, 10, and 11 (counting from the 5' end); and (ii) an antisense strand having a length of 23 nucleotides, a 2'-fluoro modification at positions 2, 6, 8, 9, 14, and 16 (counting from the 5' end), a phosphorothioate internucleotide linkage between the 21st and 22nd nucleotides and between the 22nd and 23rd nucleotides (counting from the 5' end), and optionally a duplex heat-destabilizing modification at position 7 (counting from the 5' end), wherein the dsRNA molecule has a 2-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0144] In some other embodiments, the dsRNA molecule comprises: (i) a sense strand having a length of 21 nucleotides, a linker attached to the 3'-end, 2'-fluoro modifications at positions 7, 9, 10, and 11 (counting from the 5'-end), and phosphorothioate internucleotide linkages between the first and second nucleotides and between the second and third nucleotides (counting from the 5'-end); and (ii) an antisense strand having a length of 23 nucleotides, 2'-fluoro modifications at positions 2, 6, 14, and 16 (counting from the 5'-end), and phosphorothioate internucleotide linkages between the first and second nucleotides, the second and third nucleotides, the 21st and 22nd nucleotides, and the 22nd and 23rd nucleotides (counting from the 5'-end), and optionally a duplex heat-destabilizing modification at position 7 (counting from the 5'-end), wherein the dsRNA molecule has a 2-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

[0145] In yet another embodiment, the dsRNA molecule comprises (i) a sense strand having a length of 21 nucleotides and a linker attached to its 3'-end, 2'-fluoro modifications at positions 7, 9, 10, and 11 (counting from the 5'-end), and phosphorothioate internucleotide linkages between the first and second nucleotides and between the second and third nucleotides (counting from the 5'-end), and (ii) an antisense strand having a length of 23 nucleotides and 2'-fluoro modifications at positions 2, 14, and 16 (counting from the 5'-end), and phosphorothioate internucleotide linkages between the first and second nucleotides, the second and third nucleotides, the 21st and 22nd nucleotides, and the 22nd and 23rd nucleotides (counting from the 5'-end), and optionally a duplex heat-destabilizing modification at positions 6 or 7 (counting from the 5'-end), wherein the dsRNA molecule has a two-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

[0146] In some embodiments, the dsRNA molecule comprises (i) a sense strand having a linker attached to the 3' end and phosphorothioate internucleotide linkages between the first and second nucleotides and between the second and third nucleotides (counting from the 5' end), and (ii) an antisense strand having 2'-fluoro modifications at positions 2, 14, and 16 (counting from the 5' end) and, optionally, a duplex heat-destabilizing modification at position 6 or 7 (counting from the 5' end).

[0147] In another embodiment, the dsRNA molecule comprises (i) a sense strand having a linker attached to its 3'-end and a phosphorothioate internucleotide linkage between the first and second nucleotide and between the second and third nucleotide (counting from the 5'-end), and (ii) an antisense strand having 2'-fluoro modifications at positions 2, 14, and 16 (counting from the 5'-end) and phosphorothioate internucleotide linkages between the first and second nucleotide, between the second and third nucleotide, between the 21st and 22nd nucleotide, and between the 22nd and 23rd nucleotide (counting from the 5'-end), and optionally a duplex heat-destabilizing modification at position 6 or 7 (counting from the 5'-end), wherein the dsRNA molecule has a two-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

[0148] In some embodiments, the dsRNA molecule comprises (i) a sense strand having a length of 21 nucleotides, a linker attached at three ends, and a 2'-fluoro modification at positions 7, 10, and 11 (counting from the 5' end), and (ii) an antisense strand having a length of 23 nucleotides, a 2'-fluoro modification at positions 2, 14, and 16 (counting from the 5' end), a phosphorothioate internucleotide linkage between the 21st and 22nd nucleotides and between the 22nd and 23rd nucleotides (counting from the 5' end), and optionally a duplex heat-destabilizing modification at positions 5, 6, or 7 (counting from the 5' end), wherein the dsRNA molecule has a 2-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0149] In some other embodiments, the dsRNA molecule comprises: (i) a sense strand having a length of 21 nucleotides, a linker attached to the 3'-end, 2'-fluoro modifications at positions 7, 9, 10, and 11 (counting from the 5'-end), and phosphorothioate internucleotide linkages between the first and second nucleotides and between the second and third nucleotides (counting from the 5'-end); and (ii) an antisense strand having a length of 23 nucleotides, 2'-fluoro modifications at positions 2, 14, and 16 (counting from the 5'-end), and phosphorothioate internucleotide linkages between the first and second nucleotides, the second and third nucleotides, the 21st and 22nd nucleotides, and the 22nd and 23rd nucleotides (counting from the 5'-end), and optionally a duplex heat-destabilizing modification at positions 5, 6, or 7 (counting from the 5'-end), wherein the dsRNA molecule has a two-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

[0150] In some other embodiments, the dsRNA molecule comprises (i) a sense strand having a length of 21 nucleotides, a linker attached to the 3'-end, 2'-fluoro modifications at positions 7, 9, 10, and 11 (counting from the 5'-end), and at least one, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more LNA modifications; and (ii) an antisense strand having a length of 23 nucleotides, 2'-fluoro modifications at positions 2, 14, and 16 (counting from the 5'-end), phosphorothioate internucleotide linkages between the 1st and 2nd nucleotides, the 2nd and 3rd nucleotides, the 21st and 22nd nucleotides, and the 22nd and 23rd nucleotides (counting from the 5'-end), and optionally a duplex heat-destabilizing modification at positions 5, 6, or 7 (counting from the 5'-end), wherein the dsRNA molecule has a two-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

[0151] In some embodiments, the dsRNA molecule comprises: (i) a sense strand having a length of 21 nucleotides, a linker attached to the 3'-end, and a 2'-fluoro modification at positions 7, 9, 10, and 11 (counting from the 5'-end), and an LNA modification at at least one of positions 1, 2, and 3, for example, 1, 2, or 3 (counting from the 5'-end); and (ii) an antisense strand having a length of 23 nucleotides, a 2'-fluoro modification at positions 2, 14, and 16 (counting from the 5'-end), and a phosphorothioate internucleotide linkage between the 1st and 2nd nucleotides, the 2nd and 3rd nucleotides, the 21st and 22nd nucleotides, and the 22nd and 23rd nucleotides (counting from the 5'-end), and optionally a duplex heat-destabilizing modification at positions 5, 6, or 7 (counting from the 5'-end), wherein the dsRNA molecule has a 2-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

[0152] In some embodiments, the dsRNA molecule comprises (i) a sense strand having a length of 21 nucleotides, a linker attached at the 3' end, 2'-fluoro modifications at positions 7, 9, 10, and 11 (counting from the 5' end), at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more LNA modifications, and a phosphorothioate internucleotide linkage between the first and second nucleotide and between the second and third nucleotide (counting from the 5' end), and (ii) a 23 nucleotide length and a linker attached at the 3' end. The dsRNA molecule comprises an antisense strand having 2'-fluoro modifications at positions 2, 14, and 16 (counting from the 5' end), phosphorothioate internucleotide linkages between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 21st and 22nd nucleotides, and between the 22nd and 23rd nucleotides (counting from the 5' end), and optionally a duplex heat-destabilizing modification at positions 5, 6, or 7 (counting from the 5' end), wherein the dsRNA molecule has a two-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0153] In some other embodiments, the dsRNA molecule comprises: (i) a sense strand having a length of 21 nucleotides, a linker attached to the 3'-end, and a 2'-fluoro modification at positions 7, 9, 10, and 11 (counting from the 5'-end), and an LNA modification at at least one of positions 1, 2, and 3, for example, 1, 2, or 3 (counting from the 5'-end); and (ii) an antisense strand having a length of 23 nucleotides, a 2'-fluoro modification at positions 2, 14, and 16 (counting from the 5'-end), and a phosphorothioate internucleotide linkage between the 1st and 2nd nucleotides, the 2nd and 3rd nucleotides, the 21st and 22nd nucleotides, and the 22nd and 23rd nucleotides (counting from the 5'-end), and optionally a duplex heat-destabilizing modification at positions 5, 6, or 7 (counting from the 5'-end), wherein the dsRNA molecule has a 2-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

[0154] In some embodiments, the dsRNA molecule comprises: (i) a sense strand having a length of 21 nucleotides and a linker attached at the 3' end, 2'-fluoro modifications at positions 7, 9, 10, and 11 (counting from the 5' end), LNA modifications at at least one of positions 1, 2, and 3, e.g., 1, 2, or 3 (counting from the 5' end), and phosphorothioate internucleotide linkages between the first and second nucleotides and between the second and third nucleotides (counting from the 5' end); and (ii) a sense strand having a length of 23 nucleotides and a linker attached at the 3' end, 2'-fluoro modifications at positions 7, 9, 10, and 11 (counting from the 5' end), LNA modifications at at least one of positions 1, 2, and 3, e.g., 1, 2, or 3, and an antisense strand having 2'-fluoro modifications at positions 2, 14, and 16 (counting from the 5' end), phosphorothioate internucleotide linkages between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 21st and 22nd nucleotides, and between the 22nd and 23rd nucleotides (counting from the 5' end), and optionally a duplex heat-destabilizing modification at positions 5, 6, or 7 (counting from the 5' end), wherein the dsRNA molecule has a two-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0155] In yet another embodiment, the dsRNA molecule comprises (i) a sense strand having a length of 21 nucleotides and a linker attached to its 5'-end, and 2'-fluoro modifications at positions 7, 9, 10, and 11 (counting from the 5'-end), and phosphorothioate internucleotide linkages between the first and second nucleotides and between the second and third nucleotides (counting from the 5'-end); and (ii) an antisense strand having a length of 23 nucleotides and 2'-fluoro modifications at positions 2, 6, 8, 9, 14, and 16 (counting from the 5'-end), and phosphorothioate internucleotide linkages between the first and second nucleotides, the second and third nucleotides, the 21st and 22nd nucleotides, and the 22nd and 23rd nucleotides (counting from the 5'-end), and optionally a duplex heat-destabilizing modification at position 7 (counting from the 5'-end), wherein the dsRNA molecule has a two-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

[0156] In yet another embodiment, the dsRNA molecule comprises (i) a sense strand having a length of 21 nucleotides and a linker attached to its 5'-end, and 2'-fluoro modifications at positions 7, 9, 10, and 11 (counting from the 5'-end), and phosphorothioate internucleotide linkages between the first and second nucleotides and between the second and third nucleotides (counting from the 5'-end); and (ii) an antisense strand having a length of 23 nucleotides and 2'-fluoro modifications at positions 2, 6, 9, 14, and 16 (counting from the 5'-end), and phosphorothioate internucleotide linkages between the first and second nucleotides, the second and third nucleotides, the 21st and 22nd nucleotides, and the 22nd and 23rd nucleotides (counting from the 5'-end), and optionally a duplex heat-destabilizing modification at position 7 (counting from the 5'-end), wherein the dsRNA molecule has a two-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

[0157] In some embodiments, the dsRNA molecule comprises: (i) a sense strand having a length of 21 nucleotides, a linker attached to the 5' end, and 2'-fluoro modifications at positions 7, 10, and 11 (counting from the 5' end); and (ii) an antisense strand having a length of 23 nucleotides, a 2'-fluoro modification at positions 2, 6, 8, 9, 14, and 16 (counting from the 5' end), a phosphorothioate internucleotide linkage between the 21st and 22nd nucleotides and between the 22nd and 23rd nucleotides (counting from the 5' end), and optionally a duplex heat-destabilizing modification at position 7 (counting from the 5' end), wherein the dsRNA molecule has a 2-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0158] In some other embodiments, the dsRNA molecule comprises: (i) a sense strand having a length of 21 nucleotides, a linker attached to the 5'-end, 2'-fluoro modifications at positions 7, 9, 10, and 11 (counting from the 5'-end), and phosphorothioate internucleotide linkages between the first and second nucleotides and between the second and third nucleotides (counting from the 5'-end); and (ii) an antisense strand having a length of 23 nucleotides, 2'-fluoro modifications at positions 2, 6, 14, and 16 (counting from the 5'-end), and phosphorothioate internucleotide linkages between the first and second nucleotides, the second and third nucleotides, the 21st and 22nd nucleotides, and the 22nd and 23rd nucleotides (counting from the 5'-end), and optionally a duplex heat-destabilizing modification at position 7 (counting from the 5'-end), wherein the dsRNA molecule has a two-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

[0159] In yet another embodiment, the dsRNA molecule comprises (i) a sense strand having a length of 21 nucleotides and a linker attached to its 5'-end, and 2'-fluoro modifications at positions 7, 9, 10, and 11 (counting from the 5'-end), and phosphorothioate internucleotide linkages between the first and second nucleotides and between the second and third nucleotides (counting from the 5'-end), and (ii) an antisense strand having a length of 23 nucleotides and 2'-fluoro modifications at positions 2, 14, and 16 (counting from the 5'-end), and phosphorothioate internucleotide linkages between the first and second nucleotides, the second and third nucleotides, the 21st and 22nd nucleotides, and the 22nd and 23rd nucleotides (counting from the 5'-end), and optionally a duplex heat-destabilizing modification at position 6 or 7 (counting from the 5'-end), wherein the dsRNA molecule has a two-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

[0160] In some embodiments, the dsRNA molecule comprises (i) a sense strand having a linker attached to its 5' end and phosphorothioate internucleotide linkages between the first and second nucleotides and between the second and third nucleotides (counting from the 5' end), and (ii) an antisense strand having 2'-fluoro modifications at positions 2, 14, and 16 (counting from the 5' end) and, optionally, a duplex heat-destabilizing modification at position 6 or 7 (counting from the 5' end).

[0161] In another embodiment, the dsRNA molecule comprises (i) a sense strand having a linker attached to its 5'-end and phosphorothioate internucleotide linkages between the first and second nucleotides and between the second and third nucleotides (counting from the 5'-end), and (ii) an antisense strand having 2'-fluoro modifications at positions 2, 14, and 16 (counting from the 5'-end) and phosphorothioate internucleotide linkages between the first and second nucleotides, the second and third nucleotides, the 21st and 22nd nucleotides, and the 22nd and 23rd nucleotides (counting from the 5'-end), and optionally a duplex heat-destabilizing modification at position 6 or 7 (counting from the 5'-end), wherein the dsRNA molecule has a two-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

[0162] In some embodiments, the dsRNA molecule comprises (i) a sense strand having a length of 21 nucleotides, a linker attached to the 5'-end, and 2'-fluoro modifications at positions 7, 10, and 11 (counting from the 5'-end), and (ii) an antisense strand having a length of 23 nucleotides, a 2'-fluoro modification at positions 2, 14, and 16 (counting from the 5'-end), a phosphorothioate internucleotide linkage between the 21st and 22nd nucleotides and between the 22nd and 23rd nucleotides (counting from the 5'-end), and optionally a duplex heat-destabilizing modification at positions 5, 6, or 7 (counting from the 5'-end), wherein the dsRNA molecule has a 2-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

[0163] In some other embodiments, the dsRNA molecule comprises: (i) a sense strand having a length of 21 nucleotides, a linker attached to the 5'-end, 2'-fluoro modifications at positions 7, 9, 10, and 11 (counting from the 5'-end), and phosphorothioate internucleotide linkages between the first and second nucleotides and between the second and third nucleotides (counting from the 5'-end); and (ii) an antisense strand having a length of 23 nucleotides, 2'-fluoro modifications at positions 2, 14, and 16 (counting from the 5'-end), and phosphorothioate internucleotide linkages between the first and second nucleotides, the second and third nucleotides, the 21st and 22nd nucleotides, and the 22nd and 23rd nucleotides (counting from the 5'-end), and optionally a duplex heat-destabilizing modification at positions 5, 6, or 7 (counting from the 5'-end), wherein the dsRNA molecule has a two-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

[0164] In some other embodiments, the dsRNA molecule comprises (i) a sense strand having a length of 21 nucleotides, a linker attached to the 5'-end, 2'-fluoro modifications at positions 7, 9, 10, and 11 (counting from the 5'-end), and at least one, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more LNA modifications; and (ii) an antisense strand having a length of 23 nucleotides, 2'-fluoro modifications at positions 2, 14, and 16 (counting from the 5'-end), phosphorothioate internucleotide linkages between the first and second nucleotides, the second and third nucleotides, the 21st and 22nd nucleotides, and the 22nd and 23rd nucleotides (counting from the 5'-end), and optionally a duplex heat-destabilizing modification at positions 5, 6, or 7 (counting from the 5'-end), wherein the dsRNA molecule has a two-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

[0165] In some embodiments, the dsRNA molecule comprises: (i) a sense strand having a length of 21 nucleotides, a linker attached to the 5'-end, and a 2'-fluoro modification at positions 7, 9, 10, and 11 (counting from the 5'-end), and an LNA modification at at least one of positions 1, 2, and 3, for example, 1, 2, or 3 (counting from the 5'-end); and (ii) an antisense strand having a length of 23 nucleotides, and a 2'-fluoro modification at positions 2, 14, and 16 (counting from the 5'-end), and a phosphorothioate internucleotide linkage between the 1st and 2nd nucleotides, the 2nd and 3rd nucleotides, the 21st and 22nd nucleotides, and the 22nd and 23rd nucleotides (counting from the 5'-end), and optionally a duplex heat-destabilizing modification at positions 5, 6, or 7 (counting from the 5'-end), wherein the dsRNA molecule has a 2-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

[0166] In some embodiments, the dsRNA molecule comprises (i) a sense strand having a length of 21 nucleotides, a linker attached to the 5' end, 2'-fluoro modifications at positions 7, 9, 10, and 11 (counting from the 5' end), at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more LNA modifications, and a phosphorothioate internucleotide linkage between the first and second nucleotide and between the second and third nucleotide (counting from the 5' end), and (ii) a 23 nucleotide length and a linker attached to the 5' end. The dsRNA molecule comprises an antisense strand having 2'-fluoro modifications at positions 2, 14, and 16 (counting from the 5' end), phosphorothioate internucleotide linkages between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 21st and 22nd nucleotides, and between the 22nd and 23rd nucleotides (counting from the 5' end), and optionally a duplex heat-destabilizing modification at positions 5, 6, or 7 (counting from the 5' end), wherein the dsRNA molecule has a two-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0167] In some other embodiments, the dsRNA molecule comprises: (i) a sense strand having a length of 21 nucleotides, a linker attached to the 5'-end, and a 2'-fluoro modification at positions 7, 9, 10, and 11 (counting from the 5'-end), and an LNA modification at at least one of positions 1, 2, and 3, for example, 1, 2, or 3 (counting from the 5'-end); and (ii) an antisense strand having a length of 23 nucleotides, a 2'-fluoro modification at positions 2, 14, and 16 (counting from the 5'-end), and a phosphorothioate internucleotide linkage between the 1st and 2nd nucleotides, the 2nd and 3rd nucleotides, the 21st and 22nd nucleotides, and the 22nd and 23rd nucleotides (counting from the 5'-end), and optionally a duplex heat-destabilizing modification at positions 5, 6, or 7 (counting from the 5'-end), wherein the dsRNA molecule has a 2-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

[0168] In some embodiments, the dsRNA molecule comprises: (i) a sense strand having a length of 21 nucleotides and a linker attached to the 5' end, a 2'-fluoro modification at positions 7, 9, 10, and 11 (counting from the 5' end), an LNA modification at at least one of positions 1, 2, and 3 (counting from the 5' end), e.g., 1, 2, or 3, and a phosphorothioate internucleotide linkage between the first and second nucleotide and between the second and third nucleotide (counting from the 5' end); and (ii) a length of 23 nucleotides and a linker attached to the 5' end. and an antisense strand having 2'-fluoro modifications at positions 2, 14, and 16 (counting from the 5' end), phosphorothioate internucleotide linkages between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 21st and 22nd nucleotides, and between the 22nd and 23rd nucleotides (counting from the 5' end), and optionally a duplex heat-destabilizing modification at positions 5, 6, or 7 (counting from the 5' end), wherein the dsRNA molecule has a two-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0169] In some further embodiments of the exemplary dsRNA molecules described above, the sense strand comprises at least 1, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 2'-OMe modifications.

[0170] In some other further embodiments of the exemplary dsRNA molecules described above, the antisense strand comprises at least 1, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 2'-OMe modifications.

[0171] In still some other further embodiments of the exemplary dsRNA molecules described above, the sense strand comprises at least one, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17, 2'-OMe modification and the antisense strand comprises at least one, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, 2'-OMe modification.

[0172] Dual variable domain immunoglobulins Aspects of the present invention include dual variable domain (DVD) immunoglobulin molecules (Igs). Dual variable domain immunoglobulin molecules generally contain a first variable domain that binds to a target antigen and a second variable domain that contains unique reactive residues that provide sites for covalent attachment of a linker molecule. In addition, DVD immunoglobulin molecules contain two identical light chains and two identical heavy chains. Each light chain and each heavy chain contains an N-terminus and a C-terminus. Assembly of the two light chains and two heavy chains results in the formation of a DVD immunoglobulin molecule with various inter- and intra-chain disulfide bonds that stabilize the interaction of the light and heavy chains.

[0173] Each light chain is V L 1 and V L a first variable domain and a second variable domain designated C LIn some embodiments, the light chain comprises a kappa light chain. In other embodiments, the light chain comprises a lambda light chain.

[0174] In some embodiments, the second variable domain contains only one unique reactive lysine residue that provides a site for the covalent attachment of a linker molecule.

[0175] In some other embodiments, the second variable domain contains only one unique reactive arginine residue that provides a site for the covalent attachment of a linker molecule.

[0176] In some embodiments, each heavy chain comprises a V H 1 and V H a first variable domain and a second variable domain designated C H 1, followed by a heavy chain Fc region domain. In some embodiments, the Fc region domain on the heavy chain can comprise an Fc region domain characteristic of a particular immunoglobulin type or subtype, such as, but not limited to, an Fc region from an IgG (such as IgG1, IgG2, IgG3, or IgG4), IgA (such as IgA1 or IgA2), IgM, IgE, or IgD antibody. For example, the immunoglobulin belongs to the IgG class and the heavy chain comprises a gamma heavy chain. In some embodiments, the immunoglobulin belongs to the IgG1 class and the heavy chain comprises a gamma 1 heavy chain. In other embodiments, the immunoglobulin belongs to the IgG2 class and the heavy chain comprises a gamma 2 heavy chain. In still other embodiments, the immunoglobulin belongs to the IgG3 class and the heavy chain comprises a gamma 3 heavy chain. In some embodiments, the immunoglobulin belongs to the IgG4 class and the heavy chain comprises a gamma 4 heavy chain.

[0177] In some embodiments, the immunoglobulin belongs to the IgA class. For example, the immunoglobulin belongs to the IgA class and the heavy chain comprises an a heavy chain. In some embodiments, the immunoglobulin belongs to the IgA1 class and the heavy chain comprises an a1 heavy chain. In some embodiments, the immunoglobulin belongs to the IgA2 class and the heavy chain comprises an a2 heavy chain.

[0178] In some embodiments, the immunoglobulin belongs to the IgD class and the heavy chain comprises a delta heavy chain. In some embodiments, the immunoglobulin belongs to the IgE class and the heavy chain comprises an epsilon heavy chain. In some embodiments, the immunoglobulin belongs to the IgM class and the heavy chain comprises a mu heavy chain.

[0179] In some embodiments, an immunoglobulin molecule can comprise a native polypeptide sequence found in nature.

[0180] The variable and constant domains in light chains are generally arranged as follows from the N-terminus to the C-terminus: V L 1-V L 2-C L However, in some embodiments, the structure is V from the N-terminus to the C-terminus. L 2-V L 1-C L The configuration of the variable domains in the light chain can be reversed so that, from N-terminus to C-terminus, V L 1-V L 2 or V L 2-V L In some embodiments, the light chain does not include a second variable domain. For example, the configuration of variable and constant domains in the light chain can be such that the domain sequence from N-terminus to C-terminus is V L 1-C L 1 or V L -C L It can be configured so that:

[0181] Similarly, the variable and constant domains in the heavy chain are generally arranged from the N-terminus to the C-terminus, e.g., V H 1-V H 2-C H 1. V H 1-C H 1-V H 2-C H 1. V H 1-V H2-C H 1-FC or V H 1-C H 1-V H 2-C H 1-FC, which may be modified to reflect the configuration of domains on the light chain so that appropriate domains on the light chain pair with appropriate domains on the heavy chain when the immunoglobulin molecule or fragment thereof is assembled.

[0182] In some embodiments, the variable and constant domains in the light and heavy chains are arranged such that the domains in the light chain are V, V, VH ... L 1-V L 2-C H 1, and the heavy chain domain structure is V from the N-terminus to the C-terminus. H 1-V H 2-C L -FC. This configuration is referred to as the CrossMAb configuration and is described in detail in Klein et al., mAbs 4, 653-663 (2012), the disclosure of which is incorporated herein by reference in its entirety. In some embodiments, the CrossMAb configuration can be used to generate bispecific DVD immunoglobulins, as described below.

[0183] In some embodiments, the variable and constant domains in the light chain are arranged such that the domains in the light chain are V, VB, VC, VD, VE, VF, VH, VF ... L 1-C H The heavy chain variable and constant domains can be configured so that the domain arrangement in the heavy chain is V1 from the N-terminus to the C-terminus. H 1-V H 2-C H 1. V H 1-C H 1-V H 2-C H 1. V H 1-V H 2-C H 1-FC or V H 1-C H 1-V H 2-C HIt can be configured to be 1-FC.

[0184] In some embodiments, the variable and constant domains in the light chain are arranged such that the domains in the light chain are V, VB, VC, VD, VE, VF, VH, VF ... L 1-C H The heavy chain variable and constant domains can be configured so that the domain arrangement in the heavy chain is V1 from the N-terminus to the C-terminus. H 1-C H 1-V H 2-C H 1 or V H 1-C H 1-V H 2-C H It can be configured to be 1-FC.

[0185] Note that different domains, such as two variable domains, a variable domain and a constant domain, a variable domain and an Fc domain, and / or a constant domain and an Fc domain, can be linked together via a linker. The linker can be a chemical linker, a single peptide bond (e.g., directly linked to each other), or a peptide linker containing one or more amino acid residues (e.g., an amino acid or amino acid sequence intervenes between the domains). As used herein, the term "peptide linker" refers to a peptide having an amino acid sequence (in some embodiments, of synthetic origin). Note that peptide linkers may affect the folding of a given fusion protein and may also react / bind with other proteins, and these properties can be screened by known techniques. Peptide linkers can contain 1 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, 20 or more amino acids, 25 or more amino acids, 30 or more amino acids, 35 or more amino acids, 40 or more amino acids, 45 or more amino acids, or 50 or more amino acids. Conversely, the peptide linker can contain fewer than 50 amino acids, fewer than 45 amino acids, fewer than 40 amino acids, fewer than 35 amino acids, fewer than 30 amino acids, fewer than 30 amino acids, fewer than 25 amino acids, fewer than 20 amino acids, fewer than 15 amino acids, or fewer than 10 amino acids.

[0186] In some embodiments of the various aspects described herein, the peptide linker comprises about 5 to about 40 amino acids. For example, the peptide linker can comprise about 5 to about 35 amino acids, about 5 to about 30 amino acids, or about 5 to about 25 amino acids. In some embodiments of the various aspects described herein, the peptide linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids.

[0187] Exemplary peptide linkers include those consisting of glycine and serine residues, so-called Gly-Ser polypeptide linkers. As used herein, the term "Gly-Ser polypeptide linker" refers to a peptide consisting of glycine and serine residues. In some embodiments of the various aspects described herein, the peptide linker has the amino acid sequence (Gly x Ser) n wherein x is 2, 3, 4, or 5 and n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments of the various aspects described herein, x is 3 and n is 1, 2, 3, or 4. In some embodiments of the various aspects described herein, x is 3 and n is 3 or 4. In some embodiments of the various aspects described herein, x is 4 and n is 1, 2, 3, or 4. In some embodiments of the various aspects described herein, x is 4 and n is 1, 2, or 3.

[0188] Further exemplary peptide linker sequences that can be used are listed in U.S. Pat. No. 7,612,181 and PCT Publication No. WO2017 / 049139, the contents of both of which are incorporated herein by reference in their entireties.

[0189] In some embodiments, the linker is TIFF2026041866000014.tif18160 and any combination thereof.

[0190] In some embodiments, the first variable domain and the second variable domain are linked in each light or heavy chain by a peptide linker sequence. The peptide linker sequence can be a single amino acid or a polypeptide sequence. In some embodiments, the first variable domain and the second variable domain are linked by a peptide linker, and the peptide linker sequence is TIFF2026041866000015.tif25160 or any combination thereof.

[0191] Additional peptide linker sequences that can be used to link two domains, such as the first and second variable domains of the present DVD immunoglobulins, are described in U.S. Pat. No. 7,612,181 and PCT Publication No. WO2017 / 049139, the contents of both of which are incorporated herein by reference in their entireties.

[0192] In some embodiments, the DVD immunoglobulin molecule comprises a first variable domain with antigen-binding functionality. L 1 Array and V H The V1 sequence is selected to specifically bind to a target, such as a cell surface marker or antigen. One skilled in the art will recognize that antigens are known for virtually any type of cell. Thus, the V1 sequence of the present DVD immunoglobulin molecules L 1 Array and V H One sequence is selected to specifically bind to virtually any known antigen on virtually any type of cell.

[0193] In some embodiments, the V of the DVD immunoglobulin molecule L 1 Array and V H The immunoglobulin sequence is selected to specifically bind to an antigen on tumor cells. Immunoglobulins can exert antitumor effects by inducing apoptosis, redirecting cytotoxicity, disrupting ligand-receptor interactions, or preventing the expression of proteins critical to the neoplastic phenotype. Additionally, immunoglobulins can target components of the tumor microenvironment to disrupt important structures, such as the formation of tumor-associated vasculature. Immunoglobulins can also target growth factor receptors, such as epidermal growth factor receptors, to inhibit the binding of natural cell-stimulating ligands to target tumor cells. Alternatively, immunoglobulins can induce ADCC, ADCP, or CDC.

[0194] Exemplary tumor-associated binding targets that can be targeted by the first variable domain of a DVD immunoglobulin molecule include, but are not limited to, CD138, BCMA, SLAMF7, HER2 (ERBB2), FOLR1, FOLR2, CD19, CD79A, CD79B, ROR1, ROR2, FCRM, CS1, GPA33, PSMA, Siglec-1, Siglec-4, Siglec-5, Siglec-6, Siglec-7, Siglec-8, Siglec-9, Siglec-10, MSLN, CD52, CD20, CD3, CD4, CD8, CD20, CD21, CD22, CD23, CD30, CD33, CD38, CD44, CD56, CD70, BMP6, IL12A, IL1A, IL1B, IL2, IL24, INHA, TNF, TNFSF1O, BMP 6, EGF, FGF1, FGF10, FGF11, FGF12, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF2, FGF20, FGF21, FGF22, FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, GRP, IGF1, IGF2, IL12A, IL1A, IL1B, IL2, INHA, TGFA, TGF B1, TGFB2, TGFB3, VEGF, CDK2, EGF, FGF10, FGF18, FGF2, FGF4, FGF7, IGF1, IGF1R, IL2, VEGF, BCL2, CD164, C DKN1A, CDKN1B, CDKN1C, CDKN2A, CDKN2B, CDKN2C, CDKN3, GNRH1, IGFBP6, IL1A, IL1B, ODZ1, PAWR, PLG, TGFBlII、AR、BRCA1、CDK3、CDK4、CDK5、CDK6、CDK7、CDK9、E2F1、EGFR(ERBB1)、HER3(ERBB3)、HER4(ERBB4)、ENO1、ESR1、ESR2、IGFBP3、IGFBP6、IL2、INSL4、MYC、NOX5、NR6A1、PAP、PCNA、PRKCQ、PRKD1、PRL、TP53、FGF22、FGF23、FGF9、IGFBP3、IL2、INHA、KLK6、TP53、CHGB、GNRH1、IGF1、IGF2、INHA、INSL3、INSL4、PRL、KLK6、SHBG、R1D1、R1H3、NR1I3、R2F6、R4A3、ESR1、ESR2、R0B1、R0B2、R1D2、R1H2、R1H4、R1I2、R2C1、R2C2、R2E1、R2E3、R2F1、R2F2、R3C1、R3C2、R4A1、R4A2、R5A1、R5A2、R6A1、PGR、RARB、FGF1、FGF2、FGF6、KLK3、KRT1、APOC1、BRCA1、CHGA、CHGB、CLU、COL1A1、COL6A1、EGF、ERK8、FGF1、FGF10、FGF11、FGF13、FGF14、FGF16、FGF17、FGF18、FGF2、FGF20、FGF21、FGF22、FGF23、FGF3、FGF4、FGF5、FGF6、FGF7、FGF8、FGF9、GRH1、IGF1、IGF2、IGFBP3、IGFBP6、IL12A、IL1A、IL1B、IL2、IL24、INHA、INSL3、 INSL4、KLK10、KLK12、KLK13、KLK14、KLK15、KLK3、KLK4、KLK5、KLK6、KLK9、MMP 2、MMP9、MSMB、NTN4、ODZ1、PAP、PLAU、PRL、PSAP、セルピンA3、SHBG、TGFA、TTMP3、C D44、CDH1、CDH10、CDH19、CDH20、CDH7、CDH9、CDH1、CDH10、CDH13、CDH18、CDH19 、CDH20、CDH7、CDH8、CDH9、ROB02、CD44、ILK、ITGA1、APC、CD164、COL6A1、MTSS 1、PAP、TGFBIII、AGR2、AIG1、AKAP1、AKAP2、CANT1、CAV1、CDH12、CLDN3、CLN3、 CYB5、CYC1、DAB21P、DES、DNCL1、ELAC2、EN02、EN03、FASN、FLJ12584、FLJ2553 0、GAGEB1、GAGEC1、GGT1、GSTP1、HIP1、HUMCYT2A、IL29、K6HF、KAI1、KRT2A、MIB 1、PART1、PATE、PCA3, PIAS2, PIK3CG, PPID, PR1, PSCA, SLC2A2, SLC33A1, SLC43A1, STEAP, STEAP2, TPM1, TPM2, TRPC6, ANGPT1, ANGPT2, ANPEP, ECGF1, EREG, FGF1, FGF2, FIGF, FLT1, JAG1, KDR, LAMA5, NRP1, NRP2, PGF, PLXDC1, STAB1, VEGF, VEGFC, ANGPTL3, BAI1, COL4A3, IL8, LAMA5, NRP1, NRP2, STAB1, ANGPTL4, PECAM1, PF4, PROK2, Serpin F1, TNFAIP2, CCL11, CCL2, CXCL1, CXCL10, CXCL3, CXCL5, CXCL6, CXCL9, IFNA1, IFNB1, IFNG, IL1B, IL6, MDK, EDG1, EFNA1, EFNA3, EFNB2, EGF, EPHB4, FGFR3, HGF, IGF1, ITGB3, PDGFA, TEK, TGFA, TGFB1, TGFB2, TGFBR1, CCL2, CDH5, COL18A1, EDG1, ENG, ITGAV, ITGB3, THBS1, THBS2, BAD, BAG1, BCL2, CCNA1, CCNA2, CCND1, CCNE1, CCNE2, CDH1 (E-cadherin), CDKN1B (p27Kip1), CDKN2A (p16INK4a), COL6A1, CTNNB1 (β-catenin), CTSB (cathepsin B), ESR1, ESR2, F3 (TF), FOSL1 (FRA-1), GATA3, GSN (gelsolin), IGFBP2, IL2RA, IL6, IL6R, IL6ST (glycoprotein 130), ITGA6 (α6 integrin), JUN, KLK5, KRT19, MAP2K7 (c-Jun), MKI67 (Ki-67), NGFB (NGF), NGFR, NME1 (NM23A), PGR, PLAU (uPA), PTEN, Serpin B5 (maspin), SERPFE1 (PAI-1), TGFA, THBS1 (thrombospondin-1), TIE (Tie-1), TNFRSF6 (Fas), TNFSF6 (FasL), TOP2A (topoisomerase IIa), TP53, AZGP1 (zinc-α-glycoprotein), BPAG1 (plectin), CDKN1A (p21Wapl / Cip1), CLDN7 (claudin-7), CLU (clusterin), FGF1, FLRT1 (fibronectin), GABRP (GABAα), GNAS1, ID2, ITGA6 (α6 integrin), ITGB4 (b4 integrin), KLF5 (GC Box BP), KRT19 (keratin 19), KRTHB6 (hair-specific type II keratin), MACMARCKS, MT3 (metallothionectin-III), MUC1 (mucin), PTGS2 (COX-2), RAC2 (p21Rac2), S100A2, SCGB1D2 (lipophilin B), SCGB2A1 (mammaglobin 2), SCGB2A2 (mammaglobin 1), SPRR1B (Spr1), THBS1, THBS2, THBS4, and TNFAIP2 (B94).

[0195] The amino acid sequences of the first variable domain regions that provide antigen-binding functionality can comprise chimeric, humanized, or human amino acid sequences, and the first variable domains of DVD immunoglobulin molecules can incorporate any suitable combination of such sequences.

[0196] The antigen-binding variable region sequences can be selected from a variety of monoclonal antibodies known in the art that are capable of binding to specific targets. These include anti-TNF antibodies (U.S. Pat. No. 6,258,562), anti-IL-12 and / or anti-IL-12p40 antibodies (U.S. Pat. No. 6,914,128), anti-IL-18 antibodies (US2005 / 0147610A1), anti-C5, anti-CBL, anti-CD147, anti-gp120, anti-VLA4, anti-CD11a, anti-CD18, anti-VEGF, anti-CD40L, anti-Id, anti-ICAM-1, anti-CXCL13, anti-CD2, anti-EGFR, anti-TGF-beta2, anti-E-selectin, anti-Fact VII, anti-Her2 / neu, anti-F gp, anti-CD11 / 18, anti-CD14, anti-ICAM-3, anti-CD80, anti-CD4, anti-CD3, anti-CD23, anti-beta2-integrin, anti-alpha4beta7, anti-CD52, anti-HLA Antibodies include, but are not limited to, DR, anti-CD22, anti-CD20, anti-MIF, anti-CD64 (FcR), anti-TCR alpha beta, anti-CD2, anti-HepB, anti-CA125, anti-EpCAM, anti-gp120, anti-CMV, anti-gpIIbIIIa, anti-IgE, anti-CD25, anti-CD33, anti-HLA, anti-VNR integrin, anti-IL-1 alpha, anti-IL-1 beta, anti-IL-1 receptor, anti-IL-2 receptor, anti-IL-4, anti-IL4 receptor, anti-IL-5, anti-IL-5 receptor, anti-IL-6, anti-IL-8, anti-IL-9, anti-IL-13, anti-IL-13 receptor, anti-IL-17, and anti-IL-23 (Presta LG. 2005 Selection, design, and engineering of therapeutic antibodies J Allergy Clin Immunol. 116:731-6 and Clark, M. "Antibodies for Therapeutic Applications" (Department of Pathology, University of Cambridge, UK, October 15, 2000, published online on the M. Clark homepage on the Cambridge University Department of Pathology website).

[0197] Antigen-binding variable region sequences can also be selected from various therapeutic antibodies that are approved for use, in clinical trials, or in development for clinical use, including, but not limited to, the chimeric anti-CD20 antibody RITUXAN® (IDEC / Genentech / Roche), approved for the treatment of non-Hodgkin's lymphoma (see, e.g., U.S. Pat. No. 5,736,137); HUMAX-CD20®, an anti-CD20 currently in development by Genmab; the anti-CD20 antibodies described in U.S. Pat. No. 5,500,362; AME-133 (Applied Molecular Evolution); hA20 (Immunomedics, Inc.); HumaLYM (Intracel); and PRO70769 (PCT / US2003 / 040426, entitled "Immunoglobulin Variants and Uses"). Thereof), the humanized anti-Her2 / neu antibody trastuzumab (HERCEPTF®, Genentech) approved for the treatment of breast cancer (see, e.g., U.S. Pat. No. 5,677,171 ); pertuzumab (rhuMab-2C4, OMNITARG®), currently in development at Genentech; the anti-Her2 antibody described in U.S. Pat. No. 4,753,894 ; the chimeric anti-EGFR antibody cetuximab (ERBITUX®, Imclone) in clinical trials for a variety of cancers (U.S. Pat. No. 4,943,533 ; PCT Publication No. WO 2014 / 022946 ). 96 / 40210); ABX-EGF currently being developed by Abgenix-Immunex-Amgen (U.S. Patent No. 6,235,883); HUMAX-EGFR™ currently being developed by Genmab (U.S. Application No. 10 / 172,317); 425, EMD55900, EMD62000, and EMD72000 (Merck KGaA) (U.S. Patent No. 5,558,864; Murthy et al. 1987, Arch Biochem Biophys. 252(2):549-60; Rodeck et al., 1987, J Cell Biochem. 35(4):315-20; Kettleborough et al., 1991, ProteinEng. 4(7):773-83); ICR62 (Institute of Cancer Research) (PCT WO95 / 20045, Modjtahedi et al., 1993, J. Cell Biophys. 1993, 22(1-3):129-46, Modjtahedi et al., 1993, Br J Cancer. 1993, 67(2):247-53; Modjtahedi et al., 1996, Br J Cancer, 73(2):228-35, Modjtahedi et al., 2003, Int J Cancer, 105(2):273-80); TheraCIM hR3 (YM Biosciences (Canada) and Centro de Immunologia Molecular (Cuba) (U.S. Patent No. 5,891,996, U.S. Patent No. 6,506,883, Mateo et al. al, 1997, Immunotechnology, 3(1):71-81; mAb-806 (Ludwig Institute for Cancer Research, Memorial Sloan-Kettering) (Jungbluth et al. 2003, Proc Natl Acad Sci USA. 100(2):639-44); KSB-102 (KS Biomedix); MR1-1 (IVAX, National Cancer Institute) (PCT WO 0162931A2); and SCIOO (Scancell) (PCT WO 01 / 88138); the humanized monoclonal antibody alemtuzumab (CAMPATH®, Millennium), currently approved for the treatment of B-cell chronic lymphocytic leukemia; the anti-CD3 antibody muromonab-CD3 (Orthoclone OKT3®), developed by Ortho Biotech / Johnson & Johnson, IDEC / Schering ibritumomab tiuxetan (ZEVALIN®), an anti-CD20 antibody developed by AG; gemtuzumab ozogamicin (MYLOTARG®), an anti-CD33 (p67 protein) antibody developed by Celltech / Wyeth; and anti-LFA-3 antibody developed by Biogen.Fc fusion alefacept (AMEVIVE®), abciximab (REOPRO®) developed by Centocor / Lilly, basiliximab (SFMULECT®) developed by Novartis, palivizumab (SYNAGIS®) developed by Mediimmune, anti-TNF alpha antibody infliximab (REMICADE®) developed by Centocor, anti-TNF alpha antibody adalimumab (HUMIRA®) developed by Abbott, anti-TNF alpha antibody HUMICADE® developed by Celltech, anti-TNF alpha Fc fusion etanercept (ENBREL®) developed by Immunex / Amgen, and anti-CD147 antibody ABX-CBL developed by Abgenix , anti-IL8 antibody ABX-IL8 being developed by Abgenix, anti-MUC18 antibody ABX-MA1 being developed by Abgenix, anti-MUC1 antibody Pemtumomab (R1549, 90Y-muHMFGl) being developed by Antisoma, anti-MUC1 antibody Therex (R1550) being developed by Antisoma, AngioMab (AS1405) being developed by Antisoma, HuBC-1 being developed by Antisoma, Thioplatin (AS1407) being developed by Antisoma, anti-alpha-4-beta-l (VLA4) and alpha-4-beta-7 antibody ANTEGREN® (natalizumab) being developed by Biogen, anti-VLA-1 integrin antibody VLA-1 being developed by Biogen mAb, anti-lymphotoxin beta receptor (LTBR) antibody LTBR mAb developed by Biogen, anti-TGF-P2 antibody CAT-152 developed by Cambridge Antibody Technology, anti-IL-12 antibody J695 developed by Cambridge Antibody Technology and Abbott, anti-TGFpi antibody CAT-192 developed by Cambridge Antibody Technology and Genzyme, Cambridge AntibodyAnti-eotaxin I antibody CAT-213 being developed by Cambridge Antibody Technology; anti-Blys antibody LYMPHOSTAT-B (registered trademark) being developed by Cambridge Antibody Technology and Human Genome Sciences, Inc.; anti-TRAIL-R1 antibody TRAIL-R1 mAb being developed by Cambridge Antibody Technology and Human Genome Sciences, Inc.; anti-VEGF antibody AVASTIN (bevacizumab, rhuMAb-VEGF) being developed by Genentech; anti-HER receptor family antibodies being developed by Genentech; anti-tissue factor antibody anti-tissue factor (ATF) being developed by Genentech; anti-IgE antibody XOLAIR (omalizumab) being developed by Genentech; anti-CD11a antibody RAPTIVA (efalizumab) being developed by Genentech and Xoma; MLN-02 antibody (formerly LDP-02) being developed by IDEC Pharmaceuticals; HUMAX CD4®, an anti-CD4 antibody being developed by Genmab; HUMAX™-IL15, an anti-IL15 antibody being developed by Genmab and Amgen; HUMAX™-Inflam, an anti-heparanase I antibody being developed by Genmab, Medarex, and Oxford GlycoSciences; HUMAX™-Cancer, an anti-heparanase I antibody being developed by Genmab, Medarex, and Oxford GlycoSciences; HUMAX™-Lymphoma, an anti-heparanase I antibody being developed by Genmab and Amgen; HUMAX™-TAC, an anti-CD40L antibody being developed by IDEC Pharmaceuticals; IDEC-131, an anti-CD4 antibody being developed by IDEC Pharmaceuticals; IDEC-151 (clenoliximab), an anti-CD4 antibody being developed by IDEC Pharmaceuticals; IDEC-114, an anti-CD80 antibody being developed by IDEC Pharmaceuticals; IDEC-152, an anti-CD23 antibody being developed by IDEC Pharmaceuticals;an anti-macrophage migration factor (MIF) antibody being developed by Imclone Pharmaceuticals, an anti-idiotypic antibody BEC2 being developed by Imclone, an anti-KDR antibody FMC-1C11 being developed by Imclone, an anti-flk-1 antibody DC101 being developed by Imclone, an anti-VE-cadherin antibody being developed by Imclone, an anti-carcinoembryonic antigen (CEA) antibody CEA-CIDE® (labetuzumab) being developed by Immunomedics, and an anti-CD22 antibody LYMPHOCIDE® (epratuzumab) being developed by Immunomedics; AFP-Cide developed by Immunomedics, MyelomaCide developed by Immunomedics, LkoCide developed by Immunomedics, ProstaCide developed by Immunomedics, anti-CTLA4 antibody MDX-010 developed by Medarex, anti-CD30 antibody MDX-060 developed by Medarex, MDX-070 developed by Medarex, MDX-018 developed by Medarex, Medarex and Immuno-Designed The anti-Her2 antibody OSIDEM® (IDM-1) being developed by Molecules, the anti-CD4 antibody HUMAX®-CD4 being developed by Medarex and Genmab, the anti-IL15 antibody HuMax-IL15 being developed by Medarex and Genmab, the anti-TNFα antibody CNTO148 being developed by Medarex and Centocor / J&J, the anti-cytokine antibody CNTO1275 being developed by Centocor / J&J, the anti-intercellular adhesion molecule-1 (ICAM-1) (CD54) antibodies MOR101 and MOR102 being developed by MorphoSys, the anti-fibroblast growth factor receptor 3 (FGFR-3) antibody MOR201 being developed by MorphoSys, the anti-CD3 antibody NUVION® (visilizumab) being developed by Protein Design Labs, and Protein Design HUZAF®, an anti-gamma interferon antibody being developed by Protein Design Labsanti-a5β1 integrin being developed by Labs, anti-IL-12 being developed by Protein Design Labs, anti-Ep-CAM antibody JNG-1 being developed by Xoma, humanized anti-IgE antibody XOLAIR® (omalizumab) being developed by Genentech and Novartis, and anti-beta2 integrin antibody MLN01 being developed by Xoma. The contents of the references cited above in this paragraph are expressly incorporated herein by reference in their entirety.

[0198] In some embodiments, a DVD immunoglobulin molecule comprises a second variable domain derived from the 38C2 antibody, which comprises a reactive lysine residue. The 38C2 antibody is described, for example, in U.S. Patent No. 8,252,902, the disclosure of which is incorporated herein by reference in its entirety. Briefly, the heavy chain variable region of the 38C2 antibody contains only one unique reactive lysine residue, which can react with a linker to provide an attachment point for conjugation with a drug moiety. Thus, an immunoglobulin molecule comprising the variable domain of the 38C2 antibody contains two such attachment points (one on each heavy chain) that can be used for conjugation with a drug moiety. Once the reactive lysine residue is conjugated to a linker, the binding functionality of the 38C2 variable domain is lost; that is, the variable domain no longer binds to the target. Thus, without being bound by any particular theory, the variable domain of the 38C2 antibody used in the present DVD immunoglobulin molecule provides an attachment point for conjugation but does not provide antigen-binding functionality. In some embodiments, the DVD immunoglobulin molecule comprises a second variable domain derived from the 38C2 antibody as described in WO2017 / 049139, the contents of which are incorporated herein by reference in their entirety.

[0199] Light chain variable (V L ) domain is as follows: TIFF2026041866000016.tif18147

[0200] Heavy chain variable (V H ) domain is as follows: TIFF2026041866000017.tif18146

[0201] The variable light chain (V) of anti-BCMA Fab (VOO1 Fab) L ) domain is as follows: TIFF2026041866000018.tif14146

[0202] VOO1 Fab heavy chain variable (V H ) domain is as follows: TIFF2026041866000019.tif14146

[0203] The variable light chain (V) of humanized VOO1 Fab L ) domain is as follows: TIFF2026041866000020.tif14146

[0204] The heavy chain variable (V) of humanized VOO1 Fab H ) domain is as follows: TIFF2026041866000021.tif14146

[0205] The variable light chain (V L , shown in bold) domain and constant (C L ) domain is as follows: TIFF2026041866000022.tif32146

[0206] The heavy chain variable (V H , shown in bold) domain and constant (C H 1. Hinge, C H 2 and C H3) Exemplary amino acid sequences of the domains are as follows: TIFF2026041866000023.tif60146

[0207] In some embodiments, the DVD immunoglobulin molecule comprises the light chain variable domain sequence of the humanized 38C2 antibody (SEQ ID NO:9 and SEQ ID NO:15) as V L In some embodiments, the DVD immunoglobulin molecule comprises a VL2 domain sequence substantially similar to SEQ ID NO:9 or SEQ ID NO:15, e.g., having at least about 80% amino acid sequence identity, or at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to SEQ ID NO:9 or SEQ ID NO:15.

[0208] In some embodiments, the DVD immunoglobulin molecule comprises the heavy chain variable domain sequence of the humanized 38C2 antibody (SEQ ID NO:10 and SEQ ID NO:16) as the V H In some embodiments, the DVD immunoglobulin molecule comprises a V domain sequence that is substantially similar to SEQ ID NO:10 or SEQ ID NO:16, e.g., has at least about 80% amino acid sequence identity to SEQ ID NO:10 or SEQ ID NO:16, or has at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to SEQ ID NO:10 or SEQ ID NO:16, and has a reactive lysine residue. H It contains a two-domain sequence.

[0209] DVD immunoglobulin molecules can include chimeric, humanized, and human immunoglobulin sequences, and in some embodiments, can contain any mixture thereof. For example, in some embodiments, a DVD immunoglobulin molecule can include a chimeric first variable domain and a human second variable domain. In some embodiments, a DVD immunoglobulin molecule can include a humanized first variable domain and a human second variable domain. The DVD immunoglobulin molecules can utilize any suitable combination of chimeric, humanized, and human immunoglobulin sequences.

[0210] In some embodiments, the DVD immunoglobulins described herein can be modified with respect to immunoglobulin effector function. This can be achieved by introducing one or more amino acid substitutions into the Fc region of the immunoglobulin. Alternatively, or in addition, cysteine ​​residues can be introduced into the Fc region to allow interchain disulfide bond formation in this region. The immunoglobulins thus generated can have improved internalization capabilities and / or increased effector function. See Caron et al., J. Exp Med. 176:1191-1195 (1992) and Shopes, BJ Immunol. 148:2918-2922 (1992). To increase the serum half-life of immunoglobulins, salvage receptor binding epitopes can be incorporated into the immunoglobulin (especially immunoglobulin fragments), as described, for example, in U.S. Patent No. 5,739,277. As used herein, the term "salvage receptor binding epitope" refers to an epitope in the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.

[0211] The DVD immunoglobulin molecules according to aspects of the present invention comprise a first variable domain that provides antigen-binding functionality and a second variable domain derived from the 38C2 antibody that contains only one unique reactive lysine residue that can be conjugated to a linker.

[0212] In certain aspects, DVD immunoglobulin molecules are bispecific in that one arm of the immunoglobulin comprises a first variable domain with binding specificity for a first binding target and the second arm comprises a first variable domain with binding specificity for a second binding target, thereby enabling binding to two different targets and thereby providing additional functionality.

[0213] In certain aspects, DVD immunoglobulin molecules are bi-paratopic in that one arm of the immunoglobulin comprises a first variable domain with binding specificity for a first binding target, and a second arm comprises a first variable domain with binding specificity for the same binding target but a different binding epitope, allowing binding to the same target and covering two different, but potentially somewhat overlapping, binding epitopes, thereby providing target cross-linking functionality and triggering lysosomal transport after internalization.

[0214] In some embodiments, the immunoglobulin molecule comprises an additional chain. For example, the immunoglobulin molecule comprises a first heavy chain, a second heavy chain, and a light chain. The first heavy chain comprises a first variable domain and a second variable domain. The second heavy chain comprises a first variable domain and a second variable domain, and the light chain comprises a variable domain and a constant domain. The first heavy chain and the second heavy chain are capable of forming a heterodimer.

[0215] In some embodiments, the variable and constant domains in the light chain are arranged such that the domains in the light chain are V, VB, VC, VD, VE, VF, VH, VF ... L 1-C HThe variable and constant domains in the first heavy chain can be configured such that the domain arrangement in the first heavy chain is V1 from the N-terminus to the C-terminus. H 1-V H 2-C H 1. V H 1-C H 1-V H 2-C H 1. V H 1-V H 2-C H 1-FC or V H 1-C H 1-V H 2-C H The variable and constant domains in the second heavy chain can be configured as V1-FC, and the domain arrangement in the second heavy chain can be V1-FC from the N-terminus to the C-terminus. H 1'-V H 2', V H 1'-V H 2'-C H 1', V H 1'-C H 1'-V H 2'-C H 1', V H 1'-V H 2'-FC', V H 1'-V H 2'-C H 1'-FC' or V H 1'-C H 1'-V H 2'-C H It can be configured to be 2'-FC'.

[0216] It should be noted that the variable domains of the first and second heavy chains can bind to the same epitope on the same antigen, or different epitopes on the same antigen. For example, the DVD-Ig described herein is a multispecific, e.g., bispecific, DVD-Ig, in which the variable domains of the first and second heavy chains can bind to different epitopes, and these different epitopes can be on the same antigen or different antigens. Techniques for generating multispecific Ig molecules include, but are not limited to, recombinant coexpression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein, C. and Cuello, A.C., Nature 305 (1983) 537-540; WO 93 / 08829; and Traunecker, A. et al., EMBO J. 10 (1991) 3655-3659) and "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can be produced by engineering electrostatic steering effects to create antibody Fc heterodimeric molecules (WO 2009 / 089004), cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980 and Brennan, M. et al., Science 229 (1985) 81-83), using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny, SA et al., J. Immunol. 148 (1992) 1547-1553), using "diabody" technology to create bispecific antibody fragments (see, e.g., HoUiger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448), and using single-chain Fv (scFv) dimers (see, e.g., Gruber, M. et al., J. Immunol. 148 (1992) 1547-1553). al, J. Immunol. 152 (1994) 5368-5374), and for example, Tutt, A. et al, J. Immunol. 147 (1991) 60-69, the contents of both of which are incorporated herein by reference in their entirety.Brinkmann et al., in "The making of bispecific antibodies" (MABS; 2017, VOL. 9, NO. 2, 182-212), the contents of which are incorporated herein by reference in their entirety, highlight the various formats and strategies that can be used to generate recombinant bispecific antibodies.

[0217] In some embodiments, the first heavy chain or the second heavy chain contains a modification in the CH3 domain that reduces the ability of the CH3 domain to interact with itself, i.e., to form a homodimer. Specifically, one or more residues that make up the CH3-CH3 interface are replaced with charged amino acids to make the interaction electrostatically unfavorable. For example, a positively charged amino acid in the interface, such as lysine, arginine, or histidine, is replaced with a negatively charged amino acid, such as aspartic acid or glutamic acid. In another embodiment, a negatively charged amino acid in the interface is replaced with a positively charged amino acid. In some embodiments, an amino acid is replaced with a non-natural amino acid with desired charge characteristics. In some embodiments, the first heavy chain and the second heavy chain contain a modification in the CH3 domain that reduces the ability of each CH3 domain to interact with itself, but increases the ability of each CH3 domain to interact with each other, i.e., to form a heterodimer. This can be achieved by replacing one or more residues that make up the CH3-CH3 interface in both CH3 domains with charged amino acids, making homodimer formation electrostatically unfavorable but heterodimerization electrostatically favorable. In certain embodiments, charged amino acids in each CH3 domain are replaced with amino acids of the opposite charge. For example, a positively charged amino acid can be replaced with a negatively charged amino acid in the first CH3 domain, and a negatively charged amino acid can be replaced with a positively charged amino acid in the second CH3 domain. Reversing the charges of amino acids reduces homodimer formation. When the replacements are properly coordinated, the reversed charges, i.e., the opposite charges in the interface, electrostatically favor heterodimer formation. Some exemplary mutations for enhancing heterodimer formation are listed in Table 1.

[0218] Table 1. A list of some possible pairwise charge residue mutations to enhance heterodimer formation. a TIFF2026041866000024.tif59160 a Combinations of the above pairwise charged residue mutations could also be used, for example, the Lys409---Asp399' interacting pair mutation could be combined with the Lys439---Asp356' pair mutation. b Histidine (His) can be added to this list of positively charged residues, but its increased side chain volume and pH dependence should be considered in the design. c To enhance heterodimer formation, these single residue mutations can be combined with other pairwise mutations listed in the table.

[0219] In some embodiments, the first CH3 domain comprises the amino acid modifications L351Y, F405A, and Y407V, and the second CH3 domain comprises the amino acid modifications T366L, K392M, and T394W. In some embodiments, the first CH3 domain comprises the amino acid modifications L351Y, F405A, and Y407V, and the second CH3 domain comprises the amino acid modifications T366L, K392L, and T394W. In some embodiments, the first CH3 domain comprises the amino acid modifications T350V, L351Y, F405A, and Y407V, and the second CH3 domain comprises the amino acid modifications T350V, T366L, K392M, and T394W. In some embodiments, the first CH3 domain comprises the amino acid modifications T350V, L351Y, F405A, and Y407V, and the second CH3 domain comprises the amino acid modifications T350V, T366L, K392L, and T394W. In some embodiments, the first CH3 domain comprises the amino acid modifications T366L, N390R, K392R, and T394W, and the second CH3 domain comprises the amino acid modifications L351Y, S400E, F405A, and Y407V. In some embodiments, the first CH3 domain comprises the amino acid modifications T350V, T366L, N390R, K392R, and T394W, and the second CH3 domain comprises the amino acid modifications T350V, L351Y, S400E, F405A, and Y407V.

[0220] In some embodiments, one of the CH3 domains comprises one or more of the following modifications, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more: K392D, K392E, N392D, N392E, R409D, R409E, K409D, K409E, D399K, D399R, E356R, E356K, D356R, D356K, Y349T, L351T, L368T, L398T, F405T, Y407T, Y407R, L234A, and L235A.

[0221] In some embodiments, the first CH3 domain comprises the amino acid modifications S345C and T366W, and the second CH3 domain comprises the amino acid modifications Y349C, T366S, L368A, and Y407V.

[0222] In certain aspects, the immunoglobulin molecule comprises a first variable region and a second variable region as described above, and a C on the light chain. L domain and heavy chain constant domain C H 1. C H 2 and C H The constant domains are intact immunoglobulin molecules, including those derived from the Fab', Fab', Fab, and Fv fragments. The constant domains can comprise native or non-native sequences or amino acid sequence variants thereof. In certain aspects, the immunoglobulin molecule can be an immunoglobulin fragment. Examples of immunoglobulin fragments include, but are not limited to, (Fab')2, Fab', Fab, and Fv fragments.

[0223] Ligand A wide variety of entities can be coupled to the DVD immunoglobulins described herein. A preferred moiety is a ligand, which is preferably covalently coupled to the DVD immunoglobulins described herein, either directly or indirectly via a linker. Ligands can include natural substances, such as peptides, polypeptides, carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid), vitamins, or lipids. Ligands can be recombinant molecules or synthetic molecules, such as synthetic polymers.

[0224] Generally, a ligand alters the distribution, targeting, or lifespan of the DVD immunoglobulin into which it is incorporated. Some ligands can have endosomolytic properties. Such ligands are also referred to herein as endosomolytic ligands. Endosomolytic ligands promote endosomal lysis and / or transport of the compositions of the present invention or their components from the endosome into the cytoplasm of the cell. Endosomolytic ligands include, but are not limited to, imidazoles, polyimidazoles or oligoimidazoles, PEI, peptides, fusogenic peptides, polycarboxylates, polycations, shielded oligo- or polycations or oligo- or polyanions, acetals, polyacetals, ketals / polyketals, orthoesters, polymers with shielded or unshielded cationic or anionic charges, and dendrimers with shielded or unshielded cationic or anionic charges.

[0225] The endosomolytic ligand can be a polyanionic peptide or peptidomimetic that exhibits pH-dependent membrane activity and membrane fusogenicity. In some embodiments, the endosomolytic ligand adopts its active conformation at endosomal pH. The "active" conformation refers to the conformation that the endosomolytic ligand adopts when promoting endosome lysis and / or transport of the composition of the present invention or its components from endosomes to the cytoplasm of cells. Exemplary endosomolytic ligands include GALA peptide (Subbarao et al., Biochemistry, 1987, 26:2964-2972, which is incorporated herein by reference in its entirety), EALA peptide (Vogel et al., J. Am. Chem. Soc., 1996, 118:1581-1586, which is incorporated herein by reference in its entirety), and their derivatives (Turk et al., Biochem. Biophys. Acta, 2002, 1559:56-68, which is incorporated herein by reference in its entirety). In some embodiments, the endosomolytic ligand can contain a chemical group (e.g., an amino acid) that undergoes a change in charge or protonation in response to a change in pH. The endosomolytic ligand can be linear or branched.

[0226] In some embodiments, the endosomolytic ligand has the amino acid sequence The peptide contains TIFF2026041866000025.tif4128.

[0227] It should be noted that a ligand, e.g., an endosomolytic ligand, can be attached to the heavy chain, light chain, or double-stranded RNA molecule of a DVD-Ig described herein. For example, the ligand, e.g., an endosomolytic ligand, can be attached to the N-terminus or C-terminus of the light chain or heavy chain. In some embodiments, the ligand, e.g., an endosomolytic ligand, is attached to the C-terminus of the light chain.

[0228] Exemplary DVD Immunoglobulins In some embodiments, the DVD immunoglobulin comprises a first variable domain that binds to CD138. For example, the DVD immunoglobulin comprises a first variable domain that binds to CD138 and a humanized 38C2 antibody variable domain as the second variable domain. The variable domains can be, for example, on each light chain and on each heavy chain: TIFF2026041866000026.tif18160. For example, DVD immunoglobulins are (i) Amino acid sequence: a light chain comprising an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to or substantially similar to TIFF2026041866000027.tif53134; and / or (ii) Amino acid sequence: a heavy chain comprising an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to, or substantially similar to, TIFF2026041866000028.tif88134; Includes:

[0229] Further exemplary DVD immunoglobulins comprising a first variable domain that binds to CD138 are described in WO2017 / 049139, the contents of which are incorporated herein by reference in their entirety.

[0230] In some embodiments, the DVD immunoglobulin comprises a first variable domain that binds to BCMA. For example, the DVD immunoglobulin comprises a first variable domain that binds to BCMA and a humanized 38C2 antibody variable domain as the second variable domain. The variable domains are on each light chain and each heavy chain, e.g., TIFF2026041866000029.tif18160. For example, DVD immunoglobulins are (i) Amino acid sequence: a light chain comprising an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to or substantially similar to TIFF2026041866000030.tif53134; and / or (ii) Amino acid sequence: a heavy chain comprising an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to, or substantially similar to, TIFF2026041866000031.tif89134; Includes:

[0231] In some embodiments, the DVD immunoglobulin comprises a first variable domain that binds to SLAMF7. For example, the DVD immunoglobulin comprises a first variable domain that binds to SLAMF7 and a humanized 38C2 antibody variable domain as the second variable domain. The variable domains can be, for example, on each light chain and on each heavy chain: TIFF2026041866000032.tif18160. For example, DVD immunoglobulins are (i) Amino acid sequence: a light chain comprising an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to or substantially similar to TIFF2026041866000033.tif53134; and / or (ii) Amino acid sequence: a heavy chain comprising an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to, or substantially similar to, TIFF2026041866000034.tif88134; Includes:

[0232] In some embodiments, the DVD immunoglobulin comprises a first variable domain that binds to HER2. For example, the DVD immunoglobulin comprises a first variable domain that binds to HER2 and a humanized 38C2 antibody variable domain as the second variable domain. The variable domains are on each light chain and each heavy chain, e.g., TIFF2026041866000035.tif18160. For example, DVD immunoglobulins are (i) Amino acid sequence: a light chain comprising an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to or substantially similar to TIFF2026041866000036.tif53133; and / or (ii) Amino acid sequence: a heavy chain comprising an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to, or substantially similar to, TIFF2026041866000037.tif88134; Includes:

[0233] Further exemplary DVD immunoglobulins comprising a first variable domain that binds to HER2, FOLR1 or CD79b are described in WO2017 / 049139, the contents of which are incorporated herein by reference in their entirety.

[0234] In some embodiments, the DVD immunoglobulin comprises a first variable domain that binds to SLAMF7, and the DVD immunoglobulin further comprises an endosomolytic ligand. For example, the DVD immunoglobulin comprises a first variable domain that binds to SLAMF7 and a humanized 38C2 antibody variable domain as the second variable domain. The variable domains can be, for example, on each light chain and on each heavy chain: TIFF2026041866000038.tif11135. For example, DVD immunoglobulins are (i) Amino acid sequence: a light chain comprising an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to or substantially similar to TIFF2026041866000039.tif53134; and / or (ii) Amino acid sequence: a heavy chain comprising an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to TIFF2026041866000040.tif88134, or an amino acid sequence substantially similar to said amino acid sequence. Includes:

[0235] In some embodiments, the DVD immunoglobulin is (i) Amino acid sequence: a first heavy chain comprising an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to or substantially similar to TIFF2026041866000041.tif67140; and / or (ii) Amino acid sequence: a second heavy chain comprising an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to or substantially similar to TIFF2026041866000042.tif102134; and / or (iii) amino acid sequence: a light chain comprising an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to, or substantially similar to, TIFF2026041866000043.tif32134; Includes:

[0236] DVD immunoglobulin production The DVD immunoglobulins of the present invention can be produced by any of several techniques known in the art. For example, for expression from a host cell, an expression vector encoding the DVD heavy chain and / or DVD light chain is transfected into the host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of foreign DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, and DEAE-dextran transfection. While the DVD immunoglobulins of the present invention can be expressed from either prokaryotic or eukaryotic host cells, expression of DVD immunoglobulins in eukaryotic cells is preferred, and expression in mammalian host cells is most preferred, because such eukaryotic cells, particularly mammalian cells, are more likely than prokaryotic cells to assemble and secrete properly folded, immunologically active DVD immunoglobulins.

[0237] Preferred mammalian host cells for expressing the recombinant immunoglobulins of the present invention include Chinese hamster ovary (CHO) cells (including, e.g., dhfr-CHO cells described by Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with the DHFR selectable marker as described by R.J. Kaufman and P.A. Sharp (1982) Mol. Biol. 159:601-621), human embryonic kidney (HEK) cells, NS0 myeloma cells, COS cells, and SP2 cells. When a recombinant expression vector encoding a DVD immunoglobulin is introduced into a mammalian host cell, the DVD immunoglobulin is produced by culturing the host cell for a period of time sufficient to permit expression of the DVD immunoglobulin in the host cell, and more preferably, secretion of the DVD immunoglobulin into the culture medium in which the host cell is grown. The DVD immunoglobulin can be recovered from the culture medium using standard protein purification methods.

[0238] In a preferred system for recombinant expression of the DVD immunoglobulins of the present invention, a recombinant expression vector encoding both the DVD heavy chain and the DVD light chain is introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the DVD heavy chain gene and the DVD light chain gene are each operably linked to a CMV enhancer / AdMLP promoter regulatory element to drive high-level transcription of the genes. The recombinant expression vector also carries a DHFR gene, which allows CHO cells transfected with the vector to be selected using methotrexate selection / amplification. Selected transformant host cells are cultured to allow expression of the DVD heavy chain and the DVD light chain, and intact DVD immunoglobulin is recovered from the culture medium. Standard molecular biology and tissue culture techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells, and recover the DVD immunoglobulin from the culture medium. Additionally, aspects of the present invention include methods for synthesizing the DVD immunoglobulins of the present invention by culturing host cells of the present invention in an appropriate culture medium until the DVD immunoglobulins of the present invention are synthesized. The method can further comprise isolating the DVD immunoglobulin from the culture medium to obtain an isolated immunoglobulin.

[0239] A feature of the DVD immunoglobulins is that they can be produced and purified in the same manner as conventional antibodies. The production of DVD immunoglobulins can result in a homogeneous single major product with the desired activity without any sequence alteration of the constant regions or any type of chemical modification.

[0240] Linker The conjugates disclosed herein may include a linker, which may comprise one or more linker components. The term "linker" refers to an organic moiety that connects two parts of a compound, such as connecting a DVD immunoglobulin to a dsRNA. A linker is typically a direct bond or an atom such as oxygen or sulfur, NR 1, C(O), C(O)O, C(O)NR 1 , SO, SO2, SO2NH, or a chain of atoms, such as substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted alkynyl, aryl alkyl, aryl alkenyl, aryl alkynyl, heteroaryl alkyl, heteroaryl alkenyl, heteroaryl alkynyl, heterocyclyl alkyl, heterocyclyl alkenyl, heterocyclyl alkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylaryl alkyl, alkylaryl alkenyl, alkylaryl alkynyl, alkenylaryl alkyl, alkenylaryl alkenyl, alkenylaryl alkynyl, alkynylaryl alkyl, alkynylaryl alkenyl, alkynylaryl alkynyl, alkylheteroaryl alkyl, alkylheteroaryl a and alkylaryl, alkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylheterocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylheteroaryl, wherein one or more methylenes are selected from the group consisting of O, S, S(O), SO, N(R 1 )2, C(O), a cleavable linking group, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted heterocyclic group, and R 1 is hydrogen, acyl, an aliphatic group, or a substituted aliphatic group.

[0241] The conjugates may include various types of linker functionalities, including, but not limited to, cleavable and non-cleavable linkers, and reversible and non-reversible linkers.

[0242] In some embodiments, the linker is a cleavable linker.Cleavable linker is a linker that the two parts that the linker connects together, for example, DVD-Ig and dsRNA, are released by a process in target cells, such as reduction in the cytoplasm, exposure to acidic conditions in lysosomes or endosomes, or cleavage by specific enzymes (for example, proteases) in cells.Therefore, cleavable linker allows the two dsRNAs to be released in their original form after the conjugate is internalized and processed in target cells.Cleavable linkers include, but are not limited to, those that have a bond that can be cleaved by enzymes (for example, peptide linkers), reduction conditions (for example, disulfide linkers), or acidic conditions (for example, hydrazones and carbonates).

[0243] Generally, a cleavable linker comprises at least one cleavable linker. A cleavable linker is a linker that is sufficiently stable outside a cell but that, upon entry into a target cell, is cleaved to release the two moieties that the linker joins together. In a preferred embodiment, the cleavable linker is cleaved at least 10 times faster, preferably at least 100 times faster, inside the target cell or under first reference conditions (which can be selected, for example, to mimic or represent intracellular conditions) than in the subject's blood or serum or under second reference conditions (which can be selected, for example, to mimic or represent conditions found in blood or serum).

[0244] Cleavable linkers are sensitive to cleavage agents, such as pH, redox potential, or the presence of degradative molecules. Generally, cleavage agents are found to be more prevalent or at higher levels or activity inside cells than in serum or blood. Examples of such degradative agents include redox agents that are selective for a specific substrate or that do not have substrate specificity, such as oxidases or reductases, or reducing agents such as mercaptans present in cells that can degrade redox-cleavable linkers by reduction; esterases; agents that can create endosomes or acidic environments, such as those that result in a pH of 5 or less; enzymes that can hydrolyze or degrade acid-cleavable linkers by acting as general acids, peptidases (which can be substrate-specific), and phosphatases.

[0245] Cleavable linkers, such as disulfide bonds, can be pH-sensitive. While the pH of human serum is 7.4, the average intracellular pH is slightly lower, ranging from about 7.1 to 7.3. Endosomes are more acidic, ranging from 5.5 to 6.0, and lysosomes have an even more acidic pH, around 5.0. Some linkers will have cleavable linkers that are cleaved at a suitable pH, thereby releasing the cationic lipid from the ligand intracellularly or to a desired compartment of the cell.

[0246] The linker can include a cleavable linker that can be cleaved by a specific enzyme. The type of cleavable linker incorporated into the linker can depend on the target cell. For example, a liver-targeting ligand can be linked to a cationic lipid via a linker containing an ester group. Because liver cells are rich in esterases, the linker will be cleaved more efficiently in liver cells than in cell types that are not rich in esterases. Other cell types that are rich in esterases include lung, renal cortex, and testicular cells. Linkers containing peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synovial cells.

[0247] In general, the suitability of a candidate cleavable tether can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate tether. It may also be desirable to test the candidate cleavable tether for its ability to resist cleavage in blood or when contacted with other non-target tissues. Thus, the relative susceptibility to cleavage can be determined between first conditions selected to indicate cleavage in target cells and second conditions selected to indicate cleavage in other tissues or biological fluids, such as blood or serum. Evaluation can be performed in a cell-free system, in cells, in cell culture, in organs or tissues, or in whole animals. It may be useful to perform initial evaluations under cell-free or cell culture conditions and confirm them with further evaluations in whole animals. In preferred embodiments, useful candidate compounds are cleaved at least 2-fold, 4-fold, 10-fold, or 100-fold faster in cells (or under in vitro conditions selected to mimic intracellular conditions) than in blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0248] One class of cleavable linkers is redox-cleavable linkers, which may be used in the dsRNA molecules of the present invention that are cleaved upon reduction or oxidation. One example of a reductively cleavable linker is a disulfide linker (-SS-). To determine whether a candidate cleavable linker is a suitable "reductively cleavable linker," or whether it is suitable for use with, for example, a particular iRNA moiety and a particular targeting agent, one can rely on the methods described herein. For example, candidates can be evaluated by incubating with dithiothreitol (DTT) or other reducing agents using reagents known in the art that mimic the cleavage rate that would be observed in cells, e.g., target cells. Candidates can also be evaluated under conditions selected to mimic blood or serum conditions. In a preferred embodiment, the candidate compound is cleaved at most 10% in blood. In a preferred embodiment, a useful candidate compound is degraded at least 2-fold, 4-fold, 10-fold, or 100-fold faster in cells (or under in vitro conditions selected to mimic intracellular conditions) than in blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of a candidate compound can be determined using standard enzyme kinetic assays under conditions selected to mimic the intracellular medium and compared to conditions selected to mimic the extracellular medium.

[0249] The phosphate-based cleavable linkers that can be used in the dsRNA molecules of the invention are cleaved by agents that degrade or hydrolyze the phosphate group. An example of an agent that cleaves phosphate groups within a cell is an enzyme such as a phosphatase within the cell. Examples of phosphate-based linking groups are -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, -OP(S)(Rk)-S-. Preferred embodiments are -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O-, -SP(S)(H)-O-, -SP(O)(H)-S-, and -OP(S)(H)-S-. One preferred embodiment is -OP(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

[0250] The acid-cleavable linker that can be used in the dsRNA molecules of the present invention is a linker that is cleaved under acidic conditions. In a preferred embodiment, the acid-cleavable linker is cleaved in an acidic environment with a pH of about 6.5 or less (e.g., about 6.0, 5.5, 5.0 or less), or by an agent such as an enzyme that can act as a general acid. In cells, specific low-pH organelles such as endosomes and lysosomes can provide a cleavage environment for the acid-cleavable linker. Examples of acid-cleavable linkers include, but are not limited to, hydrazones, esters, and esters of amino acids. Acid-cleavable groups can have the general formula -C=NN-, C(O)O, or -OC(O). In a preferred embodiment, the carbon attached to the oxygen of the ester (alkoxy group) is an aryl group, a substituted alkyl group, or a tertiary alkyl group, such as dimethylpentyl or t-butyl. These candidates can be evaluated using methods similar to those described above.

[0251] The ester-based cleavable linker that can be used in the dsRNA molecules of the present invention is cleaved in cells by enzymes such as esterases and amidases. Examples of ester-based cleavable linkers include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. Ester cleavable linkers have the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0252] Peptide-based cleavable linkers that can be used in the dsRNA molecules of the present invention are cleaved intracellularly by enzymes such as peptidases and proteases. Peptide-based cleavable linkers are peptide bonds formed between amino acids to give oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable groups do not include amide groups (-C(O)NH-). Amide groups can be formed between any alkylene, alkenylene, or alkynylene. A peptide bond is a special type of amide bond that forms between amino acids to give peptides and proteins. Peptide-based cleavable groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to give peptides and proteins, and do not include the entire amide functionality. Peptide-based cleavable linkers have the general formula -NHCHR A C(O)NHCHR B C(O)—, where R A and R B are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

[0253] Non-limiting examples of cleavable linkers are described in Figure 7 of WO2017 / 049139, the contents of which are incorporated herein by reference in their entirety.

[0254] Non-cleavable linkers utilize catabolic degradation of the immunoconjugate to release the drug moiety. The released drug moiety generally retains the linker and the amino acid residue of the immunoglobulin to which the linker was conjugated. Non-cleavable linkers include, but are not limited to, PEG linkers, hydrocarbon linkers, and thioether linkers. Non-limiting examples of non-cleavable linkers are shown in Figure 8 of WO2017 / 049139, the contents of which are incorporated herein by reference in their entirety.

[0255] Aspects of the conjugate can also include reversible and irreversible linkers. Reversible linkers utilize chemical bonds that can be easily broken or reversed using an appropriate reagent. Thus, after the formation of a reversible linker, the linker can be broken at a desired location by treating it with a reagent, thereby releasing the immunoglobulin molecule from the linker. Non-limiting examples of reversible linkers are shown in Figure 9 of WO2017 / 049139, the contents of which are incorporated herein by reference in their entirety. Irreversible linkers cannot be easily broken or reversed after their formation. Thus, after the formation of an irreversible linker, the immunoglobulin molecule cannot be easily released. Non-limiting examples of irreversible linkers are shown in Figure 10 of WO2017 / 049139, the contents of which are incorporated herein by reference in their entirety. Examples of linker reactions in which immunoglobulins are conjugated to reversible or irreversible linkers are described in Figure 13 of WO2017 / 049139, the contents of which are incorporated herein by reference in their entirety. In some embodiments, in addition to β-lactam and diketone moieties, other moieties, such as vinyl diketones and pro-vinyl diketones, can also be used for conjugation. In some embodiments, electrophilic moieties (handles) can be used alone or in combination with such moieties. Electrophilic moieties can be used for site-specific conjugation to only one uniquely reactive lysine in the h38C2 variable domain, and can also be used for non-specific conjugation after the h38C2 lysine has been conjugated to a drug moiety.Non-limiting examples of other moieties include 6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP"), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl ("PAB"), and those resulting from conjugation with the following linker reagents: N-succinimidyl 4-(2-pyridylthio)pentanoate forming linker moiety 4-mercaptopentanoic acid ("SPP"), N-succinimidyl 4-(N-maleimidomethyl)cyclohexyl Examples include the 2,5-dioxopyrrolidin-1-yl methyl cyclohexane carboxylic acid ("SMCC," also referred to herein as "MCC"), the 2,5-dioxopyrrolidin-1-yl 4-(pyridin-2-yldisulfanyl)butanoate-forming linker moiety 4-mercaptobutanoic acid ("SPDB"), N-succinimidyl (4-iodoacetyl)aminobenzoate ("SIAB"), and ethyleneoxy-CHCH- ("EO" or "PEO") as one or more repeat units. Further information is provided in Sinha et al., Nat. Protoc. 2, 449-456 (2007), the disclosure of which is incorporated herein by reference in its entirety.

[0256] In some embodiments, the linker component can include an amino acid unit. In one such aspect, the amino acid unit allows the linker to be cleaved by a protease, thereby facilitating the release of the drug from the immunoconjugate when exposed to an intracellular protease, such as a lysosomal enzyme. See, for example, Doronina et al. (2003) Nat. Biotechnol. 21:778-784. Non-limiting examples of amino acid units include, but are not limited to, dipeptides, tripeptides, tetrapeptides, and pentapeptides. Non-limiting examples of dipeptides include valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe), phenylalanine-lysine (fk or phe-lys), or N-methyl-valine-citrulline (Me-val-cit). Non-limiting examples of tripeptides include glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acid units can include naturally occurring amino acid residues as well as rare amino acids and unnatural amino acid analogs, such as citrulline. Amino acid units can be designed and optimized for their selectivity for enzymatic cleavage by specific enzymes, such as tumor-associated proteases, cathepsins B, C, and D, or plasmin proteases.

[0257] In some embodiments, the linker L can be a branched or dendritic linker for covalently attaching two or more dsRNAs to an immunoglobulin via a branched multifunctional linker moiety (Sun et al. (2002) Bioorganic & Medicinal Chemistry Letters 12:2213-2215, Sun et al. (2003) Bioorganic & Medicinal Chemistry 11:1761-1768). Non-limiting examples of branched, dendritic linkers include 2,6-bis(hydroxymethyl)-p-cresol and 2,4,6-tris(hydroxymethyl)-phenol dendrimer units (WO 2004 / 01993, Szalai et al. (2003) J. Amer. Chem. Soc. 125:15688-15689, Shamis et al. (2004) J. Amer. Chem. Soc. 126:1726-1731, Amir et al. (2003) Angew. Chem. Int. Ed. 42:4494-4499). Branched linkers can increase the RNA-to-immunoglobulin molar ratio, i.e., loading capacity, which is related to the efficacy of ADCs. Thus, for example, if an immunoglobulin has only one reactive amino acid residue for conjugation, multiple dsRNAs can be attached via a branched linker. Without limitation, the branch point of a branched linker can be at least a trivalent atom, and can also be a tetravalent, pentavalent, or hexavalent atom or group exhibiting such multiple valencies. In certain embodiments, the branch point can be -N, -N(Q)-C, -OC, -SC, -SS-C, -C(O)N(Q)-C, -OC(O)N(Q)-C, -N(Q)C(O)-C, or -N(Q)C(O)OC, where Q is, independently at each occurrence, H or an optionally substituted alkyl. In another embodiment, the branch point can be glycerol or a glycerol derivative.

[0258] Linker components, including stretcher, spacer, and amino acid units, can be synthesized by methods known in the art, such as those described in U.S. Patent Application Publication No. 2005 / 0238649 A1, which is incorporated herein by reference in its entirety.

[0259] In some embodiments, the structure of the linker is: TIFF2026041866000044.tif17128, where X is a spacer.

[0260] The spacer X is typically a direct bond or an atom such as oxygen or sulfur, NR 1 , C(O), C(O)O, C(O)NR 1, SO, SO2, SO2NH, or other units or chains of atoms, such as substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, aryl alkyl, aryl alkenyl, aryl alkynyl, heteroaryl alkyl, heteroaryl alkenyl, heteroaryl alkynyl, heterocyclyl alkyl, heterocyclyl alkenyl, heterocyclyl alkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylaryl alkyl, alkylaryl alkenyl, alkylaryl alkynyl, alkenylaryl alkyl, alkenylaryl alkenyl, alkenylaryl alkynyl, alkynylaryl alkyl, alkynylaryl alkenyl, alkynylaryl alkynyl, alkylheteroaryl alkyl, alkylheteroaryl alkenyl, alkynylaryl alkynyl, alkyl alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylheterocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylheteroaryl, wherein one or more methylenes are selected from O, S, S(O), SO, N(R 1 )2, C(O), a cleavable linking group, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted heterocyclic group, and R 1 is hydrogen, acyl, an aliphatic group, or a substituted aliphatic group.

[0261] In some embodiments, the spacer X is —O(CH2CH2O) pCH2CH2O-, where p can be 0 or an integer from 1 to 1000. For example, p can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some preferred embodiments, p is 2.

[0262] In some embodiments, the spacer X comprises at least one cleavable linker. For example, the spacer X comprises a disulfide, e.g., -SS- linkage. In some embodiments, the spacer X is -O(alkyl)-SS-(alkyl)O-, where each alkyl is optionally interrupted by one, e.g., two, three, or more groups independently selected from O, S, S(O), SO, NH, C(O), or C(O)O. For example, the spacer X is -O(CH) q O(CH2) r SS(CH2O) s (CH2) t O-, where q, r, s, and t are integers independently selected from 1 to 15. For example, each q, r, s, and t can independently be 1, 2, 3, 4, 5, 6, 7, or 8. Note that q, r, s, and t can all be the same, or all the same, or some can be the same and some can be different. For example, q and t can be the same and selected from 2, 3, 4, 5, and 6. Similarly, r and s can be the same and selected from 1, 2, 3, and 4. In some embodiments, q and t are the same, and r and s are the same but different from q and t. In some preferred embodiments, q and t are 4, and r and s are 2.

[0263] Synthesis of the conjugate The conjugates described herein can be prepared using any method known in the art for conjugating two molecules together. For example, conjugates can be prepared using the methods disclosed in the Examples section of this specification. In some embodiments, the second variable domain of a DVD-Ig contains a reactive lysine residue, and the conjugate is created using a controlled conjugation reaction in which a linker / dsRNA molecule composition is conjugated to the reactive lysine residues on each heavy chain of a naked Ig. Conditions for this reaction are described, for example, in U.S. Pat. No. 8,252,902, which is incorporated herein by reference in its entirety. Briefly, this reaction can be carried out by reacting the Ig with the linker / dsRNA moiety composition in a solution of PBS, pH 7.4, and 2% DMSO at room temperature, resulting in the attachment of one linker / dsRNA moiety to each of the reactive lysine residues on the Ig. The result is a conjugate having two dsRNA moieties attached via linkers to the reactive lysine residues on each heavy chain of the Ig.

[0264] In certain aspects, additional dsRNA molecules can be conjugated to DVD-Ig molecules using uncontrolled conjugation techniques. For example, in certain aspects, amino acid residues other than the only uniquely reactive lysine residue in the 38C2 variable domain can be used as attachment points for conjugating dsRNA molecules via linkers. The result of such uncontrolled conjugation is a conjugate with one or more dsRNA molecules attached to the other amino acid residues on the immunoglobulin molecule. Such additional conjugation can be achieved by reacting a linker / dsRNA molecule composition with, for example, a lysine residue on the immunoglobulin molecule that is not the only uniquely reactive lysine residue in the second variable domain, or a standard or engineered cysteine ​​residue on the immunoglobulin molecule, or one or more engineered selenocysteine ​​residues on the immunoglobulin molecule, or a uniquely reactive arginine residue in the second variable domain. The result of such uncontrolled conjugation is a conjugate in which the average number of dsRNA molecules is from about 1 to about 20 dsRNA molecules per antibody, depending on the number of amino acid residues available for reaction with the linker / dsRNA molecule composition. In certain aspects, the average number of dsRNA molecules per immunoglobulin molecule achieved using an uncontrolled conjugation approach is from about 1 to about 8, e.g., 2, 3, 4, 5, 6, or 7 dsRNA molecules per immunoglobulin.

[0265] Generally, immunoglobulins are composed of two identical light chains and two identical heavy chains. In one aspect, the immunoglobulin light chains include kappa light chains. In one aspect, the immunoglobulin light chains include lambda light chains. In one aspect, the immunoglobulin is an IgA immunoglobulin having a heavy chain. In one aspect, the immunoglobulin is an IgA1 immunoglobulin. In one aspect, the immunoglobulin is an IgA2 immunoglobulin. In one aspect, the immunoglobulin is an IgD immunoglobulin having a delta heavy chain. In one aspect, the immunoglobulin is an IgE immunoglobulin having an epsilon heavy chain. In one aspect, the immunoglobulin is an IgG immunoglobulin having a gamma heavy chain. In one aspect, the immunoglobulin is an IgG1 immunoglobulin. In one aspect, the immunoglobulin is an IgG2 immunoglobulin. In one aspect, the immunoglobulin is an IgG3 immunoglobulin. In one aspect, the immunoglobulin is an IgG4 immunoglobulin. In one aspect, the immunoglobulin is an IgM immunoglobulin having a μ heavy chain.

[0266] In one aspect, the immunoglobulin is an intact immunoglobulin. In one aspect, the immunoglobulin is a naked immunoglobulin. In one aspect, the immunoglobulin is an immunoglobulin fragment. In one aspect, the immunoglobulin fragment is selected from the group consisting of Fab, Fab', F(ab')2, Fv, and scFv.

[0267] In some aspects, the immunoglobulin is a dual variable domain immunoglobulin. In some aspects, the immunoglobulin comprises a native polypeptide sequence. In some aspects, the immunoglobulin comprises a non-native polypeptide sequence. In some aspects, the immunoglobulin comprises a polypeptide. In some aspects, the immunoglobulin is a monoclonal immunoglobulin. In some aspects, the immunoglobulin comprises a chimeric immunoglobulin. In some aspects, the immunoglobulin comprises a humanized immunoglobulin. In some aspects, the immunoglobulin comprises a human immunoglobulin. In some aspects, the immunoglobulin is an isolated immunoglobulin. In some aspects, the immunoglobulin comprises a polypeptide sequence that is a fusion of two or more polypeptide sequences. In some aspects, the immunoglobulin is a conjugated immunoglobulin.

[0268] In some aspects, the immunoglobulin specifically binds to or is specific for a binding target. In some aspects, the immunoglobulin has a binding affinity. In some aspects, the immunoglobulin has a K d In some aspects, the immunoglobulin binds to an epitope. In some aspects, the immunoglobulin binds to a target or binding target. In some aspects, the binding target comprises a binding region to which the immunoglobulin binds. In some aspects, the immunoglobulin binds to an antigen. In some aspects, the immunoglobulin comprises an antigen-binding site or antigen-binding region.

[0269] In some aspects, the immunoglobulin is produced in a host cell. In some aspects, the immunoglobulin is produced by a cell line or cell culture. In some aspects, the immunoglobulin is produced from a nucleic acid sequence operably linked to another nucleic acid sequence.

[0270] In some aspects, an immunoglobulin amino acid sequence has a percent amino acid sequence identity to another amino acid sequence.

[0271] Use of conjugates The conjugates described herein can be used to inhibit the expression of a target gene. Accordingly, in another aspect, a method for inhibiting the expression of a target gene is provided herein. The method comprises administering to a cell a conjugate described herein in an amount sufficient to inhibit the expression of the target gene. In a preferred embodiment, the present invention further relates to the use of the conjugates described herein for inhibiting the expression of a target gene in a target cell in vitro.

[0272] Exemplary target genes include β-catenin (CTNNB1), IRF4, factor VII, Eg5, PCSK9, TPX2, apoB, SAA, TTR, RSV, PDGF beta gene, Erb-B gene, Src gene, CRK gene, GRB2 gene, RAS gene, MEKK gene, JNK gene, RAF gene, Erk1 / 2 gene, PCNA (p21) gene, MYB gene, JUN gene, FOS gene, BCL-2 gene, hepcidin, activated protein C, cyclin D gene, VEGF gene, EGFR gene, cyclin A gene, cyclin E gene, WNT-1 gene, and beta-glucanase (BG) gene. These include, but are not limited to, mutations in the ta-catenin gene, c-MET gene, PKC gene, NFKB gene, STAT3 gene, survivin gene, Her2 / Neu gene, topoisomerase I gene, topoisomerase II alpha gene, p73 gene, p21(WAF1 / CIP1) gene, mutations in the p27(KIP1) gene, mutations in the PPM1D gene, mutations in the RAS gene, mutations in the caveolin I gene, mutations in the MIBI gene, mutations in the MTAI gene, mutations in the M68 gene, mutations in tumor suppressor genes, and mutations in the p53 tumor suppressor gene.

[0273] In some aspects, the conjugates described herein can be used to treat subjects or mammals. For example, the conjugates described herein can be used to treat a wide variety of diseases by targeting and killing cells that express specific tumor antigens. The conjugates can be used broadly to treat any of a variety of cancers. It is believed that any type of tumor and any type of tumor-associated antigen can be targeted by the conjugates. Examples of cancer types include, but are not limited to, blood cancers, carcinomas, sarcomas, melanomas, and central nervous system cancers.

[0274] Non-limiting examples of hematological cancers that can be treated with the immunoconjugates include leukemia, acute myeloid leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, plasma cell leukemia, and myelodysplastic syndrome.

[0275] Non-limiting examples of cancers that can be treated with the immunoconjugates include skin cancer, head and neck, thyroid, lung, nasopharyngeal, colorectal, liver, bladder, ovarian, cervical, endometrial, prostate, gastric, esophageal, pancreatic, renal, and breast cancer.

[0276] Non-limiting examples of sarcomas that can be treated with the immunoconjugates include angiosarcoma, chondrosarcoma, Ewing's sarcoma, fibrosarcoma, gastrointestinal stromal tumor, leiomyosarcoma, liposarcoma, malignant peripheral nerve sheath tumor, osteosarcoma, pleomorphic sarcoma, rhabdomyosarcoma, Kaposi's sarcoma, and synovial sarcoma.

[0277] Non-limiting examples of central nervous system cancers that can be treated with the immunoconjugates include glioma, meningioma, and neuroma.

[0278] Non-limiting examples of other cancers that can be treated with the immunoconjugates include melanoma.

[0279] In some cases, the method of using the conjugate involves administering the conjugate described herein to a subject together with one or more additional therapies for treating a particular cancer.Thus, the conjugate can be used alone to treat a particular cancer, or can be used in combination with or as an adjunct to other drug therapies, such as conventional treatment with anti-neoplastic agents.Immunoconjugates can generally be used in combination with any anti-neoplastic agent, such as conventional and / or experimental chemotherapeutic agents, radiation treatment, etc.

[0280] For example, in some aspects, the additional therapy can include an antibody, an anti-neoplastic agent, a cytotoxic agent, an anti-angiogenic agent, or an immunosuppressant. Non-limiting examples of additional therapeutic agents include cisplatin, carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide, doxorubicin, daunorubicin, valrubicin, idarubicin, epirubicin, actinomycin, bleomycin, plicamycin, mitomycin, bevacizumab, imatinib, erlotinib, gefitinib, ibrutinib, idelalisib, lenalidomide, vincristine, vinblastine, vinorelbine, vindesine, paclitaxel, and docetaxel.

[0281] Pharmaceutical Compositions For therapeutic use, the conjugates described herein can be formulated into pharmaceutical compositions.Therefore, in another aspect, the present invention provides pharmaceutical compositions comprising the conjugates defined herein.Pharmaceutically acceptable compositions comprise a therapeutically effective amount of one or more conjugates described herein, administered alone or formulated together with one or more pharmaceutically acceptable carriers (additives), excipients and / or diluents.

[0282] Pharmaceutical compositions can be specially formulated for administration as solids or liquids, including those suitable for the following administrations: (1) oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., intended for oral mucosal, sublingual, and systemic absorption, boluses, powders, granules, and pastes for application to the tongue; (2) parenteral administration, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection as a sterile solution or suspension or sustained-release formulation; (3) topical application, e.g., as a cream, ointment, or controlled-release patch, or as a spray applied to the skin; (4) vaginal or rectal administration, e.g., as a pessary, cream, or foam; (5) sublingual administration; (6) ophthalmic administration; (7) transdermal administration; or (8) nasal administration. Delivery using subcutaneous or intravenous methods is particularly advantageous.

[0283] As used herein, the phrase "therapeutically effective amount" means an amount of a compound, material, or composition, including a conjugate described herein, that is effective to produce some desired therapeutic effect in at least a subpopulation of cells in an animal, at a reasonable benefit / risk ratio applicable to any medical treatment.

[0284] The phrase "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit / risk ratio.

[0285] As used herein, the phrase "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, magnesium talc, calcium or zinc stearate, or steric acid), or solvent encapsulating material, necessary to carry or transport the compound from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; and (7) lubricants, such as magnesium stearate. state), sodium lauryl sulfate and talc, (8) excipients, such as cocoa butter and suppository wax, (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil, (10) glycols, such as propylene glycol, (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol, (12) esters, such as ethyl oleate and ethyl laurate, (13) agar, (14) buffers , such as magnesium hydroxide and aluminum hydroxide, (15) alginic acid, (16) pyrogen-free water, (17) isotonic saline, (18) Ringer's solution, (19) ethyl alcohol, (20) pH buffer solutions, (21) polyesters, polycarbonates, and / or polyanhydrides, (22) bulking agents, such as polypeptides and amino acids, (23) serum components, such as serum albumin, HDL, and LDL, and (22) other non-toxic compatible substances used in pharmaceutical formulations.

[0286] As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Any conventional media or agent is contemplated for use in the present compositions, except insofar as it is incompatible with the active compound. A supplementary active compound can also be incorporated into the present compositions. Pharmaceutical carriers include sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art.

[0287] The formulations can be conveniently presented in unit dosage form and can be prepared by any method known in the art of pharmacy. The amount of active ingredient that can be combined with carrier materials to produce a single dosage form will vary depending on the host being treated and the specific mode of administration. The amount of active ingredient that can be combined with carrier materials to produce a single dosage form will generally be the amount of compound that produces a therapeutic effect. Generally, this amount will range from about 0.1% to about 99% of the active ingredient, preferably from about 5% to about 70%, and most preferably from about 10% to about 30%, of one hundred percent.

[0288] In certain embodiments, the formulations of the present invention comprise an excipient selected from the group consisting of cyclodextrins, celluloses, liposomes, micelle-forming agents such as bile acids, and polymeric carriers such as polyesters and polyanhydrides, and a conjugate described herein. In certain embodiments, the formulations render the conjugates described herein orally bioavailable.

[0289] The conjugate preparation can be formulated in combination with another agent, such as another therapeutic agent or an agent that stabilizes the conjugate. Additional other agents include chelating agents, such as EDTA (e.g., Mg 2+ (for removing divalent cations such as HCl, etc.), salts, RNAse inhibitors (for example, broad-specificity RNAse inhibitors such as RNAsin), etc.

[0290] Methods of preparing these formulations or compositions include the step of bringing into association a compound of the present invention with the carriers and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0291] In some cases, it is desirable to slow the absorption of a drug from subcutaneous or intramuscular injection in order to prolong the effect of the drug. This can be achieved by using a suspension of crystalline or amorphous material with poor water solubility. In this case, the absorption rate of the drug depends on its dissolution rate, which may depend on the crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered dosage form can be achieved by dissolving or suspending the drug in an oil vehicle.

[0292] The compounds of the invention, by analogy with other pharmaceuticals, may be formulated for administration in any convenient way for use in human or veterinary medicine.

[0293] The compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and / or dispersing agents. Prevention of the presence of microorganisms can be ensured by both sterilization procedures and the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, etc. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, etc., in the compositions. Additionally, prolonged absorption of injectable pharmaceutical forms can be brought about by the inclusion of agents that delay absorption, for example, aluminum monostearate and gelatin.

[0294] The composition must be sterile and fluid to the extent that delivery of the composition by syringe is possible. In addition to water, the carrier is preferably an isotonic buffered saline solution.

[0295] Route of administration The conjugates described herein or pharmaceutical compositions comprising the same can be administered by various methods known in the art. As will be understood by those skilled in the art, the route and / or mode of administration will vary depending on the target disease or condition and the desired results. To administer the conjugates described herein by certain administration routes, it may be necessary to coat the conjugate with a material to prevent the conjugate from being inactivated, or to administer the conjugate together with a material to prevent the conjugate from being inactivated. For example, the conjugate can be administered in an appropriate carrier, such as a liposome, or a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions.

[0296] Exemplary routes of administration include, but are not limited to, intravenous, subcutaneous, intratumoral, topical, rectal, anal, vaginal, nasal, pulmonary, and ocular.

[0297] The compositions of the present invention can be administered in several ways, depending on whether local or systemic treatment is desired and on the area to be treated. Administration can be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral, or parenteral. Parenteral administration includes intravenous infusion, subcutaneous, intraperitoneal, or intramuscular injection, or intrathecal or intravenous administration.

[0298] The route and site of administration can be chosen to enhance targeting. For example, to target muscle cells, intramuscular injection into the muscle of interest would be a logical choice. Lung cells can be targeted by administering the conjugate in aerosol form. Vascular endothelial cells can be targeted by coating a balloon catheter with the conjugate and mechanically introducing the conjugate.

[0299] Dosage The actual dosage level of the active ingredient in the pharmaceutical compositions of the present invention, such as the conjugates described herein, can be varied to provide an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without causing toxicity to the patient. The selected dosage level will depend on various pharmacokinetic factors, including factors well known in the medical arts, such as the activity of the particular composition of the present invention used, the route of administration, the time of administration, the excretion rate of the particular compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular compound used, and the age, sex, weight, condition, general health, and medical history of the patient being treated.

[0300] In some embodiments, the unit dose is less than 10 mg per kg of body weight, or less than 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001 mg per kg of body weight, and less than 200 nmoles (e.g., about 4.4 x 10) of dsRNA molecules per kg of body weight. 16 copies), or less than 1500, 750, 300, 150, 75, 15, 7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, or 0.00015 nmoles of dsRNA molecules per kg of body weight.

[0301] The defined amount can be an amount that is effective for treating or preventing a disease or disorder, for example, a disease or disorder associated with the target gene.The unit dose can be administered, for example, by injection (for example, intravenous, subcutaneous or intramuscular), inhalation administration, or external application.In some embodiments, the dosage can be less than 10, 5, 2, 1, or 0.1 mg / kg body weight.

[0302] In some embodiments, the unit dose is administered less than once a day, for example, less than once every 2, 4, 8, or 30 days. In another embodiment, the unit dose is not administered with any frequency (e.g., not with a regular frequency). For example, the unit dose may be administered only once.

[0303] In some embodiments, an effective dose is administered with other conventional treatment modalities.

[0304] In some embodiments, an initial dose and one or more maintenance doses are administered to a subject. The one or more maintenance doses may be the same as the initial dose or may be less than the initial dose, e.g., half the initial dose. The maintenance regimen may involve one or more treatments of the subject with a dose ranging from 0.01 μg to 15 mg / kg body weight per day, e.g., 10, 1, 0.1, 0.01, 0.001, or 0.00001 mg / kg body weight per day. The maintenance dose may be administered no more frequently than once every 2, 5, 10, or 30 days, for example. Furthermore, the treatment regimen is continued for a period of time, which will vary depending on the nature of the particular disease, its severity, and the patient's overall condition. In certain embodiments, the dosage may be delivered no more frequently than once per day, e.g., no more frequently than once every 24, 36, or 48 hours or more, e.g., no more frequently than once every 5 or 8 days. After treatment, the patient can be monitored for changes in their condition and for improvement of the symptoms of the disease state. If the patient does not respond significantly to the current dosage level, the dosage of the compound can be increased, or if improvement of the symptoms of the disease state is observed, the disease state has disappeared, or undesirable side effects are observed, the dosage can be decreased.

[0305] An effective dose can be administered in a single dose, or in two or more doses, as desired or appropriate under the particular circumstances. If repeated or frequent infusions are desired, implantation of a delivery device, such as a pump, a semi-permanent stent (e.g., intravenous, intraperitoneal, intracisternal, or intracapsular), or a reservoir may be advisable.

[0306] In some embodiments, the composition comprises a plurality of dsRNA molecular species.In another embodiment, the dsRNA molecular species has a sequence that is not overlapping or adjacent to another species with respect to natural target sequence.In another embodiment, the plurality of dsRNA molecular species are specific to different natural target genes.In another embodiment, the dsRNA molecule is allele-specific.

[0307] The conjugates described herein can be administered to mammals, particularly large mammals, such as non-human primates or humans, in several ways.

[0308] In some embodiments, the administration of the conjugate is parenteral administration, for example, intravenous (for example, as a bolus or as a diffusible infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intravenous, intracranial, subcutaneous, transmucosal, oral mucosal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary, intranasal, urethral or ocular administration.Administration can be performed by the subject or by another person, for example, a healthcare provider.Dosage can be performed in a metered dose or by a dispenser that delivers a metered dose.

[0309] Liposomes and lipid formulations The conjugates described herein can be formulated into membrane molecular assemblies, such as liposomes or micelles, for delivery. As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one or more bilayers. Liposomes include unilamellar and multilamellar vesicles, which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the siRNA composition. The lipophilic material separates the aqueous interior from the aqueous exterior, which typically does not contain the siRNA composition, but in some cases may contain the siRNA composition. Liposomes are useful for importing and delivering active ingredients to the site of action. Because the liposome membrane is structurally similar to biological membranes, when the liposome is applied to a tissue, the liposome bilayer fuses with the cell membrane bilayer. As the liposome and cell merge, the aqueous contents, including the conjugates described herein, are delivered into the cell, where dsRNA can specifically bind to target RNA and mediate RNAi. Optionally, the liposomes are also specifically targeted, for example, to direct the conjugate to a particular cell type.

[0310] Liposomes containing the conjugates described herein can be prepared by various methods. In one example, the lipid components of the liposome are dissolved in a detergent so that the lipid components form micelles. For example, the lipid components can be amphipathic cationic lipids or lipid conjugates. The detergent can have a high critical micelle concentration and can be non-ionic. Exemplary detergents include cholic acid, CHAPS, octylglucoside, deoxycholic acid, and lauroyl sarcosine. Next, the siRNA preparation is added to the micelles containing the lipid components. The cationic groups on the lipids interact with the dsRNA of the conjugate and condense around the conjugate to form liposomes. After condensation, the detergent is removed by dialysis or the like to obtain a liposome preparation of siRNA.

[0311] If necessary, a carrier compound that aids in condensation can be added during the condensation reaction, such as by controlled addition. For example, the carrier compound can be a polymer other than a nucleic acid (e.g., spermine or spermidine). The pH can also be adjusted to favor condensation.

[0312] Further description of methods for producing stable polynucleotide delivery vehicles incorporating polynucleotide / cationic lipid complexes as components of the delivery vehicle is provided, for example, in WO 96 / 37194. Liposome formation has been described by Felgner, PLet al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987, U.S. Patent No. 4,897,355, U.S. Patent No. 5,171,678, Bangham, et al. al.Biochim.Biophys.Acta 557:9,1979, Szoka,et al.Proc.Natl.Acad.Sci.75:4194,1978, Mayhew,et al.Biochim.Biophys.Acta 775:169,1984, Kim,et al.Biochim.Biophys.Acta 728:339,1983 and Fukunaga,et The method may also include one or more aspects of the exemplary method described in (e.g., Mayer, et al., Endocrinol. 115:757, 1984, which is incorporated herein by reference in its entirety.) Techniques commonly used to prepare lipid aggregates of a size suitable for use as a delivery vehicle include sonication and freeze-thaw extrusion (see, e.g., Mayer, et al., Biochim. Biophys. Acta 858:161, 1986, which is incorporated herein by reference in its entirety). If consistently small (50-200 nm) and relatively uniform aggregates are desired, microfluidic technology can be used (Mayhew, et al., Biochim. Biophys. Acta 775:169, 1984, which is incorporated herein by reference in its entirety). These methods can be readily adapted to packaging siRNA into liposomes.

[0313] pH-sensitive or negatively charged liposomes encapsulate nucleic acids rather than forming complexes with them. Because both nucleic acid molecules and lipids are similarly charged, repulsion occurs rather than complex formation. Nevertheless, some nucleic acid molecules are encapsulated within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cultured cell monolayers. Expression of exogenous genes was detected in target cells (Zhou et al., Journal of Controlled Release, 19, (1992) 269-274, which is incorporated herein by reference in its entirety).

[0314] One major type of liposome composition contains phospholipids other than naturally occurring phosphatidylcholine. Neutral liposome compositions can be formed, for example, from dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC). Anionic liposome compositions are generally formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are primarily formed from dioleoylphosphatidylethanolamine (DOPE). Another type of liposome composition is formed from phosphatidylcholine (PC), such as soybean PC and egg PC. Another type is formed from a mixture of phospholipids and / or phosphatidylcholine and / or cholesterol.

[0315] Other exemplary methods for introducing liposomes into cells in vitro include U.S. Pat. No. 5,283,185, U.S. Pat. No. 5,171,678, WO 94 / 00569, WO 93 / 24640, WO 91 / 16024, Felgner, J. Biol. Chem. 269:2550, 1994, Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993, Nabel, Human Gene Ther. 3:649, 1992, Gershon, Biochem. 32:7143, 1993, and Strauss EMBO J. 11:417, 1992.

[0316] In some embodiments, cationic liposome is used.Cationic liposome has the advantage that it can fuse with cell membrane.Non-cationic liposome cannot fuse with plasma membrane efficiently, but it can be taken up by macrophage in vivo and can be used to deliver siRNA to macrophage.

[0317] Additional advantages of liposomes include that liposomes derived from natural phospholipids are biocompatible and biodegradable, a wide range of water-soluble and lipid-soluble drugs can be incorporated into liposomes, and liposomes can protect siRNA encapsulated in their internal compartments from metabolism and degradation (Rosoff, "Pharmaceutical Dosage Forms," ​​Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.

[0318] The positively charged synthetic cationic lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) can be used to form small liposomes that spontaneously interact with nucleic acids to form lipid-nucleic acid complexes that fuse with negatively charged lipids in the plasma membrane of tissue culture cells, resulting in the ability to deliver siRNA (for a review of DOTMA and its use with DNA, see, e.g., Felgner, PL et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987, and U.S. Pat. No. 4,897,355, which are incorporated herein by reference in their entireties).

[0319] The DOTMA analog 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with phospholipids to form DNA-complexed vesicles. Lipofectin™ (Bethesda Research Laboratories, Gaithersburg, Maryland) is an effective agent for delivering highly anionic nucleic acids into living tissue culture cells. It contains positively charged DOTMA liposomes that spontaneously interact with negatively charged polynucleotides to form complexes. If sufficient positively charged liposomes are used, the net charge of the resulting complex will still be positive. The positively charged complexes prepared in this way spontaneously adhere to negatively charged cell surfaces and fuse with the plasma membrane, efficiently delivering functional nucleic acids to, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane ("DOTAP") (Boehringer Mannheim, Indianapolis, IN), differs from DOTMA in that the oleoyl moieties are linked by ester rather than ether linkages.

[0320] Other reported cationic lipid compounds include those conjugated to a variety of moieties, such as carboxyspermine, including compounds such as 5-carboxyspermylglycine dioctaoleylamide ("DOGS") (Transfectam™, Promega, Madison, Wisconsin) and dipalmitoylphosphatidylethanolamine 5-carboxyspermylamide ("DPPES"), conjugated to one of two types of lipids (see, e.g., U.S. Pat. No. 5,171,678).

[0321] Another cationic lipid conjugate involves lipid derivatization with cholesterol ("DC-Chol") formulated into liposomes in combination with DOPE (see, e.g., Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolylysine, prepared by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta 1065:8, 1991, which is incorporated herein by reference in its entirety). For certain cell lines, these liposomes containing conjugated cationic lipids are said to exhibit lower toxicity and result in more efficient transfection than DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Md.) Other cationic lipids suitable for delivery of oligonucleotides are described in WO 98 / 39359 and WO 96 / 37194.

[0322] Liposome preparations are particularly suitable for external administration, and liposomes have several advantages compared with other preparations.These advantages include reducing the side effects associated with the high systemic absorption of administered drugs, increasing the accumulation of administered drugs in the desired target, and being able to administer siRNA to skin.In some embodiments, liposomes are used to deliver siRNA to epidermal cells and enhance the penetration of siRNA into dermal tissue, for example, into skin.For example, liposomes can be applied externally. The topical delivery of drugs formulated as liposomes has been described in detail (e.g., Weiner et al., Journal of Drug Targeting, 1992, vol. 2, 405-410 and du Plessis et al., Antiviral Research, 18, 1992, 259-265; Mannino, RJ and Fould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T. et al. Gene 56:267-276, 1987; Nicolau, C. et al. Meth. Enz. 149:157-176, 1987; Straubinger, R. M. and Papahadjopoulos, D. Meth. Enz. 101:512-527, 1983; Wang, C. Y. and Huang, L., Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987, which are incorporated herein by reference in their entireties.

[0323] Nonionic liposome systems, particularly those containing nonionic surfactants and cholesterol, have also been investigated to determine their usefulness in delivering drugs to the skin.Novasome I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) nonionic liposome formulations have been used to deliver drugs to the dermis of mouse skin.Such formulations containing siRNA are useful for treating skin disorders.

[0324] Liposomes containing the conjugates described herein can be made highly deformable. Such deformability can allow the liposomes to penetrate through pores smaller than the average radius of the liposomes. For example, transfersomes are one type of deformable liposome. Transfersomes can be prepared by adding a surface edge activator, usually a surfactant, to a standard liposome composition. Transfersomes containing siRNA can be delivered, for example, subcutaneously by infection to deliver siRNA to keratinocytes in the skin. To cross intact mammalian skin, lipid vesicles must pass through a series of pores, each less than 50 nm in diameter, under the influence of an appropriate transdermal gradient. In addition, due to the properties of lipids, these transfersomes can be self-optimizing (adaptable to the shape of pores, e.g., skin pores), self-repairing, often able to reach their respective targets without fragmentation, and often self-loading.

[0325] Other formulations suitable for the present invention are described in U.S. Provisional Application Nos. 61 / 018,616, filed January 2, 2008, 61 / 018,611, filed January 2, 2008, 61 / 039,748, filed March 26, 2008, 61 / 047,087, filed April 22, 2008, and 61 / 051,528, filed May 8, 2008. PCT Application No. PCT / US2007 / 080331, filed October 3, 2007, also describes formulations suitable for the present invention.

[0326] Surfactants. Surfactants are widely used in formulations such as emulsions (including microemulsions) and liposomes (see above). Conjugate formulations can include surfactants. In some embodiments, the conjugates described herein are formulated as emulsions containing surfactants. The most common method for classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by using the hydrophilic / lipophilic balance (HLB). The nature of the hydrophilic group provides the most useful means for categorizing the various surfactants used in formulations (Rieger, "Pharmaceutical Dosage Forms," ​​Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0327] If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants are widely used in pharmaceutical products and can be used over a wide range of pH values. Their HLB values ​​generally range from 2 to approximately 18, depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and alkanol ethers, such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated / propoxylated block polymers, are also included in this class. Polyoxyethylene surfactants are the most common members of the nonionic surfactant class.

[0328] If the surfactant molecule has a negative charge when dissolved or dispersed in water, the surfactant is classified as anionic.Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates.The most important members of the anionic surfactant class are alkyl sulfates and soaps.

[0329] If the surfactant molecule carries a positive charge when dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. Quaternary ammonium salts are the most commonly used members of this class.

[0330] If the surfactant molecule can carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines, and phosphatides.

[0331] The use of surfactants in drug products, formulations and emulsions has been reviewed (Rieger, "Pharmaceutical Dosage Forms", Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0332] Micelle and Other Membrane Formulations. Formulations containing the conjugates described herein can be provided as micellar formulations. A "micelle" is defined herein as a specific type of molecular assembly in which amphiphilic molecules are arranged in a spherical structure such that the hydrophobic portions of the amphiphilic molecules all face inward, while the hydrophilic portions remain in contact with the surrounding aqueous phase. If the environment is hydrophobic, the opposite arrangement exists.

[0333] Mixed micelle formulations suitable for delivery through transdermal membranes contain an aqueous solution of the siRNA composition, alkali metal C8-C 22 They can be prepared by mixing alkyl sulfates and micelle-forming compounds. Exemplary micelle-forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleate, monolaurate, borage oil, evening primrose oil, menthol, trihydroxyoxocholanylglycine and pharmaceutically acceptable salts thereof, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and analogs thereof, polidocanol alkyl ethers and analogs thereof, chenodeoxycholic acid, deoxycholic acid, and mixtures thereof. The micelle-forming compound can be added simultaneously with or after the addition of the alkali metal alkyl sulfate. Mixed micelles can be formed by mixing virtually any type of component, but vigorously mixing can be used to obtain smaller micelles.

[0334] In one method, a first micelle composition containing a conjugate described herein and at least an alkali metal alkyl sulfate is prepared. The first micelle composition is then mixed with at least three micelle-forming compounds to form a mixed micelle composition. In another method, a micelle composition is prepared by mixing at least one of a conjugate described herein, an alkali metal alkyl sulfate, and micelle-forming compounds, and then adding the remaining micelle-forming compounds and stirring vigorously.

[0335] Phenol and / or m-cresol may be added to the mixed micelle composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol and / or m-cresol may be added along with the micelle-forming components. A tonicity agent, such as glycerin, may also be added after the mixed micelle composition is formed.

[0336] To deliver the micelle formulation as a spray, the formulation can be placed in an aerosol dispenser, and the dispenser is filled with a propellant. The propellant under pressure is in liquid form in the dispenser. The ratio of components is adjusted so that there is one aqueous phase and one propellant phase, i.e., there is only one phase present. If there are two phases, the dispenser must be shaken before dispensing a portion of the contents, for example, through a metering valve. The dosage of the pharmaceutical agent is sprayed as a fine mist from the metering valve.

[0337] Propellants may include hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether, and diethyl ether. In certain embodiments, HFA134a (1,1,1,2 tetrafluoroethane) may be used.

[0338] The specific concentrations of the essential ingredients can be determined by relatively simple experimentation. For oral absorption, it is often desirable to increase the dosage, e.g., at least two or three times, over that administered by injection or via the gastrointestinal tract.

[0339] In some embodiments, the conjugates described herein can be incorporated into particles, e.g., microparticles. Microparticles can be produced by spray drying, but can also be produced by other methods, such as freeze-drying, evaporation, fluidized bed drying, vacuum drying, or a combination of these techniques.

[0340] kit The present invention also provides kits comprising the conjugates described herein.

[0341] In some embodiments, the kit further comprises instructions for use.

[0342] Exemplary embodiments of the various aspects described herein can be illustrated by the following numbered embodiments:

[0343] Aspect 1: (a) (i) a first variable domain that binds to a binding target; (ii) a second variable domain containing a reactive residue; a dual variable domain immunoglobulin molecule (Ig) or an antigen-binding fragment thereof, comprising: (b) a linker covalently conjugated to a reactive residue of the second variable domain of the Ig; and (c) a double-stranded RNA (dsRNA) molecule conjugated to a linker A conjugate comprising: Optionally, the dsRNA has the ability to inhibit expression of a target gene. Optionally, the dsRNA comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, and the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference. Optionally, the dsRNA comprises: (i) a melting temperature (T m ), (ii) the antisense strand contains two, three, four, five, or six 2'-fluoro modifications; (iii) the antisense strand contains one, two, three, or four phosphorothioate internucleotide linkages; (iv) the sense strand is conjugated with a linker; (v) the sense strand contains two, three, four, or five 2'-fluoro modifications; (vi) the sense strand contains 1, 2, 3, or 4 phosphorothioate internucleotide linkages; (vii) the dsRNA contains at least four 2'-fluoro modifications; (viii) the dsRNA contains a double-stranded region of 12 to 40 nucleotide pairs in length; (ix) the dsRNA has a blunt end at the 5' end of the antisense strand, and (x) dsRNA has an overhang at the 3' end of the antisense strand and

[0344] Aspect 2: dsRNA has a melting temperature (T m 2. The conjugate of embodiment 1, having

[0345] Embodiment 3: A conjugate according to any one of embodiments 1 to 2, wherein the dsRNA has a melting temperature of at least 60°C.

[0346] Embodiment 4: A conjugate according to any one of embodiments 1 to 3, wherein the dsRNA comprises at least four 2'-fluoro modifications.

[0347] Embodiment 5: A conjugate according to any one of embodiments 1 to 4, wherein the dsRNA comprises a duplex region of 12 to 40 nucleotide base pairs in length.

[0348] Embodiment 6: A conjugate according to any one of embodiments 1 to 5, wherein the dsRNA comprises a duplex region of 18 to 25 nucleotide base pairs in length.

[0349] Embodiment 7: A conjugate according to any one of embodiments 1 to 6, wherein the dsRNA comprises a blunt end at the 5' end of the antisense strand.

[0350] Embodiment 8: A conjugate according to any one of embodiments 1 to 7, wherein the dsRNA comprises an overhang at the 3' end of the antisense strand.

[0351] Embodiment 9: A conjugate according to any one of embodiments 1 to 8, wherein the dsRNA comprises an overhang of at least 2 nucleotides at the 3' end of the antisense strand.

[0352] Embodiment 10: A conjugate according to any one of embodiments 1 to 9, wherein the sense strand is covalently conjugated to the linker.

[0353] Embodiment 11: A conjugate according to embodiment 10, wherein the 5'-end of the sense strand is covalently conjugated to a linker.

[0354] Embodiment 12: A conjugate according to embodiment 10, wherein the 3'-end of the sense strand is covalently conjugated to a linker.

[0355] Embodiment 13: A conjugate according to any one of embodiments 1 to 12, wherein the sense strand is 19 to 25 nucleotides in length.

[0356] Embodiment 14: A conjugate according to any one of embodiments 1 to 13, wherein the sense strand is 21 nucleotides in length.

[0357] Embodiment 15: A conjugate according to any one of embodiments 1 to 14, wherein the sense strand comprises 2, 3, 4 or 5 2'-fluoro modifications.

[0358] Embodiment 16: A conjugate according to any one of embodiments 1 to 15, wherein the sense strand comprises three or four 2'-fluoro modifications.

[0359] Embodiment 17: A conjugate according to any one of embodiments 1 to 16, wherein the sense strand comprises 2'-fluoro modifications at positions 7, 10 and 11, counting from the 5' end.

[0360] Embodiment 18: A conjugate according to any one of embodiments 1 to 17, wherein the sense strand comprises 2'-fluoro modifications at positions 7, 9, 10 and 11, counting from the 5' end.

[0361] Embodiment 19: A conjugate according to any one of embodiments 1 to 18, wherein the sense strand comprises 0, 1, 2, 3 or 4 phosphorothioate internucleotide linkages.

[0362] Embodiment 20: A conjugate according to any one of embodiments 1 to 19, wherein the sense strand comprises a phosphorothioate internucleotide linkage between the first and second nucleotide and between the second and third nucleotide, counting from the 5' end.

[0363] Embodiment 21: A conjugate according to any one of embodiments 1 to 20, wherein the antisense strand is 19 to 25 nucleotides in length.

[0364] Embodiment 22: A conjugate according to any one of embodiments 1 to 21, wherein the antisense is 23 nucleotides in length.

[0365] Embodiment 23: A conjugate according to any one of embodiments 1 to 22, wherein the antisense comprises 2, 3, 4, 5 or 6 2'-fluoro modifications.

[0366] Embodiment 24: A conjugate according to any one of embodiments 1 to 23, wherein the antisense comprises 2'-fluoro modifications at positions 2, 14 and 16, counting from the 5' end.

[0367] Embodiment 25: A conjugate according to any one of embodiments 1 to 24, wherein the antisense comprises 2'-fluoro modifications at positions 2, 6, 9, 14 and 16, counting from the 5' end.

[0368] Embodiment 26: A conjugate according to any one of embodiments 1 to 25, wherein the antisense comprises 2'-fluoro modifications at the 2nd, 6th, 8th, 9th, 14th and 16th positions counting from the 5' end.

[0369] Embodiment 27: A conjugate according to any one of embodiments 1 to 26, wherein the antisense comprises 1, 2, 3 or 4 phosphorothioate internucleotide linkages.

[0370] Embodiment 28: A conjugate according to any one of embodiments 1 to 27, wherein the antisense comprises a phosphorothioate internucleotide linkage between the 21st and 22nd nucleotide and between the 22nd and 23rd nucleotide counting from the 5' end.

[0371] Embodiment 29: A conjugate according to any one of embodiments 1 to 28, wherein the antisense comprises a phosphorothioate internucleotide linkage between the 1st and 2nd nucleotide, between the 2nd and 3rd nucleotide, between the 21st and 22nd nucleotide, and between the 22nd and 23rd nucleotide, counting from the 5' end.

[0372] Embodiment 30: A conjugate according to any one of embodiments 1 to 29, wherein the antisense strand comprises at least one thermodestabilizing modification of the duplex within the first 9 nucleotide positions of the 5' region.

[0373] Embodiment 31: A conjugate according to embodiment 30, wherein the thermodestabilizing modification is at the 4th, 5th, 6th, 7th, 8th or 9th position counting from the 5' end of the antisense strand.

[0374] Embodiment 32: A conjugate according to embodiment 31, wherein the thermodestabilizing modification is at position 7 counting from the 5' end of the antisense strand.

[0375] Embodiment 32: A conjugate according to any one of embodiments 1 to 32, wherein the antisense comprises a 5'-vinylphosphonate nucleotide at the 5' end.

[0376] Embodiment 34: A conjugate according to any one of embodiments 1 to 33, wherein the dsRNA comprises at least one 2'-OMe modification.

[0377] Embodiment 35: A conjugate according to any one of embodiments 1 to 34, wherein the sense strand comprises at least one 2'-OMe modification.

[0378] Embodiment 36: A conjugate according to any one of embodiments 1 to 35, wherein the antisense strand comprises at least one 2'-OMe modification.

[0379] Embodiment 37: A conjugate according to any one of embodiments 1 to 36, wherein the dsRNA comprises at least one locked nucleic acid (LNA) modification.

[0380] Embodiment 38: A conjugate according to any one of embodiments 1 to 37, wherein the reactive residue is lysine.

[0381] Embodiment 39: A conjugate according to any one of embodiments 1 to 38, wherein the first variable domain of the Ig is positioned more N-terminally than the second variable domain.

[0382] Embodiment 40: A conjugate according to any one of embodiments 1 to 39, wherein the Ig is a bispecific immunoglobulin molecule.

[0383] Embodiment 41: A conjugate according to any one of embodiments 1 to 40, wherein the antigen-binding fragment comprises a first variable domain and a second variable domain of an Ig and is selected from a Fab, a Fab', a F(ab')2, an Fv or an scFv.

[0384] Embodiment 42: A conjugate according to any one of embodiments 1 to 41, wherein the antigen-binding fragment comprises a Fab.

[0385] Embodiment 43: A conjugate according to any one of embodiments 1 to 42, wherein the Ig comprises a chimeric immunoglobulin sequence.

[0386] Embodiment 44: A conjugate according to any one of embodiments 1 to 43, wherein the Ig comprises a humanized immunoglobulin sequence.

[0387] Embodiment 45: A conjugate according to any one of embodiments 1 to 44, wherein the Ig comprises a human immunoglobulin sequence.

[0388] Embodiment 46: A conjugate according to any one of embodiments 1 to 45, wherein the binding target is a tumor cell surface antigen.

[0389] Embodiment 47: A conjugate according to any one of embodiments 1 to 46, wherein the first variable domain binds to CD138, B-cell maturation antigen (BCMA), SLAMF7, HER2, FOLR1 or CD79b.

[0390] Embodiment 48: A conjugate according to any one of embodiments 1 to 47, wherein the linker L is a reversible linker.

[0391] Embodiment 49: A conjugate according to any one of embodiments 1 to 48, wherein the linker L is a non-reversible linker.

[0392] Embodiment 50: A conjugate according to any one of embodiments 1 to 49, wherein the linker L is a cleavable linker.

[0393] Embodiment 51: A conjugate according to any one of embodiments 1 to 50, wherein the linker L is a non-cleavable linker.

[0394] Embodiment 52: A conjugate according to any one of embodiments 1 to 51, wherein the linker L is a branched linker.

[0395] Embodiment 53: A conjugate according to any one of embodiments 1 to 52, wherein the linker L is a linear linker.

[0396] Embodiment 54: A conjugate according to any one of embodiments 1 to 53, wherein the Ig comprises a first heavy chain and a light chain.

[0397] Embodiment 55: A conjugate according to any one of embodiments 1 to 54, wherein the Ig comprises a first heavy chain, a second heavy chain and a light chain, and wherein the first heavy chain and the second heavy chain are different.

[0398] Embodiment 56: A conjugate according to any one of embodiments 1 to 55, wherein the Ig is capable of binding to two different epitopes.

[0399] Embodiment 57: A conjugate according to any one of embodiments 1 to 54, wherein the Ig comprises a heavy chain, a light chain and a J chain.

[0400] Embodiment 58: A conjugate according to any one of embodiments 1 to 57, wherein the Ig further comprises a ligand.

[0401] Embodiment 59: A conjugate according to embodiment 58, wherein the ligand is an endosomolytic ligand.

[0402] Embodiment 60: A conjugate according to embodiment 59, wherein the ligand is linked to the light chain.

[0403] Embodiment 61: A conjugate according to any one of embodiments 1 to 60, wherein one of the variable domains, such as the first variable domain or the second variable domain, comprises an amino acid sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and any combination thereof.

[0404] Embodiment 62: A conjugate according to any one of embodiments 1 to 61, wherein the Ig comprises a peptide linker between the two domains, such as between the first and second variable domain.

[0405] Embodiment 63: The peptide linker is 63. The conjugate of embodiment 62, comprising an amino acid sequence selected from the group consisting of: TIFF2026041866000045.tif25160, and any combination thereof.

[0406] Embodiment 64: A conjugate according to any one of embodiments 1 to 63, wherein the Ig comprises an amino acid sequence selected from the group consisting of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and any combination thereof.

[0407] Embodiment 65: A pharmaceutical composition comprising the conjugate according to any one of embodiments 1 to 64, alone or in combination with a pharmaceutically acceptable carrier or excipient.

[0408] Embodiment 66: A gene silencing kit comprising a conjugate according to any one of claims 1 to 64.

[0409] Embodiment 67: A method for silencing a target gene in a cell, comprising the step of introducing into the cell a conjugate according to any one of embodiments 1 to 64.

[0410] Embodiment 68: Use of a conjugate according to any one of embodiments 1 to 64 in the preparation of a medicament.

[0411] Excerpt from definition Certain terms used in the specification, examples, and appended claims are collected here for convenience. Unless otherwise stated or clearly indicated by context, the following terms and phrases include the meanings set forth below. Unless otherwise expressly stated or apparent from context, the following terms and phrases do not exclude the meaning that the term or phrase has acquired in the relevant art. These definitions are provided to aid in the description of particular embodiments and are not intended to limit the claimed invention, as the scope of the present invention is limited only by the claims. Furthermore, unless otherwise required by context, singular forms shall include plural forms and plural forms shall include the singular.

[0412] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any known methods, devices, and materials can be used in the practice or testing of the present invention, such methods, devices, and materials are described herein in this regard.

[0413] Furthermore, the practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are described in detail in such publications as "Molecular Cloning: A Laboratory Manual," second edition (Sambrook et al., 1989), "Oligonucleotide Synthesis" (MJ Gait, ed., 1984), "Animal Cell Culture" (RI Freshney, ed., 1987), "Methods in Enzymology" (Academic Press, Inc.), "Current Protocols in Molecular Biology" (FMA Usubel et al., eds., 1987 and regularly updated editions), "PCR: The Polymerase Chain Reaction" (Mullis et al., ed., 1994), "A Practical Guide to Molecular Cloning" (Perbal Bernard V., 1988), and "Phage Display: A Laboratory Manual" (Barbas et al., 2001).

[0414] Where a range of values ​​is provided, it is understood that each intervening value (to the tenth of that unit unless the context clearly indicates otherwise) between the upper and lower limits of that range, and any other stated or intervening value within that stated range, is encompassed within the invention. The upper and lower limits of these subranges may independently be included in the subrange and are also encompassed within the invention, except for any specifically excluded limit in the stated range. When the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also encompassed within the invention.

[0415] In this specification, certain ranges are presented by numerical values ​​preceded by the term "about." In this specification, the term "about" is used to literally support the number it precedes and a number that is near or approximately the number it precedes. In determining whether a number is near or approximately a specifically recited number, the near or approximately unrecited number may be a number that, in the context in which it is presented, is substantially equivalent to the specifically recited number.

[0416] As used herein, the terms "comprising" or "comprises" are used in reference to compositions, methods, and their respective components that are essential to the invention, but also allow for the inclusion of non-specified elements, whether essential or not.

[0417] The singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise. Furthermore, it should be noted that claims may be drafted to exclude any optional element. Accordingly, this statement shall serve as a prerequisite for the use of exclusive language such as "solely" or "only" or for the use of a "negative" limitation in connection with the recitation of claim elements.

[0418] As used herein, the terms " dsRNA ", " siRNA " and " iRNA agent " are used interchangeably to refer to the agent that can mediate the silencing of target RNA, for example, mRNA, for example, the transcription product of the gene that codes for protein.For convenience, this mRNA is also referred to herein as the mRNA that is silenced.This gene is also referred to as target gene.Generally, the RNA that is silenced is endogenous gene, exogenous gene or pathogenic gene.In addition, RNA other than mRNA, for example, tRNA and viral RNA, can also be the target.

[0419] As used herein, the phrase " mediate RNAi " refers to the ability to silence target gene (for example, mRNA) in a sequence-specific manner.Without wishing to be bound by theory, it is believed that this silencing uses RNAi mechanism or RNAi process and the antisense strand of guide RNA (for example, dsRNA), where antisense strand is 21-23 nucleotides in length.

[0420] As used herein, " specifically hybridizable " and " complementary " are terms used to indicate a sufficient degree of complementarity so that stable specific binding occurs between the present invention and target RNA molecules.Specific binding requires a sufficient degree of specificity to avoid non-specific binding of oligomeric compounds to non-target sequences under the conditions where specific binding is desired, i.e., under physiological conditions in the case of assays or therapeutic treatments, or under the conditions where assays are performed in the case of in vitro assays.Non-target sequences typically differ by at least 5 nucleotides.

[0421] In some embodiments, dsRNA molecules are "sufficiently complementary" to target RNA, for example, target mRNA, so that the dsRNA molecule silences the production of the protein encoded by the target mRNA.In another embodiment, dsRNA molecules are "exactly complementary" to target RNA, for example, when target RNA and dsRNA double-stranded agent anneal, they form a hybrid that is exclusively made up of Watson-Crick base pairs in the exact complementary region.A "sufficiently complementary" target RNA can comprise an internal region (for example, a region of at least 10 nucleotides) that is exactly complementary to the target RNA.In addition, in some embodiments, dsRNA molecules specifically discriminate between single nucleotide differences.In this case, dsRNA molecules mediate RNAi only when exact complementarity is found in the region (for example, within 7 nucleotides) where a single nucleotide differs.

[0422] As used herein, the term "oligonucleotide" refers to a nucleic acid molecule (RNA or DNA) that is less than, for example, 100, 200, 300, or 400 nucleotides in length.

[0423] The term "BNA" refers to bridged nucleic acids, often referred to as constrained or inaccessible RNA. BNAs can contain 5-, 6-, or even 7-membered bridge structures with "locked" C3'-endo sugar puckering. The bridge is typically incorporated at the 2',4' position of the ribose to give a 2',4'-BNA nucleotide (e.g., LNA or ENA). Examples of BNA nucleotides include the following nucleosides: TIFF2026041866000046.tif72145

[0424] The term "LNA" refers to locked nucleic acid, often referred to as constrained RNA or inaccessible RNA. LNA is a modified RNA nucleotide. The ribose moiety of an LNA nucleotide is modified with an additional bridge (e.g., a methylene or ethylene bridge) that connects the 2' hydroxyl to the 4' carbon of the same ribose sugar. For example, the bridge may force the ribose into a 3'-endo (North) conformation: It can be "locked" to TIFF2026041866000047.tif28128.

[0425] The term "ENA" refers to ethylene-bridged nucleic acid, often called restricted or inaccessible RNA.

[0426] As used herein, the term "cleavage site" refers to the backbone junction in the target gene or sense strand that is cleaved by the RISC mechanism when an iRNA agent is used. The target cleavage site region also includes at least one or at least two nucleotides on either side of the cleavage site. In the case of the sense strand, if the sense strand itself is the target cleaved by the RNAi mechanism, the cleavage site is the backbone junction cleaved in the sense strand. The cleavage site can be determined using methods known in the art, such as the 5'-RACE assay described in Soutschek et al., Nature (2004) 432, 173-178, which is incorporated herein by reference in its entirety. As is well understood in the art, in the case of a conical double-stranded RNAi agent containing two 21-nucleotide long strands (where the strands form a 19-contiguous base pair duplex region with a 2-nucleotide single-stranded overhang at the 3' end), the cleavage site region corresponds to positions 9-12 from the 5' end of the sense strand.

[0427] The terms "reduce," "reduce," "reduction," or "inhibit" are all used herein to refer to a statistically significant reduction. In some embodiments, "reduce," "reduction," "reduce," or "inhibit" typically refers to a reduction of at least 10% compared to a reference level (e.g., in the absence of a given treatment), and can include, for example, a reduction of at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more. As used herein, "reduction" or "inhibition" does not encompass complete inhibition or complete reduction compared to a reference level. "Complete inhibition" is 100% inhibition compared to a reference level. The decrease may preferably be down to a level that is accepted as within the normal range for individuals without the given disorder.

[0428] The terms "immunoglobulin" or "antibody" are used interchangeably herein to refer to a basic four-chain heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds, depending on the H chain isotype. Each H and L chain has an N-terminus and a C-terminus, and also contains regularly spaced intrachain disulfide bridges. Each H chain contains a variable domain (V) at its N-terminus. H ), followed by three constant domains (C H 1. C H 2 and C H 3) at the N-terminus of each L chain. L ), followed by one constant domain (C L ) continues. V L is V H It is aligned with C L is the first constant domain of the heavy chain (CH 1). Specific amino acid residues are thought to form an interface between the light-chain variable domain and the heavy-chain variable domain. H and V L Together, these pairings form a single antigen-binding site.

[0429] L chains from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of their respective constant domains. H Depending on the amino acid sequence of their amino acids, immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, which have heavy chains designated α, δ, ε, γ, and μ, respectively. The γ and a classes are further subdivided into C H They are divided into subclasses based on relatively minor differences in sequence and function; for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0430] The "variable region" or "variable domain" of an immunoglobulin refers to the N-terminal domain of an immunoglobulin heavy or light chain. The variable domain of a heavy chain is called the "V H The variable domain of the light chain can be called "V L These domains are generally the most variable parts of the immunoglobulin and contain the antigen binding sites.

[0431] The term "variable" refers to the fact that certain segments of variable domains vary significantly in sequence among immunoglobulins. V domains mediate antigen binding and define the specificity of a particular immunoglobulin for its particular antigen. However, variability is not evenly distributed throughout the 110-amino acid span of most variable domains. Instead, V regions consist of relatively invariant stretches of 15–30 amino acids called framework regions (FRs) separated by short, highly variable regions called "hypervariable regions," each 9–12 amino acids long. Native heavy and light chain variable domains each contain four FRs, largely in a beta-sheet configuration, connected by three hypervariable regions that form loops that span and, in some cases, form part of the beta-sheet structure. The hypervariable regions in each chain are held in close proximity by the FRs and, together with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of the immunoglobulin (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)). The constant domains are not directly involved in binding of the immunoglobulin to an antigen but exhibit various effector functions, such as the participation of immunoglobulins in antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC).

[0432] An "intact" immunoglobulin is one that contains not only the antigen-binding site but also the C L chain and at least the H chain constant domain, C H 1. C H and C H 3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. An intact immunoglobulin can have one or more effector functions.

[0433] A "naked immunoglobulin," as used herein, is an immunoglobulin that is not conjugated to a dsRNA molecule.

[0434] An "immunoglobulin fragment" comprises a portion of an intact immunoglobulin, preferably the antigen-binding or variable region of an intact immunoglobulin. Examples of immunoglobulin fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear immunoglobulins (see Example 2 of U.S. Pat. No. 5,641,870; Zapata et al., Protein Eng. 8(10):1057-1062 (1995)); single-chain immunoglobulin molecules; and multispecific immunoglobulins formed from immunoglobulin fragments. In some aspects, immunoglobulin fragments include all possible alternative fragment formats. In some aspects, immunoglobulin fragments can be bispecific. In some aspects, immunoglobulin fragments can be biparatopic. In some aspects, immunoglobulin fragments can be trispecific. In some aspects, immunoglobulin fragments can be multimeric. In some aspects, immunoglobulin fragments comprise the antigen-binding site of an intact immunoglobulin and therefore retain the ability to bind to an antigen. In some aspects, immunoglobulin fragments contain a single variable domain capable of binding to an antigen. In some aspects, immunoglobulin fragments are further modified (including but not limited to, peptide addition, PEGylation, HESylation, glycosylation) to adjust activity, specificity, pharmacokinetic behavior, and in vivo efficacy.

[0435] Papain digestion of immunoglobulins yields two identical antigen-binding fragments called "Fab" fragments and a residual "Fc" fragment. The "Fc" name reflects the ease with which it can be crystallized. The Fab fragment contains the entire light chain and the variable region domain (V) of the heavy chain. H) and the first constant domain of one heavy chain (C H 1). Each Fab fragment is monovalent with respect to antigen binding; i.e., each Fab fragment contains a single antigen-binding site. Pepsin treatment of immunoglobulins yields a single large F(ab')2 fragment. This fragment roughly corresponds to two disulfide-linked Fab fragments with bivalent antigen-binding activity and still retains the ability to cross-link antigen. Fab' fragments contain several additional residues, including one or more cysteines from the immunoglobulin hinge region, which are then joined to the C(ab')2 fragment. H F(ab')2 immunoglobulin fragments differ from Fab fragments in that they have a free thiol group at the carboxy terminus of one domain. Fab'-SH is the designation used herein for Fab' in which the cysteine ​​residues of the constant domains bear a free thiol group. F(ab')2 immunoglobulin fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of immunoglobulin fragments are also known.

[0436] The Fc fragment contains the carboxy-terminal portions of both heavy chains held together by disulfides. The effector functions of immunoglobulins are determined by sequences in the Fc region, which is also the region recognized by Fc receptors (FcRs) found on certain cell types.

[0437] An "Fv" is the minimum immunoglobulin fragment containing a complete antigen recognition and binding site. This fragment consists of a dimer of one heavy-chain variable domain and one light-chain variable domain tightly associated by noncovalent bonds. In single-chain Fv (scFv) species, one heavy-chain variable domain and one light-chain variable domain can be covalently linked by a flexible peptide linker, allowing the light and heavy chains to associate into a "dimeric" structure similar to that of two-chain Fv species. Folding of these two domains generates six hypervariable loops (three loops each from the heavy and light chains) that provide the amino acid residues for antigen binding and confer antigen-binding specificity to the immunoglobulin. However, even a single variable domain (i.e., half of an Fv containing only the three antigen-specific CDRs) is capable of recognizing and binding to antigen, although typically with lower affinity than the entire binding site. As used herein in reference to DVD immunoglobulin molecules, the term "Fv" refers to a binding fragment comprising both the first and second variable domains of the heavy and light chains.

[0438] "Single-chain Fv", also abbreviated as "sFv" or "scFv", refers to VFvs linked together in a single polypeptide chain. H Immunoglobulin domains and V L Preferably, the sFv polypeptide is an immunoglobulin fragment comprising an immunoglobulin domain and a V H Domains and V LThe domains further comprise a polypeptide linker that enables the sFv to form the desired structure for antigen binding. For a review of sFvs, see, e.g., Plueckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995, infra. As used herein with respect to DVD immunoglobulin molecules, the term "scFv" refers to a binding fragment comprising both the first and second variable domains of the heavy and light chains.

[0439] As used herein, the term "dual variable domain immunoglobulin" or "DVD-Ig" refers to an immunoglobulin molecule as described above in which both the heavy and light chains contain a second variable domain located adjacent to the first variable domain. Thus, the light chain of a DVD-Ig contains, from the N-terminus to the C-terminus, the following domains: V L 1-V L 2-C L Therefore, the heavy chain of DVD-Ig contains, from the N-terminus to the C-terminus, the following domains: H 1-V H 2-C H 1-C H 2-C H 3. V L 1 and V H 1 together form the first antigen-binding site. L 2 and V H 2 together form a second antigen-binding site.

[0440] Unless otherwise stated, the term "immunoglobulin" or "antibody" specifically encompasses native human and non-human IgG1, IgG2, IgG3, IgG4, IgE, IgA1, IgA2, IgD and IgM antibodies, including naturally occurring variants.

[0441] The term "native" with respect to a polypeptide (e.g., an antibody or immunoglobulin) is used herein to refer to a polypeptide having a sequence that occurs in nature, regardless of its mode of preparation. The term "non-native" with respect to a polypeptide (e.g., an antibody or immunoglobulin) is used herein to refer to a polypeptide having a sequence that does not occur in nature.

[0442] The term "polypeptide" is used herein in its broadest sense and includes peptide sequences. The term "peptide" broadly refers to a linear molecular chain of amino acids containing up to about 30, preferably up to about 60, amino acids covalently linked by peptide bonds.

[0443] As used herein, the term "monoclonal" refers to an antibody or immunoglobulin molecule (e.g., a DVD Ig molecule) obtained from a substantially homogeneous population of immunoglobulins; that is, the individual immunoglobulins comprising the population are identical except for possible minor natural mutations. Monoclonal immunoglobulins are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal immunoglobulin is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the immunoglobulin as being obtained from a substantially homogeneous population of immunoglobulins and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal immunoglobulins of the present invention can be produced by the hybridoma method first described by Kohler and Milstein (1975) Nature 256:495, or can be produced by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).

[0444] As used herein, monoclonal immunoglobulins specifically include "chimeric" immunoglobulins in which a portion of the heavy and / or light chains are identical to or homologous to corresponding sequences in antibodies from one particular species, while the remainder of the chains are identical to or homologous to corresponding sequences in antibodies from another species, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567 and Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81:6851-6855).

[0445] "Humanized" forms of non-human (e.g., rodent, e.g., mouse or rabbit) immunoglobulins are immunoglobulins that contain minimal sequences derived from the non-human immunoglobulin. Humanized immunoglobulins are largely human immunoglobulins (recipient antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-human species (donor antibody) such as mouse, rat, hamster, rabbit, chicken, cow, or non-human primate having the desired specificity, affinity, and capacity. Optionally, Fv framework region (FR) residues of the human immunoglobulin are also replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues that are not found in either the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. Generally, humanized immunoglobulins will comprise substantially all of at least one, and typically two, variable domains, with all or substantially all of the hypervariable loops corresponding to those of a non-human immunoglobulin and all or substantially all of the FR regions being those of a human immunoglobulin sequence. A humanized immunoglobulin optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al. (1986) Nature 321:522-525, Riechmann et al. (1988) Nature 332:323-329, and Presta (1992) Curr. Op. Struct. Biol. 2:593-596.

[0446] As used herein, the term "human immunoglobulin" is intended to include immunoglobulins having variable and constant regions derived from human germline immunoglobulin sequences. The human immunoglobulins of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-directed mutagenesis or by in vivo somatic mutation), for example in the CDRs, particularly CDR3. However, the term "human immunoglobulin" as used herein does not include immunoglobulins in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0447] As used herein, an "isolated" immunoglobulin refers to one that has been identified and separated and / or recovered from components of its natural environment in recombinant host cells. Contaminating components of that natural environment are substances that would interfere with the immunoglobulin's diagnostic or therapeutic use, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes, as well as undesired by-products of production. In some aspects, an isolated immunoglobulin herein will be purified (1) to greater than 95%, or greater than 98%, or greater than 99% by weight, as determined by SDS-PAGE or SEC-HPLC, (2) sufficiently to obtain at least 15 residues of N-terminal or internal amino acid sequence using an amino acid sequencer, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue staining or, preferably, silver staining. Typically, an isolated immunoglobulin will be prepared by at least one purification step.

[0448] The terms "specific binding" or "specifically binds to" or "specific for" refer to the binding of a binding moiety to a binding target, e.g., the binding of an immunoglobulin to a target antigen, e.g., an epitope on a particular polypeptide, peptide, or other target (e.g., a glycoprotein target), and mean binding that is measurably different from a non-specific interaction (e.g., a non-specific interaction can be binding to bovine serum albumin or casein). Specific binding can be measured, for example, by determining binding of a binding moiety or immunoglobulin to a target molecule compared to binding to a control molecule. For example, specific binding can be determined by competition with a control molecule that resembles the target, e.g., an excess of unlabeled target. In this case, specific binding is indicated if binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target. As used herein, the terms "specific binding" to or "specifically binds to" or "is specific for" a particular polypeptide or epitope on a particular polypeptide target means, for example, a K for that target of at least about 200 nM, alternatively at least about 150 nM, alternatively at least about 100 nM, alternatively at least about 60 nM, alternatively at least about 50 nM, alternatively at least about 40 nM, alternatively at least about 30 nM, alternatively at least about 20 nM, alternatively at least about 10 nM, alternatively at least about 8 nM, alternatively at least about 6 nM, alternatively at least about 4 nM, alternatively at least about 2 nM, alternatively at least about 1 nM, or stronger. d In certain instances, the term "specific binding" refers to binding by a molecule to a particular polypeptide or an epitope on a particular polypeptide without substantially binding to any other polypeptides or polypeptide epitopes.

[0449] "Binding affinity" refers to the strength of the aggregate of non-covalent interactions between a single binding site of a molecule (e.g., an immunoglobulin) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity," as used herein, refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an immunoglobulin and an antigen). The affinity of a molecule X for its partner Y is generally measured by the dissociation constant (K d ) can be expressed as: For example, K d The affinity can be about 200 nM, 150 nM, 100 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 8 nM, 6 nM, 4 nM, 2 nM, 1 nM, or stronger. Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind slowly to antigens and tend to dissociate quickly, whereas high-affinity antibodies generally bind more quickly and tend to remain bound longer. Various methods for measuring binding affinity are known in the art.

[0450] As used herein, "K d " or "K d "Value" refers to the dissociation constant measured by a technique appropriate for that immunoglobulin-target pair, for example, using a surface plasmon resonance assay, for example, on an immobilized antigen CM5 chip using a Biacore X100 or Biacore T200 (GE Healthcare, Piscataway, NJ) at 25°C.

[0451] The terms "conjugate," "conjugated," and "conjugation" refer to any and all forms of covalent or non-covalent linkage, including, but not limited to, direct genetic or chemical fusion, coupling via a linker or cross-linking agent, and non-covalent association.

[0452] The term "fusion" is used herein to refer to the combination of amino acid sequences of different origins in one polypeptide chain by combining nucleotide sequences encoding those amino acid sequences in frame. The term "fusion" explicitly encompasses not only fusion to one of the termini, but also internal fusion of sequences of different origins within one polypeptide chain, i.e., insertion ...

Claims

1. (a) (i) A first variable domain that binds to SLAMF7, CD138, or HER2, (ii) Second variable domain containing reactive residues and A bivariable domain immunoglobulin molecule (Ig) or its antigen-binding fragment, including; (b) Linker (L) covalently conjugated to a reactive residue of the second variable domain of Ig; and (c) A double-stranded RNA (dsRNA) molecule conjugated to a linker, wherein the dsRNA has the ability to inhibit the expression of a target gene, the dsRNA comprises a sense strand and an antisense strand, each having 18 to 35 nucleotides, the dsRNA contains a double-stranded region of 18 to 25 nucleotide base pairs in length, the antisense strand has sufficient complementarity to mediate RNA interference with the target sequence, and the dsRNA is (i) Melting temperature (T) of approximately 40°C to approximately 80°C m ), (ii) The antisense chain contains two, three, four, five or six 2'-fluoromodifications, (iii) The antisense strand contains one, two, three or four phosphorothioate nucleotide interlinking regions, (iv) The sense chain is conjugated to the linker. (v) The sense chain contains two, three, four or five 2'-fluoromodifications. (vi) The sense strand contains one, two, three, or four phosphorothioate nucleotide junctions. (vii) dsRNA contains at least four 2'-fluoro modifications, (viii) dsRNA has a blunt end at the 5' end of the antisense strand, and (ix) dsRNA has an overhang at the 3' end of the antisense strand. A dsRNA molecule having at least one of the following characteristics A conjugate that includes this.

2. dsRNA has a melting temperature of approximately 40°C to 80°C (T m The conjugate according to claim 1, having ).

3. The conjugate according to claim 1, wherein the dsRNA has a melting temperature of at least 60°C.

4. The conjugate according to claim 1, wherein the dsRNA comprises at least four 2'-fluoro modifications.

5. The conjugate according to claim 1, wherein the dsRNA comprises a double-stranded region having a length of 19, 20, 21, or 22 nucleotide base pairs.

6. The conjugate according to claim 1, wherein the dsRNA includes a blunt end at the 5' end of the antisense strand.

7. The conjugate according to claim 1, wherein the dsRNA includes an overhang at the 3' end of the antisense strand.

8. The conjugate according to claim 7, wherein the dsRNA includes an overhang of at least two nucleotides at the 3' end of the antisense strand.

9. The conjugate according to claim 1, wherein the sense chain is covalently conjugated with the linker.

10. The conjugate according to claim 9, wherein the 5' end of the sense chain is covalently conjugated with a linker.

11. The conjugate according to claim 9, wherein the 3' end of the sense chain is covalently conjugated with a linker.

12. The conjugate according to claim 1, wherein the sense strand is 19 to 25 nucleotides long.

13. The conjugate according to claim 12, wherein the sense strand is 21 nucleotides long.

14. The conjugate according to claim 1, wherein the sense chain comprises two, three, four, or five 2'-fluoromodifications.

15. The conjugate according to claim 14, wherein the sense chain comprises three or four 2'-fluoromodifications.

16. The conjugate according to claim 1, wherein the sense chain includes 2'-fluoro modifications at the 7th, 10th, and 11th positions, counting from the 5' end.

17. The conjugate according to claim 1, wherein the sense chain includes 2'-fluoro modifications at the 7th, 9th, 10th, and 11th positions, counting from the 5' end.

18. The conjugate according to claim 1, wherein the sense strand comprises zero, one, two, three, or four phosphorothioate nucleotide interlinking regions.

19. The conjugate according to claim 1, wherein the sense strand includes phosphorothioate internucleotide junctions between the 1st and 2nd nucleotides and between the 2nd and 3rd nucleotides, counting from the 5' end.

20. The conjugate according to claim 1, wherein the antisense chain is 19 to 25 nucleotides long.

21. The conjugate according to claim 20, wherein the antisense strand is 23 nucleotides long.

22. The conjugate according to claim 1, wherein the antisense chain comprises two, three, four, five, or six 2'-fluoromodifications.

23. The conjugate according to claim 1, wherein the antisense chain includes 2'-fluoro modifications at the 2nd, 14th, and 16th positions, counting from the 5' end.

24. The conjugate according to claim 1, wherein the antisense chain includes 2'-fluoro modifications at the 2nd, 6th, 9th, 14th, and 16th positions, counting from the 5' end.

25. The conjugate according to claim 1, wherein the antisense chain includes 2'-fluoro modifications at the 2nd, 6th, 8th, 9th, 14th and 16th positions, counting from the 5' end.

26. The conjugate according to claim 1, wherein the antisense chain comprises one, two, three, or four phosphorothioate nucleotide interlinking units.

27. The conjugate according to claim 1, wherein the antisense chain includes phosphorothioate internucleotide junctions between the 21st and 22nd nucleotides and between the 22nd and 23rd nucleotides, counting from the 5' end.

28. The conjugate according to claim 1, wherein the antisense chain includes phosphorothioate internucleotide junctions between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 21st and 22nd nucleotides, and between the 22nd and 23rd nucleotides, counting from the 5' end.

29. The conjugate according to claim 1, wherein the antisense strand includes at least one double-strand thermal destabilization modification within the first nine nucleotide positions of the 5' region.

30. The conjugate according to claim 29, wherein the thermal destabilization modification is located at the 4th, 5th, 6th, 7th, 8th or 9th position from the 5' end of the antisense chain.

31. The conjugate according to claim 30, wherein the thermal destabilization modification is located at the 7th position from the 5' end of the antisense chain.

32. The conjugate according to claim 1, wherein the antisense strand contains a 5'-vinylphosphonate nucleotide at its 5' end.

33. The conjugate according to claim 1, wherein the dsRNA comprises at least one 2'-OMe modification.

34. The conjugate according to claim 1, wherein the sense chain includes at least one 2'-OMe modification.

35. The conjugate according to claim 1, wherein the antisense chain includes at least one 2'-OMe modification.

36. The conjugate according to claim 1, wherein the dsRNA comprises at least one locked nucleic acid (LNA) modification.

37. The conjugate according to claim 1, wherein the reactive residue is lysine.

38. The conjugate according to claim 1, wherein the first variable domain of Ig is located closer to the N-terminus than the second variable domain.

39. The conjugate according to claim 1, wherein Ig is a bispecific immunoglobulin molecule.

40. The antigen-binding fragment contains the first and second variable domains of Ig, and is Fab, Fab', F(ab') 2 The conjugate according to claim 1, selected from Fv or scFv.

41. The conjugate according to claim 1, wherein the antigen-binding fragment comprises Fab.

42. The conjugate according to claim 1, comprising Ig and a chimeric immunoglobulin sequence.

43. The conjugate according to claim 1, comprising Ig, a humanized immunoglobulin sequence.

44. The conjugate according to claim 1, wherein Ig comprises a human immunoglobulin sequence.

45. The conjugate according to claim 1, wherein linker L is a reversible linker.

46. The conjugate according to claim 1, wherein linker L is an irreversible linker.

47. The conjugate according to claim 1, wherein linker L is a severable linker.

48. The conjugate according to claim 1, wherein linker L is a non-cuttable linker.

49. The conjugate according to claim 1, wherein linker L is a branched linker.

50. The conjugate according to claim 1, wherein the linker L is a linear linker.

51. The conjugate according to claim 1, further comprising Ig as a ligand.

52. The conjugate according to claim 51, wherein the ligand is an endosomal lytic ligand.

53. The conjugate according to claim 51, wherein the ligand is linked to a light chain.

54. The conjugate according to claim 1, wherein the second variable domain of Ig comprises an amino acid sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, and any combination thereof.

55. The conjugate according to claim 1, wherein Ig includes a peptide linker between the first variable domain and the second variable domain.

56. Peptide linkers The conjugate according to claim 55, comprising an amino acid sequence selected from the group consisting of and any combination thereof.

57. The conjugate according to claim 1, wherein Ig comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 29, and SEQ ID NO:

30.

58. A pharmaceutical composition comprising the conjugate described in claim 1, either alone or in combination with a pharmaceutically acceptable carrier or excipient.

59. A gene silencing kit comprising the conjugate described in claim 1.

60. An in vitro method for silencing a target gene in a cell, comprising the step of introducing the conjugate described in claim 1 into a cell.

61. Use of the conjugate according to claim 1 in the preparation of pharmaceuticals.

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