MODIFIED DOUBLE STRANDED OLIGONUCLEOTIDE
The disclosure is directed to dual variable domain immunoglobulin double-stranded RNA conjugates that are advantageous for inhibition of target gene expression, as well as compositions suitable for therapeutic use. The dual variable domain immunoglobulin comprises a first variable domain that binds to a binding target, and a second variable domain that comprises a reactive residue, where the linker is covalently conjugated to the reactive residue. The dsRNA is linked to the linker and is capable of inhibiting the expression of the target gene by RNA interference. The disclosure also provides pharmaceutical compositions comprising these conjugate and methods of inhibiting the expression of a target gene by administering these conjugates, e.g., for the treatment of various disease conditions.
1 . A conjugate comprising:
a. a dual variable domain immunoglobulin molecule (Ig), or an antigen-binding fragment thereof, wherein 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;
b. a linker (L) covalently conjugated to the reactive residue of the second variable domain of the Ig; and
c. a double-stranded RNA (dsRNA) molecule conjugated to the linker, where the dsRNA is capable of inhibiting the expression of a target gene, where the dsRNA comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference.
2 . The conjugate of claim 1 , wherein the dsRNA has a melting temperature (T m ) of from about 40° C. to about 80° C.
3 . (canceled)
4 . The conjugate according to claim 1 , wherein the dsRNA comprises at least four 2′-fluoro modifications.
5 . The conjugate of claim 1 , wherein the dsRNA comprises a duplex region of 12-40 nucleotide base pairs in length.
6 . The conjugate of claim 5 , wherein the dsRNA comprises a duplex region of 18-25 nucleotide base pairs in length.
7 . The conjugate of claim 1 , wherein the dsRNA comprises a blunt end at 5′-end of the antisense strand.
8 . The conjugate of claim 1 , wherein the dsRNA comprises an overhang at 3′-end of the antisense strand.
9 . (canceled)
10 . The conjugate of claim 1 , wherein the sense strand is covalently conjugated with the linker.
11 .- 14 . (canceled)
15 . The conjugate of claim 1 , wherein the sense strand comprises 2 to 5 2′-fluoro modifications.
16 .- 18 . (canceled)
19 . The conjugate of claim 1 , wherein the sense strand comprises 0 to 4 phosphorothioate internucleotide linkages.
20 .- 22 . (canceled)
23 . The conjugate of claim 1 , wherein the antisense comprises 2 to 6 2′-fluoro modifications.
24 .- 26 . (canceled)
27 . The conjugate of claim 1 , wherein the antisense comprises 2 to 4 phosphorothioate internucleotide linkages
28 .- 29 . (canceled)
30 . The conjugate of claim 1 , wherein the antisense strand comprises at least one thermally destabilizing modification of the duplex within the first 9 nucleotide positions of the 5′ region.
31 .- 32 . (canceled)
33 . The conjugate of claim 1 , wherein the antisense comprises a 5′-vinylphosphonate nucleotide at 5′-end.
34 . The conjugate of claim 1 , wherein the dsRNA comprises at least one 2′-OMe modification or the dsRNA comprises at least one locked nucleic acid (LNA) modification.
35 .- 38 . (canceled)
39 . The conjugate of claim 1 , wherein the first variable domain of Ig is positioned closer to an N-terminus than the second variable domain.
40 . The conjugate of claim 1 , wherein Ig is a bispecific immunoglobulin molecule.
41 .- 42 . (canceled)
43 . The conjugate of claim 1 , wherein:
(i) Ig comprises a chimeric immunoglobulin sequence; or
(ii) Ig comprises a humanized immunoglobulin sequence; or
(iii) Ig comprises a human immunoglobulin sequence.
44 .- 45 . (canceled)
46 . The conjugate of claim 1 , wherein the binding target is a tumor cell surface antigen.
47 . (canceled)
48 . The conjugate of claim 1 , wherein the linker L is a reversible linker, an irreversible linker, a cleavable linker, a non-cleavable linker, a branched linker, or a linear linker.
49 .- 53 . (canceled)
54 . The conjugate of claim 1 , wherein the Ig further comprises a ligand.
55 .- 56 . (canceled)
57 . The conjugate of 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, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and any combinations thereof.
58 . The conjugate of claim 1 , wherein the Ig comprises a peptide linker between the first variable domain and the second variable domain.
59 . (canceled)
60 . The conjugate of claim 1 , 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 combinations thereof.
61 . (canceled)
62 . A pharmaceutical composition comprising the conjugate of claim 1 alone or in combination with a pharmaceutically acceptable carrier or excipient.
63 . (canceled)
64 . A method for silencing a target gene in a cell, the method comprising introducing a conjugate of claim 1 into the cell.
65 . (canceled)