TARGETED DELIVERY TO LEUKOCYTES USING PROTEIN CARRIERS
Disclosed herein are is a leukocyte-selective delivery agent comprising, a targeting moiety that selectively binds LFA-I, a protein carrier moiety covalently linked to the targeting moiety, and a therapeutic agent associated with the carrier moiety. The delivery agent may be further selective for activated leukocytes, wherein the targeting moiety selectively binds LFA-I in its activated conformation. The targeting moiety comprises an antibody or functional fragment thereof, such as an scFV. Examples of antibodies or fragments thereof which selectively bind LFA-I activated conformation bind to the locked open I domain of LFA-I, or binds to the leg domain of the β2 subunit of LFA-I ((ILP2)—The antibody or functional fragment thereof may alternatively bind non-selectively to both low affinity and high affinity LFA-I. Examples of a non-protein carrier are a basic polypeptide such as protamine or a functional fragment thereof. One such fragment is RSQSRSRYYRQRQRSRRRRRRS. The therapeutic agent may comprise one or more of a nucleic acid, a small molecule, a polypeptide, and an antibody or functional fragment thereof. An example of a nucleic acid delivery agent comprises an RNA interference molecule. Examples of RNA interference molecules are siRNA, dsRNA, StRNA, shRNA, miRNA, and combinations thereof. Specific siRNAs are provided. Other examples of a nucleic acid delivery agent are a small RNA, an antagomir, an LNA, and an antisense oligonucleotide. Methods for leukocyte-selective delivery, or activated leukocyte-selective delivery in vivo, in vitro and ex vivo are also provided.
1 . (canceled)
2 . A leukocyte-selective delivery agent comprising,
(a) a targeting moiety that selectively binds LFA-I;
(b) a protein carrier moiety covalently linked to the targeting moiety; and
(c) a therapeutic agent associated with the carrier moiety.
3 . The delivery agent of claim 2 , which is further selective for activated leukocytes, wherein the targeting moiety selectively binds LFA-I in its activated conformation.
4 . The delivery agent of claim 2 , wherein the targeting moiety comprises an antibody or functional fragment thereof.
5 . The delivery agent of claim 4 , wherein the targeting moiety comprises an scFV.
6 . The delivery agent of claim 4 , wherein the antibody or functional fragment thereof binds to the locked open I domain of LFA-I, or binds to the leg domain of the β 2 subunit of LFA-I (α L β 2 ).
7 . The delivery agent of claim 5 , wherein the antibody or functional fragment thereof binds non-selectively to both low affinity and high affinity LFA-I.
8 . The delivery agent of claim 2 , wherein the protein carrier moiety comprises a basic polypeptide.
9 . The delivery agent of claim 8 wherein the basic polypeptide comprises protamine or a functional fragment thereof.
10 . The delivery agent of claim 9 , wherein the protein carrier moiety comprises the amino acid sequence RSQSRSRYYRQRQRSRRRRRRS (SEQ ID NO: 7).
11 . The delivery agent of claim 2 , wherein the therapeutic agent comprises one or more agents selected from the group consisting of a nucleic acid, a small molecule, a polypeptide, and an antibody or functional fragment thereof.
12 . The delivery agent of claim 11 wherein the nucleic acid comprises an RNA interference molecule.
13 . The delivery agent of claim 12 wherein the RNA interference molecule is selected from the group consisting of siRNA, dsRNA, stRNA, shRNA, miRNA, and combinations thereof.
14 . The delivery agent of claim 13 wherein the therapeutic agent comprises CCR5-siRNA, ku70-siRNA, CD4-siRNA or cyclin-D1-siRNA.
15 . The delivery agent of claim 12 , wherein the nucleic acid comprises a small RNA, an antagomir, an LNA, or an antisense oligonucleotide.
16 . A method for activated leukocyte-selective delivery comprising, administering to a subject an activated leukocyte-selective delivery agent comprising,
(a) a targeting moiety that selectively binds LFA-I in its activated conformation;
(b) a protein carrier moiety covalently linked to the targeting moiety; and
(c) a therapeutic agent associated with the carrier moiety;
to contact the delivery agent with activated leukocytes of the subject, to thereby selectively deliver the therapeutic agent to activated leukocytes of the subject.
17 . The method of claim 16 , wherein the subject has inappropriate leukocyte activation prior to administration of the delivery agent.
18 . The method of claim 16 wherein the targeting moiety comprises an antibody or functional fragment thereof, which binds to the locked open I domain of LFA-I better than the locked closed I domain of LFA-i, or binds to the leg domain of the β 2 subunit of LFA-I (α L β 2 ).
19 . A method for in vivo leukocyte-selective delivery of a therapeutic agent, comprising, administering to a subject a leukocyte-selective delivery agent comprising,
(a) a targeting moiety that selectively binds LFA-I;
(b) a protein carrier moiety covalently linked to the targeting moiety; and
(c) a therapeutic agent associated with the carrier moiety; to contact the delivery agent with leukocytes of the subject, to thereby selectively deliver the therapeutic agent to leukocytes of the subject.
20 . The method of claim 19 wherein the delivery agent is further selective for activated leukocytes, wherein the targeting moiety selectively binds LFA-I in its activated conformation.
21 . The method of claim 19 , wherein the targeting moiety comprises an antibody or functional fragment thereof.
22 . The method of claim 21 , wherein the antibody or functional fragment thereof comprises a scFV.
23 . The method of claim 21 , wherein the antibody or functional fragment thereof binds to the locked open I domain of LFA-I better than the locked closed I domain of LFA-I, or binds to the leg domain of the β 2 subunit of LFA-I (CILP 2 ).
24 . The method of claim 21 , wherein the antibody or functional fragment thereof binds non-selectively to both low affinity and high affinity LFA-I.
25 . The method of claim 19 , wherein the protein carrier moiety comprises a basic polypeptide.
26 . The method of claim 25 wherein the basic polypeptide comprises protamine or a functional fragment thereof.
27 . The method of claim 26 , wherein the carrier moiety comprises the amino acid sequence RSQSRSRYYRQRQRSRRRRRRS (SEQ ID NO: 7).
28 . The method of claim 19 , wherein the therapeutic agent comprises one or more agents selected from the group consisting of a nucleic acid, a small molecule, a polypeptide, and an antibody or functional fragment thereof.
29 . The delivery agent of claim 28 wherein the nucleic acid comprises an RNA interference molecule.
30 . The method of claim 29 , wherein the RNA interference molecule is selected from the group consisting of siRNA, dsRNA, stRNA, shRNA, miRNA, and combinations thereof.
31 . The method of claim 30 , wherein the siRNA comprises CCR5-siRNA, ku70-siRNA, CD4-siRNA or cyclin-D1-siRNA.
32 . A method for leukocyte-selective delivery comprising:
a) providing a leukocyte-selective delivery agent comprising,
(1) a targeting moiety that selectively binds LFA-I;
(2) a protein carrier moiety covalently linked to the targeting moiety; and
(3) a therapeutic agent associated with the carrier moiety;
b) contacting the delivery agent to a population of cells comprising leukocytes, to thereby selectively deliver the therapeutic agent to leukocytes in the population of cells.
33 . The method of claim 32 wherein the population of cells is obtained from a subject, and contacting step b) is performed in vitro.
34 . The method of claim 33 further comprising administering the population of cells contacted with the delivery agent, to the subject.