Nucleic acid molecules encoding cytotoxic conjugates that contain a chemokine receptor targeting agent
View Patent ↗Nucleic acid moleucles that encode conjugates containing as a ligand a chemokine receptor targeting agent, such as a chemokine, and a targeted agent, such as a toxin are provided. These conjugates are used to treat inflammatory responses associated with activation, proliferation and migration of immune effector cells, including leukocyte cell types, neutrophils, macrophages, and eosinophils.
1. A nucleic acid molecule, comprising a sequence of nucleotides encoding a conjugate that comprises a toxin and a chemokine receptor targeting agent, or a portion thereof sufficient for binding to a chemokine receptor and internalization of the chemokine receptor targeting agent, wherein:
the conjugate binds to a chemokine receptor resulting in internalization of the toxin in cells bearing the receptor; and
the receptor occurs on activated immune cells.
2. A nucleic acid molecule of claim 1 , wherein the encoded conjugate comprises the following components: (chemokine receptor targeting agent) n , (L) q and (toxin) m , wherein:
L is a linker for linicing the chemokine receptor targeting agent to a toxin;
chemokine receptor targeting agent is any moiety that selectively binds to a chemokine receptor;
m and n, which are selected independently, are at least 1; and
q is 0 or more as long as the resulting conjugate binds to the targeted receptor, is internalized and delivers the toxin;
the resulting conjugate binds to a receptor that interacts with and internalizes a chemokine, whereby the toxin(s) is internalized in a cell bearing the receptor, and
when the conjugate contains a plurality of toxins the toxins are the same or different, and when the conjugate contains a plurality of chemokine receptor targeting agents the targeting agents are the same or different.
3. The nucleic acid molecule of claim 2 , wherein m and n, which are selected independently, are 1–6.
4. The nucleic acid molecule of claim 2 , wherein q is 1, n is 2 and m is 1.
5. The nucleic acid molecule of claim 1 , wherein the chemokine receptor targeting agent is a chemokine, an antibody that specifically binds to a chemokine receptor or a fragment of the chemokine or antibody, wherein the fragment binds to the receptor and internalizes the toxin.
6. The nucleic acid molecule of claim 1 , wherein the chemokine receptor targeting agent specifically binds to chemokine receptors on activated leukocytes.
7. The nucleic acid molecule of claim 1 , wherein the chemokine receptor targeting agent specifically binds to chemokine receptors on cells selected from mononuclear phagocytes (MNP), leukocytes, natural killer cells, dendritic cells, T lymphocytes and B lymphocytes.
8. The nucleic acid molecule of claim 6 , wherein the leukocytes are selected from basophils, neutrophils, eosinophils, and combinations of any two or more thereof.
9. The nucleic acid molecule of claim 1 that comprises DNA.
10. The nucleic acid molecule of claim 1 , wherein the chemokine receptor targeting agent and targeting agent are joined via a linker.
11. The nucleic acid molecule of claim 10 , wherein the linker is a peptide or an amino acid.
12. The nucleic acid molecule of claim 1 , further comprising nucleic acid that encodes a non-chemokine cytokine or a receptor associated protein that binds to receptors on and/or activates one or more of the cells that promote secondary tissue damage, other than chemokine receptors.
13. The nucleic acid molecule of claim 12 , wherein the cytokine binds to a cytokine-specific receptor expressed on cells that express chemokine receptors.
14. The nucleic acid molecule of claim 13 , wherein the cytokine is selected from among interleukins, lymphokines, monokines, colony-stimulating factors and receptor associated proteins.
15. The nucleic acid molecule of claim 1 , wherein the chemokine receptor targeting agent is a monoclonal antibody, or an antigen-specific fragment thereof.
16. The nucleic acid molecule of claim 1 , further comprising nucleic acid that encodes an antibody that binds to a non-chemokine cytokine receptor and/or to a non-chemokine cytokine of claim 1 .
17. A plasmid, comprising the nucleic acid molecule of claim 1 .
18. A isolated host cell, comprising the plasmid of claim 17 .
19. A method of producing a conjugate, comprising culturing the cell of claim 18 under conditions, whereby a fusion protein comprising the conjugate is expressed, and isolating the fusion protein.
20. A nucleic acid molecule of claim 1 , wherein the chemokine is selected from the group consisting of eotaxin, stromal derived factor-1β, SDF-1α, monocyte chemotactic protein-1 (MCP-1), MCP-3, macrophage inhibitory protein-1α (MIP-1α), MIP-1β and Regulated on Activation, Normal T cell Expressed and Secreted (RANTES) protein.
21. A nucleic acid molecule of claim 1 , wherein the chemokine is selected from the group consisting of IL-8, GCP-2, GRO-α, GRO-β, GRP-γ, ENA-78, PBP, CTAP III, NAP-2, LAPF-4, MIG, IP-10, SDF-1α, SDF-1β, SDF-2, MCP-1, MCP-2, MCP-3, MCP-4, MCP-5, MIP-1α, MIP-1β, MIP-1γ, MIP-2, MIP-2α, MIP-3α, MIP-3β, MIP-4, MIP-5, MDC, HCC-1, Tim-1, eotaxin-1, eotaxin-2, I-309, SCYA17, TARC, RANTES, DC-CK-1, lymphotactin, ALP, lungkine and fractalkine.
22. A nucleic acid molecule of claim 1 wherein the encoded conjugate is selected from among OPL98104, OPL98112, OPL98108, OPL98102, OPL98110, OPL98106, OPL98101, OPL98109, OPL98105, OPL98103, OPL98111 and OPL98107 ligand-toxin fusion gene constructs.
23. The nucleic acid molecule of claim 1 , wherein the chemokine receptor targeting moiety effects binding to a receptor selected from the group consisting of CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, XCR1 and CX3CR-1, whereby the conjugate is internalized into a cell bearing the receptor.
24. The nucleic acid molecule of claim 1 , wherein the toxin is a ribosome inactivating protein, a bacterial toxin or a fragment thereof that retains the ribosome inactivating or toxin activity.
25. The nucleic acid molecule of claim 1 , wherein the chemokine receptor targeting agent specifically binds to chemokine receptors on immune effector cells.
26. The nucleic acid molecule of claim 1 , wherein the chemokine receptor targeting agent specifically binds to chemokine receptors on cells selected from among monocytes, macrophages, leukocytes and microglia.