CHIMERIC ACTIVATORS: QUANTITATIVELY DESIGNED PROTEIN THERAPEUTICS AND USES THEREOF

• US 20110274658A1
• Filed: 04/05/2008
• Published: 11/10/2011
• Est. Priority Date: 04/05/2007
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Miguez, David G., Cironi, Pablo, Silver, Pamela A.
• Granted Patent: US 11,535,673 B2
• Time in Patent Office: Days
• US Class Current: 424/85.7
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 47/642 the peptide or protein in the drug conjugate being a cytokine, e.g. IL2, chemokine, growth factors or interferons being the inactive part of the conjugate
• CPC Class Codes: A61K 47/6425 the peptide or protein in the drug conjugate being a receptor, e.g. CD4, a cell surface antigen, i.e. not a peptide ligand targeting the antigen, or a cell surface determinant, i.e. a part of the surface of a cell
• CPC Class Codes: A61K 47/6813 the drug being a peptidic cytokine, e.g. an interleukin or interferon
• CPC Class Codes: A61K 47/6849 the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
• CPC Class Codes: A61K 47/6853 Carcino-embryonic antigens
• CPC Class Codes: A61P 11/06 Antiasthmatics
• CPC Class Codes: A61P 3/04 Anorexiants; Antiobesity agents
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 7/06 Antianaemics
• CPC Class Codes: C07K 14/485 Epidermal growth factor [EGF], i.e. urogastrone
• CPC Class Codes: C07K 14/525 Tumour necrosis factor [TNF]
• CPC Class Codes: C07K 14/56 IFN-alpha
• CPC Class Codes: C07K 14/5759 Products of obesity genes, e.g. leptin, obese (OB), tub, fat
• CPC Class Codes: C07K 16/2863 against receptors for growth factors, growth regulators
• CPC Class Codes: C07K 16/2896 against molecules with a "CD"-designation, not provided for elsewhere
• CPC Class Codes: C07K 16/3007 Carcino-embryonic Antigens
• CPC Class Codes: C07K 2317/34 Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• CPC Class Codes: C07K 2317/76 : Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• CPC Class Codes: C07K 2319/30 : Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• CPC Class Codes: C07K 2319/33 : fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
• CPC Class Codes: C07K 2319/74 : containing a fusion for binding to a cell surface receptor

Abstract

Aspects of the invention provide methods for harnessing the potential of proteins that occur naturally (e.g., in humans) and that have serious but finite toxicity. Aspects of the invention relate to a quantitative systems-biological and structural approach to design a class Mof chimeric proteins that avoid the toxicity of protein drugs while retaining their desired activities. In particular, chimeric proteins containing a variant form of a natural protein fused to a targeting moiety may be administered to a subject to target a signal (e.g., induction of apoptosis) to particular cells without having a generalized toxic effect

Claims

1. An isolated nucleic acid encoding a chimeric activator, the nucleic acid comprising in frame a first sequence, a second sequence, and optionally a third sequence, wherein the first sequence encodes an activity element, the second sequence encodes a targeting element, and the third sequence encodes a linker, and wherein the first sequence includes a mutation that reduces the binding affinity of the activity element for a natural cell surface receptor.

2. An isolated chimeric activator protein encoded by claim 1.

3. An isolated nucleic acid of claim 1, wherein the first, second, and third sequences are arranged to encode a chimeric activator protein that comprises the targeting element at its N-terminal end, the activity element at its C-terminal end, and the linker connecting the C-terminal end of the targeting element to the N-terminal end of the activity element.

4. An isolated nucleic acid of claim 1, wherein the first, second, and third sequences are arranged to encode a chimeric activator protein that comprises the activity element at its N-terminal end, the targeting element at its C-terminal end, and the linker connecting the C-terminal end of the activity element to the N-terminal end of the targeting element.

5. A chimeric activator protein of claim 2, wherein said chimeric activator protein selectively binds to cancer cells relative to normal cells.

6. A nucleic acid of claim 1, wherein the activity element is a cytokine.

7. A nucleic acid of claim 1, wherein the targeting element is an antibody, an antibody fragment, or an Fc region.

10. A nucleic acid of claim 1, wherein the activity element activates apoptosis.

11. A nucleic acid of claim 1, wherein the activity element is an interferon.

12. A nucleic acid of claim 11, wherein the activity element is a mutant interferon 2 alpha.

13. A nucleic acid of claim 12, wherein the activity element is interferon 2 alpha (K133A), interferon 2 alpha (R144A) or interferon 2 alpha (R149A).

16. A nucleic acid of claim 1, wherein the targeting element is a ligand.

17. A nucleic acid of claim 16, wherein the targeting element is epidermal growth factor.

8-9. -9. (canceled)

14-15. -15. (canceled)

18-23. -23. (canceled)

24. A recombinant protein comprising

a first element that binds to a first receptor on a target cell;

a second element that binds to a second receptor on the target cell; and

optionally a linker that connects the first and second elements,

wherein the linker allows the first and second elements to bind simultaneously to the first and second receptors on the target cell, wherein the first element comprises a mutation that reduces its binding affinity for its receptors, and wherein the first element transmits a signal to the host cell when it is bound to the first receptor.

25. The recombinant protein of claim 24, wherein the first element binds to its receptor as a monomer.

26. The recombinant protein of claim 24, wherein the second element binds to its receptor as a monomer.

27. The recombinant protein of claim 24, wherein the linker is between about 25 and about 70 amino acids long.

28. The recombinant protein of claim 24, wherein the linker is flexible.

29-38. -38. (canceled)

39. A method for manufacturing a chimeric activator comprising an activity element and a targeting element joined by a linker, wherein the targeting element binds to a first binding site on a target cell, and wherein the activity element binds to a second binding site on a target cell, and wherein the linker length has been optimized for maximizing binding efficiency of the activity element to the second binding site when the targeting element is bound to the first binding site, the method comprising:

i) optimizing the linker length as a function of the concentration of the first and second binding sites, the size of the activity element and targeting element, the minimum distance between the binding sites of the targeting element and the activity element, and the affinities of the activity element and the targeting element for the first and second binding sites; and

ii) preparing a chimeric activator having an optimal linker length.

40. (canceled)

41. A method for targeted cellular regulation, the method comprising:

administering to a subject a biologically effective amount of a chimeric activator comprising an activity element and a targeting element joined by a linker, wherein the targeting element binds to a first binding site on a target cell, and wherein the activity element binds to a second binding site on a target cell, and wherein the linker length has been optimized for maximizing binding efficiency of the activity element to the second binding site when the targeting element is bound to the first binding site.

42. (canceled)