Regulated transcription of targeted genes and other biological events

• US 5,871,753 A
• Filed: 06/07/1995
• Issued: 02/16/1999
• Est. Priority Date: 02/12/1993
• Status: Expired due to Term

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation), Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation), Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Inventors: Ho, Steffan, Spencer, David M., Crabtree, Gerald R., Schreiber, Stuart L., Belshaw, Peter, Wandless, Thomas J.
• Primary Examiner(s): Ketter, James
• Assistant Examiner(s): SANDALS, WILLIAM O
• Application Number:
  US08/473,985
• Time in Patent Office: 1,350 Days
• Field of Search: 424/280.1, 435/183, 530/317, 514/27, 514/183
• US Class Current: 424/280.1
• 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 47/6425 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: C07D 498/18 Bridged systems
• CPC Class Codes: C07F 5/025 Boronic and borinic acid compounds
• CPC Class Codes: C07H 19/01 sharing oxygen
• CPC Class Codes: C07K 14/395 from Saccharomyces
• CPC Class Codes: C07K 14/7051 T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 14/71 for growth factors; for growth regulators
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C07K 2319/03 containing a transmembrane segment
• CPC Class Codes: C07K 2319/035 containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins
• CPC Class Codes: C07K 2319/09 containing a nuclear localisation signal
• CPC Class Codes: C07K 2319/20 containing a tag with affinity for a non-protein ligand
• CPC Class Codes: C07K 2319/32 fusions with soluble part of a cell surface receptor, "decoy receptors"
• CPC Class Codes: C07K 2319/42 containing a HA(hemagglutinin)-tag
• CPC Class Codes: C07K 2319/43 containing a FLAG-tag
• CPC Class Codes: C07K 2319/60 containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
• CPC Class Codes: C07K 2319/71 containing domain for transcriptional activaation, e.g. VP16
• CPC Class Codes: C07K 2319/715 containing a domain for ligand dependent transcriptional activation, e.g. containing a steroid receptor domain
• CPC Class Codes: C07K 2319/81 : containing a Zn-finger domain for DNA binding
• CPC Class Codes: C07K 2319/90 : containing a motif for post-translational modification
• CPC Class Codes: C07K 7/645 : Cyclosporins; Related peptides
• CPC Class Codes: C12N 15/62 : DNA sequences coding for fusion proteins
• CPC Class Codes: C12N 15/63 : Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12P 15/00 : Preparation of compounds containing at least three condensed carbocyclic rings gibbanes C12P27/00; naphthacenes C12P29/00

Abstract

Methods and compositions are provided for modified cells, where a chimeric protein consisting of a ligand binding domain fused to an action domain is employed which initiates a signal which activates a biological process: transcription of at least one gene, usually a second construct introduced into the host cells; exocytosis; or an extracellular process. The second construct optimally present provides for a promoter which responds to a transcriptional activation action domain to provide for transcription, when an appropriate ligand binds to the ligand binding domain. Exemplary of the system is the use of an FKBP/CD3ζ or transcription factor fusion protein, using dimeric FK506 or FK520 as the ligand and a promoter responsive to NF-AT or other transcription factor requiring two molecules for transcriptional activation.

Claims

1. A compound comprising two or more multimerized ligands each independently selected from the group consisting of FK506, FK520 and cyclosporin, or macrocyclic derivatives thereof.

4. The compound of claims 1, wherein at least one of the ligands is a macrolide derivative of FK506, FK520 or cyclosporin A.

6. A compound according to claim 1 or 2, wherein said ligands are linked through amide groups.

14. The compound of any of claims 1, 2, 7, 11 or 12, wherein the compound is membrane permeable.

15. The compound of any of claims 1, 2, 7, 11 or 12, wherein the compound has a molecular weight less than 5 Kd.

16. A compound according to claim 1, 2 or 7 consisting of 2 to 3 ligands covalently joined by a linker group.

17. A compound according to any of claims 1, 2, or 7, wherein the linker is selected from the group consisting of alkylene, azalkylene, N-alkylene azalkylene, arylene, aradialkylene, and bis-carbozamidoalkylene.

27. The compound of any of claims 1, 2, 7, 18 or 19, wherein the compound is at least 100 fold less immunosuppressive than FK506, cyclosporin A or rapamycin.

35. The compound of claim 27, wherein the compound is at least 1000 fold less immunosuppressive than FK 506, Cyclosporin A or rapamycin in a Tag Jurkat cell mitogen activation assay.

2. A compound comprising two or more multimerized ligands each independently selected from the group consisting of FK506, FK520, and macrocyclic derivatives thereof, which compound cross-links two or more FKBP proteins.

3. A compound according to claim 2, wherein said ligands are linked though any of annular carbon atoms at positions 17 to 24 or 32 or through ring substituents.

5. The compound of claim 2, wherein at least one of the ligands is a macrolide derivative of FK506 or FK520.

11. The compound of claim 2, wherein one or more of said ligands of the compound is a macrocycle derivative of a parent compound FK506 or FK520, which derivative has a reduced binding affinity for a native FKBP relative to the parent compound.

13. The compound of any of claims 11 or 12, wherein the derivative of the parent compound has a reduced immunosuppressive activity relative to the parent compound.

7. A compound comprising two or more multimerized ligands independently selected from cyclosporin A or macrocyclic derivatives thereof, which compound cross-links two or more cyclophilin proteins.

8. A compound according to claim 7, wherein said ligands are linked through amide groups.

9. A compound according to claim 7, wherein said ligands are linked through the MeBmt1 sidechain.

10. The compound of claim 7, wherein at least one of the ligands is a macrolide derivative of cyclosporin A.

12. The compound of claim 7, wherein one or more of said ligands of the compound is a macrocycle derivative of a parent compound cyclosporin, which derivative has a reduced binding affinity for a native cyclophilin relative to the patent compound.

18. A compound, having a molecular weight of less than 5 Kd and comprising from 2 to 3 ligands, each ligand of which independently comprises a macrocycle or portion thereof which binds to FKBP or cyclophilin, said units covalently joined together through a common linking group does not include an ester group in the backbone of the linker.

23. The compound claim 18 or 19, wherein each ligand independently comprises a macrolide or portion thereof which binds an FKBP or cyclophilin protein.

19. A compound, having a molecular weight less than 5 kd and comprising two or more ligands covalently joined through one or more linker groups, each ligand independently comprising a macrocycle or portion thereof which binds an FKBP or cyclophilin protein, wherein said compound has a reduced immunosuppressive effect in a Tag Jurkat cell mitogen activation assay.

20. The compound of claim 19, wherein said compound has a reduced immunosuppressive effect in a Tag Jurkat cell mitogen activation assay.

21. The compound of claim 19, consisting of 2 to 3 ligands covalently joined by the linker group.

22. The compound of claim 19, wherein the compound is membrane permeable.

24. The compound of claim 19, wherein the linker group does not include an ester or amide susceptible to cleavage under physiological conditions.

25. The compound of claim 19 or 24, wherein the linker group is resistant to cleavage by proteases.

26. The compound of any of claims 19 or 24, wherein the linker group is selected from the group consisting of alkylene, azaalkylene, N-alkylene azalkylene, arylene, ardialkylene group, and bis-carbozamidoalkylene.

28. A compound, having a molecular weight less than 5 kd and comprising two or more ligands covalently joined through one or more linker group, each ligand independently comprising a macrocycle or portion thereof which binds an FKBP or cyclophilin protein or mutagenized derivative thereof, wherein the compound is at least 100 fold less immunosuppressive than FK506, cyclosporin A or rapamycin.

29. The compound of claim 28, wherein the linker group is resistant to cleavage by proteases.

30. The compound of claim 28, consisting of 2 to 3 ligands covalently joined by the linker group.

31. The compound of claim 28, wherein the compound is membrane permeable.

32. The compound of claim 28, wherein the compound is at least 1000 fold less immunosuppressive than FK 506, cyclosporin A or rapamycin in a Tag Jurkat cell mitogen activation assay.

33. The compound of claim 28, wherein the linker group does not include an ester or amide susceptible to cleavage under physiological conditions.

34. The compound of any of claims 28 or 33, wherein the linker group is selected from the group consisting of alkylene, azaalkylene, N-alkylene azalkylene, arylene, ardialkylene group, and bis-carbozamidoalkylene.