Targeted enzymes

• US 20060134086A1
• Filed: 06/30/2004
• Published: 06/22/2006
• Est. Priority Date: 12/14/2000
• Status: Abandoned Application

• Current Assignee: Anthony G. Day, Christopher J. Murray, David A. Estell, Scott D. Powers, Volker Schellenberger, Yiyou Chen
• Original Assignee: Anthony G. Day, Christopher J. Murray, David A. Estell, Scott D. Powers, Volker Schellenberger, Yiyou Chen
• Inventors: Estell, David A., Schellenberger, Volker, Day, Anthony G., Murray, Christopher J., Power, Scott D., Chen, Yiyou
• Application Number:
  US10/881,221
• Publication Number:
  US 20060134086A1
• Time in Patent Office: Days
• US Class Current: 424/94.100
• 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 47/67 Enzyme prodrug therapy, e.g. gene directed enzyme drug therapy [GDEPT] or VDEPT
• CPC Class Codes: A61K 47/6891 Pre-targeting systems involving an antibody for targeting specific cells
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/01 containing a localisation/targetting motif
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12N 9/00 Enzymes; Proenzymes; Compositions thereof preparations containing enzymes for cleaning teeth A61K8/66, A61Q11/00; medicinal preparations containing enzymes or proenzymes A61K38/43; enzyme containing detergent compositions C11D; enzymes with nucleic acid structure, e.g. ribozymes, C12N15/113; Processes for preparing, activating, inhibiting, separating or purifying enzymes preparation of malt C12C1/00
• CPC Class Codes: C12N 9/50 Proteinases , e.g. Endopeptidases (3.4.21-3.4.25)
• CPC Class Codes: C12N 9/64 derived from animal tissue
• CPC Class Codes: C12N 9/86 acting on amide bonds in cyclic amides, e.g. penicillinase (3.5.2)
• CPC Class Codes: C12Y 305/02006 Beta-lactamase (3.5.2.6)

Abstract

The present invention provides targeted enzymes that bind to targets better than the corresponding pre-targeted enzymes bind the target under like conditions, methods of making targeted enzymes, methods of using targeted enzymes to treat diseases, and pharmaceutical compositions comprising targeted enzymes.

Claims

1. A pharmaceutical composition comprising a targeted enzyme (TE) and a pharmaceutically acceptable carrier, excipient or diluent, said TE exhibiting a catalytic activity and comprising:

a) a substrate recognition site; and

b) a targeting site that binds a target;

wherein

i) the targeting site comprises a variant sequence that is derived from a variation-tolerant sequence of a corresponding pre-targeted enzyme,

ii) the target is bound by the TE but not by the pre-targeted enzyme under like conditions,

iii) the target is not an isolated monoclonal antibody, and

iv) the variation-tolerant sequence is not in a protein binding domain of the pre-targeted enzyme.

9. The pharmaceutical composition of claim 1 wherein the targeted enzyme targeting site comprises two variant sequences targeted enzyme.

10. The pharmaceutical composition of claim 1, wherein the targeted enzyme comprises two targeting sites.

11. The pharmaceutical composition of claim 10, wherein the targeted enzyme targeting sites bind targets of different identities.

12. The pharmaceutical composition of claim 1, wherein the targeted sequence variant sequence is between about 1 and about 50 amino acid residues.

13. The pharmaceutical composition of claim 1, wherein the targeted sequence variant sequence is between about 3 and about 20 amino acid residues.

14. The pharmaceutical composition of claim 1, wherein the targeted enzyme has a molecular weight of less than about 45,000 Daltons.

15. The pharmaceutical composition of claim 1, wherein the targeted enzyme binds the target with a k_d of about 5 nM or less.

16. The pharmaceutical composition of claim 1, wherein the targeted enzyme, while bound to target, exhibits a catalytic activity of greater than about 1% relative to the catalytic activity of the pre-targeted enzyme.

17. The targeted enzyme of one of claims 1, 2, 7 or 8, wherein the pre-targeted enzyme is selected from the group consisting of:

proteases, carboxypeptidases, β

-lactamases, asparaginases, oxidases, hydrolases, lyases, lipases, cellulases, amylases, kinases, phosphatases, transferases, aldolases and reductases.

18. The pharmaceutical composition of claim 1, wherein the target is a protein or a cell.

19. A nucleic acid encoding the targeted enzyme of claim 1.

20. A plasmid comprising the nucleic acid of claim 19.

21. A cell comprising the plasmid of claim 20.

22. A composition comprising the targeted enzyme of claim 1, 2, 7 or 8 and a pharmaceutically acceptable carrier, excipient or diluent.

2. A targeted enzyme exhibiting a catalytic activity, comprising:

a) a substrate recognition site;

b) a first targeting site that binds a first target; and

c) a second targeting site that binds a second target,

wherein

i) each targeting site comprises a variant sequence derived from variation-tolerant sequences of a corresponding pre-targeted enzyme, and

ii) the affinity of the targeted enzyme for the first and second target is greater than the affinity of the pre-targeted enzyme for the first and second target under like conditions.

3. The targeted enzyme of claim 2, wherein the first target and the second target are of a different identity.

4. The targeted enzyme of claim 2, wherein the first target and second target bind targets of the same identity.

5. The targeted enzyme of claim 2, wherein at least one of the targeting sites comprises two variant sequences.

6. The targeted enzyme of claim 5, wherein at least one of the targeting sites comprises three variant sequences.

7. A targeted enzyme exhibiting a catalytic activity, comprising:

a) a substrate recognition site; and

b) a targeting site that binds a target,

wherein

i) the targeting site comprises two variant sequences derived from variation-tolerant sequences of a corresponding pre-targeted enzyme,

ii) the affinity of the targeted enzyme for the target is greater than the affinity of the pre-targeted enzyme for the target under like conditions, and

iii) the target is not an isolated monoclonal antibody.

8. A targeted enzyme exhibiting a catalytic activity, comprising:

a) a substrate recognition site; and

b) a targeting site that binds a target;

wherein

i) the targeting site comprises three variant sequences, wherein each of the variant sequences is derived from variation-tolerant sequences of a corresponding pre-targeted enzyme, and

ii) the affinity of the targeted enzyme for the target is greater than the affinity of the pre-targeted enzyme for the target under like conditions.

23. A pharmaceutical composition comprising a targeted -lactamase enzyme and a pharmaceutically acceptable carrier, excipient, or diluent, said enzyme comprising:

a) a substrate recognition site;

b) a targeting site that binds a target; and

c) a sequence KTXS at its substrate recognition site,

wherein

i) the targeting site comprises a variant sequence that is derived from a variation-tolerant sequence of a corresponding pre-targeted enzyme that does not bind the target,

ii) the target is bound by the targeted β

-lactamase enzyme but not by the pre-targeted β

-lactamase enzyme under like conditions, and

iii) the target is not an isolated monoclonal antibody.

31. The pharmaceutical composition of claim 23, further comprising a sequence VHKTGSTG.

32. The pharmaceutical composition of claim 23, wherein the targeting site comprises two variant sequences.

33. The pharmaceutical composition of claim 23, wherein the targeted β

-lactamase enzyme comprises two targeting sites.

34. The pharmaceutical composition of claim 23, wherein the variation-tolerant sequence is selected from the group consisting of loop A, loop B, loop C, loop D and loop E.

24. A targeted β

-lactamase enzyme exhibiting a catalytic activity, comprising;

a) a substrate recognition site;

b) a first targeting site that binds a first target;

c) a second targeting site that binds a second target; and

d) a sequence KTXS at its substrate recognition site,

wherein

i) each targeting site comprises a variant sequence derived from variation-tolerant sequences of a corresponding pre-targeted enzyme, and

ii) the affinity of the targeted enzyme for the first and second target is greater than the affinity of the pre-targeted enzyme for the first and second target under like conditions.

25. The targeted β

-lactamase enzyme of claim 24, wherein the first target and the second target are of a different identity.

26. The targeted β

-lactamase enzyme of claim 24, wherein the first target and second target bind targets of the same identity.

27. The targeted β

-lactamase enzyme of claim 24, wherein at least one of the targeting sites comprises two variant sequences.

28. The targeted β

-lactamase enzyme of claim 27, wherein at least one of the targeting sites comprises three variant sequences.

29. A targeted β

-lactamase enzyme exhibiting a catalytic activity, comprising;

a) a substrate recognition site;

b) a targeting site that binds a target, and

c) a sequence KTXS at its substrate recognition site,

wherein

i) the targeting site comprises three variant sequences, wherein each of the variant sequences is derived from variation-tolerant sequences of a corresponding pre-targeted β

-lactamase enzyme, and

ii) the affinity of the targeted β

-lactamase enzyme for the target is greater than the affinity of the pre-targeted β

-lactamase enzyme for the target under like conditions.

30. A targeted β

-lactamase enzyme exhibiting a catalytic activity, comprising;

a) a substrate recognition site; and

b) a targeting site that binds a target, and

c) a sequence KTXS at its substrate recognition site,

wherein

i) the targeting site comprises two variant sequences, wherein each of the variant sequences is derived from variation-tolerant sequences of a corresponding pre-targeted β

-lactamase enzyme,

ii) the affinity of the targeted β

-lactamase enzyme for the target is greater than the affinity of the pre-targeted β

-lactamase enzyme for the target, and

iii) the target is not an isolated monoclonal antibody.

35. A method of making a targeted enzyme, comprising:

a) generating a modified enzyme library by modifying a variation-tolerant sequence of an enzyme, wherein said enzyme comprises a substrate recognition site and has a catalytic activity, such that a multiplicity of modified enzymes is produced; and

b) selecting a first and second modified enzyme from the modified enzyme library that binds a target with an affinity that is greater than the affinity of the pre-modified enzyme for the target;

c) recombining a nucleic acid molecule that encodes the first modified enzyme and a nucleic acid that encodes the second modified enzyme so that a recombined nucleic acid molecule is formed that encodes a third modified enzyme; and

d) assaying the third modified enzyme for binding of the target with an affinity that is greater than the affinity of the pre-modified enzyme for the target under like conditions and for the catalytic activity while bound to the target.

36. The method of claim 35, further comprising in step b) selecting a first and second modified enzyme that binds a target with an affinity that is greater than the affinity of the pre-modified enzyme for the target and has the catalytic activity.

37. A method of making a targeted enzyme, comprising:

a) generating a modified enzyme library by modifying a variation-tolerant sequence of an enzyme, wherein said enzyme comprises a substrate recognition site and has a catalytic activity, such that a multiplicity of modified enzymes is produced;

b) identifying a modified enzyme from the modified enzyme library that binds a target with an affinity that is greater than the affinity of the pre-modified enzyme for the target and has the catalytic activity while bound to the target,

c) repeating a cycle of a) and b) as necessary to identify a modified enzyme that binds the target with an affinity that is at least 100-fold greater than the affinity of the unmodified enzyme for the target,

wherein an enzyme modified in a further cycle of a) was identified in a previous cycle of b).