Regulation of biological events using novel compounds

Details Regulation of biological events using novel compounds Regulation of biological events using novel compounds

1998-01-22

2001-02-13

Schwartzman, Robert A. (Department: 1636)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Carbohydrate doai

C435S325000, C435S355000, C435S372000, C435S372300, C435S375000

Reexamination Certificate

active

06187757

ABSTRACT:

BACKGROUND OF THE INVENTION
Rapamycin is a macrolide antibiotic produced by
Streptomyces hygroscopicus
which binds to a FK506-binding protein, FKBP, with high affinity to form a rapamycin:FKBP complex. Reported Kd values for that interaction are as low as 200 pM. The rapamycin:FKBP complex binds with high affinity to the large cellular protein, FRAP, to form a tripartite, [FKBP:rapamycin]:[FRAP], complex. In that complex rapamycin acts as a dimerizer or adapter to join FKBP to FRAP.
A number of naturally occurring FK506 binding proteins (FKBPs) are known. See e.g. Kay, 1996, Biochem. J. 314:361-385 (review). FKBP-derived domains have been incorporated in the design of chimeric proteins for use in biological switches in genetically engineered cells. Such switches rely upon ligand-mediated multimerization of the protein components to trigger a desired biological event. See e.g. Spencer et al, 1993, Science 262:1019-1024 and PCT/US94/01617. While the potent immunosuppressive activity of FK506 would limit its utility as a multimerizing agent, especially in animals, dimers of FK506 (and related compounds) can be made which lack such immunosuppressive activity. Such dimers have been shown to be effective for multimerizing chimeric proteins containing FKBP-derived ligand binding domains.
Rapamycin, like FK506, is also capable of multimerizing appropriately designed chimeric proteins. We have previously designed biological switches using rapamycin and various derivatives or analogs thereof (“rapalogs”) as multimerizing agents (see WO96/41865). In the case of rapamycin itself, its significant biological activities, including potent immunosuppressive activity, rather severely limit its use in biological switches in certain applications, especially those in animals or animal cells which are sensitive to rapamycin. Improved rapalogs for such applications, especially rapalogs with reduced immunosuppressive activity, would be very desirable.
A large number of structural variants of rapamycin have been reported, typically arising as alternative fermentation products or from synthetic efforts to improve the compound's therapeutic index as an immunosuppressive agent. For example, the extensive literature on analogs, homologs, derivatives and other compounds related structurally to rapamycin (“rapalogs”) include, among others, variants of rapamycin having one or more of the following modifications relative to rapamycin: demethylation, elimination or replacement of the methoxy at C7, C42 and/or C29; elimination, derivatization or replacement of the hydroxy at C13, C43 and/or C28; reduction, elimination or derivatization of the ketone at C14, C24 and/or C30; replacement of the 6-membered pipecolate ring with a 5-membered prolyl ring; and alternative substitution on the cyclohexyl ring or replacement of the cyclohexyl ring with a substituted cyclopentyl ring. Additional historical information is presented in the background sections of U.S. Pat. Nos. 5,525,610; 5,310,903 and 5,362,718.
U.S. Pat. No. 5,527,907 is illustrative of the patent literature. That document discloses a series of compounds which were synthesized in an effort to make immunosuppressive rapalogs with reduced side effects. The compounds are disclosed via seven generic structural formulas, each followed by extensive lists (two to five or more columns of text each) setting forth possible substituents at various positions on the rapamycin ring. The document includes over 180 synthetic examples. The many structural variants of that invention were reported to be potent immunosuppressive agents.
SUMMARY OF THE INVENTION
This invention provides methods and materials for multimerizing chimeric proteins in genetically engineered cells using improved rapalogs, preferably while avoiding the immunosuppressive effects of rapamycin.
The genetically engineered cells contain one or more recombinant nucleic acid constructs encoding specialized chimeric proteins as described herein. Typically a first chimeric protein contains one or more FKBP domains which are capable of binding to an improved rapalog of this invention. This first chimeric protein is also referred to herein as an “FKBP fusion protein” and further comprises at least one protein domain heterologous to at least one of its FKBP domains. The complex formed by the binding of the FKBP fusion protein to the rapalog is capable of binding to a second chimeric protein which contains one or more FRB domains (the “FRB fusion protein”). The FRB fusion protein further comprises at least one protein domain heterologous to at least one of its FRB domains. In some embodiments, the FKBP fusion protein and the FRB fusion protein are different from one another. In other embodiments, however, the FKBP fusion protein is also an FRB fusion protein. In those embodiments, the chimeric protein comprises one or more FKBP domains as well as one or more FRB domains. In such cases, the first and second chimeric proteins may be the same protein, may be referred to as FKBP-FRB fusion proteins and contain at least one domain heterologous to the FKBP and/or FRB domains.
The chimeric proteins may be readily designed, based on incorporation of appropriately chosen heterologous domains, such that their multimerization triggers one or more of a wide variety of desired biological responses. The nature of the biological response triggered by rapalog-mediated complexation is determined by the choice of heterologous domains in the fusion proteins. The heterologous domains are therefore referred to as “action” or “effector” domains.The genetically engineered cells for use in practicing this invention will contain one or more recombinant nucleic acid constructs encoding the chimeric proteins, and in certain applications, will further contain one or more accessory nucleic acid constructs, such as one or more target gene constructs. Illustrative biological responses, applications of the system and types of accessory nucleic acid constructs are discussed in detail below.
A system involving related materials and methods is disclosed in WO 96/41865 (Clackson et al) and is expected to be useful in a variety of applications including, among others, research uses and therapeutic applications. That system involves the use of a multimerizing agent comprising rapamycin or a rapalog of the generic formula:
wherein U is —H, —OR
1
, —SR
1
, —OC(O)R
1
, —OC(O)NHR
1
, —NHR
1
, —NHC(O)R
1
, —NHSO
2
-R
1
or —R
2
; R
2
is a substituted aryl or allyl or alkylaryl (e.g. benzyl or substituted benzyl); V is —OR
3
or (═O); W is ═O, ═NR
4
═NOR
4
, ═NNHR
4
, —NHOR
4
, —NHNHR
4
, —OR
4
, —OC(O)R
4
, —OC(O)NR
4
or —H; Y is —OR
5
, —OC(O)R
5
or —OC(O)NHR
5
; Z is ═O, —OR
6
, —NR
6
, —H, —NC(O)R
6
, —OC(O)R
6
or —OC(O)NR
6
; R
3
is H, —R
7
, —C(O)R
7
, —C(O)NHR
7
or C-28/C-30 cyclic carbonate; and R
4
is H or alkyl; where R
1
, R
4
, R
5
, R
6
and R
7
are independently selected from H, alkyl, alkylaryl or aryl, as those terms are defined in WO 96/41865. A number of rapalogs are specifically disclosed in that document.
The subject invention is based upon a system similar to that disclosed in WO 96/41865, but involves the use of improved rapalogs as the multimerizing agents. The subject invention thus provides a method for multimerizing chimeric proteins in cells which comprises (a) providing appropriately engineered cells containing nucleic acid constructs for directing the expression of the desired chimeric protein(s) and any desired accessory recombinant constructs, and (b) contacting the cells with an improved rapalog or a pharmaceutically acceptable derivative thereof as described herein. The rapalog forms a complex containing itself and at least two molecules of the chimeric protein(s). Improved rapalogs for use in this invention include the following.
One class of improved rapalogs for use in this invention consists of those compounds which comprise the substructure shown in Formula I:
bearing any number of a variety of substituents, and optionally unsaturated at one or more ca

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