Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Genetically modified micro-organism – cell – or virus
Reexamination Certificate
2000-01-12
2003-01-14
Ketter, James (Department: 1632)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Genetically modified micro-organism, cell, or virus
C424S093210, C435S320100
Reexamination Certificate
active
06506379
ABSTRACT:
BACKGROUND OF THE INVENTION
Rapamycin (I) is a natural product which binds to a FK506-bining 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 this tripartite complex rapamycin acts as a dimerizer or adapter to join FKBP to FRAP. The portion of the FRAP protein which interacts with the FKBP:rapamycin complex is referred to as the FRB domain, which is discussed in detail below. The rapamycin-dependent association of FKBP12 and a large mammalian protein termed FRAP, RAFT1 or RAPT1 and its yeast homologs DRR and TOR have been described by several research groups. See e.g., Brown et al, 1994, Nature 369:756-758; Sabatini et al, 1994, Cell 78:35-43; Chiu et al, 1994, Proc. Natl. Acad. Sci. USA 91:12574-12578; Chen et al, 1994, Biochem. Biophys. Res. Comm. 203:1-7; Kunz et al, 1993 Cell 73:585-596; and Cafferkey et al, 1993 Mol. Cell. Biol. 13:6012-6023. Chiu et al, supra, and Stan et al, 1994, J. Biol. Chem. 269:32027-32030 describe the rapamycin-dependent binding of FKBP12 to smaller subunits of FRAP containing the FRB domain. FRAP, RAFT, RAPT and the TOR proteins each contain homologous FRB domains and are considered FRAP proteins for the purpose of this document.
Numerous naturally occurring FK506 binding proteins (FKBPs) are known. See e.g. Kay, 1996, Biochem. J. 314:361-385 (review). FKBP proteins have been used for their ligand-binding properties in biological switches based on ligand-mediated multimerization of immunophilin-based recombinant proteins as disclosed e.g. in Spencer et al, 1993, Science 262:1019-1024 and in PCT/US94/01617. While the potent immunosuppressive activity of FK506 would limit its utility as a dimerizer, especially in animals, dimers of FK506 (and related compounds) lack such immunosuppressive activity and have been shown to be effective for dimerizing chimeric proteins containing ligand binding domains derived from FKBP.
While rapamycin, like FK506, is a natural dimerizer of proteins, and is capable of dimerizing appropriately designed chimeric proteins, its significant biological activities, including potent immunosuppressive activity, rather severely limit its use in engineered biological switches, particularly for use in animals. This invention harnesses the dimerizing potential of rapamycin (and related compounds) while avoiding its profound, inherent limitations.
SUMMARY OF THE INVENTION
This invention concerns new configurations for biological switches and provides new methods and materials for regulating biological events, particularly in animal cells. Those biological events include, for example, gene transcription, activation of an intracellular signal transduction pathway leading for example to gene expression or apoptotic cell death, gene knock-out, blockade of expression of a gene, and inhibition of the function of a gene product. The invention relies upon two types of chimeric proteins which when complexed through mutual binding to a common ligand, are capable of actuating, directly or indirectly, the desired event.
This invention encompasses recombinant DNA constructs encoding those chimeric proteins; DNA vectors containing one or more of those constructs; the fusion proteins encoded by the foregoing constructs; cells, especially animal cells, transformed with (i.e., containing and capable of expressing) one or more of the DNA constructs described herein; small molecules (bivalent or multivalent multimerizing agents) which bind to and are capable of inducing multimerization of the chimeric protein molecules; and, methods for preparing and using the foregoing.
More specifically, this invention provides methods and materials for making and using genetically engineered cells which are responsive to the presence of rapamycin or to the presence of an analog, mimic or derivative of rapamycin (a “rapalog”). The invention relies upon the introduction into cells of recombinant DNAs encoding a set of fusion proteins which are capable of complexing with each other in the presence of rapamycin or a rapalog. Contacting such genetically engineered cells with rapamycin or a suitable rapalog results in complexation of the fusion proteins and the initiation of a biological response. One of the fusion proteins contains one or more copies of an EKBP:rapamycin binding (FRB) domain and at least one heterologous protein domain. The second fusion protein contains one or more copies of a domain derived from an FKBP protein which is capable of binding to rapamycin or a rapalog and forming a complex with an FRB-containing protein. The second fusion protein also contains at least one heterologous domain which may be the same or different from a heterologous domain of the first fusion protein. FRB and FKBP domains for use in fusion proteins of this invention may be selected from naturally occurring proteins and may be variously modified, as is discussed in detail below. While FRB, FKBP and heterologous domains derived from various species may be used, human peptide sequences or variants thereof are preferred for human gene therapy applications. Operationally, the FRB and FKBP domains serve as receptor (or “ligand-binding”) domains and direct the complexation of the fusion proteins under the mediation of rapamycin or rapalog molecules. The nature of the biological response triggered by rapamycin- or rapalog-mediated complexation is determined by the heterologous domains of the fusion proteins. The heterologous domains are therefore also referred to as “action” domains.
Various heterologous protein domains may be used in these fusion proteins. In one aspect of the invention, the two fusion proteins (one of which contains at least one FRB domain, the other contains at least one FKBP domain) each contain at least one different heterologous domain, i.e., a heterologous domain not contained in the other fusion protein. For example, in certain embodiments, one of the fusion proteins contains at least one DNA binding domain and the other fusion protein contains at least one transcription activation domain. Ligand-mediated association of the fusion proteins represents the formation of a transcription factor complex and leads to initiation of transcription of a target gene linked to a DNA sequence recognized by (i.e., capable of binding with) a DNA-binding domain on one of the fusion proteins. In other embodiments, one of the fusion proteins contains at least one domain capable of directing the fusion protein to a particular cellular location such as the cell membrane, nucleus, etc. Localization domains which target the cell membrane include domains such as a myristoylation site or a transmembrane region of a receptor protein or other membrane-spanning protein. The other fusion protein contains a signalling domain capable, upon membrane localization and/or clustering, of activating a cellular signal transduction pathway. Examples of signalling domains include an intracellular domain of a growth factor or cytokine receptor, an apoptosis triggering domain such as the intracellular domain of FAS or TNF-R1, and domains derived from other intracellular signalling proteins such as SOS, Raf, Ick, ZAP-70, etc. A number of signalling proteins are disclosed in PCT/US94/01617 (see e.g. pages 23-26). In still other embodiments, each of the fusion proteins contains at least one FRB domain and at least one FKBP domain, as well as one or more heterologous domains. Such fusion proteins are capable of homodimerization in the presence of rapamycin or a rapalog. In general, domains containing peptide sequence endogenous to the host cell are preferred. Thus, for human gene therapy applications, domains of human origin are of particular interest Recombinant DNA molecules encoding the fusion proteins are also provided, as are vectors capable of directing their expression, particularly in eukaryotic cells, of which yeast and animal cells are of particula
Clackson Timothy P.
Gilman Michael
Holt Dennis
Ariad Gene Therapeutics, Inc.
Berstein David L.
Ketter James
Li Janice
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