JP2002508971A - Regulation of biological events using multimeric chimeric proteins - Google Patents

Abstract

  (57) [Summary]   Materials and methods are disclosed for target gene transcription and modulation of biological events such as the growth, proliferation or differentiation of genetically engineered cells.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION Rapamycin is derived from Streptomyces hygroscopica
opicus, and binds with high affinity to FK506-binding protein FKBP (FK506-binding protein) to form a rapamycin: FKBP complex. Reported Kd values for the interaction are as low as 200 pM. The rapamycin; FKBP complex binds with high affinity to the large cell protein FRAP and binds the three-segment [FK
BP: rapamycin]: [FRAP] complex. Within the complex, rapamycin acts as a dimerizer or adapter, binding FKBP to FRAP.

[0002]

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[0003] A number of naturally occurring FK506 binding proteins (FKBPs) are known.
See, for example, Kay, 1996, Biochem. J. , 314, 361-385 (
(Commentary). FKBP for use as a biological switch in genetically engineered cells
The induction domain was employed in the design of the chimeric protein. Such switches trigger the desired biological event by ligand-mediated multimerization of the protein component. See, for example, Spencer et al., 1993, Science, 262, 101.
See 9-1024 and PCT / US94 / 01617. The potent immunosuppressive activity of FK506 would limit its effectiveness as a multimerizing agent, especially in animals, but dimers of FK506 lacking such immunosuppressive activity (and related compounds) Can be produced. Such a dimer was found to be effective in multimerizing a chimeric protein containing an FKBP-derived ligand binding domain.

[0004] Rapamycin, such as FK506, also has the ability to multimerize appropriately designed chimeric proteins. Previously, we have used rapamycin and its various derivatives or analogs ["rapalogs"] as multimerizing agents,
I have designed a biological switch (see WO 96/41865). By itself, its significant biological activity, including potent immunosuppressive activity, severely limits its use in biological switches in certain applications, particularly in animals or animal cells that are sensitive to rapamycin. . Rapalogs improved for such applications, particularly those with reduced immunosuppressive activity, would be highly desirable.

[0005] Numerous rapamycin structural variants have been reported, typically as alternative fermentation products or resulting from synthetic efforts, to improve the therapeutic index of compounds as immunosuppressants. For example, the extensive literature on analogs, derivatives, and other compounds structurally related to rapamycin ("rapalog") includes, among other things, the following modifications related to rapamycin: demethylation, C7 Removal or substitution of methoxy at C42 and / or C29; removal, derivatization or substitution of hydroxy at C13, C43 and / or C28; reduction, removal or derivatization of ketones at C14, C24 and / or C30; Substitution of a pipecolate six-membered ring with a ring; as well as alternative substitution on a cyclohexyl ring or substitution of a cyclohexyl ring with a substituted cyclopentyl ring. For additional historical information, see U.S. Patent Nos. 5,525,610; 5,310,903 and 5,362,7.
No. 18 in the background section.

[0006] US Patent No. 5,527,907 is illustrative of the patent literature. The document is
Disclosed are a series of compounds that have been synthesized in an effort to create immunosuppressive rapalogs with reduced side effects. The compounds are disclosed through seven general structural formulas, followed by an extensive list (2-5 or more columns of text), listing possible substitutions at various positions on the rapamycin ring. The document contains 180
The above synthesis examples are included. Numerous structural variants of the invention are reported to be potent immunosuppressants. SUMMARY OF THE INVENTION The present invention provides methods and materials for multimerizing chimeric proteins in engineered cells, preferably using an improved rapalog that has nullified the immunosuppressive effects of rapamycin.

[0007] Genetically engineered cells contain one or more recombinant nucleic acid constructs encoding a particular chimeric protein as described herein. Typically, the first chimeric protein comprises one or more FKBP domains capable of binding to the improved rapalogs of the invention. Also, the first chimeric protein is referred to herein as an "FKBP fusion protein" and further includes at least one protein domain that is non-homologous to the FKBP domain. The complex formed by binding of the FKBP fusion protein to the rapalog is capable of binding to a second chimeric protein comprising one or more FRB domains ("FRB fusion protein"). In addition, an FRB protein includes at least one protein domain that is heterologous to its FRB domain. In some embodiments, the FK
The BP fusion protein and the FRB fusion protein are different from each other. However, in other embodiments, the FKBP fusion protein is also a FRB fusion protein. In those embodiments, the chimeric protein comprises one or more FKBs.
It contains a P domain as well as one or more FRB domains. In such a case, the first and second chimeric proteins can be the same protein, and the FKBP-
FRB fusion proteins may also be referred to as FKBP and / or FRB
It contains at least one domain that is heterologous to the domain.

[0008] Chimeric proteins can be readily designed such that their multimerization elicits one or more of a wide range of desired biological responses, based on employing appropriately selected heterologous domains. Can be. The type of biological response elicited by rapalog-mediated complexation is dictated by the choice of heterologous domains within the fusion protein. Therefore, heterologous domains are called "action" or "effector" domains. The genetically engineered cells used in the practice of the present invention comprise one or more recombinant nucleic acid constructs encoding a chimeric protein, and in certain applications, further comprise:
It will include one or more accessory nucleic acid constructs, such as one or more target gene constructs. Illustrative biological responses, system applications and types of accessory nucleic acid constructs are discussed in detail below.

A system including related materials and methods is described in WO 96/41865 (Clac
kson et al.) and, among other things, are expected to be useful in a variety of applications, including research and therapeutic applications. The system is either rapamycin or the general formula

[0010]

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[Wherein, U is -H, -OR¹, -SR¹, -OC (O) R¹, -C (O) NHR¹, −
NHR¹, -NHC (O) R¹, -NHSO_Two-R¹Or -R^TwoAnd R^TwoThe ant
Or aryl or alkylaryl (eg, benzyl or substituted benzyl)
V) is OR^ThreeOr (＝ O); W is ＝ O, ＮＲNR^Four, = NOR ^Four , = NNHR^Four, -NHOR^Four, -NHNHR^Four, -OR^Four, -OC (O) R^Four, -O
C (O) NR^FourOr -H; Y is -OR^Five, -OC (O) NHR^FiveAnd Z
Is = O, -OR⁶, -NR⁶, -H, -NC (0) R⁶, -OC (0) R⁶, Or-
OC (0) NR⁶And R^ThreeIs H, -R⁷, -C (O) R⁷, -C (O) NHR⁷Also
Is a C-28 / C-30 cyclic carbonate; and R^FourIs H
Or alkyl; R¹, R^Four, R^Five, R⁶And R⁷Is independently selected from H, alkyl, alkylaryl or aryl; such terms are described in WO9
6/41865] as described in US Pat. No. 6,41,865]. Many rapalogs are listed above
It is specifically disclosed in the document.

The subject of the invention is based on a system similar to that disclosed in WO 96/41865, but involves the use of improved rapalogs such as multimerizing agents.
Accordingly, the subject of the invention is to provide (a) a suitable genetically engineered cell comprising a nucleic acid construct that directs the expression of a desired chimeric protein and any desired accessory recombinant construct; and (b) herein. Contacting a cell with an improved rapalog or a pharmaceutically acceptable derivative thereof as described in (1) above. Rapalog forms a complex that includes itself and at least two chimeric protein (s) molecules. The improved rapalog used in the present invention includes the following.

One class of improved rapalogs used in the present invention is represented by Formula I

[0014]

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[0015] As shown, those compounds containing a substructure having any number of various substituents, wherein one or more of the CC bonds are unsaturated if desired without departing from the invention. And has a substantially reduced immunosuppressive effect as compared to rapamycin. "
"Substantially reduced immunosuppressive effect" means that rapalog is found in an equimolar amount of rapamycin when measured either clinically or in a suitable surrogate of human immunosuppressive activity in vitro or in vivo. Or 0.1 times less than the expected immunosuppressive effect, preferably less than 0.01 times, more preferably 0.005 times
Having less than a 2-fold immunosuppressive effect, or alternatively, rapalog is at least 10-fold, preferably at least 100-fold, more than the EC50 value observed for rapamycin in such an in vitro assay. Preferably, an EC50 value of at least 250 times greater is obtained.

One suitable surrogate for immunosuppression of human patients in vitro includes inhibition of human T cell proliferation in vitro. This is a conventional assay approach that can be performed in a number of well-known variations, using a variety of human T cells or cell lines, including human PBLs and Jurkat cells, among others.
Thus, rapalog can be assayed for human immunosuppressive activity and compared to rapamycin. A decrease in immunosuppressive activity for rapamycin, as measured in a suitable in vitro assay, predicts a decrease in human immunosuppressive activity for rapamycin. Such in vitro assays can be used to assess the relative immunosuppressive activity of rapalog.

Various examples of such rapalogs are disclosed herein. Improved rapalogs of this class include, among others, human FKB, within the range of results obtained with rapamycin in any conventional FKBP binding or rotamase assay.
Those that bind to P12 or inhibit its rotamase activity are included.

Another class of improved rapalogs used in the present invention is the compound of formula II

[0019]

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[Wherein a is

[0021]

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And R^C7aAnd R^C7bIs H, the other is -H, halo, -R^Two , -OR¹, -SR¹, -OC (O) R¹Or -OC (O) NHR¹, -NHR¹, −
NR¹R^Two, -NHC (O) R¹Or -NH-SO_Two-R¹Where R^TwoIs
Aliphatic, heteroaliphatic, aryl, heteroaryl, alkylaryl (e.g.,
Benzyl or substituted benzyl); R^C30Is halo, -OR^ThreeOr (= O); R^C24Is = O, = NR^Four, = NOR^Four, = NNHR^Four, -NHOR^Four, -NHNH R^Four, -OR^Four, -OC (O) R^FourOr -OC (O) NR^Four, Halo or -H; R^C13And R^C28Is independently H, halo, -OR^Three, -OR^Five, -OC (O) R^Five , -OC (O) NHR^Five, -SR^Five, -SC (O) R^Five, -SC (O) NHR^Five, -NR^Five R^Five'Or -N (R^Five) (CO) R^Five'R^C14Is = O, -OR⁶, -NR⁶, -H, -NC (O) R⁶, -OC (O) R⁶Or -OC (O) NR⁶R^ThreeIs H, -R⁷, -C (O) R⁷Or -C (O) NHR⁷Or C28 and C3
A cyclic moiety bridging 0 (eg, carbonic acid); R^C29Is H or OR¹¹(Eg, OH or OMe); individual substituents are present in any stereochemical direction, unless otherwise specified.
R¹, R^Four, R^Five, R⁶, R⁷, R⁹, R^TenAnd R¹¹Each of the
Independently selected from H, aliphatic, heteroaliphatic, aryl and heteroaryl
R^FiveIs H, halo, -CN, = 0, -OH, -NR⁹R^Ten, OSO_TwoCF_Three,
OSO_TwoF, OSO_TwoR^Four', OCOR^Four', OCONR^Four'R^Five', Or OCON (OR ^Four' ) R^Five'Is defined with reference to the structure represented by

Improved rapalogs useful in the practice of the present invention, including the rapalogs of Formula II, can contain substituents in any of the possible stereoisomeric orientations and are substantially free of other stereoisomers ( (> 90%, preferably> 95% free of other stereoisomers on a molar basis) may contain one stereoisomer or a mixture of stereoisomers.

One class of improved paralogs used in the present invention of particular interest is the class of formula II wherein one or both of R ^C13 and R ^C28 are independently H, halo, —OR ³ , —O ^{R 5, -OC (O) R} 5, -OC (O) NHR 5, -SR 5, -SC (O) R 5, -SC (O
) NHR ^5, a -NR ⁵ R ^{5 'or -N (R 5) (CO)} R 5', each halo moiety, rapalog of the independently, F, Cl, selected from Br and I) It is. One subset of such compounds differs structurally from rapamycin only in one or both of R ^C13 and R ^C28 . Another subset of such compounds comprises structurally rapamycin at one or more additional positions, as described above for Formula II or for any of the other classes of improved rapalogs noted herein. And different. Compounds of both subsets of particular interest are those wherein one or both of R ^C13 and R ^C28 are ^halo substituents independently selected from F, Cl, Br and I, or substituted or unsubstituted amino moieties. Or an acylated derivative thereof. These compounds include 13-halolapamycin, 28-halorapamycin, 13,28-dihalolapamycin, and one or more other moieties (eg, one or both substituents on C7) and C13 and C28 Related compounds are included wherein the substituents are different from the relevant portions of rapamycin.

Another class of improved rapalogs for use in the present invention of particular interest is the rapalog of formula II, wherein both R ^C24 and R ^C30 are other than ＯO. This class includes 24,30-tetrahydrorapamycin and its mono and diethers,
And 24,30-dihalolapamycin. One subset of such compounds differs structurally from rapamycin only in R ^C24 and R ^C30 . Another subset of such compounds comprises one or more additional positions (e.g., one of C7, One or both substituents) and structurally different from rapamycin.

Another class of improved rapalogs of particular interest for use in the present invention is the improvement of formula II wherein R ^C7a and R ^C7b are moieties other than substituted or unsubstituted allyl groups or methoxy moieties. Rapalog. This class includes ^{rapalogs wherein} one of R ^C7a and R ^C7b is H and the other is a phenyl, di- or tri-substituted phenyl, or mono- or di-substituted heterocyclic moiety. Illustratively, among others, the o, p-dialkoxyphenyl substituent [eg,
o, p-dimethoxyphenyl, o-methoxy-p-ethoxyphenyl, o-ethoxy-p-methoxyphenyl, o, p-diethoxyphenyl, o, p-di (n-
Or mono-substituted heterocycles such as iso-) propoxyphenyl and the like, trialkoxyphenyl substituents, methylthiophene and the like. One subset of such compounds differs structurally from rapamycin only in R ^C24 and R ^C30 .
Another subset of such compounds comprises, structurally, rapamycin at one or more additional positions, as described above for Formula II or for any other class of improved rapalogs noted herein. different.

Another class of modified rapalogs of particular interest for use in the present invention is where n is 1
Is a rapalog of Formula II. This class of rapalogs includes rapalogs that contain a prolyl ring system in place of the picicolate ring system. One subset of such compounds differs structurally from rapamycin only with respect to the picpicolate ring system.
Another subset of such compounds are as described above for Formula II, or for any other class of improved rapalogs noted herein.
It differs structurally from rapamycin in one or more additional structural forms (eg, one or both substituents on C7).

Another class of improved rapalogs of particular interest for use in the present invention is the part "
a "is the formula

[0029]

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Other than the above, is a rapalog of formula II. One subset of such compounds is
Only the ring system "a" differs structurally from rapamycin. Another subset of such compounds comprises one or more additional structural forms (e.g., as described above in connection with Formula II or any of the other classes of improved rapalogs noted herein) With respect to one or both substituents of C7)
Different from rapamycin. Rapalogs of this class include that the hydroxyl moiety at position 43 has the opposite stereochemical orientation to that shown immediately above, is a stereoisomeric mixture of 43-hydroxyl groups, or includes ethers, esters, carbamates, halides , Any of the aforementioned derivatives, and other derivatives of the aforementioned rapalog at position 43, are included in the class of 43-epi-rapalogs. In addition, this class includes rapalogs in which the cyclohexyl ring is otherwise substituted and / or contains five ring atoms in place of the characteristically substituted cyclohexyl ring of rapamycin.

On the other hand, the improved rapalog described herein is used in a method for multimerizing a chimeric protein in genetically engineered cells. The method comprises: (a) providing a suitable genetically engineered cell containing a nucleic acid construct that directs the expression of the desired chimeric protein (and any desired accessory recombinant construct); and Contacting with an acceptable derivative as

[0032] In one embodiment, at least one chimeric protein has its peptide sequence
The sequence is a naturally occurring FKBP peptide sequence, eg, the peptide sequence of human FKBP12.
And at least one F that differs by at most 10 amino acid residues in the peptide sequence
Includes KBP domain. Preferably, the number of changes in the peptide sequence is up to 5, more preferably
More preferably, it is limited to 1, 2 or 3. Rapalog is R^C28, R^C24And R ^C30 And / or R^C7aAnd / or R^C7bIn embodiments that include a structural modification associated with rapamycin at least in one embodiment, the at least one chimeric protein is also naturally occurring.
Sequences and related sequences of raw FKBP, particularly mammalian FKBP such as human FKBP12.
At least one FKBP domain comprising at least one contiguous amino acid substitution
It is particularly interesting to include Mutations of particular interest include independently selected
Human FKs with replacement amino acids such as valine, methionine, alanine or serine
Includes substitution of one or both of Phe36 and Phe99 of BP12 sequence
.

In another embodiment, the at least one chimeric protein has a peptide sequence that differs from a naturally occurring FRB peptide sequence, eg, the FRB domain of human FRAP, by a maximum of 10 amino acid residues in the peptide sequence. At least one F
Includes RB domain. Preferably, the number of changes in the peptide sequence is limited to up to 5, more preferably to 1, 2 or 3;
It may be preferable to include single amino acid substitutions related to the peptide sequence of the corresponding FRB domain of the AP or some other mammalian FRAP / TOR species.
Mutations of particular interest include independently selected replacement amino acids such as A, N, H,
One or more T2s of an FRB domain derived from human FRAP at I or S
098, D2102, Y2038, F2039, K2095.
Also, one or more F1975, F19 of an FRB domain derived from yeast TOR1 with independently selected replacement amino acids, eg, H, L, S, A or V.
76, substitutions of D2039 and N2035, or one or more of F1978, F1979, D2042 and N20 of the FRB domain derived from yeast TOR2
38 substitutions are interesting.

In one embodiment, the chimeric protein has at least one modification, preferably a maximum of at least one modification in the peptide sequence, in both one or more FKBP domains and one or more FRB domains, as compared to the naturally occurring sequence. Includes up to 3 modifications.

As mentioned above, in various embodiments of the present invention, the chimeric protein (s)
One or more "effects" that are heterogeneous with respect to the RBP and / or FRB domains.
Or contains an "effector" domain. Effector domains include DNA binding domains, transcription activation domains, cell localization domains, and signaling domains (eg, domains that have the ability to induce proliferation, differentiation, apoptosis, gene transcription, etc. during clustered or multimerized cell growth). ) Can be selected from a wide range of protein domains, including Illustrative of various effector domains that can be used in practicing the present invention are disclosed in the various chemical and patent literature listed herein.

For example, in one embodiment, one fusion protein has at least one DN
An A binding domain (eg, a GAL4 or ZFHD1 DNA binding domain) and another fusion protein includes at least one transcription activation domain (eg, a VP16 or p65 transcription activation domain). Ligand-mediated binding of the fusion protein refers to the formation of a transcription factor complex, which initiates transcription of a target gene linked to a DNA sequence recognized (eg, capable of binding) by a DNA binding domain on one fusion protein. Lead.

In another embodiment, one fusion protein has at least one ability to direct the fusion protein to a particular cell location, such as a cell membrane, nucleus, ER or other organelle, or cellular component. Including domain. Localization domains that target the cell membrane include, for example, domains such as myristoylation sites or transmembrane regions of receptor proteins or other transmembrane proteins. Another fusion protein can include a signaling domain that has the ability to activate a cellular signal transduction pathway upon membrane localization and / or clustering. Examples of signaling domains include growth factor intracellular domains or cytokine receptors, apoptosis-inducing domains such as FAS or the intracellular domain of TNF-R1, and SOS, Raf, l.
and domains derived from other intracellular signaling proteins such as ck, ZAP-70 and the like. A number of signaling proteins are available from PCT / US94 /
01617 (see, for example, pages 23-26). In yet other embodiments, each fusion protein comprises at least one FR domain and at least one FKBP domain, and one or more heterologous domains. Such fusion proteins have the ability to homodimerize and induce signaling in the presence of rapalogs. Generally, domains containing peptide sequences endogenous to the host cell are preferred for applications involving whole organisms. Thus, for human gene therapy applications, domains of human origin are of particular interest.

[0038] Recombinant nucleic acid constructs encoding fusion proteins can also be used in nucleic acid constructs capable of directing their expression and in cells, especially eukaryotic cells, of which yeast and animal cells are of particular interest. As well as vectors containing such constructs for introduction into certain, are provided. From the viewpoint of the components of the fusion protein,
Recombinant DNA molecules encoding them may be (a) FRB domains or FKBP
A DNA molecule encoding a polypeptide comprising the domain; and (b) a D encoding a protein that induces the heterologous domain or the heterologous protein to a domain.
NA molecules; ability to selectively hybridize. DNAs capable of hybridizing without the degeneracy of the genetic code are also included.

Using nucleic acid sequences encoding the fusion proteins, nucleic acid constructs that direct their expression in eukaryotic cells, and vectors or other means for introducing such constructs into cells, especially animal cells Thus, cells, especially animal cells, preferably mammalian cells, most preferably human cells, can be engineered for a number of important applications. To do so, one first directs the expression of the desired chimeric protein in eukaryotic (preferably animal) cells. An expression vector or nucleic acid construct is provided, followed by DNA uptake and introduced DNA at least in a portion of a cell.
The recombinant DNA is introduced into the cells in a manner that allows expression of the DNA. Any of a variety of methods and tools for introducing DNA into cells for heterologous gene expression can be used, and various methods and tools are well known and / or commercially available.

Accordingly, one object of the present invention is to provide a method for preparing cells, according to the methods described herein,
Preferably, it is a method of multimerizing the fusion protein in animal cells. In summary, one fusion protein is capable of binding the improved rapalog of the present invention and comprises at least one FKBP domain and at least one domain heterologous thereto. The second fusion protein comprises at least one FRB domain and at least one domain that is heterologous thereto to form a complex of the first fusion protein and one or more improved rapalog molecules with a tripartite. Have the ability. In some embodiments, one or more heterologous domains present on one fusion protein are also present on another fusion protein, eg, two fusion proteins have one or more common heterologous Have a domain. In other embodiments, each fusion protein contains one or more different heterologous domains.

The method includes contacting the appropriately engineered cells with the improved rapalog by adding the rapalog to the culture in which the cells are present, or administering the rapalog to the organism in which the cells are present. . The cell is preferably a eukaryote, more preferably an animal cell, and most preferably a mammalian cell. Primate cells,
Of particular interest are human cells. Administration of the improved rapalogs to humans or non-human animals will be effective using any pharmaceutically acceptable formulation and route of administration.
Oral administration of a pharmaceutically acceptable composition comprising a modified rapalog together with one or more pharmaceutically acceptable carriers, buffers or other auxiliaries is presently of great interest.

A specific object of the present invention is a method for inducing transcription of a target gene in a rapalog-dependent manner, as described above in other respects. Typically, a cell is a target operably linked to a DNA sequence that responds to the presence of a fusion protein complex comprising rapamycin or rapalog, in addition to the recombinant DNA encoding the two fusion proteins. A target gene construct containing the gene. The target gene construct can be recombinant, and the target gene and / or the regulatory nucleic acid sequence linked thereto can be heterologous with respect to the host cell. In one embodiment, the cell is
Shows a reaction upon contact with an improved rapalog that binds the FKBP fusion protein and associates with the host cell's endogenous FKBP and / or FRB-contact protein and is associated with a complex with the FRB fusion protein that includes a detectable selection. .

Another specific object of the present invention is a method for inducing cell death in a rapalog-dependent manner, as otherwise described above. In such cells, at least one heterologous domain on at least one fusion protein, usually two fusion proteins, will induce cell apoptosis upon clustering, such as the intracellular domain of FAS or TNF-R1. It is a domain.

Another specific object of the present invention is a method for inducing cell growth, differentiation or proliferation in a rapalog-dependent manner, as otherwise described above. In such a cell, at least one heterologous domain of the at least one fusion protein can be a signaling domain, such as, for example, an intracellular domain of a hormone receptor that mediates cell growth, differentiation or proliferation, or such a signaling domain. It is a downstream mediator of the receptor. Cell growth, differentiation and / or proliferation occurs following clustering of such signaling domains. Such clustering occurs spontaneously following hormone binding and within the engineered cells of the invention following contact with the improved rapalog.

Although cell types of various origins (human or other species) can be used, cells of human origin are preferred for human gene therapy applications and, if desired, human patient It can also be encapsulated in a biocompatible material for use in.

Also provided are materials and methods for making the above-described genetically engineered cells.
The purpose of this is to provide a recombinant nucleic acid, typically a DNA molecule, encoding the fusion protein with any desired auxiliary recombinant nucleic acid, such as a target gene construct, and permit uptake of the nucleic acid by the cell. It is accomplished under conditions by introducing the recombinant nucleic acid into a host cell. Such transfection can be effective ex vivo, using host cells maintained in culture. next,
Cells that have been genetically engineered in culture can be introduced into a host organism, for example, for application to ex vivo gene therapy. Thus, by doing so, a host organism, preferably a human or non-human mammal, that responds (as described herein) to the presence of the improved rapalog as provided herein. A method for providing is configured. Alternatively, if host cells are present in a human or non-human host organism, transfection can also be effective in vivo. In such cases, the nucleic acid molecule is introduced directly into the host organism under conditions that permit uptake of the nucleic acid by one or more host organism cells. Thus, this approach has led to the presence of improved rapalogs (as described herein).
An alternative method of providing a responsive host organism, preferably a human or non-human mammal, constitutes. DNA and R are added to cells in culture or cells in the whole organism.
Various materials and methods for NA are known in the art and are suitable for use in practicing the present invention.

[0047] Other objects are achieved by using the genetically engineered cells described herein. For example, a fusion protein of the invention can be multimeric by contacting a cell that has been genetically engineered according to the methods described herein with an effective amount of an improved rapalog that allows the rapalog to form a complex with the fusion protein. A method is provided for optimizing. In embodiments where multimerization of the fusion protein triggers transcription of the target gene, this constitutes a method of activating expression of the target gene.
In embodiments where the fusion protein comprises one or more signaling domains, this constitutes a method of activating a cell signaling pathway. In embodiments where the signaling domain is selected based on their ability to induce cell growth, proliferation, differentiation, or cell death following clustering, the improved rapalog-mediated clustering may comprise cell growth, proliferation, differentiation or cell growth. A method for activating cell death is provided. These methods can be performed in cell culture or in whole organisms, including human patients. In the former case, rapamycin or rapalog is added to the culture. In the latter case, rapamycin or rapalog (which may be present in the form of a pharmaceutical composition or a composition for treating livestock) is administered to the whole organism, for example orally, parenterally. Preferably, the dose of the modified rapalog administered to the animal is lower than the dose level that would cause immunosuppression in the recipient.

[0048] Kits for use in genetically engineered cells or in human or non-human animals are also disclosed. One such kit includes one or more recombinant nucleic acid constructs encoding a fusion protein of the invention. Recombinant nucleic acid constructs generally comprise
It will be in the form of a eukaryotic expression vector suitable for introduction into animal cells and capable of directing the expression of the fusion protein therein. Such a vector can be a viral vector, as described elsewhere herein. The kit can also include a sample of the improved rapalog of the present invention capable of forming a complex with the encoded fusion protein. further,
The kit includes a multimerizing antagonist, such as FK506, and several other compounds capable of binding to one of the fusion proteins but not forming a complex with both. In one embodiment, the recombinant nucleic acid construct encoding the fusion protein includes a cloning site in place of the DNA encoding one or more heterologous domains, which allows the practitioner to encode the selected heterologous domains. It becomes possible to introduce DNA. In some embodiments, the kit can include a target gene construct comprising a target gene or cloning site linked to a DNA sequence responsive to the presence of the conjugated fusion protein, as described in more detail elsewhere. The kit can include a package insert for identifying the encapsulated nucleic acid construct, and / or a device for introducing the construct into a host cell or organism. Detailed Description of the Invention Definitions The following definitions and adaptation information will assist in a thorough understanding of this document.

The FRB domain is a polypeptide region (protein “domain”), typically at least about 89 amino acid residues, that forms a three-segment complex with the FKBP protein and rapamycin (or the improved rapalog of the present invention). Have the ability to F
RB domains are FRAP proteins from humans and other species (also referred to in the literature as "RAPTI" or "RAFT"); Tor1 and Tor
2, as well as Candida FRAP homologs; Information on nucleotide sequences, cloning and other aspects of these proteins is already known in the art, and the synthesis or cloning of DNA encoding the desired FRB peptide sequence has also been described, for example, in known methods and published. Using sequence-based PCR primers is possible.

[0050]

[Table 1]

Generally, the FRB domain used in the present invention contains at least about 89-100 amino acid residues. FIG. 2 of Chiu et al., Supra, shows that human FRAP, murine FRAP, S. cerebisiae TOR1 and S
. cerevisiae TOR2 shows 160 amino acids in length. Typically, the FRB sequences selected for use in the fusion proteins of the invention have at least 89 amino acid sequences (Glu-39 to Lys / Arg-1 at the numbers in FIG. 2).
27). For reference, using the numbers in Chen et al. Or Sabitani et al., The 89-amino acid sequence is Glu-2025 to Lys-2113 for human FRAP and Glu-1965 to Lys-2113 for Tor2.
053, and in the case of Tor1, Glu-1962 to Arg-2050. The FRB domain used in the fusion proteins of the present invention can be used to detect such binding (eg, as used in the references related to FRAP / RAFT / RAPT and Tor listed herein). The ability to bind to a complex of rapamycin or an FKBP protein that binds to rapamycin or an improved rapalog of the present invention, as can be determined by any means, direct or indirect, for detecting such binding, including: Will have. Such FRB domain peptide sequences include (a) the relevant region of a naturally occurring peptide or homologous protein spanning at least the indicated 89-amino acid region of the above protein; (b) at most about the naturally occurring peptide sequence. Up to 10 (preferably 1-5, more preferably 1-3)
A variant of the naturally occurring FRB sequence, wherein in some embodiments just one amino acid has been replaced by a deleted, inserted or substituted amino acid; or (c) selecting a DNA molecule encoding a naturally occurring FRB domain. A peptide sequence encoded by a DNA sequence capable of specifically hybridizing, or capable of selectively hybridizing to a DNA molecule encoding a naturally-occurring FRB domain but excluding the degeneracy of the genetic code: Is included.

FKBPs (FK506 binding proteins) are cytosolic receptors for macrolides such as FK506, FK520 and rapamycin and are well conserved across species strains. For the purposes of this disclosure, FKBPs are proteins or protein domains that have the ability to bind to rapamycin or an improved rapalog of the present invention and also form a three-segment complex with an FRB-containing protein. The FKBP domain is also called “rapamycin binding domain”. Information on nucleotide sequence, cloning and other aspects of the various FKBP species is already known in the art, for example, using well known methods and PCR primers based on published sequences to obtain the desired FKBP peptide It allows the synthesis or cloning of DNA encoding the sequence. For example, Staenda
rt et al., 1990, Nature, 346, 671-674 (human FKBP12
Kay, 1996, Biochem. J. , 314, 361-385 (commentary). Also, FKBP proteins homologous in other mammalian species, yeast, and other organisms are known in the art and can be used in the fusion proteins disclosed herein. See, for example, Kay, 1996, Bio.
chem. J. , 314, 361-385 (commentary). The size of the FKBP domain used in the present invention varies depending on the FKBP protein used. The FKBP domain of a fusion protein of the invention has the ability to bind to rapamycin or an improved rapalog of the invention (as determined by any direct or indirect means for detecting such binding). ) FR
Form a three-syllable complex with the B-containing protein. The peptide sequence of the FKBP domain of the FKBP fusion protein of the present invention preferably contains (a) human FKBP
A naturally occurring FKBP peptide sequence derived from 12 proteins, or from another human FKBP, from a murine or other mammalian FKBP, or from some other animal, yeast or fungal FKBP derived peptide sequence; (B) up to about 10 (preferably 1-5, more preferably 1-3, and in some embodiments even exactly 1) amino acids of the naturally occurring peptide sequence are deleted, inserted or substituted amino acids; A variant of a naturally occurring FKBP sequence that has been replaced; or (c) a DNA sequence capable of selectively hybridizing to a DNA molecule encoding a naturally occurring FKBP, or a native sequence, except for the degeneracy of the genetic code. A DNA sequence that will have the ability to selectively hybridize to a DNA molecule encoding a developing FKBP. Peptide sequence encoded by: contains.

As used herein, the term “selectively hybridizable” refers to the presence of other DNA molecules under hybridization conditions that can be readily selected or empirically determined by one skilled in the art. In any case, it means that two DNA molecules are susceptible to hybridization with each other. Such treatments include very harsh conditions (e.g., containing from 0.2 to 6 x SSC at temperatures ranging from room temperature to 65 to 75 C) and / or
Washing frequently with a buffer containing 0.1% to 1% SDS). For example, FMAusube
ed., Short Protocols in Molecular Biology, Units 6.3 and 6.4 (John
See Wiley & Sons, New York, Third Edition, 1995).

The terms “protein”, “polypeptide” and “peptide” are used interchangeably herein.

As used herein, the term “nucleic acid construct” is defined below, but generally
Recombinant, can exist in free (i.e., not covalently linked to other nucleic acid sequences) form, and can be a larger molecule or genetically modified DNA vector, retrovirus or other viral vector. A nucleic acid (usually DNA, but also including, for example, RNA in a retroviral delivery system) used in the practice of the present invention that can be present in the chromosome of a host cell that has been processed to And Particularly important nucleic acid constructs include those that encode the fusion proteins of the invention or those that contain target genes and expression control elements. This construct further comprises transcription, translation, and / or
Or a nucleic acid protein containing one or more of the following elements involved in the regulation of the processing of the coding region or other of its gene products: transcriptional promoter and / or enhancer sequences, ribosome binding sites, introns, and the like.

As used herein, the terms “recombinant”, “chimera” and “fusion” are other components that are heterogeneous with each other in the sense that they do not occur together in the same sequence in nature. It shall refer to a sequence or a substance comprising various component domains. In particular, this component is not found in the same continuous polypeptide or nucleotide sequence or molecule in nature and at least in the same order or orientation or in the same cell present in the chimeric protein or recombinant DNA molecule of the present invention. not exist. The term "transcription control element" refers to regulatory DNA sequences that direct or regulate the transcription of a protein coding sequence operably linked thereto (eg, initiation signals, enhancers, and promoters). The term "enhancer" is intended to include regulatory elements that can enhance, stimulate, or promote transcription from a promoter. Such transcriptional regulatory elements may be present upstream of the coding region or, in certain cases (eg, enhancers), elsewhere (eg, introns, exons, coding regions, and 3 ′ flanking sequences).

The terms “dimerization,” “oligomerization” and “multimerization” are used interchangeably herein and are interposed by binding an agent to at least one of the proteins. It refers to the association or clustering of one or more proteins. In a preferred embodiment, multimerization is mediated by binding two or more of such protein molecules to a conventional divalent or multivalent agent. Together with one or more molecules of an FKBP ligand that is at least bivalent (e.g., FK1012 or AP1510), the formation of a complex comprising two or more proteins, each containing one or more FKBP domains, comprises such a conjugate. Or it is an example of clustering. If at least one of the proteins contains more than one drug binding domain, for example, if at least one of the proteins contains three FKBP domains, more than two protein molecules will be clustered due to the presence of the bivalent drug. I do. Even in embodiments in which the drug has more than two valencies (eg, trivalent) in its ability to bind to a protein having a drug-binding domain, clustering of more than two protein molecules can be obtained. The formation of a tripartite complex comprising a protein containing at least one FRB domain, a protein containing at least one FKBP domain and a molecule of rapamycin is another example of such protein clustering. In certain aspects of the invention, the fusion protein comprises a number of
Contains FRB and / or FKBP domains. Such a protein complex is formed by combining one or more of rapamycin or a derivative or other dimerizing agent thereof with the constituent protein.
It can contain more than one copy. Further, such multimeric complexes are referred to herein as tripartite complexes that exhibit the presence of three component building molecules, even when one or more are represented by multiple copies. The formation of a complex containing at least one bivalent drug and at least two protein molecules, each of which contains at least one drug binding domain, is "oligomerization" or "multimerization", or simply "dimerization". , "" Clustering "or" combination. "

“Dimerizer” refers to an improved rapalog of the present invention that binds together more than one protein in a multimeric complex.

As used herein, the term “activate” as applied to gene expression or transcription,
Represents a direct or indirect observable increase in the production of a gene product.

The term “genetically engineered cell” refers to a recombinant or heterogeneous nucleic acid (eg, one or more DNA constructs or their constructs).
Refers to cells that have been modified (transduced) by the introduction of (RNA complement) and further include the progeny of such cells that retain some or all of such genetic modifications.

A “therapeutically effective dose” of an improved rapalog of the present invention refers to a treatment- or prophylactic-effective dose, eg, transcription or growth or proliferation of a target gene detectable in genetically engineered cells. , Or a dose that is predicted to be treatment- or prophylactic-effective by extrapolation from data obtained in animal or cell culture media models. A therapeutically effective dose is generally preferred for treatment of human or non-human mammals.

The present invention relates to methods and materials for multimerizing chimeric proteins in cells genetically processed using improved rapalogs. The design and completion of various dimerization-based biological switches has been reported, inter alia, in Spencer et al. And in the various international patent applications referred to herein. Evaluations of successful applications of this general approach have also been reported. A chimeric protein containing the FRB domain fused to the effector domain has also been described by Rivera et al., 1996, Nature Medicine 2, 1028-1032.
And WO 96/41865 (Clackson et al.) And WO 95/33052 (Berlin et al.)
). As already described, fusion proteins can be prepared by binding the effector domain by ligand-mediated "dimerization" or "multimerization" of the fusion protein containing them to a desired biological event (e.g., transcription of the desired gene). , Cell death, cell proliferation, etc.). For example, clustering of a chimeric protein containing an action domain derived from the intracellular portion of the T cell receptor CD3 zeta domain involves transcription of the gene under the transcriptional control of the IL-2 promoter or a promoter element derived therefrom. Trigger. In other embodiments, the domain of action is a domain derived from the intracellular portion of the protein, such as FAS or TNF-α receptor (TN
Fα-R1). In yet other embodiments, the action domain is a DNA domain in which the oligomerization of the chimeric protein is recognized by the DNA binding domain (and is capable of specific binding interactions).
A DNA-binding domain (e.g., GAL4 or ZFHD1) and a transcriptional activation domain (e.g., VP16 or p65) are paired to represent the assembly of a transcription factor complex that triggers transcription of the gene bound to the NA sequence. Including.

For example, a chimeric protein containing one or more ligand-binding domains and one or more action domains to induce transcriptional activation of target genes, cell death or other signal transduction pathways, cell localization Is PCT / US94 / 01617, PCT / US
94/08008 (Spencer et al.). The design and use of such chimeric proteins for ligand-mediated gene-knockout and for ligand-mediated blockade of gene expression or inhibition of gene product function is disclosed in PCT / US95 / 10591. Novel DNA binding domains and DNA sequences to which they bind, useful in embodiments involving regulated transcription of the target gene, are disclosed in Pomeranz et al., 1995, Science 267: 93-96. These references provide substantial information, guidance and implementation information relating to the design, construction and use of DNA constructs encoding similar chimeras, target gene constructs and other embodiments that may also be useful to the practitioner of the present invention. Provide an example.

By suitably selecting the chimeric protein, activates the transcription of the desired gene in cells processed in a rapalog-dependent manner similar to the systems described in the patent literature and in the above-referenced literature according to the invention; Trigger cell proliferation, proliferation, differentiation or apoptosis; or can trigger other biological events. The engineered cells, preferably animal cells, can be grown or maintained in culture as in human gene therapy, transgenic animals, and other such uses, or can be present throughout the organism. it can. In some cases, the FKBP fusion protein and the FRB fusion protein are multimerized (as can be indirectly determined by monitoring target gene transcription, apoptosis or other biological processes so induced). The rapalog is administered to the organism containing the cell culture medium or the processed cells in such an effective amount. When administered to an entire organism, rapalog can be administered in a composition containing rapalog and one or more acceptable verterinary or pharmaceutical diluents and / or excipients.

Compounds that bind to one of the chimeric proteins but do not form a tripartite complex with any of the chimeric proteins can be used as multimerizing antagonists.
Therefore, in an amount effective to mask or reverse the effect of rapalog, i.e., to prevent, inhibit or prevent multimerization of the chimera (preferably in a composition as described above when administered to an entire animal), It can be administered to the processed cells, or to organisms containing them. For example, FK that does not form a tripartite complex with FKBP and FRAP
Either 506, FK520 or many of the synthetic FKBP ligands can be used as antagonists.

In one important aspect of the present invention, there are provided materials and methods for rapalog-dependent, direct activation of transcription of a desired gene. In one such embodiment, there is provided a series of two or more different chimeric proteins, and corresponding DNA constructs capable of directly expressing them. One such chimeric protein contains one or more transcriptional activation domains as its one or more action domains. Other chimeric proteins contain one or more DNA-binding domains as one or more action domains. The rapalogs of the present invention can bind to any chimera to form a dimer or multimer complex and thus contain at least one DNA binding domain and at least one transcription activation domain. The formation of such a complex allowed the DNA-binding domain to bind to a transcriptionally controlled DNA sequence, as can be observed by monitoring, directly or indirectly, the presence or concentration of the target gene product. The target gene is transcriptionally activated.

The DNA binding domain and chimeras containing it can be used to create other, often numerous, other DNA
Despite the presence of the sequence, binding to the selected DNA sequence (directly or indirectly)
) Binds to its recognition DNA sequence with sufficient selectivity as observed. Binding of the chimera containing the DNA-binding domain to the selected DNA sequence can be selected relative to any other DNA sequence.
By measuring the level or relative rate of transcription of a gene that binds to a DNA sequence, or by measuring in vitro binding studies, at least 2, more preferably 3, and even more, than binding to any other DNA sequence. Preferably, the size is more than 4 orders.

Applications Involving Ligand-Mediated, Coordinated Triggering of Desired Biological Events Using Cells Genetically Processed to Contain a Series of Constructs, With Any Desired Collateral Constructs And can induce regulated transcription of a desired gene, modulation of a signal transduction pathway (eg, induction of apoptosis, or other events). For example, cells that have been processed for regulatable expression of a target gene can be used for regulated production of a desired protein (or other gene product) encoded by the target gene. Such cells can be grown in culture by conventional methods. Addition of the rapalog to the medium containing the cells causes expression of the target gene by the cells and production of the protein encoded by such genes. Gene expression and protein production are stopped by removing further multimerizing agent from the medium, by removing residual multimerizing agent from the medium, or by adding a multimerizing antagonist reagent to the medium. Can be done.

For example, cells processed according to the present invention may modify an entire organism, preferably an animal, especially a human, so that the cells produce or contain the desired protein in an animal containing them. Can also be produced and / or used in vivo. Such applications include gene therapy applications.

In embodiments that include a regulatable trigger of apoptosis, engineered cells that are susceptible to rapalog-inducible cell death are provided. Such engineered cells can be used for their desired purpose (e.g., where they are no longer useful, safe, or undesirable), even though they have or exhibit adverse properties. Production of the desired protein or other product) and then removed from the cell culture medium or host organism. Rapalogs can be removed by adding to the medium or administering it to the host organism. In such cases, the chimeric action domain is a protein domain, such as the intracellular domain of FAS or TNF-R1, a downstream component of its signaling pathway, or another protein domain that induces apoptosis upon oligomerization.

Thus, the present invention provides substances and methods for obtaining a biological effect in cells by adding a rapalog of the present invention. The methods of the invention include providing cells that have been processed as described herein and exposing such cells to a rapalog.

For example, the present invention provides a method for activating transcription of a target gene in a cell. The method of the invention comprises (a) a DNA construct encoding a series of chimeric proteins of the invention capable of initiating transcription of a target gene upon rapalog-mediated multimerization, and (b) an associated syngeneic DNA sequence susceptible to the multimerization event. (E.g., a cell comprising a target gene bound to a recognized, i.e., DNA sequence capable of binding to the DNA-binding domain of the chimeric protein). The method of the invention comprises exposing the cells to a rapalog capable of binding an effective amount of the chimeric protein to express the target gene. If the cells are growing in the medium, exposure of the cells to the rapalog can be performed by adding the rapalog to the medium. When the cells are present in a host organism, exposing the host organism to the rapalog is performed by administering the rapalog to the host organism. For example, if the host organism is human or non-human, rapalog may be administered to the host by inhalation orally, buccally, sublingually, transdermally, subcutaneously, intramuscularly, intravenously, intraarticularly, or in a vehicle suitable therefor. It can be administered to an organism. Furthermore, depending on the design of any ancillary constructs and of the construct for the chimeric protein, a rapalog-mediated biological event may lead to cell function, such as cell growth, cell proliferation, gene transformation, or apoptosis. Signal transduction; deletion of key genes, blocking expression of key genes, or inhibiting function of key genes; activation of direct transformation of key genes.

The present invention further encompasses a pharmaceutical composition comprising a rapalog of the present invention mixed with a pharmaceutically acceptable carrier and optionally one or more pharmaceutically acceptable excipients. Using such pharmaceutical compositions, the multimerization of the chimeras of the present invention can be performed on cells processed throughout the animal, for example, in human gene therapy applications to achieve any of the objectives disclosed herein. Can be promoted.

In other words, the present invention comprises the engineered cells of the present invention in an animal, preferably a human patient, in need of the cells, for the purpose of the present invention, for example, a target gene (usually a heterologous gene for a therapeutic protein). Gene activation), cell growth or proliferation, cell death or any of several other selected biological events. The method of the invention comprises administering to a animal a pharmaceutical composition containing a rapalog in an amount and by an administration route effective to cause multimerization of the chimeric protein in at least a portion of the engineered cells. Multimerization occurs in the expression of the target gene;
Cell growth, proliferation or death; or chimeras can be designed and detected indirectly by detecting other targets for which cells are genetically processed.

The present invention further provides for inhibiting or reducing the degree of multimerization of a chimeric protein in a treated cell of the present invention, wholly or partially, in a patient, and inactivating the transcription of a target gene. A pharmaceutical composition comprising a multimerizing antagonist of the invention mixed with a pharmaceutically acceptable carrier and, optionally, one or more pharmaceutically acceptable excipients to arrest another biological effect of Is included. Thus,
The production of a pharmaceutical composition and the use of a multimerized antagonist reagent and a multimerized rapalog for obtaining its pharmacological effects are included in the present invention.

Also disclosed are methods for providing a host organism, preferably an animal, usually a non-human mammal or a human patient, susceptible to a rapalog of the present invention. The method of the present invention comprises introducing into a biological cell processed according to the present invention, ie, including one or more nucleic acid constructs encoding a chimeric protein. The processed cells can be encapsulated using any of a variety of materials and methods before introduction into the host organism. Alternatively, the nucleic acid constructs of the present invention can be used in a host organism (e.g., under conditions that allow the construct to be included in one or more cells of the host organism, e.g., using a viral vector, introduction of DNA by injection or a catheter, etc. Mammals).

Also provided are kits for producing cells susceptible to a rapalog of the present invention. Such kits, upon rapalog-mediated oligomerization, can direct the expression of a chimera that elicits the desired biological response and contain one or more encoding nucleic acid constructs. The kit can contain a specific amount of rapalog capable of multimerizing the chimeric protein molecule encoded by the kit (s), and further contain a specific amount of a multimerizing antagonist. be able to. The kit of the present invention further comprises a cognate
It contains a nucleic acid construct encoding a target gene (or cloning site) linked to a cognate DNA sequence that can be recognized by a dimerized chimeric protein that transcribes the gene linked to the DNA sequence. The construct is a transfectant
One or more selectable markers for convenient selection, as well as prokaryotic replication,
It can be linked to other conventional vector elements useful for eukaryotic expression and the like. The selectable marker may be the same or different for each different construct, and cells containing each of such construct (s) may be selected.

Ancillary constructs for introducing a target gene into a cell in cooperation with a syngeneic DNA sequence can contain a cloning site in place of the target gene. Kits containing such constructs can process cells for regulatable expression of the gene to be provided by the skilled worker.

Other kits of the invention may contain one or two (or more) nucleic acid constructs for a chimeric protein, one or more of which includes a cloning site, instead of a transcription activator or DNA binding protein. You can insert any domain you want. Such kits can optionally include other elements described above, for example, a nucleic acid construct for the target gene optionally including a cognate DNA sequence for the preselected DNA binding domain.

[0080] Both kits can be used to express a reporter gene according to the present invention in response to exposing the cells to a rapalog (for CAT, beta-galactosidase or any conventional detectable gene product). Positive control cells that have been reliably transformed with the constructs can also be included. A reagent for detecting and / or quantifying the expression of a reporter gene can also be provided.

For further information and guidance on the design, construction and use of such systems or components thereof that can be applied in practicing the present invention, see the following references: Spencer et al., 1993, supra; Riversa et al., 1996 supra; Spencer et al., 1996, Current Biology 6, 839-
847; Luo et al., 1996, Nature, 383, 181-185; Ho et al., 1996, Nature 382, 822-826.
Belshaw et al., 1996, Proc. Natl. Acad. Sci. USA 93, 4604-4606; Spence.
r, 1996, TIG 12 (5), 181-187; Spencer et al., 1995, Proc., Natl. Acad. Sci.
USA 92, 9805-9809; Holsinger et al., 1995, Proc. Natl. Acad. Sci. USA.
Pruschy et al., 1994, Chemistry & Biology 1 (3), 163-172; and published PCT International Publication Nos. WO94 / 18317, WO95 / 02684, WO95 / 33052, WO96 /. See No. 20951 and WO 96/41865.

An important aspect of the present invention is that FKBP as described above or elsewhere herein
And use of the improved rapalogs as mediators of protein-protein interaction in applications using FRB fusion proteins. Improved rapalogs are present throughout organisms, including humans and other mammals, or include a fundamental effect, including inducing biological events in genetically engineered cells grown or maintained in culture. Dimerization-
It can be used in various applications of the base method. Improved rapalogs have been used as research reagents in in vitro biological experiments, experiments performed on animals containing genetically engineered cells, and in humans in patients containing genetically engineered cells. It is useful as a health care and preventive or therapeutic agent for animals. Rapalogs The term "rapalogs" as used herein refers to rapamycin (r
apamycin) is intended to refer to a class of compounds, including various analogs, homologs and derivatives, as well as other compounds structurally related to rapamycin. "Rapalogs" refers to carrying any number of various substituents, and optionally being unsaturated at one or more carbon-carbon bonds, unless otherwise specified herein. Compounds other than rapamycin having the structure shown in Formula I are included.

[0083]

Embedded image

As rapalogs, inter alia, in connection with rapamycin, the following variants: demethylation, removal or substitution of methoxy at C7, C42 and / or C29; removal of C13, C43 and / or C28 with hydroxy, Derivatization or substitution; reduction, removal or derivatization of ketones at C14, C24 and / or C30; substitution of the pipecolate ring with a 6-member with a 5-membered prolyl ring; and one or more substituents on the cyclohexyl ring Of rapamycin having one or more of the following: removal, derivation or substitution, or substitution of a cyclohexyl ring with a substituted or unsubstituted cyclopentyl ring. The term rapalogs as used herein does not include rapamycin itself,
Preferably, it does not contain a ~ C30 oxygen bridge. Representative examples of rapalogs are disclosed in the documents listed in Table I. Table II shows examples of rapalogs modified in C7.

[0085]

[Table 2]

[0086]

[Table 3]

[0087]

[Table 4]

Other representative rapalogs include those shown in Table III.

[0089]

[Table 5]

[0090]

[Table 6]

Rapalogs of particular interest in the practice of various embodiments of the present invention include those of formula II:

[0092]

Embedded image

[Wherein

[0094]

Embedded image

^And one of R ^C7a and R ^C7b is H, and the other is —H, halo, —R ² , —OR ¹ , —SR ¹ , —OC (O) R ¹ , —OC (O ) NHR ¹ , -NHR ¹ , -NR ¹ R ² , -NHC (O) R ¹ , or -NH-SO ₂ -R ¹ , provided that R ² = aliphatic, heterocyclic aliphatic, aryl, Heteroaryl or alkylaryl (
For example, a benzyl or substituted benzyl), R ^C30 is halo, -OR ³ or (= O), R ^{C24 is, = O, = NR 4,} = NOR 4, = NNHR 4, -NHOR 4, - NHNHR ⁴ , -OR ⁴ , -OC (O) R ⁴ or -O
C (O) NR ⁴ , halo or -H; R ^C13 and R ^C28 are independently H, halo, -OR ³ , -OR ⁵ , -OC (O) R ⁵ , -OC (O) NHR ⁵ , -SR ^5-
, -SC (O) R ⁵ , -SC (O) NHR ⁵ , -NR ⁵ R ^{5 '} or -N (R ⁵ ) (CO) R ^5' , and R ^C14 is = O, -OR ⁶ ,- NR ⁶ , -H, -NC (O) R ⁶ , -OC (O) R ⁶ or -OC (O) NR ⁶ , wherein R ³ is H, -R ⁷ , -C (O) R ⁷ or- C (O) NHR ⁷ or a cyclic moiety bridging C28 and C30 (e.g., carbonate), R ^C29 is H or OR ¹¹ (e.g., OH or OMe), provided that each substituent is Unless otherwise stated, they can be in any stereochemical orientation, provided that R ¹ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁹ , R ¹⁰ and R ¹¹ are each independently H is selected from H, aliphatic, heterocycloaliphatic, aryl and heteroaryl; and R ⁸ is H, halo, —CN, 3O, —OH, —NR ⁹ R ¹⁰ , OSO ₂ CF ₃ , OSO ₂ R ^{4 ′} , OCOR ^{4 ′} , OCONR ^{4 ′} R ^{5 ′} or OCON (OR ^{4 ′} ) R ^{5 ′} ].

[0096] Some rapalog such of formula II, R ^C13 is -OMe, R ^C14 are located at H; R ^C14 is -OH
, -O (CO) NHMe, -O -CH 2 ( i.e., spiro epoxide), = NCH ₂ CH be ₂ -OH or -O- phenyl; R ^C13 is an -NHC (O) Me; R ⁴ is = NR or -NHR, where R is -OH, O-
Alkyl (methyl, ethyl, isobutyl, benzyl), - NH- alkyl or, be O- carboxymethyl oxime or O- carboxamide methyloxime in C24; R ^C24 is an _{-O (CO) NHCH (CH 3} ) 2; R ^C24 is -O (CO) NC (O) CH ₂ CH ₂ C (O) and R ^C28 is O-TBDMS; R ^C28 or R ^C30 is -OC (O) R or R ^C28 And R ^C30 together form —OC (O) O— linking C 28 and C 30 of the 6-membered ring; or R ^C7a or R ^C7b is isopropoxyl, —S-phenyl, 2-thiophene -Il, 3-indole
-Differs from rapamycin in that it is -yl or allyl or methallyl. Table III and Liberles et al., 1997, Proc Natl Acad Sci USA 94: 7825-7830.
Please refer to. Rapalogs other than those described above, ie, those that include another variation or combination of variations with respect to the structure of rapamycin, are preferred for use in the practice of the present invention. Thus, the improved rapalogs of the present invention are rapalogs other than those shown in Table III.

For rapamycin, R ^C7a is —OMe; R ^C7b is H; R ^C14 , R ^C24 and R ^C30 are each (= O); R ^C13 and R ^C28 are each —OH R ^C29 is OMe; R ³ and R ⁴ are each H, all having stereoisomers are shown on page 1; Rapalogs useful in the practice of the present invention include any possible stereoisomeric orientation (stereoi
one stereoisomer that can contain substituents (someric orientations) and is substantially free of other stereoisomers (> 90%, preferably> 95%, free of other stereoisomers on a molar basis) Or a mixture of stereoisomers.

Also included are pharmaceutically acceptable derivatives of the above compounds, where “pharmaceutically acceptable derivatives” are all pharmaceutically acceptable salts, esters or such esters of such compounds. Salt, or any other adduct or derivative, or metabolite or residue thereof, that, when administered to a patient, is capable of providing (directly or indirectly) the rapalog described herein. . Pharmaceutically acceptable derivatives include, among others, pro-drugs of rapalogs. Pro-drugs are usually derivatives of compounds with considerably less pharmacological activity, contain additional moieties that are easy to remove, and produce the parent molecule as a pharmacologically active species in vivo. An example of a pro-drug is an ester that is cleaved in vivo to produce a key compound. Various pro-drugs of rapamycin and other compounds, and materials and methods for derivatizing the parent compound to create pro-drugs, are known and can be adapted to the present invention.

The term “aliphatic,” as used herein, refers to a saturated and unsaturated, straight-chain (ie, unbranched), branched, cyclic or optionally substituted with one or more functional groups. It shall include any of polycyclic aliphatic hydrocarbons. Unless otherwise stated, alkyl, other aliphatic, alkoxy and acyl groups preferably contain 1 to 8, often 1 to 6, adjacent aliphatic carbon atoms. Representative examples of aliphatic groups, For example, capable of holding one or more substituents, methyl, ethyl, n- propyl, isopropyl, cyclopropyl, -CH ₂ - cyclopropyl, allyl, n- butyl, sec- butyl, isobutyl, tert- butyl, cyclobutyl, -CH ₂ - cyclobutyl, n- pentyl,
sec- pentyl, isopentyl, tert- pentyl, cyclopentyl, -CH ₂ - cyclopentyl, n- hexyl, sec- hexyl, cyclohexyl, -CH ₂ - cyclohexyl unit content and the like.

Examples of the substituent include —OH, —OR^{2 '}, -SH, -SR^{2 '}, -CHO, = O, -COOH (or S
Ter, carbamate, urea, oxime or its carbonate), -NH_Two(Also
Is a substituted amine, amide, urea, carbamate or guanidino derivative thereof), halo, trihaloalkyl, cyano, -SO_Two-CF_Three, -OSO_TwoF, -OS (O)_TwoR¹¹, -SO_Two-NHR ¹¹ , -NHSO_Two-R¹¹, Sulfate, sulfonate, aryl and heteroaryl moieties
Minutes. Aryl and heteroaryl substituents may themselves be substituted or unsubstituted.
(Eg, mono-, di- and tri-alkoxyphenyl; methylenedioxyphenyl)
Or ethylenedioxyphenyl; halophenyl; or -phenyl-C (Me)_Two-C
H_Two-O-CO- [C3-C6] alkyl or alkylamino).

The term “aliphatic” is intended to include alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties.

The term “alkyl” as used herein includes straight chain, branched and cyclic alkyl groups. Similar conventions apply to other generic terms, eg, “alkenyl”, “alkynyl”, and the like. Further, as used herein, "alkyl", "alkenyl",
"Alkynyl" and the like include both substituted and unsubstituted groups.

The term “alkyl” refers to a group usually having 1 to 8, preferably 1 to 6, carbon atoms. For example, `` alkyl '' refers to methyl, ethyl, n-propyl, isopropyl, cyclopropyl, butyl, isobutyl, sec-butyl, tert-butyl, cyclobutyl, pentyl, isopentyl, tert-pentyl, cyclopentyl, hexyl, isohexyl, cyclohexyl, etc. Point to. Suitable substituted alkyls include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2-fluoroethyl, 3-fluoropropyl, hydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl, benzyl, substituted And benzyl.

The term “alkenyl” refers to a group usually having 2 to 8, preferably 2 to 6, carbon atoms. For example, “alkenyl” refers to prop-2-enyl, but-2-ethyl, but-3
-Enyl, 2-methylprop-2-enyl, hex-2-enyl, hex-5-enyl, 2,3-
Refers to dimethylbut-2-enyl and the like. The term "alkynyl", which refers to a group having 2-8, preferably 2-6 carbons, includes, but is not limited to, prop-2.
-Inyl, but-2-ynyl, but-3-ynyl, pent-2-ynyl, 3-methylpent-4
-Inyl, hex-2-ynyl, hex-5-ynyl and the like.

The term “cycloalkyl” as used herein refers in particular to groups having 3 to 7, preferably 3 to 10, carbon atoms. Suitable cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and in the case of other aliphatic or heterocycloaliphatic or heterocyclic moieties, optionally It may be substituted.

The term “heteroaliphatic” as used herein refers to an aliphatic moiety that contains, for example, one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms instead of carbon atoms. The heterocyclic aliphatic moiety may be branched, unbranched or cyclic;
Heterocycles (eg, morpholino, pyrrolidinyl, and the like).

As used herein, the term “heterocycle” refers to a cyclic heterocyclic aliphatic group, preferably having a total of 3 to 10 ring atoms, oxetane, tetrahydrofuranyl, tetrahydropyranyl , Aziridine, azetidine, pyrrolidine, piperidine, morpholine, piperazine and the like, but are not limited thereto.

As used herein, the terms “aryl” and “heteroaryl” are stable or unsubstituted, monocyclic or multicyclic having 3 to 14 carbon atoms. Refers to cyclic, heterocyclic, polycyclic, and polyheterocyclic unsaturated moieties. Examples of the substituent include any of the above substituents. Non-limiting examples of useful aryl ring groups include phenyl, halophenyl, alkoxyphenyl, dialkoxyphenyl, trialkoxyphenyl, alkylenedioxyphenyl, naphthyl, phenanthryl, anthryl, phenanthro, and the like. Examples of typical heterocyclic groups include 5-membered monocyclic groups such as thienyl, pyrrolyl, imidazolyl, pyrazolyl, furyl, isothiazolyl, furazanyl, isoxazolyl, thiazolyl and the like; 6-membered monocyclic groups such as Pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl and the like; polycyclic heterocyclic groups such as benzo [b] thienyl, naphtho [2,3-
b] thienyl, thianthrenyl, isobenzofuranyl, chromenil, xanthenyl, phenoxathienyl, indolizinyl, isoindolyl, indolyl, indazolyl, purinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl,
Quinoxalinyl, quinazolinyl, benzothiazole, benzimidazole, tetrahydroquinolinecinnolinyl, peridinyl (pteridinyl), carbazoyl, beta-carbolinyl, phenanthridinyl, acridinyl, perimidinyl, phenanthronyl, phenazinyl, isothiazolyl, phenothazinyl, phenoxazinyl, etc. , Katrizky, Handbook of Heterocyclic Chemistry). The aryl or heterocyclic moiety can be hydroxy, C1-C8 alkoxy, C1-
It may be substituted with 1 to 5 C8 branched or straight chain alkyl, acyloxy, carbamoyl, amino, N-acylamino, nitro, halo, trihalomethyl, cyano and carboxyl. Thus, aryl moieties include, for example, phenyl; halo (e.g., chloro or fluoro), hydroxy, C1-C6 alkyl, acyl, acyloxy, C1-C6 alkoxy (e.g., methoxy or ethoxy, especially dialkoxyphenyl moieties, e.g., 2 , 3-, 2,4-, 2,5-, 3,4- or 3,5-dimethoxy or diethoxyphenyl or, for example, methylenedioxyphenyl, or 3-methoxy-5-ethoxyphenyl) Substituted phenyl bearing one or more selected substituents; or trisubstituted phenyl, such as trialkoxy (eg, 3,4,5-trimethoxy or ethoxyphenyl), 3,5-dimethoxy-4-chlorophenyl, amino , -SO ₂ NH ₂ , -SO ₂ NH (aliphatic), -SO ₂ N (aliphatic)
) ₂ , -O-aliphatic-COOH, and -O-aliphatic-NH ₂ (one or two N-aliphatic or N-
Which may contain an acyl substituent).

A “halo” substituent of the present invention may be a fluoro, chloro, bromo or iodo substituent. Fluoro is often the preferred halogen.

Of particular interest are the compounds of formula II, except for all the compounds listed in Table III, which constitute an important class of new compounds. Compounds of this type differ from rapamycin in 1, 2, 3, 4, 5, 6 or 7 substituent moieties. This class includes, inter alia, C7 and C13; C7 and C14; C7 and a ; C7 and C43;
C7 and C28; C7 and C30; C7, C13 and C14; C7 and a ; C7, C13 and C43;
C7, C13 and C24; C7, C13 and C28; C7, 13 and C30; C7, C14 and a; C7, C14 and
C43, C7, C14 and C24; C7, C14 and C28; C7, C14 and C30; C7, a and C24; C7, a and C28; C7, a and C30; C7, C24, C30 and a ; C30 and C13; C7, C24,
C30 and C14; C24, C30 and C13; C24, C30 and a; C24, C30 and C14; and C24,
Rapalogs modified at C30, C13 and a are included, except for all compounds listed in Table III, unless otherwise stated.

A subset of improved rapalogs of particular interest for carrying out the method of the present invention include:
, R^C7aA compound of formula II wherein is a moiety other than OMe (or a pharmaceutically acceptable derivative thereof)
Body). As a subset of this (C7 rapalogs), R^C7aAnd R^C7bIs one of H
The other is substituted or unsubstituted alkenyl, aryl, heteroaryl or -Z
-Aliphatic, Z-aryl, -Z-heteroaryl, or Z-acyl, provided that Z and Z 'are independently O, S or NH, and acyl is -CHO,-(C = O) -Aliphatic-, (C = O)-
Aryl,-(C = O) -heteroaryl,-(C = O) -Z'-aliphatic,-(C = O) -Z'-aryl,-
(C = O) -Z'-heteroaryl. In certain aspects of this subset, R^C7aAnd R^{C7 b} Is independently the following groups: H, substituted or unsubstituted, straight-chain, branched or unsubstituted 2 to 8 carbon atoms.
Or cyclic alkenyl, alkoxy or alkyl mercapto; substituted or unsubstituted
Substituted aryl, heteroaryl, aryloxy or heteroaryloxy,
It is selected from aryl mercapto or heteroaryl mercapto. This subordinate
Among the compounds of the assembly, among others, R^C7aIs H; (R^C7bAlkoxy; Alkyl mercapto; Amino (1 ゜, 2 ゜ or 3 ゜); Amide; Carbamate; Aryl or substituted aryl; Phenyl or substituted phenyl;
Or unsubstituted heteroaryl, for example, substituted or unsubstituted thiophenyl, furyl
, Indolyl and the like; or benzyloxy or substituted benzyloxy
You. Other exemplary C7 rapalogs and C7s that can be used in practicing the methods of the present invention.
As species of rapalogs, R^C7aAnd R^C7bOne is H and the other is -OEt-, -O-propyl, -O-butyl, -OCH_TwoCH_Two-OH, -O-benzyl, -O-substituted benzyl (e.g., 3-nitro-, 4-chloro-, 3-iodo-4-diazo-, 3,4-dimethoxy-, and 2-methoxy-
), -S-Me, -S-phenyl, -O (CO) Me-, allyl, -CH_TwoC (Me) = CH_Two, -OCH_Two-CCH, -OCH _Two -CC-Me, -OCH_Two-CC-Et, -OCH_Two-CC-CH_Two-OH or -2,4-dimethoxyphenyl, 2,4,
6-trimethoxyphenyl, furanyl, thiophen-yl, methyliophen-yl
, Pyrrolyl and indolyl. Above kind
In rapalogs, the hydroxy substituent at C43 may be in a stereochemical orientation or may be modified as described elsewhere herein. C7 rapalog
The class further differs from rapamycin at 1, 2, 3, 4, 5 and more other positions. Table I
C7 rapalogs other than those listed in II are new and essentially essential compositions.
Included in the invention.

Another subset of improved rapalogs of particular interest in practicing the various methods of the present invention are C30, C24 rapalogs of Formula II, ie, rapalogs of Formula II, provided that R ^C30 and R ^{C30 C24} is other than (= O)). In particular, C30, C where R ^C7a is a part other than OMe
24 rapalogs are important. In certain embodiments of this subset, R ^C7a and R ^C7b are independently -H, -OR ¹ , -SR ¹ , -OC (O) R ¹ or -OC (O) NHR ¹ , -NHR ¹ , -NHC (O) R ¹ , -NH-
R ^{2 is} selected from SO ₂ —R ¹ and —R ² , provided that R ^C7a and R ^C7b are H, R ² = substituted aryl or allyl or alkylaryl (eg, benzyl or substituted benzyl). In certain embodiments of this subset, R ^C30 and R ^C24 are both, for example, —OH in the [S] configuration. In another embodiment, R ^C30 and R ^C24 are independently selected from OR ³ . This subset includes, among other ^things , R ^C30 and R ^C24 are OH, and one of R ^C7a and R ^C7b contains any alternative substituent at the position specified in Formula II, and is identified by the compounds in Table II or III. All rapalogs containing any of the C7 substituents described above are included. This subset includes, among other things, other rapalogs that differ from rapamycin in part a . For example, as a subset of this, the expression:

[0113]

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[ ^Wherein at least one of R ^C7a and R ^C7b is other than -OMe]. ^Additional substituents for R ^C7a and / or R ^C7b include those described ^elsewhere herein. Of particular ^interest are ^{those compounds} in which one of R ^C7a and R ^C7b is an optionally substituted cycloaliphatic, aryl, heterocyclic or heteroaryl. Other compounds included in this subset include the hydroxyl groups 1, 2,
Three, four or five may be epimerized, fluorinated, alkylated, acylated, or modified by other ester, carbamate, carbonate or urea formation. Representative examples of compounds include compounds of formula III wherein the hydroxyl group at C43 is epimerized and the hydroxyl groups at C28 and C30 are linked by alkylation, acylation or carbonate formation.

Another subset of improved rapalogs of particular interest are those compounds of formula II, wherein one or both of R ^C13 and R ^C28 are F. In various embodiments of this subset, one, two, three, four, or five other substituents of Formula II differ from those found in rapamycin. For example, in this subset, the following structure:

[0116]

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[0117]

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^Wherein R ^C7a and R ^C7b are as defined above, and C13 fluororapalogs, C28 fluororapalogs, and C13, C28-difluororapalogs.

The 13-fluororapalogs, including certain 13-fluororapamycins and analogs, and derivatives thereof containing various substituents that do not impair the immunosuppressive activity of rapamycin itself, are important as immunosuppressants.

Some interesting aspects of the above subset of compounds are that of the formula II wherein R ^C24 and R ^C30 are both other than ( ^OO) and one or both of R ^C13 and R ^C28 Is F]
Or a series of improved rapalogs comprising a pharmaceutically acceptable salt thereof.
This series of rapalogs includes, in particular, 24,30-tetrahydro-13-F rapalogs, 24,30-tetrahydro-28-F rapalogs, and 24,30-tetrahydro-13,28-diF rapalogs, as well as those described above. Any C7 variant, provided that R ^C7a is other than OMe. This series of rapalogs has the following structure:

[0121]

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^Wherein R ^C7a and R ^C7b are as defined above. These compounds relates C14 and C43 substituents of rapamycin itself, for example, it can be further derivatized by deformation of one or both of R ^{C 14} and R ^C43.

Another subset of improved rapalogs of particular interest includes compounds of formula II wherein R ^C14 is O, OH
Or other than H], for example, wherein R ^C14 is -OR ⁶ , -NR ⁶ , -NC (O) R ⁶ , —OC (O) R ⁶ or —OC (O) NR ⁶ .

Another subset of important improved rapalogs includes a compound of formula II wherein R ^C13 is a C1-C4 alkyl wherein one or more other variations are optional at other positions relative to rapamycin. Other than an alkoxy group containing a moiety]. For example, these subsets include (a) a substituent R ^C13 other than a C1-C4 alkoxy group in place of OH at ^C13 , and (b) a substituent R as defined above instead of MeO at C7. ^Rapalogs that differ from rapamycin by treatment with ^C7a and R ^C7b are included.

Another subset of important modified rapalogs includes those of formula II wherein R ^C24 is other than OO, in which one or more other variants have been made to rapamycin at other positions. There are compounds.

Another subset of improved rapalogs of particular interest in practicing the methods of the present invention include compounds of formula II wherein R ^C7a is —OMe, wherein R ^C30 is a chemical isomer of rapamycin. Is not = O, R ^C24 is not = O, R ^C13 is not -OH, R ^C14 is not = O and / or R ³ and / or R ⁴ is not H].

Other improved rapalogs of interest include compounds of formula II wherein R ^C14 is OH.

In addition, the present invention relates to the use of carbon at position 1, 2, 3, 4, 5, or 6 of rapamycin in a molecule.
One or more of the carbon double bonds are saturated, e.g., C7, C13, C43, C24, C2
Includes improved rapalogs in combination with 8 and / or C30 variants at one or more other locations. In any of the compounds disclosed herein, using methods known in the art, C3, C4 double bond may be epoxidized to give; C6 methyl group -CH ₂ OH or -CH ₂ OM
It is also contemplated that C43 hydroxy may be converted to F, Cl or H or other substituents; and that the C42 methoxy moiety may be demethylated. Similarly, the part " a " is:

[0129]

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Can be replaced by any of It is known to produce a rapamycin by guidance fermentation or total synthesis of the synthesis. It is also known to produce various rapalogs as fermentation products. Examples of these include, among others, rapalogs that carry another moiety on the characteristic cyclohexyl or pipecolate ring of rapamycin, and C7-desmethyl-rapamycin, C29-desmethyl-rapamycin and C29-desmethoxyrapamycin.

As well as methods for obtaining rapamycin and various rapalogs by fermentation, methods and materials for performing various chemical modifications of rapamycin and structurally related macrolides are known. Various such chemical variants of rapamycin and various rapalogs are disclosed in the patents listed in Table I above, which were provided to show those skilled in the art the chemical synthesis and product recovery, purification and formulation. And may be applied in the practice of the present invention. The following representative variations and / or references that can be used to produce the desired rapalogs are exemplary.

[0132]

[Table 7]

An approach to the synthesis of various fluoro and difluororapalogs is outlined below.

[0134]

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[0135]

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An approach to the synthesis of various 24,30-tetrahydrorapalogs is shown below.

[0137]

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As a further example, starting with 13-fluororapamycin instead of rapamycin gives the corresponding 13-fluoro-24,30-tetrahydroC7 rapalog.

One approach for the synthesis of other C13 derivatives is shown below.

[0140]

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Further, rapalogs for use in the present invention and intermediates for producing such rapalogs are described, for example, in Katz et al., PCT International Publication No. WO 93/13663 and Cane et al., WO 97/02358. Can be produced by the direct biosynthesis disclosed in US Pat. For further biological methods, see also Khaw et al., 1998, J. Bacteriology 180 (4): 809-814.

The novel rapalogs of the present invention can be manufactured by those skilled in the art by methods and materials known to those skilled in the art as guided by the disclosure set forth herein. For example, the methods and materials can be adapted to known methods referenced or indicated in the above-cited references, the contents of all such references being incorporated herein by reference. Further guidance and examples are provided herein for explanation and as guidance to practitioners. Chemists of ordinary skill in the art can easily modify the above description, for example, by adding suitable protecting groups to sensitive moieties during synthesis and then removing such protecting groups when no longer needed. And other synthetic approaches could be readily determined. FKBP domain and fusion protein The FKBP fusion protein is composed of at least one FKBP domain containing all or a part of the peptide sequence of the FKBP domain, and at least one heterologous action domain. Chimeric proteins can be measured by direct binding assays (e.g., fluorescence quenching), competitive binding assays (e.g., against FK506), inhibition of FKBP enzyme (rotamase) activity, or other assays. Preferably a Kd value of less than about 100 nM, more preferably a Kd value of less than about 10 nM, even more preferably a Kd value of less than about 1 nM
The d value must be able to bind to the improved rapalog of the present invention. Typically, a chimeric protein contains one or more protein domains that include a peptide sequence selected from a native FKBP protein (eg, human FKBP12 disclosed in PCT / US94 / 01617). Such peptide sequences can have altered binding characteristics by substitution, insertion or deletion of less than 10, preferably less than 5, amino acid residues. Such a variant may have the following binding profile (a) to the modified FKBP domain, or to a chimera containing that domain, than to FKBP or FKBP12 endogenous to the host cell to be processed (by any measure B.) Binding the improved rapalog, preferably in at least one, more preferably at least two, even more preferably three or more, large orders; and (b) other endogenous endogenous host cells to be processed. Preferably, the FRB fusion protein has a greater order of magnitude than the FRB-containing protein or FRAP (by any measure), more preferably at least 2, more preferably at least 2, and even more preferably at least 3.
Binding KBP rapalog complex is selected in a particular manner to obtain one or both.

The FKBP chimera also contains at least one heterologous action domain, ie, a protein domain containing a non-FKBP peptide sequence. Such action domains include, for example, DNA-binding domains, transcriptional activation domains, cell localization domains, as disclosed elsewhere herein or in PCT / US94 / 01617 or other references. It may be an intracellular signal transduction domain or the like. Typically, an action domain directs a chimeric protein to a selected cellular location, for example, when multimerizing proteins containing the same or different action domains, or initiates a biological action upon binding or aggregation with another action domain. can do.

A recombinant nucleic acid encoding such a fusion protein can be a parent FKBP protein (eg, human FKBP protein).
Such hybridization is possible if it can selectively hybridize to the DNA encoding KBP12) or if there is no degeneracy of the genetic code. These chimeric proteins have action domains derived from other proteins (e.g., Ga1
4, VP16, FAS, CD3 zeta chain, etc.), the recombinant DNA encoding the chimeric protein can selectively hybridize to DNA encoding other proteins, Such hybridization is possible if there is no denaturation.

The FKBP fusion proteins of the invention, as well as the FRB fusion proteins discussed in detail below, may contain one or more copies of one or more action domains and one or more copies of one or more different ligand binding domains. it can. The ligand binding domain (s) (i.e., FKBP and FRB domains) may be N-terminal, C-terminal, or interspersed with the active domain (s). . Embodiments containing multiple copies of the ligand binding domain typically include 2, 3, or
There are four copies. For example, an FKBP fusion protein can contain 2, 3 or 4 FKBP domains. The various domains of the FKBP-derived protein (and the FRB fusion proteins discussed below) are optionally separated by binding peptide regions that can be derived from one of the adjacent domains or can be heterologous.

Representative examples of FKBP fusion proteins useful in the practice of the present invention include PCT / US94 / 01617 (S
tanford & Harvard), PCT / US94 / 08008 (Stanford & Harvard), Spencer et al. (supra), P.
FKBP fusion protein disclosed in CT / US95 / 10591 (ARIAD), PCT / US95 / 06722 (Mitotix, Inc) and other references cited herein; FKBP fusion protein disclosed in the following Examples Any variant of the above FKBP fusion protein which contains 10 or less (preferably 1-5) amino acid insertions, deletions or substitutions in one or more of the FKBP domains and is capable of binding to rapamycin or rapalog; Any variant of the above FKBP fusion protein comprising one or more copies of an FKBP domain encoded by a DNA sequence capable of selectively hybridizing to a DNA sequence encoding the domain and capable of binding to rapamycin or rapalog Any of the foregoing, wherein one or more heterologous domains have been deleted, replaced or supplemented with a different heterologous domain; FKBP domains derived from a non-human source capable of binding rapamycin or rapalog; FKB containing in
Any variant of the P fusion protein; and human FK, wherein one or more of its amino acid residues is replaced by a different amino acid and the variant is capable of binding to rapamycin or rapalog.
Any variant of the above FKBP fusion protein containing one or more amino acid residues corresponding to Tyr26, Phe36, Asp37, Arg42, Phe46, Phe48, Glu54, Val55 or Phe99 of BP12.

For example, in many cases, FKBP fusion proteins are encoded by ACTAGA
Containing multiple copies of the FKBP domain containing amino acids 1-107 of human FKBP12 separated by a 2-amino acid linker Thr-Arg, the ligation products of DNA are digested with restriction endonucleases SpeI and Xbal, respectively. The following table shows a representative subset of mutant FKBP domains based on the above FKBP12 sequences.

[0148]

[Table 8]

Note: Entries identify the natural amino acid by letter code one letter and sequence position, followed by amino acid substitutions with the variant. Thus, F36V represents the human FKBP12 sequence in which the phenylalanine at position 36 has been replaced by valine. F36V / F99A is a double mutant in which phenylalanine at positions 36 and 99 has been replaced by valine and alanine, respectively. FRB Domains and Fusion Proteins An FRB fusion protein is at least one heterologous to at least one FRB domain (which may include all or a portion of a peptide sequence of a FRAP protein described elsewhere herein or a variant thereof). Contains effector domains.

Generally, a FRB domain, or a chimeric protein comprising the same, is a DNA molecule encoding a protein comprising a native FRB domain, such as a human or other mammalian FRAP protein or yeast protein, Tor-1 or Tor. -2 or a DNA molecule capable of selectively hybridizing to a DNA molecule encoding the above Candida FRB-containing protein. Examples of the FRB domain of the present invention include those that can bind to a complex of the FKBP protein and the improved rapalog of the present invention.

The FRB fusion protein must be able to bind to the complex formed by the improved rapalog of the present invention and the FKBP fusion protein. Preferably, the FRB fusion protein binds to such a complex with a Kd value of less than 200 μM, more preferably less than 10 μM, as determined by conventional methods. The FRB domain is of sufficient length and composition to maintain high affinity for the complex of the FKBP fusion protein and rapalog. FRB domains can be less than about 150 amino acids in length, but can be less than about 100 amino acids. One such region includes the 133 amino acid region of human FRAP extending from Val2012 to Tyr2144. C
See hiu et al., 1994, Proc. Natl. Acad. Sci. USA 91: 12574-12578. Particularly important FRB regions span from Glu2025 to Gln2114 of human FRAP and retain affinity for FKBP12-rapamycin or FKBP-rapalog complexes. In some embodiments, Q2214 is removed from the 90-amino acid sequence, leaving it an 89-amino acid FRB domain. FRB peptide sequences can be modified to adjust binding properties, usually by substitution, insertion or deletion of less than 10, preferably less than 5, amino acids. Rather than forming a complex of a heterologous FKBP and FRAP protein with a rapalog, in a particular embodiment, the complex comprises one or more molecules of an FKBP fusion protein, an FRB fusion protein, and an improved rapalog. Select a transformation. Preferably, the preference is at least one, more preferably at least two, even more preferably three orders of magnitude (measured in any way).

Recombinant DNA encoding such a protein can selectively hybridize to DNA encoding a FRAP species or, without the genetic code, can perform such hybridization. These chimeric proteins include, for example, Gal4, VP16, Fas, C
Since it contains an effector domain derived from another protein such as D3 zeta,
Recombinant DNA encoding a chimeric protein can selectively hybridize to DNA encoding another protein, or can undergo such hybridization if the genetic code is not altered.

Representative examples of FRB chimeras useful in practicing the present invention include those disclosed in the Examples below, wherein one or more of the heterologous domains has been replaced by another heterologous domain, or one or more additional A variant thereof in which a heterologous domain has been supplemented, wherein one or more of the FRB domains has a domain derived from a non-human peptide sequence (e.g., Tor2 or Candid
a) and amino acids substitutions, substitutions or insertions as described herein, as long as the chimera can bind to the complex formed by the FKBP protein and the improved rapalog of the present invention. Mutants in which the FRB domain has been modified. A typical example of an FRB fusion protein is at least 89-amino acids, comprising a sequence spanning at least residues 2025-2113 of human FRAP, separated by a linker THr-Arg formed by the binding of SpeI-Xbal sites as described above. One or more
Contains Fed. Such restriction sites or linkers in any of the fusion proteins of the present invention can be deleted, replaced or extended using conventional methods such as site-specific mutagenesis. Mixed Chimeric Proteins A third type of chimeric protein includes one or more FKBP domains, one or more heterologous effector domains, and one or more FRB domains as described for FRB fusion proteins.

[0154] Mixed chimeric protein molecules can form homodimeric or homomultimeric protein complexes in the presence of the improved rapalog to which they are attached. Embodiments involving mixed chimeras have the advantage that a single recombinant nucleic acid construct must be introduced into the cell instead of two recombinant nucleic acid constructs, otherwise the FKBP fusion protein and FRB fusion Expression of any of the proteins must be induced.

[0155] The recombinant DNA encoding the mixed chimera protein is composed of a DNA encoding FKBP protein, FR
Whether it can selectively hybridize to the DNA encoding the AP and a heterologous DNA sequence encoding a protein from which one or more effector domains are derived (e.g., Gal4, VIP6, Fas, CD3 zeta chains, etc.) Such hybridization can be performed if the code is not altered. Heterologous domains As described above, the heterologous effector domains of the FKBP and FRB fusion proteins are capable of inducing (or inhibiting) DNA-binding and / or transcription of a target gene upon mutual binding of the chimeric proteins carrying them; A protein domain that can initiate differentiation, proliferation or apoptosis; direct proteins to specific cell locations; or initiate other biological events.

[0156] Embodiments involving regulatable gene transcription include those that control the transcription of DNA elements that are sensitive to binding or multimerization of the heterologous domains of the first and second chimeric proteins (polypeptides, antisense RNAs, ribozymes). (Encoding) the use of a target gene construct comprising the target gene.

In embodiments of the invention that involve direct activation of transcription, the heterologous domains of the first and second chimeric proteins may be a DNA binding domain (eg, GAl4) or a chimeric DNA as discussed below.
A binding domain (e.g., ZFHD1), and a transcription activation domain (e.g., VP16 or p6
5). When the chimeric protein containing the transcriptionally active domain is multimerized, the transcriptional activator is targeted to the promoter element to which the DNA binding domain binds, and the transcription of the target gene bound to the promoter element is regulated. Activate. Elimination of the transcriptional activation domain or substitution of the transcriptional activation domain with a repressor domain (see PCT / US94 / 01617) yields similar chimeras useful for inhibiting transcription of the target gene. The complex DNA binding domain and DNA sequence to which they bind are disclosed in Pomerantz et al., Supra, 1995, the contents of which are incorporated herein by reference. Such a complex DNA binding domain can be used as a DNA binding domain in the practice of the present invention, together with a target gene construct containing a cognate DNA sequence to which the complex DBD binds.

In embodiments involving indirect activation of transcription, the heterologous domain of the chimera is the effector domain of a signaling protein that, upon aggregation or multimerization, initiates transcriptional activity under the control of an individual promoter. For example, the signaling domain, upon aggregation, the IL-2 promoter or a derivative thereof (e.g., repeat N
It may be an intracellular domain of the zeta subset of the T cell receptor that induces transcription of the gene bound to the (F-AT binding site).

In another aspect of the invention, the heterologous domains are protein domains that upon binding can induce cell death. Examples of such domains include the intracellular domain of Fas antigen or TNF R1. The chimeric protein containing the Fas domain is PCT / US94 / 0161
It can be designed and manufactured in a manner similar to that disclosed in No. 7. Engineered receptor domains As described above, the FKBP and FRB domains can include a peptide sequence selected from the peptide sequences of the native FKBP and FRB domains. The natural sequence is human
There are the FRB domain of FRAP and the sequence of human FKBP12. Alternatively, the peptide sequence can be derived from such a naturally occurring peptide sequence, but usually contains less than 10, preferably 1 to 5 mutations in one or any of such peptide sequences. As disclosed elsewhere herein, such mutations provide a number of important features. For example, an FKBP domain binds to a rapalog by an unmodified FKBP domain, whereas it may preferentially bind to an improved rapalog at least 1, preferably 2, more preferably 3 or 4 or more orders of magnitude in size. Can be transformed as follows. The FRB domain is at least 1, preferably 2, more preferably 3 orders of magnitude larger than the unmodified FRB domain, so that it can preferentially bind to the (modified or unmodified) FKBP rapalog complex. Can be deformed so as to be able to be combined more efficiently. The FKBP and FRB domains should be at least 1, preferably 2, and more preferably 3 orders of magnitude, relative to the unmodified FKBP and FRB domain, so that they can preferentially form the improved rapalog and tripartite complex. It can be deformed so that it can be formed more efficiently with the size of. (a) FKBP A method for identifying FKBP mutations that confer high ability to bind derivatives of FK506 containing various substituents (bumps) was disclosed in PST / US94 / 01617. Similar methods can be used to obtain modified FKBPs that preferentially bind bumped rapamycin derivatives (ie, rapalogs). The structure of the complex between rapamycin and FKBP12 is known (see, for example, Van Duyne et al., J. Am. Chem. Soc. 1991, 1
13, 7433-7434). Such data can be used to identify amino acid residues that are inconsistent with various rapalog substituents. In this approach, molecular modeling can be used to identify candidate amino acid substitutions in the FKBP domain that match the rapalog substituent (s) and the corresponding FKBP domain, using site-directed mutagenesis. The protein mutants thus identified can be processed. The mutant is expressed by standard methods, and if the mutant has suitable activity / affinity, the binding affinity for rapalogs can be, for example, inhibition of rotamase activity or It is measured by competing for binding to molecules such as FK506.

In particular, Applicants have recognized that certain improved rapalogs of the present invention (eg, those modified with C-13 or C-14 relative to rapamycin) include the residues Tyr26, Phe36, Asp37,
It is believed that one or more of Tyr82 and Phe99 preferentially binds to FKBP substituted with amino acids having smaller side chains (eg, Gly, Ala, Val, Met and Ser). Examples of mutant FKBPs modified at position 26 or 36 are described above under "Representative mutant FKBPs". Similarly, Applicants believe that C20-modified rapalogs (i.e., those in which R4 is other than -H) have Tyr82 and / or Ile56 in which other amino acids, especially smaller side chains, It is considered that it preferentially binds to FKBP substituted with an amino acid. In another example, Applicants report that C24-deformed rapalogs (i.e., W =
(Other than O) are considered to preferentially bind FKBP in which one or more of Phe46, Phe48 and Val55 have been replaced with other amino acids, especially amino acids with smaller side chains.
In addition, Applicants have reported that rapalogs modified at C28 and / or C30 (ie, where R3 is other than H and / or V is other than OO), that Glu54 is another amino acid , Particularly preferentially bind FKBPs that are substituted with amino acids having smaller side chains. In all of the above examples, single or multiple amino acid substitutions can be made. Specific examples are shown in the preceding table.

Another method for iteratively processing and testing single or multiple mutants is
The co-randomize of structurally identified residues that are in or contact with one or more rapalog or rapamycin substitutes. A population of polypeptides containing a FKBP domain randomized at the identified location (eg, as described in the preceding paragraph) is produced using conventional synthetic or genetic methods. Such a population represents a series of FKBP domains that contain substitution amino acids at one or more of such positions. The population is screened and FKBP variants are selected that retain the desired rapalog binding properties. Usually, randomizing several residues at the same time produces mutants with high affinity and specificity for a given raised rapalog to maximize the potential for beneficial cooperative interactions between side chains It is predicted that. Methods for producing randomized libraries at other sites are known and include primer-induced mutagenesis using altered oligonucleotides, PCR using altered oligonucleotides, and cassette mutations using altered oligonucleotides. Mutagenesis (e.g., Lowman, HB,
And Wells, JA Methods: Comp. Methods Enzymol. 1991. 3, 205-216;
Dennis, MS and Lazarus, RA 1994, J. Biol. Chem. 269, 22129-22136;
And references therein).

Applicants further note that in many cases, randomization of only a few residues at a given position or almost directly in contact with rapamycin will result in FKBPs that can optimally accommodate a rapalog substituent (bulge) We believe that it is not possible to completely explore possible deformations in conformation. Thus, random libraries containing random substitutions that are not based on structural considerations are also anticipated to identify the few mutations or combinations that preferentially bind to the raised rapalog. Alanine-scanning mutagenesis (
For example, Cunningham and Wells, 1989, Science 244, 1081-1085), PCR misincorporation mutagenesis (e.g., Cadwell and Joyce, 1992, PCR Meth.
Applic. 2, 28-33) and "DNA Shuffling" (Stemmer, 1994, Natur
Several suitable mutagenesis schemes have been described, such as e 370, 389-391 and Crameri et al., 1996, Nature Medicine 2, 100-103). With these methods,
A library of random mutants, or a series of single mutants, is produced and then studied by a screening or selection approach.

In many cases, an effective way to identify the best mutant that binds preferentially to a given ridge is a combination of a structure-based approach and a randomized approach. Clackson and Wells, 1994, Trends and Biothechnology 12, 173-1
See 84 (Review). For example, Applicants have predicted a library construct in which key contact residues have been randomized by degenerate oligonucleotides, but have used error-promoting conditions to introduce additional mutations at random sites. Amplification was performed. Another example is the construction of component DNA fragments from structure-based and random random libraries using DNA shuffling.

Screening the library for the desired mutation is performed using the yeast two-hybrid system.
(Fields and Song, 1989, Nature 340, 245-246). For example, a FRB-VP16 fusion can be introduced into one vector and a library of randomized FKBP sequences cloned into another GAL4 fusion vector. The yeast co-transformant is treated with rapalog, and the harboring complementing FKBP mutant is essentially, for example, beta-galactosidase or luciferase production (sieving), or if the vector used is suitable, On plates lacking various nutrients (selection). FKBP
The requirement for raised rapamycin to bind to the -FRAP interaction is a false positive (po
It is a useful sieve for eliminating sitives).

Another method for isolating modified ligand-binding domains from a library of FKBP (or FRB) mutants is described in Hu et al. (Hu, JC, et al. 1990, Science. 250: 1400-1403; review). (See Hu, JC 1995. Structure. 3: 431-433) for the use of gene selection for functional dimer formation. This method demonstrates that bacteriophage lambda repressor cI binds to DNA as a homodimer, and that binding of the homodimer to operator DNA prevents transcription of phage genes involved in the lysin pathway of the phage life cycle. Use. These bacterial cells expressing a functional lambda repressor are immune to lysis by superinfection with phage lambda. The repressor protein contains an amino-terminal DNA binding domain (amino acids 1-92) linked to a carboxy-terminal dimerization domain by a 40 amino acid flexible linker. The isolated N-terminal domain binds to low affinity DNA due to poor dimer formation. High affinity DNA binding can be restored with a heterodimerization domain (eg, GCN4 leucine zipper). Hu et al. Described a system that uses phage immunity as a gene selection to isolate GCN4 leucine zipper mutants that can mediate lambda repressor dimer formation from numerous sequences (Hu et al., 1990). ).

For example, to use the lambda repressor system to identify a FRAP mutant that is complementary to a raised rapalog, a lambda repressor that carries the mutant FRAP sequence
The FRAP library is transformed into E. coli cells expressing the wild-type lambda repressor-FKBP protein. Plasmids expressing FRAP mutants are isolated from persistently lysed colonies on bacterial plates containing "raised" rapamycin compounds and high titers of lambda phage. Alternatively, to isolate the FKBP mutant,
The above method was repeated with the lambda repressor-FKBP library carrying the mutant FKBP sequence transformed into E. coli cells expressing the wild-type lambda repressor-FRAP protein.

As a further alternative, the randomized FKBP sequence can be cloned into a vector for phage display and a mutant that optimally binds to rapalog selected in vitro. In vitro affinity selection can be performed in various ways. For example, rapalogs are mixed with a library phage pool in solution in the presence of recombinant FRAP tagged with an affinity handle (e.g., a hexa-histidine tag, or GST). The resulting complex is captured on a suitable affinity matrix to increase the amount of phage displaying FKBP harboring complementation mutation. Although phage display methods have been described, other in vitro selection systems are also contemplated (eg, display on lambda phage, display on plasmid, display on baculovirus). In addition, selection and sieving methods can be used to improve other properties that are beneficial in the use of the present invention, such as high in vivo stability. For a review, see Clackson, T. & Wells, JA 1994, Trends.
See Biotechnol. 12, 173-184. (B) FRAP A similar consideration is the generation of mature FRB dominant species that preferentially bind to an improved rapalog containing (ie, “bumped”) a mutation to rapamycin in the FRAP binding portion of the macrocycle. Applies to For example, a human FRAP FRB peptide having a substituent other than -OMe substituted with FRB at the C7 position based on the peptide sequence, but having a residue T
A dominant bond can be obtained using a rapalog having one or more amino acid substituents of ry2038, Phe2039, Thr2098, Gln2099, Trp2101 and Asp2101. Examples of mutants include Y2038, Y2038L, Y2038V, Y2038A, F23039H, F23039V, D2102A, T
2098A, T2098N and T2098L. Rapalogs with substituents other than -OH at C28 and / or with substituents other than = O at C30 can be used to obtain dominant binding to FRAP with amino acid substitutions for Ghu2032. Examples of mutants include E2032A and E20325. Proteins, proteins containing FRBs having one or more amino acid substitutions at the above positions, libraries of proteins or proteins randomized at these positions (ie having various substitution amino acids at these residues), whole protein domain A library or random combination of these mutants is created using the methods described above to identify mutant FRAPs that bind predominantly to the bumped rapalog.

The affinity of the candidate mutant FRB for the FKBP protein in complex with the rapalog can be determined by a number of techniques, such as binding to GST-FKBP in the presence of an in vitro translated FRB mutant agent (Chen et al. Natl. Acad. Sci. USA 92, 4947-4951) or by a yeast two-hybrid assay with the ability to participate in a rapalog-dependent transcriptionally active complex with an appropriate FKBP fusion protein. Can be tested.

[0169] FRB variants with the desired binding ability can be isolated from libraries displayed on phage using a variety of classification strategies.

An additional feature of the FRB fusion proteins that may vary in various embodiments of the present invention is the exact sequence of the FRB domain used. In some applications, it is preferable to use a protein of the FRB that is larger than the minimum (89 amino acids) FRB domain. These include the N-terminal residue Glu2025 (preferably at least Arg2018 or
2174 or more) as well as a C-terminal extension beyond position 2113, such as to position 2113, 2141 or 2174 or more, and in some cases the stability of the folded FRB domain and / or Improve the efficiency of expression. Another use where different FRB sequence ends may be used is where long linkers are required at one or both sites of the FRB domain for steric use, e.g., at the cell membrane or on DNA. Includes those required to accommodate the curvature of the polypeptide chain required for mediator protein-protein binding. Conversely, in other applications, short linkers on one or both sites of the FRB domain are preferred or required to provide a foreign effector domain appropriate for biological function. In human gene therapy applications, the naturally occurring human FRAP sequence of such a linker is:
It is generally preferred to introduce foreign sequences or to reduce the risk of nullifying the immune response in the host organ.

[0171] Some rapalogs, particularly those that have denatured or substituted groups (for rapamycin) at positions believed to be located at the boundary between the FKBP binding domain and the FRAP binding domain, such as C28, C30, C7 and C24, The ability of rapamycin to form complexes with both mammalian FKBP and FRB domains, particularly those with these domains having naturally occurring human peptide sequences, is reduced. Reduced ability is defined as decreased binding affinity as measured by the direct or indirect assay means described herein or an immunosuppressive activity measured in a suitable assay, such as a T cell proliferation assay. Expressed as reduced. In such cases, iterative methods can be used to identify mutant FKBP and mutant FRB pairs that can complex with a more efficient rapalog than the corresponding domain containing the naturally occurring human peptide sequence. For example, first, a complemented modified FKBP domain capable of binding the complex is identified as described above, and then the mutated FKBP domain is used as an affinity matrix in complexation with the rapalog to use the complementary modified FRKB domain capable of binding the rapalog.
The B domain may be selected. Several cycles of such mutagenesis and screening may be performed to optimize the protein pair.

For some embodiments, it may be desirable to use FRB and / or FKBP domains that contain mutations that can affect protein-protein interactions. For example, a mutant FKBP domain that can complex with an endogenous FRB when bound to a rapalog that is significantly less effective than a mutant FRB is of great interest. Also of interest is a mutant domain that can bind to a complex of mutant FKBP and a given rapalog that is much more effective than a complex of endogenous FKBP and rapalog. Using selection and screening approaches similar to those described above, (i) amino acid substitutions in the FKBP domain that are significantly different from the ability of the domain to form a tripartite complex with a given rapalog and endogenous FRB; Identifying deletions or additions, (ii) identifying amino acid substitutions, deletions or additions to the FRB domain that are significantly different from the ability of the domain to form a tripartite complex of a given rapalog and endogenous FKBP; and (iii
) Competitive mutants in partner proteins can be selected and / or otherwise identified. Preferred mutant FKs with reduced efficacy in tripartite complex formation
As an example of a BP, one or both of His87 and Ile90 are amino acids, such as Arg containing large side groups.
, Trp, Phe, Tyr or Lys, comprising a mammalian, preferably human FKBP; preferably a mammalian, more preferably a FRB comprising a human peptide sequence, followed by mutagenesis as described above, A competing variant having the following rapalogs may be generated. Representative FRB mutants that are effective with H87W or H87R hFKBP12 include Y2038 replaced with V, S, A or L; F2039 replaced with A and / or R2042 replaced with L, A or S Human FRB. A representative FRB mutant that is effective using I90W or hFKB12 is a human FRB in which K2095 has been replaced with L, S, A or T.

In addition, in optimizing the receptor domain of the present invention, it is contemplated that the immunity of the polypeptide will require binding of the peptide by MHC proteins and recognition of the given peptide as an endogenous T-cell receptor. You should understand that At least for human gene therapy applications, it is preferred to adapt a given heterologous peptide sequence, including the connecting peptide sequence, to minimize the likelihood of it being immunologically presented to humans. For example, a peptide that binds to a human MHC class I molecule has stringent requirements for certain residues at key "anchor" positions in the bound peptide. That is, HLA-A2 requires leucine, methionine or isoleucine at position 2 and leucine or valine at the C-terminus (for a review, see Stern and Wiley (1994) Structure 2, 145-251). Therefore, when manipulating proteins in the practice of the present invention, cycles of these residues are preferably avoided, especially in human gene therapy applications. The foregoing applies to all protein engineering aspects of the invention, including, but not limited to, the manipulation of point mutagenesis into the receptor domain and the selection and design of boundaries between the various protein domains. Other components, design features and uses The chimeric protein is a cell localization domain as a heterologous domain,
For example, it may include a membrane retention domain. See PCT / US94 / 01617, especially pages 26-27. Briefly, membrane retention domains can be isolated from accessible membrane-associated proteins, whether endogenous to the host cell or not. The membrane-retaining cell domain can be a transmembrane domain, ie, an amino acid sequence that extends across the membrane as in the case of cell surface proteins that contain numerous receptors. The transmembrane peptide sequence can be extended to span some or all of the transmembrane and / or intramembrane domains. Instead, the membrane retention domain is
It may be a lipid retention domain, such as myristylation or palmitylation, which binds the cell surface membrane. A lipid membrane retention domain is usually added at the 5 'end, which encodes the sequence for N-terminal attachment to the membrane, and at the base of the 3 terminal for C-terminal attachment. Peptide sequences with post-transcriptional processing to provide lipid binding are described in Carr, et. Al., PNAS US
A (1988) 79, 6128: Aitken et al., FEBS Lett. (1982) 150, 314; Henderson,
et al., PANAS USA (1983) 80, 319; Schulz, et.al., Virology (1984), 123
, 2131; Dellman el al., Nature (1985) 314, 374;
n Rev. of Biochem (1988) 57, 69. Interesting amino acid sequences include the sequence MGGSSKSKPKDPSQR. Various DNA sequences can be used to encode such sequences in various chimeric proteins of the invention. Other localization domains are particularly useful for organelle-targeting domains and sequences that target proteins that carry them to the endoplasmic reticulum, such as -KDEL- and -HDEL- and chimeric proteins intended for (direct) transcriptional regulation. Contains certain nuclear localization sequences. Various cell localization sequences and signals are well known in the art.

A chimeric tan containing a cytoplasmic signal reprogramming domain, eg, various effector domains including the CD3 zeta chain, a nuclear transcription factor domain including VP16 and GAL3, a domain capable of inducing apoptosis including a Fas cytoplasmic domain, and the like. Details that can be used in the practice of the subject invention with respect to the design, assembly and use of the structure encoding the protein are described in PCT / US94 / 01917 and PCT / US95 / 10591. Have been. The latter international patent application further discloses additional features that are particularly applicable to the production of genetically engineered animals that can be used as disease models in biomedical research. These features are flanked by the use of cell-specific control elements in the construct for the chimeric protein and loxP sequences.
Includes the use of controlled transcription for the expression of Cre recombinase as a target gene leading to the elimination of an interesting gene. Alternatively, flp and its cognate recognition sequence can be used in place of Cre and lox. These features may be compatible with the subject invention.

In various cases, particularly in embodiments involving all animals including cells engineered in accordance with the present invention, it is often preferred to derive the various domains of the chimeric protein from the same species of protein as the host cell. And in some cases it is necessary. Thus, it is often preferred for genetic manipulation of human cells that the heterologous domains (as well as the FKBP and FRB domains) be human organs rather than microbial, yeast or non-human sources.

We also note that epitope tags have been introduced into the chimeric proteins of the invention to facilitate their determination. Tissue Specific or Cell Type Specific Expression In certain embodiments, it is preferred that the chimeric protein be expressed cell-specifically or tissue-specifically. The specificity of such expression can be determined by combining one or more DNA sequences encoding the chimeric protein with a cell type-specific transcription control sequence (promoter / promoter).
(Enhancer). Numerous cell type-specific transcription control sequences are known. Others can be obtained from genes expressed in a cell-specific manner. See PCT / US95 / 10591, especially pages 36-37.

For example, a structure that expresses a chimeric protein may include regulatory sequences derived from known genes for specific expression in a selected tissue. Representative examples are shown in the table.

[0178]

[Table 9]

[0179]

[Table 10]

Target Gene Composition In embodiments of the invention where the chimeric proteins are designed such that their multimerization activates transcription of the target gene, an appropriate target gene composition is also used for the engineered cells. . Suitable target gene constructs are those that include the target gene and cognate transcription regulators, such as promoters and / or enhancers that are responsive to multimerization of the chimeric protein. In embodiments involving the direct activity of transcription, the responsiveness is dependent upon the target of one or more DNA sequences recognized by the DNA-binding domain of the chimeric protein of the invention (ie, the DNA sequence to which the chimeric protein binds). This can be achieved by the presence of the genetic makeup. In embodiments involving indirect activity of transcription, the responsiveness may be achieved by the presence of a promoter and / or enhancer target gene construct that is activated by intracellular signals generated by the multimerization of the chimeric protein. For example, if the chimeric protein contains a TCR zeta chain intracellular domain, the target gene binds under expression control of the IL-2 promoter region.

The present invention also relates to (a) DNA-binding and (b) which are capable of binding cognate gene sequences, such as the DNA-binding chimeric proteins of the invention (or are susceptible to indirect activation as described above).
) DNA flanking from the locus of the desired target gene, which is inscribed in the host cell
Also provided is a target DNA construct comprising the sequence. These constructs allow for heterologous recombination of cognate DNA sequences in the host ligated to the endogenous target gene. In other embodiments, the construct may include a cognate DNA sequence, or the cognate DNA sequence may be provided by a locus.

A target gene in any of the above embodiments may be, for example, a surface membrane protein (eg, a receptor protein), a secretory protein, a cytoplasmic protein, a nucleoprotein,
It may encode a recombinase such as Cre, ribozyme or antisense RNA. PCT / US94 / 01617 for general construction and design details and for various applications including gene therapy, and PCT / IS95 for application to animal models of disease.
See / 10591.

The present invention includes various configurations related to the chimeric protein. However,
In all cases involving activation of target gene transcription, the chimeric protein divides key characteristic cells containing the chimeric and target gene structure-encoding structures,
More than 1-fold, preferably 2 times, in the presence of the multimerizing ligand than in its absence
It expresses a large amount of 3 times or more, more preferably 3 times or more. Most desirably, the selected gene is not observed unless the cell is or has not been exposed to the multimerizing ligand.

To regenerate, the chimeric protein is capable of initiating the detection of a detectable level of the target gene in a genetically engineered cell following cell rapalog, ie, multimerization of the chimera. Thus, transcription of the target gene is activated in the engineered cells of the invention following exposure of the cells to an improved rapalog capable of multimerizing the chimeric protein. The unique engineered cells of the invention contain chimeric proteins as described above and are responsive to the presence and / or concentration of an improved rapalog capable of multimerizing these chimeric protein molecules. . Its responsiveness is represented by the transcriptional activity of the target gene. Such transcriptional activity is easily detected by conventional assays for transcription of the target gene. In another embodiment, the biological response to ligand-mediated multimerization of the chimera is cell death or other biological event rather than direct activation of transcription of the target gene. Design and assembly of DNA constructs The constructs can be designed in accordance with the principles, i.e., representative examples and materials and methods disclosed in the patent and scientific literature described herein, with the specification and literature changing and Further examples are incorporated herein by reference. The components of the components can be prepared by conventional methods, wherein the coding sequence and control regions are ligated and cloned in an appropriate cloning host, if desired, and analyzed by restriction or sequencing or other convenient methods. Is done. In particular, PCR is used to isolate individual fragments containing all or some of the functional units and to kill one or more mutations as desired, including "primer repair", ligations, ex vivo sudden mutations. It can be introduced using mutagenesis or the like. In the case of a DNA construct encoding a fusion protein, a fusion protein capable of translating the DNA sequences encoding the individual domains and subdomains into a single polypeptide that conceals all component domains in a cell or cell lysates Construct an open reading frame. The DNA construct encoding the fusion protein can then be placed in a vector that directs the expression of the protein in a suitable cell type. For biological analysis of the encoded chimera, it is desirable to construct a plasmid that directs the expression of the protein in a microorganism or protein in a reticulocyte-lysate system. For use in producing proteins in mammalian cells, the protein-encoding sequence is introduced into an expression vector that directs expression in these cells. Expression vectors suitable for such uses are well-known in the art. Various types of such vectors are commercially available.

The constructs encoding the chimeric proteins and target genes of the present invention can be introduced into cells as one or more DNA molecules or constructs, and in many cases, one or more markers. And selecting a cell host containing the construct. Once completed and shown to have the proper sequence, the construct is then introduced by any convenient method. The construct can be introduced into a vector capable of episomal replication (eg, a BPV or EBV vector) or into a vector designed to integrate into the chromosome of the host cell. The construct can be integrated and packed into a non-replication-defective virus genome, such as an adenovirus containing a retroviral vector, adeno-associated virus (AAV) or herpes simplex virus (HSV), for infection or correction. The virus delivery system is discussed in detail below. Instead, the composition is changed to DNA, etc.
biolistics), calcium phosphate transfection, lipofection, microinjection. The host cells are grown and expanded in some cases prior to introducing the construct, and then undergoing appropriate processing to introduce the construct and effecting host integration. Various markers that can be used successfully include hprt, neomycin resistance, thymidine kinase, hygromycin resistance, and the like, and include various cell surface markers such as Tac, CD8, CD3, Thy1 and NGF receptor.

In some cases, it may be possible to have a target site for heterologous recombination, wherein the organization is integrated at a particular locus. For example, a heterologous gene (at the same locus or elsewhere) can be deleted and / or replaced by a recombinant target configuration of the invention. For heteromorphic recombination, generally Ω or O-vector can be used. For example, Thomas and Capecchi, Cell (198
7) 51, 503-212; Mansour, et al., Nature (1988) 336, 348-352 and Joyner.
el al., Nature (1989) 338, 153-156.

The construct can be introduced as a single DNA molecule encoding all genes or as different genes having one or more genes. The configurations can be introduced simultaneously or sequentially using the same or different markers, respectively.

[0188] Vectors containing active factors, such as replicating microbial or yeast organs to produce stocks of constituent DNA and perform transfection,
Selectable and / or expandable markers for expression in rytos) or eukaryotes, promoter / enhancer elements and mammalian expression control elements are well known in the art and are commercially available. Nucleic acid transport: In vitro and in vivo Any means of introducing heterologous nucleic acids into host cells, especially eukaryotic cells, animal cells, preferably human or non-human mammalian cells, can be applied in the practice of the present invention. For the purposes of this discussion, the various nucleic acid constructs described herein are referred to as transgenics. In vitro studies of DNA transport include calcium phosphate precipitation, electrophoresis, lipofection and viral vector-mediated infection. Two general in vivo gene therapy approaches include (a) delivery of "naked" lipid complex or liposome formulations or other formulated DNA and (b) delivery of heterologous nucleic acids via viral vectors. In a first approach, plasmids with genetically transformed transformants carrying the desired DNA structure can first be experimentally optimized for expression (eg, 5 ′ untranslated) prior to DNA formulation, eg, lipid formulation. Intron incorporation into region and deletion of unnecessary sequences (Felgner, et al., Ann NY Acad Sci 126-139,
1995). The incorporation of the DNA, for example, with various lipid or liposome materials, can then be accomplished using known methods and materials and transported to the recipient mammal.

Although various viral vectors can be used in the practice of the present invention, retrovirus AAV- and adenovirus-based approaches are of particular interest. For example, Dunb
ensky et al., (1984) Proc Natl Acad Sci. USA 81, 7529-7533; Kaneda et al.
., (1989) Science 243, 375-378; Hiebert et al., (1989) Proc Natl Acad Sc
i. USA 86, 3584-3598; Hatzoglu et al., (1990) J. Biol. Chem. 265, 17285-
See 17293 and Ferry et al., (1991) Proc Natl Acad Sci. USA 88, 8377-8381. The following additional guidance on the selection and use of viral vectors:
Useful for professionals.

Retrovirus Vectors Retroviruses are a type of RN whose RNA genome is reverse transcribed into DNA in infected cells.
A virus. The retroviral genome can integrate into the host cell genome and requires three viral genes, gag, pol and env, and the viral long terminal repeat (LTR). The packaging sequence of the virus (ψ) is
Differentiate NA from other RNAs in cells (Verma et al., Nature 389: 239-242, 1997)
). For expression of the foreign gene, the viral protein is replaced with the gene of interest in the viral vector, which is then transfected into a packaging line containing the viral packaging components. The packaged virus is secreted from the packaging line into the culture medium, which can then be used to infect cells in the culture. Since retroviruses cannot infect non-dividing cells, they have been used initially for in vitro gene therapy.

AA Vectors Vectors based on adeno-associated virus (AAV) are of interest as delivery vehicles to various tissues, including muscle and lung. AAV vectors infect cells and frequently and stably integrate with the cytoplasmic genome. AAV can infect and integrate into growth-suppressing cells (eg, lung epithelium) and is non-toxic.

AAV-based expression vectors that are typically used include direct blunting of the restriction endonuclease, followed by excision of the transformant with restriction enzymes, or blunting the appropriate DN.
A flanking restriction site that can be used for subcloning of transgenic transformants either by ligation of the A linker, restriction digestion and ligation to sites between ITRs14
Contains a 5-nucleotide AAV inverted terminal repeat (ITP). The capacity of the AAV vector is about 4.4 kb. The following proteins have been expressed using various AAV-based vectors and various promoters / enhancers. That is, neomycin phosphotransferase, chloramphenicol acetyltransferase,
Fanconi anemia gene, vesicle fibrosis transmembrane conductance regulator and granulocyte macrophage colony-stimulating factor (Kotin, RM Human Gene Thera
py 5: 793-801, 1994, Table I). Genetic transformants into which various DNA structures of the present invention are introduced are similarly included in AAV-based vectors. Instead of incorporation of a constitutive promoter that induces expression of the recombinant DNA encoding the fusion protein, eg, CMV, an AAV promoter can be used (ITR itself or AAV p5 (Flotte
, et al., J. Biol. Chem. 268: 3781-3790, 1993)).

[0193] Such vectors can be packaged in AAV virions by the methods reported. For example, a human cell line, eg, 293, is transfected with an AAV-based expression vector and other plasmids with open reading frames encoding AAV rep and cap under the control of an endogenous AAV promoter or a heterologous promoter. Can be used. Rep protein R in the absence of helper virus
Ep68 and Rep78 prevent the accumulation of replicative forms, but upon superinfection with adenovirus or herpes virus, these proteins recognize replication from the ITR (only present in the construct containing the transgenic transformant) and The expression of the capside protein is observed. This system packages transgenic DNA into AAV virions (Carter, BJ Current Opinion in Biotechnology 3: 533-539, 1992;
Kotin, RM, Human Gene Therapy 5: 793-801, 1994). Genetic transformants can be expressed in AAV virions using methods that improve AAV titers. Such a strategy is a method of directing the viral package by transfection with a second plasmid following stable expression of the ITR-flanked genetic transformant in the cell line; cell lines that induce and express AAV proteins, such as Including but not limited to the use of temperature sensitive induced expression or pharmaceutical induced expression. Additionally,
The efficiency of AAV transduction can be increased by treating cells with an agent that promotes the conversion of single-stranded to double-stranded form as disclosed in Wiloson et al, mWO 96/39530.

Concentration and purification of the virus can be performed by reported methods, such as banding on a cesium chloride gradient as used for initial reports of in vivo AAV vectors (Flot
te et al., J. Biol. Chem. 268: 3781-3790, 1993) or O'Riordan et al., WO 97/08298.

Methods and Materials for Introducing Genetic Transformants, Genetic and Purification of AAV Vectors Containing Genetic Transformants, and AAV Techniques Effective in the Practice of the Subject of the Invention Including Uses to Transfect Cells and Mammals For additional detailed guidance, see, eg, Carter et al., US Pat. No. 4,797,368 (Jan. 10, 1989), M.
uzyczka et al., US Pat. No. 5,139,941 (August 18, 1992), Lebkowski
et al., U.S. Patent No. 5,173,414 (December 22, 1992), Strivastava, U.S. Patent No. 5,252,479 (October 12, 1993), Lebkowski et al., U.S. Patent No. 5,3.
No. 54,678 (October 11, 1994); Shenk et al., US Pat. No. 5,436,146 (July 25, 1995); Chatterjee et al., US Pat. No. 5,454,935 (1
December 12, 995), Carter et al., WO 93/24641 (published December 9, 1993) and Flotte et al., US Pat. No. 5,658,776 (August 19, 1997). See also.

Adenovirus Vectors A variety of adenovirus vectors have been shown to be effective in delivering genes to mammals, including humans. Expression of marker proteins and CFTR in lung epithelium using copy-deficient adenovirus vectors. The first development E1a-deleted adenovirus vector is improved by a second development that induces a temperature-sensitive E2a protein that expresses few viral proteins and thus reduces the number of virus-infected cells targeted to the immune system (Goldman et al. al., Human Gene Therapy
6: 839-851, 1995). More recently, viral vectors in which the open reading frame of all viruses have been deleted have been reported (Fisher et al., Virology 217: 11-12,1).
996). Furthermore, it has been reported that expression of viral IL-10 inhibits immune responsiveness to adenovirus antigens (Qin et al., Human Gene Therapy 8: 1356-1374).
.

A number of adenovirus-type DNA sequences are available from the gene bank. The adenovirus DNA sequence can be obtained from any of the 41 human adenovirus types currently identified. Various adenovirus strains are available from the American Type Culture Collection (Rockville, Maryland) or upon request from commercial or academic sources. The transformants described herein can be used in adenovirus vectors and in the same manner as used to insert CFTR or other genes into vectors for delivery protocols (restriction digestion, linker ligation or terminal Filling and ligating). Flanking selected portions of adenovirus sequences, ie, transgenics or other conventional vector control elements
Hybrid adenovirus-AAV vector capsids containing 5 'and 3' AAV ITR sequences can also be used. For example, Wilson 1 et al., International Patent Application Publication W
See O96 / 13598. For additional detailed guidance on adenovirus and hybrid adenovirus-AAV technology useful in practicing the subject of the present invention,
See Wilson1 et al., WO94 / 28938, WO96 / 13597 and WO96 / 26285, and the references cited therein.

Generally, the DNA or virus particles are transferred to a biologically compatible solution or pharmaceutically acceptable transport vehicle, such as sterile saline or other aqueous or non-aqueous isotonic sterile infusion solution or suspension. However, those examples involving Ringer's phosphate buffer or other similar media are well known in the art.

Preferably, in gene therapy applications, DNA or recombinant virus is administered in sufficient amounts to transfect cells at a level that produces a therapeutic effect without adverse effects. Optimal dosages of DNA and virus will depend on various factors, as described elsewhere, and can vary somewhat from patient to patient. Again, therapeutically effective doses of virus can range from about 1 x 10 ₇ to about 1 A range of about 20 to about 50 ml of a saline solution containing × 10 ₁₀ pfu virus / mg, for example, about 1 × 10 ₈ to about 1 × 10 ₉ pfu virus / mg is contemplated. Host Cells The present invention is very useful for the manipulation of animal cells and for applications involving the use of such engineered animal cells. Animal cells can be insects, insects or mammals. A variety of animal cells can be used, including, for example, horses, cows, sheep, dogs, cats, rats and non-human primate cells, but human cells are of particular interest. Among various species, for example, reticuloendothelium, nerve, glial, mesenchyme, skin, muscle (including smooth muscle cells), spleen, hematopoietic tissue, epithelium, endothelium, liver, kidney, gastrointestinal tract, Various types of cells can be used, such as lung, fibroblasts and other types of cells. Of particular interest are reticuloendothelial cells, including erythroid, lymphoid or myeloid mononuclear cell junctions and either nucleated cells that may be associated with myoblasts and fibroblasts.
Pedigree and progenitor cells such as hematopoietic tissue, nerves, stromal, muscle, liver, lung, gastrointestinal tract, and interfilling pedicle cells.

The cells can be autologous cells, adherent cells, allogenic cells, and in some cases, heterologous cells for the host organ of interest. Cells can be denatured by altering the major histocompatibility complex ("MHC") profile, inactivating β2-microglobulin to prevent the formation of functional class IMHC molecules, and class II Inactivation of one or more MH
Increases or inactivates cytotoxic performance by providing expression of C molecules and increasing or inactivating expression of genes associated with cytotoxic activity.

In some instances, specific clones or oligoclones are of interest when the cells have T cells and B cells with a particular specificity, eg, a particular antigen specificity or a resulting target partial specificity. Introducing the structure into animals The cells that have been modified in vitro with the DNA structure are grown in culture under selective conditions, and the cells selected as having the desired structure are then expanded and treated, e.g., with a host cell. Further analysis can be performed using the polymerase chain reaction to determine the presence of structures therein and / or assays for the production of the desired gene product. Once the denatured host cells have been identified, they can then be used as designed, for example, for growth in culture or for introduction into host organs.

[0202] Depending on the nature of the cell, the cell can be introduced into the host organ, eg, a mammal, in a variety of ways. Hematopoietic tissue cells can be administered by inoculation into the tubing,
There are usually about 10 ₄ or more cells, and generally 10 ₉ or less cells. The number of cells to be used depends on the number of situations, the purpose of introduction, the life span of the cells, the protocol used, e.g.
It will depend on the ability of the cells to proliferate, the stability of the therapeutic, the physiological requirements of the therapeutic, and the like.
Generally, for myoblasts or fibroblasts, for example, the number of cells is
The following is generally injected as a dispersion at or near a site of interest.
The cells are in a physiological application medium.

[0203] Cells engineered according to the present invention can also be encapsulated prior to implantation into a host organ or patient for the production of a therapeutic protein, for example, using conventional biocompatible materials and methods. Hguyen et al., Tissue Implant System and Met
hods for Sustaining viable High Cell Densities within a Host, U.S. Patent No.
5,314,471 (Baxter International, Inc); Uludag and Sefton, 1993, J
Biomed. Nater. Res. 27 (10): 1213-14 (HepG2 cells / hydroxyethyl methacry
late-methyl methacrylate membranes); Chang et al., 1993, Hum Gene Ther 4
(4): 433-40 (mouse Ltk-cell expressing hGH / immunoprotective perm-select
ive alginate microcapsules; Reddy et al., 1993, J Infect Dis 168 (4): 10
82-3 (alginate); Tai and Sun, 1993, FASEB J 7 (11): 106-9 (mouse fibrobla
st expressing hGH / alginate-poly-L-lysibe-alginate membrane); Ao et al.,
1995, Transplantation Proc. 27 (6): 3349, 3350 (alginate); Rajotte et al
., 1995, Transplantation Proc. 27 (6): 3389 (alginate); Lakey et al., 19
95, Transplantation Proc. 27 (6): 3266 (alginate); Korbett et al., 1995,
Transplantation Proc. 27 (6): 3212 (alginate); Dorian et al., U.S. Patent No. 5,429,821 (alginate); Emerich et al., 1993, Exp Neurol 122 (1):
37-47 (polymer-encapsulated PC12 cells); Sagen et al., 1993, J. Neeuros
ci 13 (6): 2415-23 (bovine chromaffin celss encapsulated in semipermeable
polymer membrane and implanted into rat spinal subarachnoid soace); Aebi
scher et al., 1994, Exp Neurol 126 (2): 151-8 (polymer-encapsulated rat
PC12 cells implanted into monkeys; (see also WO92 / 19595 by Aebischer et al.)
); Savelkoul et al., 1994, J Immunol Methods 170 (2): 185-96 (encapsulated
hybdridomas producing antibodies; encapsulated transfected cell lines e
xpressing various cytokines); Winn et al., 1994, PNAS USA 91 (6): 2324-8
(engineered BHK cells expressing human nerve growth factor encapsulated
in an immunoisolation polymeric device and transplanted into rats); Emer
ich et al., 1994, Prog Neuropsychopharmacol Biol Psychiatry 18 (5): 935-
46 (polymer-encapsulated engineered BHK cells implanted into rats); Kord
ower et al., 1994, PNAS USA 91 (23); 10898-902 (polymer-encapsulated engi
neered BHK cells expressing hNGF implanted into monkeys) and Bulter et
See al WO95 / 04521 (encapsulated device). The cells are then introduced in encapsulated form into an animal host, preferably a mammalian and more preferably a human subject, on demand. Preferably, the encapsulating material is semi-permeable, allowing the host to release secreted proteins produced by the encapsulated cells.
In many embodiments, semi-permeable encapsulation provides encapsulation that is immunologically isolated from the host organ into which the encapsulated cells are introduced. In these embodiments, the cells to be encapsulated express one or more chimeric proteins having component domains derived from host species proteins and / or viral proteins or proteins from a species different from the host species. . For example, in such a case, the chimera can include elements derived from GAL4 and VP16. The cells can be derived from one or more individuals different from the recipient and can be derived from a different species than the recipient organ or patient.

[0204] Instead of ex vivo degeneration of cells, it is desirable in many situations to denature in vivo. For this purpose, various techniques for denaturing target tissues and cells in vivo have been developed. Numerous viral vectors have been developed that recognize transfection and, in some cases, integrate the virus into the host, such as adenoviruses, adeno-associated viruses and retroviruses as described above. For example,
Dunbensky et al. (1984) Proc. Natl. Acad. Sci. USA 81, 7529-7533; Kaneda
et al., (1989) Science 243, 375-378; Hiebert et al., (1989) Proc Natl A
cad Sci. USA 86, 3584-3598; Hatzoglu et al., (1990) J. Biol. Chem. 265,
17285-17293 and Ferry et al., (1991) Proc Natl Acad Sci. USA 88, 8377-8
See 381. Vectors can be administered by injection, for example, intravenously or intramuscularly, by inhalation or other parenteral modes. Non-viral delivery methods such as administration or infusion of DNA via a complex with liposomes, catheters or biolistics can also be used.

According to in vivo gene denaturation, the method of denaturation depends on the properties of the tissue, the required efficiency of cell degeneration, the accessibility of the cells to the DNA composition to be introduced, and the like. By utilizing a weakened or modified retrovirus having a targeted translation initiation region, one can optionally use one of the translation factor structures of interest for the purpose of producing the virus and transfecting neighboring cells. It can activate the virus.

The DNA transfer does not need to be integrated in all cases. In some situations, the temporary hold introduced is sufficient. In this method, a short-term effect may be determined after a period of time following the introduction of the cells into the host, eg, after the cells have returned to a particular site.
Binding, detection Rapamycin binds to the human protein, FKBP12, and binds hFKBP12 and FRA
It is known that P forms a three-part complex with human transcripts for the yeast proteins TOR1 and TOR2. Rapalog binds to human protein, FKBP12 (or a fusion protein or a fragment comprising an FRB domain) and / or binds to human FKBP1
It can be characterized for its ability to form a three-part complex with 2 and human FRAP and compared to rapamycin. See WO96 / 41865 (Clackson et al.). The specification states that human FKBP12 and FR each bind to human FKBP12 or human FRAP, respectively.
Various materials and methods are disclosed that can form a ternary complex (ie, "heterodimerize") with a protein that includes a B domain. Such assays include fluorescence polarity assays that measure binding. Further, the ability of the compound to form a three-part complex
Also included are cell-based translation assays that measure indirectly by the observed level of receptor gene product produced by a mammalian cell that has been genetically engineered in the presence of the compound. Corresponding cell-based assays can also be performed in genetically engineered yeast cells. See, for example, WO 95/33052 (Berlin et al.).

The rapalogs of the present invention can be made physiologically compatible (eg, lacking inappropriate toxicity to the cells or organs to be used) and orally administrable by animals (ie, orally for use in all animals, including therapy). Active) and / or can be capable of crossing cells or other membranes as required for a particular application.

In addition, preferred rapalogs are mutants, including, but not limited to, amino acids that differ in Phe36, preferably amino acids with fewer bulking R groups, such as valine or alanine, over native or naturally occurring immunophilins. Human FKBP). For example, such compounds can bind dominantly to mutant FKBP to a greater or lesser degree than bind to human FKBP12 as determined by a scientifically justified or art-recognized assay. Mutated FKBP is more than two or three times more likely to bind to human FKBP12
Can be predominantly combined.

The binding affinities of the various rapalogs of the invention for human FKBP12, its variants or other immunophilins can be measured by applying known methods used in the case of FKBP. For example, one skilled in the art can determine the ability of a compound of the invention to compete with the binding of a known ligand to a protein of interest. For example, Sierkierka et al., 1989, Nature 341, 755-757 (test compound competes
with binding of labeled FK506 derivative to FKBP).

[0210] One set of preferred rapalogs of the invention is a direct binding assay (eg, fluorescence quenching), a competitive binding assay (eg, versus FK506), inhibition of FKBK enzyme activity (rotamase), or other assay. A human FKBP12 having a Kd value of less than about 200 nM, more preferably less than about 50 nM, even more preferably less than about 10 nM, and even more preferably less than about 1 nM;
It binds to the above mutant or a fusion protein containing such FKBP domain. In one subset of such compounds, the FKBP domain is one in which the alanine at position 36 has been replaced with an amino acid having a less bulky side chain, such as alanine, valine, methionine or serine.

The competitive binding FP assay is described in detail in the Examples below. The assay recognizes in vivo measurements of IC50 values for compounds that reflect its ability to bind FKBP protein in competition with labeled FKBP ligand, for example, FK506.

[0212] A preferred group of compounds of the invention are those that have up to 10, preferably up to 5 amino acid substitutions in a competitive binding FP assay by a given FKBP domain and ligand pair, eg, human FKBP12 or fluorescent FK506. 1000nM or more for that variant having
It is a rapalog having an IC50 value of preferably 300 nM or more, more preferably 10 nM or more. In one subset of the group, the FKBP domain has any of the above modifications at position 36.

The ability of rapalogs to multimerize chimeric proteins can be measured in a cell-based assay by measuring the occurrence of an event that caused such multimerization. For example, an expression DNA encoding a first chimeric protein comprising one or more FKBP domains and one or more effector domains and a FRB domain and can motivate a biological response upon multimerization Cells that contain and can express a second chimeric protein containing one or more effectors are used. Further, it is preferable to use a cell further containing a receptor under the transcriptional control of a regulatory element (ie, a promoter) responsive to multimerization of the chimeric protein. The design and preparation of representative compounds and their use in engineered cells is described in WO 96/41865.
And other international patent applications cited in this section and the sections above. The cells are grown or retained in the cells. Rapalog is added to the culture medium and after a suitable incubation time (for gene expression and secretion,
For example, several hours or overnight), the presence of the receptor gene product is measured. A positive result is obtained. That is, multimerization is associated with receptor gene transcription as observed by the appearance of the receptor gene product. The receptor gene product is
It can be a protein that is conveniently detectable (eg, by ELISA) or can catalyze the formation of a product (eg, color) that is conveniently detectable. Materials and methods for producing suitable cell lines for performing such assays are disclosed in the above-mentioned International Patent Application in this section. Examples of typically used target genes include SEAP, hGH, beta-galactosidase, green fluorescein protein and luciferase, and convenient assays are commercially available.

Another preferred group of the present invention is a 2-group based fusion protein comprising an FKBP domain.
It can include a detection signal in a hybrid transcription assay. Preferably, the FKBP domain is an FKBP domain other than wild-type human FKBP12.

Other assays that measure the ability of rapalogs to multimerize chimeric proteins, such as FKBP-based transcription assays, are cell-based assays that measure the occurrence of events induced by such multimerization. In this case, cells that structurally express the detectable product are used. The cell encodes a chimeric protein comprising one or more immunophilin-derived ligand binding domains and one or more effector domains capable of inducing cell death upon multimerization, such as a FAS intracellular domain. And can express it. Designs and preparations typically used for such genetically engineered cells are described in WO 95/02684. See also WO 96/41865.
The cells are maintained and cultured in a cell growth or continuous viable culture medium. The cells or medium are assayed for the presence of structural fiber products, thus establishing a baseline level of the receptor. Cells engineered for structural products of HGH or other convenient detectable products that act as receptors can be used. The test compound is added to the medium, the cells are incubated, and the cell lysate or medium is tested at one or more time points for the presence of the receptor. Decreased receptor production indicates cell death and is an indirect measure of multimerization of the fusion protein.

Another preferred group of the invention is one that is capable of eliciting a detection signal in such an FKBP / FRB-based apoptosis assay. FKBP domain is wild-type human FK
Preferably, it is an FKBP domain other than BP12. In some cases, FKBP
Denature the domain as described above. The FRB domain also contains the wild-type F from human FRAP.
Preferably, it is an FRB domain other than RB. In some examples, the FRB domain is modified at position 2098 as described above.

Having performed such an assay, one skilled in the art would be able to determine the desired IC50 value and / or wild-type human FKBP
It will be possible to select rapalogs with binding advantages for twelve mutant FKBPs. A competitive binding FP assay allows one to select a desired IC50 value and / or control, eg, a monomer or rapalog that has the binding advantage of mutant FKBP or wild-type FKBP over FK506. Uses rapalog, WO94 / 18317, WO95 / 02684, WO96 / 20951, WO95 / 41865, for example, to activate the transcription of the desired gene under control, delete the target gene,
It can be used to motivate apoptosis or to induce other biological matters to grow engineered cells in culture or in whole organs, including gene therapy applications. The application of the object of the present invention will be described below, but is not limited thereto. 1. Controlled Gene Therapy In many instances, the ability to switch on / off or accurately titrate expression of a therapeutic gene as desired is important for therapeutic efficiency. The present invention is well suited to achieving controlled expression of therapeutic target genes for human gene therapy. One example is to use a pair of chimeric proteins, one containing at least the FRB domain and the other containing at least the FKBP domain, wherein the improved rapalog of the present invention comprises a chimeric and target to be expressed. Gene composition can be dimerized. One of the chimeric proteins comprises a DNA-binding domain, preferably a complex DNA-binding domain as described in Pomerantz et al, supra, as a heteromorphic effector domain. The second chimeric protein contains a transcriptional activation domain as a heteromorphic effector domain. The improved rapalogs can bind to both chimeras and thus crosslink the chimeras efficiently. Can encode and direct the expression of these chimeric proteins
The DNA molecule is introduced into the cells to be genetically modified. When the engineered cells or progeny thereof are contacted with the improved rapalog (by administering it to an animal or patient), the transcription factor complex is assembled and the target gene is thus expressed. The design and use of similar components is described in PCT / US93 / 01617 and WO96 / 41865 (Cla
ckson et al). Indeed, the level of target gene expression should be a function of the number and concentration of the chimeric transcription factor complex, and in turn should be a function of the concentration of the improved rapalog. The dose of response gene expression (of the improved rapalog) is typically observed.

An improved rapalog can be administered to a patient as desired to activate transcription of a target gene. Improved binding affinity of rapalog, desired response, method of administration, biological half-life of rapalog and / or target gene mRNA,
Various protocols are available, depending on the number of genetically engineered cells present. The improved rapalog can be administered parenterally or orally by various routes. The number of doses will depend on the factors described above. The improved rapalog can be inoculated by oral inoculation, buccal, sublingual, intravenous, intraperitoneal, intramuscular, subcutaneous injection, inhalation or the like as a tablet, powder or dispersion. Improved rapalogs (and monomeric antagonist compounds) can be formulated using conventional methods and materials well known in the art for various routes of administration. The exact dosage and particular method of administration will depend on the factors described above, and can be determined by the attending physician or human or animal health care provider. For the most part, the manner of administration will be empirically determined.

[0219] Monomer compounds that compete with the improved rapalog in terms of supplementing or terminating transcriptional activity by the improved rapalog may be administered. Thus, in the event of a reverse reaction or a requirement to terminate the therapeutic effect, an antagonist to the dimerizing agent can be administered in a convenient manner, particularly by intravenous inoculation, where rapid reversal is required. Alternatively, an inactivation domain (or transcription silencer) can be present with the ligand binding domain. In another approach, it can be removed by apoptosis via signaling through Fas or TNF as described above. See International Patent Applications PCT / US94 / 01617 and PCT / US94 / 08008.

The specific dosage of the improved rapalog for any application can be determined according to the method used for therapeutic dosage monitoring, where the specific level of expression is prolonged. Over time, individual or repeated doses of the improved rapalog may be taken over a short period of time, for example for two weeks or more, if needed or if there is repeated treatment, for example for more than two weeks. An improved dose of rapalog within a predetermined range is given and monitored for response so as to provide a time expression level and observe the therapeutic response. Depending on the time interval and the level observed during the therapeutic response, more or less doses will be provided over time according to the response. The method is iteratively repeated until a therapeutic range of dose is obtained. If the improved rapalog is administered for an extended period of time, the maintenance dose of the improved rapalog will be determined at extended intervals to ensure that the cell system provides adequate response and levels of expressed product.

The system is based on a number of variables, such as the cellular response to an improved rapalog, the efficiency of expression and, if desired, the level of secretion, the activity of the expressed product, the specific needs of the patient, which can vary with time and environment, individual cells. Alternatively, it depends on the rate of loss of cell activity resulting from the loss of expression activity, and the like. 2. Production of Recombinant Proteins and Viruses Production of recombinant therapeutic proteins for commercial and research purposes is often achieved through the use of mammalian cell lines that have been genetically engineered to express proteins at a premium. The use of mammalian cells, rather than microorganisms or yeast, is indicated where proteins of the appropriate function j require post-translational denaturation that is not commonly performed in homologous heterologous cells. Examples of proteins produced industrially by this method include erythrocyte proteins, tissue plasminogen activator, coagulation factors such as factor VIIIc, antigens and the like. The cost of producing proteins in this way is directly related to the level of expression achieved in the engineered cells. A second limitation on the production of such proteins is their toxicity to host cells. That is, protein expression prevents cells from growing at high density, and production levels are significantly reduced as described for controlled gene therapy. Thus, the ability to tightly control protein expression allows cells to grow at high density in the absence of protein production. Only after the optimal cell density has been reached, the expression of the gene is activated and the protein product is subsequently harvested.

[0222] Similar problems are encountered in the construction and use of "packaging lines" for the production (eg, gene therapy) of recombinant viruses industrially or for experimental use. These cell lines produce the viral proteins required for the assembly of infectious virions that are genetically engineered to hide the defective recombinant genome. Such packaging line dependent viral vectors include retroviruses, adenoviruses, adeno-associated viruses. In the latter case, the titer of the viral stock obtained from the packaging line is directly related to the level of viral rep or nucleoprotein production. However, these proteins are very toxic to host cells. Therefore, it was found that it was difficult to generate high-titer recombinant AAV virus. The present invention provides a solution to this problem by constructing a packaging line that places rep and nuclear genes under the control of a controllable transcription factor of the design described herein. Packaging cells can be grown at high density, infected with a helper virus, and transfected with the recombinant viral genome. The expression of the viral protein encoded by the packaging cells is then induced by the addition of a dimerizing agent to produce the virus at a high titer. 3. Biological Research The present invention is applicable to a wide range of biological experiments where precise control over target genes is desired. These include: (1) expression of proteins or RNAs of interest for biochemical purification; (2) interest in tissue culture cells (or in vivo via genetically engineered cells) for the purpose of evaluating their biological function. Controlled expression of a protein or RNA; (3) animal genetics for the purpose of evaluating its biological function; regulated expression of a protein or RNA of interest in transgenic animals; (4) evaluating the biological function of the gene. It involves controlling the expression of genes encoding other regulatory proteins, ribosomes or assistance molecules that act on the transfectant for the purpose. Transgenic animal models and other uses to which the components of the present invention may be applied include those described in PCT / US95 / 10591.

[0223] The present invention further provides kits that are effective for the above uses. Such a kit comprises a DNA structure encoding the chimeric protein of the invention and capable of directing the expression of the chimeric protein (and may include additional components as described above), and with control of gene transcription. The present invention includes a target gene construct comprising a target gene bound to one or more transcription factors activated by multimerization of a chimeric protein. Alternatively, the target gene construct may include a cloning site for introducing a desired target gene by one skilled in the art. Such a kit may include a sample of a dimerizing agent that dimerizes the two recombinant proteins and activates transcription of the target gene. Formulation, Dosage and Administration Due to the ability to promote protein-protein interactions, the rapalogs of the invention can be used in pharmaceutical compositions, chimeric proteins of the invention in human and non-human mammals containing the engineered cells of the invention. May be used in a method for promoting the compounding of the composite.

A preferred method of such treatment or prevention is to promote the significant incorporation of such a complex in a genetically engineered cell of the invention in a mammal or, preferably, to treat a desired biological event elicited by such a complexation. By administering an effective amount of the compound that promotes significant motivation, for example, transcription of the target gene, apoptosis of genetically engineered cells, and the like. Therapeutic / Prophylactic Administration and Pharmaceutical Compositions Rapalog can exist in free form or, if desired, in the form of a salt. Pharmaceutically compatible salts of many types of compounds and their preparation are well known in the art. Pharmaceutically compatible salts of the compounds of the present invention include the conventional non-toxic or quaternary ammonium salts of the compounds of the present invention formed, for example, from inorganic or organic bases.

The compounds of the present invention can form hydrates or sorbates. It is known to those skilled in the art that charged compounds form hydrated species when lipidated with water or form sorbate species when concentrated in solution with a suitable organic solvent.

The invention also relates to pharmaceutical compositions comprising a therapeutically (or prophylactically) effective amount of a compound and one or more pharmaceutically compatible carriers and / or excipients. Carriers include, for example, saline, buffered saline, dextrose, water, glycerol, ethanol, and mixtures thereof, and are described in detail below. The composition, if desired, can contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation or powder. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as reagent grade mantol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. The formulation involves mixing, granulating, and compressing or dissolving the ingredients as appropriate to the desired preparation.

The pharmaceutical carrier employed can be, for example, a solid or liquid. Representative solid carriers include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium phosphate, stearic acid, and the like.
Solid carriers can include flavoring agents, lubricants, solubilizing agents, suspending agents, fillers, lubricants, compression aids, binders or disintegrants, and may be encapsulating agents. In powders, the carrier is a finely divided solid, which is in a mixture with the finely divided active component. In tablets, the active component is mixed with the carrier having the necessary compression properties in suitable proportions and compacted in the shape and size desired. 99% powder and tablets
It is preferred to contain up to an active ingredient. Suitable solid carriers include, for example, calcium phosphate, magnesium stearate, talc, sugar, lactose, dextrin, starch, gelatin, cellulose, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, low melting waxes and ion exchange resins.

[0228] Representative liquid carriers include syrup, peanut oil, olive oil, water, and the like. Liquid carriers are used in preparing solutions, suspensions, emulsions, syrups, elixirs and compressed compositions. The active ingredient is dissolved or suspended in a pharmaceutically compatible liquid carrier such as water, organic solvents, mixtures of both or pharmaceutically compatible oils or fats. The liquid carrier may contain other suitable pharmaceutically compatible additives, such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavorants, suspending agents, thickeners, colorants, viscosity modifiers, stabilizers. Or an osmotic pressure adjusting agent. Examples of liquid carriers suitable for oral or parenteral administration include water (partially including the additives described above, for example, cellulose derivatives, preferably sodium carboxymethylcellulose solution), alcohols (monohydric and polyhydric alcohols, such as glycols). And its derivatives, oils such as fractionated coconut and peanut oils. For parenteral administration, the carrier can also be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid carriers are effective in the form of sterile liquids for parenteral administration. The liquid carrier for the compressed composition can be a halogenated hydrocarbon or other pharmaceutically compatible propellant. Liquid pharmaceutical compositions, which are sterile solutions or suspensions, can be used, for example, by intramuscular, intraperitoneal or subcutaneous inoculation. Sterile solutions can also be administered intravenously. The compounds can be administered orally in either liquid or solid composition form.

The carrier or elixir may contain the long acting materials well known in the art, for example, glyceryl monostearate or gericeryl distearate alone or with a wax, ethyl cellulose, hydroxypropyl methyl cellulose, methyl methacrylate and the like. PHOSAL PG when formulated for oral administration
0.001% Tween 80 in -50 (phospholipid concentrate with 1,2-propylene glycol A Na
Ttermann & Cie. GmbH) has been identified as producing compatible oral formulations of other compounds, and can be adapted for the formulation of various compounds of the present invention.

A variety of pharmaceutical forms can be utilized. If a solid carrier is used, the preparation may be tableted, placed in a hard gelatin capsule in powder or tablet form or in a troche or drop form. The amount of solid carrier will vary widely but preferably will be from about 25 mg to about 1 g. If a liquid carrier is used, the preparation will be in the form of a syrup, emulsion, soft gelatin capsule, sterile injectable solution or suspension in an ampoule or vial or non-aqueous suspension.

To obtain a suitable water-soluble dosage form, the pharmaceutically compatible salt of the multimerizing agent can be dissolved in an aqueous solution of an organic or inorganic acid, such as a 0.3 M solution of succinic or citric acid. Alternatively, the acid derivative can be dissolved in a suitable basic solution. If a soluble salt form is not available, the compound can be dissolved in a suitable co-solvent or a combination thereof. Such suitable co-solvents include alcohols, propylene glycol, polyethylene glycol at concentrations ranging from 0 to 60% of the total volume.
300, polysorbate 80, glycerin polyoxyethylated fatty acid, fatty alcohol or glycerin hydroxy fatty acid ester, but are not limited thereto.

A variety of delivery systems are known, including multimerizing agents or tablets, capsules,
They can be used to administer injection solutions, encapsulation in liposomes, these formulations, including microparticles, microcapsules and the like. Methods of introduction include, but are not limited to, skin, endothelium, intramuscular, intradermal, intravenous, subcutaneous, intranasal, lung, epidermal, ocular and (preferably preferably) oral routes. The compounds can be administered by convenient or suitable routes, such as by injection or pill inoculation, by absorption through the epithelial or mucosal skin (eg, oral mucosa, rectum or intestinal mucosa, etc.), and other biologically active agents It can be administered together. Administration can be systematic or local. For the treatment or prevention of nasal, bronchial or pulmonary disease, the preferred route of administration is oral, nasal or bronchial air oven sol or nebulizer.

In certain embodiments, it may be desirable to administer the compound locally to the area in need of treatment, for example, by local injection, topical application, inoculation, catheterization during surgery, by suppository or by skin The implant can be, but is not limited to, a patch or implant, such as a porous, non-porous or gelatinous material containing fibers.

In certain embodiments, the compositions can be formulated in accordance with conventional procedures as suitable pharmaceutical compositions for intravenous administration to actual humans. Typically,
Compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic to ease pain at the site of the inoculation. Generally, these ingredients are supplied separately or in unit dosage form, for example, as a water-free concentrate in a lipophilic powder or a sealed container, for example an ampoule or perfume bag, indicating the amount of active agent. When administering the composition by infusion, it can be administered in an infusion bottle containing sterile reagent grade water or saline. Where the composition is administered by inoculation, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

Administration of an effective amount of the compound to an individual can also be accomplished by administering the compound directly and locally to the infected area of the individual. For this purpose, the compounds may be converted into pharmaceutically compatible topical carriers, such as, but not limited to, gels, ointments, carriers containing carriers such as water, glycerol, alcohol, propylene glycol, fatty alcohols, triglycerides, fatty acid esters or mineral oils. Administered or applied in compositions including lotions or creams.

Other topical carriers include liquid petroleum, isopropyl palmitate, polyethylene glycol, ethanol (95%), polyoxyethylene monolaurate (5%) in water, sodium lauryl sulfate (5%) in water. No. Other materials such as antioxidants, humectants, viscosity stabilizers and similar agents may be added as needed. A percutaneous penetration enhancer, such as Azone, may be included.

In addition, in certain instances, the compound may be located in a device that is located on, in, or below the skin. Such devices include patches with passive or active release mechanisms, implantation, and injections that release the compound to the skin.

Materials and methods for making various formulations are well known in the art and are suitable for practicing the subject of the present invention. For example, U.S. Pat.
2,293 and 4,837,311 (tablets, capsules and other oral and intravenous formulations) and EP 0 649 959 (19
Published April 25, 1995; typical administration for IV administration) and 0648494 (19
Published 25 April 1995; typical dosing for oral administration).

An effective dose of a compound is typically from about 0.01 to about 50 mg / kg, preferably from about 0.1 to about 10 mg / kg, of mammalian weight in one or more doses.
kg range. Generally, the compound will be about 1 to 200 mg per patient per day for those in need of such treatment.

The amount of a compound that is effective in the treatment or prevention of a particular disease or indication will depend, in part, on the characteristics of the fusion protein to be multimerized, the characteristics and location of the engineered cell, and will be determined by standard therapeutic techniques. It depends on the nature of the disease or sign that can be done. In addition, in vivo or in vitro assays may optionally be employed to help identify optimal dosage ranges. Effective doses can be inferred from dose-response curves derived from in vitro or animal model test systems. The exact dosage level should be determined by the attending physician or other health care provider, including the route of administration and age, weight, gender and general health of the individual, the use (or no use) of co-treatment, and the number of cells in the patient. Depend on well-known factors, including the nature and extent of genetic engineering of

The present invention also provides a pharmaceutical pack or kit comprising one or more containers containing one or more components of the pharmaceutical composition of the present invention. If necessary,
In connection with such a container, it can be a notice in the form provided by an administrative agency for the manufacture, use or sale of a pharmaceutical or biological product, this notice comprising:
It reflects the regulatory approval of manufacture, use or sale for human administration.
The notice or package insert includes usage notes for the improved rapalog of the present invention consistent with the disclosure herein.

EXAMPLES Example 1 Representative C-24 Modified Rapalog 1.1. Rapamycin purification Rapamycin was obtained by fermentation. Rapamycin production organ, Streptomyces hygroscopicus (ATCC # 29253), was cultured on a complex inner ground in a 15 L or 30 L fed batch fermenter. Biomass was harvested after 9 to 14 days by centrifugation. The supernatant is allowed to react for 1-2 hours with a nonionic polymer absorbing resin, XAD-16 (Rhom and
Hass). The adsorbent was recovered by centrifugation, combined with the biomass and repeatedly extracted with methylene chloride. The solvent was removed in vacuo and the resulting residue was extracted with acetonitrile, which was coagulated in a similar manner. Chromatographic purification of the crude rapamycin was achieved by flash chromatography on silica gel (40% acetone / hexane), followed by C-18 reverse phase HPLC (70% CH3CN / H2O). The resulting rapamycin is identical to a reliable sample of rapamycin.
The HPLC spectrum and biological characteristics were shown. 1.2. Rapamycin (E and Z) -24- (O-Methyloxime) (5,6) (General Method) A solution of rapamycin (60 mg 65.6 mmol) in MeOH (2 mL) was treated with NaOAc (22 mg 262 mmol, 4.0 eq.) Followed by methoxylamine hydrochloride (22 mg 262 mmol, 4.0 eq.) And stirred at room temperature for 48 h. After this time, the reaction mixture was washed with H2O (1
(0 mL) and extracted with EtOAC (3 × 10 mL). The combined organic extracts were washed with saturated NaC
Washed with II solution (2 x 10 mL), dried over Na2SO4, filtered, and the solution was concentrated in vacuo. The resulting residue was subjected to flash chromatography on silica gel (10% MeOH / dichloromethane) to give a mixture of isomers. This isomer mixture was subjected to HPLC (Kromasi C-1
8 Separation by 250 x 20 mm column, 35% AE 25% H2O / MeCN passing through 12 mL / min to give 13 mg (21%) of the first eluting Z isomer and 7.6 mg (12%) of the E isomer. Z isomer:
High resolution mass spectrum (FAB) m / z 965.579 ((M + Na) + calculated C52H82N2O13Na 965.
5710]. E isomer: high resolution mass spectrum (FAB) m / z 965.510 [(M + Na) + calculated C5
2H82N2O13Na 965.5710]. 1.3. Rapamycin (E and Z) -24- (O-ethyloxime) (7,8) Prepared in a similar manner to rapamycin (E and Z) -24- (O-methyloxime). The isomer mixture was separated by HPLC (Kromasi C-18 250 × 20 mm column, 30% H2O / MeCN passing 12 mL / min) with 7.7 mg (25%) of the first eluting Z isomer and 0.5 mg (12%) of E The isomer was obtained. Z isomer: high resolution mass spectrum (FAB) m / z 979.5902 [(M + Na) +
Calculated value for C53H84N2O13Na 979.5871]. 1.4. Rapamycin (E and Z) -24- (O-isobutyl oxime) (9,10) Prepared in a similar manner to rapamycin (E and Z) -24- (O-methyl oxime). The isomer mixture was separated by HPLC (Kromasi C-18 250 × 20 mm column, 15% H2O / MeCN passing 12 mL / min) and 28 mg (65%) of the first eluting Z isomer and 3.0 mg (7%) of the E Sexual body was obtained. Z isomer: high resolution mass spectrum (FAB) m / z 1007.6146 [(M + Na) +
Calculated C55H88N2O13Na 1007.6184]. E isomer: high resolution mass spectrum (FAB) m / z 1007.6157 [(M + Na) + calculated C55H88N2O13Na 1007.6184]. 1.5. Rapamycin (E and Z) -24- (O-benzyloxime) (11,12) Prepared in a similar manner to rapamycin (E and Z) -24- (O-methyloxime). The isomer mixture was separated by HPLC (Kromasi C-18 250 × 20 mm column, 15% HO / MeCN passing 12 mL / min) with 19.6 mg (44%) of the first eluting Z isomer and 6.1 mg (14%) of E
The isomer was obtained. Z isomer: high resolution mass spectrum (FAB) m / z 1041.6033 [(M + Na)
+ Calculated for C58H86N2O13Na 1041.6028]. E isomer: High resolution mass spectrum (FAB) m /
z 1041.5988 [(M + Na) + calcd. C58H86N2O13Na 1041.6028]. 1.6. Rapamycin (E and Z) -24- (O-carboxymethyl oxime) (13,14) Prepared in a similar manner to rapamycin (E and Z) -24- (O-methyl oxime). The isomer mixture was separated by HPLC (Kromasi C-18 250 × 20 mm column, 45% H2O / MeCN passing 12 mL / min) to 4.6 mg (11%) of the first eluting Z isomer and 1.0 mg (2%) of E The isomer was obtained. Z isomer: high resolution mass spectrum (FAB) m / z 1009.5664 [(M + Na) +
Calculated value for C53H82N2O15Na 1009.5613]. E isomer: high resolution mass spectrum (FAB) m / z 1009.5604 [(M + Na) + calculated C53H82N2O15Na 1009.5613]. 1.7. Rapamycin (E and Z) -24- (O-carboxamidomethyl oxime) (15,16) Prepared in a similar manner to rapamycin (E and Z) -24- (O-methyl oxime). The isomer mixture was separated by HPLC (Kromasi C-18 250 × 20 mm column, 35% HO / MeCN passing 12 mL / min) to 6.2 mg (10%) of the first eluting Z isomer and 1.4 mg (2%) of E The isomer was obtained. Z isomer: high resolution mass spectrum (FAB) m / z 1008.5790 [(M + Na) +
Calculated for C53H83N3O14Na 1008.5768]. E isomer: high resolution mass spectrum (FAB) m / z 1008.5753 [(M + Na) + calculated C53H83N3O14Na 1008.5768]. Example 2 Assay for Binding of Rapamycin C24 Derivatives to FKBP The affinity of the rapamycin C24 analog for FKBP was measured using a competition assay based on fluorescence polarization (FP). A fluorescein-labeled FK506 probe (AP1491) was synthesized and its fluorescence polarization was used as a direct readout of the percentage of bound probe in equilibrium binding experiments containing subsaturated FKBP and a variable amount of rapamycin analog as a competitor. Synthesis of fluorescein-labeled FK506 probe (AP1491)

[0243]

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2.1. 24,32-Bis (tert-butyldimethylsilyl) ether of FK506 FKB506 (18 mL, 470 μmol) of tert-butyldimethylsilyltrifluoromethanesulfonate in dichloromethylene (3 mL) at 0 ° C. 103 mg 128 μmol) and
It was added dropwise to a stirred solution of 2,6-lutidine (89.5 μL, 768 μmol). The resulting solution was stirred at 0 ° C. for 2 hours, then treated with MeOH (0.5 mL) and ether (15 mL).
The mixture was washed with 10% aqueous NaHCO3 (3 mL) and brine (3 mL). The organic phase was decanted, dried over anhydrous Na2SO4 and concentrated to a yellow oil. Column chromatography (silica gel, hexane-EtOAc 3: 1) gave the title compound as a colorless oil (104 mg). 2.2. Intermediate 1 24,32-bis (tert-butyldimethylsilyl) ether (100%) of FK506 in THF (2.5 ml)
mg, 97 μmol) was added morpholine N-oxide (68 mg, 580 μmol) followed by an aqueous solution of water (60 μL) and 4% osmium tetroxide (123 μL, 20 μmol). The resulting mixture mixture was stirred at room temperature for 4.5 hours. Then, this is 50% M
Treatment with aqueous eOH (0.5 mL) and sodium peroxide (207 mg, 970 μmol), and the suspension was stirred for an additional hour. The mixture was diluted with ether (10 mL) and washed with saturated aqueous NaHCO3 (2x4 mL). The organic phase was decanted, dried over anhydrous sodium sulfate containing a small amount of sodium sulfite, filtered and concentrated. The residue was dissolved in anhydrous THF (2.8 mL), cooled to -78 ° C under nitrogen, and
(282 μl) was treated with a 0.5 M solution of lithium tris [(3-ethyl-3-phenyl) oxy] aluminum hydride. The resulting solution was stirred at -78 ° C for 1.75 hours, then ether (
(6 mL) and a saturated ammonium chloride solution (250 μL). The mixture was cooled to room temperature and treated with anhydrous sodium sulfate. Filtration and concentration under reduced pressure gave a pale yellow oil (97 mg), which was subjected to column chromatography.
(Silica gel, hexane-EtOAc 3: 1) to give 1 as a colorless oil. 2.3. Intermediate 2 A solution of the above alcohol (300 mg, 290 μmol) in acetonitrile (10 mL) was treated with 2,6-lutidine (338 mg, 2.9 μmol) and N, N′-succinimidyl carbonate (371 mg).
, 1.45 mmol). The resulting suspension was stirred at room temperature for 14.5 hours, then concentrated under reduced pressure. The residue was chromatographed (silica gel, hexane-EtOAc 2: 1
To 100% EtOAc) and mixed carbonate 2 (127 m2) as a pale yellow oil.
g) was obtained. 2.4. Intermediate 3 The above carbonate (30 mg, 26 μmol) and triethylamine (36 μL, 34 μmol) in acetonitrile (1 mL) were converted to 4 ′-(aminomethyl) fluorescein (371 mg, 1.45 mL).
Mol). The resulting bright orange suspension was stirred at room temperature for 1 hour and then concentrated under reduced pressure. The residue was chromatographed (silica gel, hexane-EtOAc 1: 1 to 100% EtOAc and gradient to EtOAc 1: 1) to give 3 (2
0.5 mg). 2.5. Compound 4 Bis-silyl ether (35 mg, 25 μmol) in acetonitrile (1 mL) was added to 48
% (w / w) HF. The resulting mixture was stirred at room temperature for 5.5 hours. It was then diluted with dichloromethane (10mL) and washed with water (2x2mL). The organic phase was decanted, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was chromatographed (silica gel, 100% EtOAc) to give 4 (13m
g) was obtained. 2.6. Measurement of binding affinity (IC50s) of rapalogs using FP A series of 10-fold dilutions of each analog were prepared in 100% ethanol in glass bottles and stored on ice. All other operations were performed at room temperature. Recombinant pure FKBP (prepared by standard methods. Wiederrecht, G. et al. 1992. J. Biol. Chem. 26
7,21753-21760) is diluted to about nM in 50 mM potassium phosphate pH 7.8 / 150 mM NaCl / 100 μg / ml bovine gamma globulin (“FP” buffer: low fluorescent reagent from Panvera) and a 98 μ aliquot is Transferred to the wells of a Tech Micro four-neck 96-well fluorescent plate. A 2.0 μl sample of the rapamycin analog was then transferred to the wells with agitation. Finally, a probe solution containing 10 nM AP149 in 0.1% ethanol / FP buffer was prepared and 100 μl was added to each well with stirring. Duplicate control wells contained ethanol instead of rapamycin analog (for 100% probe binding) or contained ethanol instead of rapamycin and FP buffer (0% binding) instead of FKBP.

The plate was covered and stored in the dark for approximately 30 minutes to equilibrate, and then the fluorescence polarization of the sample in each well was determined using a Jolly FPM-2 FP plate reader (Jolly Consulting and Rese
arch, Inc., Grayslake, Ill.) according to the manufacturer's recommendations.

[0246]

[Table 11]

[0247]

[Table 12]

The average polarization (mP units) for each competitor compound concentration is usually changed to a% total binding by referring to a control value, and the logarithm of the final molar concentration of the competitor compound (% total binding). (y) plotted against x). The curve was fitted using a non-linear least squares analysis and the IC50 was derived according to the following equation:

Y = M1 + (M4-M1) / (1 + exp (M2 * (M3-x))) In the above equation, M3 is IC50. For incomplete curves, the IC50 was determined by interpolation. In each case, rapamycin and C14-desoxo-rapamycin were included as reference standards (C14-desoxo-rapamycin was described by Luengo, JI et al.
94 Tet Lett. 35, 6469-6472 ") 2.7 Results of Binding Analysis of Rapamycin C24 Oxime Affinity is described as IC50 and as fold reduction of affinity (= IC50 / (IC50 of rapamycin)) [Comparative binding data between C24 rapalog and rapamycin and against C24 rapalog and desoxo-rapamycin against human FKBP12 are plotted in PCT / US86 / 09848.] Example 3. C7 rapalog synthesis of; C7 rapalog -FKBP complex and binding assays straight and branched chain alkoxy with FRAP, arylalkyloxy, -NHCO-O Al kill, are selected from -NHSO ₂ alkyl, substituted aryl moiety, and substituted heteroaryl moiety A series of C7 rapalogs with various C7 substituents were synthesized using chemistry commonly described in other literature (see, for example, Luengo et al.
95, Chemistry and Biology 2, 471-481 "and references cited in Table II for additional background.) See also the following tables.

[0250] 3.1 Compound ^{27, 28 - (R C7 =} Et) is, "Chemistry & Biology July by Luengo et al
1995, 2: 471-481 ".

[0251] 3.2 Compound 29 - (R ^C7 = iPr) rapamycin at room temperature (60 mg, 0.066 mmol) of 2-
A solution obtained by dissolving in propanol (3 ml) was treated with p-toluenesulfonic acid (75 mg, 0.394 mmol) and stirred for 4 hours. Thereafter, the reaction mixture was poured into a biphasic solution of a saturated aqueous solution of NaHCO ₃ (20 ml) and EtOAc (30 ml). The organic layer was further washed with a saturated aqueous solution of NaHCO ₃ (2 × 20 ml), then with a saturated aqueous solution of NaCl (2 × 10 ml), dried over Na ₂ SO ₄ , filtered and the solvent was evaporated off. 65% acetonitrile / water as eluent,
Purification by HPLC at 55 C using a Kromasil C-18 column (20 × 250 mm) gave AP1700 (25 mg). MS (FAB): (M + Na) ⁺ calc: 964.5762, found: 964.57
533.3.3 Compound 30- (R ^C7 = benzyl) is described in Chemistry & Biology July 1995, 2:47
Synthesized as described in 1-481 ".

3.4 Compounds 31 , 32- (R ^C7 = -NH-CO-OMe) are described in "Chemistry & Biology July 1995, 2:
471-481 ".

[0253] 3.5 Compound ^{33 - (R C7 = -NH-} SO 2 -Me) rapamycin at -40 ℃ (75mg, 0.082 mmol) and methanesulfonamide (312 mg, 3.282 mmol) was dissolved in dichloromethane (3 ml) The resulting solution was treated dropwise with trifluoroacetic acid (126 μl, 1.636 mmol) and stirred for 3 hours. The reaction mixture was then combined with a saturated aqueous solution of NaHCO ₃ (20 ml) and EtOAc.
Poured into a biphasic solution with c (10 ml). The organic layer was washed with a saturated aqueous solution of NaCl (2 × 10 ml), dried over Na ₂ SO ₄ , filtered, the solvent was evaporated off, silica gel (dichloromethane: hexane: EtOAc: MeOH, 200: 50: 42.5: 7.5) Was used for flash chromatography. The semi-purified material thus obtained was purified by HPLC at 55 C using a Chromasil C-18 column (20 × 250 mm) using 65% acetonitrile / water as eluent to obtain AP17.
05 (24 mg) was obtained. MS (FAB) :( M + Na ) + calcd: 999.5246, Found: 999.5246 3.6 Compound 34, 35 - (R ^C7 = furanyl) These compounds "Chemistry & Biology July 1995, 2 : 471-481" in It can be synthesized as described.

[0254] 3.7 Compound ^{36, 37 -. (R C7} = methylthiophene) These compounds "J. Org Che
m. 1994 , 59, 6512-6513 ".

[0255] 3.8 Compound 39, 38 - (R ^C7 = ethyl thiophene) rapamycin (50 mg, 0.055 mmol) and 2-ethyl-thiophene (248μl, 2.188 mmol) was dissolved in dichloromethane (1.5 ml) at the -40 ℃ The resulting solution was treated dropwise with trifluoroacetic acid (84 μl, 1.094 mmol) and stirred for 3 hours. Thereafter, the reaction mixture was washed with a saturated aqueous solution of NaHCO ₃ (15
ml) and EtOAc (10 ml). The organic layer was washed with a saturated aqueous solution of NaCl (2 × 10 ml
), Dried over Na ₂ SO ₄ , filtered, evaporated off and flash chromatographed on silica gel (MeOH: dichloromethane, 2:98 then 5:95). The semi-purified material thus obtained was purified by HPLC at 55 C using a Chromasil C-18 column (20 × 250 mm) using 80% acetonitrile / water as eluent to obtain AP1859 (28 m
g) was obtained. MS (ES +) :( M + NH 4) + 1016; MS (ES -) :( MH) - 992

[0256]

[Table 13]

[0257] 3.9 Compound ^{40, 41 - (R C7 =} tert- butyl thiophene) rapamycin (50 mg, 0.05
(5 mmol) and 2-tert-butylthiophene (276 mg, 2.188 mmol) were dissolved in dichloromethane (1.5 ml) at -40 ° C, and trifluoroacetic acid (84 μl, 1.094 mmol) was added dropwise. And stirred for 3 hours. Afterwards the reaction mixture was poured into a biphasic solution of a saturated aqueous solution of NaHCO ₃ (15 ml) and EtOAc (10 ml). Wash the organic layer with saturated aqueous NaCl (2 × 10 ml), dry over Na ₂ SO ₄ , filter, evaporate off the solvent and use silica gel (MeOH: dichloromethane, 2:98 then 5:95) And subjected to flash chromatography. The semi-purified material thus obtained was purified by HPLC at 55 C using a Chromasil C-18 column (20 × 250 mm) with 80% acetonitrile / water as eluent.
AP1856 (4 mg) and AP1857 (14 mg) were obtained. MS (ES +) :( M + Na) + 1045; MS (ES -) :( MH) - 10
21 3.10 Compound ^{43, 42 - (R C7 =} o, p- dimethoxyphenyl) These compounds "Chem
istry & Biology July 1995, 2: 471-481 ".

3.11 Compound44,45-(R^C7= Indolyl) rapamycin (50mg, 0.055mmol)
And indole (64 mg, 0.547 mmol) were dissolved in dichloromethane (2.0 ml) at -40 ° C.
Treat the solution obtained by dissolving trifluoroacetic acid (84 μl, 1.094 mmol) dropwise,
Stir for 3 hours. Thereafter, the reaction mixture was washed with NaHCO_ThreeSaturated aqueous solution (15 ml) and EtOAc (10 ml)
And into a two-phase solution. The organic layer was washed with a saturated aqueous solution of NaCl (2 × 10 ml),_TwoSO _Four , Filter and evaporate off the solvent, silica gel (dichloromethane H: hexane).
: EtOAc: MeOH, 200: 50: 42.5: 7.5). The semi-purified material thus obtained was purified by chromatography on 65% acetonitrile / water
Purified by HPLC using a Masil C-18 column (20 × 250 mm) to obtain AP1701 (12 mg) and AP1
702 (7.6 mg) was obtained. MS (FAB): (M + Na)⁺ Calculated: 1021.5765, Observed: 1021.5788 (A
P1701) and 1021.5797 (AP1702) 3.12 Compound46-(R^C7= o, p-diethoxyphenyl) rapamycin (108 mg, 0.118
Mmol) and 1,3-diethoxybenzene (783 mg, 4.72 mmol) at −40 ° C.
Trifluoroacetic acid (154 μl, 2.01 mM)
And the mixture was stirred for 3 hours. Thereafter, the reaction mixture was washed with NaHCO_ThreeSaturated aqueous solution
Poured into a biphasic solution of the liquid (15 ml) and EtOAc (15 ml). The organic layer was washed with a saturated aqueous solution of NaCl (2 × 10 ml),_TwoSO_Four, Filter and evaporate off the solvent.
(Chloromethane: hexane: EtOAc: MeOH, 200: 50: 42.5: 7.5).
Chromatographically processed. The material obtained in this way is treated with (dichloromethane: hexa).
Lanin silica column (a Rainin: EtOAc: MeOH, 210: 65: 65: 10).
Purification by HPLC using a silica column) (20 × 250 mm) to give AP20808 (20 mg)
. MS (ES +): (M + Na)⁺ 1065.95 3.13 Compound47-(R^C7= Methylthiophene) rapamycin (105 mg, 0.115 mmol
) And 3-methylthiophene (445 μl, 4.60 mmol) at −40 ° C. in dichloromethane (
Trifluoroacetic acid (150 μl, 1.96 mmol) was added dropwise to the solution obtained by dissolving
And stirred for 3 hours. Thereafter, the reaction mixture was washed with NaHCO_ThreePoured into a biphasic solution of a saturated aqueous solution (15 ml) and EtOAc (15 ml). The organic layer was washed with a saturated aqueous NaCl solution (2 × 10 ml).
Wash and Na_TwoSO_Four, Filter, evaporate the solvent and flash chromatograph using silica gel (dichloromethane: hexane: EtOAc: MeOH, 200: 50: 42.5: 7.5).
Ruffy processed. The material thus obtained was purified by adding (dichloromethane: hexane: EtOAc
: MeOH, 210: 65: 65: 10) as an eluent and purified by HPLC using a linin silica column (20 x 250 mm) to give AP20809 (60 mg). MS (ES +): (M + Na)⁺ 1002.96 3.14 Compound48-(R^C7= N-methylpyrrole) Rapamycin (51 mg, 0.056 mmol) and N-methylpyrrole (198 μl, 2.23 mmol) in dichloromethane (2.0 ml) at 0 ° C
) Was treated with zinc chloride (76 mg, 0.557 mmol) and allowed to stand overnight.
To warm to room temperature. Thereafter, the reaction mixture was washed with NaHCO_ThreeSaturated aqueous solution (15 ml) and EtOAc (15
ml). The organic layer was washed with a saturated aqueous solution of NaCl (2 × 10 ml), _Two SO_Four, Filter and evaporate off the solvent, silica gel (dichloromethane: hexa).
Flash chromatography using (EtOAc: MeOH, 100: 150: 150: 10)
did. The material thus obtained was purified by adding (dichloromethane: hexane: EtOAc: MeOH, 210: 6
5:65:10) as eluent, and a Rainin silica column (20 × 2
Purification by HPLC using 50 mm) provided AP20810 (10 mg). MS (ES +): (M + NH_Four)⁺ 9
81.05; MS (ES-) :( M-H)^- 961.69 C7 rapalog was characterized by having accurate mass and NMR spectra.

3.15 Assay of FKBP Binding Affinity of C7 Rapalog The affinity of various C7 rapalogs for FKBP was assessed using competitive FP as described above for the C24 rapalog. Rapamycin and C14-desoxo-rapamycin (produced as described by Luengo et al. In "1994. Tetrahedron Lett. 35, 6469-6472") were incorporated as controls.

Affinities are described as IC50 and fold reduction of affinity (= IC50 / (IC50 of rapamycin)). See the "Example C7 Rapalog" table below. These data indicate that large C7 substituents do not necessarily cause a large decrease in the affinity of rapalog for human FKBP.

[0261]

[Table 14]

Preparation of - [tetrahydropyran rapa Ma leucine (53) 24 (S), 30 (S) ] [0262] Example 4 R ^C24 and rapalog which R ^C30 are modified

[0263]

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Rapamycin (46 mg, 0.050 mmol) was dissolved in 2.0 ml of methanol at −78 ° C.
And cerium (III) chloride heptahydrate (46 mg, 0.123 mmol) was added. The solution was stirred for 15 minutes and sodium borohydride (7.6 mg, 0.20 mmol) was added. After 30 minutes, the reaction mixture was partitioned between ethyl acetate (15ml) and 5% hydrochloric acid (2ml). Organic phase in water (2 ml)
And brine (1 ml), dried over anhydrous magnesium sulfate and concentrated. Flash chromatography (silica gel, 15: 75: 50: 200 methanol: ethyl acetate: hexane: dichloromethane) provided 35 mg (76%) of the desired product as a white foam. Mass spectral data: (ES + / NaCl / NH 3) m / z 942.21 (M + Na) +, 935.83 (
M + NH ₄ ) +; (ES- / NaCl) m / z 963.04 (M + Cl)-, 917.34 (MH)-Article: Luengo, JI; R
ozamus, LW; and Holt, DA, "Tetrahedron Lett. 1994, 35, 6469-647.
2 " Example 5 Preparation of C24, C30, and C7 Modified Rapalog 24 (S), 30 (S) -Tetrahydrorapamycin ( 53 ) prepared in Example 4 was obtained in the aforementioned C7 rapalog Example. Denaturation can be performed at C7 using the approach shown, for example: 5.1 7 (S)-(2 ', 4'-dimethoxy) benzyl-7-demethoxy-24 (S), 30 (S)- Tetrahydro-rapamycin

[0265]

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24 (S), 30 (S) -tetrahydro-rapamycin (20 mg, 0.022 mmol) was dissolved in dichloromethane (1.0 ml). 1,3-Dimethoxybenzene (0.20 ml, 1.5 mmol) was added and the solution was cooled to -60 ° C. Trifluoroacetic acid (0.030 ml, 0.39 mmol) was added and the reaction mixture was stirred at -60 ° C for 1 hour, then partitioned between ethyl acetate (10 ml) and saturated aqueous sodium bicarbonate (1 ml). The organic phase was washed with water (2ml) and brine (1ml), dried over anhydrous magnesium sulfate, filtered and concentrated. Flash chromatography (silica gel, 15: 75: 50: 200 methanol: ethyl acetate: hexane: dichloromethane) provided 8 mg (35%) of the desired product as a white solid. Mass spectrum data: (ES + / NaCl / NH ₃ ) m / z 1046.96 (M + Na) +, 1042.15 (M + NH ₄ ) +; (ES- /
NaCl) m / z 1069.09 (M + Cl)-Article: Luengo, JI; Konialian-Beck, A .; Rozamus
And J. Org. Chem. 1994, 59, 6512-6513 by Holt, DA, by analogous methods, to other C7 substituents described herein (eg, other substituted aryl groups, 24 (S), 30 (S) -tetrahydro-rapamycin having a heteroaryl group, an -O-aliphatic group, a thioether group, or any of the other types of moieties described above for R ^C7a or R ^C7b. It can also be manufactured. These compounds have the appropriate C
It can be obtained by reduction of the 7 rapalog at C24 and C30, or by conversion of the appropriate C24, C30-tetrahydrorapalog at C7. Representative examples are shown below.

The rapalogs modified at C24, C30, and C7 can also be used at various positions described herein (eg, R ^C13 , R ^C43 , R ^C28 , R ^C29 , R ⁴ , and “a (Eg, with respect to one or more such as "). For example, starting with 13-F-rapamycin instead of rapamycin gives the 13-fluoro analog of compound 53-79.

[0268] 5.2 Compound ^{54, 55 - (R C7 =} Et) is Ru can be synthesized as described in Example 4.1, but using each compound 27 and 28 in place of rapamycin.

[0269] 5.3 Compound 56 - but (R ^C7 = iPr) can be synthesized as described in Example 4.1, using the compound 29 in place of rapamycin.

[0270] 5.4 Compound 57 - (R ^C7 = benzyl) is cut at be synthesized as described in Example 4.1, using the compound 30 in place of rapamycin.

[0271] 5.5 Compound 58, 59 - is ^{(R C7 = -NH-CO-} OMe) may the synthesized child as described in Example 4.1, using the respective compounds 32 and 31 in place of rapamycin.

[0272] 5.6 Compound ^{60 - (R C7 = -NH-} SO 2 -Me) is can be synthesized as described in Example 4.1, using the compound 33 in place of rapamycin.

[0273] 5.7 Compound 61, 62 - (R ^C7 = furanyl) may can be synthesized as described in Example 4.1, using the respective compounds 34 and 35 in place of rapamycin.

[0274] 5.8 Compound 63, 64 - (R ^C7 = methylthiophene) is can be synthesized as described in Example 4.1, using the respective compounds 36 and 37 in place of rapamycin.

[0275] 5.9 Compound 65, 66 - (R ^C7 = ethyl thiophene) is can be synthesized as described in Example 4.1, using the respective compounds 38 and 39 in place of rapamycin.

5.10 Compounds 67 , 68- (R ^C7 = tert-butylthiophene) can be synthesized as described in Example 4.1, but using compounds 41 and 40 instead of rapamycin, respectively.

[0277] 5.11 Compound ^{69, 70 - (R C7 =} o, p- dimethoxyphenyl) is can be synthesized as described in Example 4.1, using the respective compounds 43 and 42 in place of rapamycin.

[0278] 5.12 Compound 71, 72 - (R ^C7 = indolyl) may can be synthesized as described in Example 4.1, using the 45 and 44 respectively in place of compound rapamycin.

[0279] 5.13 Compound ^{73 - (R C7 = o,} p- di-ethoxyphenyl) is can be synthesized as described in Example 4.1, using the compound 46 in place of rapamycin.

[0280] 5.14 Compound 74 - but (R ^C7 = methylthiophene) can be synthesized as described in Example 4.1, using the compound 47 in place of rapamycin.

[0281]

[Table 15]

[0282] 5.15 Compound 75 - (R ^C7 = N- methylpyrrole) can be synthesized as described in Example 4.1, but using Compound 48 in place of rapamycin.

[0283] 5.16 Compound ^{75, 76 - (R C7 =} 2,4,6- trimethoxyphenyl) is can be synthesized as described in Example 5.1, 1 instead of 1,3-dimethoxybenzene, 3,5-trimethoxybenzene is used. Example 6 Preparation of Fluoro-rapalog 6.1 C13-Fluoro-rapalog A new class of rapalog, C13-fluoro-rapalog, can be prepared by the following reaction scheme.

[0284]

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In this example, the hydroxyl moiety at positions 28 and 43 is protected prior to treatment with DAST.
We used the bis-triethylsilyl (shown above) protecting group and the bis-triisopropyl protecting group. Various alternative protecting groups may be used based on the preferences or convenience of the user, and taking into account the reaction conditions for the conversion prior to or simultaneously with the removal of the protecting group. Can also be used. The protected compound is then treated with a DAST reagent to introduce a 13-fluoro substituent. The DAST reaction can be performed, for example, at -42 ° C, as described above, or at 0 ° C.

13-Fluoro- having any of the various moieties described above for R ^C7a or R ^C7b
Modifications can be made at the 7 position of 13-fluororapamycin to produce a series of C7-rapalogs. For example, 7- (o, p-dimethoxy) -13-fluoro-rapalog ( 96 and 97 ) can be obtained by converting 78 with C7 and then removing the protecting group, or, as shown below, from 78. It can be prepared by removing the protecting group and then converting at C7 (and can be recovered separately if necessary).

[0287]

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To obtain the corresponding 13-F analogs, various other chemical transformations as disclosed or cited herein for 13-F-rapamycin instead of rapamycin
(E.g., fluorination at C28, reduction at C24 and C30, fluorination at C24 and C30, and modification at C43, as well as modification at C7 or as an alternative to modification at C7) You can also.

[0289] 6.2 Compound ^{81, 82 - (R C7 =} Et) is Ru can be synthesized as described in Example 3.1 but, 13-F- use of rapamycin (79) in place of rapamycin.

[0290] 6.3 Compound 83 - (R ^C7 = iPr) is can be synthesized as described in Example 3.2, 13-F- use of rapamycin (79) in place of rapamycin.

[0291] 6.4 Compound 84 - (R ^C7 = benzyl) is cut at be synthesized as described in Example 3.3, 13-F- use of rapamycin (79) in place of rapamycin.

[0292] 6.5 Compound 85, 86 - the ^{(R C7 = -NH-CO-} OMe) is can the synthesis child as described in Example 3.4, 13-F- rapamycin in place of rapamycin (79) use.

[0293]

[Table 16]

[0294] 6.6 Compound ^{87 - (R C7 = -NH-} SO 2 -Me) is can be synthesized as described in Example 3.5, using 13-F- rapamycin (79) in place of rapamycin .

[0295] 6.7 Compound 88, 89 - (R ^C7 = furanyl) may can be synthesized as described in Example 3.6, using 13-F- rapamycin (79) in place of rapamycin.

6.8 Compound 90 , 91- (R ^C7 = methylthiophene) can be synthesized as described in Example 3.7, but using 13-F-rapamycin ( 79 ) instead of rapamycin.

6.9 Compounds 92 , 93- (R ^C7 = ethylthiophene) can be synthesized as described in Example 3.8, but using 13-F-rapamycin ( 79 ) instead of rapamycin.

[0298] 6.10 Compound ^{68, 69 - (R C7 =} tert- butyl thiophene) is can be synthesized as described in Example 3.8, using 13-F- rapamycin (79) in place of rapamycin.

[0299] 6.11 Compound ^{94, 95 - (R C7 =} o, p- dimethoxyphenyl) is capable of good urchin synthesis described in Example 3.10, using 13-F- rapamycin (79) in place of rapamycin I do.

[0300] 6.12 Compound 96, 97 - (R ^C7 = indolyl) may can be synthesized as described in Example 3.11, using 13-F- rapamycin (79) in place of rapamycin.

[0301] 6.13 Compound ^{98 - (R C7 = o,} p- diethoxy phenyl) can be synthesized as described in Example 3.12, but for 13-F- use of rapamycin (79) in place of rapamycin I do.

[0302] 6.14 Compound 99 - (R ^C7 = methylthiophene) is able you to synthesized as described in Example 3.13, using 13-F- rapamycin (79) in place of rapamycin.

6.15 Compound 100- (R ^C7 = N-methylpyrrole) can be synthesized as described in Example 3.14, but using 13-F-rapamycin ( 79 ) instead of rapamycin.

6.20 Preparation of 28-F-Rapamycin (107) DAST (21 ml, 0.156 mmol) was added to a solution of rapamycin (71 mg, 0.078 mmol) dissolved in methylene chloride (1 ml) at −78 ° C. The reaction mixture was stirred for 2 hours before MeOH was added to quench the reaction. The reaction mixture was warmed to room temperature and stirred for 30 minutes. It was poured into a biphasic solution of a saturated aqueous solution of NaHCO ₃ (20 ml) and EtOAc (30 ml). The organic layer was washed with a saturated aqueous solution of NaHCO ₃ (2 × 20 ml) and then with a saturated aqueous solution of NaCl (2 × 1
0 ml), dried over Na ₂ SO ₄ , filtered and the solvent was evaporated off. The resulting material was flash chromatographed on silica gel (hexane: EtOAc, 1: 1 to 1: 2). MS and fluorine NMR indicated that C28 fluorinated rapamycin had been formed. The stereoisomer is subjected to reverse phase chromatography (C18 column, MeOH: H ₂ O
, 80:20) and can be used in place of rapamycin for the synthesis of various F-28 rapalogs.

[0305]

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6.21 Compound 108- (13-F, 28-F-rapamycin) can be synthesized as described above for 28-F-rapamycin, but at twice the volume at higher temperatures (−40 ° C.). DAST
(41 ml). Example 7: Constructs Encoding Chimeric Transcriptional Regulator A. Unless otherwise specified, all DNA manipulations described in this and other examples are by standard methods (e.g., by FMAusubel et al.). "Eds., Current Protocols in M
olecular Biology (John Wiley & Sons, New York, 1994) ").

B. Plasmids Human FKBP12, yeast GAL4 DNA binding domain, HSV VP16 activation domain, human
Constructions encoding T cell CD3 zeta chain intracellular domains or fusions with the intracellular domain of human FAS are disclosed in PCT / US94 / 01617.

A further DNA vector for directing the expression of fusion proteins related to the present invention was derived from the mammalian expression vector pCGNN (Attar, RM and Gilman, MZ).
1992. MCB 12: 2432-2443). The insert, cloned into pCGNN as an Xbal-BamHI fragment, was transcribed (nucleotides -522 to +72 relative to the cap site) under the control of the promoter and enhancer sequences of human CMV and any epitope tag (monoclonal antibody 12CA5 With the H. influenza hemagglutinin gene recognized by H. influenza hemagglutinin gene) and, in the case of the transcriptional regulator domain, with the N-terminal nuclear localization sequence (NLS; from SV40T antigen).

Unless otherwise specified, all fragments cloned into pCGNN were inserted as an XbaI-BamHI fragment containing a SpeI site just upstream of the BamHI site. XbaI and SpeI generate compatible ends, which result in XbaI-Bam
The HI fragment could be further inserted downstream of the initial insert, and the stepwise assembly of a multi-component protein was facilitated. SpeI stop codon
Inserted between the site and the BamHI site. The vector pCGNN-GAL4 was further used for the initial construction, in which case the codons 1-94 of the gene for the GAL4 DNA binding domain were replaced with the XbaI site Cloned. Thus, the XbaI-BamHI fragment could be cloned into the vector to generate a GAL4 fusion and subsequently recovered.

(A) Construction Encoding the GAL4 DNA Binding Domain-FRAP Fusion To obtain a portion of the human FRAP gene, MMLV reverse transcriptase and random hexamer primer (Clontech first strand) Human thymus total RNA (Clontech # 64028-1) was reverse transcribed using a synthesis kit). Primers 1 and 2 and Pfu polymerase (Stratagene
)] Was used directly in the PCR reaction containing The primers are human FRAP [ie, Chen et al., "Proc. Natl. Acad. Sci. USA (1995) 92, 4
947-4951 ", an amino acid 2025 including the 89 amino acid region essentially corresponding to the minimal 'FRB' domain identified as necessary and sufficient for FKBP-rapamycin binding (hereinafter referred to as the FRB). The coding sequence was designed to amplify the coding sequence for -2113.The appropriately ordered bands were purified, digested with XbaI and SpeI, and
Ligation was performed on pCGNN-GAL4 digested with aI and SpeI. This configuration is a restriction analysis (restriction
analysis) (to confirm the correct orientation) and DNA sequencing, and was shown to be pCGNN-GAL4-1FRB.

The construct encoding the FRB multimer was isolated from the FRB XbaI-BamHI fragment, which was then ligated back into pCGNN-GAL4-1FRB digested with SpeI and BamHI and pCGNN-GAL4-2FRB ( (Confirmed by restriction analysis). Repeat this procedure for the new configuration in the same way as for pCGNN-GAL4-3FR
B and pCGNN-GAL4-3FRB were obtained.

A vector encoding a larger fragment of FLAP (surrounding the minimal FRB domain (amino acids 2025-2113) but extending beyond it) was also constructed. PCR primers that amplify various FLAP regions flanked by 5 'XbaI and 3' SpeI sites were designed as follows.

[0313]

[Table 17]

First, the fragment FRAPi was amplified by RT-PCR as described above, with XbaI and Spe
Digested with I and ligated into XbaI-SpeI digested pCGNN-GAL4. This configuration (p
CGNN-GAL4-FRAPi) was analyzed by PCR to confirm the insertion orientation and verified by DNA sequencing. It was then used as a PCR substrate and other fragments were amplified using the indicated primers. The new fragment was cloned as a GAL4 fusion as described above to obtain constructs such as pCGNN-GAL4-FRAPa and pCGNN-GAL4-FRAPb (confirmed by DNA sequencing).

A vector encoding a concatenate of two larger FRAP fragments (FRAPd and FRAPe) was generated by a method similar to that used above. The XbaI-BamHI fragment encoding FRAPd and FRAPe was transformed into pCGNN-GAL4-F
Isolated from RAPd and pCGNN-GAL4-FRAPe, and religated into the same vector digested with SpeI and BamHI to generate pCGNN-GAL4-2FRAPd and pCGNN-GAL4-2FRAPe. Repeat this procedure for the new configuration in the same way for pCGNN-GAL4-3FRAPd, pCGNN-GAL
4-3FRAPe, pCGNN-GAL4-4FRAPd, and pCGNN-GAL4-4FRAPe were obtained. All configurations were confirmed by restriction analysis.

(B) Construction Encoding the FRB-VP16 Activation Domain Fusion In order to generate an N-terminal fusion between the FRB domain and the activation domain of the herpes simplex virus protein VP16, the FRB 1,2 X encoding 3, 3 and 4 copies
The baI-BamHI fragment was recovered from the GAL4 fusion vector, and ligated to pCGNN digested with XbaI-BamHI to obtain pCGNN-1FRB, pCGNN-2FRB, and the like. These vectors were then digested with SpeI and BamHI. XbaI-B encoding amino acids 414-490 of VP16
The amHI fragment was isolated from plasmid pCG-Gal4-VP16 (Das, G., Hinkley, CS
And Herr, W. (1995) Nature 374, 657-660). The composition was confirmed by restriction analysis and / or DNA sequencing.

(C) Construction Encoding ZFHD1 DNA Binding Domain-FRB Fusion An expression vector for directing expression of the ZFHD1 coding sequence in mammalian cells was prepared as follows. The Zif268 sequence was amplified from a cDNA clone by PCR using primers 5'Xba / Zif and 3'Zif + G. Primer 5'NotOctHD and Spe
The Oct1 homeodomain sequence was amplified from a cDNA clone by PCR using / Bam3'Oct. The Zif268 PCR fragment was cut with XbaI and NotI. The OctIPCR fragment was cut with NotI and BamHI. Both fragments were ligated with the XbaI site of pCGNN and the Ba
Ligating with the mHI site in a three-way ligation (Attar and Gilman, 1992) pCGNNZFH
D1 was manufactured. In pCGNNZFHD1, the cDNA insert is transcriptionally regulated by the human CMV promoter and enhancer sequences and is linked to a nuclear localization sequence from the SV40T antigen. Plasmid pCGNN also contains a gene for ampicillin antimicrobial activity that can function as a selectable marker. with the SpeI site of pCGNNZFHD1
Derivatives containing one or three parallel repeats of human FKBP12 linked as a XbaI-BamHI fragment between the BamHI site (pCGNNZFHD1-FKBP × 1 and pCGNNZFHD1-F
KBP × 3) was manufactured. Am sample of pCGNNZFHD1-FKBP × 3 in ATCC registration No. 97399
Deposited with erican Type Culture Collection. The sequence of the primer is shown in WO96 / 41865.

To generate a C-terminal fusion of the FRB domain with the chimeric DNA binding protein ZFHD1, the XbaI-BamHI fragment encoding 1, 2, 3 and 4 copies of the FRB was
Recovered from the AL4 fusion vector and ligated to pCGNN-ZFHD1 digested with Spe-BamHI,
NN-ZFHD1-1FRB and pCGNN-ZFHD1-2FRB were obtained. The configuration is restricted analysis and / or
Confirmed by DNA sequencing.

To investigate the effect of introducing an additional 'linker' polypeptide between ZFHD1 and the C-terminal FRB domain, a FRAP fragment encoding an additional sequence, N-terminal-FRB, was cloned into ZFHD1 Cloned as a fusion. FRAPa, FRAPb, FRA
The XbaI-BamHI fragment encoding Pc, FRAPd, and FRAPe was ligated with pCGNN-
Vectors such as GAL4-FRAPa and pCGNN-GAL4-FRAPb were cut out and ligated to pCGNN-ZFHD1 digested with SpeI-BamHI to obtain vectors such as pCGNN-ZFHD1-FRAPa and pCGNN-ZFHD1-FRAPb. In addition, pCGNN-GAL4-2FRAPe, pCGNN-GAL4-3FRAPe, and pCGNN-GAL4
XbaI-BamHI fragments encoding 2FRAPe, 3FRAPe, and 4FRAPe were isolated from -4FRAPe, and these fragments were digested with SpeI-BamHI into pCGNN-ZFHD.
Linked to 1 for pCGNN-ZFHD1-2FRAPe, pCGNN-ZFHD1-3FRAPe, and pCGNN-ZFHD1-4FR
By obtaining APe, vectors encoding fusions of ZFHD1 to 2, 3 and 4 C-terminal copies of FRAPe were also constructed. All configurations were confirmed by restriction analysis.

A vector encoding an N-terminal fusion between the FRB domain and ZFHD1 was also constructed. XbaI-BamHI fragments encoding 1, 2, 3 and 4 copies of FRAPe were isolated from pCGNN-GAL4-1FRAPe and pCGNN-GAL4-2FRAPe, etc., and ligated to XbaI-BamHI digested pCGNN to form Plasmids such as 1FRAPe and pCGNN-2FRAPe were obtained. These vectors are then digested with SpeI and BamHI, ligated to the XbaI-BamHI fragment encoding ZFHD1 (isolated from pCGNN-ZFHD1) and ligated into pCGNN-1FRAPe-ZFHD1 or pCGN1.
A configuration such as N-2FRAPe-ZFHD1 was obtained. All of these configurations were confirmed by restriction analysis.

(D) Construction Encoding the FRB-p65 Activation Domain Fusion In order to generate a fusion between the activation domain of the human NF-kB p65 subunit (hereinafter referred to as p65) and the FRB domain. First, two fragments were obtained from plasmid pCG-p65.
Amplified by PCR. Primers 9 (p65 / 5'Xba) and 11 (p65 3'Spe / Bam) are amino acids
Amplify the coding sequence for 450-550, primers 10 (p65 / 361Xba) and 11 amplify the coding sequence for amino acids 361-550, both flanked by 5 'XbaI and 3' SpeI / BamHI sites. You. Digest the PCR product with XbaI and BamHI,
Cloned into pCGNN digested with I and pCGNN-p65 (450-550) and pCGNN-p65 (361-55
0). These configurations were confirmed by restriction analysis and DNA sequencing.

A DNA sequence encoding a p65 transcription activation sequence of 100 amino acids, and a more expanded p65 transcription activation domain (351-550) are shown in WO 96/41865.

To generate an N-terminal fusion between a part of the p65 activation domain and the FRB domain, plasmids such as pCGNN-1FRB and pCGNN-2FRB were digested with SpeI and BamHI. p65 (450
The XbaI-BamHI fragment encoding (-550) was isolated from pCGNN-p65 (450-550), ligated into a vector digested with SpeI-BamHI and ligated into pCGNN-1FRB-p65 (450-550).
) And pCGNN-2FRB-p65 (450-550). Using the XbaI-BamHI fragment isolated from pCGNN-p65 (361-550), pCGNN-1FRB-p65 (361-550) was similarly assembled. These configurations were confirmed by restriction analysis.

To investigate the effect of introducing an additional 'linker' polypeptide between the p65 activation domain and the N-terminal FRB domain, an additional sequence, the C-terminal-FRB, was coded. The existing FRAP fragment was cloned as a p65 fusion. FRAPa,
The XbaI-BamHI fragment encoding FRAPb, FRAPf, FRAPg, and FRAPh was excised from vectors such as pCGNN-GAL4-FRAPa and pCGNN-GAL4-FRAPb, and XbaI-B
Vectors such as pCGNN-FRAPa and pCGNN-FRAPb were obtained by ligating with pCGNN digested with amHI. These plasmids were then digested with SpeI and BamHI, and p65 (amino acids 450-550).
) Is ligated to the XbaI-BamHI fragment encoding pCGNN-FRAPa-p65 or pCGN
Five pectors such as N-FRAPb-p65 were obtained. These were confirmed by restriction analysis.
Vectors encoding the fusion of p65 to the 1 and 3 N-terminal copies of FRAPe were produced by digesting pCGNN-1FRAPe and pCGNN-3FRAPe with SpeI and BamHI. Then, p65 (450-550) and p65 (361-550) (pCGNN-p65 (450-550) and pCGNN-p65 (361-550)
550), and ligating the XbaI-BamHI fragment encoding pCGNN-
1FRAPe-p65 (450-550), pCGNN-3FRAPe-p65 (450-550), pCGNN-1FRAPe-p65 (361-550
) And pCGNN-3FRAPe-p65 (361-550). All configurations were confirmed by restriction analysis.

A vector encoding a C-terminal fusion between a portion of the p65 activation domain and the FRB domain was also constructed. Plasmids such as pCGNN-p65 (450-550) and pCGNN-p65 (361-550) were digested with SpeI and BamHI, and 1 and 3 of FRAPe (isolated from pCGNN-GAL4-1FRAPe and pCGNN-GAL4-3FRAPe) PCGNN-p65 (450-550) -1FRAPe, pCGNN-p65 (450-550) -3FRAPe linked to an XbaI-BamHI fragment encoding a copy as well as one copy of the FRB (isolated from pCGNN-GAL4-1FRB) , PCGNN-p65 (361-550) -1FRAPe, pCGNN-p65 (361-55)
Plasmids such as 0) -3FRAPe, pCGNN-p65 (450-550) -1FRB, and pCGNN-p65 (361-550) -1FRB were obtained. All configurations were confirmed by restriction analysis.

(E) Further Construction Another assembly was carried out in the same manner as described above, using the FRB domain containing the peptide sequence which had been subjected to a substitution or modification of the FRAP sequence. For example, primers 12 and 13 are used to amplify the entire coding region of FRAP. Primers 1 and 13, 6
13, and 5 and 13 surround the FRB domain and are used to amplify three fragments (including the lipid kinase homology domain) extending at the C-terminus of the protein. The three fragments differ by encoding different parts of the protein N-terminal to the FRB domain. In any case, RT-P
CR was used as described above to amplify a region from human thymus RNA, the PCR product was purified, digested with XbaI and SpeI, and ligated into XbaI-SpeI digested pCGNN. The composition was confirmed by restriction analysis and DNA sequencing.

[0327] (f) Primer sequences 1 5'GCATGTCTAGAGAGATGTGGCATGAAGGCCTGGAAG 2 5'GCATCACTAGTCTTTGAGATTCGTCGGAACACATG 3 5'GCACATTCTAGAATTGATACGCCCAGACCCTTG 4 5'CGATCAACTAGTAAGTGTCAATTTCCGGGGCCT 5 5'GCACTATCTAGACTGAAGAACATGTGTGAGCACAGC 6 5'GCACTATCTAGAGTGAGCGAGGAGCTGATCCGAGTG 7 5'CGATCACCTAGTGGAAACATATTGCAGCTCTAAGGA 8 5'CGATCAACTAGTTGGCACAGCCAATTCAAGGTCCCG 9 5'ATGCTCTAGACTGGGGGCCTTGCTTGGCAAC 10 5'ATGCTCTAGAGATGAGTTTCCCACCATGGTG 11 5'GCATGGATCCGCTCAACTAGTGGAGCTGATCTTGACTCAG 12 5'ATGCTCTAGACTTGGAACCGGACCTGCCGCC 13 5'GCATCACTAGTCCAGAAAGGGCACCAGCCAATAT Restriction sites are underlined (XbaI = TCTAGA, SpeI = ACGAGT, BamHI = GGATCC).

(G) DNA sequence of a representative final construct: pCGNN-ZFHD1-1FRB encoding the 12CA5 epitope-SV40 NLZ-ZFHD1-FRB fusion is shown in WO 96/41865.

(H) Bicistronic Constructs An internal ribosome translocation sequence from Insepharomyocarditis virus (In
internal ribosome entry sequence (IRES) was amplified by PCR from pWZL-Bleo. The resulting fragment was pBS-S
It was cloned into K + (Stratagene) and contains an XbaI site and a stop codon upstream and downstream of the IRES sequence, an NcoI site surrounding the ATG, followed by a SpeI and BamHI site. To enable cloning, pCGNN was used with the oligonucleotides shown in WO96 / 41865.
The starting ATG of -ZFHD-3FKBP was mutated to an NcoI site and XbaI
The site was mutated to a NheI site. Nco containing ZFHD1-3FKBP
The I-BamHI fragment was cloned downstream of pBS-IRES and pBS-I
RES-ZFHD1-3FKBP was produced. XbaI derived from this plasmid
The -BamHI fragment was then digested with SpeI / BamHI cut pCGNN-1FRB-p
65 (361-550) and cloned into pCGNN-1FRB-p65 (
361-550) -IRES-ZFHD1-3FKBP was produced.

C. Retroviral Vector for Expression of Chimeric Protein The retroviral vector used to express a transcription factor fusion protein derived from a stably integrated low copy number gene is pSRaMSVtkNeo (Mu
ller et al. MCB 11: 1785-92, 1991) and pS
RaMSV (XbaI) (Swyers et al. J. Exp. Med. 1)
81: 307-313, 1995). Single BamHI for both vectors
The sites were digested with BamHI, blunted with Klenow, and ligated again to create pSMTN2 and pSMTX2, respectively. pSM
TN2 expresses the Neo gene from an internal thymidine kinase promoter. Z
The eocin gene (Invitrogen) was converted to pSMTX as a NheI fragment.
2 is cloned into a single XbaI site downstream of the internal thymidine kinase promoter. This Zeocin fragment was synthesized using the following primers.
By introducing a NheI site surrounding the coding region, pZeo / SV (Inv
(Itrogen). Primer 1 5'-GCCATGGTGGCTAGCCTATAGTGAG Primer 2 5'-GGCGGTGTTGGCTAGCGTCGGTCAG pSMTN2 contains a single EcoRI and HindIII site downstream of the LTR. To enable the cloning of transcription factor fusion proteins synthesized as XbaI-BamHI fragments, the following sequences were EcoRI and HindIII:
Inserted between the sites to create pSMTN3.

[0331]

Embedded image

An equivalent fragment was inserted into a single EcoRI site of pSMTZ and 3 ′ HindI
PSMT with changes only replacing the II site with an EcoRI site
Z3 was made.

The pSMTN3 and pSMTZ3 chimeric transcription factors can be cloned downstream of the 5 ′ viral LTR as an XbaI-BamH1 fragment, selecting for stable integrators by their ability to confer resistance to the antibiotics G418 or Zeocin, respectively. enable.

To make the retroviral vector SMTN-ZFHDI-3FKBP, pCGNN-ZFHD1-3FKBP was initially mutated to add an EcoRI site upstream of the first amino acid of the fusion protein. ZFHD1-3F
EcoRI-BamHI (KBP) such that KBP is expressed from the retroviral LTR.
The blunted) fragment was EcoRI-HindIII (blunted) pSRaMSVtkN
cloned into eo.

Example 8 Rapamycin-Dependent Activation of Transcription In preliminary experiments, three copies of FKBP fused to a Gal4 DNA binding domain or a transcription activation domain resulted in only one or two FKBP domains Activated a more stably incorporated or transiently transformed reporter gene than the corresponding fusion protein containing. To assess this parameter with the FRB fusion protein, an effector plasmid containing a Gal4 DNA junction fused to one or more copy numbers of the FRB domain was constructed using three F
Plasmids encoding the KBP domain and the p65 activation domain (3xFK
(BP-p65) along with transient transformation. In this system, four copies of the FRB domain fused to the Gal4 DNA binding domain were found to activate the stronger and more stably incorporated reporter gene than the corresponding fusion protein with fewer FRB domains.

Method: HT1080 B cells were grown in MEM supplemented with 10% bovine serum. Approximately 4 × 10 5 per well in a 6-well plate (Falcon)
Cells were transiently transformed by the lipofectin method as recommended by the vendor (GIBCO, BRL). The DNA: Lipofectamine ratio used in this experiment corresponds to 1: 6. Cells in each well received 500 ng of pCGNNF3-p65, 1.9 ug of PUC118 plasmid as a carrier, and 100 ng of any of the following plasmids. pCGNNGal4-4FRB, pCGN
N Gal4-2FRB, pCGNN Gal4-3FRB or pCGNGNG
al4-4FRB. Following transformation, 2 ml of fresh culture was added and the indicated concentrations of rapamycin were added. After 24 hours, 100 ul of culture was tested for SEAP activity as described (Spencer et al, 1993).

To test whether multiple FRB domains fused to the p65 activation domain result in increased transcriptional activation of the reporter gene, HT1080 B cells were fused to Gal4-3xFKBP and the p65 activation domain. 1,2
, 3, or 4 copies of FRB.
Surprisingly, unlike the DNA binding domain-FRB fusion, one copy of the FRB fused to the p65 activation domain is significantly stronger than the corresponding fusion protein with more than one copy of the FRB. Was activated.

Method: HT1080 B cells were grown in MEM supplemented with 10% bovine serum. Approximately 4 × 10 5 per well in a 6-well plate (Falcon)
Cells were transiently transformed by the lipofectin method according to GIBCO, BRL recommendations. The DNA: Lipofectamine ratio used corresponds to 1: 6. Cells in each well were loaded with 1.9 ug of PUC118 plasmid, 100 ng of pCGNNGal4F3, and 500 n of one of the following plasmids as carriers:
g was received. pCGNN1, 2, 3, or 4FRB-p65. Following transformation, 2 ml of fresh culture was added and the indicated concentrations of rapamycin were added. Twenty-four hours later, 100 ul of culture was written as described (Spencer
et al, 1993) SEAP activity was tested.

DNA encoding a 5xGal4-IL2-SEAP reporter gene and a fusion protein comprising a Gal4 DNA binding domain and 3 copies of FKBP
Similar experiments were also directed with another stable cell line (HT1080B14) that stably incorporates and contains the sequence. These cells have one or more copy numbers of F
Transiently transformed with an effector plasmid expressing the p65 activation domain fused to the RB domain. Similar to our observations with HT1080 B cells, in these experiments, the effector plasmid expressing one copy of the FRB-p65 activation domain fusion protein was modified to contain two or more copies of the FRB. Activated the reporter gene more strongly than the others.

Example 9 A. Rapamycin-dependent transcriptional activation in transiently transformed cells:
ZFHD1 and p65 fusion SEAP target gene and representative ZFHD-FKBP- and FRB-p6
Human fibrosarcoma cells transiently transformed with a plasmid encoding the 5-containing fusion protein showed rapamycin-dependent and dose-responsive secretion of SEAP into cell culture. See FIG. 4A. When one or both transcription factor fusion protein plasmids were omitted, no SEAP production was detected in the cells, and no rapamycin was added (Figure 4B). When all components were present, however, even at rapamycin concentrations as low as 0.5 nM, SEA
P secretion was detectable (FIG. 4A). A peak of SEAP secretion was observed at 5 nM. Similar results were obtained when the same transcription factor was used for rapamycin-dependent activation of the SEAP reporter gene stably incorporated by infection with the hGH reporter gene or retroviral vector. Because of the undetectable SEAP secretion in the absence of drug, it is difficult to determine how many times it is activated in response to rapamycin, but in this system at least 1000 background levels in the absence of rapamycin. It is clear that there is a double amplification. Thus, this system exhibits undetectable background activity and high dynamic range.

Several different arrangements of the transcription factor fusion protein were tried (WO96 / 41
865, see FIG. 5). When the FKBP domain is fused to ZFHD1 and the FRB is fused to p65, multiple FKBPs are fused to ZFHD1 and a small number of FRBs are fused to p65.
The optimal level of rapamycin-induced activation occurred when fused. The preference for multiple drug binding domains on DNA binding proteins may reflect the ability of these proteins to recruit multiple activation domains and induce higher promoter activity. The presence of a single drug binding domain on the activation domain may allow FKBP on each ZFHD to be recruited to one p65. Any increase in the number of FRBs on p65 may increase the likelihood that fewer activation domains can be recruited to ZFHD, each linked by multiple FRB-FKBP interactions.

Methods: HT1080 cells from human fibrosarcoma (ATCC CCL-121) were grown in MEM supplemented with non-essential amino acids and 10% fetal calf serum. 24-
Well culture dishes (falcon, 6 × 10 4 cells / well) were transformed with lipofectamine under the conditions recommended by the manufacturer (GIBCO / BRL). Each well was transformed with a total of 300 ng of the following DNA: 100 n
g ZFHDx12-CMV-SEAP reporter gene, 2.5 ng pCG
NN-ZFHD1-3FKBP or other DNA binding domain fusion, 5n
gpCGNN-1FRB-p65 (361-550) or other activation domain fusion and 192.5 ng pUC118. If the DNA binding or activation domain was omitted, an equivalent amount of the empty pCGNN expression vector was replaced. Following lipofection (5 hours), 500 ul containing the indicated amount of rapamycin was added to each well. After 24 hours, the culture was removed
At an excitation wavelength of 350 nm and an emission wavelength of 450 nm, Luminescence S
Spectrometer (PerkinElmer) as described (Spencer et al, Science 262: 1019-24,).
1993) SEAP activity was tested. Background SEAP activity measured from mock transformed cells was subtracted from each value. To prepare transiently transformed HT1080 cells for injection into mice (see below), cells in 100 mm culture dishes (2 × 10 6 cells / culture dish) were transformed by calcium phosphate precipitation with the following DNA for 16 hours. Converted (Gat
z, C.I. , Kaiser, A, & Wendenburg, R .; , 1991, M
ol. Gen. Genet. 227, 229-237): 10 mg of pZHWT
x12-CMV-fGH, 1mg pCGNN-ZFHD1-3FKBP, 2m
g pCGNN-1FRB-p65 (361-550) and 7 mg pUC1
18. Transformed cells were rinsed with phosphate buffered saline (PBS) and given a fresh culture for 5 hours. Cells were collected 10 m from the culture dish for harvesting for injection.
Transferred into Hepes buffer solution containing M EDTA, PBS / 0.1% BSA /
Washed with 0.1% glucose and suspended in the same at a concentration of 2 × 10 7 cells / ml.

Plasmids: Construction of transcription factor fusion plasmids is described above. Copy of the parallel ZFHD1 binding site of pZHWTx12-CMV-SEAP12 (Pomerantz et al.
. This reporter gene contains the basal promoter from the human cytomegalovirus immediate-early gene, which drives the expression of secreted alkaline phosphatase (SEAP). 12 Z shown in 96/41865
NheI-XbaI containing the FHD binding site and XbaI-Hind containing the minimal CMV promoter (-54 to +45) indicated in WO 96/41865
It was adjusted by replacing with the III fragment.

PZHWTx12-CMV-hGH Activation of this reporter gene results in production of hGH. This is pZHWT
HindIII-B of x12-CMV-SEAP (including SEAP coding sequence)
The amHI (blunt) fragment was cloned from the (hGH genomic clone; Selden et al.
BamHI and Ec, MCB 6: 3171-3179, 1986;
HindIII (blunt) from p0GH (both oRI sites are blunted)
It was constructed by replacement with an EcoRI fragment.

PZHWTx12-IL2-SEAP This reporter gene is an XbaI-HindI containing the minimal CMV promoter.
The II fragment comprises the following XbaI-HindI containing the minimal IL2 gene promoter:
II is identical to pZHWTx12-CMV-SEAP except that it has been replaced by a II fragment (-72 to +45 relative to the start site; Siebenlist et al., MCB 6: 3042-3049, 1986) (WO 96
/ 41865).

The following retroviral vectors were constructed to allow stable incorporation of the pLH reporter gene at single or low copy numbers. A pLH (LTR-hph) containing a hygromycin B resistance gene and a single internal ClaI site governed by the Moloney murine leukemia virus LTR was constructed as follows: pBabe H
ygro (Morganstern and Land, NAR 18: 358)
7-96, 1990), the hph gene was transformed into a BamHI-ClaI truncated pBabe Bleo (resulting in a bleo gene deletion: both BamHI and HindIII sites were blunted). Cloned.

PLH-ZHWTx12-IL2-SEAP A copy of the reporter gene containing the ZFHD1 binding site of 12 parallel copies and the basal promoter from the IL2 gene that governs the expression of the SEAP gene was pLH-ZHWTx12-IL2-SEAP.
PZHWTx12-IL for cloning into an H retroviral vector
The MluI-ClaI fragment from 2-SEAP (with added ClaI linker) was cloned into the ClaI site of pLH. The orientation was such that the directions of transcription from the viral LTR and the internal ZFHD-IL2 promoter were the same.

PLH-G5-IL2-SEAP Five Gs embedded in the minimal IL2 promoter that direct SEAP gene expression
To construct a retroviral vector containing an al4 site, a ClaI-BstBI fragment consisting of the following was inserted into the ClaI site of pLH such that transcription from the viral LTR and the internal Gal4-IL2 promoter was identical. : 5 Gal4 sites and -324 to -294 and -72 of the IL2 gene
To ClaI-Hin containing the +45 region (shown in WO 96/41865)
A dIII fragment and a HindIII-BstBI fragment containing the SEAP gene coding sequence (shown in WO 96/41865) mutated to add a BstBI site immediately after the stop codon (Berger et al., Gene 66: 1-). 10, 1988).

B. Rapamycin-dependent transcriptional activation in stably transformed cells The following experiments confirmed that this system exhibited similar properties in stably transformed cells. We have generated stable cell lines by a series of transformations with expression vectors for the SEAP target protein and ZFHD1-3FKBP and 1FRB-p65, respectively. A few dozen stable clones resulting from the final transformation showed rapamycin-dependent SEAP production. From this pool we analyzed several individual clones, many of which produced high levels of SEAP in response to rapamycin. The result from one such clone was
It is shown in FIG. 4C of WO 96/41865. This clone produced about 40 times more levels of SEAP than the pool, which was significantly more than the transiently transformed cells. In an attempt to unambiguously quantify background SEAP production and induction in this clone, we determined that the length of the SEAP test was approximately 50 times greater to detect any SEAP activity in untreated cells. A second group of augmented tests was performed. Under these conditions, mock-transformed cells show 47 in any fluorescent unit, whereas transformed cells show 54 units in the absence of rapamycin and more than 90,000 units in the presence of 100 nM rapamycin. Was. Therefore, in this cell line, background gene expression was negligible, and the induction rate was at least four orders of magnitude larger.

To simplify the task of stable transformation, an internal ribosome translocation sequence (IRES
), A bicistronic expression vector was used that directs the production of ZFHD1-3FHBP and 1FRB-p65. This expression plasmid was combined with a retrovirus-transformed SEAp
After transformation into the reporter gene, 50 drug-resistant clones were selected and amplified. This pool of clones also showed, without detectable background, rapamycin-dependent SEAP production showing a dose-dependent curve very similar to that observed in transiently transformed cells. This pool is WO96 / 4
It can be expected to include individual clones with similar capabilities as the clone studied in FIG. 4 of 1865. Thus, rapamycin responsive gene expression can be readily obtained in both transiently and stably transformed cells. In both cases, the ability to regulate is characterized by a very low background and a high induction rate.

A stable cell line. Helper-free retroviruses containing a reporter gene or DNA binding region fusion were generated by transient co-transformation into 293T cells (Pear WS, Nolan GP, Scott M. L. & Baltimore). D., 1993, Production of high titer helper-free retroviruses by transient transformation Proc Natl.
d. Sci. USA 90, 8392-8396), each of which is a Psi
(-) Broad host packaging vector and retroviral vector pLH-ZHWTx1
Performed with 2-IL2-SEAP or SMYN-ZFHD1-3FKBP. To generate a clonal cell line that contains a stably integrated reporter gene, HT1080 cells infected with the retroviral stock were diluted to 300 m
Selected in the presence of g / ml hygromycin B. Each clone described below, from this and other cell lines, was screened after transient transformation of the missing contents by addition of rapamycin as described above. All 12 clones analyzed were inducible and had little or no basal activity. The most responsive clone, HT1080L, was selected for further study.

The stably integrated pLH-ZHWTx12-IL2-SEAP reporter gene, ZFHD1-3FKBP DNA binding region and 1FRB-p65 (361
550) HT20-6 cells with the activation region are initially HT1080L
Cells were infected with SMTN-ZFHD1-3FKBP packaging retrovirus and 50
Created by selection in media containing 0 mg / ml G418. HT1080L3, a strongly responsive clone, was transformed with the linearized pCGNN-1FRB.
Infect p65 (361-550) and pZeoSV (Invitrogen),
Selections were made in media containing 250 mg / ml Zeocin. Each clone first
The presence of 1FRB-p65 (361-550) was tested by Western. Eight positive clones were analyzed by the addition of rapamycin. All eight had weak basal activity, of which gene expression was induced in at least two orders of magnitude in intensity. HT2, the clone that gave the strongest response
0-6 was selected for further analysis.

The HT23 cells were pCGNN-1FRB-p linearized to HT1080L cells.
65 (361-550) -IRES-ZFHD1-3FKBP and pZeoSV
Was created by co-transformation and selection in media containing 250 mg / ml zeocin. About 50 clones were pooled for analysis.

For analysis, cells were seeded in a 96-well dish (1.5 × 10 4 cells / well) and 200 μl of medium containing the indicated amount of rapamycin (or solvent) was added to each well. Eighteen hours later, the medium was removed and SEAP activity was analyzed. In some cases, media was diluted prior to analysis and multiplexed relative SEAP units obtained by fold dilution. Background SEAP activity measured from untransformed HT1080 cells was subtracted from each value. Example 10: Rapamycin-dependent hGH production in mice In vivo methods: Animal, animal husbandry and general procedures. Male nu / nu mice were obtained from the Charles River lab (Wilmington, MA) and were acclimated for 5 days prior to experiments. They were raised under aseptic conditions, had free access to sterile food and water throughout the experiment, and were operated through aseptic techniques. The immunocompromised animals did not show any external infection or disease as a result of breeding, livestock or laboratory techniques.

To implant the transiently transformed cells into mice, 2 × 10 6 transformed HT1080 cells were suspended in 100 ml of PBS / 0.1% BSA / 0.1% glucose buffer, The animals were injected (approximately 25 ml per field) into four intramuscular fields on the hips and sides. Control mice received only the same amount of buffer.

Rapamycin is dissolved in an equal volume of a mixture of N, N-dimethylacetamide and polyethylene glycol (average molecular weight 400) and polyoxyethylene sorbitan monooleate 9: 1 (v: v) for in vivo administration. A capsule was prepared. The concentration of rapamycin in the complete capsule is 2.0 ml / kg
Sufficient in vivo administration of the appropriate amount of introduction. Solution volume accuracy was determined by HPLC analysis prior to vascular injection into the tail vein. Some control mice are
It had untransformed HT1080 cells and received 10.0 mg / kg rapamycin. In addition, other control mice have the transformed cells,
Only rapamycin solvent was received.

Blood was collected by anesthetizing or sacrificing mice by CO2 inhalation and collected. Anesthetized mice were used to collect 100 ml of blood by puncturing the heart. The mice were revived and recovered, and blood was subsequently collected. The sacrificed mice were exsanguinated immediately. Blood samples were clotted at 4 ° C. for 24 hours, and serum was collected by subsequent centrifugation at 1000 × g for 15 minutes. Serum hGH is Boe
hringer Mannheim nonisotopic sandwich ELISA (catalog number 1585878). The analysis had a lower detection limit of 0.0125 ng / ml and a dynamic range extended to 0.4 ng / ml. The recommended analysis instructions are as follows: Absorption was read at 405 nm with a reference wavelength of 490 nm on a Molecular Device microtiter plate reader.
Antibody reagents in the ELISA did not show cross-reactivity with endogenous mouse hGH in diluted serum or non-denatured specimens. HGH expression in vivo. For testing dose-dependent stimulation of rapamycin-induced hGH expression, mice were administered rapamycin approximately one hour following injection of HT1080 cells. Rapamycin doses are 0.01, 0.03, 0.1, 0.3, 1
. 0, 3.0, or 10.0 mg / kg. Seventeen hours after rapamycin administration, mice were sacrificed for blood collection.

To show the time course of hGH expression in vivo, mice received 10.0 mg / ml rapamycin one hour after cell injection. Mice were treated with rapamycin4.
They were sacrificed after 8, 17, 24 and 42 hours.

The ability of rapamycin to sustainably express hGH from transplanted HT1080 was tested by repeated administration of rapamycin. Mice are transformed HT10
Eighty cells were administered as described above. Approximately one hour after injection of the cells, the mice received the first of five 10 mg / kg doses of rapamycin vascular administration. The remaining four doses were given 16, 32, 48, and 64 hours later, with immediate and continuous blood collection under anesthesia conditions. 72,80 from first rapamycin administration
Additional blood collections were also performed at, 88 and 96 hours. Control mice received cells but received vehicle alone at various times during rapamycin administration. The experimental animals and their controls were each assigned as one of two populations. Each of the two experimental populations and the two control populations received the same drug or solvent treatment, respectively. Two populations that differed in the final blood time alternated between the two populations to reduce the frequency of blood collection in each animal. Results Rapamycin induced dose-dependent hGH production in these animals (W
FIG. 6 of O96 / 41865). The hGH concentration in animals treated with rapamycin is favorably compared to normal circulating levels in humans (0.2-0.3 ng / ml). In these experiments, no plateau in hGH production was observed, suggesting that the maximal hGH producing capacity of the transformed cells had not been reached. Control animals that received transformed cells but no rapamycin, and that received rapamycin but no cells, showed no detectable hGH in serum. Thus, hGH production in these animals was entirely dependent on the presence of both the engineered cells and rapamycin.

The presence of a significant degree of hGH in serum 17 hours after rapamycin administration is important because hGH is eliminated by circulation with a half-life of less than 4 minutes in these animals. This observation suggests that the designed cells continue to secrete hGH for a long time after rapamycin treatment. To confirm the kinetics of rapamycin control of hGH production, we treated animals with a single dose of rapamycin and then measured hGH levels at different times. Serum hGH was observed within 4 hours after rapamycin treatment, peaked at 8 hours (more than 100 times the detection limit of the hGH ELISA), and remained detectable for 42 hours after treatment. The hGH concentration decayed from its peak with a half-life of about 11 hours. In these animals, this half-life is hundreds of times longer than the half-life of hGH itself, about twice the half-life of rapamycin (4.6 hours). The slow decay of serum hGH compared to rapamycin could reflect the presence of higher tissue rapamycin concentrations around the implanted cells. Alternatively, the sustained hGH production from the engineered cells is
It may also enhance the stability of H mRNA.

Interestingly, a second administration of rapamycin to these animals at 42 hours resulted in a second peak of serum hGH, which was attenuated with similar kinetics, indicating that the designed cells were not exposed to rapamycin. The ability to respond to it for at least 2 days. Therefore, to determine the ability of this system to raise and maintain circulating hGH levels, we performed experiments in animals that received multiple doses of rapamycin at 16 hour intervals. This interval corresponds to the time required for hGH levels to peak and then decay about half. According to this plan,
The rapamycin concentration was designed to reach a steady state through a concentration of 1.7 μg / ml after two doses (as indicated by the dotted line in FIG. 8 of WO 96/41865). hGH levels also reached a steady state in concentration after the second dose. The treated animals actually responded to repeated doses of rapamycin and maintained relatively stable levels of circulating hGH. After the last dose, hGH levels remained constant for 16 hours and declined with a similar half-life as rapamycin (hGH was 6.
8 hours versus rapamycin 4.6 hours). These data suggest that, in multiple doses, circulating rapamycin tightly regulates hGH secreted by transplanted cells in vivo. In particular, it is clear that protein production is rapidly terminated when no drug is given. Discussion These experiments are performed on the possibility of controlling the production of therapeutic proteins secreted by genetically engineered cells using low molecular weight drugs. This system is
It has many features required for use in human gene and cell therapy. It is characterized by a very low background activity and a high induction rate. It functions independently of host physiology or cell type-specific factors. It is composed entirely of human proteins. Control agents are in good condition for in vivo and oral bioavailability.

With this genetically designed system, it is possible to produce stable and accurate titers of therapeutic proteins from engineered cells in vivo. Fragmented and periodic administration is also feasible. A considerable advantage of partitioning proteins from engineered cells under the control of small molecules is that the rate of protein production at any given time is a function of the circulating concentration of the small molecule drug. for that reason,
The apparent pharmacological kinetics of therapeutic proteins such as hGH is dramatically enhanced. In our experiments, for example, the kinetics of circulating hGH distributed from engineered cells after a single dose of rapamycin is significantly different from that observed after a single dose of the recombinant protein . HGH injected vascularly into mice disappears with a half-life of a few minutes, while hGH from engineered cells induced by rapamycin.
H levels decayed with a half-life of about 11 hours. Even in humans with a half-life of hGH extinction of 20 minutes, injections must be performed every other day and serum hGH levels fluctuate dramatically. Under precise pharmacological control, it is likely that proteins distributed from engineered cells will lead to more effective therapies, especially in proteins with poor pharmacological kinetics or low therapeutic effect.

The use of small molecule drugs linked to DNA binding and activation regions is an effective strategy for controlling gene expression in vivo. One particularly attractive feature is
The system is entirely modular, and each element can be fully utilized and independently designed. In contrast to bacterial repressors, which rely on relatively subtle allosteric molecular interactions to control DNA binding activity, dimerization strategies can adapt to virtually all DNA binding and activation domains. is there. We used here a defined structure of the DNA binding domain, which simply indicates the rational design of DNA binding affinity and novel recognition properties. Similarly, the activation area allows for the design of maximum potential and other suitable features. In fact, the designed transcription factors used in these experiments elicit very high levels of gene expression compared to the mediocre promoter / enhancer system, and can easily introduce further enhancement in any region .
The ability to introduce engineered transcription factors dedicated to the transcription of a single target gene offers the opportunity to allow for lower background and substantially higher gene expression than a mediocre expression vector.

We have also selected our regulated transcription factors from human proteins that minimized their ability to be recognized by the immune system. Autologous T cells expressing a fusion protein of bacterial hygromycin phosphotransferase and herpesvirus thymidine kinase are effectively recognized and removed by host cytotoxic T cells, even in AIDS patients with compromised immune systems. (Riddell, S.
. R. , Et al. Removal of genetically modified HIV-specific cytotoxic T cells via T cells in HIV infected patients. Nature Med. 2, 216
-223 (1996)). This observation suggests that the danger of immunorecognition of the heteroprotein in the engineered cells is real and therefore the use of human proteins to perform regulatory functions in human cells is conservative. Although the individual elements of our transcription factor fusion proteins are human in sequence, each protein contains a binding peptide that is potentially recognized as foreign. However, these conjugates are designed and selected to minimize presentation to the immune system, as discussed previously.

A fundamental limitation of rapamycin-dependent systems is the natural biological activity of rapamycin, which blocks cell cycle progression that leads to immunosuppression in vivo through inhibition of FRAP activity. However, introducing substituents or modifying the structure of rapamycin to eliminate or eliminate binding to FKBP and / or FRAP provides access to rapalogs lacking independent immunosuppressive activity. The use of such rapalogs, especially the advanced rapalogs of the present invention, along with the correspondingly designed FKBP and / or FPB regions, has been widely used in laboratory animal and human gene therapy as well as in controlling the production of designed proteins. It has proven to be widely useful in the control of various biological processes. Example 11: FR analysis of rapalog binding to FKBP The affinity of rapalog for FKBP protein can be determined using fluorescence polarity (FP) based competition analysis. Fluorescently labeled FK506 probe (AP
1941) was synthesized as described in WO 96/41865 (see Example 6 of that specification), and the increase in the polarity of its fluorescence included quasi-saturated FKBP and varying amounts of a rapamycin analog as a competitor Used as a direct readout of the percentage of bound probe in equilibrium binding experiments. Determination of Rapalog Binding Affinity (IC50s) Using FP A 10-fold dilution series of each analog is prepared in 100% ethanol in glass vials and stored on ice. All other operations are performed at room temperature. Stock of pure recombinant FKBP (purified by standard methods, Wiederrecht,
G. FIG. et al. 1992. J. Biol. Chem. 267, 2
1753-21760) is 50 mM potassium phosphate pH 7.8 / 15.
0 mM NaCl / 100 μg / ml bovine gamma globulin (“FP buffer”)
: Prepared using only the low fluorescent reagent from Panvera)
Dilute to 5 nM and aliquot 98 μl with Dynatech Microflow Black 9
Transfer to a 6-well fluorescent plate. The 2.0 μl sample of rapalog is then repeatedly transferred to the wells with mixing. Finally, the probe solution was 0.1% ethanol / FP
Prepare to contain 10 nM AP1491 in buffer and add 100 μl to each well with mixing. Multiple control wells contained either ethanol for rapalog (for 100% probe binding) or ethanol for rapalog and FKBP (0%
(For probe binding).

The plate was covered and stored under dark conditions for about 30 minutes to equilibrate, and the fluorescence polarity of each well was determined using a Jolley FPM-2 FP plate reader (Jolley Conn).
salting and Research, Inc. , Grayslake,
IL) according to the manufacturer's recommendations. The average polarity (mP value) for each competitor is usually converted and plotted as a percentage of total binding referenced to a control value (y) and the log of the competitor
The final molarity corresponds to this (x). Nonlinear least squares analysis corresponds to a curve,
The following equation was used to derive the IC50: y = M1 + (M4-M1) / (1 + exp (M2 * (M3-x))) where M3 is the IC50. Because of the incomplete curve, the IC50 is determined by interpolation. Rapamycin and C14-desoxo-rapamycin are included in each case as controls (C14-deoxo-rapamycin is described in Luengo, J.I.
. et al. 1994 Tetrahedron Lett. 35,
6469-6472). Example 12: Rapalog-dependent transcriptional activation in transiently transformed cells Rapalog was analyzed for its dimerization ability of FKBP and FRB fusion proteins using the following transcriptional readout. A construct encoding a rapalog-dependent transcription factor fusion protein is introduced into a cell that contains, or is designed to contain, a reporter gene linked to expression of a transcriptionally regulated DNA-licensed reporter gene with rapalog-dependent dimerization of the transcription factor fusion protein. did. The transcription factor fusion protein comprises (a)
It includes a FKBP fusion protein containing a DNA binding region (DBD) as a hetero effector region, and (b) an FRB fusion protein containing a transcription activation region as a hetero effector region. The FKBP and / or FRB region can include naturally occurring or non-naturally occurring peptide sequences. The presence of a rapalog capable of mediating dimerization of the two fusion proteins leads to expression of the reporter gene. The degree of reporter observed indicates the activity of rapalog as a dimerization factor. The use of a target gene of interest in place of a reporter gene results in the use of a regulated gene expression system in all of the cells or individuals growing it in culture.

We used this system as follows: HT10 from human fibrosarcoma
80 cells (ATCC CCL-121) were grown in MEM medium supplemented with non-essential amino acids and 10% fetal calf serum. Cells (Falcon, 6 × 10 4 cells / well) seeded on a 24-well dish were prepared by the manufacturer (GIBCO / BRL).
) Were transformed using Lipofectamine under the conditions recommended by). The following total 300 ng DNA was transformed into cells in each well: (a) 100 ng ZEHDx12-CMV-SEAP reporter gene (1
(B) a reporter gene linked to two ZFHD1 DNA binding region recognition sequences), (b) 2.5 ng of pCGNN-ZFHD1-3FKBP or other DN
A-binding region fusion (fusion protein consisting of three FKBPs and one ZFHD1 region), (c) 5 ng of pCGNN-1FRB-p65 (361-550) (fusion protein consisting of FRB region and p65 transcription activation region) and (D) 192.5 ng of pUC118. In some experiments, pCGNN-1FKB (T2098L) -p65 (3
61-550) was used to produce a FRB fusion protein containing the designed FRB region instead of pCGMM-1FKRB-p65 (361-550). In control experiments lacking a DNA binding or activation region, the same amount of empty pCGNN expression vector was substituted. For detailed information on assembly and use of constructs, including those mentioned herein, see WO 96/41865.
(Clackson et al), especially the examples in that specification, which are specifically incorporated by reference herein. Following lipofection (5 hours), 500 μl of medium containing the indicated amount of rapalog was added to each well. Twenty-four hours later, the medium was removed and as described (Spencer et a).
1, Science 262: 1019-24, 1993) SEAP activity was measured. SEAP target genes and expressed ZFHD-FKBP- and FRB-
Human fibrosarcoma cells transiently transformed with a plasmid encoding a p65-containing fusion protein have been shown to mediate rapalog-dependent and dose-responsive SEAP secretion into the medium. SEAP production was not detected in cells lacking one or both transcription factor fusion plasmids, nor was it detected without rapalog addition. As shown in FIG. 1, cells transformed with the wild-type FKBP and FRB border constructs exhibited SEAP expression when the concentration of the dimerization factor was as low as about 1 nM. FIG. 2 shows preferential stimulation of SEAP expression in cells expressing mutant FRB (T2098L, FIGS. 2B and 2D; T2098F, FIG. 2E) compared to wild type (FIGS. 2A, 2C). Similar results indicate that the same transcription factor is linked to the hGH target gene or
This was observed when a stably integrated form of the SEAP reporter gene created by retroviral infection was used to induce rapalog-dependent activation. Example 13: Mutation for generating complementary modified ligand binding domain to various rapalogs (rapalog) induction and phage display A. Engineering FKBP and FRB Domains We designed and prepared recombinant DNA constructs encoding fusion proteins summarized below with exemplary modified ligand binding domains. Unless otherwise stated, mutants were prepared by standard methods (Kunkel, TA, Beben).
ek, K .; And McClary, J.M. 1991. Meth Enzy
mol. 204, 235-139) and was confirmed by dideoxy sequencing using oligonucleotide-mediated site-directed mutagenesis.

[0368]

[Table 18]

We have also prepared constructs encoding the following FRB fusion proteins:

[0370]

[Table 19]

[0370] Yeast and Candida FRBs that are similarly modified as the modified hFRAP FRB domains discussed herein also have two conserved Phe residues and a conserved Asp in each of their FRB domains. And one of Asn residues
Alternatively, one or more may be prepared by substituting different amino acid codons. By way of example, modified FRB domains from TOR1 and TOR2 include:

[0372]

[Table 20]

B. Using a rational design FKBP mutant test standard for binding to rapalog, a hexahistidine tag, and monoclonal antibody 12CA5
PET20b (Novag), which is preceded by a part of the H. influenza hemagglutinin protein, which is an epitope of
en). The sequence of the protein encoded by this vector is as follows:

[0374]

Embedded image

To generate an expression vector for FKBP mutated at the rapamycin contact residue, as described (Kunkel, TA, Bebenek, K
. And McClary, J.M. 1991. Meth Enzymol.
204, 235-139) and oligonucleotide-mediated site-directed mutagenesis was performed on the single-stranded form of the vector prepared from E. coli CJ236. Mutants were confirmed by dideoxy sequencing. As described (Wiederere
cht, G .; Et al., 1992. J. Biol. Chem. 267,
21753-21760), and the mutant protein was transformed into E. coli BL21 (DE3).
(Novagen) and as described (Cardenas,
M. E. FIG. Et al., 1994. EMBO J.C. 13, 5944-5957) It was purified to homogeneity.

Using this protocol, the following mutant human FKBP12 protein was generated using the indicated oligonucleotide primers (mutant bases are capitalized; 5 ′ → 3 ′): Bind to C24 rapalog Mutants designed for

[0377]

Embedded image

Mutants designed to bind to the C13 / C14 rapalog:

[0379]

Embedded image

Mutants designed to bind to C28 / C30 rapalogs:

[0381]

Embedded image

To assay the relative binding affinity of rapamycin and rapalog for FKBP mutants, FK506 (and thus probe) with the mutants
A competitive fluorescence polarization (FP) assay that relies on retention of binding affinity is used. The method comprises:
The same as described in Example 12, except that a direct binding assay is first performed and the dilution (concentration) of mutant FKBP used in the competition reaction is determined to obtain subsaturation. F at a volume of 100 μl in a Dynatech microfluorescence plate
Make serial dilutions of mutant FKBP in P buffer (Example 12) and then 10 nM AP1491 (probe) in [FP buffer + 2% ethanol]
Add 100 μl to each well. Equilibration and plate reading were performed as in Example 1.
2 A plot of mP units versus FKBP mutant concentration is plotted using the following equation:

[0383]

Embedded image

And the final mutant concentration / at which 90% of the probes specifically bind
Dilution is determined by interpolation. This final concentration is then replaced by the 11.25 nM FKBP in the protocol of Example 12 with the 2X mutant final concentration and used in the competitive FP assay performed as in Example 12. For higher sensitivity of the competition assay, 75% may be selected instead of 90% saturation. Serial dilutions of rapamycin analog were used as competitors, and the results are expressed as IC50s for each rapalog binding to each mutant.

C. Rational design for binding to the FKBP-rapalog complex PCR of FRB mutants using test primers 28 and 29 (below) resulted in NcoI-BamHI encoding residues 2021-2113 (inclusive) of human FRAP. PET20b (+), generating a fragment and mutating the NdeI site to NcoI
(Novagen) and cloned into pET-FRAP (2021-2).
113). As described above, in the site-directed mutagenesis method, a single-stranded DNA of the present vector was used as a template to generate a vector encoding FRAP mutated at a rapamycin contact residue. Mutants were confirmed by dideoxy sequencing. The mutant was then amplified by PCR using primers (30 and 31) to add Xbal and Spel sites and cloned into Xbal-Spel digested pCGNN-FRB-p65 (361-550) (Example 7). , E-N-mutant FRAP (2021-2113)-
A series of constructs have been generated that direct mammalian expression of chimeric proteins of the type p65 (361-550). Where E indicates the HA epitope tag and N indicates the nuclear localization sequence. Constructs were verified by restriction digestion and dideoxy sequencing.

Using this method, the following constructs encoding the candidate mutant FRAP for binding to the C7 rapalog, each fused to the p65 (361-550) activation domain, were constructed using the oligonucleotide primers shown ( Mutated bases are shown in capital letters; generated using 5 '→ 3'):

[0387]

Embedded image

To assay the relative binding affinity of these mutants for the complex of FKBP with rapamycin and various rapalogs, each construct was transiently transferred to human HT1080B14 cells as described in Example 8. Co-transfect.
After transfection, serial dilutions of rapamycin or rapalog are added to the medium. After 24 hours, SEAP activity is measured as described in Example 8; the intensity of SEAP activity at various rapalog concentrations is proportional to the affinity of the FRAP mutant for the complex between FKBP and rapalog .

PCR primers (restriction sites are shown in upper case; 5 ′ → 3 ′):

[0390]

Embedded image

D. Functional data of FKBP and FRB domains on filamentous bacteriophage Isupure: as an alternative to rational design of modified domains, phage for display and selection of one approach mutant FKBP and FRB domains for selection The display system includes vector construction, preparation of His6-flag-FKBP, pCANTAB-AP-FKBP, binding enrichment, primers, pCANTAB-AP-FRAP (2015-2114) and pC
Sequence of ATAB-AP-FKBP, biotinylated FK50 for affinity enrichment studies
6, functional FKBP by competitive ELISA using biotinylated FK506
Display, including generation of a mutant FKBP library on phage, targeting library positions C13 and C14 of rapamycin, and library sorting.
O 96/41865 (Clackson et al.). Example 14: Rapamycin-dependent signaling activation Many cell receptors can be activated by aggregation with their physiological ligands or anti-receptor antibodies. In addition, aggregation of two different proteins can often trigger intracellular signals. Using rapamycin and its analogs, oligomerization of a chimeric protein, one containing one or more FKBP and effector domains, and the other containing one or more FRAP and effector domains, It is possible to trigger activation of the receptor effector domain. This plan is illustrated in FIG. T1 (a). Both proteins are anchored and shown on the membrane,
One is membrane immobilized, and the second protein may be attracted to the membrane via dimerization by the addition of rapamycin or an analog. Membrane immobilization may be achieved through transmembrane protein anchors or through lipid modification of the protein, such as myristoylation. The same effector domain may be present on both proteins, or different functionally interacting protein domains may be used, such as protein kinases and protein kinase substrates. Or,
The second effector may serve to inhibit the activity of the first effector.

We note that, in some embodiments, the chimeric protein is a mixed chimera and contains a heterologous effector domain along with the FKBP and FRAP domains, as discussed above. Oligomerization of a single mixed chimera may also be used to activate signaling, as shown in Figure T1 (b). The repetition of the FKBP and FRAP domains generates a greater number and is subject to geometric constraints.

[0393] Two examples of the use of rapamycin in signaling are inducing receptor tyrosine kinase activation and inducing apoptosis via Fas activation, both of which are discussed below. Unless otherwise stated, all DNA manipulations were performed using standard methods (FM Ausubel et al., Current Pr.
otocols in Molecular Biology, John W
iley & Sons, New York, 1994), and all protein protocols are standard (Harlow, E. and Lane).
, D. 1988, Antibodies, a Laboratory
Manual. Cold Spring Harbor Laboratory
y, Cold Spring Harbor). All PCR products used to generate the construct were confirmed by sequencing.

A. Rapamycin-inducible receptor tyrosine kinase activation 1. Expression vector containing myristylation signal, Xba obtained by annealing construction oligonucleotides 1 and 2 of pCM
The I-Myr-BamHI cassette was digested with XbaI / BamHI and pC
G expression vectors (Tanaka, M. and Herr, W. 1990.
Cell 60: 375-386) at the XbaI / BamHI site to generate pCGM. (See (7) below for oligonucleotide sequences). The oligonucleotide cassette contains a sequence encoding the first 15 amino acid residues of the c-Src tyrosine kinase, which has been shown to allow for myristoylation and target the protein to the cell membrane, following an in-frame XbaI site ( Cross et al., 1984. MCB.
). The myristoylated domain is followed by an in-frame SpeI site and a stop codon. The XbaI site in the pCG vector is positioned so that it adds two amino acids between the starting Met and the sequence to be cloned. Start M
The spacing between et and myristylated Gly is important for c-Src membrane localization (Pellman et al., 1985. PNAS. 82: 1623-1627).
Therefore, the XbaI site following the ATG in pCGM was deleted by site-directed mutagenesis (Muta-Gene, BioRad) according to the manufacturer's protocol. The SpeI site in the myristylation cassette was mutated to XbaI to facilitate an additional cloning step. Single-chain uracil-D of pCGM
NA is prepared and both oligonucleotide 3 (deleting the XbaI site following the ATG and adding EcoRI 5 ′ of the ATG) and oligonucleotide 4 (changing the SpeI site following the myristylation domain to an XbaI site) Was used for mutagenesis. The sequence around the ATG of the resulting pCM vector was confirmed by sequencing using oligonucleotide 5 (see sequence 1 in (8) below).

[0395] 2. FKBP and an epitope tag were added to pCM, and pCMF1 / 2/3. The SpeI-HA-BamHI cassette was prepared by annealing complementary oligonucleotides that produce HA (oligonucleotides 6 and 7). This cassette has an in-frame SpeI site followed by the 9 amino acids of the Haemophilus influenzae hemagglutinin gene recognized by monoclonal antibody 12CA5, a stop codon and a BamHI site. Insert the SpeI-HA-BamHI cassette into pCGGNNF
1, subcloned into pCGGNNF2 and pCGGNNF3 at the Spel / BamHI site. Subsequently, 1/2/3 copy of FKB fused to the HA epitope
P was subcloned into pCM as an XbaI / BamHI fragment. The resulting plasmid (pCMF1 / 2 / 3.HA) has the following characteristics: myristylation domain; in-frame XbaI site; 1/2/3 copy of FKBP; in-frame SpeI site; HA epitope tag; and stop codon.

[0396] 3. FRB and epitope tag were added to pCM, and pCMFR1 / 2/3. By annealing the complementary oligonucleotides that generate the Flag (oligonucleotides 8 and 9), it is possible to prepare a SpeI-Flag-BamHI cassette. This cassette contains an anti-FLAG. M2 (Kodak Scientific Imaging)
Systems), eight amino acids (DYKDDDDY) (Hop
p et al., 1988. Biotech. 6: 1205-1210), except that it has the same characteristics as the SpeI-HA-BamHI cassette described above, except that the sequence coding for SpeI-HA-BamHI is followed. The SpeI-Flag-BamHI cassette was inserted into pCGNN-1FRB, p
Subclones into the SpeI / BamHI sites of CGNN-2FRB and pCGNN-3FRB. Subsequently, a 1/2/3 copy of the FRB domain-Flag epitope fusion is subcloned into pCM as an XbaI / BamHI fragment. The resulting plasmid (pCMFR1 / 2 / 3.Flag) has the following characteristics: myristylation domain; in-frame XbaI site; 1/2/3 copy of FR
B; In-frame SpeI site; Flag epitope tag; and stop codon.

[0397] 4. Fusion of FKBP and FRB constructs to the receptor tyrosine kinase cytoplasmic domain Selected receptor tyrosine kinases (eg, EGFR, erbB-2, PDGF
The cytoplasmic domain of R, KDR / Flk-1, Flt-1) is PCR amplified to have in-frame 5 'XbaI and 3' SpeI sites. The PCR product may be subcloned into an in-frame XbaI site to restore an XbaI site in a pCMFR series or pCMF series vector (see above), or in-frame to restore a SpeI site. It may be subcloned at the SpeI site. As a result, the FKBP / FRB domain may be located at the C-terminus or NH2-terminus of the cytoplasmic domain of the receptor tyrosine kinase.
The vector may (i) fuse the cytoplasmic domain of a given receptor to both FKBP and FRB (eg, EGFR fused to either FKBP or FRB).
(Ii) the cytoplasmic domains of two different receptors are fused to FKBP and FRB (eg, the EGFR cytoplasmic domain fused to FKBP and the erbB-2 fused to FRB). Cytoplasmic domain). In the former (i), the addition of the drug, rapamycin, will induce the formation of homodimers (eg, EGFR / EGFR), while in the latter (ii), the addition of the drug, heterodimer ( For example, EGFR / erbB-2
) And will result in activation of the signaling cascade.

[0398] 5. Testing Constructs To test whether rapamycin or an analog induces FKBP and FRB receptor cytoplasmic domain fusion dimerization, select a construct (eg, pCMEGE).
R-FR1 and pCMEGFR-F1) were lipofected (Gibco B
(RL) to co-transfect Cos-1 cells. Three days after transfection, cells are induced with rapamycin and lysis buffer (1% Trit
on X-100; 50 mM Tris. Cl pH 8.0; 150
mM NaCl; 5 mM NaF; 1 mM sodium orthovanadate; 10 μg / ml aprotinin; 10 μg / ml leupeptin). Anti-Flag fusion protein from rapamycin-treated and untreated cell lysates
And 12CA5 antibody and immunoblot with anti-phosphotyrosine antibody. The choice of cell type; the amount of DNA to be transfected; the concentration of rapamycin used and the time of drug treatment are varied to achieve optimal results.

[0399] 6. Rapamycin-induced cell proliferation Select mammalian cell lines (e.g., NIH3T3) into constructs encoding FRB and FKBP fusion proteins (e.g., pCMEGFR-FR1 and pCME).
GFR-F1) is co-transfected and a stable cell line expressing the fusion protein is established. To determine if rapamycin-induced receptor cytoplasmic domain activation will induce cell proliferation, stable cell lines expressing the fusion protein are grown in the presence or absence of rapamycin, and the cell growth rate Changes are determined by routine methods (eg, by monitoring cell number; by measuring the rate of 3H thymidine incorporation, etc.). Selection of receptor tyrosine kinase; the type of receptor activation (homo-dimer versus hetero-dimer) can be selected for optimal results.

[0400] 7. Oligonucleotide sequence

[0401]

Embedded image

[0402] 8. Sequence 1:

[0403]

Embedded image

The modified sequence is shown in lowercase bold and the starting ATG is underlined. The uppercase sequence is from the parent pCG backbone.

B. The ability to modulate rapamycin-induced apoptotic Fas activation and induce apoptosis via small molecules has applications in both gene therapy, which may be used to selectively remove engineered cells, and in experimental systems. The proteins described herein are anchored to the membrane via a low affinity NGF receptor, also called p75. However, it should be recognized that other protein anchors can easily be substituted. p75 is experimentally useful because antibodies against its extracellular domain are available and lack high-affinity interactions with any identified ligand (Botwell, M. 1).
995. Annu. Rev .. Neurosci. 18: 223-253
).

1.2—Protein Rapamycin Controlled Fas Activation (a) Construction of p75 Vector Expression of the FRAP-Fas fusion protein containing the extracellular and transmembrane domains of the low affinity NGF receptor (also known as p75) The indicated vector was modified using standard methods to replace the pUC scaffold with the pUC scaffold, mammalian pJ7W (Morgenstern, JP and Land,
H. 1990. Nucleic Acids Res. 18: 1068)
Obtained from. We call this vector pA7W. The insert cloned into the polylinker site of this plasmid is transcribed under the control of the monkey CMV promoter and enhancer sequences. The polylinker follows the CMV sequence, and HindIII-S
alI-XbaI-BamHI-SmaI-SstI-EcoRI-ClaI-
It has KpnI-BglII. Any mammalian expression vector containing a suitable cloning site and promoter may be substituted.

The sequence of p75 (Johnson, D., Lanahan, A., Bu
ck, C.I. R. , Shegal, A .; Morgan, C .; , Me
rcer, E.C. , Bothwell, M .; , Chao, M .; 198
6. Based on Cell 47: 545-554), a restriction fragment encoding a fragment of p75 flanked by HindIII and XbaI sites was generated by PCR using primers J1 (5 ′) and J2 (3 ′). The original source of the PCR template was a clone from a human brain library using primers similar to J1 and J2 but with different restriction sites. The 5 'end of the resulting fragment contains a HindIII site followed by an EcoRI site, a Kozak sequence and the start of the p75 coding sequence (amino acid 1). The 3 'end generated is the receptor sequence up to amino acids including amino acid 274, 2 amino acids past the predicted transmembrane sequence, followed by X
Encodes a baI site. Similar portions of other transmembrane receptors may be substituted for the fragment. The PCR product was converted to a HindIII-XbaI fragment,
It was subcloned into XbaI cut pA7W to generate pA7Wp75. The construct was verified by restriction analysis and DNA sequencing.

(B) Addition of Fas to pA7Wp75 Fas amino acids 206-304 (FasS) and Fas amino acids 206-3
An XbaI-SpeI fragment encoding 19 (FasL) was generated by PCR,
Then, it was subcloned into pA7Wp75 cut with the same enzyme. Primers used were J3 (5 ') and J4 or J5 (3'). J5 generates a Fas fragment that ends before the stop codon; when cut with SpeI, the terminal 1 of Fas
The nucleotides encoding 5aa are removed, resulting in a truncated form of intracellular Fas called FasS. Removal of these 15aas may result in F in some cell types.
as activity (Itoh, N., and Nagata, S. 19).
93. J. Biol. Chem. 268: 10932.) Primer J
4 replaces the natural stop codon of Fas with a SpeI site and also mutates the original SpeI site contained in Fas, resulting in FasL. Plasmids generated by subcloning these fragments were pA7W
p75-FasS and pA7Wp75-FasL. These constructs
Matched by restriction analysis and DNA sequencing. To attach epitope tags to these inserts, the XbaI-SpeI Fas fragment was isolated and SpeI
Plasmid pC as described above encoding a BamHI site following the 9 amino acid epitope tag from the Haemophilus influenzae hemagglutinin protein (E) 3 'of the I site.
MG1 / 2/3. It was ligated to the XbaI-SpeI cut backbone of HA. Cleavage of the resulting plasmid with XbaI and BamHI produced fragments encoding Fas followed by an epitope tag (these constructs are referred to as E).

(C) p75-FRAP-Fas-epitope fusion protein: p75-FR
PA7Wp75-FasSE and pA7Wp75-FasL to produce APx-FasS or LE and p75-FasS or L-FRAPxE
Addition of FRAP-Containing Fragment to E XbaI-SpeI fragments containing portions of FRAP have been described earlier in this document. These XbaI-SpeI fragments can be used to generate p75-FRAPx-FasS or LE at the XbaI site immediately after the p75 coding sequence, or p75-FaS.
It was inserted into either the SpeI site immediately after the Fas fragment to generate sS or L-FRAPxE. Alternatively, as a FRAPn fragment or as a first FRAP
Becomes available after subcloning into XbaI or SpeI.
By successively subcloning the XbaI-SpeI fragment into the site,
One or more FRAP fragments may be subcloned. Thus, a series of final vectors comprises one or more FRAPs fused N- or C-terminal to the p75 extracellular and transmembrane sequences, one or more Fas intracellular domains (from N to C-terminus). Encodes a domain of origin, and an epitope tag.

(D) p75-FKBP-Fas fusion protein: p75-FKBPn-Fa
for producing sS or L or p75-FasS or L-FKBPn,
Addition of FKBP-Containing Fragments to pA7Wp75-FasSE and pA7Wp75-FasLE XbaI-SpeI fragments containing one or more FKBPs are described elsewhere in this document. These fragments were inserted either into the XbaI site immediately after the p75 coding sequence to generate p75-FKBPn-FasS or L, or into the SpeI site immediately after the Fas fragment to generate p75-FasS or L-FKBPn. Thus, a series of final vectors comprise one or more FKBPs fused N- or C-terminal to the p75 extracellular and transmembrane sequences, one or more Fas intracellular domains (from N to C-terminus). , And an epitope tag.

(E) Rapamycin-Mediated Fas Activation Assay Proteins Containing Fas and FRAP Domains and Fas and FKBP
The ability of the expression of the protein containing the domain to activate Fas and induce cell death upon addition of rapamycin may be tested in either transient or stable transfected cells.

For transient transfection, the two plasmids to be tested are co-transfected into a cell line such as HT1080 by standard methods such as lipofection, calcium phosphate precipitation or electroporation. One or more days after transfection, the cells are treated with no or one or more concentrations of rapamycin, or one or more concentrations of a dimerizing agent such as FK1012. FK1012 serves as a positive control that the FKBP-Fas construct functions. After a few hours to a day, by one of several methods,
Monitor cells for response. A cell lysate is prepared by conventional means and
Probe with antibodies directed against A or against the extracellular domain of p75 (p
used to generate a western blot. Alternatively, by collecting in an isotonic solution supplemented with 10 mM EDTA, staining with an anti-p75 monoclonal antibody and a labeled secondary antibody, and measuring positive cells by FACS,
Cells may be assayed. Western blot signal or FA for processing
Decreased CS signal indicates induction of cell death (or decreased protein expression)
Indicates success. In addition, apoptosis may be monitored using commercially available kits.

[0413] Stable transfected cells are co-transfected with a vector encoding a selectable marker such as neomycin resistance, along with the plasmids described. Two or three days after transfection, cells are plated in G418 and resistant populations or clones are isolated by standard means. These populations may then be treated with a dimerizing agent and then monitored directly for apoptosis induction by cell counting or other measures of cell viability.

Another means of generating a stable cell line that expresses the construct of interest is to subclone the insert into a retroviral vector. The insert can be excised with EcoRI to facilitate this subcloning. Then, using the vector, transduction (transduction) with a packaging cell is performed using a conventional method.
) Make supernatant.

[0415] 2. Single protein rapamycin-regulated Fas activation (a) FK for rapamycin-dependent homodimerization of Fas intracellular domain
Construction of BP-FRAP chimeric fragment FKBP-FRAP fusion construct i. Structure-assisted design To design molecules containing both FRAP and FKBP domains that are capable of rapamycin dependent homodimerization, a triple complex between human FKBP12, rapamycin, and a portion of human FRAP containing a minimal FRB domain May be considered. The requirements for homodimerization of two molecules of the fusion protein containing FRAP, FKBP and Fas moieties include (i) FRA between membrane-tethered molecules.
The length and flexibility of the polypeptide sufficient to provide the necessary distortion for the P-FKBP interaction to occur, while retaining the ability of aggregated Fas to transmit information; and (ii) the chelating effect For example, the prevention or minimization of intramolecular dimerization by rapamycin, an event that is expected to be very entropically favorable and thus expected to prevent desirable intermolecular dimerization.

[0416] Structure considerations lead to the following design preferences for fusion constructs:
(I) FRB and FKBP should be joined by a polypeptide linker that is short enough such that intramolecular dimerization is sterically hindered. The currently preferred arrangement is F
FR such that the C-terminus of RAP and the N-terminus of FKBP are separated and still prevent intramolecular dimerization, but allow a long linker (> 10 amino acids) that provides flexibility.
AP-FKBP. (Ii) the present FRAP-FKBP "cassette" can be membrane proximal (i.e., adding a Fas domain to the C-terminus), or membrane distal (Fas domain is membrane proximal, and the FRAP-FKBP cassette can be C-terminal). Accompanying). (Iii) to allow for structural distortion associated with dimerization in the membrane, or FRAP
If a -FKBP domain is added at the C-terminus, there should be a long linker at the N-terminus of the FRAP-FKBP domain. Again, the location of FRAP at the N-terminus is preferred because it allows the long linker to include the native FRAP sequence from the N-terminal region of the FRB domain, minimizing the immunogenicity of the chimeric protein. (Iv) The optimal linker length and fusion position for a given protein should be confirmed experimentally.

A series of 12 fusions of FKBP and FRAP, designated T1-T12, were designed. Nine are N-FRAP-FKBP-C fusions, comprising 13, 23 or 33 amino acids N-terminal to Arg2018 (N-terminal linker) and between 4, 7 or 10 residues separating the two proteins . The remaining three are N-FKBP-FRA
P-C fusion, FKBP Glu107 and FRAP Arg2018
Intervened by 3, 0 or -4 residues of the FRAP sequence. (Ii) Construction Twelve fusions were prepared using a three primer PCR splicing method (Yon, J. and Fried, M. 1989. Nucleic Acids Res.
17, 4895) as an XbaI-BamHI cassette which can be cloned directly as a single fragment. By cloning in this manner, it was avoided to introduce restriction sites between the genes that would encode foreign sequences and alter the length of the linker. A mixture of 1 ng each of pCGNN-1FRAPi and pCANTAB-AP-FKBP was combined with a single “splice” oligo (B) 0.01 μM complementary to both genes, indicating the desired fusion.
Were amplified using 1 μM of each of the two external primers (A and C) and Pfu polymerase. The primers used are summarized below:

[0418]

[Table 21]

The PCR product was purified, digested with XbaI and BamHI, and XbaI-
Ligation was performed on BamHI digested pCM. The construct was verified by restriction analysis and DNA sequencing.

[0420] Primer sequences and a representative FRB-FKBP construct: FRAP (200
The sequence of the fusion T6 of 5 →) -FKBP is as described in WO 96/41865 (p. 109).
Is disclosed.

(B) F to pA7Wp75-FasSE and pA7Wp75-FasLE
Addition of RAP-FKBP chimeric insert pA linearized with XbaI from T1 to T12 as XbaI-SpeI fragment
Subcloning into 7Wp75-FasSE and pA7Wp75-FasLE to generate p75TFasS or LE. PA7Wp linearized with SpeI
Subcloning into 75-FasLE generates p75FasS or LT-E. These constructs are described in the table ((d) et seq.).

(C) Alternative FRAP-Fas-FKBP Constructs Instead of the format of the chimeric fragment T1-T12, the single-chain strategy may require differently arranged domains for optimal activity. To this end, another series of constructs was made in which FKBP and FRB were separated by a Fas fragment. The starting points for these constructs were pCMF1HA, pCMF2HA, and pCMF3H
A. Similar to the strategy described above for the construction of chimeric transcription factors, FKBP and FKBP
The RB fragment (described elsewhere in this document) was constructed using SpeI just upstream of the BamHI site.
The site was cloned into the pCM backbone as an XbaI-BamHI fragment. Xb
aI and SpeI generate compatible ends, which allows the insertion of an additional XbaI-BamHI fragment downstream of the initial insert. further,
Cloning of the XbaI-SpeI fragment adds a fragment to the 5 'end of the construct. The final p75-fixed construct was the XbaI-S shown in Table ((d) and below).
The peI fragment was created by subcloning into pA7Wp75-FasSE. A similar series is created by subcloning into pA7Wp75-FasLE. Insertion into the vector cut with XbaI resulted in the addition of the insert 3 'of the p75 fragment. Insertion into this vector cut with SpeI resulted in the addition of the insert 3 'of the Fas fragment. Insertion into this vector cut with XbaI and SpeI resulted in the addition of the p75 fragment 3 ′ and the removal of the Fas fragment originally in the vector. By using these three subcloning strategies, the following series of constructs was generated. The subscript number defines the number of times the domain has been repeated.

(D) Table Abbreviations: N = p75 NGF receptor aal-274 Fas = Fas aa 206-304 Fas L = Fas aa 206-319 K = FKBP aa 2-108 R = FRAP 2012-2113, but at another boundary E = pCMF1 / 2/3. HA epitope followed by a stop codon as described for HA

[0424]

[Table 22]

[0425]

[Table 23]

(E) For the construction of inserts cloned 3 ′ of the myristoylated signal sequence as XbaI-BamHI or XbaI-SpeI fragments, see WO 96 for fragment end / junction sequences, oligos and other details. / 41865. (F) Stable transfected rapamycin-controlled apoptosis of human HT1080 cells in culture XbaI-BamHI fragments from constructs A30 and A31 (d, Table) are cloned into pCM and constructs that direct the expression of MT5FasSE and MT6FasSE, M30 and M31 was generated. Where M indicates the myristoylated domain (see Examples section A.1 and A.8) and other abbreviations are d, as described in the Table. The EcoRI-BamHI fragment containing these expression cassettes was then transformed into the retroviral vector pSMTN3 (
Example 7). Using this construct and the Psi (-) broad host packaging vector, 293T cells (Pear, WS et al., 1993.
Proc. Natl. Acad. Sci. USA, 90, 8392
-8396) by transient co-transfection to generate a helper-free retrovirus containing this DNA. HT1080 cells were infected with the virus stock and selected with G418.

To assay apoptosis of the stable transfection pool of cells in response to rapamycin, cells were plated at 10,000 cells / well in a 96-well culture plate. After overnight incubation, serial dilutions of rapamycin were added with 50 ng / ml (final) actinomycin D, and incubated at 37 ° C and 5 ° C.
Incubation continued for approximately 20 hours with% CO2. 10% except medium
The medium was replaced with 100 μl of medium containing Alamar Blue dye. Plates are incubated as before and 570 on a microtiter plate reader.
Cell viability was evaluated periodically by spectroscopic measurements of the OD at nm and 600 nm. Typically, after the control (untreated) wells have reduced blanks, the OD0
. Reading was continued until it reached 2-0.4.

The survival of cells stably transfected with (a) M30 and (b) M31 expression constructs was strongly reduced in the presence of rapamycin in a dose dependent manner. The degree of cell death was determined by the myristoylated (FKBP x 2) -Fas construct (PCT / US94 / 08008) treated with the synthetic FKBP homodimerizing agent AP1428.
As disclosed in US Pat. The system preferably employs one or more mutations in the FKBP and / or FRB domains and may be adapted for use in the improved rapalogs of the present invention.

The complete disclosure of each of the patent documents and scientific articles cited herein is hereby incorporated by reference. These documents serve to illustrate the state of the art in various aspects of the invention. Many modifications and variations of the present invention should be apparent to those skilled in the art. Such modifications and variations may lead to design choices in selecting heterologous action domains,
It is intended to be within the scope of the present invention and the appended claims, including improved rapalogs, fusion protein designs, DNA formulations, viral vectors or other DNA delivery means, modes and routes of transgene administration, and the like.

[Brief description of the drawings]

FIG. 1. Secreted alkaline phosphatase (“SEAP”) in HT1080 cells manipulated according to the method described in Example 7.
Shows the ability of 13-F-rapalog (compounds 79 and 108, respectively, synthesized according to the methods described in Examples 6.1 and 6.21) to enhance expression of a DNA sequence encoding .

FIG. 2 shows the results of a transcription assay using rapalogs 42, 53, 69 and 96 as dimerizers, synthesized according to the methods described herein. Intracellular expression of wild type FRB (FIGS. 2A and 2C) and Th
Rapalog was tested in r2098 with Leu (FIGS. 2B and 2C) or with Phe (FIG. 2E) in cells expressing the mutated FRB.

[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission date] December 16, 1999 (Dec. 16, 1999)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

Embedded image Wherein n is 1 or 2 and has one or more substituents, and may be unsaturated at one or more carbon-carbon bonds connecting the 1- to 8-position carbons. .

Embedded image (Where a is

Embedded imageAnd R^C7aAnd R^C7bIs H and the other is -H, halo, -R^Two, -OR¹ , -SR¹, -OC (O) R¹, -OC (O) NHR¹, -NHR¹, -NR¹R^Two,-NHC (O) R¹Or -NH-SO_Two-R¹Where R^TwoIs an aliphatic, Hete
R is aliphatic, aryl, heteroaryl, or alkylaryl;^C30Is halo, -OR^ThreeOr (= O); R^C24Is = O, = NR^Four, = NOR^Four, = NNHR^Four, -NHOR^Four, -NHN HR^Four, -OR^Four, -OC (O) R^Four, -OC (O) NR^Four, Halo or -H; R^C14Is = O, -OR⁶, -NR⁶, -H, -NC (O) R⁶, -OC (O) R⁶Or -OC (O) NR⁶R^Three0 is H, -R⁷, -C (O) R⁷Or -C (O) NHR⁷Or C28 and
A cyclic moiety bridging C30; R^C28Is halo or -OR^ThreeAnd R^C29Is H, OH or OMe; individual substituents are present in any stereochemical direction, unless otherwise indicated
Can be R¹, R^Four, R^Five, R⁶, R⁷, R⁹, R^TenAnd R¹¹Each of
Independently selected from H, aliphatic, heteroaliphatic, aryl and heteroaryl
Selected; R^FiveIs H, halo, -CN, = 0, -OH, -NR⁹R^Ten, OSO_TwoCF_Three, OSO _Two F, OSO_TwoR^Four', OCOR^Four', OCONR^Four'R^Five', Or OCON (OR^Four') R ^Five Where; R^C13And R^C28One or both are halo substituents; R^C24And R^C30 Are both other than = O; R^C7aAnd R^C7bOne is H and the other is phenyl, di- or tri-substituted phenyl or a mono- or di-substituted heterocyclic moiety;
n is 1; and / or part "a" is

Embedded image ).

31. The method of any of claims 28-30, wherein the improved rapalog is administered orally.

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[Submission date] March 6, 2001 (2001.3.6)

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FIG.

FIG. 2

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[Submission date] March 14, 2001 (2001. 3.14)

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FIG.

FIG. 2

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07K 14/00 C07K 19/00 19/00 (C07D 498/18 // (C07D 498/18 273: 00 273 : 00 311: 00 311: 00 221: 00) 221: 00) C12N 15/00 ZNAA (31) Priority claim number 60 / 072,219 (32) Priority date January 22, 1998 (1998. 22) (33) Priority claim country United States (US) (31) Priority claim number 09 / 012,097 (32) Priority date January 22, 1998 (1998.1.22) (33) Priority claim Country United States (US) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM) AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM , HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (72 ) Inventor Gilman, Michael Zee, United States Massachusetts 02168, Newton, Chestnut Street 550 (72) Inventor Holt, Dennis A. Massachusetts, United States 01718, Acton, Tambrain Hawk 516 (72) Invention Keenan, Terrence P. 02141, Massachusetts, United States 02141, Cambridge, Spring Street 62, Apartment 1 El 02167, Massachusetts, United States, Chestnut Hill, Hammond Pond Parkway Number 3,34

Claims (1)

 [Claims] 1. A method for multimerizing a chimeric protein in a cell, comprising the steps of:
Consisting of: (A) providing a cell comprising:     (i) at least one FKBP domain that binds to rapamycin or a homolog thereof; And a first key comprising at least one protein domain heterologous thereto.
A first recombinant nucleic acid encoding a melaprotein, wherein the FKBP domain is
Including the selected peptide sequence:       (1) Natural FKBP       (2) 10 or less amino acid residues are deleted, inserted or substituted Acid-substituted variant of natural FKBP       (3) It can selectively hybridize to DNA encoding FKBP of (i) or (ii). FKBP encoded by the DNA sequence;     (ii) both (a) rapamycin or a homolog thereof and (b) the first chimeric protein;
Form a complex with at least one FRB domain and heterologous thereto. A second recombinant nucleus encoding a second chimeric protein comprising at least one domain
An acid, wherein the FRB domain comprises a peptide sequence selected from:       (1) Natural Fed       (2) 10 or less amino acid residues are deleted, inserted or substituted Variants of natural FRB domain replaced by acid       (3) It can selectively hybridize to DNA encoding (iv) or (v) FRB
FRB domain encoded by the DNA sequence; And (b) combining the cell with itself and at least one of each of the first and second chimeric proteins; Contacting with an improved rapalog forming a complex comprising the two molecules, wherein the complex comprises
The improved rapalog is a substantially pure stereoisomer or a mixture of stereoisomers,
Or less than 0.01 times the amount of rapamycin as a pharmaceutically acceptable derivative thereof
Having an immunosuppressive effect of the formula I: Embedded image(Wherein n has one or more substituents of 1 or 2 and has 1 to 8 carbon atoms (Which may be unsaturated at one or more carbon-carbon bonds).
. 2. A method for multimerizing a chimeric protein in a cell, comprising the steps of:
How to become: (A) providing a cell comprising:     (i) at least one FKBP domain that binds to rapamycin or a homolog thereof; And a first key comprising at least one protein domain heterologous thereto.
A first recombinant nucleic acid encoding a melaprotein, wherein the FKBP domain is
Including the selected peptide sequence:       (1) Natural FKBP       (2) 10 or less amino acid residues are deleted, inserted or substituted Acid-substituted variant of natural FKBP       (3) It can selectively hybridize to the DNA encoding FKBP of (i) or (ii). FKBP encoded by the DNA sequence;     (ii) both (a) rapamycin or a homolog thereof and (b) the first chimeric protein;
Form a complex with at least one FRB domain and heterologous thereto. A second recombinant nucleus encoding a second chimeric protein comprising at least one domain
An acid, wherein the FRB domain comprises a peptide sequence selected from:       (1) Natural Fed       (2) 10 or less amino acid residues are deleted, inserted or substituted Variants of natural FRB domain replaced by acid       (3) It can selectively hybridize to DNA encoding (iv) or (v) FRB
FRB domain encoded by the DNA sequence; And (b) combining the cell with itself and at least one of each of the first and second chimeric proteins; Contacting with an improved rapalog forming a complex comprising the two molecules, wherein the complex comprises
The improved rapalog is a substantially pure stereoisomer or a mixture of stereoisomers,
Or as a pharmaceutically acceptable derivative thereof, Embedded image (Where a is Embedded image And   R^C7aAnd R^C7bIs H and the other is -H, halo, -R^Two, -OR¹ , -SR¹, -OC (O) R¹, -OC (O) NHR¹, -NHR¹, -NR¹R^Two, − NHC (O) R¹Or -NH-SO_Two-R¹Where R^TwoIs an aliphatic, Hete
B is aliphatic, aryl, heteroaryl, or alkylaryl;   R^C30Is halo, -OR^ThreeOr (= O);   R^C24Is = O, = NR^Four, = NOR^Four, = NNHR^Four, -NHOR^Four, -NHN HR^Four, -OR^Four, -OC (O) R^Four, -OC (O) NR^Four, Halo or -H; R^C14Is = O, -OR⁶, -NR⁶, -H, -NC (O) R⁶, -OC (O) R⁶Ma Or -OC (O) NR⁶Is;   R^Three0 is H, -R⁷, -C (O) R⁷Or -C (O) NHR⁷Or C28 and
A cyclic moiety bridging C30;   R^C28Is halo or -OR^ThreeIs;   R^C29Is H, OH or OMe;   Individual substituents exist in any stereochemical direction, unless otherwise indicated
Can be R¹, R^Four, R^Five, R⁶, R⁷, R⁹, R^TenAnd R¹¹Each of
Independently selected from H, aliphatic, heteroaliphatic, aryl and heteroaryl
Selected; R^FiveIs H, halo, -CN, = 0, -OH, -NR⁹R^Ten, OSO_TwoCF_Three, OSO _Two F, OSO_TwoR^Four', OCOR^Four', OCONR^Four'R^Five', Or OCON (OR^Four') R ^Five Is;   Where R^C13And R^C28One or both are halo substituents; R^C24And R^C30 Are both other than = O; R^C7aAnd R^C7bOne is H, the other is phenyl, A di- or tri-substituted phenyl or a mono- or di-substituted heterocyclic moiety;
n is 1; and / or part "a" is Embedded image ). 3. R^C133. The method of claim 2, wherein is halo. 4. R^C134. The method of claim 3, wherein is fluoro. 5. R^C285. The method of claim 2, 3 or 4, wherein is halo. 6. R^C284. The method of claim 3 wherein is fluoro. 7. R^C24And R^C307. Any of claims 2 to 6, wherein both are other than = O
That way. 8. R^C24And R^C30One or both are -OH, -OR¹Or a halo
The method of claim 7. 9. R^C7aAnd R^C7bAt least one is a part other than -OMe
Any of 2-8. 10. R^C7aAnd R^C7bOne is H and the other is phenyl, di or 10. The method of claim 9 which is a tri-substituted phenyl or a mono- or di-substituted heterocyclic moiety.
Law. 11. R^C7aAnd R^C7bOne is H and the other is o, p-dialkylphenyl
10. The method of claim 9 which is phenyl or tri-alkylphenyl. 12. R^C7aAnd R^C7bOne is H and the other is o, p-dimethoxyphenyl
, O-methoxy-p-ethoxyphenyl, o-ethoxy-p-methoxyphenyl,
o, p-diethoxyphenyl, trimethoxyphenyl or triethoxyphenyl 10. The method of claim 9, wherein 13. The improved rapalog has less than 0.01-fold immunity to rapamycin.
The method according to any one of claims 1 to 12, which has a suppressing effect. 14. The chimeric protein encoded by the first recombinant nucleic acid is reduced.
It contains at least one FKBP domain, the peptide sequence of which is identical to the native FKBP peptide sequence.
14. Including 1-4 amino acid substitutions compared to 1-4, 6, 8, or 10-13.
Either way. 15. The chimeric protein encoded by the first recombinant nucleic acid is reduced.
It contains at least one FKBP domain, the peptide sequence of which is identical to the native FKBP peptide sequence.
6. The method of claim 5, comprising one amino acid substitution in comparison. 16. The method according to claim 16, wherein the chimeric protein encoded by the first recombinant nucleic acid is low.
It contains at least one FKBP domain, the peptide sequence of which is identical to the native FKBP peptide sequence.
8. The method of claim 7, comprising one amino acid substitution in comparison. 17. The method according to claim 17, wherein the chimeric protein encoded by the first recombinant nucleic acid is small.
At least one FKBP domain, the peptide sequence of which is a native FKBP peptide sequence
10. The method of claim 9, comprising one amino acid substitution as compared to 18. The method according to claim 18, wherein the chimeric protein encoded by the first recombinant nucleic acid is low.
It contains at least one FKBP domain, the peptide sequence of which is identical to the native FKBP peptide sequence.
15. The method of claim 14, comprising one amino acid substitution in place of phenylalanine-36.
Law. 19. The method according to claim 19, wherein the chimeric protein encoded by the first recombinant nucleic acid is low.
It contains at least one FKBP domain, the peptide sequence of which is identical to the native FKBP peptide sequence.
Claim 15-1 comprising one amino acid substitution in place of phenylalanine-36.
7. The method according to any of 7. 20. The method according to claim 20, wherein the chimeric protein encoded by the second recombinant nucleic acid is low.
It contains at least one FRB domain, the peptide sequence of which differs from the native FRB peptide sequence.
Claims 1-4, 6, 8, 10-13 or including all three or fewer amino acid substitutions.
Any one of 15-18. 21. The method according to claim 21, wherein the chimeric protein encoded by the second recombinant nucleic acid is low.
It contains at least one FRB domain, the peptide sequence of which differs from the native FRB peptide sequence.
6. The method of claim 5, comprising all one amino acid substitutions. 22. The chimeric protein encoded by the second recombinant nucleic acid has a low content.
It contains at least one FRB domain, the peptide sequence of which differs from the native FRB peptide sequence.
9. The method of claim 7, comprising all one amino acid substitutions. 23. The chimeric protein encoded by the second recombinant nucleic acid has a low content.
It contains at least one FRB domain, the peptide sequence of which differs from the native FRB peptide sequence.
10. The method of claim 9, comprising all one amino acid substitutions. 24. The chimeric protein encoded by the second recombinant nucleic acid has a low content.
Contains at least one FRB domain, the peptide sequence of which is in the native FRB peptide sequence
Instead of one or more of Tyr2038, Phe2039, Thr2098, Gln2099, Trp2101 or Asp2102
15. The method of claim 14, comprising one amino acid substitution. 25. At least one of the chimeric proteins has a DNA-binding domain, a transcription An activation domain or other containing at least one signal domain such as:
Dimerization with other proteins triggers growth, proliferation, differentiation or apoptosis
25. The method of any of claims 1-24, comprising an action domain that is a cell signaling domain for
Method. 27. The method wherein cells are grown in a medium and contact with the improved rapalog
The method according to any one of claims 1 to 25, wherein the step is performed by adding the prepared rapalog to a medium.
That way. 28. The cell according to claim 26, wherein said cell is present throughout the body of an organism and has an improved rapalog.
2. The method of claim 1, wherein the contacting is performed by administering the improved rapalog to the organism.
-25. 29. The cell according to claim 2, wherein the cell is of a mammal and the organism is a mammal.
Method 8. 30. The cell of claim 29, wherein the cell is of a primate and the organism is a primate.
Method. 31. The method of claim 30, wherein the primate is a human. 32. Any of claims 29-31 wherein the improved rapalog is administered orally.
That way.