JP2002503667A - Novel dimerizing agents, their production and use - Google Patents

Abstract

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

Description

DETAILED DESCRIPTION OF THE INVENTION

(Related Information) Attachment A containing materials related to the present application is attached. The entire contents of this attachment are cited here.

BACKGROUND OF THE INVENTION Rapamycin binds with high affinity to the PK506-binding protein FKBP to form a Streptomyces hy that forms a rapamycin: FKBP complex.
groscopicus is a macrolide antibiotic. Kd values reported for that interaction are as low as 200 pM. Rapamycin: FK
The BP complex binds with high affinity to the large cellular protein FRAP to form a tripartite [FKBP: rapamycin]: [FRAP] complex. In the complex, rapamycin acts as a dimerizing agent or adapter to convert FKBP into F
Bound to RAP.

[0003]

[Formula 6] Rapamycin A number of naturally occurring FK506 binding proteins (FKBPs) are known.
See, for example, Kay, 1996, Biochem. J. 314: 361-385 (review). FKBP-derived domains have been incorporated into the design of chimeric proteins for use in biological switches in genetically engineered cells. Such a switch can trigger a desired biological event by liganding the protein component-
Relies on mediated multimerization. See, for example, Spencer et al., 1993, Science 262: 1019-1024 and PCT / US94 / 01617. Although the excellent immunosuppressive activity of FK506 limits its utility as a multimerizing agent, particularly in animals, it is possible to make dimers of FK506 (and related compounds) that lack such immunosuppressive activity. Such dimers have been shown to be effective in multimerizing chimeric proteins containing FKBP-derived ligand binding domains.

[0004] Like FK506, rapamycin can multimerize appropriately designed chimeric proteins. Rapamycin or various derivatives or analogs thereof (
Biological switches using "rapalog") as a multimerizing agent have been disclosed (
WO 96/41865). In the case of rapamycin itself, its effective biological activity, including excellent immunosuppressive activity, rather severely limits the biological switch in certain applications, especially in animals or animal cells that are sensitive to rapamycin. Improved rapalogs for such applications, especially those with reduced immunosuppressive activity, would be highly desirable.

[0005] A large number of structural variants of rapamycin have been reported, typically from synthetic efforts to improve the therapeutic index of compounds as animated fermentation products or as immunosuppressants. . For example, many references to analogs, homologs, derivatives and other compounds structurally related to rapamycin include, inter alia, variants of rapamycin having one or more of the following modifications with respect to rapamycin:
Demethylation, removal or substitution at C7, C42 and / or C29 methyl; Removal, derivatization or substitution of C13, C43 and / or C28 hydroxy; Reduction, removal or derivation of ketone at C14, C24 and / or C30 Substitution of a 6-membered pipecolate ring with a 5-membered phenyl ring; and another substitution on the cyclohexyl ring or substitution of a cyclohexyl ring with a substituted cyclopentyl ring. For further historical information see US Patent Nos. 5,525,610, 5,310,9.
03 and in the background section of 5,362,718.

US Pat. No. 5,527,907 shows a patent document. This reference discloses a series of compounds synthesized in an effort to create immunosuppressive rapalogs with reduced side effects. The compounds are disclosed via a number of lists (two to five or more columns of each of the text) describing the general structural formula, each followed by possible substituents at various positions on the rapamycin ring. You. The document contains over 180 synthetic examples. Many structural variants of the invention have been reported to be excellent immunosuppressants.

SUMMARY OF THE INVENTION As described above in several laboratories of new classes of rapamycin leading pharmaceutical companies and academic facilities, decades of rather focused and competitive research have led to a wide variety of rapalogs. Extensive literature has been created describing As reported in that document,
Each part of rapamycin, which is considered to be sensitive to chemical modification, was modified, and the resulting rapalog was evaluated for its utility as an immunosuppressant.

[0008] We have opposed the broad background of making completely unexpected discoveries that led to the present invention. Briefly, while repeating the previously described substitution of the C7 methoxy group of rapamycin with an allyl or methallyl site and trying to restore the desired C7-substituted rapalog, we instead proceed to the prior art. Re-encompassed unintentionally synthesized members of the unknown family and found that these compounds possess useful and in fact important biochemical properties. The reagents and conditions used to introduce substitutions at C3 cannot be used with rapamycin as a starting material, except for a wide variety of rapalogs that can be obtained as separate fermentation products or by synthetic modification of rapamycin. In addition, C3-substituted rapalogs can themselves be subjected to a wide variety of subsequent chemical modifications as reported for rapamycin. Thus, our discovery of the method for C3 substitution provides access to a whole new and diverse family of rapalogs.

[0009] Our first new rapalog of this series is 3-allyl-rapamycin (R
= H) and 3-methyl-rapamycin (R = methyl).

[0010]

Embedded image These compounds are particularly notable for their ability to form a tripartite complex with FKBP12 and a genetically engineered FRB domain or their fusion protein containing the FKBP and FRB domains. This binding property and its significance will be discussed later. As noted above, substitutions at C3 can be combined with one or more other modifications to the rapamycin structure, of which many examples are known in the art. Among other things, these are as described above (a
A) a substitution site at any two or more of positions 13, 14, 20, 24, 28 and 30; (b) a prolyl site instead of the otherwise characteristic pipecolate site; and / or (c) pipecoline Includes C3 rapalogs containing a prolyl on the substituted cyclohexyl ring above or in place of the site. These are disclosed generally below.

[0011] These new rapalogs ("C3 rapalogs") thus have one or more optional substitutions, generally as substantially pure stereoisomers or mixtures of stereoisomers, Formula I unsaturated at one or more carbon-carbon bonds ranging from 1 to 8 carbons:

Embedded image Wherein R ^C3 is other than H, or a pharmaceutically acceptable derivative thereof (this term is defined below). For example, in various embodiments of the present invention, R ^C3 is a substituted or unsubstituted aliphatic, heteroaliphatic, aromatic or heteroaromatic moiety.

C3 rapalogs useful in practicing the present invention can contain any of the possible stereoisomeric orientations, one stereoisomer substantially free of other stereoisomers (> 90%, preferably 95% free from other stereoisomers on a molar basis) or a mixture of stereoisomers.

One preferred class of such compounds has a substantially reduced immunosuppressive effect compared to rapamycin. By "substantially reduced immunosuppressive effect" we mean that rapalog is a chemically or appropriately in vitro or in vitro test of human immunosuppressive activity performed on tissue, preferably of lymphoid origin. Has less than 0.1, preferably less than 0.01, and even more preferably less than 0.005 fold of immunosuppressive activity observed or predicted with equimolar amounts of rapamycin, as measured by either vivo surrogate Or that the rapalog is at least 10-fold, preferably at least 100-fold, more preferably at least 250-fold greater than the EC50 value observed with rapamycin in the same assay, as in an in vitro assay.
Means producing a C50 value.

One suitable in vitro surrogate for immunosuppression in human patients is the inhibition of human T cell proliferation in vitro. This is a common assay approach that can be performed in a number of well-known variants using a variety of human T cells or cell lines, including other human PBLs and jacket cells, among others. Thus, rapalogs can be assayed for human immunosuppressive activity and compared to rapamycin. A decrease in immunosuppressive activity against rapamycin as measured by a suitable in vitro assay indicates a decrease in immunosuppressive activity in humans against rapamycin. Such in vitro assays can be used to assess the relative immunosuppressive activity of rapalogs.

[0015] Various illustrative examples of such rapalogs are disclosed herein. Notably, this class of C3 rapalog binds to human FKBP12, or
Includes those that inhibit its rotamase activity within the order of magnitude of the results obtained with rapamycin in KBP binding or rotamase assays.

Of particular note, one class of C3 rapalogs (exemplified by our first two C3 rapalogs) are either substantially pure stereoisomers or mixtures of stereoisomers. ^Rc3 having one or more optional substituents as:

Embedded image(Where R¹, R⁴, R⁵, R⁶And R⁷Is each H or substituted or unsubstituted
Which is an aliphatic, aromatic, heteroaliphatic, or heteroaromatic moiety) or a pharmaceutically acceptable derivative thereof. Some specific examples
In the above, the C3 substituent is, for example, R¹And R⁴, R⁴And R⁵Or R ⁵ And R⁶May comprise a cyclic moiety covalently linked together to form a ring.

Another class of C3 rapamycins of particular interest is the compound of formula II as a substantially pure stereoisomer or a mixture of stereoisomers:

Embedded image Wherein R ^C3 is other than H;

Embedded image Is a; ^{R C30} is halo, -OR ³ or (= O), ^{R C24} is ^{= O, = NR 4, =} NOR 4, = NNHR 4, -NHOR 4, -N
NHHR ⁴ , —OR ⁴ , —OC (O) R ⁴ , —OC (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 ′; R ^C14 represents —OR ⁶ , —NR ⁶ , —H, NC (O) R ⁶ , —OC (O ) it is ^{R 6} or ^{-OC (O) NR 6; R} 3 is ^H, -R 7, -C (O) ^{R 7} or -C (O) cyclic group bridging NHR ⁷ or C28 and C30 (e.g., R ^C29 is H or OR ¹¹ (eg, OH or OMe); unless otherwise specified, each substituent can be present in any stereochemical orientation;
Wherein each occurrence of R ¹ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁹ , R ¹⁰ and R ¹¹ is independently selected from H, aliphatic, heteroaliphatic, aryl and heteroaryl; And R ⁸ is H, halo, —CN, ＯO, —OH, —NR ⁹ R ¹⁰ , OSO ₂ CF ₃ ,
OSO ₂ F, OSO ₂ R ⁴ ′, OCOR ⁴ ′, OCONR ⁴ ′ R ⁵ ′, or O
CON (OR ′) R ⁵ ′] or a pharmaceutically acceptable derivative thereof.

Another class of C3 rapalogs of particular interest is R^C13And R^{C2 8} Are independently H, halo, -OR³, -OR⁵, -OC (O) R ⁵ , -OC (O) NHR⁵, -SR⁵, -SC (O) R⁵, -SC (O) NHR ⁵ , -NR⁵R⁵’Or -N (R⁵) (CO) R⁵’Where each halo group
Is independently selected from F, Cl, Br and I.

Another class of C3 rapalogs of particular interest is the formula I rapalog where n is 1. This class replaces 24,30-tetrahydro-C3, which replaces rapamycin.
, Their mono- and diethers and their 24-halo, 30-halo, 24,30-nihalo derivatives.

Another class of C3 rapalogs of particular interest are those in which one or both of R ^C24 and R ^C30 are other than ＯO. This class of rapalogs includes rapalogs that contain a pyrrolyl ring system instead of a pipecolate ring system.

Another class of C3 rapalogs of particular interest is the group “a”

Embedded image Is a rapalog of Formula II that is other than This class of analogs has a stereochemical orientation in which the hydroxyl site at position 43 is opposite that shown immediately above,
A class of 43-epi-rapalog which is a mixture of stereoisomers of the 3-hydroxyl group, or any other of the foregoing, including ethers, esters, carbamates, halides and other derivatives of any of the aforementioned position 43 rapalogs. Containing a derivative of This class further includes rapalogs where the cyclohexyl ring is substituted and / or contains 5 ring atoms in place of the characteristic substituted cyclohexyl ring of rapamycin.

The subset of said classes of C3 rapalogs is further described above in relation to Formula II or in relation to any of the other classes of C3 rapalogs described herein,
The structure differs from rapamycin with respect to one or more additional structural features (eg, one or both substituents at C7).

Methods for Producing C3 Rapalog The present invention further comprises subjecting the rapamycin or rapalog starting material to reagents and conditions that allow for the replacement of the C3 hydrogen with the desired group. The product can be recovered from the reaction mixture and purified from unreacted starting materials and by-products. The product can be subjected to one or more further transformations prior to final purification. Methods and materials for C3 displacement and product recovery are described in detail below.

Method for Multimerizing Chimeric Protein The present invention further preferably further comprises avoiding the immunosuppressive effect of rapamycin,
Provided are methods and materials for multimerizing chimeric proteins in cells genetically created using the novel rapalogs disclosed herein.

Genetically engineered cells contain one or more recombinant nucleic acid constructs encoding a specialized chimeric protein as described herein. Typically, the first chimeric protein comprises one or more FKBs capable of binding to a C3 rapalog of the invention.
Contains the P domain. This first chimeric protein is also referred to herein as a "FKBP fusion protein" and further comprises at least one protein heterologous to at least one of its FKBP domains. The complex formed by binding the FKBP fusion protein to the C3 rapalog can bind to a second chimeric protein containing one or more FRB domains ("FRB fusion protein").
. The FRB fusion protein further comprises at least one protein domain that is heterologous to at least one of the FRB domains. In some embodiments, the FK
The BP fusion protein and the FRB fusion protein are different from each other. However, in one embodiment, the FKBP fusion protein is a FRB fusion protein. In those embodiments, the chimeric protein comprises one or more FKBP domains as well as one or more FRB domains. In such a case, the first and second chimeric proteins may be the same protein and may be referred to as a FKBP-FRB fusion protein, containing at least one domain that is heterologous to FKBP and / or FRB. .

[0027] Chimeric proteins can be readily designed based on appropriately selected heterologous domains, such that multimerization triggers one or more of a wide variety of desired biological responses. The nature of the biological response triggered by rapalog-mediated complexation is determined by the choice of heterologous domains in the fusion protein. Thus, a heterologous domain is referred to as an "effect" or "effector" domain. The genetically engineered cells used in practicing the present invention contain one or more recombinant nucleic acid constructs encoding a chimeric protein, and in certain applications, further include one or more target gene constructs, Contains one or more accessory nucleic acid constructs.
Exemplary biological responses, uses of the system, and types of accessory nucleic acid constructs are discussed in detail below.

A system including related materials and methods is described in WO 96/418665 (Clacks).
on et al.) and are expected to be useful in a variety of applications, including research and therapeutic applications, among others. The system has the general formula:

Embedded image (Where U is -H, -OR ¹ , -SR ¹ , -OC (O) R ¹ , -OC (O) NH
R ¹ , —NHR ¹ , —NHC (O) R ¹ , —NHSO ₂ —R ¹ or —R ² ; R ² is a substituted aryl or allyl or an alkylaryl (eg, benzyl or substituted benzyl) by and; V is an -OR ³ or (= ^{O); W} is ^{= O, = NR 4, =} NOR 4, = NNHR 4, -NHOR 4, -NH
^{NHR 4, -OR 4, -OC (} O) R 4, be -OC (O) NR ⁴ or -H; Y is ^{-OR 5, -OC (O) R} 5 or -OC (O) NHR ⁵ Yes; Z =
^{O, -OR 6, -NR 6,} -H, -NHC (O) R 6, be a -OC (O) ^{R 6} or ^{-OC (O) NR 6; R} 5 is ^H, -R ^7, -C ^{(O) R 7, -C (} O) N
HR ⁷ or a C-28 / C-30 cyclic carbonate; and R ⁴ is H or alkyl; wherein R ¹ , R ⁴ , R ⁵ , R ⁶ and R ⁷ are independently H,
Alkyl, alkylaryl or aryl (the terms are described in WO 96/418)
65)), comprising the use of a multimerizing agent comprising rapamycin or rapalog. A number of rapalogs are specifically disclosed in that document.

The present invention is based on a system similar to that disclosed in WO 96/41865 and involves the use of C3 rapalog as a multimerizing agent. Thus, the present invention provides suitably prepared cells containing (a) a nucleic acid construct for detecting expression of a desired chimeric protein and a desired accessory recombinant construct, and (b) providing the cells with a C3 rapalog. Alternatively, there is provided a method for multimerizing a chimeric protein construct, comprising contacting with a pharmaceutically acceptable derivative thereof described herein. The rapalog itself forms a complex comprising at least two molecules of the chimeric protein.

In one embodiment, at least one of the chimeric proteins has at least 10 amino acid residues in the peptide sequence at which the peptide sequence naturally occurs in FK.
It differs from the BP peptide sequence, for example, the peptide sequence of human FKBP12. Preferably, the number of changes in the peptide sequence is limited to 5, more preferably 1, 2 or 3. Preferably, the change is a substitution rather than a simple deletion or insertion. In embodiments where the rapalog differs from rapamycin at one or more positions in addition to the modification at C3, in at least one of the chimeric proteins, loss of C7 methoxy and loss of triene conjugation is due to the loss of naturally occurring FKBP, especially human FKBP12. At least one FKBP containing at least one amino acid substitution relative to the sequence of a mammalian FKBP such as

In another embodiment, at least one of the chimeric proteins has a peptide sequence wherein the peptide sequence has up to 10 amino acid residues in the peptide sequence and a naturally occurring FRB peptide sequence, eg, the FKB domain of human FRAP. Contains at least one different FRB domain. The number of changes in the peptide sequence is preferably limited to 5, but is more preferably 1, 2 or 3. Mutations of particular interest include T2098, D in the FKB domain derived from human FRAP.
2102, Y2038, Y2039, K2095, including substitution with independently selected substituted amino acids, eg, A, N, H, L or S. Also of interest are the FRB domains F1975, F19 derived from yeast TOR1.
76, D2039 and N2035, or F1978, F1979, D2042 and N203 of the FRB domain derived from yeast TOR2.
One or more of eight independently selected substituted amino acids, eg, H, L, S,
A or V substitution.

In certain embodiments, the chimeric protein contains at least one modification, preferably up to three, in the peptide sequence relative to the naturally occurring sequence in both one or more FKBP domains and one or more FRB domains. I do.

As noted above, in various embodiments of the invention, the chimeric protein contains one or more “effect” or “effector” domains that are heterologous with respect to the FKBP and / or FRB domains. Effector domains include DNA binding domains, transcriptional activation domains, cell localization domains and signaling domains (eg, domains capable of cell growth, proliferation, differentiation, apoptosis, gene transcription, etc. upon clustering or multimerization). One can choose from a variety of protein domains. Various exemplary effectors in which the invention can be practiced are disclosed in the various scientific and patent documents cited herein.

For example, in certain embodiments, one fusion protein has at least one D
NA binding domain (eg, GAL4 or ZFHD DNA-binding domain)
And another fusion protein comprises at least one transcription activation domain (
For example, VP16 or p65 transcription activation domain). Ligand-mediated association of the fusion protein indicates the formation of a transcription factor complex and one of the fusion proteins
This leads to the start of transcription of the target gene bound to the DNA sequence recognized by (ie, capable of binding to) the DNA-binding domain immediately above.

In another embodiment, one fusion protein contains at least one domain that can direct the fusion protein to a particular cellular location, such as a cell membrane, nucleus, ER or other organelle or cell component. Localization domains that target the cell membrane include, for example, domains such as myristylation sites or transmembrane regions of receptor proteins or proteins across other membranes. Another fusion protein can contain a signaling domain that can activate a cell signal transduction pathway upon membrane localization and / or clustering. Examples of signaling domains are the intracellular domain of growth factor or cytokine receptors, the apoptosis triggering domain such as the intracellular domain of FAS or TNT-R1, and other intracellular signaling such as SOS, Raf, lck, ZAP-70, etc. Contains domains from proteins. A number of signaling proteins are disclosed in PCT / US94 / 01617 (eg, 23
-26 page). In yet another embodiment, each of the derived proteins contains at least one FRB domain and at least one FKBP domain, and one or more heterologous domains. Such derived proteins can trigger multimerization or signaling in the presence of rapalogs. Generally, domains containing peptide sequences that are endogenous to the host cell are preferred for applications involving whole organisms. Thus, in human gene therapy applications, domains of human origin are of particular interest.

A nucleic acid construct capable of directing its expression and a vector containing such a construct for introducing it into a cell, especially a fusion protein such as a eukaryotic cell, of which yeast and animal cells are of particular interest. There is provided a recombinant nucleic acid construct encoding Considering the components of the fusion protein, the recombinant DN
The A molecule is capable of selectively hybridizing to (a) a DNA molecule encoding a polypeptide comprising a FRB or FKBP domain and (b) a heterologous domain or a protein from which a heterologous protein domain is derived. Also included are DNAs that can be so hybridized without the degeneracy of the genetic code.

Using nucleic acid sequences encoding the fusion protein, nucleic acid constructs for directing its expression in eukaryotic cells, and vectors and other means for introducing such constructs into cells, especially animal cells, Genetically cells, especially animal cells, preferably mammalian cells, most preferably human cells, can be created for a number of important applications. To do so, one first provides an expression vector or nucleic acid construct for directing expression of the desired chimeric protein in eukaryotic (preferably animal) cells, and then transforms the recombinant DNA into at least a portion of the cells. The cells are introduced so as to allow ingestion and expression of the introduced DNA. Various methods and materials for introducing DNA into cells for heterologous gene expression can be used, various of which are well known and / or commercially available.

Thus, one object of the present invention is a method for multimerizing a fusion protein as described herein in a cell, preferably an animal cell. To recap, one of the fusion proteins can bind to the C3 rapalog of the invention,
It contains at least one FKBP domain and at least one domain heterologous thereto. The second fusion protein contains at least one FRB domain and at least one heterologous domain, and is capable of forming a tripartite complex with the C3 rapalog fusion protein and one or more molecules. In some embodiments, one or more of the heterologous domains present on the fusion protein is also present on another fusion protein, ie, the two fusion proteins have one or more common heterologous domains. In another embodiment, each fusion protein has 1
It contains these different heterologous domains.

The method comprises contacting the appropriately prepared cells with a C3 rapalog by adding the rapalog to the culture medium in which the cells are located, or by administering the rapalog to the organism in which the cells are located. The cells are preferably eukaryotic cells, more preferably animal cells, most preferably mammalian cells. Primate cells, especially human cells, are of particular interest. Administration of the C3 rapalog to humans or non-human animals can be performed using pharmaceutically acceptable compositions and administration routes. Oral administration of a pharmaceutically acceptable composition containing C3 rapalog together with one or more pharmaceutically acceptable carriers, buffers or other excipients is currently of greatest interest.

A particular object of the present invention is a method for inducing the transcription of a target gene in a rapalog-dependent manner, as described above. The cells typically contain, in addition to recombinant DNA encoding the two fusion proteins, a target gene operably linked to the DNA sequence in response to the presence of the complex of the fusion protein with rapamycin or rapalog. Containing target gene constructs. The target gene construct may be recombinant, and the target gene and / or the regulatory nucleic acid sequence linked thereto may be heterologous with respect to the host cell. In certain embodiments, the cells bind to the FKBP fusion protein and participate in a complex with the FRB fusion protein with detectable preference over binding to the host cell's endogenous FKBP and / or FRB-containing protein. Responsive to contact with the C3 rapalog.

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

Another particular object of the present invention is a method for inducing cell growth, differentiation or proliferation in a rapalog-dependent manner, as described above. In such cells, at least one of the at least one heterologous domain of the fusion protein is, for example, an intracellular domain of a receptor for a hormone that mediates cell growth, differentiation or proliferation, or a signaling domain such as a downstream mediator of such signaling. is there. Cell growth, molecules and / or proliferation follows such signaling. Such clustering naturally occurs following hormone binding and, in the created cells of the invention, contact with the C3 rapalog.

While cells of human origin are preferred for human gene therapy applications, cell types of various origins (human or other species) can be used, and if desired, within biocompatible materials used in human subjects. Can be encapsulated.

Also provided are substances or methods for producing the cells of the above-mentioned generation. The purpose of this is to provide any desired auxiliary recombinant nucleic acid, such as a target gene construct, and primarily a recombinant nucleic acid, typically a DNA molecule encoding a fusion protein, and then allow uptake of the nucleic acid by cells. The adaptation is carried out by introducing the recombinant nucleic acid into the host cell under the following conditions. Such transfection can be performed ex vivo using host cells maintained in culture. Cells created in culture are subsequently
For example, it can be introduced into a host organism in ex vivo gene therapy applications. Thus, doing so constitutes a method of providing a host organism, preferably a human or non-human mammal, that is responsive to the presence of the C3 rapalog provided herein (as described herein). I do. Alternatively, it can be performed in vivo using host cells present in a human or non-human host organism. In such cases,
The nucleic acid molecule is introduced directly into the host organism under conditions that allow uptake of the nucleic acid by cells of one or more host organisms. Thus, this approach constitutes another method of providing a host organism, preferably a human or non-human mammal, that is responsive to the presence of the C3 rapalog (as described herein). Various materials and methods for introducing DNA and RNA into cells in culture or in whole organisms are known in the art and can be adapted for use in the practice of the present invention.

[0044] Other objects are achieved by using cells generated as described herein.
For example, a method of multimerizing a fusion protein of the invention by contacting a cell produced as described herein with an effective amount of C3 rapalog that allows the rapalog to form a complex with the fusion protein. Is provided. 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 contains one or more signaling domains, this constitutes a method of activating the cell signal transduction pathway. In particular embodiments where the signaling domain is selected based on its ability to trigger cell growth, proliferation, differentiation or cell killing, C3 rapalog-mediated clustering may be applied to cell growth, proliferation, A method for activating differentiation or cell killing is provided.
These methods can be performed in cell culture or whole organisms, including human patients. In the former case, rapamycin or rapalog is added to the culture medium. In the latter case, rapamycin or rapalog (which may be in the form of a pharmaceutical or animal composition) is administered to the whole organism, for example, orally, parenterally or the like. Preferably, the dose of C3 rapalog administered to the animal is below the dose level that would cause excessive immunosuppression at the receptor.

Also provided are kits for use in the genetic production of cells or human or non-human animals as described herein. One such kit contains one or more recombinant nucleic acid constructs encoding a fusion protein of the invention. The recombinant nucleic acid construct is
Generally, 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 may be a viral vector described elsewhere herein. The kit may also contain a sample of the C3 rapalog of the present invention capable of forming a complex with the encoded fusion protein. The kit can further bind to one or more fusion proteins, but cannot form a complex with both.
It can contain a multimerizing antagonist such as K506 or some other compound. In certain embodiments, the recombinant nucleic acid construct encoding the fusion protein contains a cloning site in place of the DNA encoding one or more heterologous domains, and thus the DNA encoding the heterologous domain selected by the practitioner.
Can be introduced. In some embodiments, the kit comprises a target gene or cloning site linked to a DNA sequence that is reactive to the presence of the complexed fusion protein, as described in more detail elsewhere herein. And a target gene construct containing The kit can include a package insert that identifies the included nucleic acid construct, and / or instructions for introducing the construct into a host cell or organism.

Detailed Description of the Invention Definitions The following definitions and orientation information will assist in a thorough understanding of the present specification.

The FRB domain is a polypeptide region (typically a protein “domain”) of at least about 89 amino acid residues capable of forming a tripartite complex with the FKBP protein and rapamycin (or the C3 rapalog of the present invention). "). F
The RB domain is derived from human and other species ("RAPT1" or "RAFT"
It is present in a number of naturally occurring proteins, including FRAP proteins; yeast proteins, including Tor1 and Tor2; and Candida FRAP homologs. Information on nucleotide sequences, cloning, and other aspects of these proteins is already known in the art, for example, using the PCR primers based on well-known methods and published sequences to obtain the desired FRB protein. It allows the construction or cloning of the DNA encoding the sequence.

[0048]

[Table 1] The FRB domains used in the present invention generally will have at least about 89-10
Contains 0 amino acid residues. FIG. 2 of Chiu et al., Supra, contains a human FRAP, murine FRAP, S. cerevisiae TOR1
And S.I. 1 shows a 160 amino acid span of cerevisiae TOR2. Typically, the FRP sequences selected for use in the fusion proteins of the invention will have at least an 89-amino acid sequence G, as the sequences are numbered in the figure.
Will range from lu-39 to Lys / Arg-127. For reference, C
Using the numbering of Hen et al. or Sabitini et al., the 89-amino acid sequence is Glu-2025 to Lys-2113 in the case of human FRAP,
In the case of r2, they are numbered Glu-1965 to Lys-2053, and in the case of Tor1, they are numbered Glu-1962 to Arg-2050. The FRB domains used in the fusion proteins of the present invention may include (for example, F
The rapamycin of the present invention or as determined by either direct or indirect methods for detecting such binding, including the means for detecting such binding used in RAP / RAFT / RAPT and Tor-related literature). It could bind to a complex of FKBP protein bound to C3 rapalog. The peptide sequence of such an FRB domain may be (a) a naturally occurring peptide sequence that spans at least the indicated 89-amino acid region of the aforementioned protein or a corresponding region of a homologous protein; Is 1
-5, more preferably 1-3) a variant of a naturally occurring FRB sequence in which amino acids have been deleted, inserted or substituted with alternative amino acids; or (c) a DNA molecule encoding a naturally occurring FRB domain. Depending on the DNA sequence that can be selectively hybridized, the degeneracy of the genetic code can be eliminated or the naturally occurring FRB
D that can selectively hybridize to DNA molecules encoding the domain
Includes the peptide sequence encoded by the NA sequence. Preferred FRB triple mutants are disclosed in the experimental examples below.

FKBP (FK506 binding protein) is a cytoplasmic receptor for macrolites such as FK506, FK520 and rapamycin and is highly conserved across species systems. For the purposes of this disclosure, FKBP is a protein or protein domain that binds to a rapamycin or C3 rapalog of the invention and is capable of forming a tripartite complex with an FRB-containing protein. An FKBP domain can also be referred to as a "rapamycin binding domain." Information on the nucleotide sequences, cloning, and aspects of the various FKBP species is already known in the art, and includes, for example, PPS based on well-known methods and popular sequences.
The CR primer allows the synthesis or cloning of DNA encoding the desired FKBP peptide sequence. For example, Staendart et al., 1990,
Nature 346, 671-674 (human FKBP12);
6, Biochem, J .; 314, 361-385 (Review). Homologous FKBP proteins in other mammalian species, yeast, and other organisms are also known in the art and can be used in the fusion proteins disclosed herein. See, for example, Kay, 1996, Biochem. J. 314, 361-385 (Review). The size of the FKBP domain used in the present invention will vary depending on which FKBP protein is used. FKBP of fusion protein of the present invention
The domain binds to the rapamycin or C3 rapalog of the present invention and participates in a tripartite complex with the FRB-containing protein (as can be measured by either direct or indirect means to detect such binding). Will be able to do that. The peptide sequence of the FKBP domain of the FKBP fusion protein of the present invention may be (a) a naturally occurring FKBP peptide sequence derived from a human FKBP12 protein (exemplified below) or a murine or other mammalian FKB.
A peptide sequence from another human FKBP from P or from some other animal, yeast or fungal FKBP; (b) up to about 10 (preferably 1-5, more preferably Preferably 1-3, and in some embodiments just 1) a variant of a naturally occurring FKBP sequence in which amino acids have been deleted, inserted or substituted with alternative amino acids; or (c) a naturally occurring FKBP A DNA sequence encoding a naturally occurring FKBP by a DNA sequence capable of selectively hybridizing to the encoding DNA molecule or by removing the degeneracy of the genetic code.
Includes a peptide sequence encoded by a DNA sequence capable of selectively hybridizing to the A molecule.

As used herein, the phrase “selectively hybridizable” refers to hybridization conditions that can be selected by one of ordinary skill in the art or readily determined empirically. Means that the two DNA molecules are susceptible to hybridization with each other despite the presence of other DNA molecules. Such a treatment may comprise 0.2 to 6 × SSC and / or 0.1% to 1% SD at a temperature ranging from room temperature to 65-750 ° C.
Includes high stringency conditions, such as thorough washing with S-containing buffers. For example, F. M. Aududel et al. Ed., Short Protocols i
n Molecular Biology, units 6.3 and 6.4 (J
ohn Wiley and Sons, New York Third Edition, 199.
See 5).

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

As used herein, the term “nucleic acid construct” refers to the free form (ie, other forms) that are generally recombinant, as the term is defined below. (Not covalently linked to a nucleic acid sequence) or present in a larger molecule, such as a chromosome of a DNA vector, retrovirus or other viral vector or a genetically engineered host cell. (Usually DNA, but eg RNA in a retroviral delivery system)
). Of particular interest are nucleic acid constructs that encode the fusion proteins of the invention or that include target genes and expression control elements. The construct may further comprise one of the following elements involved in the regulation of transcription, translation and / or other processing of the coding region or its gene product: transcriptional promoter and / or enhancer sequences, ribosome binding sites, introns, etc. A nucleic acid portion containing the above can be included.

As used herein, the terms “recombinant”, “chimeric” and “chimeric”
"Fusion" refers to materials that include various component domains, sequences or other components that are heterogeneous with respect to one another in the sense that they do not naturally occur together in the same arrangement.
More specifically, the component proteins are at least not in the same cell or order or orientation, or at the same intervals present in the chimeric protein or recombinant DNA molecule of the invention, naturally identical and continuous polypeptides or The component part is not found in the nucleotide sequence or molecule.

“Transcription control elements” refers to regulatory DNA sequences, such as initiation signals, enhancers and promoters, that direct or control the transcription of a protein coding sequence to which they are operably linked. The term "enhancer" is intended to include regulatory elements that can increase, stimulate, or enhance transcription from a promoter. Such transcriptional regulatory components can be located upstream of the coding sequence or, in some cases (eg, enhancers), at other positions, such as introns, exons, coding regions and 3 'flanking sequences.

“Dimerization”, “oligomerization” and “multimerization” are used interchangeably herein and are mediated by the binding of a drug to at least one of two or more proteins. Refers to the association or clustering of protein molecules. In a preferred embodiment, multimerization is mediated by the binding of two or more such protein molecules to a common divalent or multivalent drug. With one or more molecules of a FKBP ligand that is at least bivalent (eg, FK1012 or AP1510), the formation of a complex comprising two or more protein molecules, each of which contains one or more FKBP domains, involves such association or clustering. This is an example. 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, the presence of a bivalent drug will lead to clustering of more than two protein molecules. Embodiments in which a drug is more than divalent (eg, trivalent) in its ability to bind to a protein bearing a drug binding domain can result in clustering of more than two protein molecules. Protein containing at least one FRB domain, at least one FKBP
The formation of a tripartite complex containing a protein containing a domain and a molecule of rapamycin is another example of clustering of such proteins. In certain embodiments of the invention, the fusion protein contains multiple FRB and / or FKBP domains. Such a complex of proteins can contain more than one molecule of rapamycin or a derivative or other dimerizing agent and more than one copy of one or more constituent proteins. Again, such multimeric complexes are still referred to herein as tripartite complexes, even though one or more are represented by multiple copies, to indicate the presence of three types of constituent molecules. The formation of a complex containing at least one bivalent drug and at least two protein molecules, each containing at least one drug binding domain, is "oligomerization" or "multimerization" or simply "bimerization". It can be referred to as "merization", "clustering" or "association".

“Dimerizing agent” refers to a C3 rapalog of the present invention that links two or more proteins together in a multimeric complex.

“Activate” as applied herein to the expression or transcription of a gene refers to a directly or indirectly observable increase in the production of a gene product.

“Genetically created cells” are modified by the introduction of recombinant or heterologous nucleic acids (eg, one or more DNA constructs or their RNA counterparts) (
"Transduced") cells, and further includes the progeny of such cells that carry some or all of such genetic modification.

A “therapeutically effective amount” of a C3 rapalog of the invention is a therapeutically or prophylactically effective amount, eg, transcription of a target gene detectable in genetically created cells or cell growth, proliferation. , A dose that is expected to be therapeutically or prophylactically effective by extrapolation from data obtained in animal or cell culture models. A therapeutically effective amount is usually preferred for human or non-human mammals.

[0060] The present invention includes methods and materials for multimerizing chimeric proteins in cells genetically created using the C3 rapalog. The design and implementation of various dimerization-based biological switches are reported, inter alia, in Spencer et al. And the various international patent applications cited herein. Other explanations of the successful application of this general approach have also been reported. Chimeric proteins containing an FRB domain fused to an effector domain have also been described by Rivera et al., 1996,
Nature Medicine 2, 1028-1032 and WO9
6/41865 (Clackson et al.) And WO 95/33052 (Bell
in et al.). As noted above, fusion proteins may be those in which the association of effector domains is desired via ligand-mediated "dimerization" or "multimerization" of the fusion proteins containing them, such as transcription, cell killing, cell proliferation, etc. of the desired gene. It is designed to trigger a desired biological event such as For example, clustering of chimeric proteins containing an action domain derived from the intracellular portion of the T cell receptor CD3 zeta domain triggers gene transcription under the transcriptional control of the IL-2 promoter or promoter element derived therefrom. In another embodiment, the action domain is a domain derived from the intracellular portion of a protein, such as FAS or TNF-alpha receptor (TNFalpha-R1), that can trigger cell apoptosis upon oligomerization. Including. In yet another embodiment, the action domain is a transcription factor complex in which oligomerization of the chimeric protein triggers transcription of a gene bound to a DNA sequence recognized by the DNA binding domain (which can specifically bind and interact therewith). It contains a DNA-binding domain such as GAL4 or ZFHD1 and a transcriptional activation domain such as VP1-16 or P65, paired to represent body assembly.

For example, one or more ligand-binding domains and one or more action domains for triggering cell killing or other signal transduction pathways, cell localization, etc., for activation of target gene transcription. The contained chimeric protein was PCT / US94
No./01617, PCT / US94 / 08008 and Spencer et al., Supra.
Is disclosed. The design and use of such chimeric proteins for ligand-mediated gene-knockout and for ligand-mediated block of gene expression or inhibition of gene product function is disclosed in PCT / US95 / 10591. The DNA sequences to which they bind, useful in embodiments involving regulated transcription of novel DNA binding domain target genes, are described, for example, in Tomeranz et al., 1995,
Science 267: 93-96. These documents provide substantial information, guidance and examples relating to the design, construction and use of similar chimeras, DNA constructs encoding target gene constructs, and other aspects that may be useful to practitioners of the present invention. provide.

By appropriate selection of the chimeric gene, the present invention provides for the transcription of the desired gene in the generated cells in a rapalog-dependent manner, as well as in the systems described in the patent documents and other references cited therein. To activate cell growth, proliferation, differentiation or apoptosis; or to trigger other biological events. The generated cells are preferably capable of growing or maintaining animal cells in culture, or can be present in whole organisms, as in the case of human gene therapy, transgenic animals, and other such applications. Rapalog is known to multimerize FKBP and FRB fusion proteins (which can be indirectly monitored by monitoring target gene transcription, apoptosis or other biological processes so triggered). An effective amount, as applicable, is administered to the cell culture or organism containing the produced cells. For whole organism administration, rapalog can be administered in a composition containing rapalog and one or more acceptable animals or pharmaceutical diluents and / or excipients.

Compounds that bind to one of the chimeric proteins but do not form a tripartite complex with both chimeric proteins can be used as multimerization antagonists. As such, it is effective to block or reverse the effects of rapalog, ie, to prevent, inhibit or destroy multimerization of the chimera (preferably when administered to all animals). (In the compositions described above) can be administered to engineered cells or organisms containing them. For example, FK506, FK520 or FK520
Any of a number of synthetic FKBP ligands that do not form a tripartite complex with KBP and FRAP can be used as antagonists.

One important aspect of the present invention provides materials and methods for rapalog-dependent direct activation of transcription of a desired gene. In one such embodiment, 2
A set of these different chimeric proteins and corresponding DNA constructs that can direct their expression are provided. One such chimeric protein contains one or more transcriptional activation domains as its working domain. Other chimeric proteins contain one or more DNA-binding domains as their working domains. The rapalogs of the invention can bind to both chimeras, thus forming a dimeric or multimeric complex containing at least one DNA binding domain and at least one transcriptional activation domain. The formation of such a complex, as can be observed by monitoring, directly or indirectly, the presence or concentration of the target gene product, the DN to which the DNA-binding domain can bind
Under the transcriptional control of the A sequence, the transcription of the target gene linked to the DNA sequence is activated.

Preferably, the DNA binding domain, and the chimeras containing it, are selected (directly or indirectly) despite the presence of other, often numerous, other DNA sequences. Binds to the recognized DNA sequence with sufficient selectivity so that binding to the recognized DNA sequence can be observed. Preferably, the binding of the chimera comprising the DNA-binding domain to the selected DNA sequence is determined by in vitro binding experiments or by comparing the selected DNA with any other DNA sequence.
At least two, more preferably three, and even more preferably more than four orders of magnitude greater than the binding to any one other DNA sequence as measured by the relative rate or level of transcription of the gene associated with the A sequence. The size is big.

With any desired accessory construct, a cell that has been genetically engineered to contain such a set of constructs will be treated as if it were regulated transcription, apoptosis or triggering of the desired gene. Regulated triggering of signal transduction pathways, another event, can be used in applications involving ligand-mediated modulation of desired biological events. Cells created for regulatable expression of a target gene can be used, for example, in the regulated production of a desired protein (or other gene product) encoded by the target gene. Such cells can be grown in culture by conventional means. The addition of rapalog to the culture medium containing the cells leads to the expression of the target gene by the cell and the production of the protein encoded by that gene. Gene expression and protein production can be switched off by removing additional multimerizing agent from the medium and removing the remaining multimerizing agent from the medium, or by adding a multimerizing antagonist reagent to the medium. Can be.

The generated cells of the present invention are produced and / or used in vivo so that the cells produce the desired protein or other result in an animal containing them, preferably an animal, , Especially humans.

Embodiments involving the regulatable operation of apoptosis provide cells that have been made susceptible to rapalog-induced cell killing. Such engineered cells may be useful if they have or produce undesirable properties, or if they are no longer useful, safe or desired, for their intended purpose (eg, the desired protein or Other products) can be removed from the cell culture or the host organism after achieving it. Removal is accomplished by adding the rapalog to the medium or administering it to the host organism. In such a case, the working domain of the chimera is a protein domain downstream from a component of its signaling pathway, such as the intracellular domain of FAS or TNF-R1, or another protein domain that triggers apoptosis upon oligomerization.

Thus, the present invention provides materials and methods for achieving a biological effect in a cell in response to the addition of a rapalog of the present invention. The method includes providing a cell made as described herein and exposing the cell to a rapalog.

For example, the present invention provides a method for activating transcription of a target gene in a cell. The method comprises: (a) a DNA construct encoding a set of chimeric proteins of the invention capable of initiating transcription of a target gene upon rapalog-mediated multimerization; and (b) in response to a multimerization event. A cell containing a target gene (e.g., a recognized DNA sequence capable of binding to the DNA-binding domain of the chimeric protein) linked to the dissected cognate DNA sequence of E. coli. The method includes exposing the cells to a rapalog capable of binding the chimeric protein in an amount effective to result in expression of the target cells. If the cells grow in culture, exposure of the cells to rapalogs can be performed by adding rapalogs to the culture medium. If the cells are present in a host organism, their exposure to rapalogs is performed by administering the rapalog to the host organism. For example, if the host organism is human or non-human, the rapalog may be administered to the host organism orally, buccally, sublingually, transdermally, subcutaneously, intramuscularly, intravenously, intraarticularly, or by inhalation in a suitable vehicle therefor. Can be administered. Again, depending on the structure for the chimeric protein and the design of any accessory constructs, rapalog-mediated biological events can be cell growth, cell proliferation, gene transcription, or apoptosis; Blocking gene expression or inhibiting the function of the gene product of interest; activation of cellular functions such as signal transduction leading to direct transcription of the gene of interest or the like.

The present invention further includes pharmaceutical compositions comprising a rapalog of the present invention in admixture with a pharmaceutically acceptable carrier, together with one or more pharmaceutically acceptable excipients. Such pharmaceutical compositions can promote the multimerization of the chimera of the present invention in the resulting cells in whole animal, eg, human gene therapy applications, to achieve any of the objects disclosed herein.

Alternatively, the present invention may be used in animals, preferably human patients in need thereof and containing the engineered cells of the present invention, for any of their purposes, such as target genes (typically therapeutics). A method is provided for activating transcription of a heterologous gene for a protein, for growing or proliferating cells, for killing cells or for some other selected biological event. The method comprises administering to the animal a pharmaceutical composition containing rapalog by an administration route and amount effective to cause multimerization of the chimeric protein in at least a portion of the engineered cells. Multimerization can be detected jointly by detecting target gene expression; cell growth, proliferation or death; or a chimera designed for that purpose and other genetically generated cells. it can.

The present invention further provides for inhibiting or reducing the extent of multimerization of a chimeric protein in a subject cell of the invention completely or partially, and thus deactivating transcription of a target gene. For example, to turn off another biological result of the invention, mixed with a pharmaceutically acceptable carrier, optionally with one or more pharmaceutically acceptable excipients. And pharmaceutical compositions comprising the activating antagonist. Thus, the use of multimerizing rapalogs and multimerizing antagonist reagents to prepare a pharmaceutical composition and achieve its pharmacological results is included in the present invention.

Also disclosed are methods of providing a host cell, preferably an animal, typically a non-human mammal or human subject, responsive to a rapalog of the invention. The method comprises introducing into a biological cell made according to the present invention one containing one or more nucleic acid constructs encoding a chimeric protein. The producing cells can be encapsulated using any of a variety of materials and methods before being introduced into a host organism. Alternatively, the nucleic acid construct of the present invention can be prepared by introducing the nucleic acid construct into one or more cells of a host mammal using, for example, a viral vector, by injection or by catheterization of the DNA.
Can be introduced into a host organism, for example, a mammal, under conditions that allow introduction into the host organism.

Also provided is a kit for producing cells responsive to a rapalog of the present invention. One such kit includes one or more nucleic acid constructs capable of encoding and directing the expression of a chimera that triggers a desired biological response upon rapalog-mediated oligomerization. The kit can contain a certain amount of rapalog capable of multimerizing the chimeric protein molecule encoded by the construct of the kit, and can additionally contain a certain amount of a multimerizing antagonist. . The kit may further comprise a target gene (or or Cloning site). The construct may be associated with one or more selectable markers for convenient selection of transfectants, as well as other common vector elements useful in eukaryotic replication for expression in eukaryotes. it can. The selectable marker may be the same or different for each different construct, allowing for the selection of cells containing each such construct.

An accessory construct for introducing a target gene associated with a cognate DNA sequence into a cell can contain a cloning site in place of the target gene. Kits containing such constructs allow the generation of cells for regulatable expression of the gene to be provided by the practitioner.

Other kits of the invention can contain one or two (or more) nucleic acid constructs for a chimeric protein, one or more of which contains a cloning site, instead of a transcription activator or DNA binding protein. Allows the user to insert a domain whenever desired. Such kits can include other elements as described above, for example, a nuclear construct for the target gene with or without cognate DNA sequences for the preselected DNA binding domain, if desired.

[0078] Any of the kits, in response to exposing the cells to the rapalog, they express a reporter gene (for CAT, SEAP, beta-galactosidase or any conveniently detectable gene product). Thus, positive control cells stably transformed with the constructs of the present invention can be included. Reagents for detecting and / or quantifying reporter gene expression can also be provided.

For further information and guidance on the design, construction and use of such systems or components thereof that can be adapted to be used in practicing the present invention, reference is made to the following publications: It is. Spencer et al, 1993, s, the contents of each of which are incorporated herein by reference.
upra; Rivera et al, 1996, supra; Spence
er et al, 1996, Current Biology 6,839-
847; Luo et al, 1996, Nature, 383, 18
1-185; Ho et al, 1996, Nature 382, 822.
-826; Belshaw et al, 1996, Proc. , Natl
. Acad. Sci. USA 93, 4604-60; Spencer 1996, TIG12 (5), 181-187; Spencer et al., 1995, Proc.
. Natl. Acad. Sci. ScL USA 92, 9805-9809; Holsinger et al, 1995, Proc. Natl. Acad. Sc
i. USA 92, 9810-9814; Pruschy et al, 1994.
, Chemistry & Biology 1 (3), 163-172; and published international patent applications WO94 / 18317, WO95 / 02684, WO9.
5/33052, WO 96/20951, WO 96/41865 and WO 98/02441.

A key focus of the present invention is to focus on proteins-in applications using FKBP and FRB fusion proteins, as described above and elsewhere herein.
Use of C3 rapalog as a mediator of protein interactions. C3 rapalogs include triggering biological events in genetically created cells grown or maintained in culture or present in all organisms, including humans and other mammals. It can be used in various applications of the underlying dimerization-based technology. Thus, C3 rapalog is useful as a research reagent in in vitro biological experiments in experiments performed on animals containing genetically engineered cells, and in animals containing genetically engineered cells. And may be useful as prophylactic or therapeutic agents in human health care.

Rapalog, C3 Rapalog and Pharmaceutically Acceptable Derivatives As used herein, “rapalog” refers to the class of compounds, including various analogs, homologs and derivatives of rapamycin, and the structure of rapamycin. Other compounds that are closely related are shown.

Rapalogs are, inter alia, the following modifications to rapamycin: demethylation, elimination or substitution of C7, C42 and / or C29 methoxy; elimination, derivatization or substitution of C13, C43 and / or C28 hydroxy. C14, C24
And / or reduction, elimination or derivatization of a ketone at C30; substitution of a 6-membered pipecolate ring with a 5-membered pyrrolyl ring; and elimination, derivatization or substitution of one or more substituents of the cyclohexyl ring or substitution of the cyclohexyl ring. Or a variant of rapamycin having one or more of the substitutions on the substituted cyclopentyl ring. As the term is used herein, rapalog does not include rapamycin itself, and preferably does not contain an oxygen bridge between C1 and C30. Illustrative examples of rapalogs are disclosed in the documents listed in Table I.

[0083]

[Table 2] Other exemplary rapalogs include those shown in Table II

[Table 3]

[Table 4] "C3 rapalog" is defined above with reference to Formula I and is exemplified by the various classes and subsets of the compounds disclosed herein.

The phrase “pharmaceutically acceptable derivative” refers to a pharmaceutically acceptable salt, ester, or salt of such an ester, or a C3 rapalog or metabolite thereof described herein upon administration to a patient. Or any other adapt or derivative that can provide a residue (directly or indirectly). Thus, pharmaceutically acceptable derivatives include, inter alia, prodrugs of rapalog. Prodrugs are derivatives of compounds that are susceptible to in vivo elimination and that contain additional sites that give rise to the parent molecule as a pharmacologically active species, usually having significantly reduced pharmacological activity. Examples of prodrugs are esters that yield the compound of interest cleaved in vivo. Various prodrugs of rapamycin and other compounds, as well as materials and methods for derivatizing the parent compound that produces the prodrug, 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. Including polycyclic aliphatic hydrocarbons. Unless otherwise specified, alkyl, other aliphatic,
Alkoxy and acyl groups preferably contain 1-8, often 1-6, continuous aliphatic carbon atoms. Exemplary aliphatic groups, thus, for example, 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 - comprises a cyclohexyl sites and the like, which can again, carrying one or more substituents .

Examples of the substituent are —OH, —OR²’, -SH, -SR²', -CHO, = O,-
COOH (or its ester, carbamate, urea, oxime or carbon
), -NH₂(Or its substituted amines, amides, ureas, carbamates
Or guanidide derivative), halo, trihaloalkyl, cyano, -SO₂-CF ₃ , -OSO₂F, -OS (O)₂R¹¹, -SO₂-NHR¹¹, -NHSO ₂ -R¹¹, Sulfate, sulfonate, aryl and heteroaryl moieties
including. Aryl and heteroaryl substituents may themselves be substituted.
May be unsubstituted (eg, mono-, di- and tri-alkoxy
Nil; methylenedioxyphenyl or ethylenedioxyphenyl; halophenyl
Or -phenyl-C (Me)₂-CH₂-O-CO- [C3-C6] al
Kill or alkylamino).

Thus, the term “aliphatic” is intended to include alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl groups.

As used herein, the term “alkyl” includes straight chain, branched and cyclic alkyl groups. An analogous convention applies to other generic terms such as "alkenyl", "alkynyl" and the like. Further, as used herein, the term "alkyl",
“Alkenyl”, “alkynyl” and the like include both substituted and unsubstituted groups.

The term “alkyl” usually refers to groups 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, ter
Refers to t-butyl, cyclobutyl, pentyl, isopentyl, tert-pentyl, cyclopentyl, hexyl, isohexyl, cyclohexyl and the like. Suitable substituted alkyls include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2-fluoroethyl, 3-fluoropropyl, hydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl, benzyl , Substituted benzyl and the like.

The term “alkenyl” refers to a group having 2 to 8, preferably 2 to 6, carbon atoms. For example, “alkenyl” refers to prop-2-enyl, but-2-enyl, but-3-enyl, 2-methylprop-2-ethyl, hexa-2-enyl, hexa-5-enyl, 2,3-dimethylbuta -2-enyl and the like. The term "alkynyl", which also refers to groups having 2 to 8 groups, preferably 2 to 6 carbons, includes, but is not limited to, prop-2-ynyl, but-2-ynyl, buta-3 -Inyl, penta-2-ynyl, 3-methylpenta-4
-Inyl, hexa-2-ynyl, hexa-5-ynyl and the like.

As used herein, the term “cycloalkyl” refers specifically to groups having 3 to 7, preferably 3 to 10, carbon atoms. A suitable cycloalkyl is
Including, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexyl, and the like, which may be optionally substituted as in other aliphatic or heteroaliphatic or heterocyclic groups.

As used herein, the term “heteroaliphatic” refers to an aliphatic group that contains, for example, one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms instead of carbon atoms. Heteroaliphatic groups can be branched, unbranched or cyclic and include heterocycles such as morpholino, hydridinyl and the like.

As used herein, the term “heterocycle” refers to a cyclic heteroaliphatic group, preferably 3 to 10 total ring atoms, including, but not limited to, oxetane, tetrahydrofuran Nyl, tetrahydropyranyl, aziridine, azetidine, pyrrolidine, piperidine, morpholine, piperazine and the like.

As used herein, the terms “aryl” and “heteroaryl”
A stable monocyclic, polycyclic, heterocyclic, polycyclic and polyheterocyclic unsaturated group having 3 to 14 carbon atoms which may be substituted or unsubstituted.
The substituents include any of the substituents already mentioned. Non-limiting examples of useful aryl ring groups are phenyl, halophenyl, alkoxyphenyl, dialkoxyphenyl,
Including trialkoxyphenyl, alkylenedioxyphenyl, naphthyl, phenanthryl, anthryl, phenanthro and the like. Examples of typical heteroaryl rings include 5-membered monocyclic groups such as thienyl, pyrrolyl, imidazolyl, pyrazolyl, furyl, isothiazolyl, flazanyl, isoxazonyl, thiazolyl, etc .; 6-membered monocyclic groups such as pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, etc. A ring group; and benzo [b] thienyl, naphtho [2,3-b] thienyl, thiantrenyl,
Isobenzofuranyl, chromenil, xanthenyl, phenoxanthienyl, indolizinyl, isoindolyl, indolyl, indazolyl, prenyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, benzothiazole, benzimidazole, carbimidazole, carberidinyl, pteridinyl , Beta-carbolinyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl, phenandinyl, isothiazolyl, phenothiadinyl, phenoxazinyl and the like (for example, Katritzky, Handbook of Heterology).
click Chemistry). The aryl or heteroaryl group is selected from the group consisting of hydroxy, C1-C8 alkoxy, C1-C8 branched or straight chain alkyl, acyloxy, carbamoyl, amino, N-acylamino, nitro, halo, trihalomethyl, cyano and carboxyl. May be substituted with 1 to 5 members. Thus, an aryl group is one or more substituents selected from groups including, for example, halo, hydroxy, C1-C6 alkyl, acyl, acyloxy, C1-C6 alkoxy (such as methoxy or ethoxy), such as chloro or fluoro. A substituted phenyl bearing
3-, 2,4-, 2,5-, 3,4- or 3,5-dimethoxy or diethoxyphenyl, or methylenedioxyphenyl or 3-methoxy-5
A dialkoxyphenyl group such as ethoxyphenyl; or trialkoxy (
For example, 3,4,5-trimethoxy or ethoxyphenyl), 3,5-dimethoxy-4-chlorophenyl, etc.), _{amino, -SO 2 NH 2, -SO 2} NH ( aliphatic), - _SO 2 N (aliphatic ) _2, trisubstituted phenyl, such as -O- aliphatic -COOH, and -O- aliphatic -NH ₂ (they may contain one or two N- aliphatic or N- acyl substituent) including.

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

The C3 rapalog further differs from rapamycin in addition to the modifications at C3 in 0, 1, 2, 3, 4, 5, 6 or 7 substituent sites. This class includes, inter alia, C3 and C13; C3 and C14; C3 and a; C3 and C4.
C3 and C28; C3 and C30; C3, C13 and C14; C3, C13 and a; C3, C13 and C43;
And C24; C3, C13 and C28; C3, C13 and C30;
C14 and Ca; C3, C14 and C43; C3, C14 and C24; C
3, C14 and C30; C3, a and C24; C3, a and C24; C3
, A and C28; C3, a and C30; C3, C24 and C30;
C3, C24, C30 and a; C3, C24, C30 and C13;
And other rapalogs with modifications to rapamycin at C30 and C14; and at C3, C24, C30, C13 and a.

One subset of C3 rapalogs that are of interest in the practice of the various methods of the invention are C3 rapalogs where R ^C30 and R ^C24 are both other than (（O). In certain embodiments of this subset, R ^C30 and R ^C24 are both, for example, “S
"-OH configuration. In another embodiment, R ^C30 and R ^C24 are independently selected from OR ³ . This subset includes, inter alia, rapalogs further different from rapamycin with regard to group a. For example, this subset has the formula II
I:

Embedded image Wherein R ^C3 is other than -H. Alternative substituents for R ^C3 are as disclosed elsewhere herein. Other compounds within this subset are 1, 2, 3, 4 or 5
Hydroxyl groups are epimerized, fluorinated, alkylated, acylated or modified via other ester, carbamate, carbonate or urea formation. Exemplary further compounds are, for example, the hydroxyl groups at C43 are in the opposite stereochemical orientation and / or the hydroxyl groups at C28 and C30 are alkylated, acylated, or linked via carbonate formation. Others are compounds as shown in Formula III.

Another subset of C3 rapalogs of particular interest is R^C13And R ^C28 Is one or both of which are F. Various examples of this subset
Wherein 1, 2, 3, 4, or 5 substituents in formula II are rapamycin
Different from the substituents found in For example, this subset is C13 fluoro-
C3-rapalog, C28 fluoro-C3-rapalog and C13, C28-diff
Including Ruoro-C3-rapalog.

[0099] Particularly Another subset of C3 rapalog of interest as ^{R C14} is other than O, OH or H, for example, or without one or more other modifications with respect to ^{rapamycin, R C14} is -OR ^{6 , -NR 6, -NC (O)} R 6, -OC (
O) is ^{R 6} or -OC (O) C3 rapalog is NR ^6.

Another subset of C3 rapalogs of interest is again that R ^C24 is other than ＯO, with or without one or more other modifications at other positions with respect to rapamycin.

Another subset of C3 rapalogs of particular interest in practicing the methods of the present invention has the stereoisomerism of rapamycin, where R ^C7a is —OMe and where R ^C30 is ^Includes where ^{な く} O is not R ^C24 is ＝ O, R ^C13 is not —OH, R ^C14 is not ^ＯO and / or R ³ and / or R ⁴ is not H.

Other C3 rapalogs of interest include those where R ^C14 is OH.

Furthermore, the present invention relates to the invention wherein one or more of the carbon-carbon double bonds at the 1,2,3,4 or 5,6 position in rapamycin, alone or in combination with other modifications of the molecule, for example C3 rapalogs that are saturated in one or more of C13, C43, C24, C28 and / or C30. Further, C3, C4 double bond that may be epoxidized; it C6 methyl group may be substituted by _CH 2 OH or _-CH 2 OMe; C43 hydroxy F, Cl or H or other And the C42 methoxy group may be demethylated in any of the compounds disclosed herein using methods known in the art. Should be recognized. Similarly, group "a" may be substituted with any of the following:

[0104]

Embedded image Synthetic Guidance The production of rapamycin by fermentation and by total synthesis is known. The production of a large number of "other than C3 rapalogs" by fermentation or the synthetic modification of fermentation products is also known. These include, inter alia, other sites for the characteristic cyclohexyl or pipecolate ring of rapamycin and C29-desmethyl-rapamycin and C29-desmethoxyrapamycin and various other rapalogs as described herein. Includes rapalogs.

Rapamycin can be converted to C3 rapalog using silyl ethers as disclosed in the experimental examples below.

The C3 rapalog can be manufactured using various techniques. These techniques can be synthetic processes involving nucleophiles and catalysts. The first rapalog is the desired C
3 can be treated with a nucleophile under conditions that allow for the formation of rapalogs. The first rapalog may be a C7 modified rapalog, for example, C7-methoxy lapalog.
These conditions include factors such as the type of nucleophile, the type of catalyst, and the choice of purification techniques used to isolate or purify the final C3 rapalog.

For example, the substituted rapalogs of the present invention are prepared by treating a rapalog having a substituent attached to the macrocycle, for example, at the C7 position, with a nucleophile, such as a silyl ether, in the presence of a catalyst. Can be. In another embodiment, rapamycin can be converted to C3 rapalog using silyl enol ethers known to those skilled in the art.

Suitable substituents include those recognized by those skilled in the art and include halogen, tosylate, mesylate, ester and alkoxide, preferably lower alkyl alkoxide. Treatment of a rapalog having a substituent at the C7 position with an appropriate nucleophile was found to form a rapalog modified at the C3 position by the nucleophile.

Suitable nucleophiles include those recognized by those skilled in the art and include silyl ethers, silyl enol ethers, Grignard reagents, enolates, carbanions, and the like. Preferred nucleophiles are silyl ethers, preferably trialkylsilyl ethers, especially substituted and unsubstituted allyl groups, for example methallyl trialkyl ethers.

The term “silyl ether” is known in the art and includes (2) R, R ′ and R
Has the formula RC3-SiRR'R "wherein" is an aliphatic, aromatic, heteroaliphatic or heteroaromatic group and RC3 is as defined above, preferably a substituted or unsubstituted allyl group. It is intended to include compounds. In addition, silyl ethers are commercially available and can be purchased from chemical suppliers such as ALDRICH and SIGMA.

The term “silyl enol ether” is known in the art, where R, R ′ and R ″ are aliphatic, aromatic, heteroaliphatic or heteroaromatic groups and R ^C3 is as defined above. An expression that is the same:

Embedded image A compound having the formula: Silyl enol ethers are also commercially available and can be purchased from chemical suppliers such as ALDRICH and SIGMA.

Suitable catalysts include Lewis acid catalysts such as AlCl ₃ , ZnCl ₂ , P-toluenesulfonic acid, preferably BF ₃ etherate.

The reaction scheme described below:

Embedded image Provides examples of nucleophilic displacement of a leaving group by a nucleophile, such as a silyl ether. Treatment of compound 1 rapalog with silyl ether 2 under conditions that allow the formation of a compound having formula 3 can be achieved by treating a rapalog bearing a C7 heteroatom, such as OMe, in the presence of a catalyst, preferably BF ₃ / etherate. In combination with a trialkylsilyl ether. Generally, the reaction conditions are below ambient temperature, preferably about -4
Performed at 0 ° C.

C3 rapalogs containing further modifications with respect to the structure of rapamycin can be prepared similarly, starting with the corresponding rapalog in place of rapamycin. Alternatively, one or more further transformations can be performed on the C3 rapalog intermediate.

Methods and materials for performing various chemical transformations of rapamycin and structurally related macrolides are known in the art, as are methods for obtaining rapamycin and various rapalogs by fermentation. Many such chemical transformations of rapamycin and various rapalogs are described in Table I above, which describes the level of skill and knowledge in the field of chemical synthesis and the recovery, purification, and formation of products applicable in the practice of the present invention. In the patent documents identified in The following representative transformations and / or literature that can be used to produce the desired rapalog are illustrative:

[Table 5] In addition, rapalogs used in the present invention and intermediates for the preparation of such rapalogs are described, for example, by Katz et al., WO 93/13663, and by Cane.
It can be prepared by directed biosynthesis as described by WO9702358.

The novel rapalogs of the present invention can be prepared by those skilled in the art depending on methods and materials known in the art, as guided by the disclosure provided herein. For example, the methods and materials can be adapted to known methods described or mentioned in the cited documents, which are hereby incorporated by reference in their entirety. Further guidance and examples are provided herein to the practitioner by way of explanation and further guidance. A chemist of ordinary skill in the art will be able to make modifications to the above, for example, by adding suitable protecting groups to the sensitive site during the synthesis, so that the protecting groups are no longer needed or desired. Can be removed and other synthetic approaches can be determined.

Purification of the mixture of rapalogs isolated from the above conditions employs techniques that allow the isolation or recovery of the desired C3 rapalog, eg, the desired purity or form, eg, the absence of the C7 rapalog. Can be achieved. An example of such a technique is the recycling HPLC technique described in the examples below. Recycling HPL
The use of C leads to the discovery that, under said conditions, a nucleophile is added at the C3 position while simultaneously displacing the C7 group, resulting in a C3 rapalog. Structural confirmation that the rapalog is functionalized at the C3 position was determined by mass spectroscopy and / or NMR spectroscopy.

As a non-limiting example, an unpurified reaction mixture of a rapalog product can be eluted through an HPLC column and recycled through the column to separate components and thus increase the final isolate . HPLC systems are known to those skilled in the art and can be adapted to include such recycling features. Generally, the eluate and components are recycled until the desired separation and purity of the components is achieved, for example, up to 15 to 20 cycles. One skilled in the art can determine how many cycles are required, determine the required solvent, column type, etc. to achieve the desired purity.

For example, a polystyrene size exclusion column can be used. Polystyrene columns can be functionalized, for example, to contain hydroxyl functionality to facilitate separation of components. In one particular preferred embodiment, JAIG
The components can be separated using an EL GS-310 column.

One aspect of the present invention relates to the purity of the isolated rapalog. The purity of the isolate rapalog can be determined by techniques known to those skilled in the art, mass spectrometry, ¹ HN
Includes MR and ¹³ NMR. In one embodiment, the isolated C3 of the present invention is
The rapalog is substantially free of contaminants, for example, substantially free of C7 rapalog. One example of determining the purity of an isolated C3 rapalog is by ¹ H NMR. The purity of a compound can be measured by analyzing the peak high ratio of the desired product to that of the contaminants. In one embodiment, the ratio is at least about 5: 1 (product / contaminant). In another embodiment, the ratio is at least about 10: 1, more preferably 50: 1, most preferably 100: 1,
Most preferably, no contaminants are detected by NMR.

In another embodiment, the purity of the isolated rapalog is determined by determining the starting material C7 relative to that of the peak height associated with the group attached to the C3 position in the resulting product.
¹ HN by analyzing the ratio between the peak heights associated with the groups attached to
It can be measured by MR. The purity of the C3 rapalog can be determined by analyzing the peak height ratio of the desired product to that of the peak associated with the starting material containing the C7 group. In one embodiment, the ratio is at least about 5: 1 (isolated C3 rapalog product / C7 starting material). Another one
In one embodiment, the ratio is at least about 10: 1, more preferably 50: 1.
, Most preferably 100: 1, and most preferably no contaminants are detected by NMR.

In another embodiment, the isolated C3 rapalog is at least about 90% as determined by techniques known in the art, for example, capillary gas chromatography, analytical HPLC, and the like. In a more preferred embodiment, the isolated C3 rapalog is at least about 95%. In another embodiment, the isolated C3 rapalog is about 98% pure. In a most preferred embodiment, the isolated C3 rapalog is about 100% pure.

FKBP Domains and Fusion Proteins FKBP fusion proteins comprise all or part of the peptide sequence of the FKBP domain and at least one heterologous domain. This chimeric protein is
Direct binding assays (eg, fluorescence quenching), competitive binding assays (eg, versus FK5)
06), inhibition of FKBP enzymatic activity (rotamers), or as determined by other assay methods, the C3 rapalog of the invention preferably has less than about 100 nM, more preferably less than about 10 nM, and even more preferably less than about 1 nM. It can be combined with the Kd value. Typically, chimeric proteins are described, for example, in International Patent Application P
As described in CT / US94 / 01617, one comprising a peptide sequence selected therefrom of a naturally occurring FKBP protein such as human FKBP12.
It will contain the above peptide domains. The peptide sequence usually has 10
Modification by substitution, insertion or deletion of not more than 5, preferably not more than 5, amino acid residues can adjust the binding specificity. Such modifications are selected in certain embodiments to provide the following binding profiles: (a) preferably at least 1, more preferably (by any measurement), than for FKBP12 or FKBP endogenous to the host cell to be produced. Binding of the C3 rapalog to the modified FKBP domain, or a chimera containing it, which is at least better than 2, even more preferably on the order of 3 or more; and (b) endogenous to FRAP or the host cell to be produced. Of the binding of the FKBP: rapalog complex to the FRB fusion protein is preferably at least 1, more preferably at least 2, and even more preferably 3 better than by a specific FRB-containing protein. Produce one or both of them.

Also, the FKBP chimera has at least one heterologous action domain, ie, a non-
It contains a protein domain containing the FKBP peptide sequence. The action domain can be, for example, a DNA-binding domain, a transcriptional activation domain, a cell localization domain, an intracellular signal transduction domain, etc., as described herein or in PCT / US94 / 01617 or other references. . Generally speaking, an action domain directs a chimeric protein to a selected cellular location or upon association or aggregation with another action domain, for example, upon multimerization of a protein containing the same or a different action domain. Biological effects can be triggered.

A recombinant nucleic acid encoding such a fusion protein can selectively hybridize to a parent FKBP protein, for example, DNA encoding human FKBP12, or can undergo such hybridization except for the degeneracy of the genetic code. It is possible. These chimeric proteins comprise another protein, such as Gal4
, VPI6, FAS, CD3 zeta chain, etc., the recombinant DNA encoding the chimeric protein can be used as a recombinant DNA encoding other proteins.
It will be able to selectively hybridize to NA, or will be capable of such hybridization without the degeneracy of the genetic code.

The FKBP fusion proteins of the invention, described in further detail below, as well as the discussed FRB fusion proteins, contain one or more copies of one or more different ligand binding domains and one or more copies of one or more action domains. be able to. The ligand binding domains (ie, FKBP and FRB domains) are N-terminal,
It can be C-terminal or interspersed with respect to the action domain. Embodiments containing multiple copies of the ligand binding domain typically have 2, 3 or 4 such copies. For example, an FKBP fusion protein can contain 2, 3 or 4 FKBP domains. The various domains of the FKBP fusion protein (and of the FRB fusion protein discussed below) may be separated by a linking peptide region, which may be derived from one of the adjacent domains, or heterologous, as desired.

An example of a FKBP fusion protein useful in the practice of the present invention is PCT / US94 /
01617 (Stanford & Harvard), PCT / US94 / 080
08 (Stanford & Harvard), Spencer et al., Supra, PC
T / US95 / 10591 (ARIAD), PCT / US95 / 06722 (M
itorix, Inc. ) And the FKs disclosed in other references cited therein
A BP fusion protein; a FKBP fusion protein disclosed in the Examples below; containing up to 10 (preferably 1-5) amino acid insertions, deletions or substitutions in one or more FKBP domains and still containing rapamycin or rapalog A variant of any of the foregoing FKBP fusion proteins that is capable of binding to a DNA sequence encoding one of the FKBP domains that encodes a naturally occurring FKBP domain. A variant of the FKBP fusion protein of any of the foregoing FKBP fusion proteins that is still capable of binding to rapamycin or rapalog; one or more heterologous domains are deleted and replaced or supplemented with a different heterologous domain Any of the above mutants It can bind to rapamycin or rapalog, or variants of the FKBP-derived protein containing the FKBP domain derived from a non-human source; and human FKBP12 Tyr26, Phe36, Asp37
, Arg42, Phe46, Phe48, Glu54, Val55 or Phe
99. A variant of any of the above FKBP fusion proteins comprising one or more amino acid residues corresponding to 99, wherein one or more of the amino acid residues has been replaced by a different amino acid, wherein the variant binds to rapamycin or rapalog. can do)
including.

For example, in many cases, the FKBP fusion protein is human FKBP12, separated by the 2-amino acid linker Thr-Arg, encoded by the ligation products of DNA digested with ACTAGA, restriction endonucleases SpeI and XbaI, respectively. Contains multiple copies of the FKBP domain containing amino acids 1-107. The following table provides an exemplary subset of mutant FKBP domains based on the FKBP12 sequence.

Exemplary Mutant FKBP

[Table 6] Note: Entries identify natural amino acids by one letter code and sequence position, followed by the replacement amino acid in the mutant. Thus, F36V is at position 3
FKBP12 in which phenylalanine in SEQ ID No. 6 has been replaced by villin
Shows the sequence. F36V / F99A shows a double mutation in which phenylalanine at positions 36 and 99 has been replaced by valine and alanine, respectively.

FRB Domain and FRB Fusion Protein The FRB fusion protein comprises at least one FRB domain (as described elsewhere,
And may comprise all or part of the peptide sequence of the RAP protein or a variant thereof) and at least one heteroeffector domain.

Generally speaking, the FRB domain, or a chimeric protein comprising the FRB domain, is, for example, a human or other mammalian FRAP protein or one of the yeast proteins, Tor-1 or Tor-2, or It is encoded by a DNA molecule capable of selectively hybridizing to a DNA molecule encoding a naturally occurring FRB domain-containing protein, such as a DNA molecule encoding a Candida FRB-containing protein described above. The FRB domain of the present invention includes an FRB domain capable of binding to a complex of the FKBP protein and the C3 rapalog of the present invention.

The FRB fusion protein must be able to bind to the complex formed by the C3 rapalog of the invention and the FKBP fusion protein. Measured in the traditional way,
The FRB fusion protein preferably binds to the complex with a Kd value of 200 μM or less, and more preferably binds to the complex with a Kd value of 10 μM or less. The FRB domain will be of sufficient length and composition to maintain high affinity for the complex of FKBP fusion protein and rapalog. In certain embodiments, the FRB domain is less than about 150 amino acids in length, and in some cases, less than about 100 amino acids in length. One such region encompasses a 133 amino acid region extending from Val2012 to Tyr2144 of human FRAP. Chiu et al, 1994, Proc. Natl. Acad. Sci. USA 91:
See 12574-12578. FRB regions of particular interest are Glu2025 to Gln2114 of human FRAP.
And retains affinity for the FKBP12-rapamycin complex or FKBP-rapalog complex. In one embodiment, Q2214 has been removed from the 90 amino acid sequence to provide an 89 amino acid FRB domain. The FRB peptide sequence can be modified to modulate binding specificity, usually by substitution, insertion, or deletion of 10 or less, preferably 5 or less amino acids. To selectively form a complex comprising one or more molecules of a FKBP fusion protein, a FRB fusion protein, and a C3 rapalog, rather than forming a complex of an endogenous FKBP and FRAP protein with a C3 rapalog, In some embodiments, such modifications are selected. The preference is preferably at least 10 times, more preferably 10 ² times, more preferably 10 ³ times (by any indicator).

Recombinant DNA encoding such a protein can selectively hybridize to DNA encoding a FRAP species or, if there is no degeneracy of the genetic code. Hybridization will be possible. In addition, these chimeric proteins include effector domains from other proteins, such as, for example, Gal4, VP16, Fas, CD3 zeta (ξ) chain, etc., and thus encode the chimeric protein. Recombinant DNA is DNA that encodes another protein
May hybridize selectively to, or such hybridization would be possible if, without the degeneracy of the genetic code.

As a specific example of an FRB chimera useful in the practice of the present invention, one or more heterodomains may be substituted for the heterologous heterologous domain as long as the chimera can bind to the complex formed by the FKBP protein and the C3 rapalog of the present invention. A variant thereof, wherein the variant has been replaced by a domain or supplemented by one or more additional heterodomains, wherein one or more FRB domains is a domain derived from a non-human peptide sequence (eg, Tor2 or Candida), And as described herein, including those disclosed in the examples such as variants in which the FRB domain has been modified by amino acid exchanges, substitutions or insertions. An illustrative FRB fusion protein comprises at least a sequence spanning residues 2025-2113 of human FRAP, separated by a Thr-Arg linker formed by a SpeI-XbaI site ligation reaction as described above. Includes one or more FRBs consisting of 89 amino acids. Conventional techniques such as site-directed mutagenesis, for example, should be used to evaluate the ability to delete, replace, or extend such restriction sites or linkers in any of the fusion proteins of the invention. is there.

Mixed Chimeric Proteins As described for FRB fusion proteins, a third type of chimeric protein is
Includes one or more FKBP domains, one or more heteroeffector domains, and one or more FRB domains.

[0136] Mixed chimeric protein molecules can form homodimeric or homomultimeric protein complexes in the presence of the C3 rapalog to which they bind. An example involving a mixed chimera is one single set instead of the two recombinant nucleic acids required to direct the expression of both FKBP and FRB fusion proteins in a different way. This has the advantage that the recombinant nucleic acid may be introduced into cells.

[0137] The recombinant DNA encoding the mixed chimeric protein includes the DNA encoding the FKBP protein, the DNA encoding the FRAP, and the protein from which one or more effector domains are derived. (E.g., Gal4, VP16, Fas, CD3 zeta (ξ) chain, etc.) can selectively hybridize to a heterologous DNA sequence or, if there is no degeneracy in the genetic code. Hybridization will be possible.

Heterodomain As described above, the heteroeffector domains of the FKBP fusion protein and the FRB fusion protein are capable of inducing (or inhibiting) DNA binding and / or transcription of a target gene when chimeric proteins having them are mutually bound. Act on cell growth, differentiation, proliferation, or apoptosis; direct proteins to specific cell locations; or activate other biological events; protein domains.

[0139] Examples involving regulatable gene transcription are directed to the transcription of DNA elements responsible for the association or multimerization of the first and second chimeric protein heterodomains, under the transcriptional control of the target gene (poly- (Encoding peptides, antisense RNAs, ribozymes, etc.).

In embodiments of the invention that include activating direct transcription, the first and second chimeric protein heterodomains may be a DNA such as Gal4 or a chimeric DNA binding domain such as, for example, ZFHD1 discussed below. Includes a binding domain, and a transcriptional activation domain as derived from VP16 or p65, respectively. Such multimerization of a transcriptional activation domain-containing chimeric protein to a DNA-binding domain-containing chimeric protein targets a transcriptional activator to a promoter element to which the DNA-binding domain binds, and therefore, promotes the promoter. Activates transcription of the target gene linked to the tar element. When the target gene precedes the transcriptional activation domain, or when the transcriptional activation domain is replaced with a repressor domain (see PCT / US94 / 01617), a similar chimera is provided that is useful for inhibiting transcription of the target gene. Synthetic DNA binding domains and the DNA sequences to which they bind are described in Pomerantz et al, 1995, supra
, The contents of which are incorporated herein by reference. Such synthetic DNA binding domains include indirect transcriptional activation that can be used as a DNA binding domain in the practice of the present invention, along with a target gene construct that includes the cognate DNA sequence to which it binds. In an embodiment, the heterodomain of the chimera is the effector domain of a signal protein that, when aggregated or multimerized, causes transcriptional activation under the control of a response promoter. For example, the signal domain is
Intracellular zeta (ξ) subunit of the T cell receptor that when aggregated causes transcription of a gene linked to the IL-2 promoter or a derivative thereof (eg, a repeated NF-AT binding site) Can be a domain.

In another context of the invention, a heterodomain is a protein domain that can bind to and induce cell death. Examples of such domains are Fas antigen or T
And the intracellular domain of NF R1. Chimeric proteins that include a Fas domain can be designed and prepared by analogy to those disclosed in PCT / US94 / 01617.

Recombinant Receptor Domain As described above, the FKBP and FRB domains may include a peptide sequence selected from the peptide sequences of naturally occurring FKBP and FRB domains. Naturally occurring sequences include the FKBP domain of human FKBP12 and the FRB domain of human FRAP. Alternatively, the peptide sequence may be derived from such a naturally occurring peptide sequence, but usually contains up to 10 mutations in one or both of such peptide sequences, more preferably 1-5 Including mutations. As disclosed in more detail elsewhere herein, such mutations can confer a number of important features. For example, FKBP domains than rapalog binding by FKBP domain of unmodified, e.g., at least 10 fold, preferably 10 ² times, more preferably 10 ³ fold or 10 ⁴ higher than Ueno efficiency, selected C3 rapalog Can be modified so as to be able to bind together. The FRB domain is, for example, at least 10 times, preferably 1
0 ² times, more preferably 10 ³ times higher efficiency, (modified or unmodified) FKB
P: Can be modified to selectively bind to the rapalog complex. FKBP and FRB domains, than FKBP and FRB domains of unmodified, such as at least 10 fold, preferably 10 ² times, more preferably 10 ³ times higher efficiency, forming a double coalescence consisting C3 rapalog and 3 persons Can be modified to

(A) FKBP Methods for identifying FKBP mutations that enhance binding to FK506 derivatives containing various substituents (“bumps”) have been disclosed in PCT / US94 / 01617. The method can be used to obtain a modified FKBP that selectively binds to bumped rapamycin derivatives.The structure of the complex of rapamycin and FKBP12 is known (eg, Van Duyne). et al., J. Am. Chem
Soc. (1991) 113,7433-7434). Such information can be used to identify amino acids that collide with various rapalog substituents. In this approach, molecular modeling was used to identify candidate amino acid substitutions in the FKBP domain that would fit into the rapalog substituent, and further, a sudden mutation at a specific site was attempted to attempt the identified protein mutation. Mutagenesis can be used. The mutants are expressed by standard methods and if the mutants retain the appropriate activity / affinity, their binding affinity for rapalogs can be determined, for example, by inhibiting rotamase activity. Or by competition for binding to a molecule such as FK506.

More specifically, without sticking to the theory, for example, the C-13 or C-
C3 rapalogs of the present invention, such as rapalogs modified at 14, are Tyr26, Phe36
, Asp37, Tyr82, and Phe99 residues preferentially bind to FKBPs that have been replaced by amino acids with smaller side chains (eg, Gly, Ala, Val, Met, and Ser). I have. Examples of mutated FKBPs in which amino acids at positions 26 or 36 have been modified are mentioned in the "Illustrative Mutated FKBPs" table above. Similarly, rapalogs modified at C20 (e.g., rapalogs where R4 is other than -H) are relative to FKBP where Tyr82 and / or Ile56 are replaced with other amino acids, especially amino acids with small side chains. Believes that they are selectively combined. In another illustration, a rapalog modified at C24 (e.g., W is other than = O) has one or more of Phe46, Phe48, and Val55 replaced with another amino acid, again an amino acid having a particularly small side chain. Are thought to selectively bind to FKBP.
Furthermore, rapalogs modified at C28 and / or C30 (eg, where R3 is other than H and / or V is other than OO) are those in which Glu54 is another amino acid, especially an amino acid having a smaller side chain. It is believed that it selectively binds to the substituted FKBP. In all of the above examples, single or multiple amino acid substitutions may be made.
Specific examples are shown in the above table. Instead of repeatedly designing and testing single or multiple mutations, structurally in contact with or adjacent to, or likely to be in contact with, one or more rapalog or rapamycin substituents And randomizing the residues specified at the same time. A polypeptide sample containing a FKBP domain randomized at the specified location (as referred to in the preceding paragraph) is prepared using conventional synthetic or genetic methods. Such a sample represents a collection of FKBP domains containing amino acid substitutions at one or more specified positions. The sample is examined and the FKBP mutant that results in the desired rapalog binding is selected. To provide compensatory mutants with higher affinity and specificity for certain bumped rapalogs that maximize the potential for beneficial cooperative interactions between side chains, It is expected that multiple residues will be randomized simultaneously. Techniques for preparing randomized libraries at discrete locations are known, and primer-directed mutagenesis (primer-dire
cted mutagenesis), PCR using degenerate oligonucleotides, and cassette mutagenesis using degenerate oligonucleotides (eg, Lowman, HBand Wells, JA
.Methods: Comp. Methods Enzymol. 1991.3, 205-216; Dennis, MSand Lazarus, R.
A. 1994, J. Biol. Chem. 269, 22129-22136; and the references therein).

Furthermore, in many cases, direct contact at a particular position in rapamycin or randomization of only a few adjacent residues will result in a rapalog substituent (bump)
It is not possible to fully explore all possible mutants in the FKBP form that can be optimally matched to ()). Therefore, bump-rapalog (bum
In order to identify subtle mutations or combinations thereof that selectively bind to ped rapalogs), the structure is a library containing unbiased random substitutions that are not based on structural considerations. I believe. Alanine-scanning mutagenesis (Cunningham and Wells (1989) Science 24
4,1081-1085), PCR misincorporation mutagenesis (PCR misincorporation)
mutagenesis) (see Cadwell and Joyce, 1992 PCR Meth. Applic. 2, 28-33), and DNA shuffling (Stemmer, 1994, Nature 370, 38-391a).
Several suitable mutagenesis schemes have been proposed, including nd Crameri et al, 1996, Nature Medicine 2,100-103). These techniques create random mutant libraries, or collections of single mutants, which are then explored by screening or sorting.

In many cases, an efficient way to identify the best mutant to selectively bind to a particular bump is to combine a structure-based approach and an unbiased approach. is there. See Clackson and Wells, 1994, Trends Biotechnology 12,173-184 (review). Consider, for example, a library structure in which important contact residues are randomized by PCR using degenerate oligonucleotides but amplified using error-promoting conditions to induce further mutations at random sites. ing.
Another example combines DNA fragments from a library based on structure and a library that is unbiasedly randomized using DNA shuffling.

Using a two-hybrid system (Fields and Song (1989) Nature 340, 245-246),
Screening of the library to obtain preferred mutants can be performed. For example, a FB-VP16 fusion is introduced into a vector, and further randomized FK
The library of BP sequences is cloned into individual GAL4 fusion vectors. The yeast co-transformants are treated with a rapalog and, for example, production of β-galactosidase or luciferase (one screen) or deletion of essential nutrients as appropriate for the vector used. Transformants containing the complementary FKBP mutant are identified by survival on one plate (one selection). Bump-rapamycin (bum
The requirement that ped rapamycin) bridge the FKBP-FRAP interaction would be a useful screen to eliminate false positives.

Another method for isolating modified ligand binding domains from a library of FKBP (or FRB) mutants utilizes genetic selection for functional dimer formation (Hu , JC, et al. 1990, Science 250: 1400-1403; Hu, JC 1995, Structure
3: 431-433). In this method, the cI repressor of lambda (λ) bacteriophage binds to DNA as a homodimer, and when such homodimer binds to operator DNA, it is included in the lysis pathway of the phage life cycle. It takes advantage of the fact that it inhibits the transcription of phage genes. Thus, bacterial cells expressing a functional lambda (λ) repressor are not affected by lysis by superinfection with lambda (λ) phage. The repressor protein contains an amino-terminal DNA binding domain (amino acids 1-92) and is joined to the carboxyl-terminal dimerization domain by a flexible 40 amino acid linker. The isolated N-terminal domain is
Binds to DNA with low affinity due to inefficient dimer formation. High affinity DNA binding can be restored with heterodimerization domains such as GCN4 "leucine zipper". Hu et al. Can mediate lambda (λ) repressor-dimer formation from multiple sequences
A system using phage immunity as a gene selection to isolate GCN4 leucine zipper mutants is shown (Hu et. Al., 1990).

For example, to use a lambda (λ) repressor system to identify FRAP mutants complementary to bumped rapalogs, a lambda (λ) repressor-FRAP with a mutated FRAP sequence The library is transformed into E. coli cells expressing the wild-type lambda (λ) repressor-FKBP protein. Plasmids expressing FRAP mutants have high titers of lambda (λ
3.) Isolate from those colonies that have lysed and survived on bacterial plates containing phage and "bumped" rapamycin compounds. Furthermore, to isolate the FKBP mutant, the above method was repeated to obtain a lambda (λ) having a mutated FKBP sequence.
Repressor-FKBP library-is replaced with wild type lambda (λ) repressor-FRAP
Transform into E. coli cells expressing the protein.

As a further alternative, FKBP sequences randomized to vectors for phage display that allow in vitro selection of variants that bind best to rapalogs can be cloned. Be converted. Affinity sorting in vitro can be performed in a number of ways. For example, in a solution in which a recombinant FRAP with an affinity handle (eg, hexa-histidine tag, or GST) is present, the rapalog is mixed with the library's phadipool and has a complementary mutation. To increase the purity of the phage displaying FKBP, the reaction complex is captured on a suitable affinity substrate. Far
The didisplay technology has already been described and further in vitro selection systems are conceivable (eg display on lambda (λ) phage, display on plasmid, display on baculovirus). Furthermore, screening and screening methods can be used to improve other advantages in the present application, such as enhanced stability in vivo. For summary, Clackson, T. & Wells, JA1994, Trends Bi
See otechnol. 12,173-184.

(B) FRAP In the FRAP-binding portion of the macrocycle, bind selectively to the C3 rapalog containing the modification (eg, being “bumped”), as compared to rapamycin. Similar ideas apply to create different mutated FRB domains. For example, based on the human FRAP FRB peptide sequence but Tyr2038, Phe2039, Thr2
098, Gln2099, Trp2101, Ser2035, Tyr2105, Pro2095, and the FRB in which one or more of rapamycin triene, a residue near the rapamycin triene and any other residue approaching Asp2102 has been substituted with one or more amino acids. A preferential bond can be obtained using a rapalog having a substituent other than -OMe at the C7 position. Exemplary mutants include Y2038H, Y2038L, Y2038V, Y2038A, F2039H, F2039L, F2039A, F
2039V, including D2102A, T2098A, T2098N, and T2098S. Rapalogs substituted at C28 other than -OH and / or substituted at C30 other than = O can selectively bind to FRAP proteins in which Glu2032 has an amino acid substitution. Exemplary mutants include E2032A and E2032S. Bumped rapalo
gs) to identify those mutated FRAPs that selectively bind to the protein, including those FRBs containing one or more amino acid substitutions at the aforementioned locations, at those locations (eg, at those residues). Using the method described above, a protein or peptide library randomized by various methods (including various substituted residues), a library in which entire protein domains are randomized, or a combination of these mutants. did.

The affinity of a candidate mutated FRB for a FKBP protein complex complexed with a rapalog can be measured by a number of techniques; for example, in vitro translated in the presence of a drug. Binding of FRB mutant to GST-FKBP (Che
USA et al. 1995, Proc. Natl. Acad. Sci. USA 92, 4947-4951);
Ability to participate in a rapalog-dependent transcriptional activation complex with the appropriate FKBP fusion protein in a 2- or 3-hybrid assay.

Using various classification methods, FRBs with favorable binding properties can be isolated from phage-expressed libraries. For example, in a solution in which a recombinant FRAP with an affinity handle (eg, a hex-histidine tag, or GST) is present, the rapalog is mixed with the library's phage pool, The reaction complex is captured on a suitable affinity substrate to increase the purity of the phage displaying FKBP with the mutant.

An additional feature of the FRB fusion proteins that differs in various embodiments of the present invention is the exact sequence of the FRB domain used. In some applications, the minimum (8
It may be preferable to use a portion of the FRB that is larger than the (9 amino acids) FRB domain. They are N-terminally extended to Glu2025 residues (preferably at least Ar
g2018 or Ile2021), and the C-terminus is, for example, 2113, 2141, or 2
174 or beyond, extending beyond position 2113, and in some cases, their extension can improve the stability and / or expression efficiency of the folded FRB domain. Other applications in which different FRB sequence endpoints may be used include, for example, stereochemistry on one or both sides of the FRB domain to adjust for polypeptide chain distortions required for FRB-mediated protein-protein binding in cell membranes or DNA. Includes those where long linkers are preferred for structural reasons. Conversely, in other applications, short linkers on one or both sides of the FRB domain are preferred or required to properly present the heteroeffector domain for biological function. In human gene therapy applications, the use of naturally occurring human FRAP sequences as such linkers is commonly used to introduce heterologous sequences or reduce the risk of causing an immune response in the host organism. Would be preferred.

[0155] Some rapalogs have modifications or substitutions at positions believed to be near the border between the FKBP binding domain and the FRAP binding domain, such as C28, C30, C7 and C24 (compared to rapamycin). Rapalogs have a reduced ability to complex with both mammalian FKBP and FRB domains, and especially those domains that include naturally occurring human peptide sequences, as compared to rapamycin. The reduction in capacity is determined as reduced binding affinity as determined by any of the direct or indirect measurement means described herein, or as determined by an appropriate assay such as a T cell proliferation assay. As demonstrated immunosuppressive activity. In such cases, identify a set of mutated FKBPs and a mutated FRB that can form a complex with the rapalog more efficiently than the domain encompassing the corresponding naturally occurring human peptide sequence. To be repeated. For example, as described above, first a complementarily modified FKBP domain capable of binding to a rapalog is identified, and then the mutated FKBP domain is identified as an affinity complexed with a rapalog. By using as a sexual substrate, a complementarily modified FRB domain that can bind to the complex can be selected. By repeating such mutagenesis and screening several times, a protein pair can be optimized.

In certain embodiments, it may be preferable to use FRB / and / or FKBP domains that contain mutations that can affect protein-protein interactions. For example, mutated FKBP domains that are capable of forming a complex with the endogenous FRB at a reasonably lower efficiency than the mutated FRB when bound to a particular rapalog are of particular interest. Also of interest are mutated FRB domains that can bind to mutated FKBP-specific rapalog complexes with a reasonably high efficiency over endogenous FKBP-rapalog complexes. Using selection and screening methods similar to those described above, (i) the domain may have a modest reduction in its ability to form a tripartite complex with a particular rapalog and endogenous FRB. An FRB domain that identifies amino acid substitutions, deletions, or insertions in the FKBP domain, and (ii) moderately reduces the ability of the domain to form a triple complex with a particular rapalog and endogenous FKBP. (Iii) identifying amino acid substitutions, deletions or insertions with respect to
Mutating mutations in the partner protein can be selected and / or identified. As examples of suitable mutated FKBPs that form triplicate complexes with reduced efficiency, one or both of His87 and Ile90 have Arg, Trp, Phe, Tyr, or Tyr containing large side chain groups. Mammalian, preferably human FKBP, which is substituted with an amino acid such as Lys; preferably a FRB domain comprising a peptide sequence of a mammal, more preferably human, comprising a mutated FKBP and a specific rapalog Mutations are further made as described above to generate complementary mutants that can form a complex. Illustrative F that may be useful with H87W, or H87R hFKBP12
RB mutants include human FRBs in which Y2038 has been replaced with V, S, A, or L; F2039 has been replaced with A; and / or R2042 has been replaced with L, A, or S. ing. Illustrative FRB mutants that may be useful with I90W or I90R hFKBP12 include human FRBs in which K2095 has been replaced with L, S, A, or T.

Furthermore, in optimizing the receptor domain of the present invention, the immunogenicity of the polypeptide sequence is determined by the fact that the peptide binds to the MHC protein and the peptide is recognized as a foreign substance by the endogenous T cell receptor. I think it is necessary. At least for human gene therapy applications, it is preferable to prepare a specific foreign peptide sequence including a connecting peptide sequence according to requirements in order to minimize the possibility of immunogenicity in humans. For example, a peptide that binds to a human MHC class I molecule is:
There are stringent requirements for specific residues at key "anchor" sites in the attached peptide; for example, HLA-A2 requires that site 2 be leucine, methionine, or isoleucine and the C-terminus is leucine. Or valine (S
trem and Wiley (1994) Structure 2,145-251 for summary). Therefore, in protein engineering in the practice of the present invention, and especially in human gene therapy applications, it is preferable to avoid the periodicity of such residues. The foregoing applies, without limitation, to all protein engineering situations of the present invention, including partial mutations to the receptor domain, and to the selection or design of boundaries between various protein domains.

Other Components, Design Features, and Applications The chimeric protein may include a cell localization domain as a heterodomain, such as a membrane-retaining domain. See, for example, PCT / US94 / 01617, especially pages 26-27. In particular, the membrane retention domain, whether endogenous to the host cell or not, can be isolated from any convenient membrane bound protein. The membrane retention domain may be a transmembrane retention domain, such as an amino acid sequence extending through a membrane, as in the case of cell surface proteins containing many receptors. The transmembrane peptide sequence may extend into the span part or all extracellular and / or intracellular domains. Further, the membrane retention domain may be a lipid membrane retention domain, such as a myristoylated or palmitoylated site capable of binding cell surface membrane lipids. A lipid membrane-retaining domain is usually added at the 5 'end of the coding sequence for N-terminal attachment to the membrane and proximate the 3' end for C-terminal attachment. Carr is a peptide sequence that requires post-translational processing for lipid membrane binding
, et al, PNAS USA (1988) 79,6128; Aitken, et al., FEBS Lett. (1982) 150, 314; Hen
derson et al., PNAS USA (1983) 80, 319; Schulz, et al., Virology (1984), 123, 2131; Dellman, et al., Nature (1985) 314, 374;
v. of Biochem, (1988) 57,69. Amino acid sequences of interest include the sequence MGGSKPKDPSQR. Various DNA sequences can be used to encode such sequences in various chimeric proteins of the invention. Other localization domains, as well as nuclear localization sequences useful in chimeric proteins designed specifically for (direct) transcriptional regulation, may be organelle targeting domains.
ella-targeting domains) as well as sequences such as KDEL and HDEL that target proteins that carry them to the endoplasmic reticulum. Various cell localization sequences and signals are well known in the art.

[0159] Cytoplasmic signal initiation domains such as the CD3 zeta (核酸) chain, nucleic acid transcription factor domains including VP16 and GAL4 among others, apoptotic domains including the Fas cytoplasmic domain, etc. More detailed descriptions that can be used in the practice of the present invention with respect to designing, assembling and using structures encoding chimeric proteins including various effector domains including PCT / US94 / 01617 and PCT / US95 / 10591
Is disclosed. The latter international application further discloses additional features that are particularly applicable to the generation of genetically engineered animals that can be used as disease models in biopharmaceutical research. Their features include the use of tissue-specific regulatory elements within the construct for chimeric protein expression, and the expression of Cre recombinant enzyme as a target gene, leading to the release of the gene of interest flanked by LoxP sequences. Including the application of transcriptional regulation. Alternatively, flp and its cognate recognition sequence may be used instead of Cre and lox. Those features may be adapted to the subject invention.

In the case of species, especially in embodiments involving whole animals having cells designed in accordance with the present invention, it is often preferred that the various domains of the chimeric protein be derived from a protein of the same species as the host cell. And in some cases it may be needed. Thus, for genetic manipulation of human cells, heterodomains (as well as FKBP and FRB domains) are preferably derived from humans, rather than from bacteria, yeast, or other non-humans . Also, for convenience in detection, epitope tags may be incorporated into the chimeric proteins of the present invention.

Tissue-Specific or Cell-Type Specific Expression In certain embodiments, it may be desirable for the chimeric protein to be expressed in a cell-specific or tissue-specific manner. Such expression specificity is achieved by operably linking one or more DNA sequences encoding the chimeric protein to a cell type-specific transcriptional regulatory sequence (eg, a promoter / enhancer). . Numerous cell type-specific transcriptional regulatory sequences are known. Other sequences are obtained from genes that are expressed in a cell-specific manner. See PCT / US95 / 10591, especially pages 36-37.

For example, a construct for expressing a chimeric protein may include a regulatory sequence from a known gene for specific expression in a selected tissue.

The following are representative examples:

[Table 7]

[Table 8] Target Gene Structure In embodiments of the present invention in which a chimeric protein is designed such that multimerization activates transcription of the target gene, an appropriate target gene structure is used in recombinant cells. Suitable target gene structures are those that include cognate transcriptional regulatory elements such as promoters and / or enhancers that are sensitive to multimerization of the target gene and chimeric protein. In embodiments involving direct activation of transcription, the sensitivity is targeted to one or more DNA sequences recognized by the DNA binding domain of the chimeric protein of the invention (eg, the DNA sequence to which the chimeric protein binds). It is brought about by being within the genetic structure. In embodiments involving indirect activation of transcription, the reactivity may be due to the presence of promoter and / or enhancer sequences in the target gene structure that are activated by intracellular signals resulting from the multimerization of the chimeric protein. It is brought by doing. For example, a chimeric protein
If the target gene includes the intracellular domain of the TCR zeta (標的) chain, the target gene is linked to the IL-2 promoter site, and is further regulated under the expression control of the IL-2 promoter site. I will

The present invention also provides (a) a cognate DNA sequence to which a DNA binding chimeric protein of the invention can bind (or is susceptible to indirect activation as described above), and (b) The flanking DN of a preferred target locus that is endogenous to the host cell
Provide a target DNA structure including the A sequence. These structures, in association with the endogenous target gene, cause homologous recombination of the cognate DNA sequence to the host cell. In another embodiment, the structure includes a preferred gene and adjacent DNA sequences from the target locus that cause homologous recombination of the target gene at the preferred locus. Such a target structure may also include a cognate DNA sequence, or the locus may provide cognate DNA.

In any of the foregoing embodiments, the target gene may encode, for example, a surface membrane protein (eg, a receptor protein), a secreted protein, a cytoplasmic protein, a nucleoprotein, a recombinant enzyme such as Cre, a ribozyme or an antisense RNA. it can. PCT / US for general design and detailed construction, and for various applications including gene therapy
See PCT / US95 / 10591 for application to animal models of disease.

The present invention includes various configurations of chimeric proteins. In all cases involving the transcriptional activation of the target gene, however, the chimeric protein is an important feature: the structure encoding the chimeric protein, and the cell containing the target gene structure, is capable of undergoing the action in the presence of a multimerizing ligand. at least 10 times than that but not present, preferably at least 10 ² times, even more preferably at least 10 ³ fold or 10 ⁴ fold or 10 ⁵ fold higher, expressing the target gene: sharing. Optimally, no expression of the selected gene is observed if the cells are not or are not exposed to the multimerizing ligand. In summary, the chimeric protein may initiate detectable levels of transcription of the target gene in the recombinant cell following exposure of the cell to, for example, a C3 rapalog, following multimerization of the chimeric protein. Then, when the cells are exposed to the C3 rapalog so that the chimeric protein molecule can be multimerized, transcription of the target gene is subsequently activated in the recombinant cells of the invention. In other words, the recombinant cells of the present invention include chimeric proteins, as described above, and are responsive to the presence and / or concentration of C3 rapalogs capable of multimerizing those chimeric protein molecules. Its reactivity is manifested by the transcriptional activation of the target gene. Such transcriptional activation can be readily detected by any conventional measure of target gene transcription. In another embodiment, the biological response to ligand-mediated multimerization of the chimeric protein is cell death, or other biological phenomenon, rather than direct transcriptional activation of the target gene.

Design and Assembly of DNA Structures Structures can be designed according to principles, examples, or materials or methods disclosed in the patent or scientific literature cited therein, each of which As described herein, along with modifications and other illustrations, are incorporated herein by reference. The structural components can be isolated in a conventional manner such that the coding sequence and regulatory sites can be isolated, ligated, cloned into a suitable host, and analyzed by restriction or sequencing or other convenient means. Can be prepared. In particular, using PCR to isolate individual fragments containing all or part of a functional unit capable of introducing one or more mutations by "primer repair", ligation, in vitro mutagenesis, etc. can do. In the case of a DNA structure encoding a fusion protein, the fusion protein encoding unit is such that the DNA sequences encoding the individual domains and subdomains can be translated into a single polypeptide containing all the component domains in a cell or cell lysate. They are connected so as to form an open reading frame. The DNA structure encoding the fusion protein can be further inserted into a vector that directs the expression of the protein in an appropriate cell type. In bacteria, for biochemical analysis of the encoded chimeric protein,
Alternatively, it may be preferable to construct a plasmid that directs the expression of the protein in a reticulocyte lysate system. For use in producing proteins in mammalian cells, sequences encoding the protein are introduced into expression vectors that direct expression in those cells. Expression vectors suitable for such use are well known in the art. A variety of such vectors are commercially available.

[0168] The structure encoding the chimeric protein and the target gene of the invention are often linked to one or more markers to allow for the selection of host cells containing the structure, resulting in one or more markers. They can be introduced into cells as DNA molecules or structures. Once completed,
The structure, which has been proved to have proper sequence, can then be introduced into the host cell by any convenient means. The structure may be incorporated into a vector capable of episomal replication (eg, a BPV or EBV vector) or a vector designed to integrate with the chromosome of the host cell. The structure can be used to infect or transduce cells, such as adenovirus, adeno-associated virus (AAV), or herpes simplex virus (HSV), or other viruses containing retroviral vectors. It may be integrated and packed into the genome of the non-replication defective virus. The following more detailed description discusses a virus delivery system. Alternatively, the structure may be introduced by protoplast fusion, electroporation, biolistics, calcium phosphate, lipofection, microinjection, or similar methods. In some cases,
Prior to introducing the structure, the host cells are cultured and grown and expanded, and then appropriate treatment is performed to introduce the structure and integrate the structure. The cells are grown further and sorted by the effect of the markers present in the structure. Various markers that can be suitably used include hprt, neomycin resistance, thymidine kinase, hygromycin resistance, etc., and various cell surface markers such as Tac, CD8, CD3, Thyl and NGF receptors. Including.

In one embodiment, the structure has a target site for homologous recombination, preferably integrated at a particular locus. For example, one endogenous gene can be deleted and / or replaced with a recombinant target structure of the invention (at the same locus or elsewhere). For homologous recombination, generally either the 1/2 vector or the O-vector can be used. For example, Thomas and Capecchi, Cell (1987) 51,503
-512: Mansour, et al., Nature (1988) 336,348-352; Joyner, et al., Nature (198
9) See 338,153-156.

[0170] The structure can be introduced as one single DNA molecule encoding all genes, or as different DNA molecules having one or more genes. The structures may each be introduced with the same or different markers, simultaneously or sequentially.

Replication / selectable and / or amplifiable markers from bacteria or yeast, promoter / enhancer elements for expression in prokaryotes or eukaryotes, and mammalian expression control elements Vectors that can be used to prepare stocks of structural DNA containing useful elements, such as, as well as to transfect, are well known in the art and many are commercially available.

Transport of Nucleic Acids: Any means for introducing ex vivo and in vivo heteronucleic acids into host cells, especially eukaryotic cells, especially animal cells, preferably human or non-human mammalian cells, is contemplated in the practice of the present invention. Can be adapted. For the purpose of examining this, the various nucleic acid structures described herein can be referred to as transgenes. Ex vivo methods for DNA transport include the calcium phosphate method, electroporation, lipofection, and viral vector-mediated infection. Two common methods of in vivo gene therapy include (a) D complexed with “naked” lipids.
There is the transport of DNA shaped in the form of NA or liposomes, or of DNA shaped elsewhere, and (b) the transport of heteronucleic acids via viral vectors. In the former method, for example, a plasmid containing a transgene having the desired DNA structure can be first experimentally optimized for expression (eg, 5 ′ untranslated) before forming DNA with lipids. (Including introns at sites and removing unwanted sequences) (Felgner, et al., Ann NY Acad Sci 126-139, 1995). Using well-known methods and materials, the DNA can be formed, for example, from various lipids or liposomal materials, and transported to a further implanted mammal.

Although a variety of viral vectors may be used in the practice of the present invention, methods based on retroviruses, AAA- and adenovirus are of particular interest. For example, Dubensky et al. (1984
Acad. Sci. USA 81, 7529-7533; Kaneda et al., (1989) Science 243, 37).
5-378; Hiebert et al., (1989) Proc. Natl. Acad. Sci. USA 86, 3594-3598; Hatzogl.
u et al., (1990) J. Biol. Chem. 265, 17285-17293 and Ferry et al., (1991) Proc. N.
See atl.Acad.Sci.USA 88, 8377-8381. The following additional guidance on the selection and use of viral vectors may be helpful to practitioners.

Retroviral Vectors—Retroviral vectors are a group of RNA viruses whose RNA genome is reversibly transcribed into DNA in infected cells. The retroviral genome integrates into the host cell genome and requires three genes, gag, pol, and env, similar to viral long terminal repeats (LTRs). LTRs also act as enhancers and promoters for viral genes. The viral packaging sequence (Y) distinguishes viral RNA from other RNAs in cells (Verma et al., Nature 389: 239-242, 1997). For expression of the foreign gene, the viral protein is replaced in the viral vector by the gene of interest, and the gene of interest is further transfected into a packaged cell line containing the package components of the virus. The package virus is secreted into the medium from the packaged cell line and can be used to further infect cultured cells. Because retroviruses cannot infect non-dividing cells, they have been used primarily for ex vivo gene therapy.

AAV Vectors-Vectors based on adeno-associated virus (AAV) are of general interest as carriers for various tissues, including muscle and lung. AAV vectors infect cells and frequently integrate tightly into the cell genome. AAV can infect and integrate with growth arrest cells (eg, lung epithelial cells) and is non-pathogenic.

[0176] Commonly used AAV-based expression vectors are available using direct available restriction enzyme sites, or blunting their ends following truncation of the transgene with a restriction enzyme to provide an appropriate DNA linker. 145 nucleotide AAV inverted terminal repeats (ITRs) adjacent to a restriction enzyme recognition site that can be used for subcloning of the transgene either by ligation, restriction digestion, and ligation to a site between ITRs Is included. The maximum capacity of the AAV vector is about 4.4 kb. The following proteins are expressed using various AAV-based vectors and various promoters / enhancers: neomycin phosphotransferase, chloramphenicol acetyltransferase, Fancon anemia gene, cystic fibrosis. Transmembrane conductivity regulatory proteins and granulocyte macrophage colony-stimulating factors (Kotin, RM, Human Gene Therap
y 5: 793-801, 1994, Table 1). Transgenes incorporating the various DNA structures of the present invention can likewise be included in AAV-based vectors. Instead of including a structural promoter such as CMV to express recombinant DNA encoding the fusion protein, an AAV promoter can be used (ITR itself or AAV p5 (Flottr, e
etal., J. Biol. Chem. 268: 3781-3790, 1993)).

[0177] Such vectors can be packed into AAV virions by the reported methods. For example, a human cell line such as 293 may be transformed into an AAV-based expression vector and an AAV rep (cap) encoding cap under the control of an endogenous AAV promoter or heteropromoter. Co-transfection can be performed with another plasmid, including a packaging frame. In the absence of the helper virus, the rep proteins Rep68 and Rep78 prevent the accumulation of replicative molecules, but upon superinfection with adenovirus or herpes virus, these proteins are reduced to ITRs (in the structure containing the transgene). ) And expression of the viral capsid protein. This system results in a transgene
Packing DNA into AAV virions (Carter, BJ, Current Opinion in Biotechnology
3: 553-539, 1992; Kotin, RM, Human Gene Therapy 5: 793-801, 1994). In addition, in order to express the transgene in the AAV virion, a method for improving the titer of AAV can be used. Such methods include, but are not limited to: stably expressing the ITR flanking transgene in a cell line, followed by transfection with a second plasmid to direct viral packing; temperature-sensitive inducible expression or This involves using a cell line that is induced to express the AAV protein, such as drug-induced expression. Further, as described in Wilson et al., WO 96/39530, the efficiency of AAV transduction can be improved by treating cells with an agent that promotes the conversion of single-stranded molecules to double-stranded molecules. Can be improved.

The cesium chloride density gradient centrifugation method used in the first report of AAV vector expression in vivo (Flotte, et al., J. Biol. Chem. 268: 3781-3790, 1993), or O'Rio
Viruses can be concentrated and purified by reported methods, such as the chromatography-purification method described in edan et al., WO 97/08298.

[0179] The invention may be useful in practicing the subject invention, including methods and materials for integration of the transgene, propagation and purification of the recombinant AAV vector containing the transgene, and its use in transfected cells and mammals. For more detailed guidance on AAV technology, see, for example, Carter et al., U.S. Pat.No. 4,797,368 (10 Jan 1989).
Muzyczka et al., U.S. Patent No. 5,139,941 (18 Aug 1992); Lebkowski et al., U.S. Patent No. 5,173,414 (22 Dec 1992); Srivastava, U.S. Patent No. 5,252,479 (12
Oct 1993); Lebkowski et al., U.S. Patent No. 5,354,678 (11 Oct 1994); Shenk et
al., U.S. Pat.No. 5,436,146 (25 July 1995); Charterjee et al., U.S. Pat.
454,935 (12 Dec 1995), Carter et al WO93 / 24641 (9 Dec 1993 published), and Flo
See tte et al., U.S. Patent No. 5,658,776 (19 Aug 1997).

Adenovirus Vectors-Various adenovirus vectors have been found to be useful in delivering genes to mammals, including humans. Incompletely replicating adenovirus vectors have been used to express marker proteins and CFTR in lung epithelium. The first-generation E1a-deficient adenovirus vector is improved in the second generation, which contains a temperature-sensitive E2a virus protein, expresses less viral protein, and thus makes virus-infected cells less likely to be targeted by the immune system (Glodman et al., Human
Gene Therapy 6: 839-851, 1995). More recently, all virus open
A viral vector lacking the ding frame has been reported (Fisher e
tal., Virology 217: 11-22, 1996). Furthermore, it has been shown that expression of viral IL-10 inhibits the immune response to adenovirus antigens (Qin Jiang Tal., HumanGen.
eTherapy 8: 1365-1374, 1997).

A number of adenovirus-type DNA sequences are available from Genbank. The adenovirus DNA sequence can be obtained from 41 human adenovirus types identified to date. Various adenovirus species are available from the American Type Culture Collection, Rockville, Maryland, or by applying to a number of commercial and academic sources. The transgene as described herein can be optionally ligated by the same methods used to insert CFTR or other genes into the vector (restrictive digestion, linker ligation or filling into ends, and ligation). Adenovirus vectors, and transport protocols. Hybrid adenovirus-AA represented by an adenovirus capsid including selected sites in adenovirus sequences, 5 'and 3' AAV ITR sequences flanking the transgene, and other conventional vector-regulatory elements
The A vector may be used. For example, see Wilson et al., International Patent Application Publication No.
See / 13598. Adenoviruses and hybrids useful in the practice of the present invention, including methods and materials for transgene integration, propagation and purification of recombinant AAV vectors containing the transgene, and their use in transfected cells and mammals For further guidance on adenovirus-AAV technology, see Wilson et al., WO 94/28938, WO 96/13597 and WO 96/26285, and the references cited therein.

In general, the DNA or virus particles may be in sterile saline, many examples of which are well known in the art, or other water-soluble or water-insoluble, including Ringer-phosphate buffers or other similar solvents. Transfer to a biologically compatible liquid such as an isotonic sterile injectable solution or suspension or a pharmaceutically acceptable transport solvent.

Preferably, in gene therapy applications, the DNA or recombinant virus is injected in an amount sufficient to transfect cells at a level that produces a therapeutic effect without undue side effects. The optimal dosage of DNA or virus, as discussed elsewhere, depends on a variety of factors and may therefore vary somewhat from patient to patient. Also, the amount of therapeutically effective virus can be from about 1 × ^{10 7} to about 1 × ^{10 10} pfu virus / ml, such as 1
From x10 ⁸ the saline solution at a concentration of 1x10 ⁹ pfu virus / ml is believed to approximately 20ml in the range of about 50 ml.

Host Cells The present invention is particularly useful in animal cell engineering, and in applications utilizing such recombinant animal cells. The animal cell can be an insect, parasite, or mammalian cell. As an example, a variety of mammalian cells can be used, including horses, cows, sheep, dogs, cats, rats, and non-human primate cells, with human cells being of particular interest. Among various species, hematopoietic cells, nerve cells, glial cells, mesenchymal cells, skin cells, mucosal cells, stromal cells, muscle cells (including smooth muscle cells), spleen cells, reticuloendothelial cells,
Various types of cells such as epithelial cells, endothelial cells, hepatocytes, kidney cells, gastrointestinal cells, lung cells, fibroblasts and the like can be used. Cells of particular interest are hematopoietic cells, including myoblasts and fibroblasts, as well as any agglutinating cells that may be involved in the red blood cells, lymph, or myeloid lineage. Also of interest are stem cells and progenitor cells such as hematopoietic cells, nerve cells, stromal cells, muscle cells, hepatocytes, lung cells, gastrointestinal cells, and mesenchymal stem cells.

The cells can be autologous, syngeneic, allogeneic, and optionally heterologous to the organism of interest. Inactivate β2-microglobulin to prevent the formation of functional MHC class I molecules, inactivate class II molecules, and
To provide the expression of C and enhance or inhibit the expression of genes associated with cytotoxicity to enhance or inactivate cytotoxicity, thereby altering the characteristics of major histocompatibility complex (“MHC”) Can modify cells.

In certain embodiments, certain clones, such as T cells and B cells, which have unique antigen specificity or homing target site specificity, such that the cells have specific specificity,
Or interested in oligoclonal cells.

Introduction of Structures into Animals Cells ex vivo modified with DNA structures can be cultured and grown under selective conditions, and cells selected as having the desired structure can be For example, using a polymerase chain reaction to determine the presence of the structure in the host cell and / or using an assay for production of the desired gene product, and further analyzed. Once the modified host cells are identified, they can be used as designed, for example, in culture or introduced into a host organism.

Depending on the nature of the cells, the cells can be introduced into the host organism, eg, a mammal, in a wide variety of ways. Hematopoietic cells can usually be administered by injecting at least about 10 ⁴ cells, and generally about 10 ¹⁰ cells or less, into the vascular system. The number of cells used depends on the situation, the purpose of the transfer, the lifetime of the cells, the protocol used, such as the number of doses, the growth potential of the cells, the stability of the therapeutic agent, the physiological requirements for the therapeutic agent. It will depend on gender etc. Generally, for example, for myoblasts or fibroblasts, the number of cells is from about 10 ^{4 to} about 10 ⁹ , used in a dispersed manner and generally injected at or near the site of interest. The cells are usually in a physiologically preferred medium.

Cells engineered in accordance with the present invention may also be encapsulated, eg, using conventional biocompatible materials and methods, prior to implantation into a host organism or patient to produce a therapeutic protein. Good. For example, guyen et al. Tissue Implant
Systems and Methods for Sustaining viable High Cell Densities within a H
ost, U.S. Patent No. 5,314,471 (Baxter International, Inc.); Uludag and Sefton
1993, J. Biomed.Master.Res. 27 (10): 1213-24 (HepG2 cells / hydroxyethyl metha
crylate-methyl methacrylate membrane); Chang et al., 1993, Hum Gene Ther 4
(4): 433-40 (mouse Ltk-cells expressing hGH / immunoprotective perm-selectiv
e alginate microcapsules; Reddy et al., 1993, J. Infect Dis 168 (4): 1082-3 (
alginate): Tai and Sum, 1993, FASEB J7 (11): 1061-9 (mouse fibroblasts express
ing hGH / alginate-poly-L-lysine-alginate membrane); Ao, et al., 1995, Trans
plantation Proc. 38 (6): 3349, 3350 (alginate); Rajotte, et al., 1995, Transpla
ntation Proc. 27 (6): 3389 (alginase); Lakey, et al., 1995, Transplantation Pro
c.27 (6): 3266 (alginate); Korbutt, et al, 1995, Transplantation Proc.27 (6): 32
12 (alginate); Dirian, et al., US Pat. No. 5,429,821 (alginate); Emerich, et.
al., 1993, Exp Neurol 122 (1): 37-47 (polymer-encapsulated PC12 cells); Sagen, et al., 1993, J. Neurosci 13 (6): 2415-23 (bovine chromaffin cells enca psulated in Semipermeable polymer membrane and implanted into rat spinal subarachnoid space); Aebischer, et al., 1994, Exp. Neurol 126 (2): 151-8 (polymer-encapsulated rat PC12 cells implanted into monkeys; Albischer, WO9 2/19595 Savelkoul, et al., 1994, J. Immunol Methods 170 (2): 185-96 (encapsulated hybridomas producing antibodies; encapsulated transfected cell lines expressing various cytokines); Winn, et al., 1994, PNAS USA. 91 (6): 2324-8 (engineered NHK cells expressing human nerve growth factor encap sulated in an immunoisolation polymeric device and transplanted into rat s); Emerich, et al., 1994, Prog. Neuropsychopharmacol Biol. Psychiatry 18 (5): 935-46 (polymer-encapsulated PC12 cells implanted into rats); Kordower, et al., 1994, PNAS USA 91 (23): 10898-902 (polymer-encapsulated engineered BHK cells expressing hNGF implanted into monkeys). And Butler, et al., WO 95/04521 (encapsulated device). The cells can then be introduced in encapsulated form into an animal host, preferably a mammal, more preferably a patient in need thereof. Preferably, the encapsulant is semi-permeable and capable of releasing secreted proteins produced by the encapsulated cells to the host. In many embodiments, semipermeable encapsulation allows the encapsulated cells to be immunologically isolated from the host organism into which they are introduced. In these embodiments, the encapsulated cells can express one or more chimeric proteins that include structural domains from protein and / or viral proteins of the host species or from species other than the host species. For example, in such a case, the chimeric protein can include elements from GAL4 and VP16. The cells may be from one or more individuals other than the transplant recipient, and may also be from a species other than the organism or patient being transplanted.

As an alternative to ex vivo modification of cells, in many situations, it may be desirable to modify cells in vivo. To this end, various techniques for modifying cells in vivo have been developed. As mentioned above, adenovirus, adeno-associated virus,
Numerous viral vectors have been developed that allow transfection, and in some cases, link the virus to a host, such as and retroviruses. For example, Dubensky, et al., (1984) Proc. Natl. Acad. USA 81, 7529-1533; Kaneda.
, et al., (1989) Science 243, 375-378; Hiebert, et al., (1989) Proc. Natl. Acad.
Sci. USA 86, 3594-3598; Hatzoglu, et al., (1990) J. Biol. Chem. 265, 17285-17293 and Ferry, et al., (1991) Pro. Natl. Acad. Sci. USA 88, See 8377-8381. For example, the vector can be administered by vascular or intramuscular injection, or by inhalation, or other parenteral methods. Non-viral delivery methods may also be used, such as administering the DNA in complex with the liposome or by injection, catheter or biolistics.

Based on in vivo gene modification, the mode of modification depends on the nature of the tissue, the required efficiency of cell modification, the opportunity to modify particular cells, the accessibility of the tissue to the DNA structure to be introduced, etc. . By using an attenuated or modified retrovirus carrying the transcription initiation region, if desired, the virus can be produced using one of the transcription factor structures of interest such that the virus is produced and transfected into adjacent cells. Can be activated.

DNA transfer does not require integration in all cases in all cases. In some situations, it may be sufficient to keep the introduced DNA temporarily. In this case, there is a short-term effect that the cells are introduced into the host and then act after a certain period of time, for example, after the cells can be present at a particular location.

Binding Properties, Assay Rapamycin is known to bind to the human protein FKBP12 and form a triad with hFKBP12 and FRAP, the human counterparts of yeast TOR1 and TOR2. Rapalog has binding ability to human FKBP12 and / or human FKBP12 and human
The ability to form a tripartite complex with FRAP (or a fusion protein or a fragment encompassing the FRB domain thereof) can be characterized and compared to rapamycin. See WO96 / 41865 (Clackson, et al.). The patent application discloses the ability of the compound to bind to human FKBP12 or to form a ternary complex with a protein comprising human FKBP12 and the FRB domain of human FRAP (eg, a "heterodimerize") protein. Disclosed are materials and methods that can be used to quantify Such assays include fluorescence polarimetry to measure binding. Also,
The ability of a compound to form a tripartite complex is cell-based, as measured indirectly by correlation with the observed levels of gene products produced by engineered mammalian cells in the presence of the compound. Transcription assays are also included. A corresponding cell-based assay has also been performed on engineered yeast cells. For example, WO95 / 33052 (Berlin, et al.) The rapalogs of the invention are physiologically preferred (without undue side effects on the cells or organisms in which they are used) and can be administered orally to animals (eg, gene therapy It will often be preferable to be able to cross cell and other membranes, as required for certain applications, and / or active for oral administration in applications including animals).

Further, preferred rapalogs are immunophilins that are more mutated than natural or naturally occurring immunophilins (by way of non-limiting example, amino acids in which Phe36 differs, preferably large amino acids such as valine, alanine). It is a rapalog that selectively binds to human FKBP (where the R group is replaced with fewer amino acids). For example, such compounds can be mutated to FKBP mutated at least 10-fold more efficiently than they bind to human FKBP12, as determined by any scientifically sound or technically accepted assay. can be selectively coupled against, in some cases even at 10 ² times or more, or 10 ³ times or more efficient than binding to human FKBP2, it can bind to the mutated FKBP. The binding affinity of various rapalogs of the present invention to human FKBP12, its variants or other immunophilin proteins can be determined by adapting known methods used for FKBP. For example, a practitioner can measure the ability of a compound of the invention to compete for binding of a known ligand to the protein of interest. Sierkierka, et al., 1989, Nature 341,755-757 (te
st compound competes with binding of labeled FK506 derivative to FKBP).

One set of preferred rapalogs of the present invention is a direct binding assay for human FKBP12, or a mutant thereof as described above, or a fusion protein comprising such an FKBP domain ( Less than about 200 nM, more preferably less than about 50 nM, as measured by competitive binding assays (eg, against FK506), inhibition of FKBP enzyme activity (rotamase), or other assays. More preferably, it binds with a Kd value of about 10 nM or less, more preferably about 1 nM or less. In one subset of such compounds, the FKBP domain has the phenylalanine at position 36 replaced with an amino acid that does not contain much large side chains, such as alanine, valine, methionine, or serine. Competitive binding FP assays are described in detail in WO96 / 41865. The method allows for an in vitro measurement of the IC50 value of a particular compound, reflecting the ability to compete with a labeled FKBP ligand, eg, FK506, to bind to FKBP protein.

One preferred class of compounds of the present invention is specific FKBP domains and ligands, such as, for example, human FKBP12 or a variant thereof having at least 10 amino acids, preferably at least 5 amino acids, and a fluorescently labeled FK506. 10 pairs in a competitive binding FP assay
00 nM or less, preferably 300 nM or less, more preferably 100 nM or less, more preferably 10 nM or less.
Rapalog with an IC50 of nM or less. In one subset of that class, at position 36, the FKBP domain has one of the modifications described above.

The ability of rapalogs to multimerize chimeric proteins can be measured by measuring the occurrence of events induced by such multimerization, using a cell-based assay (cel).
l-based assay). For example, DNA encoding a first chimeric protein comprising one or more FKBP domains and one or more effector domains, and one FRB domain and one or more effectors capable of multimerizing and activating a biological reaction.
Cells containing and capable of expressing both DNAs encoding the second chimeric protein encompassing the domain can be used. It is preferable to use cells that further contain a receptor gene that is under transcriptional control of a regulator (eg, a promoter) that responds to multimerization of the chimeric protein. The design and preparation of specific compounds, and their use in engineered cells, is described in WO 96/41865 and other international patent applications referenced in this and the preceding paragraphs. The cells are cultured for growth or maintenance. After the rapalog is added to the medium and incubated for a suitable period of time (allowing expression and secretion of the gene, eg, one hour or one day and night), the gene product of the reporter gene is measured. Positive results, such as multimerization, are correlated with reporter gene transcription as observed by the appearance of the reporter gene product. The reporter gene product is a conveniently detectable protein (eg, by ELISA) or can catalyze the production of a conveniently detectable product (eg, colored). Materials and methods for generating suitable cell lines for performing such assays are disclosed in the international patent applications cited earlier in this paragraph. Commonly used target genes include, by way of example, SEAP, hGH, beta (β) galactosidase, green fluorescent protein, and luciferase, for which convenient assays are commercially available. Can be used

Another preferred class of compounds of the present invention are those that are capable of inducing a detectable signal in a two-hybrid transcription assay based on a fusion protein that includes an FKBP domain. Preferably, the FKBP domain is an FKBP other than wild-type human FKBP12.
Domain.

Other assays that measure the ability of rapalogs to multimerize chimeric proteins include:
It is a cell-based assay that measures the occurrence of events induced by such multimerization. In this case, cells that structurally express the detectable product are used. The cell may also contain a DNA encoding a chimeric protein that includes one or more immunophilin-derived ligand binding domains and one or more effector domains, such as the intracellular domain of FAS that can multimerize and induce cell death. Can include and manifest. The design and preparation of specific components and their use in engineered cells is described in WO 95/02684. See WO96 / 41865. Cells are maintained or cultured in a medium that allows for cell growth or continued viability. The structural cell products in the cells or medium are measured and subsequently a baseline level reporter is established. Engineered cells can be used to constructively produce hGH or any other conveniently detectable product that acts as a reporter. The compound to be tested is added to the medium, the cells are incubated, and the presence of the reporter in the cell lysate or medium is tested at multiple time points. Decreased reporter production indicates cell death and is an indirect measure of multimerization of the fusion protein.

Another preferred class of compounds of the present invention is the FKBP / FRB-based apopto-
It can induce a signal detectable in a cis assay. Preferably, the FKBP domain is an FKBP domain other than wild-type human FKBP12. In some cases,
The FKBP domain is modified as described above. Also preferably, the FRB domain is a FRB domain other than the wild-type FRB of human FRAP. In some cases, the FRB domain is modified at position 2098 as described above.

Performing such an assay allows the practitioner to select a rapalog that has a preferred IC50 and / or binds preferentially to FKBP mutated over wild-type human FKBP12. I do. Competitive binding FP assays allow selection of monomers or rapalogs that have a preferred IC50 and / or that bind selectively to mutated or wild-type FKBP relative to a control such as FK506. And

Uses As described in WO94 / 18317, WO95 / 02684, WO96 / 20951, WO95 / 41865,
Rapalog includes gene therapy applications, for example, in engineered cells growing in culture or in an intact organism, regulatablely activates transcription of the desired gene, causing the target gene to be deleted, It can be used to activate apoptosis or cause other biological events. The following are non-limiting applications of the present invention.

1. Regulated Gene Therapy In many instances, being able to freely turn on or off a therapeutic gene or being able to accurately measure expression is important for therapeutic efficacy. The invention is particularly well-suited for achieving regulated expression of target therapeutic genes in the context of human gene therapy. In one example, a pair of chimeric proteins (one containing at least one FRB domain and another containing at least one FKBP domain), a C3 rapalog of the invention that is capable of dimerizing with the chimeric protein, And the target gene structure to be expressed. One of the chimeric proteins comprises a DNA binding domain as a heteroeffector domain, preferably a synthetic DNA binding domain as described in Pomerantz, et al, suppra. The second chimeric protein includes a transcription activation domain as a heteroeffector domain. The C3 rapalog binds to both chimeric proteins and can efficiently crosslink the chimeric proteins. DNA molecules encoding and capable of expressing these chimeric proteins are introduced into the cells to be manipulated. In addition, a target gene linked to the DNA sequence so that the DNA binding domain can bind is also introduced. Contacting the engineered cells or their progeny with a C3 rapalog (by administration to an animal or patient) causes the assembly of the transcription factor complex and, consequently, the expression of the target gene. For similar component designs and specifications, see PCT / US93 / 01617 and WO96 / 41865 (Clackson, et al.
). In practice, the level of target gene expression should depend on the number or concentration of the chimeric transcription factor complex, which should depend on the C3 rapalog concentration. Dose-responsive (C3 rapalog) responsive gene expression is commonly observed.

[0204] The C3 rapalog can be administered to a patient to be activated for transcription of a target gene. C3 rapalog binding affinity, desired response, mode of administration, rapalog and / or target gene mR
Various protocols can be used depending on the biological half-life of the NA and the number of engineered cells present. C3 rapalog can be administered by various routes including parenteral or oral. The number to be administered depends on the factors mentioned above. C3 rapalog can be administered orally as a tablet, powder, or dispersion; buccal administration; sublingual administration; intravascular, intraperitoneal, intramuscular, subcutaneous injection; inhalation and the like. C3 rapalogs (and monomeric antagonist compounds) can be formed using conventional methods and materials well known in the art for various routes of administration. The exact dosage and particular mode of administration will depend on the factors and will be specified by the attending physician or human or veterinary medical personnel. Primarily, the mode of administration is specified empirically.

If transcriptional activation by C3 rapalog is to be abolished or terminated, a monomeric compound capable of competing with C3 rapalog can be administered. Thus, if side effects occur or if one wishes to end the therapeutic effect, the antagonist to the dimerizing agent can be administered in any convenient way, especially if intravenous injection is desired if it is to be quickly reversed. Alternatively, an inactivation domain (or transcription silencer) may be provided in the ligand binding domain. Alternatively, cells may be removed by apoptosis via a signal via the Fas or TNF receptor as described elsewhere. See International Patent Applications PCT / US94 / 01617 and PCT / US94 / 08008.

Any such that a specific level of expression is required for an extended period of time, eg, about 2 weeks or more, or that repetitive treatment with individual or repeated amounts of C3 rapalog is performed over a short period of time, eg, 2 weeks or more. The specific dosage of C3 rapalog for the application of is specified according to the procedure used for therapeutic dosage monitoring. Administer a dose of C3 rapalog that is within the expected range and monitor the response to obtain a time-expression level correlation, as well as observe the therapeutic response. Higher or lower doses can then be administered, depending on the drug levels and therapeutic response observed during that period. This process is repeated until a dosage within the therapeutic range is obtained. Once C3 rapalog is administered chronically, once a maintenance dose of C3 rapalog is identified, it should be assayed at long intervals to ensure that the cell line produces the appropriate response and level of expression product. Just fine.

The system is based on the cell response to C3 rapalog, the efficiency of expression, and, in particular, the level of secretion, the activity of the expressed product, the special needs of the patient, which may vary with time and circumstances, cell loss or individual loss. It can be seen that many variables are affected, such as the rate of cell activity loss as a result of the loss of cell expression activity.

2. Production of recombinant proteins and viruses.

Production of recombinant therapeutic proteins for commercial and research purposes is accomplished by using mammalian cell lines that have been engineered to express high levels of the protein. The use of mammalian cells rather than bacteria or yeast indicates that proper functioning of the protein requires post-translational modifications not commonly performed in heterologous cells. Examples of proteins commercially produced by this method include erythropoitin, tissue plasminogen activator, coagulation factors such as factor VIII: c, antibodies, and the like. The cost of producing a protein in this manner is directly related to the level of expression in the engineered cells. A second limitation in the production of such proteins is toxicity to host cells: protein expression can prevent cells from growing at high density and suddenly reducing production levels. Thus, the ability to properly regulate protein expression, as described for regulated gene therapy, allows cells to grow at high density in the absence of protein products. Gene expression is only activated after optimal cell density, followed by harvesting of the protein product.

[0210] Similar problems are encountered in the construction and use of "package cell lines" for producing recombinant viruses for commercial (eg, gene therapy) and experimental use. These cell lines have been engineered to produce the required viral proteins for the assembly of infectious virions containing the defective recombinant genome.
Viral vectors depending on such packaged cell lines include retroviruses, adenoviruses, and adeno-associated viruses. In the latter case, the package
The titer of storage virus obtained from dicell lines is directly related to the rep of the virus and the level of core protein production. However, these proteins are highly toxic to host cells. Therefore, it has been found that it is difficult to produce a high titer recombinant AAV virus. The present invention provides for a packaged cell line in which the rep and core genes are placed under the control of a regulatable transcription factor designed as described herein. It offers a solution to this problem. The packaged cell line is grown to high density, infected with a helper virus,
Further, a recombinant virus genome can be transfected. Thus, the addition of a dimerizing agent to produce a high-density virus induces the expression of viral proteins encoded by the packaged cells.

3. Biological Studies The present invention is applicable to a wide range of biological experiments that require precise regulation of target genes. These include (1) expression of a protein or RNA of interest for biochemical purification; (2) in tissue culture cells of interest (or by engineered cells) for the purpose of assessing its biological function. (3) regulated expression of a protein or RNA of interest for the purpose of assessing its biological function in transgenic animals; (4) And / or) regulating the expression of a gene encoding an antisense molecule that acts on an endogenous gene for the purpose of assessing the biological function of another regulatory protein, a ribozyme or gene. Transformed animal models and other applications to which the structures of the present invention may be applied include PCT / US95 / 10591
Including those disclosed in US Pat.

[0212] The present invention further provides kits useful for the above applications. Such a kit may comprise a DNA structure encoding a chimeric protein of the present invention and capable of directing expression (and which may include additional domains as described above), and an embodiment comprising regulated gene transcription. And a target gene structure including a target gene linked to one or more transcriptional regulatory elements activated by multimerization of the target gene. Alternatively, the target gene structure can include a cloning site for the practitioner to insert the desired target gene. Such kits may also include a dimerizing agent that can dimerize the two recombinant proteins and further activate transcription of the target gene.

Formulation, Dosage, and Administration Due to the ability to promote protein-protein interactions, the rapalogs of the present invention can be used in humans or mammals other than humans, including the genetically engineered cells of the present invention. It can be used in a pharmaceutical composition for promoting complex formation and a pharmaceutical method.

Preferred such treatment or prevention methods are to promote such complex formation in the engineered cells, or preferably, such as for transcription of the target gene, apoptosis of the engineered cells. It is done by administering to a mammal an effective amount of a compound to promote the proper operation of the desired biological event induced by such complex formation.

Therapeutic / Prophylactic Administration and Formulation Composition Rapalog can be present in free form or, where appropriate, in salt form. Pharmaceutically preferred salts of many types of compounds and their preparation are well known to those skilled in the art.
Pharmaceutically preferred salts of the compounds of the present invention include the conventional non-toxic salts or the quaternary ammonium salts of such compounds, formed, for example, from the inorganic or organic acids of the base.

The compounds of the present invention can form hydrates or solvates. It is well known to those skilled in the art that charged compounds form hydrates when lyophilized with water or form solvates when concentrated with a suitable organic solvent.

The present invention also relates to pharmaceutical compositions comprising a therapeutically (or prophylactically) effective amount of a compound, and one or more pharmaceutically acceptable carriers and / or other excipients. Carriers include, for example, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof, and are discussed in further detail below. If desired, the composition will also include small amounts of surfactants or emulsifiers, or pH buffers. Its composition is
It can be a solution, suspension, emulsion, tablet, pill, capsule, sustained release form, or powder. The composition can be formed as a suppository, with traditional binders and carriers such as triglycerides. For oral preparations, pharmaceutical grade mannite-
, Lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carboxane, and the like. Forming includes mixing, granulating, compacting, or dissolving the ingredients, as desired.

The formulation carrier used is, for example, either solid or liquid.

Specific solid carriers include lactose, gypsum, sucrose, talc, gelatin, agar, pectin, gum acacia, magnesium stearate, stearic acid and the like. Solid carriers include flavoring agents, lubricants, solubilizers, suspending agents, fillers, compression aids, binders or tablet disintegrants; which can also be encapsulating materials. The carrier in powder is a finely divided solid that is in admixture with the finely divided active ingredient. 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. Powders and tablets preferably contain up to 99% of the active ingredient. Suitable solid carriers are, for example, calcium phosphate, magnesium stearate, talc, sugar, lactose, dextrin, starch, gelatin, cellulose, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidine, low solubility waxes And an ion exchange resin.

[0220] Specific liquid carriers include syrup, peanut oil, olive oil and the like. Liquid carriers include solutions, suspensions, emulsions, syrups, elixirs and pressurized ingredients. The active ingredient is dissolved or suspended in water, an organic solvent, a mixture of water and an organic solvent, or a pharmaceutically acceptable liquid carrier such as a pharmaceutically acceptable oil or fat. Liquid carriers include solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickeners, pigments, viscosity modifiers, stabilizers or other suitable osmotic agents. Pharmaceutical additives can be included. Suitable examples of liquid carriers for oral and parenteral administration include water (including some of the aforementioned additives such as cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohol ( Monohydric alcohols and polyhydric alcohols such as glycols) and their derivatives, and oils (eg, coconut oil and peanut oil). For parenteral administration,
The carrier may be an oily ester such as methyl oleate, isopropyl myristate. Sterile liquid carriers are useful in sterile liquid form for parenteral administration.
The liquid carrier for pressurized configurations may be halogenated hydrocarbon or other pharmaceutically preferred propellant. Liquid formulation preparations, which are sterile solutions or suspensions, can be used, for example, for intramuscular, intraperitoneal, or subcutaneous injection. Sterile solutions can also be administered intravascularly. The compounds can also be administered orally, either liquid or solid.

The carrier or excipient can be a time delay material well known in the art, such as glyceryl monostearate or glyceryl distearate, along with wax, ethyl cellulose, hydroxypropylmethyl cellulose, methyl methacrylic acid, and the like. May be included. Upon molding for oral administration, 0.01% Tween 80 in PHOSAL PG-50 (a phospholipid concentrate with 1-2-propylene glycol, Nattermann & Cie. GmbH) is a preferred oral dosage form for other compounds. It has been found to result in molding and is applicable to the formulation of the various compounds of the present invention.

A wide variety of formulation forms can be used. If a solid carrier is used, it may be tabletted or enclosed in a hard gelatin capsule in powder or pellet form.
It can be formed into a helix or rhombus shape. The amount of solid carrier varies widely but preferably will be in the range of about 25 mg to 1 g. If a liquid carrier is used, it can be formed into a syrup, emulsion, soft gelatin, capsule, sterile injectable solution or suspension in an ampoule or vial, or non-aqueous suspension.

To obtain a stable, water-soluble dosage form, a pharmaceutically acceptable salt of a multimer such as 0.3 M succinic or citric acid can be dissolved in an aqueous solution of an organic or inorganic acid.
Alternatively, the acidic derivative can be dissolved in a suitable base solution. If a soluble salt is not available, the compound is dissolved in a suitable cosolvent or a combination thereof. Examples of such suitable cosolvents include, but are not limited to, alcohol, propylene glycol, polyethylene glycol 300, polysorbate in a concentration range of 0-60% of the total volume. G, glycerin, fatty acid polyoxyethylene, fatty alcohol or glycerin, glycerin hydroxy fatty acid ester and the like.

Various delivery systems are known for administering multimerizing agents or those formed into tablets, capsules, injectable solutions, liposomes, microparticles, microcapsules and the like. These transport systems can be used. Methods of introduction include, but are not limited to, skin, intradermal, intramuscular, intraperitoneal, intravascular, subcutaneous, intranasal, lung, epidural, ocular, and (generally preferably) oral routes It is. The compounds can be administered by any convenient or suitable route, such as by infusion or an intravenous bolus, for example, by absorption through the epithelium or the lining of the skin and mucous membranes (eg, oral mucosa, rectal and small intestinal mucosa, etc.); Can be co-administered with other biologically active agents. Administration can be systemic or local. Preferred routes of administration for the treatment or prevention of nasal, bronchial or pulmonary conditions are oral, nasal or via a bronchial aerosol or nebulizer.

In certain embodiments, it is preferable to administer the compound locally to the specific area in need of treatment; this includes, but is not limited to, local infusion during surgery,
Alternatively, by injection, using a catheter, using a suppository, or a skin patch (s
It can be achieved by topical application with kin patches, or by implants made of porous or non-porous or gelatinous materials containing membranes or fibers, such as sialastic membranes.

In a specific embodiment, the composition is shaped according to routine procedures as a pharmaceutical composition adapted for intravascular administration to humans. Generally, compositions for intravascular 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 injection. Generally, the components are provided separately or in a unit dose, for example as a lyophilized powder or a water-free concentrate, in a sealed container such as an ampoule or pouch indicating the quantity of active agent. Is done. If the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, sterile water for injection or saline is provided in an ampoule so that the ingredients may be mixed prior to administration.

By administering a compound directly to an individual skin lesion, an effective amount of the compound for an individual can be administered locally. For this purpose, the compounds may include, but are not limited to, gels, ointments, salves, carriers including carriers such as water, alcohols, propylene glycol, fatty alcohols, triglycerides, fatty acid esters, or mineral oils. Administration or use in a composition comprising a pharmaceutically preferred topical carrier such as a cream or cream.

Other topical carriers include mineral oil, isopropyl palmitic acid, polyethylene glycol, ethanol (95%), aqueous polyethylene monolaurate (5%), or aqueous sodium lauryl sulfate (5%). Other materials, such as antioxidants, wetting agents, viscosity stabilizers, can be added as needed. A transdermal penetration enhancer, such as Azone, may also be included.

[0229] Further, in some examples, it is required that the compound be able to be treated on the skin, in the skin or in a device placed under the skin. Such devices include patches, implants, and infusions that release the compound into the skin by a passive or active release mechanism.

Materials and methods for producing various forms are well known in the art and can be applied to practice of the subject invention. U.S. Patent Nos. 5,182,293 and 4,837,311 (tablets, capsules and other oral dosage forms as well as intravascular dosage forms) and European Patent Application Publication No. 0 649 659 (published April 26, 1995; IV Specific forms for administration) and
See 648 494 (published April 19, 1995; specific forms for oral administration).

An effective amount of the compound will generally be in the range from about 0.01 to about 50 mg / kg. Typically, the compounds can be administered to patients in need of such treatment in a daily dosage range of about 1 mg to 2000 mg per patient.

The amount of a compound that is effective in the treatment or prevention of a particular disease or condition will depend on the characteristics of the fusion protein being multimerized, the characteristics and distribution of the engineered cells, and the disease or condition that can be identified by standard clinical techniques. It will depend in part on the nature of the state. In addition, in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. Effective doses can be extrapolated from dose-response curves obtained in in vitro or animal model evaluation systems. The exact dosage level should be specified by the attending physician or other healthcare professional, and should be determined by the route of administration and the age, weight, sex, and overall physical health of the individual; the nature, severity, and clinical stage of the disease. The use (or absence) of an attending therapist; the nature and extent of genetic manipulation of the cells in the patient;

The present invention also provides a pharmaceutical pack or kit consisting of one or more containers containing one or more components of the pharmaceutical composition of the present invention. Precautions in the form prescribed by governmental agencies governing the manufacture, use or sale of chemicals or biological products are optionally associated with such containers, and such precautions may be taken for administration to humans. Reflects accreditation by the agency controlling production, use or sales. The notice or package insert contains instructions for using the C3 rapalog of the present invention, consistent with those disclosed herein.

The present invention is not intended to constitute a further limitation, but is further described by the following examples. The contents of all references, pending patent applications and published patents cited throughout this application are hereby expressly incorporated by reference.

Experimental Example Synthesis of C3-methallyl-rapamycin and C3-allyl-rapamycin

Embedded image The synthesis technique we currently use is Liberl for C7 rapalog adjustment.
es et al., July 1997, Proc. Natl. Acad. Sci.
USA. 94: 7825 7830. (C7 and C3 referred to herein are, respectively, in the nomenclature of Liberles et al.,
Ring positions C16 and C20). 22 mg of rapamycin is placed in a 3 ml flame dried Wheaton vial equipped with a stir bar. 200 μl of rapamycin
And cooled to -40 ° C. It has been found that low temperatures do not work effectively. 50 μl of methallyl (or allyl) trimethylsidan (12
Eq) followed by 40 μl of neat BF ₃ etherate (13 eq). 2
After time, the reaction is terminated as determined by TLC, and quenched by addition of saturated NaHCO _3. The reaction mixture is then washed with brine and dried over sodium sulfate (the aqueous wash is back-extracted with methylene chloride and combined with the reaction mixture). Filter the sample through a 0.45 micron nylon filter and evaporate the solvent in vacuo.

The crude product was 100 μl for injection on a JAI recycling HPLC described below.
Dissolve in chloroform. After approximately 15-20 cycles, three prominent peaks are obtained that can be separated at baseline resolution (under certain conditions a fourth peak eluting before the other three appears). The first of the three dominant peaks is C7-5 methallyl rapamycin as measured by ¹ H-NMR. The middle peak is the compound of interest, C3-methallyl rapamycin, and the fourth peak consists of by-products from hypersilylation of rapamycin. Following this procedure, we obtained 4.2 mg of pure C3-methallyl rapamycin. It is very important that unmodified rapamycin coexists with C3-methallyl rapamycin, is not effectively removed by JAI, and that rapamycin is completely consumed during the reaction. Excess addition products are removed much more easily than unreacted starting materials.

[0237] JAI-purified methallyl rapamycin can be demonstrated by UV, NMR, and mass spectrometry and biological activity. The presence of contaminants such as rapamycin can be easily measured by UV as described below.

Using this same approach, C3 alkyl-rapamycin is synthesized and easily extended to synthesize various other C3 derivatives.

Analytical Considerations One particularly convenient diagnosis is UV spectroscopy. Consistent with the disappearance of triene,
The UV spectrum of the C3 compound has the maximum absorption at lambda 238 rather than 274-282, typically observed with the C7 (R) (S) compound. C3
UV analysis is a good indicator of sample purity and the presence of any toxic contaminants since the compound has a baseline absorption in the region where rapamycin best absorbs.

Also, the loss of triene in C3-methallyl rapamycin is reflected in ¹ HNMR. ¹ H-NM methallyl rapamycin taken in CDCL3
R reveals a diagnostic peak of C5 at 6.0 ppm. The rest of the olefinic protons are all upfield between 5 and 5.6 ppm.

Chromatographic Recovery of C3 Rapalog Japan Analytical Industry Co. Ltd. (J
AI) to efficiently purify the device C3 rapalog produced. The instrument we used was "LC-908 recycling preparative HPLC" and the column we used was "JAIGEL GS 310" with dimensions of "2 x 500 mm".

Functional Features of C3 Rapalog The toxicity of the lead compound is IL-2-dependent and its ability to inhibit the growth of CTLL-2 cells that is highly sensitive to rapamycin (IC50 <1 nM) Tested by. Incubation of CTLL-2 cells with 1 μm C3-methallyl rapamycin had no detectable effect on proliferation.

Similarly, C3-allyl rapamycin and C3-methallyl rapamycin have G
Al4-FKBP3, wild-type FRB-VP16, and its ability to activate transcription in transfected Phuket cells with UAS-SEAP were lost (these constructs are described in Liberles et al., supra; The contents of which are expressly considered part of this specification). FRB # -VP16 (T2098
L, W2101F, and K2095P) displace wild-type FRB-VP16 and reporter gene activity can detect EC50 near 10 nM C3-methallyl rapamycin, C3-allyl rapamycin; likewise, the amplitude of reporter gene activity is It is higher than that induced by the rapamycin / wild-type FRB-VP16 system. The C3 derivative of rapamycin can be effectively dimerized with improved toxicity (immunosuppressive activity) together with the generated FRB domain.

Preparation of 24 (S), 30 (S) -tetrahydro-C3-rapalog

Embedded image 24,30-tetrahydrorapamycin is prepared as described elsewhere,
It is converted to the desired C3-substituted rapalog by the method described above.

Preparation of 13-Fluoro-C3-rapalog

Embedded image The title compound is prepared as described above, substituting 13-fluororapamycin for rapamycin.

Preparation of 28-Fluoro-C3-rapalog

Embedded image The title compound is prepared as described above, substituting 28-fluororapamycin for rapamycin.

Preparation of 43-epi-C3-rapalog

Embedded image Replace rapamycin with 43-epirapamycin and prepare the title compound as described above.

Equivalents One skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Will be able to. Such equivalents are intended to be included in the following claims.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07K 1/14 C07K 1/14 4H045 19/00 19/00 C12N 15/00 C12P 21/02 C C12P 21 / 02 (C07D 498/18 // (C07D 498/18 311: 00 311: 00 273: 00 273: 00 211: 00) 211: 00) C12N 15/00 (72) Inventor Club Tree, Gerald Earl United States of America California 94062 Woodside Durham Road 7 (72) Inventors Rivers, Stephen Dee, Massachusetts, United States 02143 Summerville, Dane Street 68 R F-term (reference) 4B024 AA01 BA21 BA80 DA02 EA02 GA11 HA17 4B064 AE01 AG01 CA03 CA10 CC24 CD12 DA01 4C072A03 CC01 CC12 UU01 4C084 AA13 BA32 BA50 CA18 CA53 CA56 C A59 NA14 ZB081 ZB082 ZB261 ZB262 4C086 AA01 AA02 AA03 AA04 CB22 MA01 NA14 ZB08 ZB26 4H045 AA10 AA20 BA51 CA11 DA01 EA20 FA73 FA74

Claims (20)

[Claims] 1. One or more optionally unsaturated, at one or more carbon-carbon bonds, optionally spanning 1 to 8 carbons, as substantially pure stereoisomers or a mixture of stereoisomers. A structure having the substituent: embedded image [Wherein R ^C3 is other than H] or a pharmaceutically acceptable derivative thereof. 2. As substantially pure stereoisomers or a mixture of stereoisomers.
Formula: embedded image[Wherein, R^C3Is other than H, and a is:R^C30Is halo, -OR³Or (= O), R^C24Is = O, = NR⁴, = NOR⁴, = NNHR⁴, -NHOR⁴, -N
Hnhr⁴, -OR⁴, -OC (O) R⁴, -OC (O) NR⁴, Halo or-
H; R^C13And R^C28Is independently H, halo, -OR³, -OR⁵, -OC (
O) R⁵, -OC (O) NHR⁵, -SR⁵, -SC (O) R⁵, -SC (O)
NHR⁵, -NR⁵R⁵’Or -N (R⁵) (CO) R⁵’; R^C14Is O, -OR⁶, -NR⁶, -H, NC (O) R⁶, -OC (O) R ⁶ Or -OC (O) NR⁶R³Is H, -R⁷, -C (O) R⁷Or -C (O) NHR⁷Or C28
And a cyclic group bridging C30 (eg, carbonate); R^C29Is H or OR¹¹(Eg, OH or OMe); wherein, unless otherwise specified, each substituent can be in any stereochemical orientation;
Where R¹, R⁴, R⁵, R⁶, R⁷, R⁹, R¹⁰And R¹¹Each occurrence of
Independently selected from H, aliphatic, heteroaliphatic, aryl and heteroaryl
And R⁸Is H, halo, -CN, = 0, -OH, -NR⁹R¹⁰, OSO₂CF₃,
OSO₂F, OSO₂R⁴’, OCOR⁴’, OCONR⁴'R⁵’Or O
CON (OR⁴’) R⁵'Or a pharmaceutically acceptable derivative thereof. 3. The method according to claim 1, wherein R ^C3 is of the formula: Wherein R ¹ , R ⁴ , R ⁵ , R ⁶ and R ⁷ are substituted aliphatic, aromatic, heteroaliphatic or heteroaromatic groups. 3. The compound according to 1 or 2. (4) The compound according to claim 2, wherein the compound is selected from the group consisting of: 5. The compound according to claim 1, wherein R ^C3 is allyl or methallyl. 6. A process comprising reacting a compound containing group (1) with a silyl ether (2) under conditions which allow the formation of a compound of formula (3), wherein R, R ′ and R ″ Is an aliphatic, aromatic, heteroaliphatic, or heteroaromatic group, and R ^C3 is a substituted or unsubstituted allyl group. 7. The method according to claim 6, further comprising a step of separately recovering the compound (3) from other substances in the reaction mixture. 8. (a) (i) a first recombinant nucleic acid encoding a first chimeric protein that binds to rapamycin or an analog thereof, which comprises at least one FKBP domain and at least one heterologous protein domain. The FKBP domain includes: (1) a naturally occurring FKBP, (2) a naturally occurring FKBP variant in which 10 or more amino acid residues have been deleted, inserted, or substituted with alternative amino acids. (3) comprising a peptide sequence selected from FKBP encoded by a DNA sequence capable of selectively hybridizing to the DNA encoding FKBP of (i) or (ii); (ii) (a) rapamycin Alternatively, a rapamycin analog and (b) a first chimeric protein forming a complex with both A second recombinant nucleic acid encoding a chimeric protein of SEQ ID NO: 1, which comprises at least one FRB domain and at least one domain heterologous thereto, wherein the FRB domains are: (1) a naturally occurring FRB domain; A) a variant of a naturally occurring FRB domain in which 10 or more amino acid residues have been deleted, inserted or substituted with an alternative amino acid; (3) a DNA encoding the FRB of (iv) or (v); Comprising a peptide sequence selected from a FRB domain encoded by a DNA sequence capable of selectively hybridizing; and (b) itself and the first and second chimeric proteins. 6. The rapalog of claim 1 which forms a complex containing at least one molecule of each. A method for multimerizing a chimeric protein in a cell, which comprises contacting the cell with a cell. 9. The chimeric protein encoded by the first recombinant nucleic acid, wherein the peptide sequence contains up to three amino acid substitutions with respect to the naturally occurring FKBP peptide sequence. the method of. 10. The chimeric protein encoded by the first recombinant nucleic acid, wherein the peptide sequence contains one amino acid substitution relative to a naturally occurring FKBP peptide sequence. Method. 11. The chimeric protein encoded by the second recombinant nucleic acid, wherein the peptide sequence comprises at least one FRB containing up to four amino acid substitutions relative to a naturally occurring FRB peptide sequence. Claim 8
11. The method according to any one of claims 10 to 10. 12. The chimeric protein encoded by the second recombinant nucleic acid, wherein the peptide sequence comprises at least one FRB containing three amino acid substitutions relative to a naturally occurring FRB peptide sequence. The method of claim 11, wherein 13. The chimeric protein encoded by the second recombinant nucleic acid comprises a Thr209 in the FRB peptide sequence whose peptide sequence occurs naturally.
13. The method of claim 12, comprising at least one FRB containing a substituted amino acid for one or more of Trp2101 and Lys2095. 14. The chimeric protein encoded by the second recombinant nucleic acid has a Thr209 in the FRB peptide sequence whose peptide sequence is naturally occurring.
2. The method according to claim 1, wherein at least one of 8Leu, Trp2101Phe and Lys2095Pro contains at least one FRB containing a replacement amino acid.
3. The method according to 3. 15. A DNA-binding domain for triggering growth, proliferation, differentiation or apoptosis upon multimerization with at least one such protein containing at least one such signaling domain, transcription, 15. The method according to any one of claims 8 to 14, wherein the method comprises an activation domain or an action domain that is a cell signaling domain. 16. The method according to claim 8, wherein the cells are grown in a culture medium, and the contact with the rapalog is carried out by adding the improved rapalog to the culture medium. Method. 17. The method according to claim 8, wherein the cells are present in an intact organism, and the contact with the improved rapalog is effected by administering the rapalog to the organism. Method. 18. The method according to claim 17, wherein the cell is a mammal and the organism is a mammal. 19. The method of claim 18, wherein said cells are of primate origin and said organism is a primate. 20. The method according to claim 19, wherein the primate is a human.