KR19990022651A - Rapamycin-Based Control of Biological Events - Google Patents

Abstract

The present invention relates to an array method for biological conversion and provides novel methods and materials useful for modulating biological events in animal cells. The present invention includes recombinant DNA constructs comprising DNA sequences derived from sequences encoding proteins FRAP, Tor1, Tor2, and other proteins capable of binding FKBP: rapamycin, DNA sequences encoding some or all of FKBP. Other recombinant DNA constructs, proteins encoded by these constructs, cells transfected with one or more constructs (especially animal cells), small molecules that bind to chimeric proteins and induce multiple bonds of chimeric proteins (multiple multiple binders) And methods for making and using them.

Description

Rapamycin-Based Control of Biological Events

Rapamycin of formula (1) is a natural product that binds to a FK506-binding protein, ie, FKBP, with high affinity to form a rapamycin: FKBP complex. The reported Kd value for the interaction is at least 200 pM. The rapamycin: FKBP complex binds to the large cellular protein FRAP with high affinity to form a tripartite, [FKBP: rapamycin]: [FKAP], complex. In these three-part complexes, rapamycin acts as a dimer or adapter to bind FKBP to FRAP. The portion of the FRAP protein that interacts with the FKBP: rapamycin complex is referred to as the FRB domain described in detail below. The rapamycin dependency of large mammalian proteins, called FKBP12 and FRAP, and the relationship between RAFT1 or RAFT1 and its yeast analogues DRR and TOR have been discussed by several research groups (see, for example, Brown et al., 1994, Nature 369: 756-758; Sabatini et al, 1994, Cell 78: 35-43; Chiu et al, 1994, Biochem. Biophys. Res. Comm. 203: 1-7; Kunz et al, 1993 Cell 73: 585-596; and Cafferkey et al, 1993 Mol. Cell. Biol. 13: 6012-6023. See Chiu et al, supra, and Stan et al, 1994, J. Biol. Chem. 269: 32027-32030, describe rapamycin dependent binding of FKBP12 to small subunits of FRAP containing FRB domains. The FRAP, RAFT, RAPT and TOR proteins each contain homologous FRB domains and are believed to be FRAP proteins of the invention:

A wide range of natural FK506 binding proteins (FKBPs) are known proteins [see, eg, Kay, 1996, Bioche, m. J. 314: 361-385. FKBP proteins are biologically based on ligand mediated multibinding of immunophilin based recombinant proteins as disclosed in Spencer et al, 1993, Science 262: 1019-1024 and patent application PCT / US94 / 01617. Their ligand binding properties are used in the switch. On the other hand, immunosuppressive activity of FK506 may be limited in its usefulness as a dimerizing agent, especially in animals, but dimers of FK506 (and related compounds) lack such immunosuppressive activity, and contain chimeric containing ligand binding proteins derived from FKBP. It has been found to be effective in dimerizing proteins.

Rapamycin, such as FK506, is a natural protein dimerization agent and can dimerize suitably designed chimeric proteins, but its significant biological activity, including potent immunosuppressive activity, has been shown in its use in engineered biological switches, particularly in animals. Further restricts the use of. The present invention allows for the efficient dimerization of rapamycin (and related compounds) while suppressing serious and inherent disadvantages.

1A is a diagram showing rapamycin dependent dimerization of FKBP chimera containing a DNA binding domain with FRAP chimera containing a transcriptional activation domain.

1B shows the plasmid used in certain experimental examples. Transcription factor fusion proteins are produced under the conditions of the human cytomegalovirus (hCMV) pseudo early promoter and enhancer. E represents the epitope tag, N represents the SV40T antigen nuclear localization sequence, and both are fused to the N-terminus of the transcription factor. The receptor gene consists of a minimal hCMV promoter lacking an enhancer sequence by the site of repeated binding to ZFHD1.

FIG. 2 depicts the various FRAP derived and FKBP derived domains of the fusion proteins of the invention shown in schematic form with rapamycin and rapamycin analogs.

FIG. 3 is a comparison of the results obtained in transcriptional assays using constructs of various multimers and forms of FRAP and FRBP domains.

4 is a dose for rapamycin dependent gene transcription as measured by the generation of alkaline phosphatase activity secreted at various rapamycin concentrations in transiently transfected cells (A, B) and stably transfected cells (C, D). It is a figure which shows a response curve. (A) Plasmids encoding ZFHD1-3FKBP and 1FRB-p65 (361-450) are transfected into HT1080 cells along with the pZHWTx12-CMV-SEAP receptor gene. SEAP activity secreted into growth medium is measured at the indicated concentration of rapamycin after culture. (B) The indicated plasmid (-) is removed and replaced with a coexpressed vector alone. SEAP activity is measured with or without rapamycin at the indicated 10 nM rapamycin after incubation. Transfections were performed in triplicate and plotted mean values (relative units) ± standard deviation. Rapamycin regulated genes are expressed in stable cell lines. (C) The pLH-ZHWTx12-IL2-SEAP receptor gene, ZFHD1-3FKBP DNA binding domain and 1FRB-p65 (361-550) activation domain were stably integrated into HT1080 cells in three steps, producing a clonal cell line HT20-6 Let's do it. SEAP activity secreted into the growth medium is measured with the indicated concentrations of rapamycin after culturing HT20-6 cells. The assay was performed in triplicate and plotted the mean values (relative units) ± standard deviation. (D) Top: IRES containing plasmid used to express 1FRB-p65 (361-550) and ZFHD1-3FKBP by single transcription. Bottom: HT1080 already containing pLH-ZHWTx12-IL2-SEAP receptor gene safely integrated with pCGNN-1FRB-p65 (361-550) -IRES-ZFHD1-3FKBP with plasmids resistant to zeocin The cells were transfected. A pool of zeocin resistant clones, HT23 cells, was incubated with the indicated concentrations of rapamycin and the SEAP activity secreted in the growth medium was measured.

5 shows the results of rapamycin dependent gene transcription experiments on various forms of transcription factor fusion proteins. The experiment aims to determine the optimal form for the rapamycin binding domain in the system of the present invention. HT1080 cells were transfected with plasmids encoding the indicated ZFHD1-FKBP and FRB-p65 (450-550) fusion proteins. The transfected cells were cultured in medium containing 10 nM rapamycin and the SEAP activity was measured. Transfection was performed in triplicate and plotted mean values (relative units) ± standard deviation. Immune plot analysis showed that all fusions were expressed at similar levels except ZFHD1-1FKBP, which was expressed at high levels. The same result was obtained even when the amount of each plasmid transfected varied over a wide range. The results are shown in four separate experiments.

FIG. 6 is a diagram demonstrating that when a dimerization agent is administered in vivo to an animal into which an engineered cell is inserted, a regulated gene is expressed and gene product is produced and secreted. Transfected HT1080 cells (total 2 × 10 ⁶ per animal, at 4 different sites) were injected intramuscularly into male nu / nu mice. After about an hour, the animals received rapamycin at the indicated concentrations intravenously. Blood samples were collected 17 hours after rapamycin administration and assayed for hGH concentration. Rapamycin treatment showed a dose dependent increase in serum hGH (X ± SEM; n = 5 or higher at each dose). * Indicates statistical significance from low rapamycin doses respectively, + indicates statistical significance from rapamycin that is 10 times or less (p <0.05, one analysis of changes and Turkish-Cremer multiple comparison test: Tukey-kramer multiple comparison testing).

FIG. 7 demonstrates the ability of HT1080 to be manipulated as described in Example 4 (see also FIG. 6) and implanted into mice to produce hGH for a delayed period following intravenous rapamycin administration. Mice were treated with 10.0 mg / kg of rapamycin after transplantation of transfected HT1080 cells. Blood samples were collected at 4, 8, 17, 24 and 42 hours after rapamycin administration and serum hGH levels were measured (X ± SEM, n = 4 per point). The final half life of the hGH response was calculated to be 11.4 hours.

8 demonstrates the ability of transplanted engineered cells to be restimulated by a second rapamycin administration after the effect of the first dose has disappeared. Rapamycin (10.0 mg / kg) was administered in many cases (arrows). Dates are plotted with X ± SEM per point, n = 4-9 mice (solid line). The concentration of rapamycin presumed to circulate throughout the experiment was plotted (dashed line). The estimated flat horizontal line through the rapamycin concentration showed a steady state in the concentration of rapamycin calculated by rapamycin pharmacokinetics. The downline starting from 80 hours represents an estimated circulating rapamycin concentration representing an elimination half-life of 4.6 hours.

FIG. 9 shows analysis of various rapamycin C24 oximes (prepared in Example 5) for FKBP binding affinity using the competitive FP assay described in FIG. 6. Assays show mean ± SD for two separate experiments (C24 methyl oxime plot represents single experiment).

FIG. 10 shows the specific enrichment of phagemids showing FRAP after incubation with His6-labeled FKBP in the presence of indicator, followed by capture and elution on Ni-NTA agarose. The results are expressed as the ratio of FRAP phagemid to non-display control phagemid, with a ratio of 1 before sorting. The experiment is detailed in Example 7.

11: FKBP display phage was prepared and its binding characteristics were analyzed by ELISA as described in Example 7. Aliquots of FKBP phage particles were incubated with various concentrations of FK506 and added to wells coated with biotinylated FK506 bound to streptavidin. Phage binding was visualized with anti-phage polyclonal antibodies conjugated to HRP. Error bars represent SD for triple measurements. The results indicate that FKBP displayed on phage interacts with FK506 at essentially the same value as the value of soluble protein (IC50 = 0.65nM).

FIG. 12 shows an example of a form useful for the rapamycin dependent control of signal hemostasis introduction. M / E represents a myristoylated motif or extracellular receptor domain that places the chimeric construct on the membrane; EFF represents an effector domain that induces a cellular response upon oligomerization (eg, a polypeptide comprising the lethal domain of FAS). Both forms can be used to single dimerize or heterodimerize effector domains (heterodimerization is illustrated in (a) by EFF and EFF '). It should be noted that many other possible forms can be observed: in particular, it should be noted that the order and number of each domain can be changed as appropriate.

FIG. 13 expresses a mixed chimeric protein (a, b) comprising a portion of the myristolisation domain as described in FIG. 8, the domain from human FRBP, the FKBP domain from hFKBP12, and the intracellular domain of Fas. Decrease in cells. Example 8 (c) illustrates a control experiment using a chimera containing a myristoylation domain, two FKBP domains, and a Fas intracellular domain that dimerize in the presence of AP1428 but not dimerize in the presence of rapamycin, have.

FIG. 14 shows gel mobility change assays for binding reactions containing nearly equal amounts of Lex-FKBP and Lex-FRAP proteins incubated with DNA containing Lex binding sites. No specific binding is detected in the absence of rapamycin (FIG. 14, lane 1). Increasing amounts of rapamycin in binding reactions result in specific complexes involved in mobile migration gels (Figure 14, lanes 2-4). These results support the conclusion that rapamycin promotes the formation of Lex-FKBP / Lex-FRAP heterodimers that can bind to DNA containing LexA target sequences (see Example 9 for details).

The definitions and descriptions set forth below will help to understand the invention in more detail.

The FRB domain is a polypeptide region (protein domain) of at least about 89 amino acid residues capable of forming a three part complex with the FKBP protein and rapamycin (or rapalog). FRB domains are FRAP proteins from human or other species (referred to in literature as RAPT1 or RAFT); Yeast proteins including Tor1 and Tor2; And candida FRAP homologs. Information on nucleotide sequences, cloning and other features of these proteins is already known in the art, for example, by using PCR primers based on known methods and known sequences to encode the desired FRB peptide sequences. DNA can be synthesized and cloned.

Protein sources Reference / sequence accession number Person frap Brown et al, 1994, Nature 369, 756-758: GenBank Accession No. L34075, NCBI Seq ID 508481: Chiu et al, 1994 PANS USA 91, 12574-12578: Chiu et al, 1995 PANS USA 92, 4947-4951 Rat RAPT1 Chiu et al, Yeast Tor1 Helliwell et al, 1994, Mol Cell Biol 5, 105-118: EMBL Accession No. X74857, NCBI Seq Id # 468738 Yeast Tor2 Kunz et al, 1993, Cell 73, 585-596; EMBL Accession No. 71416, NCBI Seq ID 298027 Candida homologue WO95 / 33052

The FRB domain used in the present invention generally contains about 89-100 amino acid residues. FIG. 2 of Chiu et al. Shows human FRAP, rat FRAP, S. comprising conserved FRB regions. S. cerevisiae TOR1 and S. Celeb2 shows a 160 amino acid span of TOR2. Typically the FRB sequence selected for use in the fusion protein of the invention may be at least 89 amino acid sequences Glu-39 to Lys / Arg-127. Sequences are numbered as shown in the figure. For example, using the numbering method of Chen et al. Or Sabitini et al., The 89-amino acid sequence is Glu-2025 to Lys-2113 for humans and Glu-1965 to Lys-2053 for Tor2. , And Tor1, are numbered Glu-1962 to Arg-2050. The FRB peptide sequences used in the fusion proteins of the invention can be bound to rapamycin or a complex of FRBP bound to the rapalog (measured by any means that detects such binding directly or indirectly) and the indicated 89- of the protein. May comprise a native peptide sequence spanning an amino acid region or a corresponding region of a homologous protein; May contain up to about 10 (preferably 1 to 5) amino acid substituents, inserts, or deletions within a region related to the native sequence; May be a peptide sequence encoded by a DNA sequence that can optionally hybridize to a DNA molecule encoding a native FRB region; It may be selectively hybridized to a DNA molecule encoding a native FRB region but encoded by an NDA sequence that is not denaturation of the genetic code.

FKBP (FK binding protein) is a cytosol receptor for macrolides such as FK506, FK520 and rapamycin and is well conserved through species cell lines. For the purposes of the present invention, FRBP is a protein or protein domain capable of binding to rapamycin or rapalog of the present invention and forming a three part complex with an FRB containing protein. Information related to nucleotide sequences, cloning, and other features for various FKBP species is known in the art, such as, for example, using a PCR primer based on known methods and known sequences to determine the desired FKBP peptide sequence. DNA encoding can be synthesized and cloned (see, eg, Staendart et al, 1990, Nature 346, 671-674 (human FKBP12); Kay, 1996, Biochem. J. 314, 361-385. 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, eg, Kay, 1996, Biochem. J. 314, 361-385. The size of the FKBP domain used in the present invention may vary depending on the FKBP protein used. The FKBP peptide sequence used in the fusion protein of the invention can bind to rapamycin or rapalog and form a three-part complex with an FRB containing protein (measured by any means that detects such binding directly or indirectly), and the human Natural peptide sequences derived from FKBP12 protein (illustrated below), or other human FKBPs, murine or other mammalian FKBPs, or peptide sequences derived from any animal, yeast or fungal FKBPs; May contain up to about 10 (preferably 1 to 5) amino acid substituents, inserts, or deletions within a region related to the native sequence; It may be a peptide sequence encoded by a DNA sequence that may be selectively hybridized to a DNA molecule encoding a native FKBP region, or may be selectively hybridized to a DNA molecule encoding a native FKBP region, but not a denaturation of the gene coat. Can be encoded by a DNA sequence.

As used herein, an expression selectively hybridizable is capable of hybridizing two DNA molecules to one another despite the presence of DNA molecules, under hybrid conditions that can be selected or readily determined experimentally by those skilled in the art. Means sensitive. Such treatments include very stringent conditions, such as washing exclusively with buffers containing 0.2-6 × SSC and / or 0.1% -1% SDS in the temperature range from room temperature to 65-75 ° C. [Reference: F.M. Ausubel et al., Eds, Short Protocols in Molecular Biology, Units 6.3 and 6.4 (John Wiley and Sons, New York, 3d Edition, 1995).

As used herein, the terms recombination, chimeric and fusion refer to heterologous structures that are mutually different in the sense that the various component domains or sequences do not occur together in essentially the same arrangement. More specifically, the component parts are not found at all in the same contiguous polypeptide or nucleotide sequence or molecule, and are not at least in the same order or orientation or at equal intervals in the chimeric protein or recombinant DNA molecule of the invention.

As used herein, the terms dimerization, oligomerization and multimerization refer to the binding of two or more proteins mediated by binding each of said proteins to a common ligand in the practice of the present invention. The formation of a tripartite complex comprising a protein containing the FRB domain, a protein containing the FKBP domain, and rapamycin is an example of dimerization. In certain embodiments of the invention, the fusion protein contains multiple copies of the FRAP and / or FRB domains. Complexes of such proteins may contain one or more molecules of rapamycin or rapalog, and one or more copies of one or more constituent proteins. Even if one or more is represented by multiple copies, the multimeric complex is still referred to herein as a three-part complex to indicate the presence of three forms of constituent molecules.

Raphalogs are compounds other than rapamycin, preferably having a molecular weight of 5 kD or less, more preferably 2.5 kD or less, which compounds can bind to FKBP fusion proteins and are complexes with FKBP fusion proteins and FRB fusion proteins. Can be formed. Particularly important raphalogs include compounds other than rapamycin, of compounds of the formula:

Where

U is -H, -OR ¹ , -OC (O) R ¹ , -OC (O) NHR ¹ , -SR ¹ , -NHR ¹ , -NHC (O) R ¹ , -NH-SO ₂ -R ¹ or -R ² ; V is -OR ³ or (= O); W is = O, = NR ⁴ , = NOR ⁴ , = NNHR ⁴ , -NHOR ⁴ , -NHNHR ⁴ , -OR ⁴ , -OC (O) R ⁴ , -OC (O) NR ⁴ , or -H; Y is -OR ⁵ , -OC (O) R ⁵ or -OC (O) NHR ⁵ ; Z is ═O, —OR ⁶ , —NR ⁶ , —H, —NC (O) R ⁶ , —OC (O) R ⁶ or —OC (O) NR ⁶ ; R ² is substituted aryl or allyl or alkylaryl (eg benzyl or substituted benzyl); R ³ is H, —R ⁷ , —C (O) R ⁷ , —C (O) NHR ⁷ or C-28 / C-30 cyclic carbonate; R ¹ , R ⁴ , R ⁵ , R ⁶ and R ⁷ are independently selected from H, alkyl, alkylaryl or aryl, including individually dissolved stereoisomers and mixtures thereof. Particularly important raphalogs are as low as measurable than those in proteins containing the corresponding FKBP and FRAP domains, where one or two receptor domains contain one or more amino acid substitutions, deletions or additions, as described herein. It forms a complex with a protein comprising native human FKBP and FRB domains with affinity.

As mentioned above, a chimeric protein contains one or more receptor or ligand binding domains comprising peptide sequences derived from FRAP or FKBP proteins, and heterologous, one or more functional domains associated with the receptor domains, but of chimeric protein molecules The oligomerization may initiate a cellular or biological response. The functional domain of the chimeric protein may be selected from a wide range of protein domains that may affect the desired biological outcome upon oligomerization or clustering of the chimeric protein. For example, one domain of action is derived from a particular cell site (membrane, nucleus, other organelles, etc.), or, for example, the intracellular domain of a growth factor receptor or cytokine receptor, for clustering or Upon multimerization, the signaling domain is capable of initiating intracellular signal transduction pathways that can grow or proliferate cells, transcribe desired genes, kill cells, or cause other desired outcomes. Inducible localization domains may be included. For example, clustering of chimeric proteins containing functional domains derived from the intracellular portion of the T cell receptor CD3 zeta domain initiates transcription of the gene under transcriptional control of the IL-2 promoter or derivatives thereof. In another embodiment, the functional domain comprises a domain derived from a protein such as a FAS antigen or a TNF-alpha receptor (TNFalpha-R1), wherein the functional domain can initiate apoptosis of cells upon oligomerization. . In yet another embodiment, the functional domain represents the association of a transcription factor complex where oligomerization of the chimeric protein initiates the transcription of a gene bound to the DNA sequence recognized by the DNA binding domain (which can specifically bind to interact with). DNA-binding domains, such as GAL4, and transcriptional active domains, such as VP16, which are paired to a degree.

DNA constructs that encode the expression of these chimeric proteins (and accessory constructs, such as DNA constructs that encode a target gene) are provided for use in genetic techniques of the host cell. To generate the genetically engineered cells of the present invention, the above DNA constructs can be incorporated into host cell recombinant DNA molecules capable of inducing expression of the desired chimera. In addition, any desired accessory structures are introduced. Such a method can be accomplished using conventional methods and materials, and various examples thereof are achieved on a large scale. As long as desired, the modified cells can be selected and isolated from other cells and cultured again by conventional methods. The treated cells of the present invention may contain and express one or more DNA constructs that encode the chimeric protein. In some embodiments, the cells can contain and express DNA encoding a pair of chimeric proteins of the invention that can multimerize in the presence of a suitable ligand. In yet another embodiment, the cell further contains a target gene construct containing the target gene under the control of expression of the DNA element responsive to the multimerized chimeric protein. The treated cells can be transiently transfected or stably transformed with one or more transgenic DNA molecules.

Useful multimerization ligands include rapamycin and rapalog, which are capable of dimerizing the FRB and FKBP domains of the invention. Such compounds are divalent ligands. That is, the compound can bind and multimerize two or more chimeric protein molecules containing FRAP and FRB, respectively.

Many embodiments of the present invention comprise two recombinant DNA molecules (or two or more DNA sequences within the same DNA molecule) encoding a pair of different chimeric proteins. The first recombinant DNA molecule comprises a chimeric protein comprising (i) at least one FKBP domain capable of binding to rapamycin or raphalog and (ii) at least one heterologous (working) domain of heterologous in relation to at least one such FKBP domain. Code it. The chimeric protein (simply referred to as FKKBP chimeras) can bind to rapamycin or raphalog to form a complex, similar to the complex formed by binding a natural FKBP protein such as FKBP12 to rapamycin. The second recombinant DNA molecule comprises (i) at least one FRB domain capable of binding to the first chimeric protein and a complex formed by rapamycin or raphalog and (ii) a heterologous protein in relation to at least one said FRB domain. The chimeric protein comprising the domain is encoded. These chimeric proteins are simply referred to as FRAP chimeras. Pairs of FKBP chimeras and FRAP chimeras can form three-part complexes with rapamycin or rapalog that can be detected by various means, including, for example, coimmunoprecipitation. Embodiments involving multiple FKBP and / or FRB domains for chimeras can form higher grade multimers in the presence of rapamycin or raphalog. Some embodiments include recombinant DNA molecules encoding mixed chimeric proteins containing heterologous one or more FKBP domains, one or more FRB domains, and one or more functional domains with respect to one or more receptor domains. Mixed chimeras can form protein homodimers or homomultimers by mutually binding the mixed chimeric protein molecules to rapamycin or raphalog.

PCT / US94 / 01617, PCT / US94 / 08008, to Spenser et al., Disclose one or more ligands for, for example, transcriptional activity of target genes, cell death or other signal transduction pathways, cell localization, and the like. Chimeric proteins containing a binding (ie receptor) domain and one or more functional domains are described. The design and use of such chimeric proteins for ligand mediated gene knockout and for ligand mediated blockade of gene expression or for inhibition of gene product action is described in PCT / US95 / 10591. Pomeranz et al, 1995, Science 267: 93-96 describe novel DNA binding domains and DNA sequences that participate in the binding used in embodiments involving controlled transcription of target genes. The above references provide substantial information, guidance and examples relating to the design, structure, and similar chimeras, target gene constructs, use of DNA constructs to encode multivalent ligands, and other aspects that may also be useful to the practitioner of the present invention. (Reference: PCT / US95 / 06722, Applicant to Mititox, Inc.). The entire contents of the above reference examples are incorporated herein by reference.

By appropriate selection of chimeric proteins, the present invention activates the desired gene, performs apoptosis, or the systems described in PCT / US94 / 01617 and PCT / US94 / 08008 and other references cited above. Other biological events are disclosed in cells that have been treated in a rapamycin- or raphalog-dependent method similar to. The treated cells, preferably animal cells, may be grown or maintained in culture or may be present in all organs, such as in human gene therapy, transgenic animals, and other such applications. Rapamycin or raphalog multimerization agents are administered to cell cultures or, optionally, to organisms containing the cells that have been treated, in an effective amount to multimerize the chimeric protein containing the corresponding ligand binding domain (target gene transcription). Apoptosis, or other biological method), which can be observed indirectly by monitoring). When administered to the whole organism, rapamycin or rapalog may be administered in the form of a composition containing a multimerizing agent and one or more acceptable veterinary or pharmaceutical diluents and / or excipients.

Compounds that bind to one of the chimeric proteins but do not form a complex with the chimeric protein can be used as the multimerization antagonist. As such, it is described in an effective amount to inhibit or convert the effect of the multimerizing agent (preferably in the form of a composition as described above when administered to the whole animal), ie to inhibit or inhibit the multimerization of chimeric proteins. Administered to the treated cells, or the organism containing them. For example, when rapamycin is used as a multimerizing agent, any number of synthetic FKBP ligands that do not form a trident complex with FK506, FK520 or FKBP and FRAP can be used as an antagonist.

One aspect of the invention provides materials and methods for the transcriptional activity of a desired gene according to the ligand. In one such embodiment, a set of two or more different chimeric proteins and corresponding DNA constructs that cause their expression are provided. Such chimeric proteins contain one or more transcriptional active domains as functional domain (s). Other chimeric proteins contain one or more DNA-binding domains (FIG. 1) as functional domains. Rapamycin or rapalog of the invention contain one or more DNA binding domains and transcriptional active domains by binding to both chimeras to form a dimeric or multisynthetic complex. As observed by direct or indirectly monitoring the presence or concentration of the target gene product, this complex formation results in the transcriptional activity of the target gene linked to the DNA sequence to which the DNA-binding domain can bind, under transcriptional regulation. .

Preferably, the DNA binding domain, and the chimera containing the same, have sufficient selectivity such that binding to the selected DNA sequence can be observed (directly or indirectly) despite the presence of other, often multiple, DNA sequences. Bound to its recognized DNA sequence. Preferably, binding a chimera comprising a DNA-binding domain to a selected DNA sequence is determined by in vivo binding studies, or by measuring the rate or level of transcription of a gene associated with the selected DNA sequence compared to all alternative DNA sequences. Likewise, it is at least two, preferably at least three and even more preferably at least four sizes larger than the size of binding to all alternative DNA sequences.

Cells that have been genetically engineered to contain the set of constructs, along with all desired accessory constructs, signal traits such as ligand mediated and regulated actions of a desired biological event, apoptosis, even if transcription of the desired gene is regulated. It may be used in applications including controlled initiation of the route of entry and the like. Cells that have been treated for the controllable expression of the target gene can be used, for example, to regulate the production of the desired protein (or other gene product) encoded by the target gene. The cells can be cultured in culture using conventional means. The addition of ligands to the culture medium containing the cells results in expression of the target genes by the cells and proteins encoded by the target genes. Gene expression and protein production can be blocked by removing residual multimerization agent from the medium or withholding additional multimerization agent from the medium by adding a multimerization antagonist reagent to the medium.

Techniques of the invention The treated cells can be produced and / or used in the body to modify the whole organism, preferably the animal, especially humans, such that the cells produce the desired proteins or other results in the animal containing them. Such uses include gene therapy applications.

Embodiments that include a modulatory action of apoptosis provide for technology treated cells capable of ligand induced cell death. After the cells treated with the above techniques have undesired properties or develop or are no longer useful, safe or undesirable, they are used for intentional purposes (eg, production of the desired protein or other product), Cells treated as described above may be removed from the cell culture or host organism. Removal is performed by adding rapamycin or rapalog to the medium or to the host organism. In this case, the functional domain of the chimeras is a protein domain, such as the FAS antigen or the intracellular domain of TNF-R1 upon oligomerization trigger apoptosis.

As such, the present invention provides materials and methods that seek to achieve a biological effect in the nest against the addition of a multimerization ligand. The method comprises providing a cell that has been treated as described herein and exposing the cell to a ligand.

For example, the present invention provides a method for activating transcription of a target antigen of a cell. The method is linked to (a) a DNA construct encoding a set of chimeric proteins of the invention capable of initiating transcription of a target gene upon ligand mediated multimerization and (b) an associated cognate DNA sequence in response to the multimerization event. Providing a cell containing a target gene (eg, the DNA sequence recognized, ie bindable, DNA binding domain of the aforementioned chimeric protein). The method includes exposing the cell to a multimerization ligand capable of binding the chimeric protein in an effective amount to express the target gene. When cells grow in culture, exposing the cells to the ligand can be performed by adding the ligand to the culture medium. When the cells are present in the host organism, exposing the cells to the ligand is performed by administering the ligand to the host organism. For example, where such host organism is human or non-human, the ligand is administered to the host organism by oral, buccal, sublingual, epithelial, subcutaneous, intramuscular, intravenous, intraarticular or inhalation administration in the form of a suitable excipient. Again, in accordance with the design of the constructs for chimeric proteins and the design of all accessory constructs, ligand mediated biological events may include activation of cellular actions such as signal transduction that causes cell growth, cell proliferation, gene transcription, or apoptosis; Deletion of important genes, inhibition of important gene expression, or inhibition of the action of important gene products; Direct transduction of important genes, and the like.

The invention further comprises a pharmaceutical composition comprising a rapamycin or rapalog of the invention in admixture with a pharmaceutically acceptable carrier and one or more pharmaceutically acceptable excipients. Any of the objectives described herein can be achieved if the pharmaceutical composition is used to facilitate the multimerization of the chimeras of the invention of whole animals, eg, cells treated in the field of human gene therapy.

Contrary to the above, the present invention is directed to the transcription of any of the above targets, eg, target genes (typically heterologous genes for therapeutic proteins) in animals, preferably human patients containing the cells of the present invention as required. Provided are methods in which activity, cell growth or proliferation, cell death or some other selected biological event can be achieved. The method comprises administering to the animal a pharmaceutical composition containing rapamycin or rapalog. Such methods include administering to the animal a pharmaceutical composition containing rapamycin or raphalog in an amount effective to cause multimerization of the chimeric protein by one of the methods of administration and in at least some of the cells treated with the technique. . Multimerization is the development of expression of target genes; Cell growth, proliferation and disappearance; Or can be detected directly by detecting other objects in which the chimeras are designed and the cells genetically engineered.

The present invention inhibits, or otherwise reduces, the degree of multimerization of chimeric proteins in whole or in part in the cells treated with the techniques of the present invention to deactivate the transcription of the target gene, for example, or another of the present invention. Also included is a pharmaceutical composition comprising a pharmaceutically acceptable carrier and, optionally, a multimerization antagonist of the invention in admixture with a pharmaceutically acceptable excipient to block biological consequences. As such, the use of multimerization reagents and multimerization antagonist reagents to prepare pharmaceutical compositions and achieve their pharmacological results is encompassed by the present invention.

The invention also provides a method for providing a host organism, preferably an animal, typically a non-human mammal, or a human subject, which responds to the multimerization ligand of the invention. The method comprises introducing into an organism cell that has been treated according to the invention, ie, containing one or more DNA constructs that encode chimeric proteins and the like. Techniques Treated cells can be encapsulated using a variety of materials and methods prior to introduction into the host organism. Alternatively, the DNA constructs of the present invention may be prepared under conditions that permit, for example, the incorporation of DNA into one or more cells of a host mammal using viral vectors, and the introduction of DNA by injection or via a catheter, and the like. For example, it can be introduced into a mammal.

Also provided are kits for cell introduction in response to rapamycin or rapalog of the present invention. The kit contains one or more DNA constructs that can encode and express chimeras that initiate the desired biological response upon ligand mediated oligomerization. The kit may contain a large amount of oligomerizing ligand (rapamycin or rapalog) capable of multimerizing chimeric protein molecules encoded by the kit's DNA structure (s), and in addition may contain large amounts of multimerization antagonists. have. The kit comprises a DNA construct encoding a target gene (or cloning site) linked to a cognate DNA sequence recognized by a multimerized chimeric protein that allows transcription of a gene linked to a cognate DNA sequence in the presence of the multimerized chimeric protein molecule. Additionally included. The DNA constructs will preferably be mixed with one or more selection markers for expression in prokaryotes to facilitate selection of transfectants as well as other conventional vector elements used for replication in eukaryotes. The selection marker may be the same or different for each different DNA construct and will allow for cell selection containing each said DNA construct (s).

An accessory construct for introducing a target gene mixed with a cognate DNA sequence into a cell may contain a cloning site instead of the target gene. Kits containing the constructs allow for the articulation of cells for the controllable expression of genes by those skilled in the art.

Other kits of the present invention may contain one or two (or more) DNA constructs for chimeric proteins containing one or more cloning sites in place of transcriptional activators or DNA binding proteins. The kit optionally includes a target gene DNA construct in the presence or absence of other elements as described above, eg, cognate DNA sequences for preselected DNA binding domains.

Any kit can be used to generate positive control cells stably transformed with a construct of the present invention that expresses a reporter gene (for CAT, beta-galactosidase or any readily detectable gene product) in response to cell exposure to a ligand. It may also contain. Reagents for detecting and / or quantifying expression of reporter genes may also be provided.

FKBP Chimeras

FKBP chimeric proteins include one or more ligand binding domains containing all or part of the peptide sequence of FKBP, and one or more heterologous domains. Such chimeric proteins are Kd measured using direct binding measurements (eg, fluorescence quenching), competitive binding measurements (eg, as compared to FK506), inhibition of FKBP enzyme activity (rotamases), or other assays. The value should be able to bind to rapamycin or raphalog, which is preferably less than about 100 nM, more preferably less than about 10 nM and even more preferably less than 1 nM. Typically, chimeric proteins will contain one or more protein domains comprising a peptide sequence corresponding to the sequence of FKBP12, as described, for example, in International Patent Application PCT / US94 / 01617. The peptide sequence can be modified to adjust binding specificity by replacement, insertion or deletion of amino acid residues of 10 or less, preferably 5 or less. This modification is selected in certain embodiments to provide one or both of the following binding profiles: (a) preferably one or more, more preferably, better than in FKBP12 or FKBP endogenous to the host cell to be treated Binds the raphalog to a modified FKBP domain, or chimer containing it, that is at least two, more preferably three or four sizes in size; And (b) more preferably (by any measurement) more preferably one or more, more preferably two or more, even more preferably than FRAP or other FRB-containing proteins endogenous to the host cell to be treated by the technique. Binding of the complex formed by the FKBP chimera with rapamycin or raphalog to the second chimera (containing one or more FRB domains as described below) in three or more sizes.

FKBP chimeras also contain one or more heterologous functional domains, ie, protein domains containing non-FKBP peptide sequences. The functional domain is, for example, as described herein or in PCT / US94 / 01617 or other cited references. DNA-binding domain, transcriptional activation domain, cell localization domain, intracellular signal transduction domain. Generally speaking, a functional domain may induce a chimeric protein at a selected cell site or initiate a biological effect upon association or aggregation with another functional domain, eg, upon multimerization of a protein containing the same or different functional domains. Can be.

Recombinant DNA encoding the protein may selectively hybridize the parent FKBP protein, eg, DNA encoding human FKBP12. These chimeric proteins contain functional domains derived from another protein, such as Gal1, VP16, FAS, CD3 zeta chains, etc., and the recombinant DNA encoding the chimeric protein is optionally selectively added to the DNA encoding the other protein. Hybridization would be possible, and hybridization would have been possible without genetic code denaturation.

In addition to the FRAP chimeric proteins described in the further detailed description below, the FKBP chimeric proteins of the invention may contain one or more copies of one or more different ligand binding domains and one or more copies of one or more functional domains. The ligand binding domain (s) can be N-terminal, C-terminal, or can be interspersed with respect to the functional domain (s). Embodiments involving multiple copies of the receptor domain will generally have 2, 3 or 4 such copies. For example, FKBP chimeras may contain 2, 3 or 4 FKBP domains. The various domains of the FKBP chimeras (and FRAP chimeras described below) are optionally separated by binding peptide regions, which may be derived or heterologous from one of the contiguous domains.

Examples of FKBP chimeras useful in practicing the present invention include PCT / US94 / 01617, Applicant's Stanford Harvard, PCT / US94 / 08008, Applicant's Stanford & Harvard, Spencer et al. FKBP fusion proteins described in PCT / US95 / 10591, ARIAD) and PCT / US9506722, Applicant of Mitotix, Inc .; FKBP fusion proteins described in the Examples below; Variants of all of the aforementioned FKBP fusion proteins, which contain less than 10 (preferably 1-5) amino acid insertions, deletions or replacements in one or more FKBP domains and which are still able to bind rapamycin or rapalog; Variants of all of the aforementioned FKBP fusion proteins containing one or more copies of the FKBP domain encoded by a DNA sequence that can optionally hybridize to a DNA sequence encoding a native FKBP domain and still bind to rapamycin or a rapalog; All previous variants wherein one or more heterologous domains are deleted, replaced, or supplemented with a different heterologous domain; Variants of all the aforementioned FKBP fusion proteins capable of binding to rapamycin or rapalog and containing FKBP domains derived from non-human sources; Tyr26, Phe36, Asp37, Arg42, Phe46, Phe48, Glu54, Val55, or one or more amino acid residues corresponding to Phe99 of FKBP12 in humans in which one or more amino acid residues are replaced by different amino acids, and in rapamycin or raphalog It includes all the aforementioned variants of the FKBP fusion protein that can bind.

For example, in many cases, the FKBP fusion protein comprises multiple copies of the FKBP domain containing amino acids 1-107 of human FKBP12, separated by a 2-amino acid linker The-Arg encoded by ACTAGA. The following table is provided to illustrate the subgroups of the variant FKBP domains based on the FKBP12 sequences mentioned above:

F36A Y26A F48H F36V / F99A F36V Y26V F48L F36V / F99G F36M Y26S F48A F36M / F99A F36S D37A E54A F36M / F99G F99A 190A F54K F99G 191A V55A F46H W59A F46L H87W F46A H87R Note: The entry identifies the native amino acid by single letter code and sequence position followed by amino acid substitution in the mutant. Thus, F36V represents the human FKBP12 sequence where the phenylaniline at position 36 is substituted by valine. F36V / F99A represents a double mutation in which phenylalanine at positions 36 and 99 is substituted with valine and alanine, respectively.

FRAP chimeric protein

A second type of chimeric protein, referred to as a FRAP chimeric protein, comprises one or more FRB domains (which may include the peptide sequence of all or part of the FRAP protein or variants thereof, as mentioned elsewhere) and one or more heterologous proteins ( Action) contains a domain.

Generally, a FRB domain or chimeric protein comprising the same is a DNA molecule encoding a protein comprising a native FRB domain, eg, a human or other mammalian FRAP protein or any yeast protein, ie Tor-1 or Tor- 2 or a DNA molecule capable of selectively hybridizing to a DNA molecule encoding a Candida FRB-containing protein already mentioned. FRB domains of the invention include those capable of binding to a complex of FKBP protein and rapamycin or rapalogue. As described in more detail herein, the rapalogs comprise a compound of the formula:

Where

U is -H or -OR ¹ , -OC (O) R ¹ or -OC (O) NHR ¹ , -SR ¹ , -NHR ¹ , -NHC (O) R ¹ , -NHSO ₂ -R ¹ or -NHSO ₂ -R ² , where R ² is substituted aryl degree or allyl degree or alkylaryl (eg, benzyl or substituted benzyl),

V is -OR ³ or (= O),

W is = O, = NR ⁴ = NOR ⁴ or = NNHR ⁴ , -NHOR ⁴ or -NHNHR ⁴ , -OR ⁴ , -OC (O) R ⁴ or -OC (O) NR ⁴ , or -H,

Y is -OR ⁵ , -OC (O) R ⁵ or -OC (O) NHR ⁵ ,

Z is = O, -OR ⁶ , -NR ⁶ , -H, -NC (O) R ⁶ , or OC (O) R ⁶ or -OC (O) NR ⁶ , where R ³ is H, -R ⁷ , -C (O) R or -C (O) NHR ⁷ or C-28 / C-30 cyclic carbonate, R ⁴ is H or alkyl,

Wherein R ¹ , R ⁴ , R ⁵ , R ⁶ and R ⁷ are independently selected from H, alkyl, alkylaryl or aryl.

The FRAP chimeric protein must be able to bind to the complex formed by the FKBP chimera and rapamycin or raphalog. Preferably the FRAP chimera binds to a complex having a Kd value of less than 200 μM, preferably less than 10 μM as measured by conventional methods. The FRB domain will be of sufficient length and composition to maintain high affinity for rapamycin or a complex of rapamycin and FKBP chimera. In some embodiments, the FRB domain is less than about 150 amino acids in length and in some cases less than about 100 amino acids. One such region includes the 133 amino acid region of human FRAP, ranging from Val ²⁰¹² to Tyr ^2144. Chiu et al., 1994, Proc, Natl. Acad. Sci. USA 91: 12574-12578. Particularly valuable FRB regions range from Glu ²⁰²⁵ to Gln ²¹¹⁴ of human FRAP and have affinity for FKBP12-rapamycin. In some embodiments Q2214 is removed from the 90-amino acid sequence giving rise to the 89-amino acid FRB domain. FRB peptide sequences can generally be modified to control binding specificity by substitution, insertion or deletion of up to 10, preferably up to 5 amino acids. Such modifications are selected in certain embodiments to preferentially achieve formation of a complex comprising one or more FKBP chimeras, FRAP chimeras and rapamycin or raphalog molecules relative to the formation of a complex of endogenous FKBP and FRAP proteins with rapamycin / lapalog do. Preferably, one or more, more preferably two or more, very preferably three, prevail in size order (by measurement).

Recombinant DNA encoding such proteins can selectively hybridize DNA encoding FRAP species, or hybridize in the absence of denaturation of the genetic code. In other words, since such chimeric proteins contain functional domains derived from other proteins, such as Gal14, VP16, Fas, CD3 zeta chains, and the like, recombinant DNA encoding chimeric proteins may be incorporated into the DNA encoding these other proteins. Selectively hybridized to This hybridization can be enabled without denaturation of the genetic code.

Illustrative examples of FRB chimeras useful in the practice of the present invention include those described in the Examples below, one or more of these, as long as the chimera can bind to FKBP protein and complexes synthesized by rapamycin or rapalogue. Variants wherein the domain is substituted with an optional heterologous domain or supplemented with one or more additional heterologous domains, variants wherein one or more of these FRB domes is a domain of non-human peptide sequence origin (eg, Tor or Candida), and FRB domains Variants modified by amino acid substitutions, substitutions, or insertions as described below are included. Exemplary FRB fusion proteins contain one or more 89-amino acid FRBs containing residues 2025-2113 of human FRAP separated by linker Thr-Arg formed by linkage of the SpeI-XbaI site, as already mentioned. Such restriction sites or linkers in the fusion proteins of the invention can be deleted, substituted or extended using conventional techniques such as site-induced mutagenesis.

Heterogeneous domain

As mentioned above, the heterologous domains of FKBP and FRAP chimeras induce (or inhibit) DNA-binding and / or transcription of target genes, or induce cell growth, differentiation, and proliferation in relation to the mutual binding of chimeric proteins containing Ir. Or a protein domain capable of activating apoptosis, inducing proteins at specific cell locations or activating other biological events.

Embodiments that include controllable gene transcription include target genes (which are valuable polypeptides, antisense RNAs, ribozymes, etc.) under the transcriptional control of DNA components that are responsive to binding or multiple binding of heterologous domains of the first and second chimeric proteins. Encoding target gene construct).

In embodiments of the invention in which transcription relates to direct activation, the heterologous domains of the first and second chimeric proteins are derived from DNA binding domains such as Gal4 or chimeric DNA binding domains such as ZFHD1 or VP16 or p65 respectively. A transcriptional activation domain such as that shown. Multiplexing of chimeric proteins containing such transcriptional activation domains to chimeric proteins containing DNA binding domains targets transcriptional activators to the promoter component to which the DNA binding domain binds, thus targeting genes linked to the promoter component Activates transcription. Omitting the transcriptional activation domain or substituting the inhibitory domain instead of the transcriptional activation domain allows to provide similar chimeras useful for inhibiting transcription of the target gene. Complex DNA binding proteins and the DNA sequences to which they bind are described in the above Pomeranz et al. (1995), incorporated herein by reference. Such complex DNA binding domains may be used as DNA binding domains in the practice of the present invention in conjunction with target gene constructs containing cognate DNA sequences to which complex DBDs bind.

In embodiments related to indirect activity of transcription, the heterologous domain of the chimera is the effector domain of a signal protein that induces the activity of transcription under the control of a reactive promoter upon aggregation or multibinding. For example, the signal domain may be a zeta subunit intracellular domain of the T cell receptor, which, upon aggregation, is responsible for the IL-2 promoter or genes to which they are linked to derivatives (eg, repeated NF-AT binding sites). Induce transcription.

In another aspect of the invention, the heterologous domain is a protein domain capable of inducing cell death upon mutual binding. Examples of such domains are Fas antigens or intracellular domains of TNF R1. Chimeric proteins containing Fas domains can be designed and produced similarly to the contents of PCT / US94 / 01617.

Engineered Receptor Domain

As already mentioned, the FKBP and FRB domains may contain peptide sequences selected from the peptide sequences of native FKBP and FRB domains. Naturally occurring sequences comprise the sequence of human FKBP12 and the FRB domain of human FRAP. Alternatively, peptide sequences may be derived from such native peptide sequences, but generally comprise one or two such peptide sequences, generally no more than 10, preferably 1-5 mutants. As described in more detail herein and shown in FIG. 2, such mutants can confer many important properties. For example, the FKBP domain preferentially prioritizes the raplogs, i.e. one or more, preferably two, more preferably three or four (more effective in order of size) for the unmodified FKBP domain. It can be modified to engage. FRB domains preferentially (ie, at least one, preferably two, more preferably three (more in order of size) FKBP raphalogs (modified or unmodified) over unmodified FRB domains Can be modified to bond. The FKBP and FRB domains can preferentially bind three-part complexes of rapalogues with rapamycin, namely one or more, preferably two, more preferably three (more effective in order of size). Can be modified to be. 2A-E are for illustrative purposes only, a combination of several related modified rapalogs and a receptor domain containing a reward mutant is also encompassed by the present invention and may be applied for various purposes.

(a) FKBP

Methods for identifying FKBP mutants that confer enhanced binding to derivatives of FK506 containing several substituents (bumps) are described in PCT / US94 / 01617. Similar methods can be used to obtain preferentially bumped rapamycin derivatives, ie modified FKBPs that bind rapalogues. The complex structure between rapamycin and FKBP12 is known. See, eg, Van Duyne et al., J. Am. Chem. Soc. (1991) 113, 7433-7434. This data can be used to identify amino acid residues that may conflict with various Rapalog substituents. In this approach, molecular modeling is used to identify candidate amino acid substitutions within the FKBP domain that can accept Rapalog substituents, and then site-directed mutagenesis is used to manipulate the protein mutants thus identified. Used to. Mutants are expressed by standard methods, and their binding affinity to the rapalogs is, for example, by inhibition of rotamase activity, or when a mutant possesses sufficient activity / affinity with a molecule such as FK506. It is measured by the binding contention of.

In particular, the rapamycin derivatives modified at C-13 or C-14 (ie, rapalogues where Y is a substituent other than -OH and / or Z is not = 0) are one or more residues, ie Tyr26, Phe36, Asp37, It is believed that Tyr82 and Phe99 bind preferentially to FKBP substituted with amino acids having smaller side chains (eg, Gly, Ala, Val, Met and Ser). Examples of mutant FKBPs modified at position 26 or 36 are described in the table Exemplary Mutant FKBPs above. Similarly, altered rapalogs (ie, rapalogs in which R ⁴ is not -H) are believed to bind preferentially to FKBP where Tyr82 and / or Ile56 are substituted by other amino acids, especially those with smaller side chains. . In a further embodiment, the rapalog containing the altering moiety at C24 (ie, the rapalog where W is not = 0) is substituted by one or more Phe46, Phe48 and Val55 by other amino acids, especially those with smaller side chains. It is believed to bind preferentially to FKBP.

In addition, the raphalogs containing a modification at C28 and / or C30 (ie, raphalogs in which R is not H and / or V is not = O) are substituted by Glu54 having another amino acid, especially a smaller side chain. It is believed to bind preferentially to the FKBP. Specific examples are listed in the preceding table.

An alternative to repeat manipulations and testing of single or complex mutants is to jointly randomize structurally identified residues that are in contact with or adjacent to one or more rapalogues or rapamycin periods. Collections of polypeptides containing randomized FKBP domains at identified locations (as described in the preceding paragraph) are prepared, for example, using conventional synthetic or genetic methods. This collection represents a series of FKBP domains containing substituted amino acids at one or more such positions. The collections are screened and FKBP variants are selected that have the desired rapalog binding properties. In general, several randomized residues are expected to produce compensating mutants of higher affinity and specificity for a given bumped rapalog by maximizing the likelihood of advantageous cooperative interactions between the side chains at the same time. Techniques for preparing randomized libraries at discrete positions are known and include primer induced mutagenesis using denatured oligonucleotides, PCR with denatured oligonucleotides, and mutagenesis with denatured oligonucleotides. , HB and Wells, J. A. Methods: Comp. Methods Enzymol. 1991.3, 205-216; Dennis, M.S. and Lazarus, R. A. 1994. J. Biol. Chem. 269, 22129-22136; and cited herein.

In many cases, it is also possible to fully investigate all possible variations in the FKBP structure that can accommodate random optimally rapalogue substituents (bumps) of only a few residues within or adjacent to a predetermined position in rapamycin. It is not possible. Thus, the structure is observed with an equilibrium library containing random substituents that are not based on structural considerations to identify sensitive mutants or combinations thereof that preferentially bind to bumped raphalogs. Some suitable mutagenesis outlines have been described, alanine-injection mutagenesis (Cunningham and Wells (1989) Science 244, 1081-1085), PCR misincorporation mutagenesis (see Cadwell and Joyce, 1992, PCR Meth. Applic. 2, 28-33), and DNA shuffling (Stemmer, 1994, Nature 370, 389-391 and Crameri et al, 1996, Nature Medicine 2, 100-103). Such techniques are investigated by screening or selection after generating a library of random mutants or a series of single mutants.

In many cases, an effective method for identifying optimal mutants for preferential binding of a given bump is a combination of structure-based and equitable approaches. Clackson adn Wells, 1994, Trends Biotechnology 12, 173- 184 (review)]. For example, although the major contact residues are randomized by PCR with denaturing oligonucleotides, there is a construct in the library that is accompanied by amplification performed using error-promoting conditions that introduce additional mutants at random sites. Is considered.

Screening of libraries for mutants of interest can be performed using a yeast two-hybrid system (Fields and Song (9189) Nature 340, 245-246). For example, the FRBVP16 melt can be introduced into one vector and a library of randomized FKBP sequences can be cloned into separate GAL4 fusion vectors. Yeast co-transformants are treated with bumped rapamycin (ie, rapalogue), and the inclusion of a compensating FKBP mutant, for example, in the production of beta-galactosidase or luciferase (screen), or This is confirmed by survival (selection) on a plate that lacks essential nutrients for the vector used. A prerequisite for the bumped rapamycin that bridges the FKBP-FRAP interaction is a useful screen to remove false positives.

Another method for isolating modified ligand-binding domains from a library of FKBP (or FRB) is described in Hu et al., Hu, JC, et al. 1990, Science, 250: 1400-1403; for reexamination. See, Hu, J. C. 1995. Structure. 3: 431-433), a gene selection method for functional dimer formation is used. This method takes advantage of the fact that the bacteriophage lambda inhibitor cI binds to DNA as a homodimer and that binding of the homodimer to the operator DNA inhibits the transcription of the phage gene associated with the cell lysis pathway of the phage's survival cycle. Thus, bacterial cells expressing functional lambpa inhibitors are immunized and lysed as phage lambta as a superinfection sieve. The inhibitory protein comprises an amino acid terminal DNA binding domain (amino acids 1-92) bound to the carboxy terminal dimerization domain by a 40 amino acid soft linker. Isolated N-terminal domain binds to low affinity DNA due to ineffective dimer formation. High affinity DNA binding can be restored to heterologous dimerization domains such as GCN4 leucine zippers. Hue et al. Describe a system for isolating GCN4 leucine zipper mutants capable of using phage immunity as gene selection to mediate large populations of sequencer lambda inhibitor dimers (Hu et al., 1990).

For example, to use a lambda inhibitor system to identify FRAP mutants that are complementary to bumped rapalogs, a lambda inhibitor-FRAP library containing a mutated FRAP sequence may be transformed into E. coli expressing the wild-type lambda inhibitor-FKBP protein. Let's do it. Plasmids expressing FRAP mutants are isolated from colonies that survive in lysis on bacterial plates containing high titers of lambda phage and bumped rapamycin compounds. Alternatively, to isolate the FKBP mutant, the method is repeated with a lambda inhibitor-FKBP library containing the mutant FKBP sequence transduced into Escherichia coli expressing the wild type lambda inhibitor-FRAP protein.

Another alternative is to clone the randomized FKBP sequence into a vector for phage display, allowing the selection of variants that optimally bind to the rapalog in vitro.

In vitro affinity selection can be performed in a number of ways. For example, the rapalogs are mixed into a library phage pool in solution in the presence of recombinant FRAP tagged with an affinity handle (eg, hexa-histidine tag, or GST), and the resulting complex is mixed. Capture on suitable affinity matrix to enrich for phage display FKBP containing supplemental mutants. Phage display is described in the literature and other in vitro selection systems are also contemplated (eg, display on lambda phage, display on plasmid, display on baculovirus). In addition, screening and screening methods can also be used to improve other advantageous properties in the present application, such as enhanced stability in vivo. See, eg, Clackson, T. and Wells, J. A. 1994. Trends Biotechnol. 12, 173-184.

(b) FRAP

Similar considerations apply to the generation of mutant FRB domains that preferentially bind to rapalogues containing modifiers (ie, bumped) with respect to rapamycin in the FRAP-binding effector domains. For example, a substituent other than -OMe in C7 with FRB based on a human FRAP FRB peptide sequence, except that at least one contains amino acid substitutions for residues Tyr2038, Phe2039, Thr2098, Gln2099, Trp2101 and Asp2102. It can bind preferentially using the containing rapalog. Exemplary mutants include Y2038H, Y2038L, Y2038V, Y2038A, F2039H, F2039L, F2039A, F2039V, D2021A, T2098A, T2098N and T2098. Raphalogs containing substituents other than -OH at C28 and / or substituents other than = O at C30 can be used to preferentially bind to FRAP proteins containing amino acid substitutions for Glu2032. Exemplary mutants include E2032A and E2032S. At least one amino acid substitution at that position, a library of proteins or peptides randomized at this position (ie, containing multiple amino acids substituted at these residues), a library that randomizes the entire protein domain, or a series of such mutations The combination is made using the procedure described above to identify mutant FRAP that binds preferentially bumped raphalogs.

The affinity of the Candidate mutant FRB for the complex of the Rapalog and the complexed FKBP protein can be assayed by several methods; For example, in vitro transformed FRB mutants to GST-FKBP in the presence of a drug bind (Chen et al. 1995. Proc. Natl. Acad. Sci. USA 92, 4947-4951); Or ability to involve rapalog-dependent transcriptional activity complexes with a suitable FKBP fusion protein in a yeast double hybrid assay.

FRB mutants with the desired binding specificity can be isolated from libraries displayed on phage using various sorting methods. For example, the rapalogs are mixed into a library phage pool in solution in the presence of recombinant FRAP tagged with an affinity handle (eg, hexa-histidine tag, or GST), and the resulting complex is mixed. Capture on suitable affinity matrix to enrich for phage display FKBP containing supplemental mutants.

A further feature of the FRB fusion proteins, in which the invention may be varied in various embodiments, is the exact sequence of the FRB domain used. For some uses, it may be desirable to use a portion of FRB that is larger than the minimum (89 amino acid) FRB domain. These include the N-terminal extension to residue Glu2025 (preferably at least to Art2018 or Ile2021) as well as the C-terminal extension past position 2113 (eg, positions 2113, 2141 or 2174 or beyond), which in some cases It may improve the stability and / or efficiency of expression of overlapping FRB domains. Other applications in which different FRB sequence termini can be used include either or both sides of the FRB domain such that the long linker accommodates modification of the polypeptide chain required for FRB-mediated protein-protein binding, eg, on cell membranes or on DNA. Suitable for three-dimensional basis is included in the image. In contrast, in other applications short linkers on one or both sides of the FRB domain may be desirable or required to provide heterologous functional domain (s) suitable for biological function. In human gene therapy applications, the use of native human FRAP sequences for such linkers will generally reduce the risk of introducing an heterologous sequence or causing an immune response in the host organism.

Rapa with altering substances or substituents (relative to rapamycin) at positions believed to be adjacent to the boundary between the FKBP binding domain and the FRAP binding domain, such as those on some rapalogues, particularly those on C28, C30, C7 and C24. The log has a reduced ability to form a complex having two mammalian FKBP and FRB domains, especially a domain containing native human peptide sequences, compared to rapamycin. Such reduced complex-forming ability can be evident with reduced immunoaffinity activity measured in suitable assays such as reduced binding affinity or T cell proliferation assays measured by the direct or indirect assay methods mentioned herein. In such cases, repeat procedures can be used to identify mutant FKBP and mutant FRB pairs that can be complexed with rapalogs more effectively than the corresponding domains containing native human peptide sequences. For example, first, a modified FKBP domain of a supplement capable of binding to rapalog can be identified as described above, and then using the mutant FKBP domain as an affinity matrix in the complex with rapalog to bind to the complex. One can choose to supplement the altered FRB domain. Several cycles of such mutation generation and screening can be used to optimize protein pairs.

In some embodiments, it will be desirable to use FRB and / or FKBP domains containing mutants that can affect protein-protein interactions (see, eg, FIG. 2E). For example, mutant FKBP domains that are capable of complexing with endogenous FRBs to some extent less effectively than mutant FRBs are particularly valuable when bound to rapamycin or a given rapalogue. It is also worth noting that a mutant FRB domain capable of binding to a complex of mutant FKBP and rapamycin or a given paralog more effectively than the complex of endogenous FKBP and rapamycin. Selection and screening approaches similar to those already described (i) identify amino acid substituents, deletions, or inserts with FKBP domains that somewhat reduce the ability of rapamycin or a domain to form a three-part complex with raplog and endogenous FRB. To identify (ii) amino acid substituents, deletions, or inserts with a rapamycin or FKBP domain that reduces the ability of the domain to form a three-part complex with rapamycin and endogenous FKBP to (iii) a relative protein. Supplementary mutants within can be used to select and / or otherwise identify. Suitable mutant FKBPs with reduced efficiency in the formation of a three-part complex are examples of mammals in which one or two His87 and Ile90 are substituted with amino acids such as Arg, Trp, Phe, Tyr or Lys containing large side chain groups. Let FKBP; FRB domain containing preferably a FRB domain of a mammal, more preferably a human, wherein the peptide sequence can be mutated as described above to form a three-part complex with the mutant FKBP and rapamycin or a given rapalogue Complementary variants can be generated. Exemplary FRB mutants that may be useful with H87W or H87R hFKBP12 include human FRBs with Y2038 replaced with V, S, A or L, F2039 replaced with A and / or R2042 replaced with L, A or S. Included. Exemplary FRB mutants that may be useful with I90W or I90R hFKBP12 include human FRBs where K2095 has been replaced with L, S, A or T.

In addition, in optimizing the receptor domain of the present invention, it should be appreciated that immunogenicity of the polypeptide sequence requires binding of the peptide by the MHC protein and recognition of the peptide as foreign by the endogenous T-cell receptor. For at least human gene therapy applications, it may be desirable to fit certain foreign peptide sequences, including peptide sequence linkages, ie to minimize the possibility of being immunologically presented to humans. For example, a peptide that binds to a human MHC class 1 molecule is essential for a residue specific to an important 'fixed' position in the bound peptide. For example, HLAA2 requires leucine, methionine or isoleucine at position 2 and leucine or valine at the C-terminus (see, eg, Stern and Wiley (1994) Structure 2, 145-251). Thus, in manipulating proteins in the practice of the present invention, the periodicity of such residues is preferably avoided, especially in human gene therapy applications. The application applies to selecting or designing the boundaries between various protein domains and embodiments of engineering all proteins of the invention, including but not limited to engineering point mutations in the receptor domain.

Other ingredients, design features and applications

Chimeric proteins may contain a cell localization domain, such as a membrane-bearing domain, as a heterologous domain (see, eg, PCT / US94 / 01617, in particular pages 26-27). In brief, membrane-bearing domains can be isolated from easy membrane binding proteins, whether or not endogenous to the host cell. The membrane-bearing domain may be a transmembrane and retention domain containing many receptors, ie amino acids in which cell surface proteins extend across the membrane as in the case. The transmembrane peptide sequence may also extend to some or all of the extracellular and / or intracellular domains. Alternatively, the membrane bearing domain can be a lipid membrane bearing domain such as a myristoylated or palmitoylated site that allows binding to lipids of the cell surface membrane. Lipid membrane retention domains will generally be added at the 5 'end of the coding sequence for N-terminal binding to the membrane and adjacent to the 3' end for C-terminal binding. Peptide sequences comprising post-translational processes provided for lipid membrane binding are described in the literature (Carr, et al., PNAS USA (1988) 79, 6128; Aitken, et al., FEBS Lett. (1982) 150, 314; Henderson, et al., PNAS USA (1983) 80, 319; Schulz, et al., Virology (1984), 123, 2131; Dellman, et al., Nature (1985) 314, 374; Ann. Rev. of Biochem. (1988) 57, 69]. A valuable amino acid sequence comprises the sequence M-G-S-S-K-S-K-P-K-D-P-S-Q-R. Various DNA sequences can be used to encode such sequences in the various chimeric proteins of the invention. Other localization proteins are particularly useful for chimeric proteins designed for (direct) transcriptional regulation as well as organelle-target domains and sequences such as -KDEL and -H-DEL (they target proteins containing them in the endoplasmic reticulum). It includes. Several cell localization sequences and signals are well known in the art.

Chimeric proteins containing various functional domains, including cytoplasmic signal initiation domains, such as the CD3 zeta chain, among others, nuclear transcription factor domains including VP16 and GAL4, Fas cytoplasmic domains, and other domains capable of apoptosis Further details that can be used in the embodiments of the present invention in connection with the design, assembly, and use of coded components are described in PCT / US94 / 01617 and PCT / US95 / 10591. The latter international application further describes additional properties that are particularly applicable to the generation of genetically engineered animals that can be used as disease models in herbal medicine research. These properties include transcription regulated for expression of Cre recombinase as a target gene that induces the use of tissue specific regulatory components in constructs for the expression of chimeric proteins and the removal of genes of interest flanked by lox P sequences. This includes applying. This property can be applied to the present invention.

In many cases, especially in embodiments involving whole animals containing cells treated according to the invention, in some cases where desired, it is often desirable for the various domains of the chimeric protein to be derived from a protein of the same species as the host cell. . As such, in gene processing of human cells, it is often desirable that the heterologous domains (as well as the FKBP and FRB domains) are of human origin rather than bacteria, yeast or other non-human sources.

The inventors also mention that the epitope tag can be mixed into the chimeric protein of the invention and conveniently detected.

Tissue Specificity of Cell Type Specificity Expression

In some embodiments, the chimeric protein is preferably expressed in a cell specific or tissue specific manner. Such specificity of expression can be achieved by operatively binding one or more DNA sequences encoding a chimeric protein to a cell type specific transcriptional regulatory sequence (eg, promoter / enhancer). Numerous cell type specific transcription control sequences are known. Other sequences can be obtained from genes expressed in a cell specific manner. See for example PCT / US95 / 10591, in particular pages 36 and 37.

For example, constructs expressing chimeric proteins may contain regulatory sequences derived from known genes for specific expression in selected tissues. Representative examples are described below:

Target gene constructs

In embodiments of the invention in which the chimeric protein is designed such that multiple binding thereof activates the transcription of the target gene, suitable target gene constructs are also used in the treated cells. Suitable target gene constructs are those containing cognate transcriptional regulatory elements such as promoters and / or enhancers that respond to multiple binding of target genes and chimeric proteins. In embodiments involving direct activation of transcription, the response may be achieved by the presence of one or more target gene constructs of the DNA sequence recognized by the DNA binding domain of the chimeric protein of the invention (ie, the DNA sequence to which the chimeric protein binds). Can be. In embodiments involving indirect activation of transcription, the response can be achieved by the presence of a promoter and / or enhancer sequence in the target gene construct that is activated by intercellular signals generated by multiple binding of chimeric proteins. . For example, if the chimeric protein contains a TCR zeta chain intercellular domain, the target gene is bound under the control of expression of the IL-2-promoter region.

The invention also relates to (a) a cognate DNA sequence, eg, a DNA sequence to which the DNA binding chimeric protein of the invention can bind (or a DNA sequence that is prone to indirect activation as described above), and (b) A target DNA construct is provided that contains flanking DNA sequences from the locus of a desired target gene that is endogenous to the host cell. Such constructs allow homologous recombination of cognate DNA sequences in host cells associated with endogenous target genes. In other embodiments the construct contains a flanking DNA sequence from a target locus that allows homologous recombination of the desired gene and target gene into the desired locus. The target construct may also contain cognate DNA sequences, or cognate DNA sequences may be provided by loci.

The target gene of any of the above embodiments may encode, for example, surface membrane proteins (eg, receptor proteins), secretory proteins, cytoplasmic proteins, nuclear proteins, recombinants such as Cre, ribozyme or antisense RNA. See PCT / US94 / 01617 for detailed descriptions of general designs and constructs and for various applications including gene therapy and PCT / US95 / 10591 for applications related to animal models of disease.

The present invention includes various arrangements for chimeric proteins. However, in all cases involving activation of target gene transcription, the chimeric protein has the following important features: Cells containing constructs encoding chimeric and target gene constructs target genes in their presence rather than in the absence of multiple binding ligands. One or more, preferably two or more, more preferably three or four or more sizes. Bestly, expression of the selected gene is not observed when the cell is or is not exposed to multiple binding ligands.

To regenerate, the chimeric protein can initiate a detectable level of transcription of the target gene in the treated cells upon exposure of the cells to rapamycin or rapalogue ligands, ie after multiple binding of the chimeric. As such, transcription of the target gene can be activated in the genetically treated cells of the present invention following exposure of the cells to rapamycin or rapalogue ligands capable of multiplexing chimeric protein molecules. In other words, the genetically treated cells of the present invention respond to the presence and / or concentration of rapamycin or rapalogue ligands that contain chimeric proteins and are capable of multiplexing the chimeric protein molecules as described above. The response is evidenced by the activation of transcription of the target gene. The transcriptional activity can be readily detected by all conventional assays for the transcription of target genes. In other embodiments, the biological response to ligand mediated multiple binding of the chimera is cell death or other biological event rather than direct activation of transcription of the target gene.

Injection of components into cells

The invention is particularly useful for applications involving the treatment of animal cells and the use of such treated animal cells. Animal cells may be insect, worm or mammalian cells. Human cells are of particular interest, although various mammalian cells can be used, including horses, cows, sheep, dogs, cats, mice, and nonhuman primate cells. Among various species, various types of vesicles, such as hematopoietic, nervous system, glial, mesenchymal, skin, mucous membranes, epilepsy, muscle (including smooth muscle cells), spleen, reticular, epithelial, endothelial, liver, kidney, gastrointestinal, lung, Fibroblasts, and other cell types, can be used. Cells of particular interest are hematopoietic cells which may comprise any of the nucleated cells associated with myoblasts and fibroblasts in addition to the erythroid, lymphocyte or myeloid monocyte lineage. Also of interest are household and progenitor cells such as hematopoietic, nervous system, epilepsy, muscle, hepatic, lung, gastrointestinal and hepatitis household cells.

The cells may be self cells, consensus gene cells, allogeneic cells, and in some cases may be heterologous cells associated with the intended host organism. The cells are provided for expressing one or more MHC molecules, which change the major histocompatibility complex (MHC) profile and increase or inactivate cell disruption potential by increasing or inhibiting expression of genes associated with cell disruption activity, etc. It can be modified by inactivating β ₂ -microglobulin to prevent the formation of functional class I MHC molecules, inactivation of class II molecules.

In some cases specific clone or oligoclonal cells may be of interest, in which case the cells have specific specificities, such as T cells and B cells having specific antigen specificity or homing target site specificity.

Constructs encoding the chimeric proteins and target genes of the invention may be injected intracellularly, such as one or more DNA molecules or constructs, in many cases involving one or more markers that allow for the selection of host cells containing the constructs. Constructs can be prepared in a conventional manner, and coding sequences and regulatory regions can be isolated, appropriately ligated, cloned in appropriate cloning hosts, and analyzed by definition or sequencing, or other convenient means. In particular, using PCR, individual fragments comprising all or a portion of the functional units can be isolated and one or more mutations can be injected using appropriate primer repair, ligation, in vitro mutation induction. Once the construct is complete and marked to have the proper sequence, it can be injected into the host cell by any convenient means. The construct may be included in a vector capable of episomal replication (eg, a BPV or EBV vector) or a vector designed for incorporation into a stain of host cells. The constructs are integrated and packaged into a nonreplicating, defective virus genome, similar to adenovirus, adeno-associated virus (AAV), or herpes simplex virus (HSV) or others, including retroviruses, for infection or intracellular transfection. Can be. Alternatively, the construct can be injected by protoplasm fusion, electroporation, biolithography, calcium phosphate transformation, lipofection, microinjection, such as DNA. The host cell will in some cases be grown and developed in culture prior to injection of the construct and then appropriately processed for infusion of the construct and integration of the construct. The cells will then be developed and screened by markers present in the construct. Various markers that can be used successfully include hprt, neomycin resistant materials, thymidine kinase, hygromycin resistant materials, and the like, and there are various cell surface markers such as Tac, CD8, CD3, Thy1 and NGF receptors.

In some cases, the construct may have a target site for homologous recombination and it is desirable for the construct to be integrated at a particular locus. For example, constructs may delete and / or replace endogenous genes having recombinant target constructs of the invention (done at the same locus or at other locations). For homologous recombination, cells can generally use Ω or O-vectors. See, eg, Thomas and Capecchi, Cell (1987) 51, 503-512; Mansour, et al., Nature (1988) 336, 348-352; And Joyner, et al., Nature (1989) 338, 153-156.

Constructs can be injected with a single DNA molecule encoding all of the genes, or with different DNA molecules having one or more genes. The constructs can be injected simultaneously or sequentially, each with the same or different markers.

Vectors containing useful elements such as bacterial or yeast origin of replication, selectable and / or amplifiable markers, promoters / enhancer elements for expression in prokaryotic or eukaryotic cells, and mammalian expression control elements, etc. Can be used to prepare and perform transformation) are known in the art and are commercially available.

Injection of components into the animal

Cells that are ex vivo modified with the DNA construct can be grown in culture under selected conditions, and the cells selected to have the desired construct can be developed, for example, after the polymerase chain reaction to determine the presence of the construct in the host cell, and And / or using assays for the production of desired gene products. Modified host cells can be identified and used as planned, for example, grown in culture or injected into the host organism.

Depending on the nature of the cell, the cell can be injected into the host cell, for example a mammal, in a number of ways. Hematopoietic cells may be administered by injection into a vascular system, which typically has about 10 ⁴ or more cells and generally about 10 ¹⁰ or less cells. The number of cells used will vary depending on many circumstances, the purpose of the infusion, the life of the cells, the protocol used, for example the number of doses used, the ability of the cells to proliferate, the stability of the therapeutic agent, the physiological demand for the therapeutic agent, and the like. Generally, for example, for myoblasts, fibroblasts, the number of cells will be at least about 10 ⁴ and up to about 10 ⁹ and may be applied as a dispersion and generally injected at or near the site of interest. The cells will generally be a physiologically acceptable medium.

Cells treated according to the invention can also be encapsulated using conventional biocompatible materials and methods prior to infusion into the host organism or patient for the production of therapeutic proteins. See, eg, Hguyen et al, Tissue Implant Systems and Methods for Sustaining viable High Cell Densities within a Host, US Pat. No. 5,314,471 to Baxter International, Inc .; Uludag and Sefton, 1993, J Biomed. Mater. Res. 27 (10): 1213-24 (HepG2 cells / hydroxyethyl methacrylate-methyl methacrylate membrane); Chang et al, 1993, Hum Gene Ther 4 (4): 433-40 (mouse Ltk-cells expressing hGH / immunoprotective perm-selective alginate microcapsules; Reddy et al, 1993, J Infect Dis 168 (4): 1082-3 ( alginate); Tai and Sun, 1993, FASEB J 7 (11): 1061-9 (mouse fibroblast expressing hGH / alginate-poly-L-lysine-alginate membrane); Ao et al, 1995, Transplanation Pro. 27 (6) : 3349, 3350 (alginate); Rajotte et al, 1995, Transplantation Proc. 27 (6): 3389 (alginate); Lakey et al, 1995, Transplantation Proc. 27 (6): 3266 (alginate); Korbutt et al, 1995, Transplantation Proc. 27 (6): 3212 (alginate); Dorian et al, US Pat. No. 5,429,821 alginate; Emerich et al, 1993, Exp Neurol 122 (1): 37-47 (polymer0 encapsulated PC12 cells); Sagen et al, 1993, J Neurosci 13 (6): 2415-23 (bovine chromaffin cells encapsulated 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. See: Aebischer, WO 92/19595); Savelkoul et al, 1994, PNAS USA 91 (6): 2324-8 (engineered BHK cells expressing human nerve growth factor encapsulated in an immunoisolation polymeric device and transplanted into rats); Emerich et al, 1994, Prog Neuropsychopharmacol Biol Psychiatry 18 (5): 935-46 (polymer-encapsulated PC 12 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 injected in an encapsulated form into animal hosts, preferably mammals and more preferably humans in need of the cells. Preferably the encapsulating material is semipermeable and allows release of the secreted protein produced by the encapsulated cells into the host. In many embodiments, semipermeable encapsulation immunologically isolates the encapsulated cells from the host organism into which the encapsulated cells are injected. In such embodiments, the cell to be encapsulated may express one or more chimeric proteins containing component domains derived from proteins of the host species and / or viral proteins or proteins from other species. For example, in this case the chimera may contain elements derived from GAL4 and VP16. The cell may be derived from a recipient or one or more other individuals and may be derived from a species other than that of the recipient organism or patient.

Instead of ex vivo expression of a cell, in many situations the inventor may wish to modify the cell in vivo. For this purpose, various techniques have been developed for modification of target tissues and cells in vivo. Numerous viral vectors such as adenoviruses, adeno-associated viruses and retroviruses are considered for transformation, and in some cases, for the integration of viruses into the host. See, eg, Dubensky et al. (1984) Proc. Natl. Acad. Sci. USA 81, 7529-7533; Kaneda et al., (1989) Science 243, 375-378; Hiebert et al. (1989) Proc. Natl. Acad. Sci. USA 86, 3594-3598; Hatzoglu et al. (1990) J. Biol. Chem. 265, 17285-17293 and Ferry, et al. (1991) Proc. Natl. Acad. Sci. USA 88, 8377-8381. The vector may be administered by injection, eg, by endovascular injection or by intramuscular injection, inhalation, or other parenteral mode. Non-viral delivery methods can be used, for example administration of DNA through injections with liposomes, injection, catheters or by virolytics.

Depending on the genetic modification in vivo, the manner of modification may vary depending on the characteristics of the tissue, the efficiency of the required cell type modification, the number of times to modify the particular cell, and the influence of the tissue on the DNA construct to be injected. By using a virus that has attenuated or modified toxins that carry the target transcription initiation region, the virus can be produced and transformed with surrounding cells, if desired, by activating the virus using one of the superficial transcription factor constructs.

DNA injection does not need to cause integration in all cases. In some situations, instantaneous maintenance of the introduced DNA may be sufficient. In this way, the inventor can obtain a short cycle effect, the cells can be introduced into the host, and then the cells can return after a predetermined time, for example after they can be returned at a particular site.

Multiple binders

Ligands suitable for use in the present invention include rapamycin and rapamicin analogs, derivatives and pseudo (rapalogs) that bind to the FKBP chimera and FRAP chimera to form a tripatitic complex. Preference is given to the rapalogues of the invention that contain one or more substituents (bumps) that reduce binding to, and preferably significantly interfere with, endogenous FKBP and / or FRAP proteins in the treated cells. Variant FKBPs of the present invention can be obtained and screened for binding to the provided rapalogs as described in PCT / US94 / 01617 and PCT / US94 / 08008. Some rapalogs bind to or complex with (variant) FKBP chimeric proteins, (variant) FRAP chimeras (see FIG. 2), but have any substituents that reduce binding to the FRAP and preferably significantly interfere with the FKBP complex. . The rapalog containing the pump can more selectively bind to mutant FKBP chimeras and / or mutant FRAP chimeras without being affected by the endogenous congestion of FKBP12 or FRAP. This is desirable for many applications, especially for use in the whole organism. A further advantage of reduced binding to endogenous FRAP is that the multibinding agents, either alone or in combination with endogenous FKBP12 or chimeric containing FKBP domains, have correspondingly reduced immunosuppressive activity or mechanism based toxicity with respect to rapamycin. Also contemplated by the present invention is that a variant of the FKBP domain capable of binding in a ligand-substrate manner to chimeric proteins containing one or more complementary mutations, although mutations due to surface residues significantly interfere with ligand attachment to endogenous FRAP. It is a chimeric protein containing (see Fig. 2-E).

In addition to monovalent monovalent ligands, such as those described in PCT / US94 / 01617, the inducible compounds described herein can be bound to one of the chimeric proteins, but are either double bonds or higher multiple bonds (monovalent properties of the ligand). It is a multiple binding antagonist that does not affect).

Raphalogs of particular interest bind to human FKBP12 and / or inhibit its rotamase activity beyond a less potent size than any of FK506, FK520 or rapamicin. Such assays are well known in the art. See, eg, Holt, et al., J. Amer. Chem. Soc., 1993, 115, 9925-9938. The reduction in inhibition type can be as large as about 2 in size and in some cases will exceed about 3 in size. Useful raphalog substituents include, among others, alkyl, aryl, -O-alkyl, -O-aryl, substituted or unsubstituted amines, amides, carbamides, and ureas, wherein double alkyls and aryls are other moieties herein. As defined in. See, eg, PCT / US94 / 01617 and PCT / US94 / 08008.

Specifically, the multiple binding ligands of the present invention include rapamycin and rapalogue of the following formula.

In the above formula,

U is -H, -OR ¹ , -SR ¹ , -OC (O) R ¹ or -OC (O) NHR ¹ , -NHR ¹ , -NHC (O) R ¹ , -NH-SO ₂ -R ¹ or -NHSO ₂ R ² , wherein R ² is substituted aryl or allyl or alkylaryl (eg benzyl or substituted benzyl),

V is -OR ³ or (= O),

W is = O, = NR ⁴ = NOR ⁴ or = NNHR ⁴ , -NHOR ⁴ or -NHNHR ⁴ , -OR ⁴ , -OC (O) R ⁴ or -OC (O) NR ⁴ , or -H,

Y is -OR ⁵ , -OC (O) R ⁵ or -OC (C) NHR ⁵ ,

Z is = O, -OR ⁶ , -NR ⁶ , -H, -NC (O) R ⁶ , or -OC (O) R ⁶ or -OC (O) NR ⁶ ,

R ³ is H, -R ⁷ , -C (O) R ⁷ or -C (O) NHR ⁷ or C-28 / C-30 cyclic carbonate,

R ⁴ is H or alkyl, wherein R ¹ , R ⁴ , R ⁵ , R ⁶ and R ⁷ are individually selected from H, alkyl alkylaryl or aryl.

In rapamicin, U is -OMe, V is = O, W is = O, Y is -OH, Z is = O, and R ³ and R ⁴ are the steric solids shown in formula (1) on page 1 H with isomers. The rapalogs of the present invention may contain substituents in any of the possible stereoisomeric orientations.

As used herein, alkyl refers to saturated and unsaturated, straight, branched, cyclo, and polycyclo aliphatic hydrocarbons (usually containing 1 to 8 consecutive aliphatic carbon atoms, for example methyl, ethyl, n-propyl, iso -Propyl, cyclopropyl, n-butyl, iso-butyl, secondary-butyl, t-butyl, cyclobutyl, n-pentyl, iso-pentyl, secondary-pentyl, cyclopentyl and the like), which is hydroxy , C ₁ -C ₈ alkoxy, acyloxy, carbamoyl, amino, N-acylamino, keto, halo (chloro, bromo, fluoro or urethra), trihalomethyl, cyano, carboxyl, alkyl, cyclo One or more functional groups selected from the group consisting of alkyl, aryl and heteroaryl (with the exception of hydroxy, halo and cyano groups themselves possess one or more of these functional groups) are optionally substituted. Preferred hydrocarbons are alkyl, alkoxy and acyl groups containing 1 to 6 consecutive aliphatic carbon atoms, which may bear substituents.

As used herein, aryl is a stable cyclo, heterocyclo, polycyclo, and polyheterocyclo unsaturated C ₃ -C ₁₄ moiety, including but not limited to phenyl, biphenyl, naphthyl, pyridyl, furyl, thiophenyl, Imidazoyl, pyrimidinyl and oxazoyl), which include hydroxy, C ₁ -C ₈ alkoxy, C ₁ -C ₈ branched or straight chain alkyl, acyloxy, carbamoyl, amino, N- A functional group selected from the group consisting of acylamino, nitro, halo, trifluoromethyl, cyano, and carboxyl (except for hydroxy, halo and cyano groups, itself has one or more of these functional groups) It may be substituted with 1 to 5.

As used herein, heteroalkyl and heteroaryl refer to alkyl and aryl moieties each containing one or more oxygen, sulfur, or nitrogen in place of one or more carbon atoms.

In some embodiments the ligand of particular interest is a reduced form of rapamycin having a hydroxyl group at position 14 or a U-C ₂ -C ₈ straight chain, branched or cycloaliphatic or alkoxy moiety; Aryl or heteroaryl substituted alkyl or alkoxy moieties; Or rapamicin itself, which is a rapamycin derivative that is an aryl, aryloxy, heteroaryl or heteroaryloxy moiety (the aryl and heteroaryl moieties may not be substituted or unsubstituted).

Examples of various such compounds, or related compounds comprising such substituents, are known in the art, as are their synthesis. For illustrative purposes, the following synthetic schemes represent transformations that can be used to generate the desired raphalogs:

Preparation of C-7 Bumps

[Ref. Luengo, et al. J.Org. Chem, 59,6512 (1995). Luengo, et al. Chem & Biol 2 (7), 471-481 (1995)]

Preparation of C-13 Bumps

[References: Luengo et al. Tet. Lett. 35,6469 (1994)]

Preparation of C-14 Bumps

[Reference Document: Schubert, et al. Angew Chem Int Ed Engl 23,167 (1984)]

Preparation of C-20 Bumps

[Reference: Nelson, US Patent 5,387,680]

Preparation of C-24 Bumps

[References: Failli, et al. US Patent 5,373,014. Kao, et al. US Patent 5,378, 386. Lane, et al. Synthesis 1975, p 136.]

Preparation of C-28 Bumps

Manufacture of C-30 Bumps

[References: Luengo et al. Tet. Lett. 35,6469 (1994)]

Variation at C-14

[Ref. Luengo, et al. Tetrahedron Letters 35: 6469 (1994)]

[References: Fisher, et al. J. Org. Chem. 56: 2900 (1991)]

[References: Fisher, et al. J. Org. Chem. 56: 2900 (1991)]

[Ref. Luengo, et al. Tetrahedron Letters 35: 6469 (1994). Finet Chemical Reviews 89: 1487 (1989)]

Variation on C-13

[Reference: Donald, et al. Tetrahedron Letters 1375 (1991)]

Variation on C-24

[Ref. Luengo, et al. Tetrahedron Letters 35: 6469 (1994)]

Formulations, Dosages and Administration

By the efficacy of these abilities to promote protein-protein interactions, rapamycin or a rapalogue of the present invention is a tree of pharmaceutical compositions and chimeric proteins of the present invention in mammals containing the genetically engineered cells of the present invention. It can be used in a method for promoting the formulation of the patite complex.

Preferred methods of such treatment or prevention are those desired to facilitate measurable formulation of such complexes in genetic cells, or preferably initiated by such complexes, for example transcription of target genes, apoptosis of genetic cells, and the like. An effective amount of a composition for promoting measurable actuation of a biological event is to be administered to a mammal.

Therapeutic / Prolactic Administration and Pharmaceutical Compositions

Rapamycin and various rapalogs may be present in free form or in suitable salt form. Pharmaceutically acceptable salts and their preparation are well known in the art. Pharmaceutically acceptable salts of such compositions include conventional non-toxic salts or quaternary ammonium salts of such compositions formed from, for example, inorganic or organic acids of bases.

The composition of the present invention may form a hydrate or a solvent compound. It is known in the art to form hydrate species when the charged compound is freeze-dried with water or to form a solvent compound when concentrated in a solution with a suitable organic solvent.

The present invention also relates to pharmaceutical compositions containing a therapeutically (or prophylactically) effective amount of the composition and a pharmaceutically acceptable carrier or excipient. Carriers include, for example, saline, buffered saline, glucose, water, glycerol, ethanol and combinations thereof, and are discussed in more detail below. The composition may also contain a minimum amount of a wetting or emulsifying agent, or pH buffer, as desired. The composition may be in the form of a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. The composition may be formulated as a suppository with a conventional binder or carrier such as triglycerides. Oral formulations may also include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharide, cellulose, magnesium carbonate, and the like. The formulation may comprise mixing, granulating, and compressing or dissolving the ingredients suitable for the desired preparation.

The pharmaceutical carrier used may, for example, be either solid or liquid.

Illustrative solid carriers include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and analogs thereof. Solid carriers can include one or more substances that can also serve as flavors, lubricants, solubilizers, suspension formulations, fillers, glycans, compression aids, binders, or tablet-disintegrants; It can also be a capsule material. In powders, the carrier is a finely divided solid present in a mixture with the finely divided active ingredient. In tablets, the active ingredient was mixed with the carrier having the necessary compression properties in suitable ratios 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, sugars, lactose, dextrin, starch, gelatin, cellulose, methyl cellulose, carboxymethylsodium cellulose, polyvinylpyrrolidine, low melt wax and ion exchange Resin.

Illustrative liquid carriers include syrup, peanut oil, olive oil, water and the like. Liquid carriers are used in the preparation of solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The active ingredient may be dissolved or suspended in a pharmaceutically acceptable liquid carrier such as water, an organic solvent, a mixture of the two or a pharmaceutically acceptable oil or fat. Solution carriers may contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preventives, sweeteners, spices, suspending agents, thickening agents, colors, viscosity regulators, stabilizers or osmo-modulators. Suitable examples of liquid carriers for oral and parenteral administration include water (for example partially containing additives as the cellulose derivative, preferably carboxymethylsodium cellulose solution), alcohols (monohydric and polyhydric alcohols, eg Containing glycols) and derivatives thereof, and oils (eg, fractionated coconut oil and arachis oil). For partial administration, the carrier may also be an oily ester such as ethyl oleate and isopropyl mipistate. Sterile liquid carders are useful in sterile liquid form compositions for parenteral administration. The liquid carrier for the pressurized composition may be a halogenated hydrocarbon or other pharmaceutically acceptable propellant. Liquid preparation compositions, which are sterile solutions or suspensions, can be used, for example, for infusion intramuscularly, intraperitoneally or subcutaneously. Sterile solutions can also be administered intravenously. The composition may also be administered orally in the form of a solution or solid composition.

Carriers or excipients may include time delay materials known to those skilled in the art such as glyceryl monostearate or glyceryl distearate with or without wax, ethylcellulose, hydroxypropylmethylcellulose, methylmethacrylate and analogs thereof. Can be. When formulated for oral administration, 0.01% Tween 80 in PHOSAL PG-50 (phospholipid enriched with 1,2-propylene glycol, A.Nattermann & Cie.GmbH) provides an acceptable oral formulation for rapamycin. Has been recognized and may be suitable for formulation for various rapalogs.

A wide variety of pharmaceutical forms can be used. If a solid carrier is used, the preparation may be a tablet and may be placed in a solid gelatin capsule in powder or implantable form or in a pill or lozenge tablet. The amount of solid carrier varies widely but will preferably range from about 25 mg to 1 g. If a liquid carrier is used, the preparation will be in the form of injectable sterile solutions or suspensions in syrups, soft gelatin capsules, injection bottles or potions, or non-aqueous liquid suspensions.

To recover a stable water soluble dosage form, a pharmaceutically acceptable salt of rapamycin or rapalogue may be dissolved in an aqueous solution of an organic or inorganic acid, such as a 0.3 M succinic or citric acid solution. Alternatively, the acidic derivatives can be dissolved in a suitable base solution. If a soluble salt form is not available, the composition is dissolved in a suitable cosolvent or combinations thereof. Examples of such suitable cosolvents are alcohols, propylene glycol, polyethylene glycol 300, polysorbate 80, polyoxyethylated fatty acids, fatty alcohols or glycerine hydroxy fatty acid esters in the concentration range from about 0% to 60% of the total volume, and Analogues thereof, but are not limited to these examples.

Various delivery systems are known and can be used to administer rapamycin or rapalog or their various formulations, including tablets, capsules, injectable solutions, capsules in liposomes, microparticles, microcapsules and the like. Methods of infusion include, but are not limited to, the dermal, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, pulmonary, dural, eye, and oral (generally preferred) routes. The compound may be administered by any convenient or suitable route, for example, by infusion or large pill infusion, by absorption through the epithelial or mucosal skin gut (eg, oral mucosa, rectal and intestinal mucosa, etc.) And other biologically active agents.

In some embodiments, it may be desirable to administer the compound locally to the area in need of treatment; This is for example achieved (but not limited to) by local infusion during treatment, ie during local application, by infusion, by catheterization, by suppository, or by skin patching or implantation, It is a substance of gelatin comprising membranes or fibers, such as grafts or sialastic membranes.

In certain embodiments, the composition is formulated according to a predetermined method as a pharmaceutical composition suitable for human intravenous administration. Typically, the composition for intravenous administration is a solution in sterile isotonic aqueous buffer. Where necessary, the composition may also include solubilizing agents and local anesthesia to relieve pain at the site of injection. Generally, the ingredients are provided in unit dosage form, e.g., separated or mixed together in lyophilized powder, or in free concentration in a sealed container such as an ampoule or bag indicating the amount of active agent. . An infusion bottle containing a formulated grade of salt or salt is unnecessary where the composition is administered by infusion. Where the composition has been administered by infusion, an ampoule of sterile water or saline for infusion is provided so that the components can be mixed before administration.

Individual administration of an effective amount of the compound can be effected locally by administering the compound directly to the affected area of the individual skin. For this purpose, the compound is administered or used in a composition comprising a pharmaceutically acceptable local carrier, such as gels, ointments, lotions, creams, wherein the composition is without limitation water, glycerol, alcohol, propylene, fatty alcohol, triglycerides Carriers such as fatty acid esters or mineral oils.

Other local carriers include liquid petroleum, isopropyl palmitate, polyethylene glycol, ethanol (95%), polyoxyethylene monolaurate in water (5%) or sodium lauryl sulfate in water (5%). Other materials can be added as needed, such as anti-oxidants, wetting agents, viscosity stabilizers, and similar agents. Enhancement of penetration through the skin, such as azone, may also be included.

Also in certain instances, the compound may be placed in a device located on, in, or inside the skin. Such devices include patches, implants, and infusions that release the composition to the skin by a passive or active release mechanism.

Materials and methods for producing various formulations are well known in the art and the invention can be adapted to subject performance. [Reference Document: US Patent Nos. 5,182,293 and 4,837,311 (tablets, capsules, and other oral formulations as well as intravenous formulations), and European Patent Application Publication Nos. 0 649 659 (published April 26, 1995; rapamycin formulation for IV administration) and 0 648 494 (published April 19, 1995: rapamycin formulation for oral administration).

Effective doses of the compound administered in single or multiple doses are typically about 0.01 to 50 mg / kg of mammal weight, preferably about 0.1 to 10 mg / kg. In general, the compounds are administered to patients in need of such treatment in an amount of about 1 to 2000 mg per patient per day. In embodiments in which the compound is some residual rapalog or rapamycin with an immunosuppressive effect, it is preferred that the single dose be less than the amount associated with the severe immunosuppressive effect.

The amount of compound effective for treating or preventing a particular disorder or condition will depend somewhat on the nature of the disorder or condition and can be measured by standard clinical techniques. In addition, ex vivo or in vivo assays can optionally be used to help identify optimal dosage ranges. Effective amounts can be estimated from single dose-response curves derived from ex vivo or animal model test systems. The correct level of dosage should be measured by the attending physician or other health care professional, including well known information including age of administration, age, weight, sex and personal health status; Nature, severity and clinical stage of the disease; The use of concomitant (or non-concomitant) treatment; It will depend on the nature and scope of the patient's genetically engineered cells.

The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more components of the pharmaceutical composition of the present invention. Anything related to such a container may be informed in a form prescribed by a governmental agency that regulates the manufacture, use, or sale of a pharmaceutical or biological product, which notification may be approved by the manufacturer, use, or vendor for human administration. Reflect.

Applications

The present invention is applicable to any condition that directs the expression of exogenous-inserted genes inserted into large genomes. The desired level of expression can be preadjusted very high or very low. Alternatively, the system can also be processed to achieve regulated or titratable expression. [Reference: PCT / US93 / 01617]. In most cases, accidental activity of genes in unrelated cells is undesirable. The following is not intended to limit the examples of application of the subject matter of the present invention.

1. Regulated gene therapy. In many instances, the ability to switch on and off therapeutic genes where desired or to properly titrate expression is important for therapeutic efficacy. The present invention is particularly well suited for performing controlled expression of therapeutic target genes in the context of human gene therapy. One embodiment includes a pair of chimeric proteins (one containing a FRAP-derived receptor domain, another containing a FKBP-derived receptor domain), a dimer preparation capable of dimerizing a chimeric and an expressed target Use genetic constructs. The first chimeric protein comprises the hybrid DNA binding domain described in Pomerantz et al. As a heterologous activity domain. The second chimeric protein comprises a transcriptional active domain as a heterologous activity domain. Rapamycin or rapalog dimerization agents can bind both chimeras and thus can chimera or dimerize chimeras. The expression of these chimeric proteins can be directed and the DNA molecules encoding are engineered into cells. Also inserted into the cell is a target gene linked to a DNA sequence to which the mixed DNA-binding domain can bind. Contact with their progeny or engineered cells with a dimeric preparation (by administering the preparation to an animal or patient) induces a combination of transcription factor complexes to induce the expression of the target gene. The use and design of similar components is described in PCT / US93 / 01617. These include the use of mixed DNA-binding domains in alternative DNA-binding domains and DNA sequences encoding them; FRAP-induced receptors on one of the chimeras in the FRAP-induced receptor domain; And monomeric rapamycin form dimer preparations in dimeric dimer preparations, such as those recorded in the reference patent document. In practice, the level of target gene expression may be the action of the concentration or number of chimeric transcription factor complexes, which in turn may be the action of the concentration of dimeric ligands. The experimental data discussed below are evidence of this dose (of dimer ligand) -responsive gene expression.

Dimeric ligands can be administered to a patient as an activated transcription of a desired target gene. Depending on the binding affinity of the ligand, the desired response, the mode of administration, the half-life, and the number of cells present, various protocols can be used. The ligand can be administered parenterally or orally. The number of administrations depends on the factors described above. Ligands are orally as pills, powders or dispersions; By mouth; By the sublingual gland; And may be injected by inhalation or similarly intravenously, intraperitoneally, intramuscularly, subcutaneously. Ligands (and monomeric antagonist compounds) can be formulated using conventional methods and materials well known in the art for the various routes of administration. The dose and specific method of quantification for administration will depend on such factors and are measured by the attending physician or human or animal health manager. In most cases, the mode of administration will be determined experimentally.

In this result, the transcriptional activity by the ligand can be reversed or terminated and a monomeric compound capable of competing with the dimeric ligand can be administered. Thus, in the case of adverse reactions or termination of the desired therapeutic efficacy, the antagonists for dimer preparations can be administered by conventional routes, and especially intravascularly if desired for rapid conversion. Alternatively, the presence of an inactive domain (or transcriptional silencer) with a ligand binding domain can be provided. In another approach, cells can be eliminated through apoptosis via signaling via Fas or TNF receptors as described elsewhere. [Reference: International Patent Aplications PCT / US94 / 01617 and PCT / US94 / 08008]

Specific doses of ligands for any application may be measured depending on the method used for therapeutic dose monitoring, and maintenance of a particular level of expression may require an extended time, for example longer than about two weeks or Or repeat treatment with individual or repetitive doses of ligand at extended periods of time, eg, short periods of time with at least about 2 weeks. Doses of ligands within a precalculated range are provided and monitored for response to obtain the relationship between time and expression level as well as therapeutic response. Depending on the time and the level observed during the therapeutic response, the next time more or less dose is given and then the reaction occurs. This method is repeated until the appropriate amount within the therapeutic range is obtained. Where the ligand is administered long term, the maintenance dose of the ligand is measured and then the cellular system is verified to ensure that it provides the appropriate level of response and expression product at extended intervals.

The system is susceptible to many variations, such as expression efficacy, ligands for appropriate secretion levels, activity of expression products, and the specific needs of the patient, and what is specifically needed by the patient is the cell as a result of time and environment, i.e., loss of cells. Loss ratio of activity or individual expression activity and the like.

2. Generation of recombination protein light virus. The production of recombination therapeutic proteins for commercial and research purposes is often accomplished through the use of engineered mammalian cell systems to express the protein at high levels. The use of mammalian cells rather than bacteria or yeast appears where the function of proteins requires post-translational modifications that are not normally performed by heterologous cells. Examples of proteins produced commercially in this way include coagulation factors such as erythropoiesis, tissue plasminogen activator, factor VII: c, antigens and the like. In this method the cost of protein production is directly related to the level of expression achieved in the engineered cell. A second limitation in the production of such proteins is toxicity to host cells: protein expression can prevent cells from growing to high densities, ie, a sharp decrease in product levels. Thus, the ability to tightly control protein expression as described for regulated gene therapy allows to grow to high density in the absence of protein production. After reaching the optimal cell density, the expression of the activated gene occurs and the protein product is obtained.

Similar problems arise in packaging and packaging lines for the production of recombination viruses for commercial (eg gene therapy) and experimental use. This cell line is engineered to produce the viral proteins required for the combination of mucosal viral particles that bury the defective recombination gene. Virus carriers that depend on this packaging line include retroviruses, adenoviruses and adeno-bound viruses. In later cases, the appropriate concentration of virus accumulation obtained from the packaging line is directly related to the level of viral replication and production of key proteins. However, these proteins are very toxic to host cells. Thus, it has been demonstrated that high-titration concentration recombination AAV virus production is difficult. The present invention provides a solution to this problem that allows for the construction of a packaging line where replication and key genes are located under the control of the controllable transcription factors of the designs described herein. Packaged cell lines can grow at high density and are infected with the helper virus and with the recombination virus genome. The expression of viral proteins encoded by the packaging cells is then induced by the addition of dimer preparations that allow the production of virus at high titer concentrations.

3. Biological Research. The present invention is suitable for a wide range of biological experiments that require precise control of the target gene. These include: (1) expression of RNA or proteins important for biochemical purification; (2) regulation of important RNA or protein expression in tissue culture cells (or in vivo, through engineered cells) for the purpose of evaluating their biological action; (3) regulated expression of RNA or protein of interest in transgenic animals for the purpose of evaluating their biological action; (4) regulation of the expression of a gene encoding another regulatory protein, ribozyme or antisense molecule that acts on a gene that is resistant to the purpose of evaluating the biological action of the gene. Transgenic animal models and other applications in the components of the invention may be applied, including those described in PCT / US95 / 10591.

The invention also provides kits useful for the above applications. Such kits contain a DNA construct encoding, in an embodiment containing a target gene construct containing a regulated gene transcription, a target gene linked to one or more transcriptional control elements activated by multiple binding of chimeric proteins, and comprising the present invention (and the May contain an additional domain as described in (C). Alternatively, the target gene construct may contain a cloning site for insertion of a desired target gene by one of skill in the art. This kit also includes a sample of dimeric preparations capable of dimerizing two recombined proteins and activating the transcription of a target gene.

Example

Example 1 Configuration of Coding Processing Transfer Factors

A. Unless otherwise noted, all DNA manipulations described in this and other examples were performed using standard procedures. See FM Ausubel et al., Eds., Current Protocols in Molecular Biology (John Wiley Sons, New York, 1994).

B. Plasmid

The construct encoding the fusion of human FKBP12 (hereinafter referred to as FKBP) using the yeast GAL4 DNA binding domain, HSV VP16 active domain, human T cell CD3 zeta chain intracellular domain or intracellular domain of human FAS is PCT / US94. / 01617.

Additional DNA vectors to direct expression of the fusion proteins associated with the present invention were derived from the mammalian expression vector pCGNN (Attar, R. M. and Gilman, M. Z. 1992, MCB 12: 2432-2443). Inserts cloned into pCGNN as XbaI-BamHI fragments are transcribed under the control of human CMV v promoter and enhancer sequences (nucleotides -522 to +72 associated with the cap site) to be recognized by any epitope tail (monoclonal antibody 12CA5). H. influenza hemagglutinin gene 16 amino acid position), in the case of the transcription factor domain is expressed as the N-terminal nuclear locus sequence (NLS; from the SV40 T antigen)

Unless otherwise noted, all fragments cloned into pCGNN were inserted as XbaI-.BamHI fragments containing the SpeI site immediately upstream of the BamHI site. As XbaI and SpeI produce suitable ends, additional XbaI-BamHI fragments can be inserted downstream of the initial insert and promote stepwise assembly of proteins including multiple components. Stop codons were placed between SpeI and BamHI. For initial construction, a vector pCGNN-GAL4 was further used, wherein by cloning codons 1-94 of the GAL4 DNA binding domain gene into the XbaI site of pCGNN, the XbaI site was regenerated only at the 3 ′ end of the fragment. Thus, the XbaI-BamHI fragment could be cloned into the vector to cause GAL4 fusion and then regenerated.

(a) a construct that encodes a GAL4 DNA binding domain-FRAP fusion

To obtain part of the human FRAP gene, human thymus total RNA (Clontech # 64028-1) was transcribed using MMLV converting transcriptase and random hexamer primer (Clontech 1st strand synthesis kit). The cDNA was used directly for PCR reactions containing primers 1 and 2 and Pfu polymerase (Stratagene). These primers correspond essentially to the 'FRB' domain of mammals identified by amino acids 2025-2113: Chen et al. (Proc. Natl. Acad. Sci. USA (1995) 92, 4947-4951), including human FRAP. Designed as needed and sufficient for FKBP-rapamycin binding (hereinafter referred to as FRB) to amplify the coding sequence for the 89 amino acid region. Bands of appropriate size were purified, digested with XbaI and SpeI, and ligated with XbaI-SpeI digested pCGNN-GAL4. The construct was confirmed by restriction analysis and DNA sequencing and designated pCGNN-GAL4-1FRB.

The construct encoding the FRB multiplex was made by isolating the FRB XbaI-BamHI fragment and then ligation back to pCGNN-GAL4-1FRB digested with SpeI and BamHI to generate pCGNN-GAL4-2FRB, which was confirmed by restriction analysis. . This process was similarly repeated for the new construct to yield pCGNN-GAL4-3FRB and pCGNN-GAL4-4FRB.

In addition, the vector was constructed to encode a larger fragment of FRAP that encompassed but extended below the mammalian FRB domain (amino acids 2025-2113). PCR primers were designed to amplify the various regions of the FRAP adjacent to the 5'XbaI and 3'SpeI sites as shown below.

designation amino acid 5 'primer 3 'primer FRAP _a FRAP _b FRAP _c FRAP _d FRAP _e FRAP _f FRAP _g FRAP _h FRAP _i 2012-21271995-21411945-21131995-21132012-21132025-21272025-21412025-21741945-2174 653561113 782227844

First, fragment FRAB _i was amplified by RT-PCR as described above and digested with XbaI and SpeI and ligated with XbaI-SpeI digested pCGNN-GAL4. This configuration, pCGNN-GAL4-FRAP _i , was analyzed by PCR to confirm insert orientation and verified by DNA sequencing. It was then used as a PCR substrate to amplify other fragments using primers on the list. The new fragments were cloned into GAL4 fusions as described above to yield constructs such as pCGNN-GAL4-FRAP _a , pCGNN-GAL4-FRAP _b and confirmed by DNA sequencing.

A vector encoding the linkage of two larger FRAP fragments of FRAP _d and FRAP _e was generated in a similar manner to the one used above. FRAP _d and FRAP _e code crystallized XbaI-BamHI fragment of pCGNN-GAL4-FRAP _d and pCGNN-GAL4-FRAP _e and thereby isolated from the re-ligated into a vector, such as in the above was digested with SpeI and BamHI pCGNN-GAL4-2FRAP _d and pCGnn-GAL4-2FRAP _e were generated. This process was similarly repeated for the new construct to yield pCGNN-GAL4-3FRAP _d , pCGNN-GAL4-3FRAP _e , pCGNN-GAL4-4FRAP _d and pCGNN-GAL4-4FRAP _e . All constructs were verified by restriction analysis.

(b) constructs encoding FRAP-VP16 active domain fusions

To generate an N-terminal fusion of FRB domain (s) with the active domain of VP16, the herpes simplex virus protein, XbaI-BamHI fragments encoding 1, 2, 3 and 4 copies of FRB were recovered from the GAL4 fusion vector. Ligation with XbaI-BamHI digested pCGNN yielded pCGNN-1FRB, pCGNN-2FRB and the like. The vector was then digested with SpeI and BamHI. XbaI-BamHI fragment encoding amino acids 414-490 of VP16 was isolated from plasmid pCG-Gal4-VP16 (Das, G., Hinkley, CS and Herr, W. (1995) Nature 374, 657-660) SpeI Ligation with -BamHI digested vector regenerated pCGNN-1FRB-VP16, pCGNN-2FRB-VP16 and the like. The configuration was verified by restriction analysis and / or DNA sequencing.

(c) a construct encoding the ZFHD1 DNA binding domain-FRAP fusion

Expression vectors for directing the expression of ZFHDI encoded sequences in mammalian cells were prepared as follows. Zif268 sequences were amplified from cDNA clones by PCR using primers 5'Xba / Zif and 3'Zif + G. Oct1 homeodomain sequences were amplified from cDNA clones by PCR using primers 5'NotOctHD and Spe / Bam3'Oct. Zif268PCR fragments were digested with XbaI and NotI. OctIPCR fragments were cleaved using NotI and BamHI. The two fragments were ligated with 3-way ligation between the XbaI and BamHI sites of pCGNN (Attar and Gilman, 1992), whereby cDNA insertion was performed under the transcriptional control of human CMV promoter and enhancer sequences, resulting in nuclear positioning sequences from the SV40 T antigen. PCGNNZFHD1 was associated with. Plasmids also contain genes for ampicillin resistance that can act as selective markers. (Derivatives containing one or three tandem repeats of human FKBP12 ligated as XbaI-BamHI between the SpeI and BamHI sites of pCGNNZFHD1, pCGNNZFHD1-FKBP × 1 and pCGNNZFHD1-FKBP × 3, were prepared. Samples were deposited into the American Type Culture Collection under ATCC Accession No. 97399.)

To generate C-terminal fusions of FRB domain (s) using the mutagenic DNA binding protein ZFHD1, XbaI-BamHI fragments encoding 1, 2, 3 and 4 copies of FRB were recovered from the GAL4 fusion vector and Spe Ligation into -BamHI digested pCGNNZFHD1 yielded pCGNN-ZFHD1-1FRB, pCGNN-ZFHD1-2FRB and the like. The configuration was verified by restriction analysis and / or DNA sequencing.

To test the effect of further introduction of the chain polypeptide between ZFHD1 and the C-terminal FRB domain, FRAP fragments encoding the N-terminal sequence for FRB were cloned into ZFHD1 fusions. XbaI-BamHI fragments encoding FRAP _a , FRAP _b , FRAP _c , FRAP _d and FRAP _e were excised from vector pCGNN-GAL4-FRAP _a , pCGNN-GAL4-FRAP _b and the like and ligated into SpeI-BamHI digested pCGNN-ZFHD1 Vector pCGNN-ZFHD1-FRAP _a , pCGNN-ZFHD1-FRAP _b and the like were calculated. Further, the solidifying vector encoding a fusion of ZFHD1 to 2, 3 and 4 copies of FRAP _e C- terminus is pCGNN-GAL4-2FRAP _e, pCGNN-GAL4-3FRAP _e and pCGNN-GAL4-4FRAP _e from _e 2FRAP, 3FRAP isolated and _e and _e 4FRAP code crystallized XbaI-BamHI fragment and, to configure them by ligation into the SpeI-BamHI digested pCGNN-ZFHD1, vector pCGNN-ZFHD1-2FRAP _e, pCGNN-ZFHD1-3FRAP _e and pCGNN-ZFHD1-4FRAP _e was calculated. All configurations were verified by restriction analysis.

The vector was also configured to encode the N-terminal fusion of FRB domain (s) with ZFHD1. XbaI-BamHI fragments encoding 1, 2, 3, and 4 copies of FRAP _e were isolated from pCGNN-GAL4-2FRAP _e , pCGNN-GAL4-2FRAP _e and the like and ligated into XbaI-BamHI digested pCGNN to form plasmid pCGNN. -1FRAP _e , pCGNN-2FRAP _e and the like. These vectors were then digested with SpeI and BamHI and ligated XbaI-BamHI fragments encoding ZFHD1 (isolated from pCGNN-ZFHD1) to yield pCGNN-1FRAP _e -ZFHD1, pCGNN-2FRAP _e -ZFHD1 and the like. The composition was verified by restriction analysis.

(d) constructs encoding FRAP-p65 active domain fusion

Two fragments were amplified by PCR from plasmid pCG-p65 to generate fusion of the FRB domain (s) into the active domain of the human NF-kB p65 subunit (hereinafter referred to as digested p65). Primers 9 (p65 / 5'Xba) and 11 (p653'Spe / Bam) amplify the coding sequence for amino acids 450-550, and primers 10 (p65 / 361Xba) and 11 encode the coding sequence for amino acids 361-550. Amplify, both of which are flanked by the 5'XbaI and 3'SpeI / BamHI sites. PCR products were digested with XbaI and BamHI and cloned into XbaI-BamHI digested pCGNN, yielding pCGNN-p65 (450-550) and pCGNN-p65 (361-550). The configuration was verified by restriction analysis and DNA sequencing.

The 100 amino acid P65 transcriptional active sequence is encoded by the following linear sequence:

Further extended p65 transcriptional active domains 361-550 are encoded by the following linear sequence:

To generate N-terminal fusion of the FRB domain (s) using a portion of the p65 active domain, plasmids pCGNN-1FRB, pCGNN-2FRB, etc. were digested with SpeI and BamHI. XbaI-BamHI, encoding p65 (450-550), was isolated from pCGNN-p65 (450-550) and ligated into SpeI-BamHI digested vector to plasmid pCGNN-1FRB-p65 (450-550), pCGNN- 2FRB-p65 (450-550) and the like were calculated. Constitutive pCGNN-1FRB (450-550) was similarly prepared using XbaI-BamHI fragment isolated from pCGNN-p65 (361-550). The configuration was verified by restriction analysis.

To test the effect of further introduction of the linker polypeptide between the p65 active domain and the N-terminal FRB domain, the FRAP fragment encoding the C-terminal sequence for FRB was cloned as a p65 fusion. XbaI-BamHI fragments encoding FRAP _a , FRAP _b , FRAP _f , FRAP _g and FRAP _h were excised from vector pCGNN-GAL4-FRAP _a , pCGNN-GAL4-FRAP _b and the like and ligated into XbaI-BamHI digested pCGNN, Vectors pCGNN-FRAP _a , pCGNN-FRAP _b and the like were calculated. The plasmid was then digested with SpeI and BamHI and ligated with an XbaI-BamHI fragment encoding p65 (amino acids 450-550) to give five vectors pCGNN-FRAP _a -p65, pCGNN-FRAP _b -p65 and the like. Was calculated. The configuration was verified by restriction analysis.

In addition, vectors encoding the fusion of p65 to the 1 and 3 N-terminal copies of FRAP _e were prepared by digesting pCGNN-1FRAP _e and pCGNN-3FRAP _e with SpeI and BamHI. The XbaI-BamHI fragment encoding the p65 (450-550) and p65 (361-550) (pCGNN-p65 (450-550) and pCGNN-p65 (361-550)) was then ligated to vector pCGNN-1FRAP _e -p65 (450-550), pCGNN-3FRAP _e -p65 (450-550), pCGNN-1FRAP _e -p65 (361-550) and pCGNN-1FRAP _e -p65 (361-550) were calculated. All configurations were verified by restriction analysis.

The vector was also configured to encode the C-terminal fusion of the FRB domain (s) using a portion of the p65 active domain. Plasmid pCGNN-p65 (450-550) and pCGNN-p65 (361-550) were SpeI And XbaI-BamHI fragments digested with BamHI and encoding one and three copies of FRAP _e (isolated from pCGNN-GAL4-1FRAP _e ) and one copy of FRB (isolated from pCGNN-GAL4-1FRB). Plasmid pCGNN-p65 (450-550) -1FRAP _e , pCGNN-p65 (450-550) -3FRAP _e , pCGNN-p65 (361-550) -3FRAP _e , pCGNN-p65 (450-550)- 1FRB and pCGNN-p65 (361-550) -1FRB were extracted. All configurations were verified by restriction analysis.

(e) additional configuration

Other constructs can be made by replacing part of the FRAP sequence and using this procedure. For example, primers 12 and 13 are used to amplify the entire coding region of FRAP. Primers 1 and 13, 6 and 13 and 5 and 13 are used to amplify three fragments that extend throughout the C-terminal end of the protein (including the lipid kinase homology domain) surrounding the FRB domain. These fragments differ in that they encode different portions of the protein N-terminus for the FRB domain. In each case, RT-PCR is used to amplify the region from human thoracic RNA, purify the PCR product, digest with XbaI and SpeI, ligation into XbaI-SpeI digested pCGNN, and analyze its configuration. Proven by DNA sequencing.

(f) primer sequences

Restriction sites are underlined (XbaI = TCTAGA, SpeI = ACGAGT, BamHI = GGATCC).

(g) DNA sequence of a representative final construct: pCGNN-ZFHD1-1FRB

Non-coded nucleotides are shown below.

(S / X) and (X / S) show the result of ligation between compatible products digested with XbaI and SpeI, yielding sequences that cannot be cleaved by either enzyme.

* Indicates a stop codon.

(h) Bistronic Configuration

Internal ribosomal inlet sequence (IRES) from cerebral myocardial virus was amplified by PCR from pWZL-Bleo. The formed fragments were cloned into pBS-SK + (STratagene), which contained stop codons upstream and downstream of the XbaI site and the IRES sequence, containing the NcoI site surrounding the ATG followed by the SpeI and BamHI sites. do. To facilitate cloning, the sequence around the starting ATG of pCGNN-ZFHD1-3FKBP was denatured to the NcoI site and the XbaI site was used with oligonucleotides of 5'-GAATTCCTAGAAGCGACCATGGCTTCTAGC-3 'and 5'-GAAGAGAAAGGTGGCTAGCGAACGCCCATAT-3', respectively. Degeneration with NheI site. The NcoI-BamHI fragment containing ZFHD1-3FKBP was then cloned downstream of pBS-IRES to yield pBS-IRES-ZFHD1-3FKBP. The XbaI-BamHI fragment from this plasmid was cloned into SpeI / BamHI-cleaved pCGNN-1FRB-p65 (361-550) to yield pCGNN-1FRB-p65 (361-550) -IRES-ZFHD1-3FKBP. .

C. Retroviral vectors for expressing mutant proteins

Retroviral vectors were used to stably integrate the transcription factor fusion proteins, and the lower copy genes were pSRαMSVtkNeo (Muller et al., MCB 11: 1785-92, 1991) and pSRαMSV (XbaI) (Sawyers et al., J.). Exp. Med. 181: 307-313, 1995). The only BamHI site in the two vectors was digested with BamHI and packed and ligated therein with Klenow, resulting in pSMTN2 and pSMTX2, respectively. pSMTN2 expresses the neo gene from internal thymidine kinase promoters. The eosin gene (Invitrogen) will be cloned into the only XbaI site at the bottom of the internal thymidine kinase promoter in pSMTX2 as a NheI fragment to yield pSNTZ. This zeocin fragment was generated by denaturing pZeo / SV (Invitrogen) using the following primers to introduce the NheI site flanked by the keosin coding sequence.

Primer1 5'-GCCATGGTGGCTAGCCTATAGTGAG

Primer2 5'-GGCGGTGTTGGCTAGCGTCGGTCAG

pSMTN2 contains unique EcoRI and HindIII sites downstream of LTR. To facilitate cloning of transcription factor fusion proteins synthesized as XbaI-BamHI fragments, the following sequence was inserted between the EcoRI and HindIII sites to yield pSMTN3:

The same fragment was inserted into the only EcoRI site of pSMTZ, yielding pSMTZ3 with the 3'HindIII site replaced by the EcoRI site.

pSMTN3 and pSMTZ3, as XbaI-BamHI fragments, allow for mutagenic transcription factors to be cloned downstream of the 5 'viral LTR, and because of their ability to select stable integrators, thereby enhancing resistance to either antibiotic G418 or zeocin Grant.

To generate the retroviral vector SMTN-ZFHD1-3FKBP, pCGNN-ZFHD1-3FKBP was first denatured by adding an EcoRI site downstream of the first amino acid of the fusion protein. ZFHD1-3FKBP was then expressed from retroviral LTR by cloning EcoRI-BamHI (blunted) into EcoRI-HindIII pSRαMSVtkNeo (ref. 51).

Example 2: Rapamycin-dependent Transcriptional Activity

The experiment showed three complexes of FKBP fused to a Gal4 DNA binding domain or a transcriptional activation domain that activated a report gene that was transiently transiently or stably integrated stronger than the corresponding fusion protein containing only one or two FKBP domains. To assess this parameter with the FRB fusion protein, the main plasmid containing the Gal4 DNA binding domain fused to one or more copies of the FRB domain was subjected to transient transcription to the three FKBP domains or the p65 active domain (3 × FKBP−). p65) was inter-infected with the plasmid encoded. The data shown in FIG. 3A shows four copies of the FRB domain fused to a Gal4 DNA binding domain that activated a report gene that is more stably integrated with the smaller FRB domain and other corresponding fusion proteins.

Methods: HT1080 B cells were grown in MEM supplemented with 10% calf serum. About 4 × 10 ⁵ cells / well in a 6 well plate (Falcon) were temporarily infected by lipofection procedure as recommended by the supplier (GIBCO, BRL). DNA: The ratio of lipofectamine used in this experiment is 1: 6. The cells in each well received 500 ng of pCGNN F3-p65, 19 ug of PUC 118 plasmid and 100 ng of plasmid as carrier: pCGNN Gal4-1FRB, pCGNN Gal4-2FRB, pCGNN Gal4-3FRB Or pCGNN Gal4-4FRB. Following transcription, 2 ml of fresh medium was added and supplemented with rapamycin at the disclosed concentration. After 24 hours, SEAP activity was assayed as described for 100 μl of medium (Spencer et al., 1993).

To test whether multiple FRB domains fused to the p65 active domain will result in increased electronic activity of the report gene, we have identified Gal, 1, 2, 3 or 4 copies of the FRB fused to the p65 active domain. HT1080 B cells were inter-infected using plasmids expressing -3x FKBP. Surprisingly, unlike in the DNA binding domain-FRB fusion, a single copy of the FRB fused to the p65 active domain activated the report gene significantly stronger than the corresponding fusion protein containing two or more copies of the FRB. 3B.

Methods: HT1080 B cells were grown in MEM supplemented with 10% calf serum. About 4 × 10 ⁵ cells / well in 6-well plates were temporarily infected by the lipofection process as recommended by GIBCO, BRL. DNA: The ratio of lipofectamine used in this experiment is 1: 6. Cells in each well received 19 ug of PUC 118 plasmid, 100 ng of pCGNN Gal4-F3 and 500 ng of the following plasmid as carrier: pCGNN 1, 2, 3 or 4FRB-p65. Following transcription, 2 ml of fresh medium was added and supplemented with rapamycin at the disclosed concentration. After 24 hours, SEAP activity was assayed as described for 100 μl of medium (Spencer et al., 1993).

In addition, using another stable cell line (HT1080 B14) containing a DNA sequence encoding a 5 × Gal4-IL2-SEAP report gene and a Gal4 DNA binding domain and a fusion protein containing three copies of the stably integrated FKBP Similar experiments were performed. These cells were transiently infected with major plasmids expressing the p65 active domain fused to one or more copies of the FRB domain. Similar to that observed in HT1080 B cells, the major plasmids expressing a single copy of the FRB-p65 active domain fusion protein activated the genes reported more strongly than others with two or more copies of FRB.

Example 3

A. Rapamycin-dependent transcriptional activity in transiently infected cells: ZFHD1 and p65 fusions

Human fibrosarcoma cells transiently infected with plasmids encoding SEAP target genes and representative ZFHD-FKBP- and FRB-p65-containing fusion proteins were subjected to dose-responsive secretion and rapamycin-dependency of SEAP in cell culture medium. Indicated. 4A, SEAP production was not detected in cells omitting one or two transcription factor fusion plasmids, and not in cells without rapamycin added (FIG. 4B). However, when all components were present, SEAP secretion could be detected even at rapamycin concentrations as low as 0.5 nM (FIG. 4A). Maximum SEAP secretion was observed at 5 nM. Similar results were obtained when using the same transcription factor to induce a stable integrated version of the SEAP report gene or rapamycin-dependent activity of the hGH report gene by infection with a retroviral vector. Since the SEAP secretion level in the presence of the drug cannot be detected, it is difficult to determine the wall activity for rapamycin, but it is evident in this system that it is at least 1000 times stronger than the background level in the presence of rapamycin. Thus, the system shows undetectable background activity and high dynamic range.

Several different structures for the transcription factor fusion proteins were found (FIG. 5). When the FKBP domain fused to ZFHD1 and FRBs fused to p65, the optimal level of rapamycin-induced activity occurs when multiple FKBPs fused to ZFHD1 and smaller FRBs fused to p65. More preferred multiple drug-binding domains on the DNA-binding protein can propagate multiple active domains and thus affect the dose of the protein to elicit higher levels of promoter activity. If there is only one drug-binding domain on the active domain, each FKBP must be allowed on ZFHD to replenish one p65. All numerical increases in FRBs on p65 increase the chance that smaller active domains can spread to ZFHD.

Way:

HT1080 cells (ATCC CCL-121) derived from human fibrosarcoma were grown in MEM supplied with non-essential amino acids and 10% calf serum. Cells spread out in 24-well dishes (Falcon, 6 × 10 ⁴ cells / well) were infected using lipofectamine under conditions recommended by the manufacturer (GIBCO / BRL). A total of 300 ng of the following DNA was infected into each well: 100 ng ZFHD × 12-CMV-SEAP report gene, 2.5 ng pCGNN-ZFHD1-3FKBP or DNA binding domain fusion, 5 ng pCGNN-1FRB-p65 (361-550) or Other active domain fusions and 192.5 ng pUC118. When the DNA binding domain or active domain was omitted, the equivalents lacking the pCGNN expression vector were replaced. 500 μl media of lipofection (for 5 hours) containing the indicated amount of rapamycin was added to each well. After 24 hours, the medium was removed and assayed for SEAP activity as disclosed using a luminescence spectrometer (Perkin Elmer) at 350 nm excitation and 450 nm emission (Spencer et al., Science 262: 1019-24, 1993). Assay. Background SEAP activity measured from false-infected cells was subtracted from each value.

To prepare transiently infected HT1080 cells for intra-mouse injection (see below), cells in 100 mm dishes (2 × 10 ⁶ cells / dish) were infected for 16 hours by calcium phosphate precipitation using the following DNA: (Gatz, C., Kaiser.A Wendenburg, R. 1991, Mol. Gen. Genet. 227, 229-237): 100 μg ZHWT × 12-CMV-hGH, 1 μg pCGNN-ZFHD1-3FKBP, 2 μg PCGNN-1FRB-p65 (361-550) and 7 μg pUC118. Infected cells were rinsed twice with phosphate buffered saline (PBS) and fresh media was provided for 5 hours. To recover the injections, cells were removed from dishes in a Hepes buffered saline solution containing 10 mM EDTA, washed with PBS / 0/1% BSA / 0.1% glucose and concentrated at 2 × 10 ⁷ cells / ml. Resuspend in solution.

Plasmid:

The construction of the transcription factor fusion plasmid has been described above.

pZHWT × 12-CMV-SEAP

This report gene is ZFHD! Contains 12 tandem copies of binding sites (Pemerantz et al., 1995) and basal promoters from genes directly adjacent to human cytomegalovirus (Boshart et al, 1985) that express genes encoding secreted alkali phosphates. And, the NheI-HindIII fragment of pSEAP promoter (Clontech) contains the following NheI-XbaI fragments containing 12 ZFHD binding sites:

And the following XbaI-HindIII fragments containing minimal CMV promoter (-54 to +45):

pZHWT × 12-CMV-hGH

The activity of this report gene is derived from the production of hGH. This report gene is derived from HindIII-BamHI (bulky) of ZHWT × 12-CMV-SEAP (containing the SEAP coding sequence) p0GH (containing the hGH genomic clone; Selden et al., MCB 6: 3171-3179, 1986). Construct by replacement with HindIII (Blug) -EcoRI fragment from BumHI and EcoRI site blunt together).

pZHWT × 12-IL2-SEAP

This report gene contains the XbaI-HindIII fragment containing the minimum CMV promoter and the minimum IL2 gene promoter (-72 to +45 in relation to the starting site; Siebenlist et al., MCB 6: 3042-3049, 1986). Same as pZHWT × 12CMV-SEAP except for replacing with the following XbaI-HindIII fragment:

pLH

The following retroviral vectors were constructed to facilitate stable integration of single or slight copies of the report genes. The pLH (LTR-hph) containing the hygromycin B resistance gene driven by leukemia virus LTR and the only internal ClaI site in Moroni rats was constructed as follows: The hph gene was constructed as pBabe Hygro (Morganstern and Land, NAR 18). : 3587-96, 1990) and cloned into BamHI-ClaI digested pBabe Bleo (resulting in loss of Bleo gene; blunting BamHI and HindIII sites together) from HindIII-ClaI fragments.

pLH-ZHWT × 12-IL2-SEAP

PZHWT × 12-IL2-SEAP (with a ClaI linker) to clone a copy of the report gene containing 12 tandem copies of the ZFHD1 binding site and a basal promoter from the IL2 gene expressing the SEAP gene into the pLH retroviral vector. MluI-ClaI fragments from were cloned into the ClaI site of pLH. Orientation was performed such that the direction of transcription from the virus LTR and the internal ZFHD-IL2 promoter was the same.

pLH-G5-IL2-SEAP

To construct a retroviral vector containing a 5 Gal4 site embedded in the lowest IL2 promoter expressing the SEAP gene, the same direction of transcription from the viral LTR and the internal Gal4-IL2 promoter is:

The ClaI-BstBI fragment consisting of was inserted into the ClaI site of pLH: ClaI- containing 5 Gal4 site (underlined) and -324 to -294 (in bold) and -72 to +45 of region IL2 gene (italic). HindIII-BstBI fragments containing HindIII fragments and SEAP gene coding sequences (Berger et al., Gene 66: 1-10, 1988) were added to the 5'-CCCGTGGTCCCGCGTTGCTTCGAT sequence (containing the BstBI site) immediately after the stop codon. Individual mutation.

B. Rapamycin-dependent Transcriptional Activity in Stably Infected Cells

We conducted the following experiments to ensure that our system exhibited similar characteristics in stably infected cells. We generated stable cell lines by subsequent infection of the expression vector against the SEAP target gene and ZFHD1-3FKBP and 1FRB-p65, respectively. The dozens of stable clones resulting from the final transcription showed rapamycin-dependent SEAP production. From this pool, we found that several individual clones produced high levels of SEAP in response to rapamycin. One such clone resulting from this result is shown in FIG. 4C. The clone produced about 40 times higher than the pool and significantly higher levels of SEAP than transiently infected cells. In an attempt to accurately quantify the background SEAP production and introduction rate in these clones, the inventors increased the length of the SEAP assay to about 50 factors twice in order to detect all SEAP activity in untreated cells. The assay was performed. Under these conditions, the false infected cells produced 47 random fluorescent units, whereas the infected clones produced 54 units and more than 90,000 units in 100 nM rapamycin in the presence of rapamycin. Thus, for this stable cell line, background gene expression was negligible and the rate of introduction (7 units for 90,000 units) was greater than 4 powers.

To simplify the task of stable infection, we used bistronic expression vectors that direct the production of both ZFHD1-3FKBP and 1FRB-p65 using the internal ribosomal entry sequence (IRES). The expression plasmid was transfected with the zeocin-resistant marker plasmid into a cell line carrying the reverse-transformed SEAP report gene, and selected where about 40 drug-resistant clones were plagued. Expanded. FIG. 4D shows the rapamycin-dependent SEAP production where the clone was clogged and without detection of background and shows a dose-response curve similar to that observed in transiently infected cells. It is expected that the clones will contain individual clones with similar performance as the clones studied in FIG. 4C. Thus, rapamycin-responsive gene expression can be readily obtained in both transiently and stably infected cells. In both cases, very low background and high introduction rates were shown.

Stable cell lines. The helper-free retrovirus containing the report gene or DNA binding domain fusions can be expressed in 293T cells using Psi (-) Amphoropic packing vector and retroviral vector pLH-ZHWT × 12-IL2-SEAP or SMTN-ZFHD1-3FKBP, respectively. Pear, WS, Nolan, GP, Scott, ML Baltimore D., 1993). To generate clonal cell lines containing stably integrated report genes, HT1080 cells infected with retroviral stocks were diluted and selected from the presence of 300 μg / ml hygromycin B. Each clone from this and other cell lines disclosed below were selected for transient infection of the missing components followed by the addition of rapamycin as described above. All 12 clones analyzed could be derived and had little or no basal activity. The most representative clone, HT1080L, was selected for further study.

HT20-6 cells containing a pLH-ZHW × 12-IL2-SEAP report gene, a ZFHD1-3FKBP DNA binding domain and a stably integrated 1FRB-p65 (361-550) active domain were first identified as SMTN-ZFHD1- It was generated by infection with 3FKBP-packed retrovirus and selecting from media containing 500 μg / ml G418. The strong responsive clone, HT1080L3, was then infected with linearized pCGNN-1FRB-p65 (361-550) and pZeoSV (Invitrogen) and selected from media containing 250 μg / ml zeosin. First, each clone was tested for the presence of 1FRB-p65 (361-550). Rapamycin was added to 8 positive clones for analysis. All eight had low basal activity and in six of them gene expression was induced by at least power. HT20-6, a clone that showed strong response, was selected for further analysis.

HT1080L cells were inter-infected with pCGNN-1FRB-p65 (361-550) -IRES-ZFHD1-3FKBP and pZeoSV linearized and selected from media containing 250 μg / ml zeosin.

For analysis, cells were plated in 96-well dishes (1.5 × 10 ⁴ cells / well) and 200 μl of medium containing the indicated amount of rapamycin (or basal) was added to each well. After 18 hours, the medium was removed and assayed for SEAP activity. In some cases, media was diluted prior to analysis and the corresponding SEAP units obtained were doubled by doubling. Background SEAP activity measured from uninfected HT1080 cells was subtracted from each value.

Example 4: Rapamycin-dependent product of hGH in mice

In vivo methods: animals, farming and general processes. Male nu / nu mice were obtained from Charles River Laboratories (Wilington, Mass.) And tamed for 5 days prior to the experiment. Mice were allowed to grow in an sterile environment, freely eaten sterile food and sterile water during the experiment, and treated with thorough sterilization techniques. There were no immuno-resolved animals exhibiting external infections or apparent diseases as a result of housing, tillage techniques or experimental techniques.

To transplant transiently infected cells into mice, 2 × 10 ⁶ infected HT1080 cells were suspended in 100 μl PBS / 0.1% glucose buffer and administered to four intramuscular sites on the hip and flank of the animal (site). About 125 μl per). Control mice were injected with only equivalent volume of buffer.

Rapamycin was formulated for in vivo administration by dissolving polyglycol glycol (average molecular weight 400) and polyoxyethylene sorbitan monooleate in a back N, N-dimethylacetamide and 9: 1 (volume ratio) mixture. In the finished formulation, the concentration of rapamycin was sufficient to allow an appropriate single dose in vivo administration at 2.0 ml / kg injection. The precision of the solution to be taken once was assured by HPLC analysis prior to intravenous administration into the tail vein. Some control mice that had no infected HT1080 cells received 10.0 mg / kg rapamycin. In addition, other control mice with infected cells received only rapamycin basal.

CO₂ Blood was collected from anesthetized or sacrificed mice via inhalation. Anesthetized mice were used to collect 100 μl of blood from cardiac puncture. Mice were resuscitated and restored for subsequent blood collection. The mice sacrificed immediately drained blood. Blood samples were condensed at 4 ° C. for 24 hours and serum collected and centrifuged at 1000 × g for 15 minutes. Serum hGH was measured by the Charinger Mannheim non-isotropic sandwich ELISA (Cat No. 1 585 878). The assay had a lower detection limit of 0.0125 ng / ml and a dynamic range extended to 0.4 ng / ml. Recommended test instructions are as follows: Absorbance was read at 405 nm using a 490 nm standard wavelength on a molecular device microtiter plate reader. Antibody reagents in ELISA showed no reactivity to endogenous rat hGH in diluted serum or natural samples.

HGH expression in vivo. For the assay of single dose-dependent rapamycin-induced stimulation of hGH expression, mice were administered rapamycin about 1 hour after HT1080 cell injection. The single dose of rapamycin was 0.01, 0.03, 0.1, 0.3, 1.0, 3.0 or 10.0 mg / kg. Several hours after rapamycin administration, mice were sacrificed and blood was collected.

To determine the time course of hGH expression in vivo, mice received 10.0 mg / kg of rapamycin 1 hour after cell injection. Mice were sacrificed 4, 8, 17, 24 and 42 hours after rapamycin administration.

With repeated administration of rapamycin, the ability of rapamycin to induce sustained expression of hGH from transplanted HT1080 cells was tested. Infected HT1080 cells were administered to mice as described above. About 1 hour after cell injection, 1/5 of 10.0 mg / kg intravenous dose of rapamycin was shown. The remaining 4 minutes of one dose were given as blood collection under anesthesia and immediately at 16, 32, 48 and 64 hours. Additional blood collection was performed at 72, 80, 88 and 96 hours following the first rapamycin dose. Cells were administered to control mice, but only basal agent was also accepted for multiple administrations of rapamycin. Experimental animals and their counterparts were each assigned to one of two groups. Each of the 2 experimental and 2 control groups received the same drug or base agent treatment separately. The two groups differ in that blood is collected alternately between the two groups, reducing the frequency of blood collection for each animal.

result

Rapamycin elicited single dose-responsive production of hGH in these animals (FIG. 6). HGH concentrations in animals treated with rapamycin were comparable to normal blood circulation levels (0.2-0.3 ng / ml) in humans. In this experiment, no high plateau was observed in hGH production, meaning that the maximum dose of infected cells for hGH production was not reached. Control animals-infected cells but not rapamycin, rapamycin but not cells-showed no detectable serum hGH. Thus, the production of hGH in these animals was absolutely dependent on the presence of both treated cells and rapamycin.

At 17 hours after rapamycin administration, the level of hGH in the serum was remarkable, as it is evident that the hGH circulated with a half-life of up to 4 minutes in the animal. This observation indicated that treated cells continued to secrete hGH for several hours after rapamycin treatment. To test the kinetic of the rapamycin control of hGH production, we treated the animals with a single dose of rapamycin and then measured hGH levels several times. Serum hGH was observed within 4 hours after rapamycin treatment, peaked at 8 hours, and detected for 42 hours after treatment (FIG. 7). The hGH concentration collapsed from its peak with a half-life of about 11 hours. This half-life is several hundred times greater than the half-life of hGH itself in the animal and about two times longer than the half-life of rapamycin (4.6 hours). Late breakdown of serum hGH compared to rapamycin may affect higher tissue concentrations of rapamycin in the vicinity of transplanted cells. In other words, the duration of hGH production from treated cells can be enhanced by the stability of hGH mRNA.

Interestingly, administration of the second single dose of rapamycin to the animal at 42 hours results in a second peak of serum hGH, which collapses into a similar kinetic, in which treated cells are treated for rapamycin for at least two days. Indicates that the ability to respond continues. Thus, to characterize the system's ability to assess and maintain circulating hGH concentrations, we conducted a test in which animals received multiple doses of rapamycin at 16 hour intervals. The interval corresponds to the time required to decay by about half after the hGH level reaches its highest. According to this curing method, rapamycin concentration is expected to approach a steady-state structural concentration of 1.7 μg / ml after two doses (indicated by the dashed line in FIG. 8). hGH levels also allow access to steady-state structural concentrations following the second dose. 8 shows that treated animals maintain relatively stable levels of circulating hGH in response to repeated doses of rapamycin. After the last dose, hGH levels remain constant for 16 hours and then decline with a half-life similar to that for rapamycin (6.8 hours for hGH versus 4.6 hours for rapamycin). This data means that, at multiple doses, circulating rapamycin provides a tight control over hGH secretion from infected cells in vivo. In fact, it is evident that protein production terminates quickly upon discontinuation of drug dosing.

Review

This experiment demonstrates the possibility of modulating the production of therapeutic proteins secreted from genetically treated cells with low molecular weight drugs. This system has many aspects that are required for use in human gene and cell therapy. The system is characterized by very low background activity and high introduction rate. In addition, it is not affected for host physiology or for all cell-type-specific factors. In addition, it consists entirely of human proteins. The control drug works well in vivo and can be orally bioactive.

In the above system of general design, it is possible to provide in vivo a stable and correctly titrated dose of therapeutic protein secreted from treated cells. Temporary interruptions and irregular doses can also occur. A significant advantage of protein delivery from treated cells under low molecular weight controls is that the rate of protein production affects the circulating concentration of low molecular weight drugs at any given time. Thus, the clear pharmacological kinetic of therapeutic proteins such as hGH can change dramatically. For example, in this experiment, the kinetic of circulating hGH delivered from treated cells following a single administration of rapamycin is significantly different from the kinetic observed following a single administration of the recombinant protein. Intravenously administered hGH for mice is eliminated with a half-life of several minutes, while hGH levels from treated cells are induced with rapamycin that has collapsed with a half-life of about 7 hours. Even in humans with a half-life of about 20 minutes for hGH removal, injection only is required daily, and serum hGH levels change dramatically. Under precise pharmacological control, protein delivery from treated cells, particularly poor pharmacological kinetics or protein with low therapeutic index, will lead to more effective treatment.

The use of low-molecular weight drugs for linking DNA-binding domains and active domains is an effective tactic for ordering gene expression in vivo. One particularly important feature is that the system can be adjusted as a whole, with each component separately optimized and processed. Compared to bacterial repressors, which rely on relatively sparse allosteric intermolecular interactions to control DNA-binding activity, a bidivision strategy can be accommodated for virtually all DNA-binding and active domains. We used a limited structure of DNA-binding domains in this experiment to facilitate the rational processing of DNA-binding affinity and new cognitive specificity. Similarly, active domains can handle maximum potential and other suitable properties. Indeed, the treated transcription factors used in this experiment elicited very significant levels of gene expression compared to conventional promoter / enhancer systems and further enhanced the two domains for easy mixing. The ability to introduce treatment transcription factors for the transcription of a single target gene offers the opportunity to achieve substantially higher levels of gene expression and lower background in vivo than conventional expression vectors.

We also chose to construct regulated transcription factors from human proteins in order to minimize the possibility of being caused by the immune system. Effective recognition of autolytic T cells expressing a fusion protein consisting of bacterial hygromycin phosphate convertase and herpes viral thymidine kinase and eliminated by host cytotoxic T cells, even in AIDS patients with weakened immune systems (Riddell, SR, et al., T-cell mediated rejection of genemodified HTV-specific cytotoxic T lymphocytes in HTV-infected patients, Nature Med. 2, 216-223 (1996)). This observation implies that there may actually be an immune cognitive risk of the heterologous protein in the treated cells and, therefore, that human proteins must be used with caution in order to perform the chorus function in human cells. Although each individual component of the present transcription factor fusion protein is from a human, each protein contains a linking protein that may be recognized as heterologous. However, the linkages can be designed or selected to minimize their positioning with respect to the immune system as disclosed above.

Example 5 Synthesis of Representative C-24 Modified Rapalogs

Rapamycin tablets. Rapamycin was obtained by fermentation. Rapamycin, which prepares the organism Streptomyces hygroscopicus (ATCC # 29253), was cultivated on complex media in 15L or 30L fed-batch fermentations. Biomass was harvested after 9-14 days by centrifugation. The supernatant was contacted with XAD-16 (Rohm and Haas), a nonionic polymer adsorption resin, for 1-2 hours. The adsorbent was recovered by centrifugation, combined with biomass and extracted repeatedly with methylene chloride. The solvent was removed under reduced pressure and the resulting residue was extracted with acetonitrile and then condensed in a similar manner. Crude rapamycin was chromatographed purified by flash chromatography on silica gel (40% acetone / hexane) followed by C-18 converted-phase HPLC (70% CH ₃ CN / H ₂ O). The obtained rapamycin showed the same HPLC, spectroscopic and biological properties as the original rapamycin sample.

Rapamycin (E and Z) -24- (O-methyloxime) (AP1731 and 1732) General Course

A solution of rapamycin (60 mg, 65.6 mmol) in MeOH (2 mL) was treated with NaOAc (22 mg, 262 mmol, 4.0 eq) followed by methyloxyamine hydrochloride (22 mg, 262 mmol, 4.0 eq) and room temperature It was stirred for 48 hours. The reaction mixture was then quenched with H ₂ O (10 mL) and extracted with EtOAc (3 × 10 mL). The combined organic extracts were washed with saturated NaCl solution (2 × 10 mL), dried over Na ₂ SO ₄ , filtered and the solution was concentrated in vacuo. The residue formed was flash chromatographed on silica gel (10% MeOH / dichloromethane) to give an isomer mixture. The isomeric mixture was separated by HPLC (Kromasil C-18 250x20 mm column passed 12 ml per minute, 35% AE 25% H ₂ O / MeCN) to give 13 mg (21%) of the faster eluted Z isomer and 7.6 mg (12%) of the E isomer was obtained. Z isomer: high-resolution mass spectrum (FAB) m / z 965.5749 [(M + Na) ⁺ _, calc. C ₅₂ H ₈₂ N ₂ O ₁₃ Na 965.5710]. E isomer: high-resolution mass spectrum (FAB) m / z 965.5701 [(M + Na) ⁺ _, calc. C ₅₂ H ₈₂ N ₂ O ₁₃ Na 965.5710].

Rapamycin (E and Z) -24- (O-ethyloxime) (AP1688 and 1689)

Prepared in the same manner as in rapamycin (E and Z) -24- (O-methyloxime). The isomeric mixture was separated by HPLC (Kromasil C-18 250x20 mm column passed 12 mL per minute, 30% H ₂ O / MeCN), yielding 7.7 mg (25%) of the eluted Z isomer and 0.5 mg (2%) ) Isomer of E was obtained. Z isomer: high-resolution mass spectrum (FAB) m / z 979.5902 [(M + Na) ⁺ _, calc. C ₅₃ H ₈₄ N ₂ O ₁₃ Na 979.5871]. E isomer: high-resolution mass spectrum (FAB) m / z 965.5701 [(M + Na) ⁺ _, calc. C ₅₂ H ₈₂ N ₂ O ₁₃ Na 965.5710].

Rapamycin (E and Z) -24- (O-isobutyloxime) (AP1684 and 1685)

Prepared in the same manner as in rapamycin (E and Z) -24- (O-methyloxime). The isomeric mixture was separated by HPLC (Kromasil C-18 250x20 mm column passed 12 mL per minute, 15% H ₂ O / MeCN), yielding 25 mg (65%) of the faster eluted Z isomer and 3.0 mg (7%). ) Isomer of E was obtained. Z isomer: high-resolution mass spectrum (FAB) m / z 1007.6146 [(M + Na) ⁺ _, calc. C ₅₅ H ₈₈ N ₂ O ₁₃ Na 1007.6184]. E isomer: high-resolution mass spectrum (FAB) m / z 1007.6157 [(M + Na) ⁺ _, calc. C ₅₅ H ₈₈ N ₂ O ₁₃ Na 1007.6184].

Rapamycin (E and Z) -24- (O-benzyloxime) (AP1682 and 1683)

Prepared in the same manner as in rapamycin (E and Z) -24- (O-methyloxime). The isomeric mixture was separated by HPLC (Kromasil C-18 250 × 20 mm column passed 12 ml / min, 15% H ₂ O / MeCN), yielding 19.6 mg (44%) of the faster eluted Z isomer and 6.1 mg (14%). ) Isomer of E was obtained. Z isomer: high-resolution mass spectrum (FAB) m / z 1041.6033 [(M + Na) ⁺ _, calc. C ₅₈ H ₈₆ N ₂ O ₁₃ Na 1041.6028]. E isomer: high-resolution mass spectrum (FAB) m / z 1041.5988 [(M + Na) ⁺ _, calcd C ₅₈ H ₈₆ N ₂ O ₁₃ Na 1041.6028].

Rapamycin (E and Z) -24- (O-carboxymethyloxime) (AP1686 and 1687)

Prepared in the same manner as in rapamycin (E and Z) -24- (O-methyloxime). The isomeric mixture was separated by HPLC (Kromasil C-18 250x20 mm column passed 12 mL per minute, 45% H ₂ O / MeCN), yielding 4.6 mg (11%) of the eluted Z isomer faster and 1.0 mg (2%). ) Isomer of E was obtained. Z isomer: high-resolution mass spectrum (FAB) m / z 1009.5664 [(M + Na) ⁺ _, calc. C ₅₃ H ₈₂ N ₂ O ₁₅ Na 1009.5613]. E isomer: high-resolution mass spectrum (FAB) m / z 1009.5604 [(M + Na) ⁺ _, calc. C ₅₃ H ₈₂ N ₂ O ₁₅ Na 1009.5613].

Rapamycin (E and Z) -24- (O-carboxamidomethyloxime) (AP1729 and 1730)

Prepared in the same manner as in rapamycin (E and Z) -24- (O-methyloxime). The isomeric mixture was separated by HPLC (Kromasil C-18 250x20 mm column passed 12 mL per minute, 35% H ₂ O / MeCN), yielding 6.2 mg (10%) of the eluted Z isomer and 1.4 mg (2%) ) Isomer of E was obtained. Z isomer: high-resolution mass spectrum (FAB) m / z 1008.5709 [(M + Na) ⁺ _, calc. C ₅₃ H ₈₃ N ₃ O ₁₄ Na 1008.5768]. E isomer: high-resolution mass spectrum (FAB) m / z 1008.5753 [(M + Na) ⁺ _, calcd C ₅₃ H ₈₃ N ₃ O ₁₄ Na 1008.5768]

Example 6

A. Binding Assay of Rapamycin C24 'Bump' Analogs to FKBP

The affinity of rapamycin C24 analogues to FKBP was determined using a competition assay based on fluorescence polarity (FP). Direct readout of the% border probe in an equilibrium binding experiment in which a fluorescence-labeled FK506 probe (AP1491) was synthesized and contained an increase in its fluorescence polarity as an unsaturated FKBP and varying amounts of rapamycin analog. It was used as.

(i) Synthesis of Fluorescent FK506 Probe (AP1491)

24, 32-bis (tert-butyldimethylsilyl) ether of FK506

tert-Butyldimethylsilyl trifluoromethanesulfonate (108 μl, 470 μmol) was added to a solution of 2, 6-Lutidine (89.5 μl, 768 μmol) in FK50-6 (103 mg, 128 μmol) and dichloromethane (3 mL). It was added dropwise at 0 ° C. with stirring. The forming solution was stirred at 0 ° C. for 2 hours and then treated with MeOH (0.5 mL) and ether (15 mL). The mixture was washed with 10% aqueous NaHCO ₃ (3 mL) and brine (3 mL). The organic layer was decanted, dried over anhydrous Na ₂ SO ₄ , filtered and concentrated to give a yellow oily phase. Column chromatography (silica gel, hexane-EtOAc = 3: 1) gave the title compound (104 mg) as a colorless oil.

Intermediate 1

To 24, 32-bis (tert-butyldimethylsilyl) ether solution of FK506 (100 mg, 97 μmol) in THF (2.5 mL) was added morpholine (68 mg, 580 μmol), followed by water (60 μL) and 4% 4 An aqueous osmium oxide solution (123 µl, 20 µmol) was added. The formation mixture was stirred at rt for 4.5 h. It was then treated with 50% aqueous MeOH (1.5 mL) and sodium periodate (207 mg, 970 μmol) and the suspension was stirred for an additional hour. The mixture was diluted with ether (10 mL) and washed with saturated aqueous NaHCO ₃ (2 × ₄ mL). The organic layer was decanted, dried over anhydrous sodium sulfate containing a small amount of sodium sulfite, filtered and concentrated. The residue was dissolved in anhydrous THF (2.8 mL), cooled to −78 ° C. under nitrogen and treated with 0.5 M tris [(3-ethyl-3-petyl) oxy] ammonium hydride solution in THF (282 μl). It was. The forming solution was stirred at -78 ° C for 1.75 hours and then quenched by addition of ether (6 mL) and saturated ammonium chloride solution (250 μL). The temperature of the mixture was raised to room temperature and treated with anhydrous sodium sulfate. Filtration and concentration under reduced pressure gave a pale yellow oily phase (97 mg), which was subjected to column chromatography (silica gel, hexane-EtOAc = 3: 1) to give intermediate 1 as a colorless oil.

Intermediate 2

The solution of alcohol (300 mg, 290 μmol) in acetonitrile (10 mL) was diluted with 2, 6-lutidine (338 μl, 2.9 mmol) and N, N′-disuccinimidylcarbonate (371 mg, 1.45 mmol). Treated. The resulting suspension was stirred for 14.5 hours at room temperature and then concentrated under reduced pressure. The residue was chromatographed (silica gel, hexane-EtOAc = 2: 1 to 100% EtOAc gradient) to give mixed carbonate 2 as a pale yellow oily phase (127 mg).

Intermediate 3

The solution of carbonate (30 mg, 26 μmol) and triethylamine (36 μl, 260 μmol) in acetonitrile (1 mL) was treated with 4 ′-(aminomethyl) fluorescein (13.5 mg, 34 μmol). The light orange suspension formed was stirred at room temperature for 1 hour and then concentrated under reduced pressure. The residue was chromatographed (silica gel, hexane-EtOAc = 1: 1 to 100% EtOAc to EtOAc-MeOH = 1: 1 gradient) to give 3 as a pale yellow solid (20.5 mg).

Compound 4

A solution of bis-silyl ether 3 (35 mg, 25 μmol) in acetonitrile (2 mL) was treated with 48% (w / w) HF in water (250 μL). The resulting mixture was stirred at rt for 5.5 h. It was then diluted with dichloromethane (10 mL) and washed with water (2 × 2 mL). The organic layer was decanted and dried over anhydrous sodium sulfate. Concentrated under reduced pressure. The residue was chromatographed (silica gel, 100% EtOAc) to give 4 as a pale yellow solid (13 mg).

(ii) Determination of Binding Affinity (IC50s) of Rapamycin Analogues Using FP

Each analog was diluted 10-fold with 100% ethanol in a vial and stored on ice. All other operations were performed at room temperature. A stock solution of recombined pure FKBP (purified by standard methods, see Wiederrecht, G. et al., 1992, J. Biol. Chem. 267, 21753-21760) was added to 50 mM potassium phosphate pH7.8 / 150 mM NaCl / 100 µg / Diluted with 11.25 nM in ml calf gamma globin (FP buffer: prepared using only low-fluorescence reagents from Panvera) and 98 μl purified water transferred to the wells of a Dynethech micro-fluoro black 96-well fluorescent plate. 2.0 μl samples of rapamycin analogs were transferred to the wells with mixing. Finally, a probe solution containing 10 nM AP1491 in 0.1% ethanol / FP buffer was prepared and 100 μl was added to each well with mixing. Replication control wells contained ethanol (for 100% probe binding) instead of rapamycin analogs and FP buffer (0% binding) instead of FKBP.

After equilibration by keeping the plate covered in the dark for about 30 minutes, the fluorescence polarity of the sample in each well was determined by the manufacturer's recommended Jolley FPM-2 FP plate reader (Jolley Consulting and Research, Inc., Grayslake, IL). The mean polarity (in mP units) for each competition concentration was converted to percent total binding as a reference to the control value, plotted against the competitor's log molar final concentration (x) (y). The curves were made good using non-linear least squares analysis and the IC50 was derived using the following equation:

y = M1 + (M4-M1) / (1 + exp (M2 ^* (M3-x)))

Wherein M3 is IC50. IC50 was determined by interpolation for incomplete curves. Rapamycin and C14-desoxo-rapamycin were included as controls in each case (C14-desoxo-rapamycin was prepared as described in Luengo, JI et al., 1994 Tetrahedron Lett. 35, 6469-6472 ).

(c) Results of binding analysis of rapamycin C24 oxime

Affinity is reported as IC50 and fold loss of affinity (= IC50 / IC50 of rapamycin).

B. Binding Assay of C7 Raphalog-FKBP Complex to FRAP

A series of Rapalog ('C7 Rapalog') with bulky substituents at C7 was synthesized as described in the references (Luengo et al., 1995, Chemistry and Biology 2, 471-481). The data herein is for the following three rapalogs:

(a) synthesis

AP1700, 7S-isopropoxyrapamycin:

A solution of rapamycin (60 mg, 0.066 mmol) in 30 ml of isopropanol was treated with solid p-toluenesulfonic acid (75 mg, 0.39 mmol). The reaction mixture was stirred at room temperature for 4 hours, then diluted with 30 ml of ethyl acetate and washed with saturated aqueous sodium bicarbonate (3x20ml) and brine (2x20ml). The organic layer was dried over anhydrous magnesium sulfate, filtered and concentrated. The reverse phase was HPLC (Rainin C-18 ODS1 column, 35% acetonitrile / water, 55 ° C.) to afford the desired product (25 mg, 40%).

AP1701, 7R- (3-indolyl) rapamycin and AP1702, 7S- (3-indolyl) rapamycin:

A solution of rapamycin (50 mg, 0.055 mmol) and indole (64 mg, 0.55 mmol) in 2 mL of dichloromethane was added dropwise with trifluoroacetic acid (0.084 mL, 1.1 mmol) at −40 ° C. with stirring. . After maintaining the temperature at -40 ° C to -45 ° C for 3 hours, the reaction mixture was partitioned between ethyl acetate (10 mL) and brine (20 mL). The organic layer was further washed with brine (4x20 mL) and dried over anhydrous sodium sulfate. The reverse phase was HPLC (Rainin C-18 ODS1 column, 65% acetonitrile / water, 55 ° C.) to give 11.8 mg of 7S-indole and 7.6 mg of 7R-indole (35%).

Compounds were analyzed by mass spectra and NMR.

(b) FP assay of FKBP binding affinity of C7 rapalog

As described for the C24 rapalog in section A above, antagonistic FP was used to assay the affinity of the rapalog for FKBP. Rapamycin and C14-desoxo-rapamycin (prepared as described in Luengo et al., 1994, Tetrahedron Lett. 35, 6469-6427) were included as controls.

Affinity is expressed as IC50s and fold loss of affinity (IC50 / IC50 of rapamycin).

IC50 (nM) Pear loss of affinity Rapamycin 7.7 (One) Desokso-rapamycin 162 20.8 AP1700 17.8 2.5 AP1701 33.1 4.6 AP1702 24.5 3.0

The data indicate that upon selection of the C7 position at the rapamycin-FRAP contact surface, the large C7 substituent does not significantly reduce the affinity of the rapalog to FKBP.

(c) FP assay of FKBP binding affinity of C7 rapalog

To analyze the binding of the Rapalog-FKBP complex to FRAP in cultured cells, we present HT20-6 cells (containing ZFHD1-responsive SEAP reporters and stably express ZFHD1- (3xFKBP) and FRB-p65: Reference Example 3 (B)) was used. HT20-6 cells were plated in 96-well dishes (1 × 10 ⁴ cells / well), and 200 μl of medium containing serial dilutions of Rapalog was added for each well. After 22 hours, the medium was removed and assayed for SEAP activity as shown in Example 2.

The results are shown in FIG. 15, wherein the isopropoxy C7 substituent of AP1700 was found to adequately reduce FRAP affinity. However, the larger indolyl substituents of AP1701 and AP1702 almost completely eliminate SEAP expression, which appears to act as an obstacle to prevent binding of the Raphalog-FKBP complex to FRAP by modification.

In addition, C7 rapalogues were assayed for their ability to reduce proliferation of activated human T cells or rat splenocytes as their ability to bind FRAP was reduced. Suitable assays are well known in the art—eg, inhibition of ³ H-thymidine uptake (Luengo et al., 1995, Chemistry and Biology 2, 471-481).

Example 7: Mutagenesis and phage display to generate modified ligand-binding domains complementary to various raphalogs

A. FKBP and FRB Domains Treated

We designed and prepared recombinant DNA constructs encoding the fusion proteins listed below having denatured ligand-binding domains. Unless otherwise noted, mutants using oligonucleotide-added site-directed mutagenesis according to standard methods (Kunkel, TA, Bebenek, K. and McClary, J. 1991, Meth Enzymol, 204, 234-139) Was generated and verified by dideoxy sequencing.

We also made a construct that encodes the following FKB fusion protein:

In addition, yeast and Candida FRB modified by analogy to the denatured hFRAP FRB domain were prepared by substituting codons for different amino acids in each FRB domain instead of two conserved Phe residues and conserved Asp and Asn residues. The denatured FRB domain is derived from TOR1 and TOR2, including:

B. Testing of Rationally Designed FKBP Mutants for Binding to Raphalogs

Expression vector based on pET20b (Novagen), using standard procedures for expressing FKBP followed by a portion of H. influenza hemagglutinin protein, an antigenic element for hexahistidine tail and monoclonal antibody 12CA5 Was constructed. The protein sequence encoded by the vector is as follows:

To generate expression vectors for mutated FKBPs at rapamycin contact residues, oligonucleotide-added site-directed mutagenesis is described in Kunkel, TA, Bebenek, K. and McClary, J. 1991, Meth Enzymol, 204. , 235-139), on single-stranded foams of vectors prepared from E. Coli CJ236. Mutants were identified by dideoxy sequencing. Mutant proteins are expressed in E. coli BL21 (DE3) (Novagen) as described in Weiderrecht, G. et al., 1992, J. Biol. Chem. 267, 21753-21760, and in Cardenas, Purified by homogenization as in ME et al., 1994, EMBO J. 13, 5944-5957).

Using this protocol, the following mutant human FKBP12 protein was generated:

Designed Mutants for Binding to C24 Rapalog:

Mutants designed for binding to C13 / C14 rapalogs:

Mutants designed for binding to C28 / C30 rapalogs:

To assay the relative binding affinity of rapamycin and rapalog to the FKBP mutant, a competitive fluorescence polarity (FP) assay was used that depends on the retention of FK506 (and probe) binding affinity by the mutant. The procedure is the same as in Example 6, except that a direct binding assay was first performed to determine the dilution (concentration) of the mutant FKBP for use in competition reactions to obtain sub-saturation. Serial dilutions of mutant FKBP were placed in 100 μl volume FP buffer (Example 6) in Dynatech micro-fluorine plates, and then 100 μl of 10 nM AP1491 (probe) in [FP buffer + 2% ethanol] in each well. It is prepared by adding. Equilibrium and plate readings are as in Example 6. Plots of mP units against concentrations of FKBP mutants fit the formula:

y = M3 + (((x + M1 + M2) -SQRT (((x + M1 + M2)))-(4 ^* x ^* M1)) / (2 ^* (M1)) ^* (M4-M3)

The final mutant concentration / dilution at which 90% of the probes are specifically bound is determined by interpolation. This final concentration is used for the competition FP assay performed in Example 6, with a final concentration of 2 × mutants instead of 11.25 nM FKBP in the protocol. Instead of 90% saturation, 75% may be chosen which is more sensitive to competition assays. A series of dilutions of rapamycin analogs were used as competitors and the results were expressed as IC 50 for each rapalogue binding to each mutant.

C. FRB Mutant Test Rationally Designed for Binding to FKBP-Raphalog Complex

NcoI-BamHI fragments encoding residues 2021-2113 of human FRAP were generated by PCR using primers 28 and 29 (below) and cloned into derivatives of pET20b (+) (Novagen) where the NdeI site was mutated to NcoI. , pET-FRAP (2021-2113) was calculated. The single-stranded DNA of the vector was used as a template in the site-directed mutation process to generate a vector encoding the mutated FRAP at the rapamycin contact residue. Dideoxy sequencing identified the mutants. The mutants were then amplified by PCR using primers 30 and 31 attaching XabI and SpeI sites and cloned into XbaI-SpeI digested pCGNN-FRB-p65 (361-550) (Example 1) To direct mammalian expression of the processed protein of FRAP (2021-2113) -p65 (361-550), wherein E represents the HA antigenic element tail and N represents the nuclear locus sequence. A series of constructs was generated. The composition was verified by restriction analysis and dideoxy sequencing.

Using this procedure, the following construct was generated which encodes a mutant FRAP that binds to C7 rapalog with the oligonucleotide primers shown (uppercase mutant base; 5'-3 '), each of p65 (361-550). ) Fused to the active domain:

In order to assay the relative binding affinity of the mutant to the complex of FKBP using rapamycin and various raplogs, each construct was temporarily inter-infected with human HT1080B14 cells. Following infection, serial dilutions of rapamycin or rapalogue were added to the culture medium. After 24 hours, SEAP activity was measured as in Example 2; The efficacy of SEAP activity at various raphalog concentrations is proportional to the affinity of the FRAP mutant for the complex between FKBP and raphalog.

PCR primers (uppercase restriction sites; 5'-3 '):

D. Functional Display of FRB Domains of FRAP on Fibrous Bacteriophage: An Approach to Select as an Alternative to Rational Design of Denatured Domains

(a) vector composition

Carbenicillin-resistant pazamide display vector (Pharmacia), a derivative of pCANTAB-5E, was constructed by site-directed mutation with primer 1 in which Cys202 of fd geneIII was replaced with Tyr. Cys can disrupt the display of fused proteins in Asp198 due to the formation of mixed disulfides and the problem is alleviated by Tyr substituents (Cumningham. B. C. et al., 1994, EMBO J. 13, 2508). The construct was verified by DNA sequencing and then digested with NcoI and BamHI. The pair of oligonucleotides (2 and 3) were ligated to contain the plasmid pCANTAB-AP-poly containing the NcoI-SpeI-BamHI polylinker between the NcoI site in the geneIII signal sequence and the BamHI site at codon 198 in geneIII. Livestock. The vector is housed in a frame NcoI-BamHI fragment to provide a construct that directs the N-terminal display, which is the coding protein on pIII residues 198-406, fusion orientation is described in Cunningham et al. (1994, EMBO J. 13, 2508). It is described.

Fragments of FRAP encoding residues 2015-2114 were amplified from pCGNN-FRAP _i by PCR using Pfu polymerase (Stratagene) and primers 4 and 5. The fragment was purified, digested with NcoI and BamHI and ligated into NcoI-BamHI digested pCANTAB-AP-poly to yield the vector pCANTAB-FRAP (2015-2114). The configuration was verified by DNA sequencing.

(b) Preparation of His6-flag-FKBP

FKBP processed by hexahistidine tail (for affinity purification) and flag sequence (Kodak IBI) (for immunological detection) to provide a tracing FKBP protein for affinity enrichment of phagemids displaying FRAP An expression vector based on pET20b (Novagene) was constructed using a standard procedure to express. Proteins are expressed in E. coli BL21 (Novagen) according to Weiderrecht, G. et al. 1992, J. Biol. Chem. 267, 21753-21760, and Cardenas, ME et al. 1994, EMBO J 13, 5944-5957), and purified homogeneously using Ni-NTA agarose (Qiagen).

The sequence of the expressed protein is as follows:

(c) binding enrichment

p-CANTAB-AP-FRAP (2015-2114) was grown by growing overnight cultivation for phagemid growth in a medium containing 2% glucose and incubating the phage at 37 ° C. initially for 1 hour and then at 25 ° C. Display phage prepared by helper phage K07 based on the content described in Lowman, H. B, and Wells, JA Methods: Comp. Methods Enzymol, 1991, 3, 205-216, except that K07 was infected. And E. coli XL-1 (Stratagene). To provide a reference reagent, phagemid particles were prepared from cells converted to chloramphenicol-resistant vector pBC (Stratagene). In each case the phagemid titration concentration (colony-forming unit, cfu) was determined according to Lowman, H. B. and Wells, J. A. Metheds: Comp. Methods Enzymol. 1991, 3, 205-216.

To demonstrate specific binding enrichment of FRAP display phage on FKBP rapamycin, about 10 ⁸ cfu was mixed with about 10 ¹⁰ cfu of pBC phage in 250 μl of PBS / 3% BSA / 0.05% tween-20 ('PBSBT') It was. After addition of 1 μM (final) His6-flag-FKBP protein, only 2.5 μl of 10 μM rapamycin or FK506 or ethanol in 100% ethanol was added. After the mixture was incubated for 1 hour at room temperature, 100 μl of 1: 1 slurry of Ni-NTA agarose beads equilibrated in PBSBT was added (to capture His6-tagged FKBP and all related phagemids). The mixture was lightly ground for 15 minutes on an end-over-end mixer and then spun into pellets by spinning at 8000 rpm for 30 minutes in microfuge. The beads were washed five times with 1 ml PBS / 0.05% tween-20 and five times with 1 ml PBS / 0.05% tween-20 / 1M NaCl. 200 μl of 0.2 M glycine pH2.0 was added and incubated for 10 minutes to elute the interface phagemid from the beads. The beads were pelleted and the supernatant (containing eluted phagemid) was collected and neutralized with 26 μl 1M Tris base. Carbenicillin (carb) and chlor-amphenicol (chlor) -resistant cfu titration concentrations were determined as above and the carb / chlor ratio was used as a measure of the specific enrichment of (carbenicillin-resistant) FRAP phagemids. It was.

As a result, FRAP phage was significantly richer at 271 times compared to the starting mixture after the affinity enrichment on the rapamycin-FKBP matrix, but was 2.4 times higher than the FK506-FKBP matrix or 1.5 times higher than the FKBP alone. This experiment represents a process for purification from the library mutant FRAP that binds rapamycin analogs with obstructions on the FRAP binding side (eg C7 position) of the molecule.

(d) primer

Restriction sites are underlined (NcoI = CCATGG; BamHI = GGATCC).

(e) the sequence of pCANTAB-AP-FRAP (2015-2114)

E. Functional Display of FKBP on Fiber Bacteriophage

(a) vector composition

The coding sequence of FKBP (amino acids 1-107) was prepared using primers 1 using 5 ng of plasmid pCGNN-F1 (US Patent Application No. 08/581, 713, filed Dec. 29, 1995) as a template. Amplification by PCR using 2. The PCR product was purified, digested with NcoI and BamHI and ligated into NcoI-BamHI digested pCANTAB-AP-poly to yield a vector pCANTAB-FRAP. The configuration was verified by restriction analysis and DNA sequencing. This configuration directs the N-terminal display of FKBP on pIII residues 198-406 where Cys202 was mutated to Tyr: The orientation is described by Cunningham et al. (1994, EMBO J. 13, 2508).

(b) Synthesis of biotinylated FK506 for affinity enrichment studies

To provide a stationary phase ligand for showing the functional display of FKBP on phage, several FK506s were synthesized with biotin moieties attached through a linker to the allyl group of the main effector domain (see below). Biotin-Su (25.1 mg, 73.5 mmol) was added to a solution of N- (6-aminohexyl) FK506 (35 mg, 36.8 mmol) in ₂ mL CH ₂ Cl ₂ , followed by the addition of NEt ₃ (51.4 mL, 368 mmol). Added. DMF was added to the suspension until the reaction mixture became clear. After 10 minutes the reaction was complete. The mixture was diluted with CH ₂ Cl ₂ (25 mL), washed with water (15 mL), brine (15 mL) and dried over Na ₂ SO ₄ . Flash chromatography using 10% MeOH / CH ₂ Cl ₂ as the eluent gave 13 mg (30%) of the desired product as a white solid. The structure was confirmed by NMR and MS.

(c) Growth of FKBP Display Phage

p-CANTAB-AP-FRAP (2015-2114) was grown in Lowman, H. B, and Wells, JA Methods, except that overnight cultivation for phagemid growth was grown in a medium containing 2% glucose. : Display phage prepared by helper phage K07 and E. coli XL-1 (Stratagene) based on the content described in Comp. Methods Enzymol, 1991, 3, 205-216). Phagemid titration concentrations (colony-forming units, cfu) were determined according to Lowman, H. B. and Wells, J. A. Metheds: Comp. Methods Enzymol. 1991, 3, 205-216.

(d) Demonstration of functional FKBP display by competitive ELISA with biotinylated FK506

Wells of 96-well maxithorp plates (Nunc) were covered with 100 μl of 1 μg / ml strepvidavidine (Pierce) in PBS per well and then incubated at 4 ° C. overnight. After shielding with 1% tween-20 / PBS at room temperature, the shielding solution was removed and 10 nM biotinylated FK506 in PBS / 1% ethanol was added and incubated for a further 10 minutes. At the same time, 100 μl of incubation liquid containing FKBP phage (approximately ⁵ × 10 ⁵ cfu) and serial dilutions of FK506 in PBS / 0.02% BSA / 1% ethanol were prepared. The incubation solution was left to stand at room temperature for 1 hour to equilibrate, then the wells were coated, washed 5 times with PBS / 0.05% tween-20 and the phage-FK506 mixture was added to the wells. Plates were incubated at room temperature for 1 hour and then washed 15 times with PBS / 0.05% tween-20. To detect bound phage, 100 μl of a 1/5000 dilution of anti-M13 antibody-HRP conjugate (Pharmacia) was added to each well, incubated for 1 hour at room temperature, and the plates washed 10 times as before. Afterwards, HRP activity was quantified according to the manufacturer's instructions using a TMB substrate (Boehringer). Values were calculated as% total phage binding, based on controls that either did not coat biotinylated FK506 (0% binding 0 FK506 competitors at all (100% binding)).

As shown in the figure, this experiment shows the specific high-affinity binding of FKBP to FK506. The IC 50 for interaction in the assay was 0.65 nM and was exactly in line with the known Kd of FKBP for FK506 of 0.4-1 nM.

(e) Production of phage mutant FKBPs targeted to the C13 and C14 positions of rapamycin

To generate mutant FKBP to obtain a variant that binds a rapamycin analogue impaired at the C13 or C14 position, altered primers 3 and 4 are used to amplify a portion of the FKBP gene and to Tyr26, Phe36, Asp37, Arg42 and Phe99 A modified codon NNS was introduced at the codon's position for where N is all bases and S is G or C. The side chains of the residues were contacted using computer graphics to contact C14 carbonyl and / or C13 hydroxyl groups, or contact upon residue. Longitudinal ApaLI and BamHI sites adjacent to the disordered region were used.

Portions of FKBP were amplified from pCGNN-F1 using primers 3 and 4 and Pfu polymerase. PCR products were purified using the QIAEX II kit (Qiagen), digested with ApaLI and BamHI and ligated into ApaLI-BamHI digested pCANTAB-AP-FKBP. The ligation product was purified by phenol extraction, concentrated by ethanol precipitation and electroporated into E. coli XL-1 cells by standard techniques (Dower, WJ et al., 1988, Nucleic Acids Res. 16, 6127). . Phage were taken and titered as described above (Lowman, H. B. and Wells, J. A. Methods: Comp. Methods Enzymol, 1991, 3, 205-216).

(f) library classification

About 10 ¹⁰ -10 ¹¹ cfu phagemid particles were prepared in 250 μl of PBS / 3% BSA / 0.05% tween-20 (PBSBT). Final concentration by addition of recombinant GST-FRAP fusion protein (expressed in E. coli and purified as described in Chen et al, m 1995, Proc. Natl. Acad. Sci. USA, 92, 4947-4951) After 1 μM, 2.5 μl of 10 μM bumped rapamycin 100% ethanol stock solution was added. After the mixture was incubated for 1 hour at room temperature, 100 μl of a 1: 1 slurry of glutathione-agarose beads (Pharmacia) equilibrated in PBSBT was added (to capture GST-tagged FKBP and all related phagemids). The mixture was lightly ground for 15 minutes on an end-over-end mixer and then spun into pellets by spinning at 8000 rpm for 30 minutes in a microfuge. The beads were washed five times with 1 ml PBS / 0.05% tween-20 and five times with 1 ml PBS / 0.05% tween-20 / 1M NaCl. 200 μl of 0.2 M glycine pH2.0 was added and incubated for 10 minutes to elute the interface phagemid from the beads. The beads were pelleted and the supernatant (containing eluted phagemid) was collected and neutralized with 26 μl 1M Tris base. Carbenicillin (carb) and chlor-amphenicol (chlor) -resistant cfu titration concentrations were determined as above and the carb / chlor ratio was used as a measure of the specific enrichment of (carbenicillin-resistant) FRAP phagemids. It was.

When specific enrichment was detected throughout the background, individual FKBP phage clones were isolated and sequenced by standard techniques. The binding of clones to rapamycin bumped by competitive ELISA or fluorescence polarity assay was measured.

(g) primer sequences

Restriction sites are underlined (NcoI = CCATGG; BamHI = GGATCC; ApaLI = GTGCAC).

(h) the sequence of pCANTAB-AP-FKB

Example 8: Rapamycin-dependent Activity of Signal Transformation

By aggregating a biological ligand or anti-reporter antibody, many cell type reporters can be activated. In addition, aggregation of two different proteins can often act to induce intracellular signals. Rapamycin and its analogs induce the active action of a reporter main body domain by oligomerizing an individual mutagenic protein, one containing at least one FKBP and a main effector domain and the other containing at least one FRAP domain and a main effector domain. Can be used. This schema is illustrated in Figure T1 (a). Although two proteins appear to be hung on the membrane, one is hung on the membrane and an additional rapamycin or analog is intended to allow the second protein to diffuse into the membrane through dimerization. Membrane jamming can be effected through protein anchors on the other side of the membrane or through lipid denaturation of the protein, such as myristolation. The main effector domain may be on two proteins or on different protein domains. They can be used functionally like protein kinases and protein kinase substrates. As an alternative, a second main active may be provided to inhibit the activity of the first main active.

The inventors noted that, in some embodiments, the mutant protein contains a mixed kinase and contains FKBP and FRAP domains with heterologous main effector domains. Oligomerization of a single mixed individual variant may also be used to activate signal transduction, as shown in FIG. T1 (b). Rapamycin is shown to dimerize two identical copies of the protein. Repetition of the FKBP and FRAP domains allows for more multiplexes to the genomic body.

Two examples of the use of rapamycin in signal transduction are examples of imparting tyrosine kinase activity to reporters and examples of imparting apoptosis through Fas activity. Unless otherwise noted, all DNA manipulations were performed according to the standard procedures of FM Ausubel et al., Eds., Current Protocols in Molecular Biology, John Wiley Sons. New York, 1994, and all protein protocols are described in Harlow, E. and Lane, D. 1988, Antibodies, a Laboratory Manual.Cold Spring Harbor Laboratory, Cold Spring Harbor). All PCR products were used to make up the constructs, the constructs confirmed by sequencing.

A. Rapamycin-Induced Reporter Tyrosine Kinase Activity.

1. Construction of pCM, Expression Vectors Containing Prestillation Signals

The XbaI-Myr-BamHI cassette obtained by annealing oligonucleotides 1 and 2 was digested with XbaI / BamHI and incorporated into the XbaI / BamHI site of the pCG expression vector (Tanaka, M. and Herr, W, 1990, Cell 60; 375-386). Cloning yielded pCGM (cf. (7) below). The oligonucleotide cassette consists of an in-frame XbaI site, followed by c-Src, which seems to allow myristolation and target protein for the plasma membrane (Cross et al., 1984, MCB. 4: 1834-1842). Consisting of a sequence encoding the first 15 amino acid residues of a tyrosine kinase. The myristolation domain follows the in-frame SpeI site and stop codon. The XbaI site in the pCG vector is positioned so that two amino acids are added between the starting Met and the cloned sequence. Because the space between the starting Met and the pre-stilled Gly is important for determining the membrane position of c-Src (Pellman et al, m 1985, PNAS. 82: 1623-1627), the Aba in the XbaI site followed by pCGM is removed. It was. To facilitate the cloning step, the SpeI site in the mystilation cassette was mutated to the XbaI site. Single-stranded uracil-DNA of pCGM was prepared and oligonucleotide 3 (to remove ATG followed by XbaI site and EcoRI site 5 'to ATG) and oligonucleotide 4 (mypelation site followed by SpI site) Mutations were performed using both) to switch to the XbaI site. The forming sequence surrounding the ATG of the pCM vector was confirmed by sequencing with oligonucleotide 5 (see SEQ ID NO: 1, (8)).

2. Production of pCMF1 / 2 / 3.HA by Tagging Antigens to pCM and Addition of FKBP

Complementary oligonucleotides (6 and 7) were annealed to make the SpeI-HA-BamHI cassette. The cassette has an in-frame SpeI site following the 9 amino acids of the H. influenza hemagglutinin gene recognized by the monoclonal antibody 12CA5, stop codon and BamHI site. The SpeI-HA-BamHI cassette was subcloned into the SpeI / BamHI site of pCGNNF1, pCGNNF2 and pCGNNF3. Subsequently, 1/2/3 copies of FKBP fused to HA antigen elements were subcloned as XbaI / BamH1 in pCM. The formed plasmid (pCMF1 / 2 / 3.HA) has the following aspects: myristylation domain; Inframe XbaI site; 1/2/3 copy of FKBP; Inframe SpeI site; HA antigen element tag; And stop codons.

3. Generation of pCMF1 / 2 / 3.Flag by Tagging Antigens to pCM and Addition of FRB

Complementary oligonucleotides (8 and 9) were annealed to make the SpeI-HA-BamHI cassette. The cassette is designed for the 8 amino acid (DYKDDDDY) (Hopp et., 1988, Biotech. 6: 1205-1210) in which the in-frame SpeI site is recognized by the monoclonal antibody anti-FLAG.M2 (Kodak Scientific Imaging Systems). It has the same aspect as in SpeI-HA-BamH1 except following the sequence which is the code. The SpeI-Flag-BamHI cassette was subcloned into the SpeI / BamHI sites of pCGNN-1FRB, pCGNN-2FRB and pCGNN-3FRB. Subsequently, 1/2/3 copies of the FRB of the FRB domain-Flag antigenic fusion were subcloned as XbaI / BamH1 in pCM. The formed plasmid (pCMFR1 / 2 / 3.Flag) has the following aspects: myristylation domain; Inframe XbaI site; 1/2/3 copy of FRB; Inframe SpeI site; Flag antigen element tag; And stop codons.

4. Configuration for Reporter Tyrosine Kinase Cytoplasmic Domains of FKBP and FRB Fusions

Cytoplasmic domain selection (eg EGFR, erbB-2, PDGFR, KDR / Flk-1, Flt-1) of reporter tyrosine kinase was PCR amplified with in-frame 5′XbaI and 3′SpeI sites. PCR products can be subcloned into the frame SpeI site so that the SpeI site is stored in the pCMFR series or pCMF series vector, or subcloned into the in-frame XbaI site so that the XbaI site is stored. As a result, the FKBP / FRB domain (s) can position the C-terminus or NH ₂ -terminus to the cytoplasmic domain of the reporter tyrosine kinase. (i) the cytoplasmic domain of a given reporter is fused for both FKBP and FRB (eg, the erbB-2 cytoplasmic domain fused to FRB and the EGFR cytoplasmic domain fused to FKBP) (ii ) Vectors such that the cytoplasmic domains of two different reporters can be fused to FKBP and FRB (eg, the EGFR cytoplasmic domain fused to FKBP and the erbB-2 cytoplasmic domain fused to FRB) Was constructed. In the former (i), the addition of the drug, rapamycin, causes the formation of homodimers (eg EGFR / EGFR), whereas in the latter (ii), the addition of the drug is heterodimer (eg For example, EGFR / erbB-2) is induced to cause activation of the signal transduction cascade.

5. Component test

To test the ability of rapamycin or an analog to induce dimerization of FKBP- and FRB-reporter cytoplasmic domain fusions, selected constructs (eg, pCMEGFR-FR1 and pCMEGFR-F1) were lipofected (Gibco). BRL) was transfected into Cos-1. Three days after infection, cells were induced with rapamycin and Lysis buffer (1% Triton X-100; 500 mM Tris. Cl pH8.0; 150 mM NaCl; 5 mM NaF; 1 mM sodium ortho vanadate; 10 μg / ml aprotinin 10 µg / ml lupeptin). Fusion proteins from rapamycin-treated and untreated cell lysates were immunoprecipitated with anti-Flag and 12CA5 antibodies and immunodepleted with anti-phosphotyrosine antibodies. To achieve optimal results, cell type; DNA infection amount; The concentration of rapamycin used and the persistence of drug treatment were varied.

6. Rapamycin-Induced Cell Growth

The selected mammalian cell line (eg NIH3T3) is transfected with a construct encoding the FRB and FKBP fusion proteins (eg pCMEGFR-FR1 and pCMEGFR-F1) and establishes a stable cell line that expresses the fusion protein. It was. To determine if the rapamycin-induced activity of the cytoplasmic domain we report causes cell proliferation, a stable cell line expressing a fusion protein is grown in the presence or absence of rapamycin and subjected to a process (eg, cell number). Cell growth rate was measured by measuring the ³ H thymidine incorporation rate and the like. Report tyrosine kinases to ensure optimal results; Reporter activity (heterodimer vs. homodimer) was selected.

7. Oligonucleotide Sequence

8. SEQ ID NO: 1

Denatured sequences are shown in the lower boldface, and the starting ATG is underlined. The top sequence is the pCG backbone from the parent.

B. Construct Encoding Mutagenic Proteins Containing Domain (s) of Fas

The ability to control Fas activity and induce apoptotic action via small molecules has been applied to gene therapy and laboratory systems that can be used to selectively remove treated cells. The protein here is anchored to the membrane via a low affinity NGF reporter, called p75. However, it is expected that another protein anchor may be substituted. p75 is laboratory useful because of the applicability of the antibody to the extracellular domain and the lack of high affinity interactions with all identified ligands (Bothwell, M. 1995. Annu. Rev. Neurosci. 18: 223-253).

1. 2-protein Rapamycin-Modulated Fas Activity

(a) Composition of p75 vector

A vector for directing the expression of the FRAP-Fas fusion protein containing the transmembrane domain and the extracellular domain of the low-affinity NGF reporter (also known as p75) was selected from the mammalian expression vector pJ7W (Morgenstern, JP and Land, H. 1990). , Nucleic Acids Res. 18: 1068), was modified by replacing the pUC backbone with the original pBR backbone using standard methods. This is called vector pA7W. Inserts cloned into the polylinker site of the plasmid were transcribed under the control of the apes CMV promoter and enhancer sequences. The polylinker is followed by the CMV sequence with HindIII-SalI-XbaI-BamH1-SmaI-SstI-EcoRI-ClaI-KpnI-BgIII. All mammalian expression vectors with the appropriate cloning site and promoter can be substituted.

Restriction fragments encoding fragments of p75 bound by the HindIII and XbaI sites include sequences of p75 (Johnson, D., Lanahan, A., Buck. CR, Shegal, A., Morgan, C., Mercer, E., PCR was generated using primers J1 (5 ') and J2 (3') based on Bothwell.M., Chao.M. 1986. Cell 47: 545-554. The original source of PCR templates was clones derived from human brain libraries using primers similar to J1 and J2 but with different restriction sites. The 5 'end of the formed fragment contains the HindIII site followed by the EcoRI site, Kozak sequence and p75 coated sequence start (amino acid 1). The resulting 3 ′ end is encoded following the XbaI site beyond the expected membrane spanning sequence, including amino acids 274, 2 amino acids, to the reporter sequence. PCR products were subcloned into HindIII-XbaI digested pA7W as HindIII-XbaI fragments, resulting in pA7Wp75. The construction was verified by restriction analysis and DNA sequencing.

(b) addition of Fas to pA7Wp75

XbaI-SpeI fragments encoding Fas amino acids 206-304 (Fas _S ) and Fas amino acids 206-319 (Fas _L ) were prepared by PCR and subcloned into pA7Wp75 digested with the enzyme. Primers used were J3 (5 ') and J4 or J5 (3'). J5 produces a fragment of Fas that terminates under its terminal codon: When cleaved with SpeI, the nucleotides encoding terminal 15aa of Fas were removed to obtain a truncated intracellular Fas (called Fas _S ). Removal of 15aa increased the activity of Fas in some cell types (Itoh, N., and Nagata, S. 1993, J. Bilo. Chem 268: 10932). Primer J4 replaces the native terminal codon of Fas with the SpeI site and mutates the original SpeI site contained in Fas to produce Fas _L. The plasmids generated by subcloning the fragment are pA7Wp75-Fas _S and pA7Wp75-Fas _L, respectively. This configuration was verified by restriction analysis and DNA sequencing.

(c) p75-FRAP-Fas-epitope fusion protein: p-75-FRAPx-Fas _SirK E and p75-Fas _SORL -FRAPxE due to the addition of FRAP-containing fragments to pA7Wp75-Fas _S E and pA7Wp75-Fas _L E Generation of

XbaI-SpeI fragments containing portions of FRAP have been described. The XbaI-SpeI fragment was inserted into the XbaI site immediately after the p75 coating sequence to generate p-75-FRAPx-Fas _SirK E or into the SpeI site immediately after the Fas fragment to generate p75-Fas _SORL -FRAPxE. Alternatively, one or more FRAP fragments were subcloned. The vector of the final series thus encodes the p75 extracellular and transmembrane sequences, one or more FRAP-derived domains and epitope fused to the N- or C-terminus for one or more Fas intracellular domains.

(d) p75-FKBP-Fas fusion protein: production of p75-FKBPn-Fas _SirK or p75-Fas _SORL -FKBPn due to the addition of FRAP-containing fragments to pA7Wp75-Fas _S E and pA7Wp75-Fas _L E

XbaI-SpeI fragments containing portions of FKBP have been described. The XbaI-SpeI fragment was inserted into the XbaI site immediately after the p75 coating sequence to generate p-75-FKBPn-Fas _SirK or inserted into the SpeI site immediately after the Fas fragment to generate p75-Fas _SORL -FKBPn. The vector of the final series thus encodes the p75 extracellular and transmembrane sequences, one or more FKBP-derived domains and epitope tags that have been N- or C-terminally fused to one or more Fas intracellular domains.

(e) Assay of Rapamycin-Interposed Fas Activity

The addition of rapamycin to proteins containing Fas and FRAP domains and proteins containing Fas and FKBP domains was tested in cells that transiently or stably infected the ability to express Fas by activating Fas and causing cell limbs.

To stably infect cells, vectors encoding selective markers, such as neomycin resistance, were transfected with plasmids. Two to three days after infection, cells were plated into G418 and resistant populations or clones were isolated by standard means. The population was treated with dimers followed by cells and monitored to induce apoptosis directly.

Subclones the insert into a retroviral vector as an alternative to forming stable cell lines that express important constructs. The insert can be excised with Eco RI to facilitate subcloning. The vector was used to transform the supernatant by packing cells using conventional methods.

2. Single Protein Rapamycin-Controlled Fas Activity

(a) Construction of FKBP-FRAP mutant fragment FKBP-FRAP fusion constructs for rapamycin-dependent homodimerization of Fas intracellular domain

i. Structure-designed design

To design molecules containing both rapamycin-dependent homodimerizable FRAP and FKBP domains, the three-dimensional structure of the four complexes between human FKBP12, rapamycin and a portion of the human FRAP surrounding the minimum FRB domain was Can be considered FR10-FKBP interactions occurring between molecules bound to the membrane, while maintaining the ability of the aggregated Fas to convert signals to (i) convert the signal to homodimerize two-molecule fusion proteins containing the FRAP, FKBP and Fas moieties. Sufficient length and flexibility of the polypeptide to accommodate the modifications needed for functioning; And (ii) preservation or minimization of intramolecular dimerization by rapamycin.

In designing a fusion protein, it is desirable to consider the following:

(i) FRB and FKBP must be linked with a linker having a polypeptide that is sufficiently short that intramolecular dimerization is maintained stericly. The preferred orientation is FRAP-FKBP as the C-terminus of FRAP and the N-terminus of the FKBP should be distanced to allow long (10 amino acid) linkers so that intramolecular dimerization still confers flexibility.

(ii) The FRAP-FKBP cassette may be present in the membrane-near (ie with a Fas domain added at the C-terminus) or at the membrane-terminus.

(iii) Long linkers must be present at the N-terminus to the FRAP-FKBP domain to allow for structural modifications implied by the dimerization in the membrane. The N-terminal position of the FRAP is preferably such that the long linker can include native FRAP sequences from the region N-terminus to the FRB domain, thereby reducing the immunity of the mutant protein.

(iv) The optimal linker length and fusion position for a given protein should be conservatively convincing.

A series of 12 fusions of FKBP and FRAP representing T1-T12 were designed. 9 was the N-FRAP-FKBP-C fusion contained between 13, 23 or 33 amino acid N-terminus and 4, 7 or 10 residues separating the two proteins for Arg2018 (N-terminal linker). The remaining 3 were N-FKBP-FRAP-C fusions with 3, 0 or -4 residues of the FRAP sequence between FKBP Glu107 and FRAP Arg2018.

(ii) composition

12 fusions were prepared as XbaI-BamH1 cassettes that can be cloned directly as single fragments using a 3-primer PCR slicing method (Yon, J. and Fried, M. 1989, Nudcleic Acids Res. 17, 4895). Cloning in this manner avoids the introduction of restriction sites between genes that encode heterologous sequences and change the length of the linker. Using 1 ng of each pCGNN-1FRAP _i and pCANTAB-AP-FKBP mixtures with Pfu polymerase with 2 outer primers (A and C) of 1 μM each, a single complementary to two genes indicating the desired fusion 'Slice' oligos (B) were amplified in the presence of 0.01 μM. The primers used are listed below:

PCR products were purified, digested with XbaI and BamHI and ligated into XbaI-BamHI digested pCM. The configuration was verified by restriction analysis and DNA sequencing.

Primer sequence

Restriction sites are underlined (XbaI = TCTAGA, SpeI = ACGAGT, BamHI = GGATCC).

Sequence of representative construct: fusion T6

(b) Addition of FRAP-FKBP mutant insert to pA7Wp75-Fas _S E and pA7Wp75-Fas _L E

P75TFas _SorL E was generated by subcloning T1 to T2 into pA7Wp75-Fas _S E and pA7Wp75-Fas _L E linearized with XbaI as XbaI-SpeI fragments. P75TFas _SorL TE was generated by subcloning into pA7Wp75-Fas _L E linearized with SpeI. The configuration is shown in Table 1 ((d)).

(c) FRAP-Fas-FKBP Configuration

Instead of forming the mutagenic fragments T1-T12, a single chain may be required to orient the domains differently for optimal activity. FKBP and FRB made up another series of constructs separated by Fas fragments. The starting point for this configuration is pCMF1HA, pCMF2HA and pCMF3HA. Similar to the same strategy as in the construction of mutant transcription factors, FKBP and FRB fragments were cloned into pCM as XbaI-BamHI fragments with SpeI sites immediately upstream of the BamHI site. As XbaI and SpeI produce complementary ends, additional XbaI-BamHI fragments may be inserted downstream of the initial insert. Incidentally, cloning of the XbaI-SpeI fragment adds the fragment at the 5 'end of the construct. The final p75-anchored construct was prepared by subcloning the XbaI-SpeI fragment shown in Table 1 (d) into pA7Wp75-Fas _S E. Similar moorings were made by subcloning into pA7Wp75-Fas _L E. Insertion into the vector cleaves XbaI due to the addition of Insert 3 'to the Fas fragment. Insertion into the vector cleaves SpeI due to the addition of insert 3 'to the Fas fragment. Insertion into the vector cleaves XbaI and SpeI due to addition of Insert 3 'to the p75 fragment and removal of the original Fas fragment in the vector. Using these three subcloning strategies, the following series of constructs were created. Subscript numbers indicate the number of times a domain is repeated.

(d) Table 1

(e) the ends of the fragments used (FKBP and FRB fragments are shown throughout this specification)

i. p75 extracellular and transmembrane fragments

ii. Intracellular domain of Fas:

(f) oligos used

For constructs that use myristoylation sequences to immobilize proteins to the membrane, use pCM (described herein) and clone the insert at 3 ′ to the myristoylation signal sequence as an XbaI-BamHI or XbaI-SpeI fragment. I was.

(g) Rapamycin-regulated apoptosis of stable transfected human HT1080 cells in culture

Cloning XbaI-BamHI from constructs A30 and A31 (d, Table 1) into pCM to represent MT5FasSE and MT6FasSE (where M represents the myristoylation domain (see Examples section A.1 and A.8. Above). The remaining abbreviations, d, described in Table 1) resulted in M30 and M31 constructs for expression. EcoRI-BamHI fragments containing these expression cassettes were cloned into retroviral factor pSMTN3 (Example 1). The helper-free retrovirus containing the DNA was constructed using 293T cells (Pear, WS et al., 1993. Proc. Natl. Acad. Sci. USA, 90, 8392-8396) by construct and by Psi (-) amphoteric packaging factor. ) By transient co-transfection. HT1080 cells were infected with viral stock and selected with G418.

To assay apoptosis of stably transfected pools of cells in response to rapamycin, cells were plated in 96-well culture plates at 10000 cells / well. After balsamin incubation, a series of dilutions of rapamycin were added with 50 ng / ml (final) antinomycin D and continued incubation at 37 ° C. and 5% CO 2 for about 20 hours. The medium was removed and replaced with 100 μl of medium containing 10% Alamar Blue Dye. Plates were incubated as above and cell viability was assessed periodically by spectroscopic measurement of OD at 570 nm and 600 nm on a microtiter plate reader. Typically, reading was continued until control (untreated) wells were at OD 0.2-0.4 after deletion of blank.

FIG. 13 shows that the survival of cells stably transfected with (a) M30 and (b) M31 expression constructs can be reduced in the presence of rapamycin in a dose dependent manner. As demonstrated from FIG. 13 (c), the degree of cell death expressed myristoylated (FKBP × 2) -Fas construct (described in PCT / US94 / 08008) treated with synthetic FKBP homodimerizer AP1428. Comparable to the degree of cell death that causes

AP1428 is compound 49 described in PCT / US95 / 10559. It should be noted that rapamycin has a minimal effect on the survival of cells that do not express mixed chimeric proteins.

Example 9: Rapamycin Dependent DNA Binding

The ability to modulate transcription factor function at the level of DNA binding is another approach to small molecule regulation of therapeutic gene expression. One approach for generating transcription factors that are dependent on rapamycin for DNA binding utilizes that many transcription factors bind their cognate sequences as dimers. The DNA binding domains of such transcription factors can often be subdivided into regions involved in direct contact of DNA, and regions involved in mediating dimer production. As such, by replacing the native transcription factor dimer and domain with FKBP or FRAP, the chimeric transcription factor exhibits rapamycin dependent DNA binding.

Any human dimer transcription factor can follow this approach. Examples include HNF-1, SRF or other MADS box containing proteins, any member of the helix-loop-helix transcription factor family, eg MyoD (myogenin), any member of the bZIP transcription factor family, eg FOS , JUN, ATF, CREB, etc.

In order to prevent unwanted activation of the therapeutic gene, the DNA binding specificity of the chimeric electronic factor can be treated differently from the specificity of the mother protein. This can be accomplished by the reaction modification of the site space of the palindromic target DNA sequence so that the rapamycin mediated chimeric protein binds but the naturally occurring parent dimer cannot bind.

In vitro rapamycin dependent DNA binding was demonstrated by derivatives of the Valteria Refresher Protein LexA.

LexA binds to its target operator as a dimer and includes an amino terminal domain that interacts directly with DNA and a carboxy terminal domain that mediates dimerization. A chimeric protein is constructed wherein the LexA dimerization domain has been replaced with FKBP or FRAP. FIG. 14 shows gel shift shift assays of binding reactions containing approximately equal amounts of Lex-FKBP and Lex-FRAP incubated with DNA containing Lex binding sites. In the absence of rapamycin, no specific binding can be detected (FIG. 14, lane 1). Inclusion of increasing amounts of rapamycin in the binding reaction results in a contaminated appearance of specific complexes in the mobile shift gel (FIG. 14, lanes 2-4). This result is supported by the finding that rapamycin promotes the production of Lex-FKBP / Lex-FRAP heterodimers that can bind to DNA containing LexA target sequences.

Figure 14.

Rapamycin dependent DNA binding by Lex-FKBP / Lex-FRAP heterodimer Lex-FKBP and Lex-FRAP proteins was translated in vitro and mixed together in a binding reaction with radiolabeled LexCon oligonucleotides. Rapamycin is involved in binding reactions at the concentrations shown (lanes 1-4).

The arrow points to the location of the rapamycin dependent DNA complex. The sequences of LexCon probes are described below and the LexA binding sites are underlined.

Plasmid construct

Plasmids p19BL87, p19BL87G6FKBP and p19BL87FRB were constructed among the modified pET19BHA, pET-19B RLWO factors so that all expressed proteins contained amino-terminal His. The tag is followed by the hematoglutin epitope tag.

p19BL87 encodes the Lex DNA binding domain (aa 1-87). Lex encoding sequences are amplified by PCR from pCGNNLex202 with primers LexA Xba and LexA87Spe / Bam. The PCR product is cleaved by XbaI and BamHI and ligated between the XbaI and BamHI sites of p19BHA.

p19BL87G6FKBP encodes LexA (aa 1-87) fused to FKBP (aa 2-108) in the backbone via six glycine soft linkers. The FKBP coding sequence is amplified by PCR from pCGNNF1 by primers 5'XG6FKBP and FKBP 3 'Spe / Bam. pCGNNZFHD1-FKBPx3 (ATCC Accession No. 97399) can also be used as a source for FKBP encoded DNA (USSN 08 / 581,713, filed Dec. 29, 1995). The PCR product is cleaved by XbaI and BamHI and ligated between the SpeI and BamHI sites of p19BL87.

p19BL87FRB encodes LexA (aa 1-87) fused to FRAP (aa 2025-2113) in the backbone. The FRAP coding sequence is isolated as an XbaI-BamHI fragment from pCGNN-GAL4-1FRB and ligated between the SpeI and BamHI sites of p19BL87.

PCR primers

Binding reaction

p19BL87G6FKBP and p19BL87FRB plasmids were translated ex vivo using a TNT coupled reticulosite system (Promega) according to the manufacturer's instructions.

The binding reaction was 15 ml of binding buffer (10 mM Tris pH 7.5 mM DDT, 0.1 mM EDTA, 10% v / v glycerol, 5 mM MgCl 2, 60 mM NaCl), 50 mg / ml bovine serum albumin (NED); 200 ng poly (dIdC) from Pharmacia; 1 ml of each L87G6FKBP and L87FRB programmed reticulocyte lysate and 0.2 ng g-32P-dATP labeled LexCon probe (total volume: 20 ml). Rapamycin is added at a suitable concentration and the reaction is incubated for 30 minutes at room temperature.

Gel mobility shift assay

The complex is dissolved in a 4% 40: 1 crosslinked polyacrylamide gel in 0.5X TBE buffer, treated at 10-15 V / cm for 2 hours.

Summary of the Invention

The present invention relates to forms for biological switches, and in particular to novel methods and materials for controlling biological events in animal cells. Such biological events include, for example, activation of intracellular signal transduction pathways leading to gene transcription, gene expression or apoptotic cell death, gene knockout, gene expression blockade, and inhibition of gene production. The present invention relates to two types of chimeric proteins, which can be activated either directly or indirectly as a preferred event when complexed via mutual binding to a common ligand.

The present invention provides a recombinant DNA construct encoding a chimeric protein; A DNA vector containing one or more recombinant DNA constructs; Fusion proteins encoded by the construct; Cells, particularly animal cells, transformed (ie, containing and capable of expressing) by one or more of said DNA constructs; Small molecules (bivalent or multivalent multiplexing agents) capable of multiplexing and binding to chimeric protein molecules; And methods of making and using those described above.

More particularly, the present invention provides methods and materials for preparing and using genetically engineered cells that respond to the presence of rapamycin of the present invention or to the presence of analogs, pseudo or derivatives of the rapamycin (rapalog). . The present invention encompasses the introduction of recombinant DNA into cells which encode sets of fusion proteins that can be complexed with one another in the presence of rapamycin or rapalog. Contacting these genetically engineered cells with rapamycin or a suitable rapalog complexes the fusion protein and initiates a biological response. One of these fusion proteins contains one or more replicas of FKBP: rapamycin binding (FRB) domains and one or more heterologous protein domains. The second fusion protein contains one or more copies of a domain derived from FKBP protein capable of binding to rapamycin or rapalog and complexing with the FRB containing protein. Such a second fusion protein also contains one or more heterologous domains which may be the same or different from the heterologous domain of the first fusion protein. The FRB and FKBP domains used in the fusion proteins of the invention can be selected from natural proteins and can be variously modified as described below. FRB, FKBP and heterologous domains derived from various species can be used, but human peptide sequences or variants thereof are preferred for human gene therapy. In operation, the FRB and FRBP domains act as receptor (ligand binding) domains and induce the complexation of fusion proteins under the mediation of rapamycin or raphalog molecules. The biological response properties of rapamycin or rapalog mediated complexation are measured by the heterologous domain of the fusion protein. The heterologous domain is thus called the functional domain.

Various heterologous protein domains can be used for such fusion proteins. In one aspect of the invention, two fusion proteins, one of which contains one or more FRB domains and the other containing one or more FKBP domains, each of one or more different heterologous domains, i.e. It contains a heterologous domain that does not contain an outer fusion protein. For example, in certain embodiments, one fusion protein contains one or more DNA binding domains and the other fusion protein contains a transcriptional activation domain. Ligand mediated relevance of the fusion protein indicates the formation of a transcription factor complex and initiates the transcription of a target gene bound to a DNA sequence recognized (ie capable of binding) by a DNA-binding domain on one fusion protein. In one embodiment, one of the fusion proteins contains one or more domains capable of directing the fusion protein to a particular cellular region, such as a cell membrane or a nucleus. Localization domains that target cell membranes include domains such as myristoylation sites or transmembrane regions of receptor proteins or membrane spanning proteins. Other fusion proteins contain a signaling domain capable of activating cell signaling transduction pathways in membrane localization and / or clustering. Examples of signaling domains include apoptotic initiation domains, such as the intracellular domains of growth factors or cytokine receptors, the intracellular domains of FAS or TNFR1, and other signaling proteins derived from SOS, Raf, lck, ZAP-70, and the like. Domain is included. Many signaling proteins are disclosed in PCT / US94 / 01617 (Reference Examples 23-26). In another embodiment of the invention, each fusion protein contains one or more FRB domains and one or more FRKB domains as well as one or more heterologous domains. Such fusion proteins may be single dimerized in the presence of rapamycin or rapalog. Generally, domains containing peptide sequences of host cell endogenous are preferred. Thus, human domains are particularly preferred for human gene therapy.

Also provided are recombinant DNA molecules encoding fusion proteins, which are vectors that can induce expression, particularly in eukaryotic cells. Of these cells, yeast and animal cells are particularly preferred. In view of the components of a fusion protein, a recombinant DNA molecule encoding a fusion protein is a DNA molecule and a heterologous or heterologous protein encoding a ligand binding domain (FRB domain or FKBP domain) of a given fusion protein or a protein containing such domain. The domain may be selectively hybridized to a DNA molecule encoding a protein from which it is derived. Also included in the present invention are DNAs that can hybridize without denaturing the genetic code.

Using DNA sequences encoding chimeric proteins of the invention and vectors capable of inducing their expression in eukaryotic cells, genetically engineered cells can be obtained that can be used in a variety of ways. To do so, it forms an expression vector or DNA construct that induces expression in the eukaryotic cell (preferably an animal) of the desired chimeric protein, and then introduces the recombinant DNA into the cell in such a way that the DNA is taken up and at least in part of the cell. Express the DNA. Various methods and materials for introducing DNA into cells to express heterologous genes can be used, and such methods and materials are known and commercially available materials.

It is therefore an object of the present invention to provide animal cells containing recombinant DNA encoding the two fusion proteins described above. One such fusion protein is capable of binding rapamycin or rapalog and contains one or more FKBP domains and one or more domains heterologous thereto. The second fusion protein contains one or more FRB domains and one or more domains heterologous thereto and form a three part complex with the first fusion protein and one or two rapamycins or raphalogs. In some embodiments, one or more heterologous domains present in one fusion protein are also present on another fusion protein. That is, the two fusion proteins have one or more common heterologous domains. In other embodiments, each fusion protein contains one or more different heterologous domains.

It is a particular object of the present invention to provide animal cells engineered to transcribe target genes when the cells are contacted with rapamycin or raphalog. Such cells contain a target gene construct comprising a target gene operably linked to a DNA sequence in addition to the recombinant DNA encoding the two fusion proteins. The DNA sequence is reactive to the presence of a complex of a fusion protein with rapamycin or raphalog. In certain embodiments, the cell is responsive to contact of the FKBP fusion protein with a rapalog that binds to the FRB fusion protein that is detectable for binding to the endogenous FKBP or FRB containing protein of the host cell.

Another object of the present invention is to provide an animal cell that has been engineered to initiate cell death when the cell is contacted with rapamycin or raphalog. In such cells, one or more heterologous domains on one or more fusion proteins are domains such as the intracellular domain of FAS or TNF-R1 that, when clustered, initiate apoptosis of the cell.

Another object of the present invention is to provide animal cells engineered to stimulate the growth, differentiation, or proliferation of cells when the cells are contacted with rapamycin or raphalog. In such cells, one or more heterologous domains on one or more fusion proteins are domains such as the intracellular domain of a receptor for hormones that mediate cell growth, differentiation or proliferation. Cell growth, differentiation and / or proliferation clusters the receptor intracellular signaling domains. Such clustering occurs naturally by hormonal binding and occurs in engineered cells of the invention following contact with rapamycin or rapalog.

Various cell types (human or otherwise) can be used and encapsulated in biocompatible materials for use in humans, if desired, but human cells are preferred for human gene therapy.

It is still another object of the present invention to provide a material and a method for producing a cell which is engineered as described above. This object can be achieved by providing recombinant DNA encoding the fusion protein with any auxiliary recombinant DNA, such as a target gene construct, and introducing the recombinant DNA into a host cell under conditions where the DNA is taken up by the cell. Such transfection can be performed by extracellular methods using host cells maintained in culture. Cells engineered in culture can be introduced into the host, for example in gene therapy in vivo. Doing so constitutes a method of providing a host, preferably a human or non-human mammal, which responds (as described above) to the presence of rapamycin or rapalog. In addition, transfection can be performed in vivo using host cells present in human or non-human hosts. In such cases, the DNA molecule is introduced directly into the host under conditions where the DNA is taken up by one or more host cells. This method is another method of providing a host, preferably a human or non-human mammal, which responds (as described above) to the presence of rapamycin or rapalog. Various materials and methods for introducing DNA into cultures or into cells in whole organisms are known in the art and can be engineered to be used to carry out the present invention.

Another object is achieved using the cells engineered as above. For example, one method is to multiplex the fusion protein of the invention by contacting the engineered cells as described above with an effective amount of rapamycin or raphalog such that the rapamycin or rapalogue complexes with the fusion protein. . In embodiments in which multiplexing of the fusion protein initiates transcription of the target gene, a method of activating a target gene is constructed. In embodiments in which the fusion protein contains one or more signaling domains, a method of activating a cellular signal transduction pathway is constructed. In embodiments in which the fusion protein contains one or more clusterable domains that initiate apoptotic cell death, a method of promoting cell death is constructed. Such methods can be performed in whole organisms, including cell cultures or human patients. When performed in cell culture, rapamycin or raphalog is added to the culture medium. When performed in whole organisms, rapamycin or rapalog (which may be a pharmaceutical or veterinary composition) is administered to the whole organism, for example orally or parenterally. Preferably, the dose of rapamycin or rapalog administered to the animal is below the dose level at which unwanted immunosuppression can be induced in the doser.

It is still another object of the present invention to provide a kit for use in manipulating genes of cells or human or non-human animals as described above. Such kits contain a recombinant DNA construct that encodes a pair of fusion proteins of the invention. Recombinant DNA constructs may be in the form of eukaryotic expression vectors suitable for introduction into animal cells and capable of inducing expression of fusion proteins in cells. Such kits may also contain a sample of rapamycin or rapalog that can form a complex with the encoded fusion protein. Such a kison may also contain multiplexing antagonists such as FK506 or any other compound that can bind to one of the fusion proteins but cannot form a complex with both. In certain embodiments, the recombinant DNA construct encoding the fusion protein contains a cloning site instead of the DNA encoding one or more heterologous domains, allowing the user to introduce DNA encoding the heterologous domain. In some embodiments, the kit may also contain a target gene construct containing a target gene or cloning site bound to a DNA sequence responsive to the presence of a complexed fusion protein as described in detail below.

Claims (45)

An animal cell containing two or more recombinant DNAs, The first recombinant DNA is capable of binding rapamycin or an analog thereof, and encodes a first chimeric protein comprising at least one rapamycin binding domain and at least one protein domain heterologous thereto, wherein the rapamycin binding The domain comprises (i) a naturally occurring FKBP or portion thereof, (ii) a variant of naturally occurring FKBP which is deleted, inserted, or substituted with amino acid substituents of up to 10 amino acid residues, and (iii) a substance. a peptide sequence selected from the group consisting of FKBPs encoded by DNA sequences that can optionally hybridize to the DNA sequences encoding FKBPs of (i) or (ii); The second recombinant DNA may form a complex with a rapamycin or rapamycin analogue and a first chimeric protein and encode a second chimeric protein comprising at least one FRB domain and at least one domain heterologous thereto, Wherein the FRB domain is (iV) a naturally occurring FRB domain, (V) a variant of the naturally occurring FRB in which up to 10 amino acid residues are deleted, inserted, or substituted with amino acid substituents, and (Vi) substances An animal cell comprising a peptide sequence selected from the group consisting of a FRB domain encoded by a DNA sequence that can optionally hybridize to a DNA sequence encoding (iV) or (V) the FRB. The method of claim 1, wherein the first chimeric protein comprises: a peptide sequence of human FKBP12; Variants of the sequence containing up to 5 amino acid substituents; Or an animal cell comprising a peptide sequence selected from peptide sequences encoded by a DNA sequence that can optionally hybridize to a DNA sequence encoding human FKBP12. 3. The FKBP domain of claim 2, wherein the first chimeric protein comprises one or more amino acid residues corresponding to Tyr26, Phe36, Asp37, Arg42, Phe46, Phe48, Glu54, Val55, and Phe99 of human FKBP12, or substituted with an amino acid substituent. Containing animal cells. The method of claim 1, wherein the second chimeric cells comprise Glu-2025 to Lys-2113 of human or murine FRAP; Glu-1962 to Arg-2050 of Tor1 (S. cerevisiae); Peptide sequences Glu-1962 to Arg-2050 of Tor1 (S. cerevisiae); Glu-1965 to Lys-2053 from Tor2 (S. cerevisiae); A variant of any one of the above sequences containing up to 10 amino acid substituents, deletions or insertions; Or an animal cell comprising a peptide sequence selected from the group consisting of peptide sequences encoded by DNA that can optionally hybridize to a DNA sequence encoding any of the peptide sequences. The animal cell of claim 4, wherein the second chimeric cell comprises a FRB domain wherein one or more amino acid residues corresponding to Tyr2038, Phe2039, Thr2098, Gln2099, TrP2101 and Asp2102 of a human FRAP are deleted or substituted with amino acid substituents. The animal cell of claim 1, wherein at least one of the chimeric proteins contains a membrane localization domain. The animal cell of claim 1, wherein at least one of the chimeric proteins contains a heterologous domain comprising a human peptide sequence. The animal cell of claim 1, wherein the first chimeric protein contains two or more FKBP domains. The animal cell of claim 1, wherein the second chimeric protein contains two or more FRB domains. The method of claim 1, wherein one of the chimeric proteins contains at least one DNA binding domain, the remaining chimeric protein contains at least one transcriptional activity and domain, and the cell acts on a DNA sequence capable of binding a DNA-binding domain. An animal cell further containing a target gene that is coupled to its target. The animal cell of claim 1, wherein at least one of the chimeric proteins comprises a heterologous domain comprising a heterologous domain capable of initiating intracellular signal stimulating cell growth, differentiation, proliferation, apoptosis or gene expression upon multiple binding. The animal cell of claim 11, wherein the heterologous domain comprises a peptide sequence derived from an intracellular domain of FAS or TNF-R1 or a fragment thereof. The animal cell of claim 1, wherein the two chimeric proteins each contain one or more common heterologous domains. The animal cell of claim 13, wherein the two chimeric proteins each contain a domain capable of initiating intracellular signal stimulating cell growth, differentiation, proliferation, apoptosis or gene expression upon multiple binding. The animal cell according to any one of claims 11 to 14, wherein the two chimeric proteins are identical and stimulate intracellular signals upon multiple binding. The animal cell according to any one of claims 1 to 15, which is derived from a human. The animal cell of claim 1, encapsulated in a biocompatible material. Non-human cell containing an animal cell according to any one of claims 1 to 17. 19. A method of producing an animal cell according to any one of claims 1 to 18, comprising introducing into a host animal cell at least one DNA molecule encoding a chimeric protein under conditions providing DNA uptake by the cell. 20. The method of claim 19, further comprising introducing a DNA gene encoding a target gene bound to DNA binding into the cell, thereby transcribing the target gene in response to binding of the chimeric protein. An animal cell according to any one of claims 1 to 16 comprising introducing into the animal body at least one DNA molecule encoding a chimeric protein under conditions providing uptake of the DNA by at least one animal cell. How to generate. 18. A method of producing an animal containing a cell according to any one of claims 1 to 17, comprising introducing a cell according to any one of claims 1 to 17 into an animal body. 18. A method of multiplexing a chimeric protein in a cell according to any one of claims 1 to 17, which comprises contacting the cell with an effective amount of mapamycin or a suitable rapamycin analog, under conditions capable of producing a complex with the chimeric protein. How to combine. The method of claim 23, wherein the cell is contacted with a compound different from rapamycin of the formula: Where U is -H, -OR1, -SR1, -OC (O) R1, -OC (O) NHR1, -NHR1, -NHC (O) R1, -NH-SO2-R1 or -NH-SO2-R2 (here R 2 is substituted aryl, allyl or alkylaryl, V is -OR3 or (= O), W is = O, = NR4, = NOR4, = NNHR4, -NHOR4, -NHNR4, -OR4, -OC (O) R4, -OC (O) NR4 or -H, Y is -OR5, -O (O) R5 or -OC (O) NHR5, Z is = O, -OR6, -NR6, -H, -NC (O) R6, -OC (O) R6 or -OC (O) NR6, R3 is H, -R7, -C (O) NHR7 or C-28 / C-30 cyclic carbonate, R 4 is H or alkyl, Wherein R 1, R 4, R 5, R 6 and R 7 are independently selected from the group consisting of H, alkyl, alkylaryl or aryl. A method of activating the transcription of a target gene in a cell according to any one of claims 10, 11 or 14 containing the target gene construct responsive to dimerization of chimeric proteins, the cell providing gene expression. A method comprising contacting rapamycin or a rapamycin analog capable of forming a complex with a chimeric protein in an amount effective below. The method of claim 25, wherein the cell is contacted with a compound different from rapamycin of the formula: Where U is -H, -OR1, -SR1, -OC (O) R1, -OC (O) NHR1, -NHR1, -NHC (O) R1, -NH-SO2-R1 or -NH-SO2-R2 (here R 2 is substituted aryl, allyl or alkylaryl, V is -OR3 or (= O), W is = O, = NR4, = NOR4, = NNHR4, -NHOR4, -NHNR4, -OR4, -OC (O) R4, -OC (O) NR4 or -H, Y is -OR5, -O (O) R5 or -OC (O) NHR5, Z is = O, -OR6, -NR6, -H, -NC (O) R6, -OC (O) R6 or -OC (O) NR6, R3 is H, -R7, -C (O) NHR7 or C-28 / C-30 cyclic carbonate, R 4 is H or alkyl, Wherein R 1, R 4, R 5, R 6 and R 7 are independently selected from the group consisting of H, alkyl, alkylaryl or aryl. A signal transduction in a cell according to any one of claims 11 to 14, comprising contacting the cell with a rapamycin or rapamycin analog capable of complexing with the chimeric protein in an amount effective to form the complex. Method for acting on the pathway. The method of claim 27, wherein the cell is contacted with a compound different from rapamycin of the formula: Where U is -H, -OR1, -SR1, -OC (O) R1, -OC (O) NHR1, -NHR1, -NHC (O) R1, -NH-SO2-R1 or -NH-SO2-R2 (here R 2 is substituted aryl, allyl or alkylaryl, V is -OR3 or (= O), W is = O, = NR4, = NOR4, = NNHR4, -NHOR4, -NHNR4, -OR4, -OC (O) R4, -OC (O) NR4 or -H, Y is -OR5, -O (O) R5 or -OC (O) NHR5, Z is = O, -OR6, -NR6, -H, -NC (O) R6, -OC (O) R6 or -OC (O) NR6, R3 is H, -R7, -C (O) NHR7 or C-28 / C-30 cyclic carbonate, R 4 is H or alkyl, Wherein R 1, R 4, R 5, R 6 and R 7 are independently selected from the group consisting of H, alkyl, alkylaryl or aryl. Performing cell death in cells according to any one of claims 11, 12, 14 and 15 containing one or more chimeric proteins containing heterologous domains that can stimulate apoptosis upon multiple binding A method comprising: contacting a cell with rapamycin or a rapamycin analog capable of forming a complex with a chimeric protein in an amount effective to form a complex with the chimeric protein. 30. The method of any one of claims 23-29, wherein the cells are grown in the incubator and the contacting is performed by adding rapamycin or rapamycin analog to the incubator. The method of any one of claims 23-29, wherein the cells are in the host organism and the contacting is performed by administering rapamycin or rapamycin analog to the host organism. 32. The method of claim 31 wherein the host organism is a mammal and the rapamycin type compound is administered by oral, buccal, sublingual, transdermal, subcutaneous, intramuscular, intravenous, intraarticular or inhaled administration in a suitable pharmaceutical or veterinary excipient thereto. How to be. A method of providing an animal cell that is reactive to rapamycin or a rapamycin analog, including introducing two or more recombinant DNAs into a host animal cell, The first recombinant DNA is capable of binding rapamycin or an analog thereof, and encodes a first chimeric protein comprising at least one rapamycin binding domain and at least one protein domain heterologous thereto, wherein the rapamycin binding The domain comprises (i) a naturally occurring FKBP or portion thereof, (ii) a variant of naturally occurring FKBP which is deleted, inserted, or substituted with amino acid substituents of up to 10 amino acid residues, and (iii) a substance. a peptide sequence selected from the group consisting of FKBPs encoded by DNA sequences that can optionally hybridize to the DNA sequences encoding FKBPs of (i) or (ii); The second recombinant DNA may form a complex with a rapamycin or rapamycin analogue and a first chimeric protein and encode a second chimeric protein comprising at least one FRB domain and at least one domain heterologous thereto, Wherein the FRB domain is (iV) a naturally occurring FRB domain, (V) a variant of a naturally occurring FRB in which up to 10 amino acid residues are deleted, inserted, or substituted with amino acid substituents, and (iii) substances A peptide comprising a peptide sequence selected from the group consisting of FRB domains encoded by a DNA sequence that can optionally hybridize to a DNA sequence encoding (iV) or (V) the FRB. A method of providing a mammal that is responsive to rapamycin or a rapamycin analog, comprising introducing two or more recombinant DNAs into a host mammal under conditions that provide for transfection of one or more cells of the host animal. The first recombinant DNA is capable of binding rapamycin or an analog thereof, and encodes a first chimeric protein comprising at least one rapamycin binding domain and at least one protein domain heterologous thereto, wherein the rapamycin binding The domain comprises (i) a naturally occurring FKBP or portion thereof, (ii) a variant of naturally occurring FKBP which is deleted, inserted, or substituted with amino acid substituents of up to 10 amino acid residues, and (iii) a substance. a peptide sequence selected from the group consisting of FKBPs encoded by DNA sequences that can optionally hybridize to the DNA sequences encoding FKBPs of (i) or (ii); The second recombinant DNA may form a complex with a rapamycin or rapamycin analogue and a first chimeric protein and encode a second chimeric protein comprising at least one FRB domain and at least one domain heterologous thereto, Wherein the FRB domain is (iv) a naturally occurring FRB domain, (v) a variant of a naturally occurring FRB in which up to 10 amino acid residues are deleted, inserted, or substituted with amino acid substituents, and (vi) substances or (v) a peptide sequence selected from the group consisting of FRB domains encoded by a DNA sequence that can optionally hybridize to a DNA sequence encoding a FRB of (v). A method of providing a mammal responsive to rapamycin or a rapamycin analog comprising introducing a cell containing two or more recombinant DNAs into a host mammal, The first recombinant DNA is capable of binding rapamycin or an analog thereof, and encodes a first chimeric protein comprising at least one rapamycin binding domain and at least one protein domain heterologous thereto, wherein the rapamycin binding The domain comprises (i) a naturally occurring FKBP or portion thereof, (ii) a variant of naturally occurring FKBP which is deleted, inserted, or substituted with amino acid substituents of up to 10 amino acid residues, and (iii) a substance. a peptide sequence selected from the group consisting of FKBPs encoded by DNA sequences that can optionally hybridize to the DNA sequences encoding FKBPs of (i) or (ii); The second recombinant DNA may form a complex with a rapamycin or rapamycin analogue and a first chimeric protein and encode a second chimeric protein comprising at least one FRB domain and at least one domain heterologous thereto, Wherein the FRB domain is (iv) a naturally occurring FRB domain, (v) a variant of a naturally occurring FRB in which up to 10 amino acid residues are deleted, inserted, or substituted with amino acid substituents, and (vi) substances or (v) a peptide sequence selected from the group consisting of FRB domains encoded by a DNA sequence that can optionally hybridize to a DNA sequence encoding a FRB of (v). A kit comprising a protein capable of binding rapamycin or an analog thereof and comprising at least one rapamycin binding domain and at least one protein domain heterologous thereto, Rapamycin binding domains include (i) naturally occurring FKBPs or portions thereof, (ii) variants of naturally occurring FKBPs having up to 10 amino acid residues deleted, inserted, or substituted with amino acid substituents, and ( iii) a peptide sequence selected from the group consisting of FKBP encoded by a DNA sequence that can optionally hybridize to a DNA sequence encoding a FKBP of substance (i) or (ii); The second recombinant DNA may form a complex with a rapamycin or rapamycin analogue and a first chimeric protein and encode a second chimeric protein comprising at least one FRB domain and at least one domain heterologous thereto, Wherein the FRB domain is (iv) a naturally occurring FRB domain, (v) a variant of naturally occurring FRB in which up to 10 amino acid residues are deleted, inserted, or substituted with amino acid substituents, and (iv) substances A kit comprising a peptide sequence selected from the group consisting of FRB domains encoded by a DNA sequence that can optionally hybridize to a DNA sequence encoding a FRB of (v) or (vi). The kit of claim 36, further comprising a rapamycin or rapamycin analog capable of forming a complex with the chimeric protein. The kit of claim 36, further comprising multiple binding antagonists. 37. The kit of claim 36, further comprising at least one target gene construct containing a target gene bound to a chimeric protein and a transcriptional regulatory element responsive to the complex formed by the rapamycin or rapamycin analog. 36. The kit of claim 35, wherein the one or more DNA constructs contain cloning elements instead of heterologous domains. The kit of claim 36, wherein the target gene construct contains a cloning site in place of the target gene. A recombinant DNA construct encoding a fusion protein containing at least one FRB domain and at least one domain containing a peptide sequence selected from the peptide sequences of the FAS. A recombinant DNA construct that encodes a fusion protein containing two or more FRB domains and one or more domains heterologous thereto. A recombinant DNA construct encoding a fusion protein containing at least one FRB domain and a cell localization domain. A recombinant DNA construct encoding a fusion protein containing at least one FRB domain that, together with a FKBP domain containing protein, preferentially forms a tricomponent complex with rapamycin analogs.