Note: Descriptions are shown in the official language in which they were submitted.
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REGULATION OF BIOLOGICAL EVENTS USING MULTIMERIC CHIMERIC PROTEINS
Background of the Invention
Rapamycin is a macrolide antibiotic produced by Streptomyces hygroscopicus
which
binds to a FK506-binding protein, FKBP, with high affinity to form a
rapamycin:FKBP
complex. Reported Kd values for that interaction are as low as 200 pM. The
rapamycin:FKBP complex binds with high affinity to the large cellular protein,
FRAP, to
form a tripartite, [FKBP:rapamycin]:[F$~], complex. In that complex rapamycin
acts as a
dimerizer or adapter to join FKBP to FRAP.
A number of naturally occurring FK506 binding proteins (FKBPs) are known. See
e.g. Kay,
1996, Biochem. J. 314:361-385 (review). FKBP-derived domains have been
incorporated in
the design of chimeric proteins for use in biological switches in genetically
engineered cells.
Such switches rely upon ligand-mediated multimerization of the protein
components to
trigger a desired biological event. See e.g. Spencer et al, 1993, Science
262:1019-1024 and
PCT/US94/01617. While the potent immunosuppressive activity of FK506 would
limit its
utility as a multimerizing agent, especially in animals, dimers of FK506 (and
related
compounds) can be made which lack such immunosuppressive activity. Such dimers
have
been shown to be effective for multimerizing chimeric proteins containing FKBP-
derived
ligand binding domains.
Rapamycin, like FK506, is also capable of multimerizing appropriately designed
chimeric proteins. We have previously designed biological switches using
rapamycin and
various derivatives or analogs thereof ("rapalogs") as multimerizing agents
(see
W096/41865). In the case of rapamycin itself, its significant biological
activities, including
potent immunosuppressive activity, rather severely limit its use in biological
switches in
certain applications, especially those in animals or animal cells which are
sensitive to
rapamycin. Improved rapalogs for such applications, especially rapalogs with
reduced
immunosuppressive activity, would be very desirable.
1
Rapamycin
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A large number of structural variants of rapamycin have been reported,
typically
arising as alternative fermentation products or from synthetic efforts to
improve the
compound's therapeutic index as an immunosuppressive agent. For example, the
extensive
literature on analogs, homologs, derivatives and other compounds related
structurally to
rapamycin ("rapalogs") include, among others, variants of rapamycin having one
or more of
the following modifications relative to rapamycin: demethylation, elimination
or
replacement of the methoxy at C7, C42 and/or C29; elimination, derivatization
or
replacement of the hydroxy at C13, C43 and/or C28; reduction, elimination or
derivatization
of the ketone at C14, C24 and/or C30; replacement of the 6-membered pipecolate
ring with a
5-membered prolyl ring; and alternative substitution on the cyclohexyl ring or
replacement
of the cyclohexyl ring with a substituted cyclopentyl ring. Additional
historical
information is presented in the background sections of US Patent Nos.
5,525,610; 5,310,903
and 5,362,718.
US Patent No. 5,527,907 is illustrative of the patent literature. That
document discloses
a series of compounds which were synthesized in an effort to make
immunosuppressive
rapalogs with reduced side effects. The compounds are disclosed via seven
generic structural
formulas, each followed by extensive lists (two to five or more columns of
text each) setting
forth possible substituents at various positions on the rapamycin ring. The
document includes
over 180 synthetic examples. The many structural variants of that invention
were reported
to be potent immunosuppressive agents.
Summary of the Invention
This invention provides methods and materials for multimerizing chimeric
proteins in
genetically engineered cells using improved rapalogs, preferably while
avoiding the
immunosuppressive effects of rapamycin.
The genetically engineered cells contain one or more recombinant nucleic acid
constructs
encoding specialized chimeric proteins as described herein. Typically a first
chimeric
protein contains one or more FKBP domains which are capable of binding to an
improved
rapalog of this invention. This first chimeric protein is also referred to
herein as an "FKBP
fusion protein" and further comprises at least one protein domain heterologous
to at least one
of its FKBP domains. The complex formed by the binding of the FKBP fusion
protein to the
rapalog is capable of binding to a second chimeric protein which contains one
or more FRB
domains (the "FRB fusion protein"). The FRB fusion protein further comprises
at least one
protein domain heterologous to at least one of its FRB domains. In some
embodiments, the
FKBP fusion protein and the FRB fusion protein are different from one another.
In other
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embodiments, however, the FKBP fusion protein is also an FRB fusion protein.
In those
embodiments, the chimeric protein comprises one or more FKBP domains as well
as one or
more FRB domains. In such cases, the first and second chimeric proteins may be
the same
protein, may be referred to as FKBP-FRB fusion proteins and contain at least
one domain
heterologous to the FKBP and/or FRB domains.
The chimeric proteins may be readily designed, based on incorporation of
appropriately
chosen heterologous domains, such that their multimerization triggers one or
more of a wide
variety of desired biological responses. The nature of the biological response
triggered by
rapalog-mediated complexation is determined by the choice of heterologous
domains in the
fusion proteins. The heterologous domains are therefore referred to as
"action" or "effector"
domains.The genetically engineered cells for use in practicing this invention
will contain one
or more recombinant nucleic acid constructs encoding the chimeric proteins,
and in certain
applications, will further contain one or more accessory nucleic acid
constructs, such as one or
more target gene constructs. Illustrative biological responses, applications
of the system and
types of accessory nucleic acid constructs are discussed in detail below.
A system involving related materials and methods is disclosed in WO 96/41865
(Clackson et al) and is expected to be useful in a variety of applications
including, among
others, research uses and therapeutic applications. That system involves the
use of a
multimerizing agent comprising rapamycin or a rapalog of the generic formula:
wherein U is -H, -OR1, -SR1, -OC(O)R1, -OC(O)NHR1, -NHR1, -NHC(O)R1, -NHS02-R1
or
-R2; R2 is a substituted aryl or allyl or alkylaryl (e.g. benzyl or
substituted benzyl); V is-
OR3 or (=O); W is =O, =NR4 =NOR4, =NNHR4, -NHOR4, -NHNHR4, -OR4, -OC(O)R4,-
OC(O)NR4 or -H; Y is -0R5, -OC(O)R5 or -OC(O)NHRS; Z is =O, -OR6, -NR6, -H;
NC(O)R6, -OC(O)R6 or -OC(O}NR6; R3 is H, -R~, -C(O)RD, -C(O)NHR~ or C-28 / C-
30 cyclic
carbonate; and R4 is H or alkyl; where Rl, R4, R5, R6 and R~ are independently
selected from
H, alkyl, alkylaryl or aryl, as those terms are defined in WO 96/41865. A
number of
rapalogs are specifically disclosed in that document.
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The subject invention is based upon a system similar to that disclosed in WO
96/41865,
but involves the use of improved rapalogs as the multimerizing agents. The
subject invention
thus provides a method for multimerizing chimeric proteins in cells which
comprises (a)
providing appropriately engineered cells containing nucleic acid constructs
for directing the
expression of the desired chimeric proteins) and any desired accessory
recombinant
constructs, and (b) contacting the cells with an improved rapalog or a
pharmaceutically
acceptable derivative thereof as described herein. The rapalog forms a complex
containing
itself and at least two molecules of the chimeric protein(s). Improved
rapalogs for use in this
invention include the following.
One class of improved rapalogs for use in this invention consists of those
compounds
which comprise the substructure shown in Formula I:
n=lor2
bearing any number of a variety of substituents, and optionally unsaturated at
one or more
carbon-carbon bonds unless specified to the contrary herein, which have a
substantially
reduced immunosuppressive effect as compared with rapamycin. By a
"substantially reduced
immunosuppressive effect" we mean that the rapalog has less than 0.1,
preferably less than
0.01, and even more preferably, less than 0.005 times the immunosuppressive
effect observed
or expected with an equimolar amount of rapamycin, as measured either
clinically or in an
appropriate in vitro or in vivo surrogate of human immunosuppressive activity,
preferably
carned out on tissues of lymphoid origin, or alternatively, that the rapalog
yields an EC50
value in such an in vitro assay which is at least ten times, preferably at
least 100 times and
more preferably at least 250 times larger than the EC50 value observed for
rapamycin in the
same assay.
One appropriate in vitro surrogate of immunosuppression in a human patient is
inhibition of human T cell proliferation in vitro. This is a conventional
assay approach that
may be conducted in a number of well known variations using various human T
cells or cells
lines, including among others human PBLs and Jurkat cells. A rapalog may thus
be assayed
for human immunosuppressive activity and compared with rapamycin. A decrease
in
immunosuppressive activity relative to rapamycin measured in an appropriate in
vitro
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assay is predictive of a decrease in immunosuppressive activity in humans,
relative to
rapamycin. Such in vitro assays may be used to evaluate the rapalog's relative
immunosuppressive activity.
A variety of illustrative examples of such rapalogs are disclosed herein. This
class of
improved rapalogs includes, among others, those which bind to human FKBP12, or
inhibit
its rotamase activity, within an order of magnitude of results obtained with
rapamycin in
any conventional FKBP binding or rotamase assay.
Other classes of improved rapalogs for use in this invention are defined with
reference
to the structure shown in Formula II:
O RC24~ ,RC28
Rc~ 4
.r~~z O RC29
Q
wherein
R~z g
R8 I a
a = H3CO'vT ~ a'
H3C ~~~ H C R~
HCO
and,
one of RC~a and RCS is H and the other is -H, halo, -R2, -ORl, -SRl, -OC(O)Rl
or-
OC(O)NHRl, -NHRl, -NR1R2, -NHC(O)Rl, or -NH-S02-Rl where R2 = aliphatic,
heteroaliphatic, aryl, heteroaryl or alkylaryl (e.g. benzyl or substituted
benzyl);
RCS is halo, -0R3 or (=O);
R~4 is =O, =NR4 =NOR4, =NNHR4, -NHOR4, -NHNHR4, -OR4, -OC(O)R4 or
-OC(O)NR4, halo or -H;
RCls ~d RC2s are independently H, halo, -OR3, -ORS, -OC(O)R5, -OC(O)NHRS, -
SRS;
SC(O)R5, -SC(O)NHRS, -NR5R5~or -N(R5)(CO)RS~;
RC14 is =O, -OR6, -NR6, -H, -NC(O)R6, -OC(O)R6 or -OC(O)NR6;
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R3 is H, -R~, -C(O)RD or -C(O)NHR~ or a cyclic moiety (e.g., carbonate)
bridging C28 and
C30; and,
RC29 is H or ORll (e.g., OH or OMe);
where each substituent may be present in either stereochemical orientation
unless otherwise
indicated, and where each occurrence of Rl, R4, R5, R6, R~, R9, Rl~ and Rll is
independently
selected from H, aliphatic, heteroaliphatic, aryl and heteroaryl; and Rs is H,
halo, -CN,
=O, -OH, -NR9R1~ , OSO2CF3, OSO2F, OSO2R4~, OCOR4~, OCONR4~R5~, or
OCON(OR4~)R5~..
Improved rapalogs useful in practicing this invention, including rapalogs of
Formula II,
may contain substituents in any of the possible stereoisomeric orientations,
and may comprise
one stereoisomer substantially free of other stereoisomers (>90%, and
preferably >95%, free
from other stereoisomers on a molar basis) or may comprise a mixture of
stereoisomers.
One class of improved rapalogs for use in this invention which are of
particular interest
are rapalogs of Formula II wherein one or both of RW3 and Rc2s is are
independently H,
halo, -OR3, -ORS, -OC(O)R~, -OC(O)NHRS, -SRS, -SC(O)R5, -SC(O)NHR5, -NR5R5~or-
N(R~)(CO)R~~, where each halo moiety is independently selected from F, Cl, Br
and I. One
subset of such compounds differs in structure from rapamycin only at one or
both of R~13 and
Rte. Another subset of such compounds differs in structure from rapamycin at
one or more
additional positions, as set forth above in connection with Formula II or in
connection with
any of the other classes of improved rapalogs noted herein. Compounds of both
subsets
which are of particular note are those in which one or both of R~13 and RC2s
is a halo
substituent, independently selected from F, Cl, Br and I, or a substituted or
unsubstituted
amino moiety or acylated derivative thereof. These compounds include the 13-
halo
rapamycins, 28-halo rapamycins, 13, 28-dihalo rapamycins and related compounds
in which
one or more other moities (e.g. one or both substituents at C7, for instance),
in addition to the
C13 and C28 substituents, differ from the corresponding moiety(ies) in
rapamycin.
Another class of improved rapalogs for use in this invention which are of
particular
interest are rapalogs of Formula II wherein both R~24 and RC3o are other than
=O. This class
includes 24, 30-tetrahydro rapamycin and mono and diethers thereof and the
24,30-dihalo
rapamycins. One subset of such compounds differs in structure from rapamycin
only at R~4
and Rte. Another subset of such compounds differs in structure from rapamycin
at one or more
additional positions (e.g. one or both substituents at C7, for instance), as
set forth above in
connection with Formula II or in connection with any of the other classes of
improved
rapalogs noted herein.
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Another class of improved rapalogs for use in this invention which are of
particular
interest are rapalogs of Formula II wherein Rc~a and Rc~' are moieties other
than a
substituted or unsubstituted allyl group or a methoxy moiety. This class
includes rapalogs in
which one of R~~a and Rte' is H and the other is phenyl, di- or tri-
substituted phenyl or a
mono- or di-substituted heterocyclic moiety. Illustrative examples include
among others,
o,p-dialkoxyphenyl substituents (e.g., o,p-dimethoxyphenyl, o-methoxy-p-
ethoxyphenyl,
o-ethoxy-p-methoxyphenyl, o,p-diethoxyphenyl, o,p-di (n- or iso-
)propoxyphenyl, etc.),
trialkoxyphenyl substituents, monosubstituted heterocycles such as
methylthiophene, etc.
One subset of such compounds differs in structure from rapamycin only at R~24
and Rte.
Another subset of such compounds differs in structure from rapamycin at one or
more
additional position, as set forth above in connection with Formula II or in
connection with
any of the other classes of improved rapalogs noted herein.
Another class of improved rapalogs for use in this invention which are of
particular
interest are rapalogs of Formula II wherein n is 1. This class of rapalogs
includes rapalogs
comprising a prolyl ring system in place of a pipicolate ring system. One
subset of such
compounds differs in structure from rapamycin only with respect to the
pipicolate ring
system. Another subset of such compounds differs in structure from rapamycin
with respect to
one or more additional structural features (e.g. one or both substituents at
C7, for instance), as
set forth above in connection with Formula II or in connection with any of the
other classes of
improved rapalogs noted herein.
Another class of improved rapalogs for use in this invention which are of
particular
interest are rapalogs of Formula II wherein moiety "a" is other than
~,
Me0 " J'
One subset of such compounds differs in structure from rapamycin anly with
respect to the
ring system, "a". Another subset of such compounds differs in structure from
rapamycin with
respect to one or more additional structural features {e.g. one or both
substituents at C7, for
instance), as set forth above in connection with Formula II or in connection
with any of the
other classes of improved rapalogs noted herein. This class of rapalogs
include the class of
43-epi-rapalogs in which the hydroxyl moiety at position 43 has the opposite
stereochemical orientation with that shown immediately above, is a mixture of
stereoisomers of the 43-hydroxyl group or contains derivatives of any of the
foregoing,
including ethers, esters, carbamates, halides and other derivatives of any of
the foregoing
position 43 rapalogs. This class further includes rapalogs in which the
cyclohexyl ring is
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otherwise substituted and/or contains 5 ring atoms in place of the
characteristic substituted
cyclohexyl ring of rapamycin.
Again, the improved rapalogs as described herein are used in a method for
multimerizing chimeric proteins in genetically engineered cells. The method
involves (a)
providing appropriately engineered cells containing nucleic acid constructs
for directing the
expression of the desired chimeric proteins (and any desired accessory
recombinant
constructs), and (b) contacting the cells with an improved rapalog or a
pharmaceutically
acceptable derivative thereof.
In one embodiment, at least one of the chimeric proteins contains at least one
FKBP
domain whose peptide sequence differs from a naturally occurring FKBP peptide
sequence,
e.g. the peptide sequence of human FKBP12, at up to ten amino acid residues in
the peptide
sequence. Preferably the number of changes in peptide sequence is limited to
five, and more
preferably to 1, 2, or 3. In embodiments in which the rapalog comprises a
structural
modification relative to rapamycin at R~28, at R~24 and RC3o ~ ~d/or at R~~a
and/or R~~',
it is also of special interest that at least one of the chimeric proteins
contains at least one
FKBP domain comprising at least one amino acid replacement relative to the
sequence of a
naturally occurring FKBP, especially a mammalian FKBP such as human FKBP12.
Mutations of particular interest include replacement of either or both of
Phe36 and Phe99 of
human FKBP12 sequence with independently selected replacement amino acids,
e.g. valine,
methionine, alanine or serine.
In another embodiment, at least one of the chimeric proteins contains at least
one FRB
domain whose peptide sequence differs from a naturally occurring FRB peptide
sequence, e.g.
the FRB domain of human FRAP, at up to ten amino acid residues in the peptide
sequence.
Preferably the number of changes in peptide sequence is limited to five, and
more preferably
to 1, 2, or 3. in many cases it will be preferred that the FRB domain contains
a single amino
acid replacement relative to the peptide sequence of the corresponding FRB
domain of
human FRAP or some other mammalian FRAP/TOR species. Mutations of particular
interest
include replacement of one or more of T2098, D2102, Y2038, F2039, K2095 of an
FRB domain
derived from human FRAP with independently selected replacement amino acids,
e.g. A, N,
H, L, or S. Also of interest are the replacement of one or more of F1975,
F1976, D2039 and
N2035 of an FRB domain derived from yeast TOR1, or the replacement of one or
more of
F1978, F1979, D2042 and N2038 of an FRB domain derived from yeast TOR2, with
independently selected replacement amino acids, e.g. H, L, S, A or V.
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In certain embodiments the chimeric proteins) contain at least one
modification in
peptide sequence, preferably up to three modifications, relative to naturally
occurring
sequences, in both one or more FKBP domains and one or more FRB domains.
As mentioned previously, in the various embodiments of this invention, the
chimeric
proteins) contain one or more "action" or "effector" domains which are
heterologous with
respect to the FKBP and/or FRB domains. Effector domains may be selected from
a wide
variety of protein domains including DNA binding domains, transcription
activation
domains, cellular localization domains and signaling domains (i.e., domains
which are
capable upon clustering or multimerization, of triggering cell growth,
proliferation,
differentiation, apoptosis, gene transcription, etc.). A variety of
illustrative effector
domains which may be used in practising this invention are disclosed in the
various
sscientific and patent documents cited herein.
For example, in certain embodiments, one fusion protein contains at least one
DNA
binding domain (e.g., a GAL4 or ZFHD1 DNA-binding domain) and another fusion
protein
contains at least one transcription activation domain (e.g., a VP16 or p65
transcription
activation domain). Ligand-mediated association of the fusion proteins
represents the
formation of a transcription factor complex and leads to initiation of
transcription of a target
gene linked to a DNA sequence recognized by (i.e., capable of binding with)
the DNA-
binding domain on one of the fusion proteins.
In other embodiments, one fusion protein contains at least one domain capable
of
directing the fusion protein to a particular cellular location such as the
cell membrane,
nucleus, ER or other organelle or cellular component. Localization domains
which target the
cell membrane, for example, include domains such as a myristoylation site or a
transmembrane region of a receptor protein or other membrane-spanning protein.
Another
fusion protein can contain a signaling domain capable, upon membrane
localization and/or
clustering, of activating a cellular signal transduction pathway. Examples of
signaling
domains include an intracellular domain of a growth factor or cytokine
receptor, an
apoptosis triggering domain such as the intracellular domain of FAS or TNF-Rl,
and
domains derived from other intracellular signaling proteins such as SOS, Raf,
lck, ZAP-70,
etc. A number of signaling proteins are disclosed in PCT/US94/01617 (see e.g.
pages 23 - 26).
In still other embodiments, each of the fusion proteins contains at least one
FRB domain and
at least one FKBP domain, as well as one or more heterologous domains. Such
fusion proteins
are capable of homodimerization and triggering signaling in the presence of
the rapalog. In
general, domains containing peptide sequence endogenous to the host cell are
preferred in
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applications involving whole organisms. Thus, far human gene therapy
applica~~ns,
domains of human origin are of particular interest.
Recombinant nucleic acid constructs enrnding the fusion proteins are also
provided, as are
nucleic acid constructs capable of directing their expression, and vectors
containing such
constructs for introducing them into cells, particularly eukaryotic cells, of
which yeast and
animal cells are of particular interest. In view of the constituent components
of the fusion
proteins, the recombinant DNA molecules which encode them are capable of
selectively
hybridizing (a) to a DNA molecule encoding a polypeptide comprising an FRB
domain or
FKBP domain and (b) to a DNA molecule encoding the heterologous domain or a
protein from
which the heterologous protein domain was derived. DNAs are also encompassed
which
would be capable of so hybridizing but for the degeneracy of the genetic code.
Using nucleic acid sequences encoding the fusion proteins, nucleic acid
constructs for
directing their expression in eukaryotic cells, and vectors or other means for
introducing such
constructs into cells, especially animal cells, one may genetically engineer
cells, particularly
animal cells, preferably mammlian cells, and most preferably human cells, for
a number of
important uses. To do so, one first provides an expression vector or nucleic
acid construct for
directing the expression in a eukaryotic (preferably animal) cell of the
desired chimeric
proteins) and then introduces the recombinant DNA into the cells in a manner
permitting
DNA uptake and expression of the introduced DNA in at least a portion of the
cells. One
may use any of the various methods and materials for introducing DNA into
cells for
heterologous gene expression, a variety of which are well known and/or
commercially
available.
One object of this invention is thus a method for multimerizing fusion
proteins, such as
described herein, in cells, preferably animal cells. To recap, one of the
fusion proteins is
capable of binding to the improved rapalog of this invention and contains at
least one FKBP
domain and at least one domain heterologous thereto. The second fusion protein
contains at
least one FRB domain and at least one domain heterologous thereto and is
capable of forming
a tripartite complex with the first fusion protein and one or more molecules
of the improved
rapalog. In some embodiments one or more of the heterologous domains present
on one of the
fusion proteins are also present on the other fusion protein, i.e., the two
fusion proteins have
one or more common heterologous domains. In other embodiments, each fusion
protein
contains one or more different heterologous domains.
The method comprises contacting appropriately engineered cells with the
improved
rapalog by adding the rapalog to the culture medium in which the cells are
located or
administering the rapalog to the organism in which the cells are located. The
cells are
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preferably eukaryotic cells, more preferably animal cells, and most preferably
mammalian
cells. Primate cells, especially human cells, are of particular interest.
Administration of the
improved rapalog to a human or non-human animal may be effected using any
pharmaceutically acceptable formulation and route of administration. Oral
administration
of a pharmaceutically acceptable composition containing the improved rapalog
together
with one or more pharmaceuticaly acceptable carriers, buffers or other
excipients is
currently of greatest interest.
A specific object of this invention is a method, as otherwise described above,
for inducing
transcription of a target gene in a rapalog-dependent manner. The cells
typically contain, in
addition to recombinant DNAs encoding the two fusion proteins, a target gene
construct
which comprises a target gene operably linked to a DNA sequence which is
responsive to the
presence of a complex of the fusion proteins with rapamycin or a rapalog. The
target gene
construct may be recombinant, and the target gene and/or a regulatory nucleic
acid sequence
linked thereto may be heterologous with respect to the host cell. In certain
embodiments the
cells are responsive to contact with an improved rapalog which binds to the
FKBP fusion
protein and participates in a complex with a FRB fusion protein with a
detectable
preference over binding to endogenous FKBP and/or FRB-containing proteins of
the host cell.
Another specific object of this invention is a method, as otherwise described
above, for
inducing cell death in a rapalog-dependent manner. In such cells, at least one
of the
heterologous domains on at least one fusion protein, and usually two fusion
proteins, is a
domain such as the intracellular domain of FAS or TNF-Rl, which, upon
clustering, triggers
apoptosis of the cell.
Another specific object of this invention is a method, as otherwise described
above, for
inducing cell growth, differentiation or proliferation in a rapalog-dependent
manner. In
such cells, at least one of the heterologous domains of at least one of the
fusion proteins is a
signaling domain such as, for example, the intracellular domain of a receptor
for a hormone
which mediates cell growth, differentiation or proliferation, or a downstream
mediator of
such a receptor. Cell growth, differentiation and/or proliferation follows
clustering of such
signalling domains. Such clustering occurs in nature following hormone
binding, and in
engineered cells of this invention following contact with an improved rapalog.
Cells of human origin are preferred for human gene therapy applications,
although cell
types of various origins (human or other species) may be used, and may, if
desired, be
encapsulated within a biocompatible material for use in human subjects.
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Also provided are materials and methods for producing the foregoing engineered
cells.
This object is met by providing recombinant nucleic acids, typically DNA
molecules,
encoding the fusion proteins, together with any desired ancillary recombinant
nucleic acids
such as a target gee construct, and introducing the recombinant nucleic acids
into the host
cells under conditions permitting nucleic acid uptake by cells. Such
transfection may be
effected ex vivo, using host cells maintained in culture. Cells that are
engineered in culture
may subsequently be introduced into a host organism, e.g. in ex vivo gene
therapy
applications. Doing so thus constitutes a method for providing a host
organism, preferably a
human or non-human mammal, which is responsive (as described herein) to the
presence of
an improved rapalog as provided herein. Alternatively transfection may be
effected in
vivo, using host cells present in a human or non-human host organism. In such
cases, the
nucleic acid molecules are introduced directly into the host organism under
conditions
permitting uptake of nucleic acids by one or more of the host organism's
cells. This approach
thus constitutes an alternative method for providing a host organism,
preferably a human or
non-human mammal, which is responsive (as described herein) to the presence of
an
improved rapalog. Various materials and methods for the introduction of DNA
and RNA
into cells in culture or in whole organisms are known in the art and may be
adapted for use in
practicing this invention.
Other objects are achieved using the engineered cells described herein. For
instance, a
method is provided for multimerizing fusion proteins of this invention by
contacting cells
engineered as described herein with an effective amount of the improved
rapalog permitting
the rapalog to form a complex with the fusion proteins. In embodiments in
which
multimerization of the fusion proteins triggers transcription of a target
gene, this constitutes
a method for activating the expression of the target gene. In embodiments in
which the
fusion proteins contain one or more signaling domains, this constitutes a
method for
activating a cellular signal transduction pathway. In specific embodiments in
which the
signaling domains are selected based on their ability following clustering to
trigger cell
growth, proliferation, diffeentiation or cell death, improved rapalog-mediated
clustering
constitutes a method for actuating cell growth, proliferation, diffeentiation
or cell death, as
the case may be. These methods may be carried out in cell culture or in whole
organisms,
including human patients. In the former case, the rapamycin or rapalog is
added to the
culture medium. In the latter case, the rapamycin or rapalog (which may be in
the form of a
pharmaceutical or veterinary composition) is administered to the whole
organism, e.g.,
orally, parenterally, etc. Preferably, the dose of the improved rapalog
administered to an
animal is below the dosage level that would cause undue immunosuppression in
the
recipient.
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WO 99/36553 PCT/US99/00178
Also disclosed are kits for use in the genetic engineering of cells or human
or non-human
animals as described herein. One such kit contains one or more recombinant
nucleic acid
constructs encoding fusion proteins of this invention. The recombinant nucleic
acid constructs
will generally be in the form of eukaryotic expression vectors suitable for
introduction into
animal cells and capable of directing the expression of the fusion proteins
therein. Such
vectors may be viral vectors as described elsewhere herein. The kit may also
contain a
sample of an improved rapalog of this invention capable of forming a complex
with the
encoded fusion proteins. The kit may further contain a multimerization
antagonist such as
FIC506 or some other compound capable of binding to one of the fusion proteins
but incapable
of forming a complex with both. In certain embodiments, the recombinant
nucleic acid
constructs encoding the fusion proteins will contain a cloning site in place
of DNA encoding
one or more of the heterologous domains, thus permitting the practitioner to
introduce DNA
encoding a heterologous domain of choice. In some embodiments the kit may also
contain a
target gene construct containing a target gene or cloning site linked to a DNA
sequence
responsive to the presence of the complexed fusion proteins, as described in
more detail
elsewhere. The kit may contain a package insert identifying the enclosed
nucleic acid
construct(s), and/or instructions for introducing the constructs) into host
cells or organisms.
Brief Description of the Figures
Figure 1 demonstrates the ability of 13-F-rapalogs (compounds 7~ and 1~$,
synthesized
as described in Examples 6.1 and 6.21, respectively) to stimulate expression
of a DNA
sequence encoding ~gcreted ~.lkaline ~hosphatase ("SEAP") in I-IT1080 cells
engineered as
described in Example 7.
Figure 2 depicts the results of transcription assays using rapalogs ~, ~, ~Q
and 9~,
synthesized as describedherein, as dimerizer. Rapalog s were tested in cells
expressing
wild-type FRB (Figs. 2A and 2C) as well as in cells expressing a mutant FRB in
which Thr
2098 was replaced by Leu {Figs 2B and 2D) or by Phe (Fig 2E).
Detailed Description of the Invention
Definitions
The definitions and orienting information below will be helpful for a full
understanding
of this document.
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WO 99/36553 PCT/US99/00178
FRB domains are polypeptide regions (protein "domains"), typically of at least
about 89
amino acid residues, which are capable of forming a tripartite complex with an
FKBP
protein and rapamycin (or an improved rapalog of this invention). FRB domains
are present
in a number of naturally occurring proteins, including FRAP proteins (also
referred to in the
literature as "RAPTl" or RAFT'S from human and other species; yeast proteins
including
Torl and Tor2; and a Candida FRAP homolog. Information concerning the
nucleotide
sequences, cloning, and other aspects of these proteins is already known in
the art,
permitting the synthesis or cloning of DNA encoding the desired FRB peptide
sequence, e.g.,
using well known methods and PCR primers based on published sequences.
pmtein source reference/sequence aaession numbers
human FRAP Brown et al, 1994, Nahxre 369, 756-758;
GenBank accession # L34075, NCBI Seq ID 508481;
Chiu et al, 1994, PNAS USA 91, 12574-12578;
Chen et al, 1995, PNAS USA 92, 4947-4951
marine RAP'Tl Chiu et al, supra.
yeast Torl Helliwell et al, 1994, Mol Cell Biol 5, 105-118;
EMBL Accession #X74857, NCBI Seq Id #468738
yeast Tor Kunz et al, 1993, Cell 73, 585-596;
2
EMBL Accession #X71416, NCBI Seq ID 29$027
Candida TOR W095/33052 (Berlin et al)
FRB domains for use in this invention generally contain at least about 89 -100
amino acid
residues. Fig.2 of Chiu et al, supra, displays a 160-amino acid span of human
FRAP, marine
FRAP, S. cerevisiae TORT and S. cerevisiae TOR2 encompassing the conserved FRB
region.
Typically the FRB sequence selected for use in fusion proteins of this
invention will span at
least the 89-amino acid sequence Glu-39 through Lys/Arg-127, as the sequence
is numbered in
that figure. For reference, using the numbering of Chen et al or Sabitini et
al, the 89-amino
acid sequence is numbered Glu-2025 through Lys-2113 in the case of human FRAP,
Glu-1965
through Lys-2053 in the case of Tort, and Glu-1962 through Arg-2050 in the
case of Torl. An
FRB domain for use in fusion proteins of this invention will be capable of
binding to a
complex of an FKBP protein bound to rapamycin or an improved rapalog of this
invention (as
may be determined by any means, direct or indirect, for detecting such
binding, including, for
example, means for detecting such binding employed in the FRAP/RAFT/RAPT and
Tor-
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WO 99/36553 PCT/US99/00178
related references cited herein). The peptide sequence of such an FRB domain
comprises (a) a
naturally occurring peptide sequence spanning at least the indicated 89-amino
acid region of
the proteins noted above or corresponding regions of homologous proteins; (b)
a variant of a
naturally occurring FRB sequence in which up to about ten (preferably 1-5,
more preferably 1-
3, and in some embodiments just one) amino acids of the naturally-occurring
peptide sequence
have been deleted, inserted, or replaced with substitute amino acids; or (c) a
peptide
sequence encoded by a DNA sequence capable of selectively hybridizing to a DNA
molecule
encoding a naturally occurring FRB domain or by a DNA sequence which would be
capable,
but for the degeneracy of the genetic code, of selectively hybridizing to a
DNA molecule
encoding a naturally occurring FRB domain.
FKBPs {FK506 binding proteins) are the cytosolic receptors for macrolides such
as FK506,
FK520 and rapamycin and are highly conserved across species lines. For the
purpose of this
disclosure, FKBPs are proteins or protein domains which are capable of binding
to
rapamycin or to an improved rapalog of this invention and further forming a
tripartite
complex with an FRB-containing protein. An FKBP domain may also be referred to
as a
"rapamycin binding domain". Information concerning the nucleotide sequences,
cloning, and
other aspects of various FKBP species is already known in the art, permitting
the synthesis
or cloning of DNA encoding the desired FKBP peptide sequence, e.g., using well
known
methods and PCR primers based on published sequences. See e.g. Staendart et
a1,1990,
Nature 346, 671-674 (human FKBP12); Kay, 1996, Biochem. J. 314, 361-385
(review).
Homologous FKBP proteins in other mammalian species, in yeast, and in other
organsims are
also known in the art and may be used in the fusion proteins disclosed herein.
See e.g. Kay,
1996, Biochem. J. 314, 361-385 {review). The size of FKBP domains for use in
this invention
varies, depending on which FKBP protein is employed. An FKBP domain of a
fusion protein
of this invention will be capable of binding to rapamycin or an improved
rapalog of this
invention and participating in a tripartite complex with an FRB-containing
protein (as may
be determined by any means, direct or indirect, for detecting such binding).
The peptide
sequence of an FKBP domain of an FKBP fusion protein of this invention
comprises (a) a
naturally occurring FKBP peptide sequence, preferably derived from the human
FKBP12
protein (exemplified below) or a peptide sequence derived from another human
FKBP, from
a murine or other mammalian FKBP, or from some other animal, yeast or fungal
FKBP; (b) a
variant of a naturally occurring FKBP sequence in which up to about ten
(preferably 1-5,
more preferably 1-3, and in some embodiments just one) amino acids of the
naturally-
occurring peptide sequence have been deleted, inserted, or replaced with
substitute amino
acids; or (c) a peptide sequence encoded by a DNA sequence capable of
selectively
hybridizing to a DNA molecule encoding a naturally occurring FKBP or by a DNA
sequence
CA 02318402 2000-07-14
WO 99/36553 PCT/US99/00178
which would be capable, but for the degeneracy of the genetic code, of
selectively
hybridizing to a DNA molecule encoding a naturally occurring FKBP.
"Capable of selectively hybridizing" as that phrase is used herein means that
two
DNA molecules are susceptible to hybridization with one another, despite the
presence of
other DNA molecules, under hybridization conditions which can be chosen or
readily
determined empirically by the practitioner of ordinary skill in this art. Such
treatments
include conditions of high stringency such as washing extensively with buffers
containing 0.2
to 6 x SSC, and/or containing 0.1% to 1% SDS, at temperatures ranging from
room
temperature to 65-75°C. See for example F.M. Ausubel et al., Eds, Short
Protocols in
Molecular Biology, Uruts 6.3 and 6.4 (John Wiley and Sons, New York, 3d
Edition, 1995).
The terms "protein", "polypeptide" and "peptide" are used interchangeably
herein.
"Nucleic acid constructs", as that term is used herein, denote nucleic acids
(usually
DNA, but also encompassing RNA, e.g. in a retroviral delivery system) used in
the practice
of this invention which are generally recombinant, as that term is defined
below, and which
may exist in free form (i.e., not covalently linked to other nucleic acid
sequence) or may be
present within a larger molecule such as a DNA vector, retroviral or other
viral vector or a
chromosome of a genetically engineered host cell. Nucleic acid constructs of
particular
interest are those which encode fusion proteins of this invention or which
comprise a target
gene and expression control elements. The construct may further include
nucleic acid portions
comprising one or more of the following elements relevant to regulation of
transcription,
translation, and/or other processing of the coding region or gene product
thereof:
transcriptional promoter and/or enhancer sequences, a ribosome binding site,
introns, etc.
"Recombinant", "chimeric" and "fusion", as those terms are used herein, denote
materials comprising various component domains, sequences or other components
which are
mutually heterologous in the sense that they do not occur together in the same
arrangement,
in nature. More specifically, the component portions are not found in the same
continuous
polypeptide or nucleotide sequence or molecule in nature, at least not in the
same cells or
order or orientation or with the same spacing present in the chimeric protein
or recombinant
DNA molecule of this invention.
"Transcription control element" denotes a regulatory DNA sequence, such as
initiation
signals, enhancers, and promoters, which induce or control transcription of
protein coding
sequences with which they are operably linked. The term "enhancer" is intended
to include
regulatory elements capable of increasing, stimulating, or enhancing
transcription from a
16
CA 02318402 2000-07-14
WO 99136553 PCT/US99/00178
promoter. Such transcription regulatory components can be present upstream of
a rcxiing .I
region, or in certain cases (e.g. enhancers), in other locations as well, such
as in introns, exons,
coding regions, and 3' flanking sequences.
"Dimerization", "oligomerization" and "multimerization" are used
interchangeably
herein and refer to the association or clustering of two or more protein
molecules, mediated
by the binding of a drug to at least one of the proteins. In preferred
embodiments, the
multimerization is mediated by the binding of two or more such protein
molecules to a
common divalent or multivalent drug. The formation of a complex comprising two
or more
protein molecules, each of which containing one or more FKBP domains, together
with one or
more molecules of an FKBP ligand which is at least divalent (e.g. FK1012 or
AP1510) is an
example of such association or clustering. In cases where at least one of the
proteins contains
more than one drug binding domain, e.g., where at least one of the proteins
contains three
FKBP domains, the presence of a divalent drug leads to the clustering of more
than two
protein molecules. Embodiments in which the drug is more than divalent (e.g.
trivalent) in
its ability to bind to proteins bearing drug binding domains also can result
in clustering of
more than two protein molecules. The formation of a tripartite complex
comprising a protein
containing at least one FRB domain, a protein containing at least one FKBP
domain and a
molecule of rapamycin is another example of such protein clustering. In
certain embodiments
of this invention, fusion proteins contain multiple FRB and/or FKBP domains.
Complexes of
such proteins may contain more than one molecule of rapamycin or a derivative
thereof or
other dimerizing agent and more than one copy of one or more of the
constituent proteins.
Again, such multimeric complexes are still referred to herein as tripartite
complexes to
indicate the presence of the three types of constituent molecules, even if one
or more are
represented by multiple copies. The formation of complexes containing at least
one divalent
drug and at least two protein molecules, each of which contains at least one
drug binding
domain, may be referred to as "oligomerization" or "multimerization", or
simply as
"dimerization", "clustering" or association".
"Dimerizer" denotes an improved rapalog of this invention which brings
together two or
more proteins in a multimeric complex.
"Activate" as applied herein to the expression or transcription of a gene
denotes a
directly or indirectly observable increase in the production of a gene
product.
"Genetically engineered cells" denotes cells which have been modified
("transduced")
by the introduction of recombinant or heterologous nucleic acids (e.g. one or
more DNA
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WO 99/36553 PCT/US99/00178
constructs or their RNA counterparts) and further includes the prog~y of such
cellc which
retain part or all of such genetic modification.
A "therapeutically effective dose" of an improved rapalog of this invention
denotes a
treatment- or prophylaxis-effective dose, e.g., a dose which yields detectable
target gene
transcription or cell growth, proliferation, differentiation, death, etc. in
the genetically
engineered cell, or a dose which is predicted to be treatment- or prophylaxis-
effective by
extrapolation from data obtained in animal or cell culture models. A
therapeutically
effective dose is ususally preferred for the treatment of a human or non-human
mammal.
This invention involves methods and materials for multimerizing chimeric
proteins in
genetically engineered cells using improved rapalogs. The design and
implementation of
various dimerization-based biological switches has been reported, inter alia,
in Spencer et
al and in various international patent applications cited herein. Other
accounts of successful
application of this general approach have also been reported. Chimeric
proteins containing
an FRB domain fused to an effector domain has also been disclosed in Rivera et
al, 1996,
Nature Medicine 2,1028-1032 and in WO 96/41865 (Clackson et al) and WO
95/33052 (Berlin
et al). As noted previously, the fusion proteins are designed such that
association of the
effector domains, through ligand-mediated "dimerization" or "multimerization"
of the
fusion proteins which contain them, triggers a desired biological event such
as transcription
of a desired gene, cell death, cell proliferation, etc. For example,
clustering of chimeric
proteins containing an action domain derived from the intracellular portion of
the T cell
receptor CD3 zeta domain triggers transcription of a gene under the
transcriptional control of
the IL-2 promoter or promoter elements derived therefrom. In other
embodiments, the action
domain comprises a domain derived from the intracellular portion of a protein
such as FAS
or the TNF-alpha receptor (TNFalpha-Rl), which are capable, upon
oligomerization, of
triggering apoptosis of the cell. In still other embodiments, the action
domains comprise a
DNA-binding domain such as GAL4 or ZFHDl and a transcription activation domain
such as
VP16 or p65, paired such that oligomerization of the chimeric proteins
represents assembly
of a transcription factor complex which triggers transcription of a gene
linked to a DNA
sequence recognized by (capable of specific binding interaction with) the DNA
binding
domain.
Chimeric proteins containing one or more ligand-binding domains and one or
more action
domains, e.g. for activation of transcription of a target gene, triggering
cell death or other
signal transduction pathway, cellular localization, etc., are disclosed in
PCT/US94/01617,
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WO 99/36553 PCT/US99/00178
PCT/US94/08008 and Spencer et al, supra. The design and use of such chimeric
prntPins for
ligand-mediated gene-knock out and for ligand-mediated blockade of gene
expression or
inhibition of gene product function are disclosed in PCT/US95/10591. Novel DNA
binding
domains and DNA sequences to which they bind which are useful in embodiments
involving
regulated transcription of a target gene are disclosed, e.g., in Pomeranz et
a1,1995, Science
267:93-96. Those references provide substantial information, guidance and
examples relating
to the design, construction and use of DNA constructs encoding analogous
chimeras, target
gene constructs, and other aspects which may also be useful to the
practitioner of the subject
invention.
By appropriate choice of chimeric proteins, this invention permits one to
activate the
transcription of a desired gene; actuate cell growth, proliferation,
differentiaion or
apoptosis; or trigger other biological events in engineered cells in a rapalog-
dependent
manner analogous to the systems described in the patent documents and other
references cited
above. The engineered cells, preferably animal cells, may be growing or
maintained in
culture or may be present within whole organisms, as in the case of human gene
therapy,
transgenic animals, and other such applications. The rapalog is administered
to the cell
culture or to the organism containing the engineered cells, as the case may
be, in an amount
effective to multimerize the FKBP fusion proteins and FRB fusion proteins (as
may be
observed indirectly by monitoring target gene transcription, apoptosis or
other biological
process so triggered). In the case of administration to whole organisms, the
rapalog may be
administered in a composition containing the rapalog and one or more
acceptable verterinary
or pharmaceutical diluents and/or excipients.
A compound which binds to one of the chimeric proteins but does not form
tripartite
complexes with both chimeric proteins may be used as a multimerization
antagonist. As such
it may be administered to the engineered cells, or to organisms containing
them (preferably
in a composition as described above in the case of administration to whole
animals), in an
amount effective for blocking or reversing the effect of the rapalog, i.e. for
preventing,
inhibiting or disrupting multimerization of the chimeras. For instance, FK506,
FK520 or any
of the many synthetic FKBP ligands which do not form tripartite complexes with
FKBP and
FRAP may be used as an antagonist.
One important aspect of this invention provides materials and methods for
rapalog-
dependent, direct activation of transcription of a desired gene. In one such
embodiment, a set
of two or more different chimeric proteins, and corresponding DNA constructs
capable of
directing their expression, is provided. One such chimeric protein contains as
its action
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WO 99/36553 PCT/US99/00178
domains) one or more transcriptional activation domains. The other chimeric
protein
contains as its action domains) one or more DNA-binding domains. A rapalog of
this
invention is capable of binding to both chimeras to form a dimeric or
multimeric complex
thus containing at least one DNA binding domain and at least one
transcriptional activating
domain. Formation of such complexes leads to activation of transcription of a
target gene
linked to, and under the transcriptional control of, a DNA sequence to which
the DNA-
binding domain is capable of binding, as can be observed by monitoring
directly or indirectly
the presence or concentration of the target gene product.
Preferably the DNA binding domain, and a chimera containing it, binds to its
recognized
DNA sequence with sufficient selectivity so that binding to the selected DNA
sequence can
be observed (directly or indirectly) despite the presence of other, often
numerous other, DNA
sequences. Preferably, binding of the chimera comprising the DNA-binding
domain to the
selected DNA sequence is at least two, more preferably three and even more
preferably more
than four orders of magnitude greater than binding to any one alternative DNA
sequence, as
measured by in vitro binding studies or by measuring relative rates or levels
of transcription
of genes associated with the selected DNA sequence as compared with any
alternative DNA
sequences.
Cells which have been genetically engineered to contain such a set of
constructs, together
with any desired accessory constructs, may be used in applications involving
ligand-
mediated, regulated actuation of the desired biological event, be it regulated
transcription
of a desired gene, regulated triggering of a signal transduction pathway such
as the
triggering of apoptosis, or another event. Cells engineered for regulatable
expression of a
target gene, for instance, can be used for regulated production of a desired
protein (or other
gene product) encoded by the target gene. Such cells may be grown in culture
by conventional
means. Addition of the rapalog to the culture medium containing the cells
leads to
expression of the target gene by the cells and production of the protein
encoded by that gene.
Expression of the gene and production of the protein can be fumed off by
withholding further
multimerization agent from the media, by removing residual multimerization
agent from
the media, or by adding to the medium a multimerization antagonist reagent.
Engineered cells of this invention can also be produced and/or used in vivo,
to modify
whole organisms, preferably animals, especially humans, e.g. such that the
cells produce a
desired protein or other result within the animal containing them. Such uses
include gene
therapy applications.
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Embodiments involving regulatable actuation of apoptosis provide engineered
cells
susceptible to rapalog-inducible cell death. Such engineered cells can be
eliminated from a
cell culture or host organism after they have served their intended purposed
(e.g. production
of a desired protein or other product), if they have or develop unwanted
properties, or if
they are no longer useful, safe or desired. Elimination is effected by adding
the rapalog to
the medium or administering it to the host organism. In such cases, the action
domains of the
chimeras are protein domains such as the intracellular domains of FAS or TNF-
Rl,
downstream components of their signaling pathways or other protein domains
which upon
oligomerization trigger apoptosis.
This invention thus provides materials and methods for achieving a biological
effect in
cells in response to the addition of a rapalog of this invention. The method
involves
providing cells engineered as described herein and exposing the cells to the
rapalog.
For example, this invention provides a method for activating transcription of
a target
gene in cells. The method involves providing cells containing (a) DNA
constructs encoding a
set of chimeric proteins of this invention capable upon rapalog-mediated
multimerization of
initiating transcription of a target gene and {b) a target gene linked to an
associated cognate
DNA sequence responsive to the multimerization event (e.g. a DNA sequence
recognized, i.e.,
capable of binding with, a DNA-binding domain of a foregoing chimeric protein.
The
method involves exposing the cells to a rapalog capable of binding to the
chimeric proteins
in an amount effective to result in expression of the target gene. In cases in
which the cells
are growing in culture, exposing the cells to the rapalog may be effected by
adding the
rapalog to the culture medium. In cases in which the cells are present within
a host
organism, exposing them to the rapalog is effected by administering the
rapalog to the host
organism. For instance, in cases in which the host organism is a human or non-
human, the
rapalog may be administered to the host organism by oral, bucal, sublingual,
transdermal,
subcutaneous, intramuscular, intravenous, infra joint or inhalation
administration in an
appropriate vehicle therefor. Again, depending on the design of the constructs
for the
chimeric proteins and of any accessory constructs, the rapalog-mediated
biological event
may be activation of a cellular function such as signal transduction leading
to cell growth,
cell proliferation, gene transcription, or apoptosis; deletion of a gene of
interest, blockade of
expression of a gene of interest, or inhibition of function of a gene product
of interest; direct
transcription of a gene of interest; etc.
This invention further encompasses a pharmaceutical composition comprising a
rapalog
of this invention in admixture with a pharmaceutically acceptable carrier and
optionally
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with one or more pharmaceutically acceptable excipients. Such pharmaceutical ;
compositions can be used to promote multimerization of chimeras of this
invention in
engineered cells in whole animals, e.g. in human gene therapy applications to
achieve any
of the objectives disclosed herein.
Said differently, this invention provides a method for achieving any of those
objectives,
e.g. activation of transcription of a target gene (typically a heterologous
gene for a
therapeutic protein), cell growth or proliferation, cell death or some other
selected
biological event, in an animal, preferably a human patient, in need thereof
and containing
engineered cells of this invention. That method involves administering to the
animal a
pharmaceutical composition containing the rapalog by a route of administration
and in an
amount effective to cause multimerization of the chimeric proteins in at least
a portion of
the engineered cells. Multimerization may be detected indirectly by detecting
the occurrence
of target gene expression; cell growth; proliferation or death; or other
objective for which
the chimeras were designed and the cells genetically engineered.
This invention further encompasses a pharmaceutical composition comprising a
multimerization antagonist of this invention in admixture with a
pharmaceutically
acceptable carrier and optionally with one or more pharmaceutically acceptable
excipients
for inhibiting or otherwise reducing, in whole or part, the extent of
multimerization of
chimeric proteins in engineered cells of this invention in a subject, and thus
for de-activating
the transcription of a target gene, for example, or turning off another
biological result of this
invention. Thus, the use of the multimerizing rapalogs and of the
multimerization
antagonist reagents to prepare pharmaceutical compositions and achieve their
pharmacologic results is encompassed by this invention.
Also disclosed is a method for providing a host organism, preferably an
animal,
typically a non-human mammal or a human subject, responsive to a rapalog of
this
invention. The method involves introducing into the organism cells which have
been
engineered in accordance with this invention, i.e. containing one or more
nucleic acid
constructs encoding the chimeric proteins, and so forth. The engineered cells
may be
encapsulated using any of a variety of materials and methods before being
introduced into
the host organism. Alternatively, one can introduce the nucleic acid
constructs of this
invention into a host organism, e.g. a mammal, under conditions permitting
incorporation
thereof into one or more cells of the host mammal, e.g. using viral vectors,
introduction of
DNA by injection or via catheter, etc.
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Also provided are kits for producing cells responsive to a rapalog of this
invention. One .
such kit contains one or more nucleic acid constructs encoding and capable of
directing the
expression of chimeras which, upon rapalog-mediated oligomerization, trigger
the desired
biological response. The kit may contain a quantity of a rapalog capable of
multimerizing
the chimeric protein molecules encoded by the constructs) of the kit, and may
contain in
addition a quantity of a multimerization antagonist. The kit may further
contain a nucleic
acid construct encoding a target gene (or cloning site) linked to a cognate
DNA sequence
which is recognized by the dimerized chimeric proteins permitting
transcription of a gene
linked to that cognate DNA sequence in the presence of multimerized chimeric
protein
molecules. The constructs may be associated with one or more selection markers
for
convenient selection of transfectants, as well as other conventional vector
elements useful for
replication in prokaryotes, for expression in eukaryotes, and the like. The
selection markers
may be the same or different for each different construct, permitting the
selection of cells
which contain each such construct(s).
The accessory construct for introducing into cells a target gene in
association with a
cognate DNA sequence may contain a cloning site in place of a target gene. A
kit containing
such a construct permits the engineering of cells for regulatable expression
of a gene to be
provided by the practitioner.
Other kits of this invention may contain one or two (or more) nucleic acid
constructs for
chimeric proteins in which one or more contain a cloning site in place of the
transcriptional
activator or DNA binding protein, permitting the user to insert whichever such
domain s/he
wishes. Such a kit may optionally include other elements as described above,
e.g. a nucleic
construct for a target gene with or without a cognate DNA sequence for a pre-
selected DNA
binding domain.
Any of the kits may also contain positive control cells which were stably
transformed
with constructs of this invention such that they express a reporter gene {for
CAT, beta-
galactosidase or any conveniently detectable gene product) in response to
exposure of the
cells to the rapalog. Reagents for detecting and/or quantifying the expression
of the reporter
gene may also be provided.
For further information and guidance on the design, construction and use of
such systems
or components thereof which may be adapted for use in practising the subject
invention,
reference to the following publications is suggested: Spencer et al, 1993,
supra; Rivera et al,
1996, supra; Spencer et x1,1996, Current Biology 6, 839-847; Luo et al, 1996,
Nature, 383,
181-185; Ho et al, 1996, Nature 382, 822-826; Belshaw et al, 1996, Proc. Natl.
Acad. Sci. USA
23
CA 02318402 2000-07-14
WO 99/36553 PC'T/US99/00178
93, 4604-4607; Spencer,1996, TIG 12(5),181-187; Spencer et a1,1995, Proc.,
Natl. Acad. Sci.
USA 92, 9805-9809; Holsinger et al, 1995, Proc. Natl. Acad. Sci. USA 92, 9810-
9814; Pruschy
et al, 1994, Chemistry & Biology 1(3),163-172; and published international
patent
applications WO 94/18317, WO 95/02684, WO 95/33052, WO 96/20951 and WO
96/41865.
A key focus of the subject invention is the use of improved rapalogs as
mediators of
protein-protein interactions in applications using FKBP and FRB fusion
proteins such as
described above and elsewhere herein. The improved rapalogs may be used in the
various
applications of the underlying dimerization-based technology, including
triggering
biological events in genetically engineered cells grown or maintained in
culture or present in
whole organisms, including humans and other mammals. The improved rapalogs may
thus
be useful as research reagents in biological experiments in vitro, in
experiments conducted on
animals containing the genetically engineered cells, and as prophylactic or
therapeutic
agents in animal and human health care in subjects containing genetically
engineered cells.
Rapalogs
"Rapalogs" as that term is used herein denotes a class of compounds comprising
the
various analogs, homologs and derivatives of rapamycin and other compounds
related
structurally to rapamycin. "Rapalogs" include compounds other than rapamycin
which
comprise the substructure shown in Formula I, bearing any number of a variety
of substituents,
and optionally unsaturated at one or more carbon-carbon bonds unless specified
to the
contrary herein.
n=1 nr2
I
Rapalogs include, among others, variants of rapamycin having one or more of
the
following modifications relative to rapamycin: demethylation, elimination or
replacement
of the methoxy at C7, C42 and/or C29; elimination, derivatization or
replacement of the
hydroxy at C13, C43 and/or C28; reduction, elimination or derivatization of
the ketone at
24
CA 02318402 2000-07-14
WO 99/36553 PCTNS99/00178
C14, C24 and/or C30; replacement of the 6-membered pipecolate ring with a 5-
membered
prolyl ring; and elimination, derivatization or replacement of one or more
substituents of the
cyclohexyl ring or replacement of the cyclohexyl ring with a substituted or
unsubstituted
cyclopentyl ring. Rapalogs, as that term is used herein, do not include
rapamycin itself, and
preferably do not contain an oxygen bridge between Cl and C30. Illustrative
examples of
rapalogs are disclosed in the documents listed in Table I. Examples of
rapalogs modified at
C7 are shown in Table II.
Table I
W09710502 W09418207 W09304680 US5527907 US5225403
W09641807 W09410843 W09214737 US5484799 US5221625
W09635423 W09409010 W09205179 US5457194 US5210030
W09603430 W094/04540 US5604234 US5457182 US5208241
W09600282 W09402485 US5597715 US5362735 US5200411
W09516691 W09402137 US5583139 US5324644 US5198421
W09515328 W09402136 US5563172 US5318895 US5147877
W09507468 W09325533 US5561228 US5310903 US5140018
W09504738 W09318043 US5561137 US5310901 US5116756
W09504060 W09313663 US5541193 US5258389 US5109112
W09425022 W09311130 US5541189 US5252732 US5093338
W09421644 W09310122 US5534632 US5247076 US5091389
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WO 99/36553 PCT/US99/00178
Table II: Illustrative C7 rapalog structures
0
""' H oMe '~.. ~ A~ Ar = phenyl, 3-nitrophenyl 4-chlorophenyl
.., p;propyl ,I, ~ 3iodo-4-diazophenyl, 3,4-dirnethoxyphenyl,
-OMe or 2-methoxyphenyi
oMe
Ol.p.~. oi"~8 ~ O~A~ Ar = 3,4-dimethoxyphenyl
~~~~ OEt
~t ~ - CH2COPhenyl
OMe
..,.OCH2CHzOH ~
~~ OCHzCHzOH -CHzCHZOH
,~~ SMe ~ pMe - CHzCH(OH)ChIzOH
-SMe ~ -CHzCH20CHzCH20CH2CHzOCH3
~~~~~ SPhenyl
- SPhenyl '[",~ X= O or NH
,.... NHCOxMe ~, I ,'I O
- NHC02Me x ~''' O~CH3
-~ Oacetyl ~h~~ ~'" O ~ CH2CH3
".. O-~wyl Me CHzOH
-~ellyl ,~ H ~ H
\ ~ / \ ~ /
.../
26
CA 02318402 2000-07-14
WO 99/36553 PCTNS99/00178
Table II (conYd):
O
.,.,... H
...
"
- OMe
fee e~$~~
Luengo et aI, Chemistry & Biology, 1995, 2 (7):471-481; JOC, 1995, 59(22):6512-
13
WO 94/02136 (SmithKline Beecham)
W095/16691 (Sandoz)
US 5583139 (Abbott)
Grinfeld et al, 1994, Tett Letters 35(37):6835-6838
W0 96/41865 (ARIAD) -
27
7-oxorepartiycrc~
CA 02318402 2000-07-14
WO 99/36553 PCT/US99/00178
Other illustrative rapalogs include those depicted in Table III:
Table III
HO."
HO""
M ~ Me
i
~O O H (~p I
cz.
q '' H
H O ~~
O M~, O tt''~~ o O
p ~
I
Ru. _ ,+, '~" ~
~
NHR ~N N
OO~'' ~'~f \H
R = -OH, -O-alkyl,
-NH-alkyl
Mi Me0
O O H
1 T O OC(OIR
00 O M
O
H
~O O a
N T I
O
'
O O O Mv0 O O
O I
O Oy, O O O Me O
H
Ha.N
Me ~
i H HO
.. ~~
( H
O O O O
'
F;c" p H l~~ (MeIH~
O ~
~~,
MeO
'O QMe
/ / /
28
CA 02318402 2000-07-14
WO 99/36553 PCT/US99/00178
Table III (cont'd) ~;
HO.,
V
Q O I y0 O I OH
N
~
[
O M~". O M~, O
O O
HO."
O
O OMe ~ OMe
/ / / ' / / / .
HO"
HO.,
's
I
' I ' I ~p O I OH
~O O
O
O
H O O ~"' O ~ O O Me0'
O OMe -O ~Me
/ / / ' / / / .
HO.,y
TBDM&O ",
_
o . o . ~ I H
o ~
o.~eoMs
H O O
O O MoO
H O O MeO, O
O
9 OM.
/ /
/ /
TBOMS. . tBuMh&i-
Rapalogs of particular interest for the practice of various aspects of this
invention include
compounds of formula II:
ll
29
CA 02318402 2000-07-14
WO 99!36553 PCT/US99l00178
wherein
R~z
Re Re Ra
HsCO~ H~ Ra ~''~ IiCO
and,
one of RC~a and RCS is H and the other is -H, halo, -R2, -0Rl, -SRl, -OC(O)Rl,
-OC(O)NHRl,-
NHRl, -NR1R2, -NHC(O)Rl, or -NH-S02-Rl where R2 = aliphatic, heteroaliphatic,
aryl,
heteroaryl or alkylaryl (e.g. benzyl or substituted benzyl),
RC3o ys ~lo, -0R3 or (=O),
RC24 is =O, =NR4 =NOR4, =NNHR4, -NHOR4, -NHNHR4, -OR4, -OC(O)R4 or -OC(O)NR4,
halo or -H,
RC13 and RC28 are independently H, halo, -OR3, -0R5, -OC(O)R5, -OC(O)NHRS, -
SR5,-
SC(O)R5, -SC(O)NHRS, -NR5R5~or -N(R5)(CO)R5~
RC14 ~ =p~ -OR6, -NR6, -H, -NC(O)R6, -OC(O)R6 or -OC(O)NR6
R3 is H, -R~, -C(O)RD or -C(O)NHR~ or a cyclic moiety (e.g., carbonate)
bridging C28 and C30
R~9 is H or ORll (e.g., OH or OMe)
where each substituent may be present in either stereochemical orientation
unless otherwise
indicated, and where each occurrence of Rl, R4, R5, R6, R~, R9, Rl~ and Rll is
independently
selected from H, aliphatic, heteroaliphatic, aryl and heteroaryl; and R8 is H,
halo, -CN, =O;
OH, -NR9R1° , OS02CF3, OS02F, OS02R4~, OCOR4~, OCONR4~R5~, or
OCON(OR4~)RS~.
Some rapalogs of Formula II differ from rapamycin only in that RC13 is -0Me
and RC14 is H;
RC14 is -0H, -O(CO)NHMe, -O-CH2- (i.e., a spiro epoxide), =NCH2CH2-OH, or -O-
phenyl;
RC13 ~ -NHC(O)Me; R4 is Me; RC24 is =NR or -NHR, where R is -OH, O-alkyl
(methyl, ethyl,
isobutyl, benzyl), -NH-alkyl or an O-carboxymethyloxime or O-
carboxamidomethyloxime at
C24; R~4 is -O(CO)NHCH(CH3)Z; RC24 is -0(CO)NC(O)CH2CH2C(O) and RC28 O-TBDMS;
RC28 or R~ is -OC(O)R, or RCS and RCS together comprise -OC(O)O- linking C28
and C30
in a six-membered ring; or Rya or RCS' is isopropoxyl, -S-phenyl, 2-thiophen-
yl, 3-indol-yl or
ally! or methallyl. See Table III and Liberles et al, 1997, Proc Natl Acad Sci
USA 94:7825-7830.
Rapalogs other than the foregoing, i.e., which contain alternative
modifications or
combinations of modifications relative to the structure of rapamycin, are
preferred for use in
CA 02318402 2000-07-14
WO 99/36553 PCT/US99/00178
practising the subject invention. Thus the improved rapalogs of this invention
are rapalogs
other than those depicted in Table III.
In rapamycin, RC~a is -OMe; RCS' is H; RC14, RC24 and RC3o are each (=O); RCi3
and RC28 are
each -OH; RC29 is OMe; and R3 and R4 are each H, all with the stereoisomerism
as shown on
page 1. Rapalogs useful in practicing this invention may contain substituents
in any of the
possible stereoisomeric orientations, and may comprise one stereoisomer
substantially free of
other stereoisomers (>90%, and preferably >95%, free from other stereoisomers
on a molar
basis) or may comprise a mixture of stereoisomers.
Also included are pharmaceutically acceptable derivatives of the foregoing
compounds,
where the phrase "pharmaceutically acceptable derivative" denotes any
pharmaceutically
acceptable salt, ester, or salt of such ester, of such compound, or any other
adduct or derivative
which, upon administration to a patient, is capable of providing (directly or
indirectly) a
rapalog as described herein, or a metabolite or residue thereof.
Pharmaceutically acceptable
derivatives thus include among others pro-drugs of the rapalogs. A pro-drug is
a derivative of a
compound, usually with significantly reduced pharmacological activity, which
contains an
additional moiety which is susceptible to removal in vivo yielding the parent
molecule as the
pharmacologically active species. An example of a pro-drug is an ester which
is cleaved in vivo
to yield a compound of interest. Various pro-drugs of rapamycin and of other
compounds, and
materials and methods for derivatizing the parent compounds to create the pro-
drugs, are
known and may be adapted to the present invention.
The term "aliphatic" as used herein includes both saturated and unsaturated,
straight
chain (i.e., unbranched), branched, cyclic, or polycyclic aliphatic
hydrocarbons, which are
optionally substituted with one or more functional groups. Unless otherwise
specified, alkyl,
other aliphatic, alkoxy and acyl groups preferably contain 1-8, and in many
cases 1-6,
contiguous aliphatic carbon atoms. Illustrative aliphatic groups thus include,
for example,
methyl, ethyl, n-propyl, isopropyl, cyclopropyl, -CH2-cyclopropyl, allyl, n-
butyl, sec-butyl,
isobutyl, tert-butyl, cyclobutyl, -CH2-cyclobutyl, n-pentyl, sec-pentyl,
isopentyl, tert-pentyl,
cyclopentyl, -CH2-cyclopentyl, n-hexyl, sec-hexyl, cyclohexyl, -CH2-cyclohexyl
moieties and
the like, which again, may bear one or more substituents.
Examples of substituents include: -OH, -OR2~, -SH, -SR2~,-CHO, =O, -COOH (or
ester,
carbamate, urea, oxime or carbonate thereof), -NH2 (or substituted amine,
amide, urea,
carbamate or guanidino derivative therof), halo, trihaloalkyl, cyano, -S02-
CF3, -OS02F,-
OS(O)2R11, -S02-NHRll, -NHS02-Rll, sulfate, sulfonate, aryl and heteroaryl
moieties. Aryl
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WO 99/36553 PCTNS99/00178
and heteroaryl substituents may themselves be substituted or unsubstituted
(e.g. mono-, di- and - ,
tri-alkoxyphenyl; methylenedioxyphenyl or ethylenedioxyphenyl; halophenyl; or -
phenyl-
C(Me)2-CH2-O-CO-[C3-C6] alkyl or alkylamino).
The term "aliphatic" is thus intended to include alkyl, alkenyl, alkynyl,
cycloalkyl,
cycloalkenyl, and cycloalkynyl moieties.
As used herein, the term "alkyl" includes both straight, branched and cyclic
alkyl groups.
An analogous convention applies to other generic terms such as "alkenyl",
"alkynyl" and the
like. Furthermore, as used herein, the language "alkyl", "alkenyl", "alkynyl"
and the like
encompasses both substituted and unsubstituted groups.
The term "alkyl" refers to groups usually having one to eight, preferably one
to six carbon
atoms. For example, "alkyl" may refer to methyl, ethyl, n-propyl, isopropyl,
cyclopropyl,
butyl, isobutyl, sec-butyl, tert-butyl, cyclobutyl, pentyl, isopentyl tert-
pentyl, cyclopentyl,
hexyl, isohexyl, cyclohexyl, and the like. Suitable substituted alkyls
include, but are not
limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2-fluoroethyl, 3-
fluoropropyl,
hydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl, benzyl, substituted benzyl and
the like.
The term "alkenyl" refers to groups usually having two to eight, preferably
two to six
carbon atoms. For example, "alkenyl" may refer to prop-2-enyl, but-2-enyl, but-
3-enyl, 2-
methylprop-2-enyl, hex-2-enyl, hex-5-enyl, 2,3-dimethylbut-2-enyl, and the
like. The
language "alkynyl," which also refers to groups having two to eight,
preferably two to six
carbons, includes, but is not limited to, prop-2-ynyl, but-2-ynyl, but-3-ynyl,
pent-2-ynyl, 3-
methylpent-4-ynyl, hex-2-ynyl, hex-5-ynyl, and the like.
The term "cycloalkyl" as used herein refers specifically to groups having
three to seven,
preferably three to ten carbon atoms. Suitable cycloalkyls include, but are
not limited to
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and the like,
which, as in the
case of other aliphatic or heteroaliphatic or heterocyclic moieties, may
optionally be
substituted.
The term "heteroaliphatic" as used herein refers to aliphatic moieties which
contain one
or more oxygen, sulfur, nitrogen, phosphorous or silicon atoms, e.g., in place
of carbon atoms.
Heteroaliphatic moieties may be branched, unbranched or cyclic and include
heterocycles such
as morpholino, pyrrolidinyl, etc.
The term "heterocycle" as used herein refers to cyclic heteroaliphatic groups
and
preferably three to ten ring atoms total, includes, but is not limited to,
oxetane,
32
CA 02318402 2000-07-14
WO 99/36553 PCT/US99/00178
tetrahydrofuranyl, tetrahydropyranyl, aziridine, azetidine, pyrrolidine,
piperidine,
morpholine, piperazine and the like.
The terms "aryl" and "heteroaryl" as used herein refer to stable mono- or
poiycyclic,
heterocyclic, polycyclic, and polyheterocyclic unsaturated moieties having 3 -
14 carbon atoms
which may be substituted or unsubstituted. Substituents include any of the
previously mentioned
substituents. Non-limiting examples of useful aryl ring groups include phenyl,
halophenyl,
alkoxyphenyl, dialkoxyphenyl, trialkoxyphenyl, alkylenedioxyphenyl, naphthyl,
phenanthryl, anthryl, phenanthro and the like. Examples of typical heteroaryl
rings include
5-membered monocyclic ring groups such as thienyl, pyrrolyl, imidazolyl,
pyrazolyl, furyl,
isothiazolyl, furazanyl, isoxazolyl, thiazolyl and the like; 6-membered
monocyclic groups such
as pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl and the like; and
polycyclic
heterocyclic ring groups such as benzo[b]thienyl, naphtho[2,3-b]thienyl,
thianthrenyl,
isobenzofuranyl, chromenyl, xanthenyl, phenoxathienyl, indolizinyl,
isoindolyl, indolyl,
indazolyl, purinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl,
quinoxalinyl,
quinazolinyl, benzothiazole, benzimidazole, tetrahydroquinoline cirmolinyl,
pteridinyl,
carbazolyl, beta-carbolinyl, phenanthridinyl, acridinyl, perimidinyl,
phenanthrolinyl,
phenazinyl, isothiazolyl, phenothiazinyl, phenoxazinyl, and the like (see e.g.
Katritzky,
Handbook of Heterocyclic Chemistry). The aryl or heteroaryl moieties may be
substituted with
one to five members selected from the group consisting of hydroxy, Cl-C8
alkoxy, Cl-C8
branched or straight-chain alkyl, acyloxy, carbamoyl, amino, N-acylamino,
vitro, halo,
trihalomethyl, cyano, and carboxyl. Aryl moieties thus include, e.g. phenyl;
substituted phenyl
bearing one or more substituents selected from groups including. halo such as
chloro or fluoro,
hydroxy, C1-C6 alkyl, acyl, acyloxy, Cl-C6 alkoxy (such as methoxy or ethoxy,
including
among others dialkoxyphenyl moieties such as 2,3-, 2,4-, 2,5-, 3,4- or 3,5-
dimethoxy or diethoxy
phenyl or such as methylenedioxyphenyl, or 3-methoxy-5-ethoxyphenyl; or
trisubstituted
phenyl, such as trialkoxy (e.g., 3,4,5-trimethoxy or ethoxyphenyl), 3,5-
dimethoxy-4-chloro-
phenyl, etc.), amino, -S02NH2, -S02NH(aliphatic), -S02N(aliphatic)2, -O-
aliphatic-
COOH, and -O-aliphatic-NH2 (which may contain one or two N-aliphatic or N-acyl
substituents).
A "halo" substituent according to the present invention may be a fluoro,
chloro, bromo or
iodo substituent. Fluoro is often the preferred halogen.
Compounds of formula II, exclusive of any compounds depicted in Table III, are
of special
interest and constitute an important class of novel compounds. Compounds of
this class may
differ from rapamycin with respect to one, two, three, four, five, six or
seven substituent
moieties. This class includes among others rapalogs with modifications,
relative to rapamycin,
33
CA 02318402 2000-07-14
WO 99/36553 PCT/US99/00178
at C7 and C13; C7 and C14; C7 and ~; C7 and C43; C7 and C24; C7 and C28; C7
and C'.30; C7, C13
and C14; C7, C13 and ~; C7, C13 and C43; C7, C13 and C24; C7, C13 and C28; C7,
C13 and C30; C7,
C14 and a; C7, C14 and C43; C7, C14 and C24; C7, C14 and C28; C7, C14 and C30;
C7, a and C24;
C7, ~ and C28; C7, ~ and C30; C7, C24 and C30; C7, C24, C30 and ~; C7, C24,
C30 and C13; C7, C24,
C30 and C14; C24, C30 and C13; C24, C30 and a; C24, C30 and C14; and C24, C30,
C13 and ~,
exclusive of any compounds depicted in Table III or otherwise previously
reported publicly.
One subset of improved rapalogs of special interest for practicing the methods
of this
invention are those compounds of formula II (or pharmaceutically acceptable
derivatives
thereof) in which Rya is a moiety other than OMe. This subset ("C7 rapalogs")
includes
compounds in which one of R~~a and R~~' is H and the other is selected from
substituted or
unsubstituted alkenyl, aryl, heteroaryl or -Z-aliphatic, Z-aryl, -Z-
heteroaryl, or Z-acyl,
where Z and Z' are independently O, S or NH and acyl comprises -CHO, -(C=O)-
aliphatic;
(C=O)-aryl, -(C=O)-heteroaryl, -(C=O)-Z'-aliphatic, -(C=O)-Z'-aryl, -(C=O)-Z'-
heteroaryl.
In certain embodiments of this subset, R~~a and Rte' are independently
selected from the
following groups: H; a substituted or unsubstituted two to eight carbon
straightchain, branched
or cyclic alkenyl, alkoxyl or alkylmercapto; and a substituted or
unsubstituted aryl ,
heteroaryl, aryloxy or heteroaryloxy, arylmercapto or heteroarylmercapto.
Compounds of this
subset include among others those in which R~~a is H; (together with R~~') =O;
alkoxy;
alkylmercapto; amino (1°, 2° or 3°); amido; carbamate;
aryl or substituted aryl; phenyl or
substituted phenyl; substituted or unsubstituted heteroaryl such as
substituted or unsubstituted
thiophenyl, furyl, indolyl, etc.; or benzyloxy or substituted benzyloxy. Other
illustrative C7
rapalogs and types of C7 rapalogs which may be used in practicing the methods
of this
invention include those in which one of R~~a and Rte' is H and the other is
selected from -OEt,-
O-propyl, -O-butyl, -OCH2CH2-0H, -O-benzyl, -O-substituted benzyl (including
e.g., 3-nitro-,
4-chloro-, 3-iodo-4-diazo-, 3,4-dimethoxy-, and 2-methoxy-), -S-Me, -S-phenyl,
-O(CO)Me;
allyl, -CH2C(Me)=CH2, -OCH2-CCH, -OCH2-CC-Me, -OCH2-CC-Et, -OCH2-CC-CH20H, or-
2,4-dimethoxyphenyl, 2,4,6-trimethoxyphenyl, furanyl, thiophen-yl,
methylthiophen-yl,
pyrolyl and indolyl. In the foregoing types of rapalogs, the hydroxy
substituent at C43 may be
present in either stereochemical orientation or may be modified as described
elsewhere herein.
C7 rapalogs may further vary from rapamycin at one, two, three, four, five or
more other
positions as well. C7 rapalogs other than those depicted in Table III are
novel and are
encompassed by this invention as compositions of matter per se.
Another subset of improved rapalogsof special interest in the practice of the
various
methods of the invention are C30,C24 rapalogs of formula II , i.e., rapalogs
of formula II in
34
CA 02318402 2000-07-14
WO 99/36553 PCT/US99/00178
~,"~~ RC3o ~d RC24 are both other than (=O). Of special interest are those
C30,C24 rapalogs - ,
in which R~~a is a moiety other than OMe. In certain embodiments of this
subset, R~~a and R~~'
are independently selected from -H, -ORl, -SRI, -OC(O)Rl or -OC(O)NHRl, -NHRl;
NHC(O)Rl, -NH-S02-Rl and -R2, where R2 = substituted aryl or allyl or
alkylaryl (e.g. benzyl
or substituted benzyl), so long as one of Rya and Rte' is H. In certain
embodiments of this subset,
R~ and R~4 are both -OH, e.g. in the "S" configuration. In other embodiments
R~~ and R~24
are independently selected from OR3. This subset includes among others all
rapalogs in which
R~ and Ra4 are OH and one of Rc~a and Rte' comprises any of the replacement
substituents at
that position specified for formula II, including any of the C7 substituents
identified in
compounds of Tables II or III. This subset includes among others rapalogs
which differ from
rapamycin with respect to the moiety ~. For instance, this subset includes
compounds of the
formula:
1 II
O OH~OH
~~O O MeO~~~~OH
Rc~aR~
7
III
where at least one of R~~a and R~~' is other than -OMe. Alternative
substituents for R~~a
and/or R~ are as disclosed elsewhere herein. Of special interest are compounds
in which one
of Rya and R~~' is cyclic aliphatic, aryl, heterocyclic or heteroaryl, which
may be optionally
substituted. Other compounds within this subset include those in which one,
two, three, four or
five of the hydroxyl groups is epimerized, fluorinated, alkylated, acylated or
otherwise
modified via other ester, carbamate, carbonate or urea formation. An
illustrative compound for
example is the compound of formula III in which the hydroxyl group at C43 is
epimerized and
the hydroxyl groups at C28 and C30 are alkylated, acylated or linked via
carbonate formation.
Another subset of improved rapalogs of special interest are those compounds of
formula II in
which one or both of R~13 and R~ is F. In various embodiments of this subset,
one, two, three,
CA 02318402 2000-07-14
WO 99136553 PCT/US99/00178
four or five other substituents in formula II differ from the substituents
found in ravamycin. For -
instance, this subset includes C13 fluororapalogs, C28 fluororapalogs and C13,
C28-
difluororapalogs of the following structures, where Rya and R~~' are as
previously defined:
~~ O O ~ OH ~ O
O ~
OH
as
as
F , 00 MeCJ"~ O F O
Me0"'
,e O
O
,a O QMa ~ ~
,~
C7a
,3Q -
Me Me
~O O ~ OH C~O O ~ OH
F a 00 O
MeCT"~ O F
Me0'~
OO
~
~C7a
~o
0 0Ma
i i '.
H O,, H O,,
Me v Me
24
I F 24 =
~O O I F
o
O
2 N
O ~4 O O M e O''~O
O
2
M a O'~
O
O R
H 3
' H
~ 4
c~a
~
s0 QMa
i i . ~ ~ i
H H
Me v Me
24 ,"~~ 24
' 1 O O I F
O F
2 N
O is 00 Me03 2
O O
H
O R
Me0''
H ~4
~a
3
~3Q QMe ~ = Rc~b
~ i i . ~/ i
36
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WO 99/36553 PCT/US99/00178
O O ~ F
2
MeO,° O
3
C7b
Q '/R 1
cy
C~ O O ~ F
O
14 O ~ , M80'~~ 3 O
1
7
The 13-fluoro rapalogs, including in particular 13-fluoro rapamycin and
analogs and
derivatives thereof containing various substituents which do not abolish
immunosuppressive
activity in rapamycin itself, are of interest as immunosuppressants.
A:-~ interesting intersection of some of the foregoing subsets of compounds is
the set of
improved rapalogs comprising compounds of formula II, or pharmaceutically
acceptable
derivatives thereof, in which R~24 and Rao are both other than (=O) and one or
both of R~13
and R~~ is F. This set includes, inter alia, 24,30-tetrahydro-13-F rapalogs,
24,30-tetrahydro-
28-F rapalogs and 24,30-tetrahydro-13,28-diF rapalogs, as well as C7 variants
of any of the
foregoing, in which R~~a is other than OMe. A portion of that set is
illustrated by the following
structure, where R~~a and R~~' are as previously defined:
HO.,,
MeOr v
"", ~
O OH ~ Of
,,,
F 14 O ~ ~a Me0
1 Q ~Rc~b 1
7 f' _ ~
These compounds may be further derivatized, e.g., by modifications at one or
both of R~i4 and
R~'~ relative to the C14 and C43 substituents in rapamycin itself.
37
CA 02318402 2000-07-14
WO 99/36553 PCT/US99/00178
Another subset of improved rapalogsof special interest are those compounds of
formula II in _ ~;
which RC14 ys over than O, OH or H, e.g., compounds wherein RC14 is -OR6, -
NR6, -NC(O)R6;
OC(O)R6 or -OC(O)NR6, with or without one or more other modifications relative
to
rapamycin.
Another subset of improved rapalogs of interest are those compounds of formula
II in which
Rcl3 ~ over than an alkoxyl group comprising a Cl-C4 alkyl moiety, with or
without one or
more other modifications at other positions relative to rapamycin. For
example, this subset
includes rapalogs which differ in structure from rapamycin by virtue of
possessing (a) in place of
OH at C13, a replacement substituent R~13 which is other than C1-C4 alkoxy,
and (b) in place
of Me0 at C7, replacement substituents Rya and R~ as defined above.
Another subset of improved rapalogs of interest are those compounds of formula
II in which
R~24 is other than =O, again, with or without one or more other modifications
at other positions
relative to rapamycin.
Another subset of improved rapalogs which is of special interest in practicing
the methods
of this invention include those compounds of formula II which share the
stereoisomerism of
rapamycin and in which RC~a is -0Me wherein R~ is not =O, RC24 is not =O, RC13
is not -OH,
RC14 is not =O and/or R3 and/or R4 are not H.
Other improved rapalogs of interest include compounds of formula II in which
R~14 is OH.
Furthermore, this invention encompasses improved rapalogs in which one or more
of the
carbon-carbon double bonds at the 1,2, 3,4 or 5,6 positions in rapamycin are
saturated, alone or in
combination with a modification elsewhere in the molecule, e.g. at one or more
of C7, C13, C43,
C24 C28 and/or C30. It should also be appreciated that the C3,C4 double bond
may be
epoxidized; that the C6 methyl group may be replaced with -CH20H or -CH20Me;
that the
C43 hydroxy may be converted to F, Cl or H or other substituent; and that the
C42 methoxy
moiety may be demethylated, in any of the compounds disclosed herein, using
methods known in
the art. Likewise, moiety "~ may be replaced with any of the following
Ra
~ HOOC
H~~~~~ ~ ~ N~ H3C0 C
HO'~~~ O HO~~~
HCO
38
CA 02318402 2000-07-14
WO 99/36553 PCT/US99/00178
Synthetic guidance
The production of rapamycin by fermentation and by total synthesis is known.
The
production of a number of rapalogs as fermentation products is also known.
These include among
others rapalogs bearing alternative moieties to the characteristic cyclohexyl
ring or pipecolate
ring of rapamycin, as well as C7-desmethyl-rapamycin, C29-desmethyl-rapamycin
and C29-
desrnethoxyrapamycin.
Methods and materials for effecting various chemical transformations of
rapamycin and
structurally related macrolides are known in the art, as are methods for
obtaining rapamycin
and various rapalogs by fermentation. Many such chemical transformations of
rapamycin and
various rapalogs are disclosed in the patent documents identified in Table I,
above, which serve
to illustrate the level of skill and knowledge in the art of chemical
synthesis and product
recovery, purification and formulation which may be applied in practicing the
subject
invention. The following representative transformations and/or references
which can be
employed to produce the desired rapalogs are illustrative:
--
ring position literature reference
modified
C7 Luengo, et al. JOC 59, 6512 (1995); Chem & Biol
2(7), 471-481 (1995)
C-13 C13->F: protect C28 and C43, rxn at 0
C-14 Schubert, et al. Angew Chem Int Ed Engl 23, 167
(1984).
C-20 Nelson, US Patent 5,387,680
C-24 US Patent 5,373,014; 5,378,836
Lane, et al. Synthesis 1975, p136.
C-30 Luengo et al. Tet. Lett. 35, 6469 (1994)
various positionsOr et al, US Patent Nos. 5,527,907 and 5,583,139
Luengo, WO 94/02136; Cotters et al, WO 95/16691
Approaches to the synthesis of the various fluoro and difluoro rapalogs are
presented
schematically below:
39
CA 02318402 2000-07-14
WO 99/36553 PCT/US99/00178
' O O ~ OH
28
HO 14 O O Me0''~ O
O OMe
/ /
DAST (4eq); -42 °C DAST (2.2 eq); -78 °C
43 HO. 43 _
C ~O O ~ F ~~O O ( F
14 O ~O Me0''~ O H 14 O TO~ Me0''~ O
13 O flMe
13 O QMe
~~,, ; ~ /
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WO 99/36553 PCTNS99/00178
H0,,43
TESO,,~, 43 . ,
M
Me0
OH
28 TESOTf; 2,&lutidine ~p O ~ OTES
Hpp 14 p p M8~ p 28
O OMe -78 ~C H O to O O MeO~ O
/ / 13 O 9Me
.~, ~ ~ / /
DAST (1.5 eq) -42 °C
TESO~ 43
M
~.O O , ~ OTES
ze
O O MeO~~ O
3 O ~_ Me
.~, ~ / /
HF/Py
HO,a3 THF RC7a = ~
Me0 HO ,43
Me
n_ M
p O . ~ OH
11II~~ ', 2a / OMe ~'
FO 14 O O Me0 O - i ~O o . ~ OH
13 p
.~, OMB / / TFA o t4 0 o~~s
t3 ~O _
.., / / /
1. Smith, A. B. et al, J. Am. Chem. Soa 1997, 119, 962-973.
2. Middleton,W. J, J. Org. Chem., 1875, 40, 574-578.
notes: The tri-isopropylsilyl homolog, TIPs, may be used in place of the
triethylsilyl protecting moiety, TES.
The DAST reaction on the doubly protected rapamycin may be conducted at 0 C if
desired.
41
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WO 99/36553 PCTNS99/00178
An approach to the synthesis of various 24,30-tetrahydro rapalogs is
illustrated below:
NaBH4, CeCl3~r1-lz0
MeOH, -78'C
1,3 dimethoxybenzene
TFA, CIizCl2, -60'C
By way of further example, starting with 13-fluoro rapamycin instead of
rapamycin yields the
corresponding 13-fluoro-24,30-tetrahydro C7 rapalog.
One approach for the synthesis of other C13 derivatives is illustrated below:
42
CA 02318402 2000-07-14
WO 99/36553 PCT/US99/00178
SOCI2, Pyridine 'N~-O O
~O " Me0''~ O
3 QMe
~,, ~ i i
Me
NH40H THF
HO... Ar =
OMe
Me0
Ha,. HF/pyridine
~ a
~O O ~ H .~ Me0
I
H2 O O O Me0''~ O ~ onne , _
Ar ' ~O O ~ I
TFA
.,, ~ ~ ~ H2 O O O Me0''
QMe
i i
7 / v v ""
See Donald, D. et al. WO 91/13889
Additionally, it is contemplated that rapalogs for use in this invention as
well as
intermediates for the production of such rapalogs may be prepared by directed
biosynthesis, e.g.
as described by ICatz et al, WO 93/13663 and by Cane et al, WO 9702358. See
also Khaw et al,
1998, J. Bacteriology ~$Q(4):809-814 for additional biological methods.
Novel rapalogs of this invention may be prepared by one of ordinary skill in
this art
relying upon methods and materials known in the art as guided by the
disclosure presented
herein. For instance, methods and materials may be adapted from known methods
set forth or
referenced in the documents cited above, the full contents of which are
incorporated herein by
reference. Additional guidance and examples are provided herein by way of
illustration and
further guidance to the practitioner. It should be understood that the chemist
of ordinary skill
in this art would be readily able to make modifications to the foregoing, e.g.
to add appropriate
protecting groups to sensitive moieties during synthesis, followed by removal
of the protecting
groups when no longer needed or desired, and would be readily capable of
determining other
synthetic approaches.
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WO 99/36553 PCT/US99/00178
FKBP domains and fusion proteins
The FKBP fusion protein comprises at least one FKBP domain containing all or
part of the
peptide sequence of an FKBP domain and at least one heterologous action
domain. This chimeric
protein must be capable of binding to an improved rapalog of this invention,
preferably with a
Kd value below about 100 nM, more preferably below about 10 nM and even more
preferably
below about 1 nM, as measured by direct binding measurement (e.g. fluorescence
quenching),
competition binding measurement (e.g. versus FK506), inhibition of FKBP enzyme
activity
(rotamase), or other assay methodology. Typically the chimeric protein will
contain one or
more protein domains comprising peptide sequence selected from that of a
naturally occurring
FKBP protein such as human FKBP12, e.g. as described in International Patent
Application
PCT/US94/01617. That peptide sequence may be modified to adjust the binding
specificity,
usually with replacement, insertion or deletion of 10 or fewer, preferably 5
or fewer, amino acid
residues. Such modifications are elected in certain embodiments to yield one
or both of the
following binding profiles: (a) binding of an improved rapalog to the modified
FKBP domain, or
chimera containing it, preferably at least one, and more preferably at least
two, and even more
preferably three or four or more, orders of magnitude better (by any measure)
than to FKBP12 or
the FKBP endogenous to the host cells to be engineered; and (b) binding of the
FKBP:rapalog
complex to the FRB fusion protein, preferably at least one, and more
preferably at least two,
and even more preferably at least three, orders of magnitude better (by any
measure) than to
the FRAP or other FRB-containing protein endogenous to the host cell to be
engineered.
The FKBP chimera also contains at least one heterologous action domain, i.e.,
a protein
domain containing non-FKBP peptide sequence. The action domain may be a DNA-
binding
domain, transcription activation domain, cellular localization domain,
intracellular signal
transduction domain, etc., e.g. as described elsewhere herein or in
PCT/US94/01617 or the other
cited references. Generally speaking, the action domain is capable of
directing the chimeric
protein to a selected cellular location or of initiating a biological effect
upon association or
aggregation with another action domain, for instance, upon multimerization of
proteins
containing the same or different action domains.
A recombinant nucleic acid encoding such a fusion protein will be capable of
selectively
hybridizing to a DNA encoding the parent FKBP protein, e.g. human FKBP12, or
would be
capable of such hybridization but for the degeneracy of the genetic code.
Since these chimeric
proteins contain an action domain derived from another protein, e.g. Gal4,
VP16, FAS, CD3 zeta
chain, etc., the recombinant DNA encoding the chimeric protein will also be
capable of
44
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WO 99/36553 PCT/US99/00178
selectively hybridizing to a DNA encoding that other protein, or would be
capable of such
hybridization but for the degeneracy of the genetic code.
FKBP fusion proteins of this invention, as well as FRB fusion proteins
discussed in further
detail below, may contain one or more copies of one or more different ligand
binding domains and
one or more copies of one or more action domains. The ligand binding domains)
(i.e., FKBP and
FRB domains) may be N-terminal, C-terminal, or interspersed with respect to
the action
domain(s). Embodiments involving multiple copies of a ligand binding domain
usually have 2 ,
3 or 4 such copies. For example, an FKBP fusion protein may contain 2, 3 or 4
FKBP domains. The
various domains of the FKBP fusion proteins (and of the FRB fusion proteins
discussed below)
are optionally separated by linking peptide regions which may be derived from
one of the
adjacent domains or may be heterologous.
Illustrative examples of FKBP fusion proteins useful in the practice of this
invention
include the FKBP fusion proteins disclosed in PCT/US94/01617 (Stanford &
Harvard),
PCT/US94/08008 (Stanford & Harvard), Spencer et al (supra), PCT/US95/10591
(ARIAD),
PCT/US95/06722 (Mitotix, Inc.) and other references cited herein; the FKBP
fusion proteins
disclosed in the examples which follow; variants of any of the foregoing FKBP
fusion proteins
which contain up to 10 (preferably 1-5) amino acid insertions, deletions or
substitutions in one or
more of the FKBP domains and which are still capable of binding to rapamycin
or to a rapalog;
variants of any of the foregoing FKBP fusion proteins which contain one or
more copies of an
FKBP domain which is encoded by a DNA sequence capable of selectively
hybridizing to a
DNA sequence encoding a naturally occurring FKBP domain and which are still
capable of
binding to rapamycin or to a rapalog; variants of any of the foregoing in
which one or more
heterologous action domains are deleted, replaced or supplemented with a
different
heterologous action domain; variants of any of the foregoing FKBP fusion
proteins which are
capable of binding to rapamycin or a rapalog and which contain an FKBP domain
derived from
a non-human source; and variants of any of the foregoing FKBP fusion proteins
which contain
one or more amino acid residues corresponding to Tyr26, Phe36, Asp37, Arg42,
Phe46, Phe48,
G1u54, Va155, or Phe99 of human FKBP12 in which one or more of those amino
acid residues is
replaced by a different amino acid, the variant being capable of binding to
rapamycin or a
rapalog.
For instance, in a number of cases the FKBP fusion proteins comprise multiple
copies of an
FKBP domain containing amino acids 1-107 of human FKBP12, separated by the 2-
amino acid
linker Thr-Arg encoded by ACTAGA, the ligation product of DNAs digested
respectively with
the restriction endonucleases SpeI and XbaI. The following table provides
illustrative subsets of
mutant FKBP domains based on the foregoing FKBP12 sequence:
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WO 99/36553 PCT/US99/00178
Illustrative Mutant FKBPs
F36A Y26V F46A W59A
F36V Y26S F48H H87W
F36M D37A F48L H87R
F36S I90A F48A F36V/F99A
F99A I91A E54A F36V/F99G
F99G F46H E54K F36M/F99A
Y26A F46L V55A F36M/F99G
note: Entries identify the native amino acid by single letter code and
sequence position,
followed by the replacement amino acid in the mutant. Thus, F36V designates a
human
FKBP12 sequence in which phenylalanine at position 36 is replaced by valine.
F36V/F99A
indicates a double mutation in which phenylalanine at positions 36 and 99 are
replaced by
valine and alanine, respectively.
FRB domains and fusion proteins
The FRB fusion protein comprises at least one FRB domain (which may comprise
all or part
of the peptide sequence of a FRAP protein or a variant thereof, as described
elsewhere) and at
least one heterologous effector domain.
Generally speaking, the FRB domain, or a chimeric protein encompassing it, is
encoded by a
DNA molecule capable of hybridizing selectively to a DNA molecule encoding a
protein
comprising a naturally occurring FRB domain, e.g. a DNA molecule encoding a
human or other
mammalian FRAP protein or one of yeast proteins, Tor-1 or Tor-2 or the
previously mentioned
Candida FRB-containing protein. FRB domains of this invention include those
which are
capable of binding to a complex of an FKBP protein and an improved rapalog of
this invention.
The FRB fusion protein must be capable of binding to the complex formed by the
FKBP fusion
protein with an improved rapalog of this invention. Preferably, the FRB fusion
protein binds to
that complex with a Kd value below 200 ~.M, more preferably below 10~.M, as
measured by
conventional methods. The FRB domain will be of sufficient length and
composition to maintain
high affinity for a complex of the rapalog with the FKBP fusion protein. In
some embodiments
46
CA 02318402 2000-07-14
WO 99/36553 PCT/US99/00178
the FRB domain spans fewer than about 150 amino acids in length, and in some
cases fewer than. ,
about 100 amino acids. One such region comprises a 133 amino acid region of
human FRAP
extending from Va12012 through Tyr2144. See Chiu et al, 1994, Proc. Natl.
Acad. Sci. USA
91:12574-12578. An FRB region of particular interest spans G1u2025 through
G1n2114 of human
FRAP and retains affinity for a FKBP12-rapamycin complex or for FKBP-rapalog
complex. In
some embodiments Q2214 is removed from the 90-amino acid sequence rendering
this an 89-
amino acid FRB domain. The FRB peptide sequence may be modified to adjust the
binding
specificity, usually with replacement, insertion or deletion, of 10 or fewer,
preferably 5 or
fewer, amino acids. Such modifications are elected in certain embodiments to
achieve a
preference towards formation of the complex comprising one or more molecules
of the FKBP
fusion protein, FRB fusion protein and an improved rapalog over formation of
complexes of
endogenous FKBP and FRAP proteins with the rapalog. Preferably that preference
is at least
one, and more preferably at least two, and even more preferably three, orders
of magnitude (by
any measure).
A recombinant DNA encoding such a protein will be capable of selectively
hybridizing to a
DNA encoding a FRAP species, or would be capable of such hybridization but for
the degeneracy
of the genetic code. Again, since these chimeric proteins contain an effector
domain derived from
another protein, e.g. Gal4, VP16, Fas, CD3 zeta chain, etc., the recombinant
DNA encoding the
chimeric protein will be capable of selectively hybridizing to a DNA encoding
that other
protein, or would be capable of such hybridization but for the degeneracy of
the genetic code.
Illustrative examples of FRB chimeras useful in the practice of this invention
include those
disclosed in the examples which follow, variants thereof in which one or more
of the
heterologous domains are replaced with alternative heterologous domains or
supplemented
with one or more additional heterologous domains, variants in which one or
more of the FRB
domains is a domain of non-human peptide sequence origin (such as Tor 2 or
Candida for
example), and variants in which the FRB domain is modified by amino acid
substitution,
replacement or insertion as described herein, so long as the chimera is
capable of binding to a
complex formed by an FKBP protein and an improved rapalog of this invention.
An illustrative
FRB fusion protein conkains one or more FRBs of at least 89-amino acids,
containing a sequence
spanning at least residues 2025-2113 of human FRAP, separated by the linker
Thr-Arg formed
by ligation of SpeI-XbaI sites as mentioned previously. It should be
appreciated that such
restriction sites or linkers in any of the fusion proteins of this invention
may be deleted,
replaced or extended using conventional techniques such as site-directed
mutagenesis.
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Mixed chimeric proteins
A third type of chimeric protein comprises one or more FKBP domains, one or
more
heterologous effector domains,and one or more FRB domains as described for the
FRB fusion
proteins.
Mixed chimeric protein molecules are capable of forming homodimeric or
homomultimeric
protein complexes in the presence of an improved rapalog to which they bind.
Embodiments
involving mixed chimeras have the advantage of requiring the introduction into
cells of a single
recombinant nucleic acid construct in place of two recombinant nucleic acid
constructs otherwise
required to direct the expression of both an FKBP fusion protein and a FRB
fusion protein.
A recombinant DNA encoding a mixed chimeric protein will be capable of
selectively
hybridizing to a DNA encoding an FKBP protein, a DNA encoding FRAP, and a
heterologous
DNA sequence encoding the protein from which one or more effector domains is
derived (e.g.
Gal4, VP16, Fas, CD3 zeta chain, etc.), or would be capable of such
hybridization but for the
degeneracy of the genetic code.
Heternlogous domains
As mentioned above, the heterologous effector domains of the FKBP and FRB
fusion proteins
are protein domains which, upon mutual association of the chimeric proteins
bearing them, are
capable of triggering (or inhibiting) DNA-binding and/or transcription of a
target gene;
actuating cell growth, differentiation, proliferation or apoptosis; directing
proteins to a
particular celllular location; or actuating other biological events.
Embodiments involving regulatable gene transcription involve the use of target
gene
constructs which comprise a target gene (which encodes a polypeptide,
antisense RNA,
ribozyme, etc. of interest) under the transcriptional control of a DNA element
responsive to the
association or multimerization of the heterologous domains of the 1st and 2d
chimeric proteins.
In embodiments of the invention involving direct activation of transcription,
the
heterologous domains of the 1st and 2d chimeric proteins comprise a DNA
binding domain such
as Gal4 or a chimeric DNA binding domain such as ZFHDI, discussed below,and a
transcriptional activating domain such as those derived from VP16 or p65,
respectively. The
multimerization of a chimeric protein containing such a transcriptional
activating domain to a
chimeric protein containing a DNA binding domain targets the transcriptional
activator to the
promoter element to which the DNA binding domain binds, and thus activates the
transcription
of a target gene linked to that promoter element. Foregoing the transcription
activation domain
48
CA 02318402 2000-07-14
WO 99/36553 PCT/US99/00178
or substituting a repressor domain (see PCT/US94/01617) in place of a
transcripN~n activation
domain provides an analogous chimera useful for inhibiting transcription of a
target gene.
Composite DNA binding domains and DNA sequences to which they bind are
disclosed in
Pomerantz et al,1995, supra, the contents of which are incorporated herein by
reference. Such
composite DNA binding domains may be used as DNA binding domains in the
practice of this
invention, together with a target gene construct containing the cognate DNA
sequences to which
the composite DBD binds.
In embodiments involving indirect activation of transcription, the
heterologous domains of
the chimeras are effector domains of signaling proteins which upon aggregation
or
multimerization trigger the activation of transcription under the control of a
responsive
promoter. For example, the signaling domain may be the intracellular domain of
the zeta
suburut of the T cell receptor, which upon aggregation, triggers transcription
of a gene linked to
the IL-2 promoter or a derivative thereof (e.g. iterated NF-AT binding sites).
In another aspect of the invention, the heterologous domains are protein
domains which
upon mutual association are capable of triggering cell death. Examples of such
domains are the
intracellular domains of the Fas antigen or of the TNF Rl. Chimeric proteins
containing a Fas
domain can be designed and prepared by analogy to the disclosure of
PCT/US94/01617.
Engineered receptor domains
As noted previously, the FKBP and FRB domains may contain peptide sequence
selected
from the peptide sequences of naturally occurring FKBP and FRB domains.
Naturally occurring
sequences include those of human FKBP12 and the FRB domain of human FRAP.
Alternatively,
the peptide sequences may be derived from such naturally occurring peptide
sequences but
contain generally up to 10, and preferably 1-5, mutations in one or both such
peptide sequences.
As disclosed in greater detail elswhere herein, such mutations can confer a
number of important
features. For instance, an FKBP domain may be modified such that it is capable
of binding an
improved rapalog preferentially, i.e. at least one, preferably two, and even
more preferably
three or four or more orders of magnitude more effectively, with respect to
rapalog binding by
the unmodified FKBP domain. An FRB domain may be modified such that it is
capable of
binding a (modified or unmodified) FKBP:rapalog complex preferentially, i.e.
at least one,
preferably two, and even more preferably three orders of magnitude more
effectively, with
respect to the unmodified FRB domain. FKBP and FRB domains may be modified
such that they
are capable of forming a tripartite complex with an improved rapalog,
preferentially, i.e. at
49
CA 02318402 2000-07-14
WO 99136553 PCT/US99100178
least one, preferably two, and even more preferably three orders of magnitude
more effectively;
with respect to unmodified FKBP and FRB domains.
(a) FKBP
Methods for identifying FKBP mutations that confer enhanced ability to bind
derivatives
of FK506 containing various substituents ("bumps") were disclosed in
PCT/US94/01617. Similar
strategies can be used to obtain modified FKBPs that preferentially bind
bumped rapamycin
derivatives, i.e., rapalogs. The structure of the complex between rapamycin
and FKBP12 is
known (see for example Van Duyne et al., J. Am. Chem. Soc. (1991) 113, 7433-
7434). Such data
can be used to reveal amino acid residues that would clash with various
rapalog substituents. In
this approach, molecular modelling is used to identify candidate amino acid
substitutions in
the FKBP domain that would accommodate the rapalog substituent(s), and site-
directed
mutagenesis may then be used to engineer the protein mutations so identified.
The mutants are
expressed by standard methods and their binding affinity for the rapalogs
measured, for
example by inhibition of rotamase activity, or by competition for binding with
a molecule such
as FK506, if the mutant retains appropriate activity/affinity.
More particularly, we contemplate that certain improved rapalogs of this
invention, e.g.
rapalogs with modifications relative to rapamycin at C-13 or C-14 bind
preferentially to
FKBPs in which one or more of the residues, Tyr26, Phe36, Asp37, Tyr82 and
Phe99, are
substituted with amino acids that have smaller side chains (such as Gly, Ala,
Val, Met and
Ser). Examples of mutant FKBPs with modifications at positions 26 or 36 are
noted in the
"Illustrative Mutant FKBPs" table above. Similarly, we contemplate that
rapalogs with
modifications at C20 (i.e., rapalogs in which R4 is other than -H) bind
preferentially to FKBPs
in which Tyr82 and/or I1e56 are replaced by other amino acids, especially
those with smaller
side chains. In a further example, we contemplate that rapalogs bearing
modifications at C24
(i.e., in which W is other than =O) bind preferentially to FKBPs in which one
or more of Phe46,
Phe48 and Va155 are replaced by other amino acids, again especially those with
smaller side
chains. Moreover, we envisage that rapalogs with modifications at C28 and/or
C30 (i.e., in
which R3 is other than H and/or V is other than =O) bind preferentially to
FKBPs in which
G1u54 is replaced by another amino acid, especially one with a smaller side
chain. In all of the
above examples, single or multiple amino acid substitutions may be made.
Again, specific
examples are noted in the previous table.
An alternative to iterative engineering and testing of single or multiple
mutants is to co-
randomize structurally-identified residues that are or would be in contact
with or near one or
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more rapalog or rapamycin substituents. A collection of polypeptides
containing FKBP domains
randomized at the identified positions (such as are noted in the foregoing
paragraph) is
prepared e.g. using conventional synthetic or genetic methods. Such a
collection represents a set
of FKBP domains containing replacement amino acids at one or more of such
positions. The
collection is screened and FKBP variants are selected which possess the
desired rapalog binding
properties. In general, randomizing several residues simultaneously is
expected to yield
compensating mutants of higher affinity and specificity for a given bumped
rapalog as it
maximizes the likelihood of beneficial cooperative interactions between
sidechains.
Techniques for preparing libraries randomized at discrete positions are known
and include
primer-directed mutagenesis using degenerate oligonucleotides, PCR with
degenerate
oligonucleotides, and cassette mutagenesis with degenerate oligonucleotides
(see for example
lowman, H.B, 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 references
therein).
We further contemplate that in many cases, randomization of only the few
residues in or
near direct contact with a given position in rapamycin may not completely
explore all the
possible variations in FKBP conformation that could optimally accommodate a
rapalog
substituent (bump). Thus the construction is also envisaged of unbiased
libraries containing
random substitutions that are not based on structural considerations, to
identify subtle mutations
or combinations thereof that confer preferential binding to bumped rapalogs.
Several suitable
mutagenesis schemes have been described, including alanine-scanning
mutagenesis (Cunningham
and Wells (1989) Science 244,1081-1085), PCR misincorporaHon mutagenesis (see
eg. 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). These techniques
produce
libraries of random mutants, or sets of single mutants, that are then searched
by screening or
selection approaches.
In many cases, an effective strategy to identify the best mutants for
preferential binding of a
given bump is a combination of structure-based and unbiased approaches. See
Clackson and
Wells,1994, Trends Biotechnology 12, 173-184 (review). For example we
contemplate the
construction of libraries in which key contact residues are randomized by PCR
with degenerate
oligonucleotides, but with amplification performed using error-promoting
conditions to
introduce further mutations at random sites. A further example is the
combination of component
DNA fragments from structure-based and unbiased random libraries using DNA
shuffling.
Screening of libraries for desirable mutations may be performed by use of a
yeast 2-hybrid
system (Fields and Song (1989) Nature 340, 245-246). For example, an FRB-VP16
fusion may be
introduced into one vector, and a library of randomized FKBP sequences cloned
into a separate
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GA1,4 fusion vector. Yeast co-transformants are treated with rapalog, and
those harboring
complementary FKBP mutants are identified by for example beta-galactosidase or
luciferase
production (a screen), or survival on plates lacking an essential nutrient (a
selection), as
appropriate for the vectors used. The requirement for bumped rapamycin to
bridge the FKBP-
FRAP interaction is a useful screen to eliminate false positives.
A further strategy for isolating modified ligand-binding domains from
libraries of FKBP
(or FRB) mutants utilizes a genetic selection for functional dimer formation
described by Hu et.
al. (Hu, J.C., et al. 1990. Science. 250:1400-1403; for review see Hu, J.C.
1995. Structure. 3:431-
433). This strategy utilizes the fact that the bacteriophage lambda repressor
cI binds to DNA
as a homodimer and that binding of such homodimers to operator DNA prevents
transcription of
phage genes involved in the lytic pathway of the phage life cycle. Thus,
bacterial cells
expressing functional lambda repressor are immune to lysis by superinfecting
phage lambda.
Repressor protein comprises an amino terminal DNA binding domain (amino acids
1-92), joined
by a 40 amino acid flexible linker to a carboxy terminal dimerization domain.
The isolated N-
terminal domain binds to DNA with low affinity due to inefficient dimer
formation. High
affinity DNA binding can be restored with heterologous dimerization domains
such as the
GCN4 "leucine zipper". Hu et al have described a system in which phage
immunity is used as a
genetic selection to isolate GCN4 leucine zipper mutants capable of mediating
lambda repressor
dimer formation from a large population of sequences {Hu et. al.,1990).
For example, to use the lambda repressor system to identify FRAP mutants
complementary
to bumped rapalogs, lambda repressor-FRAP libraries bearing mutant FRAP
sequences are
transformed into E. coli cells expressing wildtype lambda repressor-FKBP
protein. Plasmids
expressing FRAP mutants are isolated from those colonies that survive lysis on
bacterial plates
containing high titres of lambda phage and "bumped" rapamycin compounds.
Alternatively, to
isolate FKBP mutants, the above strategy is repeated with lambda repressor-
FKBP libraries
bearing mutant FKBP sequences transformed into E. coli cells expressing
wildtype lambda
repressor-FRAP protein.
A further alternative is to clone the randomized FKBP sequences into a vector
for phage
display, allowing in vitro selection of the variants that bind best to the
rapalog. Affinity
selection in vitro may be performed in a number of ways. For example, rapalog
is mixed with
the library phage pool in solution in the presence of recombinant FRAP tagged
with an affinity
handle (for example a hexa-histidine tag, or GST), and the resultant complexes
are captured on
the appropriate affinity matrix to enrich for phage displaying FKBP harboring
complementary mutations. Techniques for phage display have been described, and
other in
vitro selection selection systems can also be contemplated (for example
display on lambda
52
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phage, display on plasmids, display on baculovirus). Furthermore, selection
and screening
strategies can also be used to improve other properties of benefit in the
application of this
invention, such as enhanced stability in vivo. For a review see Clackson, T. &
Wells, J.A. 1994.
Trends Biotechnol. 12, 173-184.
(b) FRAP
Similar considerations apply to the generation of mutant FRB domains which
bind
preferentially to improved rapalogs containing modifications (i.e., are
'bumped') relative to
rapamycin in the FRAP-binding portion of the macrocycle. For example, one may
obtain
preferential binding using rapalogs bearing substituents other than -0Me at
the C7 position
with FRBs based on the human FRAP FRB peptide sequence but bearing amino acid
substitutions
for one of more of the residues Tyr2038, Phe2039, Thr2098, GIn2099, Trp2101
and Asp2102.
Exemplary mutations include Y2038H, Y2038L, Y2038V, Y2038A, F2039H, F2039L,
F2039A,
F2039V, D2102A, T2098A, T2098N, andT2098S. Rapalogs bearing substituents other
than -OH at
C28 and/or substituents other than =O at C30 may be used to obtain
preferential binding to
FRAP proteins bearing an amino acid substitution for G1u2032. Examplary
mutations include
E2032A and E2032S. Proteins comprising an FRB containing one or more amino
acid replacements
at the foregoing positions, libraries of proteins or peptides randomized at
those positions (i.e.,
containing various substituted amino acids at those residues), libraries
randomizing the entire
protein domain, or combinations of these sets of mutants are made using the
procedures described
above to identify mutant FRAPs that bind preferentially to bumped rapalogs.
The affinity of candidate mutant FRBs for the complex of an FKBP protein
complexed with
a rapalog may be assayed by a number of techniques; for example binding of in
vitro translated
FRB mutants to GST-FKBP in the presence of drug (Chen et al. 1995. Proc. Natl.
Acad. Sci. USA
92, 4947-4951); or ability to participate in a rapalog-dependent
transcriptionally active
complex with an appropriate FKBP fusion protein in a yeast two-hybrid assay.
FRB mutants with desired binding properties may be isolated from libraries
displayed on
phage using a variety of sorting strategies. For example, a rapalog is mixed
with the library
phage pool in solution in the presence of recombinant FKBP tagged with an
affinity handle (for
example a hexa-histidine tag, or GST), and the resultant complexes are
captured on the
appropriate affinity matrix to enrich for phage displaying FRAP harboring
complementary
mutations.
An additional feature of the FRB fusion protein that may vary in the various
embodiments
of this invention is the exact sequence of the FRB domain used. In some
applications it may be
preferred to use portions of an FRB which are larger than the minimal (89
amino acid) FRB
53
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domain. These include extensions N-terminal to residue G1u2025 (preferably
extending to at
least Arg2018 or I1e2021), as well as C-terminal extensions beyond position
2113, e.g. to position
2113, 2141 or 2174 or beyond), which may in some cases improve the stability
of the folded FRB
domain and/or the efficiency of expression. Other applications in which
different FRB sequence
termini may be used include those in which a long linker is desired for steric
reasons on one or
both sides of the FRB domain, for example to accommodate the distortions of
the polypeptide
chain required for FRB-mediated protein-protein association at the cell
membrane or on DNA.
Conversely, in other applications short linkers on one or both sides of the
FRB domain may be
preferred or required to present the heterologous effector domains)
appropriately for
biological function. In human gene therapy applications the use of naturally
occurring human
FRAP sequence for such linkers will generally be preferred to the introduction
of heterologous
sequences, or reduce the risk of provoking an immune response in the host
organism.
Some rapalogs, especially rapalogs with modifications or substituents
(relative to
rapamycin) at positions believed to lie near the boundary between the FKBP
binding domain
and the FRAP binding domain, such as those on C28, C30, C7 and C24, possess
reduced ability,
relative to rapamycin, to form complexes with both mammalian FKBP and FRB
domains, in
particular, with those domains containing naturally occurring human peptide
sequence. That
reduced ability may be manifested as a reduced binding affinity as determined
by any of the
direct or indirect assay means mentioned herein or as reduced
immunosuppressive activity as
determined in an appropriate assay such as a T cell proliferation assay. In
such cases, iterative
procedures may be used to identify pairs of mutant FKBPs and mutant FRBs that
are capable of
complexing with the rapalog more effectively than the corresponding domains
containing
naturally occurring human peptide sequence. For example, one may first
identify a
complementary modified FKBP domain capable of binding to the rapalog, as
discussed
previously, and then using this mutant FKBP domain as an affinity matrix in
complex with the
rapalog, one may select a complementary modified FRB domain capable of
associating with
that complex. Several cycles of such mutagenesis and screening may be
performed to optimize
the protein pair.
For some embodiments, it will be desirable to use FRB and/or FKBP domains
containing
mutations that can affect the protein-protein interaction. For instance,
mutant FKBP domains
which when bound to a given rapalog are capable of complexing with an
endogenous FRB
measurably less effectively than to a mutant FRB are of particular interest.
Also of interest are
mutant FRB domains which are capable of associating with a complex of a mutant
FKBP with a
given rapalog measurable more effectively than with a complex of an endogenous
FKBP with
the rapalog. Similar selection and screening approaches to those delineated
previously can be
54
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used (i) to identify amino acid substitutions, deletions or insertions to an
FKBP domain which .
measurably diminish the domain's ability to form the tripartite complex with a
given rapalog
and the endogenous FRB; (ii) to identify amino acid substitutions, deletions
or insertions to an
FRB domain which measurably diminish the domain's ability to form the
tripartite complex
with a given rapalog and the endogenous FKBP; and (iii) to select and/or
otherwise identify
compensating mutations) in the partner protein. As examples of suitable mutant
FKBPs with
diminished effectiveness in tripartite complex formation, we include
mammalian, preferably
human FKBP in which one or both of His87 and I1e90 are replaced with amino
acids such as
Arg, Trp, Phe, Tyr or Lys which contain bulky side chain groups; FRB domains,
preferably
containing mammalian, and more preferably of human, peptide sequence may then
be mutated
as described above to generate complementary variants which are capable of
forming a
tripartite complex with the mutant FKBP and a given rapalog. Illustrative FRB
mutations
which may be useful with H87W or H87R hFKBPI2s include human FRBs in which
Y2038 is
replaced by V, S, A or L; F2039 is replaced by A; and/or 82042 is replaced by
L, A or S.
Illustrative FRB mutations which may be useful with I90W or I90R hFKBPI2s
include human
FRBs in which K2095 is replaced with L, S, A or T.
A3ditionally, in optimizing the receptor domains of this invention, it should
be
appreciated that immunogenicity of a polypeptide sequence is thought to
require the binding of
peptides by MHC proteins and the recognition of the presented peptides as
foreign by
endogenous T-cell receptors. It may be preferable, at least in human gene
therapy applications,
to tailor a given foreign peptide sequence, including junction peptide
sequences, to minimize the
probability of its being immunologically presented in humans. For example,
peptide binding to
human MHC class I molecules has strict requirements for certain residues at
key 'anchoi
positions in the bound peptide: eg. HLA-A2 requires leucine, methionine or
isoleucine at
position 2 and Ieucine or valine at the C-terminus (for review see Stern and
Wiley (1994)
Structure 2,145-251). Thus in engineering proteins in the practice of this
invention, this
periodicity of these residues is preferably avoided, especially in human gene
therapy
applications. The foregoing applies to all protein engineering aspects of the
invention,
including without limitation the engineering of point mutations into receptor
domains, and to
the choice or design of boundaries between the various protein domains.
Other components, design features and applications
The chimeric proteins may contain as a heterologous domain a cellular
localization domain
such as a membrane retention domain. See e.g. PCT/US94/01617, especially pages
26-27.
Briefly, a membrane retention domain can be isolated from any convenient
membrane-bound
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protein, whether endogenous to the host cell or not. The membrane retention
domain may be a
transmembrane retention domain, i.e., an amino acid sequence which extends
across the
membrane as in the case of cell surface proteins, incluing many receptors. The
transmembrane
peptide sequence may be extended to span part or all of an extracellular
and/or intracellular
domain as well. Alternatively, the membrane retention domain may be a lipid
membrane
retention domain such as a myristoylation or palmitoylation site which permits
association
with the lipids of the cell surface membrane. Lipid membrane retention domains
will usually be
added at the 5' end of the coding sequence for N-terminal binding to the
membrane and,
proximal to the 3' end for C-terminal binding. Peptide sequences involving
post-translational
processing to provide for lipid membrane binding are described by 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; and
reviewed in Ann. Rev. of Biochem. (1988) 57, 69. An amino acid sequence of
interest includes 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 this invention. Other
localization domains include
organelle-targeting domains and sequences such as -K-D-E-L and -H-D-E-L which
target
proteins bearing them to the endoplasmic reticulum, as well as nuclear
localization sequences
which are particularly useful for chimeric proteins designed for (direct)
transcriptional
regulation. Various cellular localization sequences and signals are well known
in the art.
Further details which may be used in the practice of the subject invention
relating to the
design, assembly and use of constructs encoding chimeric proteins containing
various effector
domains including cytoplasmic signal initiation domains such as the CD3 zeta
chain, nuclear
transcription factor domains including among others VP16 and GAL4, domains
capable of
triggering apoptosis including the Fas cytoplasmic domain and others are
disclosed in
PCT/US94/01617 and PCT/US95/10591. The latter international application
further discloses
additional features particularly applicable to the creation of genetically
engineered animals
which may be used as disease models in biopharmaceutical research. Those
features include the
use of tissue specific regulatory elements in the constructs for expression of
the chimeric proteins
and the application of regulated transcription to the expression of Cre
recombinase as the target
gene leading to the elimination of a gene of interest flanked by IoxP
sequences. Alternatively,
flp and its cognate recognition sequences may be used instead of Cre and lox.
Those features may
be adapted to the subject invention.
In various cases, especially in embodiments involving whole animals containing
cells
engineered in accordance with this invention, it will often be preferred, and
in some cases
required, that the various domains of the chimeric proteins be derived from
proteins of the
56
CA 02318402 2000-07-14
WO 99/36553 PCT/US99/00178
same species as the host cell. Thus, for genetic engineering of human cells,
it is often preferred
that the heterologous domains {as well as the FKBP and FRB domains) be of
human origin,
rather than of bacterial, yeast or other non-human source.
We also note that epitope tags may also be incorporated into chimeric proteins
of this
invention to permit convenient detection.
Tissue-specific or cell-type specific expression
It will be preferred in certain embodiments, that the chimeric proteins be
expressed in a
cell-specific or tissue-specific manner. Such specificity of expression may be
achieved by
operably linking one ore more of the DNA sequences encoding the chimeric
proteins) to a cell-
type specific transcriptional regulatory sequence (e.g. promoter/enhancer).
Numerous cell-type
specific transcriptional regulatory sequences are known. Others may be
obtained from genes
which are expressed in a cell-specific manner. See e.g. PCT/US95/10591,
especially pp. 36-37.
For example, constructs for expressing the chimeric proteins may contain
regulatory
sequences derived from known genes for specific expression in selected
tissues. Representative
examples are tabulated below:
Tissue Gage Reference
lens g2-crystallinBreitman, M.L., Clapoff, S., Rossant, J.,
Tsui, L.C., Golde, L.M.,
Maxwell, LH., Bemstin, A. (1987) Genetic
Ablation: targeted
expression of a toxin gene causes microphthalmia
in transgenic
mice. Science 238:1563-1565
aA-crystallinLandel, C.P., Zhao, j., Bok, D., Evans,
G.A. (1988) Lens-specific
expression of a recombinant ricin induces
developmental defects
in the eyes of transgenic mice. Genes Dev.
2:1168-1178
Kaur, S., key, B., Stock, J., McNeish,
J.D., Akeson, R., Potter, S.S.
(1989) Targeted ablation of alpha-crystallin-synthesizing
cells
produces lens-deficient eyes in transgenic
mice. Development 105:
613-619
pituitary Growth hormoneBehringer, R.R., Mathews, L.S., Palmiter,
R.D., Brinster, R.L.
- somatrophic (1988) Dwarf mice produced by genetic ablation
of growth
cells hormone-expressing cells. Genes Dev. 2:
453-461 ... / ...
57
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WO 99/36553 PCT/US99/00178
pancreas Insulin- Ornitz, D.M., Palmiter, R.D., Hammer, R.E.,
Brins~-, R.L., 1
Elastase Swift, G.H., MacDonald, R.J. (1985) Specific
- acinar expression of an
cell specificelastase-human growth fusion in pancreatic
acinar cells of
transgeneic mice. Nature 131: 600-603
Palmiter, R.D., Behringer, R.R., Quaife,
C.J., Maxwell, F.,
Maxwell, LH., Brinster, R.L. (1987) Cell
lineage ablation in
transgeneic mice by cell-specific expression
of a toxin gene. Cell
50: 435-443
T cells lck promoterChaffin, K.E., Beals, C.R., Wilkie, T.M.,
Forbush, K.A., Simon,
M.L, Perlmutter, R.M. (1990) EMBO Journal
9: 3821'3829
B cells ImmunoglobulinBorelli, E., Heyman, R., Hsi, M., Evans,
R.M. (1988) Targeting of
kappa light an inducible toxic phenotype in animal
cells. Proc. Natl. Acad.
chain Sci. USA 85: 7572-7576
Heyman, R.A., Borrelli, E., Lesley, J.,
Anderson, D., Richmond,
D.D., Baird, S.M., Hyman, R., Evans, R.M.
(1989) Thymidine
kinase obliteration: creation of transgenic
mice with controlled
immunodeficiencies. Proc. Natl. Acad. Sci.
USA 86: 2698-2702
Schwann Po promoter Messing, A., Behringer, R.R., Hammang,
cells J.P. Palmiter, RD,
Brinster, RL, Lemke, G. ,PO promoter directs
espression of reporter
and toxin genes to Schwann cells of transgeruc
mice. Neuron 8: 507-
5201992
Myelin basicMiskimins, R. Knapp, L., Dewey,MJ, Zhang,
X. Cell and tissue-
protein specific expression of a heterologous gene
under control of the
myelin basic protein gene promoter in trangenic
mice. Brain Res
Dev Brain Res 1992 Vol 65: 217-21
spermatids protamine Breitman, M.L., Rombola, H., Maxwell, LH.,
Klintworth, G.K.,
Bernstein, A. (1990) Genetic ablation in
transgenic mice with
attenuated diphtheria toxin A gene. Mol.
Cell. Biol. 10: 474-479
lung Lung surfacantOrnitz, D.M., Palmiter, R.D., Hammer, R.E.,
Brinster, R.L.,
gene Swift, G.H., MacDonald, R.J. (1985) Specific
expression of an
elastase-human growth fusion in pancreatic
acinar cells of
transgeneic mice. Nature 131: 600-603
adipocyte Ross, S.R, Braves, RA, Spiegelman, BM Targeted
P2 expression of a
toxin gene to adipose tissue: transgenic
mice resistant to obesity
Genes and Dev 7:1318-24 1993
muscle myosin lightLee, KJ, Ross, RS, Rockman, HA, Harris,
AN, O'Brien, TX, van-
chain Bilsen, M., Shubeita, HE, Kandolf, R.,
Brem, G., Prices et alJ.
BIoI. Chem.1992 Aug 5, 267:15875-85
Alpha actin Muscat, GE., Perry, S. , Prentice, H. Kedes,
L. The human skeletal
alpha-actin gene is regulated by a muscle-specific
enhancer that
binds three nuclear factors. Gene Expression
2,111-26,1992 .../...
neurons neurofilamentReeben, M. Halmekyto, M. Alhonen, L. Sinervirta,
R. Saarma, M.
proteins Janne,J. Tissue-specific expression of
rat light neurofilament
promoter-driven reporter gene in transgenic
mice. BBRC 1993:
192: 465-70
58
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liver tyrosine
aminotransfer-
ase, albumin,
apolipoproteins
Target Gene Constructs
In embodiments of the invention in which the chimeric proteins are designed
such that
their multimerization activates transcription of a target gene, an appropriate
target gene
construct is also used in the engineered cells. Appropriate target gene
constructs are those
containing a target gene and a cognate transcriptional control element such as
a promoter and/or
enhancer which is responsive to the multimerization of the chimeric proteins.
In embodiments
involving direct activation of transcription, that responsiveness may be
achieved by the
presence in the target gene construct of one or more DNA sequences recognized
by the DNA-
binding domain of a chimeric protein of this invention (i.e., a DNA sequence
to which the
chizneric protein binds). In embodiments involving indirect activation of
transcription,
responsiveness may be achieved by the presence in the target gene construct of
a promoter and/or
enhancer sequence which is activated by an intracellular signal generated by
multimerization
of the chimeric proteins. For example, where the chimeric proteins contain the
TCR zeta chain
intracellular domain, the target gene is linked to and under the expression
control of the IL-2
promoter region.
This invention also provides target DNA constructs containing (a) a cognate
DNA sequence,
e.g. to which a DNA-binding chimeric protein of this invention is capable of
binding (or which
is susceptible to indirect activation as discussed above), and (b) flanking
DNA sequence from
the locus of a desired target gene endogenous to the host cells. These
constructs permit
homologous recombination of the cognate DNA sequence into a host cell in
association with an
endogenous target gene. In other embodiments the construct contains a desired
gene and flanking
DNA sequence from a target locus permitting the homologous recombination of
the target gene
into the desired locus. Such a target construct may also contain the cognate
DNA sequence, or the
cognate DNA sequence may be provided by the locus.
The target gene in any of the foregoing embodiments may encode for example a
surface
membrane protein (such as a receptor protein), a secreted protein, a
cytoplasmic protein, a
nuclear protein, a recombinase such as Cre, a ribozyme or an antisense RNA.
See
PCT/US94/01617 for general design and construction details and for various
applications
including gene therapy and see PCT/US95/10591 regarding applications to animal
models of
disease.
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This invention encompasses a variety of configurations for the chimeric
proteins. In all cases ~,
involving the activation of target gene transcription, however, the chimeric
proteins share an
important characteristic: cells containing constructs encoding the chimeras
and a target gene
construct express the target gene at least one, preferably at least two, and
more preferably at
least three or four or more orders of magnitude more in the presence of the
multimerizing ligand
than in its absence. Optimally, expression of the selected gene is not
observed unless the cells
are or have been exposed to a multimerizing ligand.
To recap, the chimeric proteins are capable of initiating a detectable level
of transcription
of target genes within the engineered cells upon exposure of the cells to the
an improved
rapalog, i.e., following multimerization of the chimeras. Thus, transcription
of target genes is
activated in genetically engineered cells of this invention following exposure
of the cells to an
improved rapalog capable of multimerizing the chimeric protein molecules. Said
differently,
genetically engineered cells of this invention contain chimeric proteins as
described above and
are responsive to the presence and/or concentration of an improved rapalog
which is capable of
multimerizing those chimeric protein molecules. That responsiveness is
manifested by the
activation of transcription of a target gene. Such transcriptional activity
can be readily
detected by any conventional assays for transcription of the target gene. In
other embodiments,
the biological response to ligand-mediated multimerization of the chimeras is
cell death or
other biological events rather than direct activation of transcription of a
target gene.
Design and assembly of the DNA constructs
Constructs may be designed in accordance with the principles, illustrative
examples and
materials and methods disclosed in the patent documents and scientific
literature cited herein,
each of which is incorporated herein by reference, with modifications and
further
exemplification as described herein. Components of the constructs can be
prepared in
conventional ways, where the coding sequences and regulatory regions may be
isolated, as
appropriate, ligated, cloned in an appropriate cloning host, analyzed by
restriction or
sequencing, or other convenient means. Particularly, using PCR, individual
fragments including
all or portions of a functional unit may be isolated, where one or more
mutations may be
introduced using "primer repair", ligation, in vitro mutagenesis, etc. as
appropriate. In the case
of DNA constructs encoding fusion proteins, DNA sequences encoding individual
domains and
sub-domains are joined such that they constitute a single open reading frame
encoding a fusion
protein capable of being translated in cells or cell lysates into a single
polypeptide harboring
all component domains. The DNA construct encoding the fusion protein may then
be placed into
a vector that directs the expression of the protein in the appropriate cell
type(s). For
CA 02318402 2000-07-14
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biochemical analysis of the encoded chimera, it may be desirable to construct
plasmids that
direct the expression of the protein in bacteria or in reticulocyte-lysate
systems. For use in the
production of proteins in mammalian cells, the protein-encoding sequence is
introduced into an
expression vector that directs expression in these cells. Expression vectors
suitable for such uses
are well known in the art. Various sorts of such vectors are commercially
available.
Constructs encoding the chimeric proteins and target genes of this invention
can be
introduced into the cells as one or more DNA molecules or constructs, in many
cases in association
with one or more markers to allow for selection of host cells which contain
the construct(s). The
constructs) once completed and demonstrated to have the appropriate sequences
may then be
introduced into a host cell by any convenient means. The constructs may be
incorporated into
vectors capable of episomal replication (e.g. BPV or EBV vectors) or into
vectors designed for
integration into the host cells' chromosomes. The constructs may be integrated
and packaged
into non-replicating, defective viral genomes like Adenovirus, Adeno-
associated virus (AAV),
or Herpes simplex virus (HSV) or others, including retroviral vectors, for
infection or
transduction into cells. Viral delivery systems are discussed in greater
detail below.
Alternatively, the construct may be introduced by protoplast fusion, electro-
poration, biolistics,
calcium phosphate transfection, lipofection, microinjection of DNA or the
like. The host cells
will in some cases be grown and expanded in culture before introduction of the
construct(s),
followed by the appropriate treatment for introduction of the constructs) and
integration of the
construct(s). The cells will then be expanded and screened by virtue of a
marker present in the
constructs. Various markers which may be used successfully include hprt,
neomycin resistance,
thymidine kinase, hygromycin resistance, etc., and various cell-surface
markers such as Tac,
CDB, CD3, Thy1 and the NGF receptor.
In some instances, one may have a target site for homologous recombination,
where it is
desired that a construct be integrated at a particular locus. For example, one
can delete and/or
replace an endogenous gene (at the same locus or elsewhere) with a recombinant
target construct
of this invention. For homologous recombination, one may generally use either
S2 or O-vectors.
See, for example, 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.
The constructs may be introduced as a single DNA molecule encoding all of the
genes, or
different DNA molecules having one or more genes. The rnnstructs may be
introduced
simultaneously or consecutively, each with the same or different markers.
Vectors containing useful elements such as bacterial or yeast origins of
replication,
selectable and/or amplifiable markers, promoter/enhancer elements for
expression in
procaryotes or eucaryotes, and mammalian expression control elements, etc.
which may be used
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to prepare stocks of construct DNAs and for carrying out transfections are
well known iri the art, .
and many are commercially available.
Delivery of Nuceic Acid: Ex vivo and in vivo
Any means for the introduction of heterologous nucleic acids into host cells,
especially
eucaryotic cells, an in particular animal cells, preferably human or non-human
mammalian
cells, may be adapted to the practice of this invention. For the purpose of
this discussion, the
various nucleic acid constructs described herein may together be referred to
as the transgene. Ex
vivo approaches for delivery of DNA include calcium phosphate precipitation,
electroporation, lipofection and infection via viral vectors. Two general in
vivo gene therapy
approaches include (a) the delivery of "naked", lipid-complexed or liposome-
formulated or
otherwise formulated DNA and (b) the delivery of the heterologous nucleic
acids via viral
vectors. In the former approach, prior to formulation of DNA, e.g. with lipid,
a plasmid
containing a transgene bearing the desired DNA constructs may first be
experimentally
optimized for expression (e.g., inclusion of an intron in the 5' untranslated
region and
elimination of unnecessary sequences (Felgner, et al., Ann NY Acad Sci 126-
139, 1995).
Formulation of DNA, e.g. with various lipid or liposome materials, may then be
effected using
known methods and materials and delivered to the recipient mammal.
While various viral vectors may be used in the practice of this invention,
retroviral-,
AAV- and adenovirus-based approaches are of particular interest. See, for
example, 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
following additional guidance on the choice and use of viral vectors may be
helpful to the
practitioner.
Retroviral Vectors
Retroviruses are a class of RNA viruses in which the RNA genome is reversely
transcribed
to DNA in the infected cell. The retroviral genome can integrate into the host
cell genome and
requires three viral genes, gag, pol and env, as well as the viral long
terminal repeats (LTRs).
The LTRs also act as enhancers and promoters for the viral genes. The
packaging sequence of the
virus, ('I'), allows the viral RNA to be distinguished from other RNAs in the
cell (Verma et al.,
Nature 389:239-242,1997). For expression of a foreign gene, the viral proteins
are replaced with
the gene of interest in the viral vector, which is then transfected into a
packaging line
containing the viral packaging components. Packaged virus is secreted from the
packaging line
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into the culture medium, which can then be used to infect cells in culture.
Since retroviruses are
unable to infect non-dividing cells, they have been used primarily for ex vivo
gene therapy.
AAV Vectors
Adeno-associated virus (AAV)-based vectors are of general interest as a
delivery vehicle to
various tissues, including muscle and lung. AAV vectors infect cells and
stabiy integrate into the
cellular genome with high frequency. AAV can infect and integrate into growth-
arrested cells
(such as the pulmonary epithelium), and is non-pathogenic.
The AAV-based expression vector to be used typically includes the 145
nucleotide AAV
inverted terminal repeats (ITRs) flanking a restriction site that can be used
for subcloning of the
transgene, either directly using the restriction site available, or by
excision of the transgene
with restriction enzymes followed by blunting of the ends, ligation of
appropriate DNA linkers,
restriction digestion, and ligation into the site between the ITRs. The
capacity of AAV vectors
is about 4.4 kb. The following proteins have been expressed using various AAV-
based vectors,
and a variety of promoter/enhancers: neomycin phosphotransferase,
chloramphenicol acetyl
transferase, Fanconi's anemia gene, cystic fibrosis transmembrane conductance
regulator, and
granulocyte macrophage colony-stimulating factor (Kotin, R.M., Human Gene
Therapy
5:793-801, 1994, Table 1). A transgene incorporating the various DNA
constructs of this invention
can similarly be included in an AAV-based vector. As an alternative to
inclusion of a
constitutive promoter such as CMV to drive expression of the recombinant DNA
encoding the
fusion protein(s), an AAV promoter can be used (1TR itself or AAV p5 (Flotte,
et al. J. Biol.
Chem. 268:3781-3790, 1993)).
Such a vector can be packaged into AAV virions by reported methods. For
example, a human
cell line such as 293 can be co-transfected with the AAV-based expression
vector and another
plasmid containing open reading frames encoding AAV rep and cap under the
control of
endogenous AAV promoters or a heterologous promoter. In the absence of helper
virus, the rep
proteins Rep68 and Rep78 prevent accumulation of the replicative form, but
upon superinfection
with adenovirus or herpes virus, these proteins permit replication from the
ITRs (present only
in the construct containing the transgene) and expression of the viral capsid
proteins. This
system results in packaging of the transgene DNA into AAV virions (Carter,
B.J., Current
Opinion in Biotechnology 3:533-539,1992; Kotin, R.M, Human Gene Therapy 5:793-
801,1994)).
Methods to improve the titer of AAV can also be used to express the transgene
in an AAV virion.
Such strategies include, but are not limited to: stable expression of the ITR-
flanked transgene in
a cell line followed by transfection with a second plasmid to direct viral
packaging; use of a cell
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WO 99/36553 PCT/US99/001?8
line that expresses AAV proteins inducibly, such as temperature-sensitive
inducihlP expression- ~I
or pharmacologically inducible expression. Additionally, one may increase the
efficiency of
AAV transduction by treating the cells with an agent that facilitates the
conversion of the
single stranded form to the double stranded form, as described in Wilson et
al., W096/39530.
Concentration and purification of the virus can be achieved by reported
methods such as
banding in cesium chloride gradients, as was used for the initial report of
AAV vector
expression in vivo (Flotte, et al. J. Biol. Chem. 268:3781-3790, 1993) or
chromatographic
purification, as described in O'Riordan et al., W097/08298.
For additional detailed guidance on AAV technology which may be useful in the
practice of
the subject invention, including methods and materials for the incorporation
of a transgene , the
propagation and purification of the recombinant AAV vector containing the
transgene, and its
use in transfecting cells and mammals, see e.g. Carter et al, US Patent No.
4,797,368 (10 Jan
1989); Muzyczka et al, US Patent No. 5,139,941 (18 Aug 1992); Lebkowski et al,
US Patent No.
5,173,414 (22 Dec 1992); Srivastava, US Patent No. 5,252,479 (12 Oct 1993);
Lebkowski et al, US
Patent No. 5,354,678 (11 Oct 1994); Shenk et al, US Patent No. 5,436,146 (25
July 1995);
Chatterjee et al, US Patent No. 5,454,935 (12 Dec 1995), Carter et al WO
93/24641 (published 9
Dec 1993), and Flotte et al., US Patent No. 5,658,776 (19 Aug 1997) .
Adenovirus Vectors
Various adenovirus vectors have been shown to be of use in the transfer of
genes to mammals,
including humans. Replication-deficient adenovirus vectors have been used to
express marker
proteins and CFTR in the pulmonary epithelium. The first generation Ela
deleted adenovirus
vectors have been improved upon with a second generation that includes a
temperature-sensitive E2a viral protein, designed to express less viral
protein and thereby
make the virally infected cell less of a target for the immune system (Goldman
et al., Human
Gene Therapy 6:839-851, 1995). More recently, a viral vector deleted of all
viral open reading
frames has been reported (Fisher et al., Virology 217:11-22, 1996). Moreover,
it has been shown
that expression of viral IL-10 inhibits the immune response to adenoviral
antigen (Qin et al.,
Human Gene Therapy 8:1365-1374, 1997).
DNA sequences of a number of adenovirus types are available from Genbank. The
adenovirus DNA sequences may be obtained from any of the 41 human adenovirus
types
currently identified. Various adenovirus strains are available from the
American Type Culture
Collection, Rockville, Maryland, or by request from a number of commercial and
academic
sources. A transgene as described herein may be incorporated into any
adenoviral vector and
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WO 99/36553 PCT/US99/00178
delivery protocol, by the same methods (restriction digest, linker ligation or
filling in of ends, - 1
and ligation) used to insert the CFTR or other genes into the vectors. Hybrid
Adenovirus-AAV
vectors represented by an adenovirus capsid containing selected portions of
the adenovirus
sequence, 5' and 3' AAV TTR sequences flanking the transgene and other
conventional vector
regulatory elements may also be used. See e.g. Wilson et al, International
Patent Application
Publication No. WO 96/13598. For additional detailed guidance on adenovirus
and hybrid
adenovirus-AAV technology which may be useful in the practice of the subject
invention,
including methods and materials for the incorporation of a transgene, the
propagation and
purification of recombinant virus containing the transgene, and its use in
transfecting cells and
mammals, see also Wilson et al, WO 94/28938, WO 96/13597 and WO 96/262$5, and
references
cited therein.
Generally the DNA or viral particles are transferred to a biologically
compatible solution
or pharmaceutically acceptable delivery vehicle, such as sterile saline, or
other aqueous or
non-aqueous isotonic sterile injection solutions or suspensions, numerous
examples of which are
well known in the art, including Ringer's, phosphate buffered saline, or other
similar vehicles.
Preferably, in gene therapy applications, the DNA or recombinant virus is
administered in
sufficient amounts to transfect cells at a level providing therapeutic benefit
without undue
adverse effects. Optimal dosages of DNA or virus depends on a variety of
factors, as discussed
elsewhere, and may thus vary somewhat from patient to patient. Again,
therapeutically
effective doses of viruses are considered to be in the range of about 20 to
about 50 ml of saline
solution containing concentrations of from about 1 X 10~ to about 1 X 101 pfu
of virus/ml, e.g. from
1 X 108 to 1 X 109 pfu of virus/ml.
Host Cells
This invention is particularly useful for the engineering of animal cells and
in applications
involving the use of such engineered animal cells. The animal cells may be
insect, worm or
mammalian cells. While various mammalian cells may be used, including, by way
of example,
equine, bovine, ovine, canine, feline, murine, and non-human primate cells,
human cells are of
particular interest. Among the various species, various types of cells may be
used, such as
hematopoietic, neural, glial, mesenchymal, cutaneous, mucosal, stromal, muscle
(including
smooth muscle cells), spleen, reticulo-endothelial, epithelial, endothelial,
hepatic, kidney,
gastrointestinal, pulmonary, fibroblast, and other cell types. Of particular
interest are
hematopoietic cells, which may include any of the nucleated cells which may be
involved with
CA 02318402 2000-07-14
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the erythroid, lymphoid or myelomonocytic lineages, as well as myoblasts and
fihroblasts. I
Also of interest are stem and progenitor cells, such as hematopoietic, neural,
stromal, muscle,
hepatic, pulmonary, gastrointestinal and mesenchymal stem cells
The cells may be autologous cells, syngeneic cells, allogeneic cells and even
in some cases,
xenogeneic cells with respect to an intended host organism. The cells may be
modified by
changing the major histocompatibility complex ("MHC") profile, by inactivating
Q2-
microglobulin to prevent the formation of functional Class I MHC molecules,
inactivation of
Class II molecules, providing for expression of one or more MHC molecules,
enhancing or
inactivating cytotoxic capabilities by enhancing or inhibiting the expression
of genes associated
with the cytotoxic activity, or the like.
In some instances specific clones or oligoclonal cells may be of interest,
where the cells have
a particular specificity, such as T cells and B cells having a specific
antigen specificity or
homing target site specificity.
Introduction of Constructs into Animals
Cells which have been modified ex vivo with the DNA constructs may be grown in
culture
under selective conditions and cells which are selected as having the desired
constructs) may
then be expanded and further analyzed, using, for example, the polymerase
chain reaction for
determining the presence of the construct in the host cells and/or assays for
the production of
the desired gene product(s). Once modified host cells have been identified,
they may then be
used as planned, e.g. grown in culture or introduced into a host organism.
Depending upon the nature of the cells, the cells may be introduced into a
host organism, e.g.
a mammal, in a wide variety of ways. Hematopoietic cells may be administered
by injection
into the vascular system, there being usually at least about 104 cells and
generally not more
than about 101 cells. The number of cells which are employed will depend upon
a number of
circumstances, the purpose for the introduction, the lifetime of the cells,
the protocol to be used,
for example, the number of administrations, the ability of the cells to
multiply, the stability of
the therapeutic agent, the physiologic need for the therapeutic agent, and the
like.
Generally, for myoblasts or fibroblasts for example, the number of cells will
be at least about
104 and not more than about 109 and may be applied as a dispersion, generally
being injected at
or near the site of interest. The cells will usually be in a physiologically-
acceptable medium.
Cells engineered in accordance with this invention may also be encapsulated,
e.g. using
conventional biocompatible materials and methods, prior to implantation into
the host
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organism or patient for the production of a therapeutic protein. See e.g.
Hguyen et al, Tissue
Implant Systems and Methods for Sustaining viable High Cell Densities within a
Host, US
Patent No. 5,314,471 (Baxter International, Inc.); Uludag and Sefton, 1993, J
Biomed. Mater. Res.
27(10):1213-24 (HepG2 cells/hydroxyethyl methacrylate-methyl methacrylate
membranes);
Chang et a1,1993, 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
fibroblasts expressing
hGH/alginate-poly-L-lysine-alginate membrane); Ao et al, 1995, Transplanataion
Proc.
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 Patent No.
5,429,821 (alginate);
Emerich et a1,1993, Exp Neurol 122(1):37-47 (polymer-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
also Aebischer,
WO 92/19595); Savelkoul et a1,1994, J Immunol Methods 170(2):185-96
(encapsulated
hybridomas producing antibodies; encapsulated transfected cell lines
expressing various
cytokines); Winn et al,1994, PNAS USA 91(6):2324-8 (engineered 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 PC12 cells implanted into rats); Kordower et al, 1994, PNAS USA
91(23):10898-
902 (polymer-encapsulated engineered BHK cells expressing hNGF implanted into
monkeys)
and Butler et al WO 95/04521 (encapsulated device). The cells may then be
introduced in
encapsulated form into an animal host, preferably a mammal and more preferably
a human
subject in need thereof. Preferably the encapsulating material is
semipermeable, permitting
release into the host of secreted proteins produced by the encapsulated cells.
In many
embodiments the semipermeable encapsulation renders the encapsulated cells
irnmunologically
isolated from the host organism in which the encapsulated cells are
introduced. In those
embodiments the cells to be encapsulated may express one or more chimeric
proteins containing
component domains derived from proteins of the host species and/or from viral
proteins or
proteins from species other than the host species. For example in such cases
the chimeras may
contain elements derived from GAL4 and VP16. The cells may be derived from one
or more
individuals other than the recipient and may be derived from a species other
than that of the
recipient organism or patient.
Instead of ex vivo modification of the cells, in many situations one may wish
to modify cells
in vivo. For this purpose, various techniques have been developed for
modification of target
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tissue and cells in vivo. A number of viral vectors have been developed, such
as adenovirus,
adeno-associated virus, and retroviruses, as discussed above, which allow for
transfection and,
in some cases, integration of the virus into the host. See, for example,
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, e.g. intravascularly or intramuscularly,
inhalation, or other
parenteral mode. Non-viral delivery methods such as administration of the DNA
via
complexes with liposomes or by injection, catheter or biolistics may also be
used.
In accordance with in vivo genetic modification, the manner of the
modification will
depend on the nature of the tissue, the efficiency of cellular modification
required, the number
of opportunities to modify the particular cells, the accessibility of the
tissue to the DNA
composition to be introduced, and the like. By employing an attenuated or
modified retrovirus
carrying a target transcriptional initiation region, if desired, one can
activate the virus using
one of the subject transcription factor constructs, so that the virus may be
produced and transfect
adjacent cells.
The DNA introduction need not result in integration in every case. In some
situations,
transient maintenance of the DNA introduced may be sufficient. In this way,
one could have a
short term effect, where cells could be introduced into the host and then
fumed on after a
predetermined time, for example, after the cells have been able to home to a
particular site.
Binding properties, Assays
Rapamycin is known to bind to the human protein, FKBP12 and to form a
tripartite complex
with hFKBPI2 and FRAP, a human counterpart to the yeast proteins TORT and
TOR2. Rapalogs
may be characterized and compared to rapamycin with respect to their ability
to bind to human
FKBP12 and/or to form tripartite complexes with human FKBP12 and human FRAP
(or fusion
proteins or fragments containing its FRB domain). See WO 96/41865 (Clackson et
al). That
application discloses various materials and methods which can be used to
quantify the ability
of a compound to bind to human FKBP12 or to form a tripartite complex with
(i.e.,
"heterodimerize") proteins comprising human FKBP12 and the FRB domain of human
FRAP,
respectively. Such assays include fluorescence polarization assays to measure
binding. Also
included are cell based transcription assays in which the ability of a
compound to form the
tripartite complex is measured indirectly by correlation with the observed
level of reporter
gene product produced by engineered mammalian cells in the presence of the
compound.
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Corresponding cell-based assays may also be conducted in engineered yeast
cells. See e.g. WO
95/33052 (Berlin et al).
It will often be preferred that the rapalogs of this invention be
physiologically acceptable
(i.e., lack undue toxicity toward the cell or organism with which it is to be
used), can be taken
orally by animals (i.e., is orally active in applications in whole animals,
including gene
therapy), and/or can cross cellular and other membranes, as necessary for a
particular
application.
In addition, preferred rapalogs are those which bind preferentially to mutant
immunophilins (by way of non-limiting example, a human FKBP in which Phe36 is
replaced
with a different amino acid, preferably an amino acid with a less bulky R
group such as valine
or alanine) over native or naturally-ocurring immunophilins. For example, such
compounds may
bind preferentially to mutant FKBPs at least an order of magnitude better than
they bind to
human FKBP12, and in some cases may bind to mutant FKBPs greater than 2 or
even 3 or more
orders of magnitude better than they do to human FKBPI2, as determined by any
scientifically
valid or art-accepted assay methodology.
Binding affinities of various rapalogs of this invention with respect to human
FKBP12,
variants thereof or other immunophilin proteins may be determined by
adaptation of known
methods used in the case of FKBP. For instance, the practitioner may measure
the ability of a
compound of this invention to compete with the binding of a known ligand to
the protein of
interest. See e.g. Sierkierka et al, 1989, Nature 341, 755-757 (test compound
competes with
binding of labeled FK506 derivative to FKBP).
One set of preferred rapalogs of this invention which binds, to human FKBP12,
to a mutant
thereof as discussed above, or to a fusion protein containing such FKBP
domains, with a Kd
value below about 200 nM, more preferably below about 50 nM , even more
preferably below
about 10 nM, and even more preferably below about 1 nM, as measured by direct
binding
measurement (e.g. fluorescence quenching), competition binding measurement
(e.g. versus
FK506), inhibition of FKBP enzyme activity (rotamase), or other assay
methodology. In one
subset of such compounds, the FKBP domain is one in which phenylalanine at
position 36 has
been replaced with an amino acid having a less bulky side chain, e.g. alanine,
valine,
methionine or serine.
A Competitive Binding FP Assay is described in detail in the examples which
follow. That
assay permits the in vitro measurement of an IC50 value for a given compound
which reflects its
ability to bind to an FKBP protein in competition with a labeled FKBP ligand,
such as, for
example, FK506.
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One preferred class of compounds of this invention are those rapalogs which
have an IC50 - I
value in the Competitive Binding FP Assay better than 1000 nM, preferably
better than 300 nM,
more preferably better than 100 nM, and even more preferably better than 10 nM
with respect to
a given FKBP domain and ligand pair, e.g. human FKBP12 or a variant thereof
with up to 10,
preferably up to 5 amino acid replacements, with a flouresceinated FK506
standard. In one
subset of that class, the FKBP domain has one of the abovementioned
modifications at position
36.
The ability of the rapalogs to multimerize chimeric proteins may be measured
in cell-based
assays by measuring the occurrence of an event triggered by such
multimerization. For instance,
one may use cells containing and capable of expressing DNA encoding a first
chimeric protein
comprising one or more FKBP- domains and one or more effector domains as well
as DNA
encoding a second chimeric protein containing an FRB domain and one or more
effector domains
capable, upon multimerization, of actuating a biological response. We prefer
to use cells which
further contain a reporter gene under the transcriptional control of a
regulatory element (i.e.,
promoter) which is responsive to the multimerization of the chimeric proteins.
The design and
preparation of illustrative components and their use in so engineered cells is
described in
W096/41865 and the other international patent applications referred to in this
and the
foregoing section. The cells are grown or maintained in culture. A rapalog is
added to the
culture medium and after a suitable incubation period (to permit gene
expression and secretion,
e.g. several hours or overnight) the presence of the reporter gene product is
measured. Positive
results, i.e., multimerization, correlates with transcription of the reporter
gene as observed by
the appearance of the reporter gene product. The reporter gene product may be
a conveniently
detectable protein (e.g. by ELISA) or may catalyze the production of a
conveniently detectable
product (e.g. colored). Materials and methods for producing appropriate cell
lines for
conducting such assays are disclosed in the international patent applications
cited above in this
section. Typically used target genes include by way of example SEAP, hGH,
beta-galactosidase, Green Fluorescent Protein and luciferase, for which
convenient assays are
commercially available.
Another preferred class of compounds of this invention are those which are
capable of
inducing a detectable signal in a 2-hybrid transcription assay based on fusion
proteins
containing an FKBP domain. Preferably, the FKBP domain is an FKBP domain other
than
wild-type human FKBP12.
Another assay for measuring the ability of the rapalogs to multimerize
chimeric proteins,
like the FKBP-based transcription assay, is a cell-based assay which measures
the occurrence
of an event triggered by such multimerization. In this case, one uses cells
which constitutively
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express a detectable product. The cells also contain and are capable of
expressing DNAs
encoding chimeric proteins comprising one or more immunophilin-derived ligand
binding
domains and one or more effector domains, such as the intracellular domain of
FAS, capable,
upon multimerization, of triggering cell death. The design and preparation of
illustrative
components and their use in so engineering cells is described in W095/02684.
See also
W096/41865. The cells are maintainined or cultured in a culture medium
permitting cell growth
or continued viability. The cells or medium are assayed for the presence of
the constitutive
cellular product, and a base-line level of reporter is thus established. One
may use cells
engineered for constitutive production of hGH or any other conveniently
detectable product to
serve as the reporter. The compound to be tested is addded to the medium, the
cells are
incubated, and the cell lysate or medium is tested for the presence of
reporter at one or more time
points. Decrease in reporter production indicates cell death, an indirect
measure of
multimerization of the fusion proteins.
Another preferred class of compounds of this invention are those which are
capable of
inducing a detectable signal in such an FKBP/FRB-based apoptosis assay.
Preferably, the
FKBP domain is an FKBP domain other than wild-type human FKBP12. In some
cases, the
FKBP domain is modified, as discussed above. Also preferably, the FRB domain
is an FRB
domain other than wild-type FRB from human FRAP. In some cases, the FRB domain
is
modified at position 2098, as described above.
Conducting such assays permits the practitioner to select rapalogs possessing
the desired
IC50 values and/or binding preference for a mutant FKBP over wild-type human
FKBP12. The
Competitive Binding FP Assay permits one to select monomers or rapalogs which
possess the
desired IC50 values and/or binding preference for a mutant FKBP or wild-type
FKBP relative to
a control, such as FK506.
Applications
The rapalogs can be used as described in W094/18317, W095/02684, W096/20951,
W095/41865, e.g. to regulatably activate the transcription of a desired gene,
delete a target
gene, actuate apoptosis, or trigger other biological events in engineered
cells growing in culture
or in whole organisms, including in gene therapy applications. The following
are non-limiting
examples of applications of the subject invention.
1. Regulated gene therapy. In many instances, the ability to switch a
therapeutic gene on
and off at will or the ability to titrate expression with precision are
important for therapeutic
efficacy. This invention is particularly well suited for achieving regulated
expression of a
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therapeutic target gene in the context of human gene therapy. One example uses
a pair of
chimeric proteins (one containing at least one FRB domain, the other
containing at least one
FKBP domain), an improved rapalog of this invention capable of dimerizing the
chimeras, and
a target gene construct to be expressed. One of the chimeric proteins
comprises a DNA-binding
domain, preferably a composite DNA-binding domain as described in Pomerantz et
al, supra, as
the heterologous effector domain. The second chimeric protein comprises a
transcriptional
activating domain as the heterologous effector domain. The improved rapalog is
capable of
binding to both chimeras and thus of effectively cross-linking the chimeras.
DNA molecules
encoding and capable of directing the expression of these chimeric proteins
are introduced into
the cells to be engineered. Also introduced into the cells is a target gene
linked to a DNA
sequence to which the DNA-binding domain is capable of binding. Contacting the
engineered
cells or their progeny with the improved rapalog (by administering it to the
animal or patient)
leads to assembly of the transcription factor complex and hence to expression
of the target gene.
The design and use of similar components is disclosed in PCT/US93/01617 and in
WO 96/41865
(Clackson et al). In practice, the level of target gene expression should be a
function of the
number or concentration of chimeric transcription factor complexes, which
should in turn be a
function of the concentration of the improved rapalog. Dose (of improved
rapalog)-responsive
gene expression is typically observed.
The improved rapalog may be administered to the patient as desired to activate
transcription of the target gene. Depending upon the binding affinity of the
improved rapalog,
the response desired, the manner of administration, the biological half-life
of the rapalog
and/or target gene mRNA, the number of engineered cells present, various
protocols may be
employed. The improved rapalog may be administered by various routes,
including
parenterally or orally. The number of administrations will depend upon the
factors described
above. The improved rapalog may be taken orally as a pill, powder, or
dispersion; bucally;
sublingually; injected intravascularly, intraperitoneally, intramuscularly,
subcutaneously; by
inhalation, or the like. The improved rapalog (and monomeric antagonist
compound) may be
formulated using conventional methods and materials well known in the art for
the various
routes of administration. The precise dose and particular method of
administration will depend
upon the above factors and be determined by the attending physician or human
or animal
healthcare provider. For the most part, the manner of administration will be
determined
empirically.
In the event that transcriptional activation by the improved rapalog is to be
reversed or
terminated, a monomeric compound which can compete with the improved rapalog
may be
administered. Thus, in the case of an adverse reaction or the desire to
terminate the
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therapeutic effect, an antagonist to the dimerizing agent can be administered
in anv convenient . ~,
way, particularly intravascularly, if a rapid reversal is desired.
Alternatively, one may
provide for the presence of an inactivation domain (or transcriptional
silencer) with a ligand
binding domain.1n another approach, cells may be eliminated through apoptosis
via signalling
through Fas or TNF receptor as described elsewhere. See International Patent
Applications
PCT/US94/01617 and PCT/US94/08008.
The particular dosage of the improved rapalog for any application may be
determined in
accordance with the procedures used for therapeutic dosage monitoring, where
maintenance of a
particular level of expression is desired over an extended period of times,
for example, greater
than about two weeks, or where there is repetitive therapy, with individual or
repeated doses
of improved rapalog over short periods of time, with extended intervals, for
example, two
weeks or more. A dose of the improved rapalog within a predetermined range
would be given
and monitored for response, so as to obtain a time-expression level
relationship, as well as
observing therapeutic response. Depending on the levels observed during the
time period and
the therapeutic response, one could provide a larger or smaller dose the next
time, following the
response. This process would be iteratively repeated until one obtained a
dosage within the
therapeutic range. Where the improved rapalog is chronically administered,
once the
maintenance dosage of the improved rapalog is determined, one could then do
assays at
extended intervals to be assured that the cellular system is providing the
appropriate response
and level of the expression product.
It should be appreciated that the system is subject to many variables, such as
the cellular
response to the improved rapalog, the efficiency of expression and, as
appropriate, the level of
secretion, the activity of the expression product, the particular need of the
patient, which may
vary with time and circumstances, the rate of loss of the cellular activity as
a result of loss of
cells or expression activity of individual cells, and the like.
2. Production of recombinant proteins and viruses. Production of recombinant
therapeutic
proteins for commercial and investigational purposes is often achieved through
the use of
mammalian cell lines engineered to express the protein at high level. The use
of mammalian
cells, rather than bacteria or yeast, is indicated where the proper function
of the protein
requires post-translational modifications not generally performed by
heterologous cells.
Examples of proteins produced commercially this way include erythropoietin,
tissue
plasminogen activator, clotting factors such as Factor VIII:c, antibodies,
etc. The cost of
producing proteins in this fashion is directly related to the level of
expression achieved in the
engineered cells. A second limitation on the production of such proteins is
toxicity to the host
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cell: Protein expression may prevent cells from growing to high density,
sharply reducing
production levels. Therefore, the ability to tightly control protein
expression, as described for
regulated gene therapy, permits cells to be grown to high density in the
absence of protein
production. Only after an optimum cell density is reached, is expression of
the gene activated
and the protein product subsequently harvested.
A similar problem is encountered in the construction and use of "packaging
lines" for the
production of recombinant viruses for commercial (e.g., gene therapy) and
experimental use.
These cell lines are engineered to produce viral proteins required for the
assembly of infectious
viral particles harboring defective recombinant genomes. Viral vectors that
are dependent on
such packaging lines include retrovirus, adenovirus, and adeno-associated
virus. In the latter
case, the titer of the virus stock obtained from a packaging line is directly
related to the level
of production of the viral rep and core proteins. But these proteins are
highly toxic to the host
cells. Therefore, it has proven difficult to generate high-titer recombinant
AAV viruses. This
invention provides a solution to this problem, by allowing the construction of
packaging lines in
which the rep and core genes are placed under the control of regulatable
transcription factors of
the design described here. The packaging cell line can be grown to high
density, infected with
helper virus, and transfected with the recombinant viral genome. Then,
expression of the viral
proteins encoded by the packaging cells is induced by the addition of
dimerizing agent to allow
the production of virus at high titer.
3. Biological research. This invention is applicable to a wide range of
biological
experiments in which precise control over a target gene is desired. These
include: (1) expression
of a protein or RNA of interest for biochemical purification; (2) regulated
expression of a
protein or RNA of interest in tissue culture cells (or in vivo, via engineered
cells) for the
purposes of evaluating its biological function; (3) regulated expression of a
protein or RNA of
interest in transgeruc animals for the purposes of evaluating its biological
function; (4)
regulating the expression of a gene encoding another regulatory protein,
ribozyme or antisense
molecule that acts on an endogenous gene for the purposes of evaluating the
biological function
of that gene. Transgeruc animal models and other applications in which the
components of this
invention may be adapted include those disclosed in PCT/US95/10591.
This invention further provides kits useful for the foregoing applications.
Such kits contain
DNA constructs encoding and capable of directing the expression of chimeric
proteins of this
invention (and may contain additional domains as discussed above) and, in
embodiments
involving regulated gene transcription, a target gene construct containing a
target gene linked to
one or more transcriptioal control elements which are activated by the
multimerization of the
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chimeric proteins. Alternatively, the target gene construct may contain a
cloning site for
insertion of a desired target gene by the practitioner. Such kits may also
contain a sample of a
dimerizing agent capable of dimerizing the two recombinant proteins and
activating
transcription of the target gene.
Formulations, dosage and administration
By virtue of its capacity to promote protein-protein interactions, a rapalog
of this invention
may be used in pharmaceutical compositions and methods for promoting formation
of complexes
of chimeric proteins of this invention in a human or non-human mammal
containing genetically
engineered cells of this invention.
The preferred method of such treatment or prevention is by administering to
the mammal an
effective amount of the compound to promote measurable formation of such
complexes in the
engineered cells, or preferably, to promote measurable actuation of the
desired biological event
triggered by such complexation, e.g. transcription of a target gene, apoptosis
of engineered cells,
etc.
Therapeutic/Prophylactic Administration & Pharmaceutical Compositions
The rapalogs can exist in free form or, where appropriate, in salt form.
Pharmaceutically
acceptable salts of many types of compounds and their preparation are well-
known to those of
skill in the art. The pharmaceutically acceptable salts of compounds of this
invention include
the conventional non-toxic salts or the quaternary ammonium salts of such
compounds which are
formed, for example, from inorganic or organic acids of bases.
The compounds of the invention may form hydrates or solvates. It is known to
those of skill
in the art that charged compounds form hydrated species when lyophilized with
water, or
form solvated species when concentrated in a solution with an appropriate
organic solvent.
This invention also relates to pharmaceutical compositions comprising a
therapeutically
(or prophylactically) effective amount of the compound, and one or more
pharmaceutically
acceptable carriers and/or other excipients. Carriers include e.g. saline,
buffered saline,
dextrose, water, glycerol, ethanol, and combinations thereof, and are
discussed in greater detail
below. The composition, if desired, can also contain minor amounts of wetting
or emulsifying
agents, or pH buffering agents. The composition can be a liquid solution,
suspension, emulsion,
tablet, pill, capsule, sustained release formulation, or powder. The
composition can be
formulated as a suppository, with traditional binders and carriers such as
triglycerides. Oral
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formulation can include standard carriers such as pharmaceutical grades of
mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate,
etc.
Formulation may involve mixing, granulating and compressing or dissolving the
ingredients as
appropriate to the desired preparation.
The pharmaceutical carrier employed may be, for example, either a solid or
liquid.
Illustrative solid carrier include lactose, terra alba, sucrose, talc,
gelatin, agar, pectin,
acacia, magnesium stearate, stearic acid and the like. A solid carrier can
include one or more
substances which may also act as flavoring agents, lubricants, solubilizers,
suspending agents,
fillers, glidants, compression aids, binders or tablet-disintegrating agents;
it can also be an
encapsulating material. In powders, the carrier is a finely divided solid
which is in admixture
with the finely divided active ingredient. In tablets, the active ingredient
is mixed with a
carrier having the necessary compression properties in suitable proportions
,and compacted in
the shape and size desired. The powders and tablets preferably contain up to
99% of the active
ingredient. Suitable solid carriers include, for example, calcium phosphate,
magnesium
stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, methyl
cellulose, sodium
carboxymethyl cellulose, polyvinylpyrrolidine, low melting waxes and ion
exchange resins.
Illustrative liquid carriers include syrup, peanut off, olive oil, water, etc.
Liquid carriers
are used in preparing solutions, suspensions, emulsions, syrups, elixirs and
pressurized
compositions. The active ingredient can be dissolved or suspended in a
pharmaceutically
acceptable liquid carrier such as water, an organic solvent, a mixture of both
or
pharmaceutically acceptable oils or fats. The liquid carrier can contain other
suitable
pharmaceutical additives such as solubilizers, emulsifiers, buffers,
preservatives, sweeteners,
flavoring agents, suspending agents, thickening agents, colors, viscosity
regulators, stabilizers
or osmo-regulators. Suitable examples of liquid carriers for oral and
parenteral administration
include water (partially containing additives as above, e.g. cellulose
derivatives, preferably
sodium carboxymethyl cellulose solution), alcohols (including monohydric
alcohols and
polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g.
fractionated coconut oil
and arachis oil). For parenteral administration, the carrier can also be an
oily ester such as
ethyl oleate and isopropyl myristate. Sterile liquid carriers are useful in
sterile liquid form
compositions for parenteral administration. The liquid Garner for pressurized
compositions can
be halogenated hydrocarbon or other pharmaceutically acceptable propellant.
Liquid
pharmaceutical compositions which are sterile solutions or suspensions can be
utilized by, for
example, intramuscular, intraperitoneal or subcutaneous injection. Sterile
solutions can also be
administered intravenously. The compound can also be administered orally
either in liquid or
solid composition form.
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The carrier or excipient may include time delay material well known to the
art, such as
glyceryl monostearate or glyceryl distearate along or with a wax,
ethylcellulose,
hydroxypropylmethylcellulose, methylmethacrylate and the like. When formulated
for oral
administration, 0.01% Tween 80 in PHOSAL PG-50 (phospholipid concentrate with
1,2-propylene glycol, A. Nattermann & Cie. GmbH) has been recognized as
providing an
acceptable oral formulation for other compounds, and may be adapted to
formulations for
various compounds of this invention.
A wide variety of pharmaceutical forms can be employed. If a solid carrier is
used, the
preparation can be tableted, placed in a hard gelatin capsule in powder or
pellet form or in the
form of a troche or lozenge. The amount of solid carrier will vary widely but
preferably will be
from about 25 mg to about 1 g. If a liquid carrier is used, the preparation
will be in the form of a
syrup, emulsion, soft gelatin capsule, sterile injectable solution or
suspension in an ampule or
vial or nonaqueous liquid suspension.
To obtain a stable water soluble dosage form, a pharmaceutically acceptable
salt of the
multimerizer may be dissolved in an aqueous solution of an organic or
inorganic acid, such as a
0.3M solution of succinic acid or citric acid. Alternatively, acidic
derivatives can be dissolved
in suitable basic solutions. If a soluble salt form is not available, the
compound is dissolved in a
suitable cosolvent or combinations thereof. Examples of such suitable
cosolvents include, but are
not limited to, alcohol, propylene glycol, polyethylene glycol 300,
polysorbate 80, glycerin,
polyoxyethylated fatty acids, fatty alcohols or glycerin hydroxy fatty acids
esters and the
like in concentrations ranging from 0-60% of the total volume.
Various delivery systems are known and can be used to administer the
multimerizer, or the
various formulations thereof, including tablets, capsules, injectable
solutions, encapsulation in
liposomes, microparticles, microcapsules, etc. Methods of introduction include
but are not
limited to dermal, intradermal, intramuscular, intraperitoneal, intravenous,
subcutaneous,
intranasal, pulmonary, epidural, ocular and (as is usually preferred) oral
routes. The compound
may be administered by any convenient or otherwise appropriate route, for
example by infusion
or bolus injection, by absorption through epithelial or mucocutaneous linings
(e.g., oral mucosa,
rectal and intestinal mucosa, etc.) and may be administered together with
other biologically
active agents. Administration can be systemic or local. For treatment or
prophylaxis of nasal,
bronchial or pulmonary conditions, preferred routes of administration are
oral, nasal or via a
bronchial aerosol or nebulizer.
In certain embodiments, it may be desirable to administer the compound locally
to an area
in need of treatment; this may be achieved by, for example, and not by way of
limitation, local
infusion during surgery, topical application, by injection, by means of a
catheter, by means of a
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suppository, or by means of a skin patch or implant, said implant being of a
porous, non-porous, - ,
or gelatinous material, including membranes, such as sialastic membranes, or
fibers.
In a specific embodiment, the composition is formulated in accordance with
routine
procedures as a pharmaceutical composition adapted for intravenous
administration to human
beings. Typically, compositions for intravenous administration are solutions
in sterile isotonic
aqueous buffer. Where necessary, the composition may also include a
solubilizing agent and a
local anesthetic to ease pain at the side of the injection. Generally, the
ingredients are
supplied either separately or mixed together in unit dosage form, for example,
as a lyophilized
powder or water free concentrate in a hermetically sealed container such as an
ampoule or
sachette indicating the quantity of active agent. Where the composition is to
be administered
by infusion, it can be dispensed with an infusion bottle containing sterile
pharmaceutical grade
water or saline. Where the composition is administered by injection, an
ampoule of sterile
water for injection or saline can be provided so that the ingredients may be
mixed prior to
administration.
Administration to an individual of an effective amount of the compound can
also be
accomplished topically by administering the compounds) directly to the
affected area of the
skin of the individual. For this purpose, the compound is administered or
applied in a
composition including a pharmacologically acceptable topical carrier, such as
a gel, an
ointment, a lotion, or a cream, which includes, without limitation, such
carriers as water,
glycerol, alcohol, propylene glycol, fatty alcohols, triglycerides, fatty acid
esters, or mineral
oils.
Other topical carriers include liquid petroleum, isopropyl palmitate,
polyethylene glycol,
ethanol (95%), polyoxyethylene monolaurate (5%) in water, or sodium lauryl
sulfate (5%) in
water. Other materials such as anti-oxidants, humectants, viscosity
stabilizers, and similar
agents may be added as necessary. Percutaneous penetration enhancers such as
Azone may also
be included.
In addition, in certain instances, it is expected that the compound may be
disposed within
devices placed upon, in, or under the skin. Such devices include patches,
implants, and
injections which release the compound into the skin, by either passive or
active release
mechanisms.
Materials and methods for producing the various formulations are well known in
the art
and may be adapted for practicing the subject invention. See e.g. 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;
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illustrative formulation for N administration) and 0 648 494 (published April
19, 1995;
illustrative formulation for oral administration).
The effective dose of the compound will typically be in the range of about
0.01 to about 50
mg/kgs, preferably about 0.1 to about 10 mg/kg of mammalian body weight,
administered in
single or multiple doses. Generally, the compound may be administered to
patients in need of
such treatment in a daily dose range of about 1 to about 2000 mg per patient.
The amount of compound which will be effective in the treatment or prevention
of a
particular disorder or condition will depend in part on the characteristics of
the fusion proteins
to be multimerized, the characteristics and location of the genetically
engineered cells, and on
the nature of the disorder or condition, which can be determined by standard
clinical
techniques. In addition, in vitro or in vivo assays may optionally be employed
to help identify
optimal dosage ranges. Effective doses rnay be extrapolated from dose-response
curves derived
from in vitro or animal model test systems. The precise dosage level should be
determined by
the attending physician or other health care provider and will depend upon
well known
factors, including route of administration, and the age, body weight, sex and
general health of
the individual; the nature, severity and clinical stage of the disease; the
use (or not) of
concomitant therapies; and the nature and extent of genetic engineering of
cells in the patient.
The invention also provides a pharmaceutical pack or kit comprising one or
more containers
containing one or more of the ingredients of the pharmaceutical compositions
of the invention.
Optionally associated with such containers) can be a notice in the form
prescribed by a
governmental agency regulating the manufacture, use or sale of pharmaceutical
or biological
products, which notice reflects approval by the agency of manufacture, use or
sale for human
administration. The notice or package insert may contain instructions for use
of an improved
rapalog of this invention, consistent with the disclsoure herein.
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Examples
Example 1. Synthesis of Representative C-24 modified Rapalogs
1.1. Rapamycin purification. Rapamycin was obtained by fermentation. The
rapamycin
producing organism, Streptomyces hygroscopicus (ATCC# 29253), was cultivated
on a complex
media in 15 L or 30 L fed-batch fermentations. The biomass was harvested after
9-14 days by
centrifugation. The supernatant was contacted for 1-2 hours with a nonionic,
polymeric
adsorbent resin, XAD-16 (Rohm and Haas). The adsorbent was recovered by
centrifugation,
combined with the biomass, and extracted repeatedly with methylene chloride.
The solvent
was removed in vacuo and the resulting residue extracted with acetorutrfle
which was then
condensed in a similar manner. Chromatographic purification of the crude
rapamycin was
achieved by flash chromatography on silica gel (40% Acetone/Hexanes) followed
by C-18
reversed-phase HPLC (70% CH3CN/H20). Rapamycin obtained exhibited identical
HPLC,
spectroscopic, and biological characteristics as an authentic sample of
rapamycin.
1.2. Rapamycin (E and Z)-24-(O-methyloxime) (,~, (general procedure)
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 inethoxylamine hydrochloride (22 mg 262 mmol, 4.0
eq) and stirred at
room temperature for 48 h. After this time the reaction mixture was quenched
with H20 (10
mL) and extracted with EtOAc {3x10 ml). The combined organic extracts were
washed with
saturated NaCl solution (2x10 mL), dried over Na2 S04, filtered, and the
solution concentrated
in vacuo. The resulting residue was subjected to flash chromatography on
silica gel (10%
MeOH/dichloromethane) to afford a mixture of isomers. The isomer mixture was
separated by
HPLC (35% ~E 25% H20/MeCN through a Kromasil C-18 250 x 20 mm column,12
mL/min) to
provide 13 mg (21%) of the faster eluting Z isomer and 7.6 mg (12%) of the E
isomer. Z isomer:
high-resolution mass spectrum (FAB) m/z 965.5749 [(M+Na)+, calcd for C52
H82N2013Na
965.5710]. E isomer: high-resolution mass spectrum (FAB) m/z 965.5701
[(M+Na)+, calcd for
C52 H82N2O13Na 965.5710].
1.3. Rapamycin (E and Z)-24-(O-ethyloxime) (7 ~
Prepared in an analogous manner to Rapamycin (E and Z)-24-(O-methyloxime). The
isomer
mixture was separated by HPLC (30% H20/MeCN through a Kromasil C-18 250 x 20
mm
column,12 mL/min) to provide 7.7 mg (25%) of the faster eluting Z isomer and
0.5 mg (2%) of the
E isomer. Z isomer: high-resolution mass spectrum (FAB) m/z 979.5902 [(M+Na)+,
calcd for C53
H84N2O13Na 979.5871].
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1.4. Rapamycin (E and Z)-24-(O-isobutyloxime) (,~ ~
Prepared in an analogous manner to Rapamycin (E and Z)-24-(O-methyloxime). The
isomer
mixture was separated by HPLC (15% H20/MeCN through a Kromasil C-18 250 x 20
mm
column,12 mL/min) to provide 28 mg (b5%) of the faster eluting Z isomer and
3.0 mg (7%) of the
E isomer. Z isomer: high-resolution mass spectrum (FAB) m/z 1007.6146
((M+Na)+, calcd for
C55 H88N2013Na 1007.6184]. E isomer: high-resolution mass spectrum (FAB) m/z
1007.6157
[(M+Na)+, calcd for C55 H88N2013Na 1007.6184].
1.5. Rapamycin (E and Z)-24-(O-benzyloxime) (11.12)
Prepared in an analogous manner to Rapamycin (E and Z)-24-(O-methyloxime). The
isomer
mixture was separated by HPLC (15% H20/MeCN through a Kromasil C-18 250 x 20
mm
column,12 mL/min) to provide 19.6 mg (44%) of the faster eluting Z isomer and
6.1 mg (14%) of
the E isomer. Z isomer: high-resolution mass spectrum (FAB) m/z 1041.6033
[(M+Na)+, calcd
for C58 H86N2013Na 1041.6028]. E isomer: high-resolution mass spectrum (FAB)
m/z
1041.5988 [(M+Na)+, calcd for C58 H86N2013Na 1041.6028].
1.6. Rapamycin (E and Z)-24-(O-carboxymethyloxime) (13. 14))
Prepared in an analogous manner to Rapamycin (E and Z)-24-(O-methyloxime). The
isomer
mixture was separated by HPLC (45% H20/MeCN through a Kromasil C-18 250 x 20
mm
column,12 mL/min) to provide 4.6 mg (11%) of the faster eluting Z isomer and
1.0 mg (2%) of the
E isomer. Z isomer: high-resolution mass spectrum (FAB) m/z 1009.5664
[(M+Na)+, calcd for
C53 H82N2015Na 1009.5613]. E isomer: high-resolution mass spectrum (FAB) m/z
1009.5604
[(M+Na)+, calcd for C53 H82N2015Na 1009.5613].
1.7. Rapamycin (E and Z)-24-(O-carboxamidomethyloxime) (yz,1ø)
Prepared in an analogous manner to Rapamycin (E and Z)-24-(O-methyloxime). The
isomer
mixture was separated by HPLC (35% H20/MeCN through a Kromasil C-18 250 x 20
mm
column,12 mL/min) to provide 6.2 mg (10%) of the faster eluting Z isomer and
1.4 mg (2%) of the
E isomer. Z isomer: high-resolution mass spectrum (FAB) m/z 1008.5790
[(M+Na)+, calcd for
C53 H83N3014Na 1008.5768]. E isomer: high-resolution mass spectrum {FAB) m/z
1008.5753
[(M+Na)+, calcd for C53 H83N3014Na 1008.5768].
Example 2. Assay of binding of rapamycin C24 derivatives to FKBP
Affinities of rapamycin C24 analogs for FKBP were determined using a
competitive assay based
on fluorescence polarization (FP). A fluorescein-labelled FK506 probe (AP1491)
was
synthesized, and the increase in the polarization of its fluorescence used as
a direct readout of
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bound probe in an equilibrium binding experiment containing sub-saturating
FKBP and
variable amounts of rapamycin analog as competitor.
Synthesis of fluoresceinated FK506 probe (AP1491)
s
0~
~H O~ O
O
OMA OMe ~e ~e OMe OMe
F~ 1 2
pH OH
/ ~ /
\ TH CO7Fi
/ / \ / /n \
H / ~ / H O / ~ /
O
OMe OMe OMe OMe
a
2.1. 24, 32-Bis(tent-Butyldimethylsilyl)ether of FK506
tert-Butyldimethylsilyl trifluoromethanesulfonate (108 ~L, 470 ~mol) was added
dropwise to
a stirred solution of FK506 (103 mg,128 ~mol) and 2,6-lutidine (89.5 wL, 768
~mol) in
dichloromethane (3 mL) at 0°C. The resulting solution was stirred at
0°C for 2 h, and then
treated with MeOH (0.5 mL) and ether (15 mL). The mixture was washed with 10%
aqueous
NaHC03 (3 mL) and brine (3 mL). The organic layer was decanted, dried over
anhydrous
Na2S04, filtered, and concentrated to a yellow oil. Column chromatography
(silica-gel,
hexanes-EtOAc 3:1) gave the title compound as a colorless oil (104 mg).
2.2. Intermediate 1
To a solution of 24,32-bis(tert-butyldimethylsilyl)ether of FK506 (100 mg, 97
~mol) in THF
(2.5 mL) was added morpholine N-oxide (68 mg, 580 lunol), followed by water
(60 ~L), and a 4%
aqueous solution of osmium tetroxide (123 ~L, 20 ~.mol). The resulting mixture
was stirred at
room temperature for 4.5 h. It was then treated with 50% aqueous MeOH (1.5 mL)
and sodium
periodate (207 mg, 970 Eunol), and the suspension stirred for an additional 1
h. The mixture was
diluted with ether (10 mL) and washed with saturated aqueous NaHC03 (2x4 mL).
The
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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 a 0.5 M solution
of lithium tris [(3-ethyl-
3-pentyl)oxyJaluminum hydride in THF (282~,L). The resulting solution was
stirred at -78°C for
1.75 h, and then quenched by addition of ether (6 mL) and saturated ammonium
chloride
solution (250 pL). The mixture was allowed to warm up to room temperature and
treated with
anhydrous sodium sulfate. Filtration and concentration under reduced pressure
afforded a pale
yellow oil (97 mg), which was purified by column chromatography (silica-gel,
hexanes-EtOAc
3:1) to afford 1 as a colorless oil.
2.3 Intermediate 2
A solution of the above alcohol (300 mg, 290 ~,mol) in acetorutrile (10 mL)
was treated with 2,6-
lutidine (338 pL, 2.9 mmol) and N,N'-disuccinimidylcarbonate (371 mg,1.45
mmol). The
resulting suspension was stirred at room temperature for 14.5 h, and then
concentrated under
reduced pressure. The residue was chromatographed (silica-gel, hexanes-EtOAc
2:1 to 100%
EtOAc gradient) to afford the mixed carbonate 2 as a pale yellow oil (127 mg).
2.4 Intermediate 3
A solution of the above carbonate (30 mg, 26 F,mol) and triethylamine (36 pL,
260 N,mol) in
acetonitrile (1 mL) was treated with 4'-(aminomethyl)fluorescein (13.5 mg, 34
F,mol). The
resulting bright orange suspension was stirred at room temperature for 1 h,
and then
concentrated under reduced pressure. The residue was chromatographed (silica-
gel, hexanes-
EtOAc 1:1 to 100% EtOAc to EtOAc-MeOH 1:1 gradient) to give 3 (20.5 mg) as a
bright yellow
solid.
2.5 Compound 4
A solution of bis-silyl ether 3 (35 mg, 25 pmol) in acetorutrile (2 mL) was
treated with 48%
(w/w) I-iF in water (250 ~L). The resulting mixture was stirred at room
temperature for 5.5 h. It
was then diluted with dichloromethane (10 mL) and washed with water (2x2 mL).
The organic
layer was decanted, dried over anhydrous sodium sulfate, and concentrated
under reduced
pressure. The residue was chromatographed (silica-gel,100% EtOAc) to afford 4
(13 mg) as a
bright yellow solid.
26 Determination of binding affinities (ICSOs) of rapalogs using FP
Serial 10-fold dilutions of each analog were prepared in 100% ethanol in glass
vials and stored
on ice. All other manipulations were performed at room temperature. A stock of
recombinant
pure FKBP (purified by standard methods, see eg. Wiederrecht, G. et al. 1992.
J. Biol. Chem.
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267, 21753-21760) was diluted to approximately 3 nM in 50 mM potassium
phosphate pH 7.8/150- i,
mM NaCI/ 100~g/ml bovine gamma globulin ("FP buffer": prepared using only low-
fluorescence
reagents from Panvera) and 98 ~1 aliquots transferred to wells of a Dynatech
micro-fluor black
96-well fluorescence plate. 2.0 ~l samples of the rapamycin analogs were then
transferred in
duplicate to the wells with mixing. Finally, a probe solution was prepared
containing 10 nM
AP1491 in 0.1% ethanol/FP buffer, and 100 l.~.l added to each well with
mixing. Duplicate
control wells contained ethanol instead of rapamycin analog (for 100% probe
binding) or
ethanol instead of rapamycin analog and FP buffer instead of FKBP (0%
binding).
The plates were stored covered in the dark for approximately 30 min to permit
equilibration
and then the fluorescence polarization of the sample in each well read on a
Jolley FPM-2 FP
plate reader (Jolley Consulting and Research, Inc., Grayslake, IL) in
accordance with the
manufacturer's recommendations. The mean polarization (mP units)
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Table 4
cznpd C24 isomer FKBPwt fold loss
FP in
binding affinity
assay
IC50 (nM) (vs
rapamycin)
rapamycin o 2.3 (1)
C14 desoxo ~ 63.3 27.5
17 Y Z (major) 618 269
,
HO
18 I~
E (minor) 59.1 25.7
HO'N
Y
o. Z (major) 1416 616
I
Y
6 o. E (minor) 438 190
I
Y 1287
7 Z (major) 2960
8
p-N E (minor) 1664 723
9
Z (major) >30000 >13043
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cmpd C24 isomer FKBPwt FP fold loss
in
binding assayaffinity
IC50 (nM) (vs rapamycin)
Y
o E (minor) 2048 890
Y
19 Z (major) >30000 >13043
Y
o-N E (minor) 2406 1046
,N
11 Z (major) 8342 3627
,N
~
12 E (minor) 1416 616
I
i
Y
,N
13 ~oH Z (major) 7960 3461
0
Y
14 N
q E (minor) 2351 1022
vOH
I,O
15
Z (major) 1151 500
NHz
O
16
E (minor) 204 88.7
NHp
O
for each competitor concentration was usually converted to % total binding by
reference to the
control values and plotted (y) vs. log molar final concentration of competitor
(x). Non-linear
least square analysis was used to fit the curve and extract the IC50 using the
following equation:
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y = Ml+(M4-Ml)/(1+exp(M2*(M3-x)))
where M3 is the IC50. For incomplete curves the IC50 was determined by
interpolation.
Rapamycin and C14-desoxo-rapamycin were included as controls in each case (
C14-desoxo-
rapamycin was prepared as described by Luengo, J.I. et al. 1994 Tet Lett. 35,
6469-6472).
2.7 Results of binding analysis of Rapamycin C24 oximes
Affinities are reported as IC50s and as fold loss in affinity (= IC50 / IC50
of rapamycin).
(Comparative binding data of C24 rapalogs vs rapamycin and desoxo-rapamycin
towards
human FKBP12 are plotted in PCT/US86/09848.)
Example 3. Synthesis of C7 rapalogs; Assay of binding of C7 rapalog-FKBP
complexes to FRAP
A series of C7 rapalogs containing various C7 substituents selected from
branched and
unbranched alkoxy, arylalkyloxy, -NHCO-Oalkyl, -NHS02alkyl and substituted
aryl and
heteroaryl moieties was synthesized using chemistry generally as described in
the literature
except as noetd (see e.g., Luengo et al. 1995. Chemistry, and Biology 2, 471-
481, and the
references cited in Table II for additional background). See also the table
which follows.
3.1 Compounds ~, ~$ - (R~=Et) are synthesized as described in Luengo et al,
Chemistry &
Biology July 1995, 2:471-481.
3.2 Compound ~,Q -(R~~=iPr) A solution of rapamycin (60 mg, 0.066 mmol) in 2-
propanol (3 mL) at
room temperature was treated with para-toluenesulfonic acid (75 mg, 0.394
mmol) and allowed
to stir for 4 h. After this time the reaction was poured onto a biphasic
solution of saturated
aqueous NaHC03 (20 mL) and EtOAc (30 mL). The organic layer was washed with
additional
solution of saturated aqueous NaHC03 (2 x 20 mL) followed by a saturated
aqueous solution of
NaCI (2 x 10 mL) then dried over Na2S04, filtered, evaporated. The resulting
material was
purified by HPLC on a Kromasil C-18 column (20 x 250 mm) at 55 C using 65%
acetonitrile/water
as eluant to afford AP1700 (25 mg). MS(FAB): (M+Na)+ calcd: 964.5762, found:
964.5753.
3.3 Compound ~Q -{R~~=benzyl) is synthesized as described in Chemistry &
Biology July 1995,
2:471-481.
3.4 Compounds ~ ~. - (R~= -NH-CO-0Me) may be synthesized as described in
Chemistry &
Biology July 1995, 2:471-481.
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3.5 Compound ~ - (Rc~= -NH-S02-Me) A solution of rapamycin (75 mg, 0.082
mr~~l) and
methanesufonamide (312 mg, 3.282 mmol) in dichloromethane (3 mL) at -40 ~C was
treated
dropwise with trifluoroacetic acid (126 pL,1.636 mmol) and allowed to stir for
3 h. After this
time the reaction was poured onto a biphasic solution of saturated aqueous
NaHCOg (20 mL)
and EtOAc (10 mL). The organic layer was washed with a saturated aqueous
solution of NaCI
(2 x 10 mL) then dried over Na2S04, filtered, evaporated, and flash
chromatographed on a
silica gel (dichloromethane:hexane:EtOAc: MeOH, 200:50:42.5:7.5). The
resulting
semipurified material was purified by HPLC on a Kromasil C-18 column (20 x 250
mm) at 55 C
using 65% acetorutrile/water as eluant to afford AP1705 (24 mg). MS(FAB):
(M+Na)+ calcd:
999.5246, found: 999.5246.
3.6 Compounds ~ ~ - (R~~= furanyl) These compounds may be synthesized as
described in
Chemistry & Biology July 1995, 2:471-481.
3. Compounds ~,~ - (R~= methylthiophene) These compounds may be synthesized as
described in J. Org. Chem 1994, 59, 6512-6513.
3.8 Compounds ~ ~$- (R~=ethylthiophene) A solution of rapamycin (50 mg, 0.055
mmol) and
2-ethylthiophene (248 pL, 2.188 mmol) in dichloromethane (1.5 mL) at -40 cC
was treated
dropwise with trifluoroacetic acid (84 uL, 1.094 mmol) and allowed to stir for
3 h. After this
time the reaction was poured onto a biphasic solution of saturated
aqueous NaHC03 (15 mL) and EtOAc (10 mL). The organic layer was washed with a
saturated
aqueous solution of NaCI (2 x 10 mL) then dried over Na2S04, filtered,
evaporated, and flash
chromatographed on a silica gel (MeOH:dichloromethane, 2:98 then 5:95). The
resulting
semipurified material was purified by HPLC on a Kromasil C-18 column (20 x 250
mm) at 55 C
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M~
O H
O O
Me
O
a
O ~
~
1
7~
~
Rya' Rib i gc~a
npam~d.-OMt H 41 ~~ H
teu
-OMe H 40 H
'
'
opanprda tBu
b
27 -OHt H 42 op. H
28 H -OPt 43 K .o,p.
29 -O-Ft'r H 44 H
30 -O-beryl H 45 H
32 ~1FI-(C'rO~sH 46 H
diYl
31 H .t~IH~OO~yOMa 4'l ~ H
33 NH-SOzMe H 48 / ' H
34 / H 49 i H
( a
0 r
35 H / S0 H
'
0
36 ~~ H 51 rNH-(C~O~OEtH
37 H ~ 52 H -NH-(C=Oy-0Lt
I
s
38 / ~ H
39 ~ H
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using 80% acetonitrile/water as eluant to afford AP185$ (6 mg) and AP1859 (28
mv). MS(ES+): - ;
(M+NH4)+ 1016; MS(ES-): (M-H)- 992.
3.9 Compounds ~Q, ~, - (R~=tertbutyl thiophene) A solution of rapamycin (50
mg, 0.055 mmol)
and 2-tert-butylylthiophene (276 mg, 2.188 mmol) in dichloromethane (1.5 mL)
at -40 ~C was
treated dropwise with trifluoroacetic acid (84 ~tL, 1.094 mmol) and allowed to
stir for 3 h. After
this time the reaction was poured onto a biphasic solution of saturated
aqueous NaHC03 (15
mL) and EtOAc (10 mL). The organic layer was washed with a saturated aqueous
solution of
NaCI (2 x 10 mL) then dried over Na2S04, filtered, evaporated, and flash
chromatographed on
a silica gel {MeOH:dichloro-methane, 2:98 then 5:95). The resulting
semipurified material
was purified by HPLC on a Kromasil C-18 column (20 x 250 mm) at 55 C using 80%
acetonitrile/water as eluant to afford AP1856 (4 mg) and AP1857 (14 mg).
MS(ES+): (M+Na)+
1045; MS(ES-): (M-H)- 1021.
3.10 Compounds ~,$, ~ - (R~=op-dimethoxyphenyl) These compounds may be found
in
Chemistry & Biology July 1995, 2:471-481.
3.11 Compounds øø, ~ -(R~= indolyl) A solution of rapamycin (50 mg, 0.055
mmol) and indoie
(64 mg, 0.547 mmol) in dichloromethane (2.0 mL) at -40 ~C was treated dropwise
with
trifluoroacetic acid (84 ~,L, 1.094 mmol) and allowed to stir for 3 h. After
this time the reaction
was poured onto a biphasic solution of saturated aqueous NaHC03 (15 mL) and
EtOAc (10 mL).
The organic layer was washed with a saturated aqueous solution of NaCI (2 x 10
mL) then dried
over Na2S04, filtered, evaporated, and flash chromatographed on a silica gel
(dichloromethane:hexane:EtOAc: MeOH, 200:50:42.5:7.5). The resulting
semipurified material
was purified by HPLC on a Kromasil C-18 column (20 x 250 mm) using 65%
acetorutrile/water as
eluant for AP1701 (12 mg) and AP1702 (7.6 mg). MS(FAB): (M+Na)+
calcd:1021.5765, found:
102L5788 (AP1701) and 1021.5797 (AP1702).
3.12 Compound ~ø - (R~=o,p-diethoxyphenyl) A solution of rapamycin (10$ mg,
0.118 mmol)
and 1,3-diethoxybenzene (783 mg, 4.72 mmol) in dichloromethane (2.0 mL) at -40
~C was treated
dropwise with trifluoroacetic acid (154 ~L, 2.01 mmol) and allowed to stir for
3 h. After this
time the reaction was poured onto a biphasic solution of saturated aqueous
NaHCOg (15 mL)
and EtOAc (15 mL). The organic layer was washed with a saturated aqueous
solution of NaCI
(2 x 10 mL) then dried over Na2S04, filtered, evaporated, and flash
chromatographed on a
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silica gel (dichloromethane:hexane:EtOAc:MeOH, 200:50:42.5:7.5). The resulting
material
was purified by HPLC on a Rainin silica column (20 x 250 mm) using
(dichloromethane:hexane:EtOAc:MeOH, 210:65:65:10) as eluant for AP20808 (20
mg). MS(ES+):
(M+Na)+ 1065.95.
3.13 Compound ~ - (R~= methylthiophene) A solution of rapamycin (105 mg, 0.115
mmol) and
3-methylthiophene(445 ~L, 4.60 mmol) in dichloromethane (2.0 mL) at -40 ~C was
treated
dropwise with trifluoroacetic acid (150 uL, 1.96 mmol) and allowed to stir for
3 h. After this
time the reaction was poured onto a biphasic solution of saturated aqueous
NaHC03 (15 mL)
and EtOAc (15 mL). The organic layer was washed with a saturated aqueous
solution of NaCI
(2 x 10 mL) then dried over Na2S0~, filtered, evaporated, and flash
chromatographed on a
silica gel (dichloromethane:hexane:EtOAc:MeOH, 200:50:42.5:7.5). The resulting
material
was purified by HPLC on a Rainin silica column (20 x 250 mm) using
(dichloromethane:hexane:EtOAc:MeOH, 210:65:65:10) as eluant for AP20809 (60
mg). MS(ES+):
(M+Na)+ 1002.96.
3.14 Compound ~$ -(R~= N-methylpyrrole) A solution of rapamycin (51 mg, 0.056
mmol) and
N-methylpyrrole (198 ~,L 2.23 mmol) in dichloromethane (2.0 mL) at 0 ~C was
treated with zinc
chloride (76 mg, 0.557 mmol) and allowed to warm to rt overnight. After this
time the reaction
was poured onto a biphasic solution of saturated aqueous NaHC03 (15 mL) and
EtOAc (15 mL).
The organic layer was washed with a saturated aqueous solution of NaCI (2 x 10
mL) then dried
over Na2S04, filtered, evaporated, and flash chromatographed on a silica gel
(dichloromethane:hexane:EtOAc:MeOH, 100:150:150:10). The resulting material
was purified
by HPLC on a Rainin Si column (20 x 250 mm) using
(dichloromethane:hexane:EtOAc:MeOH,
210:65:65:10) as eluant for AP20810 (10 mg). MS(ES+): (M+NH4)+ 981.05; MS(ES-
): (M-H)-
961.69.
The C7 rapalogs were characterized by exact mass spec and NMR.
3.15 Assay of FKBP binding affinity of C7 rapalogs
The affinity of a variety of the C7 rapalogs for FKBP was assayed as described
for C24
rapalogs above, using competitive FP. Rapamycin and C14-desoxo-rapamycin
(prepared as
described by Luengo et a1.1994. Tetrahedron Lett. 35, 6469-6472) were included
as controls.
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Affinities are reported below as IC50s and fold loss in affinity (= IC50 /
IC50 of rapamycin).- ':
See "Illustrative C7 lRapalogs" Table below. These data indicate that these
large C7
substituents do not necessarily cause large reductions in the affinity of the
rapalogs for human
FKBP.
Compw~ud FKBPwt FP fold loss
binding assayin
IC50 (nM) affinity
(cf
rapamycin)
rapamycin 2.3 ( 1 )
C14 desoxorap34 15
27 2.6 1.1
28 3.7 1.6
29 2.2 1.0
30 12 5.2
32 4.3 1.9
31 2.6 1.1
33 2.5 1.1
34 28 12
35 29 13
36 3.7 1.6
37 4.3 1.9
38 2.5 1.1
39 2.4 1.0
41 2.9 1.3
40 3.4 1.5
42 2.2 1.0
43 20 8.7
44 7.8 3.3
45 5.9 2.6
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Example 4 Preparation of Rapaloga modified at Ra4 and Rpa: 24(S),30(S)-
tetrahydrorapamycin (~)
L ~O O ~ NsaH.. GCIr~ii~0
~N
Oa~n O M40 MbH, -7a'C
OMe
Rapamycin (46 mg, 0.050 mmol) was dissolved in 2.0 mL of methanol, cooled to -
78'C, and cerium
(III) chloride heptahydrate (46 mg, 0.123 mmol) was added. The solution was
stirred far 0.25
h., then sodium borohydride (7.6 mg, 0.20 mrnol) was added. After 05 h, the
reaction mixture
was partitioned between ethyl acetate (15 mL) and 5% aqueous hydrochloric acid
(2 mL). The
organic phase was washed with water (2 mL) and brine (1 mL), dried over
anhydrous
magnesium sulfate, filtered, and concentrated. Flash chromatography (silica
gel,15 : 75 : 50
200 methanol : ethyl acetate : hexane : dichloromehane) yielded 35 mg (76%) of
the desired
product as a white foam. Mass spectral data: (ES+/ NaCI / NH3) m/z 942.21
{M+Na)+, 935.83
(M+NH4)+; (ES-/ NaCI) m/z 963.04 (M+CI)-, 917.34 (M-H)- lit. ref. Luengo, J.L;
Rozamus,
L.W.; Holt, D.A. Tetrahedron Lett. 1994, 35, 6469-6472.
Example 5 Preparation of Rapaloga modified at C24, C30 and C7
24(S), 30(S)-tetrahydrorapamycin (~, prepared as in Example 4, may be modified
at C7 using
approaches illustrated in the prior C7 rapalog examples. For example:
5.1 7(S)-(2',4'-dimethoxy)benzyl-~-demethoxy-24(S), 30(S)-tetrahydm-rapamycin
Me0
p O O M~~ H ~~, cHy,..eo'C oMe
HO
A RC7a a
OMB
~Ov O ~ OH t!3 as
24(S), 3D(S)-tetrahydro-rapamycin (20 mg, 0.022 mmol) was dissolved in
dichloro-methane
(1.0 mL). 1,3-dimethoxybenzene (020 mL,1.5 mmol) was added, and the solurion
was cooled
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to -60'C. Trifluoroacetic acid (0.030 mL, 0.39 mmol) was added, and the
reaction mixture
was stirred for 1 h at -60'C, then partitioned between ethyl acetate (10 mL)
and saturated
aqueous sodium bicarbonate (1 mL). The organic phase was washed with water (2
mL) and
brine (1 mL), dried over anhydrous magnesium sulfate, filtered, and
concentrated. Flash
chromatography (silica gel,15 : 75 : 50 : 200 methanol : ethyl acetate :
hexane
dichloromehane) yielded 8 mg (35%) of the desired product as a white solid.
Mass spectral
data: (ES+/ NaCI / NH3) m/z 1046.96 (M+Na)+,1042.15 (M+NH4)+; (ES-/ NaCI) m/z
1069.09 (M+Cl)- lit, ref. Luengo, J.L; IConialian-Beck, A.; Rozamus, L.W.;
Holt, D.A. J. Org.
Chem.1994, 59, 6512-6513.
By analogous means, one may produce 24(S), 30(S)-tetrahydro rapamycirLS
bearing other
C7 substituents as described elsewhere herein, e.g., containing alternatively
substituted aryl
groups, heteroaryl, -O-aliphatic groups, thioethers, or any of the other types
of moieties
designated previously for Rya or Rte'. These compounds may be obtained by
reduction at
IS C24 and C30 of the appropriate C7 rapalog, or by transformation at C7 of
the appropriate
C24, C30-tetrahydro rapalog. Illustrative examples follow.
Rapalogs modified at C24, C30 and C7 may also be differ from rapamycin at the
various
positions discussed herein, e.g. with respect to one or more of RC13~ RC43~
RC28~ RC29, R4~
"~", etc. By way of example, starting with 13-F- rapamycin in place of
rapamycin yields the
13-fluoro analogs of compounds 53-79.
5.2 Compounds ,~ø,,,~~- (R~=Et) are synthesized as described in Example 4.1,
but substituting
Compounds 2Z and Z$, respectively, for rapamycin.
5.3 Compound ~ø - (R~=iPr) is synthesized as described in Example 4.1, but
substituting
Compound ~,2 for rapamycin.
5.4 Compound ,~,Z -(RCS=benzyl) is synthesized as described in Example 4.1,
but substituting
Compound ~Q for rapamycin.
5.5 Compounds ;t~,~,Q - (RC7= -NH-CO-OMe) are synthesized as described in
Example 4.1,
but substituting Compounds ,~ and ~I, respectively, for rapamycin.
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5.6 Compound ~- (RCY= -NH-S02-Me) is synthesized as described in Example 4.1,
but
substituting Compound ~ for rapamycin
5.7 Compounds ~I and øZ (R~= furanyl) are synthesized as described in Example
4.1, but
substituting Compounds,~ø and d,~, respectively, for rapamycin.
5.8 Compounds ~, øg - (R~= methylthiophene) are synthesized as described in
Example
4.1, but substituting Compounds 3~ and ~Z, respectively, for rapamycin,
1D 5.9 Compounds ø~, øø (RCY=ethylkhiophene) are synthesized as described in
Example 4.1,
but substituting Compounds ~$ and ,3~. respectively, for rapamycin.
5.I0 Compounds øZ f$- (RC~=tertbutyl thiophene) are synthesized as described
in Example
4.1, but substituting Compounds 41 and ~Q, respectively, for rapamycin.
5.11 Compounds ~,Q, ZQ - (RCS-0,p-dimethoxyphenyl) aie synthesized as
described in
Example 4.1, but substituting Compounds 4,3 and ~, respectively, for
rapamycin.
5.12 Compounds Z1, 7~' -(RCS= indolyl) are synthesized as described in Example
4.1, but
substituting Compounds ~ and ø4, respectively, for rapamycin.
5.13 Compound J~ - (R~=o,p-diethoxyphenyl) is synthesized as described in
Example 4.1,
but substituting Compound 4ø for rapamycin.
5.14 Compound ~ - (R~= methylthiophene) is synthesized as described in Example
4.1, but
substituting Compound 4Z for rapamycin.
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M,
v
I
HO
N
O
O
M,
H
H
1
7
.v~
.
oL
t aQ~ a~ E acr. gcw
s~ ~ H 67 H
tRu
54 -0& H 6t H
s~teu
ss H -0& 69 ~,p.p~IeO~phcayH
56 .O-!Pr H 90 H ~,p.(I~tap~
s7 -O-beosy~ H 7t H
_
St -NH~QO~OM,H ~ H '
!9 H -NH~a0~ ?3 -o.t~~ H
'60-NH~O~te H 74 H -
1
6I ~ H 7s ~ H
0
6Z H ~ 76 -I,4,6.(MeO~pheoyiH
0
63 ~~ H 77 H -?,4,6-(Me0)~phenyl
64 H ~ 78 -NH-(CO)-OBtH
6s ~~ H T9 H -NH-(Ca0).pgi
_
H ~~
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5.15 Compound ~ -(R~= N-methylpyrrole) is synthesized as described in Example
4.1, but
substituting Compound ø$ for rapamycin.
5.16 Compound 7~ 1ø -(R~= 2,4,6-tria~ethoxyphenyl) is synthesized as described
in
Example 5.1, but substituting 1,3,5-trimethoxybenzene for 1,3-
dimethoxybenzene.
Example 6. Preparation of fluoro-rapalogs
6.1 C13-Pluoro-rapaiogs
A new class of rapalogs, C13-Fluora-rapalogs, may be prepared by the following
route:
Me0 ~ Me0
~~.0 O ~ O-TES ~N~.O O ~ O-TES
a O Me0'' ~ O oasr i2 ~q) ~ ~a O O M~ '~ ~ O
~O OMe -4~ ~3 O OMe
J.., ~ _ .." ~ -
a ~
1n this example, the hydroxyl moieties at positions 28 and 43 are protected
prior to
treatment with DAST. We have used bis-triethyisilyl (as shown above) and bis-
triisopropylsilyl protecting groups. Various alternative protecting groups may
be used,
based on user preference ar convenience and in consideration of the reaction
conditions of
subsequ~t transforatations prior to or concurrent with removal of protecting
groups.The
protected compound is then treated with the DAST reagent to introduce the 13-
fluoro
substituent. The DAST reaction may be conducted, e.g., at -42°C as
shown, or at 0°C.
13-Fluoro rapamycin may then be modified at position 7 as desired to produce
the family of
13-fluoro C7-rapalogs bearing any of the variety of moieties designated
previously for RC~a
or RCW For instance, the 7-(o,p-dimethoxy)-13-fluoro-rapalogs (~ø and ~ may be
prepared (and separately recovered if desired) by transformation of 1$ at C7
followed by
n~moval of protecting groups, or, as shown below, by removal of protecting
groups from 7~
followedby transformation at C7.
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HFIPy pM, ~p O OH
--- N 2e
TFA ~ to O
AC7a
~s
One may subject 13-F-rapamycin, instead of rapamycin, to various other
chemical
transformations such as are disclosed or referred to herein, including, for
instance,
fluorination at C28, reduction at C24 and C30, fluorination at C24 and C30,
modification at
C-43, etc., in addition to or as an alternative to modification at C7, in
order to obtain the
corresponding 13-F analog.
6.2 Compounds $y.~- (RCy=Et) are synthesized as descn'bed in Example 3.1, but
substiuting
13-F-rapamycin (~ for rapamycin.
6.3 Compound $,~ - (RCy=iPr) is synthesized as described in Example 3.2, but
substituting 13-
F-rapamycin (j~ for rapamycin.
6.4 Compound $ø -(RCy=benzyl) is synthesized as described in Example 3.3, but
substituting
13-F-rapamycin (J~ for rapamycin.
6.5 Compounds $;Z. $ø - (R~= -NH-CO-OMe) are synthesized as described in
Example 3.4,
but substituting 13-F-rapamycin (1~ for rapamycin.
oMe
HO~~ Rc7a
M
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N
.. O O as O
F ' I~A~
R~
t
s
f H~ i<m t~ Rar
i -08t H ~ ~~~ H
f
iZ H -~c !1 H ~op-(Ma0)~7rf
t3 ~O-~'r H ~ H .
'O'~ H 9i H
_
i5 .NH.(CO~OMeH top-orp~dpMnrlH
:6 B -NH.(ao~of~teIot H
67 .Mi.~t~teH tot H
it ~ H ta!~,~4,6.(Mep~pbmfriH
_.
i9 H ~ t0~H -?,4.~MeOhPdml~
90 ~ H t0S-NH.(C.O~.OStH
91 H ~ t06H -NH-(C.O)OEt
9Z ~ H
93 H _
a'
94
' s 'ttsu
95 H
' s- '
t8 a
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6.6 Compound $Z- (R~=-NH-SU2-Me} is synthesized as described in Example 35,
but
substituting 13-F rapamycin (j~ for rapamydn.
6.9 Compounds $$ and ~- (R~= furanyl) are synthesized as described in Example
3.6, but
substituting 13-F-rapamycin (j~ for rapamycin.
6.8 Compounds ~, $~. - (R~= methylthiophene) are synthesized as described in
Example
3.7, but substituting 13-F~rapamycin (7~ for rapamycin.
6.9 Compounds ~, ~ (R~=ethylthiophene) are synthesized as described in Example
3.8,
but substituting 13-F-rapamycin (J~ for rapamycin.
6.10 Compounds ø$ ~,2 - (RCS=tertbutyl thiophene) are synthesized as described
in Example
3.9, but substituting 13-F-rapamycin (J~] for rapamycin.
6.11 Compounds ~, $~ - (RC~=o,p-dimethoxyphenyl) are synthesized as described
in
Example 3.10, but substituting 13-F-rapamycin (j~ for rapamycin.
6.12 Compounds ~, ~ -(RCy= indolyl) are synthesized as described in Example
3.11, but
substituting 13-F-rapamycin (j~ for rapamycin.
6.13 Compound ~$ - (RCS=v,p-diethoxyphenyl) is synthesized as described in
Example 3.12,
but substituting 13-F-rapamycin (J~ for rapamycin.
6.14 Compound ~$ - (RCy= methylthiophene) is synthesized as described in
Example 3.13,
but substituting 13-F-rapamycin (7$j for rapamycin.
6.15 Compound j~ -(RCS= N-methylpyrrole) is synthesized as described in
Example 3.14,
but substituting 13-F-rapamycin (Z.9~ far rapamycin.
6.20 Preparation of 28-F-rapamycin (IQZ )
To a solution of rapamycin (71 mg, 0.078 mmol) in methylene chloride (1 mL) at
-78 oC was
added DAS'T (21 mL, 0.156 mmol) and reaction was allowed to stir for 2h before
MeOH was
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WO 99/36553 PCT/US99/00178
added bo quench the reaction. The reaction mixture was taken to room
temperature and
stirred for 30 min. It was poured onto a biphasic solution of saturated
aqueous NaHC03 (20
mL) and EtOAc (30 mL). The organic layer was washed with additional solution
of
saturated aqueous NaHC03 (2 x 20 mL) followed by a saturated aqueous solution
of NaCl (2
x 10 mL) then dried over Na2S04, filtered, evaporated. The resulting material
was flash
chromatvgraphed on a silica gel (hexane:EtOAc, 1:1 to 1:2). MS, Fluorine NMR
indicated
C28 fluorinated rapamycin. Stereoisomers can be separated by reverse phase
chromatography (C18 column, MeOH:H20, 80:20), and rnay be used in place of
rapamycin
for the synthesis of various F-28 rapalogs.
lQ1
6.21 Compound IQ$ (13-F, 28-F-rapamycin) is synthesized as described above for
28-F-
rapamycin, but with twice the volume of DAST (41 mL) at a higher temperature (-
40oC).
Example 7: Constricts encoding chimeric transcription factoa
A. Unless otherwise stated, all DNA manipulations described in this and other
examples
were performed using standard procedures (See e.g., F.M. Ausubel et al., Eds.,
Current
Protocols in Molecular Biology (John Wiley 6i Sons, New York, 1994).
B. Plasmids
Constructs encoding fusions of human FIBP12 with the yeast GAL4 DNA binding
ZS domain, the HSV VP16 activation domain, human T cell CD3 zeta chain
intracellular
domain or the intracellular domain of human FAS are disclosed in
PCT/US94/01617.
Additional DNA vectors for directing the expression of fusion proteins
relevant to this
invention were derived from the mammalian expression vector pCGNN (Attar, R.M.
and
Gilman, M.Z.1992. MCB 12: 2432-2443). Inserts cloned as XbaI-BamHI fragments
into
pCGNN are transcribed under the rnntrol of the human CMV promoter and enhancer
sequences (nucleotides -522 to +72 relative to the cap site), and are
expressed with an
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optional epitope tag (a 16 amino acid portion of the H. influer~zae
hemaglutinin gene that is
recognized by the monoclonal antibody 12CA5) and, in the case of transcription
factor
domains, with an N-t~sninal nuclear localization sequence (NLS; from SV40 T
antigen).
Except where stated, all fragments cloned into pCGNN were inserted as XbaI-
BamHI
fragments that included a Spel site just upstream of the BamHI site. As XbaI
and Spe1
produce compatible ends, this allowed further XbaI-BamHI fragments to be
inserted
downstream of the initial insert and facilitated stepwise assembly of proteins
comprising
multiple components. A stop colon was interposed between the SpeI and BamHI
sites. For
initial constructs, the vector pCGNN-GAL4 was additionally used, in which
colons 1-94 of
the GAL4 DNA binding dmnain gene were cloned into the XbaI site of pCGNN such
that a
XbaI site is regenerated only at the 3' end of the fragment. Thus XbaI-BamHI
fragments
could be cloned into this vector to generate GAL4 fusions, and subsequently
recovered.
!a) Constructs encoding GAL4 DNA binding do»win- FRAP fusions
To obtain portions of the human FRAP gene, human thymus total RNA (Clontech
#64028-I) was reverse transcribed using MMLV reverse transcriptase and random
hexamer
primer (Clontech 1st strand synthesis kit). This cDNA was used directly in a
PCR reaction
containing primers 1 and 2 and Pfu polymerise (Stratagene). The primers were
designed to
amplify the coding sequence for amino acids 2025-2113 inclusive of human FRAP:
an 89 amino
acid region essentially corresponding to the minimal 'FRB' domain identified
by Chen et al.
(Pros. Natl. Acid. Sci. USA (1995) 92, 4947-4951) as necessary and sufficient
for FKBP-
rapamycin binding (hereafter named FRB). The appropriately-sized band was
purified,
digested with XbaI and Spey and ligated into XbaI-SpeI digested pCGNN-GAL4.
This
construct was confirmed by restriction analysis (to verify the correct
orientation) and DNA
sequencing and designated pCGNN-GAL4-1PRB.
Constructs encoding FRB multimers were obtained by isolating the FRB XbaI-
BamHI
fragment, and then ligating it back into pCGNN~AL4-1PRB digested with SpeI and
BamHI to generate pCGNN-GAL4-2FRB, which was confirmed by restriction
analysis. This
procedure was repeated analogously an the new construct to yield pCGNN-GAL4-
3FRB and
pCGNN-GAL4-4FRB.
Vectors were also constructed thaYencode larger fragments of FRAP,
encompassing the
minimal FRB domain (amino acids 2025-2113) but extending beyond it. PCR
primers were
designed that amplify various regions of FRAP flanked by 5' XbaI and 3' SpeI
sites as
indicated below.
Designation amino acid! 5' primer 3' primer
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FRAPa 2012-2127 6 7
FRAPb 1995-2141 5 8
FRAPc 1945-2113 3 2
FRAPd 1995-2113 5 2
FRAPe 2012-2113 6 2
FRAPf 2025-2127 1 7
FRAPg 2025-2141 1 8
FRAPh 2025-2174 1 4
FRAPi 1945-2174 3 4
Initially, fragment FRAPi was amplified by RT-PCR as described above, digested
with
XbaI and SpeI, and ligated into XbaI-SpeI digested pCGNN-GAL4. This construct,
pCGNN-
GAL4-FRAPi, was analyzed by PCR to confirm insert orientation and verified by
DNA
sequencing. It was then used as a PCR substrate to amplify the other fragments
using the
primers listed. The new fragments were cloned as GAL4 fusions as described
above to yield
the constructs pCGNN-GAL4-FRAPa, pCGNN-GAL4-FRAPb etc, which were confirmed by
DNA sequencing.
Vectors encoding concatenates of two of the larger FRAP fragments, FRAPd and
FRAPe,
were generated by analogous methods to those used earlier. XbaI-BamHI
fragments encoding
FRAPd and FRAPe were isolated from pCGNN-GAL4-FRAPd and pCGNN-GAL4-FRAPe
and ligated back into the same vectors digested with SpeI and BamHI to
generake pCGNN-
GAL4-2FRAPd and pCGNN-GAL4-2FRAPe. This procedure was repeated analogously on
the new constructs to yield pCGNN-GAL4-3FRAPd, pCGNN-GAL4-3FRAPe, pCGNN-
GAL4-4FRAPd and pCGNN-GAL4-4FRAPe. All constructs were verified by restriction
analysis.
(b) Canst~ucts encoding FRB-VPI6 activation domain fusions
To generate N-terminal fusions of FRB domains) with the activation domain of
the
Herpes Simplex Virus protein VP16, the XbaI-BamHI fragments encoding 1, 2, 3
and 4 copies
of FRB were recovered from the GAth fusion vectors and ligated into XbaI-BamHI
digested
pCGNN to yield pCGNN-1FRB, pCGNN-2FRB etc. These vectors were then digested
with
SpeI and BamHI. An XbaI-BamHI fragment encoding amino acids 414-490 of VP16
was
isolated from plasmid pCG-Gal4-VP16 (Das, G., Hinktey, C.S. and Herr, W.
(1995) Nature
374, 657-660) and ligated into the SpeI-BamHI digested vectors to generate
pCGNN-1FRB-
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VP16, pCGNN-2FRB-VP16, etc. The constructs were verified by restriction
analysis and/or
DNA sequencing.
!c) Constructs encoding ZFHDl DNA binding domain-FRB fusions
An expression vector for directing the expression of ZFHDl coding sequence in
mammalian cells was prepared as follows. Zif268 sequences were amplified from
a cDNA
clone by PCR using primers 5'Xba/Zif and 3'Zif+G. Octl homeodomain sequences
were
amplified from a cDNA clone by PCR using primers 5'Not Oct HD and Spe/Bam
3'Oct. The
ZitZ68 PCR fragment was cut with XbaI and Notl. The OctI PCR fragment was cut
with NotI
and BamHI. Both fragments were ligated in a 3-way ligation between the XbaI
and BamHI
sites of pCGNN (Attar and Gilman, 1992) to make pCGNNZFHDl in which the cDNA
insert is under the tra~nscdpt3onal control of human CMV promoter and enhancer
sequences
and is linked to the nuclear localization sequence from SV40 T antigen. The
plasmid pCGNN
also contains a gene for ampicillin resistance which can serve as a selectable
marker.
(Derivatives, pCGNNZFHDl-FKBPxl and pCGNNZFHDl-FKBPx3, were prepared
containing one or three tandem repeats of human FKBP12 ligated as an XbaI-
BamHI
fragment between the Spel and BamHI sites of pCGNNZFHDl. A sample of
pCGNNZFHDl-FKBPx3 has been deposited with the American Type Culture Collection
under ATCC Accession No. 97399. Sequences of primers is shown in WO 96/41865.
To generate C-terminal fusions of FRB domains) with the chimeric DNA binding
protein ZFHDl, the XbaI-BamHI fragments encoding 1, Z, 3 and 4 copies of FRH
were
recovered from the GAL4 fusion vectors and ligated into Spe-BamHI digested
pCGNN-
ZFHDl to yield pCGNN-ZFHDl-1FRB, pCGNN-ZFHDl-2FRB etc. Constructs were
verified by restriction analysis and/or DNA sequencing.
To examine the effect of introducing additional 'linker' polypeptide between
ZFHDl
and a C-terminal FRB domain, FRAP fragments encoding extra sequence N-terminal
to FRB
were cloned as ZFHDI fusions. XbaI-BamHI fragments encoding FRAPa, FRAPb,
FRAPc,
FRAPd and FRAPe were excised from the vectors pCGNN-GAL4-FRAPa, pCGNN-GAL4-
PRAPb etc and ligated into SpeI-BamHI digested pCGNN-ZFHDl to yield the
vectors
pCGNN-ZFHDl-FRAPa, pCGNN-ZFHDl-FRAPb, etc. Vectors encoding fusions of ZFHDl
to 2, 3 and 4 C-terminal copies of FRAPe were also constructed by isolating
XbaI-BamHl
fragments encoding 2FRAPe, 3FRAPe and 4FRAPe from pCGNN-GALA-2FRAPe, pCGNN-
GAL~-3FRAPe and pCGNN-GAh4-4FRAPe and ligating them into SpeI-BamHI digested
pCGNN-ZFHDl to yield the vectors pCGNN-ZFHDl-2FRAPe, pCGNN-ZFHDI-3FRAPe
and pCGNN-ZFHDl-4FRAPe. All constructs were verified by restriction analysis:
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Vectors were also constructed that encode N-terminal fusions of FRB domains)
with
ZFHDl. XbaI BamHI fragments encoding 1, 2, 3 and 4 copies of FRAPe were
isolated from
pCGNN-GAL4-1FRAPe, pCGNN-GAL4-2FRAPe etc and ligated into XbaI-BamHI digested
pCGNN to yield the plasmids pCGNN-1FRAPe, pCGNN-2FRAPe etc. These vectors were
then digested with SpeI and BamHI, and an XbaI-BamHI fragment encoding ZFHDl
(isolated from pCGNN-ZFHDl) ligated in to yield the constructs pCGNN-1FRAPe-
ZFHDl, pCGNN 2FRAPe-ZFHDl etc, which were verified by restriction analysis.
(d) Constructs encoding FRB~65 activation domain fusions
To generate fusions of FRB domains) with the activation domain of the human NF-
kB
p65 subunit (hereafter designated p65), two fragments were amplified by PCR
from the
plasmid pCG-p65. Primers 9 (p65/ 5' Xba) and 11 (p65 3' Spe/Bam) amplify the
coding
sequence for amino acids 450-550, and primers 10 (p65/361 Xba) and 11 amplify
the coding
sequence for amino acids 361-550, both flanked by 5' Xbai and 3' SpeI/BamHI
sites. PCR
products were digested with XbaI and BamHI and cloned into XbaI-BamHI digested
pCGNN to yield pCGNN-p65(450-550) and pCGNN-p65(361-550). The constructs were
verified by restriction analysis and DNA sequencing.
DNA sequences encoding the 100 amino acid P65 transcription activation
sequenceand
the more extended p65 transcription activation domain (351-550) are shown in
WO
96/41865.
To generate N-terminal fusions of FRB domains) with portions of the p65
activation
domain, plasmids pCGNN-1FRB, pCGNN-2FRB etc were digested with Spel and
BamFiI.
An XbaI-BamHI fragment encoding p65 (450-550) was isolated Erom pCGNN-p65(450-
550)
and ligated into the SpeI-BamHI digested vectors to yield the plasmids pCGNN-
1FRB-
?5 p65(450-550), pCGNN-2FRB-p65(450-550) etc. The construct pCGNN-1FRB-p65{361-
550)
was made similarly using an XbaI BamHI fragment isolated from pCGNN-pb5(361-
550).
These rnnstructs were verified by restriction analysis.
To examine the effect of introducing additional 'linker' polypeptide between
the p65
activation domain and an N-terminal FRB domain, FRAP fragments encoding extra
sequence
C-tens<inal to FRB were cldned as p65 fusions. Xbal BamHI fragments encoding
FRAPa,
FRAPb, FRAPf, FRAPg and FRAPh were excised from the vectors pCGNN-GAL4-FRAPa,
pCGNN-GAL4-FRAPb etc and ligated into XbaI-BamHI digested pCGNN to yield the
vectors pCGNN-FRAPa, pCGNN-FRAPb, etc. These plasmids were then digested with
SpeI
and BamHI, and a XbaI-BamHI fragment encoding p65 (amino acids 450-550)
ligated in to
yield the five vectors pCGNN-FRAPa-p65, .pCGNN-FRAPb-p68, etc, which were
verified
by restriction analysis.
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Vectors encoding fusions of p65 to 1 and 3 N-terminal copies of FRAPe were
also prepared
by digesting pCGNN-1FRAPe and pCGNN-3FRAPe with Spel and BamHI. Xbal-BamHI
fragments encoding p65(45D-550) and p65(361-550) (isolated from pCGNN-p65(450-
550) and
pCGNN-p65(361-S50)) were then ligated in to yield the vectors pCGNN-
1FRAPtp65(450-
550), pCGNN-3FRAPe-p65(450-550), pCGNN-iFRAPe-p65(361-550) and pCGNN-3FRAPe-
p65(361-550). All constructs were verified by restriction analysis.
Vectors were also constructed that encode C-terminal fusions of FRB domains)
with
portions of the p65 activation domain. Plasmids pCGNN-p65(450-550) and pCGNN-
p65(361-550) were digested with SpeI and BamlaI, and XbaI-BamHI fragments
encoding ~
and 3 copies of FRAPe (isolated from pCGNN-GAL4-1FRAPe and pCGNN-GAL4-3FRAPe)
and 1 copy of FRB (isolated from pCGNN-GAi.,~-1FRB) ligated in to yield the
plasmids
pCGNN-p65(450-550)-1FRAPe, pCGNN-p65(450-550r3FRAPe, pCGNN-p65(361-550)-
1FRAPe, pCGNN-p65(361-550)-3FRAPe, pCGNN-p65(450-550)-1FRB and pCGNN-
p65(361-550)-1FRB. All rnnstructs were verified by restriction analysis.
(e) further constructs
Other constructs can be made analogously with the above procedures, but using
alternative portions of the FRAP sequence or FRB domains containing modified
peptide
sequence. 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
encompassing the FRB domain and extending throw to the C-terminal end of the
protein
(including the lipid lcinase homology domain). These fragments differ by
encoding different
portions of the protein N-terminal to the FRB domain. In each case, RT-PCR is
used as
described above to amplify the regions from human thymus RNA, the PCR products
are
purified, digested with Xbal and SpeI, ligated into XbaI-SpeI digested pCGNN,
and
verified by restriction analysis and DNA sequencing.
(~ Primes sequences
1 5'GCATGTCTAGAGAGATGTGGCATGAAGGCCTGGAAG
3C~ 2 5' GCATCACTAGTCTITGAGATTCGTCGGAACACATG
3 5'GCACATTCTAGAATTGATACGCCCAGACCCTTG
4 5' CGAT'CAACTAGTAAGTGTCAATTTCCGGGGCCT
5 5'GCACTATCTAGACTGAAGAACATGTGTGAGCACAGC
6 5'GCACTATCTAGAGTGAGCGAGGAGCTGAT'CCGAGTG
7 5'CGATCAACTAGTGGAAACATATTGCAGCTCTAAGGA
B 5'CGATCAACTAGTTGGCACAGCCAATTCAAGGTCCCG
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9 5' A'PGCTCTAGACTGGGGGCCITGCTPGGCAAC
S ATGCTCTAGAGATGAGTiTCCCACCATGGTG
l I S GCATGGATCCGCTCAACTAGTGGAGCTGATCTGACTCAG
12 5' A'I~CPCI'AGACTTGGAACCGGACCTGCCGCC
5 13 5'GCATCACTAGTCCAGAAAGGGCACCAGCCAATAT
Restriction sites are underlined (XbaI = TCTAGA, SpeI = ACGAGT, BamHI =
GGATCC).
(g) DNA sequence of representative final construct: pCGNN-ZFHDI-IFRB
10 encoding a 12CA5 epitope-SV40 NIS-ZFHDl-FRB fusion is shown in WO 96/41865.
(h) Bicistronic constructs
The internal ribosome entry sequence (IRES) from the en~cephalomyocarditis
virus was
amplified by PCR from pWZL-Bleo. The resulting fragment, which was cloned into
pBS-
SK+ (Stratagene), contains an XbaI site and a stop rndon upstream of the I:RES
sequence and
downstream of it, an NcoI site encompassing the ATG followed by SpeI and BamHI
sites. To
facilitate cloning, the sequence around the initiating ATG of pCGNN-ZFHDl-
3FKBP was
mutated to an NcoI site and the XbaI site was mutated to a NheI site using
oligonucleotides
shown in WO 96/41865. An NmI-BamHI fragment containing ZFHDl-3FKBP was then
cloned downstream of pBS-IItES to create pBS-IRES-ZFHDl-3FKBP. The XbaI-BamHI
fragment from this plasmid was next cloned into SpeI/BaaiH1-cut pCGNN-1FRB-
p65(361-
550) to create pCGNN-1FRB-p65(361-550)-IRES-ZFHDl-3FKBP.
C. Retroviral vectors Eor the expression of chimeric proteins
Retroviral vectors used to express transcription factor fusion proteins from
stably
integrated, low copy genes were derived from pSRaMSVtkNeo (Muller et al., MCB
11:1785-
92, 1991) and pSRaMSV(XbaI) (Sawyers et al., J. Exp. Med. 181:307-313, 1995).
Unique
HamHI sites in both vectors were removed by digesting with BamHI, filling in
with Klenow
and religating to produce pSMTN2 and pSMT?(2, respectively. pSMTN2 expresses
the Neo
gene from an internal thymidine ktnase promoter. A Zeocin gene (Invitroge~n)
is cloned as a
NheI fragment into a unique XbaI site downstream of an internal thymidine
kinase promoter
in pSMTX2 to yield pSNTZ. This Zeocin fragment was generated by mutagenizing
pZeo/SV
(Invitrogen) using the following primers to introduce NheI sites flanking the
zeocin coding
sequence.
Primer 15'-GCCATGGTGGCTAGCCTATAGTGAG
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Primer2 5'-GGCGGT GI~GCTAGCGT'CGGTCAG
pSMTN2 contains unique Fs~RI and HindllT sites downstream of the 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 create
pSMTN3:
12CA5 epitope
M A S S. y IL Y 12 Y g 12
5' gaattccagaagcgcgt ATG GCT TCT AGC TAT CCT TAT GAC GTG CCT GAC
EcoRI
SV40 T NLS
Y 8 K L ~ ~. g S. S E K IS K K K
TAT GCC AGC CTG GGA GGA CCT ~ ~ CCT AAG AAG AAG AGA AAG
Y
GTG ~ ~A TAT CGA ~ ~ Clue ~ ~
Xbal BamHI EiindII=
The equivalent fragment is inserted into a unique EcoRi site of pSMTZ to
create pSM'IZ3
with the only difference being that the 3' HindIII site is replaced by an
EcoRI site.
pSMTN3 and pSMT"Z3 permit chialeric transcription factors to be cloned
downstream of
the 5' viral LTR as XbaI-BamHI fragments and allow selection for stable
integrants by
virtue of their ability to confer resistance to the antibiotics 6418 or Zeocin
respectively.
To generate the retroviral vector SMTN-ZFHDI-3FKBP, pCGNN-ZFHDl-3FKBP was
first mutated to add an ErnRI site upstream of the first amino acid of the
fusion protein. An
EcoRI-BamHI(blunted) fragment was then cloned into EcoRI-HindIII(blunted)
pSRaMSVtkNeo so that ZFHDl-3FKBP was expressed from the retroviral LTR.
Fxampie 8: Rapamycin-dependent transcriptional activation
In preliaunary experiments, three copies of FKBP fused either to a Gal4 DNA
binding
domain or a transcription activation domain activated both the stably
integrated or
transiently transfected reporter gene more strongly than conresponding fusion
proteins
containing only one or two FKBP domains. To evaluate this parameter with FRB
fusion
proteins, effector plasmids containing Gal4 DNA binding domain fused to one or
more copies
of an FRB domain were co-tranafected with a plasmid encoding three FKBP
domains and a
p65 activation domain (3xFKBP-p65) by transient transfection. It was found
that in this
system, four copies of the FRB domain fused to the Gal4 DNA binding domain
activated the
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stably integrated reporter gene more strongly than other corresponding fusion
proteins with
fewer FRB domains.
Method: HT1080 B cells were grown in MEM supplemented with 10% Bovine Calf
Serum.
Approximately 4x105 cells/well in a 6 well plate (Falcon) were transiently
transfected by
Lipofection procedure as recommended by the supplier (GIBCO, BRL). The DNA:
Lipofectamine ratio used in this experiment correspond to 1:6. Cells in each
well recieved
500 ng of pCGNN F3-p65,1.9 ug of PUC 118 plasmid as carrier and 100 ng of one
of the
following plasmids: pCGNN Gal4-iFRB, pCGNN Gal4-2FRB, pCGNN Gal4-3FRB or
pCGNN Gal4-4FRB. Following transfection, 2 ml fresh media was added and
supplemented
with Rapamycin to the indicated concentration. After 24 hrs,100 ul of the
media was
assayed for SEAP activity as described (Spencer et al, 1993).
To test whether multiple FRB domains fused to a p65 activation domain results
in
increased transcriptional activation of the reporter gene, we co-transfected
HT1080 B cells
with plasmids expressing Gal4-3xFKBP and 1, 2, 3 or 4 copies of FRB fused to
p65 activation
domain. Surprisingly, unlike the DNA binding domain-FRB fusions, a single copy
of FRB
fused to p65 activation domain activated the reporter gene significantly more
strongly than
corresponding fusion proteins containing 2 or more copies of FRB.
Mcthod: HT1080 B cells were grown in MfiM supplemented with 10% Bovine Calf
Serum.
Approximately 4x105 cells/well in a 6 well plate were transiently transfected
by
Lipotection procedure as recommended by GIBCO, BRL. The DNA: Lipofectamine
ratio used
correspond to 1:6. Cells in each well recieved 1.9 ug of PUC 118 plasmid as
carrier ,100 ng of
pCGNNGa14F3 and 500 ng one of the following plasmids :pCGNNl, 2, 3 or 4 FRB-
p65.
Following transfectian, 2 ml fresh media was added and supplemented with
Rapamycin to
the indicated concentration. After 24 hrs, 100 ul of the media was assayed for
SEAP activity
as described (Spencer et al, 1993).
Similar experiments were also rnnducted using another stable cell line (HT1080
B14)
containing the 5xGalø1L2-SEAP reporter gene and DNA sequences encoding a
fusion protein
containing a Gal4 DNA binding domain and 3 copies of FKBP stably integrated.
These cells
were transiently transfected with effector plasmids expressing p65 activation
domain fused
to 1 or more copies of an FRB domain. Similar to our observations with HT1080
B cells, in
these experiments effector plasmids expressing a single copy of FRB-pb5
activation domain
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fusion protein activated the reporter gene more strongly than others with 2 or
more copies of
FRB.
Example 9
A. Rapamycin-dependent transariptionai activation in transiently transfected
cells: ZFl<'iDl
and p65 fusions
Human h'brosarroma cells transiently transfected with a SEAP target gene and
plasmids
encoding representative ZFFiD-FKBP- and FRB-p65-containing fusion proteins
exhibited
rapamycirrdependent and dose-responsive secretion of SEAP into the cell
culture medium.
See Fig. 4A. SEAP production was not detected in cells in which one or both of
the
transcription factor fusion plasmids was omitted, nor was it detected in the
absence of added
rapamycin (Figure 4B). When all compon~ts were present, however, SEAP
secretion was
detectable at rapamycin concentrations as low as 0.5 nM (Figure 4A). Peak SEAP
secretion
was observed at 5 nM. Similar results have been obtained when the same
transcription
factors were used to drive rapamycin-dependent activation of an hGH reporter
gene or a
stably integrated version of the SEAP reporter gene made by infection with a
retroviral
vector. It is difficult to determine the fold activation in response to
rapamycin since levels
of SEAP secretion in the absence of drug are undetectable, but it is clear
that in this system
there is at least a 1000-fold enhancement over background levels in the
absence of
rapamycin. Thus, this system exhibits undetectable background activity and
high dynamic
range.
Several different configurations for transcription factor fusion proteins were
explored
(See See WO 96/41$65 , Fig. 5). When FKBP domains were fused to ZFHD1 and FRBs
to
p65, optimal levels of rapamydn-induced activation ocurred when there were
multiple
FKBPs fused to ZFHD1 and fewer FRBs Eased to p65. The preference for multiple
drug-
binding domains on the DNA-binding protein may reflect the capacity of these
proteins to
recnut multiple activation domains and therefore to elicit higher levels of
promoter
activity. The presence of only 1 drug binding domain on the activation domain
should allow
each FKBP on ZFEID to recruit one p65. Any increase in the number of FRBs on
p65 would
increase the chance that fewer activation domains would be recruited to ZFHD,
each one
linked my multiple FRB-FKBP interactions.
Methods:
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HT1080 cells (ATCC CCL-121), derived from a human Hbmsancoma, were grown in
MEM
supplemented with non-essential amino acids and 10% Fetal Bovine Serum. Cells
plated in
24-well dishes (Falcon, 6 x 104 cells/well) were transfected using
Lipofectamine under
conditions rernmmended by the manufacturer (GIBCO/BItL). A total of 300 ng of
the
following DNA was transfrected into each well: 100 ng ZFHDxI2-CMV-SEAP
reporter gene,
2.5ng pCGNN-ZFHD1~3FKBP or other DNA binding domain fusion, 5 ng pCGNN-1FRB-
p65(361-550) or other activation domain fusion and 1925 ng pUC118. In cases
where the
DNA binding domain or activation domain were omitted an equivalent amount of
empty
pCGNN expression vector was substituted. Following lipofection (for 5 hours)
500 ~,l medium
containing the indicated amounts of rapamycin was added to each well. After 24
hours,
medium was removed and assayed for SEAP activity as described (Spencer et al,
Science
262:1019-24, 1993) using a Luminescence Spectrometer (Perkin Elmer) at 350 nm
excitation and
450 nm emission. Background SEAP activity, measured from mock-transfected
cells, was
subtracted from each value.
To prepare transiently transfected HT1080 cells for injection into mice (See
below), cells
in 100 mm dishes (2 x 106 cells/dish) were transfected by calcium phosphate
precipitation
for 16 hours (Gatz, C., Kaiser, A. dz Wendenburg, R. , 1991,Mo1. Gen. Genet.
227, 229-237)
with the following DNAs: 10 mg of ZHWfxl2-CMV-hGH, 1 mg pCGNN-ZFHDl-3FKBP, 2
mg pCGNN-1FRB-p65(361 550) and 7 mg pUC118. Transfected cells were rinsed 2
times.
with phosphate buffered saline (PBS) and given fresh medium for 5 hours. To
harvest for
injection,. cells were removed from the dish in Hepes Buffered Saline Solution
containing 10
mM EDTA, washed with PBS/0.19~° BSA/0.1°~ glucose and
resuspended in the same at a
concentration of 2 x 107 cells/ml.
Plasmids: Construction of the transcription factor fusion plasmids is
described above.
pZHWTxI2-CMV-SEAP
This reporter gene, rnntaining 12 tandem mpies of a ZFHDl binding site
(Pomerantz et
al.,1995) and a basal promoter from the immediate early gene of human
cytomegalovirus
(Boshart et. al., 1985) driving expression of a gone encoding secreted
alkaline phosphatase
(SEAP), was prepared by replacing the NheI-HindIlI fragment of pSEAP Promoter
(Cl~tech) with an NheI-XbaI fragment containing 12 ZFHD binding sites shown in
WO
96/41865 and an XbaI-HindIII fragment containing a minimal CMV promoter (-54
to +45),
also shown in WO 96/41865.
pZHWTxI2-CMV-hGH
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Activation of this reporter gene leads to the production of hGH. It was
constructed by
replacing the HindIII-BamHI (blunted) fragment of pZHWTxI2-CMV-SEAP
(containing
the SEAP coding sequence) with a HindIII (blunted) -EcoRI fragment from pOGH
(containing
an hGH genomic clone; Seld~ et al., MCB 63171-3179,1986; the BamHI and EcoRI
sites
were blunted together).
pZHWTxI2-IL2-SfiAP
This reporter gene is identical to pZHWTxI2-CMV-SEAP except the XbaI-HindIII
fragment containing the minimal CMV promoter was replaced with the following
XbaI-
HindIII fragment containing a minimal IL,2 gene promoter (-72 to +45 with
respect to the
start site; Siebenlist et al., MCB 6:3042-3049, 1986) (see WO 96/41$65)
pLH
To facilitate the stable integration of a single, or few, rnpies of reporter
gene the
following retroviral vector was constructed. pLH (LTR-hph), which contains the
hygromycin B resistance gene driven by the Moloney marine leukemia virus LTR
and a unique
internal CIaI site, was constructed as follows: The hph gene was Boned as a
HindIII-ClaI
fragment horn pBabe Hygro (Morganstem and Land, NAR 18:3587-96,1990) into
BamHI-
CIaI cut pBabe Bleo (resulting in the loss of the bleo gene; the BamHI and
HindlII sites were
blunted together).
pLH-ZHWTxI2-IL2-SEAP
To clone a copy of the reporter gene containing 12 tandem copies of the ZFHDl
binding
site and a basal promoter from the IL2 gene driving expression of the SEAP
gene into the
pLH retroviral vector, the MIuI-CIaI fragment from pZHWZ'x12-IL2-SEAP (with
CIaI
linkers added) was cloned into the CIaI site of pLH. It was oriented such that
the directions
of transcription from the viral LTR and the nnten~al ZFHD-IL2 promoters were
the same.
pLH-G5-IL2-SfiAP
To construct a retroviral vector containing 5 Gal4 sites embedded in a minimal
IL2
promoter driving expression of the SF.AP gene, a Clal-BstBI fragment
consisting of the
following was inserted into the CIaI site of pLH such that the directions of
transcription
from the viral LTR and the internal.Gal4-IL2 promoters were the same: A ClaI-
HindIII
fragment containing 5 Gal4 sites and regions -324 to -294 and -72 to +45 of
the IL2 gene (shown
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WO 99/36553 PCTIUS99/00178
in WO 96/41865) and a HindlII-BstBI fragat~tt containing the SEAP gene coding
sequence
(8erger et al., Gene 66:I-10, 1988) mutagenized to add a BstBl site
immediately after the
stop colon (shown in WO 96/41865).
B. Rapamycin-dependent 6ranscdptional activation is stably transfected cells
The following experiments c~irmed that this system exhibits similar properties
in
stably transfected cells. We generated stable cell lines by sequential
transfection of a SEAP
target gene and expr~cion vectors for ZFHDl-3FKBP and 1FRB-p65, respectively.
A pool of
several den stable clones resulting from the final transfection exhibited
rapamycin-
dependent SEAP production. From this pool, we characterized several individual
clones,
many of which produced high levels of SEAP in response t~o rapamycin. Results
from one such
clone are shown in Fig. 4C of WO 96/41865. This clone produced SEAP at levels
approximately forty times higher than the pool and significantly higher than
transiently
transfected cells. In an attempt to rigorously quantitate background SEAP
production and
induction ratio in this clone, we performed a second set of assays in which
the length of the
SEAP assay was increased by a factor of approximately 50 to detect any SEAP
activity in
untreated cells. Under these conditions, mock transfected cells produced 47
arbitrary
fluorescence units, while the tranakcted clone produced 54 units in the
absence of rapamycin
and over 90,000 units at 100 nM rapamydn. Thus, in this stable cell line,
background gene
expression was negligible and the induction ratio (7 units to 90,000 units)
was greater than
four orders of magnitude.
To simplify the task of stable transfection, we used a bicistronic expression
vector that
directs the production of both ZFHDl-3FKBP and 1FRB-p65 through the use of an
internal
ribosome entry sequence (1RES). This expression plasmid was cotransfected,
together with a
zeocin-resistance marker plasmid, into a cell line carrying a retrovirally-
transduced SEAP
reporter gene, and a pool of approximately fifty drug-resistant clones was
selected and
expanded.This pool of clones also exhibited rapamycin-dependent SEAP
production with no
detectable background and a very similar dose-response curve to that observed
in transiently
transfected cells. This pool would be expected to contain individual clones
with peiformance
similar to the clone studied in Fig. 4C of WO 96/41865. Thus, rapamycin-
responsive gene
expression can be readily obtained in both transiently and stably transfected
cells. In both
cases, regulation is characterized by very low background and high induction
ratios.
Stable cell lines. Helper-free retroviruses containing the reporter gene or
DNA binding
domain fusion were generated by transient co-transfection of 293T cells (Pear,
W.S., Nolan,
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G.P., Scott, M.L. k Baltimore D.,1993, Production of high-titer helper-free
retroviruses by
transient transfection. Proc. Natl. Acad. Sci. USA 90, 8392-8396) with a Psi(-
) amphotropic
packaging vectorand the retroviral vectors pLH-ZHVVTxI2-IL2-SEAP or SMTN-
ZFfIDI-
3FKBP, respectively. To generate a clonal cell line containing the reporter
gene stably
integrated, HT1080 cells infected with retroviral stock were diluted and
selected in the
presence of 300 mg/ml Hygromycin B. Individual Bones from this and other cell
lines
descried below wen screened by transient transfection of the missing
components followed
by the addition of rapamycin as described above. All 12 clones analyzed were
inducible and
had little or no basal activity. The most nspansive clone, HT1080L, was
selected for further
l0 study.
HT20-6 cells, which contain the pLH-ZHWTxI2-IL2-SEAP reporter gene, ZFHDl-
3FKBP DNA binding domain and 1FR&p65(361-550) activation domain stably
integrated,
were generated by first infecting HT1080L cells with SMTN-ZFHDl-3FKBP-packaged
retrovirus and selecting in medium containing 500 mg/ml 6418. A strongly
responsive clone,
HT1080I3, wan then transfected with linearized pCGNN-1FRB-p65(361-550) and
pZeoSV
(Invitrogen) and selected in medium containing 250 a~g/mI Zeocin. Individual
clones were
first tested for the presence of lFltB-p65(361-550) by weatem. Eight positive
clones were
analyzed by addition of rapamycin. All eight had low basal activity and in six
of them,
gene expression was induced by at least two orders of magnitude. The done that
gave the
strongest response, HTZO-6, was selected for further analysis.
HT23 cells were generated by co-transfecting HT1080L cells with linearized
pCGNN-
1FRB-p65(361-550)-1RES-ZFHDl-3FKBP and pZeoSV and selecting in medium
containing
250 mg/ml Zeocin. Approximately 50 clones were pooled for analysis.
For analysis, cells were plated in 96-well dishes (1.5 x 104 cells/well) and
200 pl
medium containing the indicated amounts of rapamycin (or vehicle) was added to
each well.
After 18 hours, medium was removed and assayed for SEAP activity. In some
cases, medium
was diluted before analysis and relative SEAP units obtained multiplied by the
fold-
dilutia~n. Background SEAP activity, measured from untransfected HT1080 cells,
was
subtracted from each value.
Example 10: Rapamycin-dependent Production of hGH in Mice
In Vivo Methods: Animals, husbandry, and general procedures. Male nu/nu mice
were
obtained from Charles River Laboratories (Wilmington, MA) and allowed to
acclimate for
five days prior to experimentation. They were housed under sterile conditions,
were
allowed free access to sterile food and sterile water throughout the entire
experiment, and
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WO 99/36553 PCT/U599/00178
were handled with sterile techniques throughout. No immunocoatpromised animal
demonstrated outward infection or appeared ill as a result of housing,
husbandry techniques,
or experimental techniques.
To transplant transiently transtected cells into mice, 2 x 106 transfected
HT1080 cells,
were suspended in 100 ml I'BS/0.1% BSA/0.1% glucose buffer, and administered
into four
intramuscular sites (approximately 25 ml per site) on the haunches and flanks
of the
animals. Contml mice received equivalent volume injections of buffer alone.
Rapamycin waa formulated for in vivo administration by dissolution in equal
parts of
N,N-dirnethylacetamide and a 9:1 (v:v) mixture of polyethylene glycol (average
molecular
weight of 400) and polyoxyethylene sorbitan monooleate. Concentrations of
rapamycin, in
the completed formulation, were sufficient to allow for in vivo administration
of the
appropriate dose in a 2.0 ml/kg injection volume. The accuracy of the dosing
solutions was
confirmed by HPLC analysis prior to intravenous administration into the tail
veins. Some
control mice, bearing no transfected HT1080 cells, received 10.0 mg/kg
rapamycin. In
addition, other control mice, bearing transfected cells, received only the
rapamycin vehicle.
Blood was collected by either anesthetizing or sacrificing mice via C02
inhalation.
Anesthetized mice were used to collect 100 ml of blood by cardiac puncture.
The mice were
revived and allowed to recover for subsequent blood collections. Sacrificed
mice were
immediately exsanguinated. Blood samples were allowed to clot foz 24 hours, at
4'C, and
sera were collected following centrifugation at 1000 x g for 15 minutes. Serum
hGH was
measured by the Boehringer Mannheim non-isotopic sandwich ELISA (Cat No.1585
878).
The assay had a lower detection limit of 0.0125 ng/ml and a dynamic range that
extended to
0.4 ng/ml. Recommended assay instnutiams were followed. Absorbance was read at
405 nm
with a 490 run reference wavelength on a Molecular Devices microtiter plate
reader. The
antibody reagents in the ELISA demonstrate no cross reactivity with
endogenous, murine
hGH in diluent sera or native samples.
hGH expression In Vivo. For the assessment of dose-dependent rapamycin-induced
stimulation of hGH expression, rapamycin was administered to mice
approximately one
hour following injection of HT1080 cells. Rapamycin doses were either 0.01,
0.03, 0.1, 0.3,1.0,
3.0, or 10.0 mg/kg. Seventeen hours following rapamycin administratiaan, the
mice were
sacrificed for blood collection.
To address the time course of in vivo hGH expression, mice received 10.0 mg/kg
of
rapamycin one hour following injection of the cells. Mice were sacrificed at
4, 8,17, 24, and
42 hours following rapamycin administration.
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The ability of rapamycin to induce sustained expression of hGH from
transplanted
HT1~0 cells was tested by repeatedly administering rapamycin. Mice were
administered
transfected HT1080 cells as described above. Approximately one hour following
injection of
the cells, mice received the first of five intravenous 10.0 mg/kg doses of
rapamycin. The four
remaining doses were given under anesthesia, immediately subsequent to blood
collection, at
16, 32, 48, and 64 hours. Additional blood colleckions were also performed at
72, 80, 88, and
96 hours following the first rapamycin dose. Control mice were administered
cells, but
received only vehicle at the various times of administration of rapamycin.
Experimental
animals and their control counterparts were each assigned to one of two
groups. Each of the
two experimental groups and two control groups received identical drug or
vehicle
treatments, respectively. The groups differed in that blood collection times
were alternated
between the two groups to reduce the frequency of blood collection far each
animal.
Results
Rapamycin elicited dose-responsive production of hGH in these animals (Fig. 6
of WO
96/41865). hGH concentrations in the rapamycin-treated animals compared
favorably
with normal circulating levels in humans (0.2-0.3 ng/ml). No plateau in hGH
production
was observed in these experiments, suggesting that the maximal capacity of the
transfected
cells for hGH production was not reached. Control animals--those that received
transfected
cells but no rapamycin and those that received rapamycin but no cells-
exhibited no
detectable serum hGH. Thus, the production of hGH in these animals was
absolutely
dependent ups the presence of both engineered cells and rapamycin.
The presence of significant levels of hGH in the serum 17 hours aftez
rapamycin
administration was noteworthy, because hGH is cleared from the circulation
with a half-
life of less than four minutes in these animals. This observation suggested
that the
engineered cells continued to secrete hGH for many hours following rapamycin
treatment. To
examine the kinetics of rapamycin control of hGH production, we treated
animals with a
single dose of rapamycin and then measured hGH levels at different times
thereafter. Senun
hGH was observed within four hours of rapamycin treatment, peaked at eight
hours (at over
one hundred times the sensitivity limit of the hGH ELISA), and remained
detectable 42
hours after treatment. hGH concentration decayed from its peak with a half
life of
approximately 11 hours. This half life is several hundredfold longer than the
half life of
hGH itself and approximately twice the half-life of rapamycin (4.6 hr) in
these animals.
The slower decay of senun hGH relative to rapamycin could reflect the presence
of higher
tissue concentrations of rapamycin in the vicinity of the implanted cells.
Alternatively,
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WO 99/36553 PCTNS99100178
persistence of hGH production from the engineered cells may be enhanced by the
stability of
hGH mILNA.
Interestingly. administration of a second dose of rapamycin to these animals
at 42 hr
resulted in a second peak of serum hGH, which decayed with similar kinetics
indicating
that the engineered cells retained the ability to respond to rapamycin for at
least two days.
Therefore, to ascertain the ability of this system to elevate and maintain
circulating hGH
concentrations, we performed an experiment in which animals received multiple
doses of
rapamycin at lCrhour intervals. This interval corresponds to the time required
for hGH
levels to peak and then decline approximately half way. Acrnrding to this
regimen,
rapamycin concentration is Predicted to approach a steady state trough
concentration of 1.7
~g/ml after two daces (shown as dotted line in Fig. 8 of WO 96/41865). hGH
levels should
also approach a steady state trough concentration following the second die.
Treated
animals indeed held relatively stable levels of circulating hGH in response to
repeated
doses of rapamycin. After the final dose, hGH levels remained constant for 16
hours and
then declined with a similar half-life as rapamycin (6.8 hours for hGH versus
4.6 hours for
rapamycin). These data suggest that upon multiple dosing, circvsating
rapamycin imparts
tight control over the secretion of hGH from transfected cells in vivo. In
particular, it is
apparent that protein production is rapidly terminated upon withdrawal of
drug.
Dixuseion
These experiments demonstrate the feasibility of controlling the production of
a secreted
therapeutic protein from genetically engineered cells using a small-molecule
drug. This
system has many of the features required for use in human gene and cell
therapy. It is
characterized by very low background activity and high induction ratio. It
functions
independently of host physiology or any cell-type-specific factors. It is
composed
rnmpletely of human proteins. The controlling drug is well behaved in vivo and
orally
bioavailable.
With a system of this general design, it should be possible to provide stable
and
precisely titrated doses of secreted therapeutic proteins from engineered
cells in vivo.
Intermittent and pulsatile dosing should also be feasible. A considerable
advantage of
protein delivery from engineered cells under small-molecule control is that
the rate of
protein production at any given time is a function of the circulating
concentration of the
small-molecule drug. Therefore, the apparent pharmacokinetics of a therapeutic
protein
such as hGH can be dramatically altered. In our experiments, for example, the
kinetics of
circulating hGH delivered from engineered cells following a single
administration of
rapamycin are markedly different from those observed following a single
administration of
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WO 99/36553 PCT/US99/00178
recombinant protein. hGH administered to mice intravenously is cleared with a
half time of
a few minutes, whereas hGH levels from engineered cells induced with rapamycin
decayed
with a half-time of approximately eleven hours. Even in humans, where the half-
time for
hGH clearanre is approximately twenty minutes, injections must be given every
other day,
and serum hGH levels fluctuate dramatically. It is likely that protein
delivery from
engineered cells under precise pharmacologic control will lead to more
effective therapy,
particularly for proteins with poor pharmacokinetics or low therapeutic index.
The use of a small molecule drug to link a DNA~inding domain and activation
domain
is an effective strategy for regulating gene expression in vivo. One
especially attractive
feature is that the system is entirely modular, allowing each component to be
optimized and
~gineered independently. In contrast to bacterial repwasors, which rely on
relatively subtle
allosteric intramolecular interactions to control DNA-binding activity, the
dimerization
strategy can be adapted to virtually arty DNA-binding and activation domain.
We have
used here a DNA-binding domain of defined structure which readily supports
rational
engineering of DNA-binding affinity and new recognition specificities.
Similarly,
activation domains can be engineered for maxiasal potency and other suitable
properties.
Indeed, the engineered transcription factors used in these experiments elicit
very high
levels of gene expression relative to conventional promoter/enhancersystems,
and further
enhancements in either domain can be readily incorporated. The ability to
introduce
engineered transcription factors dedicated to the transcription of a single
target gene
provides opportunities to achieve lower backgrounds and substantially higher
levels of gene
expression in vivo than conventional expression vectors.
We have also chosen to construct our regulated transcription factors from
human proteins
to minimize the potential for recognition by the immune system. It has been
reported that
autologous T cells expressing a fusion protein composed of bacterial
hygxomycin
ph~photransferase and herpes virus thymidine kinase were effectively
recognized and
eliminated by host cytotoxic T cells, even in AIDS patients with debilitated
immune
systems (Riddell, S.R., et al. T-cell mediated rejection of gene-modified HIV-
specific
cytotoxic T lymphocytes in HIV-infected patients. Nature Med. 2, 216-223
(1996)). This
observation suggests that the risk df immune rerngnition of heterologous
proteins in
engineered cells is a real one and that, therefore, the use of human proteins
for performing
regulatory functions in human cells is prudent. Although each individual
component of our
transcription factor fusion proteins is human in sequence, each protein
contains junction
peptides which could potentially be recognized as foreign. These junctions may
be designed
or selected, however, to minimize their presentation to the immune system, as
discussed
previously.
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The principal limitation of rapamycin-based systems is the native biological
activity
of rapamycin, which, through inhibition of FRAP activity blocks cell-cycle
progression
leading to immunosuppressia~n in vivo. However, the ability to introduce
substituents or
otherwise modify the structure of rapamycin to substantially reduce or abolish
binding to
FKBP and /or FRAP provides access to rapalogs devoid of undue
immunosuppressive
activity. Use of such rapalogs, especially improved rapalogs of this
invention, together
with correspondingly engineered FKBP and/or FRB domains should prove widely
useful for
the regulation of engineered protein production as well as the regulation of a
wide variety of
other biological processes in experimental animals and human gene therapy.
Example 11: FP Assay for rapalog binding to FKBP
Affinities of rapalogs for PKBP proteins may be detetanined using a
competitive assay
based on fluorescence polarization (FP). A fluorescein-labelled FK506 pmbe
(APi491) was
synthesized as described in WO 96/41865 (See Example 6 therein), and the
increase in
the polarization of its fluorescence used as a direct readout of % bound probe
in an
equilibrium binding experiment containing sub-sahuating FKBP and variable
amounts of
rapamycin analog as competitor.
Determination of binding affinities (IC50s) of rapalogs using FP
Serial 10-fold dilutions of each analog are prepared in 100% ethanol in glass
vials and
stored on ice. All other manipulations are performed at room temperature. A
stock of
recombinant pure FKBP (purified by standard methods, see eg. Wiederrecht, G.
et al.1992. J.
Biol. Chem. 267, 21753-21760) is diluted to 11.25 nM in 50 mM potassium
phosphate pH
7.8/150 mM NaCI/ 100~g/ml bovine gamma globulin ("FP buffer": prepared using
only low-
fluorescence reagents from Panvera) and 98 ltl aliquots transferred to wells
of a Dynatech
micro-fluor black 96-well fluorescence plate. 2.0 ul samples of the rapalogs
are then
transferred in duplicate to the wells with mixing. Finally, a probe solution
is prepared
containing 10 nM AP149i in 0.1% ethanol/PP buffer, and 100 N,1 added to each
well with
mixing. Duplicate control wells contain ethanol instead of rapalog (for 100%
probe bindfng)
or ethanol instead of rapalog and FP buffer instead of FKBP (0°~
binding).
The plates are stored covered in the dark for approximately 30 min to permit
equilibration and then the fluorescence polarization of the sample in each
well is read on a
Jolley FPM-2 FP plate reader Qolley Consulting and Research, lnc., Grayslake,
IL) in
accordance with the manufactures s recommendations. The mean polarization (mP
units) for
each competitor concentration is usually converted to % total binding by
reference to the
control values and plotted (y) vs. log molar final concentration of rnmpetitor
(x). Non-linear
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least square analysis was used to fit the curve and extract the IC50 using the
following
equation:
y = Ml+(M4-Ml)/(1+exp(M2*(M3-x)))
where M3 is the IC50. For incomplete curves the IC50 is determined by
interpolation.
ltapamyrin and C14-desoxo-rapamycin may be included as controls in each case (
C14-
desoxo-rapamycin was prepared as described by Luerigo, J.I. et al.1994
Tetrahedron Lett. 35,
6469-6472).
Example 12. Rapalog-dependent transcriptional activation in transiently
transfected cells:
liapalogs may be assayed for their ability to dimerize FKBP and FRB fusion
proteins using a transcription read out as follows. Consfzuc~ts encoding
rapalog-
dependent transcription factor fusion proteins are introduced into cells which
contain, or are engineered to contain, a reporter gene linked to
transeriptional
regulatory DNA permitting reporter gene expression following rapalog-dependent
dimerization of the transcription factor fusion proteins. The transcription
factor
fusion proteins include (a) an FKBP fusion protein containing, as a
heterologous
effector domain, a DNA binding domain (DBD) and (b) an FRB fusion protein
containing, as a heterologous effector domain, a transcription activator
domain. The
FKBP and/or FRB domains may contain naturally occurring or non naturally
occurring peptide sequence. The presence of a rapalog which is capable of
mediating
dimerization of the two fusion proteins leads to expression of the reporter
gene. The
level of reporter observed is indicative of the activity of the rapalog as a
dimerizer.
Use of a target gene of interest in place of a reporter gene renders this a
regulated gene
expression system for use in cells grown in culture or in whole organisms.
We have made use of such as system as follows: I-iTlO$0 cells (ATCC CCIr121),
derived from a human fibrasarrnma, were grown in MEM supplemented with
non-essential amino acids and 10~o Fetal Bovine Serum. Cells plated in 24-well
3o dishes (Falcon, 6 x 104 cel)s/well) were transfected using Lipofectamine
under
conditions recommended by the manufacturer (GIBCO/B1ZL). A total of 300 ng of
the following DNA was transfected into each well:
(a) 100 ng ZFHDxI2-CMV-SEAP reporter gene (reporter gene linked to 12
recognition sites for the ZFHDl DNA-binding domain),
{b) 2.Sng pCGNN-ZFHDl-3FICBP or other DNA binding domain fusion (fusion
protein comprising 3 FKBP domains and one ZFfiDI domain),
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(c) 5 ng pCGNN-1FRB-p65(361-550) (fusion protein coatprising an FRB domain
and and a pb5 transcription activation domain) and
(d) 192.5 ng pUC118.
In some experixn~ents, pCGNN-iFRB(T2098L)-p65(361-550) was used in puce of
pCGNN-1FRB-p65(361-550) to generate an FRB fusion protein containing an
engia>eered FRB donnain. In control experinnents where the DNA binding domain
or
activation domain were omitted, an equivalent amount of empty pCGNN expression
vector was substituted. For detailed information on the assembly and use of
counstructs including those mentioned herein, see WO 96/41865 (Clackson et
al),
especially the Examples therein (which are spedfically incorporated by
reference
herein). Following lipofection (for 5 hours) 500 ul medium containing the
indicated
amounts of rapalog was added to each well. After 24 hours, medium was removed
and assayed for SEAP activity as described (Spencer et al, Science 262:1019-
24,
1993). Human fibrosarcoma cells transiently transfected with a SEAP target
gene
and plasmids encoding representative Z~D-FKBP- and FR&p65-containing fusion
proteins exh~ited rapalog-dependent and dose-responsive secretion of SEAP into
the cell culture medium. SEAP production was not detected in cells in which
one or
both of the transcription factor fusion plasmids was omitted, nor was it
detected in
the absence of added rapalog. As shown in Figure 1, cells transfected with
wild-type
FKBP and FRB rnnstructs exhibited SEAP production at dimerizer concentrations
as
iow as 1 nM. Figure 2 illustrates preferential stimulation of SEAP production
in cells
expressing a mutant FRB (T2098L, Figures 2B and 2D; T2098F, Figure 2E) as
compared to wild-type (Figures 2A and 2C). Similar results have been obtained
when the same transcription factors were used to drive rapalog-dependent
activation
of an hGH target gene or a stably integrated version of the SEAP reporter gene
made
by infection with a retroviral vector.
Example 13: Mutageneais and phage display to generate modified Ligsnd-Binding
Domains
complementary to varioua rapalogs
A. Engineered FKBP and FRB domains
We have designed and prepared recombinant DNA constructs encoding the fusion
proteins tabulated below which bear illustrative modified ligand-binding
domains. Except
a otherwise stated, mutants were generated using oligonucleotide-mediated site-
directed
mutage~sis acrnrding to standard methods (Kunkel, T.A., Bebenek, K. and
McClary, J.1991.
Meth Enzymol. 204. 235-139), and confirmed by dideoxy sequencing.
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(F36V hFKBPI2}--p65
(F36V hFKBPI2)--(F36V hFKBPI2)---p65
(F36V hFKBPI2}-(F36V hFKBPI2)-(F36V hFKBPI2)--p65
(F36M hFKBPI2)-p65
(F36M hFKBPI2)---(F36M hFKBPI2)-p65
(F36M hFKBPI2}-(F36M hFKBPI2)-(F36M hFKBPI2}-p65
(F36V hFKBPI2}-ZFHDl
(F36V hFKBPI2)-(F36V hFKBPI2}---ZFHDl
(F36V hFKBPI2)--(F36V hFKBPI2)-(F36V hFKBPl2)--ZFHD1
(F36M hFKBPI2)---ZFHDl
(F36M hFKBPI2)-(F36M hFKBPI2)--ZFHDl
(F36M hFKBPI2)-(F36M hFKBPI2}---(F36M hFKBPI2)-ZFHDl
myr-(F36V hFKBPI2)--(F36V hFKBPl2)---Fas
myr-{F36M hFKBPI2)---(F36M hFKBPI2)--Fas
myr-(F36A hFKBPI2r--(F36A hFKBPI2)--Fas
myr-(F36S/F99A hFKBPI2)-(F36S/F99A hFKBPI2)-Fas
"hFKBPI2" indicates amino acids 1-107 of human FKBP12 referred to previously
12. "p65" indicates residues 361-550 of p65
3. "Fas" indicates residues 175-304 of human Fas
"ZFHDl" is as described elsewhere
"myr" indicates the src myristoylation sequence
mutations are indicated using the previously described convention
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We have also prepared constructs encoding the following FRB fusion proteins:
sQntainintw m~ifis~ ~A~t E8»
(T2098A PRB)-p65
(T2098N FRB)---p65
(D2102A FRB)--p65
(Y2038H FRB)---p65
(Y2038L FRB--p65
(Y2038A FRB}-p65
(F2039H FRB)---p65
(F2039L FRB)--p65
(F2039A FRB)--p65
i/D2096N/T2098N FRB}-p65
FRB)-p65
1. °p65" indicates p65 residues 361-550, as above
12. "FRB" indicates the 89 amino acid FRB of human FRAP
"TOR2 FRB" indicates amino acids 1961-2052 of S. cerevisiae TOR2
Yeast and Candida FRBs, modified by analogy to the modified hFRAP FRB domains
discussed herein, may also be prepared by substitution of a codon for a
differ~t amino acid
in place of one or more of the two conserved Phe residues and the conserved
Asp and Asn
residues within each of their FRB domains. Illustrative modified FRB domains
derived
from TOR 1 and TOR2, include the following:
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Modified TORT and TOR2
FRB Domains
TORT TOR2
F1975H F1978H
F1975L F1978L
F1975A F1978A
F1975S F1978S
F1975V F1978V
F1976H F1979H
F1976L F1979L
F1976A F1979A
F1976S F1979S
F1976V F1979V
D2039A D2042A
N2035A N2038A
N2035S N2038S
These modified TORT and
TOR2 FRBs are designed
for use with rapalogs
containing C7 substituents
B. Testing rationally designed FKBP mutants for binding to rapaloga
An expression vector based on pET20b (Novagen) was constructed using standard
procedures that expresses FKBP preceded by a hexahistidine tag and a portion
of the H.
influenza hemaglutinin protein that is an epitope for the monoclonal antibody
12CA5. The
sequence of the protein encoded by this vector is as follows:
His6 HA tag- FKBP->
MHHHF>HHYPYDVPDYAAMAHI~lGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSR
DRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDV
ELLKLE
To generate expression vectors for FKBPs mutated at rapamycin contact
residues,
oligonucleotide-mediated site-directed mutagenesis was performed on the single-
stranded
form of the vector prepared from E.coli CJ236, as described (Kunkel, T.A.,
Bebenek, K. and
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McClary, ).1991. Meth Enzymol. 204, 235-139). Mutants were confirmed by
dideoxy
sequencing. Mutant proteins were expressed in E.coli BL21{DE3) (Novagen) as
described
(Wiederrecht, G. et al. 1992. J. Biol. Chem. 267, 21753-21760), and purified
to homogeneity
as described (Cardenas, M.E. et a1.1994. EMBO J.13, 5944-5957).
Using this protocol the following mutant human FKBP12 proteins were generated,
using
the indicated oligonucleotide primers (mutated bases in upper case; 5'->3'):
Mutants designed for binding to C24 rapalogs:
Phe46His agcataaacttaTGgggcttgtttctg(1)
Phe46Leu agcataaacttTaagggcttgtttctg(2)
Phe46Ala agcataaacttaGCgggcttgtttctg(3)
Phe48His ttgcctagcataTGcttaaagggcttg(4)
Phe481xu ttgcctagcatTaacttaaagggcttg(5)
Phe48Ala ttgcctagcataGCcttaaagggcttg(6)
G1u54A1a cctcggatcaccGCctgcttgcctag(7)
Va155A1a cagcctcggatcGCctcctgcttgcc(8)
Mutants designed for binding to C13/C14 rapalogs:
Phe36Ala ccgggaggaatcGGCtttctttccatcttc (9)
Phe36V al ccgggaggaatcGACtttctttccatcttc (10)
Phe36Ser ccgggaggaatcAGAtttctttccatcttc (11)
Phe36Met ccgggaggaatcCATtttctttccatcttc (12)
(Phe36Met+Phe99Ala) aagctccacatcGGCgacgagagtggc (13) + primer 12
(Phe36Met+Phe99Gly) aagctccacatcGCCgacgagagtggc (14) + primer 12
(Phe36Ala+Phe99Ala) primer 9 + primer 13
(Phe36Ala+Phe99Gly) primer 9 + primer 14
Tyr26Ala caagcatcccggtgGCgtgcaccacgcag (15)
Asp37Ala tcccgggaggaaGCaaatttctttccatc (16)
Mutant designed for binding to C28/C30 rapalogs:
G1u54A1a cctcggatcaccGCctgcttgcctag (17)
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To assay the relative binding affinity of rapamycin and rapalogs to FKHP
mutants, a
competitive fluorescence polarization (FP) assay is used that relies on the
retention of
FIC506 (and hence probe) binding affinity by the mutants. The procedure is
identical to that
S described in fixample 12 except that a direct binding assay is first
performed to: determine
the dilution (concentration) of mutant FKBP to use in the competition
reactions in order to
obtain sub-saturation. Serial dilutions of mutant FICBP are made in FP buffer
{ Example 12}
in 1001 volumes in Dynatech micro-fluor plates, and then 1001 of 10 nM AP1491
(probe) in
[FP buffer + 2% ethanol] added to each well. Equilibration and plate reading
are as in
Example 12. A plot of mP units vs conc~tration of FKBP mutant is fit to
following equation:
y = M3+(((x+Ml+M2)-SQRT(((x+Ml+M2)~2)-(4*x*Ml)))/(2*(Ml)))*(M4-M3)
and the final mutant concentration/dilution at which 90~° of probe is
specifically bound is
determined by interpolation. This final rnncentration is then used in a
competition FP assay
carried out as in Example 12, with 2 x the final concentration of mutant
replacing 11.25 nM
FKBP in the protocol. Instead of 90% saturation, 75°~6 can be selected
to impart greater
sensitivity to the competition assay. Serial diluHons of rapamycin analogs are
used as
competitor and the results are expressed as IC50 for each rapalog binding to
each mutant.
C. Testing rationally designed FRB mutants for binding to FKBP-npalog
complexes
A NcoI-BamHI fragment encoding residues 2021-2113 (inclusive) of human FRAP
was
generated by PCR with primers 28 and 29 (below),and cloned into a derivative
of pET20b(+)
(Novagen) in which the NdeI site is mutated to NcoI, to create pET-FRAP(2021-
2113).
Single-stranded DNA of this vector was used as a template in site-directed
mutagenesis
procedures, as described above, to generate vectors encoding FRAPs mutated at
rapamycin
contact residues. Mutants were confirmed by dideoxy sequencing. Mutants were
then
amplified by PCR using primers (30 and 31) that append XbaI and SpeI sites,
and cloned into
XbaI-Spei digested pCGNN-FR&p65(361-550) ( Example 7) to generate a series of
constructs
directing mammalian expression of chimeric proteins of the form E-N-mutant
FRAP(2021-
2113)-p65(361-550), where E indicates HA epitope tag and N indicates nuclear
localization
sequence. Constructs were verified by restriction digestion and dideoxy
sequencing.
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Using this procedure the following constructs encoding candidate mutant FIZAPs
for
binding to C7 rapalogs, each fused to the p65(361-550) activation domain, were
generated
using the indicated oligonudeotide primers (mutated bases in upper case; 5'-
~3'):
Tyr2038His cctttccccaaagtGcaaacgagatgc (18)
Tyr2038Leu cctttccccaaagAGcaaacgagatgc (19)
Tyr2038A1a cctttccccaaagGCcaaacgagatgc (20)
Phe2038His gttcctftccc~cAtGgtacaaacgagatg (21)
Phe2038Leu gttcctttcccc'Taagtacaascgagatg (22)
Phe2038A1a gttcctttccccaGCgtacaaacgagatg (23)
Thr2098AIa gtcccaggcttggGCgaggtccttgac (24)
(Lys2095Ser+Asp2096Asn+Thr2098Asn) gtcccaggcttggTTgaggTTcGAgacattccctgatttc
(25) '
Thr'1098Asn gtcccaggcttgg'I'Tgaggtccttgac (26)
Asp2102A1a catgataatagaggGCccaggcttgggtg (27)
Ta assay the relative binding affinity of these mutants for complexes of FKBP
with
rapamycin and various rapalogs, each construct is transiently co-transfected
into human
HT1080B14 cells, as described in Example 8. Following transfection, serial
dilutions of
rapamycin or rapalog are added to the culture medium. After 24 bouts, SNAP
activity is
measured as described in Example 8; the potency of SEAP activation at various
rapalog
concentrations is proportional to the affinity of the FRAP mutant for the
complex between
FKBP and the rapalog.
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PCR primers (restriction sites upper case; 5'->3'):
gcatcCCATGGcaatcctctggcatgagatgtggcatgaaggcctggaag(28)
cgtgaGGATCCtactttgagattcgtcggaacac(29)
gcatcTCTAGAatcctctggcatgagatgtggcatgaaggcctggaag(30)
ggtctGGATCCctaataACTAGTctttgagattcgtcggaacacatg(31)
D. Functional display of FKBP and FRB domains on filamentons bacberiophage:
one
approach to selection as an alternative to rational design of modified domains
A phage display system for the display and selection of mutant FKBP and FRB
domains is
disclosed in detail in WO 96/41865 (Clackson et al), including vector
construction,
preparation of Iiistrflag-FKBP, pCANTAB-AP-FKBP, Binding enrichments, Primers,
the
Sequence of pCAIVI'AB-AP-FRAP(2015-2114) and pCANTAB-AP-FKBP, the synthesis of
biotinylated FIC506 for affinity enrichment studies, functional FKBP display
by competitive
BLISA using biotinylated FK506, generation of a library of mutant FKHPs on
phage
targetted to the C13 and C14 positions of rapamycin andliibsary sorting.
Example 14: liapamycin-Dependent Activation of Signal Transduction
Many cellular receptors can be activated by aggregation, either by their
physiological
ligand or by anti-receptor antibodies. Additionally, the aggregation of two
different
proteins can often trigger an intracellular signal. Rapamycin and its analogs
may be used to
trigger activation of a receptor effector domain by oligonnerizing chimeric
proteins, one of
which contains one or more FKBPs and an effector domain and the other of which
contains
one or more FRAP domains and an effector domain. This scheme is illustrated in
Figure
Tl(a). While both proteins are shown anchored to the membrane, a single one
could be
membrane anchored, and addition of rapamycin or analog would recruit the
second protein to
the membrane via dimerization. Membrane anchoring may be effected through a
transmembrane protein anchor or through lipid modification of the protein(s),
such as
myristoylation. The same effector domain may be present on both proteins, or
different
protein domains that interact functionally may be used, such as a protein
kinase and a
protein Icinase substrate. Alternatively, a second effector may serve to
inhibit the activity
of the first effector.
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We note that in some embodiatents, the chimeric proteins are mixed chimeras,
discussed
previously, and contain FKBP and FRAP domains together with the heterologous
efector
domain. Oligomerization of a single mixed chimera may also be used to activate
signal
transduction, as shown in Figure Tl(b). Here rapamycin is shown to dimerize
two identical
copies of the protein. Reiteration of the FKBP and FRAP domains permits higher
multiples
to occur, subject to geometric constraints.
Two examples of the use of rapamycin in signal tranaduction are to trigger
receptor
tyrosine kinase activation and to trigger apoptosis via Fas activation, both
of which are
discussed below. Unless otherwise mentioned all DNA manipulations were
performed
following standard procedures (F.M. Ausubel et al., Eds., Current Protocols in
Molecular
Biology, john Wiley dx Sons, New York,1994) and all protein protocols were
performed
following standard procedures (Harlow, E. and Lane, D. 1988. Antibodies, a
Laboratory
Manual. Coid Spring Harbor Laboratory, Cold Spring Harbor.). All PCR products
used to
make constructs were confirmed by sequenong.
A. Rapamycin-inducibIe receptor tyrosine kinase activation.
Construction of pCM, an expression vector containing a myristylation signal.
A XbaI-Myr-BarnHI cassette, obtained by annealing oligonucleotides 1 and 2,
was
digested with XbaI/BamHI and cloned into the XbaI/BamHI site of the pCG
expression
vector (Tanaka, M. and Herr, W. 1990. Cell 60: 375,386) to create pCGM. (For
oligonucleotide
sequences, see (~ below). This oligonucleotide cassette consists of an inframe
XbaI site
followed by sequence encoding for the first 15 amino acids residues of c Src
tyrosine kinase
that has been shown to allow myristoylation and target protein to the plasma
membrane
(Cross et al., 1984. MCB. 4:1834-1842). The myristoylation domain is followed
by an inframe
SpeI site and stop codons. The Xbal site in the pCG vector is placed such that
it adds two
amino acids between the initiating Met and the sequence cloned. Since the
spacing between
the initiating Met and the myristylated Gly is crucial for membrane
localization of c-Src
(Penman et al. 1985. PNAS. 82: 1623-162 the XbaI site following the ATG in
pCGM was
deleted by site directed mutagenesis following manufacturers protocol (Muta-
Gene, BioRad).
To facilitate future cloning steps the SpeI site in the myristylation cassette
was mutated to a
XbaI site. Single stranded uracil-DNA of pCGM was prepared and the mutagenesis
was
carried out using both oligonucleotide 3 (to delete the XbaI site following
ATG and add an
EcoRI site 5' to ATG) and oligonucleotide 4 (to change the SpeI site following
the
myristylation domain to a XbaI site). The resulting sequence surrounding the
ATG of the
pCM vector was co~rn~ed by sequencing using oligonucleotide 5 (see sequence 1,
(8) below).
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2. Addition of FKBPs and an epitope tag to pCM generates pCMFl/2/3.HA.
A Spel-HA BamHI cassette was prepared by annealing rnmplementary
oligonucleotides
(oligonucleotides 6 and ~. This cassette has an inhrame Spel site followed by
nine amino
acids of H. influenzae hemaglutinin gene that is n~cognized by the monoclonal
antibody
12CA5, stop colons and a BamHI site. The Spel-HA-BamI-iI cassette was sub
cloned into the
Spe1/BamHI site of pCGNNFl, pCGNNF2 and pCGNNF3. Subsequently, the 1/2/3
copies of
FKBP fused with HA epitope was sub cloned as an XbaI/BamHl fragment into pCM.
The
resulting plasmid (pCMFl/2/3.HA) has the following features: rnyristylation
domain; an
inframe XbaI site; one/two/three copies of FKBP; an inframe SpeI site; a HA
epitope tag;
and stop colons.
3. Addition of FRBs and an epitope tag to pCM generates pCMFRl /2/3.Flag.
A SpeI-Flag-BamHI cassette can be prepared by annealing complementary
oligonudeotides (oligonucleotides S and 9). This cassette has the same
features as the SpeI-
HA-BamHI cassette described above with the exception that the inframe Spel
site is
followed by sequence that codes for eight amino acids {DYKDDDDY) (Hopp et
al.,1988.
Biotech. 6: 1205-1210) that is recognized by a monoclonal antibody anti-
FLAG.M2 (Kodak
Scientific Imaging Systems). The SpeI-Flag-BamHI cassette is sub cloned into
the
SpeI/BamHI site in pCGNN-1FRB, pCGNN-2FRB and, pCGNN-3FRB. Subsequently 1/2/3
copies of FRB domain-Flag epitope fusions are sub cloned as a XbaI/BamHI
fragment into
pCM. The resulting piasmid (pCMFRI/2/3.Flag) has the following features:
myristylation
domain; an inhume XbaI site; one/two/three copies of FRB; an infzame SpeI
site; a Flag
epitope tag; and stop colons.
4. Fusion of FKBP and FRB constructs to receptor tyrosine kinase cytoplasmic
domain
The cytoplasmic domain of receptor tyrosine kinase of choice {e.g., EGFR, erbB-
2,
PDGFR, KDR/Flk-1, Flt-1) is PCR amplified with inframe 5'XbaI and 3' SpeI
sites. The
PCR product may be subcloned either into the inframe XbaI site such that the
XbaI site is
reston'rl, or into the inflame Spel site such that the SpeI site is restored
in pCMFR series or
pCMFseries vectors (see above). As a result, the FKBP/FRB domains) can be
placed either
C-terminal or NH2-terminal to the cytoplasmic domain of the receptor tyrosine
kinase. The
vectors are constructed such that (i) the cytoplasmic domain of a given
receptor is fused to
both FKBP and FRB (for e.g., EGFR cytoplasmic domain fused to eithtr FKBP or
FRB) or (ii)
can be constructed such that cytoplasmic domains of two different receptors
are fused to
FKBP and FRB (for e.g.,EGFR cytoplasmic domain fused to FKBP and erbB-2
cytoplasrnic
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domain fused to FRB). In the former case (i) addition of the drug, rapamycin,
will induce the
formation of homodimers (e.g., EGFR/EGFR) while, in the latter (ii) addition
of the drug
will induce heterodimer (e.g., EGFR/erbB-2 ) and result in activation of the
signal
transduction cascade.
5. Testing the constructs
To test the ability of rapamycin or analog to induce dimerization of FICBP-
and FRg_
receptor cytop)asmic domain fuaions, the constructs of choice (e.g., pCMEGFR-
FRl and
pCMEGFR-Fl) are cotranstected into Cos-1 cells by lipofection (Gibco BRL) .
Three days
af6er transfection the cells are induced with rapamycin and lysed in lysis
buffer (1% Triton
X-100; SOmM Tris.d pH8.0;150mM NaCI; 5mM NaF;1mM sodium ortho vanadate;
l0ug/ml
aprotinin; l0ug/ml leupeptin). The fusion proteins from rapamycin-treated and
untreated
cell lysates are immunoprecipitated with anti-Flag and 12CA5 antibodies and
immunoblotted with anti-phosphotyrosine antibody. The choice of cell type; the
amount of
DNA transfected; the c~centration of rapamycin used and the duration of drug
treatment
are varied to achieve optimal results.
6. Rapamycin-inducible cell growth
A selected mammalian cell line (e.g., NIH3T3) is cotransfected with constructs
encoding
for FRB and FKBP fusion proteins (e.g., pCMEGFR-FRl and pCMEGFR Fl) and stable
cell
Lines expressing the fusion proteins are established. To determine whether
rapamycin-
inducible activation of receptor cytoplasmic domain will induce cell
proliferation, stable
cell lines expressing the fusion proteins are grown either in the presence or
absence of
rapamycin and the changes in cell growth rate are determined by routine
procedures {e.g., by
monitoring cell number; by determining the 3H thymidine incorporation rate,
etc.). The
choice of receptor tyrosine kinase; the type of receptor activation (homodimer
vs.
heterodimer) may be chosen to obtain optimal results.
7. Oligonucleotide sequenccs
1: CATGTCTAGAGGGAGTAGCAAGAGCAAGCCTAAG GACCCCAGCCAG
CGCACTAGTTAAGAATTC'PGATGAT CAGCGGATCCTAGC
2: GCTAGGA'PCCGCTGATCATCAGAATTCITAACTAGTG
CGCTGGCTGGGGTCCTTAGGCTTGCTC'ITGC?'ACTCC CTCFAGACATG
3: CGCCTTGTAGAATTCGCGCGTATGGGGAGTAGCAAGA
4: CCCAGCCAGCGCTCTAGATAAGAATTCTGA
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5: AAGGG'TCCCCAAACTCAC
6: GCATGACTAGTTATCCGTACGACGTACCAGACT
ACGCATAAGAAAAGTGAGGA'DCCTACGG
7: CCGTAGGATCCTCAC~TTATGCGTAGTCT~GGT
ACGTCGTACGGATAACTAGTCATGC
8: CCGTAGGATCC'!'CACTTI'f'CT'!'AATAATCGTCATCG
TCTTTGTAGTCACTAGTCATGC
9: GCATGACTAGTGACTACAAAGACGATGACGATTA
TTAAGAAAAGTGAGGATCCTACGG
lU
8. Sequence 1:
a s s ~ s R p
CGC CTT GTA GAA ttc GCG CGT ~ ggg agt agc nag agc aag cct nag
D B S Q R S R stop stop
gac ccc agc cag cgc tct aga taa gea ttc tga tga tca gcG GAT CCT
GAG AAC T
The modified sequences are in lowercase bold and the intitiating ATG is
underlined.
Sequences in uppercase are trom the parental pCG backbone.
B. Rapamycin-inducibIe apoptosis
The ability to control Fas activation and trigger apoptosis via a small
molecule has
applications both in gene therapy, where it may be used to selectively
eliminate engineered
cells, and in experimental systems. The proteins described here an: anchored
to the
membrane via the low affinity NGF receptor, also called p75. It should be
appreciated,
however, that another protein anchor could be readily substituted. p75 is
useful
experimentally because of the availability of antibodies to its extracellular
domain, and its
lack of high affinity interaction with any identified ligand (Bothwell, M.
1995. Annu. Rev.
Neurosci. 18:223-253).
1. 2-Protein Rapamycin-Regulated Fns Activation
(a) Construction of the p75 vector
Vectors to direct the expression of FRAP-Fns fusion proteins containing the
extracellular
and transmembrane domain of the low affinity NGF receptor (also known as p75)
were
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derived from the mammalian expression vector pJ7W (Morgenstem, J.p. and hand,
H. 1990.
Nucleic Acids Res. 18:1068), modified by substitution of a pUC backbone for
the original pBR
backbone using standard methods. We call this vector pA7W. Inserts cloned into
the
polylinker sites of this plasmid are transcribed under the control of the
simian CMV
promoter and enhanoer sequences. The polylinker follows the CMV sequence with
HindIIt-
SaII-XbaI-BamHI-SmaI-SstI-EcoRI-CIaI-KpnI-BgIII. Any mammalian expression
vector
with suitable cloning sites and pra~mober could be substituted.
A restriction fragment encoding a fragment of p75 flanked by HirudIII and XbaI
sites was
generated by PCR using primers Jl (5') and J2 (3~, based on the sequ~ce of
p7.5 (Johnson, D.,
Lanahan, A., Buck, C.R., Shegal, A., Morgan, C., Mercer, E., Bothwell, M.,
Chao, M. 1986.
Cell 47:545-554). The original source of the PCR template was a clone derived
from a human
brain library, using primers similar to Ji and j2 but with different
restriction sites. The 5'
end of the resulting fragment contains a HindIII site followed by an EcoRI
site, a Kozak
sequence and the initiation of p75 coding sequence (amino acid 1). The 3' end
generated
encodes the receptor sequence up to and including amino acid 274, 2 amino ands
past the
predicted membrane spanning sequence, followed by an XbaI site. Analogous
portions of
other transmembrane receptors can be substituted for this fragment. The PCR
product was
subdoned as a HindIII-XbaI fragment into HindIII-Xbal cut pA7W, generating
pA7Wp75.
The construct was verified by restriction analysis and DNA sequencing.
(b) Addition of Fas to pA7Wp75
XbaI-Spel fragments encoding Fas amino acids 206-304 (FasS) and Fas amino
acids 206-
319 (Fast) were made by PCR and subdoned into pA7Wp75 cut with the same
enzymes. The
primers used were j3 (5') and J4 or J5 (3'). J5 generates a fragment of Fas,
that ends beyond its
termination colon; when cut with SpeI, the nucleotides encoding the terminal
15 as of Fas
are removed to give a trurucated form of intracellular Fas we call FasS.
Removal of these 15
as increases the activity of Fas in some cell types (Itoh, N., and Nagata, S.
1993. J. Biol.
Chem 268:10932). Primer J4 replaces the natural termination colon of Fas with
a Spel site,
and also mutates the original SpeI site contained in Fas, generating Fast. The
plasmids
generated from subdoning these fragments are pA7Wp75-FasS and pA7Wp75-Fast,
respectively. These construct were verified by restriction analysis and DNA
sequencing. To
attach an epitope tag to these inserts, the XbaI-SpeI Fas fragments were
isolated and
ligated into the XbaI-SpeI cut backbone of pCMFl/2/3.HA, plasmids described
above which
encode an epitope tag of 9 amino adds from the H. influenza haemagglutinin
protein (E} 3' to
the SpeI site, followed by a BamHI site. Cutting the resultant plasmid with
XbaI and
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BamHI generated fragments encoding Fas followed by the epitope tag (designated
E for
these constructs).
(c). p75-FRAP-Fas-epitope fusion proteins: addition of FRAP-containing
fragments to
pA7Wp75-FasSE and pA7Wp75-FasLE to generate p75-FRAPx-FasSorLE and p75-FasSorL-
The XbaI-SpeI fragments containing a portion of FRAP are described previously
in this
document. These XbaI SpeI fragments were inserted into either the XbaI site
directly after
the p75 coding sequence to generate p75-FRAPx-FasSorLE or into the SpeI site
directly after
the Fas fragment to generate p75-FasSorL-FRAPxE. Alternatively, more than one
FRAP
fragment is subcloned in, either as a FRAPn fragment, or by sequential
subcloning of XbaI-
SpeI fragments into the Spe I site available after subclaning the first FRAP
into either XbaI
or SpeI. Thus the final series of vectors encodes (from the N to the C
terminus) p75
extracellular and transmembrane sequence, one or more FRAP-derived domains
fused N- or
C-terminally to one or more Fas intracellular domains, and an epitope tag.
(d) p75-FKBP-Fas fusion proteins: addition of FKBP-containing fragments to
pA7Wp75-
FasSE and pA7Wp75-FasLE to generate p75-FICBPn-FasSorL or p75-FasSorL-FKBPn
The XbaI-SpeI fragments containing one or more FKBPs have been described
elsewhere
in this document. These fragments were inserted into either the XbaI site
directly after the
p75 coding sequence to generate p75-FKBPn-FasSorL or into the SpeI site
directly after the
Fas fragment to generate p75-FasSorL-FKBPn. Thus the final series of vectors
encodes (from
the N- to the C-terminus) p75 extracellular and transmembrane sequence, one or
more FKBPs
fused N- or C-terminally to one or more Fas intracellular domains, and an
epitope tag.
(e) Assay of Rapamycin-Mediated Fas Activation
The ability of expression of a protein containing Fas and FRAP domains and a
protein
rnntaining Fas and FKBP domains to activate Fas and trigger cell death upon
addition of
rapamyciii can be tested in either transiently or stably transfected cells.
For transient transfections, the two plasmids to be tested are cotransfected
into a cell
line such as HT'1080 by a standard method such as lipofection, calcium
phosphate
precipitation or electroporation. One or more days after transfection, cells
are treated with
no addition or one or more concentrations of rapamycin or one or more
concentrations of a
dimerizing agent such as FK1012. The FK1012 serves as a positive control that
the FKBP-
Fas construct is functional. Several hours to 1 day later, the cells an:
monitored for response
by one of several methods. Cell Iysates were prepared by conventional means
and used to
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generate Western blots that are pmbed with antibody directed against HA or
against true
extracellular domain of p75. Alternatively, cells can be assayed by collection
is isotonic
solution plus 10 mM EDTA, stained with anti-p75 monoclonal antibody and
labeled
~condar3, antibody, and the positive cells measured by FACS. A deacease in
either Western
blot signal or FAGS signal upon treatment indicates sucessful induction of
cell death (or
decrease in protein expression). 1n addition, commercially available kits can
be used to
monitor apoptosis.
To stably transfect cells, a vector encoding a selectable marker such as
neomycin
resistance is cotransfected along with the plasmids described. Two to three
days after
iransfection, ceps are plated into 6418 and the resistant population or clones
are isolated by
standard means. 'These populations can then be monitored directly for
induction of apoptaais
by treatment with dimerizer followed by cell counting or other measure of cell
viability.
An alternative means of generating stable cell lines expressing the constructs
of interest
is to subclone the inserts into a retroviral vector. The inserts are excisable
with Eco RI to
facilitate this subdoning. 'The vector is than used to make transducing
supernatants by a
packaging cell using rnnventional methods.
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2. Single Protein Rapamycin Regulated Fas Activation
(a). Construction of FKBP-FRAP chimeric fragments FKBP-FRAP fusi~ constructs
for
rapamycin-dependent homodimerization of Fas intracellular domain
i. Structure-assisted design
In order to design molecules containing both FRAP and FIBP domains that are
capable
of rapamycin-dependent homodimerization, the three dimensional structure of
the temuy
complex between human PKBP12, rapamycirt, and a portion of human FRAP
encompassing
the minimal FRB domain rnay be considered. Requirements for homodimerization
of two
rnoleculea of fusion proteins containing FRAP, FKBP and Fas moieties include
(i) sufficient
length and flexibility of the polypeptide to accomodate the distortions
necessary for the
FRAP FKBP interaction to occur between molecules tethered at the membrane,
while
preserving the ability of aggregated Fas to transduce a signal; and (li7
Prevention or
mininsization of intramolecular dimerization by rapamycin, an event expected
to be highly
entropically favored due to the chelate effect, and therefore to prevent the
desired
inbenmolecular molecular dimerization.
Sfrvcdual considerations led us to the following design preferences for the
fusion
constructs:
(i) FRB and FKBP should be joined with a polypeptide linker sufficiently short
that
intramolecular dimerization is aterically prevented. The currently preferred
configuration is
FRAP FKBP as the C-terminus of FRAP and the N-terminus of FKBP are distant,
allowing a
long linker (ten amino acids).. that should still prevent intramolecular
dimerization yet
afford flexibility.
(ii) This FRAP-FKSP 'cassette' can be present membrane-praxiatally (i.e. with
Fas
domains) added to the C-terminus), or membrane distal (with the Fas domain
membrane-
proximal and the FRAP-FKBP cassette appended C-terminally).
(iii) A long linker should be present N-terminal to the FRAP-FKBP domains, to
allow for
the structural distortions implied by dimerization at the membrane or if the
domains are
added C-terminally. Again a N-terminal location of FRAP is. preferred as this
long linker
can then comprise natural FRAP sequence from the region N-terminal to the FRB
domain.
minimizing the immunogenicity of the chimeric protein.
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WO 99136553 PCT/US99/00178
(iv) Optimal linker lengths and fusion positions for a given protein should be
confirn..'d
empirically.
A series of 12 fusions of FKBP and FRAP, designated Tl-T12, was designed. Nine
were N-
FRAP-FKBP-C fusions including.between 13, 23 or 33 amino acids N-terminal to
Arg2018 (the
N-terminal linker), and 4, 7 or 10 residues separating the two proteins. The
remaining three
were N-FKBP-FItAP-C fusions interposing 3, 0 or -4 residues of FItAP sequence
between FKBP
G1u107 and FRAP Arg2018.
(ii) Construction
The twelve fusions were made as XbaI-BamHI cassettes that could be cloned
directly as
a single fragment, using the three-primer' 1'CR splicing method (Yon, J, and
Fried, M.1989.
Nucleic Acids lZes.17, 4895). Cloning in this way avoided the introduction of
restriction sites
between the genes that would encode foreign sequ~ce and alter the length of
the linker, A
mixture of 1 ng each of pCGNN-1FRAPi and pCAIV'TA8-AP-FKBP was amplified using
Pfu
polynuerase with 1 pM each of two outs primers (A arid C), in the presence of
0.01 WM of a
single 'splice' ofigo (B) complementary to both genes that directs the desired
fusion. The
primers used are tabulated below:
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WO 99/36553 PCT/US99/00178
-i
# construct oligos__ N-term* linkert
A B C (aa) (aa)
T1 FRAP(1985-2116)-FKBP100 102 105 33 4
TZ FRAP(1995-2116)-FKBP93 102 105 23 4
T3 FRAP(2005-2116)-FKBP101 102 105 13 4
T4 FRAP(1985-2119)-FKBP100 103 105 33 7
T5 FRAP(1995-2119)-FKBP93 103 105 23 7
T6 FRAP(2005-2119)-FKBP101 103 105 13 7
T7 FRAP(1985-2122)-FKBP100 104 105 33 10
FRAP(1995-2122)-FKBP93 104 105 23 10
T8
T9 FRAP(2005-2122)-FKBP101 104 105 13 10
T10 FKBP-FRAP(2014-2114)106 10~ 110 - 3
Tll FKBP-FRAP(2018-2114)106 108 110 - 0
T12 FKSP-FRAP(2021-2114)106 109 110 - -4
* Number of amino acids between the Arg encoded by the 5' XbaI site and FRAP
Arg2018 (for
fusiams Tl T9)
t Number of amino acids between FRAP Sef1112 and FKBP Glyl (for fiasions T'1-
T9); or
between FKBP G1u107 and FRAP Arg 2018 (for fusions T10-T12)
PCR products were purified, digested wide XbaI and BamFiI, and ligated into
Xbal-BamHI
digested pCM. The constructs were verified by restriction analysis and DNA
sequencing.
Primer sequences and the sequence of a representative FRB-FKBP construct:
fusion T6 of
FRAP (2005->~FKBP are disclosed in WO 96/41865 (p.109).
(b) Addition of FRAP-FKBP chimeric inserts to pA7Wp75-FasSE and pA7Wp75-FasLE
Subcloning of Tl through T12 as Xbal-SpeI fragments into pA7Wp75-FasSE and
pA7Wp75-FasLE li~nearized with XbaI generates p75TFaaSorLE. Subclrnning into
pA7Wp75-
FasLE linearized with SpeI generated p75FasSorLT-E. These constructs are
listed in Table 1
((d) below).
(c) Alternative FRAP-Fas-FKBP constructs
Instead of the format of the chimeric fragments Tl-T12, the single chain
strategy could
require a different orientation of domains for optimal activity. To this end,
another series of
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constructs wan made in which FKBr and FRB are separated by a Fns Wit. The
sta.::ng
points for these constructs are pCMFIHA, pCMF2HA, and PCMF3HA. Similar to the
strategy described above for the construction of chimeric transcription
factors, FKBP and
FRB fragments (described elsewhere in this document) were cloned into the pCM
backbones
as XbaI BamHI fragments that included a SpeI site just upstream of the BamHI
site. As
XbaI and SpeI produce compatible ends, this allowed further XbaI-BamHI
fragments to be
inserted dflwnatream of the initial insert. Additionally, cloning of an XbaI-
SpeI fragment
results in the addition of the fragment at the 5' end of the construct. The
final p75-anchored
construct was made by subcloning the XbaI-SpeI fragments shown in Table 1 ((d)
below) into
pA7Wp75-FasSE. A similar series is made by subcloning into pA7Wp75-FasLE.
Insertion
into vector cut with XbaI resulted in addition of the insert 3' to the p75
fragment. Insertion
into this vector cut with SpeI resulted in addition of the insert 3' to the
Fas fragment.
Insertion into this vector cut with XbaI and SpeI resulted in addition 3' to
the p75 fragment,
and eliadnation of the Fns fragment originally in the vector. By using these
three subcloning
strategies, the following series of constructs was generated. Numerical
subscripts define the
number of times the domain is reiterated.
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(d) Table 1.
Code: N= p75 NGF receptor as 1-2~4
Fasa= Fas as 206-30~
Fast= Fas as 206-319
K=PKBP as 2-108
R=FRAP 2012-2113, but other boundaries can be substitute
E= HA epitope followed by termination coda~ns as described in pCMFl/2/3.HA
i NpMg Xba I-Spe I VECTOR SITES) CONSTRUCT
FRAGMENT USBD TO
SUBCLONED SUBCLONE INSERT
INTO pA7Wp75-
FasSE
A1 K2FasL Spe I * Xba NK2FasLE
I
p2 R Spe I NFasSRE
A3 R Xba I NRFasSE
A4 R2 Spe I NFasSR2E
A5 R2 Xba I NR2FasSE
p6 K2PasSR Spe I NFasSK2FasSRE
p7 KFasSR Spe I NFasSKFasSRE
pg K2FasSR2 Spe I NFasSK2PasSR2E
A9 KPasSR2 Spe I NFasSKFasSR2E
A10 Tl Spe I NFasSTIE
All TZ Spe I NFasST2E
A12 T3 Spe I NFasST3E
A13 T4 Spe I NFasST4E
A14 T5 Spe I NFasSTSE
A15 T6 Spe I NFasST6E
A16 T! Spe I NFasST7E
A17 T8 Spe t NFasSTBE
A18 T9 Spe I NFasST9E
A19 T10 SpeI NFasSTIOE
A20 Tll Spe I NFasSTIIE
A21 T12 . Spe I NFasSTI2E
A22 K2FasSR XbaI NK2FasSRFasSE
A23 KFasSR Xba I NKFasSRPasSE
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A24 K2FasSR2 Xba I NK2FasSR2FaaSE
~
A2,5 KFasSR2 Xba I NKFaaSR2FasSE
p26 Tl Xba I NTIFasSE
-
p27 'I~ Xba I NT2FasSE
p28 T3 Xba I NT3FasSE
p2g T4 Xba I NT4PasSE
~a I NTSFasSE
A31 T6 Xba I NT6FasSE
A32 T1 Xba I NT7FasSE
T8 Xba I NTBFasSE
T9 Xba I NT9FasSE
T10 Xba I NTIOFasSE
~1 ~a I NTIlFasSE
T12 Xba I NTl2FasSE
,fig K2PasSR Spe I + Xba NK2PasSRE
I
p39 KFasSR Spe I + Xba NKFasSRE
I
p4p K2FaaSR2 Spe I + Xba NK2FasSR2E
I
KFasSR2 Spe I + Xba NKFasSR2E
I
(e) Termini/junction sequaures of hagments, oiigos and other details for
constzuction of the
inserts which were clued in 3' to the myristoylation sigfial sequence as XbaI-
BamHI or
XbaI-Spel fragments are disclosed in detail in WC) 96/41865.
(~ ~P~Y~'i''egu~ted apoptvsis of stable transsfected human HT1080 cells in
culture
Xbal-BamHI fragments from constructs A30 and A31 (d, table 1) were cloned into
pCM to
generate M30 and M31, constructs that direct the expression of MTSFasSE and
MT6PasSE,
where M denotes a myristoylation domain (see this example sections A.1. and
A.B.) and
other abbreviations are as described in d, table 1. ErnRI BamHI fzagments
containing these
expression cassettes were then cloned into the retroviral vector pSM'TN3
(Example 7).
Helper-free retroviruses containing this DNA were generated by transient co-
transfection of
293T cells (Pear, W.S. et al. 1993. Proc. Natl. Acad. Sci. USA, 90, 8392-8396)
with the
constructs and a Psi(-) amphotropic packaging vector. H'T1080 cells were
infected with viral
stock and selected with G41B.
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To assay apoptosia of the stably aranafected pools of cells in response to
rapamyciz<, _ells
were plated in a 96-well culture plebes at 10000 cells/well. After an
overnight incubation,
serial dilutions of rapamydn were added, together with 50 ng/ml (final)
actinomycin D,
and incubation continued at 37°C and 5% C02 for approximately 20 hours.
The media was
removed and replaced with 1001 of media containing 10% alamar blue dye. Plates
were
incubated as before, and the extent of cell viabiZfty assessed periodically by
spectrophotometric determination of OD at 570nm and 600nm on a microtiter
plate reader.
Typically reading was continued until control (untreated) wells are at OD 0.2-
0.4 after
subtraction of blank
Survival of cells stably transfected with (a) M30 and (b) M31-expressing
constructs is
potently reduced in the presence of rapamycin, in a dose-dependent manner. The
extent of
cell death is comparable to that of cells expressing a myristoylated (FKBP x
2)-Fas
construct (as disclosed in PCT/US94/08008) treated with a synthetic FKBP
homodimerizer
pp1428. This system may be adapted for use with improved rapalogs of his
invention,
preferably with one or more mutations in the FKBP and/or FIZB domains used.
sk r# r# tt
The full disclosure of each of the patent documents and scientific papers
cited herein
is hereby incorporated by reference. Those documents serve to illustrate the
state of the art
in various aspects of this invention. Numerous modifications and variations of
the present
invention should be apparent to one of skill in the art. Such modifications
and variations,
including design choices in selecting a heterologous action domain, improved
rapalog, fusion
protein design, DNA formulation. viral vector m other DNA delivery means,
manner and
route of transgene administntian, etc. are intended to be encompassed by the
scope of the
invention and of the appended claims.
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