Note: Descriptions are shown in the official language in which they were submitted.
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NOVEL DIMERIZING AGENTS, THEIR PRODUCTION AND USE
Related Information
Attached hereto is Appendix A containing materials related to this
application.
The entire contents of this appendix are hereby incorporated by reference.
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
to rapamycin:FKBP complex. Reported Kd values for that interaction are as low
as 200
pM. The rapamycin:FKBP complex binds with high affinity to the large cellular
protein,
FRAP, to form a tripartite, [FKBP:rapamycin): FRAP], complex. In that complex
rapamycin acts as a dimerizer or adapter to join FKBP to FRAP.
un 2~ -
N~ O ~ ~ OH
2
HO ~~ O Me0~~0
O OMe
Rapamycin
A number of naturally occurring FK506 binding proteins (FKBPs) are known.
2o 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/LTS94/01617. 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.
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Rapamycin, like FK506, is also capable of multimerizing appropriately designed
chimeric proteins. Biological switches using rapamycin or various derivatives
or analogs
thereof ("rapalogs") as multimerizing agents have been disclosed (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.
A large number of structural variants of rapamycin have been reported,
typically
to 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 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 C 13, C43 and/or C28; reduction, elimination or
derivatization of the ketone at C 14, 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
2o 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
A new class of rapalogs
As noted above, several decades of rather focused and competitive research in
the laboratories of some of the leading pharmaceutical firms and academic
institutions
have created a rich literature describing a wide variety of rapalogs. As
reported in that
literature, each portion of the rapamycin molecule which was considered
susceptible to
chemical modification was in turn modified, and the resultant rapalogs
evaluated for
2
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utility, usually as immunosuppressive agents.
It was against that extensive background that we made the completely
unexpected discovery that led to the present invention. Briefly, while
attempting to
repeat the previously described replacement of the C7 methoxy group of
rapamycin with
an allyl or methallyl moiety and recover the desired C7-substituted rapalogs,
we instead
recovered the unintentionally synthesized members of a previously unknown
family of
rapalogs and found those compounds to possess useful and in fact quite
significant
biochemical properties. Reagents and conditions used for introducing the
substitution at
C3 may be used not just with rapamycin as the starting material, but also with
a wide
1 o variety of rapalogs which can be obtained as alternative fermentation
products or by
synthetic modification of rapamycin. Moreover, the C3-substituted rapalogs may
themselves be subjected to a wide variety of subsequent chemical modifications
such as
have been reported in the case of rapamycin. Thus, the discovery of our
methodology for
C3 substitution provides access to an entirely new and diverse family of
rapalogs.
Our first novel rapalogs of this series are 3-allyl-rapamycin (R = H) and 3-
methallyl-rapamycin (R = methyl):
2o These compounds are particularly noteworthy for their ability to form a
tripartite
complex with FKBP 12 and a genetically engineered FRB domain, or with fusion
proteins containing those FKBP and FRB domains. This binding property and its
significance is discussed in detail below. As mentioned above, the
substitution at C3
may be combined with one or more other modifications to the rapamycin
structure,
numerous examples of which are known in the art. Among others, these include
C3
rapalogs with (a) replacement moieties at any one or more of positions 13, 14,
20, 24,
28, and 30; (b) a prolyl moiety in place of the otherwise characteristic
pipicolate moiety;
and/or (c) variations on or in place of the substituted cyclohexyl ring above
the
pipicolate moiety as th:e structure is drawn above. These are disclosed in
greater detail
3o below.
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These new rapalogs ("C3 rapalogs") may thus be defined as compounds which
comprise the structure of Formula I:
n=lor2
4
O
4~ O 30
O Rcs
I
I
bearing one or more optional substituents,. and typically unsaturated at one
or more
carbon-carbon bonds spanning carbons 1 through 8, as a substantially pure
stereoisomer
or mixture of stereoisomers, or a pharmaceutically acceptable derivative
thereof (as that
~ o term is defined below), where RC3 is other than H. For example, in various
embodiments of the invention, RC3 is a substituted or unsubstituted aliphatic,
heteroaliphatic, aromatic or heteroaromatic moiety.
C3 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.
One preferred class of such compounds 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 carried 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.
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A decrease in immunosuppressive activity relative to rapamycin measured in an
appropriate in vitro 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 C3 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.
One class of C3 rapalogs which is of particular interest (and is exemplified
by
to our first two C3 rapalogs) are those compounds which comprise the structure
of formula
I in which RC3 comprises:
Ra R5
R6R~
R
bearing one or more optional substituents, whether as a substantially pure
stereoisomer
or mixture of stereoisomers, or a pharmaceutically acceptable derivative
thereof, where
R1, R4, R5, R6 and R~ are each H or a substituted or unsubstituted aliphatic,
aromatic,
heteroaliphatic or heteroaromatic moiety. In some embodiments the C3
substituent may
comprise a cyclic moiety, e.g., where R1 and R4, R4 and R5, or RS and R6 are
2o covalently linked together to form a ring.
Another class of C3 rapalogs of particular interest are those compounds of
Formula II:
a
C24I RC28
i
RC14 ~ RC29,.~ R~
RC1 w
O Rca
\ / _
II
wherein RC3 is other than H, and
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R8 Rg R~2 R8
MeO
Me0\~C Me0 R
HCO ~C/
RC30 is halo, -OR3 or (=O);
RC24 is =O, =NR4 =NOR4, =NNHR4, -NHOR4, -NHNHR4, -OR4, -OC(O)R4, -
OC(O)NR4, halo or -H;
RC 13 ~d RC28 are independently H, halo, -OR3, -ORS, -OC(O)RS, -OC(O)NHRS, -
SRS, -SC(O)R5, -SC(O)NHRS, -NRSRS~or -N(RS)(CO)RS~;
RC14 is =O, -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; and,
is
RC29 is H or OR11 (e.g., OH or OMe);
where each substituent may be present in either stereochemical orientation
unless
otherwise indicated, and where each occurrence of R1, R4, RS, R6, R~, R9, R10
and
2o R11 is independently selected from H, aliphatic, heteroaliphatic, aryl and
heteroaryl; and
> >
R8 is H, halo, -CN, =O, -OH, -NR9R10 , OS02CF3, OS02F, OS02R4 , OCOR4 ,
OCONR4~R5~, or OCON(OR4~)RS~,
as a substantially pure stereoisomer or mixture of stereoisomers, or a
pharmaceutically
acceptable derivative thereof.
25 Another class of C3 rapalogs of special interest includes those in which
one or
both of RC 13 ~d RC28 is ~.e independently H, halo, -OR3, -ORS, -OC(O)R5, -
OC(O)NHRS, -SRS, -SC(O)R5, -SC(O)NHRS, -NRSRS~or -N(RS)(CO)RS~, where each
halo moiety is independently selected from F, Cl, Br and I.
Another class of C3 rapalogs of special interest are those in which one or
both of
30 RC24 ~d RC30 ~.e other than =O. This class includes 24, 30-tetrahydro-C3
substituted
rapamycins and mono and diethers thereof and the 24-halo, 30-halo and 24,30-
dihalo
derivatives thereof.
Another class of C3 rapalogs which are of particular interest are rapalogs of
Formula I wherein n is 1. This class of rapalogs includes rapalogs comprising
a prolyl
35 ring system in place of a pipicolate ring system.
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Another class of C3 rapalogs which are of particular interest are rapalogs of
Formula II wherein moiety "a" is other than
HQ,
Me
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 otherwise substituted and/or contains 5 ring
atoms in place
of the characteristic substituted cyclohexyl ring of rapamycin.
Subsets of the foregoing classes of C3 rapalogs further differ 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 C3 rapalogs noted herein.
Methods for producing C3 rapalogs
This invention further provides methods for producing C3 rapalogs which
2o involve subjecting rapamycin or a rapalog starting material to reagents and
conditions
permitting replacement of the C3 hydrogen with the desire moiety. The product
may be
recovered from the reaction mixture, and purified from unreacted starting
materials and
side products. The product may be subjected to one or more additional
transformations
prior to final purification. Methods and materials for C3 substitution and
product
recovery are disclosed in detail below.
Methods jor multimerizing chimeric proteins
This invention further provides methods and materials for multimerizing
chimeric proteins in genetically engineered cells using the new rapalogs
disclosed
3o herein, 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 a
C3 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
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to at least one of its FKBP domains. The complex formed by the binding of the
FKBP
fusion protein to the C3 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 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
1o fusion proteins and contain at least one domain heterologous to the FKBP
andlor 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
t5 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
20 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,
25 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:
b OR3
,.
iR4~ .. .. T
O U
30 wherein U is -H, -ORI,-SRI, -OC(O)Rl, -OC(O)NHRl, -NHRI, -NHC(O)Rl, -
NHS02-Rl or -R2; R2 is a substituted aryl or allyl or alkylaryl (e.g. benzyl
or
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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 -ORS, -OC(O)RS or -
OC(O)NHRS; Z is =O, -OR6, -NR6, -H, -NC(O)R6, -OC(O)R6 or -OC{O)NR6; R3 is
H, -R7, -C(O)R7, -C(O)NHR7 or C-28 / C-30 cyclic carbonate; and R4 is H or
alkyl;
where R1, R4, R5, R6 and R7 are independently selected from H, alkyl,
alkylaryl or aryl,
as those tenors are defined in WO 96/41865. A number of rapalogs are
specifically
disclosed in that document.
The subject invention is based upon a system similar to that disclosed in WO
96/41865, but involves the use of C3 rapalogs as the multimerizing agents. The
subject
1o 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 a C3 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).
In one embodiment, at Ieast 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 FKBP 12, 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. Preferably the changes are
replacements rather
than simple deletions or insertions. In embodiments in which the rapalog
differs from
rapamycin at one or more positions in addition to the modification at C3, loss
of C7
methoxy and loss of triene conjugation, at least one of the chimeric proteins
may contain
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
FKBP 12.
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
3o peptide sequence. Preferably the number of changes in peptide sequence is
limited to
five, and more preferably to 1, 2, or 3. 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 F
197, F 1976,
D2039 and N2035 of an FRB domain derived from yeast TOR1, or the replacement
of
one or more of F 1978, F 1979, D2042 and N203 8 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 practicing this invention
are disclosed
in the various scientific 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 ZFHD 1 DNA-binding domain) and another
I5 fusion protein contains at least one transcription activation domain (e.g.,
a VP 16 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.
2o 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.
25 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
3o SOS, Raf, lck, ZAP-70, etc. A number of signaling proteins are disclosed in
PCT/LJS94/O1617 (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
35 peptide sequence endogenous to the host cell are preferred in applications
involving
whole organisms. Thus, for human gene therapy applications, domains of human
origin
are of particular interest.
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Recombinant nucleic acid constructs encoding 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
s 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
to 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 mammalian cells, and
most
15 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
20 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 a C3 rapalog of this invention and contains
at least one
2s 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 C3 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
3o 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 C3
rapalog by adding the rapalog to the culture medium in which the cells are
located or
35 administering the rapalog to the organism in which the cells are located.
The cells are
preferably eukaryotic cells, more preferably animal cells, and most preferably
mammalian cells. Primate cells, especially human cells, are of particular
interest.
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Administration of the C3 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 C3
rapalog
together with one or more pharmaceutically 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
1o 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 a
C3 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-R1,
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 signaling. Cell growth, differentiation and/or
proliferation
follows clustering of such signaling domains. Such clustering occurs in nature
following
hormone binding, and in engineered cells of this invention following contact
with a C3
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.
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 gene construct, and introducing the recombinant
nucleic
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WO 99/41258 PCT/US99/03095
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 a C3 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 a C3 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 C3
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
2o 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, differentiation or
cell death, C3
rapalog-mediated clustering constitutes a method for actuating cell growth,
proliferation,
differentiation 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
3o administered to the whole organism, e.g., orally, parenterally, etc.
Preferably, the dose of
the C3 rapalog administered to an animal is below the dosage level that would
cause
undue immunosuppression in the recipient.
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
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WO 99/41258 PCT/US99/03095
fusion proteins therein. Such vectors may be viral vectors as described
elsewhere herein.
The kit may also contain a sample of a C3 rapalog of this invention capable of
forming a
complex with the encoded fusion proteins. The kit may further contain a
multimerization
antagonist such as FK506 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
1o 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.
~5 Detailed Description of the Invention
Definitions
The definitions and orienting information below will be helpful for a full
understanding of this document.
FRB domains are polypeptide regions (protein "domains"), typically of at least
2o about 89 amino acid residues, which are capable of forming a tripartite
complex with an
FKBP protein and rapamycin (or a C3 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 "RAPT1" or RAFT") from human and other
species; yeast
proteins including Torl and Tor2; and a Candida FRAP homolog. Information
25 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.
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WO 99/41258 PCT/US99/03095
protein sourcereference/sequence accession numbers
human FRAP Brown et al, 1994, Nature 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
murine RAPT1Chiu 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, Cel173, 585-596;
2
EMBL Accession #X71416, NCBI Seq ID
298027
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, murine FRAP, S cerevisiae TOR 1 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 Tor2, 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 a C3 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-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) 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
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WO 99/41258 PCT/US99/03095
hybridizing to a DNA molecule encoding a naturally occurring FRB domain. A
preferred
FRB triple mutant is disclosed in the Experimental Examples below.
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 a C3 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
to 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 al, 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 organisms 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 a C3 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 marine or other
mammalian FKBP, or from some other animal, yeast or fungal FKBP; (b) a variant
of a
naturally occurnng 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 occurnng FKBP
or by a
3o DNA sequence which would be capable, but for the degeneracy of the genetic
code, of
selectively hybridizing to a DNA molecule encoding a naturally occurnng 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
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WO 99/41258 PCTNS99/03095
from room temperature to 65-750C. See for example F.M. Ausubel et al., Eds,
Short
Protocols in Molecular Biology, Units 6.3 and 6.4 (John Wiley and Sons, New
York, 3d
Edition, 1995).
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
1 o 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
2o 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 promoter. Such transcription regulatory components can be
present
3o upstream of a coding 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
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WO 99/41258 PCT/US99/03095
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 a C3 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 constructs or their RNA counterparts) and further includes the
progeny of such cells which retain part or all of such genetic modification.
A "therapeutically effective dose" of a C3 rapalog of this invention denotes a
3o 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 usually 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 C3 rapalogs. The design and
implementation of various dimerization-based biological switches has been
reported,
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WO 99/41258 PCT/US99/03095
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
1 o 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-RI), 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 ZFHD 1 and a
transcription
activation domain such as VP 16 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
2o 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/LJS94/01617, PCT/LJS94/08008 and Spencer et al, supra. The design and use
of
such chimeric proteins 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 al, 1995, Science 267:93-96. Those references
provide
3o 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,
differentiation 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
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WO 99/41258 PCTNS99/03095
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 veterinary or pharmaceutical diluents and/or
excipients.
A compound which binds to one of the chimeric proteins but does not form
to 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 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
CA 02319492 2000-08-02
WO 99/41258 PCT/US99/03095
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 turned off by withholding further multimerization agent from the media,
by
removing residual multimerization agent from the media, or by adding to the
medium a
I5 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.
2o 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
25 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
3o 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
35 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
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WO 99!41258 PCT/US99/03095
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,
~o intravenous, intra 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
~ 5 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 with one or more pharmaceutically acceptable excipients. Such
2o 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
25 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
3o 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
35 acceptable Garner 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,
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WO 99/41258 PCT/US99/03095
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
to 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.
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
2o 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).
3o 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
23
CA 02319492 2000-08-02
WO 99/41258 PCT/US99/03095
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, SEAP, beta-galactosidase or any conveniently detectable gene product) in
response to exposure of the cells to the rapalog. Reagents for detecting
andlor
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 practicing the
subject
1o invention, reference to the following publications is suggested: Spencer et
al, 1993,
supra; Rivera et al, 1996, supra; Spencer et al, 1996, Current Biology 6, 839-
847; Luo et
al, 1996, Nature, 383, 181-185; Ho et al, 1996, Nature 382, 822-826; Belshaw
et al,
1996, Proc. Natl. Acad. Sci. USA 93, 4604-4607; Spencer, 1996, TIG 12(5), 181-
187;
Spencer et al, 1995, Proc., Natl. Acad. Sci. USA 92, 9805-9809; Holsinger et
al, 1995,
Proc. Natl. Acad. Sci. USA 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, WO 96/41865 and WO 98/02441, the contents
of each of which is incorporated herein by reference.
A key focus of the subject invention is the use of C3 rapalogs as mediators of
protein-protein interactions in applications using FKBP and FRB fusion
proteins such as
described above and elsewhere herein. The C3 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 C3
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, C3 Rapalogs and Pharmaceutically Acceptable Derivatives Thereof
"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, 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 C 13, C43 and/or C28; reduction, elimination or
derivatization of the ketone at C 14, C24 and/or C30; replacement of the 6-
membered
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WO 99/41258 PCT/US99/03095
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 C 1 and C30. Illustrative examples of rapalogs are
disclosed in
the documents listed in Table I.
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 99/41258 PCT/US99/03095
Other illustrative rapalogs include those depicted in Table II:
Table II
ny. H _
Me Me -
~O OH
a ~z. t off
H O O MeO~ O 0
O R~~ H ~ Me0 O
O OMe
i ~ 1 i
t
Rc~ ~.
H~ H~ ~ ~ Of
G~fV1 (-TF-/~' // R = -OH, -O-alkyl ~-NH-alkyl
Me V Me v
6 off o
1 / N
O Me(Y O o ~ M ~fo~R
H
~ o flMe o oMe
MaO~ ~ ~
Me v
1
N O OC(O)R
O O O ~ O O O O
0 oMe 0 0 M
H
O OMe
1
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WO 99/41258 PCT/US99/03095
"C3 Rapalogs" are defined above with reference to Formula I and are
exemplified by the various classes and subsets of compounds disclosed herein.
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WO 99/41258 PCT/US99/03095
The phrase "pharmaceutically acceptable derivative" denotes any
pharmaceutically acceptable salt, ester, or salt of such ester, of a C3
rapalog, or any other
adduct or derivative which, upon administration to a patient, is capable of
providing
(directly or indirectly) a C3 rapalog as described herein, or a metabolite or
residue
5 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
1o 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,
15 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,
2o 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,
25 amide, urea, carbamate or guanidino derivative therofJ, halo, trihaloalkyl,
cyano, -S02-
CF3, -OS02F, -OS(O)2R1 l, -S02-NHRI l, -NHS02-Rl l, sulfate, sulfonate, aryl
and
heteroaryl moieties. Aryl 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
3o 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",
35 "alkynyl" and the like. Furthermore, as used herein, the language "alkyl",
"alkenyl",
"alkynyl" and the like encompasses both substituted and unsubstituted groups.
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WO 99/41258 PCT/US99/03095
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-
to 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 team "heteroaliphatic" as used herein refers to aliphatic moieties which
2o 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,
tetrahydrofuranyl, tetrahydropyranyl, aziridine, azetidine, pyrrolidine,
piperidine,
morpholine, piperazine and the like.
The terms "aryl" and "heteroaryl" as used herein refer to stable mono- or
polycyclic, heterocyclic, polycyclic, and polyheterocyclic unsaturated
moieties having 3
- 14 carbon atoms which may be substituted or unsubstituted. Substituents
include any
3o 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,
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WO 99/41258 PCT/US99/03095
xanthenyl, phenoxathienyl, indolizinyl, isoindolyl, indolyl, indazolyl,
purinyl,
isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, quinoxalinyl,
quinazolinyl,
benzothiazole, benzimidazole, tetrahydroquinoline cinnolinyl, 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, C1-C8 alkoxy, C1-
C8
branched or straight-chain alkyl, acyloxy, carbamoyl, amino, N-acylamino,
nitro, halo,
trihalomethyl, cyano, and carboxyl. Aryl moieties thus include, e.g. phenyl;
substituted
1o phenyl bearing one or more substituents selected from groups including:
halo such as
chloro or fluoro, hydroxy, C 1-C6 alkyl, acyl, acyloxy, C 1-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
1s ethoxyphenyl), 3,5-dimethoxy-4-chloro-phenyl, etc.), amino, -S02NH2, -
S02NH(aliphatic), -S02N(aliphatic)2, -O-aliphatic-COON, 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.
2o C3 rapalogs may further differ from rapamycin, in addition to the
modification at
C3, with respect to no, one, two, three, four, five, six or seven substituent
moieties. This
class includes among others rapalogs with modifications, relative to
rapamycin, at C3
and C13; C3 and C14; C3 and a; C3 and C43; C3 and C24; C3 and C28; C3 and C30;
C3, C 13 and C 14; C3, C 13 and a; C3, C 13 and C43; C3, C 13 and C24; C3, C
13 and
25 C28; C3, C13 and C30; C3, C14 and a; C3, C14 and C43; C3, C14 and C24; C3,
C14
and C28; C3, C14 and C30; C3, a and C24; C3, a and C28; C3, a and C30; C3, C24
and
C30; C3, C24, C30 and a; C3, C24, C30 and C13; C3, C24, C30 and C14; and C3,
C24,
C30, C 13 and a.
One subset of C3 rapalogs of interest in the practice of the various methods
of the
3o invention are C3 rapalogs in which RC30 and RC24 are both other than (=O).
In certain
embodiments of this subset, RC30 ~d RC24 ~.e both -OH, e.g. in the "S"
configuration.
In other embodiments RC30 ~d RC24 are independently selected from OR3. This
subset includes among others rapalogs which further differ from rapamycin with
respect
to the moiety a. For instance, this subset includes compounds of Formula III:
30
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WO 99/41258 PCT/US99/03095
)H
III
where RC3 is other than -H. Alternative substituents for RC3 are as disclosed
elsewhere
herein. 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.
Illustrative
additional compounds for example are those compounds otherwise as shown in
formula
III except that the hydroxyl group at C43 is in the opposite stereochemical
orientation
1o and/or the hydroxyl groups at C28 and C30 are alkylated, acylated or linked
via
carbonate formation.
Another subset of C3 rapalogs of special interest are those in which one_or
both
of RC 13 and RC28 is F. In various embodiments of this subset, one, two,
three, four or
five other substituents in formula II differ from the substituents found in
rapamycin. For
instance, this subset includes C 13 fluoro-C3-rapalogs, C28 fluoro-C3-rapalogs
and C 13,
C28-difluoro-C3-rapalogs.
Another subset of C3 rapalogs of special interest are those in which RC 14 is
other than O, OH or H, e.g., C3 rapalogs in which 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 C3 rapalogs of interest are those in which RC24 is other
than
=O, again, with or without one or more other modifications at other positions
relative to
rapamycm.
Another subset of C3 rapalogs which is of special interest in practicing the
methods of this invention include those which share the stereoisomerism of
rapamycin
and in which RC~a is -OMe wherein RC30 is not =O, RC24 is not =O, RC i 3 is
not
-OH, RC 14 is not =O and/or R3 and/or R4 are not H.
Other C3 rapalogs of interest include those in which RC 14 is OH.
Furthermore, this invention encompasses C3 rapalogs in which one or more of
3o 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
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WO 99/41258 PCT/US99/03095
one or more of 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
"a" may
be replaced with any of the following
~ N HOOC
Re HCO tJ ~ ~ CI
H,CO ~S/~~~ ~
HO''~C H'C HO ~ HCO~ HO
1o Synthetic guidance
The production of rapamycin by fermentation and by total synthesis is known.
The production of a number of rapalogs (other than C3 rapalogs) by
fermentation or
synthetic modification of 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 C29-desmethyl-rapamycin and C29-
desmethoxyrapamycin and a variety of other rapalogs such as are disclosed
herein.
Rapamycin may be converted into a C3 rapalog using silyl ethers as disclosed
in
the experimental examples which follow.
The C3 rapalogs can be produced using a variety of techniques. These
techniques can be synthetic processes involving nucleophilic reagents and
catalysts. A
first rapalog can be treated with a nucleophilic reagent under conditions
which allow for
the formation of the desired C3 rapalog. The first rapalog can be a C7
modified rapalog,
e.g. C7-methoxy rapalog. These conditions include such factors as type of
nucleophilic
reagent, type of catalysts and selection of purification technique used to
isolate or purify
the final C3 rapalog.
For example, substituted rapalogs of the invention can be prepared by treating
a
rapalog having a substituent attached to the macrocyclic ring, e.g., at the C7
position,
with a nucleophilic reagent, e.g., a silyl ether, in the presence of a
catalyst. In another
embodiment, rapamycin can be converted into C3 rapalogs using silyl enol
ethers known
3o to those skilled in the art.
Suitable substituent include those recognized in the art by skilled artisans
and
include halogens, tosylates, mesylates, esters and alkoxides, and preferably
lower alkyl
alkoxides. It was discovered that treatment of rapalogs having a substituent
at the C7
position with suitable nucleophiles formed a rapalog modified at the C3
position by the
nucleophile.
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WO 99/41258 PCTNS99/03095
Suitable nucleophilic reagents include those recognized in the art by skilled
artisans and include silyl ethers, silyl enol ethers, grignard reagents,
enolates, carbanions
and the like. A preferred nucleophilic reagent is a silyl ether, preferably
trialkylsilylethers, particularly substituted and unsubstituted allyl groups,
e.g.,
methallyltrialkylsilylether.
The term "silyl ether" is art recognized and is intended to include those
compounds having the formula RC3-SiRR'R" (2) wherein R, R' and R" are
aliphatic,
aromatic, heteroaliphatic or heteroaromatic moieties and RC3 is as defined
above,
preferably a substituted or unsubstituted allyl moiety. Moreover, silyl ethers
are
1 o commercially available and can be purchased from chemical supply companies
such as
ALDRICH~, and SIGMA~.
The term "silyl enol ether" is art recognized and is intended to include those
RC3~OS~R'R"R"'
compounds having the formula wherein R, R' and R" are
aliphatic, aromatic, heteroaliphatic or heteroaromatic moieties and RC3 is as
defined
above. Silyl enol ethers are also commercially available and can be purchased
from
chemical supply companies such as ALDRICH~, and SIGMA~.
Suitable catalysts include Lewis acid catalysts such as A1C13, ZnCl2, p-
toluene
sulfonic acid, and preferably BF3 etherate.
The following reaction scheme depicted below provides an example of
2o nucleophilic displacement of a leaving group by a nucleophilic reagent,
e.g., a silyl
ether. Treatment of compound 1, a rapalog,
n=lor2
_ n=lor2
4
ze + RC3"SiRR'R" ~ O ~ za
30 14
O OMe
O 7 Rc3
3
with silyl ether, 2, under conditions which permit the formation of a compound
having
25 formula 3 include combining a C7 heteroatom bearing rapalog, e.g., OMe,
with a
trialkylsilylether in the presence of a catalyst, preferably BF3/etherate. In
general, the
reaction conditions are performed at ambient temperature or below, preferably
at about -
40° C.
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WO 99/41258 PCT/US99/03095
C3 rapalogs comprising additional modifications with respect to the structure
of
rapamycin may be prepared analogously, starting with the corresponding rapalog
in
place of rapamycin. Alternatively, one or more additional transformations may
be
carried out on a C3 rapalog intermediate.
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
to 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 positionliterature reference
modified
C-13 C13->F: protect C28 and C43, rxn at
0C
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 Or et al, US Patent Nos. 5,527,907
and 5,583,139
positions Luengo, WO 94/02136; Cottens et al,
WO 95/16691
WO 98/02441 (Holt et al)
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 Katz et al, WO 93/13663 and by Cane et al.
WO
9702358.
2o 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
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WO 99/41258 PCTNS99/03095
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 th:e protecting groups when
no longer
needed or desired, and would be readily capable of determining other synthetic
approaches.
Purif cation of mixtures of rapalogs isolated from the above described
conditions
can be accomplished using a technique which allows for the isolation or
recovery of the
desired C3 rapalog, e.g., a desired purity or in a desired form, e.g.,
substantially free of
to C7 rapalog. An example of such a technique is the recycling HPLC technique
described
in the examples below. Use of recycling HPLC lead to the discovery, under the
conditions described above, that nucleophiles add to the C3 position with
concomitant
displacement of a C7 group to yield C3 rapalogs. Structural confirmation that
the
rapalogs were functionalized at the C3 position was determined by mass
spectral
analysis and/or NMR spectroscopy.
As a non-limiting example, an unpurified reaction mixture of rapalog products
can be eluted through an HPLC column and recycled back through the column to
increase separation of components and thus, purity of the final isolates. HPLC
systems
are known to those skilled in the art can be adapted to include such recycling
features.
2o Generally, the eluant and the components) are recycled until a desired
separation of
components and purity is achieved, e.g., 15 to 20 cycles. One skilled in the
art can
determine how many cycles are required, the solvents) required, the type of
column, etc.
to achieve the purity desired.
For example, a polystyrene size exclusion column can be used. The polystyrene
column can be functionalized, e.g., contains hydroxyl functionality, to
facilitate
separation of component. In one particularly preferred embodiment, a JAIGEL GS-
310
column can be used to effect separation of components.
One aspect of the invention pertains to the purity of the isolated rapalogs.
Purity
of the isolate rapalogs can be determined by techniques known to those skilled
in the art
3o and include mass spectrometry, 1H NMR and 13C NMR. In one embodiment, the
isolated C3 rapalogs or the invention are substantially free contaminants,
e.g.,
substantially free of C7 rapalogs. One example of determining the purity of
the isolated
C3 rapalog is by 1 H NMR. The purity of the compound can be determined by
analyzing
the peak height ratio of the desired product to that of contaminants. In one
embodiment,
the ratio is at least about S:1 (product/contaminant). In another embodiment,
the ratio is
at least about 10:1, more preferably 50:1, most preferably 100:1 with the most
preferred
being that no contaminants are detected by NMR.
CA 02319492 2000-08-02
WO 99/41258 PCT/US99/03095
In another aspect, purity of the isolated rapalog can be determined by I H NMR
by analyzing the ratio between the peak heights associated with those groups
attached at
the C7 position of the starting material to that of the peak heights
associated with the
groups attached at the C3 position in the resultant product. The purity of the
C3 rapalog
can be determined by analyzing the peak height ratio of the desired product to
that of
peaks associated with the starting material containing a C7 group. In one
embodiment,
the ratio is at least about 5:1 (isolated C3 rapalog product/C7 starting
material). In
another embodiment, the ratio is at least about 10:1, more preferably 50:1,
most
preferably 100:1 with the most preferred being that no contaminants are
detected by
NMR.
In another embodiment, the isolated C3 rapalog is at least about 90% pure, as
determined by analytical techniques known in the art, e.g. capillary gas
chromatography,
analytical HPLC, etc. In a more preferred embodiment, the isolated C3 rapalog
is at
least about 95% pure. In another embodiment, the isolated C3 rapalog is at
least about
15 98% pure. In a most preferred embodiment, the isolated C3 rapalog is about
100% pure.
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
2o domain. This chimeric protein must be capable of binding to a C3 rapalog of
this
invention, preferably with a Kd value below about 100 nM, more preferably
below about
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
25 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 FKBP 12, 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 S or
3o fewer, amino acid residues. Such modifications are elected in certain
embodiments to
yield one or both of the following binding profiles: (a) binding of a C3
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 foul or more,
orders of
magnitude better (by any measure) than to FKBP12 or the FKBP endogenous to the
host
35 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
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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
t o 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, VP 16, FAS, CD3 zeta chain, etc., the recombinant
DNA
encoding the chimeric protein will also 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.
FKBP fusion proteins of this invention, as well as FRB fusion proteins
discussed
2o 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 iigand 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
3o invention include the FKBP fusion proteins disclosed in PCT/LJS94/01617
(Stanford &
Harvard), PCT/US94/08008 (Stanford & Harvard), Spencer et al (supra),
PCT/LJS95/10591 (ARIAD), PCT/LJS95/06722 (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
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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
1o 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 I-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:
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 VSSA 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.
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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
io this invention include those which are capable of binding to a complex of
an FKBP
protein and a C3 rapalog of this invention.
The FRB fusion protein must be capable of binding to the complex formed by the
FKBP fusion protein with a C3 rapalog of this invention. Preferably, the FRB
fusion
protein binds to that complex with a Kd value below 200 ~M, more preferably
below 10
15 pM, 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 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
20 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
25 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 a C3
rapalog
over formation of complexes of endogenous FKBP and FRAP proteins with the
rapalog.
3o 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
35 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
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WO 99/41258 PCT/US99/03095
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 a C3
l0 rapalog of this invention. An illustrative FRB fusion protein contains one
or more FRBs
of at least 89-amino acids, containing a sequence spanning at least residues
202-2113
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.
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
2o FRB fusion proteins.
Mixed chimeric protein molecules are capable of forming homodimeric or
homomultimeric protein complexes in the presence of a C3 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
3o 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.
Heterologous domains
As mentioned above, the heterologous effectar 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
CA 02319492 2000-08-02
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apoptosis; directing proteins to a particular cellular 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 1 st
and 2d chimeric proteins.
In embodiments of the invention involving direct activation of transcription,
the
heterologous domains of the 1 st and 2d chimeric proteins comprise a DNA
binding
to domain such as Gal4 or a chimeric DNA binding domain such as ZFHD1,
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 or substituting a
repressor
domain (see PCT/LJS94/01617) in place of a transcription 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
2o 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 subunit 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
3o 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 RI .
Chimeric
proteins containing a Fas domain can be designed and prepared by analogy to
the
disclosure of PCT/LJS94/01617.
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Engineered receptor domains
As noted previously, the FKBP and FRB domains may contain peptide sequence
selected from the peptide sequences of naturally occurnng FKBP and FRB
domains.
Naturally occurring sequences include those of human FKBP 12 and the FRB
domain of
human FRAP. Alternatively, the peptide sequences may be derived from such
naturally
occurnng 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
elsewhere 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 a
C3
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 a
C3 rapalog,
preferentially, i.e. at 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 FKBPI2 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
modeling 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, not to be bound by theory, we contemplate that certain C3
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,
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CA 02319492 2000-08-02
WO 99141258 PCTNS99/03095
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.
l0 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 GIu54 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 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
2o 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
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WO 99/41258 PCT/US99/03095
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 misincorporation mutagenesis (see e.g. 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.
to 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 GAL4 fusion vector. Yeast co-transformants
are treated
2o 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
3o 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
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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
to 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 systems can also be
contemplated (for
2o example display on lambda 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,
I73-184.
2s (b) FRAP
Similar considerations apply to the generation of mutant FRB domains which
bind preferentially to C3 rapalogs containing modifications (i.e., are
'bumped') relative to
rapamycin in the FRAP-binding portion of the macrocycle. For example, one may
obtain
3o preferential binding using rapalogs bearing substituents other than -OMe 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,
G1n2099,
Trp2101, Ser2035, Tyr2105, Pro2095, and any other residue in the vicinity of
the
rapamycin, triene or residues near the rapamycin triene and Asp2102. Exemplary
35 mutations include Y2038H, Y2038L, Y2038V, Y2038A, F2039H, F2039L, F2039A,
F2039V, D2102A, T2098A, T2098N, and T2098S. Rapalogs bearing substituents
other
than -OH at C28 and/or substituents other than =O at C30 may be used to obtain
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preferential binding to FRAP proteins bearing an amino acid substitution for
G1u2032.
Exemplary 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 or mammalian two- three-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 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
3o 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.
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CA 02319492 2000-08-02
WO 99/41258 PCT/US99/03095
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
1o pairs of mutant FKBPs and mutant FRBs that are capable of complexing with
the
rapalog more effectively than the corresponding domains containing naturally
occurnng
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
2o 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 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
3o 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
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CA 02319492 2000-08-02
WO 99/41258 PCTNS99/03095
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.
Additionally, 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
to 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 'anchor' positions in the bound
peptide: e.g.
HLA-A2 requires leucine, methionine or isoleucine at position 2 and leucine 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
2o 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/L1S94/01617,
especially pages 26-27. Briefly, a membrane retention domain can be isolated
from any
convenient membrane-bound 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,
including many receptors. The transmembrane peptide sequence may be extended
to
3o 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
3s 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
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CA 02319492 2000-08-02
WO 99/41258 PCT/US99/03095
(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.
1o 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 loxP 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 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.
3o 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.
49
CA 02319492 2000-08-02
WO 99!41258 PCT/US99/03095
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 Gene Reference
lens g2-crystallinBreitman, M.L., Clapoff, S., Rossant,
J., Tsui, L.C.,
Golde, L.M., Maxwell, LH., Bernstin,
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
pituitaryGrowth Behringer, R.R., Mathews, L.S., Palmiter,
- R.D.,
somatrophhormone Brinster, R.L. (1988) Dwarf mice produced
by
is cells genetic ablation of growth hormone-expressing
cells.
Genes Dev. 2: 453-461
pancreas Insulin- Ornitz, D.M., Palmiter, R.D., Hammer,
R.E., Brinster,
Elastase R.L., Swift, G.H., MacDonald, R.J.
- (1985) Specific
acinar expression of an elastase-human growth
cell fusion in
specific 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
CA 02319492 2000-08-02
WO 99/41258 PCT/US99/03095
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 Immunog~obulinBorelli, E., Heyman, R., Hsi, M., Evans,
R.M. (1988)
kappa lightTargeting of an inducible toxic phenotype
chain in animal
cells. Proc. Natl. Acad. 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 promoterMessing, A., Behringer, R.R., Hammang,
J.P.
cells Palmiter, RD, Brinster, RL, Lemke,
G. ,PO promoter
directs expression of reporter and
toxin genes to
Schwann cells of transgenic mice. Neuron
8: 507-520
1992
Myelin basicMiskimins, R. Knapp, L., Dewey,MJ,
Zhang, X. Cell
protein and tissue-specific expression of a
heterologous gene
under control of the myelin basic protein
gene
promoter in trangenic mice. Brain Res
Dev Brain Res
1992 Vo165: 217-21
spermatidsprotamine 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 Ornitz, D.M., Palmiter, R.D., Hammer,
R.E., Brinster,
surfactant R.L., Swift, G.H., MacDonald, R.J.
( 1985) Specific
gene 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
51
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WO 99/41258 PCT/US99/03095
muscle myosin Lee, KJ, Ross, RS, Rockman, HA, Harris,
light AN,
chain O'Brien, TX, van-Bilsen, M., Shubeita,
HE, Kandolf,
R., Brem, G., Prices et alJ. BIoI.
Chem. 1992 Aug 5,
267: I 5875-85
Alpha actinMuscat, 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.
proteins Saarma, M. Janne,J. Tissue-specific
expression of rat
light neurofilament promoter-driven
reporter gene in
transgenic mice. BBRC 1993: 192: 465-70
fiver tyrosine
aminotransferase,
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
0 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 chimeric 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),
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WO 99/41258 PCTNS99/03095
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
o protein, a nuclear protein, a recombinase such as Cre, a ribozyme or an
antisense RNA.
See PCT/LTS94/01617 for general design and construction details and for
various
applications including gene therapy and see PCT/LJS95/10591 regarding
applications to
animal models of disease.
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
2o 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
a C3 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 a C3 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 a C3 rapalog which is capable of multimerizing those chimeric
protein
3o 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.
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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
1 o 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 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'
3o 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)
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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, Thyl
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
l0 generally use either'/Z 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 constructs 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
elerrrents,
etc. which may be used to prepare stocks of construct DNAs and for carrying
out
transfections are well known in the art, and many are commercially available.
Delivery of Nucleic 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-
3o 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.
CA 02319492 2000-08-02
WO 99/41258 PCT/US99/03095
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
to 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, (Y), 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 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
stably 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
3o 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
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5:793-801, 1994, Table I). 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 (ITR 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
1o 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 line that expresses AAV proteins inducibly,
such as
2o temperature-sensitive inducible expression 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
3o 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) .
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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
1o 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 delivery protocol, by the same methods
(restriction
2o digest, linker ligation or filling in of ends, 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
ITR
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
96/26285, 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.
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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 1010 pfu of virus/ml, e.g. from 1 X 10g 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
2o include any of the nucleated cells which may be involved with the
erythroid, lymphoid
or myelomonocytic lineages, as well as myoblasts and fibroblasts. 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 132-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
3o 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.
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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
s 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 1010 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.
2o 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 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 al, 1993, Hum Gene Ther 4(4):433-40
(mouse Ltk- cells expressing hGH/immunoprotective perm-selective alginate
microcapsules; Reddy et al, 1993, J Infect Dis 168(4):1082-3 (alginate); Tai
and Sun,
1993, FASEB J 7( 11 ):1061-9 (mouse fibroblasts expressing hGH/alginate-poly-L-
lysine-alginate membrane); Ao et al, 1995, Transplantation Proc. 27(6):3349,
3350
(alginate); Rajotte et al, 1995, Transplantation Proc. 27{6):3389 {alginate);
Lakey et al,
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
al, 1993, Exp Neurol 122( 1 ):37-47 (polymer-encapsulated PC 12 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
CA 02319492 2000-08-02
WO 99/41258 PCTNS99/03095
monkeys; see also Aebischer, WO 92/19595); Savelkoul et al, 1994, J Immunol
Methods
170(2):185-96 (encapsulated hybridomas producing antibodies; encapsulated
transfected
cell lines expressing various cytokines); Winn et al, 1994, PNAS USA 91
(6):2324-8
(engineered BHK cells expressing human nerve growth factor encapsulated in an
immunoisolation polymeric device and transplanted into rats); Emerich et al,
1994, Prog
Neuropsychopharmacol Biol Psychiatry 18(5):935-46 (polymer-encapsulated PC 12
cells
implanted into rats); Kordower et al, 1994, PNAS USA 91 (23):10898-902
(polymer-
encapsulated engineered BHK cells expressing hNGF implanted into monkeys) and
Butler et al WO 95/04521 (encapsulated device). The cells may then be
introduced in
1o 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
immunologically 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
VP 16. The cells may be derived from one or more individuals other than the
recipient
2o 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 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
3o 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
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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
turned on after a predetermined time, for example, after the cells have been
able to home
to a particular site.
Binding properties, Assays
1o Rapamycin is known to bind to the human protein, FKBP12 and to form a
tripartite complex with hFKBP 12 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
15 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 FKBP 12 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
2o 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.
Corresponding cell-based assays may also be conducted in engineered yeast
cells. See
e.g. WO 95/33052 (Berlin et al).
25 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.
3o 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-occurring immunophilins.
For
example, such compounds may bind preferentially to mutant FKBPs at least an
order of
35 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 FKBP12, as determined by any scientifically valid or art-accepted
assay
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WO 99/41258 PCTNS99/03095
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 FKBP 12,
to a mutant thereof as discussed above, or to a fusion protein containing such
FKBP
1o 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 W096/41865. 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.
One preferred class of compounds of this invention are those rapalogs which
have an IC50 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
3o 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
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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
to 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 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
maintained 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.
3o The compound to be tested is added 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
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WO 99!41258 PCT/US99/03895
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
to 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 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), a C3 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 C3 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
3o which the DNA-binding domain is capable of binding. Contacting the
engineered cells
or their progeny with the C3 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/LJS93/01617
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 C3 rapalog.
Dose (of C3
rapalog)-responsive gene expression is typically observed.
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The C3 rapalog may be administered to the patient as desired to activate
transcription of the target gene. Depending upon the binding affinity of the
C3 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 C3 rapalog may be administered by various routes,
including
parenterally or orally. The number of administrations will depend upon the
factors
described above. The C3 rapalog may be taken orally as a pill, powder, or
dispersion;
bucally; sublingually; injected intravascularly, intraperitoneally,
intramuscularly,
subcutaneously; by inhalation, or the like. The C3 rapalog (and monomeric
antagonist
o 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 C3 rapalog is to be
reversed or
terminated, a monomeric compound which can compete with the C3 rapalog may be
administered. Thus, in the case of an adverse reaction or the desire to
terminate the
therapeutic effect, an antagonist to the dimerizing agent can be administered
in any
convenient way, particularly intravascularly, if a rapid reversal is desired.
Alternatively,
2o one may provide for the presence of an inactivation domain (or
transcriptional silencer)
with a ligand binding domain. In another approach, cells may be eliminated
through
apoptosis via signalling through Fas or TNF receptor as described elsewhere.
See
International Patent Applications PCT/LJS94/01617 and PCT/US94/08008.
The particular dosage of the C3 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 C3 rapalog over short periods of time,
with
extended intervals, for example, two weeks or more. A dose of the C3 rapalog
within a
3o 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 C3 rapalog is chronically administered, once the maintenance dosage
of the
C3 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
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product.
It should be appreciated that the system is subject to many variables, such as
the
cellular response to the C3 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.
l0 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 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
3o 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.
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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 transgenic 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.
Transgenic
to 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 transcriptional
control elements
which are activated by the multimerization of the 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
2o 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
3o 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
68
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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,
1o 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 formulation can
include
standard Garners 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
3o 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 oil, 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
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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 Garner can also be an oily ester such as ethyl oleate and
isopropyl
to myristate. Sterile liquid carriers are useful in sterile liquid form
compositions for
parenteral administration. The liquid carrier 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.
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, methylmethacryiate 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 Garner 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 Garner 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
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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
1 o 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
~ 5 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 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.
2o 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
25 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.
30 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
35 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
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water, glycerol, alcohol, propylene glycol, fatty alcohols, triglycerides,
fatty acid esters,
or mineral oils.
Other topical Garners include liquid petroleum, isopropyl palmitate,
polyethylene
glycol, ethanol (95%), polyoxyethylene monolaurate (S%) in water, or sodium
iauryl
s 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
to 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
15 intravenous formulations) and European Patent Application Publication Nos.
0 649 659
(published April 26, 1995; illustrative formulation for IV 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 SO mg/kgs, preferably about 0.1 to about 10 mg/kg of mammalian body
weight,
2o 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
25 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 may be
extrapolated
from dose-response curves derived from in vitro or animal model test systems.
The
3o 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.
35 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
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WO 99/41258 PCT/US99/03095
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 a C3 rapalog of this invention, consistent
with the
disclosure herein.
The invention is further illustrated by the following examples, which should
not
be construed as further limiting. The contents of all references, pending
patent
applications and published patents, cited throughout this application are
hereby
expressly incorporated by reference.
Experimental Examples
Synthesis of C3-methallyl-rapamycin and C3-allyl-rapamycin
"" 24
O /~OH
.. ~. n.~..'~~
~O
is
The synthetic procedure we currently use is a modified version of the
procedure
described in Liberles et al., July 1997, Proc. Natl. Acad. Sci. USA 94:7825-
7830 for the
preparation of C7 rapalogs. (Note that C7 and C3 as referred to herein refer
to the ring
positions C 16 and C20, respectively, in the nomenclature of Liberles et al).
Twenty two
2o mgs of rapamycin are placed in a 3 ml flame-dried Wheaton vial equipped
with a stir
bar. The rapamycin is dissolved in 200 p,l of methylene chloride and cooled to
-40
degrees. Lower temperatures have been found to not work as efficiently. 50 ~l
of
methallyl (or allyl) trimethyl silane (12 equiv.) is added, followed by 40 pl
of neat BF3
etherate (13 equivalents). After 2 hours the reaction is over, as determined
by TLC, and
25 is quenched by addition of saturated NaHC03. The reaction mixture is then
washed
with brine and dried with sodium sulfate. (The aqueous washes can be back-
extracted
with methylene chloride and combined with the reaction mixture.) The samples
are
f ltered with a .45 micron Nylon filter and the solvent is evaporated under
vacuum.
73
R = H, Me
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The crude product is dissolved in 100 ~1 of chloroform for injection on the
JAI
recycling HPLC described below. Three predominant peaks are obtained that can
be
separated with baseline resolution after approximately 15-20 cycles. (Under
certain
reaction conditions, a fourth peak appears which elutes before the other
three.) Of the
three predominant peaks, the first is C7-S methallyl rapamycin, as determined
by 1 H-
NMR. The middle peak is the compound of interest, C3-methallyl rapamycin, and
the
fourth peak is composed of side products resulting from oversilylation of
rapamycin.
Following this procedure, we obtained 4.2 mgs of pure C3-methallyl rapamycin.
Since
unmodified rapamycin coincidentally comigrates precisely with C3-methallyl
to rapamycin, and cannot be removed efficiently with the JAI, it is critical
that rapamycin
be completely consumed during the reaction. Overaddition products are far more
easily
removed than the unreacted starting material.
The JAI-purified methallyl rapamycin can be verified by UV, NMR, and mass
spectroscopy and biological activity. The presence of contaminants, such as
rapamycin,
can be easily determined by UV, as described below.
This same procedure has been used to synthesize C3 allyl-rapamycin, and should
be easily extended to synthesize a variety of other C3 derivatives.
Analytical considerations
One particularly convenient diagnostic is UV spectroscopy. Consistent with
disappearance of the triene, the UV spectra of the C3 compounds have a maximum
absorbance at lambda 238 rather than 274-282, typically seen for the C7 (R) or
{S)
compounds. Since the C3 compounds have baseline absorbance in the region where
rapamycin absorbs best, UV analysis is a good indicator of sample purity and
the
presence of any toxic contaminants.
The loss of the triene in C3-methallyl rapamycin is also reflected in the 1H
NMR. A 1 H-NMR of methallyl rapamycin, taken in CDCL3, reveals a diagnostic
peak
from C5 at 6.0 ppm. The remainder of the olefinic protons all lie upfield
between 5 and
5.6 ppm.
Chromatographic recovery of the C3 rapalogs
An instrument made by the 3apan Analytical Industry Co., LTD. (JAI)
efficiently
purifies the C3 rapalogs. The instrument we used was a "LC-908 Recycling
Preparative
HPLC" and the column we used was "JAIGEL GS-310" with dimensions of "20 x S00
mm".
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WO 99/41258 PCT/US99/03095
Functional characteristics of C3 rapalogs
The toxicity of the lead compounds was tested by their ability to inhibit the
proliferation of CTLL-2 cells, which are IL-2 dependent and highly sensitive
to
rapamycin (IC50 < 1 nM). Incubation of CTLL-2 cells with 1 pM C3-methallyl
rapamycin had no detectable effect on proliferation.
Likewise, C3-allyl rapamycin and C3-methallyl rapamycin were impaired in
their ability to activate transcription in Jurkat cells transfected with Gal4-
FKBP3, wild
type FRB-VP16, and UAS-SEAP. (These constructs are described in Liberles et
al,
1o above, the full contents of which are incorporated herein by reference.) In
experiments
in which FRB*-VP16 (FRB harboring T2098L, W2101F, and K2095P) replaces wild
type FRB-VP16, reporter gene activity can be detected at an EC50 near 10 nM C3-
methallyl rapamycin or C3-allyl rapamycin; as well, the amplitude of reporter
gene
activity is higher than that elicited by the rapamycin/wild type FRB-VP16
system. C3
derivatives of rapamycin have impaired toxicity (immunosuppressive activity)
and can
be used efficiently as dimerizers in conjunction with engineered FRB domains.
Preparation of 24(S),30(S)-tetrahydro-C3-rapalogs
HO
v
Me0 Me0
~N~ 1~
N~O ~ I OH Ne8H4, ceci3,H2o N O H
HO~O Me0~~' O MeOH, -78 C HOO O Me
~''O OMe O OMe
HO,
Me0
O OH OH
N
O O OH
Me0~~
HO Rcs
O
~,,
24,30
-tetrahydro rapamycin is prepared as described elsewhere and is converted into
the
desired C3-substituted rapalog by the method described above.
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WO 99/41258 PCT/US99/03095
Preparation of 13-fluoro-C3-rapalogs
HO, HO,
Me V Me0
v
24
( OH p ~ ~ OH
N N
O O
F 4 O Me(y' g ~ MeO~ O
'30 ~ O ~ O Rcs
7 .. W /
The title compounds are prepared as described above, substituting 13-Fluoro
rapamycin
for rapamycin.
Preparation of 28-fluoro-C3-rapalogs
1o
HO, HO,
Me ~ Me0
v
"", 24
b ( F p ~ F
N~~ - i
O O N
4 O MeO~' 3 HO ~ Me0~ O
HO ' -"
30 ~ O ~ O Rcs
7 :. ~
The title compounds are prepared as described above, substituting 28-Fluoro
rapamycin
for rapamycin.
Preparation of 43-epi-C3-rapalogs
HO
24 = Me
N~O ~ ~ F p g I F
N
0 4 O MeO~ O O O MeO~'
HO
O ~ O ~ O Rc3
/ i, \ ~ ..
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The title compounds are prepared as described above, substituting 43-epi
rapamycin for
rapamycm.
Equivalents
Those skilled in the art will recognize, or be able to ascertain using no more
than
routine experimentation, many equivalents to the specific embodiments of the
invention
described herein. Such equivalents are intended to be encompassed by the
following
claims.
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.~nPE~IX A -
New Patent Application
entitled
Novel Dimerizing Agents, Their production and Use
by
Gerald Crabtree, Stuart Schreiber and Steven Liberles
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WO 99/41258 PCT/US99/03095
replacement of the C3 hydrogen with the desire moiety. The product may be
recovered
from the reaction mixture, and purified from unreacted starting materials and
side
products. The product may be subjected to one or more additional
transformations prior
to final purification. Methods and materials for C3 substitution and product
recovery are
disclosed in detail below.
Methods for multimerizing chimeric proteins
This invention further provides methods and materials for multimerizing
chimeric
proteins in genetically engineered cells using the new rapalogs disclosed
herein, preferably
while avoiding the immunosuppressive effects of rapamyrin.
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 a
C3 rapalog of this invention. This first chimeric protein is also referred to
herein as an
"FI~P fusion profiein" and further comprises at least one protein domain
heterologous to
at least one of its FKBP domains. The complex foamed by the binding of the
FKBP fusion
protein to the C3 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 embodiments, however, the FKBP fusion
protein is
also an FRB fusion protein. In those embodiments, the chimeric protein
comprises one or
yore PKBP 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 c proteins may be readily designed, based on incorporation of
aPPI'oP~~Y hefierologous 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 rnmplexation is detezmitued by the
choice of
heterologous domains in die fusion proteins. The heberologous domains are
therefore
referred to as "action" or Neffertor" domains.The genetically engineered cells
for use in
practicing this invention will contain one or more recombinant nucleic acid
constructs
encoding the chinreric proteins, and in certain applications, will furtiier
contain one or
more accessory nucleic acid constructs, such as one or more et gene
rnnstructs.
Illustrative biological responses, applications of tile system 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. Thatsysbem involves the
use of a
multimerizing agent comprising rapamycin or a rapalog of the generic formula:
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WO 99/41258 PCTNS99/03095
wherein U is -H, -0RI, -SRI, -OC(O)Rl, -OC(O)NHRI, -NHRI, -NHC(O)Rl, -NH502-Rl
or -Rz; R2 is a substituted aryl or all 1 or alkylaryl (e.g. benzyl or
substituted benzyl); V is
-OR3 or (=O); W is =O, =NR~ =1V01~~, =NNHR~, -NHOR~, -NFiIVHR~, -0R~, -
OC(O)R4, _
OC(O)NR~ or -H; Y is -0R5. -OC(O)R5 or -OC(O)NHRS; Z is ~, -0R6, -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 R~ is H or alkyl; where RI, R~, R5, R6 and R~ are
independently
selected from H, alkyl, alkylaryl or aryl, as those terms are defined in W0
96/41865. A
number of rapalogs are specifically disclosed in that document.
The subject invention is based upon a system similar to that disclosed in WO
96/41865, but involves the use of C3 rapalogs as the multinnerizing agents.
The subject
invention thus provides a method for multia>erizsng chin~eric proteins in
cells which
comprises (a) providing aPPmP~~Y en cells containing nucleic acid constructs
for d~ec~ng expression of the d ' chinieric proteins) and any desired
accessory
i5 recombinant constructs, and (b) contacting the cells with a C3 rapalog or a
pharrmaceutically acceptable derivative thereof as desc~ed herein. The rapalog
forms a
rnmplex containing itself and at least two molecules of the chimeric
protein(s).
In one embodiment, at least one of the chinieric proteins contains at least
one
FKBP domain whosepeptide sequence differs from a nafixrally occurring PKBP
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. Preferably the changes are
replacements rather than
simple deletions or insertions. In embodiments in which the rapalog differs
from
rapamycin at one or more positions in addition bo the modifiction at C3, loss
of C7
methoxy and loss of triene rnnjugation, at least one of the chinieric proteins
may contain
at least one FKBP do~nnain comprising at least one amino acid replacement
relative to the
sequence of a naturally occurring PKBP, espeaally a mannavalian PKBP such as
human
FKBP12.
In another embodiment, at least one of the chimeric proteins contains at least
one
3o FRB domain whose peptide sequence differs from a nahually occvning PRB
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. 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 amnw 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 TORT, or the replacement of one or more of
F1978,
F1979, D2042 and N2038 of an PRB domain derived from yeast TOR2, with
independently selected replacement amino acids, e.g. H, L, S, A or V.
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WO 99/41258
PCTNS99/03095
In certain embodiments the chimeric protein(s) contain at least one
modification in
peptide sequence, preferably up to three mo~.ficati~, ~tive to naturally
occutring
sequences, in both one or more FKBP domains and one or more FRB domains.
As mentioned previously, in the various embo~ents of this invention, the
chimeric proteins) contain one or more "action" or "effector" domains which ~e
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 si~a~n g
domains
(i.e., domains which are capable upon clustering or multimerization, oe~g 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 embodiment, one fusion protein contains at least one
DNA binding domain (e.g., a GAL4 or ZFHDl DNA-binding domain) and another
fusion
protein contains at least one transcription activation domain (e.g., a VP16 or
p65
transcription activation domain), l.igand-mediated asso~ati~ 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 DNAsequence 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. lion domains which
target
the cell membrane, for example, include domains such as a myristoylation site
or a
transmembrane region of a recepior protein yr other membrane-spanning P~~
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 (actor or
cytokine receptor,
an apoptosis triggering domain such as the intracellular domain of FAS or TNF-
Rl, and
domains derived from other intracellular signaling Prot~ such as SOS, Raf,
lck, ZAP-70,
etc. A number of signaling proteins are d;~~ ~ p~.L/U594/01617 (see e.g. ages
23 -
26). In still other embodin~nts, each of the fusion proteins contains at least
one
domain and at least one PKBP domain, as well as one or more heberologous
domains.
Such fusion proteins are capable of homodiaierization and g ~ the
presence of the rapalog In g~ral,, domains c~t~g pep~e ~e~e endogenous to
the host cell are preferred in applications involmng whole organisms. Thus,
for human
gene therapy applications, domains of human origin are of particular interest.
Recombinant nucleic acid constructs encoding the fusion proteins are also
provided, as are nucleic acid eonstrucis capable of directing thenexpression,
and vectors
~t~g sub ~ for introducing them into cells, particularly ofic cells, of
which yeast and animal cells are of particular interest. In view of the t
components of the fusion proteins, the reeombi~nant DNA molecules which encode
them
are capable of selectively hybridizing (a) to a DNA molecule encoding a
polypeptide
comparing an FRB domain or PKBP domain and (b) to a DNA molecule encoding the
heterologous domain or a protein firm which the heterologous protein domain
was
derived. DNAs are also enc~a~ gassed which would be capable of so hybridizing
but for
the degeneracy of the genetic code.
Using nucleic acid sequences enc the fusion proteins, nucleic acid constructs
for directing their expression m eukaryoti'ocas, a~ vectors or other means for
81
WO 99/41258 PCT/US99/03095
introducing such constructs into cells, espeaally animal sells, one may
genetically erwgineer
cells, particularly animal cells, preferably mammlian cells, and most
preferabl human
cells, for a number of important uses. To do so, one first provides an
expressiYon 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. ()ne 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 rnmmendally available. '
One object of this invention is thus a method for multimerizing fusion
proteins,
such as descn'bed herein, in cells, preferably animal cells. To recap, one of
the fusion
proteins is capable of binding to a C3 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
i5 capable of fonnvng a tripartite rnmplex with the first fusion protein and
one or more
molecules of the C3 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,
ie., the two fusion proteins have one or more rnmmon heterologous domains. In
other
embodiauents, each fusion protein contains one or more different heterologous
domains.
The method rnmprises contacting appropriafiely ~ cells with the C3
rapalog by adding the rapalog to the culture medium in which the cells are
located or
adm~stering'the rapalog to the organism in which tile cells are located. The
cells are
preferably e~uotic cells, more preferably animal cells, and most preferably
mammalian
cells. Primafie eel, espeQallY human cells, are of particular interest.
Administration of the
Ci rapalog to a human or non human animal may be effected using any
pharmaceutically
acceptable formulation and route of administration. Oral administration of a
pharataeeutically acceptable composition containing the Ci rapalog together
with one or
more pharrmaeeuticaly acceptable carriers, buffers or other excipients is
currently of
greatest interest.
A speafic object of this invention is a method, as otherwise descnbed above,
for
inducing tzansalption of a target gene in a rapalog-dependent manner. The
cells typically
contain, in addition to recombinant DNAs encoding the two fusion proteins,
at~get gene
construct which comprises a target gene operably linked to a DNA sequence
which is
responsive to the presence of a complex o~ the fusion proteins with rapamycin
or a
rapalog. The target gene construct may be reeombinanty and the target gene
and/or a
regulabory nucleic acid enae linked thereto may be heterologous with respect
to the
host cell. In oermin emb&x~ts the cells are a to contact with a C3 rapslog
which binds to the FKBP fusion profiein and pa~ in a complex with. a F1ZB
fusion
protein with a detectable preference over binding to endogenous FKBP and/or
FRB-
containing proteins of the host cell.
Another speciRc object of this invention is a method, as otherwise des~ed
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 proteirt, and usually two
fusion proteins,
i.~ a domain such as the intracellular domain of FAS or TNRRl, 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-depent
manner. In such cells, at least one of the heterologous domains of at least
one of a fusion
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WO 99/41258 PCT/US99/03095
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 signaling. Cell~zowth, differentiation and/or
proliferation
follows clustering of such signaling domains. uch dustering o~ in nature
following
hormone binding, and in engineered cells of this invention following contact
with a C3
rapalog. '
Cells of human origin are preferred for human gene therap applications,
although
cell types of various origins (human or other species) may be use and may, if
desired, be
encapsulated within a biocompatible material for use in human subjects.
Also provided are materials and methods for producing the foregoing engineered
cells. This object is met by providing recombinant nucleic atids, typically
DNA molecules,
encoding the fusion proteins, together with any desired ancillary recombinant
nucleic
acids such as a target gene construct, and introducing the recombinant nucleic
acids into
the host cells under conditions permitting nucleic acid uptake b cells. Such
transfection
i5 may be effected ex vivo, using host cells maintained in culture. ells 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 mamnnal, which is responsive (as de'sc~ed
herein) to
the presence of a C3 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 desc~ed
herein) to
the presence of a C3 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 desc~ed herein. For
instance,
a method is provided for multiazezizing fusion profieins of this invention by
contacting
cells engineered as des~ed herein with an effective amount of the C3 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 speciCc
embodiments in
which the s~gnalin~ domains are selected based on their ability fo~owing
clustering to
tn_gger cell growth, proliferatioxl, diffeentiation or cell death, C3 rapalog
mediated
clustering constitutes a method for actuating cell growth, pmliferatioz~,
diffeentiation or
cell dead, 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 pharnnaceutical or veterinary composition) is administered
to the
whole organism, e.g., orally, parenterally, etc. Preferably, the dose of the
C3 rapalog
administered to an aninnal is below the dosage level that would cause undue
immunosuppression in the recipient.
Also disclosed are kits for use in the genetic engineering of cells or human
or non-
human animals as desalted herein. One such kit contains one or more
recombinant
nucleic acid constructs encoding fusion roteins of this invention. The
recombu~ nucleic
and rnnstructs will generally be in the form of eukaryotic expression vectors
suibble for
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WO 99/41258 Pty'T/US99/03095
introduction into aninnal cells and capable of directing the expression of the
fusion
proteins therein Such vectors may be viral vectors as des~bed elsewhere
herein. The kit
may also rnntain a sample of a C3 rapalog of this invention capable of fornung
a complex
with the encoded fusion proteins. The kit may further contain a
multimeiization
antagonist such as FK506 or some other compound capable of binding to one of
the fusion
proteins but Incapable of forming a rnmplex 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 hetemlogous domain of choice. In some
l0 embodiments the kit may also rnntain a target gene construct containing a
target gene or
cloning site linked to a DNA sequence responsive to the presence of the
complexed fusion
pm~~~ 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.
Detailed Description of the Invention
Definitions
The definitions and orienting information below will be helpful for a full
understanding of this docualent.
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
PKBP protein and rapamycin (or a C3 rapalog of this invention). FRB domains
are
present in a number of naturally occurr proteins, including FRAP proteins
(also referred
to in the lifierature as "RAP'I'1" or ~ from human and other spec>es; yeast
proteins
including Torl and To~'1; and a Candida FRAP homolog. Information concerning
the
nucleotide enoes, cloning, and other aspects of these proteins is already
known in the
art, permi 'ttm~g the synthesis or cloning of DMA encoding the desired FRB
peptide
sequ~e, e.g., using well known methods and FCR priave~ based on published
sequences.
protein source reference/sequence accession numbers
h~ FRAp Brown et al,1994, Nature 369, 756-758:
GenBank accession # 134075, NCBI Seq ID 508481:
Chiu et al, 1994, PNAS USA 91, 12574-12578;
Chen et a1,1995, FNAS USA 92, 4947-4951
marine RAPTl Chiu et al, supra.
yeast Tori blelliwell et a1,1994, Mol Cell Biol 5,105-118;
EMBL Accession #X74857; NCBI Seq Id #468738
yeast Tor 2 Kunz et al,1993, Cell 73, 585-596;
EMBL Accession #X71416, NCBI Seq ID 298027
Candida TOR ~ W095/33052 (Berlin et al) - I
3p 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 c~human
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WO 99/41258 PCT/US99/03095
FRAP, marine FRAP, S. cerevisiae TORT and S. cerevisiae TOR2 encompassing the
conserved FRB n Typically the FRB sequence selected for use in fusion proteins
of
this invention span at least the 89-amino acid sequence Glu-39 through L / -
127
as the sequence ~s numbered in that figure. For reference using '
~ nag o C3t~en 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 Toi'L, 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 comp lex of an FKBP protein bound to
rapamycin
or a C3 rapalog of this invention (as may be defiermined by any means, d~ or
indirect,
for deflecting such binding, including, for example, means for detecting such
binding
employed in the FRAP/RAPT/RAPT and Tor-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)
amino acids
of the naturally-occurring peptide sequence have been deleted, inserted, or
replaced with
substitute amino ands; or (c) a peptide sequence encoded by a DNA sequence
capable of
selectively hybridizing to a DNA molecule encoding a naf<wally occurruig 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 ~ due.
A preferred FRB triple mutant is disclosed in the Experimental Examples below.
FKBPs (FK506 binding proteins) are the cyfiosolic receptors for macrolides
such as
FK506, FK520 and rapamycin and are highly conserved across species lines. For
the
ptrp~e of this disclosure, FKBPs are proteins or profiein domains which are
capable of
bv'iding to rapamycin or to a C3 rapalog of this invention and further forming
a ~pa~~
complex with an FRB-containing protein. An FKBP domain may also be inferred to
as a
Nra amycin binding domain". Infoiniation oonoeming flue nucleotide sequences,
claming,
another aspects of various PKBP species is aheady lmown in the art, permitting
the
synthesis or cloning of DNA encoding the desired FKBP tide
Pep sequence, e.g., using well
kcwwn methods and PCR primers based on published sequences. See e.g. Staendart
et al,
1990, Nature 346, 671-674 (human FKBP12); Kay, 1996, Biochem. j. 314, 361-385
(review). Homologous PKBP proteins in other mammalian spew, in y~st, and in
other
organsiais are a3so lrnown 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
rapamycxn or a
C3.rapalog of this invention and participating in a tripartite complex with an
FItB-
cong Protein (as may be determined by any means, direct or indirect, for
detecting
such binding). The peptide sequence of an ~~d~ of an FKBP fusion protein of
this
invention comprises (a) a naturall occurring FKBP peptide sequence, preferably
derived
fr'°n' ~ h~ ~P~ protein ~~cemplified below) or apeptide sequen derived
from
another human PKBP, from a marine or other mammalian FKBP, or from me other
aniala~, yeast or fungal FKBP; (b) a variant of a naturally occurring
FKBP~equence in
which up to about ten (preferably 1-5, more preferably 1 3, and in some
embodiments just
one) amino acsds of the naturally-occurring peptidesequence have been deleted,
inserted,
or replaced with su~st.tute amino acids; or (c) apep tide sequence encoded by
a DNA
sequence capable of selectively hybridizing to a I~1~A molecule encoding a
naturally
occurring FKBP 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 FKBP.
CA 02319492 2000-08-02
WO 99/41258 PCTNS99/03095
"Capable of selectively hybridizing" as that phrase is used herein means that
two DNA molecules are susoept~le 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, Units 6.3 and 6.4 Qohn 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 enrnmpassing 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~~~host cell. Nucleic acid
constructs of particular interest are those which en da fusion proteins of
this invention
or which rnmprise a target gene and expression contml elements. The construct
may
2o 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: transariptional promoter and/or ezlhaneer
sequences, a
n'bosome binding site, introns, etc.
"Recombinant", "chimeric" and "fusion", as those tenors are used herein,
denote
mafiet;aLs comprising various oompozuent do~a~ains, sequerwes or other
components which
are mutually heterologous in the sense that they do not occur together in the
same
arrangement, in nature. More speafically, 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, enhanoers, and promoters, which induce or control
transcription of
protein oodn~g ~qu~ with which they are operably linked. The term "enhancez"
is
intended to include regulatory elerxuents capable of increasing, stimulating,
o~ enhancing
transQiption form a pmawber. Such transQiption regulatory-companents can be
present
upstream of a coding region, or in certain cases (e.g. enhanaers), in other
locations as well,
such as in introns, exons, coding regions, and 3' flanking sequences.
"Dimerization", "oligomerization" and "multimerization" are used
intenhangeabl herein and refer to the association or clueb~ng of two or more
pmtein
4o molecules, meted by the binding of a drug to at least onethe proteins. In
prefe~d
embodiments, the multimerization is mediated by the binding of two or more
such protein
molecules to a rnmmon divalent or multivalent drug. The formation of a rnmplex
comprising two or more protein molecules, each of which containing one or more
FKBP
domains, together wig 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.,
wheat least
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WO 99/41258 PCT/US99/03095
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 profiein molecules. The
formation of a
tripartite comp lex comprising a profiein 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 profieins. 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 arerepresented by multiple copies.
The
formation of complexes containing at least one divalent drug and at least two
protein
molecules, each of which rnntains at least one drug binding domain, may be
referred to as
., .. " " " ,. ..
oligomerization or multimerization , or simply as dimerization , clustering or
association".
"Dimerizer" denotes a C3 rapalog of this invention which brings together two
or
more proteins in a multimeric rnmplex.
"Activate" as applied herein to the expression or transcription of a gene
denotes
directly or indirectly observable incsease 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 ands
(e.g. one
or more DNA constructs or their RNA counterparts) and further includes the
progeny of
such cells which retain part or all of such genetic modification
A "therapeutically effective dose" of a C3 rapalog of this invention denotes a
treatment- or praphyla~ds-effective dose, e~a dose which yields detectable
target
transcription or cell growth, proliferation, erentiation, death, etc. in the
genetics y
engineered cell, or a dose which is predicted to be treatment- or rophylaads-
effective by
extrapolation from data obtained in animal or cell culture mode. A
therapeutically.
effective dose is ususally preferred for the treatment of a human or non human
maavaial.
t
This invention involves methods and materials for multimerizin~ proteins
in genetically engineered cells C3$ra~alogs. The design and implementation of
carious dimerization~ased biolo witches has been reported, inter ells, in
Spencer et
al and in various international patent applications cited herein. Other
accounts of
successful application of this general approach have also been clad. Chiau'ric
proteins containing an PRB domain fused to an effector domain also been
disclosed in
Rivers et x1,1996, 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 li mediated
"dimerization" or
"multinterization" of the fusion proteins which contain ~~ triggers a d ssired
biological
event such as transcriplion of a desired gene, cell death, cell proliferation,
etc. For
example, clustering of chimeric pmteins containing an action domain derived
from the
intracellular portion of the T cellre~p for CD3 zeta domain triggers
transQiption of a
gene under the hansaiptional control of the ILr2 promoter or promoter
elennenerived
therefrom. In other embodiments, the action domain comprises a domain
derived~rom the
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intracellular portion of a protein such as FAS or the TNF-alpha receptor
(TNFalpha-Rl),
which are capable, upon oligomerization,, of triggering at~is of the cell. In
~ other
embodiments, the action domains comprise a DNA bmd~g domain such as GAIL or
ZFHDI 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.
Qiinieric proteins rnntaining 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 si~ transduction pathway, cellular localization, etc., are disclosed in
PC"T/US94/01617, PCT/US94/08008 and Spencer et al, supra. The design and use
of
such chimeric proteins for ligand-mediated gene-knock out and for ligand-
mediated
blockade of gene expression or inh~ition 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 x1,1995, Science 267:93-96. Those references
provide
substantial information, guidance and examples n.,lating to the design,
construction and
use of DNA constructs encoding analogous chinneras, target gene constructs,
and other
aspects which may also be useful to the practitioner of the subject invention.
By appropriate choice of chinleric profieins, this invention permits one to
activate
the transcription of a desired e, actuate cell growth, proliferation,
differentiaion or
apoptvsis: ar trigger other bio~ events in engineered sells in a rapalog-
dependent
a~aiu~er anal us tv the systems described m the pabent docuauents and other
references
dted above. e~ineered cells, preferably 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 a lications. The rapslog is administered to
the cell
culture or to the organism containing engineered sells, as the case may be, in
an
amrnmt effective to multimerize the PKBP fusion proteins and FRB fusion
proteins (as
ma be observed indirectly by nwnitoring target gene transcription. apoptvsis
or other
bio~ical process so tziggered). In the case of administration bo whohe
organisms, the
rapalog may be administered in a composition containing the rapalog and one or
more
acceptable verterinary or pharmaceutical d~luents and/or excipients.
A compound which binds to one of the cliinvexic proteins but does not form
tripartite rnmplexes with both chirrueric proteins may be used as a
multimerization
antagonist. As such it may be administered fio the eng~ed 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, inh~itin~ r disrup '~mul.ation of
the
cliiateras. For instance, FIC506, FIC520 or any of the many syn FKBP ligands
which
do not form tripartite rnmplexes with FICBI' and PRAP may be used as an
antagonist.
One important aspect of this invention ~mvides materials and methods for
rapalog-dependent, direct activation of transcription of a desired gene. In
one such
embodiment, a set of two or more diffe: :m: chinleric proteins, and co
responding DNA
constructs capable of directing their expression, is provided. One such
chimeric protein
contains as its ackion domains) one or more transcxiptional activation
domains. The
other chimeric protein contains as its action donnain(s) one or more
DNA~inding
domains. A rapalog of this invention is capable of binding to both chimeras to
fa~m a
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CA 02319492 2000-08-02
WO 99/41258 PCT/US99/03095
diaueric or multimeric rnmplex thus rnntaining at least one DNA binding domain
and at
least one transaiptional 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~inding domain is capable of binding. as
can be
observed by monitoring directly or indirectly the presence or rnncentration of
the target
gene Product.
Preferably the DNA binding domain, and a chimera containing it, binds to it<s
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
fio 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 acivation 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
negulatable expression
of a target gene, for instance, can be used for regulated roduction of a
desired protein (or
other gene pnxiuct) encoded by the target gene. Such ~ may be grov~m in
culture by
conventional means. Addition of the rapalog to the culture medium containing
the cells
leads to expression of thetarget gene by the sells and production of the
rotein encoded
by that gene. nession of the gene and production of the protein can ~ turned
eff by
withholding ftuet' 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
1 aninnals, humans, a . such that the cells
modify whole organisms, preferab y Y ~g
produce a desired protein or other result within the anmial containing them.
Such uses
include gene therapy applications.
Embodiments involving negulatable actuation of apoptosis provide engineered
cells
susceptible fio rapalog-induc~ble cell death. Such engineered cells can be
elinvnafied from a
cell culture or host organism after they have served their intended purposed
(e.g.
production of a desired protein or other nxluct), if they have or develop
unwanted
p , or if they are no longer usefu~ safe or desired. Elimination is effected
by
a~ rapalog to the medium or adavnistering it to the host organism. In such
cases,
the action domains of the chimeras are pmtein domains such as the
intracellular domains
of FAS or TNF-Rl, downstream components of their signalling pathways or other
protein
domains which upon oligomerization trigger apoptosis.
This invention thus provides materials and methods for achieving a biological
effect in c~h y in response to the addition of a rapslog of this invention.
The method
involves providing cells engineered as dherein and exposing the cells to live
rapalog.
89
CA 02319492 2000-08-02
WO 99/4125$ PCT/US99/03095
For le, this invention provides a method for activating transmption of a
target gene n~. The method involves providing cells rnntaining (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 mgnabe 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-
expn~ssion 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, intra-
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 phain~aceutical composition comprising a
rapalog of this invention in admixture with a pharmaceutically acceptable
cannier and
optionally with one or more pharn~aoeutically acceptable excipients. Such
pharmaceutical
compositions can be used tn promote multimerization of chimeras of this
invention in
engineered cells in whole aninnals, e.g. in human gene therapy applications
fio achieve any
of the objectives disclosed herein.
Said differently, this invention provides a method for achieving any of those
objectives, e.g. activation of transQiption of a target gene (typ~tally a
hefiemlogous gene
for a therapeutic protein), cell growth or proliferation, cell death or some
other selecfied
biologi~l event, m an animal, preferably a human patient, in need thereof and
containing
engineered cells of this invention. That method involves adminis6eiing to the
anin~avl a
plarn~aceutical composition containing the rapalog by a mute 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 debec6ed indirectly by detecting
the
occurrence of target gene expression; ~ proliferation or death; or other
objective
for which the chimeras were designed the sells g~eneticallY engineered. . _.
This invention furtiver encompasses a phara~aeeutical co tion comprising a
multimerization antego~ u'st of this invention in admixture with a
~p~ceutically
acceptable carrier and optionally with one or more phara~aoeutically
acoeptab1e
excipients for inheriting or otherwise reducing, in whole or part, the extent
of
multianerization 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
t<u~ning off
another biological result of this invention Thus, the use of the
multin~erizing rapalogs and
of the multimerization antagonist reagents to prepare pllarataceutical
compositions and
achieve their pharn~acologic n~ults 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 su~ject, responsive to a rapalog of
tl~s
CA 02319492 2000-08-02
WO 99/41258 PCT/US99/03095
invention. The method involves introducing into the organism cells which have
been
engineered in aoeordanae with this invention, i.e. contaic~~g one or more
nucleic acid
constructs encoding the chia~eric proteins, and so forth. The engineered ceps
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 conditi~tting
nncorporation
thereof into one or more cells of the host mammal, e.g. using v~~, ~~duction
of
DNA by injection or via catheter, etc.
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 then~sion of chimeras which, upon rapalog mediated oli
Bred gonierization,
trigger the des biological response. The kit may contain a quantity of a
rapalog
capable of multimerizing the clumeric protein molecules encoded by the
constructs) of the
kit, and may contain in addition a quantity of a multimerization antagonist.
The kit may
further rnntain a nucleic acid construct encoding a target gene (or cloning
site) linked to a
rngnate DNA sequence which is recognized by the dimerized chimeric proteins
pe~~g
h~anscription of a gene linked to that cognate DNA sequence in the presence of
multimerized chimeric protein molecules. The constructs may be assoriabed 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
assoc~afion with
a rngnate 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 fio be provided by the practitioner.
Other kits of this invention may contain one or two (or more) nucleic acid
constructs for chinieric proteins in which one or more contain a cloning site
in place of the
franscriptional activator or DNA binding protein,pera u~tting the user to
insert whichever
3o such domain s/he wishes. Such a kit may optionally include other elenienfis
as described
above, e.g. a nucleic construct for a target gene with or without a cognafie
DNA sequence
for a pre-selected DNA binding donnain.
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, SF.AP, beta-galactosidase or any conveniently detechable gene product) in
response
to exposure of the cells to the rapalog. Reagents for deflecting 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 rnmponents thereof which may be adapted for use in practising the
subject
invention, reference to the following publications is suggested: Spencer et
a1,1993, supra;
Rivera et x1,1996, supra; Spencer et x1,1996, Current Biology 6, 839-847; Luo
et al,1996,
Nature, 383,181-185; Ho et x1,1996, Nature 382, 822-826; liel.~ haw et al,
1996, Proc.
Natl. Acad. Sci. USA 93, 4604-4607; Spencer, 1996, TIG 12(5),181-187; Spencer
et al,
1995, Proc., Natl. Acad. Sci. USA 92, 9805-9809; Holsinger et x1,1995, 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,~,~VO
91
CA 02319492 2000-08-02
WO 99/41258 PCT/US99/03095
95/33052, WO 96/20951, WO 96/41865 and WO 98/02441, the contents of each of
which is inrnrporated herein by reference.
A key focus of the subject invention is the use of C3 rapalogs as mediators of
pmtein-protein interactions in a plications using FKBP and FRB fusion proteins
such as
descried above and elsewhere rein. The C3 ra slogs may be used in the various
applications of the underlying dimerization-base~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 C3 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, C3 Rapalogs and Pharmaceutically Acceptable Derivatives Thereof
"Rapalogs" as that term is used herein denotes a class of compounds comprising
the various analo , homologs and derivatives of rapamycin and other rnmpounds
related structural 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 p~pecolate ring with a 5-
mea~bered prolyl ring; and elimination, derivatization or replaeenent of one
or more
substituents of the cyclohexyl ring or replacement of ~e c~clohexyl ring with
a substituted
or unsubstituted cyclopentyl ring. Rapalogs, as that term ~s 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.
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
See also, Luengo et al, Chemist & Biology,1995, 2 (7):471-481; Luengo et al,
jOC,1995,
59(22):6512-13; WO 94/02136 ( mithKline Beecham); WO 95/16691 (Sandoz); US
5583139 (Abbott); Grinfeld et al, 1994, Tett Letters 35(37):6835-6838; WO
96/;865
(ARIAD) and WO 98/02441 (ARIAD).
92
CA 02319492 2000-08-02
WO 99/41258 PCT/US99/03095
Other illustrative rapalogs include those depicted in Table II:
Table II
M v
_ _
O ~ Me0" ~ O
00 ~ O
'O
O
~A ~ h' \
H
t
R = -0H, -O-atfcyi,
-NH-alkyl
~.o
0 o a,
o o
0 0
c o c c o 0 oYo
o
.... Ho.
1r ~ "~ "off "off
0 0
0 o wa' o "~ n~.~'o ~.~o
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CA 02319492 2000-08-02
WO 99/41258 PCT/US99/03095
Hue, - Ho...
I
t . ~ I
0 0 0 0
0 0 ~.. o Ho.,, o o ~,. o
i / ~ i /
~,~
0 0 0 0
I
00 ~ o ~ oo ~.. o
-o ow -o
i / / ~ ~ / / ~,
~d~
s ~ H
O
i I
O 180AIS
O O
O O
/ / ~ /
"C3 Rapalogs" are defined above with reference to Formula I and are
exemplified
by the various classes and subsets of compounds disclosed herein.
The phrase "pharmaceutically acceptable derivative" denotes any
pharmaceutically acceptable salt, ester, or salt of such ester, of a C3
rapalog, or any other
adduct or derivative which, upon administration to a patient, is capable of
providing
(directly or indirectly) a C3 rapalog as described herein, or a metabolite or
residue thereof.
Pharmaceutically acceptable derivatives thus include among others pro-drugs of
the
rapalogs. A pm-drug is a derivative of a compound, usually with significantly
reduced
pharnnamlogical activity, which contains an additional moiety which is
susceptible to
removal in vivo yielding the parent molecule as the pharmacolo tally active
spews. An
example of a pro-drug is an ester which is cleaved in vivo to yie~ a rnmpound
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,
strait chain (i.e., unbranched), branched, cyclic, or polycyclic aliphatic
hydrocarbons,
whi are optionally substituted with one or more functional groups. Unless
oth~vise
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CA 02319492 2000-08-02
WO 99/41258 PCT/US99/03095
specified, alkyl, other aliphatic, allcoxy and aryl grou s preferably contain
1-8,.and in
many cases 1-6, contiguous aliphatic carbon atoms. l~ustrative aliphatic
groups thus
include, for example, methyl, ethyl, n-propyl, isopropyl, cycloproPY1~ -~2-
oyclopropyl,
allyl, n-butyl, sec-butyl, isobutyl, tert-butyl, cyclobutyl, -CH2-cyclobutyl,
n-pentyl, sec_
pentyl, isopentyl, tart pentyl, cyclopentyl, -CHrryclopentyl, n-hexyl, seo-
hexyl,
cyclohexyl, -CH2-cyclohexyl moieties and the like, which again, may bear one
or more
substituenis.
Examples of substituents include: -0H, -0R2', -SH, -SRr,-CHO, =p, -Cppl..l (or
ester, carbamate, urea, oxime or carbonate thereof), -NH2 (or substituted
amine, amide,
urea, carbamate or guanidino derivative therof), halo, trihaloalkyl, cyano,
_~F~, -
pSp2F, -OS(p)2R11, -SOz-IVHRm, _NHSp~-Rll, sulfate, sulfonate, aryl and
heteroaryl
moieties. Aryl and heteroaryl substituents may themselves be substituted or
unsubstituted
(eg. mono-, di- and tri-alkoxyphenyl; methylenedio henyl or
ethylenedioxyphenyl;
halophenyl; or phenyl-C(Me)2-CHz-O-CO-(C3-C6j al ,l or alkylamino),
The term "aliphatic" is thus intended to include alkyl, alkenyl, alkynyl,
cydoalkyl,
cycloallcenyl, and cycloalkynyl moieties.
As used herein, the term "alkyl" includes both straight, branched and cyclic
alkyl
groups. An analogous rnnvention applies to other generic terms such as
"alkenyl';
"allcynyl" and the like. Furthermore, as used herein, the largosge "alkyl';
"allcenYl';
"alkynyl" and the Like encompasses both substituted and unsubsiituted 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, eth 1, n-propyl,
isopropyl,
cyclopropY~, butyl, isobutyl, sec-butyl, teat butyl, cyclobu~ pentyl,
isopentyl tart-pentyl,
cYt3'l~ ~Yl ~ ~~yl. cyclohexy~, and the like. Sui le substituted alkyls
include,
but are not limited fio, fluoromethyl, difluoromethyl, trifluommethyl, 2
fluoroethyl, 3-
fluoropropyl, h droxymethyl, 2-hydroxyethy~, 3-hydroxypropYly benzyl,
substituted
benzyl and the ~.
The term "alkenyl" refers to groups u4sually having two to eight, preferably
two to
six carbon atoms. For example, "alken 1" may refer to p 2-eny~, but 2-aryl,
but 3-aryl,
2 me~ylp p-2-aryl, hex-2-aryl, hex-~yl, 2~-dimethy~ut 2-aryl, and the like.
The
language "aDlkynyl," which also infers 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-yny~, hex-2-YnYI, hex-5-ynyl, and the like.
The term ".alley " as f~pee'cally ~ three to
seven;, preferably tie to ten caxbon atoms. Suitab cycloalkyls fn~clu~de,
but~are not
limited to cyclopropyl, cydobutyl, cyclopentyl,cyclohexyl, cyclohepty1 and the
like,
which, as in the case of other aliphatic or heteroali'phatic or heterocy~
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,
eg., in place
of carbon atoms. Hefieroaliphatic moieties may be branched, unbranched or
cyclic and
include heterocycles such as morpholino, PYrrolidi.~y;, etc.
The term "heterocycle" as used herein refers to cyclic hefieroaliphatic groups
and
preferably three to ten ring atoms total, includes, but is not limited tn,
oxetane,
tetrahydrofuranyl, tetrahydropyranyl, aziridine, azetidine, pyrrolidine,
piperidi~,
morpholine, piperazine and the like.
CA 02319492 2000-08-02
WO 99/41258 PCTNS99/03095
The terms "~ 1" and 'bet roary1" as used herein refer bo stable mono- or
PolYc3'~c~ ~~cy~c, polycyclic~,polyl~t~,clic unsaturated moieties having 3 -
14 carbon atoms which may be substituted or unsubstitubed. Substituents
include any of
the previously mentioned substituents. Non-limiting examples of useful aryl
ring groups
include phenyl, halophenyl, alkoxyphenyl, diallcoxyphenyl, ~xyphenyl,
alkylenedioxypheny naphthyl, ph~anthryl, anthryl, phenanthro and the like.
Exam les
of typical hetero 1 rings include 5-membered monocyclic ring groups such as
ti~ieay~
~lY ~ ~1, pyrazolyl, furyl, isothiazolyl, furazanyl, isoxazolyl, thiazolyl and
' monocyclic groups such as pyridyl, pyrazinyl, pyrimidinyl,
1o pyridazin-~'1, triazinyl and the like; and polycyclic heterocyclic ring
groups such as
~1~ hy~ ~phtho[2,3-b]thienyl, thianthrenyl, isobenzofuranyl, chromenyl,
y ~ tluenyl, mdolizmyl, isoindoly1, indoly~, indazolyl, purinyl, isoq~olyl,
quinolyl, p thalaziny1, naphthyridinyl, quinoxalinyl, quinazolinyl,
benzothiazole,
benzimidazole, tetrahydroquinoline cinnolinyl, pteridin 1, carbazolyl, beta-
carbolinyl,
phenanthridinyl, acridu~yl, penmidinyl, phenanthroliny~ phenyl, y~thiazolyl,
phenothiazinyl, phenoxazinyl, and the like (see eg. ICatritrky, Handbook of
Heterocyclic
C~emist~). The aryl or heteraaryl 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, nitro, halo,
trihalomethyl, cyano,
and carboxyl. Aryl moieties thus include, eg. pheny1; substituted pberyl
bearing one or
more substituents selected from groups including: halo such as chloro or
fluom, h dro
Cl-C6 allcyi, acyl, acyloxy, Cl-C6 aIkoxy (such ~ ~thox3, or ethoxy, including
among
others dialkoxyphenyl moieties such as 2,3-, 2,4-, 2,5-, 3,4- or 3,5-dimethoxy
or diethoxy
p~yl or such as methylenedioxyphenyl, or 3-methoxy 5.etho beryl; or
trisubstituted
P yl. such as trialkoxy (eg., 3,4,5-trimetho or echo beryl , 3,5-dimethoxy-4,.
chloro-phenyl, etc.), amino, -50~1~z ~~phatic , -S02N(aliphatic)z -O-
aliphatic-COOH, and -O-aliphatio-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,
bmmo or iodo substituent. Fluoro ~s often the preferred halogen.
C3 rapalogs may further differ from rapamyc~ir~, in addition to the
modification at
C3, with respect to no, otue, two, three, four, five, six or seven substituent
moieties. This
class includes among others rapalogs with modifications, relative to
rapamycin, at C3
and C13; C3 and C14; C3 and ~; C3 and C43; C3 and C24; C3 and C28; C3 and C30;
C3, C13 and C14; C3, C13 and ~; C3, C13 and C43; C3, C13 and C24; C3, C13 and
C28;
C3, C13 and C30; C3, C14 and a; C3, C14 and C43; C3, C14 and C24; C3, C14 and
C28;
C3, C14 and C30; C3, a and C24; C3, $ and C28; C3 , g and C30; C3, C24 and
C3p; .C3,
C24, C30 and ~,; C3, C24, C30 and C13; C3, C24, C30 and C14; and C3, C24, C30,
C13
and ~,.
One subset of C3 rapalogs of interest in the practice of the various methods
of the
i_nven_tion are C3 rapalogs in which R~ and R~~ are both other than (~}. In
certain
t' R~ and R~ are both -0H, eg. in the "S" configuration. In
and R~ are independently selected from OR3. This subset
includes among others .~apalogs which further differ from rapamycin with
respect to the
moiety ~. For instance, this subset includes compounds of Formula III:
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CA 02319492 2000-08-02
WO 99/41258 PCT/US99/03095
where Ra is other than -H. Alternative substituents for Ra are as disclosed
elsewhere
herein. Other compounds within this subset include those in which one, two,
three, four or
five of the hydroxyl groups is epimerized, fluorinated, alkylated, acyfated or
otherwise
modified via other ester, carbamate, carbonate or urea formation. Illustrative
additional
rnmpounds for example are those compounds otherwise as shown in formula IlI
except
that the hydroxyl group at C43 is in the opposite stereochemical orientation
and/or the
hydroxyl groups at C28 and C30 are alkylated, acylated or linked via carbonate
io formation.
Another subset of C3 rapalogs of special interest are those in which one or
both of
R~~ and R~ is F. In various embod~menis of this subset, one, two, three, four
or five
other substituents in formula II differ from the substituents found in rapam
air. For
instance, this subset includes C13 fluoro-C3-rapalogs, C28 fluoro-C.3-rapa~ogs
and C13,
C28-difluoro-C3-rapalogs.
Another subset of C3 rapalogsof special interest are those in which Rim is
other
than O, OH or H, eg., C3 rapalogs in which R~l~ is -0R6, -NR6, NC(O)R6, -
OC~O)R6 or -
OC(O)NR6, with or without one or more other nwdiflcations relative to
rapamycin.
Another subset of C3 rapalogs of interest are those in which RQ~ is other than
=O,
again, with or without one or more other modifications at other positions
relative to
rapamyain.
Another subset of C3 rapalogs which is of special interest in practicing the
methods of this invention include those which share the sfiereoisomerism of
rapamycin
and in which Ro~ is -0Me wherein R~ is not =O, RQ~ is not ~, R~ is not -OH,
R~14
is not =O and/or R3 and/or R~ are not H.
Other C3 rapalogs of interest include those in which R~~ is OH.
__. Furthermore, this invention encompasses C3 rapalogs in which one or more
of the
carbon-carbon double bonds at the 1,2, 3r4 or 5,6 positions in rapamycin are
saturated,
alone or in combination with a modification elsewhere in the molecule, eg. at
one or more
of 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 -CH2pH
or -
CH20Me; that the C~ 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
Hd'
H~~
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Synthetic guidance
The production of rapamycin by fermentation and by total synthesis is lrnown.
The production of a number of rapalogs (other than C3 rapalogs)'by
fermentation or
synthetic modification of fermentation products is also known. These include
among
others rapalogs bearing alfiernative moieties to the characteristic cyclohexyl
ring or
pipecolate ring of rapamycin, as well as C29-desmethyl-rapamycui and C29-
desmethoxyrapamycin and a variety of other rapalogs such as are disclosed
herein.
ltapamycin may be converted into a C3 rapalog using silyl ethers as
disclosed.in
the experimental examples which follow.
C3 rapalogs comprising additional modifications with respect to the structure
of
rapamycsn may be prepared analogously, starting with the corresponding rapalog
in place
of rapamycin. Alternatively, one or more additional transformations may be
carried out
on a C3 rapalog intermediate.
i5 Methods and materials for effecting various chemical transformations of
rapam~csn and structurally related macrolides are latown 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 prackic;ing the subject invention. The
following
representative transformations and/or references whip can be employed to
produce the
desired rapalogs are illustrative:
ring position literature reference
modified
C-13 C13-->P: protect C28 and C43, rxn at 0
C-14 Schubert, et al. Angew Chew 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, 64b9 (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
WO 98/02441 (Holt et al)
Additionally, it is contemplated that rapalogs for use in this invention as
well as
intermediates for the preduction of such rapalegs may be prepared by directed
biosynthesis, e.g. as ~escr~ed by ICatz et al, W0 93/13663 and by Cane et al,
WO
9702358.
3o Novel rapalogs of this invention may be prepared by one of o skill in this
art rely m' gupon methods and materials known in the art as guided by~osure
presenfied herein. For instance, methods and materials may be adapted from
kzw~m
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CA 02319492 2000-08-02
WO 99/41258 PCT/US99/03095
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
rovided
herein by way of illustration and further guidance to the practitioner.. It be
understood that the chemist of ordinary skill in this art would be readily
able to make
modifications to the foregoing, eg. to add appropriate protecting groups to
sensitive
moieties during synthesis, followed by removal of the probectzng groups
~,,rh,~ no longer
needed or desired, and would be readily capable of dete~nunmg otl~. ~~tic
approaches. ~
1o FKBp domains and fusion proteins
The FIBP fusion protein comprises at one FKBP domain containing all or
part of the peptide sequence of an FIC'BP domain and at least one hcferclogcns
action
domain. This chimenc protein must be capable of binding to a C3 rapalog of
this
invention, preferably with a Kd value below about 100 nM, more preferabl below
about
10 nM and even more preferably below about 1 nM, as measured by direct in~g
niE~asurement (e.g. fluore<soenee quenching). tion binding measurement (e.g.
versus
FIC506), inhibition of FKBP enzyme activity (rotamase), or other assay
methodology.
Typically the diinieric protein will contain one or more protein domains
compering
peptide sequence selecked from that of a naturally o~unring Fop p=,otein such
as human
FKBP12, e.g. as described in In~rnado~l Patent Application PCT/US94/01617.
That
peptide sequence may be modified to adjust the bind'mg ty~ sally with
rePlacea~ent; insertion or deletion of 10 or fewer, preferabjy 5 or feH,es.,
amino acid
residues. Such modifications are elected in certain embodiments to y~ld one or
both of the
following binding profiles: (a) binding of a Ci rapalog to the modiCed 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 hos# oel>s to be engineered; and (b)
binding of
the FKBP:rapalog complex to the PRB fusion pmtein, preferably at least one,
and more
preferably at least two, and even more preferably at least three, orders of ma
fade
etter (by any measure) than to the FRAP or other FRB-containing p=,otein
endogenous to
the host cell to be engineered,
_The FKBP chimera also contains at least one hcferclogcns action domain,,
i.e., a
~NAfi amdin d~non FKBP peptide sequence. The action domain may be a
g Win. transcription activation donnain, cellular localization domain,
intracellular signal transduction domain, etc., e.g. as dexnbed elsewhere
herein or in
Pf_T/US94/01617 or the other cited references, i~rall speaking, ~e action
domain is
capable of directing the pio~ to a ~ re ular location or of initiating a
biological effect upon association or aggregation with another action domain,
for instance,
upon multinierization of proteins containing the same or diet action domains.
A recombinant nucleic acid encoding ~~ a p~~ will be capable of
selectively hybridizing fio a DNA encodutg the anent FKBP protein, eg. human
FKBP12,
or would be capable of such hybridization but i or 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 selective) hybridizing to a DNA encoding that
other
protein, or would be capable of such hy~ri~anon 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., PKBP and FRB domains) may be N terminal, C-~~al,,~r
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CA 02319492 2000-08-02
WO 99/41258 PCT/US99/03095
interspersed withr~spect to the action domain(s). Embodiments involving
multiple rnpies
of a ligand binding domain usually have 2 , 3 or 4 such copies. For example,
an FICBp
fusion protein may contain 2, 3 or 4 FKBP domains. The various domains of ~e
PKBP
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/U594/08008 (Stanford dz 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 rnpies of an FKBP domain which
is
encoded by.a DNA sequence capable of selectively hybridizing to a DNA sequence
encoding a naturally oav~ring FKBP domain and which are still capable of
binding to
rapamycm 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 PKBP
fusion
proteins which contain one or more amino acid residues corresponding to T~26,
phe36,
Asp37, Arg42, Phe4b, 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 PKBP domain containing amino acids 1-107 of human PKBPl2,
separated by
the 2 amino acid linker 7hr-Arg encoded by ACTAGA,, the ligation product of
DNAs
digested respec ~'vely with the restriction endonucleases SpeI and XbaI. The
following
table provides illustrative subsets of mutant FKBP domains based on the
foregoing
FICBP12 sequence:
Illustrative Mutant FKBPs
F36A Y26V F46A W59A
F36V Y26S F48H
F36M D37A F48L Hg~ w
I90A F48A F36y~pg9A
F99A I91A E54A F3~/p99G
F99G P46H B54K F36M/P99A
Y26A F46L VSSA F36M/P99G
note: Entriesn ' nati
i e
ve amuio acrd by sutg a eater a an
poszt:ey followed by thereplaeement amino acid in the mutant. Thus,~~'36V
designates a human FKBP12 sequence in which phenylalanine at position 36 is
replaced by valine. P36V/F99A indicates a double mutation in which
phenylalanine at positions 36 and 99 are replaced by valine and alanine,
respectively.
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FRB domains and fusion proteins
The FRB fusion protein comprises at least one PRB domain (which may comp rise
all or part of the peptide sequence of a FRAP protein or a variant thereof, as
deed
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 t..andida FRB-containing protein. FRB
domains of this
invention include those which are capable of binding to a complex of an FKBP
protein
and a C3 rapalog of this invention.
The FRB fusion protein must be capable of binding to the comp lex formed by
the
FKBP fusion protein with a C3 rapslog of this invention. Preferably, the FRB
fusion
protein binds to that complex with a Kd value below 200 ~,~M, more referably
below
10~, as measured by conventional methods. The FRB domain will ~ of sufficient
1
and composition to maintain high affinity for a rnmplex of the rapalog with
the FKB
fusion protein. In some embodiments 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
Tyi'L144. See Chiu et a1,1994, Proc. Natl. Acad. Sci. USA 91:12574-12578. An
FRB
reg~on of particular interest spans G1u2025 through G>n2114 of human PRAP and
retains
affinity for a FKBP12-rapamycin complex or for FKBP-rapalog complex. In some
embodiments Q2214 is removed fronn the 90-amino acid sequence rendering this
an-89-
amino acid FRB domain. The FRB peptide sequence may be modified to adjust the
b specificity, usually with replacement, insertion or deletion, of 10 or
fewer,
prefers ly 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 a Ci 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 tileg~e~c code. Again, since these
chiaveric
profieins contain an effector domain derivanotiier prolein, e.g. Gal4, VP16,
Fas,
t';.'D3 zeta chain, etc., the recombinant DNA encoding the c~a~ecac 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 hefierologous domains are replaced with alternative het~erologous
domains or
supplemented with aria or more additional heberologous domains, variants in
which one
or more of the FRB domains is a domain of non-humanpeptide sequence origin
(such as
Tor 2 or Candida for example), and variants in which the FRB domain is
awdified 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 a
C~tapalog
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of this invention An illustrative FRB fusion protein contains one or more PRBs
of at least
89-amuw ands, contain~g a ce spanning at least residues 2025-2113 of human
FRAP,separat~ed by the linker formed by li,~~on of SpeI-XbaI sites as
mentioned
previously. It should be appreciatedt 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.
Mixed chimeric proteins
A third type of chimeric protein comprises one or more FKBP domains, one or
more heterologous effector domainsand one or more FRB domains as did for the
FRB fusion proteins.
Mixed chimeric protein molecules are capable of forming homodinieric or
homomultimeric protein mmpiexes in the presence of a C3 rapalog to which they
bind.
Embodiments involving mixed chimeras have the advantage of r~~uiring the
introduction
into cells of a single recombinant nucleic acid construct in place o two
recombinant
nucleic acid constructs otherwise required to direct the expn of both an FKBP
fusion
protein and a FRB fusion protein.
A recombinant DNA encoding a mixed chiateric protein will be capable of
selectively hybridizing to a DNA encoding an FKBP profiei~, a DNA encoding
PRAp, and
a heterologous DNA sequence encoding the prom ~ w~ 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.
Heterologous domains
As mentioned above, the heterolo us effector domains of the FKBP and FRB
fusion proteins are profiein domains whic~upon mutual association of the
chimesic
Pte. are capable of triggering (or u) DNA-binding and/or
transcription o a target gene, actuating cell grow, dn~tion, proliferation or
apoptosis; directing Proteins to a particular oelllular location; or actuating
other biological
events.
Embodiments involving regulatable transcription involve the use of target gene
constructs which comprise a target gene (w 'h yes a polypeptide, antisense
RNA,
ribozyme, etc. of interest) under the transaip tional control of a.DIVA
element responsive
to the association or multymerization of the heterologous domains of the 1st
and 2d
chinteric profieins.
In embodiments of the invention involving direct activation of transcription,
the
hetrrologous domains of the 1st and 2d chimeric prod ~ a DNA binding
domain such as Gal4 or a chinieric DNA binding domain such as Zg~l,
belowand a tz~ansaip tional activating domain such as those derived from VP16
or p65,
respeckively. The multinierization of a chimeric protein eontaininf~ such a
transcriptional
activating domain to a chimeric profiein containing a DNA b domain !he
kranscriptional activator to the promoter element to which the~A binding
domain
binds, and thus activates the transcription of a target gene linked to that
promofier
element. Foregoing the transcription activation domain or substituting a rep
~,~n
(see PCT/US94/0161~ in place of a transcription activation domain provides
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CA 02319492 2000-08-02
WO 99!41258 PCT/US99/03095
analogous chimera useful for inhr'biting ttansaiption of a target gene.
Composite DNA
binding domains and DNA sequences to which they bind are disclosed in
Pomerantz et
a1,1995, supra, the contents of which are intorporated herein by reference,
Such
composite DNA binding domains may be used as DNA binding domains in the
practiee
of this invention, together with a target gene construct containing the
rngnafie DNA
sequences to which ~e composite DBD binds.
In embodiments involving indirect activation of transmptic~n" the heterologous
domains of the chimeras are effector domains of signaling Proteins which upon
aggregation or multialerization trigger the activation of transcription under.
the control of a
responsive promoter. For example, tile signaling domain may be the int~~~.
domain
of the zeta subunit of the T cell receptor, which upon aggregation, triggers
iranscripLion of
a gene linked to the l:Ir2 promoter or a derivative thereof (e.g. ifierated NF
AT binding
sites).
In another aspect of the invention, the heberologous domains are protein
domains
which upon mutual association are capable of triggering cell death Examples of
such
domains are the intracellular domains of the Pas 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 FKBP and FRB domains.
Naturally occurring sequences include those of human and the FRB domain of
human FRAP. Alternatively, the peptide sequences may be derived frrnm such
naturally
occurrW g a sequences but contain generally up to 10, and preferably 1-5,
mutations
in one or bo such peptide sequences. As disclosed in greater detail elswhere
herein, such
mutations can confer a number of important features. Por , an FICBP domain may
be modified such that it is capable o~g a (~ rapalog prefenmtially, 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
preferab~~iiuee orders of magnitude more effectively, with respect to the
unmodified PRB
domain. FKBP and PRB domains may be modified such that they are capable of
forming a
tripartite complex with a C3 rapalog, preferentially, i.e. at least one,
preferably two, and
etYen moree preferably three orders of magnitude more effectively, with
respect fio
unmodifi d FKBP and FRB 'domains.
(a) FKBP
Methods for identifying FKBP mutations that confer enhanced ability to bind
derivatives of FK506 containing various substituenbs ("bumps' were disclosed
in
PCT/US94/01617. Similar strategies can be used to obtain modified FKBPs that
preferentially bind bumped rapam,,~c~m derivatives, i.e., rapalogs. The
structure of the
rnmplex between rapamycrn and P12 is known (see for example Van Duyne et al.,
j.
Am. Chew. Soc. (1991) 113, 7433-7434). Such data can be used to reveal amino
acid
residues that would clash with various rapaiog substituents. In this approach,
nt~ecular
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CA 02319492 2000-08-02
WO 99/41258 PCT/US99/03095
modelling is used to identify candidafie amino acid substitutions in the FKBP
domain that
would accommodate the rapalog substituent(s), and sihe-directed mutagen~~
be used to engm eer the protein mutations so identified. The mutants are exp y
standard methods and their binding affinity for the ra~alogs measured, for
~mple b
inh~ition of rotannase activity, or by competition for binding with a
molecule~as y
FK506, if the mutant retains appropriate activity/affinity.
More particularly, we rnntemplate that certain C3 rapalogs of this invention,
e.g.
rapalogs with modiCcations relative to rapamyan at C-13 or C-14 bind
preferentially to
PKBPs in which one or more of the residues, Tyi26, Phe36, Asp37, 2 and Phe99,
are
substituted with amino acids that have smaller side chains (such ~~y, A~~ V~~
Met
and Ser). Examples of mutant FKBPs with modifications at position 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 -I~ bind
preferentially
to FKBPs in which Tyr82 and/or I1e56 are replaced by other amino acids, y dose
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 FKgPs in
which one or more of Phe46, Phe48 and Va155 are replaced by oar ~o aa~, again
especially those with smaller side chains. Moreover, we envisage that rapslogs
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 ~ples are noted in the
pnwious
table.
An alternative to iterative e~~ ~ ~~g o f or multiple mutants is
to co-randomize structurally ideashfied ues that are or would be in contact
with or
near one or more rapalog or rapamyrin substituents. A collection of
polypeptides
containing FKBP domains randomized at the identifiedp ~'tions (such as are
noted in the
foregou~ggraPh) ~ P~~ e.g. using conveatithetic or genetic methods.
Such a collection represents a set o~FKBP domains conta~nmgrep>ac~t amino a«ds
at
one or more of such positions. The collection is screened and FKBP variants
are selected
which possess the desired rapalog binding Pte. In g,~,~ randomizing several
residues simultaneously is expected to yield compensating mutants of higher
affinity and
spea$aty for a given bumped rapalog as it maxinuzes the like,I~ood of
beneficial
cooperative interactions between sidechains. Techniques for preparing l~raries
randomized at discrefie positions are lmown and include primer-directed
mutagenesis
using degenerate oligonucleotides, PCR with dege~rafie o>ig~~otides, and
cassette
mutage~sis with degenerafie ol~~otides (see for example iowa~a~ H.B, and
Wells,
j.A. Methods: Coin Methods EnzymoL 1991. 3, 205-216; Deruus, M.S. and Lazarus,
R.A.1994. J. Biol. ~ 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
rnmpletely
explore all the possible variations in FKBP conformation that could optimally
accommodate a rapalog substituent (bump). Thus the construction is also
envisaged of
untie~,ed l~raries 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 s~ea~
have been
described, including alanine scanning niuta(C~ and Wells (1989) Scienre
244,1081-1085), PCR misincorporation mute (see eg. Cadwell and Joyce, l92,
PCR Meth Applic. 2, 28-33), and 'pNA sh~gt~r,1994, Nature 370,'$9-391
104
CA 02319492 2000-08-02
WO 99/41258 PCT/US99/03095
and Crameri et a1,1996, Nature Medicine 2,100-103). These ~u~iques produce
libraries
of random mutants, or sets of single mutants, that are then searchhx ed by
sccrening 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 lbraries in which key contact residues are
randomized
by PCR with d ate oligonudeotides, but with amplification performed using
error-
promoting con 'to introduce further mutations at random sites. A further
example is
1o the combination of component DNA fragments from structure-based and
unbiased
random libraries using DNA shuffling.
Sc~ening 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 lbrary of randomized FKBP
sequences Boned into a separate GAL4 fusion vector. Yeast co-transformants are
treated
with rapalog, and those harboring complementar~r FKBP mutants are identified
by for
example beta-galactosidase or ludferase production (a sczeen), or survival on
plates
lacking an essential nutrient (a selection), as appropriate for the vectors
used. The
requirement for bumped rapamydn to bridge the FKBP-FRAP interac6ion is a
useful
screen to eliazinate false positives.
A further strategy for isolating awdi&ed ligand~inding 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 ut0.izes the fact that the
bacberiophag~
lambda repressor cI binds to DNA as a homodinuer and that binding of such
homodimers
to operator DNA prevents trans~ip tion of phage genes involved in the lytic
pathway of
the phage life cycle. Thus, bacterial sells expressing functional lambda
repressor are
immune to lysis by superinfiecting phage lambda. Repressor protein comprises
an amino
terminal DNA binding domain (amino adds 1-92), joined. by a 40 amino acid
flexible
linker to a carboxy terminal dimerization domain. The isolated N fierminal
domain binds
to DNA with low affinity due fio inefficient dimer formation. High affinity
DNA binding
can be restored with heberologous din~xization 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 isolafie GCN4 leucinez~ per mutants capable of mediating lambda
repressor
dimer formation from a large population of sequences (Hu et. al.,1990).
For exaalple, to use the lambdab repressor system to identify PRAP mutants
complementary to bumped rapalogs, ~a~bda ressor.PRAP libraries bearing mutant
FRAP sequences are transformed into E. coli expressing wildtype lambda
repressor
FKBP protein Plasmidsexpressi~g FRAP mutants are isolated Irom those colonies
that
survive lysis on bacfierial plates containing high titres of lambda phage and
"bumped"
rapamydn compounds. Alternatively, to isolafie FKBP mutants, the above
strategy is
repeated with lambda n~pressor-FKBP Lbraries bearing mutant FKBP sequences
transformed into E. coli cells expressing wildtype lambda represso~FRAP
protein.
A further alternative is to done 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 beperformed in a number of ways . Por example,
ra aiog is
mixed with the hbrary phage pool 'm solution in the preserve of recombinant
tagged
with an affinity handle (for example a hexa-histidine tag, or GST), and the
resin t
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CA 02319492 2000-08-02
WO 99/41258 PCT/US99/03095
complexes are captured on the appropriafie affi~yity matrix to enrich for
phage displaying
FKBP harboring complementary mutations. Tedlniques for phage d;sphiy have been
described, and other in vitro selection selection i~ can also be mntemplafied
(for
example display on lambda phage, display on p ds, 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 ui
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 PRB domains which
bind
preferentially to C3 rapalogs containing modihcati~ (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 ~o
acid substitutions for one of more of the residues Tyr203g, ph~p39, Thf1098,
1n2099,
Trp2101 and Asp2102. Exemplary mutations include Y2038H, y2p3gL, y2p3gV,
Y2038A, F2039H, F2039L, F2039A, F2039V, D2102A, T2098A, T2098N, andT2098S.
Rapalogs bearing substituents other than -0H at C28 and/or substituents other
than
at C30 may be used to obtain preferential binding to FRAP pmbeins bearing an
amino
acid substitution for G1u2032. Examplary mutations include E1032A and E2p3~.
Proteins comprising an FRB containing one or more amino acid replaoenu'nts at
the
foregoing positions, libraries of protru~s or peptides randomized at those
positions (i.e.,
rnnhaining various substituted amino acids at those residues), libraries
randomizing the
entire protein domain, or rnmbinations 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 thec~mp lex of an PKBP protein
lexed with a rapalog may be assayed by a numb~~; fm. ~ple
bg of in vitro translated FRB mutants to GST FKBP in the presence of
~'ug (Chen et
aL 1995. Proc. Natl. Acad. Sci. USA 92, 4947-4951); or ability to participate
in a
3o rapalog-dependent transcriptionally active complex with an appropriate FKBP
fusion
pmbein in a yeast two-hybrid assay.
PRB mutants with desired binding ~mperd,~ ~y be ~~~d ~m ~~
~la ~ phage usutg a variety of sorting strategies. For example, a rapalog is
mixed
ha 1 in solution in the presence of recombinant FKBP tagged with
an affinity hand (~ e~~mple a hexa-histidine tag, or GS"I~, and the resultant
complexes
arr captured on the appropriate affinity matrix to enrich for phage displaying
FRAP
harboring mmpleanentary mutations.
An additional feature of the FRB fusion protein that may vary in ~ v~
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 add) FRB domain These include extensions N-terminal ~ due
G1u2025 (preferably extending to at least Arg2018 or I1e2021), as well as C-
terminal
exfiensions beyond position 2113, e.g. to position 2113, 2I4I or 2174 or
beyond), which
~Y ~ ~~ ~ prove the stability of the folder: f~itB domain and/or the effigy,
of expression. Other applications in which different PRB sequence termini nuy
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 polypeptidech~n
required for FRB-mediated protein-protein assodation at the cell membrane
orDNA.
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WO 99/41258 PCTNS99/03095
Conversely, in other applications short linkers on one or both sides of the
FRB domain
may be preferred or r~:~ired to present the heberologous effector domains)
appropriately
for biological function In human gene therapy applications the use of
naturally occurring
human FRAP sequence for such linkers willg~er ally be preferred fin the
introduction of
heterologous sequences, or reduce the risk of provoking an inimune response in
the host
organism.
Some rapalogs, espeaally rapslogs 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 donnains containing natunlly
occurring human
peptide sequence. That reduced ability may be nnanife<sted 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 PRBs that are capable of complexing with the
rapalog more
effectively than the corresponding domains containing naturally oecurring
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
rnmpleatentary 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. Por
instance, mutant
FKBP domains which when bound fin a given rapalog are capable ofrnmp lexing
with an
endogenous FRB measurably less effectively than to a mutant PItB are of
particular
interest. Also of infierest are mutant FRB domains which are capable of
associating with a-
mmplex 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 used (i) to identify amino
acid
substitutions, deletions or insertions to an FKBP domain which measurably
din~ish the
domain's ability to form the tripartifie complex wig 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 PKBP; and {iii) to select and/or otherwise
identify
compensating mutations) in the partner pmbein. As examples of suitable mutant
FKBPs
with diminished effectiveness in tripartifie complex formation, we include
mamnnalian,
preferably human FKBP in which one or both of His87 and >Ze90 are replaced
with amino
acids such as Arg, Trp, Phe, Tyr or Lys which contain side chain groups; FRB
domains, preferably con ' mammalian, and more~ly of human, peptide
sequence may then be mu~~ as described above togencrate complementary variants
which are capable of forming a tripartite complex with the mutant FKBP and a
given
rapalog. Dlustrative 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 hFhBPl2s include human FRBs in which IC2095 is replaced with
L, S,
A or T.
Additionally, in optinuzing the receptor domains of this invention, it should
be
appreciated that immunogenidty of a polypeptide sequence is thought to
require~e
107
CA 02319492 2000-08-02
WO 99/41258 PCT/US99/03095
binding ofpeptides by MHC proteins and the recognition of the presented
peptides as
foreign by endogenous T-cell reoepfiors. It mag be pre~ble, at least in human
gene
therapy applications, to tailor a given foreign peptide sequence, inclu
junction
peptide sequences, to minimize the ~robab3lity of its bein immunolo;~'gic~ty
presented in
hurans. For example, peptide binding to human MHC ~ I awlecules has strict
requirements for certain residues at key 'anchor' positions in the bound
peptide: eg. HLA-
A2 requires leucine, methionine or isoleucine at position 2 and leucine or
valine at the C-
tem~inus (for review see Stern and Wiley (1994) Structure 2,145-251). Thus in
'
g
proteins in the practice of this invention, this periodicity of these residues
is p
l0 avoided, espeaally 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.
i5 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 protein, whether endogenous to the host cell or not. The
membrane
20 retention domain may be a transmembrane retention domain, i.e., an amino
arid sequence
which extends across the membrane as in the case of cell surface proteins,
incluing many
receptors. The transnneanbranepeptide sequence may be extended to span part or
all of
an extraoellular and/or intracellular domain as well. Alfiernatively, the
membrane
retention domain may be a lipid membrane retention domain such as a
myristoylation or
25 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 se oe for N terminal binding to the membrane and, proximal fio the 3'
end for
C-trua~inamding. Peptide sequences involving post translational processi~g to
provide
for lipid membrane binding are described by Carr, et al., PNAS USA (1988) 79,
6128;
30 Aitken,, et aL, FEBS Left. (1982) 150, 314; Henden>on, et al., PNAS USA
(1983) 80, 319;
Schulz, et al., Virology (1984),123, 2131; Dellaian,, et aL, Nature (1985)
314, 374; and
re~riewed 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 diiatesic proteins of this
invention. Other
35 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 chinueric
proteins designed
for (direct) transaiptional regulation. Various cellular localization
sequences and signals
are well known in the art.
40 Further details which may be used in the practice of the subject invention
relating
to the design, assembly and use of constructs ennxling chimeric proteins
containing
various effector domains including cytoplasmic signal initiation domains such
as the CD3
zeta chain, nuclear transQiption factor ~omains including among others VP16
and GAL4,
domains capable of triggering apo~tosis including the Pas cytc~plasauc domain
and others
45 are disclosed in PCT jUS94/0161 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 speciEc regulatory elements
in i~e
108
CA 02319492 2000-08-02
WO 99/41258 PCTNS99/03095
constructs forexpcession of the chia~ric proteins and the application of
regulated
iransaiption to the expression of Cre recombinase as the target gene lea ' to
the
elimination of a gene of interest flanked by loxP sequences, pltprnativ~y~p ~d
its
rngnate recognition sequences may be used instead of Cre and lox. Those
features may be
adapted to the subject invention
In various cases, espeaally ~ e~odiments involving whole aniniaLs 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 prote~
of the 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.
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
ui a cell-speafic or tissue-specific manner. Such spec~ity of expression may
be achieved
by operably firkin one ore more of the DNA sequence;; encoding the chimeiic
proteins)
to a cell-typea~c optional regulatory sequence (e.g, promoter/enhancer).
Numerous ~-type specific >ranscripttonai 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 -
regulafiory sequences derived from known genes for specific expression in
selected tissues.
Representative examples are tabulated below:
Tissue Gene Reference
g2~~ B~~ M.L., Clapoff, S., Rossanty J., Tsui,
L.C., Golde
,
L.M., Maxweu, LH., Bernstin, A. (1987)
Genetic Ablation:
~rgeted expression of a toxin gene causes
microphthalaua in
._ transgenic mice. Science ?.38: 1563-1565
.. aA~~ ~~ C.P., Zhao, J., Bok, D., Evans, G.A.
(1988) Lens-
expression of a recombinant ricin induces
mental defects in the eyes of transgeruc
mice. Genes
Dev. 2: 1168-1178
ICaur, S., key, B., Stock, J., McNeish,
J.D., Akeson
R
Potter
,
,
,
S.S. (1989) Targeted ablation of al ha-crystallin_
p
f~izin
~
~
g re
s produces lens-de
cient eyes in transgenic
auce. Development 105: 613-619
109
CA 02319492 2000-08-02
WO 99/41258 PCT/US99/03095
pituitary Grovvtll Behringer, RR, Mathews, L.S., Palmiter,
RD., Brinster
RL.
- horawne ,
(1988) Dwarf mice produced by genetic ablation
of growth
somatrophic hormone-expressittgcells. Genes Dev. 2:
453-461
cells
pancreas Insulin ~ Ornitz, D.M., Palmiter, RD., Hammer, RE.,
Brinster, RL.,
Elastase Swift, G.H., MacDonald, RJ. (1985) Specific
- expression of
acinar cell an elastase-human growth fusion in pancreatic
acinar cells
specific of transgeneic mice. Nature 131: 600-603
Palmiter, RD., Behringer, RR, Quaife, C.j.,
Maxwell, F.,
Maxwell, LH., Brinster, RL. (1987) Cell
linesga ablation in
transgeneic mice by cell-specific expression
of a toxin gene.
Cell 50: 435-443
T cells lck promoterChaffin, K.E., Beak, C.R, Wilkie, T.M.,
Forbush, K.A.,
Simon, M.L, Perlmutter, RM. (1990) EMBO
journal 9: 3821-
3829 .../...
B cells ImmunoglobulinBorelli
E
He
man
R
H
i
M
E
RM
,
.,
y
,
,
s
,
.,
vans,
. (1988)
kappa light Targeting of an inducble toxic phenotype
chain in animal cells.
Proc. Natl. Acad. Sci. USA 85: 7572- 76
RA., Borrelli, E., Lesley, j., Anderson,
D.,
nd
D
D
Bai
d
S
M
H
.
.,
,
r
,
.
.,
yman" R, Evans, RM.
(1989) Thymidine kinase obliteration: creation
of transgenic
mice with controlled immunodeficiencies.
Fnx. Natl. Acad.
Sci. USA 86: 2698-2702
Schwann Po promoter Messing, A., Beturinger, RR, Haauman J.P.
Palmiter, RD,
save Brnnsfier, ltL, Lemke, G. ,F'0 pronwter
~ espression of
reporter and toxin genes to Schwann sells
of transgenic mice.
Neuron 8: 507 5201992
Myelin basicMisldavns, R Knapp, L., Dewey,MJ, Zhang,
X. Cell and
profiein tissue~pecific expression of a heberologous
gene under
control of five myelin basic protein gene
promoter in
trangenic mice. Brain Res Dev Brain Res
1992 Vol 65: 217-
21
spenmatids protamine Breitman,, M.L., Rombola, H., Maxwell,
LH., Klintworth,
G.K., Bernstein, A. (1990) Genetic ablation
in trapsge~uc
mice with
tt
t
d di
h
h
a
enua
e
p
t
eria fioxin A gene. MoL Cell.
Biol. 10: 474-479
hmg Lung surfacantOrnitz, D.M., Palmiter, RD., Hammer, RE.,
Brinsfier, RL.,
gee Swift, G.H., MacDonald, RJ. (1985) Spedfic
expression of
an elastase-human growth fusion in pancreatic
acinar cells
of transgene~c mice. Nature 131: 600-60;i
-
adipocyte ~~ S.R Braves, RA, Spiegelman, BM Targeted
expression
P2 of a toxin gene to adipose tissue: transgenic
mice resistant to
obesity Genes and Dev 7: 1318-24 1993
110
CA 02319492 2000-08-02
WO 99/41258 PCT/US99/03095
muscle m osin t Lee, KJ, Ross, RS, Roclmnaxt, HA, Harris,
a AN, OBrien, TX,
~
c van Bilsec~, M., Shubeita, HE, Kandolf,
in R., Brew, G., Prices
et alj. BIoI. Chew. 1992 Aug 5, 267:15875-85
Alpha actin Muscat, GE., Perry, S. , Prentioe, H.
Kedes, L. The human
skeletal alpha-actin gene is re~ted by
a muscle-specific
enhancer that binds three nu~ear factors.
Gene Expression
2, lI1-26, 1992 .../...
neurons neurofilamentReeben, M. Halnlekyto, M. Alhonen, L.
Sinervirta, R
proteins Saarma, M. janne,J. Tissue-specific expression
of rat light
neuro8lament promoter-driven reporter
gene in transgellic
mice. BBRC 1993: 192: 465-70
a
aminotransfer-
ase, albumin,
apolipoproteins
Target Gene Constructs
In embodiments of the invention in which the chin~eric proteins are designed
such
that their multimerization activates transQip tion of a target gene, an
appropriate target
gene eonshuct is also used in the engineered cells. Appt~opriabe tar gene
constructs are
those containing a target gene and a cognate tzansaiptional controement such
as a
promoter and/or enhancer which is respcmsive to the multimerization of the
chimeric
proteins. In embodiments involving direct activation of iransQiption, that
responsiveness
may be achieved by the presc~noe in the target gene construct of one or more
DNA
sequences recognized by the DNA-binding domain of a cllimeric protein of this
invention
(i.e., a DNA sequence to which the chimeric protein binds).1n embodialents
involving
indirect activation of transQiption, responsiveness may be achieved by the
presence in the
targetgena construct of a promoter and/or enhaneer sequence which is activated
by an
intracellular signal generated by multialetization of tile chinleric proteins.
For example,
where the chia>eric proteins contain the TCR zeta chain intracellular domain,
the target
gene is linked to and under the expression control of the llr2 ptnnwter
region.
. . ~ mv~d~ ~ pm~~ ~g,et DNA constructs containing (a) a cognate DNA
sequence, e.g. to which a DNA~inding chialeric protein of this invention is
capable of
binding (or which is suscept~le fio indirect activation as discussed above),
and (b)
flas~ldng DNA sequence from the locus of a desired target gene endogenous to
the host
cells. These constzucts permit homologous recombination of thecognato 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 infix the desired
locus. Such a
target construct may also contain the rngnate DNA sequence, or the cognate DNA
sequeilce may he 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 rernmbinase such as Cre, a n'bozyme or an
antisensRRNA.
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WO 99/41258 PCT/US99/03095
See PCT/US94/01617 for general design and rnnstruction details and for various
applications including gene therapy and see PCT/US95/10591 regarding
applications to
animal models of disease.
This invention enrnmpasses 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 characfieristi~ cells containing constructs
encoding the
chimeras and a target gene construct a ress the target gene at least one,
preferably at
least two, and more preferably at least or four or more orders of magnitude
more in
the presence of the multimerizing ligand than in its absence. tJptimally,
expression of the
selected gene is not observed unless the cells are or have been ex~pose~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 a
C3 rapalog, i.e., following multimerization of the chimeras. Thos,
transcription of target
genes is activated in genetically engineered cells of this invention following
exposure of the
cells to a C3 rapalog capable of multimerrzuig the chimeric protein molecules.
Said
different)y, genetically engineered cells of this invention contain chimexic
proteins as
dabove and are responsive to the presence and/or concentration of a C3 rapalog
which is capable of multimerizing those chin~eric protein molecules. That
responsiveness is
manifested by the activation of transQiption of a target gene. Such
transaiptional activity
can be readily detected by any conventional assays for 6ransaiption of the
target e. In
other embodiments, the biological response to ligand-mediated multimerization
oe
chimeras is cell death or other biological events rather than direct
activation of
transQiption 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 neferenoe,
with
modifications and further exemplification as described herein. Components of
the
constructs can be prepared in conventional ways, where the cording sequences
and
regulatory regions may be isolated, as appropriate, ligabed, cl wed in an
appropriate
cloning hosts analyzed by restriction or sequencing, or other convenient
means.
Particular)y, using PCR,, individual fragments including all or portions of a
functional unit
may be isolated, where one or more mutations may be introduced using'~rialer
repair",
ligation" in vitro mutageriesis, etc. as appropriafie. In the case of DNA
constructs
encoding fusion proteins, DNA sequences encoding individual domains and
subdomains
are joined such that they constitute a single open reading frame ending a
fusion protein
capable of being translated in cells or cell lysafies into a single
polypeptide harboring all
component domains. The DNA construct encoding the fusion protein may then be
laced
into a vector that directs the expression of the protein in the appropriate
cell pe~s). For
biochemical analysis of the encoded chimera, it may be desirable to construct
F,r~~mids
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 lawwn in the art. Various sorts of
such vectors are
comnnezrially available.
112
CA 02319492 2000-08-02
WO 99141258
PCT/US99/03095
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. BF'V or
EBV vectors) or into vectors designed for inbegrahon into the host cells'
chromosomes. The
rnnstructs may be integrated and packaged into non-replicating, defective
viral owes
like Adenovirus, Adeno-associated virus (AAV), or Herpes simplex virus
(13SV~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, caldum
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 ap mpriate treatment for introduction of the rnnstruct(s) and
integration of the
co~astruct~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, h~~romycin resistance, etc., and
various cell
surface markers such as Tac, CDB, CD3, Thyi and the NGF receptor:
In some instances, one may have a target site for homologous recombinaiion,
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
geiuerally use either ii or O-vectors. See, for example, Thomas and Capeccl>i,
Cell (198
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
ges>es, or different DNA molecules having one or more genes. The constructs
may be
introduced simultaneously or consecutively, each with the same or different
markers.
3p Vectors containing useful elements such as bacterial or yeast origins of
replication,
selectable and/or amplifiable markers, pmmober/enhancer elements for
expression in
procaryotes or eucaryotes, and maa~nnalian expression control elennents, etc.
which may
be used to prepare stocks of constrict DNAs and for carrying out transfections
are well
lrnown in the art, and many are a~ata~.ally available.
Delivery of Nuceic Acid: Ex vlvo and in vivo
Any means for the introduction of heterologous nucleic acids infix host cells,
espeaally eu otic cells, an in particular animal cells, preferably human or
non human
n;uunanalian , may be adapted to the practice of this invention. For the
purpose of this
4o discussion, the various nucleic acid constructs descnl~ed herein may
together be referred
to as the tcansgene. 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-
comptaxed 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 optinvzed for expression (e.g.,
inclusion avian
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CA 02319492 2000-08-02
WO 99/41258 PCT/US99/03095
intron in the 5' untranslated region and elimination of u:u~eaessavry
sequences (Pelgner, et
aL, Ann NY Acad Sd 126-139,1995). Pormulation 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 tile practice of five invention,
retroviral-
AAV- and adenovirus-based approaches are of particular interest. See, for
example,
Dubensky et aL (1984) Proc. Nail. Acad. Sci. USA 81, 7529-7533; ICaneda 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 Chew. 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
transcn'bed to DNA in the infected cell. The retroviral gerwme 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
gyes. The packaging sequence of the virus, (~, allows the viral RNA to be
distinguished
from other RNAs in the cell (Verma et al., Nature 389239-242,1997). For
expression of a
foreign gene, the viral proteins are replaced with the gene of interest in the
viral vector,
2o wh~h is then transfecbed into a packaging line oontaini~g the viral
pac3caging components.
Packaged virus is seaete~d from the packaging Line 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-associafied virus (AAV)-based vectors are of gezueral infierest as a
delivery
vehicle to various tissues, including muscle and lung. AAV vectors infect
cells and stably
integrate into the cellulargenome with high frequency. AAV can infect and
integrate into
growth-arresfied cells (s~wcpulmonary epiti~elium), and is non pathogenic.
The AAV~ased expression vector to be used typically includes the 145
nucleotide
AAV inverted ternninal repeats (ITRs) flanking a resbdction site that can be
used for
s~u'bof the transgene, either directly using the restr~ion site available, or
by
exciswn of the" transgene with restriction followed by blunting of the ends,
ligation of appropriate DNA linkers, restzichon digestion, and.1~gaIro~n
into.the site
between the ITRs. The capacity of AAV vectors is about 4.4 kb. The following
proteins
have been pressed using various AAV based vectors, and a variety of
promoter/en: neom~rcin phosphotransferase, chloranipheniool acetyl
transferase,
Fanooni's anemia gene, cystic fibrosis tc~nsnnembrane conductance regulafior,
and
granulocyfie macrophage colonyrstimulating factor (Kotin, R.M., Human Gene
Thempy
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 promofier f.uch as CMV to driveexpression of the
recombinant
DNA encoding the fusion prot~ein(s), an AAV promoter can be used (ITR itself
or AAV p5
(Flotte, et aL J. Biol. Chew. 268:3781-3790,1993)).
Such a vector can be packaged into AAV virions by reported methods. F~r
example, a human cell line such as 293 can be co-transfected with the AAV~a
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CA 02319492 2000-08-02
WO 99/41258 PCT/US99/03095
expression vector and another plasmid containing open reading Iraates ~ AAV re
and cap under the control of endogetwus AAV promoters or a heberologooff. Inp
the absence of helper virus, the rep proteins Rep68 and Rep78 prevent
accumulation of
the replicative foam, but upon superinfection with adeaovirus or herpes virus,
these
proteins permit replication from the TTRs (present only in the construct
containing the
transgene) and expression of the viral capsid proteins. This system results in
packaging of
the fransgene DNA into AAV virions (Carter, B.J., Current Opinion in
Biotechnology
3:533-539,1992; Kotin, RM, 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 limified 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 line that expresses AAV proteins inducibly, such as
temperatore-sensitive inducivle expression or pharmacologically induc~le
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 fio
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. Chew. 268:3781-3790,1993)
or
2o chromatographic purification, as described in O'Riordan et al., W097/08298.
For additional detailed guidance vn AAV fiechnology which may be useful in the
practice of the subject invention, including methods and materials for the
inrniporation of
a fransgene , 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 (Il Oct
1994);
Shenk et al, US Patent No. 5,436,146 (25 July 1995); Chatterjee et al, US
Patent No.
5r454,935 (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
bo mamnnals, including humans. Replication-deficient adenovirus vectors have
been used
to~cp ress marker proteins and CFTR in the pulmonary epithelium. The first
generation
Elelefied adenovirus vectors have been improved upon with a second generation
that
includes a teznperatun'-se~itive E2a viral protein, designed to express less
viral rotein
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 ILrlO 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
adtnwirus DNA sequences may be obtained from any of the 41 huanan adenovirus
types
currently identified. Various adenovirus strains are available from the
American~pe
Culture Collection, Rockville, Maryland, or by request from a number of
conuneand
academic sources. A transgene as described herein may be incorporated into and
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WO 99/41258 PCT/US99/03095
adenoviral vector and delivery protocoi, by the same mettwds (restriction
digest, linker
ligation or filling in of ends, and ligation) used to insert the CFTR or other
genes into the
vectors. Hybrid Adenovims-AAV vectors represented by an adenovirus capsid
rnn ng selected portions of the adenovirus sequence, 5' and 3' AAV ITR
sequences
flanlinsl~'ng.~e transgene and other conventional vector regulatory eleanents
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 adenavirus-
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/I3597 and WO 96/26285, and
references
cited therein.
Generally the DNA or viral particles are transferred to a biolog~tally
rnmpat~le
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 dossges of DNA or virus depends
on a
variety of factors, as discussed elsewhere, and ma)r thus vary somewhat from
patient to
patient. Again, therapeutically effective doses of vwuses 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 lOla 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 sells and
in
applications involving the use of such engineered aniazal cells. The aniazal
cells may be
insect, worm or mammalian cells. While various mannmalian cells may be used,
including,
by way of le, equine, bovine, ovine, canine, feline, marine, an~non~uman
primate
cells, human are of particular infierest. Among the various species, various
types of
dells may be used, such as hematopoieiic, neural, glial, n~nchymal, cutaneous,
mucosal,
stronnal, muscle (including smooth muscle cells), spleen, reticulo-
endothelial, epithelial,
endothelial, hepatic, kidney, gastrointestinal, Pv~la~onary, fibroblast, and
other celltypes
Of articular interest are hematopoietic cells, which may include any of
thewucleat~
which may be involved with the erythroid, lymphoid or'myelamonocyiic lineages,
as
well as myoblasts and fibroblasts. Also of interest are sfiem ~ progenitor
cells, such as
hemstopoietic, neural, stromal, muscle, hepatic, pulmonary, gastroinfiestinal
and
me'aenchymal stun cells
The cells may be autologous cells, syngeneic sells, allogeneic cells and even
in some
cases, xenogeneic cells with respect to an intended host organism. The cells
may be
modified by dianging the major histoeompat~ility complex ("M'HC'~ file, b
inactivating B2-microglobulin to prevent the formation of functional CI ~C
molecules, inactivation of Class II molecules, providing for expn~sion of one
or more
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CA 02319492 2000-08-02
WO 99/41258 PCT/US99/03095
MHC molecules, enhancing or inactivating cytotoxic capabilities by enhancing
or inh~iting
the expression of genes associated with the cytoboxic activity, or the Iike.
In some instances specific clones or olig~clonal cells may be of interest,
where the
cells have a particular speaficity, 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
poi race chain reaction for determining the pre~ce of the construct in the
host cells
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.
i5 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 y at least
about 104
cells and generally not more than about lOlo cells. The number of cells which
are
employed will depend apon a number of circums, ~ pmpoge 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 stabili~
therapeutic agent,
the phys~ologic need for the therapeutic a t, and the like. y, for myoblasts
or
fibroblasts for axan~ple, the number of will be at least about 104 and not
more than
about 109 and may be applied as a dispersion,,generally being u' ~'acted 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 biocompat~le materials and methods, prior to~mp lantation
into the
host organism or patient for the reduction of a therapeutic retain See e.g.
Hguyen et al,
Tissue a Iant Systems and Met for Sustaining viable Nigh Cell Dens ~,"it~ a
Host, U~atent No. 5,314,471 (Baxter Infiernational, Inc.); Uludag and
~on,1993, j
Biomed. Mater. Res. 27(10):1213-24 (HepG2 cells/hydm ethyl me late-methyl
methacrylafie membranes); Chang et x1,1993, Hum Gene 4(4):4~(mouse Ltk-
oells expressing hGH/imm rotective perm-selective alginafie mic~.,ap~; g~dy et
al, 1993, J Infect Dis 168(4)W-3 (alginate); Tai and Sun, 1993, FASEB J
7(11):1061-9
(mouse fibroblasts expressing hGH/alg~nate-pOly-Il.lysine-alginate m~rane); Ao
et al,
199'5, Transplanataion Proc. 27(6):3349, 3350 (alginate); Rajotte et a1,1995,
Transplantation Proc. 27(6):3389 (alginate); Lakey et a~,1995, T lantation
Proc.
27(6):3266 (alginate); Korbutt et al,1995, Transplantation Pros. 27(6 :3212
(alginate);
Dorian et aI, US Patent No. 5,429,821 (al~te); ~ et a1,1993, Exp Neurol
122(1):37-47 (polymer-encapsulated PCl~ cells); Sagen et x1,1993, j Neurosci
13(6):2415-23 (bovine chromaffm cells encapsulated in semipermeable polymer
membrane
and implanted into rat spinal subarachnoid space); Aebischer et x1,1994, Exp
Neurol
126(2):151-8 (polymer-encapsulated rat PC12 cells imnla.tted into monkeys; see
also
Aebischer, WO 92/19595); Savelkoul et a1,1994, j Immunol Methods 170(2):185-96
(encapsulated hybridomas producing antibodies; encaapsulated transfecbed cell
lines
ressing various cytokines); Wine et x1,1994, PNAS USA 91(6):?324-8 (engineered
cells expressing human nerve growth factor encapsulated in an immunoisol~ion
117
CA 02319492 2000-08-02
WO 99/41258 PCTNS99/03095
polymeric device and transplanted into rats); F.merich et a1,1994, Prog
Neuropsychophain~acol Biol Psychiatry 18(5:935-46 (polymerencapsulated PC12
cells
implanted into rats); Kordower et a1,1994, PNAS USA 91 (23):10898-902 (polymer-
encapsulated d BI-1ZC cells expressing hNGP implanted into ) and Butler
et al WO 95/04521 (encapsulated device). The cells may then be introd~in
encapsulated form into an animal host, preferably a mammal and more preferably
a
human subject in need thereof. Preferably the encapsulating material is
semipernneable,
permitting release into the host of secreted proteins produced by the
encapsulated cells. In
many embodiments the semipermeable encapsulation renders the enca ted cells
immunologically isolated from the host organism in which the encaps~u~a~ cells
are
introduced. In those eonbodiments 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 GAh4
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
Qu~i'.~cells in vivo. For this purp~e, various t~x~ have been developed for
tion of target tissue and cells in vivo. A n r 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 le, Dubensky et aL (I984) Proc. Natl. Acad. Sci. USA 81, 7529-
7533;
Kaneda et aL, (1989 293,375-378; I-iiebert et al. (1989) Proc. Natl. Acad.
Sci.
USA 86, 3594-3598; Hatzoglu et al. (1990) j. Biol. Chew. 265,17285-17293 and
Ferry, et
aL (1991) Proc. Natl. Acad. Sci. USA 88, 8377-8381. The vector may be
administen~d by
itijecbio~, e.g. intravascu>arly or intramuscularly, inhalation, or other
parenberal mode.
Non-viral delivery methods such as administration of the DNA via complexes
with
liposomes or by injection, cathefier or biolistics may also be used.
In accon3ance with in vivo genetic modification, the a~a~mer of the
modification
will depend on the nature of the tissue, the efficiency of cellular
modification required, the
nunnber of opportunities to modify theanyarticular cells, the aaessibility of
the tissue to the
DNA composition to be introduced; d the like. By employing an attenuated or
modified retrovinxs carrying a target lramsaiptional initiation region, if
desired, one can
activafie the vines using one of the subject transcription factor constructs,
so that the virus
may be produced and transfect adjacent cells.
The DNA inxroduction 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 fierm effect, where cells could be introduced into the
host and then
turned on after a predetermined time, for example, affier the sells have been
able to home
to a particular site.
Binding properties, Assays
Rapamycin is lrn ~n~~ a to bind to the human protein, FKBP12 and to form a
tripartifie complex with hFKBPI2 and FRAP, a human counterpart to the yeast
proteins
TORI and TOR2. Rapalogs may be characterized and compared to rapamycin with
respect to their ability to b'md to human PKBPI2 and/or to form tripartite
rnmplexes
with human FKBPI2 and human FRAP (or fusion proteins or fragments containi~
its FRB
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CA 02319492 2000-08-02
WO 99/41258 PCT/US99/03095
domain). See WO 96/41865 (Clackson et al). That a plication discloses various
materials and mettwds which can be used to quantity the ability of a compound
to bind
to human FKBP12 or to form a tripartite complex with (ix., Nheterodinnenze"j
proteins
rnmprising human PKBP12 and the PRB domain of human FRAP, respectivel , Such
assays include fluorescence polarization assays to measure binding. Also in~
clad are cell
based transcription assays m which the ability of a compoand to form the
tripartite
co lax is measured indirectly by rnrrelation with the observed level of
reporter gene
p uct produced by engineered nL~matalian ceps in the presence of the rnmpound.
Corresponding cell bused assays may also be conducted in engineered yeast
cells. See eg.
WO 95/33052 (Berlin et al).
It will often be preferred that the rapalogs of this invention be
physiologically
acre table (i.e., lack undue toxicity toward the cell or organism with which
it is to be
, 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
iminunophilins (by way of non-sting example, a human FKBP in which Phe36 is
replaced with a different amino acid, preferabl an amino acid with a less
bulky R group
such as valise or alanine) over native or natura~y-ocuirW g immunoph~. For
example,
such compounds may bind preferentially to mutant PKBPs at least an order of
magnitude
better than they bind to human PKBP12, 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
FKBP12, as determined by any sci~tifically valid or art accepted assay
methodology.
Binding affinities of various rapalogs of this invention with respect to human
FI~P12, variants thereof or other immunophilin profieins 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 t~e
binding.ofa
known ligand to the protein of interest. See e.g. Sierkierka et x1,1989,
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 nN~, and even more preferably below about
1 nM, as
measured by direct binding measuz~t (e.g. fluoresc~ue quenching), competition
binding measurement (e.g. versus FIC506), inhibition of FKBP e~activity
(rotamase),
or other. assay methodology. In one subset of such ac~~ounds, the FKBP domain
is one
in ~v'hich phen~lalanine at position 36 has been reply 3 wig an amino acid
having a less
bulky side chain, e.g. alanine, valise, methionine or serine.
A Compe titive Binding FP Assay is described in detail in W096/41865. That
assay permits the in vitro measurement of an IC50 value for a given rnmpoand
which
reflects its ability to bind to an FKBP protein in competition with a labeled
FKBP ligand,
such as, for example, FIC506.
One preferred class of compounds of this invention are those rapalogs which
have
an IC50 value in the Competitive Binding FP Assay better than 1000 n~M,
preferably better
than 300 nM, more preferably better than 100 nM, and even more preferably
better titan
10 nM with respect to a given PKBP domain and ligand pair, e.g. human FKBP12
or a
variant thereof with up to 10, preferably up to 5 amino acid replacements,
with, f
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WO 99/41258 PCT/US99/03095
flouresceinafied PIC506 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. Por instance, one may use cells containing and capable of
expressing
DNA encoding a first chimeric protein rnmprising one or more PKBP domains and
one or
more effector domains as well as DNA encoding a sernnd chimeric profiein
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 rnntain
a reporter
1o gene under the transQiptional 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 desc~ed 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. Ther~o rber gene
product may
be a conveniently detectable protein (e.g. by ELISA) or may catalyze the
production of a
conveniently detectable product (e.g. rnlored). Materials and methods for
producing
aPPmP~te 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 SF.AP, 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-hyb rId trans~p tion 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
chimezic
proteins, like the FKBP based transQiption assay, is a cell-based assay which
measures
the oocun~ennce of an event triggered by such multimerization. In this case,
one uses cells
which oonstitutively express a detectable product. The cells also contain and
are capable
of expressing DNAs erucoding ~ia>eriC profieins rnmprising one or more
immunophilin~lerived ligand binding domains and one or more effector domains,
such as
the intracellular domain of FAS, capable, upon multimerization,, of triggering
cell death.
The d and preparation of illustrative components and their use in so
engixu~ing cells
is des~~ri ed in W095/02684. See also W096/41865. The cells-are maintainined
or
cultured in a culture medium permitting cell growth or continued viability.
The sells or
medium are assayed for the pr~eseence 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
medium is tested for the presence of reporter at one or more time points.
in reporter production indicates cell death, an indirect measure fo
multinrerization of the fusion proteins.
Another preferred class of compo ands of this invention are those which are
capable of inducing a detectable signal 'm such an FKBP/FIZB-based apoptosis
assay.
Preferably, the FKBP domain is an FKBP domain other than wild-type human
I~BPl2.
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WO 99/41258 PCT/US99103095
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 PRAP. 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 Assa permits one to select monomers or
rapalogs
which possess the desired IC50 values and r binding preference for a mutant
FKBP or
wild-type FKBP relative to a control, such as FK506.
Applications
to The rapalogs can be used as described in W094/18317, W095/026134,
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 gn7wing 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 ~,s particularly well suified for
achieving regulated
expression of a therapeutic target gene in the context of human gene therapy.
One
example uses a pair of chimenrofieins (one containing at least one FRB domain,
the
other containing at least one FKBP domain), a C3 rapalog of this invention
capable of
dinierizing the chimeras, and a target gene construct to be expressed. One of
the chimeric
proteins rnmprises a DNA binding domain, preferably a co posito DNA~inding
domain
as described in Pomerantz et al, supra, as the heterologous ebEector domain.
The second
chia~e~ric protein comprises a transcriptional activating domain as the
heberologous
effector domain. The C3 rapalog is capable of binding to both chi~aneras and
thus of
effectively doss-linking the chimeras. DNA molecules encoding and capable of
directing
the expression of these chimeric proteins are introduced infix the cells to be
engineered.
Also introduced into the cells is a target gene linked to a DNA sequence to
which the
DNA~inding domain is capable of binding. Contacting the engineered cells or
their
progeny with the C3 rapalog (by administering it to the animas or patient)
leads to
assembly of the transcription factor complex and hence to~ re&sion of the
target gene.
The design and use of similar components is disclosed in ~/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 rn~
lexes, which
should in turn be a function of the concentration of the C3 rapalog. Dose (OI
C3 rapalog)-
responsive gene expression is typically observed.
The C3 rapalog may be administered to the patient as desired to activate
transQiption of the target gene. ding upon the binding affinity of tine C3
rapalog,
tie response desired, the manner o aiministratio~, the biological half life of
the rapalog
and/or target gene mRNA, the number of engineered cells pn~enly various
protocols may
be employed. The C3 rapalog may be administered by various mutes, including
panmterally or orally. The number of administrations will depend apon the
factors
desan~ed above. The C3 rapalog may be take. orally as a p'pow~r, or
dispersion;
bucally; sublingually; injected intravascularly, intraperitoneally,
intramuscularly,
subcutaneously; by inhalation, or the like. The C3 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'bte~od
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of administration will depend upon the above factors and be defiermined by the
atfiending
physician or human or animal healthcare provider. Por the most part, the
manner of
administration will be determined empirically.
In the event that transcriptional activation by the C3 rapclog is to be
reversed or
terminated, a monomeric compound which can compete with tC3 rapalog may be
administered. Thus, in the case of an adverse reaction or the desire to
terminate the
therapeutic effect, an antagonist to the dimerizing agent can be administered
in any
convenient way, particularly intravascularly, if a rapid reversal is desired.
Altenlatively,
one may provide for the presence of an inactivation domain (or transaiptional
silencer)
with a ligand binding domain. In another approact4 cells may be eliminafied
through
apoptosis via signalvng through Fas or TNF receptor as described elsewhere.
See
International Patent Applications PCT/US94/01617 and PCT/US94/08008.
The particular dosage of the C3 rapclog for an y~ 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 C3 rapalog over short periods o~time,
with
extended intervals, for exam le, two weeks or more. A dose of the C3 rapalog
within a
predefiernnined range would ~ 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
rnuld
provide a larger or smaller dose the next time, following the response. This
process would
be iberatively repeated until one obtained a dosage within the thera tic rage.
Where
the C'~ rapalog is cW ically administered, once the mainfienanoe ge of the C3
rapalog is determined, one rnuld then do assays at extended intervals to be
assured that
the cellular system is providing the appropriate response and level of the
expression
p~~
It should be appreciated that the~~ subject to many variables, such as the
cellular response to the C3 rapalog, the of expression and,, as appropriate,
the
level of secretwn, the activity of theexpression 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 prnteins and viruses. Production of recombinant
therapeutic proteins for coamcuerdal and investigational purposes is often
achieved
the use of mammalian cell lines ec~ineered to express the protein at high
level. The
use o ~ sells, rather than bacteria or yeast, is indicated where theproper
hutction of the protein requires post translational modifications not erally
performed
by heberologous cells. Bxaa~ples of proteins produced connaueroally way
include
er~~oietin, tissue plasminogen activator, clotting factors such as Facfior
VIII:c,
an bodies, etc. The cost of produdng 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 cell: Protein expression mayprevent
cells from
growing to hi~ density, sharply reducing production levels. Therefore, the
ability to
tightly controiprotein expression, as desc~ed for regulated gene therapy,
permits cells to
be grown to ludensity in the absence of protein production. Only after an
optimum cell
density is read, is expression of the gene activated and the protein product
subsequently harvested.
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A similar problem is encountered in the construction and use of "packaging
lines"
for the production of recombinant vauses for oommeirial (e.g., gene therapy)
and
experimental use. These cell lines are engineered to produce viral proteins
for the
assembly of infectious viral particles harboring defective recombinant genoal
vectors that are dependent on such packaging lines include i~etravirus,
adenovims, 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 preduction 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 rnnstniction of packaging lines in
which the rep
and core genes are placed under the control of regulatable transcription
factors of the
design descxzbed here. The packaging Bell 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 rnntrol over a target gene is desired. These
include: (1)
expression of a protein or RNA of interest for biochemical purification; (2)
regulated
a ression of a protein or RNA of interest in tissue culture cells (or in vivo,
via engineered
) for the purposes of evaluating its biological function; (3) regulated
expression of a
pretain or RNA of interest in transgenic animals for the purposes of
evaluating its
biological function; (4) regulating the expression of a gene encoding
anotherregu1atory
prote~, r~ozyme or antisense molecule that acts on an endogenous gene for the
purposes
of evaluating the biological Function of that gene. Transgenic animal models
and o~
applications in which the coalponents of this invention may be adapted include
those
disclosed. in..PC"T/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
profieins of this invention (and may contain additional domains as discussed
above) and,
m embodiments involving regalebed gene hanscnptio~, a target gene construct
containing a
target gene linked to one or more transaiptioal control e)~ements which are
activated by
the multimerization of the chimeric proteins. Alternatively, the target gene
rnnstruct may
contain a doping site for insertion of a desired targetgene 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 profiein-protein interactions, a rapalog
of this
invention may be used in pharnnaoeutical coinpo~tions and methods for
promoting
formation of rnmplexes of chiaieric proteins of this invention in a human or
non-human
avaaunal 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 mmplexation, e.g. transaiption of a
target gene,
apoptosis of engineered Bells, etc.
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TherapeuticlPmphylactic Administration & Pharataceutical Compositions
The rapalogs can exist in free foam or, where appropriafie, ins salt form.
Pharmaceutically acceptable salts of many t)~compounds and their preparation
are
well-known to those of skill in the art. The p ceutically acceptable salts of
compounds of this invention include the rnnventional 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 hydrafied sperie<s 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 pmphylactically) effective amount of the compound, and one
or more
phaia~aceutically acceptable carriers and/or other excipients. Carriers
include e.g. saline,
buffered saline, dextrose, water, glyceml, ethanol, and rnmbinations thereof,
and are
discussed in greater detail below. The composition, if desired, can also
contain minor
amounts of wetting or emulsifying agents, or pH bufferag~enfis. The
composition can be
a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained
release formulation,
or powder. The composition can be formulated as a suppository, with
traditional binders
and carriers such as triglycerides. Oral formulation can include standard
carriers such as
pharrmaceutical des of mannitol, lactose, starch,, ma rum sbearate, sodium
saccharine, cellu~ , magnesium carbonate, etc. Pormton may involve alixing,
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 albs, sucr~e, 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, glidanfis, 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 rnmpression properties
in suitable
pc~portions ,and compacted in the shape and size desired. Thepowders and
tablets
priably contain up to 9990 of the active ingredient. Suitable solid
carriers.include, for
example, calcium phosphate, magnesium stearate, talc, sugars, lactose,
dextrin, starch,,
gelatin, cellulose, meth 1 cellulose, sodium cari~oxyatethyl cellulose,
polyvinylpynrolidine,
low melting waxes anion exchange resins.
Illustrative liquid carriers include syrup, peanut oil,, olive oil, water,
etc. l iqu~d
carriers are used in preparing solutions, suspensions, emulsions, syrups,
elixirs and
pressurized compositions. The active ingredient can be dissolved or suspended
in a
phnara~aceutically acceptable liquid carrier such as water, an organic f
olvent, 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, agents, colors, viscosity
regulators, stabilizers or osmo-regulators. Suitable.examp of liquid carriers
fc~ypral and
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WO 99/41258 PCTJUS99I03095
parenteral administration include wafier (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). Por
parenberal
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
par~teral administration The liquid carrier for pressurized compositions can
be
halogenated hydrocarbon or other pharnnaeeutically acceptable propellant.
Liquid
pharmaceutical compositions which are sfierile 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 rnmposition form.
The carrier or excipient may include time delay material well known to the
art,
such as glyceryl monostearate or glyoeryl distearate along or wide a wax,
ethylcellul~e,
hy~iroxypropylmethylcellulose, methylmethacrylate and the like. When
formulated for
oral administration, 0.01% Tween 80 in PHOSAL PG50 (phospholipid concentrate
with
1~ P~PYI~ g1ycol, A. Natfiera~ann d= Cie. GmbI~ has been recognized as
providing an
acceptable oral formulation for other rnmpounds, 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 tablefied, placed in a hard gelatin capsule in
powder or llet
form or in the form of a troche or lozenge. The amount of solid carrier will
vary wide
but preferably will be from about 25 mg to about 1 g. If agh'quid cattier is
used, the
preparation will be in ~e form of a syrup, emulsion, soft elatm capsule,
sterile injectable
solution or suspension in an ampule or vial or nonaqueous liquid suspension.
To obtain a stable water soluble dosage form, a phara~aceuticall)r acceptable
salt
of the multialerizer may be dissolved in an aqueous solution of an organu or
inorganic
acid, such as a 0.3M solution of succinnic act od r 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.
Exam les of such suitable cosolvents include, but are not limited to, alcohol,
propylene
~yco~polyethylene glyrnl 300, polysorbabe 80, glycerin, polyoxyethylated fatty
ands,
fatty almhols 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 admituster the
maltimerizer, or the various formulations thereof, including tablets,
capsules, injectable
solutions, encapsulation in liposomes, microparticles, micro soles, etc.
Methods of
introduction include but are not lianibed to dermal, intraderma~intramuscular,
intraperitoneal, intravenous, subcutaneous, iniranasal, Pulmonary, epidural,
ocular and
(as is usually preferred) oral routes. The compound may be adminisfiered by
any
convenient or otherwise appropriate route, for example by infusion or bolus
injection, by
absorption through epithelial or mucocutaneous linings (eg., oral mucosa,
rectal and
intestinal mucosa, ctc.) and may be administered fiogether with other
biologically active
agents. Administration can be sysbemi c or local. For treatment or prophyla~ds
of nasal,
bronchial or pulmonary conditions, P,..~ferred routes of administration are
oral, nasal or
via a bronchial aerosol or nebulizer.
In certain embodiments, it may be desirable to administer the rnmpound locally
to
an area in need of treatment; this may be achieved by, for example, and not by
d~ly of
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limitation, local infusion during surgery, topical application, by injection,
by means of a
catheter, by means of a suppository, or by means of a skin patch or implant,
said implant
being of a porous, non-porous, or gelatinous mafierial," including membranes,
such as
sialastic membranes, or fibers.
In a specific embodiment, the composition is formulated in acrnrdance 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 lyophilizedpowder 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 pharn~aceutical grade
water or saline.
Where the rnmposition is administered by injection, an ampoule of sterile
water far
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 coinp ound can
also
be accomplished topically by administering the rnmpound(s) 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
wloy~ rot, alrnhol, propylene glycol, fatty alcohols, triglycerides, fatty
acid esfiers, or
n~u~eralOther ical carriers include liquid petroleum, isopropyl palmitate,
polyethylene
glycol, ethanot1~95%), pot oxyethylene monolaurafie (596) in water, or sodium
lauryl
sulfate (59~°) in water. ~er materials such as anti-oxidants,
humectants, viscosity
stabilizers, and similar agents may be added as necessary. Percutaneous
penetration
enhanoers such as Azone may also be included.
In addition, in certain instances, it is expected that the coin d may be
disposed within devices placed in, or under the skin. Such include patches,
implants, and injections which the compound into the skin, by either passive
or
active release mechanisn:LS.
Materials and methods for producing the various formulations are well known in
the art and may be adapted for practicing the subject invention. . See eg. 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; illustrative formulation for IV administration) and
0 648 494
(published Aprd 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 10mg /kg of mammalian body
weight,
administered in single or multiple doses. Generally, the compound may be
at~ered
to pa~e<:ts in need of such treatment is 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 tl~fusion
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WO 99/41258 PCT/US99/03095
pmfieins to be multimerized, the characberistic~ and location of
thegenetically engineered
cells, and on the nature of the disorder or condition, which can be deietmined
by
standard clinical ues. In addition, in vitro or in vivo assays may optionally
be
emplo ed to help iden.' optimal dosage ranges. Effective doses may be
extrapolated
from ~ose-response curves derived from in vitro or animal model fiest
sysfiems. The
precise dosega 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 rnmprising one or
more
containers containing one or more of the ingredients of the phanmaceuiical
compositions
of the invention. Optionally associated with such containers) can be a notice
in the form
pr~sc~ed by a governmental agency regulating the manufacture, use or sale of
pharmaceutical or biological products, which notice reflects approval by the
agency of
manufachu~e, use or sale for human administration The notice or package insert
may
contain instructions for use of a C3 rapalog of this invention, consistent
with the
disclosure herein.
Experimental Examples
Synthesis of t..,3-methallyl-rapamycin and 1..3-allyl-rapamycin
The synthetic procedure we currently use is a modified version of the
procedure
?5 described in L~erles et al., July 1997, Proc. NatL Acad. Sci. USA 94:7825-
7830 for the
preparation of C7 rapalogs. (Note that C7 and C3 as referred to herein refer
to the ring
positions C16 and C20, respectively, in the nomenclature of L~erles et al).
Twenty two
mgs of rapamycin are placed in a 3 ml flame-dried Wheaton vial equipped with a
stir bar.
The rapamycin is dissolved in 200 ~1 of methylene chloride and cooled to -40
degrees.
Lower temperatures have been found to not work as eftly. 50 N.1 of methallyl
(or
allyl) trimethyl silane (12 equiv.) is added, followed by 40 ~,1 of neat BF3
etherate (13
equivalents). After 2 hours the reaction is over, as determined by TLC, and is
quenched
by addition of saturated NaHC03. The reaction mixture is then washed with
brine and
dried with sodium sulfate. (The aqueous washes can be back-extracted with
methylene
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CA 02319492 2000-08-02
WO 99/41258 PCT/US99/03095
chloride and combined with the reaction mixture.) The samples are filfiered
with a .45
micron Nylon filter and the solvent is evaporated under vacuum.
The crude product is dissolved in 100 N,1 of chloroform for injeckion on the
jAI
recycling HPLC desc~ed below. Three predominant peaks are obtained that can be
separated with baseline resolution after approximately 15-20 cycles. (Under
certain
reaction conditions, a fourth peak appears which elutes before the other
three.) Of the
three predominant peaks, the first is C7-S methallyl rapamyain, as-determined
by 1H-
NMR The middle peak is the compound of interest, C3-methallyl rapamycin, and
the
fourth peak is composed of side products resulting from oversilylation of
rapamycin.
Following this procedure, we obtained 4.2 mgs of pure C3-methallyl rapamycin.
Since
unmodified rapamycin coincidently comigrates precisely with C3-methallyl
rapamycin,
and cannot be removed efficiently with the JAI, it is critical that rapamycin
be completely
consumed during the reaction. Overaddiiion products are far more easily
removed than
the unreacted starting material.
The jAI-purified methallyl rapamycin can be verified by W, NMR, and mass
spectroscopy and biological activity. The presence of contaminants, such as
rapamycin,
can be easily determined by UV, as descn'bed below.
This same procedure has been used to synthesize C3 ally)-rapamycin, and should
be easily extended to synthesize a variety of other C3 derivatives.
Analytical considerations
One particularly convenient diagnostic is iJV spectroscopy. Consistent with
disappearance of the triene, the UV spectra of the C3 compounds have a maximum
absorbance at lambda 238 rawer than 274-282, typically seen for the C7 (R) or
(S)
compounds. Since the C3 rnmpounds have baseline absorbance in the region where
rapamycin absorbs best, LJV analysis is a good indicator of sample purity and
the
presence of any toxic contaminants.
The loss of the triene in C3-methallyl rapamycin is also reflected in the 1H
NMR
A 1H-NMR of methallyl rapamycin" taken in CDCL3, reveals a diagnostic peak
from C5
at 6.0 ppm. The remainder of the olefinic protons all lie upfield between 5
and 5.6 ppm.
Chromatographic recovery of the C3 rapalogs
An instzument made by the Japan Analytical Industry Co., LTD. QA.1) efficient)
purifies the C3 rapalogs. The insizument we used was a "LC-908 ltecyclin
Preparative
HPLC" and the rnlumn we used was "JAIGEL GS-310" with dimensions o~"20 x 500
mm".
Functional characteristics of C3 rapalogs
The toxicity of the lead compounds was tested by th~:ir ability to inhibit the
proliferation of CTLL-2 cells, which are IL-2 dependent aad highly sensitive
to rapamycin
(IC50 < 1 nM). Incubation of CTLIr2 cells with 11tM C3-methallyl rapamycin had
no
detectable effect on proliferation.
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Likewise, C3-allyl rapamycin and C3-methallyl rapamycin were impaired in their
ability to activate transcription in jurkat cells transfected with Gal4-FKBP3,
wild type
FRB-VP16, and UAS-SEAP. These constructs are described in L~erles et al,
above, the
full contents of which are incorporated herein by reference.) In experiments
in which
FRB*-VP16 (FRB harboring T2098L, W2101F, and K2095P) replaces wild a FRB-
VP16, reporter gene activity can be detected at an EC50 near 10 nM C3-meyl
rapamycin or C3-allyl rapamyain; as well, the amplitude of reporter gene
activity is higher
than that elicited by the rapamycin/wild type FRB-VP16 system. - C3
derivatives of
rapamycin have impaired toxicity (immunosuppressive activity) and can be used
efficiently as dimerizers in conjunction with engineered FRB domains.
Preparation of 24(S),30(S)-tetrahydro-C3-rapalogs
Na8H4, CeCI~~FilO
MeOH, -78'C
Ha,,
Meo
~O OH ~ H
N
O O O Me0'~ H
H RC3
O
~ ~ i
24,30-tetrahydro rapamycin is prepared as desc~ed elsewhere and is converted
into the
desired C3-substituted rapalog by the method desc~ed above.
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Preparation of 13-fluoro-C3-rapalogs
The title rnmpounds are prepared as described above, substituting I3-Fluoro
rapamycin
for rapamycin.
Preparation of 2&fluoro-C3-rapalogs
0
The title compounds are prepared as described above, substituting 28-Fluoro
rapamycin
for rapamyrin.
Preparation of 43-epi-C3-rapalogs
0
The title compounds are prepared as described above, substituting 43-epi
rapamycin for
rapamycin. ~-
130
