U.S. patent application number 11/045750 was filed with the patent office on 2005-08-04 for combinatorial enzymatic complexes.
This patent application is currently assigned to Rigel Pharmaceuticals, Inc.. Invention is credited to Nolan, Garry P., Payan, Donald.
Application Number | 20050170403 11/045750 |
Document ID | / |
Family ID | 25361966 |
Filed Date | 2005-08-04 |
United States Patent
Application |
20050170403 |
Kind Code |
A1 |
Nolan, Garry P. ; et
al. |
August 4, 2005 |
Combinatorial enzymatic complexes
Abstract
The invention relates to the formation of novel in vivo
combinatorial enzyme complexes for use in screening candidate drug
agents for bioactivity.
Inventors: |
Nolan, Garry P.; (San
Francisco, CA) ; Payan, Donald; (Hillsborough,
CA) |
Correspondence
Address: |
BOZICEVIC, FIELD & FRANCIS LLP
1900 UNIVERSITY AVENUE
SUITE 200
EAST PALO ALTO
CA
94303
US
|
Assignee: |
Rigel Pharmaceuticals, Inc.
|
Family ID: |
25361966 |
Appl. No.: |
11/045750 |
Filed: |
January 28, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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11045750 |
Jan 28, 2005 |
|
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08873601 |
Jun 12, 1997 |
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Current U.S.
Class: |
435/6.16 ;
435/325; 435/7.2 |
Current CPC
Class: |
C12N 15/1034 20130101;
C12N 15/1079 20130101; C12N 15/1062 20130101; C12N 15/1044
20130101; C12N 2799/027 20130101; C12P 1/00 20130101; C12N 15/1075
20130101 |
Class at
Publication: |
435/006 ;
435/007.2; 435/325 |
International
Class: |
C12Q 001/68; G01N
033/53; G01N 033/567 |
Claims
1-26. (canceled)
27. A library of cells comprising: a) nucleic acid encoding an
exogenous scaffold comprising a first binding site and a second
binding site; and b) nucleic acid encoding a first enzyme and a
second enzyme, wherein at least one of said enzymes is heterologous
to said cells; and wherein said first enzyme binds to said first
binding site and said second enzyme binds to said second binding
site.
28. The library of cells of claim 27, wherein said cells are tumor
cells.
29. The library of cells of claim 27, wherein said cells contain an
infectious agent.
30. The library of cells of claim 27, wherein said cells are immune
system cells.
31. The library of cells of claim 27, wherein said cells contain a
virus.
32. The library of cells of claim 27, wherein said cells are
mammalian cells.
33. The library of cells of claim 27, wherein said scaffold is
linear.
Description
FIELD OF THE INVENTION
[0001] The invention relates to the formation of novel in vivo
combinatorial enzyme complexes for use in screening candidate drug
agents for bioactivity.
BACKGROUND OF THE INVENTION
[0002] Signaling pathways in cells often begin with an effector
stimulus that leads to a phenotypically describable change in
cellular physiology. Despite the key role intracellular signaling
pathways play in disease pathogenesis, in most cases, little is
understood about a signaling pathway other than the initial
stimulus and the ultimate cellular response.
[0003] Historically, signal transduction has been analyzed by
biochemistry or genetics. The biochemical approach dissects a
pathway in a "stepping-stone" fashion: find a molecule that acts
at, or is involved in, one end of the pathway, isolate assayable
quantities and then try to determine the next molecule in the
pathway, either upstream or downstream of the isolated one. The
genetic approach is classically a "shot in the dark": induce or
derive mutants in a signaling pathway and map the locus by genetic
crosses or complement the mutation with a cDNA library. Limitations
of biochemical approaches include a reliance on a significant
amount of pre-existing knowledge about the constituents under study
and the need to carry such studies out in vitro, post-mortem.
Limitations of purely genetic approaches include the need to first
derive and then characterize the pathway before proceeding with
identifying and cloning the gene.
[0004] Screening molecular libraries of chemical compounds for
drugs that regulate signal systems has led to important discoveries
of great clinical significance. Cyclosporin A (CsA) and FK506, for
examples, were selected in standard pharmaceutical screens for
inhibition of T-cell activation. It is noteworthy that while these
two drugs bind completely different cellular proteins--cyclophilin
and FK506 binding protein (FKBP), respectively, the effect of
either drug is virtually the same--profound and specific
suppression of T-cell activation, phenotypically observable in T
cells as inhibition of mRNA production dependent on transcription
factors such as NF-AT and NF-.kappa.B. Libraries of small peptides
have also been successfully screened in vitro in assays for
bioactivity. The literature is replete with examples of small
peptides capable of modulating a wide variety of signaling
pathways. For example, a peptide derived from the HIV-1 envelope
protein has been shown to block the action of cellular
calmodulin.
[0005] A major limitation of conventional in vitro screens is
delivery. While only minute amounts of an agent may be necessary to
modulate a particular cellular response, delivering such an amount
to the requisite subcellular location necessitates exposing the
target cell or system to relatively massive concentrations of the
agent. The effect of such concentrations may well mask or preclude
the targeted response.
[0006] In addition, traditional methods do not allow the creation
of completely new enzymatic pathways.
[0007] Thus, it is an object of the present invention to provide
methods and compositions for the effective introduction of
enzymatic libraries into cells to screen and create bioactive
compounds.
SUMMARY OF THE INVENTION
[0008] In accordance with the outlined objects, the present
invention provides cells containing a composition comprising an
exogeneous scaffold comprising at least a first binding site and a
second binding site; and at least a first and a second enzyme. At
least one of the enzymes is heterologous to the cell. The first
enzyme is bound to said first binding site and said second enzyme
is bound to said second binding site.
[0009] In a further aspect, the present invention provides cells
containing a composition comprising nucleic acid encoding an
exogeneous scaffold comprising at least a first binding site and a
second binding site; and nucleic acid encoding at least a first and
a second enzyme. At least one of the enzymes is heterologous to the
cell, and the first enzyme is capable of being bound to the first
binding site and the second enzyme is capable of being bound to the
second binding site.
[0010] In an additional aspect, the invention provides methods of
screening for a bioactive agent, comprising expressing in a
plurality of host cells nucleic acid encoding an exogeneous
scaffold comprising at least a first binding site and a second
binding site, and nucleic acids encoding at least a first enzyme
and a second enzyme; under conditions where the nucleic acids are
expressed, and the first enzyme binds to the first binding site and
the second enzyme binds to the second binding site. The method
further comprises screening the host cells for a cell exhibiting an
altered phenotype, wherein the altered phenotype is due to the
presence of a bioactive agent.
[0011] In a further aspect, the invention provides methods of
screening for a bioactive agent comprising expressing in a
plurality of host cells a library of nucleic acids encoding a
library of scaffolds, each scaffold comprising at least a first
binding site and a second binding site. The method further
comprises expressing in the cells a library of nucleic acids
encoding a library of enzymes; under conditions where the nucleic
acids are expressed, and at least some of the enzymes bind to the
scaffolds, followed by screening of the host cells for an altered
phenotype.
[0012] In an additional aspect, the invention provides compositions
comprising a scaffold comprising at least a first and a second
binding site; and at least a first and a second enzyme. The first
enzyme is bound to the first binding site and the second enzyme is
bound to the second binding site, wherein the enzymes do not
biologically react with said scaffold or each other.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIGS. 1A and 1B depict different compositions of the
invention. FIG. 1A depicts a linear scaffold with enzymes
containing exogeneous binding sequences (A*, B* and C*) bound to
binding sites (a', b' and c'), wherein the binding sequences are
attached to the enzymes via linkers. The scaffold is a linear
scaffold. FIG. 1B depicts a scaffold-less system, wherein the
binding sequences on the enzymes (A*, B*, C*, D* etc.) are depicted
as internal, although as will be appreciated by those in the art,
they could be exogeneous and attached via linkers as well.
[0014] FIGS. 2A, 2B, 2C, 2D, 2E and 2F depict various
scaffold-enzyme possibilities.
[0015] FIG. 3 depicts a linear scaffold.
[0016] FIGS. 4A, 4B, 4C and 4D depicts various circular
scaffolds.
[0017] FIGS. 5A and 5B depict systems utilizing transmembrane
anchoring sequences, either without a scaffold (FIG. 5A) or with a
scaffold (FIG. 5B), although as will be appreciated by those in
art, FIG. 5B does not require a scaffold if the binding sequences
associate.
[0018] FIG. 6 depicts a schematic of a retroviral construct.
DETAILED DESCRIPTION OF THE INVENTION
[0019] The present invention provides compositions of novel
mixtures of enzymes in a spatially constricted or defined manner,
i.e. by binding of the enzymes to a scaffold molecule, which allows
the enzymes to act on a precursor molecule in novel or efficient
ways to form candidate bioactive agents which may then be screened
for bioactivity.
[0020] As is known in the art, there are a number of enzymatic
pathways or cascades wherein the reactant of one enzymatic reaction
is the precursor of the next enzymatic reaction, which after
catalysis serves as the precursor for yet a third enzyme, etc. It
has been suggested that the enzymes of these pathways might be or
could be spatially oriented in an organized manner such that
productive reactions are maximized and side reactions are
minimized. However, these same mixtures of enzymes in the absence
of spatial orientation may result in the generation of no product
or a highly heterogeneous mixture of products that may be difficult
to analyze, with interesting products being made in low
concentrations. Thus, the ability to restrict the spatial
conformation of the enzyme mixture can result in a more defined
mixture of products at higher concentrations. In one embodiment,
the present invention provides such spatial constriction.
[0021] The ability to make enzyme compositions comprising any
number of enzymes from a variety of different organisms in any
number of spatially constricted conformations can result in the
generation of a large number of novel products which then may be
screened for desired biological activities. The number and type of
enzymes may be varied, as well as the orientation of the enzymes,
thus providing a combinatorial approach.
[0022] Thus, the invention generally provides for compositions of a
number of enzymes, each bound to a scaffold. A library of enzymes,
each of which binds a corresponding binding site on a scaffold, is
used. The binding sites may be randomly combined in any number of
scaffolds, in any number of orientations, providing a library of
scaffolds. Thus, for example, starting with a list of 100 enzymes,
and 100 binding sites, each of which will bind one of the enzymes,
a large number of scaffolds can be made. Thus, for example, a
linear scaffold containing seven binding sites can be configured in
100.sup.7 different ways. If only seven enzymes are included, a
library of 7.sup.7 different scaffolds, and thus 7.sup.7 different
enzyme complexes can be made, etc. In addition, non-linear
scaffolds, as are more fully described below, allow an even greater
number of orientations.
[0023] These scaffolds, and the corresponding enzymes, are then
introduced into a variety of different types of cells, generally
using retroviral introduction of the nucleic acids encoding them.
Precursor molecules may then be added, and then the cells screened
for desired phenotypes. The exact composition of the enzyme
mixture, as well as the orientation of the enzymes with respect to
both each other and the precursor upon which the enzymes act, may
be important in both eliminating undesirable reactions and products
as well as obtaining the desired reactants.
[0024] Thus the present invention provides methods of using the
novel compositions in screening methods for the synthesis,
identification and detection of bioactive agents which are capable
of altering the phenotype of cells containing the agents. The
present invention enables the production of these spatially
constricted enzymes, followed by screening of candidate agents,
within the same cells. This is different from traditional
combinatorial approaches which require the synthesis of the
candidate bioactive agents, for example synthetically, followed by
the exogeneous addition of the agent to a population of cells to
test for bioactivity. Accordingly, the present invention confers a
significant advantage since a major limitation of conventional in
vitro screens is delivery. While only minute amounts of an agent
may be necessary to modulate a particular cellular response,
delivering such an amount to the requisite subcellular location
necessitates exposing the target cell or system to relatively
massive concentrations of the agent. The effect of such
concentrations may well mask or preclude the targeted response. In
addition, delivery of the agent to the required subcellular
location, even at high extracellular concentrations, may be
poor.
[0025] Thus, the methods of the present invention provide a
significant improvement over conventional screening techniques, as
they allow the rapid screening of large numbers of candidate
bioactive agents in a single, in vivo step. In addition, the
present methods allow screening for drugs that can treat disease
conditions, in the absence of significant prior characterization of
the cellular defects per se.
[0026] Accordingly, the present invention provides compositions
comprising a scaffold and at least two enzymes. By "scaffold"
herein is meant a sequence to which a plurality of enzymes may
bind. Scaffolds may be either proteins, and bind enzymes via
protein-protein interactions, or nucleic acids, and bind enzymes
via protein-nucleic acid interactions, with protein scaffolds being
preferred. "Proteins" in this context includes proteins,
oligopeptides and peptides. "Nucleic acids" or "oligonucleotides"
in this context includes DNA, RNA, and synthetic nucleic acids.
When the scaffold is nucleic acid, it will generally contain
phosphodiester bonds, although in some cases, as outlined below, a
nucleic acid analogs are included that may have alternate
backbones, comprising, for example, phosphoramide (Beaucage et al.,
Tetrahedron 49(10): 1925 (1993) and references therein; Letsinger,
J. Org. Chem. 35: 3800 (1970); Sprinzl et al., Eur. J. Biochem. 81:
579 (1977); Letsinger et al., Nucl. Acids Res. 14: 3487 (1986);
Sawai et al, Chem. Lett. 805 (1984), Letsinger et al., J. Am. Chem.
Soc. 110: 4470 (1988); and Pauwels et al., Chemica Scripta 26: 141
91986)), phosphorothioate, phosphorodithioate,
O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides
and Analogues: A Practical Approach, Oxford University Press), and
peptide nucleic acid backbones and linkages (see Egholm, J. Am.
Chem. Soc. 114: 1895 (1992); Meier et al., Chem. Int. Ed. Engl. 31:
1008 (1992); Nielsen, Nature, 365: 566 (1993); Carlsson et al.,
Nature 380: 207 (1996), all of which are incorporated by
reference). These modifications of the ribose-phosphate backbone
may be done to increase the stability and half-life of such
molecules in physiological environments. The nucleic acids may be
single stranded or double stranded, as specified, or contain
portions of both double stranded or single stranded sequence. The
nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid,
where the nucleic acid contains any combination of deoxyribo- and
ribo-nucleotides, and any combination of bases, including uracil,
adenine, thymine, cytosine, guanine, inosine, xathanine and
hypoxathanine, etc. In a preferred embodiment, for example when
nucleic acid encoding the scaffold is introduced into cells, the
nucleic acid is DNA.
[0027] The scaffold comprises a plurality of binding sites, each of
which will bind an enzyme. Thus, for example, a scaffold with two
binding sites will bind two enzymes; a scaffold with three binding
sites will bind three enzymes; etc. That is, as is generally
depicted in the Figures, enzyme A will bind to binding site a,
enzyme B will bind to binding site b, etc. Preferably, any single
scaffold does not contain more than one binding site for a
particular enzyme; that is, the enzyme complexes of the invention
preferably contain different enzymes. Scaffolds preferably bind at
least two enzymes, with scaffolds that bind from about 2 to about
20 enzymes are preferred, and scaffolds that bind from about 3 to
about 10 enzymes being especially preferred, and from about 4 to
about 8 being particularly preferred. Generally, each binding site
comprises from about 2 to about 20 amino acid residues or from
about 2 to about 25 nucleotides.
[0028] The actual sequence of the binding sites will be determined
in any number of ways, as will be appreciated by those in the art,
and will depend on the enzyme, or the part of the enzyme, or a tag
added to the enzyme, to which it will bind. In a preferred
embodiment, desirable enzymes, as outlined below, may be run in the
yeast or mammalian two-hybrid system to determine binding
sites.
[0029] Alternatively, exogeneous binding sequences can be added to
the enzymes. A "binding sequence" is a sequence that will bind to
at least one binding site, defined above, or to another binding
sequence. Binding sequences and binding sites together form
"binding pairs", although the term "binding pair" is not meant to
exclude systems that have more than two components. Thus, rather
than determine binding sites on the basis of the wild-type sequence
of the enzyme, an exogeneous binding sequence can be added to the
enzyme, as will be appreciated by those in the art. This may be
done directly or through the use of linkers, as defined herein and
shown in FIG. 1A. Similarly, as is described below, enzymes each
containing a binding sequence to at least one other enzyme may be
generated, thus eliminating the need for the scaffold; see FIG. 1B.
Furthermore, the enzyme complex may be a mixture of these systems,
where some enzymes are bound to scaffolds and other enzymes are
associated with the bound enzymes and not to the scaffold. Suitable
binding sequences/binding site pairs (or binding sequence/binding
sequence pairs, when scaffold binding sites are not used) include
any number of known proteinaceous binding pairs including epitopes,
ligand-receptor sequences, signaling sequences, etc., which may be
used as will be appreciated by those in the art.
[0030] In addition, more than one binding site may be generated for
each enzyme. That is, binding sites for different surfaces of an
enzyme may be made, to hold the enzyme on the scaffold in a variety
of conformations. That is, binding sites may be used to different
surfaces on the enzyme. For example, binding sites which would bind
the active site of the enzyme, thus effectively sterically
hindering enzyme function, are not preferred. Similarly, one
binding site may be modified to orient the enzyme in a certain way
on the scaffold. And, as outlined herein, the binding sites may be
placed in different order within a linear or circular scaffold.
[0031] Once a binding site for each desired enzyme is determined,
the binding sites may be combined into scaffolds in any number of
ways. Generally, binding sites are joined together with linker
sequences to form scaffolds. The linker sequences may comprise
structural elements if desired. For example, when the binding sites
are proteins, linker sequences may be chosen to form alpha-helices,
.beta.-sheets, turns (i.e. proline rich areas, etc), or other known
protein structures. Similarly, when the binding sites are nucleic
acid sequences, linker sequences that form known structures such as
hairpin loops, stem-loop structures, etc. Furthermore, linkers may
be used to "channel" substrates and reaction products between
enzymes, to alter reaction kinetics, for example.
[0032] In a preferred embodiment, the binding sites may be held in
a particular structural conformation through the use of
presentation structures. By "presentation structure" or grammatical
equivalents herein is meant a sequence, which, when fused to
binding sites, causes the binding sites to assume a
conformationally restricted form. Proteins interact with each other
largely through conformationally constrained domains. Although
small peptides with freely rotating amino and carboxyl termini can
be useful, the presentation of peptides in conformationally
constrained structures will likely lead to higher affinity
interactions of the peptide with the target enzyme. This fact has
been recognized in the combinatorial library generation systems
using biologically generated short peptides in bacterial phage
systems. A number of workers have constructed small domain
molecules in which one might present randomized peptide
structures.
[0033] While the scaffolds and binding sites include nucleic acids
or peptides, presentation structures are preferably used with
peptide binding sites and scaffolds. Thus, synthetic presentation
structures, i.e. artificial polypeptides, are capable of presenting
a binding site peptide or scaffold as a conformationally-restricted
domain. Generally such presentation structures comprise a first
portion joined to the N-terminal end of the peptide, and a second
portion joined to the C-terminal end of the peptide; that is, the
peptide is inserted into the presentation structure, although
variations may be made, as outlined below. To increase the
functional isolation of the scaffold, the presentation structures
are selected or designed to have minimal biologically activity when
expressed in the target cell.
[0034] Preferred presentation structures maximize accessibility to
the peptide by presenting it on an exterior loop. Accordingly,
suitable presentation structures include, but are not limited to,
minibody structures, loops on beta-sheet turns and coiled-coil stem
structures in which residues not critical to structure are
randomized, zinc-finger domains, cysteine-linked (disulfide)
structures, transglutaminase linked structures, cyclic peptides,
B-loop structures, helical barrels or bundles, leucine zipper
motifs, etc.
[0035] In a preferred embodiment, the presentation structure is a
coiled-coil structure, allowing the presentation of the binding
site on an exterior loop. See, for example, Myszka et al., Biochem.
33: 2362-2373 (1994), hereby incorporated by reference). Using this
system investigators have isolated peptides capable of high
affinity interaction with the appropriate target. In general,
coiled-coil structures allow for between 6 to 20 randomized
positions.
[0036] A preferred coiled-coil presentation structure is as
follows: MGCAALESEVSALESEVASLESEVAALGRGDMPLAAVKSKLSAVKSKLASVKSK
LAACGPP. The underlined regions represent a coiled-coil leucine
zipper region defined previously (see Martin et al., EMBO J.
13(22): 5303-5309 (1994), incorporated by reference). The bolded
GRGDMP region represents the loop structure and when appropriately
replaced with binding sites (generally depicted herein as (X)n,
where X is an amino acid residue and n is an integer of at least 5
or 6) can be of variable length. The replacement of the bolded
region is facilitated by encoding restriction endonuclease sites in
the underlined regions, which allows the direct incorporation of
binding site oligonucleotides at these positions. For example, a
preferred embodiment generates a XhoI site at the double underlined
LE site and a HindIII site at the double-underlined KL site.
[0037] In a preferred embodiment, the presentation structure is a
minibody structure. A "minibody" is essentially composed of a
minimal antibody complementarity region. The minibody presentation
structure generally provides two binding site regions that in the
folded protein are presented along a single face of the tertiary
structure. See for example Bianchi et al., J. Mol. Biol. 236(2):
649-59 (1994), and references cited therein, all of which are
incorporated by reference). Investigators have shown this minimal
domain is stable in solution and have used phage selection systems
in combinatorial libraries to select minibodies with peptide
regions exhibiting high affinity, K.sub.d=10.sup.-7, for the
pro-inflammatory cytokine IL-6.
[0038] A preferred minibody presentation structure is as follows:
MGRNSQATSGFTFSHFYMEWVRGGEYIAASRHKHNKYTTEYSASVKGRYIVSR
DTSQSILYLQKKKGPP. The bold, underline regions are the regions which
may be replaced by binding sites. The italized phenylalanine must
be invariant in the first region. The entire peptide is cloned in a
three-oligonucleotide variation of the coiled-coil embodiment, thus
allowing two different regions to be incorporated simultaneously.
This embodiment utilizes non-palindromic BstXI sites on the
termini.
[0039] In a preferred embodiment, the presentation structure is a
sequence that contains generally two cysteine residues, such that a
disulfide bond may be formed, resulting in a conformationally
constrained sequence. As will be appreciated by those in the art,
any number of scaffold or binding site sequences, with or without
spacer or linking sequences, may be flanked with cysteine residues.
In other embodiments, effective presentation structures may be
generated by the binding site regions themselves. For example, the
regions may be "doped" with cysteine residues which, under the
appropriate redox conditions, may result in highly crosslinked
structured conformations, similar to a presentation structure.
Similarly, the randomization regions may be controlled to contain a
certain number of residues to confer .beta.-sheet or
.alpha.-helical structures.
[0040] The conformation of the scaffold may vary widely, as will be
appreciated by those in the art. Scaffolds may be linear, branched,
or cyclic. In addition, as will be appreciated by those in the art,
each scaffold may comprise more than one molecule, i.e. be
comprised of multiple scaffold segments. Any number of molecules
may be associated to form the scaffolds of the invention, as is
generally depicted in FIGS. 1 and 2.
[0041] In a preferred embodiment, the scaffolds are linear, as is
generally depicted in FIG. 3. FIG. 3 depicts a scaffold with seven
binding sites, although scaffolds with more or less binding sites
may be used as well. The binding sites are depicted with small
letters, and the associated enzymes with capital letters. As will
be appreciated by those in the art, linear scaffolds may assume a
non-linear tertiary structure in solution, determined by the
structure of the binding sites themselves, linker sequences, the
binding of enzymes or tags, the environment (including pH,
hydration, solvent, salts, proteins, cellular compartment, etc.),
or additional elements either endogeneous or exogeneous to the
environment.
[0042] In a preferred embodiment, the scaffolds are cyclic. Cyclic
scaffolds, such as are generally depicted in FIGS. 4A, 4B, 4C and
4D, may be made as will be appreciated by those in the art. Protein
scaffolds may utilize terminal or internal cysteine residues, that
form disulfide bonds under physiological conditions, to form cyclic
protein scaffolds (FIGS. 4A and 4B). Cyclic nucleic acid scaffolds
utilize regions of complementarity to form cyclic scaffolds (FIGS.
4C and 4D). Alternatively, cyclic scaffolds may be constructed
using overlapping segments, as is described below. Cyclic scaffolds
may also be linked enzymatically, either using endogeneous or
exogeneous enzymes, or chemical cross-linking processes.
[0043] In a preferred embodiment, scaffolds comprising multiple
segments are used, as is generally depicted in FIG. 2. In this
embodiment, generally a scaffold segment will comprise at least one
binding site and at least one connection site. However, as depicted
in FIGS. 2A and 2B, some segments may comprise only connection
sites. A connection site is used to connect or associate different
scaffold segments together, in a manner similar to the association
of enzymes and binding sites. Thus, when the scaffolds are nucleic
acids, each connection site may comprise areas of sequence
complementarity to other connection sites. When the scaffolds are
proteins, each connection site may be a sequence that will bind to
one or more other protein sequences.
[0044] The connection sites may be all the same, such that
aggregation of all the connection sites on all segments occurs, for
example as is shown in FIG. 2E, or may be different, for example as
is shown in FIG. 2F. As will be appreciated by those in the art, a
wide variety of different scaffolds comprising multiple segments of
binding sites and connection sites are possible.
[0045] When the novel compositions are introduced into cells as is
outlined below, the scaffolds are preferably exogeneous scaffolds.
By "exogeneous scaffold" herein is meant that the scaffold either
a) does not naturally occur within the cell, or b) does naturally
occur within the cell but is present at a either a significantly
higher concentration than is normally seen within the cell or in a
form not normally seen in the cell; e.g. is a portion of a
naturally occurring protein or nucleic acid sequence. In a
preferred embodiment, the exogeneous scaffolds are synthetic; i.e.
they do not naturally occur in nature. In some embodiments, it may
be possible to alter endogeneous scaffolds such as actin chemically
to produce novel scaffolds.
[0046] Each binding site of the scaffold binds an enzyme to form an
"enzyme complex" or "enzyme-scaffold complex". The binding or
association of the enzymes to the scaffolds is preferably
non-covalent, yet will be strong enough to cause the binding of the
enzymes to the scaffold under physiological conditions, i.e. inside
cells or subcellular compartments. That is, the affinity of the
binding sites and the enzymes will be strong enough to cause
self-aggregation or induced aggregation. Preferably, the
association is strong enough to allow purification of the whole
scaffold-enzyme complex as a unit, for example by purifying one of
the components, immunoprecipitating one or more of the enzymes.
[0047] As will be appreciated by those in the art, any number of
different enzymes will be used. The enzymes may be from any
organisms, including prokaryotes and eukaryotes, with enzymes from
bacteria, fungi, extremeophiles, animals (particularly mammals and
particularly human) and birds all possible. Suitable classes of
enzymes include, but are not limited to, hydrolases such as
proteases, carbohydrases, lipases; isomerases such as racemases,
epimerases, tautomerases, or mutases; transferases, kinases and
phophatases. Preferred enzymes include those that carry out group
transfers, such as acyl group transfers, including endo- and
exopeptidases (serine, cysteine, metallo and acid proteases); amino
group and glutamyl transfers, including glutaminases, .gamma.
glutamyl transpeptidases, amidotransferases, etc.; phosphoryl group
transfers, including phosphotases, phosphodiesterases, kinases, and
phosphorylases; nucleotidyl and pyrophosphotyl transfers, including
carboxylate, pyrophosphoryl transfers, etc.; glycosyl group
transfers; enzymes that do enzymatic oxidation and reduction, such
as dehydrogenases, monooxygenases, oxidases, hydroxylases,
reductases, etc.; enzymes that catalyze eliminations,
isomerizations and rearrangements, such as elimination/addition of
water using aconitase, fumarase, enolase, crotonase,
carbon-nitrogen lyases, etc.; and enzymes that make or break
carbon-carbon bonds, i.e. carbanion reactions. Suitable enzymes are
listed in the Swiss-Prot enzyme database.
[0048] The enzymes may be naturally occuring or variant forms of
the enzymes. As will be appreciated by those skilled in the art,
the potential list of suitable enzyme targets is quite large, and
is only limited by the ability to obtain all or part of the nucleic
acid or protein sequences, preferably the nucleic acids encoding
the enzymes.
[0049] In a preferred embodiment, the enzymes are exogeneous
(heterologous) to the host cells used. That is, the enzymes are not
normally expressed within the cell type, although as will be
appreciated by those in the art, an endogeneous copy of the nucleic
acid encoding the enzyme may be within the genome of the cell.
Generally, in a preferred embodiment, neither the nucleic acid
encoding the enzyme, or the enzyme itself, is endogeneous to the
cell.
[0050] In one embodiment, the system is chosen such that no
exogeneous scaffold is required. In one embodiment, the enzymes are
all associated through the use of binding sequences (either
endogeneous or exogeneous to the enzyme) as is shown in FIG.
1B.
[0051] Alternatively, an endogeneous structure serves as the
scaffold. Thus, for example, in the case where membrane anchoring
sequences such as all or part of a transmembrane domain, are used,
such that the enzymes are associated with a membrane, an exogeneous
scaffold may not be needed. There may be sufficient concentration
and/or association of the enzymes within the two dimensional
surface of a membrane that no additional scaffold is needed. This
may be useful due to the relatively large size, and therefore low
diffusion coefficients, of enzymes within either two or three
dimensional space. Similarly, this concentration effect may be
increased when targeting occurs to subcellular organelles, as
described below. As will be appreciated by those in the art,
systems may be generated with the enzyme active sites on the
outside (extracellular) of the cell, or on the inside
(intracellular), or, in the case of bacteria such as E. coli,
within the periplasmic space. Alternatively, the system may be
designed to have the enzymes concentrate (again, on either side of
the membrane) in a subcellular organelle membrane such as the ER,
Golgi, mitochondria, lysosome, chloroplast, etc., or in general
endocytotic vesicles.
[0052] In this embodiment, when exogeneous scaffolds are not used,
at least about two exogeneous enzymes are used, with at least about
3 being preferred, and at least about 4-10 being particularly
preferred. In this embodiment, it is preferred, but not required,
that at least one of the enzymes has a targeting sequence,
preferably a membrane anchoring sequence. If only a subset of the
enzymes have a membrane anchoring sequence, the rest of the enzymes
will have at least one exogeneous binding sequence.
[0053] In one embodiment, the binding sequence is on the same side
of the membrane as the enzyme's active site, as is generally
depicted in FIG. 5A. Alternatively, the binding sequence is on the
other side of the membrane from the enzyme's active site, as is
generally depicted in FIG. 5B.
[0054] The scaffolds and enzymes are the expression products of
nucleic acids. That is, scaffold nucleic acids encode scaffolds,
and enzyme nucleic acids encode enzymes. When the scaffold is a
nucleic acid, the scaffold is a transcription product of the
nucleic acid. When the scaffold is a protein, the scaffold is a
translation product of the nucleic acid.
[0055] Thus, the present invention provides scaffolds and enzymes,
and nucleic acids encoding them. As is more generally described
below, a nucleic acid of the invention may encode a single enzyme
or a single scaffold, or combinations of enzymes and/or scaffolds.
Thus, nucleic acids encoding two or more enzymes, an enzyme and a
scaffold, etc., can be made. Thus, the invention provides libraries
of scaffolds and libraries of enzymes. In general, as is more fully
described below, the limit on the number of components on a single
nucleic acid will be determined by the size of the nucleic acid
which may be conveniently introduced into a cell. Thus, for
example, when retroviral or adenoviral vectors are used, there may
be limits on the size of the nucleic acids which may be packaged
into viral particles.
[0056] In addition to the coding sequences for the scaffolds and
enzymes, the nucleic acids of the invention may include fusion
partners. By "fusion partner" herein is meant a sequence that is
associated either with the nucleic acid or the expression product
that confers a common function or ability. Fusion partners can be
heterologous (i.e. not native to the host cell), or synthetic (not
native to any cell). Suitable fusion partners include, but are not
limited to: 1) targeting sequences, defined below, which allow the
localization of the scaffolds and enzymes into a subcellular or
extracellular compartment; 2) rescue sequences, as defined below,
which allow the purification or isolation of either the scaffolds
and enzymes or the nucleic acids encoding them; 3) stability
sequences, which confer stability or protection from degradation to
the scaffolds and enzymes or the nucleic acids encoding them, for
example resistance to proteolytic degradation; or 4) combinations
of any of 1), 2) and 3).
[0057] In a preferred embodiment, the fusion partner is a targeting
sequence. As will be appreciated by those in the art, the
localization of proteins within a cell is a simple method for
increasing effective concentration and determining function. For
example, RAF1 when localized to the mitochondrial membrane can
inhibit the anti-apoptotic effect of BCL-2. Similarly, membrane
bound Sos induces Ras mediated signaling in T-lymphocytes. These
mechanisms are thought to rely on the principle of limiting the
search space for ligands, that is to say, the localization of a
protein to the plasma membrane limits the search for its ligand to
that limited dimensional space near the membrane as opposed to the
three dimensional space of the cytoplasm. Alternatively, the
concentration of a protein can also be simply increased by nature
of the localization. Shuttling the proteins into the nucleus
confines them to a smaller space thereby increasing concentration.
Finally, the ligand or target may simply be localized to a specific
compartment, and effectors must be localized appropriately.
[0058] Thus, suitable targeting sequences include, but are not
limited to, binding sequences capable of causing binding of the
expression product to a predetermined molecule or class of
molecules while retaining bioactivity of the expression product,
(for example by using enzyme inhibitor or substrate sequences to
target a class of relevant enzymes); sequences signalling selective
degradation, of itself or co-bound proteins; and signal sequences
capable of constitutively localizing the candidate expression
products to a predetermined cellular locale, including a)
subcellular locations such as the Golgi, endoplasmic reticulum,
nucleus, nucleoli, nuclear membrane, mitochondria, chloroplast,
secretory vesicles, lysosome, and cellular membrane; and b)
extracellular locations via either membrane anchoring sequences or
secretory signal sequences.
[0059] In a preferred embodiment, the targeting sequence is a
nuclear localization signal (NLS). NLSs are generally short,
positively charged (basic) domains that serve to direct the entire
protein in which they occur to the cell's nucleus. Numerous NLS
amino acid sequences have been reported including single basic
NLS's such as that of the SV40 (monkey virus) large T Antigen (Pro
Lys Lys Lys Arg Lys Val), Kalderon (1984), et al., Cell, 39:
499-509; the human retinoic acid receptor-.beta. nuclear
localization signal (ARRRRP); NF.kappa.B p50 (EEVQRKRQKL; Ghosh et
al., Cell 62: 1019 (1990); NF.kappa.B p65 (EEKRKRTYE; Nolan et al.,
Cell 64: 961 (1991); and others (see for example Boulikas, J. Cell.
Biochem. 55(1): 32-58 (1994), hereby incorporated by reference) and
double basic NLS's exemplified by that of the Xenopus (African
clawed toad) protein, nucleoplasmin (Ala Val Lys Arg Pro Ala Ala
Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys Leu Asp), Dingwall, et
al., Cell, 30: 449-458, 1982 and Dingwall, et al., J. Cell Biol.,
107: 641-849; 1988). Numerous localization studies have
demonstrated that NLSs incorporated in synthetic peptides or
grafted onto reporter proteins not normally targeted to the cell
nucleus cause these peptides and reporter proteins to be
concentrated in the nucleus. See, for example, Dingwall, and
Laskey, Ann, Rev. Cell Biol., 2: 367-390, 1986; Bonnerot, et al.,
Proc. Natl. Acad. Sci. USA, 84: 6795-6799, 1987; Galileo, et al.,
Proc. Natl. Acad. Sci. USA, 87: 458-462, 1990.
[0060] In a preferred embodiment, the targeting sequence is a
membrane anchoring signal sequence. This is particularly useful
since many parasites and pathogens bind to the membrane, in
addition to the fact that many intracellular events originate at
the plasma membrane. Thus, membrane-bound enzyme-scaffold complexes
are useful for both the identification of important elements in
these processes as well as for the discovery of effective
inhibitors. The invention provides methods for presenting the
enzyme complexes of the invention extracellularly or in the
cytoplasmic space. For extracellular presentation, a membrane
anchoring region is provided at the carboxyl terminus of the
expression product. The expression product (i.e. the enzyme,
scaffold, or the enzyme complex) is expressed on the cell surface
and presented to the extracellular space, such that it can bind to
other surface molecules (affecting their function) or molecules
present in the extracellular medium. Similarly, the expression
product could be contained within a cytoplasmic region, and the
transmembrane region and extracellular region remain constant or
have a defined function.
[0061] Membrane-anchoring sequences are well known in the art and
are based on the genetic geometry of mammalian transmembrane
molecules. Peptides are inserted into the membrane based on a
signal sequence (designated herein as ssTM) and require a
hydrophobic transmembrane domain (herein TM). The transmembrane
proteins are inserted into the membrane such that the regions
encoded 5' of the transmembrane domain are extracellular and the
sequences 3' become intracellular. Of course, if these
transmembrane domains are placed 5' of the variable region, they
will serve to anchor it as an intracellular domain, which may be
desirable in some embodiments. ssTMs and TMs are known for a wide
variety of membrane bound proteins, and these sequences may be used
accordingly, either as pairs from a particular protein or with each
component being taken from a different protein, or alternatively,
the sequences may be synthetic, and derived entirely from consensus
as artificial delivery domains.
[0062] As will be appreciated by those in the art,
membrane-anchoring sequences, including both ssTM and TM, are known
for a wide variety of proteins and any of these may be used.
Particularly preferred membrane-anchoring sequences include, but
are not limited to, those derived from CD8, ICAM-2, IL-8R, CD4 and
LFA-1.
[0063] Useful sequences include sequences from: 1) class I integral
membrane proteins such as IL-2 receptor beta-chain (residues 1-26
are the signal sequence, 241-265 are the transmembrane residues;
see Hatakeyama et al., Science 244: 551 (1989) and von Heijne et
al, Eur. J. Biochem. 174: 671 (1988)) and insulin receptor beta
chain (residues 1-27 are the signal, 957-959 are the transmembrane
domain and 960-1382 are the cytoplasmic domain; see Hatakeyama,
supra, and Ebina et al., Cell 40: 747 (1985)); 2) class II integral
membrane proteins such as neutral endopeptidase (residues 29-51 are
the transmembrane domain, 2-28 are the cytoplasmic domain; see
Malfroy et al., Biochem. Biophys. Res. Commun. 144: 59 (1987)); 3)
type III proteins such as human cytochrome P450 NF25 (Hatakeyama,
supra); and 4) type IV proteins such as human P-glycoprotein
(Hatakeyama, supra). Particularly preferred are CD8 and ICAM-2. For
example, the signal sequences from CD8 and ICAM-2 lie at the
extreme 5' end of the transcript. These consist of the amino acids
1-32 in the case of CD8 (MASPLTRFLSLNLLLLGESILGSGEAKPQAP; Nakauchi
et al., PNAS USA 82: 5126 (1985) and 1-21 in the case of ICAM-2
(MSSFGYRTLTVALFTLICCPG; Staunton et al., Nature (London) 339: 61
(1989)). These leader sequences deliver the construct to the
membrane while the hydrophobic transmembrane domains, placed 3' of
the random candidate region, serve to anchor the construct in the
membrane. These transmembrane domains are encompassed by amino
acids 145-195 from CD8
(PQRPEDCRPRGSVKGTGLDFACDIYIWAPLAGICVALLLSLII- TLICYHSR; Nakauchi,
supra) and 224-256 from ICAM-2 (MVIIVTVVSVLLSLFVTSVLLC-
FIFGQHLRQQR; Staunton, supra).
[0064] Alternatively, membrane anchoring sequences include the GPI
anchor, which results in a covalent bond between the molecule and
the lipid bilayer via a glycosyl-phosphatidylinositol bond for
example in DAF (PNKGSGTTSGTTR LLSGHTCFTLTGLLGTLVTMGLLT, with the
bolded serine the site of the anchor; see Homans et al., Nature
333(6170): 269-72 (1988), and Moran et al., J. Biol. Chem. 266:
1250 (1991)). In order to do this, the GPI sequence from Thy-1 can
be cassetted 3' of the variable region in place of a transmembrane
sequence.
[0065] Similarly, myristylation sequences can serve as membrane
anchoring sequences. It is known that the myristylation of c-src
recruits it to the plasma membrane. This is a simple and effective
method of membrane localization, given that the first 14 amino
acids of the protein are solely responsible for this function:
MGSSKSKPKDPSQR (see Cross et al., Mol. Cell. Biol. 4(9): 1834
(1984); Spencer et al., Science 262: 1019-1024 (1993), both of
which are hereby incorporated by reference). This motif has already
been shown to be effective in the localization of reporter genes
and can be used to anchor the zeta chain of the TCR. This motif is
placed 5' of the coding region in order to localize the construct
to the plasma membrane. Other modifications such as palmitoylation
can be used to anchor constructs in the plasma membrane; for
example, palmitoylation sequences from the G protein-coupled
receptor kinase GRK6 sequence (LLQRLFSRQDCCGNCSDSEEELPTRL, with the
bold cysteines being palmitolyated; Stoffel et al., J. Biol. Chem
269: 27791 (1994)); from rhodopsin (KQFRNCMLTSLCCGKNPLGD;
Barnstable et al., J. Mol. Neurosci. 5(3): 207 (1994)); and the
p21H-ras 1 protein (LNPPDESGPGCMSCKCVLS; Capon et al., Nature 302:
33 (1983)).
[0066] In a preferred embodiment, the targeting sequence is a
lysozomal targeting sequence, including, for example, a lysosomal
degradation sequence such as Lamp-2 (KFERQ; Dice, Ann. N.Y. Acad.
Sci. 674: 58 (1992); or lysosomal membrane sequences from Lamp-1
(MLIPIAGFFALAGLVLIVLIAYLIGRKRSHAGYQTI, Uthayakumar et al., Cell.
Mol. Biol. Res. 41: 405 (1995)) or Lamp-2
(LVPIAVGAALAGVLILVLLAYFIGLKHHHAGYEQF- , Konecki et la., Biochem.
Biophys. Res. Comm. 205: 1-5 (1994), both of which show the
transmembrane domains in italics and the cytoplasmic targeting
signal underlined).
[0067] Alternatively, the targeting sequence may be a
mitrochondrial localization sequence, including mitochondrial
matrix sequences (e.g. yeast alcohol dehydrogenase III;
MLRTSSLFTRRVQPSLFSRNILRLQST; Schatz, Eur. J. Biochem. 165: 1-6
(1987)); mitochondrial inner membrane sequences (yeast cytochrome c
oxidase subunit IV; MLSLRQSIRFFKPATRTLCSSRYLL; Schatz, supra);
mitochondrial intermembrane space sequences (yeast cytochrome c1;
MFSMLSKRWAQRTLSKSFYSTATGAASKSGKLTQKLVTAGVAAAGITASTLLYA DSLTAEAMTA;
Schatz, supra) or mitochondrial outer membrane sequences (yeast 70
kD outer membrane protein; MKSFITRNKTAILATVAATGTAIGAYYYYNQLQQQQ-
QRGKK; Schatz, supra).
[0068] The target sequences may also be endoplasmic reticulum
sequences, including the sequences from calreticulin (KDEL; Pelham,
Royal Society London Transactions B; 1-10 (1992)) or adenovirus
E3/19K protein (LYLSRRSFIDEKKMP; Jackson et al., EMBO J. 9: 3153
(1990).
[0069] Furthermore, targeting sequences also include peroxisome
sequences (for example, the peroxisome matrix sequence from
Luciferase; SKL; Keller et al., PNAS USA 4: 3264 (1987));
farnesylation sequences (for example, P21H-ras 1;
LNPPDESGPGCMSCKCVLS, with the bold cysteine farnesylated; Capon,
supra); geranylgeranylation sequences (for example, protein rab-5A;
LTEPTQPTRNQCCSN, with the bold cysteines geranylgeranylated;
Farnsworth, PNAS USA 91: 11963 (1994)); or destruction sequences
(cyclin B1; RTALGDIGN; Klotzbucher et al., EMBO J. 1: 3053
(1996)).
[0070] In a preferred embodiment, the targeting sequence is a
secretory signal sequence capable of effecting the secretion of the
translation products. There are a large number of known secretory
signal sequences which are placed 5' to the coding region of the
enzyme or scaffold, and are cleaved from the coding region to
effect secretion into the extracellular space. Secretory signal
sequences and their transferability to unrelated proteins are well
known, e.g., Silhavy, et al. (1985) Microbiol. Rev. 49,
398-418.
[0071] Suitable secretory sequences are known, including signals
from IL-2 (MYRMQLLSCIALSLALVTNS; Villinger et al., J. Immunol. 155:
3946 (1995)), growth hormone (MATGSRTSLLLAFGLLCLPWLQEGSAFPT; Roskam
et al., Nucleic Acids Res. 7: 30 (1979)); preproinsulin
(MALWMRLLPLLALLALWGPDPAAAFVN; Bell et al., Nature 284: 26 (1980));
and influenza HA protein (MKAKLLVLLYAFVAGDQI; Sekiwawa et al., PNAS
80: 3563)), with cleavage between the non-underlined-underlined
junction. A particularly preferred secretory signal sequence is the
signal leader sequence from the secreted cytokine IL-4, which
comprises the first 24 amino acids of IL-4 as follows:
MGLTSQLLPPLFFLLACAGNFVHG.
[0072] In a preferred embodiment, the fusion partner is a rescue
sequence. A rescue sequence is a sequence which may be used to
purify or isolate either the scaffolds, enzymes, or enzyme complex,
or the nucleic acids encoding them. Thus, for example, peptide
rescue sequences include purification sequences such as the
His.sub.6 tag for use with Ni affinity columns and epitope tags for
detection, immunoprecipitation or FACS (fluoroscence-activated cell
sorting). Suitable epitope tags include myc (for use with the
commercially available 9E10 antibody), the BSP biotinylation target
sequence of the bacterial enzyme BirA, flu tags, lacZ, and GST.
[0073] Alternatively, the rescue sequence may be a unique
oligonucleotide sequence which serves as a probe target site to
allow the quick and easy isolation of the retroviral construct, via
PCR, related techniques, or hybridization.
[0074] In a preferred embodiment, the fusion partner is a stability
sequence to confer stability to the expression products or the
nucleic acids encoding them. Thus, for example, peptide scaffolds
or enzymes may be stabilized by the incorporation of glycines after
the initiation methionine (MG or MGG), for protection of the
peptide to ubiquitination as per Varshavsky's N-End Rule, thus
conferring long half-life in the cytoplasm. Similarly, two prolines
at the C-terminus impart peptides that are largely resistant to
carboxypeptidase action. The presence of two glycines prior to the
prolines impart both flexibility and prevent structure initiating
events in the di-proline to be propagated into the candidate
peptide structure. Thus, preferred stability sequences are as
follows: MG-protein-GGPP.
[0075] The fusion partners may be placed anywhere (i.e. N-terminal,
C-terminal, internal) in the structure as the biology and activity
permits.
[0076] In a preferred embodiment, the fusion partner includes a
linker or tethering sequence. Linker sequences between various
targeting sequences (for example, membrane targeting sequences) and
the other components of the constructs (such as the coding regions
for the scaffolds and enzymes) may be desirable to allow the
proteins to interact with potential targets unhindered. For
example, useful linkers include glycine-serine polymers (including,
for example, (GS)n, (GSGGS)n and (GGGS)n, where n is an integer of
at least one), glycine-alanine polymers, alanine-serine polymers,
and other flexible linkers such as the tether for the shaker
potassium channel, and a large variety of other flexible linkers,
as will be appreciated by those in the art. Glycine-serine polymers
are preferred since both of these amino acids are relatively
unstructured, and therefore may be able to serve as a neutral
tether between components. Secondly, serine is hydrophilic and
therefore able to solubilize what could be a globular glycine
chain. Third, similar chains have been shown to be effective in
joining subunits of recombinant proteins such as single chain
antibodies. In addition, semi-flexible linkers, rather than fully
flexible linkers, may also be used. For example, a series of
helices, connected by joints, may be used. This may be used to
lower the entropy of the system and provide some conformational
stability as well. In addition, the linkers may include extender
sequences; the linker need not be fully flexible from the point of
contact.
[0077] In a preferred embodiment, combinations of fusion partners
are used. Thus, for example, any number of combinations may be
used, with or without linker sequences. As is described herein,
using a base vector that contains a cloning site for receiving the
enzyme and/or scaffold coding regions, one can cassette in various
fusion partners 5' and 3' of the coding region.
[0078] In addition to the coding regions of enzymes, scaffolds, and
fusion partners, the nucleic acids of the invention may also
contain enough extra sequence to effect translation or
transcription, as necessary. Thus, for enzymes or protein
scaffolds, the nucleic acids generally contain cloning sites which
are placed to allow in frame expression of the expression products
and fusion partners. When the scaffolds are nucleic acid scaffolds,
the nucleic acids encoding the scaffolds will generally be RNA for
retroviral delivery, and are generally constructed with an internal
CMV promoter, tRNA promoter or cell specific promoter designed for
immediate and appropriate expression of the RNA structure at the
initiation site of RNA synthesis. The RNA can be expressed
anti-sense to the direction of retroviral synthesis and is
terminated as known, for example with an orientation specific
terminator sequence. Interference from upstream transcription is
alleviated in the target cell with the self-inactivation deletion,
a common feature of certain retroviral expression systems. Other
orientations are possible in some vector systems.
[0079] Generally, the nucleic acids of the invention are expressed
within the cells to produce expression products of the nucleic
acids. As outlined above, the expression products include
translation products (i.e. enzymes and protein scaffolds) and
transcription products (nucleic acid scaffolds).
[0080] The nucleic acids encoding the scaffolds and enzymes are
introduced into cells in a variety of ways. By "introduced into" or
grammatical equivalents herein is meant that the nucleic acids
enter the cells in a manner suitable for subsequent expression of
the nucleic acid. The method of introduction is largely dictated by
the targeted cell type, discussed below. Exemplary methods include
CaPO.sub.4 precipitation, liposome fusion, lipofectin.RTM.,
electroporation, viral infection, etc. The candidate nucleic acids
may stably integrate into the genome of the host cell (for example,
with retroviral introduction, outlined below), or may exist either
transiently or stably in the cytoplasm (i.e. through the use of
traditional plasmids, utilizing standard regulatory sequences,
selection markers, etc.). As many pharmaceutically important
screens require human or model mammalian cell targets, retroviral
vectors capable of transfecting such targets are preferred.
[0081] In a preferred embodiment, the nucleic acids encoding the
scaffolds and enzymes are part of retroviral particles which infect
the cells. As outlined above, each retroviral particle may contain
a single construct, i.e. one enzyme or one scaffold, or more than
one, depending on the size of the vector. For example, retroviruses
allow generally 7-8 kb, adenoviruses allow up to 30 kb, and herpes
viruses can allow up to 100 kb. The constructs may also be set up
as "operon" type expression vectors, for example, when co-selection
of markers or tags are desirable. Infection can be optimized such
that each cell generally expresses a single construct, two
constructs, etc., depending on what is required, using the ratio of
virus particles to number of cells. Infection is carried out such
that preferably each cell gets nucleic acid encoding at least one
scaffold and at least some, preferably all, of the enzymes binding
to the binding sites of that scaffold. Infection generally follows
a Poisson distribution. Generally, infection of the cells is
straightforward with the application of the infection-enhancing
reagent polybrene, which is a polycation that facilitates viral
binding to the target cell.
[0082] In a preferred embodiment, the nucleic acids encoding the
scaffolds and enzymes are introduced into the cells using
retroviral vectors. Currently, the most efficient gene transfer
methodologies harness the capacity of engineered viruses, such as
retroviruses, to bypass natural cellular barriers to exogenous
nucleic acid uptake. The use of recombinant retroviruses was
pioneered by Richard Mulligan and David Baltimore with the Psi-2
lines and analogous retrovirus packaging systems, based on NIH 3T3
cells (see Mann et al., Cell 33: 153-159 (1993), hereby
incorporated by reference). Such helper-defective packaging lines
are capable of producing all the necessary trans proteins -gag,
pol, and env- that are required for packaging, processing, reverse
transcription, and integration of recombinant genomes. Those RNA
molecules that have in cis the .psi. packaging signal are packaged
into maturing virions. Retroviruses are preferred for a number of
reasons. First, their derivation is easy. Second, unlike
Adenovirus-mediated gene delivery, expression from retroviruses is
long-term (adenoviruses do not integrate). Adeno-associated viruses
have limited space for genes and regulatory units and there is some
controversy as to their ability to integrate. Retroviruses
therefore offer the best current compromise in terms of long-term
expression, genomic flexibility, and stable integration, among
other features. The main advantage of retroviruses is that their
integration into the host genome allows for their stable
transmission through cell division. This ensures that in cell types
which undergo multiple independent maturation steps, such as
hematopoietic cell progression, the retrovirus construct will
remain resident and continue to express.
[0083] A particularly well suited retroviral transfection system is
described in Mann et al., supra: Pear et al., PNAS USA 90(18):
8392-6 (1993); Kitamura et al., PNAS USA 92: 9146-9150 (1995);
Kinsella et al., Human Gene Therapy 7: 1405-1413; Hofmann et al.,
PNAS USA 93: 5185-5190; Choate et al., Human Gene Therapy 7: 2247
(1996); and WO 94/19478; and references cited therein, all of which
are incorporated by reference.
[0084] In one embodiment of the invention, the libraries of
scaffolds and enzymes are generated in a retrovirus DNA construct
backbone, as is generally described herein. Standard
oligonucleotide synthesis is done to generate the nucleic acids
encoding the scaffolds, using techniques well known in the art (see
Eckstein, Oligonucleotides and Analogues, A Practical Approach, IRL
Press at Oxford University Press, 1991. Nucleic acids encoding the
enzymes are made as is known in the art. Other viruses may also be
used, such as Semliki Forest Virus.
[0085] Thus, nucleic acid libraries of enzymes and libraries of
scaffolds are made. After generation of the DNA library, the
library is cloned into a first primer. The first primer serves as a
"cassette", which is inserted into the retroviral construct. The
first primer generally contains a number of elements, including for
example, the required regulatory sequences (e.g. translation,
transcription, promoters, etc), fusion partners, restriction
endonuclease (cloning and subcloning) sites, stop codons
(preferably in all three frames), regions of complementarity for
second strand priming (preferably at the end of the stop codon
region as minor deletions or insertions may occur), etc.
[0086] A second primer is then added, which generally consists of
some or all of the complementarity region to prime the first primer
and optional necessary sequences for a second unique restriction
site for subcloning. DNA polymerase is added to make
double-stranded oligonucleotides. The double-stranded
oligonucleotides are cleaved with the appropriate subcloning
restriction endonucleases and subcloned into the target retroviral
vectors, described below.
[0087] Any number of suitable retroviral vectors may be used.
Generally, the retroviral vectors may include: selectable marker
genes under the control of internal ribosome entry sites (IRES),
which allows for bicistronic operons and thus greatly facilitates
the selection of cells expressing peptides at uniformly high
levels; and promoters driving expression of a second gene, placed
in sense or anti-sense relative to the 5' LTR. Suitable selection
genes include, but are not limited to, neomycin, blastocidin,
bleomycin, puromycin, and hygromycin resistance genes, as well as
self-fluorescent markers such as green fluoroscent protein,
enzymatic markers such as lacZ, and surface proteins such as CD8,
etc.
[0088] Preferred vectors include a vector based on the murine stem
cell virus (MSCV) (see Hawley et al., Gene Therapy 1: 136 (1994))
and a modified MFG virus (Rivere et al., Genetics 92: 6733 (1995)),
and pBABE. A general schematic of a retroviral construct is
depicted in FIG. 6.
[0089] The retroviruses may include inducible and constitutive
promoters. For example, there are situations wherein it is
necessary to induce expression only during certain phases of the
selection process. For instance, a scheme to provide
pro-inflammatory cytokines in certain instances must include
induced expression of the peptides. This is because there is some
expectation that over-expressed pro-inflammatory drugs might in the
long-term be detrimental to cell growth. Accordingly, constitutive
expression is undesirable, and expression is only turned on during
that phase of the selection process when the phenotype is required,
and then turn off the retroviral expression to confirm the effect
or ensure long-term survival of the producer cells. A large number
of both inducible and constitutive promoters are known.
[0090] In addition, it is possible to configure a retroviral vector
to allow inducible expression of retroviral inserts after
integration of the vector in target cells; importantly, the entire
system is contained within the retrovirus. Tet-inducible
retroviruses have been designed incorporating the Self-Inactivating
(SIN) feature of 3' LTR enhancer/promoter retroviral deletion
mutant (Hoffman et al., PNAS USA 93: 5185 (1996)). Expression of
this vector in cells is virtually undetectable in the presence of
tetracycline or other active analogs. However, in the absence of
Tet, expression is turned on to maximum within 48 hours after
induction, with uniform increased expression of the whole
population of cells that harbor the inducible retrovirus,
indicating that expression is regulated uniformly within the
infected cell population. A similar, related system uses a mutated
Tet DNA-binding domain such that it bound DNA in the presence of
Tet, and was removed in the absence of Tet. Either of these systems
is suitable, and may be used when multiple retroviruses each
containing components of the enzyme complex are introduced into a
single cell.
[0091] In this manner the primers create a library of system
components, either of different enzymes or of different scaffolds.
The ligation products are then transformed into bacteria, such as
E. coli, and DNA is prepared from the resulting library, as is
generally outlined in Kitamura, PNAS USA 92: 9146-9150 (1995),
hereby expressly incorporated by reference.
[0092] Delivery of the library DNA into a retroviral packaging
system results in conversion to infectious virus. Suitable
retroviral packaging system cell lines include, but are not limited
to, the Bing and BOSC23 cell lines described in WO 94/19478;
Soneoka et al., Nucleic Acid Res. 23(4): 628 (1995); Finer et al.,
Blood 83: 43 (1994); Pheonix packaging lines such as PhiNX-eco and
PhiNX-ampho, described below; 292T+gag-pol and retrovirus envelope;
PA317; and cell lines outlined in Markowitz et al., Virology 167:
400 (1988), Markowitz et al., J. Virol. 62: 1120 (1988), Li et al.,
PNAS USA 93: 11658 (1996), Kinsella et al., Human Gene Therapy 7:
1405 (1996), all of which are incorporated by reference.
[0093] Preferred systems include PhiNX-eco and PhiNX-ampho or
similar cell lines, which are two cells lines as follows. The cell
lines are based in principle on the BING and BOSC23 cell lines
described in WO 94/19478, which are based on the 293T cell line (a
human embryonic kidney line transformed with adenovirus E1a and
carrying a temperature sensitive T antigen co-selected with
neomycin). The unique feature of this cell line is that it is
highly transfectable with either calcium phosphate mediated
transfection or lipid-based transfection protocols--greater than
50% of 293T cells can be transiently transfected with plasmid DNA.
Thus, the cell line could be a cellular milieu in which retroviral
structural proteins and genomic viral RNA could brought together
rapidly for creation of helper-defective virus. 293T cells were
therefore engineered with stably integrated defective constructs
capable of producing gag-pol, and envelope protein for either
ecotropic or amphotropic viruses. These lines were called BOSC23
and Bing, respectively. The utility of these lines was that one
could produce small amounts of recombinant virus transiently for
use in small-scale experimentation. The lines offered advantages
over previous stable systems in that virus could be produced in
days rather than months.
[0094] Two problems became apparent with these first generation
lines over the two years they have been in wide use. First, gag-pol
and envelope expression was unstable and the lines required
vigilant checking for retroviral production capacity; second the
structure of the vectors used for protein production were not
considered fully "safe" for helper virus production; and third, one
of the lines was shown to be inadvertently carrying a
hygromycin-containing retrovirus. Although the BING and BOSC23
lines are useful in the present invention, all of these potentially
problematic issues are addressed in the PhiNX second-generation
lines. These lines are based on 293T cells as well, with the
following improvements. First, the ability to monitor gag-pol
production on a cell-by cell basis was made by introducing an
IRES-CD8 surface marker expression cassette downstream of the
reading frame of the gag-pol construct (other surface markers
besides CD8 are also useful). IRES (internal ribosome entry site)
sequences allow secondary or tertiary protein translation from a
single mRNA transcript. Thus, CD8 expression is a direct reflection
of intracellular gag-pol and the stability of the producer cell
population's ability to produce gag-pol can be readily monitored by
flow cytometry. Second, for both the gag-pol and envelope
constructs non-Moloney promoters were used to minimize
recombination potential with introduced retroviral constructs, and
different promoters for gag-pol and envelope were used to minimize
their inter-recombination potential. The promoters used were CMV
and RSV. Two cell lines were created, PhiNX-eco (PHOENIX-ECO) and
PhiNX-ampho (PHOENIX-AMPHO). Gag-pol was introduced with hygromycin
as the co-selectable marker and the envelope proteins were
introduced with diptheria resistance as the co-selectable marker.
Finally, the cells were screened to find a relatively rare cell
type that produced gag-pol and env in a uniform distribution,
although this is not required. In addition, a line termed PhiNX-gp
has been produced that expresses only gag-pol. This line is
available for further pseudotyping of retroviral virions with other
envelope proteins such as gibbon ape leukemia virus envelope or
Vesicular Stomatitus VSV-G protein, Xenotropic, or retargeting
envelopes can also be added.
[0095] Both PniNX-eco and PhiNX-ampho were tested for helper virus
production and established as being helper-virus free. Both lines
can carry episomes for the creation of stable cell lines which can
be used to produce retrovirus. Both lines are readily testable by
flow cytometry for stability of gag-pol (CD8) and envelope
expression; after several months of testing the lines appear
stable, and do not demonstrate loss of titre as did the
first-generation lines BOSC23 and Bing (partly due to the choice of
promoters driving expression of gag-pol and envelope). Both lines
can also be used to transiently produce virus in a few days. Thus,
these new lines are fully compatible with transient, episomal
stable, and library generation for retroviral gene transfer
experiments. Finally, the titres produced by these lines have been
tested. Using standard polybrene-enhanced retroviral infection,
titres approaching or above 107 per ml were observed for both
PhiNX-eco and PhiNX-ampho when carrying episomal constructs. When
transiently produced virus is made, titres are usually 1/2 to 1/3
that value.
[0096] These lines are helper-virus free, carry episomes for
long-term stable production of retrovirus, stably produce gag-pol
and env, and do not demonstrate loss of viral titre over time. In
additon, PhiNX-eco and PhiNX-ampho are capable of producing titres
approaching or above 10.sup.7 per ml when carrying episomal
constructs, which, with concentration of virus, can be enhanced to
10.sup.8 to 10.sup.9 per ml.
[0097] In a preferred embodiment, the cell lines disclosed above,
and the other methods for producing retrovirus, are useful for
production of virus by transient transfection. The virus can either
be used directly or be used to infect another retroviral producer
cell line for "expansion" of the library.
[0098] Concentration of virus may be done as follows. Generally,
retroviruses are titred by applying retrovirus-containing
supernatant onto indicator cells, such as NIH3T3 cells, and then
measuring the percentage of cells expressing phenotypic
consequences of infection. The concentration of the virus is
determined by multipying the percentage of cells infected by the
dilution factor involved, and taking into account the number of
target cells available to obtain a relative titre. If the
retrovirus contains a reporter gene, such as lacZ, then infection,
integration, and expression of the recombinant virus is measured by
histological staining for lacZ exprssion or by flow cytometry
(FACS). In general, retroviral titres generated from even the best
of the producer cells do not exceed 10.sup.7 per ml, unless
concentration by relatively expensive or exotic apparatus. However,
as it has been recently postulated that since a particle as large
as a retrovirus will not move very far by brownian motion in
liquid, fluid dynamics predicts that much of the virus never comes
in contact with the cells to initiate the infection process.
However, if cells are grown or placed on a porous filter and
retrovirus is allowed to move past cells by gradual gravitometric
flow, a high concentration of virus around cells can be effectively
maintained at all times. Thus, up to a ten-fold higher infectivity
by infecting cells on a porous membrane and allowing retrovirus
supernatant to flow past them has been seen. This should allow
titres of 10.sup.9 after concentration.
[0099] The nucleic acids encoding the scaffolds and enzymes, as
part of retroviral constructs, are introduced into the cells to
screen for the production of bioactive agents capable of altering
the phenotype of a cell.
[0100] As will be appreciated by those in the art, the type of
cells used in the present invention can vary widely. Basically, any
mammalian cells may be used, with mouse, rat, primate and human
cells being particularly preferred, although as will be appreciated
by those in the art, modifications of the system by pseudotyping
allows all eukaryotic cells to be used, preferably higher
eukaryotes. As is more fully described herein, a screen will be set
up such that the cells exhibit a selectable phenotype in the
presence of a bioactive agent. As is more fully described below,
cell types implicated in a wide variety of disease conditions are
particularly useful, so long as a suitable screen may be designed
to allow the selection of cells that exhibit an altered phenotype
as a consequence of the presence of a transdominant bioactive agent
within the cell.
[0101] Accordingly, suitable host cell types include, but are not
limited to, tumor cells of all types (particularly melanoma,
myeloid leukemia, carcinomas of the lung, breast, ovaries, colon,
kidney, prostate, pancreas and testes), cardiomyocytes, endothelial
cells, epithelial cells, lymphocytes (T-cell and B cell), mast
cells, eosinophils, vascular intimal cells, hepatocytes, leukocytes
including mononuclear leukocytes, stem cells such as haemopoetic,
neural, skin, lung, kidney, liver and myocyte stem cells (for use
in screening for differentiation and de-differentiation factors),
osteoclasts, chondrocytes and other connective tissue cells,
keratinocytes, melanocytes, liver cells, kidney cells, and
adipocytes. Suitable cells also include known research cells,
including, but not limited to, Jurkat T cells, NIH3T3 cells, CHO,
Cos, etc. See the ATCC cell line catalog, hereby expressly
incorporated by reference. In one embodiment, the cells may also be
genetically engineered, that is, contain exogeneous nucleic acid,
prior to the introduction of the nucleic acids of the
invention.
[0102] Thus, the component nucleic acids, encoding scaffolds and
enzymes, are introduced into a plurality of cells, and expressed,
and the expression products or components then associate to form
enzyme-scaffold complexes within the cells. Each cell preferably
comprises a different enzyme-scaffold complex.
[0103] In a preferred embodiment, a first plurality of cells is
screened. That is, the cells into which the component nucleic acids
are introduced are screened for an altered phenotype. Thus, in this
embodiment, the effect of the bioactive agent is seen in the same
cells in which it is made; i.e. an autocrine effect.
[0104] By a "plurality of cells" herein is meant roughly from about
10.sup.3 cells to 10.sup.8 or 10.sup.9, with from 10.sup.6 to
10.sup.8 being preferred. This plurality of cells comprises a
cellular library, wherein generally each cell within the library
contains a different enzyme complex, although as will be
appreciated by those in the art, some cells within the library may
not contain a retrovirus, and some may contain more than one. When
methods other than retroviral infection are used to introduce the
component nucleic acids into a plurality of cells, the distribution
of component nucleic acids within the individual cell members of
the cellular library may vary widely, as it is generally difficult
to control the number of nucleic acids which enter a cell during
electroporation, etc.
[0105] In a preferred embodiment, the component nucleic acids are
introduced into a first plurality of cells, and the effect of the
enzyme complex is screened in a second or third plurality of cells,
different from the first plurality of cells, i.e. generally a
different cell type. That is, the effect of the bioactive agents is
due to an extracellular effect on a second cell; i.e. an endocrine
or paracrine effect. This is done using standard techniques. The
first plurality of cells may be grown in or on one media, and the
media is allowed to touch a second plurality of cells, and the
effect measured. Alternatively, there may be direct contact between
the cells. Thus, "contacting" is functional contact, and includes
both direct and indirect. In this embodiment, the first plurality
of cells may or may not be screened.
[0106] If necessary, the cells are treated to conditions suitable
for the expression of the component nucleic acids (for example,
when inducible promoters are used), to produce the component
expression products, either translation or transcription
products.
[0107] The cells may then be screened for altered phenotypes. That
is, the enzyme complex may act on a endogeneous cellular compound
to form a novel bioactive agent that is capable of altering the
phenotype of the cell. Alternatively, the bioactive agent may
already be present in the cell, but at a concentration too low to
show the bioactive effect. Optionally, precursor compounds may be
added to the cell, which then may be acted upon by the enzyme
complex to form a bioactive agent.
[0108] In a preferred embodiment, no precursor compounds are added,
and the plurality of cells is screened, as is more fully outlined
below, for a cell exhibiting an altered phenotype due to the action
of the enzyme complex on an endogeneous compound.
[0109] In a preferred embodiment, precursor compounds are added to
the cells, and the enzyme complexes either enzymatically alter the
precursor to form bioactive agents, or act on endogeneous compounds
which then interact with the precursor to form bioactive agents. By
"bioactive agent" describes any molecule, e.g., protein,
oligopeptide, small organic molecule, polysaccharide,
polynucleotide, etc., with the capability of directly or indirectly
altering a cellular phenotype.
[0110] "Candidate bioactive agents", "bioactive agent precursors",
"precursors" or grammatical equivalents encompass numerous chemical
classes, though typically they are organic molecules, preferably
small organic compounds having a molecular weight of more than 100
and less than about 2,500 daltons. Precursors generally comprise
functional groups necessary for structural interaction with
cellular components such as proteins and nucleic acids,
particularly hydrogen bonding, and typically include at least an
amine, carbonyl, hydroxyl or carboxyl group, preferably at least
two of the functional chemical groups. The candidate agents often
comprise cyclical carbon or heterocyclic structures and/or aromatic
or polyaromatic structures substituted with one or more of the
above functional groups. Precursors are also found among
biomolecules including peptides, saccharides, fatty acids,
steroids, purines, pyrimidines, derivatives, structural analogs or
combinations thereof.
[0111] Precursors are obtained from a wide variety of sources
including libraries of synthetic or natural compounds. For example,
numerous means are available for random and directed synthesis of a
wide variety of organic compounds and biomolecules, including
expression of randomized oligonucleotides. Alternatively, libraries
of natural compounds in the form of bacterial, fungal, plant and
animal extracts are available or readily produced. Additionally,
natural or synthetically produced libraries and compounds are
readily modified through conventional chemical, physical and
biochemical means. Known pharmacological agents may be subjected to
directed or random chemical modifications, such as acylation,
alkylation, esterification, amidification to produce structural
analogs.
[0112] Precursors may also include compounds with known capacities
for altering cellular phenotypes but with undesirable side effects.
For example, as is more fully described below, some
chemotherapeutic agents display unacceptable levels of toxicity.
These chemotherapeutic agents may be used as precursors such that
the enzyme complexes of the invention may alter their structure in
ways that lowers the level of toxicity, etc.
[0113] As will be appreciated by those in the art, suitable
precursor compounds include a very large number of compounds,
including, but not limited to, known pharmacophores and
pharmacophore analogs and precursors, including hydantoins (Tet.
Lett. 37(7): 937 (1996)); pyrazoles and isoxazoles (Tet. Lett.
37(7): 1003 (1996); imidazoles (Tet. Lett. 37(6): 835 (1996);
sulfonamides (Tet. Lett. 37(8): 1145 (1996); 4-thiazolidinones
(Bioorganic & Med. Chem. Let. 6(6): 707 (1996);
4-sulfamoylbenzamides (Bioorganic & Med. Chem. Let. 6(5): 559
(1996); 2,6-disubstituted quinolones (Tet. Lett. 37(16): 2757
(1996); biphenyl core compounds (Bioorganic & Med. Chem. Let.
4(5): 659 (1996); actinomycins (Bioorganic & Med. Chem. Let.
4(5): 693 (1996); other quinolones (Tet. Lett 37(27): 4815 (1996);
3-aminothiophenes and heterocyles (Tet. Lett 37(34): 6213 (1996);
benzodiazepine (Bioorganic & Med. Chem. Let. 6(19): 2299
(1996); polyazacyclophane (Tetl. Lett. 37(40: 7233 (1996); 5- and
6-membered lactams (Tet. Lett. 38(3): 359 (1997); spiroindoline
(Tet. Lett. 38(9): 1497 (1997); substituted guanidines (Tet. Lett.
38(19): 3377 (1997); and compounds described in Tetrahedron 52(13):
4527 (1996); all of which are incorporated by reference.
[0114] In a preferred embodiment, the precursors are labelled. By
"labelled" herein is meant that the precursor compound is either
directly or indirectly labeled with a label which provides a
detectable signal, e.g. radioisotope, fluorescers, enzyme,
antibodies, particles such as magnetic particles, chemiluminescer,
etc.
[0115] In addition, the precursors may include precursor targeting
sequences. Precursor targeting sequences are functionally similar
to the fusion partner targeting sequences, in that they serve to
target the precursors to a particular subcellular location.
[0116] In general, the precursors are added to the cells prior to
screening, generally added to the cell media or added to discs on
which the cells are grown, etc. The precursors will be added for a
sufficient incubation time, generally from about 0.5 to 24 hours
before screening, to allow the enzyme complexes sufficient time to
enzymatically alter the precursor into new forms. In one
embodiment, when the precursors are proteins or nucleic acids,
nucleic acids encoding the precursors may be introduced into to the
cells as outlined above for component nucleic acids. The cells are
then screened as outlined above to detect the presence of a cell
with an altered phenotype.
[0117] By "altered phenotype" or "changed physiology" or other
grammatical equivalents herein is meant that the phenotype of the
cell is altered in some way, preferably in some detectable and/or
measurable way. As will be appreciated in the art, a strength of
the present invention is the wide variety of cell types and
potential phenotypic changes which may be tested using the present
methods. Accordingly, any phenotypic change which may be observed,
detected, or measured may be the basis of the screening methods
herein. Suitable phenotypic changes include, but are not limited
to: gross physical changes such as changes in cell morphology, cell
growth, cell viability, adhesion to substrates or other cells, and
cellular density; changes in the expression of one or more RNAs,
proteins, lipids, hormones, cytokines, or other molecules; changes
in the equilibrium state (i.e. half-life) or one or more RNAs,
proteins, lipids, hormones, cytokines, or other molecules; changes
in the localization of one or more RNAs, proteins, lipids,
hormones, cytokines, or other molecules; changes in the bioactivity
or specific activity of one or more RNAs, proteins, lipids,
hormones, cytokines, receptors, or other molecules; changes in the
secretion of ions, cytokines, hormones, growth factors, or other
molecules; alterations in cellular membrane potentials,
polarization, integrity or transport; changes in infectivity,
susceptability, latency, adhesion, and uptake of viruses and
bacterial pathogens; etc. By "capable of altering the phenotype"
herein is meant that the bioactive agent can change the phenotype
of the cell in some detectable and/or measurable way.
[0118] The altered phenotype may be detected in a wide variety of
ways, as is described more fully below, and will generally depend
and correspond to the phenotype that is being changed. Generally,
the changed phenotype is detected using, for example: microscopic
analysis of cell morphology; standard cell viability assays,
including both increased cell death and increased cell viability,
for example, cells that are now resistant to cell death via virus,
bacteria, or bacterial or synthetic toxins; standard labeling
assays such as fluorometric indicator assays for the presence or
level of a particular cell or molecule, including FACS or other dye
staining techniques; biochemical detection of the expression of
target compounds after killing the cells; etc. In some cases, as is
more fully described herein, the altered phenotype is detected in
the cell in which the randomized nucleic acid was introduced; in
other embodiments, the altered phenotype is detected in a second
cell which is responding to some molecular signal from the first
cell.
[0119] An altered phenotype of a cell indicates the presence of a
bioactive agent. Preferably, the bioactive agent is a transdominant
bioactive agent. By "transdominant" herein is meant that the
bioactive agent indirectly causes the altered phenotype by acting
on a second molecule, which leads to an altered phenotype. That is,
a transdominant expression product has an effect that is not in
cis, i.e., a trans event as defined in genetic terms or biochemical
terms. A transdominant effect is a distinguishable effect by a
molecular entity (i.e., the encoded peptide or RNA) upon some
separate and distinguishable target; that is, not an effect upon
the encoded entity itself. As such, transdominant effects include
many well-known effects by pharmacologic agents upon target
molecules or pathways in cells or physiologic systems; for
instance, the .beta.-lactam antibiotics have a transdominant effect
upon peptidoglycan synthesis in bacterial cells by binding to
penicillin binding proteins and disrupting their functions. An
exemplary transdominant effect by a peptide is the ability to
inhibit NF-.kappa.B signaling by binding to I.kappa.B-.alpha. at a
region critical for its function, such that in the presence of
sufficient amounts of the peptide (or molecular entity), the
signaling pathways that normally lead to the activation of
NF-.kappa.B through phosphorylation and/or degradation of
I.kappa.B-.alpha. are inhibited from acting at I.kappa.B-.alpha.
because of the binding of the peptide or molecular entity. In
another instance, signaling pathways that are normally activated to
secrete IgE are inhibited in the presence of peptide. Or, signaling
pathways in adipose tissue cells, normally quiescent, are activated
to metabolize fat. Or, in the presence of a peptide, intracellular
mechanisms for the replication of certain viruses, such as HIV-I,
or Herpes viridae family members, or Respiratory Syncytia Virus,
for example, are inhibited.
[0120] A transdominant effect upon a protein or molecular pathway
is clearly distinguishable from randomization, change, or mutation
of a sequence within a protein or molecule of known or unknown
function to enhance or diminish a biochemical ability that protein
or molecule already manifests. For instance, a protein that
enzymatically cleaves .beta.-lactam antibiotics, a
.beta.-lactamase, could be enhanced or diminished in its activity
by mutating sequences internal to its structure that enhance or
diminish the ability of this enzyme to act upon and cleave
.beta.-lactam antibiotics. This would be called a cis mutation to
the protein. The effect of this protein upon .beta.-lactam
antibiotics is an activity the protein already manifests, to a
distinguishable degree. Similarly, a mutation in the leader
sequence that enhanced the export of this protein to the
extracellular spaces wherein it might encounter .beta.-lactam
molecules more readily, or a mutation within the sequence that
enhance the stability of the protein, would be termed cis mutations
in the protein. For comparison, a transdominant effector of this
protein would include an agent, independent of the
.beta.-lactamase, that bound to the .beta.-lactamase in such a way
that it enhanced or diminished the function of the .beta.-lactamase
by virtue of its binding to .beta.-lactamase.
[0121] In general, cis-effects are effects within molecules wherein
elements that are interacting are covalently joined to each other
although these elements might individually manifest themselves as
separable domains. Trans-effects (transdominant in that under some
cellular conditions the desired effect is manifested) are those
effects between distinct molecular entities, such that molecular
entity A, not covalently linked to molecular entity B, binds to or
otherwise has an effect upon the activities of entity B. As such,
most known pharmacological agents are transdominant effectors.
[0122] In a preferred embodiment, once a cell with an altered
phenotype is detected, the cell is isolated from the plurality
which do not have altered phenotypes. This may be done in any
number of ways, as is known in the art, and will in some instances
depend on the assay or screen. Suitable isolation techniques
include, but are not limited to, FACS, lysis selection using
complement, cell cloning, scanning by Fluorimager, expression of a
"survival" protein, induced expression of a cell surface protein or
other molecule that can be rendered fluorescent or taggable for
physical isolation; expression of an enzyme that changes a
non-fluorescent molecule to a fluorescent one; overgrowth against a
background of no or slow growth; death of cells and isolation of
DNA or other cell vitality indicator dyes, etc.
[0123] In a preferred embodiment, the component nucleic acid, the
enzyme complex and/or the bioactive agent is isolated from the
positive cell. This may be done in a number of ways. In a preferred
embodiment, primers complementary to DNA regions common to the
retroviral constructs, or to specific components of the library
such as a rescue sequence, defined above, are used to "rescue" the
sequences. The enzyme complex may then be reconstructed in vitro,
the precursor added, and the reaction products separated, tested,
and characterized chemically.
[0124] The enzyme complex may be isolated through the use of
purification sequences. Thus for example, one or all of the
components may contain a purification sequence, such as an epitope
tag or the His.sub.6 tag. The cells containing the enzyme complex
may be lysed and the complex isolated using any number of
techniques, including immunoprecipitation or affinity
chromatography.
[0125] Alternatively, the bioactive agent may be isolated using a
label present on the precursor, either by using the label directly
or by following the presence of the label in a purification scheme,
such as capillary electrophoresis and mass spectroscopy. In some
instances, as is outlined below, this may also pull out the primary
target molecule, if there is a sufficiently strong binding
interaction between the bioactive agent and the target molecule.
Alternatively, the bioactive agent may be detected using mass
spectroscopy.
[0126] Once rescued, the composition of the enzyme complex and the
identification of the bioactive agent is determined. This
information can then be used in a number of ways.
[0127] In a preferred embodiment, the bioactive agent is
resynthesized and reintroduced into the target cells, to verify the
effect. This may be done in a variety of ways, as will be
appreciated by those in the art, and may depend on the composition
of the bioactive agent. For example, proteinaceous bioactive agents
may be reintroduced using retroviruses, or alternatively using
fusions to the HIV-1 Tat protein, and analogs and related proteins,
which allows very high uptake into target cells. See for example,
Fawell et al., PNAS USA 91: 664 (1994); Frankel et al., Cell 55:
1189 (1988); Savion et al., J. Biol. Chem. 256: 1149 (1981);
Derossi et al., J. Biol. Chem. 269: 10444 (1994); and Baldin et
al., EMBO J. 9: 1511 (1990), all of which are incorporated by
reference. Simply adding the bioactive agent to target cells, in
the same way precursor molecules are added, may be sufficient.
[0128] In a preferred embodiment, the identification of a bioactive
agent is used to generate more bioactive agent precursors. For
example, analogs of the bioactive agents may be tested as
precursors. It may also be desirable to "walk" around a potential
binding site, in a manner similar to the mutagenesis of a binding
pocket, by keeping one area of the bioactive agent constant and
randomizing the other end to shift the binding of the agent
around.
[0129] In a preferred embodiment, the bioactive agent is used to
identify target molecules, i.e. the molecules with which the
bioactive agent interacts. As will be appreciated by those in the
art, there may be primary target molecules, to which the bioactive
agent binds or acts upon directly, and there may be secondary
target molecules, which are part of the signalling pathway affected
by the bioactive agent; these might be termed "validated
targets".
[0130] In a preferred embodiment, the bioactive agent is used to
pull out target molecules. For example, rescue or purification
sequences may be added to a bioactive agent, which can allow the
purification of primary target molecules via biochemical means
(co-immunoprecipitation, affinity columns, etc.). Proteinaceous
bioactive agents, when expressed in bacteria and purified, can be
used as a probe against a bacterial cDNA expression library made
from mRNA of the target cell type. Or, proteinaceous bioactive
agents can be used as "bait" in either yeast or mammalian two or
three hybrid systems. Such interaction cloning approaches have been
very useful to isolate DNA-binding proteins and other interacting
protein components. It is also possible to synthetically prepare
labeled bioactive agent and use it to screen a cDNA library
expressed in bacteriophage for those cDNAs which bind the agent.
Furthermore, it is also possible that one could use cDNA cloning
via retroviral libraries to "complement" the effect induced by the
agent. In such a strategy, the agent would be required to be
stochiometrically titrating away some important factor for a
specific signaling pathway. If this molecule or activity is
replenished by over-expression of a cDNA from within a cDNA
library, then one can clone the target. Similarly, cDNAs cloned by
any of the above yeast or bacteriophage systems can be reintroduced
to mammalian cells in this manner to confirm that they act to
complement function in the system the agent acts upon. The
bioactive agent may also be tagged with a crosslinkable tag to bind
to the target to allow purification, for example for low affinity
interactions.
[0131] Once primary target molecules have been identified,
secondary target molecules may be identified in the same manner,
using the primary target as the "bait". In this manner, signalling
pathways may be elucidated. Similarly, bioactive agents specific
for secondary target molecules may also be discovered, to allow a
number of bioactive agents to act on a single pathway, for example
for combination therapies or pathway engineering.
[0132] In addition, once a particular enzyme complex has been
identified as useful for a particular application, it may be
"evolved" using the techniques outlined herein to optimize the
system. For example, different scaffolds, different but related
enzymes or mutated enzymes, different binding sites or binding
sequences, or the same binding sites or sequences in alternative
conformations may all be used.
[0133] The screening methods of the present invention may be useful
to screen a large number of cell types under a wide variety of
conditions. As is outlined below, the libraries of enzyme complexes
may be introduced into the cells, in the presence or absence of
specific precursor compounds, and the cells tested in a variety of
ways. Generally, the host cells are cells that are involved in
disease states, and they are tested or screened under conditions
that normally result in undesirable consequences on the cells. When
a suitable bioactive agent is found, the undesirable effect may be
reduced or eliminated. Alternatively, normally desirable
consequences may be reduced or eliminated, with an eye towards
elucidating the cellular mechanisms associated with the disease
state or signalling pathway.
[0134] In a preferred embodiment, the present methods are useful in
cancer applications. The ability to rapidly and specifically kill
tumor cells is a cornerstone of cancer chemotherapy. In general,
using the methods of the present invention, enzyme complexes can be
introduced into any tumor cell (primary or cultured), and agents
identified which by themselves induce apoptosis, cell death, loss
of cell division or decreased cell growth. This may be done de
novo, or by the introduction of "biased" precursors, such as
analogs of known chemotherapeutic agents. Alternatively, the
methods of the present invention can be combined with other cancer
therapeutics (e.g. drugs or radiation) to sensitize the cells and
thus induce rapid and specific apoptosis, cell death, loss of cell
division or decreased cell growth after exposure to a secondary
agent. Similarly, the present methods may be used in conjunction
with known cancer therapeutics to screen for agonists to make the
therapeutic more effective or less toxic. This is particularly
preferred when the chemotherapeutic is very expensive to produce
such as taxol.
[0135] Known oncogenes such as v-Abl, v-Src, v-Ras, and others,
induce a transformed phenotype leading to abnormal cell growth when
transfected into certain cells. This is also a major problem with
micro-metastases. Thus, in a preferred embodiment, non-transformed
cells can be transfected with these oncogenes, and then libraries
of enzyme complexes introduced into these cells, to select for
bioactive agents which reverse or correct the transformed state.
One of the signal features of oncogene transformation of cells is
the loss of contact inhibition and the ability to grow in
soft-agar. When transforming viruses are constructed containing
v-Abl, v-Src, or v-Ras in IRES-puro retroviral vectors, infected
into target 3T3 cells, and subjected to puromycin selection, all of
the 3T3 cells hyper-transform and detach from the plate. The cells
may be removed by washing with fresh medium. This can serve as the
basis of a screen, since cells which express a bioactive agent will
remain attached to the plate and form colonies.
[0136] Similarly, the growth and/or spread of certain tumor types
is enhanced by stimulatory responses from growth factors and
cytokines (PDGF, EGF, Heregulin, and others) which bind to
receptors on the surfaces of specific tumors. In a preferred
embodiment, the methods of the invention are used to inhibit or
stop tumor growth and/or spread, by finding bioactive agents
capable of blocking the ability of the growth factor or cytokine to
stimulate the tumor cell. The introduction of libraries of enzyme
complexes into specific tumor cells with the addition of the growth
factor or cytokine, followed by selection of bioactive agents which
block the binding, signaling, phenotypic and/or functional
responses of these tumor cells to the growth factor or cytokine in
question.
[0137] Similarly, the spread of cancer cells (invasion and
metastasis) is a significant problem limiting the success of cancer
therapies. The ability to inhibit the invasion and/or migration of
specific tumor cells would be a significant advance in the therapy
of cancer. Tumor cells known to have a high metastatic potential
(for example, melanoma, lung cell carcinoma, breast and ovarian
carcinoma) can have libraries of enzyme complexes introduced into
them, and agents selected which in a migration or invasion assay,
inhibit the migration and/or invasion of specific tumor cells.
Particular applications for inhibition of the metastatic phenotype,
which could allow a more specific inhibition of metastasis, include
the metastasis suppressor gene NM23, which codes for a dinucleoside
diphosphate kinase. Thus intracellular activators of this gene
could block metastasis, and a screen for its upregulation (by
fusing it to a reporter gene) would be of interest. Many oncogenes
also enhance metastasis. Agents which inactivate or counteract
mutated RAS oncogenes, v-MOS, v-RAF, A-RAF, v-SRC, v-FES, and v-FMS
would also act as anti-metastatics. Agents which act
intracellularly to block the release of combinations of proteases
required for invasion, such as the matrix metalloproteases and
urokinase, could also be effective antimetastatics.
[0138] In a preferred embodiment, the enzyme complexes of the
present invention are introduced into tumor cells known to have
inactivated tumor suppressor genes, and successful reversal by
either reactivation or compensation of the knockout would be
screened by restoration of the normal phenotype. A major example is
the reversal of p53-inactivating mutations, which are present in
50% or more of all cancers. Since p53's actions are complex and
involve its action as a transcription factor, there are probably
numerous potential ways a bioactive agent could reverse the
mutation. One example would be upregulation of the immediately
downstream cyclin-dependent kinase p21 CIP1/WAF1. To be useful such
reversal would have to work for many of the different known p53
mutations. This is currently being approached by gene therapy; one
or more small molecules which do this might be preferable.
[0139] Another example involves screening of bioactive agents which
restore the constitutive function of the brca-1 or brca-2 genes,
and other tumor suppressor genes important in breast cancer such as
the adenomatous polyposis coli gene (APC) and the Drosophila
discs-large gene (DIg), which are components of cell-cell
junctions. Mutations of brca-1 are important in hereditary ovarian
and breast cancers, and constitute an additional application of the
present invention.
[0140] In a preferred embodiment, the methods of the present
invention are used to create novel cell lines from cancers from
patients. An enzyme complex which inhibits the final common pathway
of programmed cell death should allow for short- and possibly
long-term cell lines to be established. Conditions of in vitro
culture and infection of human leukemia cells will be established.
There is a real need for methods which allow the maintenance of
certain tumor cells in culture long enough to allow for
physiological and pharmacological studies. Currently, some human
cell lines have been established by the use of transforming agents
such as Ebstein-Barr virus that considerably alters the existing
physiology of the cell. On occasion, cells will grow on their own
in culture but this is a random event. Programmed cell death
(apoptosis) occurs via complex signaling pathways within cells that
ultimately activate a final common pathway producing characteristic
changes in the cell leading to a non-inflammatory destruction of
the cell. It is well known that tumor cells have a high apoptotic
index, or propensity to enter apoptosis in vivo. When cells are
placed in culture, the in vivo stimuli for malignant cell growth
are removed and cells readily undergo apoptosis. The objective
would be to develop the technology to establish cell lines from any
number of primary tumor cells, for example primary human leukemia
cells, in a reproducible manner without altering the native
configuration of the signaling pathways in these cells. By
introducing enzyme complexes which act to inhibit apoptosis,
increased cell survival in vitro, and hence the opportunity to
study signalling transduction pathways in primary human tumor
cells, is accomplished. In addition, these methods may be used for
culturing primary cells, i.e. non-tumor cells.
[0141] In a preferred embodiment, the present methods are useful in
cardiovascular applications. In a preferred embodiment,
cardiomyocytes may be screened for the prevention of cell damage or
death in the presence of normally injurious conditions, including,
but not limited to, the presence of toxic drugs (particularly
chemotherapeutic drugs), for example, to prevent heart failure
following treatment with adriamycin; anoxia, for example in the
setting of coronary artery occlusion; and autoimmune cellular
damage by attack from activated lymphoid cells (for example as seen
in post viral myocarditis and lupus). Enzyme complexes (and
precursors, if necessary) are inserted into cardiomyocytes, the
cells are subjected to the insult, and bioactive agents are
selected that prevent any or all of: apoptosis; membrane
depolarization (i.e. decrease arrythmogenic potential of insult);
cell swelling; or leakage of specific intracellular ions, second
messengers and activating molecules (for example, arachidonic acid
and/or lysophosphatidic acid).
[0142] In a preferred embodiment, the present methods are used to
screen for diminished arrhythmia potential in cardiomyocytes. The
screens comprise the introduction of the enzyme complexes (and
precursors, if necessary), followed by the application of
arrythmogenic insults, with screening for bioactive agents that
block specific depolarization of cell membrane. This may be
detected using patch clamps, or via fluorescence techniques.
Similarly, channel activity (for example, potassium and chloride
channels) in cardiomyocytes could be regulated using the present
methods in order to enhance contractility and prevent or diminish
arrhythmias.
[0143] In a preferred embodiment, the present methods are used to
screen for enhanced contractile properties of cardiomyocytes and
diminish heart failure potential. The introduction of the libraries
of the invention followed by measuring the rate of change of myosin
polymerization/depolymerization using fluorescent techniques can be
done. Bioactive agents which increase the rate of change of this
phenomenon can result in a greater contractile response of the
entire myocardium, similar to the effect seen with digitalis.
[0144] In a preferred embodiment, the present methods are useful to
identify agents that will regulate the intracellular and
sarcolemmal calcium cycling in cardiomyocytes in order to prevent
arrhythmias. Bioactive agents are selected that regulate
sodium-calcium exchange, sodium proton pump function, and
regulation of calcium-ATPase activity.
[0145] In a preferred embodiment, the present methods are useful to
identify agents that diminish embolic phenomena in arteries and
arterioles leading to strokes (and other occlusive events leading
to kidney failure and limb ischemia) and angina precipitating a
myocardial infarct are selected. For example, bioactive agents may
be found which will diminish the adhesion of platelets and
leukocytes, and thus diminish the occlusion events. Adhesion in
this setting can be inhibited by the libraries of the invention
being inserted into endothelial cells (quiescent cells, or
activated by cytokines, i.e. IL-1, and growth factors, i.e.
PDGF/EGF) and then screening for agents that either: 1)
downregulate adhesion molecule expression on the surface of the
endothelial cells (binding assay); or 2) block adhesion molecule
activation on the surface of these cells (signaling assay).
[0146] Embolic phenomena can also be addressed by activating
proteolytic enzymes on the cell surfaces of endothelial cells, and
thus releasing active enzyme which can digest blood clots. Thus,
delivery of the complexes of the invention to endothelial cells is
done, followed by standard fluorogenic assays, which will allow
monitoring of proteolytic activity on the cell surface towards a
known substrate. Bioactive agents can then be selected which
activate specific enzymes towards specific substrates.
[0147] In a preferred embodiment, arterial inflammation in the
setting of vasculitis and post-infarction can be regulated by
decreasing the chemotactic responses of leukocytes and mononuclear
leukocytes. This can be accomplished by blocking chemotactic
receptors and their responding pathways on these cells. Enzyme
complex libraries can be inserted into these cells, and the
chemotactic response to diverse chemokines (for example, to the
IL-8 family of chemokines, RANTES) inhibited in cell migration
assays.
[0148] In a preferred embodiment, arterial restenosis following
coronary angioplasty can be controlled by regulating the
proliferation of vascular intimal cells and capillary and/or
arterial endothelial cells. Enzyme complexes can be inserted into
these cell types and their proliferation in response to specific
stimuli monitored. One application may be intracellular agents
which block the expression or function of c-myc and other oncogenes
in smooth muscle cells to stop their proliferation. A second
application may involve the expression of libraries in vascular
smooth muscle cells to selectively induce their apoptosis.
Application of small molecules derived from these systems may
require targeted drug delivery; this is available with stents,
hydrogel coatings, and infusion-based catheter systems. Agents
which downregulate endothelin-1A receptors or which block the
release of the potent vasoconstrictor and vascular smooth muscle
cell mitogen endothelin-1 may also be candidates for therapeutics.
Agents can be isolated from these systems which inhibit growth of
these cells, or which prevent the adhesion of other cells in the
circulation known to release autocrine growth factors, such as
platelets (PDGF) and mononuclear leukocytes.
[0149] The control of capillary and blood vessel growth is an
important goal in order to promote increased blood flow to ischemic
areas (growth), or to cut-off the blood supply (angiogenesis
inhibition) of tumors. Enzyme complexes can be inserted into
capillary endothelial cells and their growth monitored. Stimuli
such as low oxygen tension and varying degrees of angiogenic
factors can regulate the responses, and agents isolated that
produce the appropriate phenotype. Screening for antagonism of
vascular endothelial cell growth factor, important in angiogenesis,
would also be useful.
[0150] In a preferred embodiment, the present methods are useful in
screening for decreases in atherosclerosis producing mechanisms to
find agents that regulate LDL and HDL metabolism. Enzyme complex
libraries can be inserted into the appropriate cells (including
hepatocytes, mononuclear leukocytes, endothelial cells) and agents
selected which lead to a decreased release of LDL or diminished
synthesis of LDL, or conversely to an increased release of HDL or
enhanced synthesis of HDL. Bioactive agents can also be isolated
from enzyme complex libraries which decrease the production of
oxidized LDL, which has been implicated in atherosclerosis and
isolated from atherosclerotic lesions. This could occur by
decreasing its expression, activating reducing systems or enzymes,
or blocking the activity or production of enzymes implicated in
production of oxidized LDL, such as 15-lipoxygenase in
macrophages.
[0151] In a preferred embodiment, the present methods are used in
screens to regulate obesity via the control of food intake
mechanisms or diminishing the responses of receptor signaling
pathways that regulate metabolism. Bioactive agents that regulate
or inhibit the responses of neuropeptide Y (NPY), cholecystokinin
and galanin receptors, are particularly desirable. Enzyme complex
libraries can be inserted into cells that have these receptors
cloned into them, and inhibitory agents selected that block the
signaling responses to galanin and NPY. In a similar manner, agents
can be found that regulate the leptin receptor.
[0152] In a preferred embodiment, the present methods are useful in
neurobiology applications. Enzyme complex libraries may be used for
screening for anti-apoptotics for preservation of neuronal function
and prevention of neuronal death. Initial screens would be done in
cell culture. One application would include prevention of neuronal
death, by apoptosis, in cerebral ischemia resulting from stroke.
Apoptosis is known to be blocked by neuronal apoptosis inhibitory
protein (NAIP); screens for its upregulation, or effecting any
coupled step could yield agents which selectively block neuronal
apoptosis. Other applications include neurodegenerative diseases
such as Alzheimer's disease and Huntington's disease.
[0153] In a preferred embodiment, the present methods are useful in
bone biology applications. Osteoclasts are known to play a key role
in bone remodeling by breaking down "old" bone, so that osteoblasts
can lay down "new" bone. In osteoporosis one has an imbalance of
this process. Osteoclast overactivity can be regulated by inserting
libraries into these cells, and then looking for bioactive agents
that produce: 1) a diminished processing of collagen by these
cells; 2) decreased pit formation on bone chips; and 3) decreased
release of calcium from bone fragments.
[0154] The present methods may also be used to screen for agonists
of bone morphogenic proteins, hormone mimetics to stimulate,
regulate, or enhance new bone formation (in a manner similar to
parathyroid hormone and calcitonin, for example). These have use in
osteoporosis, for poorly healing fractures, and to accelerate the
rate of healing of new fractures. Furthermore, cell lines of
connective tissue origin can be treated with enzyme complex
libraries and screened for their growth, proliferation, collagen
stimulating activity, and/or proline incorporating ability on the
target osteoblasts. Alternatively, enzyme complexes can be
expressed directly in osteoblasts or chondrocytes and screened for
increased production of collagen or bone.
[0155] In a preferred embodiment, the present methods are useful in
skin biology applications. Keratinocyte responses to a variety of
stimuli may result in psoriasis, a proliferative change in these
cells. Enzyme complexes can be inserted into cells removed from
active psoriatic plaques, and bioactive agents isolated which
decrease the rate of growth of these cells.
[0156] In a preferred embodiment, the present methods are useful in
the regulation or inhibition of keloid formation (i.e. excessive
scarring). Enzyme complexes are inserted into skin connective
tissue cells isolated from individuals with this condition, and
bioactive agents isolated that decrease proliferation, collagen
formation, or proline incorporation. Results from this work can be
extended to treat the excessive scarring that also occurs in burn
patients. If a common motif is found in the context of the keloid
work, then it can be used widely in a topical manner to diminish
scarring post burn.
[0157] Similarly, wound healing for diabetic ulcers and other
chronic "failure to heal" conditions in the skin and extremities
can be regulated by providing additional growth signals to cells
which populate the skin and dermal layers. Growth factor mimetics
may in fact be very useful for this condition. Enzyme candidate
libraries can be inserted into skin connective tissue cells, and
bioactive agents isolated which promote the growth of these cells
under "harsh" conditions, such as low oxygen tension, low pH, and
the presence of inflammatory mediators.
[0158] Cosmeceutical applications of the present invention include
the control of melanin production in skin melanocytes. A naturally
occurring peptide, arbutin, is a tyrosine hydroxylase inhibitor, a
key enzyme in the synthesis of melanin. Enzyme complexes can be
inserted into melanocytes and known stimuli that increase the
synthesis of melanin applied to the cells. Bioactive agents can be
isolated that inhibit the synthesis of melanin under these
conditions.
[0159] In a preferred embodiment, the present methods are useful in
endocrinology applications. The enzyme complex technology can be
applied broadly to any endocrine, growth factor, cytokine or
chemokine network which involves a signaling molecule that acts in
either an endocrine, paracrine or autocrine manner that binds or
dimerizes a receptor and activates a signaling cascade that results
in a known phenotypic or functional outcome. The methods are
applied so as to isolate an agent which either mimics the desired
hormone (i.e., insulin, leptin, calcitonin, PDGF, EGF, EPO, GMCSF,
IL1-17, mimetics) or inhibits its action by either blocking the
release of the hormone, blocking its binding to a specific receptor
or carrier protein (for example, CRF binding protein), or
inhibiting the intracellular responses of the specific target cells
to that hormone. Selection of agents which increase the expression
or release of hormones from the cells which normally produce them
could have broad applications to conditions of hormonal
deficiency.
[0160] In a preferred embodiment, the present methods are useful in
infectious disease applications. Viral latency (herpes viruses such
as CMV, EBV, HBV, and other viruses such as HIV) and their
reactivation are a significant problem, particularly in
immunosuppressed patients (patients with AIDS and transplant
patients). The ability to block the reactivation and spread of
these viruses is an important goal. Cell lines known to harbor or
be susceptible to latent viral infection can be infected with the
specific virus, and then stimuli applied to these cells which have
been shown to lead to reactivation and viral replication. This can
be followed by measuring viral titers in the medium and scoring
cells for phenotypic changes. Enzyme complexes can then be inserted
into these cells under the above conditions, and agents isolated
which block or diminish the growth and/or release of the virus. As
with chemotherapeutics, these experiments can also be done with
drugs which are only partially effective towards this outcome, and
bioactive agents isolated which enhance the virucidal effect of
these drugs.
[0161] One example of many is the ability to block HIV-1 infection.
HIV-1 requires CD4 and a co-receptor which can be one of several
seven transmembrane G-protein coupled receptors. In the case of the
infection of macrophages, CCR-5 is the required co-receptor, and
there is strong evidence that a block on CCR-5 will result in
resistance to HIV-1 infection. There are two lines of evidence for
this statement. First, it is known that the natural ligands for
CCR-5, the CC chemokines RANTES, MIP1a and MIP1b are responsible
for CD8+ mediated resistance to HIV. Second, individuals homozygous
for a mutant allele of CCR-5 are completely resistant to HIV
infection. Thus, an inhibitor of the CCR-5/HIV interaction would be
of enormous interest to both biologists and clinicians. The action
of extracellularly anchored enzyme complexes on precursors could
allow increased uptake into the cells, for example.
[0162] Viruses are known to enter cells using specific receptors to
bind to cells (for example, HIV uses CD4, coronavirus uses CD13,
murine leukemia virus uses transport protein, and measles virus
uses CD44) and to fuse with cells (HIV uses chemokine receptor).
Enzyme complexes can be inserted into target cells known to be
permissive to these viruses, and bioactive agents isolated which
block the ability of these viruses to bind and fuse with specific
target cells.
[0163] In a preferred embodiment, the present invention finds use
with infectious organisms. Intracellular organisms such as
mycobacteria, listeria, salmonella, pneumocystis, yersinia,
leishmania, T. cruzi, can persist and replicate within cells, and
become active in immunosuppressed patients. There are currently
drugs on the market and in development which are either only
partially effective or ineffective against these organisms. Enzyme
complexes can be inserted into specific cells infected with these
organisms (pre- or post-infection), and bioactive agents selected
which promote the intracellular destruction of these organisms. In
addition agents can be selected which enhance the cidal properties
of drugs already under investigation which have insufficient
potency by themselves, but when combined with a specific bioactive
agent, are dramatically more potent through a synergistic
mechanism. Finally, bioactive agents can be isolated which alter
the metabolism of these intracellular organisms, in such a way as
to terminate their intracellular life cycle by inhibiting a key
organismal event.
[0164] Antibiotic drugs that are widely used have certain dose
dependent, tissue specific toxicities. For example renal toxicity
is seen with the use of gentamicin, tobramycin, and amphotericin;
hepatotoxicity is seen with the use of INH and rifampin; bone
marrow toxicity is seen with chloramphenicol; and platelet toxicity
is seen with ticarcillin, etc. These toxicities limit their use.
Enzyme complexes can be introduced into the specific cell types
where specific changes leading to cellular damage or apoptosis by
the antibiotics are produced, and bioactive agents can be isolated
that confer protection, when these cells are treated with these
specific antibiotics.
[0165] Furthermore, the present invention finds use in screening
for bioactive agents that block antibiotic transport mechanisms.
The rapid secretion from the blood stream of certain antibiotics
limits their usefulness. For example penicillins are rapidly
secreted by certain transport mechanisms in the kidney and choroid
plexus in the brain. Probenecid is known to block this transport
and increase serum and tissue levels. Enzyme complexes can be
inserted into specific cells derived from kidney cells and cells of
the choroid plexus known to have active transport mechanisms for
antibiotics. Bioactive agents can then be isolated which block the
active transport of specific antibiotics and thus extend the serum
halflife of these drugs.
[0166] In a preferred embodiment, the present methods are useful in
drug toxicities and drug resistance applications. Drug toxicity is
a significant clinical problem. This may manifest itself as
specific tissue or cell damage with the result that the drug's
effectiveness is limited. Examples include myeloablation in high
dose cancer chemotherapy, damage to epithelial cells lining the
airway and gut, and hair loss. Specific examples include adriamycin
induced cardiomyocyte death, cisplatinin-induced kidney toxicity,
vincristine-induced gut motility disorders, and cyclosporin-induced
kidney damage. Enzyme complexes can be introduced into specific
cell types with characteristic drug-induced phenotypic or
functional responses, in the presence of the drugs, and agents
isolated which reverse or protect the specific cell type against
the toxic changes when exposed to the drug. These effects may
manifest as blocking the drug induced apoptosis of the cell of
interest, thus initial screens will be for survival of the cells in
the presence of high levels of drugs or combinations of drugs used
in combination chemotherapy. In this embodiment, the drug may also
act as the precursor.
[0167] Drug toxicity may be due to a specific metabolite produced
in the liver or kidney which is highly toxic to specific cells, or
due to drug interactions in the liver which block or enhance the
metabolism of an administered drug. Enzyme complexes can be
introduced into liver or kidney cells following the exposure of
these cells to the drug known to produce the toxic metabolite.
Bioactive agents can be isolated which alter how the liver or
kidney cells metabolize the drug, and specific agents identified
which prevent the generation of a specific toxic metabolite. The
generation of the metabolite can be followed by mass spectrometry,
and phenotypic changes can be assessed by microscopy. Such a screen
can also be done in cultured hepatocytes, cocultured with readout
cells which are specifically sensitive to the toxic metabolite.
Applications include reversible (to limit toxicity) inhibitors of
enzymes involved in drug metabolism.
[0168] Multiple drug resistance, and hence tumor cell selection,
outgrowth, and relapse, leads to morbidity and mortality in cancer
patients. Enzyme complexes can be introduced into tumor cell lines
(primary and cultured) that have demonstrated specific or multiple
drug resistance. Bioactive agents can then be identified which
confer drug sensitivity when the cells are exposed to the drug of
interest, or to drugs used in combination chemotherapy. The readout
can be the onset of apoptosis in these cells, membrane permeability
changes, the release of intracellular ions and fluorescent markers.
The cells in which multidrug resistance involves membrane
transporters can be preloaded with fluorescent transporter
substrates, and selection carried out for agents which block the
normal efflux of fluorescent drug from these cells. Enzyme
complexes are particularly suited to screening for agents which
reverse poorly characterized or recently discovered intracellular
mechanisms of resistance or mechanisms for which few or no
chemosensitizers currently exist, such as mechanisms involving LRP
(lung resistance protein). This protein has been implicated in
multidrug resistance in ovarian carcinoma, metastatic malignant
melanoma, and acute myeloid leukemia. Particularly interesting
examples include screening for agents which reverse more than one
important resistance mechanism in a single cell, which occurs in a
subset of the most drug resistant cells, which are also important
targets. Applications would include screening for inhibitors of
both MRP (multidrug resistance related protein) and LRP for
treatment of resistant cells in metastatic melanoma, for inhibitors
of both p-glycoprotein and LRP in acute myeloid leukemia, and for
inhibition (by any mechanism) of all three proteins for treating
pan-resistant cells.
[0169] In a preferred embodiment, the present methods are useful in
improving the performance of existing or developmental drugs. First
pass metabolism of orally administered drugs limits their oral
bioavailability, and can result in diminished efficacy as well as
the need to administer more drug for a desired effect. Reversible
inhibitors of enzymes involved in first pass metabolism may thus be
a useful adjunct enhancing the efficacy of these drugs. First pass
metabolism occurs in the liver, thus inhibitors of the
corresponding catabolic enzymes may enhance the effect of the
cognate drugs. Reversible inhibitors would be delivered at the same
time as, or slightly before, the drug of interest. Screening of
enzyme complex libraries in hepatocytes for inhibitors (by any
mechanism, such as protein downregulation as well as a direct
inhibition of activity) of particularly problematical isozymes
would be of interest. These include the CYP3A4 isozymes of
cytochrome P450, which are involved in the first pass metabolism of
the anti-HIV drugs saquinavir and indinavir. Other applications
could include reversible inhibitors of UDP-glucuronyltransferases,
sulfotransferases, N-acetyltransferases, epoxide hydrolases, and
glutathione S-transferases, depending on the drug. Screens would be
done in cultured hepatocytes or liver microsomes, and could involve
antibodies recognizing the specific modification performed in the
liver, or cocultured readout cells, if the metabolite had a
different bioactivity than the untransformed drug. The enzymes
modifying the drug would not necessarily have to be known, if
screening was for lack of alteration of the drug.
[0170] In a preferred embodiment, the present methods are useful in
immunobiology, inflammation, and allergic response applications.
Selective regulation of T lymphocyte responses is a desired goal in
order to modulate immune-mediated diseases in a specific manner.
Enzyme complexes can be introduced into specific T cell subsets
(TH1, TH2, CD4+, CD8+, and others) and the responses which
characterize those subsets (cytokine generation, cytotoxicity,
proliferation in response to antigen being presented by a
mononuclear leukocyte, and others) modified by members of the
library. Agents can be selected which increase or diminish the
known T cell subset physiologic response. This approach will be
useful in any number of conditions, including: 1) autoimmune
diseases where one wants to induce a tolerant state (select an
agent that inhibits T cell subset from recognizing a self-antigen
bearing cell); 2) allergic diseases where one wants to decrease the
stimulation of IgE producing cells (select an agent which blocks
release from T cell subsets of specific B-cell stimulating
cytokines which induce switch to IgE production); 3) in transplant
patients where one wants to induce selective immunosuppression
(select an agent that diminishes proliferative responses of host T
cells to foreign antigens); 4) in lymphoproliferative states where
one wants to inhibit the growth or sensitize a specific T cell
tumor to chemotherapy and/or radiation; 5) in tumor surveillance
where one wants to inhibit the killing of cytotoxic T cells by Fas
ligand bearing tumor cells; and 5) in T cell mediated inflammatory
diseases such as Rheumatoid arthritis, Connective tissue diseases
(SLE), Multiple sclerosis, and inflammatory bowel disease, where
one wants to inhibit the proliferation of disease-causing T cells
(promote their selective apoptosis) and the resulting selective
destruction of target tissues (cartilage, connective tissue,
oligodendrocytes, gut endothelial cells, respectively).
[0171] Regulation of B cell responses will permit a more selective
modulation of the type and amount of immunoglobulin made and
secreted by specific B cell subsets. Enzyme complexes can be
inserted into B cells and bioactive agents selected which inhibit
the release and synthesis of a specific immunoglobulin. This may be
useful in autoimmune diseases characterized by the overproduction
of auto antibodies and the production of allergy causing
antibodies, such as IgE. Agents can also be identified which
inhibit or enhance the binding of a specific immunoglobulin
subclass to a specific antigen either foreign of self. Finally,
agents can be selected which inhibit the binding of a specific
immunoglobulin subclass to its receptor on specific cell types.
[0172] Similarly, agents which affect cytokine production may be
selected, generally using two cell systems. For example, cytokine
production from macrophages, monocytes, etc. may be evaluated.
Similarly, agents which mimic cytokines, for example erythropoetin
and IL1-17, may be selected, or agents that bind cytokines such as
TNF-.alpha., before they bind their receptor.
[0173] Antigen processing by mononuclear leukocytes (ML) is an
important early step in the immune system's ability to recognize
and eliminate foreign proteins. Enzyme complexes can be inserted
into ML cell lines and agents selected which alter the
intracellular processing of foreign agents and sequence of the
foreign peptide that is presented to T cells by MLs on their cell
surface in the context of Class II MHC. One can look for members of
the library that enhance immune responses of a particular T cell
subset (for example, the agent would in fact work as a vaccine), or
look for a library member that binds more tightly to MHC, thus
displacing naturally occurring peptides, but nonetheless the agent
would be less immunogenic (less stimulatory to a specific T cell
clone). This agent would in fact induce immune tolerance and/or
diminish immune responses to foreign proteins. This approach could
be used in transplantation, autoimmune diseases, and allergic
diseases.
[0174] The release of inflammatory mediators (cytokines,
leukotrienes, prostaglandins, platelet activating factor,
histamine, neuropeptides, and other peptide and lipid mediators) is
a key element in maintaining and amplifying aberrant immune
responses. Enzyme complex libraries can be inserted into MLs, mast
cells, eosinophils, and other cells participating in a specific
inflammatory response, and bioactive agents selected which inhibit
the synthesis, release and binding to the cognate receptor of each
of these types of mediators.
[0175] In a preferred embodiment, the present methods are useful in
biotechnology applications. Enzym complex expression in mammalian
cells can also be considered for other pharmaceutical-related
applications, such as modification of protein expression, protein
folding, or protein secretion. One such example would be in
commercial production of protein pharmaceuticals in CHO or other
cells. Enzyme complexes resulting in bioactive agents which select
for an increased cell growth rate (perhaps agents mimicking growth
factors or acting as agonists of growth factor signal transduction
pathways), for pathogen resistance (see previous section), for lack
of sialylation or glycosylation (by blocking glycotransferases or
rerouting trafficking of the protein in the cell), for allowing
growth on autoclaved media, or for growth in serum free media,
would all increase productivity and decrease costs in the
production of protein pharmaceuticals.
[0176] Random peptides displayed on the surface of circulating
cells can be used as tools to identify organ, tissue, and cell
specific peptide targeting sequences. Any cell introduced into the
bloodstream of an animal expressing a library targeted to the cell
surface can be selected for specific organ and tissue targeting.
The bioactive agent sequence identified can then be coupled to an
antibody, enzyme, drug, imaging agent or substance for which organ
targeting is desired.
[0177] Other agents which may be selected using the present
invention include: 1) agents which block the activity of
transcription factors, using cell lines with reporter genes; 2)
agents which block the interaction of two known proteins in cells,
using the absence of normal cellular functions, the mammalian two
hybrid system or fluorescence resonance energy transfer mechanisms
for detection; and 3) agents may be identified by tethering a
random peptide to a protein binding region to allow interactions
with molecules sterically close, i.e. within a signalling pathway,
to localize the effects to a functional area of interest.
[0178] All references cited herein are incorporated by reference.
Sequence CWU 1
1
37 1 61 PRT Artificial sequence coiled-coil presentation structure
1 Met Gly Cys Ala Ala Leu Glu Ser Glu Val Ser Ala Leu Glu Ser Glu 1
5 10 15 Val Ala Ser Leu Glu Ser Glu Val Ala Ala Leu Gly Arg Gly Asp
Met 20 25 30 Pro Leu Ala Ala Val Lys Ser Lys Leu Ser Ala Val Lys
Ser Lys Leu 35 40 45 Ala Ser Val Lys Ser Lys Leu Ala Ala Cys Gly
Pro Pro 50 55 60 2 6 PRT Artificial sequence loop structure of
coiled-coil presentation structure 2 Gly Arg Gly Asp Met Pro 1 5 3
69 PRT Artificial sequence minibody presentation structure 3 Met
Gly Arg Asn Ser Gln Ala Thr Ser Gly Phe Thr Phe Ser His Phe 1 5 10
15 Tyr Met Glu Trp Val Arg Gly Gly Glu Tyr Ile Ala Ala Ser Arg His
20 25 30 Lys His Asn Lys Tyr Thr Thr Glu Tyr Ser Ala Ser Val Lys
Gly Arg 35 40 45 Tyr Ile Val Ser Arg Asp Thr Ser Gln Ser Ile Leu
Tyr Leu Gln Lys 50 55 60 Lys Lys Gly Pro Pro 65 4 7 PRT Simian
virus 40 4 Pro Lys Lys Lys Arg Lys Val 1 5 5 6 PRT Homo sapiens 5
Ala Arg Arg Arg Arg Pro 1 5 6 10 PRT Mus musculus 6 Glu Glu Val Gln
Arg Lys Arg Gln Lys Leu 1 5 10 7 9 PRT Mus musculus 7 Glu Glu Lys
Arg Lys Arg Thr Tyr Glu 1 5 8 20 PRT Xenopus laevis 8 Ala Val Lys
Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys 1 5 10 15 Lys
Lys Leu Asp 20 9 31 PRT Mus musculus 9 Met Ala Ser Pro Leu Thr Arg
Phe Leu Ser Leu Asn Leu Leu Leu Leu 1 5 10 15 Gly Glu Ser Ile Leu
Gly Ser Gly Glu Ala Lys Pro Gln Ala Pro 20 25 30 10 21 PRT Homo
sapiens 10 Met Ser Ser Phe Gly Tyr Arg Thr Leu Thr Val Ala Leu Phe
Thr Leu 1 5 10 15 Ile Cys Cys Pro Gly 20 11 51 PRT Mus musculus 11
Pro Gln Arg Pro Glu Asp Cys Arg Pro Arg Gly Ser Val Lys Gly Thr 1 5
10 15 Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala
Gly 20 25 30 Ile Cys Val Ala Leu Leu Leu Ser Leu Ile Ile Thr Leu
Ile Cys Tyr 35 40 45 His Ser Arg 50 12 33 PRT Homo sapiens 12 Met
Val Ile Ile Val Thr Val Val Ser Val Leu Leu Ser Leu Phe Val 1 5 10
15 Thr Ser Val Leu Leu Cys Phe Ile Phe Gly Gln His Leu Arg Gln Gln
20 25 30 Arg 13 37 PRT Rattus sp. 13 Pro Asn Lys Gly Ser Gly Thr
Thr Ser Gly Thr Thr Arg Leu Leu Ser 1 5 10 15 Gly His Thr Cys Phe
Thr Leu Thr Gly Leu Leu Gly Thr Leu Val Thr 20 25 30 Met Gly Leu
Leu Thr 35 14 14 PRT Homo sapiens 14 Met Gly Ser Ser Lys Ser Lys
Pro Lys Asp Pro Ser Gln Arg 1 5 10 15 26 PRT Homo sapiens 15 Leu
Leu Gln Arg Leu Phe Ser Arg Gln Asp Cys Cys Gly Asn Cys Ser 1 5 10
15 Asp Ser Glu Glu Glu Leu Pro Thr Arg Leu 20 25 16 20 PRT Rattus
norvegicus 16 Lys Gln Phe Arg Asn Cys Met Leu Thr Ser Leu Cys Cys
Gly Lys Asn 1 5 10 15 Pro Leu Gly Asp 20 17 19 PRT Homo sapiens 17
Leu Asn Pro Pro Asp Glu Ser Gly Pro Gly Cys Met Ser Cys Lys Cys 1 5
10 15 Val Leu Ser 18 5 PRT Artificial sequence lysosomal
degradation sequence 18 Lys Phe Glu Arg Gln 1 5 19 36 PRT
Cricetulus griseus 19 Met Leu Ile Pro Ile Ala Gly Phe Phe Ala Leu
Ala Gly Leu Val Leu 1 5 10 15 Ile Val Leu Ile Ala Tyr Leu Ile Gly
Arg Lys Arg Ser His Ala Gly 20 25 30 Tyr Gln Thr Ile 35 20 35 PRT
Homo sapiens 20 Leu Val Pro Ile Ala Val Gly Ala Ala Leu Ala Gly Val
Leu Ile Leu 1 5 10 15 Val Leu Leu Ala Tyr Phe Ile Gly Leu Lys His
His His Ala Gly Tyr 20 25 30 Glu Gln Phe 35 21 27 PRT Saccharomyces
cerevisiae 21 Met Leu Arg Thr Ser Ser Leu Phe Thr Arg Arg Val Gln
Pro Ser Leu 1 5 10 15 Phe Ser Arg Asn Ile Leu Arg Leu Gln Ser Thr
20 25 22 25 PRT Saccharomyces cerevisiae 22 Met Leu Ser Leu Arg Gln
Ser Ile Arg Phe Phe Lys Pro Ala Thr Arg 1 5 10 15 Thr Leu Cys Ser
Ser Arg Tyr Leu Leu 20 25 23 64 PRT Saccharomyces cerevisiae 23 Met
Phe Ser Met Leu Ser Lys Arg Trp Ala Gln Arg Thr Leu Ser Lys 1 5 10
15 Ser Phe Tyr Ser Thr Ala Thr Gly Ala Ala Ser Lys Ser Gly Lys Leu
20 25 30 Thr Gln Lys Leu Val Thr Ala Gly Val Ala Ala Ala Gly Ile
Thr Ala 35 40 45 Ser Thr Leu Leu Tyr Ala Asp Ser Leu Thr Ala Glu
Ala Met Thr Ala 50 55 60 24 41 PRT Saccharomyces cerevisiae 24 Met
Lys Ser Phe Ile Thr Arg Asn Lys Thr Ala Ile Leu Ala Thr Val 1 5 10
15 Ala Ala Thr Gly Thr Ala Ile Gly Ala Tyr Tyr Tyr Tyr Asn Gln Leu
20 25 30 Gln Gln Gln Gln Gln Arg Gly Lys Lys 35 40 25 4 PRT Homo
sapiens 25 Lys Asp Glu Leu 1 26 15 PRT unidentified adenovirus 26
Leu Tyr Leu Ser Arg Arg Ser Phe Ile Asp Glu Lys Lys Met Pro 1 5 10
15 27 19 PRT Homo sapiens 27 Leu Asn Pro Pro Asp Glu Ser Gly Pro
Gly Cys Met Ser Cys Lys Cys 1 5 10 15 Val Leu Ser 28 15 PRT Homo
sapiens 28 Leu Thr Glu Pro Thr Gln Pro Thr Arg Asn Gln Cys Cys Ser
Asn 1 5 10 15 29 9 PRT Unknown cyclin B1 destruction sequence 29
Arg Thr Ala Leu Gly Asp Ile Gly Asn 1 5 30 20 PRT Unknown signal
sequence from Interleukin-2 30 Met Tyr Arg Met Gln Leu Leu Ser Cys
Ile Ala Leu Ser Leu Ala Leu 1 5 10 15 Val Thr Asn Ser 20 31 29 PRT
Homo sapiens 31 Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe
Gly Leu Leu 1 5 10 15 Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala Phe
Pro Thr 20 25 32 27 PRT Homo sapiens 32 Met Ala Leu Trp Met Arg Leu
Leu Pro Leu Leu Ala Leu Leu Ala Leu 1 5 10 15 Trp Gly Pro Asp Pro
Ala Ala Ala Phe Val Asn 20 25 33 18 PRT Influenza virus 33 Met Lys
Ala Lys Leu Leu Val Leu Leu Tyr Ala Phe Val Ala Gly Asp 1 5 10 15
Gln Ile 34 24 PRT Unknown signal sequence from Interleukin-4 34 Met
Gly Leu Thr Ser Gln Leu Leu Pro Pro Leu Phe Phe Leu Leu Ala 1 5 10
15 Cys Ala Gly Asn Phe Val His Gly 20 35 7 PRT Artificial sequence
stability sequence 35 Met Gly Xaa Gly Gly Pro Pro 1 5 36 5 PRT
Artificial sequence linker consensus sequence 36 Gly Ser Gly Gly
Ser 1 5 37 4 PRT Artificial sequence linker consensus sequence 37
Gly Gly Gly Ser 1
* * * * *