U.S. patent application number 11/361631 was filed with the patent office on 2006-07-06 for methods and compositions for screening for altered cellular phenotypes.
Invention is credited to Peter Chu, Annabelle Friera, Yasumichi Hitoshi, Todd M. Kinsella, X. Charlene Liao, James Lorens, Esteban Masuda, Denise Pearsall.
Application Number | 20060147978 11/361631 |
Document ID | / |
Family ID | 22133216 |
Filed Date | 2006-07-06 |
United States Patent
Application |
20060147978 |
Kind Code |
A1 |
Lorens; James ; et
al. |
July 6, 2006 |
Methods and compositions for screening for altered cellular
phenotypes
Abstract
The invention relates to methods and compositions useful for
screening for altered cellular phenotypes using an inducible
expression system to enrich for and detect the altered phenotypes
and, more particularly, relates to screening libraries of candidate
bioactive agents, for example, nucleic acids and peptides, in cells
using an regulatable expression system to enrich for a
subpopulation of cells having an altered phenotype due to the
presence of a candidate bioactive agent.
Inventors: |
Lorens; James; (Portola
Valley, CA) ; Kinsella; Todd M.; (Redwood City,
CA) ; Masuda; Esteban; (Menlo Park, CA) ;
Hitoshi; Yasumichi; (Mountain View, CA) ; Liao; X.
Charlene; (Palo Alto, CA) ; Pearsall; Denise;
(Belmont, CA) ; Friera; Annabelle; (South San
Francisco, CA) ; Chu; Peter; (San Francisco,
CA) |
Correspondence
Address: |
BOZICEVIC, FIELD & FRANCIS LLP
1900 UNIVERSITY AVENUE
SUITE 200
EAST PALO ALTO
CA
94303
US
|
Family ID: |
22133216 |
Appl. No.: |
11/361631 |
Filed: |
February 24, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10096339 |
Mar 8, 2002 |
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11361631 |
Feb 24, 2006 |
|
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09076624 |
May 12, 1998 |
7001733 |
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10096339 |
Mar 8, 2002 |
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Current U.S.
Class: |
435/6.13 ;
435/6.14; 435/7.2 |
Current CPC
Class: |
C12Q 1/6897 20130101;
G01N 33/5023 20130101; C07K 14/70578 20130101; C07K 2319/00
20130101; G01N 33/5047 20130101; G01N 33/5008 20130101; C12N
2510/00 20130101; C12N 15/1079 20130101; C12N 15/63 20130101; G01N
2500/10 20130101 |
Class at
Publication: |
435/006 ;
435/007.2 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 33/567 20060101 G01N033/567 |
Claims
1-56. (canceled)
57. A screening method comprising: a) selecting from a parental
population of mammalian cells a first population of cells that
exhibit a phenotype in the presence of a test agent; b) selecting
from said first population of cells a second population of cells
that do no exhibit said phenotype in the absence of a test agent;
and c) selecting from said second population of cells a third
population of cells that exhibit said phenotype in the presence of
said test agent.
58. The screening method of claim 57, wherein said test agent is
encoded by a first recombinant nucleic acid in said cell
59. The screening method of clam 57, wherein said test agent is a
polypeptide.
60. The screening method of claim 57, wherein said test agent is a
cyclic polypeptide.
61. The screening method of claim 57, wherein the presence of said
test agent is modulated using an inducible system in which addition
of an inducer increases production of said test agent.
62. The screening method of claim 57, wherein the presence of said
test agent is modulated using a repressible system in which
addition of a repressor decreases production of said test
agent.
63. The screening method of claim 57, wherein said cells are
cultured mammalian cells.
64. The screening method of claim 57, wherein said selecting is
done by fluorescence activated cell sorting (FACS).
65. The screening method of claim 57, wherein said phenotype is
detected by detecting a marker for said phenotype.
66. The screening method of claim 57, wherein said phenotype is an
optically detectable signal produced by a reporter protein.
67. The screening method of claim 66, wherein said reporter protein
is a fluorescent protein.
68. The screening method of claim 66, wherein said reporter protein
is encoded by a second recombinant nucleic acid in said cell.
69. A screening method comprising: a) selecting from a first
population of cells a second population of cells, wherein said
first population of cells exhibit a phenotype in the presence of a
test agent and said second population of cells do no exhibit said
phenotype in the absence of said test agent, and b) selecting from
said second population of cells a third population of cells that
exhibit said phenotype in the presence of said test agent.
70. The screening method of claim 69, wherein said test agent is
encoded by a first recombinant nucleic acid in said cell
71. The screening method of clam 69, wherein said test agent is a
polypeptide.
72. The screening method of claim 69, wherein said test agent is a
cyclic polypeptide.
73. The screening method of claim 69, wherein the presence of said
test agent is modulated using an inducible system in which addition
of an inducer increases production of said test agent.
74. The screening method of claim 69, wherein the presence of said
test agent is modulated using a repressible system in which
addition of a repressor decreases production of said test
agent.
75. The screening method of claim 69, wherein said cells are
cultured mammalian cells.
76. The screening method of claim 69, wherein said selecting is
done by fluorescence activated cell sorting (FACS).
77. The screening method of claim 69, wherein said phenotype is
detected by detecting a marker for said phenotype.
78. The screening method of claim 69, wherein said phenotype is an
optically detectable signal produced by a reporter protein.
79. The screening method of claim 78, wherein said reporter protein
is a fluorescent protein.
80. The screening method of claim 78, wherein said reporter protein
is encoded by a second recombinant nucleic acid in said cell.
Description
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 09/076,624, filed May 12, 1998 (pending).
FIELD OF THE INVENTION
[0002] The invention relates to methods and compositions useful for
screening for altered cellular phenotypes using an inducible
expression system to enrich for and detect the altered phenotypes.
In particular, the invention relates to screening libraries of
candidate bioactive agents, for example, nucleic acids and
peptides, in cells using an regulatable expression system to enrich
for a subpopulation of cells having an altered phenotype due to the
presence of a candidate bioactive agent.
BACKGROUND OF THE INVENTION
[0003] Inducible expression systems have been developed to
facilitate the analysis of gene function in cells and to facilitate
the development of effective treatments using gene therapy. These
expression systems attempt to control nucleic acid expression by
using inducible eukaryotic promoters that are responsive to
inducers such as hormones (Lee et al. (1981) Nature 294:228-232;
Hynes et al. (1981) Proc. Natl. Acad. Sci. USA 78:2038-2042; Klock
et al. (1987) Nature 329:734-736; Israel & Kaufman (1989) Nucl.
Acids Res. 17:2589-2604); heavy metal ions (Mayo et al. (1982) Cell
29:99-108; Brinster et al. (1982) Nature 296:3942; Searle et al.
(1985) Mol. Cell. Biol. 5:1480-1489); or heat shock (Nouer et al.
(1991) in Heat Shock Response, e.d. Nouer, L., CRC, Boca Raton,
Fla., pp167-220). However, these expression systems are problematic
because the eukaryotic promoters can exhibit a high level of basal
expression in the non-induced state; the inducers can promote
pleiotropic effects; and the level of induction can be low.
[0004] In order to overcome these problems, inducible eukaryotic
expression systems utilizing prokaryotic regulatory elements have
been developed. The rationale for using prokaryotic regulatory
elements in a eukaryotic expression system is based on the theory
that effectors modulating the activity of such prokaryotic
regulatory elements would not be responsive to eukaryotic cellular
components. Therefore, pleiotropic effects would be eliminated. An
example of such a system is the lac operator regulatable expression
system. In this system, expression of sequences operably linked to
the lac operator is constitutively induced (or "turned on") by a
LacR-VP16 fusion protein and is repressed (or "turned off") in the
presence of isopropyl-D-thiogalactopyranoside (IPTG) (Labow et al.
(1990), cited supra). In another lac inducible system, the binding
of LacR-VP16 to the operator sequence is enhanced by increasing the
temperature of the cells: However, IPTG in eukaryotic cells is an
inefficient inducer of nucleic acid expression and must be used at
concentrations near cytotoxic levels. Furthermore, increasing the
temperature of the cells is likely to promote pleiotropic effects
in the cells. Thus, there is a need for a more efficient inducible
regulatory system that exhibits rapid and high level induction of
nucleic acid expression; is highly responsive to a specific
exogenous,inducer; and exhibits low levels of expression in the
uninduced state.
[0005] The teteracycline (Tet) inducible system utilizes entirely
prokaryotic components and, thus, pleiotropic effects are avoided
(Gossen et al. (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551;
Gossen et al. (1995) Science 268:1766-1769). In this system, the
inducer is an integral component of a transactivator that binds to
the inducible promoter and drives expression of a nucleic acid of
interest. Thus, the intermediate steps in the induction pathway are
largely eliminated and the control of expression is highly specific
and tightly controlled by contact with the inducer. The
Tet-controlled expression system therefore provides an effective
means for turning off and turning on nucleic acid expression in
cells and, thereby, allowing for the regulated expression of a
nucleic acid in cells.
[0006] With the advent of functional genomics, large numbers of
nucleic acids encoding genes of unknown function have been isolated
and cloned. Consequently, there is a critical need to develop rapid
and highly efficient methods for screening large numbers of
candidate nucleic acids for analysis of gene function and to
identify potential targets for development of therapeutic
agents.
[0007] One approach for studying gene function is to regulate the
expression of a nucleic acid in cells and look for a corresponding
alteration in cellular phenotype. Consequently, rapid and highly
efficient methods of screening diverse populations of cells for an
altered phenotype due to the inducible expression of a candidate
nucleic acid is highly desirable and useful for the analysis of
nucleic acid function and target discovery.
[0008] Thus, it is an object of the present invention to provide
rapid and highly efficient methods of screening populations of
cells for an altered cellular phenotype due to the presence of a
candidate bioactive agent, using an inducible expression system
that permits the regulated expression of candidate nucleic acids
encoding a candidate bioactive agent and permits controlled
expression of the candidate nucleic acid at a defined level.
SUMMARY OF THE INVENTION
[0009] In accordance with the objects described above, the present
invention provides methods and compositions for screening for an
altered cellular phenotype using an inducible expression system to
enrich for and detect cells having an altered phenotype due to the
presence of a candidate bioactive agent. In the methods of the
present invention, detection of cells having an altered cellular
phenotype is achieved by selection of cells responsive to the
induction and repression of expression of a nucleic acid sequence
encoding the candidate bioactive agent. The cells having an altered
cellular phenotype are enriched by multiple rounds of
selection.
[0010] The invention provides methods and compositions for
screening candidate bioactive agents useful as targets for drug
discovery, by accessing molecules and targets within living cells
and provides for the direct selection of those bioactive agents
with desired phenotypic effects. Further, the methods and
compositions of the present invention are particularly useful for
high throughput screening of candidate bioactive agents capable of
altering a cellular phenotype.
[0011] The invention provides compositions for use in the methods
of the present invention. Specifically, the invention provides
populations of cells having a parent phenotype and comprising a
nucleic acid encoding a first element expressed in the cells. The
invention further provides libraries of fusion nucleic acids, where
the fusion nucleic acids comprise a second element that is
regulatable by the first element. The fusion nucleic acids further
comprise a nucleic acid sequence operably linked to the second
element. The nucleic acid sequence encodes a candidate bioactive
agent. In addition, the invention provides a third element that
induces or represses the expression of the nucleic acid sequence
encoding the candidate bioactive agent. Thus, the invention
provides two approaches for screening for an altered cellular
phenotype: in a first approach the third element induces
expression; and in a second approach the third element represses
expression.
[0012] Using either approach, in the methods of the present
invention, the cells having an altered phenotype due to the
presence of the candidate bioactive agent are distinguished and
detected based on their responsiveness to the induction and
repression (in either order) of expression of the nucleic acid
sequence encoding the candidate bioactive agent, by the third
element. The responsive cells have the parent phenotype when
expression of the nucleic acid sequence is repressed and have an
altered phenotype when expression of the nucleic acid sequence is
induced. The nonresponsive cells can be distinguished by having a
phenotype that is not responsive to the induction and repression of
expression of the nucleic acid sequence.
[0013] Using the first approach, the invention provides methods of
screening for an altered cellular phenotype comprising the steps of
a) providing a population of cells having a parent phenotype and
comprising a nucleic acid encoding a first element inducibly or
constitutively expressed in the cells; b) introducing a library of
fusion nucleic acids into the population of cells, where the fusion
nucleic acids comprise a second element that is regulatable by the
first element and further comprise a nucleic acid sequence encoding
a candidate bioactive agent; c) inducing the expression of the
nucleic acid sequence by contacting the cells with a third element;
d) collecting a first subpopulation of cells having an altered
phenotype; e) repressing the expression of the nucleic acid by
modulating the contacting of the third element with the first
subpopulation of cells; f) collecting a second population of cells
having a parent phenotype; g) inducing the expression of the
nucleic acid sequence by contacting the third element with the
second subpopulation of cells; and g) detecting a third
subpopulation of cells.
[0014] Using the second approach, the invention provides methods of
screening for an altered cellular phenotype comprising the steps
of: a) providing a population of cells having a parent phenotype
and comprising a nucleic acid encoding a first element inducibly or
constitutively expressed in the cells; b) introducing a library of
fusion nucleic acids into the population of cells, where the fusion
nucleic acids comprise a second element that is regulatable by the
first element and further comprise a nucleic acid sequence encoding
a candidate bioactive agent; c) inducing the expression of the
nucleic acid sequence by expressing the first element; d)
collecting a first subpopulation of cells having an altered
phenotype; e) repressing the expression of the nucleic acid by
contacting a third element with the first subpopulation of cells;
f) collecting a second population of cells having a parent
phenotype; g) inducing the expression of the nucleic acid sequence
modulating the contacting the third element with the second
subpopulation of cells; and h) detecting a third subpopulation of
cells.
[0015] Using either approach, the invention provides methods of
screening for an altered cellular phenotype further comprising
collecting the responsive cells. The methods also further comprise
repeating the induction or repression of expression and detecting
the responsive cells and, further, collecting the cells to enrich
for a subpopulation of cells having the altered phenotype. The
inducing or repressing of expression, followed by detecting and,
further, by collecting a subpopulation of cells responsive to the
induction and repression can be repeated multiple times to obtain a
desired level of enrichment and detection of a subpopulation of
cells having an altered cellular phenotype due to the presence of a
candidate bioactive agent.
[0016] Examples of a first element include, but are not limited to,
a transactivator. In one aspect, the first element comprises a
tetracycline-dependent transactivator (tTA) or a reverse
tetracycline-dependent transactivator (rtTA). The first element can
be inducible; expressed constitutively, expressed stably or
transiently; and expressed in trans or in cis relative to the
nucleic acid sequence. In one aspect, the fusion nucleic acid
further comprises the nucleic acid encoding the first element.
Examples of a second element include, but are not limited to, an
operator sequence. In one aspect, the operator sequence comprises a
tetracycline operator sequence (TetO). In another aspect the second
element is an oligomer of a TetO sequence. Examples of a third
element include, but are not limited to, a molecule that induces or
represses expression of the nucleic acid sequence. In one aspect,
the molecule comprises tetracycline or analogues thereof, e.g.,
doxycycline (Dox).
[0017] In one aspect, the first element comprises a reverse
tetracycline-dependent activator (rtTA) and expression of the
nucleic acid sequence is induced by contacting the third element
with the cells, and is repressed by modulating the contacting of
the third element with the cells.
[0018] In another aspect, the first element comprises a
tetracycline-dependent activator (tTA) and expression of the
nucleic acid sequence is repressed by contacting the third element
with the cells, and is induced by modulating the contacting of the
third element with the cells.
[0019] Examples of candidate bioactive agents include, but are not
limited to, nucleic acids and polypeptides. Further examples of
bioactive agents are cyclic peptides, RNA, antisense RNA, and DNA.
Additional examples of nucleic acid sequences encoding a candidate
bioactive agent, include but are not limited to, random nucleic
acid sequences, and biased random nucleic acid sequences. Examples
of nucleic acid sequences encoding a candidate bioactive agent,
also include but are not limited to, full-length cDNA sequences,
subsequences of a full-length cDNA, and antisense sequences of a
full-length cDNA. Another example of a nucleic acid sequence
encoding a candidate bioactive agent is a nucleic acid sequence
encoding an amino acid sequence that is in-frame or out-of-frame as
compared to the open reading frame (ORF) encoded by the amino acid
sequence of a full-length cDNA.
[0020] In one aspect, the present invention provides methods
comprising the steps of: a) providing a population of cells having
a parent phenotype and comprising a nucleic acid encoding a first
element that is expressed in the cells, for example, a reverse
tetracycline-dependent transactivator (rtTA); b) introducing into
the population of cells a library of fusion nucleic acids, where
the nucleic acids each comprise a second element that is
regulatable by the first element, and a nucleic acid sequence that
is operably linked to the first element, where the nucleic acid
sequence encodes a candidate bioactive agent; c) inducing the
expression of the nucleic acid sequence by contacting a third
element with the population of cells, wherein the population of
cells is expressing the first element; d) collecting a first
subpopulation of cells having an altered phenotype; e) repressing
the expression of the nucleic acid sequence by modulating the
contacting of the third element with the first subpopulation of
cells; f) collecting a second subpopulation of cells having the
parent phenotype; g) inducing the expression of the nucleic acid
sequence by contacting the third element with the second
subpopulation of cells; and h) detecting a third subpopulation of
cells having the altered phenotype.
[0021] In an additional aspect, the method further comprises: i)
collecting the third subpopulation of cells having an altered
phenotype; j) repressing the expression of the nucleic acid
sequence by modulating the contacting of the third element with the
third subpopulation of cells; and k) detecting a fourth
subpopulation of cells having the parent phenotype.
[0022] In another additional aspect, the method further comprises:
l) collecting the fourth subpopulation of cells having the parent
phenotype; m) inducing the expression of the nucleic acid sequence
by contacting the third element with the fourth subpopulation of
cells; and n) detecting a fifth subpopulation of cells having the
altered phenotype.
[0023] In another aspect, the invention provides a method of
screening for cells having an altered phenotype comprising the
steps of: a) providing a population of cells having a parent
phenotype and comprising a nucleic acid encoding a first element,
for example, a tetracycline-dependent transactivator; b)
introducing into the population of cells a library of fusion
nucleic acids, where the nucleic acids each comprise a second
element that is regulatable by the first element, and a nucleic
acid sequence that is operably linked to the first element, where
the nucleic acid sequence encodes a candidate bioactive agent; c)
inducing the expression of the nucleic acid sequence by expressing
the first element in the population of cells; d) collecting a first
subpopulation of cells having an altered phenotype; e) repressing
the expression of the nucleic acid by contacting a third element
with the first subpopulation of cells; f) collecting a second
subpopulation of cells having the parent phenotype; g) inducing the
expression of the nucleic acid sequence by modulating the
contacting of the third element with the second subpopulation of
cells; and h) detecting a third subpopulation of cells having the
altered phenotype.
[0024] In an additional aspect, the method further comprises: i)
collecting the third subpopulation of cells having the altered
phenotype; j) repressing the expression of the nucleic acid
sequence by contacting the third element with the third
subpopulation of cells; and k) detecting a fourth subpopulation of
cells having the parent phenotype.
[0025] In a further additional aspect, the method further
comprises: l) collecting the fourth subpopulation of cells having
the parent phenotype; m) inducing the expression of the nucleic
acid sequence by modulating the contacting of the third element
with the fourth subpopulation of cells; and n) detecting a fifth
subpopulation of cells having said altered phenotype.
[0026] In one aspect, the first element comprises a reverse
tetracycline-dependent activator (rtTA); the second element
comprises an oligomer of a tetracycline operator sequence (TetO);
and the third element comprises tetracycline or doxycycline.
[0027] In another aspect, the first element comprises a
tetracycline-dependent activator (rtTA); said second element
comprises an oligomer of a tetracycline operator sequence (TetO);
and said third element comprises tetracycline (Tet) or doxycycline
(Dox).
[0028] In a another aspect, the library of fusion nucleic acids
comprises about 10.sup.3 to 10.sup.9 different said nucleic acid
sequences, or 10.sup.4 to 10.sup.8 different random nucleic acid
sequences. In a further aspect, the fusion nucleic acids are each a
component of a retroviral vector.
[0029] In another aspect, the fusion nucleic acids further comprise
a sequence encoding a reporter protein, and this sequence is
operably linked to the nucleic acid sequence encoding the candidate
bioactive agent. Examples of a reporter protein include, but are
not limited to, an autofluorescent protein, for example, a green
fluorescent protein (GFP) from Aqueorea, or a Renilla species.
[0030] In a further aspect, the cells are collected by
fluorescence-activated cell sorting (FACS)
[0031] In a further aspect, the parent phenotype of the cells is
due to the presence of a stimulator and the cells comprise a
stimulator. In a preferred embodiment, the stimulator induces the
parent phenotype. Examples of stimulators include, but are not
limited to, cytokines, ligands or antibodies against cells surface
receptors, growth factors, hormones, peptides, neuropeptides, drugs
or compounds, LPS, viruses, bacteria, and so on. In a preferred
embodiment, the stimulator is interleukin 4 (IL4), a cytokine, that
has various biological activities, for example, IL-4 induces
germline epsilon promoter (transcription) in B cells. In another
preferred embodiment, the stimulator is anti-TCR (T cell receptor),
a stimulator that activates T cell activation as monitored by CD69
upregulation.
[0032] In another aspect, the cells having an altered phenotype are
mammalian cells. Examples of mammalian cells having an altered
phenotype, include but are not limited to, rodent cells and human
cells.
[0033] In another aspect, the altered phenotype is the modulation
of a T cell surface marker, for example, CD3, CD25, CD28, CD40L,
CD69, CD95, or CD95L. Further examples of an altered phenotype,
include but are not limited to, the modulation of cell cycle
regulation, exocytosis, IgE secretion, IgE switching,
antigen-induced B cell differentiation, antigen-induced B cell
isotyping, apoptosis, angiogenesis, and T cell receptor (TCR)
activation.
BRIEF DESCRIPTION OF THE FIGURES
[0034] FIG. 1 schematically depicts the cell line BH1-4 and HBEGF
Diphtheria toxin selection in the cell line.
[0035] FIG. 2 schematically depicts the cell line BH2-A5 and HBEGF
Diphtheria toxin selection in the cell line.
[0036] FIG. 3 depicts the conventional screening method.
[0037] FIG. 4 schematically depicts five different retroviral
vectors which were constructed as positive controls for the
screening assay, and are the encoded GFP, SOCS1, STAT6.DELTA.,
and/or ires are operably linked to a promoter in the retroviral
vector: the retroviral vector cGFP contains the GFP; the retroviral
vector SOCS1-ires-GFP contains, from 5' to 3' (and operably
linked), SOCS1, internal ribosomal entry site (IRES), and GFP; the
retroviral vector GFP-SOCS1 contains, from 5' to 3' (and operably
linked), GFP-SOCS1 fusion; the retroviral vector
STAT6.DELTA.-ires-GFP contains, from 5' to 3' (and operably
linked), STAT6.DELTA., ires, and GFP; and the retroviral vector
GFP-STAT6.DELTA. contains, from 5' to 3' (and operably linked),
GFP-STAT6.DELTA. fusion.
[0038] FIG. 5 depicts the assay of the five retroviral vectors
described in FIG. 4, in BH1-4 cells.
[0039] FIG. 6 depicts the effects of SOCS1 and STAT6 on
IL4/diphtheria (IL4/dip) induced death of BH1-4 cells 6 days post
infection for each of the five retroviral vectors described in FIG.
4.
[0040] FIG. 7 depicts the same results as depicted in FIG. 6 except
that the vertical axis indicates the number of cells, and the
horizontal axis indicates the amount of GFP fluorescence.
[0041] FIG. 8 depicts a screening assaying using spiked cell.
[0042] FIG. 9 schematically depicts the IL4-dip selection assay
using BH1-4 cells and depicts the results of the assay starting
with a 1:10 dilution of the SOCS1-infected cells.
[0043] FIG. 10 depicts the results for screening assays using BH1-4
cells starting with the 1:10, 1:100, 1:1,000, and 1:10,000 dilution
of the SOCS1-infected cells.
[0044] FIG. 11 depicts the results of screening assays for BH1-4
and BH2-A5 cells starting with the 1:10, 1:100, 1:1,000, and
1:10,000 dilution of the spiked cells.
[0045] FIG. 12 depicts the results of selection beginning with
naive cells in a first round of selection with IL4-Dip and then
subjecting the surviving cells from the first round of selection to
a second round of selection with IL4-Dip.
[0046] FIG. 13 depicts a screening assay using a novel cell line
BH2-A5T with the peptide library BFP-C20 encoded by retroviral
vectors.
[0047] FIG. 14 depicts the cell line BH2-A5T (or "A5T" or "A5T-4")
which expresses a Tet regulated transactivator (tTA or Tet
transactivator) and allows for the Tet regulated expression of
candidate bioactive agents introduced into the cells.
[0048] FIG. 15 depicts histograms indicating the amount of GFP
fluorescence, in an experiment where IL4 responders were selected
in the presence and absence of Dox in BH2-A5T cells spiked with
cells infected with the retroviral construct
TRA-SOCS1-ires-GFP.
[0049] FIG. 16 depicts the round to round enrichment for cells
expressing the known inhibitor SOCS1 by induction and repression of
TRA-SOCS1-ires-GFP expression in BH2-A5T-4 cells using dox and
sorting of the altered and parental cellular phenotype.
[0050] FIG. 17 depicts histograms indicating the amount of GFP
fluorescence indicative of TRA-SOCS1-ires-GFP expression in cells
after the first round of selection.
[0051] FIG. 18 depicts histograms showing the "Sort for IL4
responders" step in FIG. 16.
[0052] FIG. 19 depicts histograms showing the "Turn inhibitor
expression back on" step in FIG. 16.
[0053] FIG. 20 depicts for the 1:10,000 dilution of cells, an 6.1%
enrichment resulting from the first round of selection; for the
second round of IL4/dip selection, a 88% enrichment from the
de-doxed Left Gate cells and a 97% enrichment from de-doxed Right
Gate cells from FIG. 19; and for the 1:100,000 dilution of cells,
an 0.3% enrichment resulting from the first round of selection; for
the second round of IL4/dip selection, a 26% enrichment from the
de-doxed Left Gate cells and 47% enrichment from de-doxed Right
Gate cells resulting from the sorting of the cells cultured in the
absence of Dox (FIG. 19).
[0054] FIG. 21 schematically depicts an example of a timeline (in
days) for a screening assay of the present invention, where the
assay involves a first round of selection and sorting; a second
round of selection and sorting; and thereafter single cell clones
are grown. The single cell clones are then subjected to selection
and FACS assays, the nucleic acid encoding the bioactive agent
(e.g., a peptide inhibitor of IL4 signaling/induced expression) is
then rescued and the phenotype is reconfirmed, e.g., by infecting
naive cells with the rescued nucleic acid and selection.
[0055] FIG. 22 schematically depicts an example of a timeline (in
days) for a screening assay of the present invention (and as
described for FIG. 21), where the complexity of the library of
candidate bioactive agents (e.g., a peptide library), and the fold
enrichment for the altered cellular phenotype, are indicated.
Further, FIG. 22 schematically depicts the histogram profile of GFP
fluorescence of false positives due to hereditable background or
stochastic non-hereditable background; as compared to the histogram
profile of GFP fluorescence of cells cultured in the presence
(+Tet) or absence (-Tet) of Tet, after a first round of
selection.
[0056] FIG. 23 schematically depicts an example of a timeline (in
days) for a screening assay of the present invention (and as
described for FIG. 21), where the complexity of the library of
candidate bioactive agents (e.g., a peptide library), and the fold
enrichment for the altered cellular phenotype, are indicated.
Further, after a second of selection, cells are single cell cloned,
aliquoted into microtiter plates, replica plated in duplicate
microtiter plates and cultured in the presence or absence of Dox,
and the single clones are contacted with IL4 for three days and
their GFP fluorescence measured by FACS.
[0057] FIG. 24 depicts the histogram profile of GFP fluorescence of
clones from a functional screen representing a BFP-peptide
inhibitor clone, CR2 (left panel); a hereditable background clone
(middle panel), and stochastic background clone (right panel),
where the histograms from the clones cultured in the presence of
Dox (+Dox) and the absence of Dox (-Dox) are overlayed.
[0058] FIG. 25 depicts the summary of the results from the peptide
screening in BH2-A5T-4 cells.
[0059] FIG. 26 depicts cell line and assay development. FIG. 26A
depicts Jurkat cells stimulated with anti-T cell receptor (TCR)
antibody C305 at 300 ng/ml and 24 hrs later, cells stained with
anti-CD69APC and analyzed on a FACSCalibur. The dashed line
indicates CD69 level before stimulation and the solid line after
stimulation. FIG. 26B depicts Jurkat clone (4D9) with optimal CD69
expression profile infected with a retroviral construct which
constitutively expresses a tetracycline transactivator protein
(tTA) and a reporter construct which expresses Lyt2 driven by a
tetracycline responsive element (TRE). The tTA-Jurkat cell clone
4D9#32 was obtained by sorting for high Lyt2 expression in the
absence of Doxycycline (Dox) and low expression of Lyt2 in the
presence Doxycycline (10 ng/ml). The solid line indicates Lyt2
level with Dox and dashed line without Dox.
[0060] FIG. 27 depicts dominant negative mutants of ZAP-70
inhibited TCR-induced CD69 expression. FIG. 27A depicts ZAP70 KI
and ZAP70 SH2 (N+C) subcloned downstream of the internal ribosome
entry site (IRES), followed by GFP in the Tet-regulated retroviral
vector (TRE). FIG. 27B depicts after infecting tTA-Jurkat cells
with retroviral constructs containing IRES-GFP, ZAP70 KI-IRES-GFP,
or ZAP70 SH2 (N+C)-IRES-GFP, cells left unstimulated or stimulated
with anti-TCR antibody for 24 hours. CD69 expression data were
analyzed after gating on the GFP.sup.+ population (infected
population, shown in R1). The dashed line and the thin line
indicate cells infected with IRES-GFP (vector) before and after TCR
stimulation, respectively, and the thick line indicates cells
infected with ZAP70 KI-IRES-GFP (top panel) or ZAP70 SH2
(N+C)-IRES-GFP (bottom panel), both after TCR stimulation. FIG. 27C
depicts after infecting tTA-Jurkat cells with retroviral constructs
containing IRES-GFP (vector) or ZAP70 SH2 (N+C)-IRES-GFP, cells
treated with Dox for 6 days, and then left unstimulated or
stimulated with anti-TCR antibody for 24 hrs. Addition of Dox
turned off GFP expression, as shown by the loss of GFP.sup.+ cells
in the region R1. CD69 expression data were analyzed on the entire
cell population. The dashed line and the thin line indicate cells
infected with IRES-GFP (vector) before and after TCR stimulation,
respectively, and the thick line indicates cells infected with ZAP
7 0 SH2 (N+C)-IRE S-GFP after TCR stimulation. FIG. 27D depicts tTA
Jurkat cells containing different retroviral constructs (shown
above the lanes) cultured in the absence (-) or presence (+) of Dox
and lysed. Whole cell lysates were loaded (100 g each lane) and
analyzed by Western blotting using anti-ZAP70 antibody (Upstate
Biotechnology catalog # 05-253).
[0061] FIG. 28 depicts a screen for inhibitors of TCR-activation
induced CD69 expression. FIG. 28A depicts a scheme of the
functional genetic screen for inhibitors of TCR-activation induced
CD69 expression. 3.5.times.10.sup.8 cells were infected with
pTRA-cDNA libraries. CD69.sup.lowCD3.sup.+ cells represent cells
expressing the lowest level of CD69 (bottom 3%) and still retaining
CD3 expression after TCR stimulation, whereas CD69.sup.high cells
are those expressing a high level of CD69 (top 10%) after grown
with Dox and after TCR stimulation. Single cell cloning took place
after at least 4 consecutive sorting of CD69.sup.lowCD3.sup.+ with
or without the placement of sorting of CD69.sup.high cells in
between. FIG. 28B depicts phenotypic enrichment via iterative cell
sorting 7.1.times.10.sup.8 cells were sorted with high-speed flow
sorters (MoFlo) after stimulation and staining with anti-CD69-APC
and anti-CD3-PE. The sort gate was set at the equivalent of 1% of
the control cells that were stimulated but were never flow-sorted
(shown as R2) to enrich for the CD69.sup.lowCD3.sup.+ phenotype.
After sorting, the desired cells were allowed to rest for a week
before another round of stimulation and sorting. With reiterative
sorting, not only the desired population was enriched (R2 cells
from 1% to 23.2%), but also the overall population demonstrated a
reduced CD69 level (shown as Y geo mean from >300 to 65). FIG.
28B depicts Dox regulation of the sorted population. Cells were
split to two populations after the third round of sorting for the
CD69.sup.lowCD3.sup.+ phenotype (shown as R2). One half of the
cells were grown in the absence of Dox (top left dot plot) while
the other half in the presence of Dox (top right dot plot). A week
later, CD69 expression was compared following anti-TCR stimulation.
The dashed line indicates CD69 level without Dox and the solid line
with Dox.
[0062] FIG. 29 depicts the identification of clones with desired
altered phenotype. FIG. 29A depicts Individual clones grown in the
presence or absence of Dox for a week and then stimulated overnight
with anti-TCR antibody. Cells were stained with anti-CD69-APC and
analyzed on a FACSCalibur. The filled peaks indicate CD69
expression level in the absence of Dox, when the cDNA hits were
expressed. The open peaks indicate CD69 expression level in the
presence of Dox, when the cDNA hits were not expressed. The
geometric means of CD69 histograms are as follows: 490.68 (+Dox)
and 28.60 (-Dox) for clone 15; 658.45 (+Dox) and 52.98 (-Dox) for
clone 24; 553.46 (+Dox) and 40.09 (-Dox) for clone 64; 433.44
(+Dox) and 82.2 (-Dox) for clone 116; 1235.77 (+Dox) and 17.68
(-Dox) for clone 157; and finally, 245.81 (+Dox) and 26.43 (-Dox)
for clone 194. The difference between filled and open peaks in any
given clone is represented by the Dox ratio of the CD69 geometric
means (using the +Dox values divided by the -Dox values), which
reflects the dependence of the altered phenotype on the cDNA
expression. FIG. 29B depicts 2,828 cell clones were assayed for
CD69 expression after stimulation in the presence or absence of
Dox; The geometric mean of CD69 fluorescent units in the presence
of Dox was divided by those in the absence of Dox to give rise to
the Dox Ratio for individual clones. A total of 1323 clones showed
a Dox ratio of >1.5. The numbers of clones showing a Dox ratio
between 1.5-10 was plotted against the Dox ratio themselves to
illustrate the population distribution. FIG. 29C depicts DNA
oligonucleotide primers specific to the library vector were
designed (BstXTRA5G and BstXTRA3D) and used in RT-PCR reactions.
The RT-PCR products were analyzed in an agarose gel followed by
ethidium blue staining. Data from representative clones were shown
along side the lkb DNA molecular weight ladder from New England
BioLabs (Catalog # N3232S).
[0063] FIG. 30 depicts the functional transfer of the phenotype of
known TCR signaling molecules. Diagrams of proteins predicted from
the cDNA inserts and those from the corresponding wild-type genes
were shown above the histograms. The left panel of histograms shows
the Dox-regulatable phenotype of the original cell clones. The
original cell clones were grown in the presence or absence of Dox
for a week and then stimulated overnight with anti-TCR antibody.
Cells were stained with anti-CD69-APC and a.eta.alyzed by FACS. The
filled peaks indicate CD69 expression level in the absence of Dox,
when the cDNA hits were expressed. The open peaks indicate CD69
expression level in the presence of Dox, when the cDNA hits were
not expressed. The Dox ratio was shown for each original mutant
clone. The right top and bottom panels of histograms show the
phenotypes after expressing the cDNA inserts (followed by IRES-GFP)
in a naive tTAJurkat population. After retroviral infection, the
tTA-Jurkat cells were either stimulated with the anti-TCR antibody
(+''-TCR, solid line) or left unstimulated (-''-TCR, dashed line),
and analyzed by FACS for CD69 induction after staining with
anti-CD69-APC. The top right histogram in each group analyzed
GFP.sup.- cells, which did not express the cDNA hit, whereas the
bottom right histogram in each group analyzed GFP.sup.+ cells,
which expressed the cDNA hit. The following cDNA hits were
analyzed: LCK (A), ZAP70 hit #1 (B), SYK (C), and PLCyl (D).
[0064] FIG. 31 depicts the functional transfer of phenotype of
unknown TCR signaling molecules. Diagrams of proteins predicted
from the cDNA inserts and those from the corresponding wild-type
genes were shown above the histograms. The left panel of histograms
shows the Dox-regulatable phenotypes of the original cell clones.
The original cell clones were grown in the presence or absence of
Dox for a week and then stimulated overnight with anti-TCR
antibody. Cells were stained with anti-CD69-APC and analyzed by
FACS. The filled peaks indicate CD69 expression level in the
absence of Dox, when the cDNA hits were expressed. The open peaks
indicate CD69 expression level in the presence of Dox, when the
cDNA hits were not expressed. The Dox ratio was shown for each
original mutant clone. The right top and bottom panels of
histograms show the phenotypes after expressing the cDNA inserts
(followed by IRES-GFP) in a naive tTAJurkat population. After
retroviral infection, the tTA-Jurkat cells were either stimulated
with the anti-TCR antibody (+''-TCR, solid line) or left
unstimulated (-''-TCR, dashed line), and analyzed by FACS for CD69
induction after staining with anti-CD69-APC. The top right
histogram in each group analyzed GFP.sup.- cells, which did not
express the cDNA hit, whereas the bottom right histogram in each
group analyzed GFP.sup.+ cells, which expressed the cDNA hit. The
following cDNA hits were analyzed: TCPTP (A), IL1 ORA (B), Integrin
''2 (C), and GG2-I (D).
[0065] FIG. 32 depicts the cDNA hits from the screening methods of
the present invention inhibited T cell activation in human primary
T lymphocytes. FIG. 32A depicts retroviral infection of primary T
lymphocytes. Primary T lymphocytes were cultured on anti-CD3 and
anti-CD28 coated wells for 3 days and then infected with the
retroviral CRU5-GFP vector, where GFP was expressed from the
constitutively active retroviral LTR promoter. Cells were stained
with anti-CD3-APC, or with anti-CD4-PE and anti-CD8-APC antibodies
and analyzed by FACS. The percentage of cells in each quadrant is
shown. FSC: forward scatter; and SSC: side scatter. FIG. 32B
depicts human primary T lymphocytes were infected with vector alone
(CRU5-GFP and CRU5-IRES-GFP or CIG) or with vector expressing the
Lck and PLC(1 dominant negative (DN) hits. The infection rate was
monitored by the percentage of GFP.sup.+ cells in M1. The geometric
mean of GFP was shown above the marker. FIG. 32C depicts IL-2
production was inhibited by LCK DN and PLC(1 DN proteins in primary
T lymphocytes. Infected primary T cells were allowed to rest and
then sorted to give rise to GFP.sup.- (filled bars) and GFP.sup.+
(open bars) populations. Equal numbers of cells were cultured in
96-well dish coated with anti-CD3 or anti-CD3+anti-CI)28
antibodies, cultured without antibodies or cultured with
PMA+ionomycin. 40 hrs later the culture supernatants were harvest
and assayed for IL-2 production by ELISA using commercial reagents
(R&D Systems).
[0066] FIG. 33 depicts an overview of identified molecular targets
as described in Example 7 and designated Table 2.
DETAILED DESCRIPTION OF THE INVENTION
[0067] The present invention provides methods and compositions for
screening for an altered cellular phenotype due to the presence of
a candidate bioactive agent. The methods of the present invention
provide a significant improvement over conventional screening
techniques because the methods allow the rapid and highly efficient
screening of large numbers of candidate bioactive agents in cells
without the need to collect or synthesize the candidate bioactive
agents. In particular, the methods of the present invention afford
significant advantages over conventional screening techniques
because the methods permit the tightly controlled and highly
specific expression of a candidate nucleic acid in cells, and rapid
detection of a subpopulation of cells highly enriched for a
cellular phenotype corresponding to the controlled expression of
the candidate nucleic acid. In particular, multiple rounds of
selection can be performed to achieve the desired enrichment of
cells having an altered phenotype corresponding to the expression
of a candidate nucleic acid. Thus, the advantages of the methods of
the present invention include the ability to rapidly and
efficiently screen diverse populations of cells for an altered
cellular phenotype corresponding to the inducible expression of a
candidate nucleic acid.
[0068] The invention provides compositions for use in the methods
of the present invention. Specifically, the invention provides
populations of cells having a parent phenotype and comprising a
nucleic acid encoding a first element expressed in the cells. The
first element is preferably a transactivator, and more preferably a
Tet-dependent transactivator. The invention further provides
libraries of fusion nucleic acids, where the fusion nucleic acids
comprise a second element that is regulatable by the first element.
The second element preferably comprises an operator sequence, and
more preferably at least one Tet operator (TetO) sequence. The
fusion nucleic acids further comprise a nucleic acid sequence
operably linked to the second element. The nucleic acid sequence
preferably encodes a candidate bioactive agent. The candidate
bioactive agent is preferably a peptide or a nucleic acid. In
addition, the invention provides a third element that induces or
represses the expression of the nucleic acid sequence encoding the
candidate bioactive agent. The third element is preferably Tet or
an analogue thereof. Thus, the invention provides two approaches
for screening for an altered cellular phenotype; in a first
approach the third element induces ("turns on" expression; and in a
second approach the third element represses ("turns off")
expression.
[0069] Thus, generally, the invention works as follows. Cells
exhibiting the parent phenotype are induced to express the
candidate agents, and the cells are screened for those exhibiting
an altered phenotype. Once identified and isolated as a first
subpopulation, the expression of the candidate agent is turned off,
and the first subpopulation is again screened for cells exhibiting
the parent phenotype. This ensures a higher level of confidence
that the altered phenotype is due to the candidate agent. This
second subpopulation is again induced (or un-repressed, as the case
may be) to express the candidate agent to result in the altered
phenotype. Thus, by screening for "off-on-off" (or, in some cases
as outlined herein, "on-off-on", etc., a more reproducible data set
with a higher level of confidence is achieved. In addition, further
reiterative rounds allow additional results. In general, this
allows the elimination of non-responsive cells and thus "false
positives".
[0070] Using either approach, in the methods of the present
invention, the cells having an altered phenotype due to the
presence of the candidate bioactive agent are distinguished and
detected based on their responsiveness to the induction and
repression of expression of the nucleic acid sequence encoding the
candidate bioactive agent, by the third element. The responsive
cells have the parent phenotype when expression of the nucleic acid
sequence is repressed and have an altered phenotype when expression
of the nucleic acid sequence is induced. The nonresponsive cells
can be distinguished by having a phenotype that is not responsive
to the induction and repression of expression of the nucleic acid
sequence. As used herein, the term "parent phenotype" refers to the
cellular phenotype of a cell in the uninduced state, i.e., the
expression of a nucleic acid sequence operably linked to a second
element is turned off or repressed in the cell. As used herein, the
term "altered phenotype" refers to the cellular phenotype of a cell
in the induced state, i.e., the expression of a nucleic acid
sequence operably linked to a second element is turned on or
induced in the cell.
[0071] In a preferred embodiment, the parent phenotype is due to
the presence of a stimulator. In another preferred embodiment, the
stimulator induces the parent phenotype. Examples of stimulators
include, but are not limited to, cytokines, ligands or antibodies
against cells surface receptors, growth factors, hormones,
peptides, neuropeptides, drugs or compounds, LPS, viruses,
bacteria, and so on. In a preferred embodiment, the stimulator is
interleukin 4 (IL-4), a cytokine, that has various biological
activities, for example, IL-4 induces germline epsilon promoter
(transcription) in B cells. In another preferred embodiment, the
stimulator is anti-TCR (T cell receptor), a stimulator that
activates T cell activation as monitored by CD69 upregulation.
[0072] In a preferred embodiment, the fusion nucleic acids further
comprise a sequence encoding a reporter protein, and this sequence
is operably linked to the nucleic acid sequence encoding the
candidate bioactive agent. Thus, the expression of the reporter
protein corresponds to the expression of the nucleic acid sequence
encoding a candidate bioactive agent. Suitable reporter proteins
are outlined below.
[0073] Cells having an altered phenotype may be sorted from cells
having a parent phenotype using standard techniques known in the
art for cell sorting. In the methods of the present invention, any
chemical, physical, or physiological markers distinguishing an
altered phenotype from a parent phenotype may be used to sort a
population or subpopulation of cells having a parent phenotype from
a subpopulation of cells having an altered phenotype. Thereby, a
subpopulation of cells having an altered phenotype can be detected
and collected. In a preferred embodiment, the fusion nucleic acids
further comprise a sequence encoding a reporter protein that is an
autofluorescent protein, for example GFP from a Renilla species,
and the cells having an altered phenotype are sorted from the cells
having a parent phenotype by fluorescence-activated cell sorting
(FACS). In this embodiment, the sequence encoding the reporter
protein is operably linked to a nucleic acid sequence encoding a
candiate bioactive agent.
[0074] Using the first approach, the invention provides methods of
screening for an altered cellular phenotype comprising the steps
of: a) providing a population of cells having a parent phenotype
and comprising a nucleic acid encoding a first element inducibly or
constitutively expressed in the cells; b) introducing a library of
fusion nucleic acids into the population of cells, where the fusion
nucleic acids comprise a second element that is regulatable by the
first element and further comprise a nucleic acid sequence encoding
a candidate bioactive agent; c) inducing the expression of the
nucleic acid sequence by contacting the cells with a third element;
d) collecting a first subpopulation of cells having an altered
phenotype; e) repressing the expression of the nucleic acid by
modulating the contacting of the third element with the first
subpopulation of cells; f) collecting a second population of cells
having a parent phenotype; g) inducing the expression of the
nucleic acid sequence by contacting the third element with the
second subpopulation of cells; and g) detecting a third
subpopulation of cells.
[0075] Using the second approach, the invention provides methods of
screening for an altered cellular phenotype comprising the steps
of: a) providing a population of cells having a parent phenotype
and comprising a nucleic acid encoding a first element inducibly or
constitutively expressed in the cells; b) introducing a library of
fusion nucleic acids into the population of cells, where the fusion
nucleic acids comprise a second element that is regulatable by the
first element and further comprise a nucleic acid sequence encoding
a candidate bioactive agent; c) inducing the expression of the
nucleic acid sequence by expressing the first element; d)
collecting a first subpopulation of cells having an altered
phenotype; e) repressing the expression of the nucleic acid by
contacting a third element with the first subpopulation of cells;
f) collecting a second population of cells having a parent
phenotype; g) inducing the expression of the nucleic acid sequence
modulating the contacting the third element with the second
subpopulation of cells; and h) detecting a third subpopulation of
cells.
[0076] Using either approach, the invention provides methods of
screening for an altered cellular phenotype further comprising
collecting the responsive cells. The methods also further comprise
repeating the induction or repression of expression and detecting
the responsive cells and, further, collecting the cells to enrich
for a subpopulation of cells having the altered phenotype. The
inducing or repressing of expression, followed by detecting and,
further, by collecting a subpopulation of cells responsive to the
induction and repression can be repeated multiple times to obtain a
desired level of enrichment and detection of a subpopulation of
cells having an altered cellular phenotype due to the presence of a
candidate bioactive agent.
[0077] In a preferred embodiment, using the first approach, the
present invention provides methods comprising the steps of: a)
providing a population of cells having a parent phenotype and
comprising a nucleic acid encoding a first element that is
expressed in the cells, for example, a reverse
tetracycline-dependent transactivator (rtTA); b) introducing into
the population of cells a library of fusion nucleic acids, where
the nucleic acids each comprise a second element that is
regulatable by the first element, and a nucleic acid sequence that
is operably linked to the first element, where the nucleic acid
sequence encodes a candidate bioactive agent; c) inducing the
expression of the nucleic acid sequence by contacting a third
element with the population of cells, wherein the population of
cells is expressing the first element; d) collecting a first
subpopulation of cells having an altered phenotype; e) repressing
the expression of the nucleic acid sequence by modulating the
contacting of the third element with the first subpopulation of
cells; f) collecting a second subpopulation of cells having the
parent phenotype; g) inducing the expression of the nucleic acid
sequence by contacting the third element with the second
subpopulation of cells; and h) detecting a third subpopulation of
cells having the altered phenotype.
[0078] In another preferred embodiment, using the first approach,
the method further comprises: i) collecting the third subpopulation
of cells having an altered phenotype; j) repressing the expression
of the nucleic acid sequence by modulating the contacting of the
third element with the third subpopulation of cells; and k)
detecting a fourth subpopulation of cells having the parent
phenotype.
[0079] In another additional preferred embodiment, using the first
approach, the method further comprises: l) collecting the fourth
subpopulation of cells having the parent phenotype; m) inducing the
expression of the nucleic acid sequence by contacting the third
element with the fourth subpopulation of cells; and n) detecting a
fifth subpopulation of cells having the altered phenotype.
[0080] In another preferred embodiment, using the second approach,
the invention provides a method of screening for cells having an
altered phenotype comprising the steps of: a) providing a
population of cells having a parent phenotype and comprising a
nucleic acid encoding a first element, for example, a
tetracycline-dependent transactivator; b) introducing into the
population of cells a library of fusion nucleic acids, where the
nucleic acids each comprise a second element that is regulatable by
the first element, and a nucleic acid sequence that is operably
linked to the first element, where the nucleic acid sequence
encodes a candidate bioactive agent; c) inducing the expression of
the nucleic acid sequence by expressing the first element in the
population of cells; d) collecting a first subpopulation of cells
having an altered phenotype; e) repressing the expression of the
nucleic acid by contacting a third element with the first
subpopulation of cells; f) collecting a second subpopulation of
cells having the parent phenotype; g) inducing the expression of
the nucleic acid sequence by modulating the contacting of the third
element with the second subpopulation of cells; and h) detecting a
third subpopulation of cells having the altered phenotype.
[0081] In another embodiment, using the second approach, the method
further comprises: i) collecting the third subpopulation of cells
having the altered phenotype; j) repressing the expression of the
nucleic acid sequence by contacting the third element with the
third subpopulation of cells; and k) detecting a fourth
subpopulation of cells having the parent phenotype.
[0082] In another embodiment, using the second approach, the method
further comprises: l) collecting the fourth subpopulation of cells
having the parent phenotype; m) inducing the expression of the
nucleic acid sequence by modulating the contacting of the third
element with the fourth subpopulation of cells; and n) detecting a
fifth subpopulation of cells having said altered phenotype.
[0083] Accordingly, the invention provides methods for screening
cells having an altered phenotype as compared to a parent
phenotype. As used herein, the term "parent phenotype" refers to
the cellular phenotype of a cell in the uninduced state, i.e., the
expression of a nucleic acid sequence operably linked to a second
element is turned off or repressed in the cell. As used herein, the
term "altered phenotype" refers to the cellular phenotype of a cell
in the induced state, i.e., the expression of a nucleic acid
sequence operably linked to a second element is turned on or
induced in the cell.
[0084] In a preferred embodiment, the parent phenotype is due to
the presence of a stimulator. A stimulator as used herein is an
agent that can cause phenotypic changes. In another preferred
embodiment, the stimulator induces the parent phenotype. Examples
of stimulators include, but are not limited to, cytokines, ligands
or antibodies against cells surface receptors, growth factors,
hormones, peptides, neuropeptides, drugs or compounds, LPS,
viruses, bacteria, and so on (see e.g., Lorens et al. (2001)
Pharmaceutical Biotechnology, pp. 613-421). In a preferred
embodiment, the stimulator is interleukin 4 (IL4), a cytokine, that
has various biological activities, for example, IL-4 induces
germline epsilon promoter (transcription) in B cells. In another
preferred embodiment, the stimulator is anti-TCR (T cell receptor),
a stimulator that activates T cell activation as monitored by CD69
upregulation.
[0085] The methods provide populations of cells. 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 below, 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 because
the methods of the present invention can be used to enrich for and
detect cells that exhibit an altered phenotype as a consequence of
the presence of a bioactive agent within the cell.
[0086] Accordingly, suitable 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.
[0087] In one embodiment, the cells may be genetically engineered,
that is, contain exogeneous nucleic acid, for example, to contain
target molecules.
[0088] By a "plurality of cells" or a "population 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 member of the retroviral molecular
library, i.e., a different candidate nucleic acid, 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 candidate nucleic acids into a plurality of cells, the
distribution of candidate nucleic acids within the individual cell
members of the cellular library may vary.
[0089] The methods rely on regulatable expression systems. Suitable
regulatable expression systems for use in the methods of the
present event are those having the following properties: a low
level of basal expression in the non-induced state; inducers that
do not promote pleiotropic effects; high levels of expression in
the induced state; highly specific induction of expression of a
candidate nucleic acid of interest; and modulation of the level of
induced expression. Examples of regulatable expression systems
having such properties include, but are not limited to: a Tet
inducible system (see e.g., Gossen et al. (1992) Proc. Natl. Acad.
Sci. USA 89:5547-5551; Gossen et al. (1995) Science 268:1766-1769);
a FK506/rapamycin inducible system (see e.g., Spencer et al. (1993)
Science 262:1019-1024; Belshaw et al. (1996) Proc. Natl. Acad. Sci.
USA 93:4604-4607); a RU486/mifepristone inducible system; and an
ecdysone inducible system (for review, see Rossi et al. (1989)
Curr. Op. Biotech. 9:451-456).
[0090] In a preferred embodiment, a Tet inducible expression system
is used in the methods of the present invention (see e.g., Gossen
et al. (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Gossen et
al. (1995) Science 268:1766-1769). In a Tet inducible expression
system, a Tet-dependent transactivator binds to a Tet operator
(TetO) sequence and activates the expression of a nucleic acid
sequence operably linked to the TetO sequence. Various
Tet-dependent transactivators are known and either bind to the
operator sequence in the presence or the absence of Tet or-an
analogue thereof. Thus, Tet or an analogue thereof, acts as an
agent mediating the binding of a transactivator to the operator
sequence and the expression of an operably linked nucleic acid
sequence. An example of a tetracycline-dependent transactivator
that binds to a TetO sequence in the presence of Tet, and not in
the absence of Tet, is the reverse tetracycline-dependent
transactivator (rtTA). An example of a tetracycline-dependent
transactivator that binds to the Tet) sequence in the absence of
Tet, and not in the presence of Tet, is the tetracycline-dependent
transactivator (tTA).
[0091] In the methods of the present invention, the first element
regulates a second element that is operably linked to a nucleic
acid sequence encoding a candidate bioactive agent. Preferably, the
first element is a transactivator. In a preferred embodiment, the
first element comprises the rtTA. In another preferred embodiment,
the first element comprises the tTA. The first element can be
expressed inducibly, constitutively, stably, transiently; or in
trans or in cis relative to a nucleic acid sequence that is
operably linked to a second element. In another preferred
embodiment, the transactivator is a polypeptide, and even more
preferably, the transactivator is a fusion protein. Thus, one
aspect of the invention relates to fusion proteins and nucleic
acids encoding fusion proteins. The term "fusion protein" as used
herein refers to at least two polypeptides which are operably
linked, either directly or indirectly, using a linker.
[0092] In a preferred embodiment, the transactivator fusion protein
comprises a first polypeptide that binds to a Tet operator sequence
in the presence of tetracycline (Tet) or an analogue thereof; and a
second polypeptide that activates expression of a nucleic acid
sequence operably linked to an operator sequence. The first
polypeptide of the transactivator fusion protein is preferably a
mutated Tet repressor.
[0093] The wild-type Tet repressor (TetR) is a component of the E.
coli tetracycline (Tc) resistance system. Wild-type TetR binds to
TetO sequences in the absence of Tet or an analogue thereof and
represses expression of nucleic acid sequences operably linked to
the TetO sequences (Gatz, C. et al. (1992) Plant J. 2:397-404).
[0094] The term "mutated TetR" or "mutant TetR" as used herein
includes polypeptides having an amino acid sequence which is
similar to a wild-type TetR but which has at least one amino acid
difference from the wild-type TetR. The term "wild-type TetR" as
used herein describes a naturally occurring protein which represses
transcription from TetO sequences in prokaryotic cells in the
absence of Tet or an analogue thereof. The amino acid difference(s)
between a mutated TetR and a wild-type TetR may be a substitution
of one or more amino acids, deletion of one or more amino acids, or
addition of one or more amino acids.
[0095] A suitable mutated TetR for use in the transactivator fusion
proteins of the present invention binds to a TetO sequence, i.e.,
it retains the DNA binding specificity of a wild-type Tet
repressor; and regulates expression in a reverse or opposite manner
as compared to a wild-type TetR, i.e., the mutated TetR binds to a
TetO sequence only in the presence of Tet or analogue thereof,
rather than in the absence of Tet.
[0096] A mutated TetR having the functional properties described
above can be constructed by substitution of amino acid residues in
the sequence of a wild-type TetR. For example, a Tn10-derived TetR
having amino acid substitutions at amino acid positions 71, 95, 101
and 102 has the desired functional properties and thus can be used
as the first polypeptide in the transactivator fusion protein of
the invention. These and other amino acid substitutions, deletions
or additions at these or other amino acid positions which retain
the desired functional properties of the mutated TetR are known in
the art (see, e.g., U.S. patent Ser. No. 6,136,954).
[0097] Further, the crystal structure of a TetR-Tet complex, as
described in Hinrichs, W. et al. (1994) Science 264:418-420, can be
used for rational design of mutated Tet repressors. Amino acid
positions 95, 101 and 102 are located within the conserved Tet
binding pocket. Thus, the Tet binding pocket of a TetR may mutated
to generate a mutated TetR suitable for inclusion in a
transactivator fusion protein of the present invention.
[0098] Additional suitable mutated TetR can be constructed
according to the teachings of the invention and in the references
cited herein. A number of different classes of TetR have been
described, e.g., A, B, C, D and E (of which the Tn10-encoded
repressor is a class B repressor). The amino acid sequences of the
different classes of TetR share a high degree of homology (i.e.,
40-60% across the length of the proteins), including in the region
encompassing the above-described mutations. The amino acid
sequences of various classes of TetR are described in Tovar, K. et
al. (1988) Mol. Gen. Genet. 215:76-80. Accordingly, equivalent
mutations to those described above for the Tn10-derived TetR can be
made in other classes of TetR for inclusion in a transactivator
fusion protein of the invention. Suitable equivalent mutations will
be apparent to those skilled in the art and can be constructed and
tested for functionality by procedures described herein or in the
cited references. Nucleotide and amino acid sequences of Tet
repressors of the A, C, D and E classes are disclosed in Waters, S.
H. et al. (1983) Nucl. Acids Res. 11:6089-6105, Unger, B. et al.
(1984) Nucleic acid 31:103-108, Unger, B. et al. (1984) Nucl. Acids
Res. 12:7693-7703 and Tovar, K. et al. (1988) Mol. Gen. Genet.
215:76-80, respectively. These wild-type TetR sequences can be
mutated according to the teachings herein and in the cited
references, for inclusion in the transactivator fusion protein of
the present invention.
[0099] Additional suitable mutated Tet repressors (i.e., having the
desired functional properties described above) can be constructed
by mutagenesis of a wild-type TetR using methods known in the art.
The nucleotide and amino acid sequences of wild-type class B Tet
repressors are disclosed in Hillen, W. and Schollmeier, K. (1983)
Nucl. Acids Res. 11:525-539 and Postle, K. et al. (1984) Nucl.
Acids Res. 12:4849-4863. The nucleotide and amino acid sequences of
wild-type class A, C, D and E type repressors are cited above. A
mutated TetR can be created and selected, for example as follows: a
nucleic acid (e.g., DNA) encoding a wild-type TetR is subjected to
random mutagenesis and the resultant mutated nucleic acids are
incorporated into an expression vector and introduced into a host
cell for screening. A screening assay is used which allows for
selection of a TetR which binds to a Tet operator sequence only in
the presence of Tet or an analogue thereof. For example, a library
of mutated nucleic acids in an expression vector can be introduced
into an E. coli strain in which TetO sequences control the
expression of a nucleic acid encoding a Lac repressor and the Lac
repressor controls the expression of a nucleic acid encoding an
selectable marker (e.g., drug resistance). Binding of a TetR to
TetO sequences in the bacteria will inhibit expression of the Lac
repressor, thereby inducing expression of the selectable marker
gene. Cells expressing the marker nucleic acid are selected based
upon the selectable phenotype (e.g., drug resistance). For
wild-type TetR, expression of the selectable marker nucleic acid
will occur in the absence of Tet. A nucleic acid encoding a mutated
TetR is selected using this system based upon the ability of the
nucleic acid to induce expression of the selectable marker nucleic
acid in the bacteria only in the presence of Tet.
[0100] As mentioned above, the first polypeptide of the
transactivator fusion protein (e.g., the mutated TetR) has the
property of binding specifically to a TetO sequence. Each class of
TetR has a corresponding target TetO sequence. Accordingly, the
term "Tet operator sequence" or A TetO sequence" as used herein
encompasses all classes of TetO sequences, e.g., class A, B, C, D,
and E. Nucleotide sequences of these five classes of TetO sequences
are described in Waters, S. H. et al. (1983) cited supra, Hillen,
W. and Schollenmeier, K. (1983) cited supra, Stuber, D. and Bujard,
H. (1981) Proc. Natl. Acad. Sci. USA 78:167-171, Unger, B. et al.
(1984) cited supra and Tovar, K. et al. (1988) cited supra. In a
preferred embodiment, the mutated TetR is a Tn10-encoded repressor
(i.e., class B) and the TetO sequence is a class B Tet operator
sequence. Alternatively, a mutated class A TetR can be used with a
class A TetO sequence, and so on for the other classes of TetR and
TetO sequences.
[0101] Another approach for creating a mutated TetR which binds to
a class A Tet operator is to further mutate the already mutated
Tn10-derived TetR described herein (a class B repressor) such that
it no longer binds efficiently to a class B operator but instead
binds efficiently to a class A operator as taught in the art (see,
e.g., Wissman et al. (1988) J. Mol. Biol. 202:397-406; Altschmied
et al. (1988) EMBO J. 7:4011-4017). Accordingly, one can alter the
binding specificity of the mutated Tn10-derived TetR as described
herein by additionally changing amino acid residue 40 from Thr to
Ala by standard molecular biology techniques (e.g., site directed
mutagenesis).
[0102] A mutated TetR having specific mutations can be constructed
by introducing nucleotide changes into a nucleic acid encoding a
wild-type repressor by standard molecular biology techniques, e.g.
site directed mutagenesis or PCR-mediated mutagenesis using
oligonucleotide primers incorporating the nucleotide mutations.
Alternatively, when a mutated TetR is identified by selection from
a library, the mutated nucleic acid can be recovered from the
library vector. To construct a transactivator fusion protein
suitable for use in the methods of the present invention, a nucleic
acid encoding a mutated TetR is then ligated in-frame to another
nucleic acid encoding a transcriptional activation domain and the
fusion construct is incorporated into a recombinant expression
vector. The transactivator fusion protein can be expressed by
introducing the recombinant expression vector into a host cell.
[0103] The first polypeptide of the transactivator fusion protein
is operably linked to a second polypeptide. The second polypeptide
directly or indirectly activates expression in eukaryotic cells of
a nucleic acid sequence operably linked to a TetO sequence. To
operably link the first and second polypeptides, the nucleic acid
sequences encoding the first and second polypeptides are ligated to
each other in-frame to create a chimeric nucleic acid encoding a
transactivator fusion protein, although the first and second
polypeptides can be operably linked by other means that preserve
the function of each polypeptide (e.g., chemically crosslinked). In
a preferred embodiment, the second polypeptide of the
transactivator fusion protein itself possesses transcriptional
activation activity (i.e., the second polypeptide directly
activates expression). In another embodiment, the second
polypeptide activates expression by an indirect mechanism, through
recruitment of an activation protein to interact with the fusion
protein.
[0104] Polypeptides that function to directly or indirectly
activate gene expression in eukaryotic cells are well known in the
art, and are suitable for use in the construction of the second
polypeptide of the transactivator fusion protein. In particular,
transcriptional activation domains of many DNA binding proteins
have been described and have been shown to retain their activation
function when the domain is transferred to a heterologous protein.
A preferred polypeptide for use in the transactivator fusion
protein of the invention is the herpes simplex virus virion protein
16 (referred to herein as VP16, the amino acid sequence of which is
described in Triezenberg, S. J. et al. (1988) Genes Dev.
2:718-729). In one embodiment, the second polypeptide of the
transactivator fusion protein comprises about 127 of the C-terminal
amino acids of VP16 are used. In another embodiment, at least one
copy of about 11 amino acids from the C-terminal region of VP16
which retain transcriptional activation ability is used as the
second polypeptide. Preferably, a dimer of this region (i.e., about
22 amino acids) is used. Suitable C-terminal peptide portions of
VP16 are described in Seipel, K. et al. (EMBO J. (1992)
13:4961-4968).
[0105] Other polypeptides with the ability to activate expression
of nucleic acid sequences in eukaryotic cells can be used for
construction of the second polypeptide of the transactivator fusion
protein of the invention. Transcriptional activation domains found
within various proteins have been grouped into categories based
upon similar structural features. Types of transcriptional
activation domains include acidic transcription activation domains,
proline-rich transcription activation domains,
serine/threonine-rich transcription activation domains and
glutamine-rich transcription activation domains. Examples of acidic
transcriptional activation domains include the VP16 regions already
described and amino acid residues 753-881 of GAL4. Examples of
proline-rich activation domains include amino acid residues 399-499
of CTF/NF1 and amino acid residues 31-76 of AP2. Examples of
serine/threonine-rich transcription activation domains include
amino acid residues 1-427 of ITF1 and amino acid residues 2-451 of
ITF2. Examples of glutamine-rich activation domains include amino
acid residues 175-269 of Oct1 and amino acid residues 132-243 of
Sp1. The amino acid sequences of each of the above described
regions, and of other useful transcriptional activation domains,
are disclosed in Seipel, K. et al. (EMBO J. (1992) 13:4961-4968).
In addition, novel activation domains can be identified and
constructed using methods known in the art, and are within the
scope of the invention.
[0106] In another embodiment, the second polypeptide of the
transactivator fusion protein indirectly activates transcription by
recruiting a transcriptional activator to interact with the fusion
protein. For example, a mutated tetR of the invention can be fused
to a polypeptide domain (e.g., a dimerization domain) capable of
mediating a protein-protein interaction with a transcriptional
activator protein, such as an endogenous activator present in a
host cell. It has been demonstrated that functional associations
between DNA binding domains and transactivation domains need not be
covalent (see e.g., Fields and Song (1989) Nature 340:245-247;
Chien et al. (1991) Proc. Natl. Acad. Sci. USA 88:9578-9582; Gyuris
et al. (1993) Cell 75:791-803; and Zervos, A. S. (1993) Cell
72:223-232).
[0107] Accordingly, the second polypeptide of the fusion protein
may not directly activate transcription but rather may form a
stable interaction with an endogenous polypeptide bearing a
compatible protein-protein interaction domain and transactivation
domain. Examples of suitable interaction (or dimerization) domains
include leucine zippers (Landschulz et al. (1989) Science
243:1681-1688), helix-loop-helix domains (Murre, C. et al. (1989)
Cell 58:537-544) and zinc finger domains (Frankel, A. D. et al.
(1988) Science 240:70-73). Interaction of a dimerization domain
present in the fusion protein with an endogenous nuclear factor
results in recruitment of the transactivation domain of the nuclear
factor to the fusion protein, and thereby to a Tet operator
sequence to which the fusion protein is bound.
[0108] A nucleic acid encoding a transactivator fusion protein of
the invention can be incorporated into a recombinant expression
vector in a form suitable for expression of the fusion protein in a
host cell. That is, the recombinant expression vector includes one
or more regulatory sequences operably linked to the nucleic acid
encoding the fusion protein in a manner which allows for
transcription of the nucleic acid into mRNA and translation of the
mRNA into the fusion protein. The term "regulatory sequence" is
art-recognized and intended to include promoters, enhancers and
other expression control elements (e.g., polyadenylation signals).
Such regulatory sequences are known to those skilled in the art and
are described in Goeddel, Nucleic acid Expression Technology:
Methods in Enzymology 185, Academic Press, San Diego, Calif.
(1990). It should be understood that the design of the expression
vector may depend on such factors as the choice of the host cell to
be transfected and/or the amount of fusion protein to be
expressed.
[0109] When used in mammalian cells, a recombinant expression
vector's control functions are often provided by viral genetic
material. For example, commonly used promoters are derived from
polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. Use of
viral regulatory elements to direct expression of the fusion
protein can allow for high level constitutive expression of the
fusion protein in a variety of host cells. In a preferred
recombinant expression vector, the sequences encoding the fusion
protein are flanked upstream (i.e., 5') by the human
cytomegalovirus IE promoter and downstream (i.e., 3') by an SV40
poly(A) signal. The human cytomegalovirus IE promoter is described
in Boshart et al. (1985) Cell 41:521-530. Other ubiquitously
expressing promoters which can be used include the HSV-Tk promoter
(disclosed in McKnight et al. (1984) Cell 37:253-262) and
.beta.-actin promoters (e.g., the human-actin promoter as described
by Ng et al. (1985) Mol. Cell. Biol. 5:2720-2732).
[0110] Alternatively, the regulatory sequences of the recombinant
expression vector can direct expression of the fusion protein
preferentially in a particular cell type, i.e., tissue-specific
regulatory elements can be used. Non-limiting examples of
tissue-specific promoters which can be used include the albumin
promoter (liver-specific; Pinkert et al. (1987) Genes Dev.
1:268-277), lymphoid-specific promoters (Calame and Eaton (1988)
Adv. Immunol. 43:235-275), in particular promoters of T cell
receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and
immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and
Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g.,
the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl.
Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund
et al. (1985) Science 230:912-916), and mammary gland-specific
promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and
European Application Publication No. 264,166).
[0111] Developmentally-regulated promoters are also encompassed,
for example the murine hox promoters (Kessel and Gruss (1990)
Science 249:374-379) and the -fetoprotein promoter (Campes and
Tilghman (1989) Genes Dev. 3:537-546).
[0112] Alternatively, a self-regulating construct encoding a
transactivator fusion protein can be constructed. To accomplish
this, nucleic acid encoding the fusion protein is operably linked
to a minimal promoter sequence and at least one Tet operator
sequence. When this nucleic acid is introduced into a cell (e.g.,
in a recombinant expression vector), a small amount of basal
transcription of the transactivator nucleic acid is likely to occur
due to "leakiness". In the presence of Tet or analogue thereof this
small amount of the transactivator fusion protein will bind to the
Tet operator sequence(s) upstream of the candidate nucleic acid
sequence encoding the transactivator and stimulate additional
transcription of the nucleic acid sequence encoding the
transactivator, thereby leading to further production of the
transactivator fusion protein in the cell.
[0113] It will be appreciated by those skilled in the art that such
a self-regulating promoter can also be used in conjunction with
other tetracycline-regulated transactivators, such as the wild-type
Tet repressor fusion protein (tTA) described in Gossen, M. and
Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551, which
binds to Tet operators in the absence of Tet When used in
conjunction with this transactivator, self-regulated transcription
of the candidate nucleic acid sequence encoding this transactivator
is stimulated in the absence of Tet. The plasmid pUHD15-3, which
comprises candidate nucleic acid sequences encoding the tTA
described in Gossen and Bujard (1992), cited supra, operably linked
to a self-regulating promoter, has been deposited on Jul. 8, 1994
under the provisions of the Budapest Treaty at the Deutsche
Sammlung Von Mikroorganismen und ZellKulturen GmbH (DSM) in
Braunschweig, Germany and assigned deposit number DSM 9280.
[0114] In one embodiment, the recombinant expression vector
containing the transactivator fusion protein is a plasmid.
Alternatively, the recombinant expression vector can be a virus, or
portion thereof, which allows for expression of a nucleic acid
introduced into the viral nucleic acid. For example, replication
defective retroviruses, adenoviruses and adeno-associated viruses
can be used. Protocols for producing recombinant retroviruses and
for infecting cells in vitro or in vivo with such viruses can be
found in Current Protocols in Molecular Biology, Ausubel, F. M. et
al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14
and other standard laboratory manuals. Examples of suitable
retroviruses include pLJ, pZIP, pWE and pEM which are well known to
those skilled in the art. Examples of suitable packaging virus
lines include .psi.Crip, .psi.Cre, .psi.2 and .psi.Am. The genome
of adenovirus can be manipulated such that it encodes and expresses
a transactivator fusion protein but is inactivated in terms of its
ability to replicate in a normal lytic viral life cycle. See for
example Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et al.
(1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell
68:143-155. Suitable adenoviral vectors derived from the adenovirus
strain Ad type 5 dl324 or other strains of adenovirus (e.g., Ad2,
Ad3, Ad7 e Tet.) are well known to those skilled in the art.
Alternatively, an adeno-associated virus vector such as that
described in Tratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260
can be used to express a transactivator fusion protein.
[0115] The fusion nucleic acids of the present invention comprise a
second element that is regulatable by the first element. The second
element is preferably an operator sequence, and more preferably a
TetO sequence. In a preferred embodiment, the second element
comprises an oligomer of a TetO sequence. The term "Tet operator
sequence" as used herein encompasses all classes of Tet operators
(e.g., A, B. C, D and E). A nucleic acid sequence can be operably
linked to a single TetO sequence, or for an enhanced range of
regulation, it can be operably linked to multiple TetO sequences
(e.g., two, three, four, five, six, seven, eight, nine, ten or more
operator sequences). In a preferred embodiment, the candidate
nucleic acid sequence is operably linked to seven TetO
sequences.
[0116] In a preferred embodiment the transactivator fusion protein
of the invention is used to regulate the expression of a nucleic
acid sequence encoding a candidate bioactive agent. This nucleic
acid sequence is operably linked to an operator sequence to which
the transactivator fusion protein binds. In a preferred embodiment,
the transactivator fusion protein regulates expression of a
candidate nucleic acid sequence operably linked to at least one
TetO sequence. Accordingly, another aspect of the invention relates
to nucleic acid sequences that are operably linked to at least one
TetO sequence. Such nucleic acid sequences are also referred to
herein as "tet-regulated transcription units" or "transcription
units". In a preferred embodiment, the transcription unit comprises
a minimal promoter and candidate nucleic acid sequence. The minimal
promoter sequence is operably linked to the candidate nucleic acid
sequence in a 5' to 3' direction by phosphodiester bonds.
Accordingly, as used herein, the terms "candidate nucleic acid
sequence" or "nucleic acid sequence" comprise a nucleic acid
sequence encoding a candidate bioactive agent and may include
operably linked functional elements. Examples of functional
elements, include but are not limited to, promoters (e.g., minimal
promoters), enhancers, restriction enzyme sites, and nucleic acid
sequences encoding a peptide or an RNA.
[0117] The term "minimal promoter" as used herein refers to a
partial promoter sequence which defines the start site of
transcription for an operably linked nucleic acid sequence but
which by itself is not capable of initiating transcription
efficiently. Thus, the activity of such a minimal promoter is
dependent upon the binding of a transcriptional activator to an
operably linked operator sequence (e.g., one or more TetO
sequences). In one embodiment, the minimal promoter is from the
human cytomegalovirus (as described in Boshart et al. (1985) Cell
41:521-530). Preferably, nucleotide positions between about +75 to
-53 and +75 to -31 are used. Other suitable minimal promoters are
known in the art or can be identified by standard techniques.
[0118] Within a transcription unit, the candidate nucleic acid
sequence (including an upstream minimal promoter sequence) is
operably linked to at least one TetO sequence. For example, the
TetO sequence(s) is operably linked upstream or 5=of the minimal
promoter sequence through a phosphodiester bond at a suitable
distance to allow for transcription of the candidate nucleic acid
sequence upon binding of a transactivator (e.g., rtTA or tTA) to
the TetO sequence. That is, the transcription unit is comprised of,
in a 5' to 3' direction: TetO sequence(s); a minimal promoter; and
a candidate nucleic acid sequence. It will be appreciated by those
skilled in the art that there is some flexibility in the
permissible distance between the TetO sequence(s) and the minimal
promoter, although typically the TetO sequences will be located
within about 200-400 base pairs upstream of the minimal promoter.
Suitable promoter sequences are known in the art and described for
example, in Gossen, M. and Bujard, H. (1992) Proc. Natl. Acad. Sci.
USA 89:5547-5551.
[0119] Alternatively, since regulatory elements have been observed
in the art to function downstream of sequences to be transcribed,
it is likely that the TetO sequence(s) can be operably linked
downstream (i.e., 3') of the candidate nucleic acid sequence. Thus,
in this conFiguration, the transcription unit is comprised of, in a
5' to 3' direction: a minimal promoter; a candidate nucleic acid
sequence; and TetO sequence(s). Again, it will be appreciated that
there is likely to be some flexibility in the permissible distance
downstream at which the TetO sequence(s) can be linked.
[0120] A tet-regulated transcription unit can further be
incorporated into a recombinant vector (e.g., a plasmid or viral
vector) by standard recombinant DNA techniques. The transcription
unit, or recombinant vector in which it is contained, can be
introduced into a host cell by standard transfection techniques,
such as those described herein. It should be appreciated that,
after introduction of the transcription unit into a population of
host cells, it may be necessary to select a host cell clone which
exhibits low basal expression of the candidate nucleic acid
sequence operably linked to the TetO sequence(s) (i.e., selection
for a host cell in which the transcription unit has integrated at a
site that results in low basal expression of the TetO-linked
candidate nucleic acid sequence).
[0121] In one preferred embodiment, the candidate nucleic acid
sequence of the tet-regulated transcription unit encodes a
candidate bioactive agent that is a peptide, e.g., a cyclic
peptide. Upon induction of expression of the candidate nucleic acid
sequence and translation of the resultant mRNA, the peptide of
interest is produced in a host cell. In another preferred
embodiment, the candidate nucleic acid sequence of the
tet-regulated transcription unit encodes a candidate bioactive
agent that is an RNA, e.g., an antisense RNA or ribozyme. Upon
induction of expression of the candidate nucleic acid sequence, the
RNA of interest is produced in the host cell.
[0122] Thus, examples of candidate bioactive agents include, but
are not limited to, nucleic acids and polypeptides. Further
examples of bioactive agents are cyclic peptides, RNA, antisense
RNA, and DNA. Additional examples of nucleic acid sequences
encoding a candidate bioactive agent, include but are not limited
to, random nucleic acid sequences, and biased random nucleic acid
sequences. Examples of nucleic acid sequences encoding a candidate
bioactive agent, also include but are not limited to, full-length
cDNA sequences, subsequences of a full-length cDNA, and antisense
sequences of a full-length cDNA. Another example of a nucleic acid
sequence encoding a candidate bioactive agent is a nucleic acid
sequence encoding an amino acid sequence that is in-frame or
out-of-frame as compared to the open reading frame (ORF) encoded by
the amino acid sequence of a full-length cDNA.
[0123] Alternatively, a transactivator of the invention can
regulate transcription of an endogenous nucleic acid sequence to
which a TetO sequence(s) is operably linked. An "endogenous"
nucleic acid sequence is a nucleic acid sequence which is present
within the genome of a host cell. An endogenous nucleic acid
sequence can be operably linked to a TetO sequence(s) by homologous
recombination between a TetO-containing recombination vector and
the endogeneous nucleic acid sequence. For example, a homologous
recombination vector can be prepared which includes at least one
TetO sequence and a miminal promoter sequence flanked at the 3' end
by sequences representing the coding region of the endogenous
nucleic acid sequence and flanked at the 5' end by sequences from
the upstream region of the endogenous nucleic acid sequence by
excluding the actual promoter region of the endogenous nucleic acid
sequence. The flanking sequences are of sufficient length for
successful homologous recombination of the vector DNA with the
endogenous gene. Preferably, upon homologous recombination between
the vector DNA and the endogenous nucleic acid sequence in a host
cell, a region of the endogenous promoter is replaced by the vector
DNA containing one or more TetO sequences operably linked to a
minimal promoter. Thus, expression of the endogenous nucleic acid
sequence is no longer under the control of its endogenous promoter
but rather is placed under the control of the TetO sequence(s) and
the minimal promoter.
[0124] In another embodiment, TetO sequences can be inserted
elsewhere within an endogenous gene, preferably within a 5' or 3'
regulatory region, via homologous recombination to create an
endogenous nucleic acid whose expression can be regulated by a tet.
An endogenous nucleic acid sequence having TetO sequences inserted
into a non-critical regulatory region will retain the ability to be
expressed in a normal constitutive and/or tissue-specific manner
but, additionally, can be downregulated by a
tetracycline-controlled transcriptional inhibitor protein
(described infra) in a controlled manner. For example, constitutive
expression of such a modified endogenous nucleic acid sequence can
be inhibited by in the presence of Tet or analogue thereof using an
inhibitor fusion protein that binds to TetO sequences in the
presence of Tet or analogue thereof.
[0125] In the present invention, the expression of a candidate
nucleic acid operably linked to a second element (e.g., an operator
sequence) that is regulatable by a first element (e.g., a
transactivator). Thus, these components, the first element and the
candidate nucleic acid operably linked to an operator sequence, are
present in a host cell, and can be contained on the same nucleic
acid or separate nucleic acids. The presence of these components in
the same host cell can be achieved in a number of different ways.
For example, a host cell can be transfected with a first nucleic
acid encoding, e.g., a first element, stably transfected cells can
be selected, and then the transfected cells can be re-transfected
(also referred to as "supertransfected") with a second nucleic acid
comprising, e.g., the second element operably linked to a candidate
nucleic acid. Two distinct selectable markers can be used for
selection, e.g., uptake of the first nucleic acid can be selected
with G418 and uptake of the second nucleic acid can be selected
with hygromycin. Alternatively, a single population of cells can be
transfected with nucleic acid corresponding to both components of
the system.
[0126] Accordingly, in one aspect, the invention provides a first
nucleic acid encoding a transactivator fusion protein and a second
nucleic acid comprising a candidate nucleic acid sequence operably
linked to at least one TetO sequence. The transactivator of the
first nucleic acid comprises a first polypeptide and second
polypeptide. The first polypeptide binds to a TetO sequence in the
presence of Tet or analogue thereof and is operably linked to a
second polypeptide which activates transcription in eukaryotic
cells.
[0127] In one embodiment, the two nucleic acids are two separate
molecules (e.g., two different vectors). Thus, in this embodiment a
host cell is cotransfected with the two nucleic acid molecules or
successively transfected first with one nucleic acid molecule and
then the other nucleic acid molecule. In another embodiment, the
two nucleic acids are linked (i.e., colinear) in the same nucleic
acid molecule (e.g., a single vector). Thus, in this embodiment a
host cell is transfected with the single nucleic acid molecule.
Further, the host cell may be a cell cultured in vitro or a cell
present in vivo.
[0128] The third element of the present invention, induces or
represses the expression of a candidate nucleic acid. In a
preferred embodiment, the third element is an agent which induces
expression of a candidate nucleic acid, e.g., by binding to a
transactivator (e.g., rtTA). In another preferred embodiment, the
third element is an agent which represses expression of a candidate
nucleic acid, e.g., by binding to a transactivator (e.g., tTA). In
a preferred embodiment, the agent comprises tetracycline or an
analogue thereof, e.g., doxycycline (Dox).
[0129] The term "tetracycline analogue" is intended to include
compounds which are structurally related to Tet and which bind to
the TetR with a K.sub.a of at least about 10.sup.6 M.sup.-1.
Preferably, the Tet analogue binds with an affinity of about
10.sup.9 M.sup.-1 or greater. Examples of such Tet analogues
include, but are not limited to, anhydrotetracycline, Dox,
chlorotetracycline, oxytetracycline and others disclosed by Hlavka
and Boothe, "The Tetracyclines," in Handbook of Experimental
Pharmacology 78, R. K. Blackwood et al. (eds.), Springer-Verlag,
Berlin-New York, 1985; L. A. Mitscher, "The Chemistry of the
Tetracycline Antibiotics", Medicinal Research 9, Dekker, N.Y.,
1978; Noyee Development Corporation, "Tetracycline Manufacturing
Processes" Chemical Process Reviews, Park Ridge, N.J., 2 volumes,
1969; R. C. Evans, "The Technology of the Tetracyclines",
Biochemical Reference Series 1, Quadrangle Press, New York, 1968;
and H. F. Dowling, "Tetracycline", Antibiotic Monographs, no. 3,
Medical Encyclopedia, New York, 1955. Preferred Tet analogues for
high level stimulation of transcription are anhydrotetracycline and
Dox. A Tet analogue can be chosen which has reduced antibiotic
activity compared to Tet. Examples of such Tet analogues are
anhydrotetracycline, epioxytetracycline and cyanotetracycline.
[0130] In a preferred embodiment, to induce or repress nucleic acid
expression in a cell in vitro, the cell is contacted with Tet or a
analogue thereof by culturing the cell in a medium containing the
compound. Preferably, the concentration range of the Tet or
analogue thereof in the cell medium is between about 10 and about
1000 ng/ml. Tet or analogue thereof can be directly added to media
in which cells are already being cultured, or more preferably for
high levels of nucleic acid induction. Additionally, the cells can
be harvested from Tet-free media and cultured in fresh media
containing Tet, or an analogue thereof, or vice versa.
[0131] The use of different Tet analogues allows for the modulation
of the level of expression of a nucleic acid sequence operably
linked to a TetO sequence, for example, by adjusting the
concentration of Tet or an analogue thereof in contact with the
cells. For example, anhydrotetracycline and doxycycline are known
to be strong inducers of expression, e.g., in a system where a
transactivator binds to a TetO sequence in the presence of Tet or
analogue thereof. From the uninduced state to the induced state,
the increase in expression of a nucleic acid operably linked to a
TetO sequence is typically about 1000- to 2000-fold, and can be at
least about 20,000 fold. In the same system, Tet,
chlorotetracycline and oxytetracycline have been found to be weaker
inducers of expression, i.e., from the uninducec state to the
induced state, the increase in expression at least about 10-fold.
Thus, an appropriate Tet analogue can be selected, for use in the
methods of the present invention, based upon the desired level of
induction or repression of expression of nucleic acid sequence
operably linked to a TetO sequence. It is also possible to change
the level of expression over time, in a host cell, of a nucleic
acid operably linked to a TetO sequence, by changing the Tet
analogue used to induce or repress expression. For example, in some
situations it may be desirable to have a strong burst of expression
initially and then have a sustained lower level of expression.
[0132] Accordingly, a first agent (e.g., a first Tet analogue)
which stimulates a high levels of expression can be used initially
as the third element in the methods of the present invention and
then the third element can be switched to a second agent (e.g., a
second analogue) which stimulates a lower level of expression.
Moreover, when regulating the expression of multiple nucleic acid
sequences (e.g., when one sequence is regulated by a one of class
TetO sequence(s) and the other is regulated by another class of
TetO sequence(s), it may be possible to independently vary the
level of expression of each sequence depending upon which
transactivator fusion protein is used to regulate transcription and
which Tet analogue is used.
[0133] In a preferred embodiment, a host cell comprises an rtTA and
a fusion nucleic acid comprising a nucleic acid sequence operably
linked to a TetO sequence, and a high level expression of the
nucleic acid sequence does not occur in the absence of a third
element, for example, tetracycline or analogues thereof. The level
of basal expression of the candidate nucleic acid sequence may vary
depending upon the host cell and site of integration of the
sequence, but is generally quite low or even undetectable in the
absence of Tet. In order to induce expression, the host cell is
contacted with Tet or analogue thereof. In order to repress
expression, the contacting of Tet or analogue thereof with the
cells is modulated. In a preferred embodiment, the contacting is
modulated by adjusting the concentration of the Tet or analogue
thereof, for example, in the cell medium. Accordingly, another
aspect of the invention relates to methods for inducing or
repressing expression of a nucleic acid sequence operably linked to
a TetO sequence in a host cell which expresses a transactivator of
the invention, by modulating the contacting of Tet or an analogue
thereof with the host cell.
[0134] In a preferred embodiment, a host cell comprises an tTA and
a fusion nucleic acid comprising a candidate nucleic acid sequence
operably linked to a TetO sequence, high level expression of the
candidate nucleic acid sequence occurs only in the presence of the
rtTA and, expression is repressed in the presence of Tet or an
analogue thereof. The level of basal expression of the candidate
nucleic acid sequence may vary depending upon the host cell and
site of integration of the sequence, but is generally quite low or
even undetectable in the absence of tTA. In order to induce
expression, tTA is expressed in the host cell. In order to repress
expression, the cells are contacted with Tet or an analogue
thereof. In a preferred embodiment, the contacting is modulated by
adjusting the concentration of the Tet or analogue thereof, for
example, in the cell medium. Accordingly, another aspect of the
invention relates to methods for inducing or repressing expression
of a nucleic acid sequence operably linked to a TetO sequence in a
host cell comprising a transactivator of the invention, by
modulating the contacting of Tet or an analogue thereof with the
host cell.
[0135] Different transactivator fusion proteins are likely to
exhibit different levels of responsiveness to Tet analogues. Thus,
the level of induction or repression of expression by a particular
combination of transactivator fusion protein and Tet analogue can
be determined by techniques described herein or known in the art.
Additionally, the level of expression can be modulated by varying
the concentration of the Tet analogue. Thus, in the methods of the
present invention, expression of a nucleic acid operably linked to
a TetO sequence can be regulated by turning the expression on or
off, but also by modulating the level of expression at intermediate
levels (between induction and repression) depending on the type and
concentration of the Tet analogue used in the methods.
[0136] Another aspect of the invention relates to inhibitor fusion
proteins. The inhibitor fusion proteins are constructed similarly
to the transactivator fusion proteins in that the fusion proteins
comprise a first and second polypeptide, and the first polypeptide
is a mutated TetR. However, in contrast to the transactivator
fusion protein, the second polypeptide of the inhibitor fusion
protein has a domain that inhibits expression (rather than
activates expression as in the transactivator fusion protein) in
eukaryotic cell of a nucleic acid sequence operably linked to a
TetO sequence. Thus, inhibitor fusion proteins can be used to
downregulate the expression of a nucleic acid sequence operably
linked to a TetO sequence, and can be used in the methods of the
present invention to screen for an altered cellular phenotype as
described herein. For example, the level of basal, constitutive
expression of a nucleic acid sequence operably linked to an
operator sequence may vary depending upon the type of cell in which
the nucleic acid is introduced or the site of integration of the
nucleic acid. Therefore, the inhibitor fusion proteins of the
invention can, in a controlled manner, inhibit the expression of a
nucleic acid operably linked to an operator sequence.
[0137] In one embodiment, the inhibitor fusion protein comprises a
first polypeptide that binds to Tet operator sequences in the
absence, but not the presence, of Tet or an analogue thereof,
operably linked to a heterologous second polypeptide that inhibits
transcription in eukaryotic cells. In another embodiment, the
inhibitor fusion protein comprises a first polypeptide that binds
to a Tet operator sequence in the presence, but not the absence, of
Tet or analogue thereof, operably linked to a heterologous second
polypeptide that inhibits transcription in eukaryotic cells. The
term "heterologous" is intended to mean that the second polypeptide
is derived from a different protein than the first polypeptide.
Like the transactivator fusion proteins, the inhibitor fusion
proteins can be prepared using standard recombinant DNA techniques
as described herein.
[0138] Proteins and polypeptide domains within proteins which can
function to inhibit transcription in eukaryotic cells have been
described in the art (for reviews see, e.g., Renkawitz, R. (1990)
Trends in Genetics 6:192-197; and Herschbach, B. M. and Johnson, A.
D. (1993) Annu. Rev. Cell. Biol. 9:479-509) have suitable inhibitor
domains for use in the inhibitor fusion proteins of the present
invention. Such transcriptional inhibitor domains have been
referred to in the art as "silencing domains" or "repressor
domains." Although the precise mechanism by which many of these
polypeptide domains inhibit transcription is not known (and the
invention is not intended to be limited by mechanism), there are
several possible means by which repressor domains may inhibit
transcription, including: competitive inhibition of binding of
either activator proteins or the general transcriptional machinery;
prevention of the activity of a DNA bound activator; and negative
interference with the assembly of a functional preinitiation
complex of the general transcription machinery. Thus, an inhibitor
domain may have a direct inhibitory effect on the transcriptional
machinery or may inhibit transcription indirectly by inhibiting the
activity of activator proteins.
[0139] Accordingly, polypeptide containing an inhibitor domain can
act either directly or indirectly to inhibit expression. As used
herein a "reduction" in the level of expression of a nucleic acid
sequence operably linked to an operator sequence refers to a
diminution in the level or amount of expression of the nucleic acid
compared to the level or amount prior to regulation by the
transcriptional inhibitor protein. Transcriptional inhibition may
be partial or complete. The terms "silencer", "repressor" and
"inhibitor" are used interchangeably herein to describe an
inhibitor protein, or domains thereof, that can inhibit expression
of a nucleic acid sequence operably linked to an operator
sequence.
[0140] A "repressor" or "silencer" domain as used herein is a
polypeptide domain that retains its repressor function (e.g.,
repression of expression of a nucleic acid sequence operably linked
to an operator sequence) when the domain is transferred to a
heterologous protein. Proteins which have been demonstrated to have
repressor domains that can function when transferred to a
heterologous protein include the v-erbA onconucleic acid product
(Baniahmad, A. et al. (1992) EMBO J. 11:1015-1023), the thyroid
hormone receptor (Baniahmad, supra), the retinoic acid receptor
(Baniahmad, supra), and the Drosophila Krueppel (Kr) protein
(Licht, J. D. et al. (1990) Nature 346:76-79; Sauer, F. and Jackle,
H. (1991) Nature 353:563-566; Licht, J. D. et al. (1994) Mol. Cell.
Biol. 14:4057-4066). Non-limiting examples of other proteins which
have transcriptional repressor activity in eukaryotic cells include
the Drosophila homeodomain protein even-skipped (eve), the S.
cerevisiae Ssn6/Tup1 protein complex (see Herschbach and Johnson,
supra), the yeast SIR1 protein (see Chien, et al. (1993) Cell
75:531-541), NeP1 (see Kohne, et al. (1993) J. Mol. Biol.
232:747-755), the Drosophila dorsal protein (see Kirov, et al.
(1994) Mol. Cell. Biol. 14:713-722; Jiang, et al. (1993) EMBO J.
12:3201-3209), TSF3 (see Chen, et. al. (1993) Mol. Cell. Biol.
13:831-840), SF1 (see Targa, et al. (1992) Biochem. Biophys. Res.
Comm. 188:416-423), the Drosophila hunchback protein (see Zhang, et
al. (1992) Proc. Natl. Acad. Sci. USA 89:7511-7515), the Drosophila
knirps protein (see Gerwin, et al. (1994) Mol. Cell. Biol.
14:7899-7908), the WT1 protein (Wilm's tumor nucleic acid product)
(see Anant, et al. (1994) Onconucleic acid 9:3113-3126; Madden et
al., (1993) Onconucleic acid 8:1713-1720), Oct-2.1 (see Lillycrop,
et al. (1994) Mol. Cell. Biol. 14:7633-7642), the Drosophila
engrailed protein (see Badiani, et al. (1994) Genes Dev. 8:770-782;
Han and Manley, (1993) EMBO J. 12:2723-2733), E4BP4 (see Cowell and
Hurst, (1994) Nucleic Acids Res. 22:59-65) and ZF5 (see Numoto, et
al. (1993) Nucleic Acids Res. 21:3767-3775),
[0141] In additional aspects described below, the invention
provides alternative approaches to regulating the expression of one
or more nucleic acid sequences. These alternative approaches to
regulating expression of nucleic acid sequences operably linked to
TetO sequences are suitable for use in the methods of the present
invention to screen for cells having an altered phenotype due to
the presence of a candidate bioactive agent.
[0142] In addition to regulating nucleic acid expression using
either a transactivator fusion protein or inhibitor fusion protein
alone, these two types of proteins, can be used in combination to
allow for both positive and negative regulation of expression of
one or more nucleic acids in a host cell. Positive regulation of
expression refers to induction or increase in expression, whereas
negative regulation of expression refers to repression or reduction
in expression. Thus, an inhibitor protein that binds to TetO either
1) in the absence, but not the presence, of Tet; or 2) in the
presence, but not the absence, of Tet or analogue thereof, can be
used in combination with a transactivator fusion protein that binds
to TetO either 1 in the absence, but not the presence, of Tet or
analogue thereof; or 2) in the presence, but not the absence, of
Tet or analogue thereof. Transactivator proteins that bind to TetO
in the absence, but not the presence, of Tet or analogue thereof
are described herein, and in U.S. Pat. No. 5,464,758, U.S. Ser. No.
08/076,327 and U.S. Pat. No. 5,650,298. Transactivator proteins
that bind to TetO in the presence, but not the absence, of Tet are
described herein, and in U.S. Ser. No. 08/270,637 and U.S. Pat. No.
5,654,168.
[0143] When more than one TetR-containing fusion protein is
expressed in a host cell, additional steps may be taken to inhibit
heterodimerization between the different TetR-containing fusion
proteins. For example, a transactivator composed of a TetR of one
class may be used in combination with an inhibitor fusion protein
composed of a TetR of a second, different class that does not
heterodimerize with the first class of TetR. Alternatively, amino
acid residues of the TetR involved in dimerization may be mutated
to inhibit heterodimerization. However, even if some
heterodimerization between transactivator and inhibitor fusion
proteins occurs in a host cell, sufficient amounts of homodimers
should be produced to allow for efficient positive and negative
regulation as described herein.
[0144] It will be appreciated by those skilled in the art that
various combinations of activator and inhibitor proteins can be
used to regulate a single nucleic acid sequence operably linked to
a TetO sequence in both a positive and negative manner or to
regulate multiple nucleic acid sequences operably linked to TetO
sequences in a coordinated manner or in an independent manner using
the teachings described herein or known in the art. Several
non-limiting examples of how the transactivator and inhibitor
fusion proteins may be used in combination are described further
below. However, many other possible combinations will be evident to
the skilled artisan in view of the teachings herein and known in
the art.
[0145] In one embodiment expression of a nucleic acid acid sequence
operably linked to a TetO sequence in a host cell is regulated in
both a negative and positive manner by the combination of an
inhibitor fusion protein that binds to TetO in the absence, but not
the presence, of Tet or analogue thereof (referred to as a Tet
controlled silencing domain, or tSD) and an transactivator fusion
protein that binds to TetO in the presence, but not the absence, of
Tet or analogue thereof (e.g., rtTA). In addition to TetO
sequences, the nucleic acid sequence is operably linked to a
promoter, and may contain other positive regulatory elements (e.g.,
enhancer sequences) that contribute to basal level, constitutive
expression of the nucleic acid in the host cell. Binding of tSD to
the TetO sequences in the absence of Tet or analogue thereof
inhibits the basal constitutive expression of the nucleic acid
sequence, thus keeping the expression of the nucleic acid sequence
in a repressed or uninduced state until expression is desired. When
expression is desired, the concentration of Tet or analogue thereof
in contact with the host cell is increased. Upon contacting a host
cell with Tet or an analogue thereof, tSD loses the ability to bind
to TetO sequences whereas the previously unbound rtTA acquires the
ability to bind to TetO sequences. The resultant binding of rtTA to
the TetO sequences operably linked to a nucleic acid sequence of
interest thus induces expression of the nucleic acid sequence. The
level of expression may be controlled by the concentration of Tet
or analogue thereof, type of Tet analogue, duration of induction,
and type of rtTA (e.g., class of TetR and transactivator domain, as
described previously herein). It will be appreciated that the
combination of transactivator and inhibitor fusion proteins (i.e.,
where the inhibitor binds in the presence but not the absence of
Tet or analogue thereof, and the transactivator binds in the
absence but not the presence of Tet or analogue thereof can also be
used in the methods of the present invention. In this case,
expression of the nucleic acid sequence of interest is repressed by
contacting the host cell with Tet (e.g., culture with Tet or
analogue) and nucleic acid expression is activated by removal of
the drug.
[0146] In another embodiment, the activator and inhibitor fusion
proteins are used in combination to coordinately regulate, in both
a positive and negative manner, two nucleic acid sequences operably
linked to a TetO sequence but are expressed bidirectionally with
respect to each other. In this case, a first nucleic acid and a
second nucleic acid are linked to the same TetO sequence(s), but in
opposite orientations. The inhibitor fusion protein is used to
repress basal levels of expression of both the first nucleic acid
and the second nucleic acid in a coordinate manner, whereas the
transactivator fusion protein is used to stimulate expression of
the first nucleic acid and the second nucleic acid in a coordinate
manner.
[0147] In another embodiment, the activator and inhibitor fusion
proteins are used to independently regulate two or more nucleic
acid sequences each operably linked to their respective TetO
sequence, where each TetO sequence is of a different class. In one
embodiment, a transactivator fusion protein that binds to one class
of TetO sequences (e.g., class A) in the presence, but not the
absence of Tet or analogue is used in combination with an inhibitor
fusion protein that binds to a second, different class of TetO
sequences (e.g., class B) also in the presence, but not the
absence, of Tet or analogue. For example, a host cell containing a
first nucleic acid sequence operably linked to class A TetO
sequences and second nucleic acid sequence operably linked to class
B TetO sequences, both nucleic acid sequences will be expressed at
basal levels in the absence of Tet or an analogue thereof, whereas
expression of the first nucleic acid will be stimulated in the
presence of Tet or an analogue thereof and expression of the second
nucleic acid will be repressed in the presence of Tet or analogue
thereof.
[0148] Alternatively, in another embodiment, the transactivator
fusion protein binds to one class of TetO sequences (e.g., class A)
in the presence, but not the absence, of Tet or analogue thereof
and the inhibitor fusion protein binds to a second, different class
of TetO sequences (e.g., class B) in the absence but not the
presence of Tet or analogue. For example, in the host cell, the
first nucleic acid sequence will be expressed at basal levels in
the absence of Tet or an analogue thereof and will be stimulated in
the presence of Tet or an analogue thereof, whereas the expression
of the second nucleic acid sequence will be repressed in the
absence of the Tet or an analogue thereof but will have basal
levels expression in the presence of Tet or analogue thereof.
Various other possible combinations will be apparent to the skilled
artisan.
[0149] By "fusion nucleic acid" or grammatical equivalents thereof
is meant a nucleic acid comprising functional elements that may or
may not be operably linked. Examples of functional elements
include, but are not limited to, a promoter, enhancer, operator
sequence, restriction enzyme site, candidate nucleic acid, and
nucleic acid encoding a polypeptide or RNA. In a preferred
embodiment the fusion nucleic acid comprises a second element and a
candidate nucleic acid. In another preferred embodiment, the fusion
nucleic acid comprises a nucleic acid encoding a first element. As
outlined herein, fusion nucleic acids, or other components of the
system such as fusion partners as well as the vectors themselves,
can include reporter proteins.
[0150] By "candidate nucleic acid" or grammatical equivalents
thereof is meant a nucleic acid comprising a nucleic acid sequence
encoding a candidate bioactive agent. The candidate nucleic acid of
the present invention can also comprises functional elements, e.g.,
a promoter, enhancer, and restriction enzyme site. In another
preferred embodiment, the candidate nucleic acid comprises and
minimal promoter operably linked to a nucleic acid sequence
encoding the bioactive agent.
[0151] By "candidate nucleic acid sequence" or grammatical
equivalents thereof is meant a nucleic acid sequence comprising a
nucleic acid sequence encoding a candidate bioactive agent.
[0152] By "candidate bioactive agents" or "candidate drugs" or
"candidate expression products" or grammatical equivalents herein
is meant the expression product of a candidate nucleic acid
sequence which may be tested for the ability to alter the phenotype
of a cell. As is described below, the candidate bioactive agents
are the expression products of a candidate nucleic acid sequence,
and encompass several chemical classes, including peptides and
nucleic acids such as DNA, cDNA, messenger RNA (mRNA), antisense
RNA, and ribozyme components. Thus, the candidate bioactive agents
(expression products) may be either translation products of the
candidate nucleic acid sequence, i.e., peptides, or transcription
products of the candidate nucleic acid sequences, i.e., either DNA
or RNA.
[0153] In a preferred embodiment, the candidate bioactive agents
are translation products of candidate nucleic acid sequences. In
this embodiment, a library of fusion nucleic acids each comprising
a candidate nucleic acid sequence are introduced into the cells,
and the cells express the nucleic acid sequence to form peptides.
Thus, in this embodiment, the candidate bioactive agents are
peptides. Generally, peptides ranging from about 4 amino acids in
length to about 100 amino acids may be used, with peptides ranging
from about 5 to about 50 being preferred, with from about 5 to
about 30 being particularly preferred and from about 6 to about 20
being especially preferred.
[0154] In a preferred embodiment, the candidate bioactive agents
are transcription products of candidate nucleic acid sequences and,
thus, the candidate bioactive agents are also nucleic acids. The
transcription products may be either primary transcripts or
secondary translation products. That is, using the retroviral
reverse transcriptase, primary DNA is made which is later converted
into double stranded DNA. Additionally, using the primary DNA, RNA
transcripts can be generated within the cell, including mRNA,
antisense RNA and ribozymes or portions thereof.
[0155] At a minimum, the candidate bioactive agents comprise
randomized expression products of the nucleic acid sequence of the
fusion nucleic acids. That is, every candidate bioactive agent has
a randomized portion, as defined below, that is the basis of the
screening methods outlined herein. In addition, to the randomized
portion, the candidate bioactive agent may also include a fusion
partner.
[0156] In a preferred embodiment, the candidate bioactive agents
are linked to a fusion partner. By fusion partner" or "functional
group" herein is meant a sequence that is associated with the
candidate bioactive agent, that confers upon all members of the
library in that class 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: a) presentation structures, as
defined below, which provide the candidate bioactive agents in a
conformationally restricted or stable form; b) targeting sequences,
defined below, which allow the localization of the candidate
bioactive agent into a subcellular or extracellular compartment; c)
rescue sequences as defined below, which allow the purification or
isolation of either the candidate bioactive agents or the nucleic
acids encoding them; d) stability sequences, which confer stability
or protection from degradation to the candidate bioactive agent or
the nucleic acid encoding it, for example resistance to proteolytic
degradation; e) dimerization sequences, to allow for peptide
dimerization; or f) any combination of a), b), c), d), and e), as
well as linker sequences as needed.
[0157] In a preferred embodiment, the fusion partner is a
presentation structure. By "resentation structure" or grammatical
equivalents herein is meant a sequence, which, when fused to
candidate bioactive agents, causes the candidate agents 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
have potent functions as is known in the art, the conversion of
such peptide structures into pharmacological agents is difficult
due to the inability to predict side-chain positions for
peptidomimetic synthesis. Therefore the presentation of peptides in
conformationally constrained structures will benefit both the later
generation of pharmaceuticals and will also likely lead to higher
affinity interactions of the peptide with the target protein. 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.
[0158] While the candidate bioactive agents may be either nucleic
acid or peptides, presentation structures are preferably used with
peptide candidate agents. Thus, synthetic presentation structures,
i.e., artificial polypeptides, are capable of presenting a
randomized peptide as a conformationally-restricted domain.
Generally such presentation structures comprise a first portion
joined to the N-terminal end of the randomized 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 randomized expression product, the
presentation structures are selected or designed to have minimal
biologically activity when expressed in the target cell.
[0159] 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-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.
[0160] In a preferred embodiment, the presentation structure is a
coiled-coil structure, allowing the presentation of the randomized
peptide on an exterior loop. See, for example, Myszka et al.,
Biochem. 33:2362-2373 (1994), hereby incorporated by reference, and
FIG. 28). 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.
[0161] A preferred coiled-coil presentation structure is as
follows:
MGCAALESEVSALESEVASLESEVAALGRGDMPLAAVKSKLSAVKSKLASVKSKLAACGPP. 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
randomized peptides (i.e., candidate bioactive agents, generally
depicted herein as (X).sub.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 randomized 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.
[0162] In a preferred embodiment, the presentation structure is a
minibody structure. A "inibody" is essentially composed of a
minimal antibody complementarity region. The minibody presentation
structure generally provides two randomizing 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, Kd=10.sup.-7, for the
pro-inflammatory cytokine IL-6.
[0163] A preferred minibody presentation structure is as follows:
MGRNSQATSGFTFSHFYMEWVRGGEYIAASRHKHNKYTTEYSASVKGRYIVSRDTSQSILYLQKKKG
PP. The bold, underline regions are the regions which may be
randomized. The italicized phenylalanine must be invariant in the
first randomizing region. The entire peptide is cloned in a
three-oligonucleotide variation of the coiled-coil embodiment, thus
allowing two different randomizing regions to be incorporated
simultaneously. This embodiment utilizes non-palindromic BstXI
sites on the termini.
[0164] 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. This embodiment is particularly preferred
when secretory targeting sequences are used. As will be appreciated
by those in the art, any number of random 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 random regions themselves. For example, the
random regions may be "oped" 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 -helical
structures.
[0165] In a preferred embodiment, the presentation structure is a
reporter protein. In a preferred embodiment, the reporter protein
can be used as a direct label, for example a detection protein for
sorting the cells or for cell enrichment by FACS. In this
embodiment, the protein product of the reporter gene itself can
serve to distinguish cells that are expressing the reporter gene.
In this embodiment, suitable reporter genes include those encoding
green fluorescent protein (GFP; Chalfie, M. et al. (1994) Science
263: 802-05; and EGFP; Clontech-Genbank Accession Number U55762 ),
blue fluorescent protein (BFP; Quantum Biotechnologies, Inc. 1801
de Maisonneuve Blvd. West, 8th Floor, Montreal (Quebec) Canada H3H
1J9; Stauber, R. H. (1998) Biotechniques 24: 462-71; Heim, R. et
al. (1996) Curr. Biol. 6: 178-82), enhanced yellow fluorescent
protein (EYFP; 1. Clontech Laboratories, Inc., 1020 East Meadow
Circle, Palo Alto, Calif. 94303), Renilla reniformis GFP (WO
99/49019), Ptilosarcus gurneyi GFP (WO 99/49019; U.S. Ser. No.
60/164,592; U.S. Ser. No. 09/710,058; U.S. Ser. No. 60/290,287),
Renilla mulleris GFP (WO 99/49019; U.S. Ser. No. 60/164,592; U.S.
Ser. No. 09/710,058; U.S. Ser. No. 60/290,287), luciferases (for
example, firefly, Kennedy, H. J. et al. (1999) J. Biol. Chem. 274:
13281-91; Renilla reniformis, Lorenz, W. W. (1996) J Biolumin.
Chemilumin. 11: 31-37; Renilla mulleris, U.S. Pat. No. 6,232,107),
b-galactosidase (Nolan, G. et al. (1988) Proc. Natl. Acad. Sci. USA
85: 2603-07), b-glucouronidase (Jefferson, R. A. et al. (1987) EMBO
J. 6: 3901-07; Gallager, S., GUS Protocols: Using the GUS Gene as a
reporter of gene expression, Academic Press, Inc., 1992), and
secreted form of human placental alkaline phosphatase, SEAP
(Cullen, B. R. et al. (1992) Methods Enzymol. 216: 362-68). In a
preferred embodiment, the codons of the reporter genes are
optimized for expression within a particular organism, especially
mammals, and particularly preferred for human cell expression (see
Zolotukhin, S. et al. (1996) J. Virol. 70: 4646-54; U.S. Pat. Nos.
5,968,750; 6,020,192; U.S. Ser. No. 60/290,287, all of which are
expressly incorporate by reference).
[0166] In another embodiment, the reporter protein will bind a
label that can be used as the basis of the cell enrichment
(sorting); that is, the reporter protein serves as an indirect
label or detection protein. In this embodiment, the reporter
protein is a cell-surface protein. For example, the reporter
protein may be any cell-surface protein not normally expressed on
the surface of the cell, such that secondary binding agents serve
to distinguish cells that contain the reporter protein from those
that do not. Alternatively, albeit non-preferably, reporters
comprising normally expressed cell-surface proteins could be used,
and differences between cells containing the reporter construct and
those without could be determined. Thus, secondary binding agents
bind to the reporter protein. These secondary binding agents are
preferably labeled, for example with fluors, and can be antibodies,
haptens, etc. For example, fluorescently labeled antibodies to the
reporter gene can be used as the label. Similarly,
membrane-tethered streptavidin could serve as a reporter gene, and
fluorescently-labeled biotin could be used as the label, i.e. the
secondary binding agent. Alternatively, the secondary binding
agents need not be labeled as long as the secondary binding agent
can be used to distinguish the cells containing the construct; for
example, the secondary binding agents may be used in a column, and
the cells passed through, such that the expression of the reporter
gene results in the cell being bound to the column, and a lack of
the reporter gene (i.e. inhibition), results in the cells not being
retained on the column. Other suitable reporter proteins/secondary
labels include, but are not limited to, antigens and antibodies,
enzymes and substrates (or inhibitors), etc.
[0167] In a preferred embodiment, the reporter gene is a survival
gene that serves to provide a nucleic acid (or encode a protein)
without which the cell cannot survive, such as drug resistance
genes. In this embodiment, expressing the survival gene allows
selection of cells expressing the fusion nucleic acid by
identifying cells that survive, for example in presence of a
selection drug. Examples of drug resistance genes include, but are
not limited to, puromycin resistance
(puromycin-N-acetyl-transferase) (de la Luna, S. et al. (1992)
Methods Enzymol. 216: 376-85), G418 neomycin resistance gene,
hygromycin resistance gene (hph), and blasticidine resistance genes
(bsr, brs, and BSD; Pere-Gonzalez, et al.(1990) Gene, 86: 129-34;
Izumi, M. et al. (1991) Exp. Cell Res. 197: 229-33; Itaya, M. et
al. (1990) J. Biochem. 107: 799-801; Kimura, M. et al. (1994) Mol.
Gen. Genet. 242: 121-29). In addition, generally applicable
survival genes are the family of ATP-binding cassette transporters,
including multiple drug resistance gene (MDR1) (see Kane, S. E. et.
al. (1988) Mol. Cell. Biol. 8: 3316-21 and Choi, K. H. et al.
(1988) Cell 53: 519-29), multi-drug resistance associated proteins
(MRP) (Bera, T. K. et al. (2001) Mol. Med. 7: 509-16), and breast
cancer associated protein (BCRP or MXR) (Tan, B. et al. (2000)
Curr. Opin. Oncol. 12: 450-58). When expressed in cells, these
selectable genes can confer resistance to a variety of toxic
reagents, especially anti-cancer drugs (i.e. methotrexate,
colchicine, tamoxifen, mitoxanthrone, and doxorubicin). As will be
appreciated by those skilled in the art, the choice of the
selection/survival gene will depend on the host cell type used.
[0168] In a preferred embodiment, the reporter gene encodes a death
gene that causes the cells to die when expressed. Death genes fall
into two basic categories: death genes that encode death proteins
requiring a death ligand to kill the cells, and death genes that
encode death proteins that kill cells as a result of high
expression within the cell and do not require the addition of any
death ligand. Preferred are cell death mechanisms that requires a
two-step process: the expression of the death gene and induction of
the death phenotype with a signal or ligand such that the cells may
be grown expressing the death gene, and then induced to die. A
number of death genes/ligand pairs are known, including, but not
limited to, the Fas receptor and Fas ligand (Schneider, P. et al.
(1997) J. Biol. Chem. 272: 18827-33; Gonzalez-Cuadrado, S. et al.
(1997) Kidney Int. 51: 1739-46; Muruve, D. A. et al. (1997) Hum.
Gene Ther. 8: 955-63); p450 and cyclophosphamide (Chen, L. et al.
(1997). Cancer Res. 57: 4830-37); thymidine kinase and gangcylovir
(Stone, R. (1992) Science 256: 1513), tumor necrosis factor (TNF)
receptor and TNF. Alternatively, the death gene need not require a
ligand, and death results from high expression of the gene; for
example, the overexpression of a number of programmed cell death
(PCD) proteins known to cause cell death, including, but not
limited to, caspases, bax, TRADD, FADD, SCK, MEK, etc.
[0169] In a preferred embodiment, death genes also include toxins
that cause cell death, or impair cell survival or cell function
when expressed by a cell. These toxins generally do not require
addition of a ligand to produce toxicity. An example of a suitable
toxin is campylobacter toxin CDT (Lara-Tejero, M. (2000) Science,
290: 354-57). Expression of CdtB subunit, which has homology to
nucleases, causes cell cycle arrest and ultimately cell death.
Another toxin, the diphtheria toxin (and similar Pseudomonas
exotoxin), functions by ADP ribosylating the ef-2 (elongation
factor 2) molecule in the cell and preventing translation.
Expression of the diphtheria toxin A subunit induces cell death in
cells expressing the toxin fragment. Other useful toxins include
cholera toxin and pertussis toxin (catalytic subunit-A ADP
ribosylates the G protein regulating adenylate cyclase), pierisin
from cabbage butterflys (induces apoptosis in mammalian cells;
Watanabe, M. (1999) Proc. Natl. Acad. Sci. USA 96: 10608-13),
phospholipase snake venom toxins (Diaz, C. et al. (2001) Arch.
Biochem. Biophys. 391: 56-64), ribosome inactivating toxins (i.e.
ricin A chain, Gluck, A. et al. (1992) J. Mol. Biol. 226:
411-24;and nigrin, Munoz, R. et al. (2001) Cancer Lett. 167:
163-69) and pore forming toxins (hemolysin and leukocidin). When
the target cells are neuronal cells, neuronal specific toxins may
be used to inhibit specific neuronal functions. These include
bacterial toxins such as botulinum toxin and tetanus toxin, which
are proteases that act on synaptic vesicle associated proteins
(i.e. synaptobrevin) to prevent neurotransmitter release (see Binz,
T. et al. (1994) J. Biol. Chem. 269: 9153-58; Lacy, D. B. et al.
(1998) Curr. Opin. Struct. Biol. 8: 778-84).
[0170] Another preferred embodiment of a reporter molecule is a
cell cycle gene, that is, a gene that causes alterations in the
cell cycle. For example, Cdk interacting protein p21 (see Harper,
J. W. et al. (1993) Cell 75: 805-16), which inhibits cyclin
dependent kinases, does not cause cell death but causes cell-cycle
arrest. Thus, expressing p21 allows selecting for regulators of
promoter activity or regulators of p21 activity based on detecting
cells that grow out much more quickly due to low p21 activity,
either through inhibiting promoter activity or inactivation of p21
protein activity. As will be appreciated by those in the art, it is
also possible to conFigure the system to select cells based on
their inability to grow out due to increased p21 activity. Similar
mitotic inhibitors include p27, p57, p16, p15, p18 and p19, p19 ARF
(human homolog p14ARF). Other cell cycle proteins useful for
altering cell cycle include cyclins (Cln), cyclin dependent kinases
(Cdk), cell cycle checkpoint proteins (i.e. Rad17, p53), Cks1 p9,
Cdc phosphatases (i.e Cdc 25) etc.
[0171] As is described generally in WO 00/20574, expressly
incorporated by reference, the reporter gene or reporter protein
can be part of a fusion nucleic acid or fusion polypeptide, and can
be attached to a candidate agent at the B or C-terminus, or
internally, with or without the use of linkers. In addition, rather
than have the reporter gene be a fusion partner, it may be located
anywhere in the vector being used, or attached to other
components.
[0172] 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 inhibitors must be localized appropriately.
[0173] 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 signaling 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 a secretory signal. Particularly
preferred is localization to either subcellular locations or to the
outside of the cell via secretion.
[0174] 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); NFkB p50 (EEVQRKRQKL; Ghosh et al.,
Cell 62:1019 (1990); NFkB 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, 84:6795-4799, 1987; Galileo, et al., Proc. Natl. Acad. Sci.
USA, 87:458462, 1990.
[0175] 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 peptide libraries 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 randomized expression
product extracellularly or in the cytoplasmic space; see FIG. 28.
For extracellular presentation, a membrane anchoring region is
provided at the carboxyl terminus of the peptide presentation
structure. The randomized expression product region 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. The
binding of such molecules could confer function on the cells
expressing a peptide that binds the molecule. The cytoplasmic
region could be neutral or could contain a domain that, when the
extracellular randomized expression product region is bound,
confers a function on the cells (activation of a kinase,
phosphatase, binding of other cellular components to effect
function). Similarly, the randomized expression product-containing
region could be contained within a cytoplasmic region, and the
transmembrane region and extracellular region remain constant or
have a defined function.
[0176] 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.
[0177] 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.
[0178] 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
(PQRPEDCRPRGSVKGTGLDFACDIYIWAPLAGICVALLLSLIITLICYHSR; Nakauchi,
supra) and 224-256 from ICAM-2 (MVIIVTVVSVLLSLFVTSVLLCFIFGQHLRQQR;
Staunton, supra).
[0179] 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 (PNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT, 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.
[0180] 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 variable 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 p21 H-ras 1 protein
(LNPPDESGPGCMSCKCVLS; Capon et al., Nature 302:33 (1983)).
[0181] 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).
[0182] 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;
MFSMLSKRWAQRTLSKSFYSTATGAASKSGKLTQKLVTAGVAAAGITASTLLYADSLTAEAMTA;
Schatz, supra) or mitochondrial outer membrane sequences (yeast 70
kD outer membrane protein;
MKSFITRNKTAILATVAATGTAIGAYYYYNQLQQQQQRGKK; Schatz, supra).
[0183] 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).
[0184] 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, P21 H-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)).
[0185] In a preferred embodiment, the targeting sequence is a
secretory signal sequence capable of effecting the secretion of the
candidate translation product. There are a large number of known
secretory signal sequences which are placed 5' to the variable
peptide region, and are cleaved from the peptide 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. This is
particularly useful to generate a peptide capable of binding to the
surface of, or affecting the physiology of, a target cell that is
other than the host cell, e.g., the cell infected with the
retrovirus. In a preferred approach, a fusion product is conFigured
to contain, in series, secretion signal peptide-presentation
structure-randomized expression product region-presentation
structure, see FIG. 28. In this manner, target cells grown in the
vicinity of cells caused to express the library of peptides, are
bathed in secreted peptide. Target cells exhibiting a physiological
change in response to the presence of a peptide, e.g., by the
peptide binding to a surface receptor or by being internalized and
binding to intracellular targets, and the secreting cells are
localized by any of a variety of selection schemes and the peptide
causing the effect determined. Exemplary effects include variously
that of a designer cytokine (i.e., a stem cell factor capable of
causing hematopoietic stem cells to divide and maintain their
totipotential), a factor causing cancer cells to undergo
spontaneous apoptosis, a factor that binds to the cell surface of
target cells and labels them specifically, etc.
[0186] 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 IL4, which
comprises the first 24 amino acids of IL-4 as follows:
MGLTSQLLPPLFFLLACAGNFVHG.
[0187] 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 candidate agent or the nucleic acid
encoding it. 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.
[0188] Alternatively, the rescue sequence may be a unique
oligocandidate nucleic acid 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.
[0189] In a preferred embodiment, the fusion partner is a stability
sequence to confer stability to the candidate bioactive agent or
the nucleic acid encoding it. Thus, for example, peptides may be
stabilized by the incorporation of glycines after the initiation
methionine (MG or MGG0), 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(X).sub.nGGPP, where X is any amino acid and n is an
integer of at least four.
[0190] In one embodiment, the fusion partner is a dimerization
sequence. A dimerization sequence allows the non-covalent
association of one random peptide to another random peptide, with
sufficient affinity to remain associated under normal physiological
conditions. This effectively allows small libraries of random
peptides (for example, 10.sup.4) to become large libraries if two
peptides per cell are generated which then dimerize, to form an
effective library of 10.sup.8 (10.sup.4.times.10.sup.4). It also
allows the formation of longer random peptides, if needed, or more
structurally complex random peptide molecules. The dimers may be
homo- or heterodimers.
[0191] Dimerization sequences may be a single sequence that
self-aggregates, or two sequences, each of which is generated in a
different retroviral construct. That is, nucleic acids encoding
both a first random peptide with dimerization sequence 1, and a
second random peptide with dimerization sequence 2, such that upon
introduction into a cell and expression of the nucleic acid,
dimerization sequence 1 associates with dimerization sequence 2 to
form a new random peptide structure.
[0192] Suitable dimerization sequences will encompass a wide
variety of sequences. Any number of protein-protein interaction
sites are known. In addition, dimerization sequences may also be
elucidated using standard methods such as the yeast two hybrid
system, traditional biochemical affinity binding studies, or even
using the present methods.
[0193] The fusion partners may be placed anywhere (i.e.,
N-terminal, C-terminal, internal) in the structure as the biology
and activity permits.
[0194] 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 randomized
candidate agents) may be desirable to allow the candidate agents to
interact with potential targets unhindered. For example, when the
candidate bioactive agent is a peptide, useful linkers include
glycine-serine polymers (including, for example, (GS).sub.n,
(GSGGS).sub.n and (GGGS).sub.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.
[0195] In addition, the fusion partners, including presentation
structures, may be modified, randomized, and/or matured to alter
the presentation orientation of the randomized expression product.
For example, determinants at the base of the loop may be modified
to slightly modify the internal loop peptide tertiary structure,
which maintaining the randomized amino acid sequence.
[0196] In a preferred embodiment, combinations of fusion partners
are used. Thus, for example, any number of combinations of
presentation structures, targeting sequences, reporter sequences,
rescue sequences, and stability sequences may be used, with or
without linker sequences.
[0197] By "candidate nucleic acids" herein is meant nucleic acids
comprising a candidate nucleic acid sequence encoding a candidate
bioactive agent. The candidate nucleic acids can be expressed to
form a candidate bioactive agent. Therefore, the candidate nucleic
acids can further encode, for example, a fusion partner and contain
sequences to effect translation or transcription. Where the
candidate bioactive agents are randomized peptides in a peptide
library, the candidate nucleic acid generally contains cloning
sites which are placed to allow in frame expression of the
randomized peptides, and any fusion partners, if present, such as
presentation structures. For example, when presentation structures
are used, the presentation structure will generally contain the
initiating ATG, as a part of the parent vector. Where the candidate
bioactive agents are RNAs in an RNA library, the candidate nucleic
acids 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 is 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.
[0198] Generally, the candidate nucleic acids are expressed within
the cells to produce expression products of the candidate nucleic
acids. As outlined above, the expression products include
translation products, i.e., peptides, or transcription products,
i.e., nucleic acid. The candidate bioactive agents and candidate
nucleic acids are randomized, either fully randomized or they are
biased in their randomization, e.g. in nucleotide/residue frequency
generally or per position. By "randomized" or grammatical
equivalents herein is meant that each nucleic acid and peptide
consists of essentially random nucleotides and amino acids,
respectively. As is more fully described below, the candidate
nucleic acids which give rise to the candidate expression products
are chemically synthesized, and thus may incorporate any nucleotide
at any position. Thus, when the candidate nucleic acids are
expressed to form peptides, any amino acid residue may be
incorporated at any position. The synthetic process can be designed
to generate randomized nucleic acids, to allow the formation of all
or most of the possible combinations over the length of the nucleic
acid, thus forming a library of randomized candidate nucleic
acids.
[0199] The library should provide a sufficiently structurally
diverse population of randomized expression products to effect a
probabilistically sufficient range of cellular responses to provide
one or more cells exhibiting a desired response. Accordingly, an
interaction library must be large enough so that at least one of
its members will have a structure that gives it affinity for some
molecule, protein, or other factor whose activity is necessary for
completion of the signaling pathway. Although it is difficult to
gauge the required absolute size of an interaction library, nature
provides a hint with the immune response: a diversity of
10.sup.7-10.sup.8 different antibodies provides at least one
combination with sufficient affinity to interact with most
potential antigens faced by an organism. Published in vitro
selection techniques have also shown that a library size of
10.sup.7 to 10.sup.8 is sufficient to find structures with affinity
for the target. A library of all combinations of a peptide 7 to 20
amino acids in length, such as proposed here for expression in
retroviruses, has the potential to code for 20.sup.7 (10.sup.9) to
20.sup.20. Thus, with libraries of 10.sup.7 to 10.sup.8 per ml of
retroviral particles the present methods allow a "working" subset
of a theoretically complete interaction library for 7 amino acids,
and a subset of shapes for the 20.sup.20 library. Thus, in a
preferred embodiment, at least 10.sup.6, preferably at least
10.sup.7, more preferably at least 10.sup.8 and most preferably at
least 10.sup.9 different expression products are simultaneously
analyzed in the subject methods. Preferred methods maximize library
size and diversity.
[0200] It is important to understand that in any library system
encoded by oligonucleotide synthesis one cannot have complete
control over the codons that will eventually be incorporated into
the peptide structure. This is especially true in the case of
codons encoding stop signals (TAA, TGA, TAG). In a synthesis with
NNN as the random region, there is a 3/64, or 4.69%, chance that
the codon will be a stop codon. Thus, in a peptide of 10 residues,
there is an unacceptable high likelihood that 46.7% of the peptides
will prematurely terminate. For free peptide structures this is
perhaps not a problem. But for larger structures, such as those
envisioned here, such termination will lead to sterile peptide
expression. To alleviate this, random residues are encoded as NNK,
where K=T or G. This allows for encoding of all potential amino
acids (changing their relative representation slightly), but
importantly preventing the encoding of two stop residues TAA and
TGA. Thus, libraries encoding a 10 amino acid peptide will have a
15.6% chance to terminate prematurely. For candidate nucleic acids
which are not designed to result in peptide expression products,
this is not necessary.
[0201] In one embodiment, the library is fully randomized, with no
sequence preferences or constants at any position. In a preferred
embodiment, the library is biased. That is, some positions within
the sequence are either held constant, or are selected from a
limited number of possibilities. For example, in a preferred
embodiment, the nucleotides or amino acid residues are randomized
within a defined class, for example, of hydrophobic amino acids,
hydrophilic residues, sterically biased (either small or large)
residues, towards the creation of cysteines, for cross-linking,
prolines for SH-3 domains, serines, threonines, tyrosines or
histidines for phosphorylation sites, etc., or to purines, etc.
[0202] In a preferred embodiment, the bias is towards peptides or
nucleic acids that interact with known classes of molecules. For
example, when the candidate bioactive agent is a peptide, it is
known that much of intracellular signaling is carried out via short
regions of polypeptides interacting with other polypeptides through
small peptide domains. For instance, a short region from the HIV-1
envelope cytoplasmic domain has been previously shown to block the
action of cellular calmodulin. Regions of the Fas cytoplasmic
domain, which shows homology to the mastoparan toxin from Wasps,
can be limited to a short peptide region with death-inducing
apoptotic or G protein inducing functions. Magainin, a natural
peptide derived from Xenopus, can have potent anti-tumor and
anti-microbial activity. Short peptide fragments of a protein
kinase C isozyme (.beta.PKC), have been shown to block nuclear
translocation of .beta.PKC in Xenopus oocytes following
stimulation. And, short SH-3 target peptides have been used as
psuedosubstrates for specific binding to SH-3 proteins. This is of
course a short list of available peptides with biological activity,
as the literature is dense in this area. Thus, there is much
precedent for the potential of small peptides to have activity on
intracellular signaling cascades. In addition, agonists and
antagonists of any number of molecules may be used as the basis of
biased randomization of candidate bioactive agents as well.
[0203] Thus, a number of molecules or protein domains are suitable
as starting points for the generation of biased randomized
candidate bioactive agents. A large number of small molecule
domains are known, that confer a common function, structure or
affinity. In addition, as is appreciated in the art, areas of weak
amino acid homology may have strong structural homology. A number
of these molecules, domains, and/or corresponding consensus
sequences, are known, including, but are not limited to, SH-2
domains, SH-3 domains, Pleckstrin, death domains, protease
cleavage/recognition sites, enzyme inhibitors, enzyme substrates,
Traf, etc. Similarly, there are a number of known nucleic acid
binding proteins containing domains suitable for use in the
invention. For example, leucine zipper consensus sequences are
known.
[0204] Where the ultimate expression product is a nucleic acid, at
least 10, preferably at least 12, more preferably at least 15, most
preferably at least 21 nucleotide positions need to be randomized,
with more preferable if the randomization is less than perfect.
Similarly, at least 5, preferably at least 6, more preferably at
least 7 amino acid positions need to be randomized; again, more are
preferable if the randomization is less than perfect.
[0205] In a preferred embodiment, biased SH-3 domain-binding
oligonucleotides/peptides are made. SH-3 domains have been shown to
recognize short target motifs (SH-3 domain-binding peptides), about
ten to twelve residues in a linear sequence, that can be encoded as
short peptides with high affinity for the target SH-3 domain.
Consensus sequences for SH-3 domain binding proteins have been
proposed. Thus, in a preferred embodiment, oligos/peptides are made
with the following biases [0206] 1. XXXPPXPXX, wherein X is a
randomized residue.
[0207] 2. (within the positions of residue positions 11 to -2):
TABLE-US-00001 11 10 9 8 7 6 5 4 3 2 1 Met Gly aa11 aa10 aa9 aa8
aa7 Arg Pro Leu Pro Pro hyd 0 -1 -2 Pro hyd hyd Gly Gly Pro Pro
STOP atg ggc nnk nnk nnk nnk nnk aga cct ctg cct cca sbk ggg sbk
sbk gga ggc cca cct TAA1.
[0208] In this embodiment, the N-terminus flanking region is
suggested to have the greatest effects on binding affinity and is
therefore entirely randomized. "Hyd" indicates a bias toward a
hydrophobic residue, i.e.--Val, Ala, Gly, Leu, Pro, Arg. To encode
a hydrophobically biased residue, "sbk" codon biased structure is
used. Examination of the codons within the genetic code will ensure
this encodes generally hydrophobic residues. s=g,c; b=t, g, c; v=a,
g, c; m=a, c; k=t, g; n=a, t, g, c.
[0209] The candidate nucleic acids are introduced into the cells to
screen for bioactive agents capable of altering the phenotype of a
cell. 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, lipofectin7, 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.
[0210] In a preferred embodiment, the candidate nucleic acids are
part of a retroviral particle which infects the cells. 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. Infection can be
optimized such that each cell generally expresses a single
construct, using the ratio of virus particles to number of cells.
Infection follows a Poisson distribution.
[0211] In a preferred embodiment, the candidate nucleic acids are
introduced into the cells using retroviral vectors. Currently, the
most efficient nucleic acid 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 R
packaging signal are packaged into maturing virions. Retroviruses
are preferred for a number of reasons. First, their derivation is
easy. Second, unlike Adenovirus-mediated nucleic acid 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.
[0212] 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.
[0213] In one embodiment of the invention, the library is generated
in a retrovirus DNA construct backbone, as is generally described
in the examples. Standard oligonucleotide synthesis is done to
generate the random portion of the candidate bioactive agent, using
techniques well known in the art (see Eckstein, Oligonucleotides
and Analogues, A Practical Approach, IRL Press at Oxford University
Press, 1991); libraries may be commercially purchased. Libraries
with up to 10.sup.9 unique sequences can be readily generated in
such DNA backbones. 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, eTet), 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 in the random
region), etc.
[0214] 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.
[0215] 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.
[0216] 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, outlined in the examples. A general schematic of the
retroviral construct is depicted in FIG. 29.
[0217] In a preferred embodiment, a retroviral vector is designed
to allow inducible expression of retroviral inserts after
integration of a single vector in target cells. Importantly, the
expression system is contained within the single retrovirus. For
example, 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 Tet or analogues thereof.
However, when using the the transactivator tTA, in the absence of
Tet or an analogue thereof, expression is turned on, 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. Further, using the
transactivator rtTA, in the presence of Tet or an analogue thereof,
expression is turned on.
[0218] In this manner the primers create a library of fragments,
each containing a different random candidate nucleic acid sequence
that may encode a different peptide (or candidate bioactive agent).
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.
[0219] 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.
[0220] Preferred systems include PhiNX-eco and PhiNX-ampho or
similar cell lines, which are two cells lines as follows. The cell
lines are based 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 be 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 are called BOSC23 and Bing, respectively. The
utility of these lines are that one can produce small amounts of
recombinant virus transiently for use in small-scale
experimentation. The lines offer advantages over previously
developed stable expression systems in that virus can be produced
in days rather than months.
[0221] Although the BING and BOSC23 lines are useful in the present
invention, the PhiNX second-generation lines are preferred. These
lines are based on 293T cells as well, and contain the following
improvements over the first-generation lines. 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, PHEONIX-ECO and
PHEONIX-AMPHO. Gag-pol was introduced with hygromycin as the
co-selectable marker and the envelope proteins were introduced with
diphtheria 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 PHEONIX-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.
[0222] Both PHEONIX-ECO and PHEONIX-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 nucleic acid transfer
experiments. Finally, the titres produced by these lines have been
tested. Using standard polybrene-enhanced retroviral infection,
titres approaching or above 10.sup.7 per ml were observed for both
PHEONIX-eco and PHEONIX-ampho when carrying episomal constructs.
When transiently produced virus is made, titres are usually 2 to
1/3 that value.
[0223] 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
addition, 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.
[0224] 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.
[0225] 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 multiplying 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 expression 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.
[0226] The candidate nucleic acids, as part of the retroviral
construct, are introduced into the cells to screen for bioactive
agents capable of altering the phenotype of a cell, as described
herein.
[0227] In a preferred embodiment, a first plurality of cells is
screened using the methods of the present invention. That is, the
cells into which the candidate 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.
[0228] In one embodiment, the candidate nucleic acids are
introduced into a first plurality of cells, and the effect of the
candidate bioactive agents 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. Using standard
techniques, for example, the first plurality of cells may be grown
in or on one media, and the media allowed to touch a second
plurality of cells, and the effect measured. Alternatively, there
may be direct contact between the cells. Thus, "contacting" as used
herein includes both direct and indirect contact and, thus, may be
a functional contact. In this embodiment, the first plurality of
cells may or may not be screened using the methods of the present
invention.
[0229] In the methods of the present, invention, the cells are
treated to conditions suitable for inducing or repressing the
expression of the candidate nucleic acids. The expression products
of the candidate nucleic acids may be translation or transcription
products.
[0230] Thus, the methods of the present invention comprise
introducing a molecular library of randomized candidate nucleic
acids into a plurality of cells, a cellular library. Each of the
nucleic acids comprises a different, generally randomized,
candidate nucleic acid sequence. The plurality of cells is then
screened, as is more fully outlined below, for a cell exhibiting an
altered phenotype. The altered cellular phenotype is due to the
presence of a candidate bioactive agent.
[0231] By "altered cellular phenotype," "altered phenotype," or
"changed physiology" or other grammatical equivalents herein is
meant that the phenotype of the cell is altered in a-manner that is
detectable and/or measurable. In a preferred embodiment, the
cellular phenotype is altered due to the presence of a candidate
bioactive agent or corresponds to the the expression of a nucleic
acid sequence encoding a candidate bioactive agent. 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 detected using the present methods of screening.
[0232] 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,
susceptibility, latency, adhesion, and uptake of viruses and
bacterial pathogens. By "capable of altering the phenotype" herein
is meant that the bioactive agent can change the phenotype of the
cell in a detectable and/or measurable way.
[0233] The altered phenotype may be detected and selected or sorted
or collected from a parent phenotype, 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
altered phenotype is detected using, for example: microscopic
analysis of cell morphology; standard cell viability assays,
including both increased cell death and increased cell viability,
e.g., 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; and other methods of biochemical known in the
art for detection of the expression of the candidate nucleic acid
sequence.
[0234] In a preferred embodiment, the candidate nucleic acid
sequence is operably linked to a sequence encoding a reporter
protein that is an autofluorescent protein, and the cells having an
altered phenotype are sorted or detected or collected by FACs.
[0235] In preferred embodiments, the altered phenotype is detected
in the cell in which the candidate nucleic acid is introduced. In
other embodiments, the altered phenotype is detected in a second
cell which is responding to a molecular signal from a first
cell.
[0236] Exemplary assays are described in PCT US97/01019; WO
99/58663; WO 00/26241; WO 99/54494; WO 00172088; and PCT
US01/10906, all of which are expressly incorporated by
reference.
[0237] In a preferred embodiment, once a cell with an altered
phenotype is detected using the methods of the invention, the cell
can be isolated. This may be performed 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
fluoroscent one; overgrowth against a background of no or slow
growth; death of cells and isolation of DNA or other cell vitality
indicator dyes.
[0238] In a preferred embodiment, the candidate nucleic acid and/or
the bioactive agent is isolated from the positive cell. This may be
performed 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 unique random sequence.
Alternatively, the bioactive agent is isolated using a rescue
sequence. Thus, for example, rescue sequences comprising epitope
tags or purification sequences may be used to pull out the
bioactive agent, using immunoprecipitation or affinity columns. 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 peptide may be detected using mass
spectroscopy.
[0239] Once rescued, the sequence of the bioactive agent and/or
bioactive nucleic acid is determined. This information can then be
used in a number of ways.
[0240] In a preferred embodiment, the bioactive agent is
resynthesized and reintroduced into the target cells, to verify the
effect This may be done 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.
[0241] In a preferred embodiment, the sequence of a bioactive agent
is used to generate more candidate bioactive agents. For example,
the sequence of the bioactive agent may be the basis of a second
round of (biased) randomization, to develop bioactive agents with
increased or altered activities. Alternatively, the second round of
randomization may change the affinity of the bioactive agent.
Furthermore, it may be desirable to put the identified random
region of the bioactive agent into other presentation structures,
or to alter the sequence of the constant region of the presentation
structure, to alter the conformation/shape of the bioactive agent.
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 end of the ligand region constant and randomizing the
other end to shift the binding of the peptide around.
[0242] In a preferred embodiment, either the bioactive agent or the
bioactive nucleic acid encoding it 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".
[0243] In a preferred embodiment, the bioactive agent is used to
pull out target molecules. For example, as outlined herein, if the
target molecules are proteins, the use of epitope tags or
purification sequences can allow the purification of primary target
molecules via biochemical means (co-immunoprecipitation, affinity
columns, etc.). Alternatively, the peptide, 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,
peptides 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. The peptide(s) can be combined with other
pharmacologic activators to study the epistatic relationships of
signal transduction pathways in question. It is also possible to
synthetically prepare labeled peptide bioactive agent and use it to
screen a cDNA library expressed in bacteriophage for those cDNAs
which bind the peptide. Furthermore, it is also possible that one
could use cDNA cloning via retroviral libraries to "complement" the
effect induced by the peptide. In such a strategy, the peptide
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 peptide acts upon.
[0244] 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, signaling
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.
[0245] The screening methods of the present invention may be useful
to screen a large number of cell types under a wide variety of
conditions. 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 and therefore the cells have an altered
phenotype. Alternatively, with an eye towards elucidating the
cellular mechanisms associated with the disease state or signaling
pathway, normally desirable consequences may be reduced or
eliminated and therefore the cells have an altered phenotype.
[0246] 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, random libraries can be
introduced into any tumor cell (primary or cultured), and peptides
identified which by themselves induce apoptosis, cell death, loss
of cell division or decreased cell growth. This may be done de
novo, or by biased randomization toward known peptide agents, such
as angiostatin, which inhibits blood vessel wall growth.
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.
[0247] 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 random
libraries introduced into these cells, to select for bioactive
agents which reverse or correct the transformed state. One of the
signal features of onconucleic acid 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.
[0248] 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 random libraries 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.
[0249] 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 random libraries introduced into them, and
peptides 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 peptide 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. Peptides 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. Peptides 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.
[0250] In a preferred embodiment, the random libraries 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 peptide or small molecule derived from a
peptide could reverse the mutation. One example would be
upregulation of the immediately downstream cyclin-dependent kinase
p21CIP1/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.
[0251] 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 nucleic acid (APC) and the
Drosophila discs-large nucleic acid (Dlg), which are components of
cell-cell junctions. Mutations of brca-1 are important in
hereditary ovarian and breast cancers, and constitute art
additional application of the present invention.
[0252] In a preferred embodiment, the methods of the present
invention are used to create novel cell lines from cancers from
patients. A retrovirally delivered short peptide 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 Epstein-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 nucleic acids encoding peptides which inhibit
apoptosis, increased cell survival in vitro, and hence the
opportunity to study signaling 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.
[0253] 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). Candidate bioactive agents
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).
[0254] In a preferred embodiment, the present methods are used to
screen for diminished arrhythmia potential in cardiomyocytes. The
screens comprise the introduction of the candidate nucleic acids
encoding candidate bioactive agents, 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.
[0255] 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.
[0256] 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.
[0257] 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
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 peptides that either: 1) downregulate adhesion molecule
expression on the surface of the endothelial cells (binding assay);
2) block adhesion molecule activation on the surface of these cells
(signaling assay); or 3) release in an autocrine manner peptides
that block receptor binding to the cognate receptor on the adhering
cell.
[0258] 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 libraries 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.
[0259] 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. Candidate
bioactive 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.
[0260] 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. Candidate bioactive agent libraries can
be inserted into these cell types and their proliferation in
response to specific stimuli monitored. One application may be
intracellular peptides 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
peptides may require targeted drug delivery; this is available with
stents, hydrogel coatings, and infusion-based catheter systems.
Peptides 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. Peptides can be isolated from these libraries 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.
[0261] 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. Candidate bioactive agent libraries 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 peptides
isolated that produce the appropriate phenotype. Screening for
antagonism of vascular endothelial cell growth factor, important in
angiogenesis, would also be useful.
[0262] In a preferred embodiment, the present methods are useful in
screening for decreases in atherosclerosis producing mechanisms to
find peptides that regulate LDL and HDL metabolism. Candidate
libraries can be inserted into the appropriate cells (including
hepatocytes, mononuclear leukocytes, endothelial cells) and
peptides 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 candidate 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.
[0263] 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. Candidate
libraries can be inserted into cells that have these receptors
cloned into them, and inhibitory peptides selected that are
secreted in an autocrine manner that block the signaling responses
to galanin and NPY. In a similar manner, peptides can be found that
regulate the leptin receptor.
[0264] In a preferred embodiment, the present methods are useful in
neurobiology applications. Candidate 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 peptides which selectively block neuronal
apoptosis. Other applications include neurodegenerative diseases
such as Alzheimer's disease and Huntington's disease.
[0265] 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
candidate 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.
[0266] 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 candidate libraries
and screened for their growth, proliferation, collagen stimulating
activity, and/or proline incorporating ability on the target
osteoblasts. Alternatively, candidate libraries can be expressed
directly in osteoblasts or chondrocytes and screened for increased
production of collagen or bone.
[0267] 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. Candidate libraries can be inserted into cells removed from
active psoriatic plaques, and bioactive agents isolated which
decrease the rate of growth of these cells.
[0268] In a preferred embodiment, the present methods are useful in
the regulation or inhibition of keloid formation (i.e., excessive
scarring). Candidate libraries 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 peptide 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.
[0269] 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. 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.
[0270] 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. Candidate libraries 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.
[0271] In a preferred embodiment, the present methods are useful in
endocrinology applications. The retroviral peptide library
technology can be applied broadly to any endocrine, growth factor,
cytokine or chemokine network which involves a signaling peptide or
protein 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 a peptide 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 peptides which increase
the expression or release of hormones from the cells which normally
produce them could have broad applications to conditions of
hormonal deficiency.
[0272] 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. Candidate libraries can then be
inserted into these cells under the above conditions, and peptides
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.
[0273] 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
extracellular anchored constructs offer superb tools for such a
discovery. Into the transmembrane, epitope tagged, glycine-serine
tethered constructs (ssTM V G20 E TM), one can place a random,
cyclized peptide library of the general sequence CNNNNNNNNNNC or
C--(X).sub.n--C. Then one infects a cell line that expresses CCR-5
with retroviruses containing this library. Using an antibody to
CCR-5 one can use FACS to sort desired cells based on the binding
of this antibody to the receptor. All cells which do not bind the
antibody will be assumed contain inhibitors of this antibody
binding site. These inhibitors, in the retroviral construct can be
further assayed for their ability to inhibit HIV-1 entry.
[0274] 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
usesCD44) and to fuse with cells (HIV uses chemokine receptor).
Candidate libraries 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.
[0275] 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.
Candidate libraries 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 a manner analogous to intracellular "antibiotic
peptides" similar to magainins. In addition peptides 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 peptide from a candidate library, 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.
[0276] 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.
Candidate libraries 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.
[0277] 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. Candidate agents 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.
[0278] 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. Candidate libraries 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.
[0279] 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. Candidate libraries 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.
[0280] Multiple drug resistance, and hence tumor cell selection,
outgrowth, and relapse, leads to morbidity and mortality in cancer
patients. Candidate libraries 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 peptides
which block the normal efflux of fluorescent drug from these cells.
Candidate libraries are particularly suited to screening for
peptides 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 peptide 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.
[0281] 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
candidate 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.
[0282] 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.
Candidate libraries 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 a
peptide 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 peptide 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 peptide 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).
[0283] 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. Candidate libraries 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.
[0284] 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-'', before they bind their receptor.
[0285] Antigen processing by mononuclear leukocytes (ML) is an
important early step in the immune system=s ability to recognize
and eliminate foreign proteins. Candidate agents can be inserted
into ML cell lines and agents selected which alter the
intracellular processing of foreign peptides 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 peptide 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.
[0286] 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. Candidate 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.
[0287] In a preferred embodiment, the present methods are useful in
biotechnology applications. Candidate library 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. Candidate libraries
resulting in bioactive agents which select for an increased cell
growth rate (perhaps peptides 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.
[0288] 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.
[0289] 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 signaling pathway,
to localize the effects to a functional area of interest.
[0290] The following examples serve to more fully describe the
manner of using the above-described invention, as well as to set
forth the best modes contemplated for carrying out various aspects
of the invention. It is understood that these examples in no way
serve to limit the true scope of this invention, but rather are
presented for illustrative purposes. All references cited herein
are incorporated by reference in their entirety.
EXAMPLES
Example 1
[0291] Construction of BH-1-4 and BH2-A5 Cell Lines for Use in
Methods for Screening for an Altered Cellular Phenotype.
[0292] Cell line BH-1-4 The novel cell line, BH-1-4 was constructed
by engineering the B cell line BJAB to contain the cDNA encoding
the reporter protein heparin-binding epidermal-growth-factor-like
growth factor (HBEGF) operably linked to an interleukin 4 (IL4)
inducible germline promoter, the epsilon promoter (P.epsilon.) (see
FIG. 1). The epsilon promoter was activated by contacting the cells
with the stimulator IL4. In this cell line, IL4 binds to IL4
receptors (IL4R) expressed on the cell surface and the IL4R
mediates transduction and activation of the epsilon promoter by
IL4.
[0293] HBEGF functions as the receptor for diphtheria toxin ("dip"
or "dt").Diphtheria toxin is a 535 amino acid protein consisting of
three domains. The receptor binding domain (residues 387-535) binds
to BEGF. The transmembrane domain, T, comprising residues 200-378,
is responsible for creating a channel in the cell membrane. The
catalytic domain, comprising residues 1-188, is responsible for
stopping potein production in the cell nd thereby causing cell
death. Thus, in the novel BH-1-4 cell line, HBEGF confers high
sensitivity to the killing of cells by diphtheria toxin following
the induction of the expression of HBEGF by the addition of the
stimulator IL4. Thus, in this example cell death is a marker for
the expression of the reporter HBEGF in the presence of diphtheria
toxin.
[0294] Cell Line BH2-A5
[0295] The novel cell line, BH2-A5 was constructed in the same
manner as described above for the BH1-4 cell line, except that the
DNA encodes the reporter protein Green Fluorescent Protein (GFP)
operably linked to HBEGF via a cleavable peptide linker (2a) (see
FIG. 2). Basically, the BH2-A5 cell line was constructed by
engineering the B cell line BJAB to contain the DNA encoding the
reporter (HBEGF) operably linked to an interleukin 4 (IL4)
inducible germline promoter, the epsilon promoter (Pe), and the GFP
reporter, where the HBEGF and GFP are linked by the cleavable
peptide linker 2a. Similar to the BH1-4 cell line, the epsilon
promoter was activated by contacting the cells with the stimulator
IL4; IL4 binds to IL4 receptors (IL4R) expressed on the cell
surface; and the IL4R mediates transduction and activation of the
epsilon promoter by IL4. Also, in this cell line, HBEGF functions
as the receptor for diphtheria. Thus, in the novel BH2-A5 cell
line, HBEGF confers high sensitivity to the killing of cells by
diphtheria toxin following the induction of the expression of HBEGF
by the addition of the stimulator IL4.In addition, the concomitant
expression of GFP was used to monitor IL4 induction of the epsilon
promoter by fluorescence using FACS. Thus, the BH2-A5 cell line was
engineered to contain the HBEGF and GFP dual-function reporter. In
this example cell death is a marker for the expression of the
reporter HBEGF in the presence of diphtheria toxin, whereas GFP is
a marker for IL4 induced expression.
[0296] Thus, the BH1-4 and BH2-A5 were used to screen for an
altered cellular phenotype due to the presence of a candidate
bioactive agent. In this example, the candidate bioactive agent is
a member of a peptide library (with a complexity of 1.times.10e9),
where the peptide is operably linked to a GFP reporter; the parent
phenotype is the result of contacting the cells with the stimulator
IL4 and induction of expression of HBEGF; and the altered cellular
phenotype is cell survival in the presence of diphtheria toxin in
cells expressing HBEGF, where the altered cellular phenotype is due
to the presence of a bioactive agent that inhibits the IL4-induced
expression of HBEGF and thereby results in the survival of the
cells in the presence of diphtheria toxin.
[0297] FIG. 3 depicts the conventional screening method which
involved first infecting the cell lines with retroviral vectors
encoding a candidate peptide operably linked to a GFP reporter and
thereby expressing the peptide in the cells; culturing the infected
cells in the presence of the stimulator IL4 to induce expression of
HBEGF; contacting the cells with diphtheria toxin to kill cells
expressing HBEGF (i.e., selecting the cells in the presence of
diphtheria toxin); removing the IL4 and diphtheria toxin by washing
the cells, concentrating them, and removing the dead cells
("debris"); from the surviving (selected) cells, rescue the
retroviral population and infect naive cells for a new fresh round
of selection and perform clonal selection and analysis of the
cloned retroviral population. However, this step requires that the
complexity and representation of the population should be
maintained as they are infected into naive cells; and, further,
this step is technically challenging. Thus, the present invention
provides a novel approach that obviates these problems. The
following examples further illustrate this novel approach for
screening for altered cellular phenotypes.
Example 2
[0298] Screening for an Altered Cellular Phenotype Using Known
Bioactive Agents, SOCS1 and STAT6.DELTA. (a C-Terminal Truncated
Version of STAT6), as Positive Controls for the Method of
Screening.
[0299] STAT6 (signal transducer and activator of transcription 6)
mediates the response of cytokines like IL4 and SOCS1 (suppressor
of cytokine signaling) and STAT6? Are known inhibitors of IL4
signal transduction. In the BH1-4 or BH2-A5 cells lines of
the-present invention, SOCS1 and STAT6.DELTA. each inhibit the IL4
inducible expression of HBEGF (and SOCS1 is a stronger inhibitor
than STAT6.DELTA.).
[0300] As depicted in FIG. 4, the following five different
retroviral vectors were constructed as positive controls for the
screening assay, and are the encoded GFP, SOCS1, STAT6.DELTA.,
and/or ires are operably linked to a promoter in the retroviral
vector: the retroviral vector cGFP contains the GFP; the retroviral
vector SOCS1-ires-GFP contains, from 5' to 3' (and operably
linked), SOCS1, internal ribosomal entry site (IRES), and GFP; the
retroviral vector GFP-SOCS1 contains, from 5' to 3' (and operably
linked), GFP-SOCS1 fusion; the retroviral vector
STAT6.DELTA.-ires-GFP contains, from 5' to 3' (and operably
linked), STAT6.DELTA., ires, and GFP; and the retroviral vector
GFP-STAT6.DELTA. contains, from 5' to 3' (and operably linked),
GFP-STAT6.DELTA. fusion.
[0301] As depicted in FIG. 5 the five retroviral vectors described
above were assayed in BH1-4 cells using the following protocol:
BH1-4 cells were infected with the retroviral constructs and
cultured for 3 days in medium containing IL4 and diphtheria toxin;
or alternatively, the infected cells were cultured in IL4 for 2
days followed by the addition of diphtheria toxin and culturing in
the presence of IL4 and diphtheria. Three days after infections, a
1:10 dilution of the cell cultures was made with fresh medium
(without IL4 and diphtheria toxin). On the day of infection, day 3,
day 6 and day 7 GFP fluorescence of the cells was measured using
FACS.
[0302] FIG. 6 depicts the effects of SOCS1 and STAT6 on
IL4/diphtheria (IL4/dip) induced death of BH1-4 cells 6 days post
infection for each of the five retroviral vectors, where the
histogram panels under the column entitled "unstimulated" refers to
a control assay where the cells were not stimulated/cultured with
IL4; the column entitled "IL4" refers to a control assay where the
cells were stimulated with IL4 but diphtheria toxin was not added
to the medium; the column entitled "IL4/dip" refers to the assay
where the cells were cultured in both IL4 and dip together for 3
days; and the column entitled "IL4 then dip" refers to the assay
where the cells were cultured in IL4 for 2 days followed by the
addition of diphtheria toxin. The histogram in each panel
summarizes the FACS measurements for each assay. The histograms in
the panels for the control assays entitled "unstimulated," "IL4,"
and "Dip" depict the fluorescent scattering pattern indicative of
live or surviving cells, whereas the histograms in the panels for
the assays entitled "IL4/dip" and "IL4 then dip" for retroviral
vectors cGFP, STAT6-IRES-GFP, and GFP-STAT6 depict a the
fluorescent scattering pattern indicative of dead or killed cells.
In contrast, the histograms in the panels for the same assays,
i.e., "IL4/dip" and "IL4 then dip," for retroviral vectors
SOCS1-IRES-GFP and GFP-SOCS1 depict a fluorescent scattering
pattern indicative of live cells. FIG. 7 depicts the same results
except that the vertical axis indicates the number of cells, and
the horizontal axis indicates the amount of GFP fluorescence.
[0303] These results indicate that the expression of SOCS1-ires-GFP
and GFP-SOCS1 retroviral constructs inhibit IL4 signaling/induced
expression of HBEGF. Further, the results indicate that the GFP
report protein is an effective marker for monitoring SOCS1
expression in the screening assays of the present invention.
Finally, these results indicate that the expression of
GFP-STAT6.DELTA. only partially inhibits IL4 signaling/induced
expression of HBEGF.
Example 3
[0304] Enrichment of Inhibitors of IL4 Signaling in BH1-4 or BH2-A5
Cell Lines Using SOCS1.
[0305] To examine the degree of enrichment that could be achieved
by the above-described IL4-diphtheria selection assay, BH1-4 and
BH2-A5 cell populations were "spiked" with BH1-4 and BH2-A5 cells
that had been infected with the retroviral construct
SOCS1-ires-GFP, and the following dilutions of the SOCS-infected
cells with uninfected cells (i.e., BH2-A5 and BH1-4 cells not
infected with a SOCS1 retroviral construct) were made: [0306] 1:10
[0307] 1:100 [0308] 1:1000 [0309] 1:10,000.
[0310] As depicted in FIG. 8, the diluted spiked cell populations
were then subjected to the following procedure: On the day of
infection, 12.times.10e6 cells at 200 k/ml were stimulated with IL4
(at 30 units/ml) in a T175 flask; On day 1 post infection,
diphtheria toxin (dip toxin) at 20 ng/ml was added to the cell
culture (but prior to the addition of dip toxin, an aliquot of
cells was taken as a "no dip control"); On day 3 post infection,
the cell culture was washed ("washout") from IL4 and diphtheria
toxin and the cells were grown in regular media; On days 4, 6, and
8 post infection, the GFP fluorescence of the cells were measured
using FACS; On day 9 post infection, the "washout" dying cells were
subjected to ficoll treatment to recover lice cells from dead
cells; and On days 15 and 18 post infection, the GFP fluorescence
of the cells were measured using FACS.
[0311] The results of this experiment indicate that the screening
assay using L4-dip selection is validated. In particular, the
"washout" treated cells ere enriched for the cells having the SOCS1
phenotype by approximately 1000 fold at the 1:10,000 seeding, from
0.01% to 10%. FIG. 9 schematically depicts the IL4-dip selection
assay using BH1-4 cells and depicts the results of the assay
starting with a 1:10 dilution of the SOCS1-infected cells. The
results are depicted in histograms where the vertical axis
indicates the number of cells and the horizontal axis indicates the
amount of GFP fluorescence measured using FACS, the day of
infection (day 1) and on days 4, 6, and 8 post-infection. GFP
fluorescence was used to monitor SOCS1 expression. SOCS1 inhibits
IL4-induced HBEGF expression making the cells expressing it
resistant to cell death by IL4-dip treatment. The results indicate
that by day 8 the majority of the surviving cells are
SOCS1-ires-GFP expressing cells. FIG. 10 depicts the results for
the assays using BH1-4 cells starting with the 1 :10, 1:100,
1:1,000, and 1:10,000 dilution of the SOCS1-infected cells. The
results are depicted in histograms where the vertical axis
indicates the number of cells and the horizontal axis indicates the
amount of GFP fluorescence measured using FACS, the day of
infection (day 1) and on days 4, 6, and 8 post-infection. FIG. 11
depicts the results for both BH1-4 and BH2-A5 cells starting with
the 1:10, 1:100, 1:1,000, and 1:10,000 dilution of the spiked
cells. The results are depicted in histograms where the vertical
axis indicates the number of cells and the horizontal axis
indicates the amount of GFP fluorescence measured using FACS on day
15 post-infection.
[0312] FIG. 12 depicts the results of selection beginning with
naive cells in a first round of selection with IL4-Dip and then
subjecting the surviving cells from the first round of selection to
a second round of selection with IL4-Dip. The naive cells are the
spiked cells diluted 1:10 and 1:10,000 with unspiked cells. The
first round of selection was performed as described above and the
amount of fluorescence was measured using FACS on days 1, 4, and 18
post infection. For the 1:10,000 seeding, the surviving cells
selected from the first round were subjected to a second round of
selection performed in the same manner as the first round, and the
amount for fluorescence was measured using FACS on the day IL4 was
added to the medium to stimulate IL4 induced expression of HBEGF;
and on days 4 and 8 thereafter.
[0313] The results of these SOCS1-infected cells spiking
experiments demonstrate that the IL4-Dip selection in the BH1-4 and
BH2-A5 cell lines result in enrichment of inhibitors of IL4
signaling/induced expression of HBEGF. At a 1:10,000 dilution of
the spiked cells, the enrichment by the IL4-Dip selection is
approximately 1000 fold (i.e., from 0.01% to 10%). However, in
these experiments, the results indicate that most survivors of the
first selection did not respond to a second round of IL4
stimulation. Thus, in this case a second round of selection lead to
very little enrichment for the peptide inhibitors. The conventional
or usual solution following selection is to rescue the retroviral
population and infect naive cells for a new and fresh round of
selection. However, this step requires that the complexity and
representation of the population should be maintained as they are
infected into naive cells; this requirement makes this step
technically challenging. Thus, the present invention provides a
novel approach that obviates these problems, as described in the
following examples.
Example 4
[0314] Screening for an Altered Cellular Phenotype Using a
Tet-Regulatable Expression System.
[0315] This example demonstrates a method of screening for an
altered cellular phenotype where cells responsive to stimulation by
IL4 ("IL4 responders") are separated from the population of cells
that have undergone the first selection. These IL4 responders can
then undergo an additional round of highly efficient selection by
repressing (or "turning down") the expression of the peptide
inhibitors of IL4 signaling using a Tet-regulatable (or "Tet
inducible") expression system; and then sorting for the IL4
responders using a reporter protein, e.g., Green Fluorescent
Protein ("GFP").
[0316] As depicted in FIG. 13, the screening assay in this example
involves infecting. A novel cell line BH2-A5T (described below)
with the peptide library BFP-C20 encoded by retroviral vectors
(described below). Thereafter, the cells are stimulated with IL4,
selected on IL4-dip, and then the cells are washed and the
surviving cells recovered. The peptide encoded by the retroviral
construct is expressed in the absence of Dox (a Tet analogue) and
repressed in the presence of Dox. After the first round of IL4-dip
selection, Dox is added to the cell culture medium containing the
surviving cells and expression of the peptide is turned off. The
cells are then stimulated with IL4 to induce expression of
HBEGF-2a-GFP and thereafter sorted for GFP fluorescence (indicative
of IL4 induced expression) using FACS. Thereafter, expression of
the peptide is turned on by removing the Dox. The cells are then
selected on IL4-Dip, washed, aliquoted into microtiter plates,
sorted by FACS for single cell clones, replica plated in duplicate
microtiter plates and cultured in the presence or absence of Dox,
and the single clones are selected on the basis that the IL-4
induced GFP is inhibited by the presence of a BFP-peptide as
measured by the BFP and GFP fluorescence using FACS.
[0317] As depicted in FIG. 14, the BH2-A5 cell line was further
engineered to construct the cell line BH2-A5T (or "A5T" or "A5T-4")
which expresses a Tet regulated transactivator (tTA or Tet
transactivator) and allows for the Tet regulated expression of
candidate bioactive agents introduced into the cells. As in the
BH2-A5 cells, the BH2-A5T cells contain the HBEGF-2a-GFP construct
that is driven by the epsilon promoter and is expression of
HBEGF-2a-GFP is inducible by stimulation with IL4. In this example,
the candidate bioactive agents are from a library of random
sequence peptides, 20 amino acids in length, which are members of
the BFP-C20mer peptide library. The 20 mer peptides are expressed
as carboxy-terminal peptides fused to the reporter protein, Blue
Fluorescent Protein (BFP). These fusion peptides (BFP-C20mer
library member) are operably linked to an oligomer of a Tet
operator sequence (TRA or TetO sequence) and promoter as depicted
in FIG. 14. In the absence of Tet or analogue thereof (e.g., Pox)
the peptide is expressed in the BH2-A5T cells, whereas in the
presence of Tet or analogue thereof, the expression of the peptide
is repressed (or turned off).
[0318] FIG. 15 depicts histograms indicating the amount of GFP
fluorescence, in an experiment where IL4 responders were selected
in the presence and absence of Dox in BH2-A5T cells spiked with
cells infected with the retroviral construct TRA-SOCS1-ires-GFP.
This construct contains the SOCS1-ires-GFP as described above and
in addition is operably linked to a TRA and thus is regulatable by
the tTA. In this experiment, the spiked cells were selected for
over 7 days on IL4 and DIP, and 1) in the absence of Dox ("IL4/dip
selection"); and 2) in the presence of Dox ("IL4/dip
selections+Dox"). After selection, the cells were cultured in the
absence of Dox to allow for the SOCS1-ires-GFP expression.
[0319] In summary, the BH2-A5T cell line (expressing tTA) was
generated and quality controlled for Dox responsiveness,
infectability, responsiveness to IL4 stimulation/induction of
expression, and IL4/Dip induced cell death. These experiments
established the fidelity of Dox regulation (turning peptide
expression on and off) and kinetics. The results demonstrate that
peptide expression levels can be 10-17 fold higher using a TRA
containing vector in comparison to the LTR vectors described above.
Further, results from the IL4/Dip selection in the presence or
absence of Dox demonstrate the correlation of enrichment and SOCS1
expression, i.e., enrichment was regulated by the presence or
absence of Dox.
Example 5
[0320] Screening for cells having an altered cellular phenotype by
multiple rounds of sorting of cells responsive to IL4 induced
expression of HBEGF-2a-GFP using an expression system regulatable
by Dox, as depicted in FIG. 13 and further described above in
Example 4.
[0321] As described above in Example 3, FIG. 12 depicts the results
of a first round and second round of selection for cells responsive
to IL4 induced expression, showing that most cells surviving
selection fail to respond to IL4 due to the presence of false
positives (i.e., stochastic or hereditary nonresponders). The
following experiments illustrate by example how the methods of the
present invention solve these problems by the round to round
enrichment for cells expressing the peptide inhibitor using a
combination of 1) induction and repression of the expression of the
putative peptide inhibitor (or other candidate bioactive agent) via
Dox with 2) selections for inhibition or activation of the parental
phenotype.
[0322] FIG. 16 depicts the round to round enrichment for cells
expressing the known inhibitor SOCS1 by induction and repression of
TRA-SOCS1-ires-GFP expression in BH2-A5T-4 cells using dox and
sorting of the altered and parental cellular phenotype. In this
system, the expression of TRA-SOCS1-ires-GFP is turned off in the
presence of Dox; and SOCS1-ires-GFP is expressed in the absence of
Dox. In these experiments, BH2-A5T4 cells are infected with the
retroviral construct TRA-SOCS1-ires-GFP (as described above); and
the infected cells are then diluted at 1:10,000 and 1:100,000 with
uninfected naive cells. In the first round of selection (Round 1)
the cells are selected on IL4-Dip for the altered phenotype (as
described above); the cells are then contacted with Dox to repress
(or turn off) expression of TRA-SOCS1-ires-GFP and the cells are
stimulated with IL4 and sorted for the parental phenotype using GFP
fluorescence which is indicative of IL4 induced expression of
HBEGF-2a-GFP. Thereafter, the cells responsive to IL4 induced
expression are collected and cultured in medium without Dox in
order to induce (or turn on) the expression of SOCS1-ires-GFP. The
cells expressing TRA-SOCS1-ires-GFP are then subjected to a second
round of selection for the altered phenotype on IL4-Dip achieving
further efficient enrichment of SOCS1-expressing cells. FIGS. 17
and 18 show the results of these experiments.
[0323] FIG. 17 depicts histograms indicating the amount of GFP
fluorescence indicative of TRA-SOCS1-ires-GFP expression in cells
after the first round of selection. The column entitled "no
selection" is a control where IL4 and Dip were not added to the
cells; the column entitled "post selection" is a control where the
cells were selected on IL4-Dip but were not cultured in the
presence of Dox; The results of the first round selection are shown
for both the selection starting with a 1:1 0,000 dilution of the
spiked cells ("TRA-SOCS1-ires-GFP 1/10K") and 1:100,000 dilution of
the spiked cells ("TRA-SOCS1-ires-GFP 1/100K"). For the selection
starting with a 1:10,000 dilution of the spiked cells
("TRA-SOCS1-ires-GFP 1/10K"), the first round of selection resulted
in about 1000-fold enrichment (from .about.0.01% to .about.9.4%) of
cells expressing TRA-SOCS1-ires-GFP. For the selection starting
with a 1:100,000 dilution of the spiked cells ("TRA-SOCS1-ires-GFP
1/100K"), the first round of selection did not result in a
statistically measurable enrichment of cells expressing
TRA-SOCS1-ires-GFP.
[0324] FIG. 18 depicts histograms showing the "Sort for IL4
responders" step in FIG. 16. The columns entitled "no selection"
and "post selection" are the same as those in FIG. 17; the column
"post selection+dox" depicts the turning off the expression of
TRA-SOCS1-ires-GFP by contacting the cells with Dox for over 7 days
in the cells that had been selected on IL4-Dip; the column "post
selection+dox+IL4" shows the GFP profile of "post selection+dox"
cells after being contacted with IL4 and Dox for 3 days. Since the
SOCS1-ires-GFP expression is repressed by Dox, the GFP expression
in this column is indicative of the IL4-induction of HBEGF-2a-GFP
(which is the parental phenotype); thus 23% to 24% of the cells
expressing GFP are IL4 responders. From these cell populations, the
10% highest expressing GFP cells (labeled Right (R) gate) and the
next 10% (labeled Left (L) gate) were collected as depicted in FIG.
18 for subsequent steps shown in FIGS. 19 and 20.
[0325] FIG. 19 depicts histograms showing the "Turn inhibitor
expression back on" step in FIG. 16. The columns entitled "no
selection" and "post selection" are the same as those in FIG. 17
and 18; the panels "sorted-left gate" and `sorted-right gate" show
histograms of the sorted populations from FIG. 18 after they have
been cultured over 5 days in the absence of Dox (referred to as
de-doxing). In this case, the GFP expression is indicative of the
cells expressing TRA-SOCS1-ires-GFP. As depicted in FIG. 19, there
is a small enrichment of the cells expressing TRA-SOCS1-ires-GFP
resulting from the sorting of the Left Gate and Right Gate
subpopulation of cells cultured in the absence of Dox. FIG. 19
depicts for the 1:10,000 dilution of cells, an 8% enrichment
resulting from the first round of selection, a 16% enrichment from
Left Gate cells and 18% enrichment from Right Gate cells resulting
from the sorting.of the cells cultured in the absence of Dox; and
for the 1:100,000 dilution of cells, an 0.6% enrichment resulting
from the first round of selection, a 0.9% enrichment from Left Gate
cells and 1.0% enrichment from Right Gate cells resulting from the
sorting of the cells cultured in the absence of Dox. For the next
step, these cell populations are subjected to a second round of
IL4/dip selection as summarized in FIG. 20.
[0326] FIG. 20 depicts for the 1:10,000 dilution of cells, an 6.1%
enrichment resulting from the first round of selection; for the
second round of IL4/dip selection, a 88% enrichment from the
de-doxed Left Gate cells and a 97% enrichment from de-doxed Right
Gate cells from FIG. 19; and for the 1:100,000 dilution of cells,
an 0.3% enrichment resulting from the first round of selection; for
the second round of IL4/dip selection, a 26% enrichment from the
de-doxed Left Gate cells and 47% enrichment from de-doxed Right
Gate cells resulting from the sorting of the cells cultured in the
absence of Dox (FIG. 19).
[0327] A summary of the screening assay in this example is
schematically depicted in FIG. 20 where in the first round of
selection the cells are selected on IL-4-Dip; and then the
surviving cells are cultured in the presence of dox and sorted for
responsiveness to IL4 induced expression of HBEGF-GFP (i.e., sorted
for "IL4 responders"). From the population of IL4 responders, a
Left Gate and Right Gate subpopulation of cells is collected and
cultured in the absence of dox (or "dedoxing"). In the absence of
dox, the expression of TRA-SOCS1-ires-GFP is turned back on and
subjected to a second round of IL4-Dip selection as described
above.
[0328] The results of these experiments demonstrate that cells
harboring a petide causing an altered phenotype can be highly
enriched and selected for by the methods of the present invention.
Specifically, the round to round induction and repression of
expression using Dox resulted in the enrichment and selection of
cells responsive to IL4 induced expression having an altered
cellular phenotype due to the presence of TRA-SOCS1-ires-GFP.
Further, in screening experiments, the putative peptide inhibitor
is operably linked to BFP, thus avoiding any potential conflicts
between the sorting for GFP fluorescence indicative of
P.epsilon.-HBEGF-2a-GFP expression and sorting for BFP fluorescence
indicative of the expression of the putative peptide inhibitor (or
candidate bioactive agent).
Example 6
[0329] Methods for Screening for an Altered Cellular Phenotype
Using a Tet-Regulatable Expression System and a Screening Cell
Line, e.g., BH2-A5T.
[0330] FIG. 21 schematically depicts an example of a timeline (in
days) for a screening assay of the present invention, where the
assay involves a first round of selection and sorting; a second
round of selection and sorting; and thereafter single cell clones
are grown. The single cell clones are then subjected to selection
and FACS assays, the nucleic acid encoding the bioactive agent
(e.g., a peptide inhibitor of IL4 signaling/induced expression) is
then rescued and the phenotype is reconfirmed, e.g., by infecting
naive cells with the rescued nucleic acid and selection.
[0331] FIG. 22 schematically depicts an example of a timeline (in
days) for a screening assay of the present invention (and as
described for FIG. 21), where the complexity of the library of
candidate bioactive agents (e.g., a peptide library), and the fold
enrichment for the altered cellular phenotype, are indicated.
Further, FIG. 22 schematically depicts the histogram profile of GFP
fluorescence of false positives due to hereditable background or
stochastic non-hereditable background; as compared to the histogram
profile of GFP fluorescence of cells cultured in the presence
(+Tet) or absence (-Tet) of Tet, after a first round of
selection.
[0332] FIG. 23 schematically depicts an example of a timeline (in
days) for a screening assay of the present invention (and as
described for FIG. 21), where the complexity of the library of
candidate bioactive agents (e.g., a peptide library), and the fold
enrichment for the altered cellular phenotype, are indicated.
Further, after a second of selection, cells are single cell cloned,
aliquoted into microtiter plates, replica plated in duplicate
microtiter plates and cultured in the presence or absence of Dox,
and the single clones are contacted with IL4 for three days and
their GFP fluorescence measured by FACS. FIG. 23 schematically
depicts the histogram profile of GFP fluorescence of false positive
clones due to hereditable background or stochastic non-hereditable
background; as compared to the histogram profile of GFP
fluorescence of cell clones harboring a peptide inhibitor cultured
in the presence of Tet (+Tet) or absence of Tet (-Tet) (see panel
of schematically depicted histograms). FIG. 23 depicts an example
of a clone where in the presence of Tet the expression of the
peptide inhibitor is turned off and thus the GFP reporter is
expressed; and in the absence of Tet the peptide is expressed and
thus the GFP reporter is inhibited in cells having an altered
phenotype. Thus, this example demonstrates a powerful approach to
identifying and eliminating (or disregarding) background cell
clones (e.g., false positives) by selecting only those clones
responsive to IL4 stimulation (i.e., IL4 induced expression) that
is regulated by Tet.
[0333] FIG. 24 depicts the histogram profile of GFP fluorescence of
clones from a functional screen representing a BFP-peptide
inhibitor clone, CR2 (left panel); a hereditable background clone
(middle panel), and stochastic background clone (right panel),
where the histograms from the clones cultured in the presence of
Dox (+Dox) and the absence of Dox (-Dox) are overlayed. In the
presence of Dox the expression of the BFP-peptide is turned off;
and in the absence of Dox the BFP-peptide is expressed.
[0334] FIG. 25 depicts the summary of the results from the peptide
screening in BH2-A5T-4 cells. In this screen, the total number of
cells targeted with the retroviral vector peptide library was
1.25.times.1010. From the total cells targeted, the total number of
cells infected with a retroviral member of the library was
2.4.times.109. From the total cells infected, the total number of
cells sorted/cloned was 24,960. From the total number of cells
sorted/cloned, the total number of regulatable clones, was 1,525.
FIG. 25 further depicts a protocol for characterizing the
identified regulatable clones by rescuing the retroviral construct
encoding the peptide from the clones, cloning and sequencing the
nucleic acid encoding the peptide, and testing for the transfer of
the altered cellular phenotype (conferred by the presence of the
peptide) into naive cells.
Example 7
[0335] Identification and Validation of Novel Signaling Molecules
Specific for T Cell Activation and Effector Function by Screening
for an Altered Cellular Phenotype in Activated T Cells.
[0336] Abstract
[0337] T lymphocytes play crucial roles in immune responses,
including the direct killing of virus-infected cells by cytotoxic T
cells and facilitation of B-cell responses by helper T cells. The
activation of T cells is mediated by the T cell receptor (TCR),
which in turn activates specific membrane-associated and
intracellular proteins. Identifying these signaling proteins
downstream of TCR activation is crucial for developing therapeutic
agents to inhibit or regulate immune responses in autoimmune
diseases and organ transplantation. Using the methods and
compositions of the present invention novel signaling molecules
specific for T cell activation and effector function were
identified and validated. A large library (5.times.10.sup.7) of
cDNA clones were introduced into T cells from which an altered
cellular phenotype was enriched for and identified. Using the
screening methods of the present invention, 2,800 individual clones
were obtained based on a reduction in T cell receptor
activation-induced CD69 expression, and the causal relationship of
cDNA expression and altered phenotype was established. In addition
to many known signaling molecules such as LCK, ZAP70, SYK, and
PLC(I, molecules previously unknown to this pathway were also
discovered using the screening methods of the present invention.
When selected for evaluation with primary human T lymphocytes, hits
from the screen inhibited anti-CD3 and anti-CD28 stimulated IL-2
production. From these molecules, potential therapeutic targets may
be identified that are effective in modulating immune-mediated
processes.
[0338] Introduction
[0339] Activation of specific signaling pathways in lymphocytes
determines the quality, magnitude and duration of immune responses.
In transplantation, acute and chronic inflammatory diseases, and
autoimmunity, it is these pathways that are responsible for the
induction, maintenance and exacerbation of disease lymphocyte
responses: In all cases, recognition of antigens presented by the
Major Histocompatability Complex (MHC) by the T cell receptor (TCR)
complex triggers the activation of T lymphocytes. Engagement of the
TCR by antigen/MHC results in actin cytoskeleton rearrangement,
induction of cyrokine and other gene transcription, and progression
into the cell cycle.sup.1,2. The proximal events of TCR signaling
include activation of src family kinases LCK, FYN, phosphorylation
of TCR component (.) and subsequent activation of ZAP70/SYK
tyrosine kinases, as well as recruitment of adaptor
molecules(CBL-B, LAT, SLP76), which couple to more distal signaling
pathways including Ras and PLC(.sup.3-5. New components of the TCR
signaling pathway have been discovered and reported, such as the
new transmembrane adaptor PAG/CBP .sup.6, albeit with a slower
pace. It has become apparent that identifying additional signaling
molecules requires novel approaches including functional genomics.
Using the methods of the present invention, novel signaling
molecules specific for T cell activation and effector function were
identified and validated.
[0340] In this example, using the methods of the present
invention,a novel approach to identifying new targets for immune
suppressive drugs is presented. Following T cell activation,
expression of numerous cell surface markers such as CD25, CD69, and
CD40L are upregulated. CD69 has been shown to be an early
activation marker in T, B, and NK cells.sup.7-9. CD69 is a
disulphide-linked dimer. The cell surface marker is not expressed
in resting lymphocytes but appears on T, B and NK cells after
activation in vitro. The relevance of CD69 as a TCR signaling
outcome has been validated using T cells deficient in certain key
signaling molecules such as SLP76 and LAT .sup.10,11. Furthermore,
re-introducing SLP76 or LAT into the deficient cells resulted in
restoration of CD69 expression. The CD69 upregulation could then be
used to monitor TCR signal transduction. The rationale of the
functional genomics screen was to identify cell clones whose CD69
upregulation was repressed following introduction of a retroviral
cDNA library. The library members conferring such repression would
then represent immune modulators that function to block TCR signal
transduction.
[0341] The use of retrovirus-mediated gene transfer has contributed
to the cloning of T cell antigens.sup.12, tumor antigens.sup.13,
various receptors.sup.14-24, signaling molecules.sup.25 and
transcription factors.sup.26. In most of the cases, retroviral cDNA
libraries were used for expressional cloning. The unique feature of
the current study reported here, is the use of retroviruses as
pharmaceutical tools for target discovery and validation along a
whole pathway of signal transduction, from ligand-receptor
interaction on the plasma membrane, membrane-proximal signaling,
actin-cytoskeleton rearrangement to gene transcription in the
nucleus. Another feature of our screen was to build-in the
functional relevance through partial and full-length cDNAs in the
library. Expression of these library inserts was expected to have
dominant effects over their endogenous counterparts by being
competitive inhibitors of endogenous protein activity, or being
constitutively active. Integration of several key technology
innovations such as new cell lines, improved retroviral infection
efficiency, high level and regulated expression of nucleic acids
(e.g., of a nucleic acid library), and high throughput screening
tools complemented successful execution of these screens.
[0342] Results and Discussion
[0343] Experimental design. Several T cell linesj-including Jurkat,
HPB-ALL, HSB-2 and PEER were tested for the presence of surface
CD3, CD25, CD28, CD40L, CD69, CD95, and CD95L. Those that expressed
CD3 were cultured with anti-CD3 or anti-TCR to crosslink the TCR
and examined for the upregulation of CD69. A Jurkat T cell line was
selected for its ability to upregulate CD69 in response to TCR
crosslinking with kinetics mimicking that of primary T lymphocytes
(data not shown). The population of Jurkat cells was sorted for low
basal and highly inducible CD69 expression following anti-TCR
simulation. Clone 4D9 was selected because CD69 in this clone was
uniformly and strongly induced following TCR stimulation in 24
hours (FIG. 26A).
[0344] In order to regulate the expression of the retroviral
library, the Tet-Off system was used. The cDNA inserts in the
retroviral library were cloned in a manner to operably link the
inserts to the tetracycline regulatory element (TRE) and the
minimal promoter of TK. Transcription of the cDNA inserts was then
dependent on the presence of tetracycline-controlled
trans-activator (tTA).sup.27, a fusion of Tet repression protein
and the VP16 activation domain, and the absence of tetracycline or
its derivatives such as doxycycline (Dox). To shut off the cDNA
expression, one can simply add doxycycline in the medium. To obtain
a Jurkat clone that stably expresses tTA, a retroviral LTR-driven
tTA in conjunction with a TRE-dependent reporter construct was
introduced, i.e., TRA-Lyt2. Through sorting of Lyt2 positive cells
in the absence of Dox and Lyt2 negative cells in the presence of
Dox, coupled with clonal evaluation, we obtained a derivative of
Jurkat clone 4D9, called 4D9#32, that showed the best Dox
regulation of Lyt2 expression, as seen in FIG. 26B.
[0345] Positive controls. ZAP70 is a positive regulator of T cell
activation.sup.28. A kinase-inactive (KI) ZAP70 and a truncated
ZAP70 (SH2 N+C).sup.29 were subcloned into the retroviral vector
under TRE control (FIG. 27A). As seen in FIG. 27B, ZAP70 SH2 (N+C)
and ZAP70 KI both inhibited TCR-induced CD69 expression. Consistent
with the published report of dominant negative forms of ZAP70 on
NFAT activity 29, the truncated protein is also a more potent
inhibitor of CD69 induction compared to the KI protein, which had a
single point mutation in the catalytic domain. In addition, with
higher protein expression, as measured by higher levels of GFP from
the bi-cistronic ZAP70 SH2 (N+C)-1RES-GFP and ZAP70 KI-IRES-GFP
constructs, inhibition of CD69 induction was stronger (data not
shown).
[0346] The CD69 inhibitory phenotype is dependent on expression of
dominant negative forms of ZAP70. As shown in FIG. 27C, when Dox
was added before TCR was stimulated, there was no inhibition of
CD69 expression. FACS analysis of cellular GFP expression revealed
a lack of GFP.sup.+ cells, supporting the notion that the
bi-cistronic ZAP70 SH2 (N+C)-IRES-GFP mRNA was not transcribed. The
lack of ZAP70 SH2 (N+C) protein expression in the presence of Dox
was confirmed by Western (FIG. 27D). In the absence of Dox, each
cell population contained roughly 50% of GFP-cells that did not
express the retrovirally introduced ZAP70 variants. Thus, FIG. 27D
showed that it took on average 4-5 fold higher level of ZAP70 SH2
(N+C) than the endogenous ZAP70 to achieve a dominant-negative
effect.
[0347] Screening for cells lacking CD69 upregulation. FIG. 28A
diagrams the scheme to obtain cell clones with CD69 inhibitory
phenotype. Jurkat 4D9#32 cells were infected with cDNA libraries
made form primary human lymphoid organs such as thymus, spleen,
lymph node and bone marrow. The library complexity was
5.times.10.sup.7 and was built on the TRE vector. The infection
rate was 52%, as judged by infecton with TRA-dsGFP in parallel
experiment using the same host cells (data not shown). TRA-dsGFP
was another TRE-dependent reporter construct using the same vector
backbone as that used by the cDNA libraries or TRA-Lyt2. After
library infection, cells were stimulated with the antiTCR antibody
C305 overnight. A total of 7.1.times.10.sup.8 cells were stained
with anti-CD69 antibody conjugated to allophycocyanin (APC) and
anti-CD3 antibody conjugated to phycoerythrin (PE) and screened
using flow cytometry. Even though there was a significant reduction
of CD3/TCR complex on the surface due to receptor-mediated
internalization, compared to unstimulated cells (data not shown),
the CD3-population was distinguishable from the CD3+population.
Greater than 2% cells lacking the CD3-PE staining (CD3-) (>2% of
total cells had lost TCR/CD3 complex) were consistently observed,
which explained their unresponsiveness to stimulation and,
consequently, low CD69 expression. Based on this fact, only cells
with the lowest CD69 expression which still retained the CD3
expression were selected. The desired altered phenotype was termed
CD69.sup.low CD3.sup.+ (FIG. 28A), which represented 1% of the
total stained cells (FIG. 28B). The 1% sorting gate also translated
as 100-fold enrichment in the first round of sorting alone, based
on cel numbers. The recovered cells were allowed to rest in
complete medium for 5 days before being stimulated again for a new
round of sorting. In subsequent rounds of sorting, the sorting gate
was always maintained to contain the equivalent of 1% of the
control cells that were stimulated but were never flow-sorted. As
shown in FIG. 28B, an enrichment was achieved after 3 rounds of
reiterative sorting. Cells with the desired CD69.sup.low CD3.sup.+
phenotype increased from 1% to 23.2%. In addition, the overall
population's geometric mean for the CD69 fluorescent intensity was
also reduced (from >300 to 65).
[0348] In order to ascertain that the phenotype was due to
expression of the cDNA library rather than to spontaneous or
retroviral insertion-mediated somatic mutation, the cells recovered
after the third round of sorting were split into two populations.
One half of the cells were grown in the absence of Dox while the
other half in the presence of Dox for 6 days. CD69 expression was
analyzed following anti-TCR stimulation overnight.
[0349] As shown in FIG. 28C, cells with the CD69.sup.low CD3.sup.+
pherotype decreased from 24.0% to 13.0% with the addition of Dox,
demonstrating that a significant number of cells (11%) had lost the
CD69.sup.low CD3.sup.+ phenotype in the presence of Dox. These data
suggested that the CD69.sup.low CD3.sup.+ phenotypein a significant
number of cells in the population was indeed caused by the
expression of the cDNA library members. Single cell clones were
deposited in conjunction with the fourth round of CD69.sup.low
CD3.sup.+ sorting. The cells from three consecutive rounds of
CD69.sup.low CD3.sup.+ sorting were referred to as LLL, and those
from four consecutive rounds of CD69.sup.low CD3.sup.+ sorting were
referred to as LLLL. Overall, cells from up to 7 rounds of sorting
(LLLLLLL) were collected.
[0350] In order to reduce the number of cells whose phenotype was
not Dox-regulatable, the population of the cells grown in the
presence of Dox were subjected to a different round of sorting. The
purpose for this round of sorting was to enrich for cells having
normal CD69 expression when the library cDNA expression was
switched off in the presence of Dox. This sorting variation was
termed LLLH where H means CD69.sup.high. The cells recovered from
LLLH sort were cultured in the absence of Dox for subsequence
sorting of the CD69.sup.low CD3.sup.+ phenotype. Single cell clones
with the CD69.sup.low CD3.sup.+phenotype were depositd in 96-well
plates from both the LLLHL and LLLL sorting variations. Table 1
showed that indeed a higher percentage of Dox-regulatable clones
with the LLLHL sorting variation than with LLLL was achieved.
[0351] Functional Analysis of Single Cell Clones. After single cell
clones started to grow into individual colonies in 96-well plates,
their cellular phenotype was characterized with the cDNA expression
turned on or off, by growing the cells in the presence or absence
of Doxcycline (Dox). FIG. 29A shows some examples of the
Dox-regulatable phenotypes for individual clones. Dox regulation of
CD69 expression was expressed as the ratio of geometric mean
fluorescent intensity (GMFI) in the presence of Dox divided by the
CD69 GMFI in the absence of Dox after TCR stimulation. This ratio
of (+Dox)/GMFI (-Dox) was termed the Dox ratio. In uninfected
cells, Dox had little or no effect on the induction of CD69
expression so that the Dox ratio for individual clones on all
occasions was consistently 1.00.+-.0.25 (standard deviation).
Therefore, the 2x standard deviation was used as a cut-off
criterion and designated clones with a ratio above 1.5 as Dox
regulated clones. Out of 2828 clones analyzed, 1323 had a
Dox-regulatable cellular phenotype as judged by the above criteria,
representing 46.8% of analyzed clones. FIG. 29B shows the
distribution of clones with the Dox ratio between 1.5 and 10, which
contained 1186 clones. Interestingly, the majority of clones have a
Dox ratio below 10 whereas rare clones were discovered with a Dox
ratio up to 70.
[0352] RNA samples were prepared from clones with Dox-regulatable
phenotypes. Using primers specific for the vector sequence flanking
the cDNA library insert, we captured the cDNA insert of selected
clones by RT-PCR. FIG. 29C showed the pattern of RT-PCR products.
Most clones generated only one DNA band, whereas a few clones
generated two or more bands. Sequencing analysis revealed that the
additional bands were usually caused by double or multiple
insertions of retroviruses. Occasionally, the two PCR products in
the same lane represented different fragments of the same gene
product. The results of the cDNA analysis are summarized in Table
2.
[0353] Characterization of proteins critical for T cell activation.
As shown in Table 2, known TCR regulators such as LCK. ZAP70, SYK,
and PLC(1 were obtained using the methods of the present
invention.
[0354] LCK is a non-receptor protein tyrosine kinase.sup.30,31. Its
role in T cell development and activation has been widely
documented.sup.32-34. To date, dominant negative forms of LCK have
not been reported. The discovery by the present inventors that over
expression of the kinase-truncated form of LCK caused inhibition of
CD69, similar to the phenotype of Jurkat somatic mutant lacking LCK
35, suggests that kinase deletion of LCK could also work as a
dominant negative form of LCK (FIG. 30A).
[0355] The two ZAP70 hits both contained the endogenous ATG
initiation codon and ended at aa 262 and 269, respectively (FIG.
30B). They both are missing the catalytic domain. The deletions are
very close to the positive control for the screen, ZAP70 SH2 (N+C),
which ended at aa 276.sup.29. Since ZAP70 SH2 (N+C) was shown to be
a dominant negative protein.sup.29 (also see FIG. 27), the two
ZAP70 hits are believed to also behave as dominant negative
proteins of ZAP70 (FIG. 30B).
[0356] SYK is a non-receptor tyrosine kinase belonging to the
SYK/ZAP70 family of kineses.sup.36. Since it has also been shown
that the lack of SYK expression in Jurkat cells did not appear to
significantly alter the TCR-mediated responses compared with Jurkat
clones expressing SYK .sup.37, the SYK hit obtained from the
present screen is believed to function mainly in blocking ZAP70
function (FIG. 30C and data not shown). SYK's similarity to ZAP70,
its ability to associate with phosphorylated TCR; chain and its
ability to reconstitute the ZAP70-deficient Jurkat T-cell line also
support this notion.sup.38.
[0357] PLC(I plays a crucial role in coupling T cell receptor
ligation to IL-2 gene expression in activated T lymphocytes.sup.1.
TCR engagement leads to rapid tyrosine phosphorylation and
activation of PLC(I.sup.39. The activated enzyme converts
phosphatidylinositol-4, 5-bisphosphate (PIP2) to
inositol-1,3,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3
triggers intracellular Ca.sup.2+ increase and DAG is a potent
activator of protein kinase C (PKC). PLC(1 has a split catalytic
domain comprised of conserved X and Y subdomains (FIG. 30D). Single
point mutation in the catalytic X box completely abolished the
enzyme activity and also blocked IL-2 reporter gene expression when
introduced into PLC(I-deficient Jurkat cells.sup.40. The hit
contained the PH domain and the N and C terminal SH2 domains of
PLC(I (FIG. 30D). Significantly this hit also deleted the crucial
tyrosine Y783 between the SH2 (C) and SH3 domains. It was reported
that Y783 was essential for coupling of TCR stimulation to IL-2
promoter activation and that mutation of Y783 to F (phenoalanine)
generated a very potent dominant negative form of PLC(I .sup.40.
Indeed, the original clone encoding the PLC(I hit had the highest
Dox ratio for CD69 expression among all clones from the cDNA
screen, indicating the strong repression of CD69 induction by the
PLC(1 hit as well as the total derepression in the absence of the
hit expression. When introduced to naive Jurkat cells, this
fragment caused severe block of TCR-induced CD69 expression (FIG.
30D).
[0358] Other signaling molecules known to involve in TCR signaling
pathway were also discovered using the methods of the present
invention. They included PAG.sup.6, CSK.sup.41,42,43, SHP-1 .sup.44
and nucleolin .sup.45 (Table 2 and data not shown). Raf is a MAP
kinase kinase kinase. It interacts with Ras and leads to activation
of the MAP kinase pathway.sup.46. Raf-1 was reported to participate
in TCR signaling.sup.47,48. The Raf hit obtained corresponds to the
truncated form of A-raf, missing the kinase domain. It is likely
that this A-raf hit is a dominant negative form of the Raf-1 kinase
.sup.49,50. Alternatively, our result suggests that A-Raf is also
critical for TCR signaling leading to CD69 activation.
[0359] In addition to the known signaling molecules, using the
methods of the present invention the function of genes whose
identity were reported previously, but whose involvement in TCR
signaling was not documented, was discovered. For example, SH2-B
was originally identified by its ability to associate with the
immunoreceptor tyrosine-based activation motif (ITAM) of the FcgR(
chain.sup.51. It belongs to a superfamily of intracellular
signaling molecules including LNK and APS.sup.52,53. Recently, LNK
was shown to be crucial for B cell production.sup.54. It is
conceivable that SH2-B plays an analogous immune modulatory role in
T cell activation (Table 2).
[0360] TCPTP (T cell protein tyrosine phosphatase) is also called
PTPN2 for protein tyrosine phosphatase nonreceptor-2.sup.55. It was
reported to be highly expressed in T cells but its function in TCR
signal transduction was not elucidated. The hit obtained from the
using the screening methods of the present invention contained a
C-terminal truncation and an intact phosphatase domain (FIG. 31A).
Constitutively active TCPTP was reported to have a C-terminal
truncation due to protease cleavage at aa. 387.sup.56.
Interestingly, the hit ends at aa 369, just 18 aa shorter than the
protease cleavage product. It is conceivable that the hit is a
constitutively activate TCPTP and that activated TCPTP is a
negative regulator of TCR signaling.
[0361] IL-10 receptor.sup.57 and Integrin ''2.sup.58 are both
transmembrane proteins. The cytoplasmic regions of these two
receptors were identified as inhibitors of TCR-induced CD69
expression (FIG. 31B and FIG. 31C), using the methods of the
present invention. Although the mechanisms of the inhibition were
not clear, these transmembrane molecules may serve as specific
"sinks" for other positive signaling molecules. Alternatively, a
more appealing explanation is that these transmembrane molecules
specifically modulate T cell activation. For example, stimulation
of T cells via the CD3-TCR complex resulted in rapid increase in $1
integrin-mediated adhesion. Integrins are capable of inside-out
signal transduction.sup.59 and have been reported to bind to
SLP-76.sup.60. The hematopoietic-specific adaptor SLAP-130/Fyb was
also shown to be important for coupling TCR-mediated actin
cytoskeletal rearrangement with activation of integrin function,
and for T cells to respond fully to activating signals.sup.61.
Recently, it has been reported that the Tec family tyrosine kinase
ITK.sup.62 regulated the inside-out signaling events from TCR to
integrins.sup.63. Furthermore, Integrin 2$1 mediates p38"
activation and upregulation of collagen gene transcription by a
mechanism involving the "2 cytoplasmic tail, Cdc42, MKK3 and
MKK4.sup.64. Taken together, our discovery of the dominant negative
effect of the cytoplasmic tail of" integrin strengthened integrin's
role in functionally modulating T cell activation (FIG. 31C).
[0362] In addition to uncovering truncated cDNAs encoding dominant
negative mutants of the positive regulators and constitutively
active mutants of the negative regulators of T cell activation,
using the screening methods of the present invention a clone
encoding the full-length open reading frame of the gene GG2-1
(Table 2 and FIG. 31D) was also identified. GG2-1, also called SCC
S2, was independently discovered as a transcript upregulated with
TNF" .sup.65,66. GG21/SCC-S2 contained a sequence in the amino
terminus that shows a significant homology to death effector domain
II of the cell death regulatory protein FLIP. Unlike FLIP, the
GG2-1/SCC-S2 open reading frame contains only one death effector
domain and lacks the carboxyl-terminal caspase-like homology
domain. The significance of the GG2-1 hit from our T cell
activation screen awaits further studies.
[0363] Function in primary T lymphocytes. The relevance of the cDNA
hits, identified by the methods of the present invention, to the
physiological function of T cells was investigated in primary T
lymphocytes. The hits were subcloned into a retroviral vector under
a constitutively active promoter embedded in the retroviral LTR,
followed by IRES-GFP. A protocol was developed to couple successful
retroviral infection to subsequence T cell activation. Primary T
lymphocytes are at the quiescent stage when isolated from healthy
donors. In order to be infected by a retrovirus, primary
lymphocytes need to be activated to progress into the cell cycle.
As shown in FIG. 32A, fresh peripheral blood lymphocytes (PBL)
contained typically T cells and B cells. The combined CD4.sup.+ and
CD8.sup.+ cells represented total T cells, which were 81% in this
particular donor. The remaining 19% CD4.sup.+ and CD8.sup.+cells
were B cells as stained by CD19 (data not shown). Upon culturing on
anti-CD3 and anti-CD28 coated dishes, primary T lymphocytes were
expanded and primary B cells and other cell types gradually died
off in the culture. FIG. 32 showed that after infection, the
culture contained virtually all T cells. Furthermore, primary T
lymphocytes were successfully infected by retroviruses (FIG. 32A
and B). As seen with Jurkat cells (data not shown), GFP translated
by way of IRKS was not as abundant as GFP translated using the
conventional Kozak sequence (comparing GFP geometric mean from
CRU5-IRES-GFP and CRU5-GFP). Nevertheless the percentage infection
remained similar. Insertion of a gene in front of IRES-GFP further
reduced the expression level of GFP, which was observed with many
cell lines (data not shown) and here in primary T lymphocytes (FIG.
32B). After allowing cells to rest following infection, we flow
sorted cells into two populations: GFP-.sup.- and GFP.sup.+. The
sorted cells were immediately put into culture. As seen in FIG.
32C, anti-CD3 alone did not induce IL-2 production. This
observation was consistent with previous report on freshly isolated
primary T lymphocytes and confirmed the notion that prior culture
and retroviral infection did not damage the physiological
properties of these primary T lymphocytes. Addition of anti-CD28 in
conjunction with anti-CD3 led to robust IL-2 production with
vector-infected cells and the GFP.sup.- population of LckDN and
PLC(IDN-infected cells. The GFP.sup.+ cell population from LckDN
and PLC(IDN-infected cells, however, were severed impaired in IL-2
production following anti-CD3 and anti-CD28 stimulation (FIG. 32C).
As expected, the defect caused by LckDN and PLC(IDN can be
completely rescued by stimulation using PMA and ionomycin (FIG.
32C). Taken together, these results showed that LCK and PLC(1 play
crucial role in IL-2 production from primary T lymphocytes,
consistently with their involvement in membrane proximal signaling
events of T cell activation. These results also demonstrated a
quick system to further validate hits using the methods of the
present invention as a functional genetic screen in primary
cells.
[0364] In conclusion, the methods of the present invention used in
this study demonstrates a successful approach to discover and
validate in a functionally relevant context important immune
regulators on a genome-wide scale. This approach, which requires-
no prior sequence information, provides a tool for functional
cloning of regulators in numerous signal transduction pathways. For
example, B cell activation-induced CD69 expression (Holland et al.,
in press) and IL-4-induced IgE class switch are all amendable to
the genetic perturbation following introduction of retroviral cDNA
libraries. The methods of the present invention are less biased
compared to forced introduction of a handful of signaling molecules
discovered in other contexts such as growth factor signal
transduction. The present invention also open the door for
discovering peptide inhibitors of immune modulatory proteins by
screening random peptide libraries expressed from retroviral
vectors using the screening methods of the present invention.
[0365] Experimental Protocol
[0366] Preparation of cDNA libraries. mRNA extracted from human
lymph nodes, thymus, spleen and bone marrow was used to produce two
cDNA libraries; both randomly primed. One library (-ATG) inserts
were directionally cloned and the second (+ATG) non-directionally
cloned and provided with 3 exogenous ATG in 3 frames. cDNAs were
cloned into the pTRA-exs vector to give rise to robust
Doxycycline-regulatable transcription from the TRE enhancer and the
minimal promoter in cell lines expressing tTA. The total combined
library complexity was 5.times.107 independent clones.
[0367] Cell lines. Phoenix A cells were cultured in DMEM
supplemented with 10% fetal calf serum, penicillin and
streptomycin. Human T cell leukemia line Jurkat was obtained from
Novartis and was routinely cultured in RPMI 1640 medium
supplemented with 10% fetal calf serum, penicillin and
streptomycin. To obtain the clone with optimal CD69 induction,
Jurkat cells were sorted for low basal CD69 expression and high
induction of CD69 expression following TCR stimulation. To produce
the tTA-Jurkat cell line, Jurkat clone (4D9) with optimal CD69
expression profile was infected with retroviral construct which
constitutively expresses the tetracycline transactivator protein
(tTA) and a reporter construct which expresses Lyt2 driven by a
tetracycline responsive element (TRE). The tTA-Jurkat cell clone
4D9#32 was obtained by sorting for high Lyt2 expression in the
absence of Doxycycline and low expression of Lyt2 in the presence
Doxycycline (10 ng/ml).
[0368] Transfection and infection. Phoenix A packaging cells were
transfected with retroviral vectors using calcium phosphate for 6
hours following standard protocols. After 24 hours, supernatant was
replaced with complete RPMI medium and virus was allowed to
accumulate for 24 hours at 32EC. Viral supernatant was collected,
filtered through a 0.2 M filter and mixed with Jurkat cells at a
density of 5.times.10.sup.5 cells/ml. Cells were spun at room
temperature for 3 hours at 2500 rpm, followed by overnight
incubation at 37EC. Transfection and infection efficiencies were
monitored by flow cytometry. Functional analysis was carried out
2-4 days after infection.
[0369] Stimulation. For CD69 upregulation experiment, Jurkat cells
were split to 2.5.times.10.sup.5 cells/ml 24 hours prior to
stimulation. Cells were spun and resuspended at 5.times.10.sup.5
cells/ml in fresh complete RPMI medium in the presence of 300 ng/ml
C305 (anti-Jurkat clonotypic TCR) hybridoma supernatant for 20-26
hours at 37EC, and then assayed for surface CD69 expression.
[0370] Antibodies and Flow cytometry. Jurkat cells or human
peripheral blood lymphocytes were stained with FITC-conjugated
monoclonal anti-mouse CD8'' (Lyt2), APCconjugated mouse monoclonal
anti-human CD3, anti-human CD8, or anti-human CD69 antibodies, and
PE-conjugated mouse monoclonal anti-human CD3 or anti-CD4
antibodies (all from Caltag) at 4EC for 20 minutes and analyzed
using a FACSCalibur instrument (Becton Dickinson) with the
CellQuest software. Fluorescent-activated cell sortings were
performed on the MoFlo instruments (Cytomation).
[0371] cDNA library screen. Phoenix A packaging cells were
transfected with a mixture of the two tTA regulated retroviral cDNA
libraries (total complexity 5.times.10.sup.7). Supernatant
containing packaged viral particles was used to infect
3.5.times.10.sup.8 tTA-Jurkat cells with an efficiency of 52% based
on parallel infection with TRA-GFP. After 4 days of cDNA
expression, library infected cells were stimulated with 300 ng/ml
C305 for 20-30 hours, stained with APC-conjugated anti-CD69 and
PE-conjugated anti-CD3, and 1% of total cells expressing the lowest
CD69 level and still positive for CD3 expression were isolated
using a fluorescence activated cell sorter (Cytomation). Sorting
was repeated multiple rounds with a 6-day rest period between
stimulations until the population was significantly enriched for
the desired phenotype of CD69.sup.lowCD3.sup.+. Following 3
consecutive sorting for the CD69.sup.lowCD3.sup.+ phenotype after
TCR stimulation in the absence of Dox, half of the cells were
cultured and stimulated in the presence of Dox and sorted for the
top 10% of the CD69 level (so called CD69.sup.high phenotype) to
enrich for clones whose phenotype was dependent on cDNA expression.
Single cells were deposited from 8 separate rounds of sorting with
different variations of the placement of the CD69.sup.high sort.
Cell clones were expanded in the presence and absence of Dox,
stimulated and analyzed for CD69 upregulation.
[0372] Isolation of cDNA inserts. PCR primers were designed to
amplify cDNA inserts from both libraries and did not amplify Lyt2
that was also under TRE regulation. The primers used contained
flanking BstXI sites for subsequent cloning to the pTRA-IRES-GFP
and CRU5-IRES-GFP vectors. BstXTRA5G: 5'
TTGCAGAACCACCACCTTGGGCTCTTAACCTAGGCCGATC3'., BstXTRA3D:
5'TTGCAGAACCAATTTAATGGCGGCCAGTCAGGCCATCGTCG3'RT-PCR cloning was
achieved with kits from Clontech or Life Technologies. The
gel-purified RT-PCR products were sequenced. The purified RT-PCR
fragments were also digested for subcloning. Dominant negative
ZAP70 (KI) and ZAP70SH2 (N+C) as well as selected hits from cDNA
screens were subcloned to the retroviral pTRA-IRES-GFP vector.
Selected hits form cDNA screens were also subcloned to
CRU5-IRES-GFP for infection of human primary T lymphocytes.
[0373] Equivalents
[0374] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
39 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 69 PRT Artificial sequence minibody presentation
structure 2 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 3 7
PRT Simian virus 40 3 Pro Lys Lys Lys Arg Lys Val 1 5 4 6 PRT Homo
sapiens 4 Ala Arg Arg Arg Arg Pro 1 5 5 10 PRT Mus musculus 5 Glu
Glu Val Gln Arg Lys Arg Gln Lys Leu 1 5 10 6 9 PRT Mus musculus 6
Glu Glu Lys Arg Lys Arg Thr Tyr Glu 1 5 7 20 PRT Xenopus laevis 7
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 8 31 PRT Mus musculus 8 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 9 21
PRT Homo sapiens 9 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 10 51 PRT Mus
musculus 10 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 11 33 PRT Homo
sapiens 11 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 12 37 PRT Rattus sp. 12 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 13 14 PRT Homo sapiens 13 Met Gly Ser Ser
Lys Ser Lys Pro Lys Asp Pro Ser Gln Arg 1 5 10 14 26 PRT Homo
sapiens 14 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 15
20 PRT Rattus norvegicus 15 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 16 19 PRT Homo
sapiens 16 Leu Asn Pro Pro Asp Glu Ser Gly Pro Gly Cys Met Ser Cys
Lys Cys 1 5 10 15 Val Leu Ser 17 5 PRT Artificial sequence
lysosomal degredation seqeunce 17 Lys Phe Glu Arg Gln 1 5 18 36 PRT
Cricetulus griseus 18 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 19 35 PRT
Homo sapiens 19 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 20 27 PRT Saccharomyces
cerevisiae 20 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 21 25 PRT Saccharomyces cerevisiae 21 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 22 64 PRT Saccharomyces cerevisiae 22 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 23 41 PRT Saccharomyces cerevisiae 23 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 24 4 PRT Homo
sapiens 24 Lys Asp Glu Leu 1 25 15 PRT unidentified adenovirus 25
Leu Tyr Leu Ser Arg Arg Ser Phe Ile Asp Glu Lys Lys Met Pro 1 5 10
15 26 15 PRT Homo sapiens 26 Leu Thr Glu Pro Thr Gln Pro Thr Arg
Asn Gln Cys Cys Ser Asn 1 5 10 15 27 9 PRT Unknown cyclin B1
destruction sequence 27 Arg Thr Ala Leu Gly Asp Ile Gly Asn 1 5 28
20 PRT Unknown signal sequence from Interleukin-2 28 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 29 29 PRT Homo sapiens 29 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 30 27 PRT Homo sapiens 30
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 31 18 PRT
Influenza virus 31 Met Lys Ala Lys Leu Leu Val Leu Leu Tyr Ala Phe
Val Ala Gly Asp 1 5 10 15 Gln Ile 32 24 PRT Unknown signal sequence
from Interleukin-4 32 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 33
10 PRT Artificial sequence stability sequence 33 Met Gly Xaa Xaa
Xaa Xaa Gly Gly Pro Pro 1 5 10 34 5 PRT Artificial sequence linker
consensus sequence 34 Gly Ser Gly Gly Ser 1 5 35 4 PRT Artificial
sequence linker consensus sequence 35 Gly Gly Gly Ser 1 36 20 PRT
Artificial sequence consensus sequence for SH-3 domain binding
protein 36 Met Gly Xaa Xaa Xaa Xaa Xaa Arg Pro Leu Pro Pro Xaa Pro
Xaa Xaa 1 5 10 15 Gly Gly Pro Pro 20 37 63 DNA Artificial sequence
oligonucleotide consensus sequence for SH-3 domain binding protein
37 atgggcnnkn nknnknnknn kagacctctg cctccasbkc ctsbksbkgg
aggcccacct 60 taa 63 38 40 DNA Artificial sequence primer 38
ttgcagaacc accaccttgg gctcttaacc taggccgatc 40 39 41 DNA Artificial
sequence primer 39 ttgcagaacc aatttaatgg cggccagtca ggccatcgtc g
41
* * * * *