U.S. patent application number 10/057467 was filed with the patent office on 2003-03-06 for methods for screening for transdominant effector peptides and rna molecules.
This patent application is currently assigned to Board of Trustees of the Leland Stanford Junior University. Invention is credited to Nolan, Garry P..
Application Number | 20030044767 10/057467 |
Document ID | / |
Family ID | 24356630 |
Filed Date | 2003-03-06 |
United States Patent
Application |
20030044767 |
Kind Code |
A1 |
Nolan, Garry P. |
March 6, 2003 |
Methods for screening for transdominant effector peptides and RNA
molecules
Abstract
Biochemical libraries are screened for transdominant
intracellularly bioactive agents by expressing a molecular library
of randomized nucleic acids as a plurality of corresponding
expression products in a plurality of cells, each of the nucleic
acids comprising a different nucleotide sequence, detecting a cell
of the plurality of cells exhibiting a changed physiology in
response to the presence in the cell of a transdominant expression
product of the corresponding expressio products; and isolating the
cell and/or transdominant expression product.
Inventors: |
Nolan, Garry P.; (Menlo
Park, CA) |
Correspondence
Address: |
Robin M. Silva
FLEHR HOHBACH TEST
ALBRITTON & HERBERT LLP
Four Embarcadero Center, Suite 3400
San Francisco
CA
94111-4187
US
|
Assignee: |
Board of Trustees of the Leland
Stanford Junior University
|
Family ID: |
24356630 |
Appl. No.: |
10/057467 |
Filed: |
January 22, 2002 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10057467 |
Jan 22, 2002 |
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08589109 |
Jan 23, 1996 |
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6365344 |
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Current U.S.
Class: |
435/4 ;
435/320.1; 435/325; 435/455; 435/6.14; 702/20 |
Current CPC
Class: |
C12N 15/1034 20130101;
G01N 33/502 20130101; G01N 33/5041 20130101; G01N 2510/00 20130101;
G01N 33/5076 20130101; C12N 15/86 20130101; C12N 15/1079 20130101;
G01N 33/5011 20130101; C07K 1/047 20130101; G01N 33/5008 20130101;
C12Q 1/6897 20130101; G01N 33/5017 20130101; C12Q 1/70 20130101;
G01N 33/6803 20130101; C12N 2740/13043 20130101; G01N 2500/00
20130101; G01N 33/68 20130101 |
Class at
Publication: |
435/4 ; 435/6;
435/455; 435/320.1; 702/20; 435/325 |
International
Class: |
C12Q 001/00; C12Q
001/68; G06F 019/00; G01N 033/48; G01N 033/50; C12N 005/00 |
Claims
What is claimed is:
1. A method of screening for a transdominant bioactive agent, said
method comprising steps: expressing a molecular library of
randomized nucleic acids as a plurality of isolated corresponding
randomized translation products in a first plurality of cells, each
of said nucleic acids comprising a different nucleotide sequence;
screening a second plurality of cells for a cell exhibiting a
changed physiology in response to the presence of a transdominant
translation product of said plurality of isolated corresponding
randomized translation products, wherein said translation product
is expressed with a fusion partner, synthetic or heterologous to
said first plurality of cells, comprising a localizing signal
sequence capable of constitutively localizing said translation
product to a predetermined subcellular locale, secretory and
membrane-anchoring signal sequences capable of localizing said
translation product to the plasma membrane, or a secretory signal
sequence capable of effecting the secretion of said translation
product. detecting said cell; isolating at least one of said cell
and said transdominant translation product, wherein said
transdominant translation product is a transdominant bioactive
agent.
2. A method according to claim 1, wherein said translation products
are presented on the extracellular surface of said first plurality
of cells.
3. A method according to claim 1, wherein said translation products
are secreted from said first plurality of cells.
4. A method according to claim 1, wherein said first and second
plurality of cells are different.
5. A method according to claim 1, wherein said expressing step
further comprises introducing said library into said cells.
6. A method according to claim 1, wherein said expressing step
further comprises introducing said library into said cells using
retroviral vectors.
7. A method of screening for a transdominant extracellularly
bioactive agent, said method comprising steps: expressing a
molecular library of randomized nucleic acids as a plurality of
isolated corresponding randomized translation products in a first
plurality of cells, each of said nucleic acids comprising a
different nucleotide sequence; screening a second different
plurality of cells for a cell exhibiting a changed physiology in
response to the presence of a transdominant translation product of
said plurality of isolated corresponding randomized translation
products, wherein said translation product is expressed with a
fusion partner, synthetic or heterologous to said first plurality
of cells, comprising a secretory and membrane-anchoring signal
sequences capable of localizing said translation product to the
extracellular surface of the plasma membrane, or a secretory signal
sequence capable of effecting the secretion of said translation
product. detecting said cell; isolating at least one of said cell
and said transdominant translation product, wherein said
transdominant translation product is a transdominant
extracellularly bioactive agent; wherein said expressing step
comprises introducing said library into said cells using retroviral
vectors.
Description
INTRODUCTION
[0001] 1. Technical Field
[0002] The technical field of this invention is methods for
screening for transdominant effector peptides and RNA molecules
selected inside living cells from randomized pools.
[0003] 2. Background
[0004] Signaling pathways in cells often begin with an effector
stimulus that leads to a phenotypically describable change in
cellular physiology. Despite the key role intracellular signaling
pathways play in pathogenesis, in most cases, little is understood
about a signaling pathway other than the initial stimulus and the
ultimate cellular response.
[0005] Historically, signal transduction has been analyzed by
biochemistry or genetics. The biochemical approach dissects a
pathway in a "stepping-stone" fashion: find a molecule that acts
at, or is involved in, one end of the pathway, isolate assayable
quantities and then try to determine the next molecule in the
pathway, either upstream or downstream of the isolated one. The
genetic approach is classically a "shot in the dark": induce or
derive mutants in a signaling pathway and map the locus by genetic
crosses or complement the mutation with a cDNA library. Limitations
of biochemical approaches include a reliance on a significant
amount of pre-existing knowledge about the constituents under study
and the need to carry such studies out in vitro, post-mortem.
Limitations of purely genetic approaches include the need to first
derive and then characterize before proceeding with identifying and
cloning the gene.
[0006] Screening molecular libraries of chemical compounds for
drugs that regulate signaling systems has led to important
discoveries of great clinical significance. Cyclosporin A (CsA) and
FK506, for examples, were selected in standard pharmaceutical
screens for inhibition of T-cell activation. It is noteworthy that
while these two drugs bind completely different cellular proteins
--cyclophilin and FK506 binding protein (FKBP), respectively, the
effect of either drug is virtually the same--profound and specific
suppression of T-cell activation, phenotypically observable in T
cells as inhibition of MRNA production dependent on transcription
factors such as NF-AT and NF-.kappa.B. Libraries of small peptides
have also been successfully screened in assays for bioactivity. The
literature is replete with examples of small peptides capable of
modulating a wide variety of signaling pathways. For example, a
peptide derived from the HIV-1 envelope protein has been shown to
block the action of cellular calmodulin.
[0007] A major limitation of conventional in vitro screens is
delivery. While only minute amounts of an agent may be necessary to
modulate a particular cellular response, delivering such an amount
to the requisite subcellular location necessitates exposing the
target cell or system to relatively massive concentrations of the
agent. The effect of such concentrations may well mask or preclude
the targeted response.
[0008] The present invention provides methods and compositions to
create, effectively introduce into cells and screen compounds that
affect a signaling pathway. Llittle or no knowledge of the pathway
is required, other than a presumed signaling event and an
observable physiologic change in the target cell. The disclosed
methods are conceptually distinct from prior library search methods
in that it is an in vivo stratagem for accessing intracellular
signaling mechanisms. The invention also provides for the isolation
of the constituents of the pathway, the tools to characterize the
pathway, and lead compounds for pharmaceutical development.
[0009] Relevant Literature
[0010] Mann et al. (1983) Cell 33, 153-159, Pear et al. (1993)
Proc. Natl. Acad. Sci. USA 90(18):8392-6 and copending U.S. patent
application Ser. No. 08/023,909 describe the BOSC and BING
retroviral systems useful as delivery vectors for the disclosed
methods.
[0011] Scott and Craig (1994) Current Opinion in Biotechnology
5:40-48 review random peptide libraries. Hupp et al. (1995)
describe small peptides which activate the latent sequence-specific
DNA binding function of p53. Palzkill et al. (1994) report the
selection of functional signal cleavage sites from a library of
random sequences introduced into TEM-1 -lactamase.
SUMMARY OF THE INVENTION
[0012] The invention provides methods and compositions for
screening for transdominant intracellularly bioactive agents such
as pharmaceuticals. The invention accesses molecules or targets
within living cells and provides for the direct selection of those
bioactive agents with desired phenotypic effects. The general
methods involve steps: expressing a molecular library of randomized
nucleic acids as a plurality of isolated corresponding randomized
expression products in a plurality of cells, each of the nucleic
acids comprising a different nucleotide sequence, screening for a
cell of the plurality of cells exhibiting a changed physiology in
response to the presence in the cell of a transdominant expression
product of the corresponding expression products; and detecting and
isolating the cell and/or transdominant expression product,
[0013] In a particular embodiment, the expressing step comprises
translating the nucleic acids and/or corresponding transcripts, and
each of the nucleic acids encodes a peptide comprising a different
amino acid sequence. The nucleic acids may be joined to sequences
encoding polypeptide backbones of artificial design capable of
intracellularly presenting randomized peptides as structured
domains. The methods may also involve introducing the library into
the cells, such as through the use of retroviral vectors, and
particularly suitable vectors are also disclosed.
BRIEF DESCRIPTION OF THE FIGURES
[0014] FIG. 1. Creation of a library of random peptides in a
retrovirus DNA construct by PCR.
[0015] FIG. 2. Creation of a library of random peptides in a
retrovirus DNA construct by primed DNA synthesis.
[0016] FIG. 3. Presentation constructs for localizing presentation
structures to specific cellular locales.
[0017] FIG. 4. Schematic of a retroviral construct.
DETAILED DESCRIPTION OF THE INVENTION
[0018] The subject methods generally involve introducing a
molecular library of randomized nucleic acids into a population of
cells. The introduced nucleic acids are randomized and expressed in
the cells as a library of isolated randomized expression products,
which may be nucleic acids, such as message, antisense RNA,
ribozyme components, etc., or peptides. 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. Generally 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.
[0019] The introduced nucleic acids and resultant expression
products are randomized, meaning that each nucleic acid and peptide
consists of essentially random nucleotides and amino acids,
respectively. The library may be fully random or biased, e.g. in
nucleotide/residue frequency generally or per position. For
example, a biased library may encode peptides for interactions with
known classes of molecules, such as SH-3 domain proteins, as
defined by peptides containing XXXPPXPXX (where X=randomized
residues). In other embodiments, the nucleotides or residues are
randomized within a defined class, e.g. of hydrophobic amino acids,
of purines, etc. In any event, 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; more 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 if the randomization is less than perfect.
[0020] An important aspect of the invention is the functional and
structural isolation of the randomized expression products. This is
important to facilitate subsequent drug development and
optimization. Generally, isolation is effected by providing free
(not covalently coupled) expression product, though in some
situations, the expression product may be coupled to a functional
group or fusion partner, preferably a heterologous (to the host
cell) or synthetic (not native to any cell) functional group or
fusion partner. Exemplary groups or partners include signal
sequences capable of constitutively localizing the expression
product to a predetermined subcellular locale such as the Golgi,
endoplasmic reticulum, nucleoli, nucleus, nuclear membrane,
mitochondria, chloroplast, secretory vesicles, lysosome, etc.;
binding sequences capable of binding the expression product to a
predetermined protein while retaining bioactivity of the expression
product; sequences signaling selective degradation, of itself or
co-bound proteins; secretory and membrane-anchoring signals;
etc.
[0021] It may also be desirable to provide a partner which
conformationally restricts the randomized expression product to
more specifically define the number of structural conformations
available to the cell. For example, such a partner may be a
synthetic presentation structure: an artificial polypeptide capable
of intracellularly presenting a randomized peptide as a
conformation-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. Preferred presentation structures
maximize accessibility to the peptide by presenting it on an
exterior loop, for example of coiled-coils, (Myszka, D. G., and
Chaiken, I. M. Design and characterization of an intramolecular
antiparallel coiled coil peptide. Biochemistry. 1994.
33:2362-2372). To increase the functional isolation of the
randomized expression product, the presentation structures are
selected or designed to have minimal biologically active as
expressed in the target cell. In addition, the 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, while
maintaining the absolute amino acid identity. Other presentation
structures include zinc-finger domains, loops on beta-sheet turns
and coiled-coil stem structures in which non-critical residues are
randomized; loop structures held together by cysteine bridges,
cyclic peptides, etc.
[0022] Following expression of the library in the targeted cells,
cells exhibiting a changed physiology in response to the presence
in such cells of a transdominant expression product are detected
and isolated. A transdominant expression product has an effect that
is not in cis, i.e., a trans event as defined in genetic terms or
biochemical terms. A transdominant effect is a distinguishable
effect by a molecular entity (i.e., the encoded peptide or RNA)
upon some separate and distinguishable target; that is, not an
effect upon the encoded entity itself. As such, transdominant
effects include many well-known effects by pharmacologic agents
upon target molecules or pathways in cells or physiologic systems;
for instance, the .beta.-lactam antibiotics have a transdominant
effect upon peptidoglycan synthesis in bacterial cells by binding
to penicillin binding proteins and disrupting their functions. An
exemplary transdominant effect by a peptide is the ability to
inhibit NF-.kappa.B signaling by binding to I.kappa.B-.alpha. at a
region critical for its function, such that in the presence of
sufficient amounts of the peptide (or molecular entity), the
signaling pathways that normally lead to the activation of
NF-.kappa.B through phosphorylation and/or degradation of
I.kappa.B-.alpha. are inhibited from acting at I.kappa.B-.alpha.
because of the binding of the peptide or molecular entity. In
another instance, signaling pathways that are normally activated to
secrete IgE are inhibited in the presence of peptide. Or, signaling
pathways in adipose tissue cells, normally quiescent, are activated
to metabolize fat. Or, in the presence of a peptide, intracellular
mechanisms for the replication of certain viruses, such as HIV-1,
or Herpes viridae family members, or Respiratory SyncytiaVirus, for
example, are inhibited.
[0023] A transdominant effect upon a protein or molecular pathway
is clearly distinguishable from randomization, change, or mutation
of a sequence within a protein or molecule of known or unknown
function to enhance or diminish a biochemical ability that protein
or molecule already manifests. For instance, a protein that
enzymatically cleaves .beta.-lactam antibiotics, a
.beta.-lactamase, could be enhanced or diminished in its activity
by mutating sequences internal to its structure that enhance or
diminish the ability of this enzyme to act upon and cleave
.beta.-lactam antibiotics. This would be called a cis mutation to
the protein. The effect of this protein upon B-lactam antibiotics
is an activity the protein already manifests, to a distinguishable
degree. Similarly, a mutation in the leader sequence that enhanced
the export of this protein to the extracellular spaces wherein it
might encounter .beta.-lactam molecules more readily, or a mutation
within the sequence that enhance the stability of the protein,
would be termed cis mutations in the protein. For comparison, a
transdominant effector of this protein would include an agent,
independent of the .beta.-lactamase, that bound to the
.beta.-lactamase in such a way that it enhanced or diminished the
function of the .beta.-lactamase by virtue of its binding to
.beta.-lactamase.
[0024] In general, cis-effects are effects within molecules wherein
elements that are interacting are covalently joined to each other
although these elements might individually manifest themselves as
separable domains. Trans-effects (transdominant in that under some
cellular conditions the desired effect is manifested) are those
effects between distinct molecular entities, such that molecular
entity A, not covalently linked to molecular entity B, binds to or
otherwise has an effect upon the activities of entity B. As such,
most known pharmacological agents are transdominant effectors.
[0025] A wide variety of phenotypic changes may be targeted, such
as changes in the amount or distribution of cellular
differentiation markers, e.g. T/B-cell activation, cell growth or
death, membrane potentials, etc. Similarly, a wide variety of
techniques may be used to isolate cells exhibiting changed
physiology in response to the presence of a library expression
product, such as FACS, lysis selection using complement, cell
cloning, etc. Where the randomized expression product is expressed
on the extracellular surface of the host cell or is secreted by the
host cell into the extracellular medium, the methods may involve
screening for a change in the physiology of a "responder cell" in
the vicinity of the host cell, exhibiting a changed physiology in
response to the presence of the surface-expressed or secreted
expression product. Alternatively, the methods may involve
screening for a "target cell" in the vicinity of the host cell,
that specifically binds the surface-expressed or secreted
expression product.
[0026] A wide variety of cells may be used depending on the nature
of the targeted cellular pathway, so long as the cell is suitably
transfectable with the library and may be monitored for the
targeted physiological changes. The method of introduction is
largely dictated by the targeted cell type. Exemplary methods
include CaPO.sub.4 precipitation, liposome fusion, lipofectin.RTM.,
electroporation, viral infection, etc. As many pharmaceutically
important screens require human or model mammalian cell targets,
retroviral vectors capable of transfecting such targets are
frequently employed. A particularly well-suited retroviral
transfection system is described in Mann et al.(1983) Cell 33,
153-159, Pear et al. (1993) Proc. Natl. Acad. Sci. USA
90(18):8392-6 and copending U.S. patent application Ser. No.
08/023,909.
[0027] In one embodiment of the invention, the library is generated
in a retrovirus DNA construct backbone. Preferred techniques for
constructing such libraries are described in more detail below.
Libraries with up to 10.sup.9 unique sequences can be readily
generated in such DNA backbones. After generation of the library in
DNA, and its isolation from bacteria, it is converted into
infectious virus. Conversion into infectious virus requires the
delivery of the library DNA to a retrovirus packaging system. Such
systems can include BOSC23, PhiNX-eco, PhiNX-ampho, 293T+gag-pol
and retrovirus envelope, PA317, and other extant packaging systems.
Methods for high-efficiency packaging of retroviral libraries
(cDNA) are described in Kitamura et al. (1995) Efficient screening
of retroviral cDNA libraries. PNAS 92: 9146-9150. Libraries
containing up to 10.sup.7 retrovirus particles per ml were derived
in this system. Scaling up these methods conveniently yields
10.sup.9 individual and unique library components in a 100 ml
reaction. The library of retroviruses is then used to infect the
target cells by any of a number of different protocols, e.g. see
Kitamura et al supra for details on some infection protocols
[0028] Depending upon how the particular resident expression
product(s) effect the target molecule function, the physiology of
the cell will be affected in differing ways. Since extremely large
libraries of different molecular peptide or RNA shapes can be
delivered to cells via retroviral transduction, this embodiment of
the invention provides a high frequency for the isolation of cells
affected in a predetermined manner. For instance, peptides that
inhibit T cell activation processes are readily isolated using a
population of Jurkat cells, e.g. Jurkat-NFAT-dipA, engineered to
express diphtheria toxin under the control of Nuclear Factor of
Activated T cells (NFAT) regulatory elements. Unproductively
transduced cells are killed when NFAT is activated through the T
cell receptor and other signaling mechanisms specific to T cells
(Serafini AT et al. (1995). Isolation of mutant T lymphocytes with
defects in capacitative calcium entry. Immunity, 3(2):239-50). This
is because NFAT activation leads to transcription and translation
of diphtheria toxin A chain in these cells, which efficiently kills
them. However, productive mutations in the NFAT activation pathway
are rescued. This approach has already been used to isolate mutants
in T cell signaling pathways. Peptide or compound expression
libraries are also used to mimic mutation of the signaling pathway
leading to activation of NFAT, i.e. peptides which interfere with
signaling of the NFAT pathway by binding to and inhibiting the
function of a required signaling protein or factor can block
programmed cell death.
[0029] The following experimental section/examples are offered by
way of illustration and not by way of limitation.
EXPERIMENTAL
[0030] Retroviral Presentation Construct for Peptide
Expression.
[0031] A series of retroviral constructs have been designed for
expression of randomized and biased peptides within target cell
populations. The peptide is expressed from a retroviral promoter.
The translation unit has several important components. Glycine
following the initiator methionine at the amino terminus stabilizes
the peptide and enhances cytoplasmic half-life, according to
Varshavsky's N-End Rule. In some constructs, a nine amino acid flu
epitope tag has been incorporated to permit co-precipitation of the
rare peptide and any molecule to which it has affinity, by using
monoclonal antibodies to the epitope. Glycines are encoded before
and after the random/biased expression product encoding regions to
provide some molecular flexibility. Two carboxyl-terminal prolines
are encoded to confer stability to carboxypeptidase.
[0032] For construction of a large library two primers were made
(schematized in FIG. 1). The first, designated the random peptide
primer, consists of 1) a complementary region for vector priming,
2) the regions mentioned above, and 3) a random or biased
expression product region, here presented as a 30 base sequence
encoding a peptide of length 10 amino acids. In addition, we have
inserted a stop codon in all three reading frames in case of minor
deletions or insertions in the random region. The design of the
primer ensures a glycine/proline termination in most reading
frames. The second primer is downstream in the vector and primes a
region of the plasrnid that contains a unique Not I site. These
primers are used to create a library of fragments, each containing
a different nucleotide sequence that each potentially encodes a
different peptide. These families of fragments are ligated to
vector fragments containing puromycin selection sequence, a 3' LTR
, and a bacterial origin of replication. The ligation products are
then electroporated into E. coli and DNA is prepared from the
resulting library. Using this technique we have constructed
independent random libraries with up to 2.times.10.sup.8 unique
inserts. Sequencing of multiple individual inserts demonstrates
they have the structure as defined by Primer 1, and the peptides
encoded are random. Such libraries thus made contain subsets of the
total 10.sup.13 predicted peptides.
[0033] Generation of Retroviral Peptide Libraries.
[0034] A scheme for generating a peptide library in the pBabe Puro
vector is shown in FIG. 2. Primers for PCR were synthesized,
purified and deprotected according to standard protocols. Primer 1,
complementary to polylinker sequences in the pBabe Puro retroviral
construct, has the sequence 5' GCT TAG CAA GAT CTC TAC GGT GGA CCK
NNK NNK NNK NNK NNK NNK NNK NNK NNK NNC CCC ACT CCC ATG GTC CTA CGT
ACC ACC ACA CTG GG 3'. N represents any of the four bases; K is
limited to G or T. Primer 2 has the sequence 5' GCT TAG CAA GAT CTG
TGT GTC AGT TAG GGT GTG G 3' and is complementary to sequences
within the pUC18 origin of replication. PCR was carried out for 8
rounds using primer 1, primer 2, pBabe Puro as template, and a
mixture of Taq DNA Polymerase (Promega) and Deep Vent DNA
Polymerase (New England Biolabs) in a ratio of 128 Taq: 1 Deep Vent
as described in Barnes (1994) Proc. Natl. Acad. Sci. USA, 91, pp.
2216-2220. The amplified PCR product was purified, digested with
restriction enzymes Bgl II and Not I (Promega), purified again and
ligated with the corresponding Bam HI-Not I fragment of pBabe Puro.
After transformation the resulting library contained
.about.2.times.10.sup.8 clones, greater than 80% of which contained
inserts.
[0035] pMSCV-PC and pBabeMN-PC Retroviral Construct Libraries:
[0036] Oligonucleotides were synthesized and purified according to
standard protocols. The "library" oligonucleotides have the
sequence 5' CTG GAG AAC CAG GAC CAT GGG C (NNK).sub.10 GGG CCC CCT
TAA ACC ATT AAA T 3' or 5' CTG GAG AAC CAG GAC CAT GGG CNN KNN KNN
KCC TCC CNN KCC TNN KNN KGG GCC CCC TTA AAC CAT TAA AT 3'. A third
oligonucleotide ("constant"), complementary to the 3' ends of the
library oligonucleotides, has the sequence 5' TCA TGC ATC CAA TTT
AAT GGT TTA AG 3'. As shown in FIG. 3, each library oligonucleotide
is annealed to the constant oligonucleotide, converted to double
stranded DNA with Sequenase (United States Biochemical) or Klenow
(Promega), digested with restriction enzyme Bst XI (New England
Biolabs), and purified and ligated with the appropriate Bst
XI-digested retroviral construct. Transformation efficiencies are
.about.2.times.10.sup.8 clones per microgram of ligated DNA,
greater than 90% of which contain an insert. A representative
retrovirus is shown in FIG. 4; see also, retroviral nucleotide
sequence below:
[0037] Retroviral Vector With Presentation Construct.
1 TGAAAGACCCCACCTGTAGGTTTGGCAAGCTAGCTTAAGTAACGCCATTTTGCAAGGCATGGAAA
ATACATAACTGAGAATAGAGAAGTTCAGATCAAGGTTAGGAACAGAGAGACAGCAG- AATATGGGC
CAAACAGAGTATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAA- GAACAGATGGTCCCCAG
ATGCGGTCCCGCCCTCAGCAGTTTCTAGAGAACCATCAGA- TGTTTCCAGGGTGCCCCAAGGACCT
GAAAATGACCCTGTGCCTTATTTGAACTAACC- AATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCT
TCTGCTCCCCGAGCTCAATAAAAG- AGCCCACAACCCCTCACTCGGCGCGCCAGTCCTCCGATAGA
CTGCGTCGCCCGGGTACCCGTATTCCCAATAAAGCCTCTTGCTGTTTGCATCCGAATCGTGGACT
CGCTGATCCTTGGGAGGGTCTCCTCAGATTGATTGACTGCCCACCTCGGGGGTCTTTCATTTGGA
GGTTCCACCGAGATTTGGAGACCCCTGCCTAGGGACCACCGACCCCCCCGCCGGGAG- GTAAGCTG
GCCAGCGGTCGTTTCGTGTCTGTCTCTGTCTTTGTGCGTGTTTGTGCCG- GCATCTAATGTTTGCG
CCTGCGTCTGTACTAGTTAGCTAACTAGCTCTGTATCTGGC- GGACCCGTGGTGGAACTGACGAGT
TCTGAACACCCGGCCGCAACCCTGGGAGACGTC- CCAGGGACTTTGGGGGCCGTTTTTGTGGCCCG
ACCTGAGGAAGGGAGTCGATGTGGA- ATCCGACCCCGTCAGGATATGTGGTTCTGGTAGGAGACGA
GAACCTAAAACAGTTCCCGCCTCCGTCTGAATTTTTGCTTTCGGTTTGGAACCGAAGCCGCGCGT
CTTGTCTGCTGCAGCGCTGCAGCATCGTTCTGTGTTCTCTCTGTCTGACTGTGTTTCTGTATTTG
TCTGAAAATTAGGGCCAGACTGTTACCACTCCCTTAAGTTTGACCTTAGGTCACTGG- AAAGATGT
CGAGCGGATCGCTCACAACCAGTCGGTAGATGTCAAGAAGAGACGTTGG- GTTACCTTCTGCTCTG
CAGAATGGCCAACCTTTAACGTCGGATGGCCGCGAGACGGC- ACCTTTAACCGAGACCTCATCACC
CAGGTTAAGATCAAGGTCTTTTCACCTGGCCCG- CATGGACACCCAGACCAGGTCCCCTACATCGT
GACCTGGGAAGCCTTGGCTTTTGAC- CCCCCTCCCTGGGTCAAGCCCTTTGTACACCCTAAGCCTC
CGCCTCCTCTTCCTCCATCCGCCCCGTCTCTCCCCCTTGAACCTCCTCGTTCGACCCCGCCTCGA
TCCTCCCTTTATCCAGCCCTCACTCCTTCTCTAGGCGCCGGAATTccaggaccatgggcGGGCCC
CCTTAAAccattaaattggtaaaataaagGATCCGTCGACCTGCAGCCAAGCTTATC- GATAAAAT
AAAAGATTTTATTTAGTCTCCAGAAAAAGGGGGGAATGAAAGACCCCAC- CTGTAGGTTTGGCAAG
CTAGCTTAAGTAACGCCATTTTGCAAGGCATGGAAAATACA- TAACTGAGAATAGAGAAGTTCAGA
TCAAGGTTAGGAACAGAGAGACAGCAGAATATG- GGCCAAACAGGATATCTGTGGTAAGCAGTTCC
TGCCCCGGCTCAGGGCCAAGAACAG- ATGGTCCCCAGATGCGGTCCCGCCCTCAGCAGTTTCTAGA
GAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAAATGACCCTGTGCCTTATTTGAACTA
ACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCAATAAAAGAGCCC
ACAACCCCTCACTCGGCGCGCCAGTCCTCCGATAGACTGCGTCGCCCGGGTACCCGT- GTATCCAA
TAAACCCTCTTGCAGTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGG- AGGGTCTCCTCTGAGT
GATTGACTACCCGTCAGCGGGGGTCTTTCATTCGTAATCAT- GGTCATAGCTGTTTCCTGTGTGAA
ATTGTTATCCGCTCACAATTCCACACAACATAC- GAGCCGGAAGCATAAAGTGTAAAGCCTGGGGT
GCCTAATGAGTGAGCTAACTCACAT- TAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAA
CCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGC
GCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCA
GCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAG- AACATGTG
AGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTG- GCGTTTTTCCATAGGC
TCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGT- CAGAGGTGGCGAAACCCGACAGGA
CTATAAAGATACCAGGCGTTTCCCCCTGGAAGC- TCCCTCGTGCGCTCTCCTGTTCCGACCCTGCC
GCTTACCGGATACCTGTCCGCCTTT- CTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCT
GTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTT
CAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTT
ATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGG- TGCTACAG
AGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATT- TGGTATCTGCGCTCTG
CTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTG- ATCCGGCAAACAAACCACCGCTGG
TAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGAT- TACGCGCAGAAAAAAAGGATCTCAAGAAGATC
CTTTGATCTTTTCTACGGGGTCTGA- CGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTC
ATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAAT
CTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCT
CAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAA- CTACGATA
CGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACC- CACGCTCACCGGCTCC
AGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGC- GCAGAAGTGGTCCTGCAACTTTAT
CCGCCTCCATCCAGTCTATTAATTGTTGCCGGG- AAGCTAGAGTAAGTAGTTCGCCAGTTAATAGT
TTGCGCAACGTTGTTGCCATTGCTA- CAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTC
ATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGG
TTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTT
ATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTG- ACTGGTGA
GTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGC- TCTTGCCCGGCGTCAA
TACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTG- CTCATCATTGGAAAACGTTCTTCG
GGGCGAAAACTCTCAAGGATCTTACCGCTGTTG- AGATCCAGTTCGATGTAACCCACTCGTGCACC
CAACTGATCTTCAGCATCTTTTACT- TTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAA
ATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAA
TATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAA
AAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTA- AGAAACCA
TTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTT- TCGTCTCGCGCGTTTC
GGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGA- GACGGTCACAGCTTGTCTGTAAGC
GGATGCCGGGAGCAGACAAGCCCGTCAGGGCGC- GTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGC
TTAACTATGCGGCATCAGAGCAGAT- TGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCAC
AGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGA
AGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGC
GATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCA- GTGCCACG
CTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTG- AGGCCGTTGAGCACCG
CCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAAC- AGTCCCCCGGCCACGGGGCCTGCC
ACCATACCCACGCCGAAACAAGCGCTCATGAGC- CCGAAGTGGCGAGCCCGATCTTCCCCATCGGT
GATGTCGGCGATATAGGCGCCAGCA- ACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTC
CGGCGTAGAG
[0038] Peptide Library Infection of a Factor-dependent Line and
Outgrowth of an Apoptosis-Resistant Line.
[0039] The Baf/3 cell line is an IL-3 dependent cell that undergoes
rapid apoptosis in the absence of IL-3. Thus it makes an attractive
cell line for dominant effector peptides. Cells expressing a a
peptide that inhibits apoptosis are readily selected against the
background of dying cells. We chose this cell line as a model for
demonstrating peptide selection.
[0040] A retroviral library containing 5.times.10.sup.6 independent
peptide inserts was transfected into BOSC23 cells and converted
into retrovirus with an approximate titer of 5.times.10.sup.5 per
ml. Twelve ml of viral supernatant was used to infect
6.times.10.sup.6 Baf/3cells (2 ml per infection of 1.times.10.sup.6
cells in 6 independent infections). Cells were grown for 3 days
after infection in the presence of IL-3 to allow retroviral
integration and peptide expression. After three days IL-3 was
withdrawn and the cells allowed to grow for two weeks. After two
weeks, one well of six had outgrowth of cells that survive in the
absence of IL-3, indicating the presence of an apoptosis-inhibiting
peptide. Peptides derived in this manner may effect the IL-3
independence by positive dominancy (i.e. mimic or circumvent the
positive regulatory role of IL-3) or by inhibition (i.e. prevent
the apoptosis process upon IL-3 withdrawal).
[0041] Subcellular Targeting:
[0042] In some embodiments of the invention, expression products
are localized to, or preferentially concentrated in, different
subcellular compartments within cells, e.g. by using appropriate
addition of addressins to a peptide presentation construct, see,
FIG. 3. Addressins are available for a wide variety of subcellular
locales including the nucleus, Golgi, mitochondria, plasma
membranes, endoplasmic reticulum, secretory granules, secreted,
cell surface (extracellular domain with random), cell surface
(intracellular domain random), etc. For example, many proteins
whose functions require entry into the cell nucleus include nuclear
localization signal (NLS) sequences: 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, 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 LysLys 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, etal., Proc, Natl. Acad, Sci,
USA, 84:6795-6799, 1987; Galileo, et al., Proc. Natl. Acad. Sci.
USA, 87:458-462, 1990.
[0043] Secreted Peptide Structure.
[0044] In some embodiments of the invention, it is desired 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 this case
the peptide library is configured with a secretion signal sequence
at its amino terminus, usually clipped off after secretion, such
that the peptide is ultimately secreted 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. 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. 3. 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.
[0045] Surface-Expressed Peptide Structure.
[0046] In certain embodiments of the invention, it is desirable to
localize the randomized peptides to the surface of a cell. The
invention provides methods for presenting the randomized expression
product extracellularly or in the cytoplasmic space; see FIG. 3.
For extracellular presentation, a modification of the secreted
structure above is made; specifically, a membrane anchoring region
is provided at the carboxyl terminus of the peptide presentation
structure. The anchor can be a transmembrane domain, such as that
found in a variety of surface expressed molecules of the Ig family
(e.g. CD4, CD8, IgM, T cell receptor), or a lipid anchoring region
such as found in Thy-1. Other anchoring domains exist and are known
to those who practice in the art. 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.
[0047] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference. Although
the foregoing invention has been described in some detail by way of
illustration and example for purposes of clarity of understanding,
it will be readily apparent to those of ordinary skill in the art
in light of the teachings of this invention that certain changes
and modifications may be made thereto without departing from the
spirit or scope of the appended claims.
* * * * *