Note: Descriptions are shown in the official language in which they were submitted.
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TARGETED GENE DELIVERY TO CELLS BY FiLAMENTOUS
BACTERIOPHAGE
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims benefit under 35 U.S.C. ~ 119(e) of provisional
application USSN 60/082,953, filed on April 24, 1998, which is herein
incorporated by
reference in its entirety for all purposes.
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY
SPONSORED RESEARCH AND DEVELOPMENT
This work was supported, in part, by Department of Defense Grants
DAMD17-96-1-6244 and DAMD17-94-4433. The government of the United States of
America may have some rights in this invention.
FIELD OF THE INVENTION
This invention relates to the field of cell transduction and gene delivery. In
particular, this invention relates to the use of filamentous phage to deliver
heterologous
nucleic acids into a cell.
BACKGROUND OF THE INVENTION
Frequently gene transfecdon techniques require the ability to target a
therapeutic gene to an appropriate "target" cell or tissue type v~rith high
efficiency (Michael
and Curiel (1994) Gene Then. 1: 223-232). Targeting of retroviral vectors has
been reported
by inserting receptor ligands or single chain Fv (scFv) antibody fragments
into the viral
envelope protein (Kasahara et al. (1994) Science 266: 1373-1376). Targeting of
adenoviral
vectors has been achieved by use of'adapter' fusion molecules consisting of an
antibody
fragment which binds the adenoviral knob and a cell targeting molecule such as
a receptor
ligand or antibody (Douglas et al. (1996) Nat. Biotechnol. 14: 1574-1578;
Watkins et al.
1997) Gene Ther. 4{10): 1004-1012). Targeting of non-viral vectors using cell
surface
receptor ligands or antibodies has also been reported (Fominaya and Wels
(1996) J. Biol.
Chem. 271(18): 10560-10568; Michael and Curiel (1994) Gene Ther. 1: 223-232).
All of
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these approaches depend on the use of targeting molecules that bind a cell
suzface receptor -
resulting in internalization of the gene delivery vehicle with subsequent
delivery of the DNA
to the nucleus.
Identification of appropriate targeting molecules has largely been performed
by individually screening receptor ligands or antibodies. In the case of
single chain (e.g.
scFv) antibody fragments this typically requires construction of the scFv from
the V-genes
of a hybridoma, construction of the targeted gene delivery vehicle, and in
vitro evaluation of
targeting ability.
Mare recently, it has proven possible to directly select peptides and antibody
fragments binding cell surface receptors from filamentous phage libraries
(Andersen et al.
(1996) Proc. Natl. Acad Sci. USA 93(5): 1820-1824; Barry et al. (1996) Nat.
Med. 2: 299-
305; Cai and Garen (1995) Proc. Natl. Acad. Sci. USA 92(24): 6537-6541; de
Kruif et al.
(1995) Proc. Natl. Acad Sci. USA 92(6): 3938-3942; Marks et al. (1993)
BiolTechnology
11(10): 1145-1149). This has led to a marked increase in the number
ofpotential targeting
1 S molecules.
Despite the increase in the number of known potential targeting molecules,
these molecules, to date, have not been effectively exploited for transfecting
genes into
target cells.
SUMMARY OF THE INVENTION
This invention is based, in part, on the discovery that filamentous phage
displaying the an antibody that binds to an internalizing receptor (e.g. anti-
ErbB2 scFv FS)
as a genetic fusion with the phage minor coat protein pIlr can directly infect
mammalian
cells expressing the target receptor epitope (e.g., ErbB2) leading to
expression of a
heterologous gene (e.g. cDNA) contained in the phage genome. Thus, in one
embodiment,
this invention provides methods of transfecting (transducing) a target cell
(e.g., vertebrate,
invertebrate, bacteria, fungus, yeast, algal cell) with a (e.g. heterologous)
nucleic acid. The
methods preferably involve i) providing a phage externally displaying a
heterologous
targeting protein (heterologous to the phage) and containing a heterologous
nucleic acid
. (heterologous to the phage and/or to the target cell); and ii) contacting
the target cell with
the phage whereby said phage is internalized into said cell and wherein the
heterologous
nucleic acid is transcribed within the cell. While in many embodiments, the
heterologous
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nucleic acid comprises a reporter gene (or -cDNA) or a selectable marker (e.g.
an antibiotic
resistance gene or cDNA), in particularly preferred embodiment, the
heterologous nucleic
acid transcribes a gene product (e.g., antisense molecule, ribozyme,
polypeptide) other than,
or in addition to, the reporter gene or selectable marker. Typically a DNA
brought into the
cell by the methods of this invention is single stranded and, without being
bound to a
particular theory, it is believed the DNA is replicated to double stranded
prior to
transcription.
In one preferred embodiment, the phage used in the methods of this invention
are monovalent, displaying, on average, one pIII fusion protein per viral
particle, while in
other preferred embodiments, the phage used in the methods of this invention
are multi-
valent, displaying on average, at least two, more preferably at least 3, and
most preferably at
least 5, pIII fusions per viral particle. The phage used to deliver the
heterologous nucleic
acid into a target cell can be a member of a library of phage wherein said
library comprises a
number of different heterologous targeting proteins (e.g. containing, on
average, at least 105,
preferably at least 106, more preferably at least 10', and most preferably at
least 108 different
members). The methods can further involve selecting phage (e.g., from a
library) that are
internalized by the target cell. The selection can be by a variety of means
including, but not
limited to detection of a reporter gene (e.g. GFP, Fflux, luciferase, J3-gal,
etc.) or by selection
via a selectable marker (e.g. an antibiotic resistance gene). The method can
further involve
amplifying phage internalized by said cell.
In one particularly preferred embodiment, the providing step involves i)
providing an assembly cell containing the heterologous nucleic acid and a
packaging signal;
and ii) infecting the assembly cell with a phage expressing on its surface
said heterologous
targeting protein and containing the gene for the targeting protein whereby
the phage acts as
a helper phage and packages the heterologous nucleic acid. Preferred assembly
cells are
prokaryotic cells {e.g. bacterial cells). In one preferred embodiment the
heterologous
targeting protein, and/ar a DNA encoding the heterologous targeting protein,
and/or the
heterologous nucleic acid are encoded by a DNA that is a phagemid. Preferred
phage for use
in the methods of this invention are filamentous phage.
Preferred heterologous targeting proteins are antibodies, more preferably
single-chain Fv, or Fabs. The phage can be preselected for binding to a
particular
internalizing cell surface receptor (e.g., erbB2). Other preferred receptors
include, but are
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not limited to receptors far platelet-derived growth factor (PDGF), epidermal
growth factor-
(EGF), insulin-like growth factor (IGF), transforming growth factor I3 (TGF-
13), fibroblast
growth factors (FGF), interleukin 2 (IL2), nerve growth factor (NGF),
interleukin 3 (II,3),
interleukin 4 (IL4), interleukin 1 (11,1), interleukin 6 (TL,6), interleulcin
7 (IL7), interleukin
13, granulocyte/macrophage colony-stimulating factor (GM-CSF), granulocyte
colony-
stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF),
erythropoietin
TGF, transferrin, erbB2, EGF, Vegf, and the like. In one particularly
preferred embodiment,
the phage can further express an endosomal escape polypeptide and/or a nuclear
localization
signal.
In another embodiment, this invention provides a vector for (e.g., specific)
transfection of a target cell. Preferred vectors comprise a phage displaying a
heterologous
targeting protein (e.g. a single chain antibody) that specifically binds to an
internalizing
receptor whereby the phage binds to and is internalized into the target cell,
and wherein the
phage contains a heterologous nucleic acid that is transcribed inside the
target cell. In one
preferred embodiment, the heterologous nucleic acid transcribes a gene product
(e.g.,
antisense molecule, ribozyme, polypeptide) other than, or in addition to, a
reporter gene or
selectable marker. The vector can include any of the viral particles or
nucleic acids encoding
the viral particles, or cells containing the nucleic acid or viral particles
described herein.
Thus, in another preferred embodiment this invention comprises a phage
vector or phagemid vector encoding: a phage coat protein in fusion with a
heterologous
targeting protein that specifically binds to an internalizing cell surface
receptor and is
internalized into a cell bearing said receptor; and a heterologous nucleic
acid in an
expression cassette allowing transcription of the heterologous nucleic acid
inside said cell as
described herein. In one preferred embodiment, the heterologous nucleic acid
transcribes a
gene product (e.g., antisense molecule, ribozyme, polypeptide) other than, or
in addition to, a
reporter gene or selectable marker
This invention also provides a kit for transducing a target cell. The kit
preferably a phage, and/or phage DNA, and/or phagemid DNA, and/or cells)
containing
phage and/or phagemid DNA, and/or cells containing phage particles as
described herein. In
on particular preferred embodiment, the kits include a phage or phagemid
vector encoding: a
phage coat protein in fusion with a heterologous targeting protein that
specifically binds to
an internalizing cell surface receptor and is internalized into a cell bearing
said receptor; and
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a pair of restriction sites that allow insertion of a heterologous nucleic
acid into the phage or
phagemid vector. The restriction sites are preferably situated in an
expression cassette such
that a gene or cDNA inserted between said restriction sites is operably linked
to a promoter
and is transcribed, and optionally translated, when said expression cassette
is transduced into
a target cell.
DEFINITIONS
As used herein, an "antibody" refers to a protein consisting of one or more
polypeptides substantially encoded by immunoglobulin genes or fragments of
immunoglobulin genes. The recognized immunoglobulin genes include the kappa,
lambda,
alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad
immunoglobulin variable region genes. Light chains are classified as either
kappa or
lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon,
which in turn
define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
A typical immunoglobulin (antibody) structural unit is known to comprise a
tetramer. Each tetramer is composed of two identical pairs of polypeptide
chains, each pair
having one "light" (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-
terminus
of each chain defines a variable region of about 100 to 110 or more amino
acids primarily
responsible for antigen recognition. The terms variable light chain (V~) and
variable heavy
chain (VH) refer to these light and heavy chains respectively.
Antibodies exist as intact immunoglobulins or as a number of well
characterized fragments produced by digestion with various peptidases. Thus,
for example,
pepsin digests an antibody below the disulfide linkages in the hinge region to
produce
F(ab)'Z, a dimer of Fab which itself is a light chain joined to VH-CHl by a
disulfide bond.
The F(ab)'2 may be reduced under mild conditions to break the disulfide
linkage in the hinge
region thereby converting the (Fab')2 dimer into an Fab' monomer. The Fab'
monomer is
essentially an Fab with part of the hinge region (see, Fundamental Immunology,
W.E. Paul,
ed., Raven Press, N.Y. (1993), for a more detailed description of other
antibody fragments).
While various antibody fragments are defined in terms of the digestion of an
intact antibody,
one of skill will appreciate that such Fab' fragments may be synthesized de
novo either
chemically or by utilizing recombinant DNA methodology. Thus, the term
antibody, as used
herein also includes antibody fragments either produced by the modification of
whole
antibodies or synthesized de novo using recombinant DNA methodologies.
Preferred
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antibodies include single chain antibodies (antibodies that exist as a single
polypeptide.
chain), more preferably single chain Fv antibodies (scFv or scFv) in which a
variable heavy
and a variable light chain are joined together (directly or through a peptide
linker) to form a
continuous polypeptide. The single chain Fv antibody is a covalently linked
VH_V~,
heterodimer which may be expressed from a nucleic acid including VH- and VL-
encoding
sequences either joined directly or joined by a peptide-encoding linker.
Huston, et al. (1988)
Proc. Nat. Acad. Sci. USA, 85: 5879-5883. While the VH and VL are connected to
each as a
single polypeptide chain, the VH and VL domains associate non-covalently. The
first
functional antibody molecules to be expressed on the surface of filamentous
phage were
single-chain Fv's (scFv), however, alternative expression strategies have also
been
successful. For example Fab molecules can be displayed on phage if one of the
chains
(heavy or light) is fused to g3 capsid protein and the complementary chain
exported to the
periplasm as a soluble molecule. The two chains can be encoded on the same or
on different
replicons; the important point is that the two antibody chains in each Fab
molecule assemble
post-translationally and the dimer is incorporated into the phage particle via
linkage of one
of the chains to gap (see, e.g., U.S. Patent No: 5733743). The scFv antibodies
and a number
of other structures converting the naturally aggregated, but chemically
separated Iight and
heavy polypeptide chains from an antibody V region into a molecule that folds
into a three
dimensional structure substantially similar to the structure of an antigen-
binding site are
known to those of skill in the art (see e.g., U.S. Patent Nos. 5,091,513,
5,132,405, and
4,956,778). Particularly preferred antibodies include all those that have been
displayed on
phage I think preferred antibodies should include all that have been displayed
on phage (e.g.,
scFv, Fv, Fab and disulfide linked Fv (Reiter et al. (1995) Protein Eng. 8:
1323-1331).
An "antigen-binding site" or "binding portion" refers to the part of an
immunoglobulin molecule that participates in antigen binding. The antigen
binding site is
formed by amino acid residues of the N-terminal variable ("V") regions of the
heavy ("H")
and light ("L") chains. Three highly divergent stretches within the V regions
of the heavy
and light chains are referred to as "hypervariable regions" which are
interposed between
more conserved flanking stretches known as "framework regions" or "FRs". Thus,
the term
"FR" refers to amino acid sequences which are naturally found between and
adjacent to
hypervariable regions in immunoglobulins. In an antibody molecule, the three
hypervariable
regions of a light chain and the three hypervariable regions of a heavy chain
are disposed
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relative to each other in three dimensional space to form an antigen binding
"surface".- This
surface mediates recognition and binding of the target antigen. The three
hypervariable
regions of each of the heavy and light chains are referred to as
"complementarity
determining regions" or "CDRs" and are characterized, for example by Kabat et
al.
Sequences of proteins of immunological interest, 4th ed. U.S. Dept. Health and
Human
Services, Public Health Services, Bethesda, MD (1987).
As used herein, the terms "immunological binding" and "immunological
binding properties" refer to the non-covalent interactions of the type which
occur between an
immunoglobulin molecule and an antigen for which the immunoglobulin is
specific. The
strength or affinity of immunological binding interactions can be expressed in
terms of the
dissociation constant (Kd) -of the interaction, wherein a smaller Kd
represents a greater
affinity. Immunological binding properties of selected polypeptides can be
quantified using
methods well known in the art. One such method entails measuring the rates of
antigen-
binding site/antigen complex formation and dissociation, wherein those rates
depend on the
concentrations of the complex partners, the affinity of the interaction, and
on geometric
parameters that equally influence the rate in both directions. Thus, both the
"on rate
constant" (ko") and the "off rate constant" (ka~) can be determined by
calculation of the
concentrations and the actual rates of association and dissociation. The ratio
of kat~ko"
enables cancellation of all parameters not related to affinity and is thus
equal to the
dissociation constant Kd (see, generally, Davies et al. (1990) Ann. Rev.
Biochem., 59: 439-
473.
The phrase "specifically binds to a protein" or "specifically immunoreactive
with", when referring to an antibody refers to a binding reaction which is
determinative of
the presence of the protein in the presence of a heterogeneous population of
proteins and
other biologics. Thus, under designated immunoassay conditions, the specified
antibodies
bind to a particular protein and do not bind in a significant amount to other
proteins present
in the sample. Specific binding to a protein under such conditions may require
an antibody
that is selected for its specificity for a particular protein. Fox example, FS
or C1 antibodies
can be raised to the c-erbB-2 protein that bind c-erbB-2 and not to other
proteins present in a
tissue sample. A variety of immunoassay formats may be used to select
antibodies
specifically immunoreactive with a particular protein. For example, solid-
phase ELISA
immunoassays are routinely used to select monoclonal antibodies specifically
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immunoreactive with a protein. See Harlow and Lane (1988) Antibodies, A
Laboratory
Manual, Cold Spring Harbor Publications, New York, for a description of
immunoassay
formats and conditions that can be used to determine specific
immunoreactivity.
The terms "polypeptide", "peptide", or "protein" are used interchangeably
herein to designate a linear series of amino acid residues connected one to
the other by
peptide bonds between the alpha-amino and carboxy groups of adjacent residues.
The amino
acid residues are preferably in the natural "L" isomeric form. However,
residues in the "D"
isomeric form can be substituted for any L-amino acid residue, as long as the
desired
functional property is retained by the polypeptide. In addition, the amino
acids, in addition
to the 20 "standard" amino acids, include modified and unusual amino acids,
which include,
but are not limited to those listed in 37 CFR 31.822(b)(4). Furthermore, it
should be noted
that a dash at the beginning or end of an amino acid residue sequence
indicates either a
peptide bond to a further sequence of one or more amino acid residues or a
covalent bond to
a carboxyl or hydroxyl end group.
The term "binding polypeptide" refers to a polypeptide that specifically binds
to a target molecule (e.g. a cell receptor) in a manner analogous to the
binding of an antibody
to an antigen. Binding polypeptides are distinguished from antibodies in that
binding
polypeptides are not ultimately derived from immunoglobulin genes or fragments
of
immunoglobulin genes.
The term "conservative substitution" is used in reference to proteins or
peptides to reflect amino acid substitutions that do not substantially alter
the activity
(specificity or binding affinity) of the molecule. Typically conservative
amino acid
substitutions involve substitution one amino acid for another amino acid with
similar
chemical properties (e.g. charge or hydrophobicity). The following six groups
each contain
amino acids that are typical conservative substitutions for one another:
1) Alanine (A), Serine (S), Threonine (T);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (I~, Giutamine (Q);
q.) ~ginine (R), Lysine (K);
5) Isoleucine (1), Leucine (L), Methionine (M), Valine ('~; and
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (VV~.
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The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and
polymers thereof in either single- or double-stranded form. Unless
specifically limited, the
term encompasses nucleic acids containing known analogues of natural
nucleotides which
have similar binding properties as the reference nucleic acid and are
metabolized in a manner
similar to naturally occurring nucleotides. The term also includes peptide
nucleic acids.
Unless otherwise indicated, a particular nucleic acid sequence also implicitly
encompasses
conservatively modified variants thereof (e.g. degenerate codon substitutions)
and
complementary sequences and as well as the sequence explicitly indicated.
Specifically, degenerate codon substitutions may be achieved by generating
sequences in which the third position of one or more selected (or all) codons
is substituted
with mixed-base and/or deoxyinosine residues (Batzer et al. (1991) Nucleic
Acid ReS. 19:
5081; Ohtsuka et al. (I985) J. Biol. Chem. 260: 2605-2608; and Cassol et al.
(1992);
Rossolini et al., (1994) Mol. Cell. Probes 8: 91-98). The term nucleic acid is
used
interchangeably with gene, cDNA, and mRNA encoded by a gene.
The terms "isolated" or "biologically pure" refer to material which is
substantially or essentially free from components which normally accompany it
as found in
its native state. However, the term "isolated" is riot intended refer to the
components present
in an electrophoretic gel or other separation medium. An isolated component is
free from
such separation media and in a form ready for use in another application or
already in use in
the new application/milieu.
The term "expression cassette", refers to nucleotide sequences which are
capable of affecting expression of a structural gene in hosts compatible with
such sequences.
Such cassettes include at least promoters and optionally, transcription
termination signals.
Additional factors necessary or helpful in effecting expression may also be
used as described
herein.
The term "operably linked" as used herein refers to linkage of a promoter
upstream from a DNA sequence such that the promoter mediates transcription of
the DNA
sequence.
A fusion protein is a chimeric molecule in which the constituent molecules
are all polypeptides and are attached (fused) to each other through terminal
peptide bonds so
that the chimeric molecule is a continuous single-chain polypeptide. The
various
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constituents can be directly attached to each other or can be coupled through
one or more '
peptide linkers.
A "target" cell refers to a cell or cell-type that is to be specifically bound
by a
member of a phage display library or a chimeric molecule of this invention.
Preferred target
cells are cells for which an internalizing antibody or binding polypeptide is
sought. The
target cell is typically characterized by the expression or overexpression of
a target molecule
that is characteristic of the cell type. Thus, for example, a target cell can
be a cell, such as a
tumor cell, that overexpresses a marker such as c-erbB-2.
A "targeting moiety" refers to a moiety {e.g. a molecule) that specifically
binds to the target molecule. Where the target molecule is a molecule on the
surface of a cell
and the targeting moiety is a component of a chimeric molecule, the targeting
moiety
specifically binds the chimeric molecule to the cell bearing the target. Where
the targeting
moiety is a polypeptide it can be referred to as a "targeting polypeptide".
The terms "internalizing" or "internalized" when used in reference to a cell
refer to the transport of a moiety (e.g. phage) from outside to inside a cell.
The internalized
moiety can be located in an intracellular compartment, e.g. a vacuole, a
lysosome, the
endoplasmic reticulum, the golgi apparatus, or in the cytosol of the cell
itself.
An internalizing receptor or marker is a molecule present on the external cell
surface that when specifically bound by an antibody or binding protein results
in the
internalization of that antibody or binding protein into the cell.
Internalizing receptors or
markers include receptors (e.g., hormone, cytokine or growth factor receptors)
ligands and
other cell surface markers binding to which results in internalization. ]
The term "heterologous nucleic acid' refers to a nucleic acid that is not
native
to the cell in which it is found or whose ultimate origin is not the cell or
cell line in which
the "heterologous nucleic acid" is currently found.
The idiotype represents the highly variable antigen-binding site of an
antibody and is itself immunogenic. During the generation of an antibody-
mediated immune
response, an individual will develop antibodies to the antigen as well as anti-
idiotype
antibodies, whose immunogenic binding site (idiotype) mimics the antigen. Anti-
idiotypic
antibodies can also be generated by immunization with an antibody, or fragment
thereof.,
A "phage display library" refers to a collection of phage (e.g., filamentous
phage) wherein the phage express an external (typically heterologous) protein.
The external
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protein is free to interact with (bind to) other moieties with which the phage
are contacted.''
Each phage displaying an external protein is a "member" of the phage display
library.
An "antibody library" refers to phage display library that displays antibodies
(binding proteins encoded by one or more antibody genes or cDNAs). The
antibody library
includes the population of phage or a collection of vectors encoding such a
population of
phage, or cells) harboring such a collection of phage or vectors. The library
can be
monovalent, displaying on average one single-chain antibody per phage particle
or multi-
valent displaying, on average, two or more single chain antibodies per viral
particle.
Preferred antibody libraries comprise on average more than 106, preferably
more than 10',
more preferably more than 108, and most preferably more than 109 different
members (i.e.
encoding that many different antibodies).
The term "filameritous phage" refers to a viral particle capable of displaying
a
heterogenous polypeptide on its surface. Although one skilled in the art will
appreciate that a
variety of bacteriophage may be employed in the present invention, in
preferred
embodiments the vector is, or is derived from, a filamentous bacteriophage,
such as, for
example, fl, fd, Pfl, M13, etc. The filamentous phage may contain a selectable
marker such
as tetracycline (e.g., "fd-tet"). Various filamentous phage display systems
are well known to
those of skill in the art (see, e.g., , Zacher et al. (1980) Gene 9: 127-140,
Smith et al.(1985)
Science 228: 1315-1317 (1985); and Parmley and Smith (1988) Gene 73: 305-318).
A "viral packaging signal" is a nucleic acid sequence necessary and sufficient
to direct incorporation of a nucleic acid into a viral capsid.
An assembly cell is a cell in which a nucleic acid can be packaged into a
viral
coat protein (capsid). Assembly cells may be infected with one or more
different virus
particles (e.g. a normal or debilitated phage and a helper phage) that
individually or in
combination direct packaging of a nucleic acid into a viral capsid.
The term "detectable label" refers to any material having a detectable
physical
or chemical property. Such detectable labels have been well-developed in the
field of
immunoassays and, in general, any label useful in such methods can be applied
to the present
invention. Thus, a label is any composition detectable by spectroscopic,
photochemical,
biochemical, immunochemical, electrical, optical or chemical means. Useful
labels in the
present invention include magnetic beads (e.g. DynabeadsTM), fluorescent dyes
(e.g.,
fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels
(e.g., 3H, lzsh
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3sS, laC, or 3zP), enzymes (e.g., LacZ, CAT, horse radish peroxidase, alkaline
phosphatase-
and others, commonly used as detectable enzymes, either as marker gene
products or in an
ELISA), and colorimetric labels such as colloidal gold or colored glass or
plastic {e.g.
polystyrene, polypropylene, latex, etc.) beads. Those detectable labels that
can be expressed
by nucleic acids are referred to as "reporter genes" or "reporter gene
products".
It will be recognized that fluorescent labels are not to be limited to single
species organic molecules, but include inorganic molecules, mufti-molecular
mixtures of
organic and/or inorganic molecules, crystals, heteropolymers, and the like.
Thus, for
example, CdSe-CdS core-shell nanocrystals enclosed in a silica shell can be
easily
derivatized for coupling to a biological molecule {Bruchez et al. (1998)
Science, 281: 2013-
2016). Similarly, highly fluorescent quantum dots (zinc sulfide-capped cadmium
selenide)
have been covalently coupled to biomolecules for use in ultrasensitive
biological detection
(Warren and Nie {1998) Science, 281: 2016-2018).
A nuclear localization signal is a nucleic acid sequence that directly or
indirectly results in localization of the nucleic acid to the cell nucleus.
Nuclear localization
sequences (NLS) are well known to those of skill in the art. In most cases the
NLS consists
either of a short division of basic amino acids, for example as shown for the
NLS of SV40 T
antigen (PKKKRKV). Alternatively, the NLS may have a bipartite structure
comprised of
two stretches of basic residues separated by a spacer of about 10 amino acids.
(Dingwell et
al. (1991) Trends Biochem. Sci. 16: 478). In the practice of the invention,
any NLS
sequences that functions to direct the localization of PUR to the nucleus may
be incorporated
into the phage or phagemid vectors.
An endosomal escape sequence is a nucleic acid sequence that directly or
indirectly results in the transport of a molecule from the endosome into the
cytoplasm of a
cell. Endosomal escape sequences (e.g. viral escape mechanisms) are well known
to those
of skill in the art. Examples include, but are not limited to the co-
internalization system of
adenovirus (Curiel et al. (1991) Proc. Natl. Acad. Sci. USA, 8: 8850-8854),
and the
influenza viral peptides lrnown to participate in endosomal escape mechanisms
(Wiley and
Skehel (1987) Ann. Rev. Biochem. 56: 365-394; Wagner et al. (1991) Proc. Natl.
Acad. Sci.
USA, 89: 7934-7938).
The following abbreviations are used herein: AMP, ampicillin; c-erbB-2
ECD, extraceilular domain of c-erbB-2; CDR, complementarity determining
region; ELISA,
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enzyme linked immunosorbent assay; FACS, fluorescence activated cell sorter;
FR,
framework region; Glu, glucose; HBS, hepes buffered saline, 10 mM hepes, 150
mM NaCI,
pH 7.4; IMAC, immobilized metal affinity chromatography; k°",
association rate constant;
lc°~, dissociation rate constant; MPBS, skimmed milk powder in PBS;
MTPBS, skimmed
milk powder in TPBS; PBS, phosphate buffered saline, 25 mM NaH2P04, 125 mM
NaCI,
pH 7.0; PCR, polymerase chain reaction; RU, resonance units; scFv or scFv,
single-chain Fy
fragment; TPBS, 0.05% v/v Tween 20 in PBS; SPR, surface plasmon resonance; Vk,
immunoglobulin kappa light chain variable region; V~, immunoglobulin lambda
light chain
variable region; VL, immunoglobulin light chain variable region; VH,
immunoglobulin heavy
chain variable region; wt, wild type.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 illustrates the method for construction of a large human scFv phage
antibody library. The strategy for library construction involved optimizing
the individual
steps of library construction to increase both the efficiency of scFv gene
assembly and to
increase the efficiency of cloning assembled scFv genes. (A). First, mRNA from
lymphocytes was used to generate VH and VL gene repertoires by RTPCR which
were
cloned into different vectors to create VH and VL gene libraries of 8.0 x 10$
and 7.2 x 106
members respectively. The cloned V-gene libraries provided a stable and
limitless source of
VH and V~ genes for scFv assembly. DNA encoding the peptide (GaS)s was
incorporated
into the 5' end of the V~, library. This permitted generation of scFv genes by
PCR splicing 2
DNA fragments. Previously, scFv gene repertoires were assembled from 3
separate DNA
fragments consisting of VH, V~,, and linker DNA. (B) VH and VL gene
repertoires were
amplified from the separate libraries and assembled into an scFv gene
repertoire using
overlap extension PCR. The primers used to reamplify the VH and VL gene
repertoires
annealed 200 by upstream of the 5' end of the VH genes and 200 by down stream
of the VL
genes. These long overhangs ensured efficient restriction enzyme
digestion.(C.) The scFv
gene repertoire was digested with NcoI and Notl and cloned into the plasmid
pHENl as
fusions with the M13 gene III coat protein gene ( ) for phage-display.
Figures 2A 2B, and 2C show schematics illustrating antibody phage display:
Cartoon of phage displaying (2A) a single scFv (2B) a single diabody or (2C)
multiple scFv.
scFv = single chain Fv antibody fragment; Vg = Ig heavy chain variable domain;
VL = Ig
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light chain variable domain; pIII = phage minor coat protein pIII; Ag =
antigen bound by
scFv.
Figure 3 shows the effect of trypsinization on the enrichment of antigen
specific phage. A mixture of fd phage (5.0 x 1011 cfu) and C6.5 scFv phagemid
(5.0 x 10g
fu) was incubated with SKBR3 cells for 2 hours at 37°C. Washes were
performed either as
described in Table 7 (-) or cells were trypsinized prior to cell lysis (+).
Phage present in the
first stripping buffer wash (cell surface phage) and the cell lysate
(intracellular phage) were
titered in the presence of ampicillin (C6.5 phagemid) or tetracycline (fd
phage).
Figure 4 shows the effect of incubation time and chloroquine on the recovery
of antigen specific phage. SKBR3 cells were incubated in the presence (~, ~)
or absence
(O, O) of chloroquine (50 ~NI) for 2 hours prior to the addition of anti-
botulinum phagemid
(O, ~) or C6.5 scFv phagemid (O, ~) (1.5 x 109 cfu/ml). Cell samples were
taken at 0
minutes, 20 minutes, 1 hour or 3 hours after phage addition, washed as
described in Figure 4
including the trypsinization step and intracellular phages titered.
Figure 5 shows the effect of phage concentration on the recovery of
intracellular phage. Various concentrations of C6.5 scFv phagemid, C6ML3-9
scFv
phagemid, C6.5 diabody phagemid or C6.5 scFv phage (input phage titer) were
incubated
with subconfluent SKBR3 cells grown in 6-well plates for 2 hours at
37°C. Cells were
treated as described in Figure 4 including the trypsinization step and
intracellular phage were
titered (output phage titer).
Figure 6 illustrates strategies for producing anti-ErbB2 phagemids and phages
packaging a eukaryotic reporter gene. Left column: Helper phage are used to
infect TGl
containing pHEN-FS-GFP, a phagemid composed of an fl origin of replication (fl
ori), the
anti-ErbB2 FS scFv gene fused to gene III and an eukaryotic GFP reporter gene
driven by
the CMV promoter. Phage recovered from the culture supernatant display an
average of 1
scFv-pIII fusion protein and 99% of them package the GFP reporter gene. Right
column: the
anti-ErbB2 FS scFv gene is cloned into the fd phage genome for expression as a
scFv-pIII
fusion. fd-FS phages are used to infect TG1 containing a GFP reporter phagemid
vector
(pcDNA3-GFP). Phages purified from the culture supernatant display multiple
scFv-pllI
fusion protein and approximately 54% package the GFP reporter gene.
Figure 7 shows a comparison of anti-ErbB2 phagemid and phage binding on
cells. 105 ErbB2 expressing SKBR3 cells were incubated with increasing
concentrations of
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FS-phagemids (circles) or fd-FS-phages (squares) at 4°C for 1 hour.
Cell surface bound -
phages were detected with biotinylated anti-M13 and streptavidin-PE. Binding
was detected
by FRCS and the results expressed as mean fluorescent intensity (1~'I).
Figures 8A and 8B illustrate phagemid-mediated gene transfer in breast
cancer cell lines. (Fig. 8A) (1, 2, 3) 2.0 x 105 MCF7 (low ErbB2 expression)
or (4, 5, 6) 2.0
x 105 SKBR3 (high ErbB2 expression) cells grown in 6-well plates were
incubated with
either no (1,4) no phage, (2, 5) S,0 x 1012 cfu/ml of helper phage packaging
GFP or (3, 6)
5.0 x l O11 cfu/ml of FS-GFP-phagemids for 48 hrs. Cells were trypsinized and
GFP
detected by FACS. (Fig. 8B) An equal number of MCF7 and SKBR3 cells (1.0 x
105) were
grown together and incubated with 5.0 x 1011 cfu /ml of FS-GFP-phagemids for
48 hrs: Cells
were trypsinized and stained for ErbB2 expression using 4D5 antibody and
rhodamine
conjugated sheep anti-mouse Ig to discriminate SKBR3 (Region Rl} and MCF7
(Region R2)
cells. The GFP content of each subpopulation was determined by FACS.
Figures 9A, 9B, 9C, and 9D show concentration dependence and time course
of phagemid mediated GFP expression in SKBR3 cells. Figures 9A and 9B show
concentration dependence of phagemid and phage mediated GFP expression in
SKBR3 cells.
5.0 x 104 cells were grown in 24-well plates and incubated with increasing
concentrations of
FS-GFP-phagemid (squares), fd-FS-GFP-phage (diamonds) or GFP-helper phage
(circles).
After 60 hrs, the cells were trypsinized and analyzed by FACS for GFP
expression. Figures
9C and 9D show the time dependence of phagemid mediated GFP expression in
SKBR3
cells. S.0 x 104 cells were incubated with 5.1011 cfu/mL of FS-GFP-phagemid
and analyzed
for GFP expression by FACS. For incubation times greater than 48 hrs, the
phage were
added to 2.5 x 104 cells and the culture medium was replaced by fresh medium
after 48 hrs
of incubation. The results are expressed as (9A, 9C) % of GFP positive cells
and (9B, 9D))
MFI of the GFP positive cells.
DETAILED DESCRIPTION
I Transfection of cells using targeted nha~e.
This invention provides methods and matezials for transfecting cells using
targeted phage. In general the methods involve providing a phage displaying an
external
binding protein or antibody and containing a heterologous nucleic acid
(heterologous to the
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phage and/or to the cell). The external binding protein preferably
specifically binds to an y
internalizing marker (receptor/receptor epitope) which results in
internalization of the phage.
Once internalized, the phage coat protein dissociates and the single stranded
phaQe DNA
genome is optionally expressed by the host cell machinery.
The antibody phage containing the heterologous nucleic acid can be prepared
by a number of methods well known to those of skill in the art. In general
these methods
involve providing cells containing phagemid vector encoding the heterologous
targeting
protein or nucleic acid that is to be transfected into the cell and a
corresponding page (e.g.
helper phage) containing nucleic acid encoding a targeting polypeptide or the
heterologous
IO nucleic acid that is to be transfected into the target cell. A bacterial
(e.g. E. coli) cell is then
infecting with the phage or phagemid or cotransfected with the phage or
phagemid nucleic
acids which are then repackaged into phage containing the desired nucleic
acids. Several
approaches are illustrated in Table 1.
15 Table I. Strategies for the construction of phage nucleic acid delivery
vehicles (vectors) of
this invention.
E coli contains Action To Produce
Phagemid encoding nfect with helper Phage containing both heterologous
Heterologous DNA; phage. nucleic acid sequences (targeting
and
DNA encoding scFv protein and heterologous
DNA) and
expressing targeting polypeptide
(e.g. scFv) on surface.
Phagemid encoding fect with helper I ) Phage containing targeting
protein
heterologous DNA phage containing on surface and heterologous
DNA
nucleic acid encoding(from phagemid clone); and
targeting polypeptide
(scFv) on its surface2) Phage containing targeting
protein
on surface and nucleic acid
encoding
targeting protein (from
phage clone)
No phagemid or phage Co-transform cell with Phage containing both heterologous
phagemid and phage nucleic acid sequences (targeting
DNA and co-select, protein and heterologous DNA) and
e.g., with antibiotics. expressing targeting polypeptide on
surface.
No phagemid or phage Infect cell with phage Phage containing both heterologous
containing both nucleic acid sequences (targeting
heterologous DNA and protein and heterologous DNA) and
DNA encoding expressing targeting polypeptide on
targeting polypeptide. surface.
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No phagemid or phage Infect cell with phage 1 ) Phage containing targeting
containing polypeptide and heterologous~DNA;
heterologous DNA and and.
phagemid containing 2) Phage containing only
targeting polypeptide heterologous DNA.
(e.~., scFv).
*phagemid contains packaging signal.
In a preferred embodiment, phage expressing the targeting molecule on their
surface are used as helper phage (see Maniatis) to package the genome of a
phagemid
containing the heterologous DNA (e.g. a mammalian expression cassette).
In one preferred embodiment, this involves subcloning the targeting protein
gene from the pHENl vector into a phage vector (such as FdDOGI (Clackson et
al, Nature
(1991) 352: 624-628) where it is located inframe with the phage gene III. For
example,
targeting scFv can be cloned as ApaLl-NotI fragments into the ApaLl-NotI sites
of
FdDOGl. The phage genome leads to production ofphage (from bacteria) which has
the
targeting protein on its surface and the phage genome inside. These phage are
then used for
superinfection of the phagemid containing bacterial cells. The phagemid vector
DNA by
definition contains a phage origin of replication and packaging signal. As a
result, the phage
genome products direct single stranded DNA synthesis of the phagemid DNA. The
phage
1 S acts as a helper phage leading to the production of two types of phage
particles, those that
contain the phage genome and those that contain the phagemid genome. Using
standard
phage (such as Fd} as helper phage results in approximately an equal
probability of the
phage packaging either genome. All of the phage will also have the targeting
protein on
their surface as pITI fusions. This is a simple way to generate phage that
have the targeting
molecule on their surface and the heterologous expression DNA inside the
phage. While
only a fraction (50%) of phage harbor the heterologous expression DNA, this is
a large
enough fraction given the high titer with which phage can be produced, to
generate targeted
phage.
Packaging is rapid, simple and most importantly can avoid tedious and time
consuming subcloning steps required to insert the DNA sequence that is to be
delivered to
the eukaryotic cells into the phagemid or phage vector harboring the DNA
sequence of the
targeting gene. Thus this approach provides a generic method for packaging any
DNA into
the targeting phage for delivery and expression in eukaryotic cells. This
makes it simple to
deliver and study the effects of a large number of different genes in
eukaryotic cells.
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It is noted that use of phage genome as a helper phage can lead to the
probleitt
of "interference" where the titer of phage generated is lower than expected
(see Maniatis). It
is also known to those skilled in the art, that a number of phage vectors
exist which can
overcome this problem. One of these, helper phage K07, uses a phage with a
plasmid origin
of replication and a partially disabled phage origin of replication (see
Maniatis). Use of K07
as a helper phage leads to production of higher phage titers. Thus similar
alternative phage
vector backbones could be used for creation of targeted helper phage to result
in higher
phage titers.
In a variant of this embodiment, the bacteria can be co-transformed with
phage .and phagemid DNA and co-selected with antibiotics. The resulting cells
contain both
genomes and make phage containing both the heterologaus targeting protein and
the nucleic
acid that is to be delivered into the cell.
In still another embodiment, the phage contains the heterologous nucleic acid
that is to be delivered into the cell and the phagemid contains the nucleic
acid encoding the
heterologous targeting protein.
In yet another embodiment, the phagemid genome containing the targeting
molecule-pIII gene fusion is modified to contain the gene sequence that is to
be delivered to
the target eukaryotic cell {for example a mammalian expression cassette
containing a
reporter gene (or cDNA) and/or another gene or (cDNA)). Targeting phage are
produced in
the standard manner by the addition of helper phage.
In still yet another embodiment, both the heterologous nucleic acid that is to
be delivered into the cell and the heterologous nucleic acid encoding the
targeting protein
(e.g. single-chain antibody) are inserted into the phage genome. The bacteria
then need only
the page genome inside and will make phage with targeting protein on the
outside and both
genomes inside.
While it is demonstrated herein that targeted phage can be delivered via an
internalizing receptor into the endosome, it is recognized that other factors
may reduce the
efficiency of gene expression. The phage preferably from the endosome and
uncoating
facilitates exposure of the single stranded genome which then finds its way to
the nucleus.
There the single stranded DNA is replicated to double stranded DNA which is
then
transcribed and translated. It is also recognized by those skilled in the art,
that methods exist
to improve the efficiency of each of these steps. For example, endosomal
escape sequences
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are known which can be incorporated into the phage coat proteins. Co
incubation with
defective adenovirus would also provide endosome escape signals. Nuclear
localization
sequences are also known which could increase delivery to the nucleus.
Inclusion of
episomal replication sequences lead to amplification of the delivered DNA with
an increase
in the efficiency of expression.
II. Target cells.
Virtually any cell bearing an internalizing marker/receptor can be transfected
using the methods of this invention. Using the assays described above and
illustrated in the
examples, internalizing phage display library members can be optimized for
internalization
by a particular marker. Alternatively or in addition, new, previously unknown
receptors or
epitopes can be identified and targeted.
Targets can be selected that whose distribution is restricted to particular
cell
types, target tissues, organs, or cells and/or tissues and/or organs.
displaying a particular
physiological state or pathological condition. Thus, for example, targets can
be selected that
are characteristic of particular tumor types. Tumor specific targets are well
known to those
of skill in the art and include, but are not limited to c-erbB-2, the IL-13
receptor, other
growth factor receptors, and so forth.
Alternatively, internalizing targets can be selected that are present on most
or
all cell types (e.g., transferrin receptor). Also, it is possible to select a
phage library to
identify such targeting molecules. For example, a scFv phage library can be
selected
without a subtracting cell line, or sequentially on unrelated cell lines. We
in fact have
already identified scFv using this approach that bind to all cell types
tested. In this instance
the transfection methods allow generalized transfection of essentially any
and/or all cells or
an organ, tissue, or organism.
Tissue specific targets can also be identified. Thus, for example, it is noted
that Ruoslahti et al. (CT.S. Patent No: 5,622,699 have identified polypeptides
that specifically
target particular tissues (e.g. brain, kidney, etc.).
III. Transfected nucleic acids.
Using the above-described methods, virtually any heterologous nucleic acid
can be transfected into a cell. Once in the cell, the nucleic acid will
optionally be
transcribed, and optionally translated, depending on the nature of the
particular nucleic acids.
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In one embodiment, the heterologous nucleic acid can encode a polypeptide gene
product irt-
is desired to introduce into the cell. Such a polypeptide gene product may
include a reporter
gene (e.g., green fluorescent protein or ~(3-galactosidase). For killing a
cell (such as a tumor
cell) one might deliver the TK gene or the gene encoding a toxin (such as
Pseudomonas
exotoxin or subunits thereof, diphtheria toxin or subunits thereof, ricin,
abrin, etc.).
Alternatively, the nucleic acid transcript can be active in its own right
(e.g. a ribozyme, an
antisense molecule, etc.).
Where a heterologous nucleic acid encodes a protein product that is to be
expressed in the target cell, the heterologous nucleic acid preferably encodes
an expression
cassette compatible with the target cell. Thus, for example, where the target
cell is a
mammalian cell, the expression cassette preferably includes a promoter that is
inducible or
constitutive in a mammalian cell, an initiation site, and a termination site.
The cassette can
optionally include a selectable marker.
IV Identifying internalizing antibodies and/or targets (e.g. recentorsl.
~LIdentification of internalizing golynentides/antibodies.
The transduction methods of this invention rely on the use of "internalizing
antibodies", or "internalizing polypeptides". Such "internalizing" molecules
are internalized
when they bind a target cell. Methods of identifying internalizing
antibodies/target epitopes
are provided herein and illustrated in the Examples. The methods generally
involve
contacting a "target" cell with one or more members of a phage display library
displaying an
antibody or a binding polypeptide. The phage display library is preferably a
polyvalent
phage display library and it is believed that this invention provides the
first description of a
polyvalent antibody phage display library.
After a suitable incubation period, the cells are washed to remove externally
bound phage (library members) and then internalized phage are released from
the cells, e.g.,
by cell lysis. It was a discovery of this invention that the internalized
phage are still viable
(infectious). Thus the internalized phage in the cell lysate can be recovered
and expanded by
using the lysate containing internalized phage to infect a bacterial host.
Growth of infected
bacteria leads to expansion of the phage which can be used for a subsequent
round of
selection. Each round of selection enriches for phage which are more
efficiently
internalized, more specific for the target cell or have improved binding
characteristics.
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The phage display library is preferably contacted with a subtractive cell ~ine-
-
(i.e. a suti~ractive cell line is added to the target cells and culture media)
to remove members
of the phage display library that are not specific to the "target" cell(s).
The subtractive cell
line is preferably added under conditions in which members of the phage
display library are
not internalized (e.g., at a temperature of about 4°C to about
20°C, more preferably at a
temperature of about 4°C) so that non-specific binding members of the
library are not
internalized (sequestered) before they can be subtracted out by the
subtractive cell line.
After subtracting out non-specific binding antibodies, the "target" cells are
washed to remove the subtractive cell line and to remove non-specifically or
weakly-bound
phage."
The target cells are then cultured under conditions where it is possible for
internalization to occur (e.g. at a temperature of about 35°C to about
39°C, more preferably
at a temperature of about 37°C). The duration of the internalization
culture period will
determine the internalization speed of the antibodies (phage display members)
for which
selection takes place. With shorter internalization periods more rapid
internalizing
antibodies are selected while with longer internalization periods slower
internalizing
antibodies are selected. The internalization period is preferably less than
about 120 minutes,
more preferably less than about 60 minutes, and most preferably less than
about 30 minutes
or even less than about 20 minutes.
It is noted that during the internalization period the target cells are grown
under conditions in which internalization can occur. For a number of cell
lines, this involves
culturing the cells adherently on culture plates.
After internalization has been allowed to occur the target cells are washed to
remove non-internalized (e.g. surface-bound phage).
The cells can then be moved to clean media. In a preferred embodiment,
where the cells are adherent, they cells are trypsinized to free the cells
from the extracellular
matrix which may contain phage antibodies that bind the extracellular matrix.
Freeing the
cells into solution permits more through washing and moving of the cells to a
new culture
flask will leave behind any phage that may have stuck to the tissue culture
dish.
The cells can then be washed with a large volume of PBS and lysed to release
the internalized phage which can then be expanded e.g. used to infect E. coli
to produce
phage for the next round of selection. It is noted that there is no need to
actually visualize
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the internalized phage. Simple cell lysis and expansion of the formerly
internalized phage iS
sufficient for recovering internalizing phage display members.
Bl Identification of internalizine receptors.
Once an antibody or polypeptide that is internalized into a cell has been
S identified, it is possible to probe one or more cell types with the
identified antibody or
polypeptide to identify the target recognized and bound by the antibody. Since
the antibody
is an internalizing antibody it is likely that such targets are themselves
internalizing targets
(e.g. members or portions of internalizing receptors).
In one embodiment, the antibody can be labeled as described below. The
cells can then be contacted with the antibody (i.e. in vivo or in vitro) and
the cells or cellular
regions to which the antibody binds can then be isolated.
Alternatively, the antibodies can be used e.g. in an affinity matrix (e.g.
affinity column) to isolate the targets (e.g. receptor or receptor subunits)
to which they bind.
Briefly, in one embodiment, affinity chromatography involves immobilizing
(e.g. on a solid
1 S support) one or more species of the internalizing antibodies identified
according to the
methods of this invention. Cells, cellular lysate, or cellular homogenate are
then contacted
with the immobilized antibody which then binds to its cognate ligand. The
remaining
material is then washed away and the boundlisolated cognate ligand can then be
released
from the antibody for further use. Methods of performing affinity
chromatography are well
known to those of skill in the art (see, e.g., U.S. Patent Nos: 5,710,254,
5,491,096,
5,278,061, 5,110,907, 4,985,144, 4,385,991, 3,983,001, etc.).
In another embodiment, the antibodies are used to immunoprecipitate the
target from cell lysate. The precipitate is then run on an SDS-PAGE gel which
is Western
blotted onto nitrocellulose. The blot is probed with the precipitating
antibody to identify the
2S location of the target. The portion of the blot containing the target can
then be sent for N-
terminal protein sequencing. The N-terminal sequence can then be used to
identify the target
from standard databases, or DNA probes can be synthesized to probe genomic or
cDNA
libraries. This approach has been used to identify the antigen bound by a
phage antibody.
Selections of a phage antibody library were done on intact Chlamydia
trachomatis (a
bacterial like organism that causes Chlamydial diseases). Selected antibodies
were then used
as described above to identify the antigen bound.
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C) Assay Components
1~ Fhage disvlay library.
a) M alent antibody libraries and polvpentide libraries.
The ability to express polypeptide and antibody fragments on the surface of
viruses which infect bacteria (bacteriophage or phage) makes it possible to
isolate a single
binding polypeptide or antibody fragment from a library of greater than 101
nonbinding
clones. To express polypeptide or antibody fragments on the surface of phage
(phage
display), a polypeptide or an antibody fragment gene is inserted into the gene
encoding a
phage surface protein (pIIi) and the antibody fragment-pIII fusion protein is
displayed on the
phage surface (McCafferty et al. (1990) Nature, 348: 552-554; Hoogenboom et
al. (1991)
Nucleic Acids Res. 19: 4133-4137). Since the antibody fragments on the surface
of the
phage are functional, phage bearing antigen binding polypeptides or antibody
fragments can
be separated from non-binding phage by antigen affinity chromatography
(McCafferty et al.
(1990) Nature, 348: 552-554). Depending on the affinity of the antibody
fragment,
enrichment factors of 20 fold - 1,000,000 fold are obtained for a single round
of affinity
selection. By infecting bacteria with the eluted phage, however, more phage
can be grown
and subj ected to another round of selection. In this way, an enrichment of
1000 fold in one
round can become 1,000,000 fold in two rounds of selection (McCafferty et al.
(1990)
Nature, 348: 552-554). Thus even when enrichments are low (Marks et al. (1991)
J. Mol.
Biol. 222: 581-597), multiple rounds of affinity selection can lead to the
isolation of rare
phage. Since selection of the phage antibody library on antigen results in
enrichment, the
majority of clones bind antigen after four rounds of selection. Thus only a
relatively small
number of clones (several hundred) need to be analyzed for binding to antigen.
In a preferred embodiment, analysis for binding is simplified by including an
amber codon between the antibody fragment gene and gene III. The amber codon
makes it
possible to easily switch between displayed and soluble (native) antibody
fragment simply
by changing the host bacterial strain (Hoogenboom et al. (1991) Nucleic Acids
Res. 19:
4133-4137).
Human antibodies can be produced without prior immunization by displaying
very large and diverse V-gene repertoires on phage (Marks et al. (I991) J.
Mol. Biol. 222:
581-597). In the first Example, natural VH and VL repertoires present in human
peripheral
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blood lymphocytes were isolated from unimmunized donors by PCR. The V-gene - '
repertoires were spliced together at random using PCR to create a scFv gene
repertoire
which was cloned into a phage vector to create a library of 30 million phage
antibodies (Id.).
From this single "naive" phage antibody library, binding antibody fragments
have been
isolated against more than I7 different antigens, including haptens,
polysaccharides and
proteins (Marks et al. (1991) J. Mol. Biol. 222: 581-597; Marks et al. (1993).
BiolTechnology. 10: 779-783; Griffiths et al. (1993) EMBO J. 12: 725-734;
Clackson et al.
(1991) Nature. 352: 624-628). Antibodies have been produced against self
proteins,
including human thyroglobulin, immunoglobulin, tumor necrosis factor and CEA
(Griffiths
et al. (1993) EMBO J. 12: 725-734). It is also possible to isolate antibodies
against cell
surface antigens by selecting directly on intact cells. For example, antibody
fragments
against four different erythrocyte cell surface antigens were produced by
selecting directly
on erythrocytes (Marks et al. (1993). BiolTechnology. I0: 779-783). Antibodies
were
produced against blood group antigens with surface densities as low as 5,000
sites/cell. The
antibody fragments were highly specific to the antigen used for selection, and
were
fimctional in agglutination and immunofluorescence assays. Antibodies against
the lower
density antigens were produced by first selecting the phage antibody library
on a highly
related cell type which lacked the antigen of interest. This negative
selection removed
binders against the higher density antigens and subsequent selection of the
depleted phage
antibody library on cells expressing the antigen of interest resulted in
isolation of antibodies
against that antigen. With a library of this size and diversity, at least one
to several binders
can be isolated against a protein antigen 70% of the time. The antibody
fragments are highly
specific for the antigen used for selection and have affinities in the 1 :M to
100 nM range
(Marks et aL (1991) J. Mol. Biol. 222: 581-597; Griffiths et al. (1993) EMBO
J. 12: 725-
734). Larger phage antibody libraries result in the isolation of more
antibodies of higher
binding affinity to a greater proportion of antigens.
The creation of a suitable large phage display antibody library is described
in
detail in Example 1.
b) Polwalent antibody phage display libraries
The probability of selecting internalizing antibodies from a phage-display
antibody library is increased by increasing the valency of the displayed
antibody. This
approach takes advantage of normal cell-surface receptor biology. Often cell-
surface
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receptors (e.g. growth factor receptors) activate upon binding their cognate
ligand through a
process of homo- or heterodimerization (or trimerization, or tetramerization,
etc.). The
association of the receptor subunits in this process can be mediated directly
(e.g. when
bound by a bivalent ligand) or indirectly by causing a conformational change
in the receptor.
It was a discovery of this invention that polyvalent antibodies in a display
library (e.g. a phage display library) can mimic this process, stimulate
endocytosis, become
internalized and deliver their payload into the cytosol. Thus, to increase the
likelihood of
identifying internalizing antibodies or recognizing internalizing epitopes,
preferred
embodiments of this invention utilize a polyvalent phage display antibody
library. It is
believed that no multivalent phage-display antibody libraries have been
created prior to this
invention. Unlike the multivalently displayed peptide phage libraries, phage
antibody
libraries typically display monomeric single chain Fv (scFv) or Fab antibody
fragments
fused to pIII as single copies on the phage surface using a phagemid system
(Marks et al.
(1991) J. Mol. Biol. 222: 581-597; Sheets et al. (1998) Proc. Natl. Acad. Sci.
USA 95: 6157-
6162.).
As used herein, a polyvalent phage display antibody library, refers to a
library
in which each member (e.g. phage particle) displays, on average) two or more
binding
domains, wherein each 'binding domain includes a variable heavy and a variable
light region.
More generally, a multivalent phage display library displays, on average, two
or more pIII
fusions per page particle. Polyvalent phage display can be achieved by
expressing diabodies
(i.e., a protein formed by fusion or conjugation of two single chain
antibodies (e.g. scFv)} or
by display of, on average, two or more antibodies on each phage particle.- In
contrast, a
mono-valent library displays, on average, one single-chain antibody per viral
particle.
il Diabodv expression.
Diabodies are scFv dimers where each chain consists of heavy (VH) and light
(VL) chain variable domains connected using a linker (e.g. a peptide linker)
that is too short
to permit pairing between domains on the same chain. Consequently, pairing
occurs
between complementary domains of two different chains, creating a stable
noncovalent
dimer with two binding sites (Holliger et al. (1993) Proc. Natl. Acad. Sci.
90: 6444-6448).
The C6.5 diabody was constructed by shortening the peptide linker between the
Ig VH and
VL domains from 15 to 5 amino acids and binds ErbB2 on SKBR3 cells bivalently
with a Kd
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approximately 40 fold lower than C6.5 (4.0 x 10-1 M) (Adams et al. (1998)
Brit. J Cancer.
77: 1405-1412, 1998).
In Example 5, described herein, C6.5 diabody genes were subcloned for
expression as pIII fusions in the phagemid pHEN-1 (Hoogenboom et al. (1991)
Nucleic
Acids Res. 19: 4133-4137). This yielded phagemid predominantly expressing a
single scFv
or diabody-pIII fusion after rescue with helper phage (Marks et al. (1992) J.
Biol. Chem.
267: 16007-16010). Diabody phagemid display a bivalent antibody fragment
resulting from
intermolecular pairing of one scFv-pllI fusion molecule and one native scFv
molecule.
Using the teachings provided herein one of skill in the art can routinely
produce other
diabodies.
Phage displaying bivalent diabodies or multiple copies of scFv were more
efficiently endocytosed than phage displaying monomeric scFv and recovery of
infectious
phage was increased by preincubation of cells with chloroquine.
The results indicate that it is possible to select for endocytosable
antibodies,
even at the low concentrations that would exist for a single phage antibody
member in a
library of 109 members.
ii) Polwalent display of single-chain antibodies.
As an alternative to the use of diabodies, antibody phage display libraries
are
created in which each viral particle, on average, expresses at least 2,
preferably at least 3,
more preferably at least 4, and most preferably at least 5 copies of a single
chain antibody.
In principle, each copy of pIII on the page (and there is controversy as to
whether there are 3 or 5 copies of pIII per phage) should express an antibody.
However,
proteolysis occurs and the number actually displayed is typically less. Thus,
preferred
multivalent antibody libraries are constructed in a phage vector and not a
phage mid vector.
This means that helper phage need not be added to make phage. Helper phage
bring into the
E. coli wild-type pllI that competes with the scFv-pIII fusion. Thus, in
phagemid vector,
this competition leas, on average, to only 1 (ore less) antibody per phage.
To produce multivalent antibody libraries, the single chain antibodies,
typically expressed in phagemid, are subcloned from the phagemid vector into a
phage
vector. No helper phage is required and there is no competition between the
wild-type pIII
and the fusion scFv pIII fusion. thus, on average, the phage display two or
more pIII
fusions. Thus, by way of illustration, Example 5 describes the subcloning of
the C6.5 scFv
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gene into the phage vector fd-Sfi/Not. This results in phage with 3 to S
copies each of scFv-
pIII fusion protein. Other phage vectors suitable for such use are well known
to those of
skill in the art.
21 Target cells.
The target cells of this invention include any cell for which it is desired to
identify an internalizing polypeptide or antibody or for which it is desired
to identify an
internalizing marker (e.g. receptor). The cells can include cells of
multicellular eukaryotes,
uni-cellular eukaryotes, including plants and fungi, and even prokaryotic
cells. Preferred
target cells are eukaryotic, more preferably vertebrate cells, and most
preferably mammalian
cells (e.g. cells from marines, bovines, primates including humans,
largomorphs, canines,
felines, and so forth). The cells can be normal healthy cells or cells
characterized by a
particular pathology (e.g. tumor cells).
Target cells can include any cell type where it would be useful to: 1) have an
antibody specifically recognize the cell type or related cell types (for
example for cell
sorting, cell staining or other diagnostic procedures); 2) have a ligand which
is specifically
internalized into the cell type or related cell types (for example to deliver
a toxic or
therapeutic gene or protein). Additional target cells include, but are not
limited to
differentiated cells (i.e. differentiated to become a tissue, e.g. prostate,
breast). Thus an
antibody that recognized and killed prostate cells would be good for prostate
cancer even if it
killed normal prostate cells (the prostate is not an essential organ). Target
cells may include
tissue specific cells, and cells at a given developmental stage. Target cells
may also include
precursor cells, e.g. bone marrow stem cells, would be useful for isolating,
perhaps
stimulating for differentiation.
Target cells can also include cell lines transfected with a gene for a known
receptor (for example ErbB2) to which it would be useful to have internalizing
antibodies.
Many ErbB2 antibodies are not internalizing. Rather than immunizing with
recombinant protein or selecting a phage library on recombinant protein,
selection on ErbB2
transfected cells for internalization should yield precisely antibodies with
the desired
characteristics (internalization). Finally, a cDNA library could be
transfected into a cell line
(for example COS) from a desired target cell line or tissue and phage
antibodies selected for
internalization. After several rounds of selection, the phage could be used to
stain and sort
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(for example by FRCS) transfected cells. DNA can be recovered from the cells,
yielding the
sequences of internalizing receptors as well as phage antibodies that bind
them.
3y Cells of a subtractive cell line.
In a preferred embodiment of the assays of this invention, the phage display
library is contacted with cells from a "subtractive" cell line. This step is
intended to deplete
or eliminate members of the phage display library that either bind the cells
non-specifically
or that bind to targets other than the target against which it is desired to
obtain a binding
polypeptide or antibody. The contacting with the cells from a "subtractive"
cell line can
occur before, during, or after the target cells are contacted with members of
the phage
display library. However, in a preferred embodiment, the contacting with cells
of a
subtractive cell line is simultaneous with contacting of the target cells.
Thus, for example, in
a preferred embodiment the target cell line (grown adherent to a tissue
culture plate) is co-
incubated with the subtracting cell line (in suspension) in a single cell
culture flask.
Virtually any cell can act as a subtractive cell. However, in a preferred
embodiment, subtractive cells display all the markers on the target cell
except the marker
(e.g. receptor) that is to act as a target for selection of the desired
binding antibodies or
binding polypeptides. Particularly preferred cells are thus closely related to
the target cell(s),
in terms of having common internalizing cell surface receptors (such as
transferrin); for
example fibroblasts. If one was selecting on a tumor cell line (for example a
breast tumor
cell line}, than one could negatively select on a normal breast cell line.
This may, however,
deplete for antibodies that bind to overexpressed antigens, so again a
parallel path would be
to negatively select on fibroblasts. If one was using transfected cells, than
non-transfected
cell could be used as the subtractive cell line. Where the tumor is epithelial
in origin, the
preferred subtractive cell will also be epithelial and even more preferably
from the same
tissue or organ.
Particularly preferred subtractive cells include, but are not limited to, non-
differentiated cell lines, non-transfected cells, mixtures of non-
differentiated and non-
transfected cells. When selecting for internalization on tumor cells,
preferred subtractive cell
lines are preferably the non-tumor cells of the same tissue (for example,
breast tumor cells
versus normal breast epithelial cells). Also, for cDNA expression libraries,
the subtractive
cell line will be the non-transformed cell line used for library construction
(e.g. COS, CHO,
etc.).
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in one particularly preferred embodiment, the "target" cell is a cell
transformed with a gene or cN=DNA for a specific target receptor. In this
instance, the
subtractive cell line is preferably the non-transformed cell line. Thus for
example where
CHO cells are transformed with a vector containing the gene for the EGF
receptor, the EGF-
expressing cells are used as the target cell line, and the subtractive cell
line is the
untransformed CHO cells. Using this approach internalizing anti-EGF receptor
antibodies
were obtained.
The subtractive cells are more effective when provided in excess over the
target cells. The excess is preferably at least about a 2-fold to about a 1000-
fold excess,
more preferably about a 3-fold to about a 100-fold excess, and most preferably
about a 5-
fold to about a 50-fold excess. In one embodiment, a 5-fold excess is
sufficient.
4~ Washine steps.
As indicated above a variety of washing steps are used in the methods of this
invention. In particular, a "weak" washing step can be used to remove the
subtractive cells
and weakly or non-specifically binding members of the phage display library. A
second
strong washing step is preferably used after internalization of members of the
phage display
library. The "strong" washing step is intended to remove tightly- and weakly-
bound surface
phage.
Buffers and methods for performing weak and strong wash steps are well
known to those of skill in the art. For example, weak washes can be done with
standard
buffers or culture media (e.g., phosphate buffered saline {buffer) DMEM
(culture media),
etc.).
5~, culturing under internalizing conditions.
As explained above, the cells are preferably cultured under "internalizing"
conditions. Internalizing culture conditions are conditions in which the cell
when bound by
a member of a phage display library at an appropriate (e.g. internalizing)
site or receptor,
transports the bound member into the cell. This can involve transport into a
vesicle, into the
endoplasmic reticulum, the golgi complex, or into the cytosol of the cell
itself.
Internalizing conditions are most easily achieved when the cells are cultured
under conditions that mimic those of the cell in its native state. Thus many
cells, e.g.
epidermal cells, preferably grow ad adherent layers attached to a basement
membrane. Such
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cells more effectively internalize binding polypeptides and antibodies when
they are cultured
as adherent monolayers. Chloroquine and serum free medium both avoid non
specific
internalization and enhance specific internalization (ligands in the serum
that induce the
internalization of receptor of interest and take with them non specific phages
being in the
neighborhood). In addition, for internalization to occur, the cells should be
cultured at a
temperature and pH that permits internalization. Suitable temperature and pH
range from
about 35°C to about 39°C and from pH 6 to about pH 8, more
preferably from about pH 6.5
to about pH 7.5, with preferred temperature and pH being about 37°C and
pH 7.5
respectively. In a preferred embodiment, the cells are preincubated in serum
culture medium
for about two hours before adding the phages and the competitor (subtraction)
cells.
61 Identification of internalized nha~e
The internalized phage display library members can be identified directly or
indirectly. Direct identification can be accomplished simply by visualizing
the phage within
a cell e.g. via immunofluorescent or confocal microscopy. Phage
internalization can be
identified by their ability to deliver a reporter gene that is expressed
within the cell. The
reporter gene can be one that produces a detectable signal (e.g. a fluorescent
(e.g. lux, green
fluorescent protein, etc.) or colorimetric signal (e.g. HRP, ~i-galactosidase)
or can itself be a
selectable marker (e.g. an antibiotic resistance gene}. The use of both ~-
galactosidase and
GFP as reporter genes in such phage is described herein.
Alternatively, the phage display member can bear a marker (e.g. a label) and
cells containing the internalized phage can be detected simply by detection of
the label (e.g.
in a flow cytometer). The direct methods preferably used for identification of
the receptors
or cells that are hound after selections are performed. It is noted that cell
sorting approaches
(FACs) will work with identification of either surface bound or internalized
phage.
However, an additional level of specificity can be achieved if the cells are
first sorted for the
presence of internalized phage prior to lysis. Direct methods are also used
during the
analysis phase to demonstrate that the phage selected are indeed internalized.
Alternatively the internalized phage display library members can be identified
indirectly. In indirect detection methods the phage-display library members)
do not need to
be detected while they are present within the cell. It is sufficient that they
simply have been
internalized.
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Indirect identification is accomplished for example, by isolating and _ -
expanding the phage that were internalized into the cells as described below.
Indirect
identification is particularly well suited where the identified phage display
library members
are going to be used in subsequent rounds of selection or to isolate bacteria
harboring
monoclonal phage genomes for subsequent monoclonal phage characterization
(that is for
the analysis of selection results).
71 Isolation and exuansion of internalized yhaQe.
It was a discovery of this invention that phage display library members that
have been internalized into target cells (e.g. mammalian tumor cells) remain
viable and can
be recovered and expanded into a "selected" library suitable for subsequent
rounds of
selection and/or isolation and characterization of particular members.
As used herein, the term "recovery" is intended to include recovery of the
infectious phage and/or recovery of the phage antibody gene and/or recovery of
a
heterologous nucleic acid accompanying the antibody gene.
The internalized phage can be isolated and expanded using standard methods.
Typically these include lysing the cells (e.g., with 100 mM triethylamine
(high pH)), and
using the lysate to infect a suitable bacterial host, e.g., E. coli TG1. The
phage-containing
bacteria are then cultured according to standard methods (see, e.g., Sambrook
supra., Marks
et al. (1991) J. Mol. Bial. 222: 581-597).
V. Libraries and vectors.
In, another embodiment, this invention provides libraries and vectors for
practice of the methods described herein. The libraries are preferably
polyvalent libraries,
including diabody libraries and more preferably including multi-valent single
chain antibody
libraries (e.g. scFv), (e.g., expressed by phage).
The libraries can take a number of forms. Thus, in one embodiment the
library is a collection of cells containing members of the phage display
library, while in
another embodiment, the library consists of a collection of isolated phage,
and in still library
. consists of a library of nucleic acids encoding a polyvalent phage display
library. The
nucleic acids can be phagemid vectors encoding the antibodies and ready for
subcloning into
a phage vector or the nucleic acids can be a collection of phagemid already
carrying the
subcloned antibody-encoding nucleic acids.
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Other preferred vectors include the phage itself carrying expressing a
heterologous binding domain (e.g. an antibody) and containing a heterologous
nucleic acid
that is to be delivered into the target cell(s). While in some embodiments,
the heterologous
nucleic acid expresses a detectable label or is itself a label (e.g. a unique
sequence detectable
by hybridization or amplification (e.g. PCR) methods) in other embodiments,
the
heterologous nucleic acid includes a nucleic acid that encodes a molecule
other than a
detectable label (e.g. a polypeptide, an antisense molecule, a ribozyme,
etc.).
V_I Transformation of cells.
This invention provides new methods for effective transfection of cells both
in vivo and ex vivo {in vitro). Virtually any cell, eukaryotic or prokaryotic,
can be
transfected according to the methods of this invention. Particularly preferred
cells are
eukaryotic cells, more preferably vertebrate (e.g., mammalian) cells. Other
cells, however,
can also be transfected. Such cells include, but are not limited to bacterial
cells (e.g. bacteria
not typically infected by phage), fungal or yeast cells (e.g. to deliver a
cytotoxin in the
treatment of fungal or yeast infections), algal cells, insect cells, and the
like.
A virtually limitless variety of nucleic acids can be transfected into "target
cells". The nucleic acids can be selected to express particular
polypeptide(s), or the nucleic
acids can have an activity themselves (e.g. antisense molecules, ribozymes).
Such expressed
heterologous genes (or cDNAs), antisense molecules, or ribozymes are useful in
a wide
variety of applications and, for example, have been used to correct acquired
and inherited
genetic defects, cancer, and viral infection in a number of contexts.
The ability to express artificial genes in humans facilitates the prevention
and/or cure of many important human diseases, including many diseases which
are not
amenable to treatment by other therapies. As an example, in vivo expression of
cholesterol-
regulating genes, genes which selectively block the replication of HIV, and
tumor-
suppressing genes in human patients dramatically improves the treatment of
heart disease,
AIDS, and cancer, respectively. For a review of gene therapy procedures, see
Anderson
(1992) Science 256:808-813; Nabel and Felgner (1993) TIBTECH 11: 211-217;
Mitani and
Caskey {1993) TIBTECH 11: 162-166; Mulligan (1993) Science 926-932; Dillon
(1993)
TIBTECH 11: 167-175; Miller (1992) Nature 357: 455-460; Van Brunt (1988)
Biotechnology 6(10): 1149-1154; Vigne (1995) Restorative Neurology and
Neuroscience 8:
35-36; Kremer and Perricaudet (1995) British Medical Bulletin 51(1) 31-44;
Haddada et al.
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(1995) in Current Topics in Microbiology and Immunology Doerfler and Bohm
(eds} - -
Springer-Verlag, Heidelberg Germany; and Yu et al. (1994) Gene Therapy 1: 13-
26.
Delivery of the gene or genetic material into the cell is the first critical
step in
gene therapy treatment of disease, in a wide variety of research systems, in
the development
of knockout (KO) mice, in the development and modification of cell lines, and
the like. It
will be appreciated that the transfection methods of this invention greatly
facilitate the
delivery of nucleic acids into cells in these and other contexts.
Al Ex vivo transformation.
For example, ex vivo cell transformation for diagnostics, research, or for
gene
therapy (e.g., via re-infusion of the transformed cells into the host
organism) is well known
to those of skill in the art. In a preferred embodiment, cells are isolated
from the subject
organism, transfected a heterologous gene according to the methods of this
invention, and re-
infused back into the subject organism (e.g., patient). Various cell types
suitable for ex vivo
transformation are well known to those of skill in the art. Particular
preferred cells are
progenitor or stem cells (see, e.g., Freshney et al. (1994) Culture of Animal
Cells, a Manual
of Basic Technique, third edition Wiley-Liss, New York) and the references
cited therein for
a discussion of how to isolate and culture cells from patients).
In one particularly preferred embodiment, stem cells are used in ex-vivo
procedures for cell transformation and gene therapy. The advantage to using
stem cells is
that they can be differentiated into other cell types in vitro, or can be
introduced into a
mammal (such as the donor of the cells) where they will engraft in the bone
marrow.
Methods for differentiating CD34+ cells in vitro into clinically important
immune cell types
using cytokines such a GM-CSF, IFN-K and TNF-I are known (see, Inaba et al.
(1992) J.
Exp. Med. 176: 1693-1702, and Szabolcs et al. {1995) 154: 5851-5861).
Stem cells are isolated for transduction and differentiation using known
methods. For example, in mice, bone marrow cells are isolated by sacrificing
the mouse and
cutting the leg bones with a pair of scissors. Stem cells are isolated from
bone marrow cells
by panning the bone marrow cells with antibodies which bind unwanted cells,
such as CD4+
and CD8+ (T cells), CD45+ {pang cells), GR-1 {granulocytes), and Iad
(differentiated
antigen presenting cells). For an example of this protocol see, Inaba et al.
(1992) J. Exp.
Med 176: 1693-1702.
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In humans, bone marrow aspirations from iliac crests are performed e.g.,
under general anesthesia in the operating room. The bone marrow aspirations is
approximately 1,000 ml in quantity and is collected from the posterior iliac
bones and crests.
If the total number of cells collected is less than about 2 x 108/kg, a second
aspiration using
the sternum and anterior iliac crests in addition to posterior crests is
performed. During the
operation, two units of irradiated packed red cells are administered to
replace the volume of
marrow taken by the aspiration. Human hematopoietic progenitor and stem cells
are
characterized by the presence of a CD34 surface membrane antigen. This antigen
is used for
purification, e.g., on affinity columns which hind CD34. After the bone marrow
is
harvested, the mononuclear cells are separated from the other components by
means of ficoll
gradient centrifugation. This is performed by a semi-automated method using a
cell
separator (e.g., a Baxter Fenwal CS3000+ or Terumo machine). The light density
cells,
composed mostly of mononuclear cells are collected and the cells are incubated
in plastic
flasks at 370C for 1.5 hours. The adherent cells (monocytes, macrophages and B-
Cells) are
discarded. The non-adherent cells are then collected and incubated with a
monoclonal anti-
CD34 antibody (e.g., the marine antibody 9C5) at 40°C for 30 minutes
with gentle rotation.
The final concentration fox the anti-CD34 antibody is 10 1g/ml. After two
washes,
paramagnetic microspheres (DynaBeads, supplied by Baxter Immunotherapy Group,
Santa
Ana, California) coated with sheep antimouse IgG (Fc) antibody are added to
the cell
suspension at a ratio of 2 cells/bead. After a further incubation period of 30
minutes at 40C,
the rosetted cells with magnetic beads are collected with a magnet.
Chymopapain (supplied
by Baxter Immunotherapy Group, Santa Ana, California) at a final concentration
of 200
U/ml is added to release the beads from the CD34+ cells. Alternatively, and
preferably, an
affinity column isolation procedure can be used which binds to CD34, or to
antibodies bound
to CD34 (see, the examples below). See, Ho et al. (1995) Stem Cells 13 (suppl.
3): 100-105.
See also, Brenner (1993) Journal of Hematotherapy 2: 7-17.
In another embodiment, hematopoietic stem cells are isolated from fetal cord
blood. Yu et al. (1995) Proc. Natl. Acad. Sci. USA 92: 699-703 describe a
preferred method
of transducing CD34+ cells from human fetal cord blood using retroviral
vectors.
Bl In vivo transformation.
The vectors of this invention (phage expressing a specific targeting antibody
and containing a heterologous nucleic acid in an expression cassette) can be
administered
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directly to the organism for transduction -of cells in vivo. Administration is
by any-of the
routes normally used for introducing a molecule into ultimate contact with
blood or tissue
cells. The phage packaged nucleic acids are administered in any suitable
manner, preferably
with pharmaceutically acceptable carriers. Suitable methods of administering
such packaged
nucleic acids are available and well known to those of skill in the art, and,
although more
than one route can be used to administer a particular composition, a
particular route can
often provide a more immediate and more effective reaction than another route.
Pharmaceutically acceptable carriers are determined in part by the particular
composition being administered, as well as by the particular method used to
administer the
composition. Accordingly, there is a wide variety of suitable formulations of
pharmaceutical
compositions of the present invention.
VII Formulations for transformation of cells.
As indicated above, particular when administered in vivo, the vectors of this
invention (targeted phage containing heterologous nucleic acid(s)) are
compounded in a
formulation in combination with a pharmaceutically acceptable excipient (i.
e., a
pharmaceutical formulation). Formulations suitable for oral administration can
consist of (a)
liquid solutions, such as an effective amount of the vectors) of this
invention suspended in
diluents, such as water, saline or PEG 400; (b) capsules, sachets or tablets,
each containing a
predetermined amount of the active ingredient, as liquids, solids, granules or
gelatin; (c)
suspensions in an appropriate liquid; and {d) suitable emulsions. Tablet forms
can include
one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn
starch, potato
starch, tragacanth, microcrystalline cellulose, acacia, gelatin, colloidal
silicon dioxide,
croscarmellose sodium, talc, magnesium stearate, stearic acid, and other
excipients,
colorants, fillers, binders, diluents, buffering agents, moistening agents,
preservatives,
flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible
carriers.
Lozenge forms can corinprise the active ingredient in a flavor, usually
sucrose and acacia or
tragacanth, as well as pastilles comprising the active ingredient in an inert
base, such as
gelatin and glycerin or sucrose and acacia emulsions, gels, and the like
containing, in
addition to the active ingredient, carriers known in the art.
The vectors of this invention, alone or in combination with other suitable
components, can be made into aerosol formulations (i.e., they can be
"nebulized") to be
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administered via inhalation. Aerosol formulations can be placed into
pressurized acceptable-
propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.
Suitable formulations for rectal administration include, for example,
suppositories, which consist of the packaged nucleic acid with a suppository
base. Suitable
S suppository bases include natural or synthetic triglycerides or paraffin
hydrocarbons. In
addition, it is also possible to use gelatin rectal capsules which consist of
a combination of
the packaged nucleic acid with a base, including, for example, liquid
triglycerides,
polyethylene glycols, and paraffin hydrocarbons.
Formulations suitable for parenteral administration, such as, for example, by
intraarticular (in the joints), intravenous, intramuscular, intradermal,
intraperitoneal, and
subcutaneous routes, include aqueous and non-aqueous, isotonic sterile
injection solutions,
which can contain antioxidants, buffers, bacteriostats, and solutes that
render the formulation
isotonic with the blood of the intended recipient, and aqueous and non-aqueous
sterile
suspensions that can include suspending agents, solubilizers, thickening
agents, stabilizers,
1 S and preservatives. In the practice of this invention, compositions can be
administered, for
example, by intravenous infusion, orally, topically, intraperitoneally,
intravesically or
intrathecally. Parenteral administration and intravenous administration are
the preferred
methods of administration. The formulations of packaged nucleic acid can be
presented in
unit-dose or mufti-dose sealed containers, such as ampules and vials.
Injection solutions and suspensions can be prepared from sterile powders,
granules, and tablets of the kind previously described. Cells transduced by
vectors of this
invention as described above in the context of ex vivo therapy can also be
administered
intravenously or parenterally as described above.
The dose administered to a patient, in the context of the present invention
2S should be sufficient to effect detectable transformation, more preferably
sufficient to effect a
beneficial therapeutic response in the patient over time. The dose will be
determined by the
efficacy of the particular vector employed and the condition of the patient,
as well as the
body weight or surface area of the patient to be treated. The size of the dose
also will be
determined by the existence, nature, and extent of any adverse side-effects
that accompany
the administration of a particular vector, or transduced cell type in a
particular patient.
In determining the effective amount of the vector to be administered in the,
the physician evaluates circulating plasma levels of the vector, vector
toxicities, progression
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of the disease, and the production of anti-vector antibodies. In general, the
dose equivalent--
of a naked nucleic acid from a vector is from about 1 p,g to 1 g for a typical
70 kilogram
patient, and doses of vectors which include a phage particle are calculated to
yield an
equivalent amount of therapeutic nucleic acid.
For administration, inhibitors and transduced cells of the present invention
can be administered at a rate determined by the LD-50 of the inhibitor,
vector, or transduced
cell type, and the side-effects of the inhibitor, vector or cell type at
various concentrations, as
applied to the mass and overall health of the patient. Administration can be
accomplished
via single or divided doses.
In a preferred embodiment, prior to infusion, blood samples are obtained and
saved for analysis. Between 1 x 108 and 1 x 1012 transduced cells are infused
intravenously
over 60-200 minutes. Vital signs and oxygen saturation by pulse oximetry are
closely
monitored. Blood samples are obtained 5 minutes and 1 hour following infusion
and saved
for subsequent analysis. Leukopheresis, transduction and reinfusion can be
repeated are
1 S repeated every 2 to 3 months. After the first treatment, infusions can be
performed on a
outpatient basis at the discretion of the clinician. If the reinfusion is
given as an outpatient,
the participant is monitored for at least 4, and preferably 8 hours following
the therapy.
Transduced cells are prepared for reinfusion according to established
methods. See, Abrahamsen et al. (1991) J. Clin. Apheresis, 6: 48-53; Carter et
al. (1988) J.
Clin. Apheresis, 4:113-117; Aebersold et al. (1988) J. Immunol. Meth., 112: 1-
7; Muul et al.
(1987) J. Immunol. Methods 101:171-181 and Carter et al. (1987) Transfusion
27: 362-365.
After a period of about 2-4 weeks in culture, the cells should number between
1 x 108 and 1
x 1012. In this regard, the growth characteristics of cells vary from patient
to patient and
from cell type to cell type. About 72 hours prior to reinfusion of the
transduced cells, an
aliquot is taken for analysis of phenotype, and percentage of cells expressing
the therapeutic
agent.
VIII Kits for transducin~ cells.
In another embodiment, this invention provides kits for practice of the
methods described herein. The kits preferably include phage expressing a
heterologous
binding domain and containing a heterologous nucleic acid (e.g., an expression
cassette) that
is to be delivered inside a target cell or a nucleic acid encoding such a
phage. The nucleic
acid can include restriction sites to facilitate insertion of a heterologous
nucleic acid into an
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expression cassette. The assay kits can additionally include any of the other
components ~ Y
described herein for the practice of the assays of this invention. Such
materials preferably
include, but are not limited to, helper phage, one or more bacterial or
mammalian cell lines,
buffers, antibiotics, labels, and the like.
In addition the kits may optionally include instructional materials containing
directions (i.e., protocols) disclosing the transformation methods described
herein. While
the instructional materials typically comprise written or printed materials
they are not limited
to such. Any medium capable of storing such instructions and communicating
them to an
end user is contemplated by this invention. Such media include, but are not
limited to
electronic storage media (e.g., magnetic discs, tapes, cartridges, chips),
optical media (e.g.,
CD ROM), and the like. Such media may include addresses to Internet sites that
provide
such instructional materials.
EXAMPLES
The following examples are offered to illustrate, but not to limit the claimed
invention.
Examnte 1 Creation of a non immune human Fab phase antibody library containing
_109-1011 members
Manipulation of previous 10' member phage display libraries revealed two
major limitations: 1) expression levels of Fabs was too low to produce
adequate material for
characterization, and 2) the library was relatively unstable. These
limitations are a result of
creating the library in a phage vector, and the use of the cre-lox
recombination system. We
therefore decided that the best approach for this project was to create a very
large scFv
library using a phagemid vector. The goal was to produce a library at least
100 times larger
than our previous 3.0 x 10' member scFv library. The approach taken was to
clone the VH
and VL library on separate replicons, combine them into an scFv gene
repertoire by splicing
by overlap extension, and clone the scFv gene repertoire into the phage
display vector
pHENl . Human peripheral blood lymphocyte and spleen RNA was primed with IgM
heavy
chain constant region and, kappa and lambda light chain constant region
primers and first
strand cDNA synthesized. 1 st strand cDNA was used as a template for PCR
amplification of
VH Vxk and V~, gene repertoires.
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The VH gene repertoires were cloned into the vector pUC119Sfi-Not as Ncol-
NotI fragments, to create a library of 8.0 x 108 members. The library was
diverse by PCR
fingerprinting. Single chain linker DNA was spliced onto the VL gene
repertoires using PCR
and the repertoire cloned as an Xhol-NotI fragment into the vector pHENIXscFv
to create a
library of 7.2 x 106 members. The VH and VL gene repertoires were amplified
from their
respective vectors and spliced together using PCR to create an scFv gene
repertoire. The
scFv gene repertoire was cloned as an NcoI-Notl fragment into the vector to
create an scFv
phage antibody library of 7.0 x 109 members. The library was diverse as
determined by
BstNl fingerprinting.
To verify the quality of the library, phage were prepared and selected on 14
different protein antigens. The results are shown in Table 2. scFv antibodies
were obtained
against all antigens used for selection, with between 3 and 15 unique scFv
isolated per
Table 2. Results of phage antibody library selections. For each antigen
(column 1), the
number and the percentage of positive clones selected (column 2) and the
number of
different antibodies isolated (column 3) is indicated
Protein antigen used for selection Percentage (number) of Number of different
ELISA positive clones antibodies isolated
FGF Receptor ECD 69 (18/26) 15
BMP Receptor Type I ECD 50 (12/24) 12
Activin Receptor Type I ECD 66 (16/24) 7
Activin Receptor Type II ECD 66 (16/24) 4
Erb-B2 ECD 91 (31/34) 14
50 (48/96) 6
VEGF
BoNT/A 28 (26/92) 14
BoNT-A C-fragment 95 (87/92) 10
BoNTB 10 (9/92) 5
BoNT/C 12 (11/92) 5
BoNT/E 9 (8/92) 3
Bungarotoxin 67 (64/96) 15
Cytochrome b5 55 (53/96) 5
Chlamydia trachomatis EB 66 (63/96) 7
antigen (average 8.7) (Table 2). This compares favorably to results obtained
from smaller
scFv libraries (1 to a few binders obtained against only 70% of antigens used
for selection).
Affinities of 4 anti-ErbB-2 scFv and 4 anti-Botulinum scFv were measured using
surface
plasmon resonance in a BIAcore and found to range from 4.0 x 10'9 M to 2.2 x
10't° M for
the anti-ErbB2 scFv and 2.6 x 10'8 M to 7.15 x 10'$ M for the anti-Botulinum
scFv (Table 3).
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scFv were highly specific for the antigen used for selection (Figure ?). The
library could_
also be successfully selected on complex mixtures of antigen.
Table 3. Affinities and binding kinetics of anti-BoNT A C-fragment and anti-
Erb-B2 seFv.
Association (lco~ and dissociation (ko f~ rate constants for purified scFvs
were measured
using surface plasmon resonance (BIAcore) and K.d calculated as (ko ff'kor~~
Specificity and ~ (x 10'9 kon (x 10~M'ls'1)koff (x 10-3s-1)
clone
ErbB-2 B7A 0.22 4.42 0.1
ErbB-2 G11D 0.48 2.19 0.11
ErbB-2 A 11 A 0.49 3.69 0.18
ErbB-2 FSA 4.03 1.62 0.65
BoNT-A 2A9 26.1 0.25 0.66
BoNT-A 2H6 38.6 2.2 8.5
BoNT-A 3F6 66.0 4.7 30.9
RnNT-A 2Rfi 71.5 1.1 7.8
For example, selection on Chlamydia trachomatis elementary bodies (the
causative organism
of Chiamydial disease) yielded seven that specifically recognized chlamydia
(Table 2). The
scFv could be successfully used in a number of immunologic assays including
ELISA,
immunofluorescence, Western blotting, epitope mapping and immunoprecipitation.
The
number of binding antibodies for each antigen, and the affinities of the
binding scFv are
comparable to results obtained from the best phage antibody libraries (Table
4). Thus the
library was established as a source of panels of human antibodies against any
antigen with
affinities at least equivalent to the secondary marine response.
Table 4. Comparison of protein binding antibodies selected from non-immune
phage-
display antibody libraries. * For library type, N = V-gene repertoires
obtained from V-genes
rearranged in vivo; SS = semi-synthetic V-genes constructed from cloned V-gene
segments
and synthetic oligonucleotides encoding Vg CDR3. ND = not determined.
Library Library size Number Average Number Range
and of
type* of proteinnumber of affinities
of for
antigensantibodiesaffinitiesprotein
studied per proteinmeasuredantigens
antigen Kd (x
10-9lVn
Marks et al 3,0 x 107 (scFv,2 2.5 1 100-2000
(1991) J. N)
Mol. Biol. 222:
581-
597
Nissim et al 1.0 x108 (scFV,15 2.6 ND ND
(1994) SS)
EMBO J. 13: 692-698
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DeKruif et al (1995) J. 3.6 x 108 (scFv, SS) 12 1.9 3 100 - 2500-
Mol. Biol. 248: 97-105
Griffiths et al (1994) 6.~ x 1010 (Fab, SS) 30 4.8 3 7 - 58
EMBO J. 13: 3245-
3260
Vau~han et al (1996) 1.4 x 1010 (scFv, N) 3 7-0 3 4.2 - 8.0
Nature Biotechnology.
14: 309-314
Present Examples 6.7 x 109 (scFv, 14 8.7 8 0.22 - 7I .5
These experiments demonstrate the creation of a high complexity human scFv
phage antibody library from which a panel of high affinity human scFv can be
generated
against any purified antigen. Such a library is ideal for probing the surface
of cells to
identify novel cell surface markers.
am le Z: LJ take of scFV into ell by rece for mediated endocvtosis and
subseauent recovery
The 7.0 x 109 member scFv phage antibody library described above was
selected on the malignant breast tumor cell lines MB231 and ZR-7~-1, both with
and without
negative selections on the normal breast cell line HBL100. Similar results
were obtained as
described in section above. scFv were isolated that could not distinguish
malignant from
non-malignant cell lines.
To increase the specificity of selections, it was hypothesized that phage
binding cell surface receptors could be taken up into cells by receptor
mediated endocytosis
and could then be recovered from cells by lysing the cells. This assumed: 1)
that phage
could be internalized by receptor mediated endocytosis and 2) that phage could
be recovered
in the infectious state from within cells prior to lysosomal degradation. The
ability to select
for internalized phage antibodies would have two major benefits: 1) the
identification of
antibodies that bind to receptors capable of internalization and 2) an added
level of
specificity in the selection process. Identif cation of antibodies which are
internalized would
be highly useful for many targeted therapeutic approaches where
internalization is essential
(e.g. immunotoxins, targeted liposomes, targeted gene therapy vectors and
others).
girl Receptor mediated internalization of FS or Cl uba.ge
To determine proof of principle, we utilized C6.5 phage and C6.5 diabody
phage (see, copending application USSN 08/665,202). We have previously shown
that C6.5
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scFv is internalized, but at a slow rate, and that the C6.5 diabody is
somewhat better
internalized (probably because it causes receptor dimerization). C6.5 phage,
C6.5 diabody
phage or an irrelevant anti-Botulinum phage were incubated with SKBR3 cells
(ErbB2
expressing breast tumor cell Line) at either 37° C or 4° C and
non-internalized phage
removed by sequential washing with PBS and low pH glycine buffer. The cells
were then
permeabilized and biotinylated anti-M13-antibody added followed by
streptavidin Texas
Red. Cells were then examined by using a confocal microscope. Both C6.5 phage
and C6.5
diabody phage were observed within the cytoplasm). Approximately 1% of cells
had
internalized C6.~ phage and 20% of the cells had internalized C6.5 diabody
phage. There
was no internalization of the anti-Botulinum phage.
To determine if infectious phage could be specifically taken up and recovered
from within cells, C6.5 phage or C6.5 diabody phage were incubated with SKBR3
cells at
37° C. Non bound phage were removed by washing with PBS and phage bound
to the cell
surface were eluted by washing twice with low pH glycine. The cells were then
lysed and
each fraction (the first and second glycine washes and the cytopiasmic
fraction) used to
infect E. coli TG1. Twenty times (C6.~) or 30 times (C6.~ diabody) more phage
were bound
to the cell surface than the anti-Botulinum phage (glycine 1 wash) (Table 5).
After the
second glycine wash, the titre of infectious phage from the cell surface
decreased, indicating
that washing was effective at removing surface bound phage (Table 5). After
cell lysis, the
titer increased more than 10 fold (C6.5 phage) or 50 fold (C6.5 diabody phage)
from the
second glycine wash. We believe this titre represents phage recovered from
inside the cell.
Recovery of phage from inside the cell was 100 times higher for ErbB2 binding
C6.5 than
for anti-Botulinum phage and 200 fold higher for C6.5 diabody phage (Table 5).
Table 5. Titer of cell surface bound phage and internalized phage. 5.0 x 1011
phage (anti-
Botulinum or anti-ErbB2) were incubated with approximately 1.0 x 105 ErbB2
expressing
SKBR3 cells at 37°C. Cells were washed 10 times with PBS and surface
bound phage
eluted with two low pH glycine washes. The cells were then washed once with
PBS and the
cells lysed to release internalized phage. The phage titer was then determined
for each of the
glycine washes and for the lysed cell fraction by infection of E. coli TGl.
Phage specificity 1st glycine wash 2nd glycine wash Lysed cell
fraction
anti-Botulinttm 6.0 x 105 1.0 x 105 6.0 x 105
Anti-ErbB2 (C6.5 scFv) 1.2 x 107 5.2 x lOb 6.8 x 107
Anti-ErbB2 (C6.5 diabody) 1.8 x 107 2.8 x i06 1.7 x 107
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Taken together, the results indicate that: 1) phage binding cell surface
receptors can be taken up by cells and the infectious phage recovered from the
cytoplasm.
The amount of uptake is significantly greater than uptake of non-binding
phage, and the 100
to 200 fold difference is well within the range that would allow enrichment
from a library.
What is unlrnown from the results is whether the phage antibodies are
mediating receptor
mediated internalization or whether they are merely taken up after binding by
membrane
turnover.
B election and characterization of internalizing antibodies from a nhaae
antibody
library
The results described above encouraged us to attempt selection of the phage
antibody library described above to identify new phage antibodies that were
internalized.
Phage antibodies were rescued from the library and selected on SKBR3 cells.
For selection,
phage were incubated with cells at 37°C, non-binding phage removed by
washing cells with
PBS and phage bound to cell surface antigens removed by sequential washes with
low pH
glycine. Cells were then lysed to release internalized phage and the lysate
used to infect E.
coli TG1 to prepare phage for the next round of selection. Three rounds of
selection were
performed. One hundred clones from each round of selection were analyzed for
binding to
SKBR3 cells and to ErbB2 extracellular domain by ELISA. We hypothesized that
we were
likely to obtain binders to ErbB2 since SKBR3 cells are known to express high
levels and
ErbB2 is a receptor which is known to be internalized. After each round of
selection, the
titer of phage recovered from the cytoplasm increased {Table ~. After the
third round, 45%
of the clones were positive SKBR3 cell binding and 17% bound ErbB2 (Table ~.
Table 6. Results of selection of a phage antibody library for internalization.
For each round
of selection, the titer of phage in lysed cells, number of cells lysed and
number of phage per
cell is indicated. After the third round, individual clones were analyzed for
binding to
SKBR3 cells by ELISA and to ErbB2 ECD by ELISA.
Round of # of phage # of cells # of % SKBR3 % ErbB2
in
selection lysate lvsed phagelcellbinders binders
cell
1 !~ _ 2.8x106 0.013 ND ND
3.5x104 .
1.2 x 1 O5 2.8 x 106 0.03 8 ND ND
~
3 7 5 x 1 n6 2.8 x 106 3.75 45% 17%
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_øø_
To estimate the number of unique binders, the scFv gene from ELISA _ '
positive clones was PCR amplified and fingerprinted by digestion with BstNl.
Two unique
restriction patterns were identified. The scFv genes were sequenced and 2
unique ErbB2
binding scFv identified. Similar analysis of SKBR3 ELISA positive clones that
did not bind
S ErbB2 identified an additional 11 unique scFv.
To verify that phage antibodies were specific for SKBR3 cells, phage were
prepared from each unique clone and analyzed for binding to SKBR3 cells (high
ErbB2
expression) as well as 2 other epithelial tumor cell lines (SK-OV-3, moderate
ErbB2
expression and MCF7, low ErbB2 expression) and a normal breast cell line
(FiS578B). Each
unique clone specifically stained tumor cell lines but not the normal breast
cell line.
SKBR3 and MCF7 cells were incubated with phage antibodies C6.5 (positive
control), 3TF5 and 3GH7. The latter two clones were isolated from the library,
with 3TF5
binding ErbB2 and the antigen bound by 3GH7 unknown. All 3 phage antibodies
intensely
stain SKBR3 cells (the selecting cell line and high ErbB2 expresses. C6.5
phage weakly
stain MCF7 cells (low ErbB2 expressor). The anti-ErbB2 clone 3TF5 from the
library stains
MCF7 cells much more intensely then C6.5, as does 3GH7.
SKBR3, SK-OV-3, MCF7 and HST578 cells were studied using native
purified scFv 3TF5 and 3GH7. For these studies, the scFv genes were subcloned
into a
vector which fuses a hexahistidine tag to the scFv C-terminus. scFv was then
expressed,
harvested from the bacterial periplasm and purified by immobilized metal
affinity
chromatography. The two scFv intensely stain SKBR3 cells, and do not stain the
normal
breast cell line HST578. There is minimal staining of the low ErbB2 expressing
cell line
MCF7 and intermediate staining of SK-OV-3 cells (moderate ErbB2 expresses). In
general,
the intensity of staining is less than seen with phage. This is to be expected
since the
secondary antibody for phage staining recognizes the major coat protein (2500
copies/phage)
resulting in tremendous signal amplification.
The anti-ErbB2 phage antibody 3TF5 was studied further to determine if it
was indeed internalized. This antibody was selected for initial study since
its internalization
could be compared to ErbB2 binding C6.5. S.0 x1011 3TF5 or C6.5 phage were
incubated
with SKBR3 cells at 37°C or at 4°C. After washing with PBS, 3TF5
phage stained cells
more intensely than C6.5 phage. After washing with low pH glycine, confocal
microscopy
revealed that 3TF5 phage were internalized by greater than 95% of cells, while
C6.5 was
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internalized by only a few percent of cells. Incubation of either antibody at
4°C led to no
internalization.
The native purified 3TF5 scFv was similarly analyzed and was also
efficiently internalized by SKBR3 cells. It should be noted that the native
3TF5 scFv existed
only as a monomer with no appreciable dimerization or aggregation as
determined by gel
filtration.
These experiments demonstrate that phage antibodies can be internalized by
cells and recovered from the cytoplasm. Phage that bind an internalizing cell
surface
receptor can be enriched more than 100 fold over non-binding phage. This level
of
enrichment is greater than that achieved by selecting on the cell surface. We
have applied
this approach to library selection and isolated phage antibodies that bind and
are internalized
by SKBR-3 cells. Several of these antibodies bind to ErbB2, but are more
efficiently
internalized than antibodies such as C6.5 that were generated by selecting on
pure antigen.
Many other antibodies have been isolated that bind specifically to SKBR-3 and
other breast
tumor cell lines and are efficiently internalized. These antibodies should
prove useful for
tumor targeting and for identifying potentially novel internalizing tumor cell
receptors.
EYampIe 3 Increasing the affinity of antibody fragments with the desired
binding
characteristics by creating mutant phase antibody libraries and selectin_ø on
the
anproDriate breast tumor cell line.
Phage display has the potential to produce antibodies with affinities that
cannot be produced using conventional hybridoma technology. Ultra high
affinity human
antibody fragments could result in excellent tumor penetration, prolonged
tumor retention,
and rapid clearance from the circulation, leading to high specificity. We
therefore undertook
a series of experiments to develop methodologies to generate ultra high
affinity human
antibody fragments. Experiments were performed to answer the following
questions: 1)
What is the most effective way to select and screen for rare higher affinity
phage antibodies
amidst a background of lower affinity binders; 2 What is the most effective
means to
remove bound phage from antigen, to ensure selection of the highest affinity
phage
antibodies; 3) What is the most efficient techniques for mal~ng mutant phage
antibody
libraries (random mutagenesis or site directed mutagenesis; 4) What region of
the antibody
molecule should be selected for mutagenesis to most efficiently increase
antibody fragment
affinity.
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PCT/US99/07398
To answer these questions, we studied the human scFv -C6.5, which binds the
extracellular domain (ECD) of the tumor antigen ErbB-2 (32) with a Kd of 1.6 x
10-8 M and
koff of 6.3 x 10'3 s't (Schier et al. (1995) Immunotechnology, 1: 63-71).
Isolation and
characterization of C6.5 is described briefly below and in detail in copending
application
USSN 08/665,202).
Despite excellent ttunor:normal tissue ratios in vivo, quantitative delivery
of
C6.5 was not adequate to cure tumors in animals using radioimmunotherapy
(Schier et al.
(1995) Immunotechnolagy, 1: 63-71). To improve the quantitative delivery of
antibody to
tumor, the affinity of C6.5 was increased. First, techniques were developed
that allowed
selection of phage antibodies on the basis of affinity, rather than
differential growth in E.
toll or host strain toxicity (Schier et al. (1996) J Mol. Biol. 255: 28-43;
Schier et al. (1996)
Gene 169: 147-155; Schier et al. (1996) Human antibodies and hybridomas 7: 97-
105).
Next, we determined which locations in the scFv gene to mutate to achieve the
greatest
increments in affinity (Schier et al. (1996) J. Mol. Biol. 255: 28-43; Schier
et al. (1996)
Gene; Schier et al. (1996) J. Mol. Biol. 263: 551-567). Random mutagenesis did
not yield as
great an increment in affinity as site directed mutagenesis of the
complementarity
determining regions (CDRs) that contain the amino acids which contact antigen.
Results
from diversifying the CDRs indicated that: 1) the greatest increment in
affinity was achieved
by mutating the CDRs located in the center of the binding pocket (VL and VH
CDR3); 2)
half of the CDR residues have a structural role in the scFv and when mutated
return as wild-
type; and 3) these structural residues can be identified prior to library
construction by
modeling on a homologous atomic crystal structure. These observations led to
development
of a generic strategy for increasing antibody affinity where mutations are
randomly
introduced sequentially into VH and VL CDR3, with conservation of residues
postulated to
have a structural role by homology modeling (Schier et al. (1996) J. Mol.
Biol. 263: 551-
567). Using this approach, the affinity of C6.5 was increased 1200 fold to a
Kd of 1.3 x 10-
t t M (Id.).
Biodistribution studies revealed a close correlation between affinity and the
percent injected dose of scFv/grarn of tumor (%ID/g) at 24 hours {Adams et al.
(1998)
Cancer Res. 58: 485-490). The greatest degree of tumor retention was observed
with t25I_
C6ML3-9 (1.42 %Ip/g, RCt =1.0 x 10-9 M). Significantly less tumor retention
was achieved
with t 25I_C6.5 (0.80 %ID/g, ~ =1.6 x to-g) and C6G98A (0.19 %m/g, Kd = 3.2 x
10-7 M).
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The tumor:normal organ ratios also reflected the differences in affinity, e.g.
tumor:blood
ratios of 17.2, 13.3, 3.5 and 2.6, and tumor to liver ratios of 26.2, 19.8,
4.0 and 3.1 for
C6ML3-9, C6.5 and C6G98A respectively at 24 hours. Studies of the higher
affinity scFv
are pending. The results demonstrate our ability to increase antibody affinity
to values not
achievable from hybridoma technology and confirm the importance of affinity in
tumor
targeting
E_xamole 4 Preclinical development of C6 5 based breast can~pr therapies
Two approaches have been collaboratively pursued to develop C6.5 based
breast cancer therapies. In one collaboration, C6.5 based molecules are being
engineered for
radioimmunotherapy. To increase quantitative tumor delivery and retention of
antibody
fragment, dimeric scFv'diabodies' were created by shortening the linker
between the VH and
VL domains from 15 to 5 amino acids. Consequently, pairing occurs between
complementary domains of two different chains, creating a stable noncovalently
bound
dimer with two binding sites. In vitro, diabodies produced from the V-genes of
C6.5 have a
significantly higher apparent affinity and longer retention on the surface of
SK-OV-3 cells
compared to C6.5 scFv (T1/2 > 5 hr vs. 5 min) (Adams et al. (1998) Brit. J.
Cancer.).
Biodistribution studies of C6.5 diabody revealed 6.5 %ID/g tumor at 24 hours
compared to
only 1 %m/g for C6.5 scFv. When diabody retentions were examined over 72 hours
and
cumulative area under the curve (AUC) values determined, the resulting
tumor:organ AUC
ratios were greater than reported for other monovalent or divalent scFv
molecules. The
therapeutic potential of these molecules is being examined in
radioimmunotherapy studies in
nude mice. Since in vivo characterization of c6.5 based molecules was not
formally one of
the technical objectives, we are continuing to use the affinity mutants of
C6.5 and C6.5
based diabodies to study the relationship between antibody affinity, size and
valency and
specific tumor targeting as part of NTH RO1 1 CA65559-OlAl.
In another collaboration, C6.5 based molecules are being used to target
doxorubicin containing stealth liposomes to ErbB2 expressing breast cancers
(Kirpotin et al.
(1997) Biochemistry. 36: 66-75). To facilitate chemical coupling of the scFv
to liposomes,
the C6.5 gene was subcloned into an E. toll expression vector resulting in
addition of a free
cysteine residue at the C-terminus of the scFv. Purified C6.Scys scFv was
conjugated to
liposomes and in vitro uptake determined using SKBR3 cells. Total uptake was
3.4 mmol
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phospholipid/106 cells at 6 hour, with 70% of the uptake internalized. The
uptake is
comparable to that achieved using the 4D5 anti-HER2 Fab' from Genentech. There
was no
uptake of unconjugated liposomes. The results indicate that C6.5 binds to a
ErbB2 epitope
that results in internalization at a rate comparable to the best internalizing
antibody produced
from hybridomas (4D5). In vivo therapy studies in scid mice indicated that
C6~.5 targeted
liposomes caused a greater degree of tumor regression and a higher cure rate
than untargeted
liposomes or a combination of untargeted liposomes and systemic 4D5 antibody..
conclusions
The experiments described herein establish that A large (7.0 x 109 member)
phage antibody library has been created which can provide panels of human
antibodies to
purified antigens with affinities comparable to the affinities of antibodies
produced by
marine immunization. The phage antibodies binding cell surface receptors can
be can be
internalized by cells and recovered in an infectious state from within the
cell.
Methodologies were developed which permit enrichment of internalizing phage
antibodies
over non-internalizing antibodies more than 100 fold. These methodologies were
then
applied to select new scFv antibodies that bind to internalizing receptors on
SKBR-3 cells.
Several of these antibodies bind to ErbB2, but are internalized more
efficiently than C6.5
based scFv. Many more antibodies bind to unknown internalizing receptors. All
of these
scFv bind specifically to SKBR-3 cells or related tumor cell lines. The
results indicate that
this selection approach is a powerful approach to generate antibodies that can
distinguish one
cell type (malignant) from another (non-malignant). Moreover, we have
demonstrated that it
is not only possible to select for binding, but to select for function
(internalization). In the
near term, we will further characterize the antibodies isolated with respect
to specificity, and
in the case of ErbB2 binding scFv, affinity. In the longer term we will use
these reagents to:
1) study the effect of affinity and valency on the rate of internalization;
and 2) identify the
antigens bound using immunoprecipitation. It is likely that the results will
lead to the
identification of novel internalizing tumor cell surface receptors which will
be useful
therapeutic targets. If this approach proves useful, we plan on applying it to
primary tumor
cells and DCIS. We also intend to evaluate 3TF5 (ErbB2 binding scFv which is
internalized
faster than C6.5) fox liposome targeting. It is possible that it will be more
effective than
C6.5
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In addition, the experiments demonstrate that methodologies for increasing- - -
antibody affinity in vitro to values not previously achieved in vivo. We have
applied these
methodologies to generate novel ErbB2 binding scFv.
Example 5 Selection of internalizing antibodies from phase libraries
Antibodies that bind cell surface receptors in a manner whereby they are
endocytosed are useful molecules for the delivery of drugs, toxins or DNA into
the cytosol
of mammalian cells for therapeutic applications. Traditionally, internalizing
antibodies have
been identified by screening hybridomas. In this example, we studied a human
scFv (C6.5)
that binds ErbB2 to determine the feasibility of directly selecting
internalizing antibodies
from phage libraries and to identify the most efficient display format. Using
wild type C6.5
scFv displayed monovalently on a phagemid, we demonstrate that anti-ErbB2
phage
antibodies can undergo receptor mediated endocytosis. Using affinity mutants
and dimeric
diabodies of C6.5 displayed as either single copies on a phagemid or multiple
copies on
phage, we define the role of affinity, valency, and display format on phage
endocytosis and
identify the factors that lead to the greatest enrichment for internalization.
Phage displaying
bivalent diabodies or multiple copies of scFv were more efficiently
endocytosed than phage
displaying monomeric scFv and recovery of infectious phage was increased by
preincubation
of cells with chloroquine. Measurement of phage recovery from within the
cytosol as a
function of applied phage titer indicates that it is possible to select for
endocytosable
antibodies, even at the low concentrations that would exist for a single phage
antibody
member in a library of 109.
Al Introduction
Growth factor receptors are frequently overexpressed in human carcinomas
and other diseases and thus have been utilized for the development of targeted
therapeutics.
The HER2/neu gene, for example, is amplified in several types of human
adenocarcinomas,
especially in tumors of the breast and the ovary (Slamon et al. (19$9) Science
244: 707-712)
leading to the overexpression of the corresponding growth factor receptor
ErbB2. Targeting
of ErbB2 overexpressing cells has been accomplished primarily using anti-ErbB2
antibodies
in different formats, including conjugation to liposomes containing
chemotherapeutics
(Kirpotin et al. (1997). Biochem. 36: 66-75), fusion to DNA carrier proteins
delivering a
toxic gene (Forminaya and Wels (1996) J. Biol. Chem. 271: 10560-1056$), and
direct fusion
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to a toxin {Altenschmidt et al. (1997) Int. J. Cancer 73: 117-124). For many
of these
targeted approaches, it is necessary to deliver the effector molecule across
the cell membrane
and into the cytosol. This can be accomplished by taking advantage of normal
growth factor
receptor biology; growth factor binding causes receptor activation via homo-
or
S heterodimerization, either directly for bivalent ligand or by causing a
conformational change
in the receptor for monovalent ligand, and receptor mediated endocytosis
(Ullrich and
Schlessinger (1990) Cell 61: 203-212). Antibodies can mimic this process,
stimulate
endocytosis, become internalized and deliver their payload into the cytosol.
In general, this
requires a bivalent antibody capable of mediating receptor dimerization
(Heldin (1995) Cell
80: 213-223; Yarden (1990) Proc. Natl. Acad. Sci . USA 87: 2569-2573). In
addition, the
efficiency with which antibodies mediate internalization differs significantly
depending on
the epitope recognized (Yarden (1990) Proc. Natl. Acad. Sci . USA 87: 2569-
2573; Hurwitz
et al. (1995) Proc. Natl. Acad. Sci. USA 92: 3353-3357.). Thus for some
applications, such
as iiposomal targeting, only antibodies which bind specific epitopes are
rapidly internalized
and yield a functional targeting vehicle.
Currently, antibodies which mediate internalization are identified by
screening hybridomas. Alternatively, it might be possible to directly select
internalizing
antibodies from large non-immune phage libraries (Marks et al. (1991) J. Mol.
Biol. 222:
581-597; Sheets et al. {1998) Proc. Natl. Acad. Sci. USA 95: 6157-6162) by
recovering
infectious phage particles from within cells after receptor mediated
endocytosis, as reported
for peptide phage libraries (Hart et al. (1994) J. Biol. Chem. 269: 12468-
12474; Barry et al.
(1996) Nat. Med~ 2: 299-305). Unlike the multivalently displayed peptide phage
libraries,
however, phage antibody libraries typically display monomeric single chain Fv
(scFv) or Fab
antibody fragments fused to pIII as single copies on the phage surface using a
phagemid
system (Marks et al. (1991) J. Mol. Biol. 222: 581-597; Sheets et al. (1998)
Proc. NatL
Acad. Sci. USA 95: 6157-6162.). We hypothesized that such monovalent display
was
unlikely to lead to efficient receptor crosslinking and phage internalization.
To determine
the feasibility of selecting internalizing antibodies and to identify the most
efficient display
format, we studied a human scFv (C6.5) which binds ErbB2 (13). Using wild type
C6.5
scFv, we demonstrate that anti-ErbB2 phage antibodies can undergo receptor
mediated
endocytosis. Using affinity mutants and dimeric diabodies of C6.5 displayed as
either single
or multiple copies on the phage surface, we define the role of affinity,
valency, and display
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format on phage endocytosis and identify the factors that lead to the greatest
enrichment for
internalization. The results indicate that it is possible to select for
endocytosable antibodies,
even at the low concentrations that would exist for a single phage antibody
member in a
library of 109 members.
Al Material and methods
ly Cells
The SKBR3 breast tumor cell line was obtained from ATCC and grown in
RPMI media supplemented with 10% FCS (Hyclone) in 5% C02 at 37°C.
Antibodies and antibody nha~e preparations
The C6.5 scFv phage vector was constructed by subcloning the C6.5 gene as
a Sfi IINot I fragment from scFv C6.5 pHENI (Schier et al. (1995)
Immunotechnology 1: 63-
71) into the phage vector fdlSfi IINot I (a gift of Andrew Griffiths, MRC
Cambridge, UK).
The C6.5 diabody phagemid vector was constructed by subcloning the C6.5
diabody gene
(Adams et al. (1998) Brit. J. Cancer. 77: 1405-1412, 1998) as a NcoI/NotI
fragment into
pHENl (Hoogenboom et al. (1991) Nucleic Acids Res. 19: 4133-4137). The anti-
botulinum
scFv phagemid (clone 3D12) (Amersdorfer et al. (1997) Infection and Immunity .
65: 3743-
3752) C6.5 scFv phagemid (Schier et al. (1995) Immunotechnology 1: 63-71) and
scFv
C6ML3-9 scFv phagemid (Schier et al. (1996) J. Mol. Biol. 263: 551-567) in
pHENl have
been previously described. Phage were prepared (Sambrook et al. (1990).
Molecular
cloning- a laboratory manual., New York: Cold Spring Harbor Laboratory) from
the
appropriate vectors and titered on E. coli TG1 as previously described (Marks
et al. (/991) J.
Mol. Biol. 222: 581-597) using ampicillin (I00 ~g/ml) resistance for titration
of constructs in
pHENl and tetracyline (50 ~g/ml) for titration of constructs in fd. Soluble
C6.5 scFv, C6.5
diabody and anti-botulinum scFv were expressed from the vector pUCl l9mycHis
(Schier et
~ al. (1995) Immunotechnology 1: 63-71) and purified by immobilized metal
affinity
chromatography as described elsewhere (Id.)).
3) Detection of internalized native antibody fra~nents and phase antibodies
SKBR3 cells were grown on coverslips in 6-well culture plates (Falcon) to
50% of confluency. Culture medium was renewed 2 hours prior to the addition of
5.1011
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cfu/ml of phage preparation (the phage preparation representing a maximum of
1/10 o~the
culture medium volume) or 20 ~g/ml of purified scFv or diabody in phosphate
buffered
saline, pH 7.4 (PBS). After 2 hours of incubation at 37°C, the wells
were quickly washed 5
times with ice cold PBS and 3 times for 10 minutes each with 4 mL of stripping
buffer (50
mM glycine pH 2.8, 0.5 M NaCI, 2M urea, 2% polyvinylpyrrolidone) at RT. After
2
additional PBS washes, the cells were fixed in 4% paraformaldehyde (10 minutes
at RT),
washed with PBS, permeabilized with acetone at -20°C (30 seconds) and
washed again with
PBS. The coverslips were saturated with PBS-1% BSA (20 min. at RT). Phage
particles
were detected with biotinylated anti-M13 immunoglobulins (5 Prime-3 Prime,
Inc, diluted
300 times) (45 min. at RT) and Texas red-conjugated streptavidin (Amersham,
diluted 300
times) (20 min. at RT). Soluble scFv and diabodies containing a C-terminal myc
peptide tag
were detected with the mouse mAb 9E10 (Santa Cruz Biotech, diluted 100 times)
(45 min. at
RT), anti-mouse biotinylated immunoglobulins (Amersham, diluted 100 times) and
Texas
red-conjugated streptavidin. Optical confocal sections were taken using a Bio-
Rad MRC
1024 scanning laser confocal microscope. Alternatively, slides were analyzed
with a Zeiss
Axioskop UV fluorescent microscope.
4) Recovery and titration of cell surface bound or internalized nhage
Subconfluent SKBR3 cells were grown in 6-well plates. Culture medium was
renewed 2 hours prior to the experiment. Cells were incubated for varying
times with
different concentrations of phage preparation at 37°C. Following PBS
and stripping buffer
washes, performed exactly as described above for detection of internalized
native antibody
fragments and phage antibodies, the cells were washed again twice with PBS and
lysed with
1 mL of 100 mM triethylamine (TEA). The stripping buffer washes and the TEA
lysate
were neutralized with 1/2 volume of Tris-HCl 1M, pH 7.4. For some experiments,
cells
were trypsinized after the three stripping buffer washes, collected in a 15 ml
Falcon tube,
washed twice with PBS and then lysed with TEA. In experiments performed in the
presence
of chloroquine, SKBR3 cells were preincubated for two hours in the presence of
complete
medium containing 50 pM chloroquine prior to the addition of phage.
Corresponding
control samples in the absence of chloroquine were prepared at the same time.
For all
experiments, phage were titered on E. coli TGl as described above.
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81 Results
I) The model system utilized to study phase antibody internalization
The human anti-ErbB2 scFv C6.5 was obtained by selecting a human scFv
phage antibody library on recombinant ErbB2 extracellular domain (13). C6.5
scFv binds
ErbB2 with a Kd =1.6 x 10-8 M and is a stable monomeric scFv in solution with
no tendency
to spontaneously dimerize or aggregate (Schier et al. (1995) Immunotechnology
1: 63-7I).
To determine the impact of affinity on internalization, we studied a scFv
(C6ML3-9) which
differs from C6.5 by 3 amino acids (Schier et al. (1996) J. MoL Bzol. 263: 551-
567).
C6ML3-9 scFv is also a stable monomer in solution and binds the same epitope
as C6.5 scFv
but with a 16 fold lower Kd (1.0 x 10-9 M) (Schier et al. (1996) J. Mol. Biol.
263: 551-567;
Adams et al. (1998) Cancer Res. 58: 485-490). Since receptor homodimerization
appears to
typically be requisite for antibody internalization we also studied the
dimeric C6.5 diabody
(Adams et al. (1998) Brit. J. Cancer. 77: 1405-1412, 1998). Diabodies are scFv
dimers
where each chain consists of heavy (VH) and light (VL) chain variable domains
connected
using a peptide linker which is too short to permit pairing between domains on
the same
chain. Consequently, pairing occurs between complementary domains of two
different
chains, creating a stable noncovalent dimer with two binding sites (Holliger
et al. (1993)
Proc. Natl. Acad Sci. 90: 6444-6448). The C6.5 diabody was constructed by
shortening the
peptide linker between the Ig VH and VL domains from 15 to 5 amino acids and
binds ErbB2
on SKBR3 cells bivalently with a Kd approximately 40 fold lower than C6.5 (4.0
x 10-lo M)
(Adams et al. (1998) Brit. J. Cancer. 77: 1405-1412, 1998).
Native C6.5 scFv and C6.5 diabody was expressed and purified from E. coli
and analyzed for endocytosis into ErbB2 expressing SKBR3 breast tumor cells by
immunofluorescent confocal microscopy. As expected, monomeric C6.5 scFv is not
significantly internalized whereas the dimeric C6.5 diabody can be detected in
the cytoplasm
of all cells visualized.
For subsequent experiments, the C6.5 and C6ML3-9 scFv and C6.5 diabody
genes were subcloned for expression as pIII fusions in the phagemid pHEN-1
(Hoogenboom
et al. (1991) Nucleic Acids Res. 19: 4133-4137). This should yield phagemid
predominantly
expressing a single scFv or diabody-pIII fusion after rescue with helper phage
(Marks et al.
(1992) J. Biol. Chem. 267: 16007-16010) (Figures 2A and 2B). Diabody phagemid
display a
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bivalent antibody fragment resulting from intermolecular pairing of one scFv-
pIII fusion -
molecule and one native scFv molecule (Figure 2B). The C6.5 scFv gene was also
subcloned into the phage vector fd-Sfi/Not. This results in phage with 3 to 5
copies each of
scFv-pIII fusion protein (Figure 2C). The human breast cancer cell line SKBR3
was used as
a target cell line for endocytosis. Its surface ErbB2 density is approximately
1.0 x 106 per
cell (Hypes et al. (1989) J. Cell. Biochem 39: 167-173).
2) C6 5 uhaQemids are end0cvtosed bvhuman cells
C6.5 scFv phagemids were incubated for 2 hours with SKBR3 cells grown on
coverslips at 37°C to allow active internalization. Cells were
extensively washed with PBS
to remove non specific binding and washed an additional three times with high
salt and low
pH (stripping) buffer to remove phage specifically bound to cell surface
receptors.
Internalized phagemid were detected with a biotinylated M13 antiserum
recognizing the
major coat phage protein pVIII. An anti-botulinum toxin phagemid was used as a
negative
control. Staining was analyzed by using immunofluorescent microscopy.
Approximately
1% of the cells incubated with C6.5 scFv phagemid showed a strong
intracellular staining
consistent with endosomal localization while no staining was observed for anti-
botulinum
phagemid. Furthermore, no staining was seen if the incubation was performed
for 2 hours at
4°C instead of 37°C (data not shown). Staining performed after
the PBS washes but before
washing with stripping buffer showed membrane staining of all the cells,
indicating that
multiple washes with stripping buffer is necessary to remove surface bound
phagemids. The
results also indicate that only a fraction of the cell bound phage are
endocytosed.
3) Increased affinity and bivalenc~lead to increased vhage endocvtosis
We compared the internalization of C6.5 scFv, C6ML3-9 scFv and C6.5
diabody phagemid and C6.5 scFv phage using immunofluorescence. Both C6ML3-9
scFv
2S and C6.S diabody phagemid as well as C6.5 scFv phage yielded increased
intensity of
immunofluorescence observed at the cell surface compared to C6.5 scFv
phagemid. For
C6ML3-9 scFv phagemid, approximately 10% of the cells showed intracellular
fluorescence
after 2 hours of incubation. This value increased to approximately 30% of
cells for the
dimeric C6.5 diabody phagemid and 100% of cells for multivalent C6.5 scFv
phage.
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3? --Infectious oha~e can be recovered from within the cell and their titre
correlates with the level of uptake observed using immunofluorescence
To determine if infectious phage antibody particles could be recovered from
within the cell, we incubated approximately 5.0 x 10~ SKBR-3 cells for 2 hours
at 37°C with
3.0 x 1011 cfu of the different phagemid or phage. Six PBS washes were used to
remove
non-specifically bound phage and specifically bound phage were removed from
the cell
surface by three consecutive washes with stripping buffer (washes I, II and
III respectively,
Table 7) . The cells were then lysed with 1 mL of a 100 mM triethylamine
solution (TEA)
(representing the intracellular phage). The three stripping washes and the
cell lysate were
neutralized and their phage titer was determined by infection of E. coli TG 1.
The titers of
phage recovery are reported in Table 7.
Table 7: Titration of membrane bound and intracellular phage. 3.0 x IOI 1 cfu
of
monovalent C6.5 scFv phagemid, 16 fold higher affinity monovalent C6ML3-9 scFv
I5 phagemid, bivalent C6.5 diabody phagemid or multivalent C6.5 fd phage were
incubated
with sub confluent SKBR3 cells for 2 hours at 37'C. Cells were washed 6 times
with PBS, 3
times with stripping buffer and then lysed to recover intracellular phage. The
various
fractions were neutralized and the phage titered. The total number of cfu of
each fraction is
reported. Non specific anti-botulinum phagemid were used to determine non
specific
recovery.
Phage Antibody Cell Surface Phage Titer (x 10-5) Intracellular
Phage Titer (x 10-5)
1st Wash 2nd Wash 3rd Wash
Anti-botulinum 280 36 2.8 15
phagemid
C6.5 scFv phagemid600 96 7.6 52
C6ML3-9 scFv 2500 140 32 270
phagemid
C6.5 diabody phagemid1800 120 I3 450
C6.5 scFv hate 2300 620 56 2200
Considerable background binding was observed in the first stripping wash for
the anti-botulinum phage even after 6 PBS washes (2.8 x 107 cfu, Table 7).
This value likely
represents phage non-specifically bound to the cell surface as well as phage
trapped in the
extracellular matrix. The amount of surface bound phage increased only 2.1
fold above this
background for C6.5 scFv phagemid (Tables 7 and 9). With increasing affinity
and avidity
of the displayed C6.5 antibody fragment, the titer of cell surface bound
phagemid or phage
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increased (Table 7). The titer of phage in the consecutive stripping washes
decreased _ -
approximately 10 fold with each wash. These additional stripping washes led to
a minor
increase in the titer of specific phage eluted compared to the background
binding of the anti-
botulinum phage (2.7 fold for C6.5 scFv phagemid to 20 fold for C6.5 scFv
phage, Table 9).
The only exception was the titer of the C6.5 diabody phagemid, where the ratio
actually
decreased from 6.4 fold to 4.6 fold. This is likely due to the fact that in
the diabody the VH
and VL domains that comprise a single binding site are not covalently attached
to each other
via the peptide linker. This increases the likelihood that a stringent eluent
(like glycine)
could dissociate VH from VL and abrogate binding to antigen.
Table 9: Specific enrichment of anti-ErbB2 phage compared to anti-botulinum
phage.
*The titers of anti-ErbB2 phage are divided by the titers of the anti-
botulinum phage (Table
7) to derive an enrichment ratio for specific vs nonspecific binding or
internalization. **The
titer of intracellular phage is divided by the titer of cell surface bound
phage (Table 7) to
derive the ratio of internalized phage vs surface bound phage.
Phage Antibody Anti-ErbB2 /Anti-Botulmum Intracellular/
Phage Titer Ratio* Cell Surface
Phage Ratio**
Cell surface Cell surface Intracellular
(1st Wash) (3rd Wash)
C6.5 scFv phagemid2.14 2.7 3.5 6.8
C6ML3-9 scFv phagemid8.9 11.4 18 8.4
C6.5 diabody phagemid6.4 4.6 30 35
C6.5 scFv phage 8.2 20 146 39
Three stripping washes were required to ensure that the titer of phage
recovered after cell lysis was greater than the titer in the last stripping
wash (Table 7). We
presumed that after three stripping washes, the majority of the phage eluted
represented
infectious particles from within the cell rather than from the cell surface.
In fact, since the
cell lysate titer observed with non-specific anti-botulinum phage was
considerable (1.5 x
106) and greater than observed in the last stripping wash, it is likely that
many phage remain
trapped within the extracellular matrix and relatively inaccessible to the
stripping buffer
washes. Some anti-botulinum phage might also be non-specifically endocytosed
by cells,
but this is likely to be a small amount given the immunofluorescence results.
The titer of
phage in the TEA fraction increased with increasing affinity and avidity of
C6.5, with the
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highest titers observed for the dimeric C6.S diabody phagemid and the
multivalent C6.S scFv
phage (Table 7). The values represent a 30 fold (C6.S diabody phagemid) and
146 fold
(C6.S scFv phage) increase in titer compared to the anti-botulinum phage
(Table 7). We
have presumed that the increase in the phage titer in the cell lysate compared
to the last
S stripping wash is due to endocytosed phage. in fact, some of these phage
could have come
from the cell surface or intracellular matrix. While this could be true for a
fraction of the
phage from the cell lysate, the immunofluorescence results indicate that at
least some of the
phage are endocytosed. One indicator of the relative fraction of endocytosed
phage for the
different C6.5 molecules is to compare the amount of phage remaining on the
cell surface
prior to cell lysis (last stripping wash) With the amount recovered after cell
lysis. This ratio
shows only a minor increase for monovalent C6.S scFv or C6MI,3-9 scFv phagemid
(6.8 and
8.4 fold respectively) compared to anti-botulinum phagemid (S.4) (Table 9). In
contrast the
ratios for dimeric C6.S diabody phagemid and multivalent C6.S scFv phage
increase to a
greater extent (35 and 39 respectively) compared to anti-botulinum phagemid.
1 S 4) Increasing the enrichment ratios of specifically endocvtosedohage
The results above indicate that phage antibodies can undergo receptor
mediated endocytosis and remain infectious in a cell lysate. Selection of
internalized phages
from a phage library requires the optimization of the method to increase
enrichment of
specifically internalized phages over non-internalized phage. Two parameters
can be
improved: (1) reduction of the recovery of non-specific or non-internalized
phage and (2)
preservation of the infectivity of internalized phage. To examine these
parameters, we
studied wild-type C6.5 scFv phagemid. We chose this molecule because it was
clearly
endocytosed based on confocal microscopy, yet was the molecule undergoing the
least
degree of specific endocytosis. C6.S scFv phagemid also represents the most
commonly
2S utilized format for display of non-immune phage antibody libraries (single
copy pill in a
phagemid vector) and has an affinity (16 nM~ more typical of Kd's of scFv from
such
libraries than the affinity matured C6ML3-9 scFv (Sheets et al. (1998) Proc.
Natl. Acad. Sci.
USA 9S: 6157-6162; Vaughan et al. (1996) Nature Biotech. 14: 309-314).
a? Reducing the background of non-internalized haEe_
To reduce the background of non-specific phage recovery, we studied the
effect of trypsinizing the cells prior to TEA lysis. This should remove phage
trapped in the
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extracellular matrix. Trypsinization also dissociates the cells from the cell
culture flask, -
permitting transfer to a new vessel and elimination of any phage bound to the
cell culture
flask. For these experiments, C6.5 scFv phagemid (5.0 x 108 ampicillin
resistant cfu) were
mixed with a 1000 fold excess of wild type fd phage (5.0 x 1011 tetracylcine
resistant cfu).
After incubation of phagemid with SKBR-3 cells for 2 hours at 37°C,
cells were washed
with PBS and three times with stripping buffer. Cells were then directly lysed
with TEA or
treated with trypsin, washed twice with PBS and then lysed with TEA. Phagemid
in the first
stripping wash and the cell lysate were titered by infection of E. coli TG1
and plated on
ampicillin and tetracycline plates. The titer of fd phage and C6.5 scFv
phagemid recovered
from the cell surface was comparable for the two experimental groups (Figure
3). The ratio
of fd phage/C6.5 scFv phagemid in the cell surface fractions (160/1 and 250/1)
yields a 4 to
6 fold enrichment achieved by specific cell surface binding from the initial
1000 fold ratio.
Without trypsinization, the ratio of fd phage /C6.5 scFv phagemid in the cell
lysate increases
only 6.1 fold; in contrast, the ratio increases 209 fold with trypsinization
(Figure 3). This
results from a 60 fold reduction in non-specific binding with only a minor
reduction in the
amount of specific phage recovery (Figure 3).
bl Imorovin the recover~of infectious internalized vha~e
To increase the recovery of infectious internalized phage, we studied whether
prevention of lysosomal acidification through the use of chloroquine would
protect
endocytosed phages from endosomal degradation (Barry et al. (1996) Nat. Med.
2: 299-305).
SKBR3 cells were incubated with chloroquine and either C6.5 scFv phagemid or
anti-
botulinum phagemid. Cell lysates were titered at various time points to
determine the
number of intracellular phagemid. C6.5 scFv phagemid were present at the 20
minute time
point and the amount of phagemid was comparable with or without the addition
of
chloroquine. At later time points, approximately twice as much infectious
phagemid was
recovered with the use of chloroquine. In contrast, much lower amounts of anti-
botulinum
phage were present and chloroquine had no effect on the titer, suggesting that
the phagemid
result from non-specific surface binding rather than non-specific endocytosis
into
endosomes. Overall, the results indicate that prevention of lysosomal
acidification increases
the amount of infectious phage recovered for incubations longer than 20
minutes.
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S1 Recovery of internalized nha~e at low nha~e concentrations
Only very large phage antibody libraries containing more than 5.0 x 109
members are capable of generating panels of high affinity antibodies to all
antigens (10, 23,
24). Since phage can only be concentrated to approximately 1013 cfu/ml, a
typical phage
preparation from a large library will only contain 104 copies of each member.
Thus
selection of libraries for endocytosis could only work if phage can be
recovered when
applied to cells at titers as low as 104. We therefore determined the recovery
of infectious
phage from within SKBR3 cells as a function of the phage titer applied. SKBR3
cells were
incubated with C6.5 scFv, C6ML3-9 scFv or C6.5 diabody phagemids or C6.5 scFv
phage
for 2 hours at 37°C. Cells were washed three times with stripping
buffer, trypsinized and
washed twice with PBS. Cells were lysed and intracellular phage titered on E.
coli TG1.
Phage recovery increased with increasing phage titer for all phage studied
(Figure 5). For
monovalently displayed antibodies, phagemid could not be recovered from within
the cell at
input titers less than 3.0 x 105 (C6.5 scFv) to 3.0 x 106 (C6ML3-9 scFv) This
threshold
decreased for bivalent and multivalent display {3.0 x 104 for C6.5 diabody
phagemid and
C6.5 scFv phage).
C7 Discussion
We demonstrate for the first time that phage displaying an anti-receptor
antibody can be specifically endocytosed by receptor expressing cells and can
be recovered
from the cytosol in infectious form. The results demonstrate the feasibility
of directly
selecting internalizing antibodies from large non-immune phage libraries and
identify the
factors that will lead to successful selections. Phage displaying anti-ErbB2
antibody
fragments are specifically endocytosed by ErbB2 expressing SKBR3 cells, can be
visualized
within the cytosol and can be recovered in an infectious form from within the
cell. When
monovalent scFv antibody fragments were displayed monovalently in a phagemid
system,
recovery of internalized phage was only 3.5 to 18 fold above background.
Display of
bivalent diabody or multivalent display of scFv in a phage vector increased
recovery of
internalized phage to 30 to 146 fold above background. This result is
consistent with our
studies of native monomeric C6.5 scFv and dimeric C6.5 diabody as well as
studies of other
monoclonal anti-ErbB2 antibodies where dirneric IgG but not monomeric Fab
dimerize and
activate the receptor and undergo endocytosis (Yarden (1990) Proc. Natl. Acad.
Sci . USA
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87: 2569-2573; Hurwitz et al. (1995) Proc. Natl. Acad. Sci. USA 92: 3353-
3357). In.fact it
is likely that endocytosis of C6.5 and C6ML3-9 scFv phagemids reflect.the
small percentage
of phage displaying two or more scFv (Marks et al. (1992) J. Biol. Chem. 267:
16007-
16010). The importance of valency in mediating either high avidity binding or
receptor
crosslinking and subsequent endocytosis is confirmed by the only other report
demonstrating
specific phage endocytosis. Phage displaying approximately 300 copies of a
high affinity
Arg-Gly-Asp integrin binding peptide on pVIII were efficiently endocytosed by
mammalian
cells (Hart et al. (1994) J. Biol. Chem. 269: 12468-12474). Recovery of phage
after
endocytosis also increases the specificity of cell selections compared to
recovery of phage
from the cell surface. Thus enrichment ratios for specific vs non-specific
surface binding
range from 2 to 20 fold. These values are comparable to the approximately 10
fold
enrichment reported by others for a single round of cell surface selection
(Pereira et al.
(1997) J. Immunol. Meth. 203: 11-24; Watters et al. (1997) Immunotechnology 3:
21-29).
In contrast our enrichment ratios for specific vs non-specific endocytosis
range from 3.5 to
146 fold.
Based on these results, selection of internalizing antibodies from phage
antibody libraries would be most successful with either homodimeric diabodies
in a
phagemid vector or multivalent scFv using a phage vector. While no such
libraries have
been published, there are no technical barriers preventing their construction.
Multivalent
libraries would present the antibody fragment in the form most likely to
crosslink receptor
and undergo endocytosis. Antibodies from such libraries would need to be
bivalent to
mediate endocytosis. Alternatively, monomeric receptor ligands can activate
receptors and
undergo endocytosis, either by causing a conformational change in the receptor
favoring the
dimeric form or by simultaneously binding two receptors. Monomeric scFv that
bound
receptor in a similar manner could also be endocytosed. Thus selection of
libraries of
monovalent scFv in a phagemid vector could result in the selection of ligand
mimetics that
activate receptors and are endocytosed as monomers. Such scFv could be
especially useful
for the construction of fusion molecules for the delivery of drugs, toxins or
DNA into the
cytoplasm. Since antibodies which mediate receptor internalization can cause
receptor down
regulation and growth inhibition (Hurwitz et al. (1995) Proc. Natl. Acad. Sci.
USA 92:
3353-3357; Hudziak et al. (1989) Mol. Cell. Biol. 9: 1165-1172; Stancovski et
al. (1991)
Proc. Natl. Acad. Sci. USA 88: 8691-8698; Lewis et al. (1993) Cancer Immunol.
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Immunother. 37: 255-263), selection for endocytosable antibodies may also
identify
antibodies which directly inhibit or modulate cell growth.
Example 6~ Transfection of Cells.
The FS scFv gene was removed from pHENI-FS by digestion of phagemid
DNA with the restriction enzymes SfiI and NotI. A phage vector based on FdDOGl
(See
prior Ref.), but modified to insert an SfiI site into the gene III leader
sequence, was digested
with SfiI and Notl and the digested FS gene ligated into digested phage Fd
vector DNA.
Recombinant transformant were identified. E. coli containing the F~
recombinant phage
were grown in culture to produce FS-Fd phage (see Maniatis for phage
preparation). FS
phages were then used to infect E. coli harboring a phagemid which contains a
mammalian
promoter (CMV) followed by either the gene for ~-galactosidase
(pcDNA3.1/HisB/LacZ, In
Vitrogen) or the gene for the enhanced green fluorescent protein (pN2EGFP,
Clonetch
plasmid) and a eucaryotic polyadenylation sequence. Bacteria were grown
overnight in the
presence of tetracycline 15 ug/mL and either ampicillin 100 ug/mL
(pcDNA3.1/HisB/LacZ
containing bacteria) or Kanamycine 30 ug/mL (pN2EGFP containing bacteria). The
phage
prepared from the supernatant a mixture of FS-Fd coat contains either the
reporter gene
(about SO% of the phages) in a single strand format or the FS-Fd phage genome
(about 50%
of the phages). Incubation of ErbB2 positive cells 5.105 SKBR3 with 10' pfu
the phage mix
(Filtered twice through a 0.45 nm filter to sterility) allowed expression of
the reporter gene
in 1% of the cells. Cells incubated with an 10 time fold more negative control
phage, i.e.
reporter gene packaging in wild type Fd, showed no expression of the reporter
genes. In an
experiment where a mixed population of ErbB2 high (SKBR3) and ErbB2 low cells
(MCF7)
(Lewis et al. (1993) Cancer Immunol Immunother 37: 255-263) were incubated
with the FS-
Fd-EGFP phages for two days, we obtained the expression of the reporter gene
only in
erbB2 positive cells, cells being differentiated by their ErbB2 level by FACS.
Example 7 Targeted gene delivery to mammalian cells by filamentous
bacteriopha~e
In this example we show that prokaryotic viruses can be re-engineered to
infect eukaryotic cells resulting in expression of a portion of the
bacteriophage genome.
Phage capable of binding mammalian cells expressing the growth factor receptor
ErbB2 and
undergoing receptor mediated endocytosis were isolated by selection of a phage
antibody
library on breast tumor cells and recovery of infectious phage from within the
cell. As
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determined by Immunofluorescence, FS phage were efficiently endocytosed into
100% 9f
ErbB2 expressing SKBR3 cells. To achieve expression of a portion of the phage
genome,
FS phage were engineered to package the green fluorescent protein (GFP)
reporter gene
driven by the CMV promoter. These phage when applied to cells underwent ErbB2
mediated endocytosis leading to GFP expression. GFP expression occurred only
in cells
overexpressing ErbB2, was dose dependent reaching 4% of cells after 60 hours
and was
detected with phage titers as low as 2.0 x 10~ cfu/ml (500 phage/cell). The
results
demonstrate that bacterial viruses displaying the appropriate antibody can
bind to
mammalian receptors and utilize the endocytic pathway to infect eukarotic
cells resulting in
viral gene expression. This represents a novel method to discover targeting
molecules
capable of delivering a gene intracellularly into the correct trafficking
pathway for gene
expression by directly screening phage antibodies. This should significantly
facilitate the
identification of appropriate targets and targeting molecules for gene therapy
or other
applications where delivery into the cytosol is required. This approach can
also be adapted
to directly select, rather than screen, phage antibodies for targeted gene
expression. The
results also demonstrate the potential of phage antibodies as an in vitro or
in vivo targeted
gene delivery vehicle.
Bl Materials and Methods
1) Anti-ErbB2 FS scFv
An anti-ErbB2 scFv (FS) in the vector pI-iEN-1 (Hoogenboorn et al. (1991)
Nucleic Aczds Res. 19(15): 4133-4137) (pHEN-FS) was obtained by selecting a
non-immune
phage antibody library (Sheets et al. (1998) Proc. Natl. Acad. Sci. USA
95(11): 6157-6162)
on ErbB2 expressing SKBR3 cells followed by screening for binding on
recombinant ErbB2
extracellular domain (ECD). The native FS scFv binds ErbB2 ECD with a Kd =1.6
x 10-~
M as determined by surface plasmon resonance in a BIAcore as previously
described (Schier
et al. (1996) J. Mol. Biol. 255(1}: 28-43).
2) Phase and phagemid vectors
pcDNA3-GFP (6.1 Kbp) was obtained by subcloning the Hind IIIlNot I
fragment of pN2EGFP (4.7 Kbp) (Clontech) into the pcDNA3-HisB/LacZ
(Invitrogen) Hind
IIIlNot I backbone. A fd-FS-phage vector was constructed by subcloning the Sfi
IlNot I
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scFv-F5 insert from pHEN-1 into the Sfi IlNot I sites of fd-Sfi/Not
(constructed from fd-.tet- -
DOG (Clackson et al. (1991) Nature 352(6336): 624-628) by changing the ApaLl
cloning
site in the gene III leader to SfiI. The pHEN-F5-GFP phagemid vector (6.8 Kbp)
was
obtained by subcloning the 1.6 Kbp pN2EGFP blunted Ase IlAfl II fragment into
the blunted
EcoR I site of pHEN-F5. The orientation of the insert was analyzed by Not I
restriction
digest.
3) Cell line culture and transfection
SKBR3 and MCF7 were grown in RPMI complemented with 10% fetal
bovine serum (FBS) (Hyclone). 50 % confluent SKBR3 cells grown in 6-well
plates were
transfected with 1 p.g of DNA per well using Lipofectamine (GIBCO BRL) as
recommended
by the manufacturer. pN2EGFP dsDNA was prepared by alkaline lysis using the
Maxiprep
Qiagen Kit (Qiagen Inc.). ssDNA was extracted from 500 ~1 of phagemid
preparation (see
below) by 2 phenol extractions followed by ethanol precipitation. DNA was
quantified by
spectophotometry with 1.0 A26o nrn equal to 40 pg/ml for ssDNA or 50 p.g/ml
for dsDNA.
For GFP detection, cells were detached using a trypsin-EDTA mix (GIBCO BRL)
and
analyzed on a FACScan (Becton Dickinson).
4) Pha~emid and~pha~e preparation
pHEN-F5, pHEN-F5-GFP, pcDNA3-GFP or pN2EGFP phagemids were
prepared from E. coli TG1 by superinfection with VCS-M13 helper phage
(Stratagene) as
previously described (Marks et al. (1991) J. Mol. Biol. 222(3): 581-597). Fd-
F5-phage were
prepared from E. coli TG1 as previously described (McCafferty et al. (1990)
Nature
348(6301): 552-554). FS-GFP-phage and F5-LacZ-phage were prepared by
superinfection
of E coli TG1 containing pcDNA3-GFP with fd-F5-phage. Virus particles were
purified
from the culture supernatant by 2 polyethylene glycol precipitations (Sambrook
et al. (1990).
Molecular cloning a laboratory manual, Cold Spring Harbor Laboratory, New
York)
resuspended in phosphate buffered saline, pH 7.4 (PBS), filtered through a
0.45 ~m filter
and stored at 4°C. Alternatively, the preparations were submitted to an
additional CsCI
ultracentrifugation step (Smith and Scott (1993) Meth. Enzymol. 217: 228-257).
The ratio
of packaged helper phage DNA versus phagemid DNA was determined by titering
(Sambrook et al., supra.) the phage for ampicillin and kanamycin resistance
(for helper
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phage rescued pHEN-F5) or ampicillin and tetracycline resistance (for fd-FS
phage rescued
pcDNA3-GFP)
Pha"ge FACS
Cells were trypsinized, washed with PBS containing 1% FBS (FACS buffer)
S and resuspended at 106 cells/ml in the same buffer. The staining procedure
was performed
on ice with reagents diluted in FACS buffer. One hundred ~1 aliquots of cells
were
distributed in conical-96-well plate (Nunc), centrifuged at 3008 and the cell
pellets
resuspended in 100 p.l of serial dilutions of phage or phagemid preparation
and incubated for
1 hr. Cells were centrifuged and washed twice, the cell pellets resuspended in
100 pl of anti-
M13 antibody (5 Prime, 3 Prime'Inc.) (diluted 1/400) and incubated for 45 min.
Cells were
washed as above, resuspended in 100 ~1 of streptavidin-Phycoerythrin (Jackson
Inc.) (diluted
1/400) and incubated for 20 min. After a final wash, the cells were analyzed
by FACS.
61 Immunotluorescence
Cells were grown on coverslips to 50% confluency in 6 well-plates. Phage
preparation (less than 10% of the culture medium) was added and the cells were
incubated
for 16 hours. The coverslips were washed 6 times with PBS, 3 times for 10 min
with Glycine
buffer (50 mM glycine, pH 2.$, NaCI 500 mM), neutralized with PBS and fixed
with PBS-
4% paraformaldehyde for 5 min at room temperature. Cells were permeabilized
with cold
acetone for 30 sec, saturated with PBS-1% BSA and incubated with anti-M13
antibody (d:
1/300 in the saturation solution) followed by streptavidin-Texas Red
(Amersham) (d: 1/300
in the saturation solution). Coverslips were analyzed with an Axioskop
fluorescent
microscope (Zeiss).
','~ Bacteriouhage mediated cell infection
CsCI phage preparations were diluted at least 10 fold in cell culture medium,
filtered through a 0.45 pm filter and added to 30% to $0% confluent cells.
After incubation,
the cells were trypsinized, washed with FAGS buffer and analyzed for GFP
expression by
FACS. In the experiments where MCF7 and SKBR3 were co-cultured, ErbB2
expression
was quantitated by FACS using the anti-ErbB2 mouse mAb 4D5 which binds ErbB2
ECD
(10 p.g/ml) (1 hr), biotinylated sheep anti-mouse immunoglobulins (Amersham)
and
streptavidin-Phycoerythrin.
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Cl Results - -
11 Internalization of ErbB2 binding monovalent and multivalent F5 nha~e
particles by ErbB2 expressing cells
We isolated the anti-ErbB2 scFv-FS from a library of scFv displayed on the
surface of bacteriophage as fasions to pIII (Sheets et al. (199$) Proc. Natl.
Acad. Sci. USA
95(11): 6157-6162) by selection on ErbB2 expressing SKBR3 breast tumor cells
and
recovery of infectious phage from within the cell (M. Poul et al., manuscript
in preparation).
This selection strategy was employed to select scFv capable of undergoing
endocytosis upon
receptor binding. When the pHEN-FS phagemid vector is rescued with VCS-M13
helper
phage, the resulting virus particles (FS-phagemid) display an average of 1
copy of scFv-pIII
fusion protein and 3 to 4 copies of the wild type pill minor coat protein from
the helper
phage (Marks et al. (1992) J. Biol. Chem. 267(23): 16007-16010). As a result,
the
phagemid bind monovalently. To improve the binding of the virus particles to
ErbB2
expressing cells, multivalent phage antibodies were created by subcloning the
FS scFv DNA
into the phage vector fd-Sfi/Not for fusion with the pIII protein. Virus
particles, referred to
as fd-FS phage, display 4 to 5 copies of scFv-pIII fusion protein (Id.).
To determine whether FS phage antibodies could be internalized by
mammalian cells, SKBR3 cells overexpressing ErbB2 were incubated for 16 hrs
with fd-FS
phage (109 colony forming unit/ml, cfu/ml), FS phagemid (1011 cfu/ml), or with
phagemids
displaying an irrelevant anti~botulinum scFv-pITI fusion protein (1012 cfu/ml)
(Amersdorfer
et al., 1997) as a negative control. The cell surface was stripped of phage
antibodies using
low pH glycine buffer, the cells pezmeabilized and fixed, and intracellular
phage detected
with anti-M13 antibody. Remarkably, all cells showed strong intracellular
staining when
incubated with fd-FS phage or with FS phagemid but not when incubated with the
anti-
botulinum phagemid. This demonstrates the dependence of phage entry on the
specificity of
the scFv fused to pill.
~~ Preparation of ErbB2 bindin~,pha~es and phagemids uacka~ing a reporter
gene for expression in eukarvotic cells
Two strategies were used to investigate whether FS phage could deliver a
reporter gene to mammalian cells leading to expression. To make monovalent
phage
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containing a reporter gene, we cloned the gene for green fluorescent protein
(GFP) driven by
the CMV promoter into the phagemid vector pHEN-F5 generating the vector pHEN-
FS-GFP
(Figure 6, left panel). Escherichia. coli TG1 containing pHEN-FS-GFP
(ampicillin resistant)
were infected with helper phage (kanamycin resistant) and high titers of
monovalent FS-GFP
S phagemids were obtained (5.0 x 101° ampicillin resistant cfu/ml of
culture supernatant). The
ratio of packaged phagemid DNA versus helper phage DNA (ampicillin versus
kanamycin
resistant cfu) was determined to be 100:1. To make multivalent phage
containing a reporter
gene, fd-F~-GFP phage were generated by infecting E. coli TGl carrying the
pcDNA3-GFP
phagemid (ampicillin resistant) with fd-FS phage (tetracycline resistant),
thus using fd-FS
phage as a helper phage. The fd-FS-GFP phage titer was approximately 5.0 x 108
ampicillin
resistant cfu/ml of culture supernatant. Lower phage titers result when fd is
used as a helper
phage because it lacks a plasmid origin of replication leading to interference
from the
phagemid fI origin (Cleary and Ray (1980) Proc. Natl. Acad. Sci. USA 77(8):
4638-4642).
The ratio of packaged reporter gene DNA versus phage DNA (ampicillin versus
tetracycline
resistant cfu) was 1:1. The lower ratio of reporter gene/helper genome when
using fd as a
helper phage is due to the presence of a fully functional packaging signal on
the fd genome
compared to the mutated packaging signal in VCS-M13 (Vieira and Messing (1987)
Meth.
Enzymol. I53: 3-11). Both phage and phagemid preparations were assessed for
SKBR3 cell
binding (Figure 7). While both preparations bound SKBR3 cells, binding could
be detected
with as little as 108 cfu/ml of fd-FS-GFP phage cfu/ml (160 femtomolar)
compared to 1010
cfu/ml of FS-GFP phagemids (15 picomolar). Thus multivalent binding leads to
an increase
in the apparent binding constant of virus particles.
3? Targeted nha~emid and phage mediated ene transfer into ErbB2
expressing breast cancer cells
2S To determine if ErbB2 binding phagemids were capable of targeted gene
delivery, 2.0 x 105 SKBlt3 cells (a breast tumor cell line expressing high
levels of ErbB2) or
2.0 x 105 MCF7 cells (a low ErbB2 expressing breast tumor cell line) were
incubated with
5.0 x 1011 cfu/ml FS-GFP phagemids at 37°C. Cells were analyzed for GFP
expression by
FRCS after 48 hrs (Figure 8A}. 1.37% of the SKBR3 cells expressed GFP after
incubation
with FS-GFP phagemids (Figure 8A6). GFP expression was identical regardless of
the
orientation of the fl packaging signal (data not shown), indicating that
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transcription/translation -was proceeding via synthesis of the complementary
DNA strand. - -
GFP expression was not detected in SKBR3 cells incubated with no phage or with
helper
phage packaging the reporter gene (Figure 8A4 and 8A5). Expression was also
not seen in
MCF7 cells incubated with no phage, helper phage or pHEN-F5-GFP, indicating
the
requirement of ErbB2 expression for targeted gene delivery (Figure 8A1, 8A2
and 4A3).
Since gene transfer applications are likely to involve targeting of specific
cells in an
heterogeneous cell population, we performed the same experiment on a co-
culture of SKBR3
and MCF7 cells (Figure 8B). Cells were stained for ErbB2 expression to
discriminate MCF7
from SKBR3 cells and the GFP expression of each subpopulation was analyzed by
FACS.
Only SKBR.3 cells (1.91%) expressed GFP. Similar results were found using F5-
GFP
phages instead of F5-GFP phagemids (data not shown). These data confirm that
fd-F5-GFP
phage and F5-GFP phagemid mediated gene delivery is restricted to ErbB2
overexpressing
cells and can be targeted to such cells in the presence of non-expressing
cells.
41 Characterization of phase mediated gene transfer
To determine the dose-response characteristics of phage mediated gene
transfer, SKBR3 cells were incubated for 60 hrs with increasing amounts of fd-
F5-GFP
phage or F5-GFP phagemids and the percent of GFP positive cells determined
(Figure 9A
and 9B). The minimal phage concentration required for detection of a
significant number of
GFP positive cells (Figure 9A) was approximately 4.0 x 10~ cfu/ml for fd-F5-
GFP phage
(0.13%) and 1.0 x 101 cfu/ml for F5-GFP phagemid (0.12%). The values correlate
closely
with the binding curves (Figure 7) and indicate that multivalent phage are 100
to 1000 time
more efficient than phagemids in terms of gene expression. No significant
number of
positive cells were observed with up to 4.0 x 1013 cfulml of helper phage
packaging the
reporter gene. For both phage and phagemid, the percent of GFP positive cells
increased
with phage concentration with no evidence of a plateau. The maximum values
achieved
were 2% of cells for fd-F5-GFP phage and 4% for F5-GFP phagemids and appear to
be
limited by the phage titer applied (1.5 x 109 cfu/ml and 4.0 x lOlz cfu/rnl
respectively). The
amount of GFP expressed per cell (estimated by the mean fluorescent intensity
{1VIF~,
Figure 9B) also increased with phage concentration, with a small number of
cells showing
expression with phage titers as low as 2.0 x 10~ cfu/ml (fd-F5-GFP phage) to
1.0 x 1010
cfu/ml (F5-GFP phagemid).
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To compare the yield of gene expression obtained with phage to traditional -._
transfection methods, single stranded (ssDNA) or double stranded (dsDNA) was
transfected
into SKBR3 using lipofectamine. Per p.g of ss DNA, efficiency of phagemid
mediated gene
delivery (approximately 1%) was comparable to lipofectamine transfection of
ssDNA
(0.98%) and dsDNA (1.27%) (Table 10). Efficiency was approximately 500 fold
higher for
phage mediated transfection, with 2.25 ng of ss DNA resulting in transfection
of 0.87% of
cells.
Table 10. Transfection efficiencies in SKBR3 cells.
Transfection Reporter plasmid Amount of reporter % of GFP
method plasmid DNA positive
cells*
FS-phagemid 15 ~g 3.84
Mediated pHEN-FS-GFP 3.1 pg 1.44
0.78 p,g 0.64
fd-FS-phage 5 ng 1.69
mediated pcDNA3-GFP 2.25 ng 0.87
1.25 ng 0.57
Helper phage 100 ~g 0.12
mediated pN2GFP 20 ~g 0.07
5 ~g 0.06
Lipofectamine pN2GFP dsDNA 1 p,g 1.27
ssDNA 1 ~,g 0.98
*Cells were analysed 48 hours after transfection for GFP expression using
FACS. Results
are expressed in % of GFP positive cells. **For phage, the amount of reporter
plasmid was
calculated from the plasmid size and the number of ampicillin (pHEN-FS-GFP or
pcDNA3-
GFP) or kanamycin (pN2GFP) resistant colonies. Mock transfected cells
contained an
average of 0.05% GFP positive cells.
To determine the time course of gene expression, 5.0 x 1011 cfu/ml of FS-GFP
phagemid were incubated with SKBR3 cells. After 48 hrs, the culture medium was
replaced
by fresh medium. GFP expressing cells can be detected within 24 hrs after
phage are applied
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and the percentage of positive cells increases linearly with increasing time
to a maximum of
4.5% by 120 hours (Figure 9C). The GFP content of the positive cells, as
estimated by the
MFI, increases up to 96 hrs (Figure 9D). After 96 hrs, the number of GFP
positive cells
continues to increase but the MFI decreases, probably due to the repartition
of GFP
molecules to daughter cells during cell division.
C~ Discussion
We demonstrate that filamentous phage displaying an anti-ErbB2 scFv
antibody fragment as a genetic fusion with the minor coat protein pIII can be
directly
targeted to mammalian cells expressing the specificity of the scFv. Such phage
undergo
receptor mediated endocytosis and enter an intracellular trafficking pathway
which
ultimately leads to reporter gene expression. This is a remarkable fording
demonstrating that
prokaryotic viruses can be re-engineered to infect eukaryotic cells resulting
in expression of
a portion of the bacteriophage genome. Gene expression was detected with as
few as 2.0 x
107 cfu of phage and increased with increasing phage titer up to 4% of cells.
Multivalent
display decreased the threshold for detectable gene expression approximately
500 fold
compared to monovalent display, most likely due to an increase in the
functional affinity and
an increased rate of receptor mediated endocytosis from receptor crosslinking.
The
maximum percent of cells transfected, however, was higher for monovalent
display
(phagemid) due to the significantly higher phage titer generated. The lower
titer of
multivalent phage is due to interference of the fl origin of replication on
the reporter
phagemid with the fd phage antibody origin of replication (Cleary and Ray
(1980) Proc.
Natl. Acad. Sci. USA 77(8): 4638-4642).
Targeted infection of mammalian cells using phage which bind endocytosable
receptors is likely to be a general phenomenon. For example, fusing an anti-
transferrin
receptor scFv to gene III of pHEN-GFP results in GFP expression in 10% of MCF7
cells,
4% of SKBR3 cells, 1% of LNCaP cells and 1% of primary melanoma cells.
Similarly,
targeted GFP gene delivery to FGF receptor expressing cells using biotinylated
phage and a
streptavidin FGF fusion molecule was recently reported (Larocca et al. (1998)
Hum. Gene
Ther. 9: 2393-2399). However, direct genetic fusion of the targeting molecule
vi.a gene III
may be more efficient than using adapter molecules. Thus while the maximum
percent of
cells transfected using the FGF-adapter molecule was not reported, we estimate
it to be only
0.03% ofFGF expressing L6 rat myoblasts based on the number of cells infected,
the time
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after infection to the measurement of gene expression and the number of cells
expressing
GFP. While a greater frequency of expression (0.5%) was seen in COS-1 cells,
this results
from the presence of large T antigen and SV40 mediated DNA replication and
thus is not
generalizable to most cells.
The approach we describe represents a novel method to discover Iigands for
targeted intracellular drug or gene delivery. Phage antibody or peptide
libraries are first
selected for endocytosis by mammalian cells (Barry et al. {1996) Nat. Med. 2:
299-305) or
for binding to purified antigen, cells, tissues or organs. After subcloning
the selected scFv
genes into the pHEN-GFP vector, phage produced from individual colonies can be
directly
screened for gene expression. This is possible since expression can be
detected with as little
as 1.0 x 101 cfu of phagemids. This permits not only direct identification of
endocytased
scFv but also the subset of receptor antibodies which undergo proper
trafficking for gene
expression. If multivalent display is necessary for efficient endocytosis, the
scFv genes can
be subcloned into fd-Sfi-Not which is then used to rescue the reporter
phagemid. Use of
scFv-fd phage also allows the targeting of a large number of different
reporter genes m
various expression vectors since many commercially available mammalian vectors
contain
fl origins of replication. As such, antibody targeted phage might prove useful
transfection
reagents, especially for cells difficult to transfect by standard techniques.
It may also prove possible to use this approach to directly select, rather
than
screen, antibodies for targeted gene delivery. For example, mammalian cells
are incubated
with a phage antibody library containing the GFP gene, and then sorted based
on GFP
expression using FACS. Phage antibody DNA would be recovered from the
mammalian
cytoplasm by cell lysis and used to transfect E. coli and prepare more phage
for another
round of selection. If the quantities of recoverable phage DNA are inadequate,
inclusion of
the neomycin gene in the pHEN-GFP vector would permit selection of GFP
expressing
mammalian cells using 6418 (Larocca et al. supra).
Finally, this system has promise as a targetable in vitro or in vivo gene
therapy vehicle. The main limitations are infection efficiency,
pharm.acokinetics and
immunogenicity. With respect to infection efficiency, values achieved by
targeted phage in
this report (8.0 x 104/m1 of phage preparation) are not dissimilar to values
reported for
targeted retrovirus (103-105/ml of virus) (Kasahara et al. (1994) Science 266:
1373-1376;
Somia et al. (1995) Proc. Natl. Acad. Sci. USA 92(16): 7570-7574) but less
than reported
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for adenovinus targeting strategies (Douglas et al. (1996) Nat. Biotechnol.
14: 1574-1578;
Watkins et al. 1997) Gene Ther. 4(10): 1004-1012). The factors limiting higher
infection
efficiencies, however, are likely to differ between the systems. Thus while
the percentage of
cells infected by retrovirus is significantly higher than observed for
bacteriophage, infection
is limited by the problems encountered producing large numbers of virus which
can enter the
cell. Since all cells take up the targeted phage, gene expression is limited
by one or several
post-uptake events (e.g. degradation of phage to release DNA, endosomal
escape, nuclear
targeting or transcription). More detailed study of the fate of the phage and
its DNA is likely
to suggest where the block lies permitting engineering of phage to increase
infection
efficiency. For example, endosomal escape could be enhanced by co-
administering
replication defective adenovirus (Carrel et al. (1991) Proc. Natl. Acad. Sci.
USA 88(19):
8850-8854) or incorporating endosomal escape peptides (Wagner et al. (1992)
Proc. Natl.
Acad. Sci. USA 89(17): 7934-7938) or proteins (Fominaya and Wels (1996) J.
Biol. Chem.
271(18): 10560-10568) into the phage major coat protein pVTII. Alternatively,
infection
efficiency could be increased combinatorially by creating scFv targeted
libraries of pVIII
mutants and selecting for increased gene expression. With respect to
pharmacokinetics,
though not extensively studied, it is likely that the biodistribution of phage
is limited to the
intravascular space. This would not affect in vitro phage gene therapy, but
might limit in
vivo uses to those targeting the vasculature. This still leaves numerous
applications
including those where neovascularization plays a role, such as cancer. With
respect to
immunogenicity, it is likely that phage will be immunogenic, thus limiting the
number of
times that phage could be administered in vivo. Alternatively, it might prove
possible to
evolve the major coat protein pViB to reduce or eliminate immunogenicity for
example by
negatively selecting a pVIII library on immune serum (Jenne et al. (I998) J.
Immunol.
161(6): 3161-3168).
It is understood that the examples and embodinnents described herein are for
illustrative purposes only and that various modifications or changes in light
thereof will be
suggested to persons skilled in the art and are to be included within the
spirit and purview of
this application and scope of the appended claims. All publicarions, patents,
and patent
applications cited herein are hereby incorporated by reference in their
entirety for all
purposes.
