Note: Descriptions are shown in the official language in which they were submitted.
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PRODUCTION OF ANTIBODIES IN TRANSGENIC PLASTIDS
RELATED APPLICATIONS
This patent application claims the benefit of U.S. Provisional Application No.
60/185661, filed 2/2912000. This application is herein incorporated by
reference.
TECHNICAL FIELD
This invention relates to compositions and methods for production of
multimeric
roteins, including antibodies, in plants containing transformed plastids.
BACKGROUND
Using transgenic plants to produce industrial or therapeutic biomolecules is
one of the
fastest' developing areas in biotechnology. Recombinant proteins like
monoclonal antibodies,
vaccines, hormones, growth factors, neuropeptides, cytotoxins, serum proteins
and enzymes have
been expressed in nuclear transgenic plants (May et al., 1996).
Plants provide several advantages for the production of therapeutic proteins,
including
lack of contamination with animal pathogens, relative ease bf genetic
manipulation, eukaryotic
protein modification machinery and economical production. Plant genetic
material is
indefinitely stored in seeds, which require little or no maintenance. In
particular, transgenic
plants offer a number of advantages for production of recombinant/monoclonal
antibodies.
Plants have no immune system, therefore only one antibody species is
expressed, and the
absence of mammalian viruses and other pathogens provides maximum safety for
humans and
animals. Some types of monoclonal antibodies, such as secretory IgA (SIgA) can
be produced in
large quantities only in plants (Ma et al., 1995).
The first report of antibodies produced in plants (plantibodies) was published
by Hiatt in
1989 (Hiatt et al., 1989) and subsequently by many others (During et al.,
1990; Ma et al., 1998;
i
Ma et al., 1995; Ma et al., 1994; Verch et al., 1998; Zeitlin et al., 1998).
Sexual crosses between
plants individually expressing immunoglobulin heavy and light chains are the
classical method
to obtain.transgenic plants expressing full length assembled antibody. This
method, however, is
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time consuming. An alternative method is co-transformation with two different
Agt~obacte~ium
strains, one carrying heavy and one carrying light chain, along with two
different selectable
markers, although efficiency of co-transformation is low (De Neve, et al.,
1993). Expression and
assembly of a full-length monoclonal antibody (mAb) in NicotiafZa benthami~aa
plants using a
plant virus vector has also been reported (Verch et al., 1998).
Despite the many attractive features of current plant expression systems,
however, a
maj or limitation in producing antibodies in plants has been their generally
low level of
expression. The highest accumulation levels reported for full-size antibodies
in plants are less
than 1% of total soluble protein (DeNeve et al., 1999; Ma et al., 1994; van
Engelen et al., 1994).
Levels as high as 5% to 6% have been reported for secretory IgA (SIgA) (Ma et
al., 1995) and
for single chain antibodies (ScFv) (Artsaenko et al., 1995; Fiedler et al.,
1997). However, these
numbers probably include non-functional antibody. Our experience with SIgA-
producing plants
(Ma et al, 1995) has taught us that levels of functional antibody in a
recoverable form are much
lower than the total amount of antibody that can be detected by western
blotting. The highest
yield of soluble, functional antibody from transgenic tobacco was 10-80 mg/kg
fresh weight of
transgenic leaves (Ma et al., 1998). This may reflect, in part, an
insolubilization of antibody in
the apoplastic space when secreted from the plant cell. In addition, a
phenomenon known as
post-transcriptional gene silencing may place an upper limit on the expression
of nuclear
transgenes in plants, including antibody genes (Vaucheret et al., 1998; De
Neve et al., 1999;
Wycoff, unpublished results). Novel means of generating very high antibody
expression in
plants are likely to make the commercial use of transgenic plants highly
attractive and
competitive.
Another impediment to producing antibodies in plants is the environmental
concerns of
nuclear genetic engineering. Despite the widespread planting of genetically
engineered crops in
the U.S. (nearly 50% of corn, cotton and soybean planted in the U.S. are now
genetically
modified), environmental concerns have led to wariness and a lack of
acceptance by part of the
public of genetically modified (GM) crops around the world (Daniell, 1999a-d).
One common
environmental concern is the escape of foreign genes through pollen or seed
dispersal, thereby
creating super weeds or causing genetic pollution among other crops. If
significant rates of such
gene flow are generally shown from crops to wild relatives (as high as 38% in
sunflower and
50% for strawberries) there may be cause for serious concern. In addition,
allegations of genetic
pollution among crops have resulted in several lawsuits and shrunk the
European market for
organic produce from Canada from 83 tons in 1994-1995 to 20 tons in 1997-1998
(Hoyle, 1999).
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Another environmental concern expressed recently is the possibility of
toxicity of transgenic
pollen from plants modified to express the insecticidal protein of Bacillus
thuringensis (B.t.) to
non-target insects, including Monarch butterflies (Losey et al., 1999),
although more recent
studies indicate this is not a significant problem (Killer, 1999). Yet another
environmental
concern has been the development of insects resistant to the insecticidal
protein B.t., due to low
levels (sub-lethal) of nuclear expression in transgenic plants (Gould, 1998).
An alternative to nuclear transformation of plants that may address both
productivity and
environmental concerns is the expression of proteins such as antibodies in
plastids. The
advantages of plastids over nuclear transformants have been summarized in
several recent
reviews (Daniell, 1999A-D). Plastids are maternally inherited and axe not
transferred through
pollen (Scott and Wilkinson, 1999). This has been clearly demonstrated using a
herbicide
resistance gene introduced via plastid genetic engineering (Daniell et al.,
1998). Thus gene flow
due to the presence of a transgene in pollen, is not a problem with plastid
transformation. The
plastid is also a protein factory par excellence: most of the protein in a
typical leaf cell is found
in plastids. Hyper-expression of foreign proteins (up to 47% of total soluble
protein) has been
accomplished via plastid genetic engineering (DeCosa et al., 2001).
Comparisons between
nuclear and plastid expression of the same transgene have shown that
expression in plastids
exceeds, by many-fold that from the nucleus. For example, biologically active
recombinant
human somatotropin, including the appropriate disulfide bonds, has recently
been expressed in
plastids at levels of up to 7% of total soluble protein (Staub et al., 2000).
This level of
somatotropin in plastids was 300-fold higher than levels in the best
transgenic plants expressing
somatotropin from a nuclear transgene.
Early investigations in plastid genetic engineering involved introduction of
isolated
plastids expressing foreign genes into protoplasts (Carlson, 1973, DanieIl et
al., 1986, Daniell
and McFadden, 1987). However, after discovery of the Gene Gun, transient
foreign gene
expression in dicots (Daniell et al., 1990, Ye et al., 1990) and monocots
(Daniell et al., 1991)
was followed by stable foreign gene expression. Plants resistant to B.t.
resistant insects (up to
40,000 fold) were obtained by hyperexpression of the cryIIA gene (Kota et al.,
1999). Plants
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were also genetically engineered via the plastid genome to confer herbicide
resistance;
introduced foreign genes were maternally inherited, overcoming the problem of
out-cross with
weeds or other crops (Daniell et al. I998). Plastid genetic engineering has
been used to produce
pharmaceutical proteins (Guda et al., 1999). Plastid genetic engineering is
now extended to
other useful crops (Sidorov et al., 1999; Daniell, I999E). Nevertheless there
has, until now, not
been a demonstration of expression and assembly of an antibody in transgenic
plastids.
Compartmentalization of foreign proteins in plastids facilitates their
purification. Intact
plastids are easy to isolate from crude homogenates by low-speed
centrifugation and may be
burst open by osmotic shock to release foreign proteins that are
compartmentalized within
(Daniell and McFadden, 1987). Another advantage of plastids is that they can
efficiently
translate polycistronic messages (Daniell et al., 1994). Antibody heavy and
Light chains (and
ofiher proteins if desired) can be introduced into a single site in the
plastid genome, although
functional expression of multimeric proteins have not been shown until the
present invention.
Plastids do not glycosylate their proteins. Although glycosylation is required
for
complement binding and effector function for some antibodies in serum, the
effectiveness of
antibodies at mucosal surfaces does not appear to involve glycosylation. Many
single chain Ab
fragments (scFv) and Fab's entirely lacking the constant regions of Ab
molecule where
glycosylation occurs bind to their appropriate antigen with the same affinity
as the native Ab
(Owen et al., 1992; Skerra et al., 1991; Skerra and Pluckthun, 1988). Non-
glycosylated full-
length antibodies bind to their appropriate antigen with the same affinity as
the native Ab {Boss
et al., 1984). Antibodies made in plastids may have advantages for parenteral
(injectable) uses,
since they will not carry the potentially immunogenic plant N-linked glycans
found on nuclear-
encoded plantibodies.
In summary, he plastid genome is thus an attractive target for introduction
and expression
of antibody genes. The reasons include: 1) capacity for extraordinarily high
levels of foreign
protein expression, 2) ability to fold, process and assemble eukaryotic
proteins, 3) simpler
purification, 4) containment of foreign genes through material inheritance and
5) no
glycosylation.
Despite the potential advantages of plastids for antibody production, it was
~zot obvious
that antibodies expressed i~t plastids would assemble in tl:is orga~zelle.
Assembled antibody
was detected in plastids of transgenic tobacco (During et al., 1990), but the
plastids themselves
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were not transformed and neither heavy nor light chain of the antibody could
be recovered from
the cell. Prior to this patent application there were no published reports of
expression of
antibodies in plastids, and there were valid reasons to suggest that it would
be problematic. In
mammalian plasma cells the immunoglobulin light and heavy chains, encoded by
nuclear genes,
are synthesized as precursor proteins containing an amino-terminal signal
peptide that guides the
chains into the lumen of the endoplasmic reticulum (ER). The signal peptide is
cleaved off in the
ER and stress proteins such as BiP/GRP78 and GRP94, which function as
chaperonins, bind to
unassembled light and heavy chains and direct their folding and assembly
(Gething and
Sambrook., 1992; Melnick et al., 1992). Disulfide bond formation is catalyzed
by protein
disulfide isomerase and N-linked glycans are attached in the ER and further
processed in the
Golgi, before the antibody is secreted from the cell.
This process appears to be broadly similar in nuclear transgenic plants (Hiatt
et al.,
1989), where homologues to the chaperonins BiP and GRP94 have been reported
(Fontes et al.,
1991; Walther-Larsen et al., 1993). Even so, there was no certainty that
antibody heavy and light
chains would assemble normally in plastids, or that they would retain their
antigen-binding
activity. There might have been unforeseen deleterious effects of high-level
expression of
antibodies in plastids on plant growth or development that were not apparent
from the
experiences with other transgenes. The pH and oxidation state of the plastid
differs from that of
the ER in ways that might inhibit or prevent antibody folding and assembly.
On the other hand, it has been known for some time that disulfide bonds exist
both within
(Ferri et al., 1978) and between some plastid proteins (Ranty et al., 1991;
Schreuder et al., 1993;
Drescher et al., 1998). Both nuclear and plastid encoded proteins are
activated by disulfide bond
oxidation/reduction cycles using the plastid thioredoxin system (Ruelland and
Miginiac-Mallow,
1999) or plastid protein disulfide isomerase (Kim and Mayfield, 1997).
Chaperonin molecules
of the HSP70 and HSP60 families, including the rubisco binding protein, have
also been reported
in plastids (Roy, 1989; Vierling, 1991). These molecules function in the
folding 'and assembly of
eukaryotic (nuclear) and~prokaryotic (plastid) proteins. We hypothesized that
they would be able
to assist in the proper assembly of immunoglobulin chains in plastids.
There are examples of protein complexes in the plastid in which all the
subunits are
native to the plant, the ribosome being an example. However, the expression
and assembly in
transformed plastids of heterologous proteins into multi-protein complexes has
not been reported
until the present invention. There is a single example in the literature of an
inter-chain disulfide
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bond in plant plastids, and that is between neighboring large subunits of the
enzyme ribulose-1,
5-biphosphase carboxylase/oxygenase (Ranty et al., 1991). The expression and
assembly in
transformed plastids of functional proteins consisting of different protein
chains, including
disulfide bonds between different subunits, as represented by expression and
assembly of a
mammalian antibody has never been demonstrated until the present invention.
SUMMARY OF THE INVENTION
The present invention provides compositions and methods for the transformation
of
plastids of plant cells with multiple genes, and proper association or
assembly of multimeric
proteins that are heterologous to be plastids of plant cells. A plasmid
construct encoding all of
the individual polypeptide components of the multimeric protein is used.
Typically, the plasmid
used in the invention is made as an "expression cassette" which includes
regulatory sequences.
For example an expression cassette might include, operationally joined, DNA
sequences coding
for immunoglobulin heavy and light chains separated by a small linker
containing an intervening
stop codon and ribosome binding site, and control sequences positioned
upstream from the 5'
and downstream from the 3' ends of the coding sequences to provide expression
of the coding
sequences in the plastid genome. Flanking each side of this expression
cassette would be DNA
sequences that are homologous to a sequence of the target plastid genome.
Stable integration of
the heterologous coding sequences into the plastid genome of the target plant
is accomplished
through homologous recombination. The present invention achieves assembly of
immunoglobulin heavy and light chains, with covalent bonding between the
chains, into
immunologically active irmnunoglobulins in the plastid.
Alternatively, the expression cassette may include, operationally joined, DNA
sequences
coding for J chain and Secretory Components separated by a small linker
containing an
intervening stop codon and ribosome binding site, and control sequences
positioned upstream
from the 5' and downstream from the 3' ends of the coding sequences to provide
expression of
these coding sequences in the plastid genome. Homologous flanking sequences
that rnay be the
same as or different than the ones provided for the expression cassette
containing the
immunoglobulin heavy and light chains are similarly provided for this
cassette. In addition to
assembly of the immunologically active immunoglobulins in the plastid,
Secretory Component
and J chain are also assembled with the immunoglobulin, when the heavy chain
is an oc (alpha)
chain thereby producing secretory immunoglobulin A (SIgA).
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The antibodies produced by the present invention are antibodies which are
useful for
mammals, including animals and human, where it is generally accepted in the
art to use
antibodies in therapy.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Construction of the pLD-TP-Guy's 13 vector and PCR analysis of
spectinomycin-resistant tobacco clones transformed with pLD-TP-Guy's 13. A.
PCR analysis
to show integration of the aadA gene, using the 3P and 3M primer pair. B. PCR
analysis to
show integration of the H and L immunoglobulin genes, using the SP and 2M
primer pair. C.
The plastid vector pLD-TP-Guy's 13 and primer annealing sites. Lane 1, I kb
ladder; Lane 2,
negative control without template; Lane 3, negative control untransformed
plant; Lanes 4-6,
transformed plants; Lane 7, the plasmid pLD-TP-Guy's 13.
Figure 2A: Construction of the pZS-TP-Guy's 13 vector and PCR analysis of
spectinomycin resistant clones transformed with pZS-TP-Guy's 13. A. PCR
analysis of
spectinomycin-resistant tobacco clones using 8P and 8M primer pair. B. PCR
analysis of
spectinomycin-resistant tobacco clones using 7P and 8M primer pair. C. The
plastid pZS-TR
Guy's I3 and primer annealing sites. Lane l, 1 kb ladder; Lane 2, negative
control without
template; Lane 3, negative control untransformed plant; Lane 4, positive
control previously
' characterized pZS-transformed plant; Lane 5, mutant clone; Lanes 6-10,
transformed clones;
Lane 11, the plasmid pZS-TP-Guy's 13.
Figure 3. Western blot analysis of antibody light chain expression in E. coli
by the
tobacco and universal vectors: Lane 1, molecular weight markers; Lane 2,
negative control
(insert in the wrong orientation); Lane 3A, XL1-Blue cells transformed with
the pZS-TP-Guy's
13 vector; Lane 4A, negative control (untransformed XL1-Blue cells); Lane 3B,
positive control
Human IgA; Lane 4B, XL1-Blue cells transformed with the pLD-TP-Guy's 13
vector. Blots
were probed with AP-conjugated goat anti-human kappa antibody.
Figure 4. Western, blot analysis of antibody heavy chain expression in E. coli
by the
tobacco vector. Lane 1, molecular weight markers; Lane 2, negative control
(insert in the wrong
orientation); Lane 3, negative control (untransformed XLI-Blue cells); Lane 4,
XL1-Blue cells
transformed with the pZS-TP-Guy's 13 vector. Samples in blot A were sonicated,
and those in
blot B were boiled. Blots were probed with AP-conjugated goat anti-human IgA
antibody.
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Figure 5. Steps in plastid transformation and regeneration of plastid
transgenic plants.
Figure 6. Western blot analysis of antibody expression in Tobacco plastids. A.
Lane 1,
molecular weight markers; Lanes 2-4, extracts from different transgenic
plants; Lanes 5 and 7,
blank, Lane 6, negative control extract from an untransformed plant; Lane 8,
positive control
human IgA. The gels were run under non-reducing conditions. Blot A was
developed with AP-
conjugated goat anti-human kappa antibodies. Blot B was developed using AP-
conjugated goat
anti-human IgA antibodies.
Figure 7. Western blot analysis of transgenic lines showing the assembled
antibody.
Lanes 1 and 2, extracts from transgenic plants; Lane 3, negative control
extract from an
untransformed plant; Lane 4 positive control human IgA. The gel was run under
non-reducing
conditions, and the blot was developed with AP-conjugated goat anti-human
kappa antibody.
Figure 8. Southern blot analysis of the clones transformed with the pZS-TP-
Guy's 13
vector. Lane C, control untransformed Petit Havana; Lanes 1-6, transgenic
lines.
Figure 9. Southern blot analysis of the clones transformed with the pLD-Guy's
13
vector. Lane C, control untransformed Petit Havana; Lanes 1-6, transgenic
lines.
Figure I0. Northern Blot analysis of light chain transcripts in the transgenic
lines
transformed with the pZS-TP-Guy's 13 and the pLD-TP Guy's 13 vectors A. RNA
gel before
transfer. B. RNA blot probed with radiolabelled light chain DNA probe. Lane 1,
RNA ladder;
Lane 2, control untransformed Petit Havana; Lanes 3-5, transgenic lines
transformed with pZS-
TP-Guy's 13; Lanes 6 and 7, transgenic lines transformed with pLD-TP-Guy's 13;
Lane 8, post-
transcriptionally silenced nuclear transformant CAR8841; Lane nine, expressing
nuclear
transformant CARS I7.
Figure 11. Northern Blot analysis of heavy chain transcripts in the transgenic
lines
transformed with the pZS-TP-Guy's 13 and pLD-TP Guy's 13 vectors. A. RNA gel
before
transfer. B. RNA blot probed with radiolabelled heavy chain DNA probe. Lane 1,
RNA ladder;
Lane 2, control untransformed Petit Havana; Lanes 3-5, transgenic lines
transformed with pZS-
TP-Guy's 13; Lanes 6 and 7, transgenic lines transformed with pLD-TP-Guy's 13;
Lane 8, post-
transcriptionally silenced nuclear transformant CAR8841; Lane 9, expressing
nuclear
transformant CAR517; Lane 10, expressing nuclear transformant CAR532.
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MODES FOR CARRYING OUT THE INVENTION
Throughout this disclosure, various publications, patents and published patent
specifications are referenced by an identifying citation. The disclosures of
these publications,
patents and published patent specifications are hereby incorporated by
reference into the present
disclosure to describe more fully the state of the art to which this invention
pertains.
Definitions
The practice of the present invention will employ, unless otherwise indicated,
conventional techniques of immunology, molecular biology, microbiology, cell
biology and
recombinant DNA, which are within the skill of the art. See, e.g., Sambrook,
Fritsch and
Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989);
CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F.M. Ausubel, et al, eds., (1987));
the
series METHODS IN ENZYMOLOGY (Academic Press, Inc.); PCR 2: A PRACTICAL
APPROACH (M.J. MacPherson, B.D. Hams and G.R. Taylor eds. (1995)); Harlow and
Lane,
eds (1988) ANTIBODIES: A LABORATORY MANUAL, and METHODS IN MOLECULAR
BIOLOGY vol. 49, "PLANT GENE TRANSFER AND EXPRESSION PROTOCOLS," H.
Jones (1995).
As used in the specification and claims, the singular form "a," "an," and
"the" include
plural references unless the context clearly dictates otherwise. For example,
the term "a cell"
includes a plurality of cells, including mixtures thereof.
A "variable region" of an antibody refers to the variable region of the
antibody's light
chain or the variable region of the heavy chain either alone or in
combination.
As used herein, a "polynucleotide" is a polymeric form of nucleotides of any
length
which contain deoxyribonucleotides, ribonucleotides, and/or their analogs. The
terms
"polynucleotide" and "nucleotide" as used herein as used interchangeably.
Polynucleotides may
have any three-dimensional structure and may perform any function, known or
unknown. The
term "polynucleotide" includes double-, single-stranded, and triple-helical
molecules. Unless
otherwise specified or required, any embodiment of the invention described
herein that is a
polynucleotide encompasses both the double-stranded form and each of two
complementary
single-stranded forms known or predicted to make up the double stranded form.
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The term "polypeptide" is used in its broadest sense to refer to a compound of
two or
more subunit amino acids. The subunits may be linked by peptide bonds. As used
herein the
term "amino acid" refers to natural and/or unnatural or synthetic amino acids,
including glycine
and both the D and L optical isomers. A peptide of three or more amino acids
is commonly
called an oligopeptide if the peptide chain is short. If the peptide chain is
long, the peptide is
commonly called a polypeptide or a protein.
A "multimeric protein" as used herein refers to a globular protein containing
more than
one separate polypeptide or protein chain associated with each other to form a
single globular
protein in vitro or ih vivo. The multimeric protein may consist of more than
one polypeptide of
the same kind to form a homodimeric or homotrimeric protein; the multimeric
protein may also
be composed of more than one polypeptide having distinct sequences to form,
e.g., a heterodimer
or a heterotrimer. Non-limiting examples of multimeric proteins include
immunoglobulin
molecules, receptor dimer complexes, trimeric G-proteins, and any enzyme
complexes.
An "immunoglobulin molecule" or "antibody" is a polypeptide or multimeric
protein
containing the immunologically active portions of an immunoglobulin heavy
chain and
immunoglobulin light chain covalently coupled together and capable of
specifically combining
with antigen. The immunoglobulins or antibody molecules are a large family of
molecules that
include several types of molecules such as IgD, IgG, IgA, secretory IgA
(SIgA), IgM, and IgE.
The term "immunoglobulin molecule" includes for example hybrid antibodies or
altered
antibodies and fragments thereof, including but not limited to Fab fragments)
and single-chain
variable fragments (ScFv).
An "Fab fragment" of an immunoglobulin molecule is a multimeric protein
consisting of
the portion of an immunoglobulin molecule containing the immunologically
active portions of an
immunoglobulin heavy chain and an immunoglobulin light chain covalently
coupled together
and capable of specifically combining with an antigen. Fab fragments can be
prepared by
proteolytic digestion of substantially intact immunoglobulin molecules with
papain using
methods that are well known in the art. However, a Fab fragment may also be
prepared by
expressing in a suitable host cell the desired portions of immunoglobulin
heavy chain and
immunoglobulin light chain using methods disclosed herein or any other methods
known in the
art.
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An "ScFv fragment" of an immunoglobulin molecule is a protein consisting of
the
immunologically active portions of an immunoglobulin heavy chain variable
region and an
immunoglobulin light chain variable region covalently coupled together and
capable of
specifically combining with an antigen. ScFv fragments are typically prepared
by expressing a
suitable host cell the desired portions of immunoglobulin heavy chain variable
region and
immunoglobulin light chain variable region using methods described herein
and/or other
methods known to artisans in the field.
"Secretory component" is a fragment of an immunoglobulin molecule comprising
secretory IgA as defined in US Patent No. 5,202,422 and US Patent No.
5,959,177, incorporated
here by reference.
"J chain" is a polypeptide that is involved in the polymerization of
immunoglobulins and
transport of polymerized immunoglobulins through epithelial cells. J chain is
found in
pentameric IgM and dimeric IgA and typically attached via disulfide bonds.
A "protection protein" is a fragment of an immunoglobulin molecule comprising
secretory IgA as defined in US Patent No. 6,046,037, incorporated herein by
reference.
"Heterologous" means derived from a genotypically distinct entity from that of
the rest of
the entity to which it is compared. For example, a polynucleotide introduced
by genetic
engineering techniques into a different cell is a heterologous polynucleotide
(and, when
expressed, can encode a heterologous polypeptide). In particular, the term
"heterologous" as
applied to a multimeric protein means that the multimer is expressed in a host
cell that is
genotypically distinct from the host cell in which the multimer is normally
expressed. For
example, the exemplified human IgA multimeric protein is heterologous to a
plant cell.
The term "immunologically active," as used herein, refers to an immunoglobulin
molecule having structural, regulatory, or biochemical functions of a
naturally occurring
molecule expressed in its native host cell. For instance, an immunologically
active
immunoglobulin produced in a plant cell by the methods of this invention has
the structural
characteristics of the naturally occurring molecule, and/or exhibits antigen
binding specificity of
the naturally occurring antibody that is present in the host cell in which the
molecule is normally
expressed.
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A "gene" refers to a polynucleotide containing at least one open reading frame
that is
capable of encoding a particular protein after being transcribed and
translated.
As used herein, "expression" refers to the process by which polynucleotides
are
transcribed into mRNA and/or the process by which the transcribed mRNA is
subsequently
translated into polypeptides or proteins.
The term "construct" or "vector" refers to an artificially assembled DNA
segment to be
transferred into a target plant tissue or cell. Typically, the construct will
include the gene or
genes of a particular interest, a marker gene and appropriate control
sequences. The term
"plasmid" refers to an autonomous, self replicating extrachromosomal DNA
molecule. In a
preferred embodiment, the plasmid constructs of the present invention contain
sequences coding
for heavy and light chains of an antibody. Plasmid constructs containing
suitable regulatory
elements are also referred to as "expression cassettes." In a preferred
embodiment, a plasmid
construct can also contain a screening or selectable marker, for example an
antibiotic resistance
gene.
The term "selectable marker" is used to refer to a gene that encodes a product
that allows
the growth of transgenic tissue on a selective medium. Non-limiting examples
of selectable
markers include genes encoding for antibiotic resistance, e.g., ampicillin,
kanamycin, or the like.
Other selectable markers will be known to those of skill in the art.
A "glycosylation signal sequence" is a three-amino acid sequence within a
polypeptide,
of the sequence N-X-S/T, where N is asparagine, X is any amino acid (except
proline), S is
serine, and T is threonine. The presence of this amino acid sequence on
secreted proteins
normally results, within the endoplasmic reticulurn, in the covalent
attachment of a carbohydrate
group to the asparagine residue.
A "primer" is a short polynucleotide, generally with a free 3' OH group, that
binds to a
target or "template" potentially present in a sample of interest by
hybridizing with the target, and
thereafter promoting polymerization of a polynucleotide complementary to the
target. A
"polymerise chain reaction" ("PCR") is a reaction in which replicate copies
are made of a target
polynucleotide using a "pair of primers" or a "set of primers" consisting of
an "upstream" and a
"downstream" primer, and a catalyst of polymerization, typically a thermally-
stable DNA
polymerise enzyme. Methods for PCR are well known in the art and taught for
example in
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MacPherson, et al. PCR: A Practical Approach (IRL Press at Oxford University
Press (1991)).
All processes of producing replicate copies of a polynucleotide such as PCR or
gene cloning are
collectively referred to herein as "replication."
"Hybridization" refers to a reaction in which one or more polynucleotides
react to form a
complex that is stabilized via hydrogen bonding between the bases of the
nucleotide residues.
The hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein
binding, or in any
other sequence-specific manner. The complex may comprise two strands forming a
duplex
structure three or more strands forming a mufti-stranded complex, a single
self hybridizing
strand, or any combination of these. A hybridization reaction may constitute a
step in a more
extensive process, such as the initiation of a PCR reaction or the enzymatic
cleavage of a
polynucleotide by a ribozyme.
When hybridization occurs in an antiparallel configuration between two single-
stranded
polynucleotides, the reaction is called "annealing" and those polynucleotides
are described as
"complementary." A double-stranded polynucleotide can be "complementary" or
"homologous"
to another polynucleotide if hybridization can occur between one of the
strands of the first
polynucleotide and the second.
As used herein, "homologous recombination" refers to a process whereby two
homologous double-stranded polynucleotides recombine to form a novel
polynucleotide.
A "transgenic plant" refers to a genetically engineered plant or progeny of
genetically
engineered plants. The transgenic plant usually contains material from at
least one unrelated
organism, such as a virus, another plant or animal.
A "control" is an alternative subject or sample used in an experiment for
comparison
purpose. A control can be "positive" or "negative." For example, where the
purpose of the
experiment is to determine the presence of an exogenously introduced plasrnid
or the expression
of a polypeptide encoded by such plasmid in a plant transformant or its
progenies, it is generally
preferable to use a positive control (a plant or a sample from a plan,
carrying such plasmid and/or
expressing the encoded protein), and a negative control (a plant or a sample
from a plant lacking
the plasmid of interest and/or expression of the polypeptide encoded by the
plasmid).
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"Guy's 13" is a monoclonal antibody against the surface antigen I/II of
Streptococcus
mutans and is described in US Patent No. 5,518,721 and PCT/US95/16889
incorporated herein
by reference.
The term "Humanized," as used herein, refers to a construct in which coding
sequences
for heavy and light chain variable regions from a species other than human
have been fused, via
genetic engineering to the coding sequences of the respective constant regions
of human heavy
and light chains. It also refers to the resulting antibodies.
"Colon optimization" is the process of customizing a transgene so that it
matches the
bias of highly expressed genes in the genome in which it is to be expressed.
For most amino
acids there are two or more (up to six) different colons that can be used in
mRNA. Every
genome has a "bias" in the colons it uses, especially for highly expressed
proteins. Changing
the colon usage of a heterologous gene has been shown in many systems to
increase the
expression of that gene.
As used herein an "operative ligand" is a polypeptide sequence that
functionally interacts
with or binds to another protein, polypeptide, carbohydrates or nucleic acid
for a preferred
function. Non-limiting examples of an operative ligand would be ICAM-1, which
binds to
human rhinovirus, or an ScFv that binds to a particular epitope.
Usefulness of the Invention:
Treatment of disease with antibodies is known as passive immunotherapy. This
is
distinguished from active immunotherapy, where vaccination stimulates the
body's own
antibody response. The efficacy of passive immunotherapy has been demonstrated
in treatment
of a number of infectious diseases, in both animals and humans. A major
impediment to the
commercialization of many types of passive immunotherapy is the need for
repetitive delivery of
large amounts of antibody to the site of the disease to overcome rapid
clearing of the antibodies
from the body. The production of antibodies by traditional methods is much too
expensive to be
practical for many types of passive immunotherapy. This is why production in
plastids is such
an attractive alternative.
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For topical, enteric and mucosal use, secretory IgA (SIgA) is the preferred
antibody
isotype. SIgA is the most abundant immunoglobulin found in the body and the
most important
form found in mucosal secretions, such as saliva, tears, breast milk and mucus
of the bronchial,
genitourinary, and digestive tracts (Kerr, 1990). It is composed of 10
polypeptides: 4 light
chains, four IgA heavy chains, a J chain and a secretory component (SC),
resulting in a total
molecular weight of 400 kDa. Binding of SIgA to bacterial and viral surface
antigens prevents
attachment of pathogens to the mucosal cells, and, once attachment is blocked,
viral infection
and bacterial colonization is inhibited.
SIgA has demonstrated superiority over other antibodies for use in passive
mucosal
immunotherapy. It is more protease resistant than IgG or IgA, thus making it
more stable in the
gastrointestinal tract (Brown et al., 1970; Crottet and Corthesy, 1998,
Renegar et al., 1998) and
buccal mucosa (Ma et al., 1998). Recent work at Planet demonstrated that in
the presence of
pepsin at pH 2.5, antigen binding of an IgG antibody lasted 5 minutes versus 5
hours for the
same antibody prepared as an SIgA plantibody. Such stability will be an
important feature of
antibodies used for the treatment of gastrointestinal tract infections, such
as rotavirus and
Clostf°idium difficile. SIgA has twice as many binding sites than IgG,
thus giving it an additional
advantage where avidity is important. The superiority of SIgA over IgG or IgA
has been
demonstrated in a number of studies: 1) SIgA protected mice against group A
Streptococci, but
serum did not, even though the IgG had a higher titer by ELISA and opsonized
cells more
effectively in a mouse model (Bessen and Fischetti., 1988); 2) Mice were
protected against
influenza virus by intravenous injection of polymeric IgA (which was
transported into nasal
secretions as SIgA) while IgGl and monomeric IgA were ineffectual (Renegar and
Parker,
1991); and 3) Anti gp160 SIgA blocked transcytosis of HIV in human cells
better than IgG,
despite having lower specific activity (Hocini et al., 1997).
Plastid Transformation Vectors:
Antibody expression in transgenic tobacco was accomplished using two plastid
expression vectors pLD and pZS, as shown in Figures 1C and 2C. Both plastid
vectors contain
the 16S rRNA promoter (Pf°rh) driving the selectable marker gene aadA
(aminoglycoside
adenylyl transferase, conferring resistance to spectinomycin) followed by the
psbA 3' region
(the terminator from a gene coding for photosystem II reaction center
components) from the
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tobacco plastid genome. The only difference between these two plastid vectors
is the site of
integration of foreign genes into the plastid genome. The tobacco vector (pZS)
integrates the
aadA gene into the spacer region between rbcL (the gene for the large subunit
of RuBisCo) and
orf512 (the accD gene) of the tobacco plastid genome. This vector is useful
for integrating
foreign genes specifically into the tobacco plastid genome; this gene oxder is
not conserved
among other plant plastid genomes. On the other hand, the universal plastid
expression/integration vector (pLD) uses tr>zA and tr>zI genes (plastid
transfer RNAs coding for
alanine and isoleucine), from the inverted repeat region of the tobacco
plastid genome, as
flanking sequences for homologous recombination. This vector can be used to
transform plastid
genomes of several other plant species (Daniell et al. 1998) because the
flanking sequences are
highly conserved among higher plants. Because the universal vector integrates
foreign genes
within the Inverted Repeat region of the plastid genome, it should double the
copy number of
antibody genes (from 5,000 to 10,000 copies per cell in tobacco). Furthermore,
it has been
demonstrated that homoplasmy is achieved even in the first round of selection
in tobacco
probably because of the presence of a plastid origin of replication within the
flanking sequence
in the universal vector (thereby providing more templates for integration).
Because of these and
several other reasons, foreign gene expression was shown to be much higher
when the universal
vector was used instead of the tobacco vector (Guda et al. 2000).
EXAMPLES
The following examples are intended to illustrate, but not limit, the scope of
the
invention.
Example #1. An IAA Antibody Against a Bacterial Surface Protein Expressed in
Plastids
A. Preparation of Antibody Heavy and Light Chain Expression Cassette
For the first antibody to be expressed in plastids, we chose to use the
binding region of a
murine Mab known as "Guy's 13" (discovered at Guy's Hospital, London), which
recognizes
the 185 kDa surface antigen of Streptococcus niuta>zs, the bacteria that
causes cavities (Smith
and Lehner, 1989). Short-term passive immunotherapy with Guy's 13 was shown to
eliminate
these cariogenic bacteria for periods of up to two years (Ma and Lehner,
1990). The potential
worldwide market for this ozze antibody zzzay approach several billion dollars
per year, azzd
require azztibody produced izzexpezzsively azzd izz large quantities.
Planet Biotechnology scientists have recently constructed humanized versions
of the Guy's 13
antibody for plant nuclear expression. The preferred heavy chain construct
consists of the
Guy's 13 heavy chain variable region fused to the human IgA2m(2) constant
region. This
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heavy chain sub-isotype is resistant to the bacterial proteases that
specifically target IgAl (I~err,
1990). The light chain construct is a fusion of the Guy's 13 kappa chain
variable region and the
human kappa constant region. Expression of these two immunoglobulin chains,
along with
human J chain and human SC have resulted in the assembly in transgenic tobacco
of a
humanized Guy's 13 SIgA plantibody, which we call CaroRx.
To prepare the humanized Guy's 13 heavy and light chain genes for plastid
transformation, coding sequences were amplified, using PCR, from expression
cassettes
designed for nuclear expression. To facilitate sub-cloning, primers were
engineered to
incorporate a ribosome binding site utilized by the plastid protein
translation machinery, and a
methionine codon (in place of the signal peptides found in the nuclear
expression constructs). H
and L chain PCR products were individually cloned into the vector pCR-Script
(Stratagene) and
and their sequences verified.
Both clones were cut with BafnH I, creating cohesive ends at the 3' end of the
H chain
and at the 3' and S' ends of the L chain, resulting in excision of the L
chain. The L chain
fragment was ligated adjacent to the 3' end of the H chain (with an
intervening stop codon and
ribosome binding site) yielding a vector, pCR-ScriptGuy's 13, that contained
both, H and L
chain fragments.
The sequence of the expression cassette between the two Xba I sites in pLD-TP-
Guy's 13
is shown in Table 1. Nucleotides 1-16 comprise linker sequences and a ribosome
binding site.
Nucleotides 17-1381 comprise a sequence encoding a mouse heavy chain
variable/human
IgA2m(2) constant hybrid with linker sequences. The native mouse signal
peptide has been
2S replaced with methionine (nt 17-19). The heavy chain variable region (nt 20-
358) is from the
murine monoclonal Guy's 13 (Smith and Lehner, 1989; US Patent No. S,S 18,721
and 5,352,446,
herein incorporated by reference). The sequence of the human IgA2m(2) constant
region (nt
359-1381) has been previously published (Chintalacharuvu et al 1994).
Nucleotides 1382-1408
comprise stop codon, linker sequences and a ribosome binding site. Nucleotides
1409-2050
comprise a sequence encoding a mouse light chain variable/human kappa constant
hybrid with
linker sequences. The native mouse signal peptide has been replaced with
methionine (nt 1409-
1411). The light chain variable region (nt 1412-1731) is from the murine
monoclonal Guy's 13
(Smith and Lehner 1989; US Patent No. 5,S 18,721 and S,3S2,446). The sequence
of the human
kappa constant region (nt 1732-2050) has been previously published (Hieter et
al. 1980).
3S
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The pCR-ScriptGuy's 13 vector was digested with Xba I to excise the H/L chain
insert,
and the insert was ligated with Xba I-digested and dephosphorylated pLD vector
(CTniversal
vector). The resulting plasmid was designated as pLD-TP-Guy's 13 (Figure 1).
The sequences
encoded are chimeric, consisting of mature variable regions from Guy's 13
heavy and light
chains fused to the constant regions of human IgA2m(2) heavy chain and kappa
light chain, A
separate sample of the pCR-ScriptGuy's 13 vector was digested with Spe I to
excise the H/L
chain insert, and the insert was ligated with Spe I-digested and
dephosphorylated pZS vector
(Tobacco vector; Figure 2). The resulting plasmid was designated as pZS-TP-
Guy's 13.
B. Expression of pLD-TP-Guy's 13 and pZS-TP-Guy's 13 in E. coli:
Since the transcriptional and translational machinery of the plastid is
similar to the
transcriptional and translational machinery of E. coli (Brixey et al., 1997),
it is possible to check
the expression of Guy's 13 construct in E. coli. The transcriptional
efficiency of the 16S
promoter is as good as the transcriptional efficiency of the T7 promoter in E.
coli (Brixey et al.,
1997, Guda et al., 2000). E. coli XLl Blue MRF TC cells were transformed with
pLD-TP-
Guy's 13 and pZS-TP-Guy's 13 vectors, and were selected on LB medium with
ampicillin (100
~g/mL). Transformed colonies were tested for the presence of the correct
coding sequence
insert by plasmid isolation and restriction digestion.
In one set of experiments, E. coli cells were lysed in TBS buffer (20 mM Tris-
HCI, pH 8,
150 mM NaCI) containing 2mM PMSF by sonication. Lysates were boiled for 5 min
with an
equal volume of 2X sample buffer [3.55 mL deionized water, 1.25 mL 0.5 M Tris-
HCI, pH 6.8,
2.5 mL glycerol, 2.0 mL 10% (w/v) SDS, 0.2 mL 0.5% (w/v) bromophenol blue] and
electrophoresed on 12% polyacrylamide gels according to the standard
procedure. In the other
set of experiments, aliquots of cells were centrifuged in micro-centrifuge
tubes at 14,000 rpm for
2 min and pellets were washed with TBS buffer. Pellets were re-suspended in
equal volumes of
TBS buffer containing 2mM PMSF and 2X sample buffer, boiled for 5 min and
electrophoresed
on 12% polyacrylamide gels according to the standard procedure. The gels were
blotted onto
nitrocellulose membranes. The unoccupied binding sites on the blots were
blocked by
incubating them in blocking buffer [10 mM Tris-HCI, 0.5 M NaCI, 0.05% Twin 20
(v/v), and 5%
non-fat dry milk (w/v)] at room temperature for 1h. After blocking, blots were
incubated with an
appropriate antibody labeled with alkaline phosphatase at room temperature for
2 h. Blots were
washed three times at room temperature in blocking buffer without non-fat dry
milk. After
washing, blots were developed using the Alkaline Phosphatase Conjugate
Substrate Kit
according to the manufacturer's instructions (Bio-Rad, Hercules, CA).
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Results of Western blot analysis indicated that the Mr of the heavy chain was
approximately 55 kDa (Figure 4). The Mr of the light chain was approximately
26 kDa (Figure
3). It was also noticed that the heavy chain protein tended to form aggregates
with very low
mobility on the gel, which were detected at the top, above the 200 kDa protein
marker band.
Aggregates of heavy and light chains were also confirmed by the presence of
smears above the
55 and 26 kDa bands of heavy and light chain respectively.
C. Bombardment and Regeneration of Plastid Transgenic Plants:
After confirming the presence of the Guy's 13 insert in both vectors, and
testing the
constructs in E. coli, plasmid DNA was purified and used for bombardment.
Tobacco (Nicotia~a
tabacum cv. Petit Havana) plants were grown aseptically by germination of
seeds on MSO
medium containing MS salts (4.3 g/liter), BS vitamin mixture (myo-inositol,
100 mg/liter;
thiamine-HCI, 10 mg/liter; nicotinic acid, 1 mg/liter; pyridoxine-HCI, 1
mg/liter), sucrose
(30g/liter) and phytagar (6 g/Iiter) at pH 5.8 (Ye et al., 1990).. Fully
expanded, dark green
leaves of about two month old plants grown under sterile conditions were used
for bombardment.
Leaves were placed abaxial side up on a Whatman No. 1 filter paper laying on
RMOP
medium (Daniell, 1993) in standard petri plates (100 x 15 mm) for bombardment.
Tungsten (1
Vim) or Gold (0.6 Vim) microprojectiles were coated with plasmid DNA (plastid
vectors) and
bombardments were performed with the biolistic device PDS 1000/He (Bio-Rad) as
described by
Daniell (1997). Following bombardment, petri plates were sealed with Parafilm
and incubated at
24°C in the dark. Two days after bombardment, leaves were cut into
small pieces of ~5 mm2 in
size and placed on selection medium (RMOP containing 500 ~g/mL of
spectinomycin
dihydrochloride) with the abaxial side touching the medium in deep (100 x 25
mm) petri plates
(~6 pieces per plate). The regenerated spectinomycin-resistant shoots were cut
into small pieces
(~2mm2) and subcloned into fresh deep petri plates (~5 pieces per plate)
containing the same
selection medium. Resistant shoots resulting from this second round of
selection were then
tested for the presence of the Guy's 13 construct (integration) using PCR (see
below) and only
transgenic shoots were transferred to rooting medium (MSO medium supplemented
with IBA, 1
mg/L and spectinomycin dihycrochloride, 500 mg/L). These plants are designated
TO plants.
Rooted plants were transferred to soil and grown at 26°C under
continuous lighting conditions
for further analysis (Figure 5). Seed collected from TO plants were germinated
on specinomycin,
and then transferred to soil. These plants are designated T1 plants.
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Spectinomycin/streptomycin resistant clones were observed within 3-6 weeks
after
bombardment. Total DNA from unbombarded and transgenic plants was isolated
using DNeasy
Plant Mini Kit (Qiagen, Valencia, CA), PCR was performed in order to
distinguish: a) true
transformants from spontaneous mutants and b) plastid transformants from
nuclear
transformants. DNA was amplified using Taq PCT core kit (Qiagen, Valencia,
CA), using
standard protocols (Sambrook et al., 1989). Samples were amplified in the
Perkin ElmerTM 92s
GeneAmp PCR system 2400. PCR products were analyzed by electrophoresis on 0.8%
agarose
gels.
For TO plants transformed by pLD-TP-Guy's 13, two primers (3P and 3M) were
used to
confirm integration of the spectinomycin resistance gene (aad A) into the
proper location in the
plastid (to distinguish transformants from mutants). Primer 3P anneals to the
16S rRNA gene
and primer 3M binds to the aadA coding region (Figure 1 C). The 3P primer
anneals only with
the plastid genome, so no PCR product can be obtained with nuclear transgenic
plants. Figure 1A
shows that the expected size PCR product (1.65 kb) was obtained with the 3P
and 3M primers,
confirming integration of foreign genes into the plastid genome. To determine
that the genes)
of interest (antibody H and L genes) have been integrated without
rearrangement, primers SP and
2M were used. One primer anneals to the aadA coding sequence and the other
anneals to the
trnA region to confirm integration of the entire gene cassette (Figure 1C).
The presence of the
expected size PCR product (3.6 kb, Figure 1B) confirmed that the entire gene
cassette was
integrated and that there were no internal deletions or loop outs during
integration via
homologous recombination.
For TO plants transformed by the l3pZS-TP-Guy's 13 vector, two primers were
used in
order to test the integration event (i.e., to distinguish transformants from
mutants). One primer
(7P) anneals to the rbcL 3' region and the other (8M) anneals to the aadA gene
to test
integration of the aadA gene in transgenic plants (Figure 2C). Figure 2B shows
that the
expected size PCR product (0.9 kb) was obtained with this primer pair,
confirming integration
of foreign genes into the genome. No PCR product was obtained with
specintomycin-resistant
mutant plants using this set of primers, In order to test integration of genes
into the plastid
genome, two primers were used. One primer (8P) anneals to the rbcL 5' gene
while another
anneals to the aadA gene (8M). Because the rbcL 5' primer anneals only with
the plastid
genome, no PCR product was obtained with nuclear transgenic plants and mutant
plants using
this set of primers. The presence of the expected size PCR product (2.1 kb)
confirmed plastid
integration of both foreign genes (Figure 2A). Plastid transgenic plants
containing the antibody
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H and L chain genes were subjected to a second round of selection in order to
achieve
homoplasmy.
D. Southern Blot Analysis:
Southern blotting was used to test homoplasmy. That is, it establishes that
the
transformed genome (with antibody genes inserted) is the only one present.
Total DNA was
extracted from leaves of transformed and wild-type (control) plants using the
DNeasy Plant Kit
(Qiagen Inc.). Total DNA was digested with Bgl II, electrophoresed on 0.7%
agarose gels and
transferred to Duralon-UV membranes (Stratagene, CA). A 1.8 kb Bgl II/EcoR V
fragment
containing flanking sequences of the pZS vector was used as a probe for the
lines transformed
with the pZS-TP-Guy's 13 vector (Figure 8). A 0.81 kb Bgl IIIBamH I fragment
containing
flanking sequences of the pLD vector was used as a probe for the lines
transformed with the
pLD-TP-Guy's 13 vector (Figure 9). The probes were labeled with 3zP-dCTP using
the Ready
To Go kit (Pharmacia Biotech, N~. The blots were prehybridized using Quickhyb
prehybridization solution (Stratagene, CA). The blots were hybridized and
washed according to
the manufacturer's instructions.
The native size fragment present in the non-transformed control should be
absent in the
transgenics. The presence of a large fragment (due to insertion of foreign
genes within the
flanking sequences) and absence of the native small fragment establishes the
homoplasmic
nature of our transformants (Daniell et al., 1998; Kota et al., 1999; Guda et
al., 2000). In the
case of TO lines transformed with the pLD-TP-Guy's 13 vector, 4.47 kb and 7.87
kb bands were
observed (Figure 9, lanes 4-6). In the case of control (untransformed) Petit
Havana, only the
4.47 kb band was observed (Figure 9, Iane C). In the case of TO Iines
transformed with the pZS-
TP-Guy's 13 vector, 2.6 kb and 6.0 kb bands were observed (Figure 8, lanes 4-
6). In case of
control (untransformed) Petit Havana only the 2.6 kb band was observed. In the
case of T1 lines
of both kinds, the wild-type bands (4.47 for the pLD and 2.6 for the pZS
transformants) were
either absent or very faint (Figures 9 and 8, lanes 1-3).
E. Northern Blot Analysis:
Northern blots were performed to test the efficiency of transcription of the
antibody
genes. Total RNA was isolated from 150 mg of frozen leaves of transformed and
untransformed plants using the "Rneasy Plant total RNA Isolation Kit" (Qiagen
Inc.,
Chatsworth, CA). RNA (9 p.g of all samples except #8841, which had 6.5 pg) was
denatured by
formaldehyde treatment, separated on a 1.2% agarose MOPS gel in the presence
of
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formaldehyde and transferred to Duralon-UV membranes (Stratagene, CA). Probe
DNAs
(antibody H and L chain coding regions) were labeled with 32P-dCTP using the
Ready To Go kit
(Pharmacia Biotech, NJ). The blots were prehybridized using Qiuckhyb
prehybridization
solution (Stratagene, CA). The blots were hybridized and washed according to
the instructional
manual (Stratagene, CA). The transcript levels were quantified using the Storm
840
phosphoimager system (Molecular Dymanics).
Abundant transcripts that hybridized to both light chain and heavy chain
probes were
detected in RNA from plastid transformants (Figures 9 and 10). These
transcripts were larger in
size than transcripts detected in nuclear transgenic plants, consistent with
the presence of
polycistronic transcripts in the transgenic plastids. The transcription levels
between the nuclear
transformants and plastids transformants were compared. The transcription
levels between the
plastid transfonnant lines transformed with the pZS-TP-Guy's 13 vector and the
lines
transformed with the pLD-TP-Guy's 13 vector were also compared. The plastid
transformants
transformed with the pLD-TP-Guy's 13 vector expressed 13/24 fold more
transcripts. The
plastid transformants transformed with the pLD-TP-Guy's 13 vector expressed
two fold more
transcripts than the plastid transformants transformed with the pZS-TP-Guy's
13.
F. Western Blot Analysis:
Two methods were used to extract proteins from the plastids. In the first
method, plant
leaves (100 mg) were ground in liquid nitrogen and resuspended in 150 ~,1 of
TBS buffer buffer
(20 mM Tris-HCI, pH 8, 150 mM NaCI). Samples were mixed well by vortexing.
Equal
volumes of the plant extracts and 2 X SDS sample buffer [10 mM TRIS-Cl, 4%
SDS, 1 mM
(Na)2EDTA, 15% glycerol (v/v) and 0.05% bromophenol blue (w/v)] were mixed,
boiled for 4
minutes, briefly centrifuged, and the supernatant loaded on polyacrylamide
gels. . In the second
method the plant leaves (100 mg) were directly ground in 2X SDS sample buffer,
boiled for 4
min, briefly spun and loaded on polyacrylamide gels. Samples treated with
reductant were
electorphoresed on 12% acrylamide gels. Non-reduced samples were
electrophoresed on 7%
acrylamide gels. The gels were electro-blotted onto nitrocellulose membranes
in a Trans-Blot
Electrophoreic transfer cell (BioRad, CA) following the manufacturer's
instructions. The
unoccupied binding sites on the blots were blocked by incubating them in
blocking buffer [10
mM Tris-HCI, 0.5 M NaCI, 0.05% Tween 20 (v/v), and 5% non-fat dry milk (w/v)]
at room
temperature for 1h. After blocking, blots were incubated for 2 hours at room
temperature with
allcaline phosphatase-conjugated goat anti-human IgA or goat anti-human kappa
antibody,
diluted 1:2000 in blocking buffer. Blots were washed three times at room
temperature in TBS.
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After washing, blots were developed using the Alkaline Phosphatase Conjugate
Substrate Kit
(Bio-Rad, Hercules, CA) according to the manufacturer's instructions.
Bands of approximately 26 Mr were detected using the alkaline phosphate (AP)
S conjugated goat anti human kappa antibody from the samples that were
electrophoresed under
reducing conditions. Bands of approximately SS Mr were detected using the AP
conjugated goat
anti human IgA antibody from the samples that were elecrophoresed under
reducing conditions
(Figure 6). Bands of approximately180 Mr were detected using the AP conjugated
goat anti
human kappa antibody from the samples that were electrophoresed under non
reducing
conditions (Figure 7). This was considered evidence of expression of both
heavy and light
chains, and assembly into an immunoglobulin.
G. ELISA Assays of Antibody Assembly
Determination of antibody concentration and detection of antibody binding
function is
performed by ELISA. Assays are done on crude extracts of leaves made by
homogenizing
small samples in two volumes of extraction buffer (2S mM Tris pH 7.5, 1S0 mM
NaCI, 10 mM
EDTA, 1% sodium citrate, 1% PVPP, 0.2% sodium thiosulfate). Homogenates are
centrifuged
in microfuge tubes for 10 minutes to pellet plastids and assays performed in
the lysed
supernatant.
The concentration of assembled antibody is determined using a double antibody
sandwich ELISA. In this assay, an antibody against kappa chain bound to the
plate captures any
plantibody in the extract, which is detected by antibody against TgA heavy
chain (to detect
assembled IgA or SIgA), or by antibody against secretory component (to detect
assembled
2S SIgA). Microtiter wells are coated overnight at 4 °C with goat anti-
human light chain-specific
antibodies (SO ~1/well at 4 ~,g/mL in PBS). Plates are washed, then blocked
with PBS + S%
non-fat dry milk 1 hour at room temperature. Supernatant is added to the
microtiter plate in
serial twofold dilutions (in PSB + S% non-fat dry milk) and the plate is
incubated 1 hour at 37
°C. Wells are washed, then incubated for 1 h at 37 °C with the
appropriate goat anti-human
chain-specific antibodies conjugated with horseradish peroxidase (Fisher
Scientific), diluted
1:2000 in PSB + S% non-fat dry milk. For plants produced in the first phase of
work
(transformed only with heavy and light chains) the detecting antibody is anti-
human IgA HRP.
For plants transformed with all the components of SIgA the detecting antibody
is anti-human
secretory component-HRP (secretory component will not assemble onto an
antibody without J
3S chain). Plates are washed with water, and antibody complexes are detected
by adding HRP
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substrate [0.1 M sodium citrate, pH 4.4 containing 0.0125% hydrogen peroxide
and 0.40
mg/mL 2.2'-azino-bis (3-Ethylbenzthiazoline-6-sulfonic acid)], and incubating
30 minutes at
room temperature. Color development (absorbance at 405 nm) is determined using
a
Benchmark Microplate Reader (Bio-Rad). Antibody concentrations in ~,g/mL) are
determined
by comparison with standard curve of human SIgA (Sigma), using a four-
parameter logistic fit
(SigmaPlot 3.0).
H. ELISA Assay of Antibody Binding Function:
The ability of plastid-produced antibody to bind to the cognate antigen,
Streptococcal
antigen I/II (SAI/II), is determined using ELISA. SA I/II is purified from
culture supernatants
of Steptococcus mutans strain IB 162 by the method of Russell et al. (1980).
Microtiter plates
are coated with purified SA I/II (50 pL/well at 2 ~g/mL) overnight at 4
°C. Plates are washed,
blocked with PBS + 5% nonfat dry milk, and probed 1 hr at 37 °C with a
dilution series of plant
extract. Bound antibodies are detected using'the appropriate HRP-conjugated
goat anti-human
second antibody, and the plates processed exactly as described above for the
double-antibody
sandwich ELISA. A reference standard lot of Guy's 13 SIgA (produced by nuclear
transgenic
plants) is always tested along with test samples to control for assay to assay
variation. Binding
titer is calculated as the dilution of test antibody (normalized to 1 mg/mL as
determined by the
double antibody sandwich ELISA) necessary to generate an ELISA signal that is
50% of the
maximum signal.
I. Purification of Antibody:
Plastids are first isolated from a crude homogenate of leaves by a simple
centrifugation
step at 1500 X g. This eliminates most of the cellular organelles and proteins
(Daniell et al.,
1983, 1986). Then plastids are burst open by re-suspending them in a hypotonic
buffer
(osmotic shock). This is a significant advantage because there are fewer
soluble proteins inside
plastids when compared to hundreds of soluble proteins in the cytosol. The
homogenate is
centrifuged at 10,000 g for 10 minutes (4 °C) and the pellet discarded.
Purification of antibody
is performed as described in Ma et al. (1998), with some modification. Plastid
homogenate is
mixed with two volumes of extraction buffer (25 mM Tris pH 7.5, 150 mM NaCI,
10 mM
EDTA, 1% sodium citrate, 1% PVPP, 0.2% sodium thiosulfate). The mixture is
centrifuged at
17,000 g for 60 min, and the supernatant filtered through a 0.2 ~,M nominal
cut-off filter.
Filtrate is concentrated by diafiltration using a 300-kD MWCO tangential flow
cassette
(Millipore Corporation), = Immunoglobulins are precipitated with 40% ammonium
sulfate,
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collected by centrifugation at 17,000 g for 15 min, and then re-suspended in
phosphate buffered
saline (PBS).
J. Inheritance of Introduced Foreign Genes:
Some of the initial tobacco transformants are allowed to self pollinate,
whereas others
are used in reciprocal crosses with control tobacco plants (transgenics as
female acceptors and
pollen donors; testing for maternal inheritance). Harvested seeds (T1) are
germinated on media
containing spectinomycin or other appropriate selective agents. Achievement of
homoplasmy
and mode of inheritance can be classified by observing germination results.
Homoplasmy is
indicated by totally green seedlings (Daniell et al., 1998) while heteroplasmy
is displayed by
variegated leaves (lack of pigmentation, Svab and Maliga, 1993). Lack of
variation in
chlorophyll pigmentation among progeny underscore the absence of position
effect, an artifact
of nuclear transformation. Maternal inheritance is demonstrated by sole
transmission of
introduced genes via seed generated on transgenic plants, regardless of pollen
source (green
seedlings on selective media). When transgenic pollen is used for pollination
of control plants,
resultant progeny do not contain resistance to 'chemical in selective media
(appear bleached;
Svab and Maliga, 1993). Molecular analyses (PCR, Southern, and Northern)
confirm
transmission and expression of introduced genes, and T2 seed is generated from
those
confirmed plants.
Example #2. Optimizing the Codon Usage of Antibody Genes to Maximize
Expression in
Plastids
Codon optimization has been used previously to successfully increase the level
of
transgenic protein in plants (McBride et al., 1995; Rouwendal et al., 1997
Horvath et al., 2000).
In the case of a j3-(1,3-1,4)-glucanase expressed in barley, codon
optimization resulted in at
least a 50-fold increase in expression (Horvath et al., 2000). Two factors
contribute to codon
bias in all organisms. One is the overall composition of the genome, which
contributes to a bias
in degenerate positions of codons (Bernardi et al., 1986). In tobacco plastid
non-coding
regions, the AT content is 69.6%. An AT-rich crylA gene (encoding a Bacillus
tlauf°i~gieyasis
toxin) accumulated to much higher levels in plastids than the same gene having
nuclear codon
preferences (McBride et al., 1995). High AT content, however, is not the whole
story. The
second factor is selection for translation efficiency, resulting in a bias for
specific codons
(Ikemura et a1.,1985). It has been proposed (Morton, 1993; Morton, 1998) that
codon use in
plastids is adapted to tRNA levels and that highly expressed genes have a
greater bias in codon
use as a result of selection for increased translation efficiency.
Modification of a transgene to
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match the codon usage of highly expressed genes should result in even higher
levels of
expression. We devised a codon optimization table (Table 2) based on published
observations
of codon useage in plastids (Morton, 1993; Morton, 1998; Morton and So 2000).
Essentially,
we hypothesized that any gene utilizing the codons found in this table, and
utilizing the rules
listed below, would express at a higher level in plastids than the native
gene.
Rule #1: The primary codon is used, unless conditions met in rules number 2
and 3 are
present.
Rule #2: If a codon ending with C is followed by a codon beginning with G, the
secondary codon is used, so as to avoid the combination NNC GNN, in which
N represents any nucleotide and NNC and GNN are adjacent codons.
Rule #3: If the same amino acid is encoded twice with four or fewer
intervening amino
acids (for example, LXXXL, where L is Leucine and X is any amino acid) the
secondary codon is used to encode one of the amino acids (either the first or
second L, in the example), being careful to avoid violating Rule #2.
Rule #3: If the same amino acid is encoded three times with four or fewer
intervening
amino acids between the first and third occurence (for example, LLXXL,
where L is Leucine and X is any amino acid) the tertiary codon is used to
encode one of the amino acids (either the first or second L, in the example),
being careful to avoid violating Rule #2.
Rule #4: If using the primary codon would result in significant secondary RNA
structure
(such as a stable stem-loop), the secondary codon is used.
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Table 2
Optimal Codons for Plastid
Expression
Amino Acid Primary Codon Secondary Codon Tertiary Codon
S Leu TTA CTT TTG
Ser TCT AGC AGT
Arg CGT AGA CGC
Pro CCT CCA
Thr ACT ACC
1O Val GTA GTT
Ala GCT GCA
Gly GGT GGA
Ile ATT ATC
His CAC CAT
1 Gln CAA CAG
S
Glu GAA GAG
Asp GAT GAC
Asn AAC AAT
Lys AAA AAG
2O Tyr TAC TAT
Cys TGT TGC
Phe TTC TTT
2S A synthetic gene was constructed that encoded a polypeptide consisting of
the variable
region of a marine anti-rotavirus monoclonal antibody fused to the constant
region of human
IgA2m(2) heavy chain (Chintalacharuvu et al 1994). The sequence of this
chimeric gene was
modified from the native mammalian gene sequences by codon optimization for
plastid
expression, using the rules in table 2. In addition, TAA was used as a stop
codon. Synthesis of
30 the gene was contracted to Entelechon GmbH. The gene was synthesized using
the overlap
extension PCR method (Rouwendal et al., 1997), but could be synthesized by
various methods
known to those skilled in the art. Another gene, encoding a polypeptide
consisting of the
variable region of a marine anti-rotavirus monoclonal antibody fused to the
constant region of
human kappa chain was synthesized by the same method, with codons optimized
for plastid
35 expression. Both synthetic genes were cloned into the vector pCR4TOP0
(Invitrogen).
The plasmid containing the heavy chain sequence was cut with Sal I, and the
plasrnid
containing the light chain sequence was cut with Sal I and Xho I. A Sal I/Xho
I fragment
containing the light chain sequence was then isolated and cloned into the Sal
I site of the
40 plasmid containing the heavy chain. The resulting bacterial clones were
screened for a clone
with the correct orientation (heavy chain followed by light chain with coding
sequences in the
same orientation). The heavy and light chain genes, with associated ribosome
binding sites
were then cut out together using Not I and ~hba I, and cloned into the pLD
vector. The sequence
between the Not I and ~'ho I sites of the heavy and light chain cassette is
shown in Table 3.
4S
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The pLD vector with codon-optimized heavy and light chain coding sequences was
used
to transform tobacco plastids as described in Example 1. Transgenic plants are
isolated and
shown to contain high levels of human IgA.
Example #3. Expression of SI~A in Plastids with al~enes on one vector
Expression of SIgA in plastids is accomplished by the simultaneous integration
of four
genes, IgA heavy chain, light chain, J chain and secretory component. These
genes are
expressed on a polycistronic message. A plasmid, based on pLD, is constructed
containing the
Guy's 13 heavy and light chains, and the mature-peptide coding regions of
human J chain and
SC genes, all downstream of the aadA gene and each having a ribosome binding
site. The total
size of this mRNA is over 4500 nt. Tobacco leaves are transformed by particle
bombardment
and transplastomic plants are selected by regeneration on antibiotic-
containing medium by
methods similar to those disclosed in Example #1. Appropriate primers are used
for PCR
analysis. Expression of J chain and SC is evaluated by western blotting, using
antisera specific
for human J chain and human secretory component. Detection of a band at 370
kDa with anti-
IgA, anti-kappa, anti-J and anti-SC antibodies is considered evidence of
assembled SIgA.
Example #4. Expression of SI~A in Plastids with J chain and Secretory Coyonent
genes on
one vector and Heavy and Light Chain Genes on another vector
Two plastid expression vectors, one containing heavy and light chain genes,
and the
other containing the J chain and secretory component genes are constructed by
methods similar
to those described in Example #1. The amino acid sequence of the J chain and
secretory
component encoded in the second vector are those described in Patent No.
5,959,177 and US
Patent No. 6,046,037, incorporated herein by reference. The two vectors use
different plastid
DNA flanking sequences, so that they integrate into the plastid chromosome in
different
locations. Tobacco leaves are transformed by particle bombardment and
transplastomic plants
are selected by regeneration on antibiotic-containing medium by methods
similar to those
disclosed in Example #1. Appropriate primers are used for PCR analysis.
Expression of J
chain and SC is evaluated by western blotting, using antisera specific for
human J chain and
human secretory component. Detection of a band at 370 kDa with anti-IgA, anti-
kappa, anti-J
and anti-SC antibodies is considered evidence of assembled SIgA.
Example #5. Expression of a chimeric heavy chain in Plastids
A fragment containing all 5 extracellular Ig-like domains of ICAM-I is
amplified from
plasmid pIgADS (a gift of T. Springer) using the primers:
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5'-AAAATCTAGAGGAGGGATTTATGCAGACATCTGTGTCCCCCTCAAAAGTC-3'
and
5'-CATACCGGGGACTAGTCACATTCACGGTCACCTCGCG-3'.
The resulting PCR product incorporates a ribosome-binding site utilized by the
plastid protein
translation machinery, and a methionine codon upstream of the first amino acid
of ICAM-1. The
PCR product is cut with Xba I and Spe I (underlined sequences) and cloned into
a vector
containing the human IgA2m(2) heavy chain constant region. The resulting
chimeric gene
encodes one continuous protein consisting of 5 domains of ICAM-1 and the
constant region of
IgA2m(2). The mature protein produced from this construct starts with the
sequence Met-Gln-
Thr-Ser-Val-, and end with the sequence -Lys-Asp-Glu-Leu. It is predicted to
have 800 amino
acids and a molecular weight of approximately 80,000. The sequence of the ICAM
gene has
been published (Staunton et al., 1988), and is incorporated herein by
reference. The entire
coding sequence of the chimeric gene is cut out with Xba I and cloned into the
pLD vector. The
resulting expression vector is used to transform tobacco plastids. The
chimeric ICAM-1/IgA
protein is expressed in transgenic plastids, and assembles into dimers. This
multimeric protein
comprises an immunoglobulin heavy chain fused to a functional ligand (ICAM-1
domains 1-5),
and binds to a site on human rhinoviruses. It is used in a therapeutic manner
to prevent
rhinovirus colds.
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