Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02388955 2002-04-24
WO 01/32714 PCT/GB00/04216
Recombinant Fusion Molecules
This invention relates to a method for increasing the production of a protein
in a cell
and more particular the production in a plant cell. This invention also
relates to
recombinant immune fusion complexes and more particularly to their expression
in
plants.
Recombinant proteins have been expressed in transgenic plants by many groups.
A
consistent problem is low levels of expression, which complicates purification
and
processing. Various approaches have been taken to overcome this, for example
using
different regulatory gene constructs or constructing synthetic genes, with
limited
success. Thus the levels of expression routinely reported are up to 0.5% total
soluble
protein, with many examples being less than 0.01%. One notable exception has
been
antibody expression in plants. Our experience here is that levels of 1% are
normally
expected, with 5-8% also achieved. The reason for these differences is
unclear, but we
believe it to be related to the inherent stability of antibody molecules in
plants, as well
as the manner in which these proteins are handled by the plant cell machinery.
Immune complexes comprise antibodies and antigens, and give rise to the
classical
immune response. Immune complexes are conventionally produced by mixing
antigen
with antibody and allowing the molecules to associate via the binding site of
the
antibody and the specific epitope of the antigen. The disadvantage of this
process is
that immune complexes in vitYO which require careful mixing of antigen with
antibody
at optimal concentrations.
The present invention provides a method of increasing the production of a
protein in a
cell comprising expressing recombinant DNA encoding a recombinant fusion
protein
comprising a desired protein fused to the constant region of a heavy chain of
an
antibody or to part of said constant region.
For convenience, the term 'heavy chain constant region' will be used
throughout the
following description to refer either to the full length immunoglobulin heavy
chain
CA 02388955 2002-04-24
WO 01/32714 PCT/GB00/04216
2
constant region or to a part (fragment) of this region, as the context
permits. The
constant region comprises a number of regions (domains) depending of the class
of the
antibody. For an IgG antibody the constant region comprises three regions
(domains)
known as CHl, CH2, and CH3; the CH3 domain being the C-terminal domain. Thus
instead of using the full length constant region, truncated versions of this
may be used
comprising the conjoined CH1/CH2, CH2/CH3 or CHl/CH3 domains.
By using the high expression of the immunoglobulin, we have found it possible
to drive
up the expression levels of the recombinant fused protein.
The desired protein can be any protein including reporter molecules such as
(3-galactosidase, luciferase and GFP. The desired protein may also be a
selectable
marker such as an antibiotic.
Preferably, the desired protein is a food protein such as a storage protein,
i.e. phaseolin
or wheat gluten.
It is also preferably that the desired protein is a therapeutic protein.
Therapeutically
useful proteins include receptors, e.g. the cystic fibrosis receptor (CFTR),
enzymes
including prodrug activating enzymes, e.g. nitroreductases, ligands,
regulatory factors,
hormones, and structural proteins. Therapeutic proteins also include sequences
encoding nuclear proteins, cytoplasmic proteins, mitochondria) proteins,
secreted
proteins, plasmalemma-associated proteins and serum proteins. The desired
protein
may also be selected from lipoproteins, glycoproteins and phosphoproteins,
hormones,
growth factors, enzymes, clotting factors such as Factor XIII and Factor IX
etc.,
apolipoproteins, receptors, erythropoietin,drugs, oncogenes and tumor
suppressors.
Specific examples of these compounds include proinsulin, growth hormone,
androgen
receptors, insulin-like growth factor I, insulin-like growth factor II,
insulin-like growth
factor binding proteins, epidermal growth factors, angiogenesis factors
(acidic
fibroblast growth factor, basic fibroblast growth factor, vascular endothelial
growth
factor and angiogenin), matrix proteins (Type IV collagen, Type VII collagen,
laminin),
phenylalanine hydroxylase, tyrosine hydroxylase, oncogenes (ras, fos, myc,
erb, src, sis,
jun), E6 or E7 transforming sequence, p~3 protein, Rb gene product, cytokine
receptor,
J
CA 02388955 2002-04-24
~J~ 0~/327~4 PCT/G~00/04226
I-1, IL-6; ITL-~, viral capsid protein, and other proteins of asefui
significance ir~ the
body. The desired protein, which can be fused, is only limited by the
availability of the
nucleic acid sequence encoding the protein or polypeptide to be incorporated.
One
skilled in the art will readily recognise that as more proteins and
polypeptides become
identified they can be integrated into the recombinant fusion protein of the
present
invention.
It is particularly preferred that the desired protein is an antigen. The term
"antigen" as
used herein refers to any protein that gives rise to an immune response in an
animal
such as a mammal, preferably a human. Preferably, the term "antigen" as used
herein
refers to a protein that stimulates a series of reaction in an animal that are
mediated by
white blood cells including lymphocytes, neutrophils and monocytes. Preferred
antigens include viral antigens, bacterial antigens, protozoal antigens,
parasitic antigens,
tumor antigens, and proteins from viral, bacterial and parasitic organisms
which can be
used to induce an immune response. It is also preferred that the antigen is
not a toxin.
A toxin is defined herein as a protein which has a direct toxic effect on a
cell and causes
cell death. It is most preferred that the antigen is human immunodeficiency
virus
gp 120, the non-toxic C-tetanus toxin fragment or bovine respiratory syncytial
virus
(BRSV) F-protein.
The desired protein may be linked to the constant region or part thereof
directly or
indirectly through an intermediate peptide linker which may include a peptide
cleavage
site. This will allow separation of the desired protein from the constant
region of part
thereof (if required) for purification purposes. Attachment of the desired
protein to the
constant region or part thereof may be made via the C-terminus of the heavy
chain or
part thereof. Alternatively, the antigen may be fused to the antibody at a
site within the
constant region or part thereof.
The method of the present invention is applicable to the production of the
desired
protein in any eukaryotic cell. Preferably the desired protein is produced in
a eukaryotic
cell which is capable of producing immunoglobulins. More preferably the
desired
protein is produced in a mammalian cell and most preferably in a plant cell.
CA 02388955 2002-04-24
WO 01/32714 PCT/GB00/04216
4
The method of the present invention has the benefit of allowing the production
of a
desired protein in a cell at a higher level than when the desired protein is
not produced
as part of a fusion protein comprising a constant region of a heavy chain of
an antibody.
Desired proteins are generally expressed at low levels in transgenic plants,
whereas
antibody molecules usually accumulate to much higher levels. By genetically
fusing the
desired protein with a component of the antibody molecule, the antibody
component
acts as a carrier and stabilising molecule and allows much higher levels of
production
and accumulation of the desired protein.
The present invention also provides a first recombinant fusion protein
comprising an
antigen fused to the constant region of the heavy chain of an antibody or to a
part of
said constant region. The recombinant fusion protein is as defined above in
connection
with the method of the present invention, except that the fusion protein
comprises an
antigen as defined above.
The present invention also provides a second recombinant fusion protein
comprising a
desired protein fused to the constant region of a heavy chain of an antibody
or to part of
said constant region, wherein the fusion protein does not comprise a
functional antigen
binding domain. The recombinant fusion protein is as defined above in
connection with
the method of the present invention, except that the fusion protein does not
comprise a
functional antigen binding domain.
A functional antigen binding domain is defined herein as the antibody domain
which
contacts and binds an antigen. The domain comprises the complementarity
determining
regions (CDRs) of the antibody, which are well known to those skilled in the
art and
can be easily identified. The domain is preferably the variable domain of the
heavy
and/or light chain of an antibody. The second recombinant fusion protein of
the present
invention can comprise an antibody which has had one or more of its variable
domains
removed. Alternatively, the second recombinant fusion protein of the present
invention
can comprise an antibody which has one or more non-functional variable
domains. The
variable domains can be made non-functional by deleting the CDRs. Other
methods for
CA 02388955 2002-04-24
WO 01/32714 PCT/GB00/04216
producing an antibody with a non-functional antigen binding domain are well
known to
those skilled in the art.
The present invention also provides a recombinant antibody molecule having an
5 antigen fused to an antibody molecule, wherein the antibody molecule has
affinity for
the antigen. The recombinant antibody molecule of the present invention can
form an
immune complex by associating with other recombinant antibody molecules of the
present invention through the antigen binding sites of the antibody. Such an
immune
complex resembles, but is not identical to, a classical immune complex. A
significant
advantage of the immune complex formed from the recombinant antibody molecules
of
the present invention is that it is easy to prepare in contrast to
conventional preparation
of immune complexes in vitro which require careful mixing of antigen with
antibody at
optimal concentrations. In our case, the expression ratio of antibody to
antigen
molecules is generally fixed at 1:1, which optimises the potential for forming
immune
complexes.
The recombinant antibody molecule of the present invention is highly
immunogenic,
especially when it is forms an immune complex, and is suitable for both
systemic and
mucosal immunisation without the need for an adjuvant. Adjuvants are generally
incorporated into vaccine compositions to improve the immune response. As the
recombinant antibody molecules of the present invention are highly
immunogenic, it is
possible to obtain immunisation without the need of an adjuvant.
The immune complex formed using recombinant antibody molecules of the present
invention will possess substantially the same enhanced immunogenic properties
as an
endogenously produced immune complex or a conventionally produced immune
complex.
The recombinant antibody molecule of the present invention can comprise any
antibody
molecule provided it is has affinity for the antigen to which it is fused. The
antibody
molecule can be a complete monoclonal antibody or an antigen binding fragment
CA 02388955 2002-04-24
WO 01/32714 PCT/GB00/04216
6
thereof, such as a Fv, Fab or F(ab')~ fragment. Preferably the antibody
molecule
comprises a constant region of a heavy chain of an antibody.
To engineer genetic constructs encoding antigen-antibody fusion proteins,
there is no
restriction as to the species of either of these components. The invention is
of especial
interest in relation to human medicine but other primate and other animal
antigen/antibody combinations may be used depending on the intended biological
application of the product. For example the antigens and the antibody heavy
and light
chains may be of human, rodent, rabbit, bovine, ovine, caprine, fowl, canine,
camel,
feline or primate origin.
To illustrate the range of application of the invention three model antigens
have been
used, namely human immunodeficiency virus gp 120, the non-toxic C-tetanus
toxin
fragment and bovine respiratory syncytial virus (BRSV) F-protein, with their
respective
specific monoclonal antibodies. For each antigen, numerous constructs are
possible, in
which the antigen is fused to either the C-terminus of a monoclonal antibody
(Mab) full
length heavy chain, or to truncated heavy chain domains consisting of 1, 2 or
3 constant
region domains. Alternatively, the antigen is fused to the constant region at
a point
within the constant region.
The present invention also provides a transgenic plant expressing a
recombinant fusion
protein comprising a desired protein fused to a constant region of a heavy
chain of an
antibody.
The present invention also provides a transgenic plant expressing the first or
second
recombinant protein of the present invention, or the recombinant antibody
molecule of
the present invention.
Methods for generating transgenic plants are well known to those skilled in
the art.
The transgenic plants of the present invention may express a number of
molecular
forms of fusion protein for each antigen, permitting the selection of the
correctly
CA 02388955 2002-04-24
WO 01/32714 PCT/GB00/04216
7
formed molecular form of fusion protein and its expression at the highest
levels and to
extract and purify the fusion protein. It is also possible to characterise the
plant derived
fusion proteins by analysis of assembled molecular forms, recognition by and
affinity
for a panel of antibodies.
The advances deriving from this work are the development of a new type of
engineered
molecule suitable for vaccination in a range of infectious diseases. By using
three
different antigens, we are able to determine the efficacy of this approach for
a standard
bacterial immunogen, the envelope protein of an important human virus and a
transmembrane fusion protein of a cattle virus that causes severe mucosal
infections.
The recombinant antibody molecule resulting from the fusion of an antigen with
an
antibody molecule has all the components required for immune complex mediated
stimulation of an immune response and our approach offers a convenient method
for
ensuring antigen/antibody complexing. The recombinant antibody molecules of
the
1 S present invention can also form larger immune complexes in planta (see
Figure 1)
which can be used for immunisation. Not all parts of the antibody molecule are
essential, such as the sites for FcR binding and complement activation that
reside in the
constant domains of IgG antibodies. Thus we also include the use of truncated
antibody
molecules which lack either the Cy3 domain (that is not involved in antigen
recognition), or the Cyl domain.
It is also possible to use the first recombinant fusion protein of the present
invention or
the second recombinant fusion protein of the present invention (wherein the
desired
protein is an antigen) for immunisation. In particular, a fusion protein
comprising an
antibody molecule which does not comprise a functional antigen binding domain
and an
antigen can be used to deliver the antigen to phagocytes and also to initiate
complement
activation. By excluding the requirement for antigen recognition, it is
possible to help
prevent the phenomenon of modulation of antigen processing that results from
antibody
masking of T cell epitopes, as the antigen will be a fusion protein with the
constant
region of an antibody.
CA 02388955 2002-04-24
WO 01/32714 PCT/GB00/04216
8
The use of plants is ideally suited for the production of the recombinant
fusion proteins
of the present invention not only because of the potential requirement for
large
quantities for vaccination purposes, but also because those complexes that
involve full
length antibody, or assembly with light chain are not readily produced in most
other
S expression systems.
To engineer genetic constructs encoding antigen-antibody fusion proteins DNA
encoding an antigen or antigenic fragment may be amplified by PCR. The genes
encoding the light and heavy chains of murine, human or other mammalian
monoclonal
antibodies specific for these antigens are also cloned. Using these DNA
sequences the
gene constructs can be made encoding the desired recombinant fusion protein.
For
example, the following gene constructs for each of the antigens can be
prepared for
plant transformation:
Genetic constructs, showing Ig domains that will be included in
each immune complex construct.
A - Heavy chain
I) Construct #1 Variable---Cyl---Cy2---Cy3---Linker peptide---antigen
II) Construct #2 Variable---Cyl---Cy2---Linker peptide---antigen
111) Construct #3 Cy2---Cy3---Linker peptide---antigen
N) Construct #4 Cy2---Linker peptide---antigen
B - Light chain
Variable---CL
These constructs are shown diagrammatically in Figure 1, which also shows the
final
assembly of products in plants.
The linker peptide as shown in Figure 2 may conveniently be (GGGGS)3. A
conventional 6xHistidine tag and a protease cleavage site (e.g. enterokinase,
Factor Xa
CA 02388955 2002-04-24
WO 01/32714 PCT/GB00/04216
9
or thrombin) may also be introduced at the C-terminus of the antigen for
purification
purposes. In addition a further different protease cleavage site (e.g.
enterokinase, Factor
Xa or thrombin) may also be introduced between the linker peptide and the
antigen.
Additional or alternative peptide tags and protease cleavage sites may be
incorporated.
The gene constructs are sequenced for confirmatory purposes and inserted into
plant
expression vectors for plant transformation.
To generate transgenic plants expressing the 12 (four for each antigen)
constructs,
Nicotiana tabacum is a convenient plant host using Agrobacterium mediated
transformation. For constructs I and II, in order to generate the recombinant
fusion
proteins , the immunoglobulin light chain genes and the heavy chain constructs
may be
introduced into separate plant lines as shown in Figure 1. Following
regeneration of
these first generation transformants, the light chain and heavy chain
transformed plants
are cross-fertilised and the second generation plants screened for production
of the final
product. For constructs III and IV, there is no requirement for the light
chain and the
final product can be produced in the first generation transformed plants.
To generate stable homozygous transgenic plant lines, transgenic plants
expressing
correctly assembled products are used to generate homozygous plant stocks. The
plants
are grown to maturity, self fertilised, and the resulting seeds screened by
back crossing
with non-transformed plants to determine those that are homozygous. Further
stocks
can be generated by self fertilisation and stored as seeds.
To characterise each molecular form of plant product and to extract and purify
material
for further study, the primary plant transformants are screened to determine
which types
of products are expressed and assembled optimally. This investigation can be
performed by Western blot analysis and ELISA of crude plant extracts, using a
range of
antisera and monoclonal antibodies that are commercially available. Extraction
and
purification of the selected recombinant products may be achieved by ammonium
sulphate precipitation, followed by filtration and affinity chromatography
using either
the immunoglobulin regions or specific peptides as affinity tags. Further
CA 02388955 2002-04-24
WO 01/32714 PCT/GB00/04216
characterisation is performed to determine molecular structure, recognition of
the
antigen moiety by a panel of antibodies and binding affinity.
The Figures show:
5
Figure 1 shows schematically immune complexes in plants and potential assembly
arrangements.
Figure 2 shows schematically the basic molecular design.
Figure 3 shows schematically the pEXGl3 principle cloning sites.
Figure 4 shows RT-PCR analysis of transgenic plants.
Figure 5 shows results of a capture ELISA for CH2-CH3-gp120.
Figure 6 shows results of a capture ELISA for recombinant gp120 expressed in
plants
Figure 7 shows a 10% SDS gel under reducing and non-reducing conditions.
EXAMPLE
Production and use of recombinant fusion molecules comprising gp120 and
constant domains of a heavy chain of an antibody.
Cloning and genetic enaineerin~of HIV gp120 constructs:
The source of DNA for the HIV gp120 antigen was an infectious cloned isolate
of HIV
IIIB. Such DNA can be obtained from the Medical Research Council AIDS Reagents
Project (NIBSC, Blanche Lane, South Mimms, Potters Bar, Herts EN6 3QG, UK).
The genes encoding the heavy and light chains of an HIV gp120 specific
monoclonal
antibody (e.g. Gorny et al., 1991, Proceedings of the National Academy of
Sciences,
CA 02388955 2002-04-24
WO 01/32714 PCT/GB00/04216
11
USA 88:3238-3242) were cloned as described (Ma, J. K-C., T. Lehner, P.
Stabila, C.I.
Fux and A. Hiatt. 1994 Assembly of monoclonal antibodies with IgGl and IgA
heavy
chain domains in transgenic tobacco plants. Eur. J. Immunol., 24:131-138). RNA
was
extracted from relevant hybridoma cells using a standard extraction kit (e.g.
Promega
SV Total RNA isolation system), cDNA was prepared by using the reverse
transcriptase
reaction. DNA encoding the antigen, or the heavy and light chains of a
monoclonal
antibodies specific to the antigen, or fragments of these antibody sequences,
was
amplified by the polymerase chain reaction using synthetic oligonucleotides
shown in
Figure 2. The oligonucleotide primers 1 and 2 corresponded to the 5' and 3'
ends of the
antibody heavy chain and the oligonucleotide primers 3 and 4 corresponded to
the 5'
and 3' ends of the antigen. The 5' primer for the antigen also includes
sequences
encoding a linker peptide and a protease cleavage site. The primers included
appropriate restriction enzyme sites (such as 5' BamHI and 3' XmaI for gp120;
5' XhoI
and 3' BamHI for Ig heavy chain) and extra sequences encoding linker peptides
(GGGSGGGSGGGS) or protease cleavage sites (e.g. Factor Xa IEGR). If a light
chain
is required, the gene can be cloned according to the methodology described by
Drake et
al., Antibody production in plants. P. Shepherd and Dean (eds). Monoclonal
Antibodies
- A practical approach. Oxford University Press. 2000)
Engineering of DNA constructs was carned out in standard cloning vectors -
pBluescript (Stratagene) and pET32 (New England Biolabs) by standard
techniques as
described in Maniatis, Fritsch and Sambrook (1982) - Molecular Cloning, A
Laboratory
Manual. Essentially, the DNA encoding the antigen was first cloned into the
vector
using the multiple cloning site, followed by the DNA encoding the heavy and
optionally
the light chain, of an antibody or antibody fragment. In some cases, the order
was
reversed, with antibody genes being cloned first. Positive transformants were
identified
by the release of a DNA fragment of the correct size following digestion with
the
appropriate restriction enzymes, or PCR using the appropriate 5' and 3'
oligonucleotide
primers. Four chimeric immunoglobulin heavy chains constructs were engineered
in
which the antigen was expressed in fusion with varying portions of the
immunoglobulin
heavy chain as shown in Figure 1.
CA 02388955 2002-04-24
WO 01/32714 PCT/GB00/04216
12
The specific example of cloning the Ig heavy chain CH2-CH3 domain fusion with
an
HIV gp120 fragment (construct III in Figure 1) is shown in Figure 2. Two PCR
products were amplified using oligonucleotide pairs 1-2 and 3-4. The HIV gp120
DNA
sequence encodes a truncated peptide starting at the N-terminus, up to and
including the
sequence KEYAL (aa147) with a stop codon immediately after. These were cloned
into
a vector based on pET32 which had previously been engineered with the key
features
of the cloning site within this vector shown in Figure 3. The gp 120 gene
fragment was
ligated into the BamHI - XmaI site of the vector, then the Ig heavy chain gene
was
cloned upstream in the NcoI - BamHI site. The entire construct was then re-
amplified
by PCR to include the downstream enterokinase cleavage site and His tag, and
to
include 5' XhoI and 3' EcoRI terminal restriction sites. This fragment was
cloned into
pBluescript for confirmatory sequencing.
The completed genetic constructs were then excised and cloned separately into
a plant
expression cassette such as that described above (Drake et al., 2000), within
a plasmid
(pMON530) suitable for initial screening in E. coli. Plasmids of this type are
widely
available, such as the pGreen system (www.p~reen.ac.uk). The plant expression
cassette in this case contains the 5' untranslated region from tobacco etch
virus which
stabilises mRNA in plant cells, as well as an upstream marine IgG leader
sequence
(Figure 3), which directs secretion of the recombinant protein out of the
plant cells.
This recombinant vector was used to transform E. coli (DHS-a). Screening of
transformed clones was by Southern blotting, using radiolabelled DNA probes
derived
from the original PCR products. Plasmid DNA was purified from positive
transformants (PromegaQiaprep kit) and used for transformation of
Agrobacterium
tumefaciens (strain LBA4404 - GibcoBRL, UK) using a freeze/thaw procedure as
follows:
1. Inoculate 50 ml Luria broth (LB) containing spectinomycin (SO~g/ml) (Sigma)
with
Agrobacterium.
2. Shake at 28°C in the dark to an OD600 of 1Ø
3. Centrifuge the agrobacteria in sterile tubes at 3000g for 15 min.
CA 02388955 2002-04-24
WO 01/32714 PCT/GB00/04216
13
4. Remove supernatant and, while keeping the tubes on ice, resuspend the
bacterial
pellet in a total of 1 ml of ice-cold l OmM CaClz.
5. Transfer 100 ml aliquots of the bacterial resuspension into sterile
microcentrifuge
tubes and then place into liquid nitrogen.
S 6. Use the frozen agrobacteria directly for DNA transformation or store at-
80°C.
7. Pipette 10 ml of binary plasmid from a DNA miniprep onto the surface of 100
ml
competent frozen agrobacteria.
8. Incubate the DNA-bacteria mixture for 5 min at 37 ° C in a water
bath.
9. Add lml of LB to the bacteria and shake at 28°C for 4 h.
10. Centrifuge the bacteria for 2 min at 12,000 x g and resuspend the pellet
in 100 ml of
LB.
11. Spread 50 ml of the bacterial suspension onto LB medium (made semi-solid
with 15
g1-' agar) containing spectinomycin required for selection of agrobacteria
carrying
binary and disarmed Ti helper plasmid. Seal the plates and incubate for 2-3
days at
28°C.
12. Re-streak resulting colonies on selective LB medium in separate Petri
dishes and
incubate at 28°C for 2 days.
Plant transformation and regeneration: All gene constructs were introduced
into
Nicotiana tabacum, var. xanthii. Tobacco transformation with A. tumefaciens
was by
standard procedures. Leaf discs are cut from surface sterilised tobacco leaves
and
incubated with a culture of the recombinant A. tumefaciens, containing cDNA
inserts.
The infected discs are transferred to culture plates containing a medium that
induces
regeneration of shoots, supplemented with kanamycin and carbenicillin (Sigma,
UK).
Shoots developing after this stage are excised and transplanted onto a root
inducing
medium, supplemented with kanamycin. Rooted plantlets are transplanted into
soil after
the appearance of roots. The detailed methodology was as follows:
1. Remove Agrobacterium (containing binary vector) from -80°C and
streak onto
semi-solid LB medium (with appropriate antibiotics) in a 9 cm Petri dish.
Incubate at
28°C for 2 d.
2. Inoculate Agrobacterium from Petri dish into 10 ml LB.
3. Shake at 28°C to an OD600 of 1Ø
CA 02388955 2002-04-24
WO 01/32714 PCT/GB00/04216
14
4. Remove 4 ml of Agrobacterium suspension and add to 16 ml of sterile
distilled water
in a sterile 9 cm Petri dish.
5. Cut 0.5 - 1.0 cm leaf discs from surface sterilised leaves and immerse for
5 min in
the diluted Agrobacterium suspension.
6. Briefly dry leaf discs on sterile filter paper.
7. Place leaf discs on shoot regeneration medium, 10-15 discs per 20 ml of
medium in
each 9 cm Petri dish. Incubate for 2 d at 25°C with a 16 h photoperiod.
8. Transfer leaf discs to shoot regeneration medium containing 500 mg/1
carbenicillin
and appropriate concentration of selective agent for transformed plant cells.
Incubate
for 21 d at 25°C with a 16 h photoperiod.
9. Transfer leaf discs to shoot regeneration medium in sterile 175 ml glass
jars
containing 500 mg/1 cefotaxime and 200 mg/1 kanamycin. Incubate at 25°C
with a 16 h
photoperiod.
10. Developing shoots are removed when they reach a convenient size (approx.
0.5 cm
in length) and transferred to rooting medium (3-4 shoots/40 ml medium/175 ml
glass
jar). Incubate for 14 d at 25°C with a 16 h photoperiod.
11. Shoots lacking roots are trimmed at the base and replaced in fresh rooting
medium
for an additional 21 d or until roots appear.
12. Rooted shoots are transferred to compost in plant pots, watered and
supplied with
nutrients. Plants are kept in seed trays and covered with a lid for 24 h after
transfer to
compost, to minimise initial water loss.
Regenerated plants were screened for expression of immunoglobulin chains and
each of
the antigens by Western blot and ELISA of crude leaf extracts using available
antisera
and monoclonal antibodies (e.g. from the MRC AmS Directed reagents programme,
address given above). Transgenic plants were self fertilised to establish
homozygous
plant lines and cross fertilised to generate antibody producing plants.
RNA extraction:
Total RNA was extracted from plants less than a month old, using the Promega
plant
RNA extraction kit (Rneasy Plant mini kit). Alternatively, leaf tissue was
frozen in
CA 02388955 2002-04-24
WO 01/32714 PCT/GB00/04216
liquid nitrogen and rapidly ground to a fine powder. This was mixed with 2
volumes of
ice cold guanidine hydrochloride buffer (8M guanidine hydrochloride , 20mM
MES,
20mM EDTA, 50mM (3-mercaptoethanol, pH7). After agitation it was added to 1
volume of phenol:chloroform:isoamyl alcohol (25:24:1), mixed thoroughly, and
5 centrifuged at 10,000 rpm for 45 minutes. The upper (aqueous) phase was
collected,
mixed with pre-cooled ethanol (0.7 volumes) and 1M acetic acid (0.2 volumes),
and
incubated at -200°C for 16 hours. After centrifugation, the precipitate
was washed three
times with 3M sodium acetate, pH5.2, and once with 70% ethanol. The pellet was
dissolved in sterile RNAse free water (Sigma, UK) containing RNAse free DNAse
10 (Promega, UK), and incubated at 37°C for 1 hour, then at 70°C
for 5 minutes.
RNA concentration and purity were assessed using the GeneQuant II RNA/DNA
Calculator (Pharmacia BioTech, UK). RNA was stored in sterile RNAse free water
at
-20°C.
RT-PCR was performed using appropriate oligonucleotide primers to determine
that the
correct RNA transcript was being made by putative transformed plants.
Characterisation and selection of recombinant immune complexes from plants:
To confirm the expression of each engineered constructs in plants, leaf
extracts were
examined by ELISA and Western blot analysis. Samples were extracted in 150mM
NaCI and 20mM tris, pH8 (TBS) with leupeptin (lOmg/ml) (Calbiochem). Capture
ELISA analyses were performed by incubating the plant extracts on microtiter
plates
that had been pre-coated with a monoclonal antibody specific for HIV gp120
(ADP401
from the MRC Aids Directed Program) and blocked with 5% non-fat dry milk in
TBS.
After overnight incubation at 4°C, the plates were washed in TBS with
0.05% Tween
20, then incubated with either a horseradish peroxidase labelled Sheep anti-
Mouse IgG
antiserum (The Binding Site, UK), or one of a panel of gp120 specific
monoclonal
antibodies (MRC Aids Directed Program), followed by the relevant horseradish
peroxidase antiserum (The Binding Site, UK). The assay was developed using the
TMB
substrate (Sigma, UK) and absorbance was read at 450nm.
CA 02388955 2002-04-24
WO 01/32714 PCT/GB00/04216
16
For Western blot analyses, the plant extracts were boiled in 75mM tris-HCl
(pH6.8) and
2% SDS under either non-reducing or reducing conditions (by addition of 0.1%
(3
mercaptoethanol). SDS-polyacrylamide gel electrophoresis (PAGE) in 10%
acrylamide
or 4-20% gradient gels was performed. The gels were blotted onto nitro-
cellulose paper
and blocked in TBS containing 0.05% Tween 20, and 1% non-fat dry milk. The
blots
were then incubated in appropriate antiserum for 2 hours at 37°C. After
washing, an
appropriate second-layer alkaline phosphatase-conjugated antiserum was applied
for 2
hours at 37°C. Antibody binding was detected by incubation with
nitroblue tetrazolium
(300mg/ml) and 5-bromo-4-chloro-3-indolyl phosphate (1 SOmg/ml) (Promega, UK).
Glycosylation is determined by Western blot, examining binding to lectins such
as
concanavalin A, or by using glycans specific antisera (kindly provided by Dr.
Loic
Faye, University of Rouen). Functional studies of antigen binding affinity to
available
monoclonal antibodies may be performed using surface plasmon resonance
techniques.
Recombinant.protein purification:
Purification was performed using a procedure which we have previously
determined for
IgG extraction from transgenic plants. Following initial precipitation from
crude plant
extract with ammonium sulphate, the recombinant antibody was concentrated by
stirred
cell filtration using a YM30 molecular weight cut-off filter (Amicon, UK).
Purification
was by affinity chromatography using agarose coupled to anti-mouse or human
IgG
antibodies (Sigma, UK) as the ligand. Elution was in O.1M glycine-HCl pH 2.5.
The recombinant heavy chains may also be designed to allow a further affinity
purification step using the 6xHis fusion peptide tag with immobilised metal
chelate
affinity chromatography, which can subsequently be cleaved by thrombin.
The expression of these fusion proteins is not limited to plants, and could be
earned out
in any other eukaryotic cells, especially cells that can produce
immunoglobulins such as
mammalian cells.
CA 02388955 2002-04-24
WO 01/32714 PCT/GB00/04216
17
The immunoglobulin chain sequences can be exchanged for sequences from other
classes of immunoglobulins.
Other sequences can be incorporated into the construct in addition to the
epitope tags
and protease cleavage sites. For example, these could code for other
functional regions,
such as markers, enzyme activity, or sites for chemical coupling to other
active
molecules.
In addition to HIV gp120, two further examples have been developed. Similar
constructs have been prepared using tetanus toxin non-toxic C-fragment (kindly
donated by Dr. Neil Fairweather at Imperial College, London) and a tetanus
toxin
specific murine monoclonal antibody (kindly donated by Dr. Claus Koch at the
State
Serum Institute, Copenhagen). In addition, we have prepared corresponding
constructs
for the F-protein of bovine respiratory syncytial virus and a specific murine
monoclonal
antibody the genes for which were kindly provided by Dr. K.G. Madsen at the
Danish
Veterinary Institute for Virus Research.
Among the benefits of the invention, the following are noteworthy:-
(i)Co-expression of antibody with antigen increases the levels of expression
of
antigen.
(ii)Antigen and whole antibody are genetically linked to form a type of immune
complex. In other constructs, antigen and antibody fragments (that may or may
not include the antigen binding site) are linked to form fusion proteins.
(iii)The recombinant antibody molecule and the antigen are automatically
expressed at 1:1 ratio. In the case of constructs involving whole IgG, larger
complexes may form spontaneously.
(iv)Antigen-antibody fusion molecules are easy to produce.
(v)The use of plants to make these molecules significantly reduces the cost,
particularly if production is increased to agricultural scale.
(vi)For oral vaccines, consumption of edible transgenic plants offers an
alternative, safe mode of delivery for immune complex vaccines.
CA 02388955 2002-04-24
WO 01/32714 PCT/GB00/04216
18
(vii)There is potentially no need for addition of an adjuvant as the
engineered
molecule is already highly immunogenic. The CH2 and CH3 immunoglobulin
domains include the site for complement activation and opsonisation of
phagocytes
Results:
Results have been obtained for two of the described constructs - CH2-gp120 and
CH2-CH3-gp 120. RT-PCR confirmed that specific mRNA of the desired size was
being made by transgenic plants (Figure 4).
Analysis by capture ELISA demonstrated that both constructs are being
expressed by
plants. We have gone on to characterise CH2-CH3-gp120 more fully and
demonstrated
that by ELISA that it contains IgG2a epitopes as well as gp120 epitopes as
shown in
Figures 5 and 6. Moreover, expression of the recombinant gp 120 is greater in
plants
expressing the CH2-CH3-gp120 construct, than those expressing either gp120
alone,
gp 120 with a C-terminal HDEL tag, or CH2-gp 120. Preliminary results indicate
an
expression level of gp120 of approximately 0.8%.
Purification of plant derived material has been carried out and Western blot
analysis
demonstrates the presence of protein bands that are recognised by specific
antibodies to
gp120 and murine IgG2a (Figure 7). Under reducing conditions, the most
prominent
band has a molecular size consistent with that expected of approximately SOKd.
Under
non-reducing conditions, higher molecular weight forms approaching 100Kd are
detected. This is consistent with the formation of homodimers, through
association of
the CH2 and CH3 domains of the heavy chain.
A preliminary investigation of immunogenicity has been carned out in a single
rhesus
monkey. Following 2 administrations of purified plant derived CH2-CH3-gp 120
(150~g per dose) covalently linked with the adjuvant mycobacterial heat shock
protein
70 using the SPDP bifunctional reagent, specific antibody and cellular immune
responses were detected. After 3 immunisations, very significant T-cell
proliferative
responses were detected, not only to the plant CH2-CH3-gp120 construct, but
also to
CA 02388955 2002-04-24'
WO 01/32714 PCT/GB00/04216
19
purified recombinant gp120 expressed in Chinese hamster ovary cells. This
indicates
that the plant recombinant gp120 is correctly expressed and folded in an
immunogenic
conformation. Furthermore, four macaques have been immunised by the mucosal
intra-vaginal route, and four by the targeted iliac lymph node route (Lehner
et al.,
Nature Medicine, 2:767-775, 1996). After two immunisations, induction of
interferon-y
and interleukin-4 has been detected, indicating that both THl and TH2
responses are
elicited. Further immunisation studies are being carned out in rhesus monkeys
as well
as mice.
