Note: Descriptions are shown in the official language in which they were submitted.
RECOMBINANT PLANT-DERIVED ANTIBODIES AND FC VARIANTS AND
RELATED METHODS
FIELD
This disclosure relates generally to recombinant plant-derived proteins, and
more
specifically to recombinant antibodies, and Fc variants thereof, and methods
of producing the
same. The disclosure also relates to methods of preventing or reducing
colonization of
Escherichia coil in a mammal. The disclosure also relates to methods of
detecting the presence
of E. coil in a sample.
BACKGROUND
Food borne pathogens such as Escherichia coil have consistently been one of
the
foremost foodborne pathogen threats worldwide. While there are many strategic
interventions
meant to prevent E. coil transmission to humans, conservative estimates
indicate that the
pathogen still causes 2.8 million acute illnesses annually (Majowicz et al.
2014).
Evading a host organism's defences, E. coil colonizes at mucosal sites
primarily in the
gastrointestinal tract (GI) in an animal. E. coil is ultimately transmitted to
humans through
consumption of contaminated foods, such as undercooked or raw meat, milk, or
vegetables, for
example, and may cause severe gastrointestinal illness with life-threatening
consequences in
some cases. The availability of effective therapeutics and diagnostics for
treatment or prevention
of E. coil contamination remains a problem.
One of the challenges in delivering such therapeutics and diagnostics is the
lack of cost-
effective production strategies. Over the past twenty years, plants have
become a preferred
platform of choice for complex immunoglobulin proteins and related synthetics,
including those
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Date Recue/Date Received 2021-02-08
that require glycosylation and disulfide bond formation for proper folding and
assembly.
However, low yield and the resulting high cost of production is arguably the
greatest barrier for
pushing these products to market (Wycoff et al. 2005). There are many
strategies for improving
the recombinant yield of plant-based biologics that include, for example,
affecting the amount
and stability of the transcript, affecting translation rates and
susceptibility to gene silencing, the
choice of host system/tissue, and affecting the physiological state of the
plant to slow
degradation of the accumulated recombinant protein through exogenous
application of hormones
or chemicals or changing environmental conditions. However, the improvements
in yield
provided by these strategies have been too modest to overcome the yield
barrier to advancing
these products to market and many require tailored optimization on a case-by-
case basis.
While the use of a plant platform for folding and assembly of recombinant
proteins and
other synthetics in the ER is well established, some ER-targeted recombinant
proteins have been
associated with issues such as unfolded proteins (De Wilde et al. 2013), ER-
associated
degradation, and misfolding, potentially limiting the proper folding in
certain antibodies, thus
reducing antibody yield.
Accordingly, in addition to a need for therapies effective in curtailing E.
coil
contamination of food and water supply, there is an associated need for
strategies for improved
recombinant protein yields to deliver plant produced therapeutics and
diagnostics in a cost
effective manner.
SUMMARY
Various embodiments of the claimed invention relate to a polypeptide
comprising:
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a single domain antibody, or an antigen binding fragment thereof, which
specifically binds to
intimin on an Escherichia coil cell. In some embodiments, the antibody binds
to an epitope
having at least 80%, 85%, 90%, 95%, 97%, or 100% amino acid sequence identity
to the
sequence set forth in SEQ ID NO: 53. In some embodiments, the antibody
comprises
complementarity determining regions (CDR) having at least 80%, 85%, 90%, 95%,
97%, or
100% amino acid sequence identity to the sequence as set forth in: (i) SEQ ID
NO: 12 (CDR1),
SEQ ID NO: 13 (CDR2), and SEQ ID NO: 14 (CDR3), (ii) SEQ ID NO: 15 (CDR1), SEQ
ID
NO: 16 (CDR2), and SEQ ID NO: 17 (CDR3), (iii) SEQ ID NO: 18 (CDR1), SEQ ID
NO: 19
(CDR2), and SEQ ID NO: 20 (CDR3), (iv) SEQ ID NO: 21 (CDR1), SEQ ID NO: 22
(CDR2),
and SEQ ID NO: 23 (CDR3), (v) SEQ ID NO: 24 (CDR1), SEQ ID NO: 25 (CDR2), and
SEQ
ID NO: 26 (CDR3), or (vi) SEQ ID NO: 27 (CDR1), SEQ ID NO: 28 (CDR2), and SEQ
ID NO:
29 (CDR3). In some embodiments, the antibody, or antigen binding fragment
thereof, comprises
a heavy chain variable (VHH) domain. In some embodiments, the VHH domain has
at least 80%,
85%, 90%, 95%, 97%, or 100% amino acid sequence identity to the sequence as
set forth in any
one of SEQ ID NOs: 1 to 11. In some embodiments, the VHH domain is linked to
an Fc chain. In
some embodiments, the VHH domain is linked to a bovine Fc chain. In some
embodiments, the
antibody neutralizes intimin on the E. coil cell from binding to an epithelial
cell. In some
embodiments, the epithelial cell is from the gastrointestinal tract of a
mammal. In some
embodiments, the polypeptide is targeted to the chloroplast thylakoid via the
Sec pathway. In
some embodiments, the polypeptide is targeted to the chloroplast thylakoid via
the Tat pathway.
In some embodiments, the polypeptide is targeted to the chloroplast stroma. In
some
embodiments, the polypeptide comprises a Sec-targeted peptide, a Tat-targeted
peptide, a
stroma-targeted peptide, or an ER-targeted peptide. In some embodiments, the
E. coil cell is a
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Shiga toxin-producing E. coil (STEC) cell. In some embodiments, the E. coil
cell is 026:H11,
0111:Hnm, 0145:Hnm, or 0157:H7. In some embodiments, the antibody is an IgA
antibody. In
some embodiments, the antibody comprises four VHH-Fc subunits, one secretory
component
(SC), and one joining chain (JC).
Various embodiments of the claimed invention relate to an antibody, or antigen
binding
fragment thereof, that competes for specific binding to intimin with the
polypeptide claimed
herein.
Various embodiments of the claimed invention relate to a nucleic acid encoding
the
polypeptide claimed herein.
Various embodiments of the claimed invention relate to an expression vector
comprising
the nucleic acid claimed herein.
Various embodiments of the claimed invention relate to a host cell comprising
the
expression vector claimed herein. In some embodiments, the host cell is a
bacterial cell. In some
embodiments, the bacterial cell is Agrobacterium tumefaciens . In some
embodiments, the host
cell is a plant cell. In some embodiments, the plant cell is a Nicotiana plant
cell. In some
embodiments, the plant cell is a Nicotiana benthamiana plant cell or a
Nicotiana tabacum plant
cell. In some embodiments, the plant cell is a Lactuca plant cell.
Various embodiments of the claimed invention relate to a non-viable harvested
plant
material comprising the host cell claimed herein. In some embodiments, the non-
viable plant
harvested material comprises a leaf or a stem.
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Date Recue/Date Received 2021-02-08
Various embodiments of the claimed invention relate to a non-viable edible
product
comprising the host cell claimed herein. In some embodiments, the non-viable
edible product
comprises a leaf or a stem.
Various embodiments of the claimed invention relate to a tobacco product
comprising the
host cell claimed herein. In some embodiments, the tobacco product is cut,
shredded, powdered,
loose, ground, granulated, or extruded.
Various embodiments of the claimed invention relate to an animal feed
comprising the
host cell claimed herein.
Various embodiments of the claimed invention relate to a pharmaceutical
composition
comprising the polypeptide claimed herein, and a pharmaceutically acceptable
carrier.
Various embodiments of the claimed invention relate to a diagnostic kit for
detecting the
presence of E. coil in a sample comprising the polypeptide claimed herein. In
some
embodiments, the sample is a food sample, an environmental sample, or a sample
from an animal
or a microorganism. In some embodiments, the sample is a fecal sample, a
carcass swab sample,
a water sample, a sample from a packaged meat, a sample from a vegetable, a
soil sample, or a
sample from a food-contacting surface.
Various embodiments of the claimed invention relate to a method of preventing
or
reducing colonization of E. coil in the gastrointestinal tract of a mammal,
comprising:
administering to the mammal the polypeptide claimed herein. In some
embodiments,
administering the polypeptide to the mammal comprises causing the mammal to
ingest plant
material from a plant that produces the polypeptide. In some embodiments, the
plant material is
for oral administration. In some embodiments, the plant material is for rectal
administration. In
Date Recue/Date Received 2021-02-08
some embodiments, the plant material is from a Nicotiana plant or a Lactuca
plant. In some
embodiments, the plant material is from a Nicotiana benthamiana plant or a
Nicotiana tabacum
plant. In some embodiments, the plant is harvested at a stage of harvest in
which accumulation of
the assembled VHH-Fc polypeptide or assembled sIgA in the plant is maximal.
Various embodiments of the claimed invention relate to a method of producing
the
polypeptide claimed herein, the method comprising transforming an organism
with a nucleic acid
molecule encoding the antibody. In some embodiments, the organism is a plant.
In some
embodiments, the plant is a Nicotiana plant or a Lactuca plant. In some
embodiments, the plant
is a Nicotiana benthamiana plant or a Nicotiana tabacum plant. In some
embodiments,
transforming the organism with the nucleic acid molecule encoding the IgA
antibody comprises
preparing Agrobacterium strain cultures comprising VHH-Fc subunits, SC, and JC
at optical
densities (OD) of about 0.57, 0.14, and 0.14, respectively, for infiltration
in the plant. In some
embodiments, the plant is harvested after infiltration. In some embodiments,
the plant is
harvested more than 3 days post infiltration (dpi). In some embodiments, the
plant is harvested
from between about 4 dpi to about 12 dpi. In some embodiments, the plant is
harvested at about
12 dpi. In some embodiments, the plant is harvested at a stage of harvest in
which accumulation
of the assembled VHH-Fc polypeptide or assembled sIgA in the plant is maximal.
Various embodiments of the claimed invention relate to a method of detecting
the
presence of E. coil in a sample, comprising: contacting the sample with the
polypeptide claimed
herein, to detect the presence of intimin in the sample, and detecting binding
between intimin
and the antibody. In some embodiments, the sample is a food sample,
environmental sample, or a
sample from an animal or microorganism. In some embodiments, the sample is a
fecal sample, a
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Date Recue/Date Received 2021-02-08
carcass swab sample, a water sample, a sample from a packaged meat, a sample
from a
vegetable, a soil sample, or a sample from a food-contacting surface.
Various embodiments of the claimed invention relate to use of the polypeptide
claimed
herein, for preventing or reducing E. coil cell colonization of the
gastrointestinal tract of a
mammal. In some embodiments, the antibody is for administration to a mammal.
In some
embodiments, the antibody is for oral administration. In some embodiments, the
antibody is for
rectal administration. In some embodiments, the antibody is produced in a
Nicotiana plant or a
Lactuca plant. In some embodiments, the antibody is produced in a Nicotiana
benthamiana plant
or a Nicotiana tabacum plant. In some embodiments, the plant is harvested at a
stage of harvest
in which accumulation of the assembled VHH-Fc polypeptide or assembled sIgA in
the plant is
maximal.
Various embodiments of the claimed invention relate to use of the polypeptide
claimed
herein, in preparation of a medicament for preventing or reducing E. coil cell
colonization of the
gastrointestinal tract of a mammal. In some embodiments, the antibody is for
administration to a
mammal. In some embodiments, the antibody is for oral administration. In some
embodiments,
the antibody is for rectal administration. In some embodiments, the antibody
is produced in a
Nicotiana plant or a Lactuca plant. In some embodiments, the antibody is
produced in a
Nicotiana benthamiana plant or a Nicotiana tabacum plant. In some embodiments,
the plant is
harvested at a stage of harvest in which accumulation of the assembled VHH-Fc
polypeptide or
assembled sIgA in the plant is maximal.
Various embodiments of the claimed invention relate to use of the polypeptide
claimed
herein, for neutralizing an E. coil cell. In some embodiments, the antibody is
for administration
to a mammal. In some embodiments, the antibody is for oral administration. In
some
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embodiments, the antibody is for rectal administration. In some embodiments,
the antibody is
produced in a Nicotiana plant or Lactuca plant. In some embodiments, the
antibody is produced
in a Nicotiana benthamiana plant or a Nicotiana tabacum plant. In some
embodiments, the plant
is harvested at a late stage of harvest in which accumulation of the assembled
VHH-Fc
polypeptide or assembled sIgA in the plant is maximal.
Various embodiments of the claimed invention relate to use of the polypeptide
claimed
herein, in preparation of a medicament for neutralizing an E. coil cell. In
some embodiments, the
antibody is for administration to a mammal. In some embodiments, the antibody
is for oral
administration. In some embodiments, the antibody is for rectal
administration. In some
embodiments, the antibody is produced in a Nicotiana plant or Lactuca plant.
In some
embodiments, the antibody is produced in a Nicotiana benthamiana plant or a
Nicotiana
tabacum plant. In some embodiments, the plant is harvested at a late stage of
harvest in which
accumulation of the assembled VHH-Fc polypeptide or assembled sIgA in the
plant is maximal.
Various embodiments of the claimed invention relate to use of the polypeptide
claimed
herein, for detecting the presence of E. coil in a sample. In some
embodiments, the sample is a
food sample, environmental sample, or a sample from an animal or
microorganism. In some
embodiments, the sample is a fecal sample, a carcass swab sample, a water
sample, a sample
from a packaged meat, a sample from a vegetable, a soil sample, or a sample
from a food-
contacting surface.
Various embodiments of the claimed invention relate to a polypeptide
comprising: a
variant Fc chain that exhibits enhanced accumulation in an organism. In some
embodiments, the
organism is a plant. In some embodiments, the plant is a Nicotiana plant or
Lactuca plant. In
some embodiments, the plant is a Nicotiana benthamiana plant or a Nicotiana
tabacum plant. In
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Date Recue/Date Received 2021-02-08
some embodiments, the Fc chain has at least one amino acid substitution
selected from N9D
(SEQ ID NO: 32), N84D (SEQ ID NO: 33), N131D (SEQ ID NO: 34), Q175E (SEQ ID
NO:
35), and Q195E (SEQ ID NO: 36). In some embodiments, the Fc chain has a de
novo disulfide
from an amino acid substitution G196C/R219C (SEQ ID NO: 37). In some
embodiments, the Fc
chain comprises the following amino acid substitutions: N9D, N84D, and N131D
(SEQ ID NO:
38). In some embodiments, the Fc chain comprises the following amino acid
substitutions: N9D,
N84D, N131D, Q175E, and Q195E (SEQ ID NO: 39). In some embodiments, the Fc
chain
comprises the following amino acid substitutions: N9D, N84D, N131D, Q175E,
Q195E, and
G196C/R219C (SEQ ID NO: 40). In some embodiments, the polypeptide exhibits at
least a 3-
fold increase in accumulation. In some embodiments, the polypeptide exhibits
up to about a 22-
fold increase. In some embodiments, the polypeptide exhibits about a 22-fold
increase. In some
embodiments, the Fc chain is a bovine Fc chain. In some embodiments, the Fc
chain is linked to
a bioactive moiety. In some embodiments, the bioactive moiety is an enzyme,
cytokine,
antibody, antibody fragment, peptide, signalling molecule, receptor, or
ligand. In some
embodiments, the polypeptide is an antibody. In some embodiments, the
polypeptide is an IgA
antibody. In some embodiments, the antibody binds to an epitope having at
least 80%, 85%,
90%, 95%, 97%, or 100% amino acid sequence identity to the sequence set forth
in SEQ ID NO:
53. In some embodiments, the antibody comprises complementarity determining
regions (CDR)
having at least 80%, 85%, 90%, 95%, 97%, or 100% amino acid sequence identity
to the
sequence as set forth in: (i) SEQ ID NO: 12 (CDR1), SEQ ID NO: 13 (CDR2), and
SEQ ID
NO: 14 (CDR3), (ii) SEQ ID NO: 15 (CDR1), SEQ ID NO: 16 (CDR2), and SEQ ID NO:
17
(CDR3), (iii) SEQ ID NO: 18 (CDR1), SEQ ID NO: 19 (CDR2), and SEQ ID NO: 20
(CDR3),
(iv) SEQ ID NO: 21 (CDR1), SEQ ID NO: 22 (CDR2), and SEQ ID NO: 23 (CDR3), (v)
SEQ
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Date Recue/Date Received 2021-02-08
ID NO: 24 (CDR1), SEQ ID NO: 25 (CDR2), and SEQ ID NO: 26 (CDR3), or (vi) SEQ
ID NO:
27 (CDR1), SEQ ID NO: 28 (CDR2), and SEQ ID NO: 29 (CDR3). In some
embodiments, the
Fc chain is linked to a VHH domain. In some embodiments, the VHH domain has at
least 80%,
85%, 90%, 95%, 97%, or 100% amino acid sequence identity to the sequence as
set forth in any
one of SEQ ID NOs 1 to 11. In some embodiments, the polypeptide is targeted to
the chloroplast
thylakoid via the Sec pathway. In some embodiments, the polypeptide is
targeted to the
chloroplast thylakoid via the Tat pathway. In some embodiments, the
polypeptide is targeted to
the chloroplast stroma. In some embodiments, the polypeptide comprises a Sec-
targeted peptide,
a Tat-targeted peptide, a stroma-targeted peptide, or an ER-targeted peptide.
Various embodiments of the claimed invention relate to a method of producing a
variant
Fc chain of a native Fc chain comprising: determining solvent accessibility of
an amino acid
residue in the Fc chain, and selecting a polar, solvent-exposed amino acid
residue for mutation to
its negatively charged counterpart. In some embodiments, selecting the polar,
solvent-exposed
amino acid residue for mutation to its negatively charged counterpart
comprises selecting an Asn
or Gln residue for mutation to an Asp or Glu residue, respectively. In some
embodiments, at least
one of the following Asn or Gln residues are for mutation to Asp or Glu,
respectively: N9D
(SEQ ID NO: 32), N84D (SEQ ID NO: 33), N131D (SEQ ID NO: 34), Q175E (SEQ ID
NO:
35), and Q195E (SEQ ID NO: 36). In some embodiments, the variant Fc chain is
an IgA Fc
chain.
Various embodiments of the claimed invention relate to a method of producing a
variant
Fc chain comprising: selecting a first amino acid and a second amino acid in
the Fc chain, the
second amino acid in a proximate distance from the first amino acid, wherein
the first amino acid
and the second amino acid are not involved in native disulfide bonding, and
mutating the first
Date Recue/Date Received 2021-02-08
and second amino acids to cysteines to form a disulfide bond between the first
and second amino
acids. In some embodiments, mutating the first and second amino acids to
cysteines to form the
disulfide bond stabilizes the tertiary structure of the Fc chain. In some
embodiments, mutating
the first and second amino acids to cysteines to form the disulfide bond
connects at least two beta
sheets within the Fc chain together. In some embodiments, the disulfide bond
is between amino
acid substituted residues G196C/R219C. In some embodiments, the Fc chain is an
IgA Fc chain.
In some embodiments, the proximate distance is less than 5 A.
Various embodiments of the claimed invention relate to a method of enhancing
accumulation of a protein, comprising: transforming a plant, or a portion
thereof, with a
recombinant expression vector comprising a nucleic acid molecule encoding a
genetically
modified variant Fc chain. In some embodiments, the Fc chain has at least one
amino acid
substitution selected from N9D (SEQ ID NO: 32), N84D (SEQ ID NO: 33), N131D
(SEQ ID
NO: 34), Q175E (SEQ ID NO: 35), and Q195E (SEQ ID NO: 36). In some
embodiments, the Fc
chain has a de novo disulfide from an amino acid substitution G196C/R219C (SEQ
ID NO: 37).
In some embodiments, the Fc chain comprises the following amino acid
substitutions: N9D,
N84D, and N131D (SEQ ID NO: 38). In some embodiments, the Fc chain comprises
the
following amino acid substitutions: N9D, N84D, N131D, Q175E, and Q195E (SEQ ID
NO: 39).
In some embodiments, the Fc chain comprises the following amino acid
substitutions: N9D,
N84D, N131D, Q175E, Q195E, and G196C/R219C (SEQ ID NO: 40). In some
embodiments,
the Fc chain accumulation in the plant, or plant portion thereof, is enhanced
up to about 22-fold.
In some embodiments, the Fc chain is linked to a bioactive moiety. In some
embodiments, the
bioactive moiety is an enzyme, cytokine, antibody, antibody fragment, peptide,
signalling
molecule, receptor, or ligand. In some embodiments, the Fc chain is linked to
a heavy chain
11
Date Recue/Date Received 2021-02-08
variable (VHH) domain. In some embodiments, the VHH-Fc accumulation in the
plant, or plant
portion thereof, is enhanced up to about 16-fold. In some embodiments, the Fc
chain is targeted
to the chloroplast thylakoid via the Sec pathway. In some embodiments, the Fc
chain is targeted
to the chloroplast thylakoid via the Tat pathway. In some embodiments, the Fc
chain is targeted
to the chloroplast stroma. In some embodiments, the Fc chain comprises a Sec-
targeted peptide,
a Tat-targeted peptide, a stroma-targeted peptide, or an ER-targeted peptide.
Various embodiments of the claimed invention relate to a method of enhancing
expression of a recombinant protein in a plant, or a portion thereof,
comprising: transforming the
plant, or plant portion thereof, with a recombinant expression vector
comprising a nucleic acid
molecule encoding the recombinant protein, wherein the recombinant protein is
targeted to the
chloroplast. In some embodiments, the recombinant protein is targeted to the
chloroplast
thylakoid via the Sec pathway. In some embodiments, the recombinant protein is
targeted to the
chloroplast thylakoid via the Tat pathway. In some embodiments, the
recombinant protein is
targeted to the chloroplast stroma. In some embodiments, the recombinant
peptide comprises a
Sec-targeted peptide, a Tat-targeted peptide, a stroma-targeted peptide, or an
ER-targeted
peptide. In some embodiments, the recombinant protein comprises an Fc chain.
In some
embodiments, the Fc chain has at least one amino acid substitution selected
from N9D (SEQ ID
NO: 32), N84D (SEQ ID NO: 33), N131D (SEQ ID NO: 34), Q175E (SEQ ID NO: 35),
and
Q195E (SEQ ID NO: 36). In some embodiments, the Fc chain has a de novo
disulfide from an
amino acid substitution G196C/R219C (SEQ ID NO: 37). In some embodiments, the
Fc chain
comprises the following amino acid substitutions: N9D, N84D, and N131D (SEQ ID
NO: 38). In
some embodiments, the Fc chain comprises the following amino acid
substitutions: N9D, N84D,
N131D, Q175E, and Q195E (SEQ ID NO: 39). In some embodiments, the Fc chain
comprises
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Date Recue/Date Received 2021-02-08
the following amino acid substitutions: N9D, N84D, N131D, Q175E, Q195E, and
G196C/R219C (SEQ ID NO: 40). In some embodiments, the recombinant protein
comprises a
bioactive moiety linked to the Fc chain. In some embodiments, the recombinant
protein
comprises a heavy chain variable (VHH) domain linked to the Fc chain. In some
embodiments,
the Fc chain is an IgA Fc chain. In some embodiments, the Fc chain is a bovine
IgA Fc chain. In
some embodiments, the recombinant protein is an antibody. In some embodiments,
the
recombinant protein is an IgA antibody. In some embodiments, the antibody
comprises
complementarity determining regions (CDR) having at least 80%, 85%, 90%, 95%,
97%, or
100% amino acid sequence identity to the sequence as set forth in: (i) SEQ ID
NO: 12 (CDR1),
SEQ ID NO: 13 (CDR2), and SEQ ID NO: 14 (CDR3), (ii) SEQ ID NO: 15 (CDR1), SEQ
ID
NO: 16 (CDR2), and SEQ ID NO: 17 (CDR3), (iii) SEQ ID NO: 18 (CDR1), SEQ ID
NO: 19
(CDR2), and SEQ ID NO: 20 (CDR3), (iv) SEQ ID NO: 21 (CDR1), SEQ ID NO: 22
(CDR2),
and SEQ ID NO: 23 (CDR3), (v) SEQ ID NO: 24 (CDR1), SEQ ID NO: 25 (CDR2), and
SEQ
ID NO: 26 (CDR3), or (vi) SEQ ID NO: 27 (CDR1), SEQ ID NO: 28 (CDR2), and SEQ
ID NO:
29 (CDR3). In some embodiments, the antibody, or antigen binding fragment
thereof, comprises
a heavy chain variable (VHH) domain. In some embodiments, the VHH domain has
at least 80%,
85%, 90%, 95%, 97%, or 100% amino acid sequence identity to the sequence as
set forth in any
one of SEQ ID NOs: 1 to 11.
Various embodiments of the claimed invention relate to a method of producing a
recombinant protein in a plant, or a portion thereof, comprising: transforming
the plant, or
portion thereof, with a recombinant expression vector comprising a nucleic
acid molecule
encoding the recombinant protein, wherein the recombinant protein is targeted
to the chloroplast.
In some embodiments, the recombinant protein is targeted to the chloroplast
thylakoid via the
13
Date Recue/Date Received 2021-02-08
Sec pathway. In some embodiments, the recombinant protein is targeted to the
chloroplast
thylakoid via the Tat pathway. In some embodiments, the recombinant protein is
targeted to the
chloroplast stroma. In some embodiments, the recombinant peptide comprises a
Sec-targeted
peptide, a Tat-targeted peptide, a stroma-targeted peptide, or an ER-targeted
peptide. In some
embodiments, the recombinant protein comprises an Fc chain. In some
embodiments, the Fc
chain has at least one amino acid substitution selected from N9D (SEQ ID NO:
32), N84D (SEQ
ID NO: 33), N131D (SEQ ID NO: 34), Q175E (SEQ ID NO: 35), and Q195E (SEQ ID
NO: 36).
In some embodiments, the Fc chain has a de novo disulfide from an amino acid
substitution
G196C/R219C (SEQ ID NO: 37). In some embodiments, the Fc chain comprises the
following
amino acid substitutions: N9D, N84D, and N131D (SEQ ID NO: 38). In some
embodiments, the
Fc chain comprises the following amino acid substitutions: N9D, N84D, N131D,
Q175E, and
Q195E (SEQ ID NO: 39). In some embodiments, the Fc chain comprises the
following amino
acid substitutions: N9D, N84D, N131D, Q175E, Q195E, and G196C/R219C (SEQ ID
NO: 40).
In some embodiments, the recombinant protein comprises a bioactive moiety
linked to the Fc
chain. In some embodiments, the recombinant protein comprises a heavy chain
variable (VHH)
domain linked to the Fc chain. In some embodiments, the Fc chain is an IgA Fc
chain. In some
embodiments, the Fc chain is a bovine IgA Fc chain. In some embodiments, the
recombinant
protein is an antibody. In some embodiments, the recombinant protein is an IgA
antibody. In
some embodiments, wherein the antibody comprises complementarity determining
regions
(CDR) having at least 80%, 85%, 90%, 95%, 97%, or 100% amino acid sequence
identity to the
sequence as set forth in: (i) SEQ ID NO: 12 (CDR1), SEQ ID NO: 13 (CDR2), and
SEQ ID
NO: 14 (CDR3), (ii) SEQ ID NO: 15 (CDR1), SEQ ID NO: 16 (CDR2), and SEQ ID NO:
17
(CDR3), (iii) SEQ ID NO: 18 (CDR1), SEQ ID NO: 19 (CDR2), and SEQ ID NO: 20
(CDR3),
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Date Recue/Date Received 2021-02-08
(iv) SEQ ID NO: 21 (CDR1), SEQ ID NO: 22 (CDR2), and SEQ ID NO: 23 (CDR3), (v)
SEQ ID NO: 24 (CDR1), SEQ ID NO: 25 (CDR2), and SEQ ID NO: 26 (CDR3), or (vi)
SEQ
ID NO: 27 (CDR1), SEQ ID NO: 28 (CDR2), and SEQ ID NO: 29 (CDR3). In some
embodiments, the antibody, or antigen binding fragment thereof, comprises a
heavy chain
variable (VHH) domain. In some embodiments, the VHH domain has at least 80%,
85%, 90%,
95%, 97%, or 100% amino acid sequence identity to the sequence as set forth in
any one of SEQ
ID NOs: 1 to 11.
BRIEF DESCRIPTION OF THE DRAWINGS
Exemplary embodiments are illustrated in referenced figures of the drawings.
It is
intended that the embodiments and figures disclosed herein are to be
considered illustrative
rather than restrictive.
FIG. lA is a chart showing the amino acid sequence of MBP-Int277 fusion
protein. MBP
sequence is underlined and TEV protease cleavage site (ENLYFQG) is shown in
grey.
FIG. 1B is an image showing an SDS-PAGE (4-20% gradient) stained with
Coomassie
Brilliant Blue. Lane 1, BSA; Lane 2, MBP-Int277.
FIG. 1C is an image showing a Western blot of MBP-Int277 using either anti-6
His
antibody (Lane 3) or anti-MBP antibody (Lane 4).
FIG. 1D is a graph showing the size exclusion profile of MBP-Int277 on a
SuperdeXim 75
10/300 GL column showing monodisperse behavior.
FIG. lE is a graph showing the binding of polyclonal goat anti-intimin
antibody to MBP-
Int277 in ELISA and detected with HRP-conjugated donkey anti-goat IgG.
Date Recue/Date Received 2021-02-08
FIG. 2A is a schematic showing all produced subunits fully assembled into an
antibody
intended for secretory IgA functionality. It notably differs from the
structure of native secretory
IgA by the replacement of the Fab region with a camelid-derived variable heavy
chain fragment
(VHH).
FIG. 2B is a schematic showing translated regions cloned into a plant
expression vector
used for Agrobacterium-mediated transient expression in N. benthamiana leaves.
VHHx-Fc,
fusion of a camelid-derived VHH to a bovine Fc where x, is either 1, 3, 9, or
10, corresponding
to the isolated VHHs; SC, bovine secretory component; JC, bovine JC; c-Myc,
FLAG, HA,
detection tags; KDEL, endoplasmic reticulum retrieval tetra-peptide. Schematic
not drawn to
scale.
FIG. 2C-E are images showing Western blots of extract from leaves of N.
benthamiana
harvested at 6 dpi expressing VHH1, 3, 9, and 10-Fc along with p19, a
suppressor of gene
silencing, secretory component (SC), and joining chain (JC).
FIG. 3A is an image of a Western blot of extract from N. benthamiana leaf
tissue
infiltrated with Agrobacterium mixtures showing accumulation over 4, 6, and 8
days post-
infiltration (dpi) of all three protein subunits (VHH-Fc/SC/JC), each lane
loaded with ten
micrograms of total soluble protein (TSP) and separated by SDS-PAGE under
reducing
conditions. Extract from p19-infiltrated N. benthamiana leaf tissue was used
as a negative
control.
FIG. 3B is a graph showing quantities of all three protein subunits, JC (light
gray), VHH-
Fc (black), and SC (dark gray), from the Western blot of Figure 3A.
16
Date Recue/Date Received 2021-02-08
FIG. 3C is an image of a Western blot of extract from N. benthamiana leaf
tissue
infiltrated with Agrobacterium mixtures showing accumulation over 6, 8, 10,
and 12 dpi of all
three protein subunits (VHH-Fc/SC/JC).
FIGs. 4A-D are images of Western blots of extract from N. benthamiana leaf
tissue co-
infiltrated with all VHH3-sIgA subunits collected at 6 dpi.
FIG. 5A is an image of a Western blot of extract from N. benthamiana leaf
tissue co-
infiltrated with a mixture of the indicated Agrobacterium strain collected
from 4 to 12 dpi
detected with anti-HA antibodies.
FIG. 5B is an image of a Western blot of extract from N. benthamiana leaf
tissue co-
infiltrated with a mixture of the indicated Agrobacterium strain collected
from 4 to 12 dpi
detected with anti-c-Myc antibodies.
FIG. 6A is an image of a Western blot of extract from N. benthamiana leaves
vacuum
infiltrated with a mixture of VHH9-Fc/SC/JC and p19, collected at 12 dpi,
purified with peptide
M agarose and detected with anti-c-Myc antibody.
FIG. 6B is an image of a Western blot of extract from N. benthamiana leaves
vacuum
infiltrated with a mixture of VHH9-Fc/SC/JC and p19, collected at 12 dpi,
purified with anti-
FLAG agarose and detected with anti-FLAG antibody.
FIG. 7A are graphs showing surface plasmon resonance (SPR) binding of VHH9-
sIgA
purified using peptide M. Plant-produced VHH9-sIgA or E. coil-produced VHH9
monomer was
immobilized on CM5 Series S sensor chips via amine coupling and MBP-Int277 was
flowed
over the resulting surfaces at concentrations ranging from 0.3 to 5 nM.
17
Date Recue/Date Received 2021-02-08
FIG. 7B are graphs showing ELISA binding of plant-produced VHH9-sIgA purified
using either peptide M (left) or anti-FLAG antibody (right) and detected using
either anti-bovine
IgA antibody (top) or anti-FLAG antibody (bottom).
FIG. 8 are confocal images showing the binding of the seven most prevalent
strains of E.
coil with VHH10-sIgA. Binding is visualized by DAPI that stains E. coil
bacterial cells and a
FITC-conjugated antibody that hybridizes to the Fc chain of VHH10-sIgA.
FIG. 9 are confocal images showing plant-produced VHH1O-Fc chain binding to E.
coil
0157:H7 by itself as well as when co-expressed with the JC and SC as a sIgA
complex. Images
show co-localization of a FITC-conjugated antibody that hybridizes to the Fc
chain of either
VHH10-sIgA (VHH1O-Fc/SC/JC) or VHH1O-Fc as well as DAPI that stains E. coil
0157:H7
bacterial cells. E. coil cells treated with PBS-T instead of antibody were
used as a negative
control.
FIG. 10A are confocal images showing immunolabelled E. coil cells with a
donkey anti-
rabbit secondary antibody as well as the actin cytoskeleton of HEp-2 cells
using rhodamine
phalloidin. Images show HEp-2 cells incubated with E. coil alone (left panel)
or with E. coil and
VHH10-sIgA (right panel).
FIG. 10B is a graph showing relative fluorescence of E. coil strains that have
been
immunolabeled, are adherent on HEp-2 cells, and either incubated on HEp-2
cells alone or in
combination with VHH10-sIgA. As a negative control, HEp-2 cells were incubated
with PBS
instead of a bacterial strain or antibody.
FIG. 10C are confocal images showing the previously used E. coil strain 0145
(C483)
and the subsequently obtained E. coil strain 0145 (C625) incubated with HEp-2
cells, in the
18
Date Recue/Date Received 2021-02-08
presence of PBS as a control, with VHH9-Fc or VHH10-sIgA. Cells that are
immunolabelled
(white) are intimately adherent on HEp-2 cells after repeated washes and their
absence suggests
neutralization. Size bar = 20 gm.
FIG. 10D is a chart showing a phylogenetic tree using a neighbor-joining
method to
cluster the aligned Int277 sequences for E. coil strains 0157, 0111, 0145,
026, 045, 0103 and
0121 based on similarity.
FIG. 11A is a schematic showing the amino acid sequence of the bovine IgA Fc
sequence. Boxes indicate positions of the candidates for supercharging and
circles indicate the
positions of the candidates for de novo disulfide bonds.
FIG. 11B is a schematic showing the Greek key connectivity of the Fc's beta
barrel
structure. Arrows indicate beta strands; S indicates the positions of
supercharging candidates and
DB indicates the positions of de novo disulfide bond candidates.
FIG. 11C is a wire diagram of a dimerized Fc with native intra- and inter-
chain
disulfides and the de novo disulfide.
FIG. 11D is an image representing the surface of the bovine Fc chain and
circles indicate
the positions of the supercharging candidates.
FIG. 12A is a graph showing accumulation levels of native Fc compared to the
supercharging Fc mutants at 4, 6, and 8dpi. Letters denote significantly
different treatments as
determined by one way ANOVA and post-hoc Tukey HSD test. P<0.05, n=3-5
biological
replicates. Error bars shown are standard error of the mean.
19
Date Recue/Date Received 2021-02-08
FIG. 12B is a graph showing accumulation levels of native Fc compared to the
de novo
disulfide Fc mutant at 4, 6, and 8dpi. * represents statistically significant
difference from native
as determined by a T-test.
FIG. 12C is a graph showing accumulation levels of native Fc compared to
various
combination Fc mutants at 4, 6, and 8dpi. Letters denote significantly
different treatments as
determined by one way ANOVA and post-hoc Tukey HSD test. P<0.05, n=3-5
biological
replicates. Error bars shown are standard error of the mean.
FIG. 12D is a graph showing accumulation levels of native VHH-Fc compared to
the
supercharging and de novo disulfide VHH-Fc mutants at 4, 6, and 8dpi. Letters
denote
significantly different treatments as determined by one way ANOVA and post-hoc
Tukey HSD
test. P<0.05, n=3-5 biological replicates. Error bars shown are standard error
of the mean.
FIG. 12 E is a graph showing accumulation levels of native VHH-Fc compared to
various
combination VHH-Fc mutants at 8dpi. Letters denote significantly different
treatments as
determined by one way ANOVA and post-hoc Tukey HSD test. P<0.05, n=3-5
biological
replicates. Error bars shown are standard error of the mean.
FIG. 13A-D are images of Western blots probed with either anti-cmyc (A,B),
anti-HA
(C,D) which correspond to differently tagged subunits VHH-Fc and JC
respectively. Leaf issue
was transformed with constructs of each subunit individually and also with
combinations of
VHH-Fc/SC/JC and VHH-(5+1) Fc/SC/JC for intended co-expression and assembly.
Detection
was done for both crude leaf extract (A,C) and for the eluent after the
extract had been co-
immunoprecipitated using an anti-FLAG column (B,D).
Date Recue/Date Received 2021-02-08
FIG. 14 are confocal images showing the seven most prevalent E. coil strains
incubated
with either VHH-Native Fc or VHH(5+1) Fc. DAPI has been used to visualize E.
coil cells and a
FITC-conjugated antibody has been used to immunolabel the Fc specifically.
Size bar=10 gm.
FIG. 15 are confocal images of the seven most prevalent E. coil strains that
have been
incubated with HEp-2 cells in the presence of PBS as a control, with VHH-Fc or
with VHH-
(5+1)Fc immunolabelled, are adherent on HEp-2 cells and either incubated on
HEp-2 cells in
PBS, with VHH-Fc or with VHH-(5+1)Fc. As a control against nonspecific Fc
binding,
0157:H7 was incubated with Fc only to confirm that neutralization was mediated
through the
VHH. Size bar = 20gm.
FIG. 16 is a graph showing the relative fluorescence of the seven most
prevalent E. coil
strains that have been immunolabelled, are adherent on HEp-2 cells and either
incubated on
HEp-2 cells in PBS, with VHH- Fc or with VHH-(5+1)-Fc and quantified by
fluorometry. As a
negative control, HEp-2 cells were incubated with PBS instead of a bacterial
strain or antibody.
Letters indicate a significant difference of the amount of immunolabelled
adherent bacteria as
determined by a one-way ANOVA with a post-hoc Tukey HSD test (p<0.05, N=3
biological
replicates). Error bars indicate standard error.
FIG. 17 is a schematic showing expected scenarios of targeting the antibody
with Sec,
Tat and stromal signal peptides.
FIG. 18 is a schematic of the thylakoid expression vector. Triangles indicate
predicted
cleavage sites after entry into stromal (black triangles) and thylakoid (white
triangles)
compaiiments. 2x355: double-enhanced promoter from Cauliflower Mosaic Virus
35S gene;
tCUP: translational enhancer from a tobacco cryptic upstream promoter;
attBliattB2: cloning
21
Date Recue/Date Received 2021-02-08
sites used for GatewayTM cloning; nosT: nopaline synthase transcription
terminator; Xpress/C-
Myc: detection/purification tags.
FIG. 19A is a graph showing VHH-Fc accumulation levels across cytoplasm, Sec,
Tat,
stromal and ER extracted in reducing (left) or non-reducing conditions
(right).
FIG. 19B is an image of a Western blot showing relative accumulation of VHH-Fc
across
comparnnents in reducing (left) and non-reducing conditions (right).
FIG. 20 are confocal images showing GFP-tagged VHH-Fc targeted to the
chloroplasts
with either Sec, Tat or stromal signals. Chlorophyll indicates the locations
of the thylakoid grana.
Fluorescence was sequentially captured, and the merged images show co-
localization of GFP
and chlorophyll. Size bar = 10jim.
FIG. 21A is a schematic of the Greek key connectivity showing the relative
positions of
native disulfides and the introduced disulfide in the Fc. X's indicate
cysteines involved in
interchain disulfide formation, circles indicate cysteines involved in
intrachain disulfide
formation, and triangles indicate introduced cysteines for de novo disulfide
formation.
FIG. 21B is a graph showing accumulation of Sec-targeted native VHH-Fc and a
VHH-
Fc with an added disulfide. *indicates statistical significance as determined
by a T-test with
p<0.05, n=3 biological replicates. Error bars shown are standard error of the
mean.
FIG. 22 are confocal images showing VHH-Fc targeted with either Sec, Tat or
stromal
signals incubated with E. coil 0157:H7. DAPI has been used to visualize E.
coil cells and a
FITC-conjugated antibody (green) has been used to immunolabel the Fc
specifically. Size
bar=10 gm.
22
Date Recue/Date Received 2021-02-08
FIG. 23 are confocal images showing 0157:H7 that has been incubated with HEp-2
cells
in the presence of either VHH-Fc targeted to Sec, Tat and stromal compai
intents or Fc as a
negative control targeted to the same compai intents. Size bar = 20 gm.
FIG. 24 is a schematic showing two possible mechanisms of disulfide bond
formation,
interaction with LT01 and spontaneous formation, that may account for
disulfide formation of
tat and stroma targeted antibodies.
DETAILED DESCRIPTION
The inventors Menassa and Henry are public servants within the meaning of the
Public
Servants Inventions Act, R.S.C., 1985, c. P-32.
Definitions
Terms defined herein are provided solely to aid in the understanding of the
present
disclosure and should not be construed to have a scope less than understood by
a person of
ordinary skill in the art.
As used herein, terms of degree such as "about", "approximately" and
"substantially"
refer to the indicated value and to all values that are within the
experimental error of the
indicated value (e.g. within the 95% confidence interval for the mean) or
within 10 percent of the
indicated value, whichever is greater. These terms may refer to a measurable
value such as an
amount, a temporal duration, and the like.
As used herein, unless otherwise required by context, singular terms such as
"a" and "an",
are understood to include pluralities and plural terms are understood to
include the singular. Any
examples following the term "for example" or "e.g." are not meant to be
limiting or exhaustive.
23
Date Recue/Date Received 2021-02-08
As used herein, the terms "comprises", "comprising", "include", "includes",
"including",
"contain", "contains" and "containing" are meant to imply inclusion of the
stated element or step
but not to the exclusion of other elements or steps.
As used herein, the term "polypeptide", "peptide", and "protein" may be used
interchangeably to refer to chains of amino acids of any length and may
comprise amino acids
modified naturally or by intervention, such as disulfide bond formation,
glycosylation, lipidation,
acetylation, phosphorylation, or any other manipulation or modification, such
as conjugation
with a labeling component. Also included within the definition are, for
example, polypeptides
containing one or more analogs of an amino acid (including, for example,
unnatural amino acids,
etc.), as well as other modifications known in the art.
The term "single domain antibody" defines an immunoglobulin molecule where the
antigen binding site is present on, and formed by, a single variable domain.
In conventional
immunoglobulins (ie. four chain antibodies), a heavy chain variable domain
(VH) and a light
chain variable domain (VL) interact to form an antigen binding site defined by
a total of six
complementarity determining regions (CDRs). In contrast, immunoglobulins with
a single
variable domain are capable of binding to an epitope without an additional
variable domain, with
the binding site formed by a single VH/VHH or VL domain. As such, the antigen
binding site of
a single variable domain is formed by not more than three CDRs. Thus, the
single variable
domain may be a light chain variable domain (e.g. VL sequence), or a heavy
chain variable
domain (e.g. VH or VHH sequence), or a fragment thereof capable of forming the
single antigen
binding unit, such that the single antigen binding domain does not need to
interact with another
variable domain to form a functional antigen binding unit.
24
Date Recue/Date Received 2021-02-08
The terms "specifically binds" or "binds specifically" is a term that is well
understood in
the art, and methods to determine such specific binding between an antibody
and antigen are also
well known in the art. An antibody "specifically binds" or "binds
specifically" to a target if it
binds with greater affinity, avidity, more readily, and/or with greater
duration to the target than it
binds to other present substances.
As used herein, the term "neutralizes" or "neutralizing antibody" means an
antibody that
reduces or abolishes the biological activity (eg. binding and/or infectivity)
of the target to which
it binds.
The terms "VHH domains", "VHH", or "VHH" refer to the variable domain of
"heavy
chain antibodies" (ie. antibodies without the light chain), and are used to
distinguish these
variable domains from the heavy chain variable domains (referred to as "VH" or
"VII" domains)
and the light chain variable domains (referred to as "VL" or "VL" domains)
present in
conventional four chain antibodies.
The term "competes", as used herein with regard to an antibody, means that a
first
antibody, antigen binding fragment thereof, ligand/receptor, or other protein
binds to an epitope
in a manner sufficiently similar to the binding of a second antibody, antigen
binding fragment
thereof, ligand/receptor, or other protein such that the result of binding of
the first antibody
antigen binding fragment thereof, ligand/receptor, or other protein to its
cognate epitope is
detectably decreased in the presence of the second antibody, antigen binding
fragment thereof,
ligand/receptor, or other protein compared to binding in the absence of the
second antibody,
antigen binding fragment thereof, ligand/receptor, or other protein.
Date Recue/Date Received 2021-02-08
The term "nucleic acid", "nucleic acid molecule", "oligonucleotide", or
"polynucleotide"
may be used interchangeably to refer to a polymer of nucleic acid residues in
single or double
stranded form.
The term "expression vector" includes plasmid vectors, cosmid vectors, phage
vectors,
viral vectors, or any other vectors known to the skilled person. Expression
vectors contain a
desired coding sequence and promoter sequences for the expression of the
operably linked
coding sequence in a particular host organism (e.g. higher eukaryotes, lower
eukaryotes,
prokaryotes). Among other features of vectors known to the skilled person, the
vector may also
contain features relating to expression control (e.g. inducible and
constitutive promoters) and
identification (e.g. markers suitable for identifying vector transformed cells
such as tetracycline
resistance or ampicillin resistance).
The term "animal feed" is used herein to refer to food suitable for
consumption by an
animal, in solid or liquid form, that comprise nutrients for the sustenance
and/or health of the
recipient animal and may comprise additional components and/or supplements.
As used herein, the term "sample" includes biological samples such as cell
samples,
bacterial samples, virus samples, samples of other microorganisms, samples
obtained from a
mammalian subject, such as tissue samples, cell culture samples, stool or
fecal samples, carcass
swab samples, and biological fluid samples (e.g., blood, plasma, serum,
saliva, urine, cerebral or
spinal fluid, and lymph liquid), environmental samples, such as samples from
food-contacting
surfaces, air samples, water samples, dust samples and soil samples, and food
samples, such as
from raw or undercooked meat, packaged meat, milk, or vegetables.
26
Date Recue/Date Received 2021-02-08
As used herein, the terms "transforming" or "transformation" refers to a
process whereby
exogenous or heterologous DNA (i.e., a nucleic acid construct) is introduced
into a recipient host
cell (e.g., prokaryotic cells, plant cells). The transfer of genetic
information to a host may be
heritable (ie. integrated within the host genome) and stable, or the transfer
may be non-heritable
and transient.
The term "solvent accessibility", as used herein, refers to the surface area
of a given
amino acid residue that is exposed to the surrounding solvent. There are a
variety of methods
known in the art used to measure such exposure, including determining the
average number of
neighbouring atoms per side chain atom (AvNAPSA) for a given amino acid
residue.
Unless otherwise defined herein, scientific and technical terms used in
connection with
the present disclosure have the meanings that are commonly understood by a
person of ordinary
skill in the art. Generally, nomenclature used in connection with, and
techniques of, cell and
tissue culture, molecular biology, immunology, microbiology, genetics and
protein and nucleic
acid chemistry and hybridization described herein are those well known and
commonly used in
the art.
The methods and techniques of the present invention are generally performed
according
to conventional methods well known in the art and as described in various
general and more
specific references that are cited and discussed throughout the present
specification unless
otherwise indicated. See, e.g., Sambrook J. & Russell D. Molecular Cloning: A
Laboratory
Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(2000);
Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory
Press, Cold Spring Harbor, N.Y. (1998).
27
Date Recue/Date Received 2021-02-08
In the specific experiments discussed herein, for the ease of reference and
understanding,
the resulting data are reported as an example of the disclosure according to
an embodiment.
Exemplary methods and materials are also described herein, although methods
and materials
similar or equivalent to those described therein can also be used in the
practice or testing of
aspects of the disclosure. It is to be understood that these examples,
materials, and methods are
for illustrative purposes only and should not be used to limit the scope of
the present invention in
any manner.
Anti-intimin Antibodies
Preventing or minimizing E. coil colonization in the gastrointestinal tract in
animals
reduces the risk of contamination from fecal shedding or at slaughter and
would ultimately
reduce contamination of food sources for human consumption. The adhesion
protein intimin,
expressed on the outer membrane in E. coil cells, mediates interaction between
the bacteria and,
for example, the epithelial cells lining the inner surface of the animal
host's GI tract and initiates
colonization. Colonization is initiated when intimin binds to the Translocated
intimin receptor
(Tir) located on epithelial cells. Interfering with this interaction by
effectively neutralizing
intimin activity through antibody binding of intimin and subsequent expulsion
of E. coil from an
animal's system would reduce such colonization.
Example 1
Camelid VHIls recognize E. coli 0157:H7 intimin with high affinity
To generate antibody candidates effective in targeting intimin, the C-terminal
277
residues of intimin (Int-277) were selected as the intended VHI-1-sIgA target
because this region
28
Date Recue/Date Received 2021-02-08
has previously been demonstrated to be immunogenic and to elicit IgAs (McKee
et al. 1996;
Gansheroff et al. 1999). Int-277 of E. coil 0157:H7 strain EDL933 intimin y
was fused C-
terminally to maltose-binding protein (MBP) and cloned into pMAL-p5X. The
resulting fusion
(MBP-Int277; FIG. 1A) (SEQ ID NO: 52) was expressed in E. coil BL21 (DE3)
cells by
Genscript USA, Inc. (New Jersey; FIG. 1A). E. coil BL21 (DE3) cells were
transformed with the
construct and grown overnight under IPTG induction. The next day, cells were
harvested, lysed
by sonication, centrifuged at 20,000xg for 20 min, and the MBP-Int277 fusion
protein was
purified using amylose affinity chromatography. Characterization of the MBP-
Int277 protein by
SDS-PAGE, western blotting, size exclusion chromatography and ELISA using an
anti-intimin
antibody (Carvalho et al. 2005) collectively suggested that MBP-Int277 had the
expected size
(73.8 kDa), was soluble and correctly folded (FIGs. 1B-E).
Camelid VHHs were generated against recombinant intimin as previously
described
(Henry et al. 2015; Henry et al. 2016). Briefly, a male llama (Lama glama) was
immunized
subcutaneously with 180 jig of MBP-Int277 in a total volume of 1 mL Tris-
buffered saline (50
mM Tris, 150 mM NaC1, 10% glycerol, pH 8.0) emulsified in an equal volume of
complete
Freund's adjuvant (Cedarlane, Burlington, ON, Canada) (day 1). The animal was
boosted with
the same dose of MBP-Int277 emulsified in incomplete Freund's adjuvant
(Cedarlane) on days
21, 28, 35 and 42. Serum polyclonal antibody responses against MBP-Int277 were
monitored
using direct ELISA and detected using HRP-conjugated goat anti-llama IgG
antibody
(Cedarlane, Cat. No. A160-100P). Total RNA was extracted from peripheral blood
lymphocytes
collected at days 35 and 49, reverse transcribed, and then expressed VHH genes
were amplified
via two rounds of nested PCR and cloned into the pMED1 phagemid vector. The
final size of the
29
Date Recue/Date Received 2021-02-08
phage-displayed VHH library size was 5x106 independent transformants, with an
insertion rate
of >95%. Library diversity was verified by DNA sequencing.
VHH-displaying phage were rescued from library-containing E. coil TG1 cells by
coinfection with M13K07 helper phage and purified by polyethylene glycol
precipitation.
Library phage were panned for three rounds against 20 jig of MBP-Int277
immobilized in wells
of microtiter plates. Bound phage were eluted with 0.1 M triethylamine for 10
min, neutralized
with 1M Tris-HC1, pH 7.4, and amplified in exponentially-growing E. coil TG1
cells for
subsequent panning rounds. After the final round of panning, binding of
individual phage clones
was assessed by monoclonal phage ELISA and detected using HRP-conjugated
rabbit anti-M13
antibody (GE Healthcare, Cat. No. 27-9421-01; Piscataway, NJ).
A competition ELISA using a polyclonal anti-intimin antibody (Carvalho et al.,
2005)
suggested that the polyclonal antibody response in the llama was directed
substantially towards
Int277 (SEQ ID NO: 53) rather than MBP, based on almost complete knock down of
the anti-
intimin antibody in the presence of serum from the immunized llama (FIG. 1E).
The phage-
displayed VHH library produced and panned for intimin-specific VHH sequences
yielded four
VHH sequences, VH111 (SEQ ID NO: 1), VHH3 (SEQ ID NO: 3), VHH9 (SEQ ID NO: 9),
and
VH1110 (SEQ ID NO: 10) showing low-nanomolar monovalent binding affinities for
intimin
based on surface plasmon resonance (SPR) and had no binding to MBP alone.
Table 1: Monovalent affinities and kinetics of the interaction between VHHs
and MBP-Int277 by
SPR (pH 7.4, 25 C)
VHH ka. (1/MS) ka (1/s) KD (nM)
VHH1 7.5x105 5.3x10-3 7.1
VHH3 7.1x105 3.2x10-4 0.5
VHH9 1.3x106 1.5x10-3 1.1
VHH10 6.8x105 1.4x10-4 0.2
Date Recue/Date Received 2021-02-08
Based on these results, the skilled person would understand that these
isolated VHH sequences
specifically bind to intimin such that, in their presence, other proteins such
as TIR are excluded
from binding to the C-terminal, extracellular 277 amino acids of intimin.
Example 2
Production of chimeric sIgA subunits in N. benthamiana
To produce VHH-IgAs effective against E. coil in the gastrointestinal tract of
an animal
susceptible to bacterial colonization, each VHI-1 sequence was fused to a
bovine IgA Fc (VHH-
Fc). The bovine Fc, JC and SC sequences were obtained from the NCBI public
database
(ANN46383, NP 786967 and NP 776568 respectively). Each of the VHH-Fc, SC and
JC
subunits were fused to the PR1b signal peptide and KDEL retrieval signal
peptide to enable ER
targeting and localization, as well as c-Myc, FLAG and HA tags respectively to
enable separate
detection of the subunits upon co-expression (FIG. 2A, B). For production in
plant leaf tissue,
the constructs were codon-optimized for N. benthamiana nuclear expression,
cloned separately
into plant expression vectors, and verified by DNA sequencing.
Transient expressions were performed either by injection (Miletic et al.,
2015) or by
vacuum infiltration for small-scale or large-scale transformations
respectively. Prior to vacuum
infiltration, Agrobacterium transformed with expression vectors encoding
either VHH3-Fc,
VHH9-Fc, VHH10-Fc, SC, JC or p19 were sub-cultured from starter cultures and
grown
separately in Luria-Bertani (LB) broth at 28 C overnight. Each of the
cultures bearing constructs
encoding the VHHx-Fc constructs was then combined with cultures carrying SC,
JC and p19, a
suppressor of gene silencing from Cymbidium ringspot virus (CymRSV) (Silhavy
et al., 2002;
Saberianfar et al., 2015). Trays of N. benthamiana plants were inverted and
submerged into each
31
Date Recue/Date Received 2021-02-08
of these co-cultures and placed into a vacuum chamber. To enable infiltration
into the leaves, a
pump was used to lower the pressure of the chamber to 85 kPa for 2 min and
then immediately
released. Plants were transferred back to the growth chamber until sampling.
Accumulation of each subunit at six days post-infiltration (dpi) was evaluated
by western
blotting using either anti-c-myc, anti-FLAG, or anti-HA antibodies to detect
the respective
subunits (FIG. 2C-E). All subunits were of slightly higher molecular mass than
their respective
predicted molecular weights based on amino acid residues only, due to
potential glycosylation
(see Table 2 below). In the fully assembled native sIgA complex, each of the
VHH-Fc, SC and JC
chains are predicted to have three, three and one N-glycosylation sites,
respectively (Steentoft et
al., 2013). The glycans on native sIgA have been shown to protect the
structure from proteolytic
degradation in the harsh mucosal environment and may also exhibit some
neutralization capacity
against some bacterial strains by sterically hindering attachment of sugar-
dependent receptors or
fimbriae to epithelial cells (Wold et al., 1990; Ruhl et al., 1996; Royle et
al., 2003).
Table 2: Predicted protein size and number of glycosylation sites for each
subunit
Chimeric sIgA Predicted size (kDa) Apparent size (kDa)
Predicted N-
subunit glycosylation sites
VHH-Fc 42 50 3
SC 66 70 3
JC 20 22 1
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Date Recue/Date Received 2021-02-08
Based on these results, the predicted size of each of the expressed subunits,
and the predicted N-
glycosylation sites, the skilled person would understand that each of the
subunits accumulated to
significant levels in N. benthamiana plants transformed with the appropriate
constructs.
Example 3
Optimizing co-expression of chimeric sIgA subunits
For correct assembly of the sIgA into a hetero-multimeric protein complex and
optimal
accumulation, nascent polypeptides are temporally and spatially coordinated in
a 4:1:1
stoichiometric ratio of VHH-Fc:SC:JC. To optimize the conditions for producing
the assembled
complex, a range of Agrobacterium ratios were tested for co-infiltration in N.
benthamiana
leaves. Infiltration cultures were prepared by mixing Agrobacterium strains
containing VHI-13-Fc
or VHI-19-Fc (SEQ ID NO: 31) with Agrobacterium strains containing SC, JC, and
p19 at optical
densities (OD at A600) of 0.57, 0.14, 0.14, and 0.14 respectively. The
accumulation levels of each
subunit were measured from four to eight dpi.
Four leaf discs were collected from each biological replicate. Protein
extraction and total
soluble protein quantification was performed according to previously known
methods in the art
(see Conley et al., 2009, for example). Quantification of VHH-Fc, JC, and SC
was performed by
Western blot analysis. The recombinant proteins were detected with one of the
following primary
antibodies: mouse anti-c-Myc monoclonal antibody (GenScript Cat. No. A00864),
mouse anti-
HA monoclonal antibody (Millipore Sigma, Cat. No. H3663), mouse anti-FLAG
monoclonal
antibody (Millipore Sigma, Cat. No. F3165), and HRP-conjugated goat anti-mouse
IgG
secondary antibody (Bio-Rad, Cat. No. 170-6516).
33
Date Recue/Date Received 2021-02-08
Accumulation levels of all three subunits in both the VHH3-Fc and VHH9-Fc
infiltration
mixtures peaked at eight dpi (FIG. 3A, B). VHI-19-Fc mixtures reached the
highest accumulation
levels for all three subunits with VHI-19-Fc at 0.22 g/kg, SC at 0.08 g/kg,
and JC at 0.04 g/kg,
resulting in a total of approximately 0.34 g/kg for sIgA subunits, which when
converted to molar
ratios result in 4.2:1:1.6 (VHI-19-Fc: Sc: JC). This combination was the
closest to the expected
4:1:1 molar ratio for assembly of sIgA to allow for in vivo assembly of the
subunits into a hetero-
multimeric protein complex.
Since the highest accumulation levels for VHI-13-sIgA and VHI-19-sIgA were
reached at
eight dpi, the accumulation levels of VHI-110-sIgA were monitored beyond eight
dpi to examine
if higher accumulation could be achieved. Similar Agrobacterium ODs for
infiltration mixtures
(0.57:0.14:0.14:0.14) were used as in the previous experiment, and the
accumulation of VH1110-
Fc up to 12 dpi (FIG. 3C) monitored. Over the course of the experiment, VHI-
110-Fc accumulated
well up to 12 dpi and reached 0.12 g/kg fresh weight (FW).
The skilled person would understand that while a range of different optical
densities may
be used for bacterial infiltration cultures, these results show that
Agrobacterium strains
containing VHH-Fc, SC, JC, and p19 at optical densities (OD at A600) of about
0.57, 0.14, 0.14,
and 0.14 respectively, provide increased accumulation levels of all three
subunits. Further, these
results show that accumulation levels continue to increase beyond 8 dpi and up
to 12 dpi.
Example 4
The chimeric sIgA subunits associate in vivo
Subunits of native sIgA are known to be covalently linked by disulfide bonds.
To
determine if the co-expressed subunits were physically associating, crude
extracts of leaves
34
Date Recue/Date Received 2021-02-08
infiltrated with VHI-13-Fc/ SC / JC were immunoprecipitated with the c-Myc
antibody specific to
the VHH-Fc subunit. The immunoprecipitated proteins were detected on a western
blot with
either anti-FLAG antibody specific to the SC subunit (FIG. 4A, C) or anti-HA
antibody specific
to the JC subunit (FIG. 4B, D). When proteins were separated under reducing
PAGE conditions,
the -70-kDa SC subunit was detected in the extracts containing SC only and
those containing all
three subunits. However, after co-immunoprecipitation (co-IP), SC was only
detected in the
treatment containing all three subunits (FIG. 4A), indicating that it was
associated with the VHH-
Fc subunit. Similarly, the ¨20-kDa JC was detected in extracts containing JC
only and those
containing all three subunits, but after co-IP, JC was only detected in the
treatment containing all
three subunits (FIG. 4B).
When the same samples were separated by non-reducing PAGE, SC expressed alone
appeared as a main band at 70-kDa, with a ladder of larger products presumably
representing
multimerization via non-specific disulfide bond formation. When all three
subunits were present
in the cell extract (VHI-13-Fc / SC / JC), several other intermediate products
were detected. After
co-IP, bands were only observed in extracts containing all three subunits,
including a band
running around 250 kDa, the expected size of the fully assembled sIgA (FIG.
4C). Similarly,
upon detection with anti-HA, several faint bands representing JC multimers
were observed, and
in the VHI-13-Fc / Sc / JC lane, JC monomer (shown with an arrow) and several
other
intermediate products were detected. As expected, after co-IP and detection
with anti-HA, the
same bands were only observed in the treatment containing all three subunits
(FIG. 4D). Co-IP
experiments were performed with all constructs and similar results were
consistently observed in
every case. These results confirm that leaves infiltrated with VHH-Fc/ Sc / JC
subunits associate
in vivo.
Date Recue/Date Received 2021-02-08
Example 5
Secretory IgA subunits assemble into a hetero-multimeric protein complex in
vivo
To determine how fast the subunits assemble into the sIgA complex, a time-
course
experiment was performed in which leaf tissue was collected every two days
from 4 to 12 dpi,
separated by SDS-PAGE under non-reducing conditions, and detected with anti-HA
(FIG. 5A),
anti-c-Myc (FIG. 5B), and anti-FLAG antibodies. The results indicated that
assembly of the sIgA
and intermediates was gradual and continued through 12 dpi (FIG. 5A arrows 1-
3; FIG. 5B
arrows 1-2 ), while monomeric JC and a 90-kDa intermediate (FIG. 5A, arrows 4-
5) and
monomeric \THH9-Fc and an 80-kDa intermediate (FIG. 5B, arrows 3-4) showed
diminishing
accumulation across the same period. Taken together, these data indicate that
the chimeric sIgA
assembled with time, and that a later harvest may be beneficial.
Example 6
Vacuum infiltration and purification of VH119-sIgA
There are no efficient methodologies available for purifying IgA. Therefore,
two methods
for purifying VHH9-sIgA were compared. The first method took advantage of a
peptide derived
from a surface protein of Streptococcus pyogenes, peptide M, which binds to
the Fc region of
bovine IgA. The second purification method used an affinity resin that binds
the FLAG tag fused
to SC (FIGs. 6A and B).
In the crude leaf extract, VHH9-sIgA was the main product observed on a
western blot
detected with the c-Myc antibody (FIG. 6A, extract lane, arrow 1). When
purified with peptide
M, unassembled and partially multimerized \THH9-Fc polypeptides were heavily
enriched, and
several bands were observed (FIG. 6A). The strongest bands belonged to
monomeric (-44 kDa)
36
Date Recue/Date Received 2021-02-08
and dimeric (-88 kDa) VH119-Fc (FIG. 6A, arrows 4 and 5). In addition, three
other bands were
observed that correspond to the trimeric (-132 kDa) and tetrameric (-176 kDa)
VH119-Fc, and a
fainter band representing the fully assembled chimeric sIgA (-270 kDa) (FIG.
6A, bands 3, 2
and 1, respectively). This method of purification was efficient and allowed
recovery of 0.6
mg/mL of c-Myc-reactive antibody fragments, as estimated by whole lane
densitometry against
known amounts of a standard protein. Purification with peptide M
preferentially recovered
monomeric and dimeric VH119-Fc compared with fully assembled VH119-sIgA.
Anti-FLAG agarose was also used for purification of VH119-sIgA in a second
attempt to
enrich for the fully assembled chimeric sIgA. After Western blot and detection
with anti-FLAG
antibody, several bands were observed. The strongest band belonged to free or
monomeric SC
(-66 kDa) (FIG. 6B, arrow 5), but the fully assembled VH119-sIgA band was much
more
prominent than following peptide M purification (-270 kDa) (FIG. 6B, arrow 1).
Three other
bands were also recovered which were speculated to belong to sIgA intermediate
products such
as SC/trimeric VH119-Fc !JC (¨ 206 kDa), SC/dimeric VH119-Fc (-160 kDa), and
SC/ VH119-Fc
(No. 4, ¨ 110 kDa) (Figure 6B, bands 4, 3 and 2, respectively). Whole lane
densitometry
indicated that the purification with anti-FLAG agarose recovered 0.014 mg/mL
protein.
Example 7
Plant-produced Vii119-sIgA is antigen-binding competent
Intimin binding by plant-produced VH119-IgA purified either using peptide M
(yielding
all VH119-Fc molecules regardless of the presence of JC and SC) or anti-FLAG
antibody
(yielding SC as well as secretory VH119-Fc in complex with SC, and possibly
JC) was assessed
by SPR and ELISA. Prior to SPR analyses, VHH monomers and MBP-Int277 were
purified by
37
Date Recue/Date Received 2021-02-08
size exclusion chromatography. Approximately 700-1600 response units (RUs) of
MBP-Int277
were immobilized in 10 mM acetate buffer, pH 4.5, on CM5 sensor chips using an
amine
coupling kit (GE Healthcare). Multi-cycle kinetic analyses were carried out on
a Biacore T200
instrument (GE Healthcare) at 25 C by injecting VHHs at concentrations ranging
from 0.3-400
nM, at a flow rate of 30-50 pL/min and with a contact time of 300 s, and then
allowing the
VHHs to dissociate for 600 s. Data were analyzed using BIAevaluation software
version 4.1 (GE
Healthcare) and fitted to a 1:1 binding model. The MBP-Int277 surface was
regenerated between
injections using glycine buffer, pH 1.5.
For SPR analyses of plant-produced VHH-IgAs, approximately 2200 RUs of VHH-IgA
or 100 RUs of matched VHH monomer were immobilized in 10 mM acetate buffer, pH
3.5, on
CMS Series S sensor chips using an amine coupling kit. Single-cycle kinetic
analyses were
carried out on a Biacore T200 instrument at 25 C by injecting MBP-Int277 in
HBS-EP+ buffer
at concentrations ranging from 0.3-5 nM, at a flow rate of 30 pL/min and with
a contact time of
300-600 s, and then allowing the VHHs to dissociate for 600 s. Data was
analyzed using
BIAevaluation software version 4.1 (GE Healthcare) and fitted to a 1:1 binding
model. The
antibody surface was regenerated between injections using glycine buffer, pH
1.5.
ELISAs using plant-produced VHH-sIgAs were conducted according to previously
known methods in the art (see Henry et al., 2015; Henry et al., 2016, for
example). The
following secondary and/or tertiary antibodies were used: Monoclonal mouse
anti-FLAG M2
antibody (Sigma-Aldrich Cat. No. F3165; St. Louis, MO), HRP-conjugated
polyclonal donkey
anti-mouse IgG (Jackson ImmunoResearch Cat. No. 715-035-150; West Grove, PA)
or HRP-
conjugated polyclonal sheep anti-bovine IgA (Abcam Cat. No. ab12755;
Cambridge, UK).
38
Date Recue/Date Received 2021-02-08
No loss of intimin-binding affinity was observed by SPR for peptide M-purified
VH119-
sIgA produced in planta compared with VH119 monomer produced in E. coil (FIG.
7A).
Moreover, both peptide M-purified and anti-FLAG-purified VHH9-sIgA bound
intimin with
similar half maximal effective concentrations (EC50s) in ELISAs detected with
horseradish
peroxidase (HRP)-conjugated anti-bovine IgG antibody (FIG. 7B). However, no
binding of
peptide M-purified VHH9-IgA was observed in ELISAs detected with anti-FLAG
antibody,
suggesting that little SC was present in the purified material.
Example 8
Plant-produced VHH10-sIgA binds E. coli strains 026:H11, 0145:Hnm, 0111:Hnm
and
0157:H7
In order to test binding of VHH-sIgA produced in planta to intimin on E. coil,
cells of
026:H11, 045:H2, 0103:H2, 0145:Hnm, 0121:H19, 0111:Hnm and 0157:H7 were
incubated
with VHI-110-sIgA purified using anti-FLAG (binds the Sc), then visualized
using a secondary
fluorescent antibody (rabbit anti-bovine-FITC) that binds the Fc and 4',6
diaminodino-2-
phenylindole (DAPI) that stains bacterial cells. The confocal images showed
consistent co-
localization of FITC signal with strains 026:H11, 0145:Hnm, 0111:Hnm and
0157:H7 cells
(FIG. 8). Heavily glycosylated SC has been reported to interact with some
bacterial strains, and
in order to rule out the possibility that the observed co-localization could
be a product of non-
specific glycan-mediated binding and not binding of the VHI-1 to intimin,
binding of each of
VHI-110-sIgA and VT-11110-ft expressed alone to E. coil 0157:H7 was compared.
Confocal
images showed co-localization of VHI-110-Fc with E. coil 0157:H7 cells in the
absence of SC
39
Date Recue/Date Received 2021-02-08
and JC, suggesting that binding was VHH-mediated (FIG. 9). As a negative
control, E. coil cells
were also treated with PBS containing 0.1% Tween-20 (PBS-T) instead of
antibodies and
similarly stained but did not show fluorescence under FITC-related imaging
conditions (480 nm
excitation and 520-540 nm detection) (FIG. 9). Based on these results, the
skilled person would
understand that the plant-produced VHH-sIgA binds E. coil strains 026:H11,
0145:Hnm,
0111:Hnm and 0157:H7 via VHH-mediated binding.
Example 9
Plant-produced VH1110-sIgA binds four E. coli serotypes and reduces adherence
to
epithelial cells
Since intimin mediates the intimate attachment of E. coil to epithelial cells,
the present
inventors investigated if the binding of VHI-110-sIgA to E. coil could
neutralize the ability of
bacteria to adhere to epithelial cells.
A HEp-2 inhibition assay with E. coil strains was performed according to
methods known
in the art (see, McKee et al. 1996). Alexa 647 phalloidin (Thermo Fisher
Scientific Cat.
No.A22287) was used to visualize actin in the HEp-2 cells and donkey anti-
rabbit Alexa 350
(Thermo Fisher Scientific Cat. No.A10039) used to visualize E. coil cells.
To quantify adherence inhibition by relative fluorescence, the assay was
adapted by
growing the HEp-2 cells in 96-well black fluorometry plates that had been
coated with poly D-
lysine.
Compared to the respective positive controls of HEp-2 cells and E. coil only,
the addition
of VHI-110-sIgA abrogated the adhesion of E. coil strains 026:H11, 0111:Hnm
and 0157:H7 to
HEp-2 cells, while it reduced adhesion of E. coil strain 0145:Hnm to HEp-2
cells (FIG. 10A).
Date Recue/Date Received 2021-02-08
To quantify the neutralization capacity of VHH10-sIgA, the adhesion assay was
adapted for
fluorometry and the relative fluorescence of HEp-2 cells incubated with a
culture of each of the
seven E. coil strains with and without VHI-110-sIgA was measured. The addition
of VHI-110-sIgA
afforded complete protection, that is, it reduced the relative fluorescence
caused by adherent
bacteria for strains 026:H11, 0111:Hnm and 0157:H7 to background levels, and
reduced the
relative fluorescence caused by adherent bacteria for strain 0145:nm (FIG.
10B). Based on the
somewhat unexpected results of 0145:nm, the neutralization assay was repeated
with 0145
strain from a different supplier (ATCC, C625). Both VHH10-sIgA and VHH9-Fc
abrogated the
adhesion of the new 0145 strain to HEp-2 cells and reduced the relative
fluorescence caused by
adherent bacteria for to background levels. Thus, both VHH10-sIgA and VHH9-Fc
were able to
completely neutralize the new 0145 strain (FIG. 10C).
A multiple sequence alignment was performed and a neighbor-joining tree of
Int277 was
derived across all seven strains and found that E. coil strains 0157, 0111,
0145 and 026
grouped together based on sequence similarity, while 045, 0103 and 0121 were
more disparate
in sequence (FIG. 10D). This is in accord with VHH10-sIgA being able to bind
and neutralize
0157, 0111, 0145 and 026.
Accordingly, the skilled person understands that the present disclosure
pertains to a
polypeptide that can interfere with the interaction of intimin expressed on
the outer membrane of
E. coil cells to the epithelial cells lining the inner surface of an animal
host's GI tract through
competitive binding of intimin in order to prevent and/or reduce colonization,
and facilitate
expulsion of the bacteria from the animal. The polypeptide comprises: a single
domain antibody,
or an antigen binding fragment thereof, which specifically binds to intimin on
an Escherichia
41
Date Recue/Date Received 2021-02-08
coil cell. In one embodiment, the antibody binds to an epitope having at least
80%, 85%, 90%,
95%, 97%, or 100% amino acid sequence identity to the sequence set forth in
SEQ ID NO: 53.
In various embodiments, the antibody comprises complementarity determining
regions
(CDR) having at least 80%, 85%, 90%, 95%, 97%, or 100% amino acid sequence
identity to the
sequence as set forth in: (i) SEQ ID NO: 12 (CDR1), SEQ ID NO: 13 (CDR2), and
SEQ ID NO:
14 (CDR3), (ii) SEQ ID NO: 15 (CDR1), SEQ ID NO: 16 (CDR2), and SEQ ID NO: 17
(CDR3), (iii) SEQ ID NO: 18 (CDR1), SEQ ID NO: 19 (CDR2), and SEQ ID NO: 20
(CDR3),
(iv) SEQ ID NO: 21 (CDR1), SEQ ID NO: 22 (CDR2), and SEQ ID NO: 23 (CDR3), (v)
SEQ
ID NO: 24 (CDR1), SEQ ID NO: 25 (CDR2), and SEQ ID NO: 26 (CDR3), or (vi) SEQ
ID NO:
27 (CDR1), SEQ ID NO: 28 (CDR2), and SEQ ID NO: 29 (CDR3).
In various embodiments, the antibody, or antigen binding fragment thereof,
comprises a
heavy chain variable (VHH) domain. In various embodiments, the VHH domain has
at least
80%, 85%, 90%, 95%, 97%, or 100% amino acid sequence identity to the sequence
as set forth
in any one of SEQ ID NOs: 1 to 11. The skilled person will appreciate that CDR
and variable
domain sequences may be highly homologous to the CDR and variable sequences
specified
herein and still retain antigen binding functionality.
In various embodiments, the VHH domain is linked to an Fc chain. In one
embodiment,
the VHH domain is linked to a bovine Fc chain. The skilled person will
recognize that the
present polypeptides could be adapted for use in other animals and thus
appreciate that the VHH
domain may be linked to an Fc chain suitable for use in other animals.
In various embodiments, the antibody neutralizes intimin on the E. coil cell
from binding
to an epithelial cell, for example, an epithelial cell of the gastrointestinal
tract of a mammal. In
42
Date Recue/Date Received 2021-02-08
some instances, the E. coil cell is a Shiga toxin-producing E. coil (STEC)
cell. In one
embodiment, the E. coil cell is 026:H11, 0111:Hnm, 0145:Hnm, or 0157:H7. In
one
embodiment, the antibody is an IgA antibody. In one embodiment, the antibody
comprises four
VHH-Fc subunits, one secretory component (SC), and one joining chain (JC). In
the GI tract,
once sIgA binds to its target, glycans on the secretory component facilitate
binding to the mucus
lining of the GI tract allowing clearance of the sIgA-pathogen complexes by
peristalsis
(Macpherson et. al, 2008). Thus, the skilled person will appreciate that an
sIgA directed against
intimin would prevent or reduce luminal E. coil cells from interacting with
the host epithelium,
clearing them by entrapment in the mucous layer and subsequent fecal shedding.
The disclosure also provides an antibody, or antigen binding fragment thereof,
that
competes for specific binding to intimin with the polypeptide claimed herein.
The disclosure also provides a nucleic acid encoding the polypeptide claimed
herein,
including fragments thereof, sequences hybridisable therewith, homologous
sequences,
sequences encoding similar polypeptides with different codons, sequences
altered by mutations
(either naturally occurring or human induced).
The disclosure also provides an expression vector comprising the nucleic acid
claimed
herein.
The disclosure also provides a host cell comprising the expression vector
claimed herein.
In one embodiment, the host cell is a plant cell. In one embodiment, the plant
cell is a Nicotiana
plant cell. In one embodiment, the plant cell is a Nicotiana benthamiana plant
cell or a Nicotiana
tabacum plant cell. In one embodiment, the plant cell is a Lactuca plant cell.
The skilled person
will appreciate that the expression vector claimed herein may be altered
slightly to be suitable for
43
Date Recue/Date Received 2021-02-08
transformation in other plant species, thus the present disclosure is not
limited to the plant
species recited herein.
The disclosure also provides a non-viable harvested plant material comprising
the host
cell as claimed herein. In one embodiment, the non-viable plant harvested
material is a leaf or a
stem.
The disclosure also provides a tobacco product comprising the host cell as
claimed
herein. In various embodiments, the tobacco product is cut, shredded,
powdered, loose, ground,
granulated, or extruded. The skilled person understand that the tobacco
product could be any
product processed into a form which can be consumed by an animal such as a
bovine.
The disclosure also provides an animal feed comprising the host cell as
claimed herein.
The disclosure also provides a pharmaceutical composition comprising the
polypeptide
claimed herein, and a pharmaceutically acceptable carrier.
The disclosure also provides a diagnostic kit for detecting the presence of E.
coil in a
sample comprising the polypeptide claimed herein. In one embodiment, the
sample is a food
sample, an environmental sample, or a sample from an animal or a
microorganism. In one
embodiment, the sample is a fecal sample, a carcass swab sample, a water
sample, a sample from
a packaged meat, a sample from a vegetable, a soil sample, or a sample from a
food-contacting
surface.
The disclosure also provides a method of preventing or reducing colonization
of E. coil in
the gastrointestinal tract of a mammal, comprising: administering to the
mammal the polypeptide
as claimed herein. The single domain antibody claimed herein neutralizes the
capacity of
different E. coil strains to bind to a host's cells thereby conferring cross-
serotype inhibition of
44
Date Recue/Date Received 2021-02-08
bacterial adhesion, adhesion that is a crucial step in the pathogenicity in a
host system. In one
embodiment, administering the polypeptide to the mammal comprises causing the
mammal to
ingest plant material from a plant that produces the polypeptide. In one
embodiment, the plant
material is for oral administration. In one embodiment, the plant material is
for rectal
administration. In one embodiment, the plant material is from a Nicotiana
plant or a Lactuca
plant. In one embodiment, the plant material is from a Nicotiana benthamiana
plant or a
Nicotiana tabacum plant. In one embodiment, the plant is harvested at a stage
of harvest in
which accumulation of the assembled VHH-Fc polypeptide or assembled sIgA in
the plant is
maximal.
Also provided in this disclosure is a method of producing the polypeptide as
claimed
herein, the method comprising transforming a protein-expressing system with a
nucleic acid
molecule encoding the antibody. In one embodiment, the protein-expressing
system is a plant. In
one embodiment, the plant is a Nicotiana plant or a Lactuca plant. In one
embodiment, the plant
is a Nicotiana benthamiana plant or a Nicotiana tabacum plant. Other plant
systems may be
selected for polypeptide or antibody production and expression vectors
delivered through
Agrobacterium-mediated plant transformation may be optimized accordingly.
Agrobacterium
strains are transformed with a plant-optimized expression vector comprising
nucleic acid
encoding each of the subunits of the polypeptide. Plant leaves are then co-
infiltrated with
transformed strains. In one embodiment, transforming the protein-expressing
system with the
nucleic acid molecule encoding the IgA antibody comprises preparing
Agrobacterium strain
cultures comprising VHH-Fc subunits, SC, and JC at optical densities (OD) of
about 0.57, 0.14,
and 0.14, respectively, for infiltration in the plant. In one embodiment, the
plant is harvested
after infiltration. In one embodiment, the plant is harvested more than 3 days
post infiltration
Date Recue/Date Received 2021-02-08
(dpi). In one embodiment, the plant is harvested from between about 4 dpi to
about 12 dpi. In
one embodiment, the plant is harvested at about 12 dpi. In one embodiment, the
plant is
harvested at a stage of harvest in which accumulation of the assembled VHH-Fc
polypeptide or
assembled sIgA in the plant is maximal.
The present disclosure also provides a method of detecting the presence of E.
coil in a
sample, comprising: contacting the sample with the polypeptide as claimed
herein, to detect the
presence of intimin in the sample, and detecting binding between intimin and
the antibody.
The presence of E. coil may be confirmed by Western analysis, ELISA, or any of
the
various antigen-antibody detection methods known in the art. In one
embodiment, the sample is a
food sample, environmental sample, or a sample from an animal or
microorganism. In one
embodiment, the sample is a fecal sample, a carcass swab sample, a water
sample, a sample from
a packaged meat, a sample from a vegetable, a soil sample, or a sample from a
food-contacting
surface.
The present disclosure also provides use of the polypeptide as claimed herein,
for
preventing or reducing E. coil cell colonization of the gastrointestinal tract
of a mammal.
The present disclosure also provides use of the polypeptide as claimed herein,
for
neutralizing an E. coil cell.
The present disclosure also provides use of the polypeptide as claimed herein,
for
detecting the presence of E. coil in a sample.
46
Date Recue/Date Received 2021-02-08
Variant Fc Chain
While there are many current strategies to improve the recombinant yield of
biologics,
more specifically plant-based biologics, the present disclosure provides for
structural changes to
the Fc chain itself in order to facilitate improved yield of recombinant
protein. Using rational
design strategies to stabilize the structure of the Fc chain, the present
inventors improved
recombinant protein accumulation by: 1) enhancing the net negative charge of
the Fc chain
through side chain alterations and reducing non-specific protein aggregation
during the
macromolecular crowding effect of recombinant protein production by providing
small charge-
charge repulsive forces on the protein surface, and 2) by introducing a de
novo disulfide bridge
in the Fc chain to tether portions of the chain together.
Example 10
Design of mutagenic Fc constructs in Nicotiana benthamiana
To develop a more stable Fc chain that could act as a stabilization partner
when fused to a
VHH or other fusion proteins, two different rational design strategies were
tested: 1) changing
key surface residues to give a higher net charge, a technique known as
supercharging the
molecule and 2) introducing novel intrachain disulfide bonds which may prevent
unfolding and
exposure of reactive hydrophobic areas.
Although the crystal structure of human IgA Fc has been determined and is
publicly
available (pdb: lIGA), bovine IgA Fc, which is 70% similar in sequence, has
not yet been
documented. Because the structure of Fc is generally well conserved across
species, the present
inventors used the I-TASSER online program to predict the structure of bovine
IgA Fc using the
human IgA Fc as a threading template (Wu et al 2008; Zhang et al. 2008). The
resulting
47
Date Recue/Date Received 2021-02-08
predicted structure had a high confidence score of 1.35 (given a range of -5
to 2) and was used to
determine rational design candidates. Engineering of negatively supercharged
Fc was performed
computationally by first ranking residues for solvent accessibility by their
average number of
neighboring atoms (within 10 A) per side-chain atom (AvNAPSA) and then
identifying highly
polar solvent-exposed Asn and Gln residues for mutation to their negatively
charged
counterparts, Asp and Glu respectively (Schrodinger 2010).
The present inventors identified three asparagines and two glutamines on the
surface of
the Fc chain, at residue positions 9, 84, 131, 175 and 195, and mutated them
to their conservative
but negatively charged counterparts: aspartic acid and glutamic acid
respectively (FIGs. 11A and
11D). The asparagines and glutamines were selected based on high solvent
exposure and non-
involvement in native glycosylation (nucleotides 682-684, 85-87) or Fc a
receptor (FcaR)
binding (nucleotides 70-78, 349-351,451-453, 457-459, 466-468, 472-474, 604-
606, 613-642).
The asparagines that were mutated are unlikely to be involved in N-
glycosylation considering
that the adjacent residues (residue 9: N-C-E, residue 84: N-S-G, residue 131:
N-E-L) do not
match the standard glycosylation motif (N-X-S/T (where X is a non-proline
residue) . Modelling
the substitutions to the five residues predicted an increase in net negative
charge from -5.30 to -
10.29 at pH 7 with a corresponding change in pI from 5.39 to 4.85.
For the selection of de novo intrachain disulfide bonds, based on modelling of
the
predicted Fc structure, disulfide candidates were chosen by manual inspection
of the molecule in
PyMol. The IgA Fc secondary structure includes a characteristic beta sandwich
of seven anti-
parallel beta strands for both its CH2 and CH3 domains. Both domains exhibit
Greek key
connectivity (ABED CFG) forming two distinct beta sheets that fold over each
other (FIG. 11B).
For both domains, an intra-chain disulfide in the centre of the beta sheet
stabilizes the tertiary
48
Date Recue/Date Received 2021-02-08
structure. De novo disulfide candidates, at residue positions 196 and 219,
were chosen based on
neighboring proximity (under 5 A) for tethering the C-terminal end of strand G
to the N terminal
end of strand F (FIGs. 11A and 11C). To retain the functionality of the native
Fc, native disulfide
sites were avoided (interchain: 16-18, 205-207, 199-201, 13-15; intrachain:
100-102, 271-273,
412-414, 601-603; tailpiece to JC: 718-720; 235-237; free S-H: 235-237).
Example 11
Rationally designed mutations improve Fc accumulation in transiently
transformed leaf
tissue
The native bovine IgA Fc sequence was obtained from the NCBI public database
(ANN46383) and synthesized by Bio Basic Inc. (Markham, ON, Canada). Rational
design
mutations, N9D (SEQ ID NO: 32), N84D (SEQ ID NO: 33), N131D (SEQ ID NO: 34),
Q175E
(SEQ ID NO: 35), Q195E (SEQ ID NO: 36), and G196C/R219C (SEQ ID NO: 37), were
then
individually made using an in vitro single primer site-directed mutagenesis
method (Huang et al
2017). A multi-site-directed mutagenesis method (Liang et al. 2012) was used
to combine
mutations. Genetic fusions to anti-E. co/i VHH9 (SEQ ID NO: 9) were done using
a sequence
and ligation independent cloning (SLIC) method (Li et al. 2007). All cloning
was confirmed by
sequencing.
To enable expression in leaf tissue, each construct was cloned into a pCaMGate
plant
expression vector (Pereira et al. 2014). Transient expressions were performed
by syringe
infiltration into leaf tissue of N. benthamiana plants. Plants were grown in a
growth chamber at
22 C with a 16 h photoperiod at a light density of 110 umol m-2 s-1 for 7
weeks and fertilized
with water soluble N:P:K (20:8:20) at 0.25 g/L (Plant products, Brampton, ON,
Canada).
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Date Recue/Date Received 2021-02-08
Screening of ER-targeted wild type and mutant Fc was done by semi-quantitative
Western
blotting at four, six, and eight days post-infiltration (dpi).
Plant extracts were prepared under native conditions as described above.
Purification was
performed using an anti-c-myc purification kit (MBL International Corp.,
Woburn, MA, USA).
Detection and quantification of samples was performed as described above
and/or according to
methods known in the art. Compared to the accumulation of native Fc (SEQ ID
NO: 30), each of
the supercharged Fc mutants, N9D (SEQ ID NO: 32), N84D (SEQ ID NO: 33), N131D
(SEQ ID
NO: 34), Q175E (SEQ ID NO: 35) and Q195E (SEQ ID NO: 36), showed a three- to
four-fold
improvement in accumulation across the time course (FIG. 12A).
Similarly, the de novo disulfide mutant, G196C/R219C (SEQ ID NO: 37), showed a
six
to seven-fold improvement in accumulation compared to the native Fc after six
dpi (FIG. 12B).
To test if these mutations could be combined to further improve accumulation,
the mutations
were combined in a step-wise manner and accumulation was measured in
transformed leaf
extract by Western blot. The Fc mutant containing three N-D supercharging
residues,
N9D/N84D/N131D (SEQ ID NO: 38) gave a progressive increase in accumulation
after transient
expression. The Fc mutant containing all five supercharging residues,
N9D/N84D/N131D/Q175E/Q195E (SEQ ID NO: 39), showed a ten-fold improvement in
accumulation compared to native (FIG. 12C). Adding the de novo disulfide to
these five
supercharging residues, N9D/N84D/N131D/Q175E/Q195E/G196C/R219C (5+1) (SEQ ID
NO:
40), further improved accumulation by approximately twenty-two-fold (FIG.
12C). To test if
these Fc mutants could also enhance accumulation as an Fc scaffold protein,
each Fc mutant was
fused to VHH9. Similar to the comparison using Fc alone, each of the
individual mutant VHH9-
Fc fusions, either with a supercharged residue or with a de novo disulfide,
showed a three- to
Date Recue/Date Received 2021-02-08
four-fold improvement in accumulation when compared to the native VHH9-Fc
fusions (FIG.
12D). When fused to the anti- E. coil VHH, the combined Fc mutants also
progressively
improved accumulation with five mutations showing an approximately sixteen-
fold improvement
compared to native (FIG. 12E).
The corresponding DNA sequences for mutant Fc chains are identified in the
following
SEQ ID NOs: native Fc (SEQ ID NO: 41), N9D Fc (SEQ ID NO: 43), N84D Fc (SEQ ID
NO:
44), N131D Fc (SEQ ID NO: 45), Q175E Fc (SEQ ID NO: 46), Q195E Fc (SEQ ID NO:
47),
G196C/R219C Fc (SEQ ID NO: 48), N9D/N84D/N131D Fc (SEQ ID NO: 49),
N9D/N84D/N131D/Q175E/Q195E Fc (SEQ ID NO: 50), and
N9D/N84D/N131D/Q175E/Q195E/G196C/R219C (5+1) Fc (SEQ ID NO: 51).
Based on these results, the skilled person would understand that the mutations
introduced
in the Fc chain led to multiple fold increases in accumulation for both Fc and
VHH-Fc fusions.
Example 12
Engineered VHH-(5+1)Fc assembles with other sIgA subunits in vivo
Structurally, secretory (sIgA) includes an IgA dimer linked by two additional
chains: a
15-kDa joining chain (JC) that links the Fc end-to-end (Krugmann et al., 1997)
and a 70-kDa
secretory component (SC) that coils around both Fc chains (Bonner et al.,
2007). Although the
VHH-Fc fusion lacks the light chains and CH1 domains found in native mammalian
sIgA,
assembly to the JC and SC is directed specifically via disulfide bond
formation with the Fc. To
determine if engineering of the VHH-Fc affected its ability to assemble with
the SC and JC
subunits, all three subunits were co-expressed and immunoprecipitation
experiments were
conducted.
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Date Recue/Date Received 2021-02-08
To determine if proper assembly occurs, each subunit had a different tag (VHH-
Fc-c-
myc; SC-Flag; JC-HA). Crude extracts were immunoprecipitated with the anti-
FLAG antibody
specific to the SC subunit, then separated and detected on a Western blot
probing for either anti-
cmyc (VHH -Fc subunit) or anti-HA (JC subunit). Bands matching the predicted
44kDa size of
VHH9-Fc were detected with anti-c-myc antibody in crude extract transformed
with VHH9-Fc,
VHH9-(5+1) Fc, co-expressed VHH9-Fc/SC/JC and co-expressed VHH9-(5+1)Fc/SC/JC,
but no
bands were detected in crude extract expressing only JC or SC (FIG. 13A).
After co-IP, ¨44kDa
bands were seen only in extracts co-expressing VHH9-Fc/SC/JC and VHH9-
(5+1)Fc/SC/JC
(FIG. 13B). This indicated that both SC and VHH-Fc or SC and VHH-(5+1)Fc
interact, and that
the mutations in Fc did not hinder this interaction. Similarly, detection with
anti-HA indicated
bands of ¨20kDa, matching the predicted size of JC, in crude extract
transformed with JC, VHH-
Fc/SC/JC and VHH-(5+1)Fc/SC/JC (FIG. 13C). After Co-IP, ¨20kDa bands were seen
only for
the co-expressed VHH-Fc/SC/JC and VHH-(5+1)Fc/SC/JC, indicating that SC and JC
are
present in the same complex (FIG. 13D).
Example 13
Engineered VHH-(5+1)Fc retains the ability to bind E. coli strains 0157, 026,
0145 and
0103
To determine if the rationally designed mutations impact VHH-Fc's pattern of
cross-
serotype binding against E. coil, either VHH9-Fc or VHH9-(5+1)Fc was incubated
with E. coil
026:H11, 045:H2, 0103:H2, 0145:Hnm, 0121:H19, 0111:Hnm or 0157:H7. E. coli
binding
assays were performed as described above and/or according to methods known in
the art. After
washing and fixing with paraformaldehyde, bacteria was visualized with DAPI
and VHH-Fc
binding visualized using a secondary fluorescent antibody (rabbit anti-bovine-
FITC) that binds
52
Date Recue/Date Received 2021-02-08
Fc. Consistent co-localization of FITC signal with strains 026:H11, 0145:Hnm,
0111:Hnm and
0157:H7 cells for both VHH9-Fc and VHH9-(5+1)Fc (FIG. 14) was observed
indicating multi-
serotype detection of E.coli . As a negative control, E. coil cells were also
treated with PBS
containing 0.1% Tween-20 (PBS-T) instead of antibodies and similarly stained
but did not show
fluorescence under FITC-related imaging conditions (480 nm excitation and 520-
540 nm
detection).
Example 14
Engineered VHH -(5+1)Fc retains the ability to neutralize adherence of E. coli
strains
0157, 026, 0111, and 0145 to HEp-2 cells
Intimin, the antigenic target of the VHH-Fc, mediates the intimate attachment
of E. coil
to epithelial cells. As a functional assay, the present inventors investigated
if the rationally
designed mutations impacted the VHH-Fc's ability to neutralize E. coil from
adhering to
epithelial cells by blocking intimin. HEp-2 adherence inhibition assays were
performed as
described above. HEp-2 cells were incubated with a culture of one of seven E.
coil strains
(026:H11, 045:H2, 0103:H2, 0145:Hnm, 0121:H19, 0111:Hnm and 0157:H7) in the
presence
or absence of either VHH-Fc or VHH-(5+1)Fc, washed to remove any non-adherent
bacteria and
then visualized by immunofluorescence microscopy. Compared to the respective
positive
controls of no VHH-Fc (+PBS treatment), the addition of either VHH-Fc or VHH-
(5+1)Fc
abrogated the adhesion of E. coil strains 026:H11, 0111:Hnm, 0145:Hnm and
0157:H7 to
HEp-2 cells to HEp-2 cells (FIG. 15).
To quantify the relative neutralization capacity of the VHH-Fc compared to the
VHH-
(5+1)Fc, the adhesion assay for fluorometry was adapted (as described above)
and the relative
53
Date Recue/Date Received 2021-02-08
fluorescence of HEp-2 cells incubated with a culture of each of the seven E.
coil strains with and
without either VHH-Fc or VHH-(5+1)Fc was measured. The addition of either
showed the same
pattern of reducing the relative fluorescence caused by adherent bacteria for
strains 026:H11,
0111:Hnm, 0145 and 0157:H7 to background levels (FIG. 16). Thus, VHH -(5+1)Fc
retains the
ability to neutralize adherence of E. coil strains 0157, 026, 0111, and 0145
to HEp-2 cells.
Accordingly, the skilled person understands that the present disclosure also
pertains to a
recombinant polypeptide having increased accumulation relative to accumulation
of a native
polypeptide. The polypeptide comprises: a variant Fc chain that exhibits
enhanced accumulation
in a protein-expressing system. In one embodiment, the protein-expressing
system is a plant. In
one embodiment, the plant is a Nicotiana plant or Lactuca plant. In one
embodiment, the plant is
a Nicotiana benthamiana plant or a Nicotiana tabacum plant. In one embodiment,
the Fc chain
has at least one amino acid substitution selected from N9D (SEQ ID NO: 32),
N84D (SEQ ID
NO: 33), N131D (SEQ ID NO: 34), Q175E (SEQ ID NO: 35), and Q195E (SEQ ID NO:
36). In
one embodiment, the Fc chain has a de novo disulfide from an amino acid
substitution
G196C/R219C (SEQ ID NO: 37). In one embodiment, the Fc chain comprises the
following
amino acid substitutions: N9D, N84D, and N131D (SEQ ID NO: 38). In one
embodiment, the Fc
chain comprises the following amino acid substitutions: N9D, N84D, N131D,
Q175E, and
Q195E (SEQ ID NO: 39). In one embodiment, the Fc chain comprises the following
amino acid
substitutions: N9D, N84D, N131D, Q175E, Q195E, and G196C/R219C (SEQ ID NO:
40). In
one embodiment, the polypeptide exhibits at least a 3-fold increase in
accumulation. In one
embodiment, the polypeptide exhibits up to about a 22-fold increase. In one
embodiment, the Fc
chain is linked to a bioactive moiety. In one embodiment, the bioactive moiety
is an enzyme,
cytokine, antibody, antibody fragment, peptide, signalling molecule, receptor,
or ligand. Due to
54
Date Recue/Date Received 2021-02-08
the wide variety of bioactive moieties that may be used as fusion partners
with the Fc chain, the
skilled person will recognize that these Fc-fusion molecules have numerous
biological and
pharmaceutical applications. In addition to their use in vaccines, intravenous
immunoglobulin
therapy, and drug therapies, in vitro applications may include, for example,
protein binding
assays, microarray applications, flow cytometry, and immunohistochemistry.
Fusion with the Fc
chain may also provide the bioactive moiety with a number of beneficial
biological and
pharmacological properties. For example, fusion with an Fc chain may
significantly increase
bioactive moiety's plasma half-life, facilitate interaction with immune cell
Fc receptors, and
improve solubility and stability both in vivo and in vitro.
The disclosure also provides a method of producing a variant Fc chain of a
native Fc
chain comprising: determining solvent accessibility of an amino acid residue
in the Fc chain, and
selecting a polar, solvent-exposed amino acid residues for mutation to its
negatively charged
counterpart. In some embodiments, selecting the polar, solvent-exposed amino
acid residues for
mutation to its negatively charged counterpart comprises selecting an Asn or
Gln residue for
mutation to a Asp or Glu residue, respectively. The predictive Fc structure is
modelled using the
I-TASSER and PyMol programs and solvent accessibility of a candidate is
determined by the
average number of neighbouring (within 10 A) per side-chain atom (AvNAPSA).
Other methods
of measuring solvent accessibility are known in the art and may be used as
well. In one
embodiment, at least one of the following Asn or Gln residues are for mutation
to Asp or Glu,
respectively: N9D (SEQ ID NO: 32), N84D (SEQ ID NO: 33), N131D (SEQ ID NO:
34),
Q175E (SEQ ID NO: 35), and Q195E (SEQ ID NO: 36).
The disclosure also provides a method of producing a variant Fc chain
comprising:
selecting a first amino acid and a second amino acid in the Fc chain, the
second amino acid in a
Date Recue/Date Received 2021-02-08
proximate distance from the first amino acid, wherein the first amino acid and
the second amino
acid are not involved in native disulfide bonding, and mutating the first and
the second amino
acids to cysteines to form a disulfide bond to form between the first and
second amino acids. For
selection of de novo intrachain disulfide bonds, the predicted Fc structure
was modelled in
PyMol and disulfide candidates chosen by manual inspection of the molecule
that will stabilize
the tertiary structure of the protein, for example, by tethering beta strands
in the Fc chain
together. In one embodiment, mutating the first and second amino acids to
cysteines to form the
disulfide bond stabilizes the tertiary structure of the Fc chain. In one
embodiment, mutating the
first and second amino acids to cysteines to form causing the disulfide bond
to form between the
first and second amino acids connects at least two beta sheets within the Fc
chain together. In
one embodiment, the disulfide bond is between amino acid substituted residues
G196C/R219C.
A disulfide bond between amino acid substituted residues G196C/R219C tethers
the C-terminal
end of strand G to the N-terminal end of strand F. In one embodiment, the
proximate distance is
less than 5 A.
The disclosure also provides a method of enhancing accumulation of a protein,
comprising: transforming a plant, or a portion thereof, with a recombinant
expression vector
comprising a nucleic acid molecule encoding a genetically modified variant Fc
chain. In one
embodiment, the Fc chain has at least one amino acid substitution selected
from N9D (SEQ ID
NO: 32), N84D (SEQ ID NO: 33), N131D (SEQ ID NO: 34), Q175E (SEQ ID NO: 35),
and
Q195E (SEQ ID NO: 36). In one embodiment, the Fc chain has a de novo disulfide
from an
amino acid substitution G196C/R219C (SEQ ID NO: 37). In one embodiment, the Fc
chain
comprises the following amino acid substitutions: N9D, N84D, and N131D (SEQ ID
NO: 38). In
one embodiment, the Fc chain comprises the following amino acid substitutions:
N9D, N84D,
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Date Recue/Date Received 2021-02-08
N131D, Q175E, and Q195E (SEQ ID NO: 39). In one embodiment, the Fc chain
comprises the
following amino acid substitutions: N9D, N84D, N131D, Q175E, Q195E, and
G196C/R219C
(SEQ ID NO: 40). In one embodiment, the Fc chain accumulation in the plant, or
plant portion
thereof, is enhanced up to about 22-fold.
Thylakoid Lumen Targeting
As an alternative to ER-targeted recombinant protein production, the present
inventors
sought to explore the possibility of targeting protein folding and assembly to
the chloroplast and
thylakoid lumen.
Example 15
Sub-compartment targeting influences accumulation and dimerization patterns of
the
VHH-Fc fusion
The VHH-Fc was cloned into five separate plant expression vectors that permit
targeting
of the protein to the chloroplast thylakoid via Sec or Tat pathways, the
chloroplast stroma, the
ER or the cytoplasm. Both Sec and Tat sequences (Accession #s: NP 001318791
and
NP 001321139 respectively) were obtained from a previous proteomics study that
isolated and
sequenced multiple luminal proteins in Arabidopsis thaliana (Schubert et al.
2002). The Tat
targeting sequence corresponds to the N-terminal 71 amino acids of a FKBP-type
peptidyl-prolyl
cis-trans isomerase (At1g20810). The Sec targeting sequence corresponds to the
N-terminal 75
amino acids of a thylakoid luminal 15.0 kDa protein 2 (At5g52970). Figure 17
depicts protein
import into the chloroplast and targeting to either the stroma or the
thylakoid. Cleavage sites of
the targeting peptides were predicted using the ChloroP and TargetP online
tools (Almagro et al.
2019; Emanuelsson et al. 1999) (FIG. 18). Sequences were synthesized and then
cloned using a
57
Date Recue/Date Received 2021-02-08
ligation independent method (Li et al. 2007) into a cytosolic expression
vector (Pereira et al.
2014). The VHH-Fc construct comprised an anti-E. coil VHH9 fused to a bovine
Fc
(ANN46383). The VHH-Fc was cloned into this adapted vector by Gateway cloning
and the
reading frame was confirmed by sequencing.
After transiently transforming leaves of N. benthamiana, tissue was harvested
and crude
extract separated by SDS-PAGE in either a reducing buffer or a non-reducing
buffer. Detection
by Western blot using an anti-c-myc antibody showed accumulation of the VHH-Fc
in the
thylakoid lumen via both pathways, in the stroma, and in the ER, but lacked
sufficient signal for
detection in the cytoplasm (FIGs. 19A and 19B). Under non-reducing extraction
conditions, the
VHH-Fc is detected predominantly as an 88kDa band matching the predicted size
of the VHH-Fc
dimer. Total accumulation is highest in the ER at 51.1 mg/kg fresh weight
(FW), followed by the
thylakoid via Sec-targeting at 30.5 mg/kg FW. Accumulation in the stroma and
thylakoid via
Tat-targeting are substantially lower at 6.6 mg/kg FW and 5.4 mg/kg FW
respectively. Under
reducing extraction conditions of the same samples, an enriched band at 44kDa
is detected
matching the predicted size of the VHH-Fc monomer for the ER, stromal,
thylakoid via Sec and
thylakoid via Tat compaiiments suggesting that the VHH-Fc dimer in these
compaiiments is
stabilized by an interchain disulfide bond.
Based on these results, while ER-targeting produced the highest yields in the
VHH-Fc
tested, the Sec-targeting accumulation remained significantly high, in
contrast to stromal,
cytoplasm, and Tat-import pathways.
Example 16
Sec-targeted recombinant proteins stably integrate in chloroplast genome
58
Date Recue/Date Received 2021-02-08
In addition to transient expression, the VHH-Fc was also encoded in the
chloroplast by
transforming the chloroplast genome through homologous recombination using
vector pCEC5
(Kolotilin et al. 2013). The VHH-Fc was targeted to the thylakoid within the
chloroplast using
the Sec import pathway. In this case, the following Seq transit peptide was
used:
MASSSRLSLKTSGDEENWVSRFRSKSLSLVFSGALALGLSLSGVGFADA (SEQ ID NO:
54). The DNA sequence comprising the Seq transit peptide, the VHH and the Fc
chain was
optimized for expression in the chloroplast genome.
Example 17
Sec- and Tat-targeted GFP-Fc localize in the thylakoid similarly
To verify that the Sec and Tat transit peptides indeed target the VHH-Fc to
the thylakoid
compaitment, subcellular localization of the VHH-Fc was tracked by fusing GFP
to the Fc chain
in each of the expression vectors. Visualization by confocal microscopy showed
the Sec and Tat-
targeted GFP-tagged protein to consistently colocalize with chlorophyll, which
accumulates in
the thylakoid and autofluoresces at ¨735nm (FIG. 20). On the other hand, the
construct targeting
the recombinant protein to the stroma showed a very distinct pattern
surrounding the thylakoid
grana, and into stromules. Therefore, the Sec and Tat transit peptides
identified indeed target the
recombinant protein to the thylakoid.
Example 18
Sec-targeted VHH-Fc fusions with an engineered disulfide show improved yield
To determine if the oxidative folding of the thylakoid can recapitulate the
yield-
improving effects of an engineered disulfide bond, the VHH-Fc fusion carrying
the
G196C/R219C mutation was targeted to the thylakoid lumen via the Sec pathway
and
59
Date Recue/Date Received 2021-02-08
accumulation by Western blot after agroinfiltration was measured. Similar to
expression in the
ER, the engineered VHH-Fc showed a significant yield improvement when targeted
to the Sec
pathway (FIGs. 11C, 21A, and 21B). Based on these results, introduction of a
rationally designed
de novo disulfide does not impede, but rather significantly enhances in vivo
accumulation when
introduced into the Sec-targeted Fc chain, and shows that the disulfide bridge
is introduced in the
lumen of the thylakoid after import of the unfolded polypeptide.
Example 19
Sec, Tat, and stroma- targeted VHH-Fc fusions bind 0157:H7
The present inventors demonstrated above that the ER-targeted VHH binds to
intimin, an
integral outer membrane protein of E. coli. To determine if the thylakoid-
targeted VHH-Fc
retained the ability to bind E. coli, purified VHH-Fc from each compaitment
was incubated with
the pathogen then fixed in paraformaldehyde, washed and probed for
immunofluorescence using
a FITC labelled anti-c-myc secondary antibody. Visualization by confocal
microscopy showed
consistent co-localization between DAPI-stained bacterial cells and the FITC-
labelled VHH-Fc
for the thylakoid via Sec, thylakoid via Tat, and stromal compaitments,
indicating that the
chloroplast-targeted VHH-Fc retains the ability to bind intimin on E. coli
surfaces (FIG. 22). As
a negative control, 0157:H7 cells were also treated with PBS containing 0.1%
Tween-20 (PBS-
T) instead of the VHH-Fc and similarly stained but did not show fluorescence
under FITC-
related imaging conditions (480 nm excitation and 520-540 nm detection). Based
on these
results, the VHH is folded correctly through Sec, Tat and stroma targeted
pathways regardless of
the status of the Fc chain.
Date Recue/Date Received 2021-02-08
Example 20
Sec, Tat and stroma-targeted VHH-Fc fusions can neutralize 0157:H7's adherence
to HEp-2 cells
Similar to the testing of ER-targeted VHH-Fc, the present inventors tested if
thylakoid
targeted VHH-Fc were able to neutralize intimin-mediated E. coil attachment to
epithelial cells.
HEp-2 cells were incubated with E. coil 0157:H7 in the presence or absence of
purified VHH-Fc
from each of the compai intents. Cells were then washed to remove non-
adherent bacteria, fixed
in paraformaldehyde and incubated with immunofluorescent labels. HEp-2 cells
were visualized
by fluorescent actin staining using rhodamine phalloidin and 0157:H7 cells
visualized using a
donkey anti-rabbit alexa 350 secondary antibody. The addition of purified VHH-
Fc from any of
the compai intents abrogated adhesion of any labelled E. coil 0157:H7 to
the incubated HEp-2
cells as visualized using confocal microscopy, while the addition of Fc alone
did not abrogate
adhesion of E. coli to Hep-2 cells (FIG. 23). Given that Tat and stromal
imported antibodies
retain functionality and show dimerized banding under non-reducing conditions
(Figure 19B),
this suggests disulfide formation in the stroma despite its reducing
environment. Figure 24 shows
two possible mechanisms of disulfide formation, interaction with LT01 and
spontaneous
formation, that may account for disulfide formation of tat and stroma targeted
antibodies.
These results indicate that the chloroplast-targeted VHH-Fc retains the
ability to
neutralize E. coil from colonizing epithelial cells and that the inhibition of
adhesion is mediated
by the VHH and not by non-specific interactions of the Fc chain of the
antibody.
61
Date Recue/Date Received 2021-02-08
Accordingly, the present disclosure also provides a method of enhancing
expression of a
recombinant protein in a plant, or a portion thereof, comprising: transforming
the plant, or plant
portion thereof, with a recombinant expression vector comprising a nucleic
acid molecule
encoding the recombinant protein, wherein the recombinant protein is targeted
to the chloroplast
thylakoid. In one embodiment, the recombinant protein is targeted to the
chloroplast thylakoid
via the Sec pathway. In one embodiment, the recombinant protein is targeted to
the chloroplast
thylakoid via the Tat pathway. In one embodiment, the recombinant protein is
targeted to the
chloroplast stroma. In one embodiment, the recombinant protein comprises a Sec-
targeted
peptide, a Tat-targeted peptide, a stroma-targeted peptide, or ER-targeted
peptide. In one
embodiment, the recombinant protein comprises an Fc chain.
In one embodiment, the Fc chain has at least one amino acid substitution
selected from
N9D (SEQ ID NO: 32), N84D (SEQ ID NO: 33), N131D (SEQ ID NO: 34), Q175E (SEQ
ID
NO: 35), and Q195E (SEQ ID NO: 36). In one embodiment, the Fc chain has a de
novo disulfide
from an amino acid substitution G196C/R219C (SEQ ID NO: 37). In one
embodiment, the Fc
chain comprises the following amino acid substitutions: N9D, N84D, and N131D
(SEQ ID NO:
38). In one embodiment, the Fc chain comprises the following amino acid
substitutions: N9D,
N84D, N131D, Q175E, and Q195E (SEQ ID NO: 39). In one embodiment, the Fc chain
comprises the following amino acid substitutions: N9D, N84D, N131D, Q175E,
Q195E, and
G196C/R219C (SEQ ID NO: 40). In one embodiment, the recombinant protein
comprises a
bioactive moiety linked to the Fc chain. In one embodiment, the Fc chain is an
IgA Fc chain. In
one embodiment, the Fc chain is a bovine IgA Fc chain. In one embodiment,
recombinant protein
is an IgA antibody. In one embodiment, the antibody comprises complementarity
determining
regions (CDR) having at least 80%, 85%, 90%, 95%, 97%, or 100% amino acid
sequence
62
Date Recue/Date Received 2021-02-08
identity to the sequence as set forth in: (i) SEQ ID NO: 12 (CDR1), SEQ ID NO:
13 (CDR2),
and SEQ ID NO: 14 (CDR3), (ii) SEQ ID NO: 15 (CDR1), SEQ ID NO: 16 (CDR2), and
SEQ
ID NO: 17 (CDR3), (iii) SEQ ID NO: 18 (CDR1), SEQ ID NO: 19 (CDR2), and SEQ ID
NO: 20
(CDR3), (iv) SEQ ID NO: 21 (CDR1), SEQ ID NO: 22 (CDR2), and SEQ ID NO: 23
(CDR3),
(v) SEQ ID NO: 24 (CDR1), SEQ ID NO: 25 (CDR2), and SEQ ID NO: 26 (CDR3), or
(vi) SEQ
ID NO: 27 (CDR1), SEQ ID NO: 28 (CDR2), and SEQ ID NO: 29 (CDR3). In one
embodiment,
the antibody, or antigen binding fragment thereof, comprises a heavy chain
variable (VHH)
domain. In one embodiment, the VHH domain has at least 80%, 85%, 90%, 95%,
97%, or 100%
amino acid sequence identity to the sequence as set forth in SEQ ID NOs: 1 to
11.
The present disclosure also provides a method of producing a recombinant
protein in a
plant, or a portion thereof, comprising: transforming the plant, or portion
thereof, with a
recombinant expression vector comprising a nucleic acid molecule encoding the
recombinant
protein, wherein the recombinant protein is targeted to the chloroplast
thylakoid.
In one embodiment, transforming the plant comprises targeting the recombinant
expression vector to the thylakoid within the chloroplast via the Sec import
pathway by a Sec-
targeted transit peptide and transforming the chloroplast genome through
homologous
recombination. In one embodiment, the Sec-targeted transit peptide has the
amino acid sequence
set forth in SEQ ID NO: 54.
References
1. Almagro et al. (2019). Life Science Alliance 2 pii: e201900429.
2. Bonner et al. (2007). Journal of Biological Chemistry 282:16969-16980.
3. Carvalho et al. (2005). Infect. Immun. 73, 2541-2546.
63
Date Recue/Date Received 2021-02-08
4. Conley etal. (2009). BMC Biol. 7:48.
5. De Wilde etal. (2013). Plant Physiology 161:1021-1033.
6. Gansheroff et al. (1999). Infect. Immun. 67, 6409-6417.
7. Henry et al. (2015). Protein Eng. Des. Se!. 28, 379-383.
8. Henry et al. (2016). Protein Eng. Des. Se!. 29, 439-443.
9. Kolotilin et al. (2013). Biotechnology for Biofuels 6:65.
10. Krugmann et al. (1997). Journal of Immunology 159:244-249.
11. Li etal. (2007). Nature Methods 4:251-256.
12. Majowicz etal. (2014a). Foodborne Pathogens and Disease 11:447-455.
13. Majowicz etal. (2014b). Foodborne Pathogens and Disease 11:447-455.
14. McKee et al. (1996). Infect. Immun. 64, 2225-2233.
15. Miletic etal. (2015). Front. Plant Sci. 6:1221.
16. Pereira etal. (2014). BMC Biotechnology 14:59.
17. Royle et al. (2003). J. Biol. Chem. 278, 20140-20153.
18. Ruhl et al. (1996). Infect. Immun. 64, 5421-5424.
19. Saberianfar et al. (2015). Plant Biotechnol. J. 13, 927-937.
20. Sainsbury etal. (2009). Plant Biotechnol . J. 7, 682-693.
21. Schubert et al. (2002). Journal of Biological Chemistry 277:8354-8365.
22. Schrodinger LLC (2010). The PyMOL molecular graphics system. Version,
1(5), 0.
23. Silhavy etal. (2002). EMBO J. 21, 3070-3080.
24. Steentoft et al. (2013). EMBO J. 32, 1478-1488.
25. Wold et al. (1990). Infect. Immun. 58, 3073-3077.
26. Wu et al. (2008). Proteins 72:547-556.
64
Date Recue/Date Received 2021-02-08
27. Wycoff et al. (2005). Curr. Pharm. Des. 11, 2429-2437.
28. Yang et al. (2015). Nature Methods 12:7-8.
29. Zhang Y (2008). BMC Bioinformatics 9:40.
Date Recue/Date Received 2021-02-08
