Note: Descriptions are shown in the official language in which they were submitted.
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RECOMBINANT PLANT-DERIVED ANTIBODIES AND FC VARIANTS
AND RELATED METHODS
Field
[0001] The present application relates generally to recombinant plant-derived
proteins,
and more specifically to recombinant antibodies, and Fc variants thereof, and
methods of
producing the same. The application also relates to methods of preventing or
reducing
colonization of Escherichia co//in a mammal. The application also relates to
methods of
detecting the presence of E. coil in a sample.
Background
[0002] Food borne pathogens such as E. coil have consistently been one of the
foremost foodborne pathogen threats worldwide. While there are many strategic
interventions meant to prevent E. co//transmission to humans, conservative
estimates
indicate that the pathogen still causes 2.8 million acute illnesses annually.
[0003] Evading a host organism's defenses, E. coli colonizes at mucosal sites
primarily
in the gastrointestinal (GI) tract in an animal. E. coli 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.
[0004] Preventing or minimizing E. coli 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. coli cells, mediates
interaction
between the bacteria and, for example, the epithelial cells lining the inner
surface of the
animal host's gastrointestinal tract. Colonization is initiated when intimin
binds to the
translocated intimin receptor (Tir) located on epithelial cells. Interfering
with this binding
would reduce such colonization and facilitate subsequent expulsion of E.
co/ifrom an
animal's gastrointestinal tract. However, the availability of effective
therapeutics and
diagnostics for treatment or prevention of E. co//contamination remains a
problem, and
new therapies which can prevent binding of intimin to gut epithelial cells are
desirable.
[0005] The use and efficacy of recombinant secretory immunoglobulin A (sIgA)
in
passive mucosal immunotherapy in livestock is documented to control pathogens
in the
GI tract. Because administration of sIgA can impart immediate, if transient,
protection
from a pathogen, it may be of value to beef producers and processors as a pre-
harvest
intervention for E. coll. In the GI tract, sIgA primarily functions to clear
pathogens by
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immune exclusion: after binding to its target, glycans on the secretory
component
facilitate binding to the mucus lining of the GI tract, enabling clearance of
sIgA-pathogen
complexes by peristalsis. A sIgA directed against intimin would thus be
expected to
prevent lumina! E. coli cells from interacting with the host epithelium,
clearing them by
entrapment in the mucus layer and subsequent fecal shedding. Chimeric sIgA
antibodies
which specifically recognize E. coli intim in and which comprise a single
domain camelid
antibody (VHH or VHH) fused to a bovine IgA Fc chain, have been reported by
Saberianfar et al (Frontiers in Plant Science (2019), 10: 270) to show
efficacy against
several strains of enterohemorrhagic E. coll.
[0006] One of the challenges in delivering therapeutics such as sIgA 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 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. 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.
[0007] While the use of a plant platform for folding and assembly of
recombinant
proteins and other synthetics in the endoplasmic reticulum (ER) is well
established,
some ER-targeted recombinant proteins have been associated with issues such as
unfolded proteins, ER-associated degradation, and misfolding, potentially
limiting the
proper folding of certain antibodies, thus reducing antibody yield.
[0008] 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
[0009] The present applicant has found that using rational design methods to
introduce
mutations into the native Fc chain of an antibody can improve the accumulation
of the
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antibody when recombinantly expressed in plant tissue. Therefore, one aspect
of the
invention provides an Fc variant polypeptide which is a variant of a native Fe
polypeptide
such that the variant sequence of the Fc variant polypeptide comprises one or
more
mutations of the native sequence of the native Fc polypeptide. The one or more
mutations result in one or more of an increase in a net surface negative
charge of the Fc
variant polypeptide compared to a net surface negative charge of the native Fc
polypeptide and introduction of cysteine residues adapted to form a disulfide
bridge. The
Fc variant polypeptide exhibits enhanced accumulation when expressed in a
plant cell
compared to accumulation of the native Fc polypeptide when expressed in the
plant cell.
[0010] In at least one embodiment, the native sequence of the native Fc
polypeptide is
SEQ ID NO:47 and the one or more mutations are selected from N9D, N84D, N131D,
0175E, 0195E, G196C/R219C and combinations thereof. In at least one
embodiment,
the Fc variant polypeptide has a sequence selected from SEQ ID NO:51, SEQ ID
NO:52,
SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID
NO:58 and SEQ ID NO:59.
[0011] Another aspect of the invention provides a method of producing an Fc
variant
polypeptide, the method comprising:
(a) determining solvent accessibility of one or more amino
acid residues in a native
Fc polypeptide;
(b) selecting at least one solvent-exposed amino acid residue; and
(c) mutating the at least one selected solvent-exposed amino acid residue
to a
negatively charged amino acid residue.
[0012] In at least one embodiment, selecting the solvent-exposed amino acid
residue for
mutation to a negatively charged amino acid residue comprises selecting an
asparagine
or glutamine residue for mutation to an aspartic acid or glutamic acid
residue,
respectively.
[0013] In a further aspect, the invention provides a method of producing a Fc
variant
polypeptide, the method comprising:
(a) selecting a first amino acid and a second amino acid in a native Fe
polypeptide,
the second amino acid being within a predetermined distance from the first
amino acid,
wherein the first amino acid and the second amino acid are free from
involvement in
native disulfide bonding; and
(b) mutating the first amino acid and the second amino acid respectively to
a first
cysteine residue and a second cysteine residue, such that a disulfide bond is
formable
between the first cysteine residue and the second cysteine residue.
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[0014] A further aspect of the invention provides an Fc variant fusion
polypeptide
including an Fc variant polypeptide as described herein fused to a bioactive
moiety. In at
least one embodiment, the bioactive moiety is a variable domain of an
antibody. In at
least one embodiment, the bioactive moiety is a single domain antibody. In at
least one
embodiment, the bioactive moiety is a VHH polypeptide as described herein. In
at least
one embodiment, the variable domain of an antibody, the single domain antibody
or the
VHH polypeptide specifically binds to intimin on an Escherichia coli cell.
[0015] In an additional aspect, the present invention provides an antibody or
antigen
binding fragment thereof comprising an Fc variant fusion polypeptide as
described herein
comprising a variable domain of an antibody fused to an Fc variant polypeptide
as
described herein. In at least one embodiment, the variable domain of the
antibody is a
single domain antibody. In at least one embodiment, the variable domain of the
antibody
is a VHH polypeptide as described herein. In at least one embodiment, the
antibody or
antigen binding fragment thereof specifically binds to intimin on an
Escherichia co//cell.
[0016] In another aspect, the present invention provides a VHH polypeptide
comprising a
first complementarity determining region (CDR1), a second complementarity
determining
region (CDR2) and a third complementarity determining region (CDR3), wherein:
(i) the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid
sequence identity to SEQ ID NO:4, the CDR2 has at least 80%, 85%, 90%, 95%,
97%,
99% or 100% amino acid sequence identity to SEQ ID NO:5 and the CDR3 has at
least
80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO:6;
(ii) the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid
sequence identity to SEQ ID NO:8, the CDR2 has at least 80%, 85%, 90%, 95%,
97%,
99% or 100% amino acid sequence identity to SEQ ID NO:9 and the CDR3 has at
least
80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO:10;
(iii) the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid
sequence identity to SEQ ID NO:12, the CDR2 has at least 80%, 85%, 90%, 95%,
97%,
99% or 100% amino acid sequence identity to SEQ ID NO:13 and the CDR3 has at
least
80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO:14;
(iv) the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid
sequence identity to SEQ ID NO:16, the CDR2 has at least 80%, 85%, 90%, 95%,
97%,
99% or 100% amino acid sequence identity to SEQ ID NO:17 and the CDR3 has at
least
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80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO:18;
(v) the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid
sequence identity to SEQ ID NO:20, the CDR2 has at least 80%, 85%, 90%, 95%,
97%,
5 99% or 100% amino acid sequence identity to SEQ ID NO:21 and the CDR3 has
at least
80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO:22;
(vi) the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid
sequence identity to SEQ ID NO:24, the CDR2 has at least 80%, 85%, 90%, 95%,
97%,
99% or 100% amino acid sequence identity to SEQ ID NO:25 and the CDR3 has at
least
80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO:26;
(vii) the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid
sequence identity to SEQ ID NO:28, the CDR2 has at least 80%, 85%, 90%, 95%,
97%,
99% or 100% amino acid sequence identity to SEQ ID NO:29 and the CDR3 has at
least
80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO:30;
(viii) the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid
sequence identity to SEQ ID NO:32, the CDR2 has at least 80%, 85%, 90%, 95%,
97%,
99% or 100% amino acid sequence identity to SEQ ID NO:33 and the CDR3 has at
least
80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO:34;
(ix) the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid
sequence identity to SEQ ID NO:36, the CDR2 has at least 80%, 85%, 90%, 95%,
97%,
99% or 100% amino acid sequence identity to SEQ ID NO:37 and the CDR3 has at
least
80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO:38;
(x) the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid
sequence identity to SEQ ID NO:40, the CDR2 has at least 80%, 85%, 90%, 95%,
97%,
99% or 100% amino acid sequence identity to SEQ ID NO:41 and the CDR3 has at
least
80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO:42; or
(xi) the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid
sequence identity to SEQ ID NO:44, the CDR2 has at least 80%, 85%, 90%, 95%,
97%,
99% or 100% amino acid sequence identity to SEQ ID NO:45 and the CDR3 has at
least
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80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO:46.
[0017] In another aspect, the invention provides an antibody or antigen
binding fragment
thereof, wherein the antibody or antigen binding fragment thereof comprises a
VHH
polypeptide as described herein. In at least one embodiment, the antibody or
antigen
binding fragment thereof comprises a VHH-Fc fusion polypeptide comprising a
VHH
polypeptide as described herein fused to an Fc polypeptide. In at least one
embodiment,
the Fc polypeptide is a native Fc polypeptide. In at least one embodiment, the
Fc
polypeptide is an Fc variant polypeptide as described herein. In at least one
embodiment, the antibody or antigen binding fragment thereof comprises an Fc
variant
fusion polypeptide as described herein comprising a VHH polypeptide as
described
herein fused to an Fc variant polypeptide as described herein. In at least one
embodiment, the antibody is an IgA antibody.
[0018] Another aspect of the invention provides a nucleic acid encoding a VHH
polypeptide as described herein, an Fc variant polypeptide as described
herein, a Fc
variant fusion polypeptide as described herein, or a Viild-Fc fusion
polypeptide as
described herein. In at least one embodiment, the nucleic acid further
comprises a
chloroplast targeting signal sequence or an endoplasmic reticulum targeting
signal
sequence. In at least one embodiment, the chloroplast targeting signal
sequence is a
stroma targeting signal sequence or a thylakoid targeting signal sequence. In
at least
one embodiment, the thylakoid targeting signal sequence is a Sec signaling
sequence. In
at least one embodiment, the thylakoid targeting signal sequence is a Tat
signaling
sequence.
[0019] In another aspect, the present invention provides an expression vector
comprising a nucleic acid as described herein. A further aspect of the
invention provides
a host cell comprising an expression vector as described herein. Yet another
aspect of
the invention provides a non-viable harvested plant material comprising a host
cell as
described herein. In another aspect, the invention provides a non-viable
edible product
comprising a host cell as described herein. A further aspect of the invention
provides a
tobacco product comprising a host cell as described herein. In another aspect,
the
invention provides an animal feed comprising a host cell as described herein.
[0020] A further aspect of the invention provides a method of producing a VHH
polypeptide as described herein, an Fc variant polypeptide as described
herein, an Fc
variant fusion polypeptide as described herein, or a VHH-Fc fusion polypeptide
as
described herein, the method comprising transforming a host cell with an
expression
vector including a nucleic acid as described herein.
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[0021] An additional aspect of the invention provides a method of enhancing
accumulation of a recombinant protein in a plant cell, the method comprising
transforming the plant cell with a recombinant expression vector comprising a
nucleic
acid encoding a Fc variant fusion polypeptide as described herein comprising a
bioactive
moiety as described herein. In at least one embodiment, the recombinant
protein is a
recombinant antibody and the bioactive moiety is a variable domain of the
antibody. In at
least one embodiment, the nucleic acid further encodes a chloroplast targeting
signal
sequence. In at least one embodiment, the chloroplast targeting signal
sequence is a
Sec signaling sequence.
[0022] A further aspect of the invention provides a method of producing a
recombinant
protein in a plant or portion thereof, the method comprising transforming the
plant or
portion thereof with a recombinant expression vector comprising a nucleic acid
encoding
a Fc variant fusion polypeptide as described herein comprising a bioactive
moiety as
described herein. In at least one embodiment, the recombinant protein is a
recombinant
antibody and the bioactive moiety is a variable domain of the antibody. In at
least one
embodiment, the nucleic acid further encodes a chloroplast targeting signal
sequence. In
at least one embodiment, the chloroplast targeting signal sequence is a Sec
signaling
sequence.
[0023] In another aspect, the invention provides a method of enhancing
expression of a
recombinant antibody in a plant cell, the method comprising transforming the
plant cell
with a recombinant expression vector comprising a nucleic acid encoding a
variable
domain of the antibody fused to an Fc polypeptide or to an Fc variant
polypeptide as
described herein and further encoding a chloroplast targeting signal sequence.
In at least
one embodiment, the chloroplast targeting signal sequence is a Sec signaling
sequence.
In at least one embodiment, the variable domain of the antibody is a single
domain
antibody. In at least one embodiment, the variable domain of the antibody is a
VHH
polypeptide as described herein. In at least one embodiment, the recombinant
antibody
specifically binds to intimin on an Escherichia coli cell.
[0024] In another aspect, the invention provides a method of producing a
recombinant
antibody in a plant or a portion thereof, the method comprising transforming
the plant or
portion thereof with a recombinant expression vector comprising a nucleic acid
encoding
a variable domain of the antibody fused to a Fc polypeptide or to an Fc
variant
polypeptide as described herein and further encoding a chloroplast targeting
signal
sequence. In at least one embodiment, the chloroplast targeting signal
sequence is a
Sec signaling sequence. In at least one embodiment, the variable domain of the
antibody
is a single domain antibody. In at least one embodiment, the variable domain
of the
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antibody is a VHH polypeptide as described herein. In at least one embodiment,
the
recombinant antibody specifically binds to intirnin on an Escherichia
co//cell.
[0025] Another aspect of the invention provides a pharmaceutical composition
comprising an antibody or an antigen binding fragment thereof as described
herein, and
a pharmaceutically acceptable carrier.
[0026] In another aspect, the invention provides use of an antibody or antigen
binding
fragment thereof as described herein, for preventing or reducing E. coli cell
colonization
of the gastrointestinal tract of a mammal. Yet another aspect of the invention
provides
use of an antibody, or antigen binding fragment thereof as described herein,
in
preparation of a medicament for preventing or reducing E. coli cell
colonization of the
gastrointestinal tract of a mammal. Another aspect of the invention provides a
method of
preventing or reducing colonization of E. co//in the gastrointestinal tract of
a mammal,
comprising administering to the mammal an antibody, or antigen binding
fragment
thereof as described herein.
[0027] In another aspect, the invention provides use of an antibody or an
antigen binding
fragment thereof as described herein for neutralizing the ability of an
Escherichia coli cell
to bind to a mammalian gastrointestinal epithelial cell. Another aspect of the
invention
provides use of an antibody, or antigen binding fragment thereof as described
herein, in
preparation of a medicament for neutralizing the ability of an E. coil cell to
bind to a
mammalian gastrointestinal epithelial cell. Another aspect of the invention
provides a
method of neutralizing the ability of an E. coli cell to bind to a mammalian
gastrointestinal
epithelial cell, comprising exposing the E. coli cell to an antibody, or
antigen binding
fragment thereof as described herein.
[0028] Another aspect of the invention provides a method of detecting the
presence of
E. coli in a sample, comprising:
(a) contacting the sample with a VHH polypeptide as described herein or
with a
VHH-Fc fusion polypeptide as described herein, and
(b) detecting binding between intimin and the VHH polypeptide or the VHH-Fc
fusion
polypeptide.
In still another aspect, the invention provides use of a VHH polypeptide as
described
herein or a VHH-Fc fusion polypeptide as described herein for detecting the
presence of
E. co//in a sample. In another aspect, the invention provides a diagnostic kit
for detecting
the presence of E. co//in a sample, wherein the kit comprises a VHH
polypeptide as
described herein or a VH H- Fc fusion polypeptide as described herein.
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Brief Description of the Drawings
[0029] 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. Further features of the present
invention will become
apparent from the following written description and the accompanying figures,
in which:
[0030] Figure 1 is a series of confocal images showing E. coli strain 0145
(C483) and
E. co//strain 0145 (C625) incubated with HEp-2 cells, in the presence of PBS
(phosphate buffered saline) alone as a control, with VHH9-Fc or with VHH10-
sIgA. Cells
that are immunolabelled (white) are intimately adherent on HEp-2 cells (red)
after
repeated washes. The absence of immunolabeling suggests neutralization. Size
bar = 20
urn.
[0031] Figure 2A is a graph showing accumulation levels of native Fc
polypeptide
compared to embodiments of the present Fc variant polypeptides at 4, 6, and 8
days
post infiltration (dpi). Letters denote significantly different treatments as
determined by
one way ANOVA (analysis of variance) and post-hoc Tukey HSD (honest
significant
difference) test. P<0.05, n=3-5 biological replicates. Error bars shown are
standard error
of the mean.
[0032] Figure 2B is a graph showing accumulation levels of native Fc
polypeptide
compared to another embodiment of the present Fc variant polypeptide at 4, 6,
and 8
dpi. * represents statistically significant difference from native Fc as
determined by a
T-test.
[0033] Figure 2C is a graph showing accumulation levels of native Fc
polypeptide
compared to various embodiments of the present Fe variant polypeptides at 4,
6, and 8
dpi. 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.
[0034] Figure 3A is a graph showing accumulation levels of an embodiment of
the
present VHH-Fc fusion polypeptide including a VHH polypeptide fused to a
native Fc
polypeptide compared to embodiments of the present VHH-Fc fusion polypeptide
including a VHH polypeptide fused to embodiments of the present Fc variant
polypeptides
at 4, 6, and 8 dpi. 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.
[0035] Figure 3B is a graph showing accumulation levels of an embodiment of
the
present VHH-Fc fusion polypeptide including a VHH polypeptide fused to a
native Fc
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polypeptide compared to various embodiments of the present VHH-Fc fusion
polypeptide
including a VHH polypeptide fused to embodiments of the present Fc variant
polypeptides
at 8 dpi. 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
5 shown are standard error of the mean.
[0036] Figure 4 is a collection of images of Western blots probed with either
anti-c-Myc
(panels A and B) or anti-HA (panels C and D) which correspond to differently
tagged
embodiments of the present VHH-Fc fusion polypeptide including a VHH
polypeptide
fused to a native Fc polypeptide (VHH-native Fe) or of the present VHH-Fc
fusion
10 polypeptide including a VHH polypeptide fused to an embodiment of the
present Fc
variant polypeptide (VHH-(5+1)-Fc) and JC subunits respectively. Leaf tissue
was
transformed with constructs of each subunit individually and also with
combinations of
VHH-Native-Fc/SC/JC and VHH-(5+1)-Fc/SC/JC for intended co-expression and
assembly. Detection was done for both crude leaf extract (panels A and C) and
for the
eluent after the extract had been co-immunoprecipitated using an anti-FLAG
column
(panels B and D).
[0037] Figure 5 is a collection of confocal images showing the seven most
prevalent E.
coli strains incubated with either an embodiment of the present VH H - Fc
fusion
polypeptide including a VHH polypeptide fused to a native Fc polypeptide (VHH-
Native
Fc) or with an embodiment of the present VHH-Fc fusion polypeptide including a
VHH
polypeptide fused to an embodiment of the present Fc variant polypeptide
(VHH-(5+1)-Fc). DAPI (4',6-diamidine-2'-phenylindole) has been used to
visualize E. coil
cells and a FITC (fluorescein 5(6)-isothiocyanate)-conjugated antibody has
been used to
immunolabel the Fc specifically. Size bar=10 pm.
[0038] Figure 6 is a collection of confocal images of the seven most prevalent
E. coil
strains that have been immunolabelled and incubated with HEp-2 cells in the
presence of
PBS as a control, or in the presence of an embodiment of the present VHH-Fc
fusion
polypeptide including a VHH polypeptide fused to a native Fc polypeptide (VHH-
Native-
Fc) or an embodiment of the present VHH-Fc fusion polypeptide including a VHH
polypeptide fused to an embodiment of the present Fc variant polypeptide
(VHH-(5+1)-Fc). As a control against nonspecific Fc binding, E. coil strain
0157:H7 was
incubated with a Fc polypeptide only to confirm that neutralization was
mediated through
the VHH. Cells that are immunolabelled (white) are intimately adherent on HEp-
2 cells
(red) after repeated washes. The absence of immunolabeling suggests
neutralization.
Size bar = 2011m.
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[0039] Figure 7 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 alone, with an embodiment of the present VHH-
Fc
fusion polypeptide including a VHH polypeptide fused to a native Fc
polypeptide
(VHH9-Native-Fc) or with an embodiment of the present VHH-Fc fusion
polypeptide
including a VHH polypeptide fused to an embodiment of the present Fc variant
polypeptide (VHH9-(5+1)-Fc; VHH9-Engineered 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.
[0040] Figure 8 is a schematic diagram representing the thylakoid expression
vector.
2x35S: double-enhanced promoter from Cauliflower Mosaic Virus 35S gene; tCUP:
translational enhancer from a tobacco cryptic upstream promoter; transit
peptide: a
signaling sequence targeting the expressed protein to a subcellular
compartment;
attB1/attB2: cloning sites used for GatewayTM cloning; VHH-Fc: an embodiment
of the
present VHH-Fc fusion polypeptide; nosT: nopaline synthase transcription
terminator;
Xpress/C-Myc: detection/purification tags.
[0041] Figure 9A is a graph showing accumulation levels of an embodiment of
the
present VHH-Fc fusion polypeptide including a VHH9 polypeptide fused to a
native Fc
polypeptide across the subcellular compartments cytoplasm, thylakoid lumen
(Sec
pathway), thylakoid lumen (Tat pathway), stroma and endoplasmic reticulum (ER)
extracted in reducing (left) or non-reducing conditions (right). Error bars
indicate
standard error.
[0042] Figure 9B is an image of a Western blot showing relative accumulation
of the
embodiment of the present VHH-Fc fusion polypeptide of Figure 9A across the
compartments of Figure 9A in reducing (left) and non-reducing conditions
(right).
[0043] Figure 10 is a collection of confocal images showing an embodiment of
the
present VHH-Fc fusion polypeptide tagged with green fluorescent protein (VHH-
Fc-GFP)
and targeted to chloroplasts with either Sec, Tat or stromal targeting
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 =
10 pm.
[0044] Figure 11 is a graph showing accumulation of an embodiment of the
present
VHH-Fc fusion polypeptide including a VHH polypeptide fused to a native Fc
polypeptide
(Native) targeted to the thylakoid lumen with a Sec targeting signal and an
embodiment
of the present VHH-Fc fusion polypeptide including a VHH polypeptide fused to
an
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12
embodiment of the present Fc variant polypeptide with an added disulfide
bridge
(+ Disulfide) targeted to the thylakoid lumen with a Sec targeting signal.
*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.
[0045] Figure 12 is a collection of confocal images showing an embodiment of
the
present VHH-Fc fusion polypeptide including a VHH polypeptide fused to a
native Fc
polypeptide (VHH-Fc) targeted to the chloroplast with either Sec, Tat or
stromal signals
incubated with E. coli 0157:H7. DAPI (orange) has been used to visualize E.
coh cells
and a FITC-conjugated antibody (green) has been used to immunolabel the Fc
specifically. Merged images appear yellow. Size bar=10 m.
[0046] Figure 13 is a collection of confocal images showing E. co//strain
0157:H7 that
has been incubated with HEp-2 cells in the presence of either an embodiment of
the
present VHH-Fc fusion polypeptide including a VHH polypeptide fused to a
native Fc
polypeptide (VHH-Fc) targeted to Sec, Tat and stromal compartments or a Fc
polypeptide
(Fc) as a negative control targeted to the same compartments. Cells that are
immunolabelled (white) are intimately adherent on HEp-2 cells (red) after
repeated
washes. The absence of immunolabeling suggests neutralization. Size bar = 20
m.
Detailed Description
[0047] One aspect of the invention provides an Fc variant polypeptide, which
is a variant
of a native Fc polypeptide wherein the variant sequence of the Fc variant
polypeptide
comprises one or more mutations of the native sequence of the native Fc
polypeptide. In
at least one embodiment, the native Fc polypeptide is an IgA Fe polypeptide.
In at least
one embodiment, the native Fc polypeptide is a bovine IgA Fc.
[0048] The present applicant used rational design strategies to introduce
mutations
which stabilize the structure of the Fc chain, thereby improving the
accumulation of the
Fc variant polypeptide when expressed in plant tissue. The strategies include:
1) enhancing the net surface negative charge of the Fe polypeptide chain
through
side chain alterations, also known as "supercharging". The resultant small
charge-charge
repulsive forces on the protein surface aid in reducing non-specific protein
aggregation
during recombinant protein production, and
2) introducing a de novo disulfide bridge in the Fc polypeptide chain to
tether
portions of the chain together, thereby providing further stability by
preventing unfolding
and exposure of reactive hydrophobic regions of the protein.
[0049] In at least one embodiment, the sequence of the native Fc polypeptide
is SEQ ID
NO:47 and the one or more mutations are selected from N9D, N84D, N131D, 0175E,
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Q195E, G196C/R219C and combinations thereof. In at least one embodiment, the
one
or more mutations comprise N9D, N84D and N131D. In at least one embodiment,
the
one or more mutations comprise N9D, N84D, N131D, Q175E and Q195E. In at least
one
embodiment, the one or more mutations comprise N9D, N84D, N131D, 0175E, 0195E
and G196C/R219C. In at least one embodiment, the Fc variant polypeptide has a
sequence selected from SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54,
SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58 and SEQ ID NO:59.
[0050] Another aspect of the invention provides a method of producing an Fc
variant
polypeptide, the method comprising:
(a) determining solvent accessibility of one or more amino acid residues in
a native
Fc polypeptide;
(b) selecting at least one solvent-exposed amino acid residue; and
(c) mutating the at least one selected solvent-exposed amino acid residue
to a
negatively charged amino acid residue.
[0051] In at least one embodiment, solvent accessibility of a candidate amino
acid is
determined by determining the average number of neighbouring atoms (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.
[0052] In at least one embodiment, the native Fc polypeptide is a native IgA
Fc
polypeptide. In at least one embodiment, the native IgA Fc polypeptide has the
sequence
of SEQ ID NO:47. In at least one embodiment, selecting the solvent-exposed
amino acid
residue for mutation to a negatively charged amino acid residue comprises
selecting an
asparagine (Asn, N) or glutamine (Gln, Q) residue for mutation to an aspartic
acid (Asp,
D) or glutamic acid (Glu, E) residue, respectively. In at least one
embodiment, at least
one of asparagine-9, asparagine-84 or asparagine-131 of SEQ ID NO:47 is
selected for
mutation to aspartic acid. In at least one embodiment, at least one of
glutamine-175 or
glutamine-195 of SEQ ID NO:47 is selected for mutation to glutamic acid.
[0053] In a further aspect, the invention provides a method of producing a Fc
variant
polypeptide, the method comprising:
(a) selecting a first amino acid and a second amino acid in a native Fc
polypeptide,
the second amino acid being within a predetermined distance from the first
amino acid,
wherein the first amino acid and the second amino acid are free from
involvement in
native disulfide bonding; and
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(b) mutating the first amino acid and the second amino acid
respectively to a first
cysteine residue and a second cysteine residue, such that a disulfide bond is
formable
between the first cysteine residue and the second cysteine residue.
[0054] Selection of de novo intrachain disulfide bonds can be carried out by
manual
inspection of a model of the molecule for disulfide bonds that are expected to
stabilize
the tertiary structure of the protein, for example, by tethering beta strands
in the Fc
polypeptide together, or by other means known in the art.
[0055] In at least one embodiment, the native Fc polypeptide is a native IgA
Fc
polypeptide. In at least one embodiment, the native IgA Fc polypeptide has the
sequence
SEQ ID NO:47. In at least one embodiment, the first amino acid is glycine-196
(Gly, G)
and the second amino acid is arginine-219 (Arg, R). In at least one
embodiment,
glycine-196 and arginine-219 are each mutated to cysteine (Cys, C) residues.
In at least
one embodiment, a disulfide bond forms between cysteine-196 and cysteine-219.
In at
least one embodiment, the predetermined distance is less than 5 A.
[0056] Without being bound by theory, it is contemplated that mutating the
first and
second amino acids to cysteine residues to form the disulfide bond stabilizes
the tertiary
structure of the Fc polypeptide. In at least one embodiment, such
stabilization can be
brought about by connecting at least two beta strands within the Fc
polypeptide together.
[0057] In at least one embodiment, the Fc variant polypeptide exhibits
enhanced
accumulation when expressed in a plant cell compared to accumulation of the
native Fc
polypeptide when expressed in the plant cell. In at least one embodiment, the
plant cell
is a cell of a Nicotiana plant or a Lactuca plant. In at least one embodiment,
the plant cell
is a cell of a Nicotiana benthamiana plant or a Nicotiana tabacum plant. In at
least one
embodiment, the Fc variant polypeptide exhibits at least a 3-fold increase in
accumulation compared to accumulation of the native Fc polypeptide in the
plant cell. In
at least one embodiment, the Fc variant polypeptide exhibits up to about a 22-
fold
increase in accumulation compared to accumulation of the native Fc polypeptide
in the
plant cell. In at least one embodiment, the Fc variant polypeptide exhibits
about a 22-fold
increase in accumulation compared to accumulation of the native Fc polypeptide
in the
plant cell.
[0058] It is also contemplated that the Fc variant polypeptide can act as a
stabilization
partner when fused to a variable domain of an antibody or to another bioactive
moiety,
so as to enhance accumulation and recombinant production of the fusion
protein. Thus,
a further aspect of the invention provides an Fc variant fusion polypeptide
including an
Fc variant polypeptide as described herein fused to a bioactive moiety. In at
least one
embodiment, the bioactive moiety is a protein, including but not limited to an
enzyme, a
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cytokine, an antigen, an antibody, an antibody fragment, a polypeptide, a
signalling
molecule, a receptor, or a ligand. Due to 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
5 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
10 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.
[0059] In at least one embodiment, the bioactive moiety is a variable domain
of an
antibody. In at least one embodiment, the bioactive moiety is a single domain
antibody.
In at least one embodiment, the bioactive moiety is a VHH polypeptide as
described
15 herein. In at least one embodiment, accumulation of a Fc variant fusion
polypeptide
containing a Fc variant polypeptide fused to a VHH polypeptide as described
herein,
when expressed in a plant cell, is enhanced up to about 16-fold compared to
accumulation of a VHH-Fc fusion polypeptide containing a native Fe polypeptide
fused to
a VHH polypeptide as described herein.
[0060] In another aspect, the present invention provides a VHH polypeptide
comprising a
first complementarity determining region (CDR1), a second complementarity
determining
region (CDR2) and a third complementarity determining region (CDR3), wherein:
(i) the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid
sequence identity to SEQ ID NO:4, the CDR2 has at least 80%, 85%, 90%, 95%,
97%,
99% or 100% amino acid sequence identity to SEQ ID NO:5 and the CDR3 has at
least
80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO:6;
(ii) the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid
sequence identity to SEQ ID NO:8, the CDR2 has at least 80%, 85%, 90%, 95%,
97%,
99% or 100% amino acid sequence identity to SEQ ID NO:9 and the CDR3 has at
least
80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO:10;
(iii) the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid
sequence identity to SEQ ID NO:12, the CDR2 has at least 80%, 85%, 90%, 95%,
97%,
99% or 100% amino acid sequence identity to SEQ ID NO:13 and the CDR3 has at
least
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80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO:14;
(iv) the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid
sequence identity to SEQ ID NO:16, the CDR2 has at least 80%, 85%, 90%, 95%,
97%,
99% or 100% amino acid sequence identity to SEQ ID NO:17 and the CDR3 has at
least
80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO:18;
(v) the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid
sequence identity to SEQ ID NO:20, the CDR2 has at least 80%, 85%, 90%, 95%,
97%,
99% or 100% amino acid sequence identity to SEQ ID NO:21 and the CDR3 has at
least
80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO:22;
(vi) the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid
sequence identity to SEQ ID NO:24, the CDR2 has at least 80%, 85%, 90%, 95%,
97%,
99% or 100% amino acid sequence identity to SEQ ID NO:25 and the CDR3 has at
least
80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO:26;
(vii) the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid
sequence identity to SEQ ID NO:28, the CDR2 has at least 80%, 85%, 90%, 95%,
97%,
99% or 100% amino acid sequence identity to SEQ ID NO:29 and the CDR3 has at
least
80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO :30;
(viii) the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid
sequence identity to SEQ ID NO:32, the CDR2 has at least 80%, 85%, 90%, 95%,
97%,
99% or 100% amino acid sequence identity to SEQ ID NO:33 and the CDR3 has at
least
80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO:34;
(ix) the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid
sequence identity to SEQ ID NO:36, the CDR2 has at least 80%, 85%, 90%, 95%,
97%,
99% or 100% amino acid sequence identity to SEQ ID NO:37 and the CDR3 has at
least
80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO :38;
(x) the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid
sequence identity to SEQ ID NO:40, the CDR2 has at least 80%, 85%, 90%, 95%,
97%,
99% or 100% amino acid sequence identity to SEQ ID NO:41 and the CDR3 has at
least
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80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO:42; or
(xi) the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100%
amino acid
sequence identity to SEQ ID NO:44, the CDR2 has at least 80%, 85%, 90%, 95%,
97%,
99% or 100% amino acid sequence identity to SEQ ID NO:45 and the CDR3 has at
least
80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO:46.
[0061] 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 at least one embodiment of the VHH polypeptide:
(i) the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100%
amino acid
sequence identity to SEQ ID NO:4, the CDR2 has at least 80%, 85%, 90%, 95%,
97%,
99% or 100% amino acid sequence identity to SEQ ID NO:5 and the CDR3 has at
least
80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO:6;
(iii) the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100%
amino acid
sequence identity to SEQ ID NO:12, the CDR2 has at least 80%, 85%, 90%, 95%,
97%,
99% or 100% amino acid sequence identity to SEQ ID NO:13 and the CDR3 has at
least
80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO:14;
(ix) the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid
sequence identity to SEQ ID NO:36, the CDR2 has at least 80%, 85%, 90%, 95%,
97%,
99% or 100% amino acid sequence identity to SEQ ID NO:37 and the CDR3 has at
least
80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO:38;
(x) the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid
sequence identity to SEQ ID NO:40, the CDR2 has at least 80%, 85%, 90%, 95%,
97%,
99% or 100% amino acid sequence identity to SEQ ID NO:41 and the CDR3 has at
least
80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO:42; or
(xi) the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid
sequence identity to SEQ ID NO:44, the CDR2 has at least 80%, 85%, 90%, 95%,
97%,
99% or 100% amino acid sequence identity to SEQ ID NO:45 and the CDR3 has at
least
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80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO:46.
[0062] In at least one embodiment, the VHH polypeptide has at least 80%, 85%,
90%,
95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:3, SEQ ID
NO:7,
SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:27, SEQ ID
NO:31, SEQ ID NO:35, SEQ ID NO:39 or SEQ ID NO:43. In at least one embodiment,
the VHH polypeptide has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino
acid
sequence identity to SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:35, SEQ ID NO:39 or
SEQ ID NO:43.
[0063] In at least one embodiment, the VHH polypeptide specifically binds to
intimin on
an Escherichia co//cell. In at least one embodiment, the VHH polypeptide binds
to an
epitope comprised in the C-terminal 277 residues of intimin. In at least one
embodiment,
the VHH polypeptide specifically binds to an epitope having at least 80%, 85%,
90%,
95%, 97%, 99% or 100% amino acid sequence identity to an epitope sequence
which is
a sub-sequence of SEQ ID NO:2. In at least one embodiment, the VHH polypeptide
prevents binding of intimin on the E. coil cell to an epithelial cell. In at
least one
embodiment, the epithelial cell is from the gastrointestinal tract of a
mammal. In at least
one embodiment, the mammal is bovine.
[0064] In another aspect, the present invention provides an antibody or
antigen binding
fragment thereof. In at least one embodiment, the antibody or antigen binding
fragment
thereof comprises an Fc variant fusion polypeptide as described herein
comprising a
variable domain of an antibody fused to an Fc variant polypeptide as described
herein. In
at least one embodiment, the variable domain of the antibody is a VHH
polypeptide as
described herein. In at least one embodiment, the antibody or antigen binding
fragment
thereof comprises a VHH polypeptide as described herein. In at least one
embodiment,
the antibody or antigen binding fragment thereof comprises a VHH-Fc fusion
polypeptide
comprising a VHH polypeptide as described herein fused to an Fc polypeptide.
In at least
one embodiment, the Fc polypeptide is a native Fc polypeptide. In at least one
embodiment, the Fc polypeptide is an Fc variant polypeptide as described
herein. In at
least one embodiment, the antibody or antigen binding fragment thereof
specifically
binds to intimin on an Escherichia co//cell.
[0065] In at least one embodiment, the antibody is a chimeric IgA antibody
comprising a
VHH-Fc fusion polypeptide in which a VHH polypeptide as described herein is
fused to an
IgA Fc polypeptide. In at least one embodiment, the IgA Fc polypeptide is a
bovine IgA
Fc polypeptide. In at least one embodiment, the Fc polypeptide is a native
bovine IgA Fc
polypeptide. In at least one embodiment, the Fc polypeptide is a bovine IgA Fc
variant
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polypeptide as described herein. In at least one embodiment, the antibody is a
chimeric
IgA antibody comprising an Fc variant fusion polypeptide as described herein
comprising
a variable domain of an antibody fused to an IgA Fc variant polypeptide as
described
herein. In at least one embodiment, the IgA Fc variant polypeptide is a bovine
IgA Fc
variant polypeptide as described herein. In at least one embodiment, the
variable domain
of the antibody is a VHH polypeptide as described herein. The skilled person
will
recognize that the present antibody or antigen binding fragment thereof could
be
adapted for use in other animals and thus appreciate that the variable domain
of an
antibody or VHH polypeptide may be fused to an Fc polypeptide or Fc variant
polypeptide
suitable for use in other animals.
[0066] Structurally, native sIgA antibodies include IgA subunits comprising
two heavy
chains, each including three constant domains (CH1, CH2 and CH3) and a
variable
domain (VH), and two light chains, each containing a constant domain (CL) and
a variable
domain (VL). The CL domains of the light chains are each bound to the CH1
domains of
the heavy chains by disulfide bonds. Each IgA subunit thus includes a Fc
region,
including the CH2 and CH3 constant domains of the heavy chains, and two
antigen-
binding Fab regions, including the variable domains (VH and VL) of the heavy
and light
chains and the CL and CH1 constant domains. Two such IgA units are linked at
the ends
of their respective Fc regions by a 15-kDa joining chain (JC) to form an IgA
dimer. A 70-
kDa secretory component (SC) coils around the Fc regions of both IgA subunits.
[0067] In contrast, in at least one embodiment, the present chimeric IgA
antibody
includes two VHH-Fc fusion polypeptides in place of the two heavy chains and
two light
chains. Although the VHH-Fc fusion polypeptide lacks the light chains and CH1
domains
found in native mammalian sIgA, assembly with the joining chain (JC) and
secretory
component (SC) subunits is directed specifically via disulfide bond formation
with the IgA
Fc region. Therefore, it is contemplated that the present VHH-Fc fusion
polypeptide forms
an sIgA complex with the JC and SC subunits. Thus, in at least one embodiment,
the
chimeric sIgA antibody comprises four VHH-Fc fusion polypeptides, one SC
subunit, and
one JC subunit.
[0068] Another aspect of the invention provides a nucleic acid encoding a VHH
polypeptide as described herein, an Fc variant polypeptide as described
herein, a Fc
variant fusion polypeptide as described herein, or a VHH-Fc fusion polypeptide
as
described herein. In at least one embodiment, the nucleic acid further
comprises a
chloroplast targeting signal sequence or an endoplasmic reticulum targeting
signal
sequence. In at least one embodiment, the chloroplast targeting signal
sequence is a
stroma targeting signal sequence or a thylakoid targeting signal sequence. In
at least
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one embodiment, the thylakoid targeting signal sequence is a Sec signaling
sequence. In
at least one embodiment, the thylakoid targeting signal sequence is a Tat
signaling
sequence.
[0069] In another aspect, the present invention provides an expression vector
5 comprising a nucleic acid as described herein. A further aspect of the
invention provides
a host cell comprising an expression vector as described herein. In at least
one
embodiment, the host cell is a bacterial cell. In at least one embodiment, the
bacterial
cell is Agrobacterium tumefaciens. In at least one embodiment, the host cell
is a plant
cell. In at least one embodiment, the plant cell is a Nicotiana plant cell. In
at least one
10 embodiment, the plant cell is a Nicotiana benthamiana plant cell or a
Nicotiana tabacum
plant cell. In at least one embodiment, the plant cell is a Lactuca plant
cell.
[0070] Yet another aspect of the invention provides a non-viable harvested
plant
material comprising a host cell as described herein. In at least one
embodiment, the non-
viable plant harvested material comprises a leaf or a stem. In another aspect,
the
15 invention provides a non-viable edible product comprising a host cell as
described
herein. In at least one embodiment, the non-viable edible product comprises a
leaf or a
stem. A further aspect of the invention provides a tobacco product comprising
a host cell
as described herein. In at least one embodiment, the tobacco product is cut,
shredded,
powdered, loose, ground, granulated, or extruded. In another aspect, the
invention
20 provides an animal feed comprising a host cell as described herein.
[0071] A further aspect of the invention provides a method of producing a VHH
polypeptide as described herein, an Fc variant polypeptide as described
herein, an Fc
variant fusion polypeptide as described herein, a VHH-Fc fusion polypeptide as
described
herein, or an assembled chimeric sIgA antibody comprising an Fe variant
polypeptide as
described herein or a VH H - Fc fusion polypeptide as described herein, the
method
comprising transforming a host cell as described herein with an expression
vector as
described herein including a nucleic acid as described herein. In at least one
embodiment, the host cell is a plant cell. In at least 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,
as will be understood by the skilled person.
[0072] In at least one embodiment, the expression vector is delivered through
Agrobacterium-mediated plant transformation. In such embodiments,
Agrobacterium
strains are transformed with a plant-optimized expression vector comprising a
nucleic
acid as described herein. In at least one embodiment of a method of producing
an
assembled chimeric sIgA antibody comprising an Fc variant polypeptide as
described
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herein or a VHH-Fc fusion polypeptide as described herein, transforming the
host cell
with the nucleic acid molecule comprises preparing Agrobacterium strain
cultures
comprising a VHH-Fc fusion polypeptide as described herein or a Fc variant
fusion
polypeptide as described herein, an SC subunit, and a JC subunit at optical
densities
(OD) of about 0.57, 0.14, and 0.14, respectively, for infiltration in the
plant.
[0073] Plant leaves are then co-infiltrated with transformed strains. In at
least one
embodiment, the plant is harvested after infiltration. In at least one
embodiment, the
plant is harvested more than 3 days post infiltration (dpi). In at least one
embodiment,
the plant is harvested from between about 4 dpi to about 12 dpi. In at least
one
embodiment, the plant is harvested at about 12 dpi. In at least one
embodiment, the
plant is harvested at a stage of harvest in which accumulation of the VHH
polypeptide, Fc
variant polypeptide, VHH-Fc fusion polypeptide, Fc variant fusion polypeptide
or
assembled chimeric sIgA antibody in the plant is maximal.
[0074] An additional aspect of the invention provides a method of enhancing
accumulation of a recombinant protein in a plant cell as described herein, the
method
comprising transforming the plant cell with a recombinant expression vector
comprising a
nucleic acid encoding a Fc variant fusion polypeptide as described herein
comprising a
bioactive moiety as described herein. A further aspect of the invention
provides a method
of producing a recombinant protein in a plant or portion thereof, the method
comprising
transforming the plant or portion thereof with a recombinant expression vector
comprising a nucleic acid encoding a Fc variant fusion polypeptide as
described herein
comprising a bioactive moiety as described herein. In at least one embodiment,
the plant
is transiently transformed. In at least one embodiment, the plant is stably
transformed. In
at least one embodiment, the recombinant protein is a recombinant antibody and
the
bioactive moiety is a variable domain of the antibody. In at least one
embodiment, the
nucleic acid further comprises an endoplasmic reticulum targeting signal
sequence. In at
least one embodiment, the nucleic acid further comprises a chloroplast
targeting signal
sequence. In at least one embodiment, the nucleic acid further comprises a Sec
signaling sequence. In at least one embodiment, the variable domain of the
antibody is a
VHH polypeptide as described herein. In at least one embodiment, the antibody
is an IgA
antibody. In at least one embodiment, the antibody is a chimeric IgA antibody.
In at least
one embodiment, the antibody specifically binds to intimin on an Escherichia
coli cell.
[0075] In another aspect, the invention provides a method of enhancing
expression of a
recombinant antibody in a plant cell, the method comprising transforming the
plant cell
with a recombinant expression vector comprising a nucleic acid encoding a
variable
domain of the antibody fused to a Fe polypeptide or to an Fc variant
polypeptide as
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described herein and further encoding a chloroplast targeting signal sequence.
A further
aspect provides a method of producing a recombinant antibody in a plant or a
portion
thereof, the method comprising transforming the plant or portion thereof with
a
recombinant expression vector comprising a nucleic acid encoding a variable
domain of
the antibody fused to an Fc polypeptide or to an Fc variant polypeptide as
described
herein and further encoding a chloroplast targeting signal sequence. In at
least one
embodiment, the plant is transiently transformed. In at least one embodiment,
the plant is
stably transformed. In at least one embodiment, the chloroplast targeting
signal
sequence is a stroma targeting signal sequence, a Sec signaling sequence or a
Tat
signaling sequence. In at least one embodiment, the chloroplast targeting
signal
sequence is a Sec signaling sequence. In at least one embodiment, the variable
domain
of the antibody is a VHH polypeptide as described herein. In at least one
embodiment,
the antibody is an IgA antibody. In at least one embodiment, the antibody is a
chimeric
IgA antibody. In at least one embodiment, the antibody specifically binds to
intimin on an
Escherichia coil cell.
[0076] Without being bound by theory, it is contemplated that including a
chloroplast
targeting signal and specifically a thylakoid targeting signal, such as a Sec
signalling
sequence or a Tat signalling sequence, in the expression vector will target
the expressed
protein to the thylakoid compartment of the chloroplast for folding. Because
folding of
both a VHH polypeptide and an Fc polypeptide requires the formation of
stabilizing intra-
chain disulfide bonds, and because the thylakoid compartment provides an
environment
in which such oxidative folding is facilitated, it was contemplated that the
expression and
accumulation of a VHH polypeptide as described herein, an Fc variant
polypeptide as
described herein, a Fc variant fusion polypeptide as described herein, or a
VHH-Fc fusion
polypeptide as described herein may be enhanced by targeting the expressed
polypeptides to the thylakoid lumen in transplastomic plants, compared to
targeting to
the endoplasmic reticulum in transgenic plants.
[0077] Another aspect of the invention provides a pharmaceutical composition
comprising an antibody, or antigen binding fragment thereof as described
herein, and a
pharmaceutically acceptable carrier. As used herein, the term "carrier" is
intended to
refer to a diluent, adjuvant, excipient, or vehicle with which an antibody, or
antigen
binding fragment thereof as described herein can be administered to an animal
in need
thereof. As used herein, the term "pharmaceutically acceptable" is intended to
refer to
carriers and compositions containing such carriers that are tolerable and do
not typically
produce untoward reactions to a subject being treated with or exposed to such
carriers
and compositions. Preferably, as used herein, the term "pharmaceutically
acceptable"
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23
means approved by a regulatory agency of the federal or a state government for
use in
pharmaceutical applications. Such pharmaceutically acceptable carriers are
well known
in the art and would be readily identified and used by the skilled person.
[0078] In another aspect, the invention provides use of an antibody or antigen
binding
fragment thereof as described herein, for preventing or reducing Escherichia
co//cell
colonization of the gastrointestinal tract of a mammal. Yet another aspect of
the invention
provides use of an antibody or antigen binding fragment thereof as described
herein, in
preparation of a medicament for preventing or reducing E. coli cell
colonization of the
gastrointestinal tract of a mammal. Another aspect of the invention provides a
method of
preventing or reducing colonization of E. coli in the gastrointestinal tract
of a mammal,
comprising administering to the mammal an antibody or antigen binding fragment
thereof
as described herein. In at least one embodiment, administering the polypeptide
to the
mammal comprises causing the mammal to ingest plant material from a plant as
described herein that produces the polypeptide. In at least one embodiment,
the
polypeptide or the plant material is for oral administration. In at least one
embodiment,
the polypeptide or plant material is for rectal administration.
[0079] In another aspect, the invention provides use of an antibody or an
antigen binding
fragment thereof as described herein for neutralizing the ability of an
Escherichia coil cell
to bind to a mammalian gastrointestinal epithelial cell. Another aspect of the
invention
provides use of an antibody or antigen binding fragment thereof as described
herein, in
preparation of a medicament for neutralizing the ability of an E. coli cell to
bind to a
mammalian gastrointestinal epithelial cell. Another aspect of the invention
provides a
method of neutralizing the ability of an E. coli cell to bind to a mammalian
gastrointestinal
epithelial cell, comprising exposing the E. coli cell to an antibody or
antigen binding
fragment thereof as described herein.
[0080] In at least one embodiment, the E. co//cell is a Shiga toxin-producing
E. coli
(STEC) cell or a cell of an enterohemorrhagic E. coli (EHEC) strain. In at
least one
embodiment, the E. coli cell is a cell of strain 026:H11, strain 0111:Hnm,
strain
0145:Hnm, or strain 0157:H7. In at least one embodiment, the antibody or an
antigen
binding fragment thereof as described herein thus neutralizes the capacity of
different E
coil strains to bind to a host's cells thereby conferring cross-serotype
inhibition of
bacterial adhesion crucial to pathogenicity in a host system.
[0081] Another aspect of the invention provides a method of detecting the
presence of
E. co//in a sample, comprising:
(a) contacting the sample with a VHH polypeptide as described herein or a
VHH-Fc
fusion polypeptide as described herein, and
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(b) detecting binding between intimin and the VHH polypeptide
or a VHH-Fc fusion
polypeptide.
[0082] In still another aspect, the invention provides use of a VHH
polypeptide as
described herein or a VHH-Fc fusion polypeptide as described herein for
detecting the
presence of E. co//in a sample. In another aspect, the invention provides a
diagnostic kit
for detecting the presence of E. coli in a sample comprising a VHH polypeptide
as
described herein or a VHH-Fc fusion polypeptide as described herein. The
presence of E.
coil may be confirmed by Western blotting analysis, ELISA, or any of the
various antigen-
antibody detection methods known in the art. In at least one embodiment, the
sample is
a food sample, environmental sample, or a sample from an animal or
microorganism. In
at least 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.
Definitions
[0083] 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.
[0084] As used herein, the terms "about" or "approximately" as applied to a
numerical
value or range of values, including but not limited to a measurable value such
as an
amount, a temporal duration, and the like, are intended to mean that the
recited values
can vary within an acceptable degree of error for the quantity measured given
the nature
or precision of the measurements, such that the variation is considered in the
art as
equivalent to the recited values and provides the same function or result. For
example,
the degree of error can be indicated by the number of significant figures
provided for the
measurement, as is understood in the art, and includes but is not limited to a
variation of
1 in the most precise significant figure reported for the measurement. Typical
exemplary degrees of error are within 20 percent ( /0), preferably within 10%,
and more
preferably within 5% of a given value or range of values, or within the
experimental error
of the indicated value (e.g. within the 95% confidence interval for the mean).
Alternatively, and particularly in biological systems, the terms "about" and
"approximately" can mean values that are within an order of magnitude,
preferably within
5-fold and more preferably within 2-fold of a given value. Numerical
quantities given
herein are approximate unless stated otherwise, meaning that the term "about"
or
"approximately" can be inferred when not expressly stated.
[0085] As used herein, the term "substantially" refers to the complete or
nearly complete
extent or degree of an action, characteristic, property, state, structure,
item, or result, or
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of a lack thereof. For example, an object that is "substantially" aligned
would mean that
the object is either completely aligned or nearly completely aligned. For an
additional
example, a composition that is "substantially free of" a material would either
completely
lack that material, or so nearly completely lack that material that the effect
would be the
5 same as if it completely lacked that material. In other words, a
composition that is
"substantially free or an ingredient or element may still actually contain
such an
ingredient or element as long as there is no measurable effect thereof. The
exact
allowable degree of deviation from absolute completeness may in some cases
depend
on the specific context. However, generally speaking, the nearness of
completion will be
10 so as to have the same overall result as if absolute and total
completion were obtained.
[0086] As used herein, unless otherwise required by context, the terms "a" and
"an" are
intended to mean "at least one" and include both singular and plural. Any
examples
following the term "for example" or "e.g." are not meant to be limiting or
exhaustive.
[0087] As used herein, the terms "comprises", "comprising", "include",
"includes",
15 "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.
[0088] 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,
20 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.
25 [0089] As used herein, the terms "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, including but not
limited to
deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
[0090] As used herein, the term "variant" when used in reference to a
polynucleotide is
intended to refer to a polynucleotide which differs in its nucleotide sequence
from the
sequence of a reference polynucleotide to which the variant is being compared
by one or
more nucleotide residues. The differences between the sequence of the variant
and the
sequence of the reference polynucleotide, also referred to herein as
variations or
mutations, can include substitution of one or more nucleotide residues with
different
nucleotide residues, insertion of additional nucleotide residues or deletion
of nucleotide
residues. In certain embodiments, a variant can differ from a reference
polynucleotide by
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26
substitution of one or more nucleotide residues with replacement nucleotide
residues
which do not alter the open reading frame(s) of the polynucleotide or the
amino acid
sequence of any protein(s) encoded by the polynucleotide.
[0091] As used herein, the term "variant" when used in reference to a
polypeptide is
intended to refer to a polypeptide which differs in its amino acid sequence
from the
sequence of a reference polypeptide to which the variant is being compared by
one or
more amino acid residues. The differences between the sequence of the variant
and the
sequence of the reference polypeptide can include substitution of one or more
amino
acid residues with different amino acid residues, insertion of additional
amino acid
residues or deletion of amino acid residues. In certain embodiments, a variant
can differ
from a reference polypeptide by conservative substitution of one or more amino
acid
residues with replacement amino acid residues which may have similar
properties,
including but not limited to charge, size and hydrophilicity, to the amino
acid residues
which the new residues replace. In certain embodiments, variants may
completely or
partially retain one or more biological functions of the reference
polypeptide. In certain
embodiments, variants may not retain one or more biological functions of the
reference
polypeptide.
[0092] As used herein, the term "percent identity" or " /c, identity" when
used in reference
to the sequence of a polypeptide or a polynucleotide is intended to mean the
percentage
of the total number of amino acid or nucleotide residues, respectively, in the
sequence
which are identical to those at the corresponding position of a reference
polypeptide or
polynucleotide sequence. In at least one embodiment, when the length of the
variant
sequence and the length of the reference sequence are not identical, percent
identity
can be calculated based on the total number of residues in the variant
sequence or
based on the total number or residues in the reference sequence. Percent
identity can
be measured by various local or global sequence alignment algorithms well
known in the
art, including but not limited to the Smith-Waterman algorithm and the
Needleman-
Wunsch algorithm. Tools using these or other suitable algorithms include but
are not
limited to BLAST (Basic Local Alignment Search Tool) and other such tools well
known
in the art.
[0093] In at least one embodiment, a variant polynucleotide sequence can
hybridize to a
polyribonucleotide or polydeoxyribonucleotide as described herein under at
least
moderately stringent conditions. By "at least moderately stringent
hybridization
conditions" it is meant that conditions are selected which promote selective
hybridization
between two complementary nucleic acid molecules in solution. Hybridization
may occur
to all or a portion of a nucleic acid sequence molecule. The hybridizing
portion is typically
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27
at least 15 (e.g. 20, 25, 30, 40 or 50) nucleotides in length. Those skilled
in the art will
recognize that the stability of a nucleic acid duplex, or hybrid, is
determined by the
melting temperature (Tm), which in sodium-containing buffers is a function of
the sodium
ion concentration ([Na.-1) and temperature (Tm = 81.5 C - 16.6 (Logic [Nal) +
0.41(%(G+C) - 600/1), where %G+C is the percentage of cytosine and guanine
nucleotides in the nucleic acid andl is the length of the nucleic acid in base
pairs, or
similar equation). Accordingly, the parameters in the wash conditions that
determine
hybrid stability are sodium ion concentration and temperature. In order to
identify
molecules that are similar, but not identical, to a known nucleic acid
molecule, a 1%
mismatch may be assumed to result in about a 1 C decrease in Tm. For example,
if
nucleic acid molecules are sought that have a >95% identity, the final wash
temperature
may be reduced by about 5 C. Based on these considerations those skilled in
the art will
be able to readily select appropriate hybridization conditions.
[0094] In some embodiments, stringent hybridization conditions are selected.
By way of
example the following conditions may be employed to achieve stringent
hybridization:
hybridization at 5x sodium chloride/sodium citrate (SSC)/5x Denhardt's
solution/1.0%
sodium dodecylsulfate (SDS) at Tm - 5 C based on the above equation, followed
by a
wash of 0.2x SSC/0.1% SDS at 60 C. Moderately stringent hybridization
conditions
include a washing step in 3x SSC at 42 C. It is understood, however, that
equivalent
stringencies may be achieved using alternative buffers, salts and
temperatures.
Additional guidance regarding hybridization conditions may be found in:
Current
Protocols in Molecular Biology, John Wiley & Sons, N.Y., 2002, and in:
Sambrook et al.,
Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory Press,
2001.
[0095] As used herein, the term "single domain antibody" is intended to mean
an
immunoglobulin molecule consisting of only a single variable domain which
includes the
antigen binding site. In conventional immunoglobulins (i.e. four chain
antibodies), a
heavy chain variable domain (VH) and a light chain variable domain (VL) each
contain
three complementarity determining regions (CDRs) interconnected by framework
regions
(FRs). The hypervariable CDRs vary widely in sequence and are in direct
contact with
the antigen, providing the specificity of binding between a particular
antibody and its
antigen. The FRs, in contrast, are less variable in sequence and aid in
maintaining the
structure of the variable domains so that the CDRs are positioned for binding
to the
antigen. Thus, the VH and VL regions interact to form an antigen binding site
defined by a
total of six CDRs. In contrast, single domain antibodies are capable of
binding to an
epitope without an additional variable domain, with the antigen-binding site
formed by a
single VH/VHH, VNAR or VL domain. As such, the antigen binding site of a
single variable
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domain is generally formed by not more than three CDRs in conjunction with its
associated FRs. 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, VHH or VNAR
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.
[0096] The terms "VHH domains", "VH1-I domains", "VHH", or "VHH" refer to the
variable
domain of camelid "heavy chain antibodies" (that is, antibodies which do not
include a
light chain), and are used to distinguish these variable domains from the
heavy chain
variable domains (referred to as "VH" or "VH" domains) and the light chain
variable
domains (referred to "as "VL" or "VL" domains) present in conventional four
chain
antibodies. The term "VNAR" refers to the variable domain of single domain
antibodies
(IgNAR) found in sharks.
[0097] The terms "specifically binds" or "binds specifically" are 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.
[0098] As used herein, the term "neutralizes" or "neutralizing antibody" means
an
antibody that reduces or abolishes the biological activity (for example,
binding and/or
infectivity) of the target to which it binds.
[0099] As used herein, the term "Fc polypeptide" is intended to refer to the
polypeptide
found in the constant region of an antibody. The term "native Fc polypeptide"
is intended
to mean an Fc polypeptide having a sequence substantially identical to the
sequence of
an Fc polypeptide found in nature and lacking any artificially induced
mutations.
[0100] 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.
[0101] The term "expression vector" includes plasmid vectors, cosmid vectors,
phage
vectors, viral vectors, or any other vectors known to the skilled person.
Expression
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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).
[0102] As used herein, the term "chloroplast targeting signal sequence" is
intended to
mean a nucleotide sequence encoding a chloroplast targeting transit peptide,
such that
any expressed protein fused to the chloroplast targeting transit peptide would
be
targeted to the chloroplast. As used herein, the term "endoplasmic reticulum
targeting
signal sequence" is intended to mean a nucleotide sequence encoding an
endoplasmic
reticulum targeting signal peptide, such that any expressed protein fused to
the
endoplasmic reticulum targeting signal peptide would be targeted to the
endoplasmic
reticulum.
[0103] 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.
[0104] 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.
[0105] As used herein, the terms "transforming" or "transformation" refer 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 (i.e. integrated within the host
genome) and
stable, or the transfer may be non-heritable and transient.
[0106] The term "solvent accessibility", as used herein, refers to the degree
of exposure
of a given amino acid residue of a protein to the surrounding solvent. For
example, an
amino acid residue located near the surface of the protein would be more
accessible to
solvent than would an amino acid residue located within interior folds of the
protein.
There are a variety of methods known in the art used to measure such exposure,
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including determining the average number of neighbouring atoms per side chain
atom
(AvNAPSA) for a given amino acid residue.
[0107] Unless otherwise defined herein, scientific and technical terms used in
connection with the present disclosure have the meanings that are commonly
5 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.
EXAMPLES
10 [0108] Other features of the present invention will become apparent from
the following
non-limiting examples which illustrate, by way of example, the principles of
the invention.
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
15 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).
[0109] In the specific experiments discussed herein, for the ease of reference
and
20 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
25 be used to limit the scope of the present invention in any manner.
Example 1: Chimeric VHH-secretory IgA antibodies against E. coil intimin
Preparation and characterization of variable domains of came/id heavy chain
only
antibodies (VHHs)
[0110] Camelid variable domains of heavy chain only antibodies (VHHs)
recognizing the
30 Escherichia coil intimin protein were prepared and characterized as
described in detail in
Saberianfar et al, Frontiers in Plant Science (2019), 10: 270, herein
incorporated by
reference. Briefly, a fusion protein (MBP-Int277, SEQ ID NO:1) including the C-
terminal
277 residues (SEQ ID NO:2) of E. coli 0157:H7 strain EDL933 intimin y (intimin-
277)
fused C-terminally to maltose-binding protein (MBP) was expressed in E. coli
BL21
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(DE3) cells and used to immunize a male llama (Lama glama). The resulting
expressed
VHH genes were incorporated into a phage-displayed library and panned to
identify VHH
domains which specifically recognize intimin. The amino acid sequences of
identified
VHH domains (VHH-1 to VHH-10) are listed in Table 1, along with the amino acid
sequence of an additional VHH domain (VHH-fl) isolated from pooled peripheral
blood
lymphocytes of unimmunized alpacas, camels and llamas, which also binds to
intimin.
Complementarity determining regions CDR1, CDR2 and CDR3 of each VHH domain
were identified based on comparative analysis using the international
ImMunoGeneTics
(IMGT) database, as described, for example, in Lefranc et al, Developmental &
Comparative Immunology (2003), 27(1): 55-77, and are listed in Table 1.
Table 1: Amino acid sequences of VHH domains and complementarity determining
regions
Sequence
VHH Sequence Identifier
QVQLVESGGGLVQAGGSLRLTCTASGRIFNLTDMSWY
V H1
RQAPGEMRALVAAITRDGQTSYGDSVKGRFTISRDNA E ID NO
SQ
H :3
KNTVYLQMNSLKPEDTAVYLCNADHYTYGLGHTDYW
GQGTQVTVSS
CDR1 GRIFNLTD SEQ ID
NO:4
CDR2 ITRDGQT SEQ ID
NO:5
CDR3 NADHYTYGLGHTDY SEQ ID
NO:6
QVQLVESGGGLVQAGGSLRLTCTASGRIFNLTDMSWY
RQAPGEMRALVAAITRDGQTSYGDSVKGRFTISRDNA
VH H2 SEQ ID NO:7
RNTVYLQMNSLKPEDTAVYLCNADHYTYGLGHTEYW
GOGTOVTVSS
CDR1 GRIFNLTD SEQ ID
NO:8
CDR2 ITRDGQT SEQ ID
NO:9
CDR3 NADHYTYGLGHTEY SEQ ID
NO:10
QVKLEESGGGLVQPGGSLRLSCAASGSFFEIDEMGW
YRQAPGKMRELVAGFTSGGMTNYADSVKGRFTFSRD
VHH3 SEQ ID NO:11
NAKNTVYLQMNSLSPEDTATYLCNAEIRLSAGWGLTE
YWGQGTQVTVSS
CDR1 GSFFEIDE SEQ ID
NO:12
CDR2 FTSGGMT SEQ ID
NO:13
CDR3 NAEIRLSAGWGLTEY SEQ ID
NO:14
QVKLEESGGGLVQPGGSLRLSCAASGSFFEIDEMGW
YRQAPGKMRELVAGFTSGGMTKYADSVKGRFTFSRD
VH H4 SEQ ID NO:15
NAKNTVYLQMNNLSPEDTAVYLCNAEIRLSAGWGLTD
YWGQGTQVTVSS
CDR1 GSFFEIDE SEQ ID
NO:16
CDR2 FTSGGMT SEQ ID
NO:17
CDR3 NAEIRLSAGWGLTDY SEQ ID
NO:18
QVKLEESGGGLVQPGGSLRLSCAVSGTFFEIETMAWY
RQAPDKMRELVAIISDGDSTRYGDSVKGRFTISRDNAK
VHH5 SEQ ID
NO:19
NTAFLQMNSLKYEDTAVYLCNADIHLSAGWGLTDYWG
QGTQVTVSS
CDR1 GTFFEIET SEQ ID
NO:20
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Sequence
VHH Sequence Identifier
CDR2 ISDGDST
SEQ ID NO:21
CDR3 NADIHLSAGWGLTDY
SEQ ID NO:22
QVKLE ESGGGLVQPGGSLRLSCAVSGTFFE I ETMAWY
RQAPDKMRELVAIISDGDSTRYGDSVKGRFTISRDNAK
VHH6 SEQ ID NO:23
NTAFLQMNSLKYEDTAVYLCNADIHLSAGWGLTGYWG
QGTQVTVSS
CDR1 GTFFE I ET
SEQ ID NO:24
CDR2 ISDGDST
SEQ ID NO:25
CDR3 NADIHLSAGWGLTGY
SEQ ID NO:26
EVQLVESGGGLVQAGGSLTLTCTASG RI FNLTDMNWY
RC:MPG EM RALVAAITI NGHTSYG DSVKG RFTISRDNAK
VHH7 SEQ ID NO:27
NTVYLQMNSLKPEDTAVYLCNANHYTYGLGHTDYWG
RGTQVTVSS
CDR1 GRIFNLTD
SEQ ID NO:28
CDR2 ITINGHT
SEQ ID NO:29
CDR3 NANHYTYGLGHTDY
SEQ ID NO:30
EVOLVESGGGLVQAGGSLTLTCTASGPVFNLTDMNW
YRQAPGEMHALVAAITINGHTSYGDSVKGRFTISRDNA
VHH8 SEQ ID NO:31
KNIVYLQMNSLKPEDTAVYLCNANHYTYGLGHTDYW
GRGTQVTVSS
CDR1 GPVFNLTD
SEQ ID NO:32
CDR2 ITINGHT
SEQ ID NO:33
CDR3 NANHYTYGLGHTDY
SEQ ID NO:34
QVQLVESGGGLVQSGGSLRLSCAASGNFFEVDTMDW
YROAPGKMRELVAGSYSGGSTNYGDSVKGRFTISRD
VHH9 SEQ ID NO:35
NAKNTVYLQMNSLKPEDTAVYLCNAQIRLQRDFGLTD
YWGQGTQVTVSS
CDR1 GNFFEVDT
SEQ ID NO:36
CDR2 SYSGGST
SEQ ID NO:37
CDR3 NAQIRLQRDFGLTDY
SEQ ID NO:38
QVQLVESGGGLVQAGGSLRLSCVLSRSTFTENDMGW
V H1 YRQAPGKQRELVASISSSGSASYADSVKGRFTISRDN E ID NO
SQ
H 0
AKNTAYLQMNSLKPEDTAVYYCNAVVGWAGSIANPRR
:39
EYWGQGTQVTVSS
CDR1 RSTFTFND
SEQ ID NO:40
CDR2 ISSSGSA
SEQ ID NO:41
CDR3 NAVVGWAGSIANPRREY
SEQ ID NO:42
QVQLVESRGGSVQAGGSLRLSCAAPEWTGRQFCVA
WFRQSPGKVHELVARTHIDGFDTTYTDSVKGRFTISPD
VHHn SEQ ID NO:43
KAKSTVHLQMNDLKGGDTGNYYCRAKFANYCSDNWG
RLDDFPYWGQGTQVTVSS
CDR1 EWTGRQFC
SEQ ID NO:44
CDR2 THIDGFDT
SEQ ID NO:45
CDR3 RAKFANYCSDNWGRLDDFPY
SEQ ID NO:46
[0111] Monovalent binding affinity data for intimin of selected VHH sequences
based on
surface plasmon resonance (SPR) are shown in Table 2. No binding to MBP alone
was
observed. Based on these results, the skilled person would understand that at
least
these isolated VHH sequences bind specifically to intimin.
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Table 2: Monovalent affinities and kinetics of the interaction between VHHs
and
MBP-Int277 by SPR (pH 7.4, 25 C)
VHH ka (1/MS) kd (us) 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
VHHn 5.3x105 1.1x10-3 2.1
Preparation and characterization of chimeric VHH-secretory IgA antibodies
[0112] Chimeric antibodies containing the VHH domains VHH1, VHH3, VHH9 and
VHH10
fused to the Fc region of bovine immunoglobulin A (IgA) were prepared and
characterized as described in detail in Saberianfar et al, Frontiers in Plant
Science
(2019), 10: 270. Briefly, each VHH sequence was fused to a bovine IgA Fc
sequence
(SEQ ID NO:47, derived from GenBank accession no. ANN46383) and a construct
containing each fused VHH-Fc sequence was cloned into a plant expression
vector
including a PR1b signal peptide and a KDEL retrieval signal peptide to enable
targeting
of the fused peptide to the endoplasmic reticulum (ER), as described by
Pereira et al
(BMC Biotechnol (2014), 14: 59). The amino acid sequence of native bovine IgA
Fc is:
DSSSCCVPNC EPSLSVQPPA LEDLLLGSNA SLTCTLSGLK SAEGASFTWN
PIGGKIAVQG SPKRDSCGCY SVSSVLPGCA DPWNSGQTYS CSVIHPESKS
SLTATIKKDL GNTFRPQVHL LPPPSEELAL NELVTLTCLV RGESPKEVLV
RWLQGNQELP REKYLTWGPL PEAGQSVTTF AVTSVLRVDA EVWKQGDTFS
CMVGHEALPL AFTQKTIDRL AGKPTHVNVS VVMSEVDGVC Y
(SEQ ID NO:47)
and the corresponding encoding DNA sequence is:
gatagttcca gttgctgtgt cccaaattgt gagccatctc taagtgtcca gcctccagca
ctagaagatt tgcttctggg ttctaacgct tctctaactt gtacactgag tggactgaag
agtgcagagg gtgcatcatt tacatggaac cctacaggag gtaagacagc tgtacaagga
agtccaaaga gagattcctg tggatgttac agcgtctcat cagtcttacc aggttgtgct
gatccatgga actccggaca aacgttctcc tgctctgtaa ctcatcctga gtctaagtca
tcactcacag caacaatcaa gaaggacctg ggaaatacgt tccgtcctca agtgcattta
ctccctccac cttcagagga actcgcattg aatgagctcg taacacttac ctgcttggta
agaggtttca gccctaagga ggttttggtt aggtggcttc aaggtaatca ggagcttccc
agggaaaaat atttgacctg ggggcccctt ccggaagctg gccaatctgt tactactttt
gctgttactt ctgttcttcg agttgatgct gaagtttgga aacagggcga tacttttagc
tgcatggttg ggcacgaagu ucttuugutt guutttactc agaaaaccat agatcggtta
gccgggaaac cgacccacgt taatgtgtct gtggtgatgt ctgaagtgga tggcgtgtgc
tatgg
(SEQ ID NO:48)
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[0113] As an example, the DNA sequence (SEQ ID NO:49, below) encoding the VHH9-
Fc protein was prepared by fusing a DNA sequence encoding the VHH9 protein
(SEQ ID
NO:35) to the DNA sequence (SEQ ID NO:48) encoding the Fc protein.
caagtacagc tagtagagtc cggtggtgga ttagttcagt ctggtggatc tcttagactt
tcctgtgcag ctagtggaaa tttcttcgag gttgatacga tggattggta ccgtcaggca
ccaggaaaga tgagagaact tgttgctggt tcatacagtg gaggttctac aaactacggg
gattcagtta agggcaggtt tacaatctca agggataacg ctaaaaatac cgtctatctg
cagatgaata gcctcaaacc tgaagacacc gccgtgtatt tgtgcaatgc ccaaattcgg
ttgcaacgag actttgggtt gactgattat tgggggcaag gcactcaagt gactgtctct
agcgagatag ttccagttgc tgtgtcccaa attgtgagcc atctctaagt gtccagcctc
cagcactaga agatttgctt ctgggttcta acgcttctct aacttgtaca ctgagtggac
tgaagagtgc agagggtgca tcatttacat ggaaccctac aggaggtaag acagctgtac
aaggaagtcc aaagagagat tcctgtggat gttacagcgt ctcatcagtc ttaccaggtt
gtgctgatcc atggaactcc ggacaaacgt tctcctgctc tgtaactcat cctgagtcta
agtcatcact cacagcaaca atcaagaagg acctgggaaa tacgttccgt cctcaagtgc
atttactccc tccaccttca gaggaactcg cattgaatga gctcgtaaca cttacctgct
tggtaagagg tttcagccct aaggaggttt tggttaggtg gcttcaaggt aatcaggagc
ttcccaggga aaaatatttg acctgggggc cccttccgga agctggccaa tctgttacta
cttttgctgt tacttctgtt cttcgagttg atgctgaagt ttggaaacag ggcgatactt
ttagctgcat ggttgggcac gaagcccttc cgcttgcctt tactcagaaa accatagatc
ggttagccgg gaaaccgacc cacgttaatg tgtctgtggt gatgtctgaa gtggatggcg
tgtgctatgg
(SEQ ID NO:49)
[0114] The amino acid sequence of the VHH9-Fc protein is:
QVQLVESGGG LVQSGGSLRL SCAASGNFFE VDTMDWYRQA PGKMRELVAG
SYSGGSTNYG DSVKGRFTIS RDNAKNTVYL QMNSLKPEDT AVYLCNAQIR
LQRDFGLTDY WGQGTQVTVS SDSSSCCVPN CEPSLSVQPP ALEDLLLGSN
ASLTCTLSGL KSAEGASFTW NTTGGKTAVQ GSPKRDSCGC YSVSSVLPGC
ADPWNSGQTF SCSVTHPESK SSLTATIKKD LGNTFRPQVH LLPPPSEELA
LNELVTLTCL VRGFSPKEVL VRWLQGNQEL PREKYLTWGP LPEAGQSVTT
FAVTSVLRVD AEVWKQGDTF SCMVGHEALP LAFTQKTIDR LAGKPTHVNV
SVVMSEVDGV CY
(SEQ ID NO:50)
[0115] Nicotiana benthamiana leaves were infiltrated with an Agrobacterium
tumefaciens
strain transformed with an expression vector including DNA encoding either
VHH1-Fc,
VHH3-Fc, VHH9-Fc (SEQ ID NO:50) or VHH1O-Fc, in addition to A. tumefaciens
strains
each transformed with a vector encoding the bovine joining chain sequence (JC;
NCB!
accession no. NP 786967), the bovine secretory component sequence (SC; NCB!
accession no. NP 776568) or p19, a suppressor of gene silencing from Cymbidium
ringspot virus. At six days post-infiltration, expression of each of the VHH1,
VH1-13-Fc,
VHH9-Fc, VHH1O-Fc, JO and SC subunits was detected in the leaves.
[0116] For correct assembly of secretory IgA (sIgA) subunits into a hetero-
multimeric
protein complex and optimal accumulation, the nascent subunit polypeptides
should be
temporally and spatially coordinated in a 4:1:1 stoichiometric ratio of VHH-
Fc:SC:JC.
Optimization of the infiltration conditions required to obtain accumulation of
an
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assembled VHH-sIgA complex and purification and characterization of the
assembled
VHH-sIgA complex were carried out as described in detail in Saberianfar et al,
Frontiers
in Plant Science (2019), 10: 270. The results show that, while a range of
different optical
densities may be used, the Agrobacterium strains expressing various VHH-Fc
subunits,
5 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 up
to 12 days
post-infiltration.
[0117] Furthermore, it was confirmed, using procedures described in detail in
Saberianfar et al, Frontiers in Plant Science (2019), 10: 270, that the VHH-
Fc, SC and JC
10 subunits associate in vivo after expression in the plant leaves to form
a chimeric VHH-
sIgA complex which can bind intimin, including intimin on the surface of cells
of
Escherichia coli strains 026:H11, 0145:Hnm, 0111:Hnm and 0157:H7. It was also
confirmed, using procedures described in detail in Saberianfar et al,
Frontiers in Plant
Science (2019), 10: 270, that binding of VH H10-sIgA to E. co//strains
026:H11,
15 0111:Hnm and 0157:H7 could neutralize the ability of the bacteria to
adhere to epithelial
cells. A further experiment carried out using the conditions described in
Saberianfar et al,
Frontiers in Plant Science (2019), 10: 270 confirmed that both VHH10-sIgA and
the
VHH9-Fc subunit alone abrogated the adhesion of an alternative 0145 strain
obtained
from a different supplier (ATCC, C625) to HEp-2 cells and reduced the relative
20 fluorescence caused by adherent bacteria for to background levels. Thus,
both VHH10-
sIgA and VH H9- Fc were able to completely neutralize the alternative 0145
strain
(Figure 1). As described in detail in Saberianfar et al, Frontiers in Plant
Science (2019),
10: 270, E. coli strains 0157, 0111, 0145 and 026 group together based on
sequence
similarity of the C-terminal 277 residues of intimin.
25 Example 2: Bovine IgA Fc variants
Selection of mutation sites in bovine IgA Fc
[0118] The structure of bovine IgA Fc was predicted using the I-TASSER
(Iterative
Threading Assembly Refinement) method (Wu et al. (2008). Proteins 72:547-556;
Zhang
Y (2008). BMC Bioinformatics 9:40), using the publicly available crystal
structure of
30 human IgA (Research Collaboratory for Structural Bioinformatics Protein
Data Bank
(RCSB PDB) structure 11GA), with which the bovine IgA Fc shares 70% sequence
similarity, as a threading template. The resulting 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.
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[0119] 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 asparagine (Asn, N) and glutamine (Gln, Q) residues for
mutation to
their negatively charged counterparts, aspartic acid (Asp, D) and glutamic
acid (Glu, E)
respectively. Residues involved in the interaction of sIgA molecules with the
Fc a
receptor (FcaR), which may be involved in activation of the immune response,
were
excluded, as were residues expected to be involved in native glycosylation.
Three
asparagine residues (at positions 9, 84 and 131 of SEQ ID NO:47) and two
glutamine
residues (at positions 175 and 195 of SEQ ID NO:47) on the surface of the Fc
chain
were selected for mutation to aspartic acid and glutamic acid, respectively.
Modelling the
substitutions to the five selected residues predicted an increase in net
negative charge
from -5.30 to -10.29 at pH 7 with a corresponding change in pl from 5.39 to
4.85.
[0120] 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 (Schrodinger LLC (2010). The PyMOL molecular graphics
system.
Version, 1(5), 0). To retain the functionality of the native Fc, native
disulfide sites were
avoided. 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. For both domains, an intra-chain disulfide in the centre of the beta
sheet stabilizes
the tertiary structure. De novo disulfide candidates at residue positions 196
and 219 of
SEQ ID NO:47 were chosen based on neighboring proximity (under 5 A) for
tethering the
C-terminal end of a portion of the Fc structure referred to as "strand G" to
the N terminal
end of a portion of the Fc structure referred to as "strand F". Without being
bound by
theory, it was contemplated that this additional disulfide bond could act to
stabilize the Fc
protein by tethering the end of strand G to adjacent strand F, thus preventing
access by
proteolytic enzymes to vulnerable hydrophobic regions of the unstructured
tailpiece
leading out from the end of strand G.
Expression of Fe mutants in Nicotiana benthamiana leaf tissue
[0121] The DNA sequence (SEQ ID NO:48) encoding the native bovine IgA Fc amino
acid sequence (SEQ ID NO:47) was synthesized by Bio Basic Inc. (Markham, ON,
Canada). Individual mutations N9D, N84D, N131D, 0175E, 0195E and G196C/R219C
were then made using an in vitro single primer site-directed mutagenesis
method (Huang
et al, (2017). Methods Mol. Biol. 1498: 375-383). A multi-site-directed
mutagenesis
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method (Liang et al. (2012). Anal. Biochem. 427: 99-101) was used to combine
mutations. The mutant Fe sequences prepared are listed in Table 3.
Table 3: Mutant Fc amino acid sequences
Sequence
Mutant Sequence
Identifier
DSSSCCVPDC EPSLSVQPPA LEDLLLGSNA SLTCTLSGLK
SAEGASFTWN PTGGKTAVQG SPKRDSCGCY SVSSVLPGCA
DPWNSGQTFS CSVTHPESKS SLTATIKKDL GNTFRPQVHL
N9D LPPPSEELAL NELVTLTCLV RGFSPKEVLV RWLQGNQELP SEQID
NO:51
REKYLTWGPL PEAGQSVTTF AVTSVLRVDA EVWKQGDTFS
CMVGHEALPL AFTQKTIDRL AGKPTHVNVS VVMSEVDGVC
DSSSCCVPNC EPSLSVQPPA LEDLLLGSNA SLTCTLSGLK
SAEGASFTWN PTGGKTAVQG SPKRDSCGCY SVSSVLPGCA
DPWDSGQTFS CSVTHPESKS SLTATIKKDL GNTFRPQVHL
N84D LPPPSEELAL NELVTLTCLV RGFSPKEVLV RWLQGNQELP SEQID NO:52
REKYLTWGPL PEAGQSVTTF AVTSVLRVDA EVWKQGDTFS
CMVGHEALPL AFTQKTIDRL AGKPTHVNVS VVMSEVDGVC
DSSSCCVPNC EPSLSVQPPA LEDLLLGSNA SLTCTLSGLK
SAEGASFTWN PTGGKTAVQG SPKRDSCGCY SVSSVLPGCA
DPWNSGQTFS CSVTHPESKS SLTATIKKDL GNTFRPQVHL
N131D LPPPSEELAL DELVTLTCLV RGFSPKEVLV RWLQGNQELP SEQID NO:53
REKYLTWGPL PEAGQSVTTF AVTSVLRVDA EVWKQGDTFS
CMVGHEALPL AFTQKTIDRL AGKPTHVNVS VVMSEVDGVC
DSSSCCVPNC EPSLSVQPPA LEDLLLGSNA SLTCTLSGLK
SAEGASFTWN PTGGKTAVQG SPKRDSCGCY SVSSVLPGCA
DPWNSGQTFS CSVTHPESKS SLTATIKKDL GNTFRPQVHL
0175E LPPPSEELAL NELVTLTCLV RGFSPKEVLV RWLQGNQELP SEQID NO:54
REKYLTWGPL PEAGESVTTF AVTSVLRVDA EVWKQGDTFS
CMVGHEALPL AFTQKTIDRL AGKPTHVNVS VVMSEVDGVC
DSSSCCVPNC EPSLSVQPPA LEDLLLGSNA SLTCTLSGLK
SAEGASFTWN PTGGKTAVQG SPKRDSCGCY SVSSVLPGCA
DPWNSGQTFS CSVTHPESKS SLTATIKKDL GNTFRPQVHL
0195E LPPPSEELAL NELVTLTCLV RGFSPKEVLV RWLQGNQELP SEQID NO:55
REKYLTWGPL PEAGQSVTTF AVTSVLRVDA EVWKEGDTFS
CMVGHEALPL AFTQKTIDRL AGKPTHVNVS VVMSEVDGVC
DSSSCCVPNC EPSLSVQPPA LEDLLLGSNA SLTCTLSGLK
SAEGASFTWN PTGGKTAVQG SPKRDSCGCY SVSSVLPGCA
DPWNSGQTFS CSVTHPESKS SLTATIKKDL GNTFRPQVHL
G196C/
LPPPSEELAL NELVTLTCLV RGFSPKEVLV RWLQGNQELP SEQID NO:56
R219C
REKYLTWGPL PEAGQSVTTF AVTSVLRVDA EVWKQCDTFS
CMVGHEALPL AFTQKTIDCL AGKPTHVNVS VVMSEVDGVC
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Sequence
Mutant Sequence
Identifier
DSSSCCVPDC EPSLSVQPPA LEDLLLGSNA SLTCTLSGLK
SAEGASFTWN PTGGKTAVQG SPKRDSCGCY SVSSVLPGCA
N9D/ DPWDSGQTFS CSVTHPESKS SLTATIKKDL GNTFRPQVHL
N84D/ LPPPSEELAL DELVTLTCLV RGFSPKEVLV RWLQGNQELP SEQID NO:57
N131D REKYLTWGPL PEAGQSVTTF AVTSVLRVDA EVWKQGDTFS
CMVGHEALPL AFTQKTIDRL AGKPTHVNVS VVMSEVDGVC
DSSSCCVPDC EPSLSVQPPA LEDLLLGSNA SLTCTLSGLK
N9D/ SAEGASFTWN PTGGKTAVQG SPKRDSCGCY SVSSVLPGCA
N84D/ DPWDSGQTFS CSVTHPESKS SLTATIKKDL GNTFRPQVHL
N131D/ LPPPSEELAL DELVTLTCLV RGFSPKEVLV RWLQGNQELP SEQID NO:58
0175E/ REKYLTWGPL PEAGESVTTF AVTSVLRVDA EVWKEGDTFS
Q195E CMVGHEALPL AFTQKTIDRL AGKPTHVNVS VVMSEVDGVC
N9D/ DSSSCCVPDC EPSLSVQPPA LEDLLLGSNA SLTCTLSGLK
N840/ SAEGASFTWN PTGGKTAVQG SPKRDSCGCY SVSSVLPGCA
N131D/ DPWDSGQTFS CSVTHPESKS SLTATIKKDL GNTFRPQVHL
Q175E/
LPPPSEELAL DELVTLTCLV RGFSPKEVLV RWLQGNQELP SEQID NO:59
Q195E/
G196C/ REKYLTWGPL PEAGESVTTF AVTSVLRVDA EVWKECDTFS
R219C CMVGHEALPL AFTQKTIDCL AGKPTHVNVS VVMSEVDGVC
(5+1) Y
[0122] The DNA sequences encoding the mutant Fc chains are identified in Table
4.
Table 4: Mutant Fc DNA sequences
Sequence
Mutant Sequence
Identifier
gatagttcca gttgctgtgt cccagattgt gagccatctc
taagtgtcca gcctccagca ctagaagatt tgcttctggg
ttctaacgct tctctaactt gtacactgag tggactgaag
agtgcagagg gtgcatcatt tacatggaac cctacaggag
gtaagacagc tgtacaagga agtccaaaga gagattcctg
tggatgttac agcgtctcat cagtcttacc aggttgtgct
gatccatgga actccggaca aacgttctcc tgctctgtaa
ctcatcctga gtctaagtca tcactcacag caacaatcaa
gaaggacctg ggaaatacgt tccgtcctca agtgcattta
N9D ctccctccac cttcagagga actcgcattg aatgagctcg SEQID
NO:60
taacacttac ctgcttggta agaggtttca gccctaagga
ggttttggtt aggtggcttc aaggtaatca ggagcttccc
agggaaaaat atttgacctg ggggcccott ccggaagctg
gccaatctgt tactactttt gctgttactt ctgttcttcg
agttgatgct gaagtttgga aacagggcga tacttttagc
tgcatggttg ggcacgaagc ccttccgctt gcctttactc
agaaaaccat agatcggtta gccgggaaac cgacccacgt
taatgtgtct gtggtgatgt ctgaagtgga tggcgtgtgc
tatgg
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Sequence
Mutant Sequence
Wentifier
gatagttcca gttgctgtgt cccaaattgt gagccatctc
taagtgtcca gcctccagca ctagaagatt tgcttctggg
ttctaacgct tctctaactt gtacactgag tggactgaag
agtgcagagg gtgcatcatt tacatggaac cctacaggag
gtaagacagc tgtacaagga agtccaaaga gagattcctg
tqqatqttac agcqtctcat cagtottacc aqqttqtqct
gatccatggg actccggaca aacgttctcc tgctctgtaa
ctcatcctga gtctaagtca tcactcacag caacaatcaa
gaaggacctg ggaaatacgt tccgtcctca agtgcattta
N840 ctccctccac cttcagagga act cgcattg aatgagctcg SEQID
NO:61
taacacttac ctgottggta agaggtttca gccctaagga
ggttttggtt aggtggcttc aaggtaatca ggagcttccc
agggaaaaat atttgacctg ggggcccctt ccggaagctg
gccaatctgt tactactttt gctgttactt ctgttottcg
agttgatgct gaagtttgga aacagggcga tacttttagc
tgcatggttg ggcacgaagc ccttccgctt gcctttactc
agaaaaccat agatcggtta gccgggaaac cgacccacgt
taatgtgtct gtggtgatgt ctgaagtgga tggcgtgtgc
tatgg
gatagttcca gttgctgtgt cccaaattgt gagccatctc
taagtgtcca gcctccagca ctagaagatt tgottctggg
ttctaacgct tctctaactt qtacactgag tqqactqaaq
agtgcagagg gtgcatcatt tacatggaac cctacaggag
gtaagacagc tgtacaagga agtccaaaga gagattcctg
tggatgttac agcgtctcat cagtcttacc aggttgtgct
gatccatgga actccggaca aacgttctcc tgctctgtaa
ctcatcctga gtctaagtca tcactcacag caacaatcaa
gaaggacctg ggaaatacgt tccgtcctca agtgcattta
N131D ctocctccac cttcagagga actcgcattg gatgagctcg SEQID NO:62
taacacttac ctgcttggta agaggtttca gccctaagga
ggttttggtt aggtggcttc aaggtaatca ggagcttccc
agggaaaaat atttgacctg ggggcccctt ccggaagctg
gccaatctgt tactactttt gctgttactt ctgttcttcg
agttgatgct gaagtttgga aacagggcga tacttttagc
tgcatggttg ggcacgaagc ccttccgctt gcctttactc
agaaaaccat agatcggtta gccgggaaac cgacccacgt
taatgtgtct gtggtgatgt ctgaagtgga tggcgtgtgc
tatgg
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Sequence
Mutant Sequence
Wentifier
gatagttcca gttgctgtgt cccaaattgt gagccatctc
taagtgtcca gcctccagca ctagaagatt tgcttctggg
ttctaacgct tctctaactt gtacactgag tggactgaag
agtgcagagg gtgcatcatt tacatggaac cctacaggag
gtaagacagc tgtacaagga agtccaaaga gagattcctg
tqqatqttac agcqtctcat cagtottacc aqqttqtqct
gatccatgga actccggaca aacgttctcc tgctctgtaa
ctcatcctga gtctaagtca tcactcacag caacaatcaa
gaaggacctg ggaaatacgt tccgtcctca agtgcattta
0175E ctccctccac cttcagagga actcgcattg aatgagctcg SEOID NO:63
taacacttac ctgottggta agaggtttca gccctaagga
ggttttggtt aggtggcttc aaggtaatca ggagcttccc
agggaaaaat atttgacctg ggggcccctt ccggaagctg
gcgaatctgt tactactttt gctgttactt ctgttottcg
agttgatgct gaagtttgga aacagggcga tacttttagc
tgcatggttg ggcacgaagc ccttccgctt gcctttactc
agaaaaccat agatcggtta gccgggaaac cgacccacgt
taatgtgtct gtggtgatgt ctgaagtgga tggcgtgtgc
tatgg
gatagttcca gttgctgtgt cccaaattgt gagccatctc
taagtgtcca gcctccagca ctagaagatt tgottctggg
ttctaacgct tctctaactt qtacactgag tqqactqaaq
agtgcagagg gtgcatcatt tacatggaac cctacaggag
gtaagacagc tgtacaagga agtccaaaga gagattcctg
tggatgttac agcgtctcat cagtcttacc aggttgtgct
gatccatgga actccggaca aacgttctcc tgctctgtaa
ctcatcctga gtctaagtca tcactcacag caacaatcaa
gaaggacctg ggaaatacgt tccgtcctca agtgcattta
0195E ctocctccac cttcagagga actcgcattg aatgagctcg SEQID NO:64
taacacttac ctgcttggta agaggtttca gccctaagga
ggttttggtt aggtggcttc aaggtaatca ggagcttccc
agggaaaaat atttgacctg ggggcccctt ccggaagctg
gccaatctgt tactactttt gctgttactt ctgttcttcg
agttgatgct gaagtttgga aagagggcga tacttttagc
tgcatggttg ggcacgaagc ccttccgctt gcctttactc
agaaaaccat agatcggtta gccgggaaac cgacccacgt
taatgtgtct gtggtgatgt ctgaagtgga tggcgtgtgc
tatgg
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Sequence
Mutant Sequence
Identifier
gatagttcca gttgctgtgt cccaaattgt gagccatctc
taagtgtcca gcctccagca ctagaagatt tgcttctggg
ttctaacgct tctctaactt gtacactgag tggactgaag
agtgcagagg gtgcatcatt tacatggaac cctacaggag
gtaagacagc tgtacaagga agtccaaaga gagattcctg
tqqatqttac aqcqtctcat caqtcttacc aqqttqtqct
gatccatgga actccggaca aacgttctcc tgctctgtaa
ctcatcctga gtctaagtca tcactcacag caacaatcaa
gaaggacctg ggaaatacgt tccgtcctca agtgcattta
G196C/ R219C ctccctccac cttcagagga actcgcattg aatgagctcg SEQID NO:65
taacacttac ctgottggta agaggtttca gccctaagga
ggttttggtt aggtggcttc aaggtaatca ggagcttccc
agggaaaaat atttgacctg ggggcccctt ccggaagctg
gccaatctgt tactactttt gctgttactt ctgttottcg
agttgatgct gaagtttgga aacagtgtga tacttttagc
tgcatggttg ggcacgaagc ccttccgctt gcctttactc
agaaaaccat agattgttta gccgggaaac cgacccacgt
taatgtgtct gtggtgatgt ctgaagtgga tggcgtgtgc
tatgg
gatagttcca gttgctgtgt cccagattgt gagccatctc
taagtgtcca gcctccagca ctagaagatt tgottctggg
ttctaacqct tctctaactt qtacactqaq tqqactqaaq
agtgcagagg gtgcatcatt tacatggaac cctacaggag
gtaagacagc tgtacaagga agtccaaaga gagattcctg
tggatgttac agcgtctcat cagtcttacc aggttgtgct
gatccatggg actccggaca aacgttctcc tgctctgtaa
ctcatcctga gtctaagtca tcactcacag caacaatcaa
N9D/N840/ gaaggacctg ggaaatacgt tccgtcctca agtgcattta
N131 ctocctccac cttcagagga actcgcattg gatgagctcg SEQID
NO:66
D
taacacttac ctgcttggta agaggtttca gccctaagga
ggttttggtt aggtggcttc aaggtaatca ggagcttccc
agggaaaaat atttgacctg ggggcccctt ccggaagctg
gccaatctgt tactactttt gctgttactt ctgttcttcg
agttgatgct gaagtttgga aacagggcga tacttttagc
tgcatggttg ggcacgaagc ccttccgctt gcctttactc
agaaaaccat agatcggtta gccgggaaac cgacccacgt
taatgtgtct gtggtgatgt ctgaagtgga tggcgtgtgc
tatgg
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Sequence
Mutant Sequence
Identifier
gatagttcca gttgctgtgt cccagattgt gagccatctc
taagtgtcca gcctccagca ctagaagatt tgcttctggg
ttctaacgct tctctaactt gtacactgag tggactgaag
agtgcagagg gtgcatcatt tacatggaac cctacaggag
gtaagacagc tgtacaagga agtccaaaga gagattcctg
tqqatqttac agcqtctcat cagtottacc aqqttqtqct
gatccatggg actccggaca aacgttctcc tgctctgtaa
ctcatcctga gtctaagtca tcactcacag caacaatcaa
N9D/N84D/ gaaggacctg ggaaatacgt tccgtcctca agtgcattta
N131 0/01 ctccctccac cttcagagga act cgcattg gatgagctcg SEQID NO:67
75E/0195E taacacttac ctgottggta agaggtttca gccctaagga
ggttttggtt aggtggcttc aaggtaatca ggagcttccc
agggaaaaat atttgacctg ggggcccctt ccggaagctg
gcgaatctgt tactactttt gctgttactt ctgttottcg
agttgatgct gaagtttgga aagagggcga tacttttagc
tgcatggttg ggcacgaagc cottccgott gcctttactc
agaaaaccat agatcggtta gccgggaaac cgacccacgt
taatgtgtct gtggtgatgt ctgaagtgga tggcgtgtgc
tatgg
gatagttcca gttgctgtgt cccagattgt gagccatctc
taagtgtcca gcctccagca ctagaagatt tgottctggg
ttctaacgct tctctaactt qtacactgag tqqactqaaq
agtgcagagg gtgcatcatt tacatggaac cctacaggag
gtaagacagc tgtacaagga agtccaaaga gagattcctg
tggatgttac agcgtctcat cagtcttacc aggttgtgct
N9D/N84D/ gatccatggg actccggaca aacgttctcc tgctctgtaa
N13-ID/ ctcatcctga gtctaagtca tcactcacag caacaatcaa
0175E/ gaaggacctg ggaaatacgt tccgtcctca agtgcattta
0195E/ ctocctccac cttcagagga actcgcattg gatgagctcg SEQID NO:68
G1960/ taacacttac ctgcttggta agaggtttca gccctaagga
R2190 ggttttggtt aggtggcttc aaggtaatca ggagcttccc
(5+1) agggaaaaat atttgacctg ggggcccctt ccggaagctg
gcgaatctgt tactactttt gctgttactt ctgttcttcg
agttgatgct gaagtttgga aagagtgtga tacttttagc
tgcatggttg ggcacgaagc ccttccgctt gcctttactc
agaaaaccat agattgttta gccgggaaac cgacccacgt
taatgtgtct gtggtgatgt ctgaagtgga tggcgtgtgc
tatgg
[0123] To enable expression in leaf tissue, each construct was cloned into a
pCaMGate
plant expression vector including an N-terminal PR1b tobacco signal peptide
sequence
and a C-terminal KDEL tag for targeting the expressed protein to the
endoplasmic
reticulum (ER) (Pereira et al. (2014). BMC Biotechnol 14: 59). Without being
bound by
theory, it was contemplated that targeting the constructs to the ER was
desirable
because of the requirement for disulfide formation for correct folding
assembly and
because targeting to the ER using the vector described in Example 1 above gave
robust
accumulation of VHH-Fc fusion proteins. The vectors were transformed into
Agrobacterium tumefaciens (EHA105) strains. Transient expressions were
performed by
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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 pmol
m2 s1 for
7 weeks and fertilized with water soluble N:P:K (20:8:20) at 0.25 g/L (Plant
products,
Brampton, ON, Canada). Plant extracts were prepared under native conditions as
described in Example 1 above. Purification was performed using an anti-c-myc
purification kit (MBL International Corp., Woburn, MA, USA). 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) as described in Example 1 above.
[0124] As seen from the results shown in Figure 2A, each of the supercharged
Fe
mutants, N9D (SEQ ID NO:51), N84D (SEQ ID NO:52), N131D (SEQ ID NO:53), Q175E
(SEQ ID NO:54) and 0195E (SEQ ID NO:55), showed a three- to four-fold
improvement
in accumulation across the time course, compared to the accumulation of native
Fc
(SEQ ID NO:47). Similarly, the de novo disulfide mutant, G196C/R219C (SEQ ID
NO:56), showed a six to seven-fold improvement in accumulation compared to the
native
Fc after six dpi (Figure 2B).
[0125] 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. As seen from the results shown in
Figure 2C,
the Fc mutant containing the three N D supercharging mutations, N9D/N84D/N131D
(SEQ ID NO:57) gave a progressive increase in accumulation after transient
expression.
Further, the Fc mutant containing all five supercharging mutations,
N9D/N84D/N131D/0175E/0195E (SEQ ID NO:58), showed a ten-fold improvement in
accumulation compared to native Fc. In addition, adding the mutations required
for de
novo disulfide formation to these five supercharging mutations to give the Fc
mutant
N9D/N84D/N131D/Q175E/0195E/G196C/R219C (5+1) (SEQ ID NO:59), further
improved accumulation by approximately twenty-two-fold_
Expression of VHH9-Fc mutants
[0126] To test if these Fc mutants could also enhance accumulation as an Fc
scaffold
protein, each Fc mutant was fused to VHH9. Genetic fusions of Fc mutant
sequences to
anti-E. coil VHH9 (SEQ ID NO:35) were carried out using a sequence and
ligation
independent cloning (SLIC) method (Li et al. (2007). Nature Methods 4:251-
256), and
the fused VHH9-Fc mutant constructs were expressed in Nicotiana benthamiana
leaf
tissue as described above. As seen from the results shown in Figure 3A, each
of the
individual mutant VHH9-Fc fusions, either with a supercharged residue or with
a de novo
disulfide, showed a three- to four-fold improvement in accumulation when
compared to
the native VHH9-Fc fusion, as seen in the comparison of the unfused Fc
mutants. When
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fused to VHH9, the combined Fc mutants also showed progressively improved
accumulation, with five mutations showing an approximately sixteen-fold
improvement
compared to the native VHH9-Fc fusion, as seen from the results shown in
Figure 3B.
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
VH H-Fc fusions.
Assembly of mutant VHH9-Fc sIgA
[0127] To determine if the presence of the Fc mutations in the VHH9-Fc
affected the
ability of the Fc subunit to assemble with the SC and JC subunits to form IgA,
all three
subunits (VHH9-Fc, SC and JC) were co-expressed and immunoprecipitation
experiments were conducted. Each subunit was labelled with a different tag
(VHH9-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-c-myc (VHH9-Fc subunit) or anti-HA (JC
subunit).
Bands matching the predicted 44 kDa size of VHH9-Fc were detected with anti-c-
myc
antibody in crude extract transformed with Vi1H9-Fc,
VH H9-N9D/N84D/N131D/Q175E/Q195E/G196C/R219C-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 (Figure 4, panel A).
After
co-imnnunoprecipitation, -44 kDa bands were seen only in extracts co-
expressing
VHH9-Fc/SC/JC and VHH9-(5+1)-Fc/SC/JC (Figure 4, panel B). This indicated that
both
SC and VHH9-Fc or SC and VHH9-(5+1)-Fc interact, and that the mutations in Fc
did not
hinder this interaction. Similarly, detection with anti-HA indicated bands of -
20 kDa,
matching the predicted size of JC, in crude extract transformed with JC, VHH-
Fc/SC/JC
and VHH9-(5+1)-Fc/SC/JC (Figure 4, panel C). After co-innmunoprecipitation, -
20 kDa
bands were seen only for the co-expressed VHH9-Fc/SC/JC and VHH9-(5+1)-
Fc/SC/JC,
indicating that SC and JC are present in the same complex (Figure 4, panel D).
Binding of mutant VHH9-Fc to Escherichia coli strains
[0128] To determine if the rationally designed mutations impact the cross-
serotype
pattern of VHH-Fc binding against E. coli, either VHH9-Fc or VHH9-(5+1)-Fc was
incubated with E. co/1026:H11, 045:H2, 0103:H2, 0145:Hnm, 0121:H19, 0111:Hnm
or
0157:H7. E. col/ binding assays were performed as described in Example 1
and/or
according to methods known in the art. After washing and fixing with
paraformaldehyde,
bacteria was visualized with DAPI and VHH9-Fc binding visualized using a
secondary
fluorescent antibody (rabbit anti-bovine-FITC) that binds Fc. As seen from the
results
presented in Figure 5, consistent co-localization of FITC signal with strains
026:H11,
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observed, indicating multi-serotype detection of E.coli . As a negative
control, E. coli 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
5 conditions (480 nm excitation and 520-540 nm detection).
Neutralization of adherence of Escherichia coil strains to epithelial cells by
mutant
V/41-19-Fc
[0129] To determine the ability of mutant VHH9-Fc to prevent E. coli from
adhering to
epithelial cells by blocking intimin, HEp-2 adherence inhibition assays were
performed as
10 described above. HEp-2 cells were incubated with a culture of one of
seven E. coli
strains (026:H11, 045:H2, 0103:H2, 0145:Hnm, 0121:H19, 0111:Hnm and 0157:H7)
in the presence or absence of either VHH9-Fc or VHH9-(5+1)-Fc, washed to
remove any
non-adherent bacteria and then visualized by immunofluorescence microscopy. As
seen
from the results presented in Figure 6, the addition of either VHH9-Fc or VHH9-
(5+1)-Fc
15 abrogated the adhesion of E. coli strains 026:H11, 0111:Hnm, 0145:Hnm
and 0157:H7
to HEp-2 cells to HEp-2 cells compared to the respective positive controls of
no VI IH-Fc
(+ PBS treatment).
[0130] To quantify the relative neutralization capacity of the VHH9-Fc
compared to the
VHH9-(5+1)-Fc, the adhesion assay for fluorometry was adapted as described
above and
20 the relative fluorescence of HEp-2 cells incubated with a culture of
each of the seven E.
coli strains with and without either VHH9-Fc or VHH9-(5+1)-Fc was measured.
The
addition of either VHH9-Fc or VHH9-(5+1)-Fc 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, as seen in Figure 7. Thus, VHH9-(5+1)-
Fc
25 retains the ability to neutralize adherence of E. coil strains 0157,
026, 0111, and 0145
to HEp-2 cells.
Example 3: Targeting of VHH-Fc fusion proteins to plant cell sub-compartments
Cloning and expression of targeting vectors
[0131] Plant expression vectors (schematically illustrated in Figure 8) were
adapted from
30 a cytosolic expression vector described by Pereira et al. (BMC
Biotechnol (2014), 14:
59). Transit peptide sequences were synthesized and cloned using a sequence
and
ligation independent cloning method (Li et al, Nature Methods (2007), 4: 251-
256). The
VHH9-Fc sequence described in Example 1 (SEQ ID NO:49) was then cloned into
each
vector by Gateway cloning and the reading frame for each vector was confirmed
by
35 sequencing.
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[0132] A vector designed to target the expressed construct to the chloroplast
thylakoid
by the secretory (Sec) pathway includes a bipartite transit peptide sequence
corresponding to the N-terminal 75 amino acids of the Arabidopsis thaliana
thylakoid
lumina! 15.0 kDa protein 2 (At5g52970; NCB! accession no. NP 001318791) and
shown
below.
MAMLFRPPPS QCRSFSPFVF NYSSREVSSS SRLSLKTSGD EENWVSRFRS
KSLSLVFSGA LALGLSLSGV GFADA
(SEQ ID NO:69)
[0133] This bipartite N-terminal transit peptide sequence consists of two
signal regions
in tandem. The first signal region targets the peptide to the outer double
membrane of
the chloroplast, where it is cleaved, releasing the remaining peptide into the
stroma. The
remaining second signal region targets the peptide to the thylakoid membrane,
where it
is cleaved, releasing the peptide into the thylakoid lumen. Using the ChloroP
and
TargetP online tools (Almagro Armenteros et al, Life Sci_ Alliance (2019), 2:
e201900429; Emanuelsson et al, Protein Sci. (1999), 8:978-984), the cleavage
site
between the two signals was predicted to be between serine-28 and serine-29 of
SEQ ID
NO:69 (indicated in underlined bold italics).
[0134] A vector designed to target the expressed construct to the chloroplast
thylakoid
by the twin-arginine translocation (Tat) pathway includes a transit peptide
sequence
corresponding to the N-terminal 71 amino acids of the Arabidopsis thaliana
FKBP-type
peptidyl-prolyl cis-trans isomerase (At1g20810; NCB! accession no. NP
001321139)
and shown below.
MASISSLHRW ASNQHSRLPR ITSISEADQS RPINQVVAFS VPISRRDASI
ILLSSIPLTS FFVLTPSSSE A
(SEQ ID NO:70)
[0135] Again, this bipartite N-terminal transit peptide sequence consists of
two signal
regions in tandem, the first targeting the peptide to the outer double
membrane of the
chloroplast, and the second targeting the peptide to the thylakoid membrane.
The
cleavage site between the two signals was predicted to be between alanine-38
and
phenylalanine-39 of SEQ ID NO:70 (indicated in underlined bold italics).
[0136] Vectors designed to target the expressed construct to the stroma, the
endoplasnnic reticulum or the cytoplasm were adapted from those described in
Pereira et
al., BMC Biotechnol (2014), 14: 59. The vector designed for targeting to the
cytoplasm
lacks a transit peptide sequence.
[0137] 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
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the VHH9-Fc in the thylakoid lumen via both pathways, in the stroma, and in
the ER, but
lacked sufficient signal for detection in the cytoplasm, as seen in Figures 9A
and 9B.
Under non-reducing extraction conditions, the VHH9-Fc is detected
predominantly as an
88 kDa band matching the predicted size of the VHH9-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 44 kDa is
detected
matching the predicted size of the VHH9-Fc monomer for the ER, stromal,
thylakoid via
Sec and thylakoid via Tat compartments suggesting that the VHH9-Fc dimer in
these
compartments is stabilized by an interchain disulfide bond.
[0138] Based on these results, while ER-targeting produced the highest yields
in the
VHH9-Fc tested, the Sec-targeting accumulation remained significantly high, in
contrast
to stromal, cytoplasm, and Tat-import pathways.
Expression of VHH9-Fc in chloroplasts
[0139] In addition to transient expression, the VHH9-Fc was also encoded in
the
chloroplast by transforming the chloroplast genome through homologous
recombination
using vector pCEC5 as described in Kolotilin et al. (2013), Biotechnology for
Biofuels
6:65. The VHH9-Fc was targeted to the thylakoid within the chloroplast using
the Sec
import pathway, using an N-terminally truncated Seq transit peptide having the
sequence:
MASSSRLSLKTSGDEENWVSRFRSKSLSLVFSGALALGLSLSGVGFADA
(SEO ID NO:71)
[0140] The DNA sequence comprising the truncated Seq transit peptide, the VHH9
and
the Fc chain was optimized for expression in the chloroplast genome.
Localization of VHH9-Fc in the thylakoid
[0141] To verify that the Sec and Tat transit peptides indeed target VHH9-Fc
to the
thylakoid compartment, subcellular localization of the VHH9-Fc was tracked by
fusing
GFP to the Fc chain in each of the expression vectors. As seen in Figure 10,
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 -735 nm. 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.
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Accumulation of VHH9-G196C/R219C-Fc in the thylakoid
[0142] To determine if the oxidative folding of the thylakoid can recapitulate
the yield-
improving effects of an engineered disulfide bond, the VHH9-Fc fusion carrying
the
G196C/R219C mutation was targeted to the thylakoid lumen via the Sec pathway
and
accumulation by Western blot after agroinfiltration was measured. As seen from
the
results shown in Figure 11, the G196C/R219C mutant VHH9-Fc showed a
significant
yield improvement over the native VHH9-Fc when targeted to the Sec pathway.
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. Without being bound by theory, this indicates that the disulfide
bridge is
introduced in the lumen of the thylakoid after import of the unfolded
polypeptide.
Binding of targeted VHH9-Fc to Escherichia coli strains
[0143] To determine if the thylakoid-targeted VHH9-Fc retained the ability of
ER-targeted
VHH9-Fc (Examples 1 and 2) to bind E. coli, purified VHH9-Fc from each
compartment
was incubated with the pathogen then fixed in paraformaldehyde, washed and
probed for
immunofluorescence using a FITC labelled anti-c-myc secondary antibody. As
seen in
Figure 12, visualization by confocal microscopy showed consistent co-
localization
between DAPI-stained bacterial cells and the FITC-labelled VHH9-Fc for the
thylakoid via
Sec, thylakoid via Tat, and stromal compartments, indicating that the
chloroplast-
targeted VHH9-Fc retains the ability to bind intimin on E. co//surfaces. As a
negative
control, 0157:H7 cells were also treated with PBS containing 0.1% Tween-20
(PBS-T)
instead of the VHH9-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 VHH9-Fc is folded correctly through Sec, Tat and stroma
targeted
pathways regardless of the status of the Fc chain.
Neutralization of adherence of Escherichia coil strains to epithelial cells by
targeted VHH9-Fc
[0144] HEp-2 cells were incubated with E. coli 0157:H7 in the presence or
absence of
purified VHH9-Fc from each of the compartments. 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 AlexaTM 350
secondary antibody. As seen in Figure 13, the addition of purified VHH9-Fc
from any of
the compartments abrogated adhesion of any labelled E. coli 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. Without being bound by
theory, given
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that Tat and stromal imported antibodies retain functionality and show
dimerized banding
under non-reducing conditions as seen in Figure 9B, this suggests disulfide
formation in
the stroma despite its reducing environment.
[0145] These results indicate that the chloroplast-targeted VHH9-Fc retains
the ability to
neutralize E. co//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.
[0146] The embodiments described herein are intended to be illustrative of the
present
compositions and methods and are not intended to limit the scope of the
present
invention. Various modifications and changes consistent with the description
as a whole
and which are readily apparent to the person of skill in the art are intended
to be
included. The appended claims should not be limited by the specific
embodiments set
forth in the examples, but should be given the broadest interpretation
consistent with the
description as a whole.
CA 03204634 2023-7- 10
