U.S. patent application number 14/017637 was filed with the patent office on 2014-05-08 for specific delivery of agrochemicals.
This patent application is currently assigned to AGROSAVFE N.V.. The applicant listed for this patent is Erik Jongedijk, Peter Verheesen. Invention is credited to Erik Jongedijk, Peter Verheesen.
Application Number | 20140128579 14/017637 |
Document ID | / |
Family ID | 42331002 |
Filed Date | 2014-05-08 |
United States Patent
Application |
20140128579 |
Kind Code |
A1 |
Jongedijk; Erik ; et
al. |
May 8, 2014 |
SPECIFIC DELIVERY OF AGROCHEMICALS
Abstract
Described is the specific delivery of agrochemicals to plants.
More specifically, a targeting agent has at least one binding
domain that specifically binds to a binding site on an intact
living plant. Such binding domains include a peptide having 4
framework regions and 3 complementary determining regions, or
fragment(s) thereof, wherein the binding domains bind or retain a
carrier onto a plant. Described are binding domains that
specifically bind trichomes, stomata, cuticle, lenticels, thorns,
spines, root hairs, or wax layer. Further described are methods for
delivering agrochemicals to a plant, for depositing agrochemicals
on a plant, and for retaining the agrochemicals on a plant, using
targeting agents comprising the binding domains, and to methods for
protecting a plant against stress or controlling plant growth.
Also, described are methods for manufacturing a specifically
targeting agrochemical carrier.
Inventors: |
Jongedijk; Erik; (Lokeren,
BE) ; Verheesen; Peter; (Gent, NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Jongedijk; Erik
Verheesen; Peter |
Lokeren
Gent |
|
BE
NL |
|
|
Assignee: |
AGROSAVFE N.V.
GENT
BE
|
Family ID: |
42331002 |
Appl. No.: |
14/017637 |
Filed: |
September 4, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13081435 |
Apr 6, 2011 |
8598081 |
|
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14017637 |
|
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61341930 |
Apr 6, 2010 |
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Current U.S.
Class: |
530/389.1 |
Current CPC
Class: |
C07K 2317/569 20130101;
A01N 3/00 20130101; A01N 25/00 20130101; C07K 2317/35 20130101;
A61K 47/6835 20170801; C07K 16/16 20130101; A01N 25/28 20130101;
A01N 25/28 20130101; A01N 25/24 20130101; A01N 25/24 20130101; C07K
2317/92 20130101; A01N 53/00 20130101 |
Class at
Publication: |
530/389.1 |
International
Class: |
C07K 16/16 20060101
C07K016/16 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 6, 2010 |
EP |
10159100.6 |
Claims
1. A binding domain able to bind at least one binding site on an
intact living plant.
2. The binding domain of claim 1, wherein the binding domain is
derived from an innate or adaptive immune system.
3. A binding domain able to bind at least one binding site on a
plant, wherein the binding domain is comprised in a peptide that
comprises four framework regions and three complementary
determining regions, or any suitable fragment thereof.
4. The binding domain of claim 1, wherein the binding domain is
able to bind a carrier onto a plant.
5. The binding domain of claim 1, wherein the binding domain is
able to retain a carrier onto a plant.
6. The binding domain of claim 1, wherein the binding domain binds
to a specific part of the plant.
7. The binding domain of claim 6, wherein the part of the plant is
selected from the group consisting of leaves, stem, roots, fruits,
cones, flowers, bulbs and tubers.
8. The binding domain of claim 1, wherein the binding domain binds
to a specific structure on the plant.
9. The binding domain of claim 8, wherein the specific structure is
selected from the group consisting of trichomes, stomata,
lenticels, thorns, spines, root hairs, cuticle and wax layer.
10. The binding domain of claim 1, wherein the binding domain binds
to the plant in an agrochemical formulation.
11. The binding domain of claim 1, wherein the binding domain binds
to gum Arabic.
12. The binding domain of claim 1, wherein the binding domain binds
to lectins, lectin-like domains, extensins, or extensin-like
domains.
13. The binding domain according to claim 1, wherein the binding
domain is derived from a camelid antibody.
14. The binding domain of claim 13, wherein the binding domain is
comprised in a VHH sequence.
15. The binding domain of claim 14 wherein the VHH sequence has two
stabilizing disulfide bridges.
16-50. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims is a continuation of U.S. Ser. No.
13,081,435, filed on Apr. 6, 2011, now U.S. Pat. No. ______, which
both claim the benefit under 35 U.S.C. .sctn.119(e) of U.S.
Provisional Patent Application Ser. No. 61/341,930, filed Apr. 6,
2010, and to European Patent Application Serial No. EP 10159100.6,
filed Apr. 6, 2010, the disclosure of each of which is hereby
incorporated herein in its entirety by this reference.
STATEMENT ACCORDING TO 37 C.F.R. .sctn.1.821(c) or (e)--SEQUENCE
LISTING SUBMITTED AS PDF FILE WITH A REQUEST TO TRANSFER CRF FROM
PARENT APPLICATION
[0002] Pursuant to 37 C.F.R. .sctn.1.821(c) or (e), a file
containing a PDF version of the Sequence Listing has been submitted
concomitant with this application, the contents of which are hereby
incorporated by reference. The transmittal documents of this
application include a Request to Transfer CRF from the parent
application.
TECHNICAL FIELD
[0003] The disclosure relates to specific delivery of agrochemicals
to plants. More specifically, it relates to a composition,
essentially consisting of a targeting agent comprising at least one
binding domain that specifically binds to a binding site on an
intact living plant and an agrochemical or a combination of
agrochemicals. The disclosure relates further to a binding domain
that specifically binds the binding site on an intact living plant.
More specifically, it relates to binding domains comprising an
amino acid sequence that comprises four framework regions and three
complementary determining regions, or any suitable fragment
thereof, wherein the binding domains are capable to bind or retain
a carrier onto a plant. In one embodiment, it relates to binding
domains which specifically bind trichomes, stomata, cuticle,
lenticels, thorns, spines, root hairs, or wax layer. It relates
further to a method for delivery of agrochemicals to a plant, for
improving the deposition of agrochemicals on a plant, and for
retaining the agrochemicals on a plant, using targeting agents
comprising the binding domains, and to a method for protecting a
plant against biotic or abiotic stress or controlling plant growth
using the same. Also, it relates to a method for manufacturing a
specifically targeting agrochemical carrier.
BACKGROUND
[0004] For many years, horticulturist and agronomist have applied
chemicals for weed control, plant protection and plant growth
regulation by spraying the fields. For compositions that need to be
applied on the plant, e.g., on the foliage, only a small part of
the composition is bound to and retained on the part of the plant
where it can exert its biological activity as large amounts are not
adhering to the plant surface and are lost by drip-off or washed
away by rain. Apart from giving rise to reduced efficacy of the
chemical, losses of chemicals into the soil due to dripping off the
plant while spraying or due to wash-out during rainfall may result
in groundwater contamination, environmental damage, loss of
biodiversity, and human and animal health consequences.
[0005] Several researchers have tried to solve this problem by
applying slow release particles to the plant that stick to the
leaves and release their content over a certain period of time.
U.S. Pat. No. 6,180,141 describes composite gel microparticles that
can be used to deliver plant-protection active principles. WO
2005102045 describes compositions comprising at least one
phytoactive compound and an encapsulating adjuvant, wherein the
adjuvant comprises a fungal cell or a fragment thereof. U.S.
20070280981 describes carrier granules, coated with a lipophilic
tackifier on the surface, wherein the carrier granule adheres to
the surface of plants, grasses and weeds.
[0006] Those microparticles, intended for the delivery of
agrochemicals, are characterized by the fact that they stick to the
plant by rather weak, aspecific interactions, such as a lipophilic
interaction. Although this may have advantages compared with the
normal spraying, the efficacy of such delivery method is limited,
and the particles may be non-optimally distributed over the leaf,
or washed away under naturally variable climatological conditions,
before the release of the compound is completed. For a specific
distribution and efficient retention of the microparticles, a
specific, strongly binding molecule is needed that can assure that
the carrier sticks to the plant till its content is completely
delivered.
[0007] Cellulose binding domains (CBDs) have been described as
useful agents for attachment of molecular species to cellulose
(U.S. Pat. Nos. 5,738,984 and 6,124,117). Indeed, as cotton is made
up of 90% cellulose, CBDs have proved useful for delivery of so
called "benefit agents" onto cotton fabrics, as is disclosed in
WO9800500 where direct fusions between a CBD and an enzyme were
used utilizing the affinity of the CBD to bind to cotton fabric.
The use of similar multifunctional fusion proteins for delivery of
encapsulated benefit agents was claimed in WO03031477, wherein the
multifunctional fusion proteins consist of a first binding domain
which is a carbohydrate binding domain and a second binding domain,
wherein either the first binding domain or the second binding
domain can bind to a microparticle. WO03031477 is exemplified using
a bifunctional fusion protein consisting of a CBD and an anti-RR6
antibody fragment binding to a microparticle, which complex is
deposited onto cotton treads or cut grass. However, the use of such
multifunctional fusion proteins for delivery of encapsulated
benefit agents suffers from a number of serious drawbacks. First,
although cellulose is a major component of plant cell walls and
about 33% of all plant matter consists of cellulose, cellulose is,
in intact living plants, shielded off from the outside environment
by the plant cuticle, formed by cutin and waxes, which is an
impermeable barrier with which plant cell walls are covered, making
cellulose poorly accessible for binding by CBDs. Secondly,
effective delivery of an encapsulated benefit agent to the plant
requires simultaneous binding of the first binding domain to the
plant and the second binding domain to the microparticle. As the
likelihood of both binding events occurring is determined by a
delicate equilibrium between the molar concentrations of the
binding domains and their target molecules and the molar
concentration of the bound complex, it is highly unlikely that
sufficient multifunctional fusion proteins are present in solution
to enable such simultaneous binding. Moreover, the equilibrium of a
binding event is strongly influenced by environmental parameters
such as temperature and pH, for which the optimal conditions may be
considerably different for each of the binding domains. Therefore,
it is highly unlikely that such simultaneous binding of two binding
domains of such multifunctional fusion protein would result in a
sufficiently strong binding that would retain an encapsulated
benefit agent to a plant. Thirdly, although binding of a CBD is to
a certain extent specific for cellulose, using a multifunctional
fusion protein in which CBD should bind to the plant is to be
considered as a generic binding approach, as all plants contain
cellulose, and is therefore similar to aspecific sticking with
tackifiers or stickers. A targeted approach in which specific
binding of a binding domain would allow discrimination between
binding to one plant species versus another would be of
considerably higher value. WO03031477 also suggests, without
further exemplification, that other binders to carbohydrates or
polysaccharides can be used to generate fusion proteins to deposit
microparticles onto living organisms. However, neither binding
domains other than CBDs, nor binding domains binding to intact
living plants were disclosed in WO03031477.
[0008] Molecules that are well known for their specificity and high
affinity to particular targets are antibodies. Antibodies can be
generated against a broad variety of targets, and antibodies that
were generated to study plant cell wall architecture and dynamics
have been described to bind specifically to particular plant
constituents, predominantly constituents of the plant cell wall
(Penell et al., 1989; Jones et al., 1997; Willats et al., 1998;
Willats and Knox, 1999; Willats et al., 2001). However, it is
unclear whether any of the plant cell wall constituents to which
the antibodies have been generated, would be directly accessible
for an antibody from the outside environment. Moreover, antibodies
are by their very nature as components of the adaptive immune
system construed such that they bind their targets under
physiological conditions, including tightly regulated pH,
temperature, and blood's normal osmolarity range. Should one
consider to use antibodies for targeted delivery of agrochemicals,
the antibodies should not only be capable of binding their target
on an intact living plant in an agrochemical formulation, for which
physicochemical characteristics deviate substantially from
physiological conditions, they should also be capable to bind
strongly enough to retain a carrier onto a plant. For neither of
the plant-binding antibodies earlier described, either of these two
crucial characteristics have been demonstrated.
[0009] The variable domains of camelid heavy chain antibodies (VHH)
are a particularly interesting type of antibody fragments, as they
are small, 15 kDa single-chain proteins, which can be selected for
displaying high affinity for their targets. Also, by their nature
as small single-chain molecules, VHH are easy to produce and have
superior stability characteristics over conventional antibodies.
However, so far, no plant-binding VHH have been described.
Moreover, although VHH that are covalently linked to a solid resin
particle have been shown to maintain functionality in the sense
that they are able to capture antigen from a solution (WO 0144301),
it has not been shown, nor can it be expected, that the affinity of
VHH for its target is sufficient to retain a carrier onto a solid
plant surface.
DISCLOSURE
[0010] We have isolated binding domains, more specifically binding
domains comprising an amino acid sequence (or "peptide") that
comprises four framework regions (FR) and three complementary
determining regions (CDR) (FR and CDR definitions according to
Kabat), wherein the binding domains are capable to bind a binding
site on an intact living plant and, surprisingly, in doing so, are
capable of retaining an agrochemical or a carrier containing an
agrochemical to the plant. In certain embodiments, the binding
domains remain stable and retain their binding capacity under harsh
conditions, such as variable temperature, pH, salt concentration,
availability of water or moisture. In some embodiments, the binding
domains remain stable and retain their binding capacity in an
agrochemical formulation. Binding domains comprising four FRs and
three CDRs, preferably in a sequence
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, are known to the person skilled in
the art and have been described, as a non-limiting example in
Wesolowski et al. (2009). In certain embodiments, the binding
domains are derived from camelid antibodies, preferably, from heavy
chain camelid antibodies, devoid of light chains, such as variable
domains of heavy chain camelid antibodies (VHH).
[0011] Targeting agents comprising these binding domains, can
retain agrochemicals specifically to binding sites on the plant or
plant parts and can be used to deliver and retain agrochemicals to
the plant, preferably to the intact living plant, wherein the
binding domains comprised in such targeting agents specifically
bind to binding sites on the plant, where the agrochemicals can
exert their activity. Agrochemical compositions comprising at least
one targeting agent and an agrochemical, preferably bound on or
comprised in a carrier, may be suitable to allow the use of a
reduced dose of the agrochemical and/or reduction of the frequency
of application of the agrochemical, comprised in such composition
whilst maintaining its overall efficacy. Moreover, when comprised
in a composition hereof, the agrochemical may exert its activity
over a longer period of time, eventually resulting in less
agrochemical being lost and contaminating the environment; also, by
applying an agrochemical in a composition hereof, it is possible to
introduce specificity into the activity of the agrochemical that is
otherwise not present.
[0012] Provided is a specific delivery method for agrochemicals in
which the agrochemical is delivered or deposited on or near its
site of action on an intact living plant utilizing a binding domain
that can bind specifically and strongly to the intact living plant,
and is able to retain an agrochemical or a carrier containing the
agrochemical onto the plant.
[0013] A first aspect, provided is a binding domain capable to bind
at least one binding site on an intact living plant. An "intact
living plant," as used herein, means a plant as it grows, whether
it grows in soil, in water or in artificial substrate, and whether
it grows in the field, in a greenhouse, in a yard, in a garden, in
a pot or in hydroponic culture systems. An intact living plant
preferably comprises all plant parts (roots, stem, branches,
leaves, needles, thorns, flowers, seeds . . . ) that are normally
present on such plant in nature, although some plant parts, such
as, e.g., flowers, may be absent during certain periods in the
plant's life cycle. An intact living plant excludes plant parts
that have been removed from the plant, such as leaves and flowers
that have been cut and separated from the plant. However, an intact
living plant includes plants that have been damaged by normal
natural events such as damage by weather (such as, but not limited
to wind, rain, or hail), by animals (whether by animals feeding on
the plants or by animals trampling on the plants), by plant pests
(such as, but not limited to insects, nematodes and fungi), or
damage caused by agricultural practice such as, but not limited to
pruning, harvesting of fruit, or harvesting of flowers. Plants
include gymnosperms and angiosperms, monocotyledons and
dicotyledons, trees, fruit trees, field and vegetable crops and
ornamental species. As a non-limiting example, the plants can be
cedars, cypresses, firs, junipers, larches, pines, redwoods,
spruces, yews, gingko, oilpalm, rubber tree, oak, beech, corn,
cotton, soybean, wheat, rice, barley, rye, sorghum, millet,
rapeseed, beans, peas, peanuts, sunflower, potato, tomato,
sugarcane, sugarbeet, cassaya, tobacco, banana, apple, orange,
lemon, olive, pineapple, avocado, vines, lettuce, cabbage, carrot,
eggplant, pepper, melon, rose, lilies, chrysanthemum, grass-like
weeds, or broadleaved weeds.
[0014] A "binding site," as used herein, means a molecular
structure or compound, such as a protein, a (poly)peptide, a
(poly)saccharide, a glycoprotein, a lipoprotein, a fatty acid, a
lipid or a nucleic acid or a particular region in such molecular
structure or compound or a particular conformation of such
molecular structure or compound, or a combination or complex of
such molecular structures or compounds. In certain embodiments, the
binding site comprises at least one antigen. "Antigen," as used
herein, means a molecule capable of eliciting an immune response in
an animal. In certain embodiments, the binding site is comprised in
a plant structure such as a trichome, stomata, lenticels, thorns,
spines, root hairs, cuticle or wax layer. Even more preferably, the
binding site is comprised in a plant structure such as a trichome,
stomata or cuticle. The binding site may be unique for one
particular plant structure, or it may be more generally comprised
in more than one plant structure. In certain embodiments, the
binding site is present on a particular part of the plant, such as
the leaves, stems, roots, fruits, cones, flowers, bulbs or tubers.
Even more preferably, the binding site is present on the surface of
such particular part of the plant, meaning that the binding site is
present at, for example, the leaf surface, the stem surface, the
root surface, the fruit surface, the cone surface, the flower
surface, the bulb surface or the tuber surface. The binding site
may be unique for one particular plant part, or it may be more
generally present on more than one plant part.
[0015] A "binding domain," as used herein, means the whole or part
of a proteinaceous (protein, protein-like or protein containing)
molecule that is capable of binding using specific intermolecular
interactions to a target molecule. A binding domain can be a
naturally occurring molecule, e.g., fibronectin, it can be derived
from a naturally occurring molecule, e.g., from components of the
innate or adaptive immune system, or it can be entirely
artificially designed. A binding domain can be immunoglobulin-based
or it can be based on domains present in proteins, including but
not limited to microbial proteins, protease inhibitors, toxins,
fibronectin, lipocalins, single-chain antiparallel coiled coil
proteins or repeat motif proteins. Non-limiting examples of such
binding domains are carbohydrate binding domains (CBD) (Blake et
al, 2006), heavy chain antibodies (hcAb), single domain antibodies
(sdAb), minibodies (Tramontano et al., 1994), the variable domain
of camelid heavy chain antibodies (VHH), the variable domain of the
new antigen receptors (VNAR), affibodies (Nygren et al., 2008),
alphabodies (WO2010066740), designed ankyrin-repeat domains
(DARPins) (Stumpp et al., 2008), anticalins (Skerra et al., 2008),
knottins (Kolmar et al., 2008) and engineered CH2 domains
(nanoantibodies; Dimitrov, 2009). Preferably, the binding domain
consists of a single polypeptide chain and is not
post-translationally modified. More preferably, the binding domain
is not a CBD. Even more preferably, the binding domain is derived
from an innate or adaptive immune system, preferably from a protein
of an innate or adaptive immune system. Still more preferably, the
binding domain is derived from an immunoglobulin. Most preferably,
the binding domain comprises four framework regions and three
complementary determining regions, or any suitable fragment thereof
(which will then usually contain at least some of the amino acid
residues that form at least one of the complementary determining
regions). Preferably, a binding domain is easy to produce at high
yield, preferably in a microbial recombinant expression system, and
convenient to isolate and/or purify subsequently. Also preferably,
a binding domain is stable, both during storage and during
utilization, meaning that the integrity of the binding domain is
maintained under storage and/or utilization conditions, which may
include elevated temperatures, freeze-thaw cycles, changes in pH or
in ionic strength, UV-irradiation, presence of harmful chemicals
and the like. More preferably, the binding domain is stable in an
agrochemical formulation. An "agrochemical formulation" as used
herein means a composition for agrochemical use, as further
defined, comprising at least one active substance, optionally with
one or more additives favoring optimal dispersion, atomization,
deposition, leaf wetting, distribution, retention and/or uptake of
agrochemicals. As a non-limiting example, such additives are
diluents, solvents, adjuvants, surfactants, wetting agents,
spreading agents, oils, stickers, thickeners, penetrants, buffering
agents, acidifiers, anti-settling agents, anti-freeze agents,
photo-protectors, defoaming agents, biocides and/or drift control
agents. Most preferably, the binding domain remains stable in an
agrochemical formulation when stored at ambient temperature for a
period of two years or when stored at 54.degree. C. for a period of
two weeks. Preferably, the binding domain is selected from the
group consisting of DARPins, knottins, alphabodies and VHH. More
preferably, the binding domain is selected from the group
consisting of alphabodies and VHH. Most preferably, the binding
domain is a VHH.
[0016] Binding of the binding domain to the binding site or to an
antigen comprised in the binding site occurs with high affinity.
The dissociation constant is commonly used to describe the affinity
between a binding domain and its target molecule. Preferably, the
dissociation constant of the binding between the binding domain and
the target molecule comprised in the binding site is lower than
10.sup.-5 M, more preferably, the dissociation constant is lower
than 10.sup.-6 M, even more preferably, the dissociation constant
is lower than 10.sup.-7 M, most preferably, the dissociation
constant is lower than 10.sup.-8 M. Preferably, binding of the
binding domain to the binding site is specific, meaning that the
binding domain binds to the binding site only if the target
molecule is present in the binding site and that the binding domain
does not bind, or binds with much lower affinity, to a binding site
lacking the target molecule. Specificity of binding of a binding
domain can be analyzed by methods such as ELISA, as described in
Example 2, in which the binding of the binding domain to its target
molecule is compared with the binding of the binding domain to an
unrelated molecule and with aspecific sticking of the binding
domain to the reaction vessel. Specificity can also be expressed as
the difference in affinity of a binding domain for its target
molecule versus the affinity for an unrelated molecule. Preferably,
the ratio of the affinity of the binding domain for its target
molecule versus its affinity for an unrelated molecule is larger
than 10, more preferably, the ratio is larger than 20, most
preferably, the ratio is larger than 100. Binding of the binding
domain can be specific for one particular plant structure, meaning
that the binding site, comprised in such plant structure, is not or
to a much lesser extent present in other plant structures; or the
binding can be more general to more than one plant structure, if
the binding site is present in more than one plant structure.
Binding of the binding domain can be specific for one particular
plant part, meaning that the binding site, present in or on such
plant part, possibly comprised in a plant structure on such plant
part, is not or to a much lesser extent present in other plant
parts; or the binding can be more general to more than one plant
part, if the binding site is present in more than one plant part.
Binding of the binding domain can be specific for one particular
plant species, meaning that the binding site, present in or on such
plant species, is not or to a much lesser extent present in other
plant species; or the binding can be more general to more than one
plant species, if the binding site is present in more than one
plant species. Binding of the binding domain can be specific for
one particular plant genus, meaning that the binding site, present
in or on such plant genus, is not or to a much lesser extent
present in other plant genera; or the binding can be more general
to more than one plant genus, if the binding site is present in
more than one plant genus. Binding of the binding domain can be
specific for one particular growth stage of the plant, meaning that
the binding site, present in or on such plant at a particular
growth stage, is not or to a much lesser extent present in the
plant at another growth stage; or the binding can be more general
to more than one plant growth stage, if the binding site is present
in more than one plant growth stage. All types of binding
specificity of the binding domains may have their specific use, as
will be explained below.
[0017] Preferably, the binding of the binding domain to the binding
site is still functional under harsh conditions, such as low or
high temperature, low or high pH, low or high ionic strength,
UV-irradiation, low availability of water, presence of denaturing
chemicals or the like. In one embodiment, the harsh conditions are
defined by a pH range from 4 to 9, more preferably, by a pH range
from 3 to 10, even more preferably, by a pH range from 2 to 10,
most preferably, by a pH range from 1 to 11. In another embodiment,
the harsh conditions are defined by a temperature range from
4-50.degree. C., more preferably, a temperature range from
0-55.degree. C., even more preferably, a temperature range from
0-60.degree. C. In another embodiment, the harsh conditions are
defined by the presence of an agrochemical formulation as defined
above.
[0018] Preferably, the binding of the binding domain to the binding
site is strong enough to bind, more preferably, to retain, a
carrier to the binding site; depending on the size of the carrier
and on the affinity of the binding domain, one or more binding
domains may bind to one or more binding sites and cooperate such
that the resulting avidity of the binding domains for the binding
site(s) ensures strong binding of the carrier, preferably retaining
the carrier, onto the plant. A "carrier," as used herein, means any
solid, semi-solid or liquid carrier in or on(to) which an active
substance can be suitably incorporated, included, immobilized,
adsorbed, absorbed, bound, encapsulated, embedded, attached, or
comprised. Non-limiting examples of such carriers include
nanocapsules, microcapsules, nanospheres, microspheres,
nanoparticles, microparticles, liposomes, vesicles, beads, a gel,
weak ionic resin particles, liposomes, cochleate delivery vehicles,
small granules, granulates, nano-tubes, bucky-balls, water droplets
that are part of an water-in-oil emulsion, oil droplets that are
part of an oil-in-water emulsion, organic materials such as cork,
wood or other plant-derived materials (e.g., in the form of seed
shells, wood chips, pulp, spheres, beads, sheets or any other
suitable form), paper or cardboard, inorganic materials such as
talc, clay, microcrystalline cellulose, silica, alumina, silicates
and zeolites, or even microbial cells (such as yeast cells) or
suitable fractions or fragments thereof (as further described
herein). "Retain" as used herein means that the binding force
resulting from the affinity or avidity of either one single binding
domain or a combination of two or more binding domains for its or
their binding site is larger than the combined force and torque
imposed by the gravity of the carrier, the force and torque, if
any, caused by the flow or drip off of a sprayed agrochemical
solution and the force and torque, if any, imposed by shear forces
caused by one or more external factors. In one embodiment, the
external factor is rain, irrigation, snow, hail or wind. One
particular advantage of binding a carrier by specific binding over
aspecific binding is that specific binding is more resistant to
external shear forces applied to the carrier (Cozens-Roberts et
al., 1990).
[0019] Preferably, a binding domain hereof binds to a binding site,
or to an antigen comprised in such binding site, present in or on
one or more particular parts of the intact living plant.
Preferably, the parts of the intact living plant are selected from
the group consisting of leaves, stem, roots, fruits, cones,
flowers, bulbs or tubers. More preferably, the parts of the intact
living plant are selected from the group consisting of leaves, stem
or roots. Preferably, a binding domain hereof binds to a binding
site, or to an antigen comprised in such binding site, on the
surface of the intact living plant. A "surface," as used herein,
can be any surface as it occurs on the intact living plant; or on
one or more parts of the intact living plant, however, it excludes
histological plant preparations. Preferably, the surface of the
intact living plant is the surface of a part of the intact living
plant, selected from the group consisting of leaf surface, stem
surface, root surface, fruit surface, cone surface, flower surface,
bulb surface or tuber surface; even more preferably, the surface of
the intact living plant is the surface of a part of the intact
living plant, selected from the group consisting of root surface,
stem surface and leaf surface.
[0020] Preferably, a binding domain hereof binds to a binding site,
or to an antigen comprised in such binding site, in or on a
particular structure of the intact living plant or in or on a
particular structure of a particular part of the intact living
plant; more preferably, in or on a particular structure involved or
implicated to be involved in transport of nutrients, agrochemicals
or other chemicals into the plant and/or involved or implicated to
be involved in plant defense. Preferably, the particular structure
is selected from the group consisting of trichomes, stomata,
lenticels, thorns, spines, root hairs, cuticle and wax layer, even
more preferably, the particular structure is selected from the
group consisting of trichomes, stomata and cuticle. In one
embodiment, the binding domain is binding to a binding site, or to
an antigen comprised in such binding site, in or on plant
trichomes. Plant trichomes are known to the person skilled in the
art, and include, but are not limited to glandular trichomes and
leaf hairs. Plant trichomes are active in plant defense (Lai et al,
2000), but especially non-glandular trichomes are also cited as
possible targets for infection (Cabo et al., 2006). Trichomes,
including glandular trichomes, are also implicated in the transport
of polar compounds across plant cuticles into the plant (Schreiber,
2005). This makes trichomes an ideal target for delivery of
agrochemicals, either by enhancing the natural defense or by
concentrating agrochemicals at the site of attack or by improved
delivery of (polar) agrochemicals into the plant. In another
embodiment, the binding domain binds to a binding site, or to an
antigen comprised in such binding site, in or on stomata. Stomata
are essential to allow CO.sub.2 to diffuse into the plant and to
minimize water loss. Stomata are also used as a major entry site
for pathogens, especially microbes (Underwood et al. 2007).
"Microbes," as used herein, means bacteria, viruses, fungi and the
like. Moreover they are directly implicated in plant defense via
specific signaling pathways allowing the plant to close stomata
upon microbial infection (Melotto et al., 2006). In yet another
embodiment, the binding domain binds to a binding site, or to an
antigen comprised in such binding site, in or on root hairs. Root
hairs are known to be important for microbial attachment to and
colonization of plants (Gage, 2004; Laus et al., 2005) and are,
therefore, an important target for the delivery of agrochemicals.
In another embodiment, the binding domain binds to a binding site,
or an antigen comprised in such binding site, in or on plant
cuticle. Plant cuticles are known to be important for microbial
attachment to and colonization of plants and to play an important
role in delivery and deposition of lipophilic agrochemicals into
the plant (Schreiber, 2005) and are therefore an important target
for the delivery and deposition of agrochemicals.
[0021] In one embodiment, the binding domain hereof is binding gum
arabic. In another embodiment, the binding domain is binding
lectins, lectin-like domains, extensins, or extensin-like domains;
more preferably, the binding domain is binding potato lectin.
Preferably, the binding domain comprises four framework regions and
three complementary determining regions, or any suitable fragment
thereof (which will then usually contain at least some of the amino
acid residues that form at least one of the complementary
determining regions); more preferably, the binding domain is
derived from a heavy chain camelid antibody, even more preferably,
the binding domain comprises a VHH sequence. Heavy chain camelid
antibodies, and the VHH-derived sequences are known to the person
skilled in the art. Camelid antibodies have been described, amongst
others in WO9404678 and in WO2007118670, incorporated herein by
reference. Still even more preferably, VHH comprises two disulphide
bridges. Most VHH molecules have only one disulphide bridge; the
presence of an additional disulphide bridge will give extra
stability to the antibody domain, which is an advantageous
characteristic for a binding domain that needs to be stable under
harsh conditions. Most preferably, VHH preferably consists of a
sequence selected from the group consisting of SEQ ID NO:1-SEQ ID
NO:42 (3A2, 3B4, 3B7, 3D10, 3D2, 3D8, 3E6, 3F5, 3F7, 3F9, 3G2, 3G4,
3H10, 3H8, 4A1, 5B5, 5B6, 5C4, 5C5, 5D4, 5E5, 5F5, 5G2, 5G5, 5H5,
7A2, 7C2, 7D2, 7E1.sub.--1, 7F1, 8B10, 8B12, 9A1, 9B5, 9C4, 9D5,
9E1, 9E4, 9F4, 9H1, 9H2 and 12H4), or any suitable fragment thereof
(which will then usually contain at least some of the amino acid
residues that form at least one of the complementary determining
regions) or homologues thereof. Homologues, as used here are
sequences wherein each or any framework region and each or any
complementary determining region shows at least 80% identity,
preferably, at least 85% identity, more preferably, 90% identity,
even more preferably, 95% identity with the corresponding region in
the reference sequence (i.e., FR1_homologue versus FR1_reference,
CDR1_homologue versus CDR1_reference, FR2_homologue versus
FR2_reference, CDR2_homologue versus CDR2_reference, FR3_homologue
versus FR3_reference, CDR3_homologue versus CDR3_reference and
FR4_homologue versus FR4_reference) as measured in a BLASTp
alignment (Altschul et al., 1997; FR and CDR definitions according
to Kabat).
[0022] A second aspect hereof is a targeting agent, capable to
retain an agrochemical on a plant and/or a plant part.
[0023] A "targeting agent," as used herein, is a molecular
structure, preferably with a polypeptide backbone, comprising at
least one binding domain. A targeting agent in its simplest form
consists solely of one single binding domain; however, a targeting
agent can comprise more than one binding domain and can be
monovalent or multivalent and monospecific or multispecific, as
further defined. Apart from one single or multiple binding domains,
a targeting agent can further comprise other moieties, which can be
either chemically coupled or fused, whether N-terminally or
C-terminally or even internally fused, to the binding domain. The
other moieties include, without limitation, one or more amino
acids, including labeled amino acids (e.g., fluorescently or
radio-actively labeled) or detectable amino acids (e.g., detectable
by an antibody), one or more monosaccharides, one or more
oligosaccharides, one or more polysaccharides, one or more lipids,
one or more fatty acids, one or more small molecules or any
combination of the foregoing. In one embodiment, the other moieties
function as spacers or linkers in the targeting agent.
[0024] "Agrochemical," as used herein, means any active substance
or principle that may be used in the agrochemical industry
(including agriculture, horticulture, floriculture and home and
garden uses, but also products intended for non-crop related uses
such as public health/pest control operator uses to control
undesirable insects and rodents, household uses, such as household
fungicides and insecticides and agents, for protecting plants or
parts of plants, crops, bulbs, tubers, fruits (e.g., from harmful
organisms, diseases or pests); for controlling, preferably
promoting or increasing, the growth of plants; and/or for promoting
the yield of plants, crops or the parts of plants that are
harvested (e.g., its fruits, flowers, seeds, etc.). Examples of
such substances will be clear to the skilled person and may, for
example, include compounds that are active as insecticides (e.g.,
contact insecticides or systemic insecticides, including
insecticides for household use), herbicides (e.g., contact
herbicides or systemic herbicides, including herbicides for
household use), fungicides (e.g., contact fungicides or systemic
fungicides, including fungicides for household use), nematicides
(e.g., contact nematicides or systemic nematicides, including
nematicides for household use) and other pesticides or biocides
(for example, agents for killing insects or snails); as well as
fertilizers; growth regulators such as plant hormones;
micro-nutrients, safeners, pheromones; repellants; insect baits;
and/or active principles that are used to modulate (i.e., increase,
decrease, inhibit, enhance and/or trigger) gene expression (and/or
other biological or biochemical processes) in or by the targeted
plant (e.g., the plant to be protected or the plant to be
controlled), such as nucleic acids (e.g., single-stranded or
double-stranded RNA, as, for example, used in the context of RNAi
technology) and other factors, proteins, chemicals, etc., known per
se for this purpose, etc. Examples of such agrochemicals will be
clear to the skilled person; and, for example, include, without
limitation: glyphosate, paraquat, metolachlor, acetochlor,
mesotrione, 2,4-D,atrazine, glufosinate, sulfosate, fenoxaprop,
pendimethalin, picloram, trifluralin, bromoxynil, clodinafop,
fluoroxypyr, nicosulfuron, bensulfuron, imazetapyr, dicamba,
imidacloprid, thiamethoxam, fipronil, chlorpyrifos, deltamethrin,
lambda-cyhalotrin, endosulfan, methamidophos, carbofuran,
clothianidin, cypermethrin, abamectin, diflufenican, spinosad,
indoxacarb, bifenthrin, tefluthrin, azoxystrobin, thiamethoxam,
tebuconazole, mancozeb, cyazofamid, fluazinam, pyraclostrobin,
epoxiconazole, chlorothalonil, copper fungicides, trifloxystrobin,
prothioconazole, difenoconazole, carbendazim, propiconazole,
thiophanate, sulphur, boscalid and other known agrochemicals or any
suitable combination(s) thereof. Other suitable agrochemicals will
be clear to the skilled person based on the disclosure herein, and
may, for example, be any commercially available agrochemical, and,
for example, include each of the compounds listed in Phillips
McDougall, AgriService November 2007 V4.0, Products Section--2006
Market, Product Index pp. 10-20. The agrochemical can occur in
different forms, including but not limited to, as crystals, as
micro-crystals, as nano-crystals, as co-crystals, as a dust, as
granules, as a powder, as tablets, as a gel, as a soluble
concentrate, as an emulsion, as an emulsifiable concentrate, as a
suspension, as a suspension concentrate, as a suspoemulsion, as a
dispersion, as a dispersion concentrate, as a microcapsule
suspension or as any other form or type of agrochemical formulation
clear to those skilled in the art. Agrochemicals not only include
active substances or principles that are ready to use, but also
precursors in an inactive form, which may be activated by outside
factors. As a non limiting example, the precursor can be activated
by pH changes, caused by plant wounds upon insect damage, by
enzymatic action caused by fungal attack, or by temperature changes
or changes in humidity.
[0025] "Plant part," as used herein, means any plant part whether
part of an intact living plant or whether isolated or separated
from an intact living plant, and even dead plant material can be
envisaged. Preferably, the plant parts are selected from the group
consisting of leaves, stem, roots, fruits, cones, flowers, bulbs
and tubers. More preferably, the plant parts are selected from the
group consisting of leaves, stem and roots. Even more preferably,
the plant is an intact living plant and/or the plant parts are
plant parts of an intact living plant.
[0026] In order to be capable to retain an agrochemical on a plant
or a plant part, either one single or multiple targeting agents are
either fused with or attached to the agrochemical, either by a
covalent bond, by hydrogen bonds, by dipole-dipole interactions, by
weak Van der Waals forces or by any combination of the foregoing.
"Attached," as used herein, means coupled to, connected to,
anchored in, admixed with or covering.
[0027] In one embodiment, the agrochemical is bound on or comprised
in a carrier, as defined above, wherein the targeting agent is
coupled either to the carrier or to the agrochemical. Preferably,
the binding domain is coupled to the carrier. "Coupled," as used
herein, can be any coupling allowing the retention of the
agrochemical or carrier containing the agrochemical by the
targeting agent; it can be a covalent as well as a non-covalent
binding. Preferably, the coupling is a covalent binding. It is
clear to the person skilled in the art how binding domains and/or
targeting agents can be coupled to any type of functional groups
present at the outer surface of a carrier. "Functional group," as
used herein, means any chemical group to which a protein can be
covalently bound, including but not limited to carboxyl-, amine-,
hydroxyl-, sulfhydryl-, or alkynyl group. As a non-limiting
example, coupling by forming of a carbodiimide bond between
carboxyl groups on the outer surface of the carrier and the
amine-groups of the binding domain and/or targeting agent can be
applied. Binding domains and/or targeting agents can be coupled
with our without linking agents to the carrier. In the case of a
microbial cell or phage, the targeting agent hereof may be encoded
by the microbial cell or phage genome, whereas the agrochemical is
contained in or coupled to the microbial cell or phage, either as
fusion protein or by chemical linking A "linking agent," as used
herein, may be any linking agent known to the person skilled in the
art; preferably, the linking agent is increasing the flexibility of
the targeting agent bound on the carrier, thereby facilitating the
binding of the binding domain comprised in the targeting agent to
the binding site on the plant. Examples of such linking agents can
be found in WO0024884 and WO0140310.
[0028] The carrier may be a microcarrier. A "microcarrier," as used
herein, is a particulate carrier where the particles are less than
500 .mu.m in diameter, preferably, less than 250 .mu.m, even more
preferably, less than 100 .mu.m, most preferably, less than 50
.mu.m. Microcarriers for delivery of agrochemicals are known to the
person skilled in the art, and include, but are not limited to
nanocapsules, microcapsules, nanospheres, microspheres, weak ionic
resin particles, polymer particles, composite gel particles,
particles made from artificially lignified cellulose, liposomes,
vesicles and cochleate delivery vehicles. It is also possible that
one or more agrochemicals are either present on or within a
microbial cell (e.g., a yeast cell) or a phage (for example,
because the one or more agrochemicals can be loaded into (or onto)
such cells or are biologicals that have been produced/expressed in
the microbial cell) or that the one or more agrochemicals are
associated (e.g., bound to or embedded in) with cell fragments
(e.g., fragments of cells walls or cell membranes), cell fractions
or other cell debris (for example, obtained by fractionating or
lysing the microbial cells into (or onto) which the one or more
agrochemicals have been loaded, produced or expressed) and that
therefore the microbial cells or phages are used as microcarriers.
As used herein microcarrier, microparticle, microsphere,
microcapsule, nanoparticle, nanocapsule and nanosphere can be used
interchangeably. Such microcarriers have been described, amongst
others, in U.S. Pat. No. 6,180,141, WO2004004453, WO2005102045 and
U.S. Pat. No. 7,494,526, incorporated here by reference.
Preferably, the microcarrier is a microparticle composed of a
natural polymer. Characteristics of microcarriers can be such that
they enable slow release of the agrochemical, delayed release of
the agrochemical or immediate release of the agrochemical, all
types of microcarriers have their specific use. Microcarriers may
naturally comprise cross-linkable residues suitable for covalent
attachment or microcarriers may be derivatized to introduce
suitable cross-linkable groups to methods well known in the art.
Such derivatization may occur prior to manufacturing of the
microcarrier, i.e., at the level of the raw materials that will be
used in the manufacturing process, it may occur during the
manufacturing process of the microcarrier or it may occur
subsequent to the manufacturing of the microcarrier. In one
specific embodiment, functional groups on the microcarrier may be
bound to a linking agent or spacer, which is on its turn bound to a
targeting agent as defined above.
[0029] In another embodiment, one or more binding domains comprised
in the targeting agent, bind to a binding site or to an antigen
comprised in such binding site, present in or on one or more
particular parts of the plant, preferably the intact living plant.
Preferably, the parts of the plant, more preferably of the intact
living plant, are selected from the group consisting of leaves,
stem, roots, fruits, cones, flowers, bulbs or tubers. More
preferably, the parts of the plant, preferably the intact living
plant, are selected from the group consisting of leaves, stem or
roots. More preferably, one or more binding domains comprised in
the targeting agent, bind to a binding site or to an antigen
comprised in such binding site, on the surface of the plant,
preferably the intact living plant. Preferably, the surface of the
plant, preferably the intact living plant, is the surface of a part
of the plant, preferably the intact living plant, selected from the
group consisting of leaf surface, stem surface, root surface, fruit
surface, cone surface, flower surface, bulb surface or tuber
surface; even more preferably, the surface of the plant, preferably
the intact living plant, is the surface of a part of the plant,
preferably the intact living plant, selected from the group
consisting of root surface, stem surface and leaf surface.
[0030] In another embodiment, one or more binding domains comprised
in the targeting agent, bind to binding site, or to an antigen
comprised in such binding site, in or on a particular structure of
the plant, preferably the intact living plant, or in or on a
particular structure of a particular part of the plant, preferably
the intact living plant; more preferably, in or on a particular
structure involved or implicated to be involved in transport of
nutrients, agrochemicals or other chemicals into the plant and/or
involved or implicated to be involved in plant defense. Preferably,
the particular structure is selected from the group consisting of
trichomes, stomata, lenticels, thorns, spines, root hairs, cuticle
and wax layer, even more preferably, the particular structure is
selected from the group consisting of trichomes, stomata and
cuticle. In one embodiment, the one or more binding domains
comprised in the targeting agent, bind to binding site, or to an
antigen comprised in such binding site, in or on plant trichomes.
In another embodiment, the one or more binding domains comprised in
the targeting agent, bind to binding site, or to an antigen
comprised in such binding site, in or on stomata. In yet another
embodiment, the one or more binding domains comprised in the
targeting agent, bind to binding site, or to an antigen comprised
in such binding site, in or on plant cuticle.
[0031] In yet another embodiment, one or more binding domains
hereof and comprised in the targeting agent, bind to gum arabic. In
another embodiment, one or more of the binding domains comprised in
the targeting agent, bind to lectins, lectin-like domains,
extensins, or extensin-like domains; more preferably, the binding
domain is binding potato lectin. Preferably, one or more of the
binding domains comprised in the targeting agent comprises four
framework regions and three complementary determining regions, or
any suitable fragment thereof (which will then usually contain at
least some of the amino acid residues that form at least one of the
complementary determining regions); more preferably, one or more of
the binding domains comprised in the targeting agent is derived
from a heavy chain camelid antibody, even more preferably, one or
more of the binding domains comprised in the targeting agent
comprises a VHH sequence. Still even more preferably, VHH comprises
two disulphide bridges. Most preferably, VHH preferably consists of
a sequence selected from the group consisting of SEQ ID NO:1-SEQ ID
NO:42 (3A2, 3B4, 3B7, 3D10, 3D2, 3D8, 3E6, 3F5, 3F7, 3F9, 3G2, 3G4,
3H10, 3H8, 4A1, 5B5, 5B6, 5C4, 5C5, 5D4, 5E5, 5F5, 5G2, 5G5, 5H5,
7A2, 7C2, 7D2, 7E1.sub.--1, 7F1, 8B10, 8B12, 9A1, 9B5, 9C4, 9D5,
9E1, 9E4, 9F4, 9H1, 9H2 and 12H4), or any suitable fragment thereof
(which will then usually contain at least some of the amino acid
residues that form at least one of the complementary determining
regions) or homologues thereof.
[0032] A third aspect hereof is the use of a targeting agent hereof
to deliver and retain an agrochemical or a combination of
agrochemicals to a plant or plant part.
[0033] Any plant part whether part of an intact living plant or
whether isolated or separated from an intact living plant, and even
dead plant material can be envisaged as a target to deliver and
retain an agrochemical or a combination of agrochemicals using a
targeting agent hereof. Preferably, the plant parts are selected
from the group consisting of leaves, stem, roots, fruits, cones,
flowers, bulbs and tubers. More preferably, the plant parts are
selected from the group consisting of leaves, stem and roots. Even
more preferably, the plant is an intact living plant and/or the
plant parts are plant parts of an intact living plant. Delivery is
carried out using any suitable or desired manual or mechanical
technique for application of an agrochemical or a combination of
agrochemicals, including but not limited to spraying, brushing,
dressing, dripping, coating, dipping, spreading, applying as small
droplets, a mist or an aerosol. As non-limiting examples, a
targeting agent hereof can be used to deliver and retain an
agrochemical or a combination of agrochemicals to the foliage of a
field grown crop, it can be used to deliver and retain an
agrochemical or a combination of agrochemicals to the roots of a
crop propagated by hydroculture, it can be used to deliver and
retain an agrochemical or a combination of agrochemicals to
harvested plant parts (e.g., fruits, flowers or seeds) as a
post-harvest treatment, it can be used to deliver and retain an
agrochemical or a combination of agrochemicals to living or dead
plant material present in the soil upon preparation of arable land,
which is particularly useful in combination with no tilling
agricultural practices, or it can be used to deliver and retain an
agrochemical to a substrate placed in the vicinity of a rhizosphere
to achieve distribution and prolonged retention of agrochemicals
throughout the rhizosphere. One particularly advantageous aspect
hereof is that it allows, by suitably choosing the combination of
targeting agent and agrochemical, or combination of agrochemicals
to formulate the same active substance for a variety of different
uses, for example, on different plant species or parts of plants,
for different environmental conditions (type of soil, amount of
rainfall and other weather conditions, or even different seasonal
conditions) and different end-uses (for example, in the field, in
greenhouses, in gardens, in hydroponic culture systems, for
possibly environmental dependent quick, delayed or slow release
use, for household use and for use by pest control operators).
Thus, by the use of the targeting agent to deliver and retain the
agrochemical, it is possible, starting from active agrochemical
substances or agrochemical formulations with proven efficacy, that
are environmentally acceptable, to provide a range of different and
improved plant protection products or agents or other agrochemical
products that are tailored for desired or intended end use. As a
non-limiting example, a broad spectrum herbicide can be made plant
species specific by delivering it using a targeting agent
comprising a plant species specific binding domain; on the other
hand, delivering the same herbicide using a targeting agent
comprising a binding domain that has a broad spectrum specificity
can help to reduce the amounts of herbicide needed to exert its
desired action. Also, undesired off-target activity of an
agrochemical, e.g., versus beneficial insects, can be avoided by
delivering the agrochemical using a targeting agent comprising a
binding domain that is highly specific for the targeted crop or for
specific parts of the targeted crop.
[0034] Preferably, the agrochemical or combination of agrochemicals
is selected from the groups consisting of herbicides, insecticides,
fungicides, nematicides, biocides, fertilizers, safeners,
micro-nutrients and plant growth regulating compounds.
[0035] Preferably, the method of delivery and retention of an
agrochemical or combination of agrochemicals results in improved
deposition of the agrochemical or combination of agrochemicals on
the plant or plant part. "Improved deposition," as used herein,
means that either the quantity of the agrochemical or combination
of agrochemicals that is bound to the plant or plant part is
increased and/or that the distribution of the agrochemical or
combination of agrochemicals is divided over the plant or plant
part either more equally or more concentrated in function of the
specificity of the binding domain comprised in the targeting agent,
when compared to the same agrochemical or combination of
agrochemicals applied without the use of any targeting agent.
[0036] In one embodiment, the agrochemical or combination of
agrochemicals is bound on or comprised in a carrier, preferably, a
microcarrier as defined earlier. This may, for example, be
particularly advantageous for an agrochemical or combination of
agrochemicals that are volatile or rapidly degradable by
environmental factors such as the presence of moisture or
UV-irradiation, or that pose a considerable toxicity hazard for the
person handling the agrochemical or combination of agrochemicals.
In one specific embodiment, functional groups on the carrier may be
bound to a linking agent or spacer, which is on its turn bound to a
targeting agent as defined above.
[0037] A fourth aspect hereof is a composition, comprising at least
(i) one targeting agent comprising at least one binding domain
hereof and (ii) an agrochemical or combination of
agrochemicals.
[0038] The targeting agent(s) comprised in the composition may
either be a "mono-specific" targeting agent or a "multi-specific"
targeting agent. By a "mono-specific" targeting agent is meant a
targeting agent that comprises either a single binding domain, or
that comprises two or more different binding domains that each are
directed against the same antigen present at or in the same binding
site or that form the binding site. Thus, a mono-specific targeting
agent is capable of binding to a single binding site, either
through a single binding domain or through multiple binding
domains. By a "multi-specific" targeting agent is meant a targeting
agent that comprises two or more binding domains that are each
directed against different antigens present at or in a binding site
or that form the binding site. Thus, a "bi-specific" targeting
agent is capable of binding to two different binding sites or
antigens present at or in a binding site or that form the binding
site; a "tri-specific" targeting agent is capable of binding to
three different antigens present at or in a binding site or that
form the binding site; and so on for "multi-specific" targeting
agents. Also, in respect of the targeting agents described herein,
the term "monovalent" is used to indicate that the targeting agent
comprises a single binding domain; the term "bivalent" is used to
indicate that the targeting agent comprises a total of two single
binding domains; the term "trivalent" is used to indicate that the
targeting agent comprises a total of three single binding domains;
and so on for "multivalent" targeting agents. Accordingly, in one
aspect, the above composition hereof comprises two or more
identical or different targeting agents, by which is meant two or
more targeting agents that, for identical targeting agents, each
bind to identical or different antigens present at or in the same
binding site, whereas for different targeting agents, at least one
binds to different antigens present at or in the same binding site
or in different binding sites.
[0039] Preferably, the targeting agent(s) comprised in the
composition, comprise at least one binding domain that binds to a
binding site or to an antigen comprised in such binding site,
present in or on one or more particular parts of a plant,
preferably of an intact living plant. Preferably, the parts of the
plant, more preferably, of the intact living plant, are selected
from the group consisting of leaves, stems, roots, fruits, cones,
flowers, bulbs or tubers. More preferably, the parts of the intact
living plant are selected from the group consisting of leaves,
stems or roots. More preferably, the targeting agent(s) comprised
in the composition, comprise at least one binding domain that binds
to a binding site or to an antigen comprised in such binding site,
on the surface of the intact living plant. Preferably, the surface
of the intact living plant is the surface of a part of the intact
living plant, selected from the group consisting of leaf surface,
stem surface, root surface, fruit surface, cone surface, flower
surface, bulb surface or tuber surface; even more preferably, the
surface of the intact living plant is the surface of a part of the
intact living plant, selected from the group consisting of root
surface, stem surface and leaf surface.
[0040] Preferably, the targeting agent(s) comprised in the
composition, comprise at least one binding domain that binds to a
binding site, or to an antigen comprised in such binding site, in
or on a particular structure of the plant, preferably the intact
living plant or in or on a particular structure of a particular
part of the plant, preferably the intact living plant; more
preferably, in or on a particular structure involved or implicated
to be involved in transport of nutrients, agrochemicals or other
chemicals into the plant and/or involved or implicated to be
involved in plant defense. Preferably, the particular structure is
selected from the group consisting of trichomes, stomata,
lenticels, thorns, spines, root hairs, cuticle and wax layer, even
more preferably, the particular structure is selected from the
group consisting of trichomes, stomata and cuticle. In one
embodiment, the targeting agent(s) comprised in the composition,
comprise at least one binding domain that binds to a binding site,
or to an antigen comprised in such binding site, in or on plant
trichomes. In another embodiment, the targeting agent(s) comprised
in the composition, comprise at least one binding domain that binds
to a binding site, or to an antigen comprised in such binding site,
in or on stomata. In yet another embodiment, the targeting agent(s)
comprised in the composition, comprise at least one binding domain
that binds to a binding site, or to an antigen comprised in such
binding site, in or on plant cuticle.
[0041] In yet another embodiment, the targeting agent(s) comprised
in the composition, comprise at least one binding domain that binds
to gum arabic. In preferred embodiment, the targeting agent(s)
comprised in the composition, comprise at least one binding domain
that binds to lectins, lectin-like domains, extensins, or
extensin-like domains; more preferably, the binding domain is
binding potato lectin. Preferably, the targeting agent(s) comprised
in the composition, comprise at least one binding domain that
comprises four framework regions and three complementary
determining regions, or any suitable fragment thereof (which will
then usually contain at least some of the amino acid residues that
form at least one of the complementary determining regions); more
preferably, one or more of the binding domains comprised in the
targeting agent is derived from a heavy chain camelid antibody,
even more preferably, one or more of the binding domains comprised
in the targeting agent comprises a VHH sequence. Still even more
preferably, VHH comprises two disulphide bridges. Most preferably,
VHH comprises, preferably, consists of a sequence selected from the
group consisting of SEQ ID NO:1-SEQ ID NO:42 (3A2, 3B4, 3B7, 3D10,
3D2, 3D8, 3E6, 3F5, 3F7, 3F9, 3G2, 3G4, 3H10, 3H8, 4A1, 5B5, 5B6,
5C4, 5C5, 5D4, 5E5, 5F5, 5G2, 5G5, 5H5, 7A2, 7C2, 7D2, 7E1.sub.--1,
7F1, 8B10, 8B12, 9A1, 9B5, 9C4, 9D5, 9E1, 9E4, 9F4, 9H1, 9H2 and
12H4), or any suitable fragment thereof (which will then usually
contain at least some of the amino acid residues that form at least
one of the complementary determining regions) or homologues
thereof.
[0042] In the composition hereof, the agrochemical or combination
of agrochemicals are preferably selected from the group consisting
of herbicides, insecticides, fungicides, nematicides, biocides,
fertilizers, safeners, micro-nutrients or plant growth regulating
compounds.
[0043] In the composition hereof, the agrochemical or combination
of agrochemicals may be in a liquid, semi-solid or solid form and,
for example, be maintained as an aerosol, flowable powder, wettable
powder, wettable granule, emulsifiable concentrate, suspension
concentrate, microemulsion, capsule suspension, dry microcapsule,
tablet or gel or be suspended, dispersed, emulsified or otherwise
brought in a suitable liquid medium (such as water or another
suitable aqueous, organic or oily medium) for storage or
application onto a plant. Optionally, the composition further
comprises one or more further components such as, but not limited
to diluents, solvents, adjuvants, surfactants, wetting agents,
spreading agents, oils, stickers, thickeners, penetrants, buffering
agents, acidifiers, anti-settling agents, anti-freeze agents,
photo-protectors, defoaming agents, biocides and/or drift control
agents or the like, suitable for use in the composition hereof.
[0044] In one embodiment, the agrochemical or combination of
agrochemicals is bound on or otherwise comprised in a carrier. In
the case of a combination of agrochemicals, each individual
agrochemical may be bound on or otherwise comprised in an
individual carrier, or a suitable combination of agrochemicals may
be jointly bound on or otherwise comprised in one carrier. As an
alternative to the use of a carrier, the agrochemical or
combination of agrochemicals may also be provided in the form of
(small) particles which are provided with a suitable coating or
(outside) layer to which the targeting agent is coupled or can bind
and which may also serve to stabilize or improve the physical
integrity or stability of the particles. As another alternative,
the agrochemical or combination of agrochemicals may be suitably
mixed with an excipient or binder to which the targeting agent is
coupled or can bind, and which may again also serve to stabilize or
improve the physical integrity or stability of the particles. Such
coated or composite particles are preferably in the form of a
slurry, wet cake or free-flowable powder, tablet, capsule or liquid
concentrate (such as an emulsion, suspension or dispersion).
[0045] In one embodiment, the composition hereof is for
agrochemical use. "Agrochemical use," as used herein, not only
includes the use of agrochemicals as defined above (for example,
pesticides, growth regulators, nutrients/fertilizers, repellants,
defoliants, etc.) that are suitable and/or intended for use in
field grown crops (e.g., agriculture), but also includes the use of
agrochemicals as defined above (for example, pesticides, growth
regulators, nutrients/fertilizers, repellants, defoliants, etc.)
that are meant for use in greenhouse grown crops (e.g.,
horticulture/floriculture) or hydroponic culture systems and even
the use of agrochemicals as defined above that are suitable and/or
intended for non-crop uses such as uses in private gardens,
household uses (for example, herbicides or insecticides for
household use), or uses by pest control operators (for example,
weed control, etc.).
[0046] Based on the teaching set out in the present specification,
and, for example, depending on the agrochemical(s) to be delivered,
on the part(s) to the plant to which the agrochemical(s) is to be
delivered, and the intended agrochemical action of the composition
hereof (and/or the agrochemical(s) included therein), the skilled
person will be able to suitably select the specific binding
domains/targeting agent that can/should be present in the
composition hereof (as well as the other components of the
composition, such as the carrier, the agrochemical and the
agrochemical form/formulation) in order to achieve the
desired/intended agrochemical action. Thus, with advantage, based
on the disclosure herein, it is possible for the skilled person to
suitably select a suitable combination of binding
domain(s)/targeting agent(s), agrochemical(s), carrier, further
components of the composition and the agrochemical form/formulation
of the composition in order to achieve the intended/desired
agrochemical action. In this respect, it should be noted that, as
currently contemplated, and although it is foreseen that some such
combinations will be more efficacious and/or more preferred than
others, there will likely be multiple such combinations possible
that will give the intended/desired agrochemical action to the more
or less same degree. This also allows the skilled person to take
into account other (secondary) factors when selecting the
combination to be used, such as the specific crop(s) to be
protected, the prevalent field, soil, weather and/or other
environmental conditions, the way that composition is preferably
applied, the environment in which it is applied (field, greenhouse,
etc.), the desired persistence and/or other factors that may
influence the choice of an agrochemical composition for a specific
application.
[0047] For example, and without limitation, when the composition
hereof is intended to bind to one or more specific parts of the
plant, the targeting agent (i.e., the one or more binding domains
present therein) are preferably directed towards one or more
binding sites (as defined herein) that are present (i.e., in a
sufficient amount) in/on the part(s) of the plant (it also being
possible that such binding site(s) are present in/on the part(s) of
the plant in a larger amount(s)/to a greater degree than on other
part(s) of the plant, i.e., so as to provide a binding
domain/targeting agent/composition hereof that can preferentially
bind to the intended/desired part(s) of the plant compared to one
or more other parts of the plant); and compositions hereof that
comprise such binding domains/targeting agents (i.e., such that the
compositions are directed towards binding sites present in the
desired part(s) of the plant and, preferably, such that they can
bind preferentially to the desired part(s) of the plant) form some
specific but non-limiting aspects hereof. For example, and without
limitation: [0048] for a composition hereof that is intended to
bind to the leaves of a plant, the binding domains and/or targeting
agent may be directed against one or more of the following binding
sites on (the leaves of) a plant: cutin, cuticular waxes,
arabinogalactan-proteins or lipid transfer proteins; [0049] for a
composition hereof that is intended to bind to the roots of a
plant, the binding domains and/or targeting agent may be directed
against one or more of the following binding sites on (the roots
of) a plant: extensins or pectins; [0050] for a composition hereof
that is intended to bind to the stem of a plant, the binding
domains and/or targeting agent may be directed against one or more
of the following binding sites on (the stem of) a plant: lignins,
extensins or excretion products; and each such composition hereof
forms a specific, but non-limiting aspect hereof.
[0051] A fifth aspect hereof is a composition, comprising at least
(i) one targeting agent comprising at least one binding domain
hereof and (ii) a carrier.
[0052] The targeting agent(s) comprised in the composition may
either be mono-specific targeting agents or multi-specific
targeting agents and may be either monovalent targeting agents or
multivalent targeting agents. Accordingly, in one aspect, the above
composition hereof comprises two or more identical or different
targeting agents, by which is meant two or more targeting agents
that, for identical targeting agents, each bind to identical or
different antigens present at or in the same binding site, whereas
for different targeting agents, at least one binds to different
antigens present at or in the same binding site or in different
binding sites.
[0053] In one specific embodiment, which is preferred but
non-limiting, the carrier is such that it allows the composition
hereof to be suitably applied to the intended site of action,
and/or such that it allows the composition hereof to be formulated
such that it can be suitably applied to the intended site of
action; using any suitable or desired manual or mechanical
technique such as spraying, brushing, dripping, dipping, coating,
spreading, applying as small droplets, a mist or an aerosol, etc.
Examples of such techniques, of compositions hereof that are
suitable for use in such techniques, and of methods for making and
formulating such compositions hereof will be clear to the skilled
person based on the disclosure herein. Preferably, the carrier is
such that one or more active substances can be incorporated,
encapsulated or included into the carrier, e.g., as a nanocapsule,
microcapsule, nanosphere, micro-sphere, liposome or vesicle. Even
more preferably, the carrier is such that upon such incorporation,
encapsulation, embedding or inclusion, the complex thus obtained
can be suspended, dispersed, emulsified or otherwise brought into a
suitable liquid medium (such as water or another suitable aqueous,
organic or oily medium) so as to provide a (concentrated) liquid
composition hereof that has a stability that allows the composition
hereof to be suitably stored or (where necessary after further
dilution) applied to the intended site of action. Even more
preferably, the carrier is such that the composition hereof can be
transported and/or stored prior to final use, optionally (and
usually preferably) as a suitable liquid concentrate, dry powder,
tablet, capsule, slurry or "wet cake," which can be suitably
diluted, dispersed, suspended, emulsified or otherwise suitably
reconstituted by the end user prior to final use (and such
concentrates form a further aspect hereof). Carriers, preferably
microcarriers, suitable for this purpose (and methods for
absorbing, encapsulating, embedding, etc., the active principles
therein) will be clear to the skilled person based on the
disclosure herein and/or may be commercially available. Some
non-limiting examples include solid or semi-solid microspheres or
granulates in which the active ingredients are embedded or absorbed
in a suitable matrix material or microcapsules comprising a shell
material that surround a core that contains the active ingredient
(i.e., encapsulated within the microcapsule).
[0054] Preferably, the carriers are such that they have immediate,
delayed, gradual, triggered or slow release characteristics, for
example, over several minutes, several hours, several days or
several weeks. Also, the carriers may be made of materials (e.g.,
polymers) that rupture or slowly degrade (for example, due to
prolonged exposure to high or low temperature, high or low pH,
sunlight, high or low humidity or other environmental factors or
conditions) over time (e.g., over minutes, hours, days or weeks) or
that rupture or degrade when triggered by particular external
factors (such as high or low temperature, high or low pH, high or
low humidity or other environmental factors or conditions) and so
release the active agent from the microcapsule. The carrier is
also, preferably, such that the agrochemicals are released from the
carrier when the composition hereof is applied to the intended site
of action, i.e., at a rate that is sufficient to provide the
desired action of the agrochemicals during the desired period of
time (e.g., the time between two applications of the composition
hereof).
[0055] In one particular embodiment, the carrier, preferably the
microcarrier, may be composed of polymer materials, such as, for
example, poly-urethane, poly-urea, poly-amide, poly-ethylene,
polyethylene-glycol, polyvinyl alcohols, melamine,
urea/formaldehyde, acrylic polymers, nylon, vinyl acetate or
siloxane polymers or--optionally (and usually preferably) for
agrochemical purposes--biodegradable polymers (such as, for
example, agar, gelatin, alginates, gums, pectins, poly-alcohols
such as cetyl-alcohol, oily substances such as hydrogenated palm
oil or soybean oil, starches, waxes, etc. Alternatively, and
although this is usually less preferred, non-biodegradable
materials may be used such as poly-methylacrylates,
poly-ethersulfones, metal oxides, carbon structures, etc.
[0056] Preferably, the carrier is selected from the group
consisting of nanocapsules, nanospheres, microcapsules,
microspheres, polymer particles, particles made from artificially
lignified cellulose, composite gel particles, weak ionic resin
particles, microbial cells or fragments thereof. More preferably,
the carrier is selected from the group consisting of microcapsules,
microspheres or polymer particles. Most preferably, the carrier is
a microcapsule.
[0057] In one embodiment, the targeting agent(s) comprised in the
composition, comprise at least one binding domain that comprises
four framework regions and three complementary determining regions,
or any suitable fragment thereof (which will then usually contain
at least some of the amino acid residues that form at least one of
the complementary determining regions); more preferably, one or
more of the binding domains comprised in the targeting agent is
derived from a heavy chain camelid antibody, even more preferably,
one or more of the binding domains comprised in the targeting agent
comprises a VHH sequence. Still even more preferably, VHH comprises
two disulphide bridges. Most preferably, VHH comprises, preferably,
consists of a sequence selected from the group consisting of SEQ ID
NO:1-SEQ ID NO:42 (3A2, 3B4, 3B7, 3D10, 3D2, 3D8, 3E6, 3F5, 3F7,
3F9, 3G2, 3G4, 3H10, 3H8, 4A1, 5B5, 5B6, 5C4, 5C5, 5D4, 5E5, 5F5,
5G2, 5G5, 5H5, 7A2, 7C2, 7D2, 7E1.sub.--1, 7F1, 8B10, 8B12, 9A1,
9B5, 9C4, 9D5, 9E1, 9E4, 9F4, 9H1, 9H2 and 12H4), or any suitable
fragment thereof (which will then usually contain at least some of
the amino acid residues that form at least one of the complementary
determining regions) or homologues thereof.
[0058] In another embodiment, the targeting agent and the carrier
comprised in the composition hereof are coupled to each other.
Preferably, the one single targeting agent or multiple targeting
agents are coupled to the carrier by affinity binding or by
covalent binding. More preferably, the one single targeting agent
or multiple targeting agents, are coupled to the carrier by
covalent binding. Preferably, the one single targeting agent or
multiple targeting agents are coupled, preferably covalently
coupled, to the carrier by the use of a functional group present on
the outer surface of the carrier. Preferably, the binding domain
comprised in the targeting agent(s) is coupled, preferably
covalently coupled, to the carrier. Alternatively, the one single
targeting agent or multiple targeting agents are coupled,
preferably covalently coupled, to the carrier via a moiety that is
not the binding domain comprised in the targeting agent.
[0059] In yet another embodiment, the carrier is coupled to and/or
comprises at least one agrochemical as defined above. Preferably,
the agrochemical is selected from the group consisting of
herbicides, insecticides, fungicides, nematicides, biocides,
fertilizers, micro-nutrients, safeners or plant growth regulating
compounds. In this embodiment, the composition is for agrochemical
use.
[0060] The carrier with the one or more targeting agents bound,
coupled or otherwise attached thereto or associated therewith may
be dissolved, emulsified, suspended or dispersed or otherwise
included into a suitable liquid medium (such as water or another
aqueous, organic or oily medium) so as to provide a (concentrated)
solution, suspension, dispersion or emulsion that is suitable for
storage.
[0061] For example, when the composition hereof is intended for
agrochemical use, the composition hereof may be in a liquid,
semi-solid or solid form that is suitable for spraying, such as a
solution, emulsion, suspension, dispersion, aerosol, flowable
powder or any other suitable form. In particular, such a
composition hereof for agrochemical use may comprise a
microcapsule, microsphere, nanocapsule, nanosphere, liposomes or
vesicles, etc., in which the one or more agrochemicals are suitably
encapsulated, enclosed, embedded, incorporated or otherwise
included; and one or more targeting agents that each comprise one
or more binding domains for binding to one or more antigens present
at or in the binding site or that form the one or more binding
sites on a plant or parts of a plant, such as a leaf, stem, flower,
fruit, bulb or tuber of a plant).
[0062] A sixth aspect hereof is a method for delivery of an
agrochemical or a combination of agrochemicals to a plant, the
method comprising at least one application of a composition hereof
to the plant.
[0063] "One application," as used herein, means a single treatment
of a plant or plant part. According to this method, either the
composition hereof is applied as such to the plant or plant part,
or the composition is first dissolved, suspended and/or diluted in
a suitable solution before being applied to the plant. The
application to the plant is carried out using any suitable or
desired manual or mechanical technique for application of an
agrochemical or a combination of agrochemicals, including but not
limited to spraying, brushing, dressing, dripping, dipping,
coating, spreading, applying as small droplets, a mist or an
aerosol. Upon such application to a plant or part of a plant, the
composition can bind at or to the binding site (or to one or more
antigens present at or in the binding site or that form the binding
site) via one or more binding domains that form part of the
targeting agent(s) comprised in the composition, preferably in a
targeted manner. Thereupon, the agrochemicals are released from the
carrier (e.g., due to degradation of the carrier or passive
transport through the wall of the carrier) in such a way that they
can provide the desired agrochemical action(s). A particular
advantage of delivering an agrochemical or combination of
agrochemicals to a plant using a composition hereof is that it may
lead to an improved deposition (as defined earlier) of the
agrochemical or combination of agrochemicals on the plant or plant
part and/or an increased resistance of the agrochemical or
combination of agrochemicals against loss due to external factors
such as rain, irrigation, snow, hail or wind.
[0064] In one embodiment, delivering an agrochemical or combination
of agrochemicals to a plant using a composition hereof results in
improved rainfastness of the agrochemical or combination of
agrochemicals. "Improved rainfastness," as used herein, means that
the percentage loss of agrochemical or combination of
agrochemicals, calculated before and after rain, is smaller when
the agrochemical or combination of agrochemicals is applied in a
composition hereof, compared with the same agrochemical or
combination of agrochemicals comprised in a comparable composition,
without any targeting agent. A "comparable composition," as used
herein, means that the composition is identical to the composition
hereof, apart from the absence of the targeting agent used in the
composition hereof.
[0065] In one embodiment, a suitable dose of the agrochemical or
combination of agrochemicals comprised in a composition hereof is
applied to the plant or plant part. A "suitable dose," as used
herein, means an efficacious amount of active substance of the
agrochemical comprised in the composition.
[0066] Preferably, the method comprises the application of a
meaningfully reduced dose of an agrochemical or combination of
agrochemicals to the plant, to obtain similar beneficial effects
for the agrochemical or combination of the agrochemicals, as
compared with the application of the same agrochemical or
combination of agrochemicals comprised in a comparable composition,
as defined above, without any targeting agent. The meaningful
reduction is obtained by directing the agrochemical to the plant
using targeting agents hereof. Alternatively, the method comprises
an application of a suitable dose, wherein the application
frequency is meaningfully reduced, to obtain similar beneficial
effects for the agrochemical, compared with the frequency of
application of the same dose of an encapsulated composition of the
agrochemical lacking the presence of a targeting agent hereof. Even
more preferably, the method comprises an application wherein the
suitable dose as well as the application frequency are both
significantly reduced to obtain similar beneficial effects for the
agrochemical, compared with the suitable dose and application
frequency of a an encapsulated composition of the agrochemical
lacking the presence of a targeting agent hereof.
[0067] A seventh aspect hereof is a method for protecting a plant
against external (biotic or abiotic) stress and/or to modulate the
viability, growth or yield of a plant or plant parts and/or to
modulate gene expression in a plant or plant part resulting in
alteration of (levels of) plant constituents (such as proteins,
oils, carbohydrates, metabolites, etc.), the method comprising at
least one application of a composition hereof. If needed, the
composition is dissolved, suspended and/or diluted in a suitable
solution. "Protecting a plant," as used here, is the protection of
the plant against any stress; the stress may be biotic stress, such
as, but not limited to, stress caused by weeds, insects, rodents,
nematodes, mites, fungi, viruses or bacteria, or it may be abiotic
stress, such as but not limited to drought stress, salt stress,
temperature stress or oxidative stress.
[0068] Preferably, the method comprises the application of a
meaningfully reduced dose of an agrochemical or combination of
agrochemicals to the plant, to obtain similar beneficial effects
for the agrochemical or combination of the agrochemicals, as
compared with the application of the same agrochemical or
combination of agrochemicals comprised in a comparable composition,
as defined earlier, without any targeting agent. The meaningful
reduction is obtained by directing the agrochemical to the plant
using targeting agents hereof. Alternatively, the method comprises
an application of a suitable dose, wherein the application
frequency is meaningfully reduced, to obtain similar beneficial
effects for the agrochemical, compared with the frequency of
application of the same dose of an encapsulated composition of the
agrochemical lacking the presence of a targeting agent hereof. Even
more preferably, the method comprises an application wherein the
suitable dose as well as the application frequency are both
significantly reduced to obtain similar beneficial effects for the
agrochemical, compared with the suitable dose and application
frequency of an encapsulated agrochemical lacking the presence of a
targeting agent hereof.
[0069] An eighth aspect hereof is a method for manufacturing a
specifically targeting agrochemical carrier, the method comprising
(a) packing an agrochemical in or on(to) a carrier and (b)
attaching at least one targeting agent hereof to the carrier.
[0070] "Packing," as used herein, means incorporating, including,
immobilizing, adsorbing, absorbing, binding, encapsulating,
embedding, attaching, admixing, anchoring or comprising. Methods
for packing an agrochemical, as defined above, in or on(to) a
carrier are known to the person skilled in the art and include,
without limitation, drip-casting, extrusion granulation, fluid bed
granulation, co-extrusion, spray drying, spray chilling,
atomization, addition or condensation polymerization, interfacial
polymerization, in situ polymerization, coacervation, spray
encapsulation, cooling melted dispersions, solvent evaporation,
phase separation, solvent extraction, sol-gel polymerization, high
or low shear mixing, fluid bed coating, pan coating, melting,
passive or active absorption or adsorption. In one preferred, but
not limiting, embodiment, an agrochemical is packed into a
microcarrier using suitable microencapsulation techniques, such as
interfacial polymerization, in situ polymerization, coacervation,
spray encapsulation, cooling melted dispersions, solvent
evaporation, phase separation, solvent extraction or sol-gel
polymerization. Preferred, but non-limiting examples of suitable
materials for producing such microcarriers are materials such as
alginates, agar, gelatin, pectins, gums, hydrogenated oils,
starches, waxes, polyalcohols, poly-urea, poly-urethane,
poly-amide, melamine, urea/formaldehyde, nylon and other
(optionally and usually preferred biodegradable or inert) polymers.
More preferably, at least one functional group is present at the
outer surface of the microcarrier.
[0071] At least one targeting agent hereof is attached to the
carrier, either by a covalent bond, by hydrogen bonds, by
dipole-dipole interactions, by weak Van der Waals forces or by a
combination of any of the foregoing. Attachment of the targeting
agent to the carrier may be performed while packing the
agrochemical in or on(to) the carrier, it may be performed
subsequent to packing of the agrochemical in or on(to) the carrier
or it may be performed only at the time the agrochemical containing
carrier is dissolved in a suitable solution for application.
Suitable processes for attaching the targeting agent to a carrier
will be clear to the person skilled in the art. In one embodiment,
the targeting agent and the carrier are coupled to each other.
Preferably, the targeting agent(s) are coupled to the carrier by
affinity binding or by covalent binding. More preferably, the
targeting agent(s) are coupled to the carrier by covalent binding.
Preferably, the targeting agent(s) are coupled, preferably
covalently coupled, to the carrier by the use of a functional group
present on the outer surface of the carrier. Preferably, the
binding domain comprised in the targeting agent(s) is coupled,
preferably covalently coupled, to the carrier. Alternatively, the
targeting agent(s) are coupled, preferably covalently coupled, to
the carrier via a moiety that is not the binding domain comprised
in the targeting agent. In one embodiment, the process for
attaching the targeting agent(s) to a carrier comprises (a)
reacting a linking agent with a carrier, and (b) reacting at least
one targeting agent with the linking agent.
[0072] A ninth aspect hereof is a process for attaching a targeting
agent hereof to a carrier, comprising (a) reacting a linking agent
with a carrier, and (b) reacting the targeting agent with the
linking agent. "Reacting," as used herein, means that the linking
agent is placed in conditions allowing the binding of the linking
agent to the carrier and/or the targeting agent.
[0073] A tenth aspect hereof is a specifically targeting
agrochemical carrier, obtained by the above described method.
"Specifically targeting," as used herein, means that the carrier
can bind specifically to a binding site on a plant or on a plant
part, through at least one targeting agent hereof, which is
attached, preferably coupled, most preferably covalently bound, to
the carrier.
[0074] A last aspect hereof is the use of any binding domain hereof
to isolate amino acid sequences that are responsible for specific
binding to the binding site or to an antigen comprised in the
binding site and to construct artificial binding domains based on
the amino acid sequences. Indeed, in the binding domains hereof,
the framework regions and the complementary determining regions are
known, and the study of derivatives of the binding domain, binding
to the same binding site or antigen comprised in the binding site,
will allow deducing the essential amino acids involved in binding
the binding site or antigen comprised in the binding site. This
knowledge can be used to construct a minimal binding domain and to
create derivatives thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0075] FIG. 1: Binding domains (VHH) binding to leaf surface.
[0076] FIG. 1A: VHH3E6 5 .mu.g/ml in PBS binding to native potato
leaf surface. Detection with anti-histidine antibodies directly
conjugated with Alexa-488 fluorescent dye. VHH 3E6 is binding leaf
surface, stomata, glandular trichomes, and leaf hairs.
[0077] FIG. 1B: VHH3E6 5 .mu.g/ml in PBS binding to native potato
leaf surface. Detection with anti-histidine antibodies directly
conjugated with Alexa-488 fluorescent dye; Imaging with a Leica SP5
confocal microscope system. VHH 3E6 is binding leaf surface,
stomata, glandular trichomes, and leaf hairs.
[0078] FIG. 1C: VHH5D4 5 .mu.g/ml in PBS binding to native potato
leaf surface. Detection with anti-histidine antibodies directly
conjugated with Alexa-488 fluorescent dye. VHH 5D4 is binding leaf
surface, stomata, glandular trichomes, and leaf hairs.
[0079] FIG. 1D: CBM3a 5 .mu.g/ml in PBS binding to wounded plant
tissue on the edge of a potato leaf disc. Detection with
anti-histidine antibodies directly conjugated with Alexa-488
fluorescent dye. CBM3a is not binding leaf surface, stomata,
glandular trichomes, or leaf hairs, but only binding to wounded
plant tissue on the edge of a potato leaf disc that is exposed from
preparing the sample by punching the leaf.
[0080] FIG. 1E: Without primary antibody (plain PBS) on native
potato leaf surface. Incubation with anti-histidine antibodies
directly conjugated with Alexa-488 fluorescent dye.
[0081] FIG. 1F: VHH3E6 5 .mu.g/ml in PBS binding to native black
nightshade leaf surface. Detection with anti-histidine antibodies
directly conjugated with Alexa-488 fluorescent dye. VHH 3E6 is
binding leaf surface, glandular trichomes, and leaf hairs.
[0082] FIG. 1G: VHH3E6 5 .mu.g/ml in PBS binding to native grass
leaf surface. Detection with anti-histidine antibodies directly
conjugated with Alexa-488 fluorescent dye. VHH 3E6 is binding to
leaf surface and wounded plant tissue on the edge of a potato leaf
disc that is exposed from preparing the sample by punching the
leaf.
[0083] FIG. 2: Binding of binding domains (VHH) to intact living
plant
[0084] FIG. 2A: VHH3E6 5 .mu.g/ml in PBS binding to an intact
living plant. Leaves attached to a potato pot plant were submerged
in a solution of VHH 3E6. Leaves were sampled. Detection with
anti-histidine antibodies directly conjugated with Alexa-488
fluorescent dye. VHH 3E6 is binding leaf surface, stomata,
glandular trichomes, and leaf hairs.
[0085] FIG. 2B: VHH3E6 5 .mu.g/ml in PBS binding to an intact
living plant. Leaves attached to a potato pot plant were submerged
in a solution of VHH 3E6. Leaves were sampled. Detection with
anti-histidine antibodies directly conjugated with Alexa-488
fluorescent dye. Excerpt from whole leaf labeling. VHH 3E6 is
binding leaf surface, stomata, glandular trichomes, and leaf
hairs.
[0086] FIG. 3: Coupling of binding domains to microcapsules
[0087] FIG. 3A: Microcapsules with coupled VHH3E6 through one-step
EDC coupling chemistry. Coupled microcapsules were labeled with
anti-histidine antibodies directly conjugated with Alexa-488
fluorescent dye. Imaging with a Leica SP5 confocal microscope
system. VHH 3E6 is coupled to the microcapsule surface through
one-step coupling chemistry.
[0088] FIG. 3B: Microcapsules with coupled VHH3E6 through two-step
EDC/NHS coupling chemistry. Coupled microcapsules were labeled with
anti-histidine antibodies directly conjugated with Alexa-488
fluorescent dye. Imaging with a Leica SP5 confocal microscope
system. VHH 3E6 is coupled to the microcapsule surface through
two-step EDC/NHS coupling chemistry.
[0089] FIG. 3C: Microcapsules incubated with VHH3E6 without
covalent coupling. Passively adsorbed VHH were labeled with
anti-histidine antibodies directly conjugated with Alexa-488
fluorescent dye. Imaging with a Leica SP5 confocal microscope
system. A minor fraction of VHH 3E6 is passively adsorbed to the
microcapsule surface.
[0090] FIG. 3D: Control condition with microcapsules not incubated
with VHH but only with anti-histidine antibodies directly
conjugated with Alexa-488 fluorescent dye. Imaging with a Leica SP5
confocal microscope system. A minor fraction of VHH 3E6 is
passively adsorbed to the microcapsule surface.
[0091] FIG. 4: Binding and retention of microcapsules to leaf
surface. Leaf disc binding assay on native potato leaf discs with
microcapsules containing a fluorescent tracer molecule. Binding and
retention of microcapsules coupled with specific plant-binding VHH,
coupled with unrelated control VHH, or blank microcapsules is
compared. Nine-fold more microcapsules coupled with specific VHH
are binding and retained on potato leaf discs compared to blank
microcapsules.
[0092] FIG. 5: Reduction of dosis using microcapsules coupled with
targeting agents. Leaf disc binding assay on native potato leaf
discs with microcapsules containing a fluorescent tracer molecule.
Binding and retention of microcapsules in different concentrations
and coupled with specific plant-binding VHH, coupled with unrelated
control VHH, or blank microcapsules is compared. Up to eight-fold
more microcapsules coupled with specific VHH are binding and
retained on potato leaf discs compared to blank microcapsules.
DETAILED DESCRIPTION OF THE DISCLOSURE
Examples
Example 1
Generation and Selection of VHH
[0093] Immunization of Llamas with Gum Arabic, Potato Leaf
Homogenate, or Wheat Leaf Homogenate
[0094] A solution of gum arabic was prepared by weighing 5 g of gum
arabic from acacia tree (Sigma) and dissolving in 50 ml water.
Bradford protein assay was used to determine the total protein
concentration. Aliquots were made, stored at -80.degree. C., and
used for immunization.
[0095] Homogenized leaves from potato plants (Solanum tuberosum
variety Desiree) or wheat plants (Triticum aestivum variety Boldus)
were prepared by freezing leaves in liquid nitrogen and
homogenizing the leaves with mortar and pestle until a fine powder
was obtained. Bradford protein assay was used to determine the
total protein concentration. Aliquots were made, stored at
-80.degree. C., and suspensions were used for immunization.
[0096] Llamas were immunized at weekly intervals with six
intramuscular injections of gum arabic, homogenized potato leaves,
or homogenized wheat leaves, according to standard procedures. Two
Llamas, "404334" and "Lahaiana," were immunized with gum arabic.
Three llamas, "407928," "Chilean Autumn," and "Niagara," were
immunized with homogenized potato leaves and another two llamas,
"33733" and "Organza," were immunized with homogenized wheat
leaves. Llamas "404334," "407928," and "33733" were immunized using
Adjuvant LQ (Gerbu), and llamas "Lahaiana," "Chilean Autumn,"
"Niagara" and "Organza" were immunized using Freund's Incomplete
Adjuvant (FIA). Doses for immunization of llama "404334" were 350
.mu.g for each day 0, 7, 14, 21, 28, 35, and peripheral blood
lymphocytes (PBL) were collected at day 40. Doses for immunizations
of llamas "407928" and "33733" were 1 mg for each day 0, 7, 14, 21,
28, 36, and PBL were collected at day 40. At time of PBL collection
at day 40, sera of llamas "404334," "407928," and "33733" were
collected. Doses for immunizations of llamas "Lahaiana," "Chilean
Autumn," "Niagara," and "Organza" were 100 .mu.g for day 0, and 50
.mu.g for days 7, 14, 21, 28, and 35. At day 0, day 25, and at time
of PBL collection at day 38, sera of llamas "Lahaiana," "Chilean
Autumn," "Niagara," and "Organza" were collected.
[0097] Library Construction--
[0098] From each immunized llama a separate VHH library was made.
RNA was isolated from peripheral blood lymphocytes, followed by
cDNA synthesis using random hexamer primers and Superscript III
according to the manufacturer's instructions (Invitrogen). A first
PCR was performed to amplify VHH and VH using a forward primer mix
[1:1 ratio of call001 (5'-gtcctggctgctcttctacaagg-3' (SEQ ID
NO:43)) and call001b (5'-cctggctgctcttctacaaggtg-3' (SEQ ID
NO:44))] and reverse primer call002 (5'-ggtacgtgctgttgaactgttcc-3'
(SEQ ID NO:45)). After isolation of the VHH fragments a second PCR
was performed using forward primer A6E
(5'-gatgtgcagctgcaggagtctggrggagg-3' (SEQ ID NO:46)) and reverse
primer 38 (5'-ggactagtgcggccgctggagacggtgacctgggt-3' (SEQ ID
NO:47)). The PCR fragments were digested using PstI and Eco91I
restriction enzymes (Fermentas), and ligated upstream of the pIII
gene in vector pMES4 (GenBank: GQ907248.1). The ligation products
were ethanol precipitated according to standard protocols,
resuspended in water, and electroporated into TG1 cells. Library
sizes ranged from 1E+08 to 6E+08 independent clones. Single colony
PCR on randomly picked clones from the libraries was performed to
assess insert percentages of the libraries. All libraries had
.gtoreq.90% insert percentages except for the library from
immunized llama "Organza" which had an insert percentage of 80%.
Libraries were numbered 25, 27, 28, 29, 30, 31, 32 for llamas
"404334," "407928," "33733," "Chilean Autumn," "Lahaiana,"
"Niagara," and "Organza," respectively. Phage from each of the
libraries were produced using VCSM13 helper phage according to
standard procedures.
Phage Selections Against Gum Arabic, Plant Epidermal Extracts, or
Whole Leaves.
[0099] A solution of gum arabic was prepared by weighing 5 g of gum
arabic and dissolving in 50 ml water. Aliquots were made and stored
at -20.degree. C. until use.
[0100] Extracts of potato plant cuticle and adhering epidermis were
prepared from thin strips from stems of potato plants. Extracts of
wheat plant cuticle and adhering epidermis were prepared from thin
strips from wheat sheath leaves. Extracts enriched in cell-wall
glycans and non-cellulosic polysaccharides were sequentially
extracted using CDTA and NaOH (Moller et al., 2007), respectively.
Strips were frozen in liquid nitrogen and ground with mortar and
pestle until fine powders were obtained. Cell-wall glycans-enriched
extracts were prepared by resuspending the fine powders in 50 mM
CDTA pH6.5 using 10 ml per gram of ground material and
head-over-head rotation at 4.degree. C. for 30 minutes. Extract and
insoluble material were separated using a syringe adapted with a
filter. The extracts were further cleared by centrifugation in a
micro centrifuge at 20,000 g for 5 minutes. Non-cellulosic
polysaccharide-enriched extracts were prepared from the insoluble
material after CDTA extraction in 4 M NaOH and 1% NaBH.sub.4 using
10 ml per gram of insoluble material and head-over-head rotation at
4.degree. C. for 30 minutes. Extract and insoluble material were
separated using a syringe adapted with a filter. The extracts were
further cleared by centrifugation in a micro centrifuge at 20,000 g
for 5 minutes.
[0101] First round selections against gum arabic were performed in
wells of a 96-well plate (Maxisorp, Nunc) coated with 1 mg/ml or 10
.mu.g/ml gum arabic in 0.1 M carbonate buffer pH 8.3. Coatings were
performed at 4.degree. C. overnight. Wells were washed three times
with PBS/0.05%-TWEEN.RTM.-20 and blocked with 5% skimmed milk in
PBS (5% MPBS). Phage were suspended in 2.5% MPBS and approximately
2E+11 cfu were used for each well. After binding to the wells at
room temperature for 2 hours, unbound phage were removed by
extensive washing with PBS/0.05%-TWEEN.RTM.-20 and PBS. Bound phage
were eluted at room temperature with 0.1 mg/ml trypsin (Sigma) in
PBS for 30 minutes. Eluted phage were transferred to a
polypropylene 96-well plate (Nunc) containing excess AEBSF trypsin
inhibitor (Sigma). The titers of phage from target-coated wells
were compared to titers of phage from blank wells to assess
enrichments. Phage were amplified using fresh TG1 cells according
to standard procedures.
[0102] The second selection round was performed similarly to the
first selection round except that for libraries 25 and 30 wells
were coated with 10 .mu.g/ml and 0.1 .mu.g/ml gum arabic instead of
1 mg/ml and 10 .mu.g/ml. No significant enrichments were obtained
for libraries 27, 28, 29, 31, and 32 in selection round 1. In
selection round 2, enrichments were >1000-fold for libraries 28,
31, and 32, and 25-fold and 250-fold for libraries 27 and 29,
respectively. Enrichments for libraries 25 and 30 were 50-fold and
>1000-fold in selection round 1, respectively. In selection
round 2, enrichments were 1000-fold for both libraries. Selections
against potato epidermal CDTA extract were performed similarly to
the selections against gum arabic but wells were coated with
ten-fold and 1000-fold diluted potato epidermal CDTA extract for
both the first and second selection rounds. Enrichments in
selection round 1 were 10, 1E+03, 20, 20, >1E+04, 15, and
five-fold for libraries 25, 27, 28, 29, 30, 31, 32, respectively
and >100-fold for all libraries in selection round 2. Selections
against wheat epidermal CDTA extract were performed similarly to
the selections against potato epidermal CDTA extract but wells were
coated with 20-fold and 2000-fold diluted wheat epidermal CDTA
extract for both the first and second selection rounds. Enrichments
in selection round 1 were >10, >100, >10, 1, >1E+03,
10, and five-fold for libraries 25, 27, 28, 29, 30, 31, 32,
respectively. Enrichments in selection round 2 were >ten-fold
for library 29 and >100-fold for libraries 25, 27, 28, 30, 31,
and 32. Selections against potato leaves were performed in two
consecutive selection rounds using leaf particles in round 1 and
whole leaves in round 2. Libraries 27, 28, 29, 30, 31, and 32 were
used for selections against leaves. The leaf particles for first
round selections were prepared by blending potato leaves in PBS
using an Ultra-Turrax T25 homogenizer. The leaf particles were
collected from the suspension by centrifugation. The supernatant,
called herein "homogenized leaf soluble fraction," is assumingly
enriched in intracellular components and was used in solution
during phage selection to compete out binders to intracellular
epitopes. Library phage were pre-incubated with the homogenized
leaf soluble fraction in 2% MPBS using head-over-head rotation at
room temperature for 30 minutes. The mixtures were added to leaf
particles and incubated with head-over-head rotation at room
temperature for 2 hours. Leaf particles with bound phage were
collected by centrifugation and supernatants were discarded. Leaf
particles with bound phage were washed extensively by consecutive
washes with PBS. Washes were performed by resuspending leaf
particles in PBS, spinning down leaf particles, and discarding
supernatants. Elution of phage and infection of TG1 were performed
as before. For the second selection round whole intact leaves were
used. Leaves were incubated floating upside-down on phage solutions
in 2% MPBS and phage were allowed to bind at room temperature for 2
hours. The leaves were washed extensively by transferring leaves to
fresh tubes with PBS. Elution of bound phage was performed with 100
mM TEA in water, and solutions with eluted phage were neutralized
using half of the eluted phage volume of 1 M Tris pH 7.5. Infection
of TG1 was performed as before.
[0103] Picking Single Colonies from Selection Outputs--
[0104] Individual clones were picked from first and second round
selections against gum arabic with libraries 25 and 30. From
selections against gum arabic with libraries 27, 28, 29, 31, and
32, clones were picked after second round selections but not first
round selections. A total of 208 clones was picked from gum arabic
selections. From selections against potato epidermal CDTA extract a
total of 321 clones was picked after both first and second round
selections from all libraries. From selections against wheat
epidermal CDTA extract a total of 162 clones was picked after
second round selections from all libraries. From potato leaf
selections a total of 184 clones was picked after second round
selections from libraries 27, 28, 29, 30, 31, and 32. Fresh TG1
cells were infected with serially diluted eluted phage and plated
on LB agar; 2% glucose; 100 .mu.g/ml ampicillin. Single colonies
were picked in 96-well plates containing 100 .mu.A per well
2.times.TY; 10% glycerol; 2% glucose; 100 .mu.g/ml ampicillin.
Plates were incubated at 37.degree. C. and stored at -80.degree. C.
as master plates.
Example 2
Characterization of the VHH
[0105] Single-Point Binding ELISA--
[0106] A single-point binding ELISA was used to identify clones
that bind to gum arabic or plant extracts. VHH-containing extracts
for ELISA were prepared as follows. 96-well plates with 100 .mu.l
per well 2.times.TY, 2% glucose 100 .mu.g/ml ampicillin were
inoculated from the master plates and grown at 37.degree. C.
overnight. 25 .mu.l per well of overnight culture was used to
inoculate fresh 96-well deep-well plates containing 1 ml per well
2.times.TY; 0.1% glucose; 100 .mu.g/ml ampicillin. After growing at
37.degree. C. in a shaking incubator for 3 hours, IPTG was added to
1 mM final concentration and recombinant VHH was produced during an
additional incubation for 4 hours. Cells were spun down by
centrifugation at 3,000 g for 20 minutes and stored at -20.degree.
C. overnight. Cell pellets were thawed, briefly vortexed, and 125
.mu.l per well of room temperature PBS was added. Cells were
resuspended on an ELISA shaker platform at room temperature for 15
minutes. Plates were centrifuged at 3,000 g for 20 minutes and 100
.mu.l per well of VHH-containing extract was transferred to
polypropylene 96-well plates (Nunc) and stored at -20.degree. C.
until further use.
[0107] Binding of clones from gum arabic selections was analyzed in
ELISA plates coated with 100 .mu.l/well gum arabic at 1 mg/ml in
carbonate buffer pH 8.3. Binding of clones from potato epidermal
CDTA extract selections was analyzed on both potato epidermal CDTA
extract and wheat epidermal CDTA extract using ELISA plates coated
with 100 .mu.l per well of 30-fold diluted potato and 30-fold wheat
epidermal CDTA extracts in 0.1 M carbonate pH 8.3. Binding of
clones from wheat epidermal CDTA extract selections was analyzed
using ELISA plates coated with 100 .mu.l per well of 20-fold
diluted wheat epidermal CDTA extract in 0.1 M carbonate pH 8.3.
After coating at 4.degree. C. overnight and continued coating at
room temperature for 1 hour on the next day, plates were washed
three times with PBS/0.05%-TWEEN.RTM.-20 and blocked with 5%
skimmed milk in PBS for 1.5 hours. Plates were emptied and filled
with 90 .mu.l per well 1% MPBS. 10 .mu.l of VHH-containing extract
from each clone was added to (an) antigen-coated well(s) and a
blank well. VHH were allowed to bind at room temperature for 1 hour
and unbound VHH were removed by washing three times with
PBS/0.05%-TWEEN.RTM.-20. Bound VHH were detected with sequential
incubations with monoclonal mouse anti-histidine antibodies (Abd
Serotec) in 1% MPBS/0.05%-TWEEN.RTM.-20 and rabbit anti-mouse IgG
whole molecule antibodies conjugated with alkaline phosphatase
(RaM/AP) (Sigma) in 1% MPBS/0.05%-TWEEN.RTM.-20. Unbound antibodies
were removed by washing three times with PBS/0.05%-TWEEN.RTM.-20.
The plates were washed an additional two times with PBS and 100
.mu.l pNPP disodium hexahydrate substrate (Sigma) was added to each
well.
[0108] The absorbance at 405 nm was measured and the ratio of VHH
bound to (a) target-coated well(s) and a non-target-coated well was
calculated for each clone. 23% of clones had a ratio greater than 2
and these clones were firstly picked for more detailed
characterization. A second group of clones with a ratio between
1.15 and 2, and comprising 10% of all clones, was revisited later.
Clones with a ratio less than 1.15 were not analyzed further.
[0109] For clones from whole leaf selections an adapted ELISA was
developed. Upside-down floating leaf discs were used instead of
coating wells with antigen. Incubations were similar to the
extracts ELISA. After incubation with the substrate the leaf discs
were removed from the wells using a forceps and the absorbance at
405 nm was measured. Signals obtained for each clone were compared
to signals obtained from wells with leaf discs without primary
antibody incubation and the ratios were calculated. A leaf
surface-binding antibody that was found and characterized from
epidermal extract selections was used as positive control antibody.
VHH with a ratio greater than 1.5 were analyzed further by
sequencing.
[0110] Single Colony PCR and Sequencing--
[0111] Single colony PCR and sequencing was performed on ELISA
positive clones as follows. Cultures from master plate wells with
ELISA positive clones were diluted ten-fold in sterile water. 5
.mu.l from these diluted clones were used as template for PCR using
forward primer MP57 (5'-ttatgcttccggctcgtatg-3' (SEQ ID NO:48)) and
reverse primer GIII (5'-ccacagacagccctcatag-3' (SEQ ID NO:49)). PCR
products were sequenced by Sanger-sequencing using primer MP57 (VIB
Genetic Service Facility, University of Antwerp, Belgium).
[0112] Antibody Production and Purification--
[0113] VHH antibody fragments were produced in E. coli suppressor
strain TG1 or non-suppressor strain WK6 (Fritz et al., Nucleic
Acids Research, Volume 16 Number 14 1988) according to standard
procedures. Briefly, colony streaks were made and overnight
cultures from single colonies inoculated in 2.times.TY; 2% glucose;
100 .mu.g/ml ampicillin. The overnight cultures were used to
inoculate fresh cultures 1:100 in 2.times.TY; 0.1% glucose; 100
.mu.g/ml ampicillin. After growing at 37.degree. C. in a shaking
incubator for 3 hours, IPTG was added to a 1 mM final concentration
and recombinant VHH antibody fragments were produced during an
additional incubation for 4 hours. Cells were spun down and
resuspended in 1/50.sup.th of the original culture volume of
periplasmic extraction buffer (50 mM phosphate pH 7; 1 M NaCl; 1 mM
EDTA) and incubated with head-over-head rotation at 4.degree. C.
overnight. Spheroplasts were spun down by centrifugation at 3,000 g
and 4.degree. C. for 20 minutes. Supernatants were transferred to
fresh tubes and centrifuged again at 3,000 g and 4.degree. C. for
20 minutes. Hexahistidine-tagged VHH antibody fragments were
purified from the periplasmic extract using 1/15.sup.th of the
extract volume of TALON metal affinity resin (Clontech), according
to the manufacturer's instructions. Purified VHH antibody fragments
were concentrated and dialyzed to PBS using Vivaspin 5 kDa MWCO
devices (Sartorius Stedim), according to the manufacturer's
instructions.
[0114] VHH Binding to Gum Arabic in ELISA--
[0115] Titration of VHH antibody fragments was performed on ELISA
plates (Maxisorp, Nunc) coated with 100 .mu.l per well 100 .mu.g/ml
gum arabic in 50 mM carbonate pH 9.6. Plates were coated at
4.degree. C. overnight and coating was continued at room
temperature for 1 hour on the next day. Plates were washed three
times with PBS/0.05%-TWEEN.RTM.-20 and blocked with 5% skimmed milk
in PBS for 1 hour. Four-fold serial dilutions of purified VHH
antibody fragments were prepared in 1% MPBS/0.05%-TWEEN.RTM.-20 in
polypropylene 96-well plates. Antibody concentrations ranged from 3
.mu.g/ml to 12 ng/ml. Antibody dilutions were transferred to the
gum arabic-coated plates and VHH antibody fragments were allowed to
bind for 1 hour at room temperature. Bound VHH were detected with
sequential incubations with monoclonal mouse anti-histidine
antibodies (Abd Serotec) and rabbit anti-mouse IgG whole molecule
antibodies conjugated with alkaline phosphatase (RaM/AP) (Sigma) in
1% MPBS/0.05%-TWEEN.RTM.-20. Unbound antibodies were removed by
washing three times with PBS/0.05%-TWEEN.RTM.-20 after each
antibody incubation. The plates were washed an additional two times
with PBS and 100 .mu.l pNPP disodium hexahydrate substrate (Sigma)
was added to each well. The absorbance at 405 nm was measured and
plotted as function of antibody concentration (see Table 1).
TABLE-US-00001 TABLE 1 [VHH] 3 3 0.75 0.1875 0.04688 0.0117188
(.mu.g/ml) [VHH] (nM) 200 200 50 12.5 3.125 0.78125 Gum arabic - +
+ + + + (100 .mu.g/ml) 1 2 3 4 5 6 VHH3E6 A 0.090 2.154 1.904 1.518
0.905 0.392 VHH5C4 B 0.082 2.010 1.710 1.036 0.386 0.166 VHH5D4 C
0.075 1.280 0.840 0.378 0.134 0.087 VHH5G5 D 0.077 1.966 1.611
0.906 0.317 0.125 VHH5E5 E 0.073 1.194 0.569 0.185 0.088 0.074
VHH7D2 F 0.074 1.427 0.906 0.347 0.136 0.083 VHH7C2 G 0.077 0.461
0.194 0.090 0.092 0.088 VHH5F5 H 0.090 0.959 0.476 0.191 0.100
0.093 VHH7A2 F 0.075 1.391 0.677 0.216 0.101 0.088
VHH Binding to Potato Lectin in ELISA
[0116] ELISA plates (Maxisorp, Nunc) coated with 100 .mu.l per well
100 .mu.g/ml potato lectin (Sigma) in PBS were coated at 4.degree.
C. overnight and coating was continued at room temperature for 1
hour on the next day. Plates were washed three times with
PBS/0.05%-TWEEN.RTM.-20 and blocked with 5% skimmed milk in PBS for
1 hour. VHH (3 .mu.g/ml) were transferred to the potato
lectin-coated plates and VHH antibody fragments were allowed to
bind for 1 hour at room temperature. Bound VHH were detected with
sequential incubations with monoclonal mouse anti-histidine
antibodies (Abd Serotec) and rabbit anti-mouse IgG whole molecule
antibodies conjugated with alkaline phosphatase (RaM/AP) (Sigma) in
1% MPBS/0.05%-TWEEN.RTM.-20. Unbound antibodies were removed by
washing three times with PBS/0.05%-TWEEN.RTM.-20 after each
antibody incubation. The plates were washed an additional two times
with PBS and 100 .mu.l pNPP disodium hexahydrate substrate (Sigma)
was added to each well and the absorbance at 405 nm was measured
(see Table 2).
TABLE-US-00002 TABLE 2 VHH VHH 3E6 5D4 VHH 5C4 VHH 5G5 VHH 7D2
<Blank Gum arabic 0.882 0.530 0.873 0.751 0.274 0.069 Potato
lectin 4.000 4.000 4.000 4.000 4.000 0.081 Blank 0.067 0.072 0.071
0.073 0.072 0.072
Example 3
Binding of Binding Domains to Plant Surface
[0117] VHH Binding to Leaf Discs--
[0118] VHH binding to non-fixed leaf discs of potato (variety
Desiree), black nightshade, grass, wheat or azalea was
investigated. For comparison, binding of CBM3a to non-fixed leaf
discs of potato (variety Desiree) was analyzed in parallel. Leaf
discs were prepared by punching a fresh potato leaf with a 5 mm
belt hole puncher tool. Leaf discs were put immediately in wells of
a 96-well plate containing 200 .mu.l per well 5% MPBS or PBS, and
incubated for 30 minutes. Leaf discs were transferred to solutions
containing 5 .mu.g/ml VHH antibody fragment, respectively, 5
.mu.g/ml CBM3a in 2% MPBS or PBS and incubated for 60-90 minutes.
Unbound VHH or CBM3a proteins were removed by washing three times
with 2% MPBS or PBS. Bound VHH or CBM3a proteins were detected with
incubation with monoclonal mouse anti-histidine antibodies directly
conjugated with Alexa-488 fluorescent dye (Abd Serotec) in 1% MPBS
for 1 hour. Unbound antibodies were removed by washing three times
with PBS. Leaf discs were put on glass slides, covered with cover
slips, and analyzed by microscopy or on a macrozoom microscope
system (Nikon) or a SP5 confocal microscope system (Leica). By
means of a non-limiting example, VHH antibody fragments (e.g., 3E6,
5D4) were found to be clearly binding to trichomes, stomata and
cuticle at the leaf surface of potato leaves (FIGS. 1A-C). In sharp
contrast, for CBM3a no binding at the surface of potato leaves was
detected and only faint binding to the wound tissue at the cut edge
of the potato leaf disc was observed (FIG. 1D). Some VHH hereof
(e.g., 3E6) were also shown to bind specifically to the surface of
black nightshade leaves or grass leaves or as shown in FIGS. 1F and
1G, respectively. No significant binding was observed to the leaf
surface of wheat or azalea.
[0119] VHH Binding to Intact Living Plants--
[0120] Binding of VHH to intact living plants was investigated on
potato pot plants (variety Desiree). Compound leaves of intact
living plants were submersed in solutions of hexahistidine-tagged
VHH in PBS, or PBS alone for control conditions, leaving the
compound leaves attached to the plants. VHH were allowed to bind
for 1 hour. Next, the compound leaves still attached to the plants
were washed five times in PBS in Erlenmeyer flasks. Different
leaves and petiole sections were sampled. Bound VHH were detected
by incubation with monoclonal mouse anti-histidine antibodies
directly conjugated with Alexa-488 fluorescent dye (Abd Serotec) in
PBS for 1 hour. Unbound anti-histidine antibodies were removed by
washing five times with PBS. Whole leaves, leaf discs, or petiole
sections were analyzed for bound VHH with microscopy. VHH proved to
bind leaf structures such as trichomes and stomata, leaf surface,
and petiole sections as shown in FIG. 2. No binding was observed
with unrelated control VHH, proving that the VHH hereof are capable
of specifically binding to intact living plants.
[0121] VHH Binding in Water--
[0122] Binding of VHH to leaf surfaces in water was investigated on
leaf discs cut from leaves from potato plants (variety Desiree).
Leaf discs were washed three times in ultrapure water.
Hexahistidine-tagged VHH were diluted in ultrapure water, added to
leaf discs, and allowed to bind for 1 hour. Although the stock
solutions of VHH were in PBS, the dilutions used here (200-fold for
5 .mu.g/ml, or 2000-fold for 500 ng/ml) result in significant
dilution of PBS from the stocks and can be considered sufficiently
dilute to represent binding in water. After allowing VHH to bind
for 1 hour, leaf discs were washed five times with ultrapure water.
Bound VHH were detected by incubation with monoclonal mouse
anti-histidine antibodies directly conjugated with Alexa-488
fluorescent dye (Abd Serotec) in PBS for 1 hour. Unbound
anti-histidine antibodies were removed by washing five times with
PBS. Leaf discs were analyzed for bound VHH with microscopy.
Binding of VHH in PBS was analyzed as described before as a control
condition. Detection of bound VHH with anti-histidine antibodies
conjugated with Alexa-488 fluorescent dye, washing away non bound
anti-histidine antibodies, and analyzing bound VHH with microscopy
was performed as for the VHH binding experiment in water. VHH
proved to bind in water to leaf structures such as trichomes and
stomata, and leaf surface. No binding was observed with unrelated
control VHH. The observed binding in water was similar as seen for
the parallel experiment performed in PBS. The VHH hereof are
capable of binding leaf structures and leaf surface in water.
[0123] VHH Binding Kinetics--
[0124] In order to further test applicability of VHH as binders for
greenhouse or field applications where binding supposedly needs to
be achieved quickly after application, a leaf dip VHH binding
experiment was employed to test minimum incubation times of VHH to
achieve detectable binding. .phi. 8 mm potato leaf discs (variety
Desiree) were cut using a puncher tool and washed three times in
PBS. 5 .mu.g/ml pre-dilutions of hexahistidine-tagged VHH were
prepared in PBS and incubated for different times with the leaf
discs. The times for incubation were 10 seconds, 30 seconds, 1
minute, 5 minutes, 20 minutes, or 1 hour. Unbound VHH were removed
by washing five times with PBS. Bound VHH were detected by
incubation with monoclonal mouse anti-histidine antibodies directly
conjugated with Alexa-488 fluorescent dye (Abd Serotec) in PBS for
1 hour. Unbound anti-histidine antibodies were removed by washing
five times with PBS. Leaf discs were analyzed for bound VHH with
microscopy. Specific binding was observed for each sample with
specific VHH from incubation time 10 seconds to VHH incubation time
1 hour. No binding was observed with unrelated control VHH. The VHH
hereof show detectable binding to leaf structures, such as
trichomes and stomata and leaf surface within 10 seconds after
application.
[0125] VHH Binding at Different pH--
[0126] In order to test applicability of VHH as binders for
greenhouse or field applications where binding supposedly may occur
at pH-values, deviating strongly from physiological conditions in
which antibodies naturally bind their targets, a leaf dip VHH
binding experiment was carried out in a series of solutions with
different pH. The following solutions were prepared: 50 mM glycine
pH 2.0, 50 mM sodium acetate pH 4.0, 50 mm sodium carbonate pH 9.6,
and 10 mM sodium hydroxide pH 11.0. .phi. 8 mm potato leaf discs
(variety Desiree) were cut using a puncher tool. The leaf discs
were first equilibrated to the different pH by washing three times
with solutions at different pH. Hexahistidine-tagged VHH were
diluted to 5 .mu.g/ml in solutions with different pH, added to the
corresponding equilibrated leaf discs, and binding of VHH was
allowed for 1 hour. After incubation with VHH, leaf discs were
washed three times with solutions at the corresponding different
pH. After that, all were washed two times with PBS to equilibrate
leaf discs to PBS. Bound VHH were detected by incubation with
monoclonal mouse anti-histidine antibodies directly conjugated with
Alexa-488 fluorescent dye (Abd Serotec) in PBS for 1 hour. Unbound
anti-histidine antibodies were removed by washing five times with
PBS. Leaf discs were analyzed for bound VHH with microscopy. Some
of the VHH hereof (e.g., VHH 3E6) showed detectable binding to leaf
discs over the whole range tested from pH 2 to pH 11.
[0127] VHH Binding at Different Temperatures--
[0128] In order to test applicability of VHH as binders for
greenhouse or field applications where binding supposedly may occur
at different and sometimes even extreme temperatures, a leaf dip
VHH binding experiment at different temperatures was used.
Temperatures used were 4.degree. C., room temperature, 37.degree.
C., 55.degree. C., or 70.degree. C. o 8 mm potato leaf discs
(variety Desiree) were cut using a puncher tool. The leaf discs
were equilibrated to different temperatures by washing three times
with PBS at different temperatures. Hexahistidine-tagged VHH were
diluted to 5 .mu.g/ml in PBS at different temperatures, added to
the corresponding equilibrated leaf discs, and binding of VHH was
allowed for 1 hour at different temperatures. After incubation with
VHH, leaf discs were washed five times with PBS at room
temperature. Bound VHH were detected by incubation with monoclonal
mouse anti-histidine antibodies directly conjugated with Alexa-488
fluorescent dye (Abd Serotec) in PBS for 1 hour at room
temperature. Unbound anti-histidine antibodies were removed by
washing five times with PBS at room temperature. Leaf discs were
analyzed for bound VHH with microscopy. Some of the VHH hereof
(e.g., VHH 3E6) showed detectable binding to leaf discs over a
temperature range from 4.degree. C. to 55.degree. C. Please note
that leaf discs severely suffer when submerged in PBS at 70.degree.
C. for 1 hour but that binding of VHH was still detected.
Example 4
Coupling of Targeting Agents to Microparticles
[0129] Construction, Production and Purification of Bivalent
VHH--
[0130] Bivalent VHH constructs were produced in bacteria by cloning
two VHH sequences in tandem into the pASF22 vector, creating a
fusion of two VHH with a 9 glycine-serine linker (GGGGSGGGS (SEQ ID
NO:50)) in between the two VHH. pASF22 is an in-house produced pMES
derivative. The tags that were used were C-terminal c-Myc
(EQKLISEEDLN (SEQ ID NO:51)) and hexahistidine (HHHHHH (SEQ ID
NO:52)). A triple alanine linker (AAA) was placed in between the
C-terminal end of the VHH and the c-Myc tag and a
glycine-alanine-alanine (GAA) linker was used in between the
C-terminal end of the c-Myc tag and the hexahistidine tag. The
complete sequence C-terminal of the bivalent VHH that was used:
AAA-EQKLISEEDLN-GAA-HHHHHH (SEQ ID NO:53). Fresh overnight cultures
were produced by starting from colony streaks and inoculation of
2.times.TY media supplemented with 2% glucose and 100 .mu.g/ml
ampicillin. The overnight cultures were used to inoculate fresh
cultures 1:100 in 2.times.TY media with 0.1% glucose and 100
.mu.g/ml ampicillin. After growing at 37.degree. C. in a shaking
incubator for 3 hours, IPTG was added to a 1 mM final concentration
and recombinant bivalent VHH were produced during an additional
incubation for 4 hours. Cells were spun down and resuspended in
1/50th of the original culture volume of PBS and incubated with
head-over-head rotation at 4.degree. C. for 30 minutes.
Spheroplasts were spun down by centrifugation at 3,000 g and
4.degree. C. for 20 minutes. Supernatants were transferred to fresh
tubes and centrifuged again at 3,000 g and 4.degree. C. for 20
minutes. The supernatant was collected and sodium chloride
concentration was adjusted to 500 mM and imidazole concentration to
20 mM. Hexahistidine-tagged bivalent VHH were purified from the
extracts using HisTrap FF Crude 5 ml IMAC columns (GE Lifesciences)
and HiLoad 16/60 Superdex 75 prep grade gel filtration column (GE
Lifesciences) on an AKTAxpress system (GE Lifesciences) following
standard procedures.
[0131] Coupling of VHH to Microparticles--
[0132] It was first examined whether VHH that are covalently bound
to microparticles can bind their target and provide sufficient
adhesion strength to a surface containing antigen for targeting of
the microparticle. Microparticles were coupled with gum
arabic-specific VHH antibody fragments and binding to ELISA plates
coated with gum arabic was investigated.
[0133] Different types of microparticles were prepared. Purified
VHH antibody fragments were (i) coupled to O2.8 .mu.m paramagnetic
Dynabeads M-270 carboxylic acid (Dynal, Invitrogen), using a
two-step coupling chemistry of EDC activation of the beads and
subsequent coupling of VHH antibody fragments, and (ii) coupled
using a one-step coupling chemistry to O2 .mu.m FluoSpheres
fluorescent microspheres (Molecular Probes, Invitrogen), both
according to the manufacturers' instructions.
[0134] Briefly, for coupling to Dynabeads M-270 carboxylic acid:
VHH were dialyzed to 50 mM MES buffer pH 5.0 using Vivaspin 5 kDa
spin filter devices (Sartorius Stedim). Beads were prepared by two
sequential washes with 10 mM NaOH, and three washes with water, and
activated with 0.1 M EDC (Pierce) at room temperature for 30
minutes. EDC-activated beads were washed by quick sequential washes
with ice-cold water and ice-cold 50 mM MES buffer pH 5.0. Beads
were dispensed with the last wash. 60 .mu.g of VHH antibody
fragment in 100 .mu.l 50 mM MES pH 5.0 were added to 3 mg beads and
incubated at room temperature for 30 minutes. The supernatant after
coupling was collected. By measuring protein A280 of the non-bound
fraction the amounts of coupled and non-coupled VHH were
calculated. Greater than 95% of VHH antibody fragment were coupled
to the beads. Beads were blocked with 50 mM Tris pH 7.4 and washed
three times with PBS/0.1%-TWEEN.RTM.-20 and stored at 4.degree.
C.
[0135] Briefly, for coupling to FluoSpheres fluorescent
microspheres: VHH were dialyzed to 50 mM MES buffer pH 6.0 using
Vivaspin 5 kDa spin filter devices (Sartorius Stedim). 0.8 .mu.m
PES filter devices (Sartorius Stedim) were used throughout the
procedure to isolate beads from solution. Beads were prepared by
washing with ultrapure water and re-suspension in ultrapure water.
100 .mu.l of VHH antibody fragments containing 200 .mu.g VHH were
added to 100 .mu.l beads. 0.8 mg EDC (Pierce) was added to each mix
of beads with VHH and the pH was adjusted to 6.5 with 0.1 M NaOH.
Coupling was performed at room temperature for 2 hours. Glycine was
added to a final concentration of 100 mM and incubated at room
temperature for 30 minutes to quench the reaction. By measuring
protein A280 of the non-bound fraction the amounts of coupled and
non-coupled VHH were calculated. Between 14% and 33% of different
VHH antibody fragments were coupled to the beads. Beads were washed
twice with 50 mM phosphate pH 7.4; 0.9% NaCl (50 mM PBS) and stored
in 1% BSA, 2 mM sodium azide in 50 mM PBS.
[0136] Coupling of Targeting Agents to Microcapsules Containing
Fluorescent Tracer or Active Ingredient--
[0137] Polyurea microcapsules were produced by interfacial
polymerization. With the objective to generate functionalized
polyurea microcapsules, VHH were coupled to microcapsules
containing either the insecticide lambda cyhalothrin or the
fluorescent tracer molecule Uvitex OB and a shell with incorporated
lysine to surface-expose carboxylic acid residues. Lambda
cyhalothrin was dissolved in benzyl benzoate in concentrations
between 30% and 66% before encapsulation. Alternatively, a core of
1.5% Uvitex in benzyl benzoate was used for easy fluorescent
visualization of microcapsules. Toluene diisocyanate (TDI) and
polymethylenepolyphenylene isocyanate (PMPPI) were dissolved in the
oil phase in different ratios and concentrations in the oil phase
to produce desired shell characteristics. Stirring speed for the
emulsion was varied to control droplet size and consequently
microcapsule diameter. Microcapsules with approximate diameters of
5 .mu.m, 10 .mu.m, or 50 .mu.m were successfully produced.
Bifunctional lysine and trifunctional diethylene triamine (DETA)
were used in different ratios and/or added sequentially during
encapsulation to on the one hand maximize amounts of carboxylic
acids on the microcapsules' surface and on the other hand obtain
sufficient strength of capsule shells. Microcapsules were washed
with water after production and stored as microcapsule suspensions
in water. The microcapsules were washed with 100 mM MES, 500 mM
NaCl, pH 6.0 immediately before coupling of VHH using a
vacuum-tight filter flask and P 1.6 filter funnel (Duran).
Alternatively, glass filter holders with 0.45 .mu.m disposable
membrane filters (Millipore) or 0.45 .mu.m 96-well deep-well
filtration plates (Millipore) were used. Couplings of VHH to
microcapsules were performed using carbodiimide-mediated couplings
using a one-step procedure, a two-step procedure without
N-hydroxysuccinimide (NHS), or a two-step procedure with NHS. The
major difference between one-step coupling and two-step coupling
procedures is the occurrence of cross-linking of VHH in one-step
coupling procedures. The protocols for the three procedures are
largely similar and differ as follows. For one-step couplings VHH
were added to washed microcapsules and
1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide Hydrochloride (EDC)
(Pierce) was added and coupling reaction was allowed for 2 hours at
room temperature. For two-step couplings washed microcapsules were
first activated with EDC in the presence or absence of NHS. Excess
unreacted EDC (and NHS) were removed by quick sequential washes
with ice-cold buffers and VHH were added and allowed to react with
activated carboxylic acids on microcapsule shells. For .phi.10
.mu.m microcapsules 2-20 .mu.g VHH were coupled per mg
microcapsules. For microcapsules with other diameters amounts were
scaled accordingly. After coupling of VHH the microcapsules were
washed with PBS and stored in PBS. Success of coupling of VHH was
investigated using a combination of analyzing coupling efficiency
by SDS-PAGE and analyzing bound hexahistidine-tagged VHH by
microscopy or a SP5 confocal microscope system (Leica) using
anti-histidine antibodies directly conjugated with Alexa-488
fluorescent dye. With SDS-PAGE analysis formation of multimers was
observed for one-step coupling reactions as expected. VHH-coupled
microcapsules were labeled with anti-histidine antibodies for 1
hour at room temperature. Unbound anti-histidine antibodies were
removed by washing five times with PBS using 0.45 .mu.m 96-well
deep-well filtration plates (Millipore). Microcapsules with coupled
VHH, microcapsules incubated with VHH to which no EDC was added,
and blank microcapsules were compared. Anti-histidine labeling of
microcapsules was most intense for microcapsules to which VHH had
been coupled using either one-step or two-step coupling procedures
as shown in FIG. 3. It was also observed that some VHH were
passively adsorbed to the microcapsules. VHH were successfully
coupled to microcapsules of different size using either one-step or
two-step coupling procedures.
Example 5
Binding of Targeting Agent-Coupled Micro Particles to
Antigen-Containing Surface
[0138] Binding Assays with VHH-Coupled Beads or Microcapsules--
[0139] Functionality of VHH-coupled microparticles was investigated
in ELISA plates that were coated with 100 .mu.g/ml gum arabic in 50
mM carbonate pH 9.6 or PBS. Coating was performed overnight and
plates were washed three times with PBS/0.05%-TWEEN.RTM.-20 and
blocked with 5% skimmed milk in PBS for 1.5 hours. VHH-coupled
paramagnetic beads were diluted 50-fold and incubated with
monoclonal mouse anti-histidine antibodies directly conjugated with
Alexa-488 fluorescent dye (Abd Serotec) in 1% MPBST for 1 hour.
Two-fold serial dilutions (50- to 800-fold) of VHH-conjugated
paramagnetic Dynabeads and FluoSpheres fluorescent beads were
prepared in 2% MPBS, transferred to the gum arabic-coated ELISA
plates, and incubated at room temperature for 1 hour. Unbound beads
were removed by washing five times with PBS/0.05%-TWEEN.RTM.-20.
The bottoms of ELISA plate wells were analyzed for bound beads by
microscopy. Counting beads and using the microscope's camera mask
for calculation of the analyzed surface area were used for
calculating number of bound beads per well as shown in Table 3.
Alternatively, microparticles were visualized using a macrozoom
microscope system (Nikon) and counted using Volocity image analysis
software (PerkinElmer); the number of bound Fluospheres per well is
shown in Table 4.
TABLE-US-00003 TABLE 3 Counted bound magnetic carboxylic acid
Dynabeads to wells coated with gum arabic Magnetic Carboxylic Acid
Dynabeads 2.8 .mu.m Gum (approximate numbers) Dilution arabic
Coupled with VHH 3E6 Coupled with VHH 5D4 50 + .apprxeq.1000
.apprxeq.500 100 + .apprxeq.500 .apprxeq.500 200 + .apprxeq.200
.apprxeq.200 400 + .apprxeq.100 .apprxeq.200 800 + .apprxeq.100
.apprxeq.100 50 - .apprxeq.10 .apprxeq.50
TABLE-US-00004 TABLE 4 Counted bound Fluospheres to wells coated
with gum arabic Fluospheres Number of Fluospheres coupled coupled
with Coating Fluospheres added with VHH 3E6 unrelated VHH No
coating 4.5 10.sup.6 115 198 Gum arabic 4.5 10.sup.6 1874 224 Gum
arabic 2.3 10.sup.6 1273 89 Gum arabic 1.1 10.sup.6 981 83
[0140] An ELISA-like assay setup was used to evaluate the
interaction of VHH-coupled microcapsules to antigen-containing
surfaces. ELISA plates (Maxisorp (Thermo Scientific Nunc) or high
bind half area microplates (Greiner Bio-One)) were coated with gum
arabic or potato lectin. Coatings were performed overnight with 100
.mu.g/ml gum arabic or potato lectin in PBS. Control wells included
blank wells or wells coated with unrelated antigens. Plates were
washed three times with PBS with 0.05%-TWEEN.RTM.-20 and blocked
with 5% skimmed milk in PBS for 1 to 2 hours. VHH-coupled lambda
cyhalothrin-containing or Uvitex-containing microcapsules were
diluted to appropriate densities in 1% skimmed milk in PBS with
0.05%-TWEEN.RTM.-20. Microcapsules were added to the antigen-coated
or control wells and allowed to bind for 1 hour. Unbound
microcapsules were removed by washing five times with PBS with
0.05%-TWEEN.RTM.-20. The bottoms of ELISA plate wells were analyzed
for bound microcapsules on a macrozoom microscope system (Nikon).
Microcapsules were counted using Volocity image analysis software
(Perkin Elmer). A DAPI filter was used to visualize Uvitex
microcapsules. White LED illumination and bright field pictures
were used for lambda cyhalothrin microcapsules. Controls for lambda
cyhalothrin-containing or Uvitex-containing microcapsules included
blank microcapsules and microcapsules to which unrelated VHH were
coupled.
TABLE-US-00005 TABLE 5 Bound microcapsules to wells coated with
potato lectin or unrelated antigen Counts Counts Counts Area Area
Microcapsules containing Microcapsules lambda-cyhalothrin
containing uvitex OB Surface Blank unrelated VHH unrelated coverage
microcapsules control 3E6 VHH 3E6 control no coating 100% 583 689
701 86.574 82.757 potato lectin 100% 755 828 7.910 504.839 16.676
potato lectin 20% 616 709 4.550 510.242 35.433 potato lectin 4% 408
348 798 144.955 7.529 no coating 100% n.d. n.d. 209 68.181 60.841
unrelated antigen 100% n.d. n.d. 861 84.508 94.153 unrelated
antigen 20% n.d. n.d. 601 47.906 39.218 unrelated antigen 4% n.d.
n.d. 386 23.525 18.517
[0141] In another experiment, lambda cyhalothrin amounts were also
determined analytically. 100 .mu.l/well aceton was added to washed
wells with bound microcapsules and transferred to glass vials with
10 ml of hexane containing 0.05% triphenylphosphate as internal
standard. The amount of lambda cyhalothrin was determined by
GC/MS-MS analysis in comparison with calibration solutions.
Controls for lambda cyhalothrin microcapsules included blank
microcapsules to which no VHH were coupled and microcapsules to
which unrelated VHH were coupled. Controls also included wells to
which no gum arabic or potato lectin was coated. Based on the
results of the ELISA-like assay with lambda cyhalothrin
microcapsules it was found that some of the VHH hereof (e.g.,
VHH3E6) are capable of binding and retaining microcapsules to
antigen-coated surfaces resulting in a 23-fold increase of amounts
of lambda cyhalothrin in wells coated with antigen compared to
blank microcapsules and a 27-fold increase was measured over blank
wells not coated with antigen.
[0142] Based on the results of the microcapsule binding assays, VHH
could be classified as capable or not capable of binding and
retaining microcapsules to a surface. Some of the VHH hereof (e.g.,
VHH3E6) proved capable of binding specifically to antigen-coated
surfaces when coupled to a microcapsule. No significant binding to
surfaces with unrelated antigens was observed. Moreover, the
specific binding was strong enough to retain the microcapsule at
the antigen-coated surface, as the binding force clearly resists
the shear forces that occur during the washing procedure. What is
more is that VHH are capable of binding and retaining microcapsules
containing relevant active ingredients to surfaces, as shown, for
example, with microcapsules containing the insecticide lambda
cyhalothrin.
[0143] Next, it was investigated if binding of microcapsules to
surfaces could be improved by using targeting agents comprising
multivalent VHH. A series of parallel couplings was performed with
equal amounts of monovalent VHH, bivalent VHH, and unrelated VHH.
Success of coupling of VHH and multivalent VHH were analyzed as
described in Example 4. An ELISA-like assay was performed using
high bind half area microplates (Greiner Bio-One) coated with 5
.mu.g/well potato lectin. Control wells included blank wells or
wells coated with unrelated antigens. Plates were washed three
times with PBS with 0.05%-TWEEN.RTM.-20 and blocked with 5% skimmed
milk in PBS for 1 to 2 hours. VHH-coupled Uvitex-containing
microcapsules were diluted to appropriate densities in 1% skimmed
milk in PBS with 0.05%-TWEEN.RTM.-20. Five-fold serial dilution
series were prepared and allowed to bind to the surface to compare
binding of microcapsules coupled with monovalent or bivalent VHH.
Microcapsules were added to the antigen-coated or control wells and
allowed to bind for 1 hour. Unbound microcapsules were removed by
washing five times with PBS with 0.05%-TWEEN.RTM.-20. The bottoms
of ELISA plate wells were analyzed for bound microcapsules on a
macrozoom microscope system (Nikon). Microcapsules were counted
using Volocity image analysis software (Perkin Elmer). A DAPI
filter was used to visualize Uvitex microcapsules. Bivalent VHH
proved capable of binding specifically to an antigen-coated surface
when coupled to a microcapsule and more microcapsules were retained
using bivalent VHH compared to microcapsules with monovalent VHH.
With the highest density of microcapsules applied (calculated to
fully cover the surface of the bottom of the well) it was found
that 17% more microcapsules with coupled bivalent VHH were retained
in the well compared to the same amount of microcapsules with
monovalent VHH. With an application of 25-fold less microcapsules
it was found that 160% more microcapsules were retained in the well
for microcapsules coupled with bivalent VHH compared to
microcapsules with monovalent VHH. The surface area of
microcapsules with coupled bivalent VHH was 15-fold above the
surface area of blank microcapsules applied at this microcapsule
density while the surface area of microcapsules with monovalent VHH
was only six-fold above the surface area of blank microcapsules
applied at this microcapsule density. This difference could be
explained by an increase in binding strength due to additional
avidity of the bivalent VHH compared to monovalent VHH, it could
also be that the use of bivalent VHH increases flexibility and
spacer length of the coupled targeting agents on microcapsules, or
a combination of both.
TABLE-US-00006 TABLE 6 Surface areas of bound microcapsules to
wells coated with potato lectin or unrelated antigen Surface
Monovalent Bivalent Blank coverage VHH 3E6 VHH 3E6 unrelated VHH
microcapsules no coating 100% 74.536 66.176 77.014 84.982 potato
lectin 100% 415.773 490.546 141.636 90.030 potato lectin 20%
307.478 511.303 43.452 44.024 potato lectin 4% 59.377 155.759
19.170 10.599 no coating 100% 72.036 55.841 68.109 66.509 unrelated
antigen 100% 69.503 45.677 78.205 50.965 unrelated antigen 20%
27.742 22.114 30.459 17.831 unrelated antigen 4% 5.011 15.038
19.755 6.279
[0144] A leaf disc binding assay was used to evaluate the
interaction of VHH-coupled microcapsules with potato, grass and
azalea leaves. .phi.8 mm leaf discs were sampled from the leaves of
potato pot plants (variety Desiree), from the leaves of
greenhouse-grown Lollium perenne and from the leaves of azalea pot
plants. Leaf discs were washed three times with PBS. Microcapsules
containing lambda cyhalothrin or Uvitex were diluted to appropriate
densities in 1% skimmed milk in PBS with 0.05%-TWEEN.RTM.-20.
Microcapsules were added to the leaf discs and settling of
microcapsules and binding of targeting agents allowed for 1 hour.
Unbound microcapsules were removed by washing three to five times
with PBS with 0.05%-TWEEN.RTM.-20.
[0145] For lambda cyhalothrin microcapsules, a residue analysis was
performed to measure lambda cyhalothrin amounts on potato leaf
discs. Washed leaf discs with bound microcapsules were transferred
to glass vials and microcapsules were dissolved in acetone. Samples
were diluted by addition of hexane containing 0.05%
triphenylphosphate as internal standard. The amount of lambda
cyhalothrin was determined by GC/MS-MS analysis in comparison with
calibration solutions. Controls for lambda cyhalothrin
microcapsules included blank microcapsules to which no VHH were
coupled and microcapsules to which unrelated VHH were coupled.
Based on the results of leaf disc binding assays with lambda
cyhalothrin microcapsules, it was found that some of the VHH hereof
are capable of binding and retaining microcapsules to leaf surfaces
resulting in a 3.3-fold and 2.2-fold increase of amounts of lambda
cyhalothrin on leaf discs compared to blank microcapsules to which
no VHH were coupled or microcapsules with coupled unrelated VHH,
respectively.
[0146] Leaf discs with Uvitex microcapsules were analyzed for bound
microcapsules on a macrozoom microscope system (Nikon).
Microcapsules were counted using Volocity image analysis software
(Perkin Elmer). A DAPI filter was used to visualize Uvitex
microcapsules. Controls for Uvitex microcapsules included blank
microcapsules to which no VHH were coupled and microcapsules to
which unrelated VHH were coupled. Based on the results of the leaf
disc binding assay with Uvitex microcapsules it was found that some
of the VHH (e.g., VHH 3E6) hereof proved capable of binding and
retaining microcapsules specifically to leaf surfaces.
[0147] On potato leaf discs, specific binding of the microcapsules
coupled with VHH 3E6, resulted in nine-fold more microcapsules
bound to leaf surfaces compared to blank microcapsules and in
six-fold more microcapsules bound to leaf surfaces compared to
microcapsules coupled with unrelated VHH, as shown in FIG. 4. On
grass leaf discs, specific binding of microcapsules coupled with
VHH 3E6 resulted in three-fold more microcapsules bound to leaf
surfaces compared to blank microcapsules and in two-fold more
microcapsules bound to leaf surfaces compared to microcapsules
coupled with unrelated VHH. On azalea leaf discs, no specific
binding of microcapsules coupled with VHH 3E6 could be observed,
which entirely resembles the plant-species related binding
specificity of the VHH as demonstrated in Example 3.
[0148] A titration experiment was performed to investigate what
dilution factor of microcapsules with specific VHH corresponds to
an application of microcapsules to which no VHH were coupled to
obtain similar amounts of microcapsules after an identical
treatment. Two-fold serial dilutions of microcapsules were prepared
and leaf disc binding was analyzed on potato leaf discs for these
dilution series. From the dosing experiment it was calculated that
an eight-fold lower concentration of microcapsules with specific
VHH resulted in similar amounts of microcapsules specifically bound
to the leaf discs compared to non-functionalized microcapsules as
shown in FIG. 5. From this experiment, it will be clear that a
meaningful reduction of a suitable dose of an agrochemical can be
achieved, by coupling one of the VHH hereof, to a microcarrier
containing the agrochemical.
Example 6
Deposition and Retention of Targeting Agent-Coupled Microcapsules
on Intact Living Plant Surface
[0149] Effects on deposition and retention of carriers with coupled
targeting agents were investigated in experiments with whole potato
pot plants (variety Desiree) grown in greenhouses. Microcapsules
coupled with specific VHH, coupled with unrelated control VHH, or
blank microcapsules were applied to multiple whole compound leaves
from different plants. In total 15 plants were used for different
treatments. Microcapsule suspensions were calculated to apply 6.4%
coverage of microcapsules on leaf surfaces. Compound leaves were
submerged in microcapsule suspensions in the same way as for
microcapsule leaf disc binding assays (see above) with the
modification that settling of microcapsules and binding of VHH was
allowed for only 15 minutes. Plants were allowed to dry up for 1
hour after application of microcapsules. One of each pair of
opposite leaves from within each compound leaf was sampled and
analyzed without any further treatment.
[0150] The effects of specific VHH coupled to microcapsules on
microcapsule deposition could be analyzed with these leaves from
different applications. The whole plants missing only the sampled
leaves were treated further to investigate the effect of specific
VHH coupled to microcapsules on retention after a rainfall event
and the combined effects of deposition and retention. A rain
simulation with fine droplets (SSCOTFVS2 nozzle type) of 1 L/m2 in
45 seconds was used to investigate retention effects. The opposite
leaves of already sampled leaves were sampled after the rain
simulation. Whole leaves with Uvitex microcapsules were analyzed
for bound microcapsules on a macrozoom microscope system (Nikon).
Microcapsules were counted using Volocity image analysis software
(Perkin Elmer). A DAPI filter was used to visualize Uvitex
microcapsules. From the leaves that were sampled before the
rainfall event it was calculated that already 2.7-fold more
microcapsules were deposited for microcapsules with specific
targeting agent compared to blank microcapsules. Leaves with
microcapsules with unrelated control targeting agent contained only
a 0.8 fraction of microcapsules compared to blank microcapsules.
This shows that specific VHH already have a beneficial effect on
the deposition of microcapsules on plants. On average 69 (.+-.8) %
of microcapsules with specific VHH was retained after the rainfall
event while only 35 (.+-.17) % and 39 (.+-.4) % of microcapsules
was retained for microcapsules coupled with unrelated control VHH
and blank microcapsules, respectively. The combination of effects
of deposition and retention resulted in five-fold and 0.9-fold in
the amount of microcapsules on leaves on whole plants for
microcapsules with specific VHH or unrelated control VHH, compared
to blank microcapsules, respectively.
[0151] From this experiment, it will be clear that specific VHH are
superior targeting agents that enable delivery and specific binding
of carriers to whole intact living plants. As a consequence of
improved deposition and improved retention targeting agents hereof
coupled to carriers containing an agrochemical or a combination of
agrochemicals hold great promise to deliver the agrochemicals
specifically to plant surfaces and hereby either increase amounts
of the agrochemicals deposited on the plant surface, or enable
reduced application rates while maintaining similar efficacy, or
enable reduced application frequencies while maintaining similar
efficacy or enable improved rainfastness of the agrochemicals or
induce a certain specificity for the agrochemicals or any
combination of the foregoing.
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Sequence CWU 1
1
531123PRTLama glamaMISC_FEATURE3A2 1Gln Val Gln Leu Gln Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Lys Val Ala Cys
Ala Ala Ala Gly Phe Ser Leu Arg Tyr Tyr 20 25 30 Gly Ile Gly Trp
Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Ala Val 35 40 45 Ser Cys
Thr Ser Ala Lys Asp Gly Ser Thr Tyr Tyr Arg Asp Ser Val 50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Asp Asp Gly Lys Asn Thr Val Tyr 65
70 75 80 Leu Gln Met Asn Arg Leu Lys Pro Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Thr Thr Asp Cys Asp Ala Thr Ser Trp Gly Thr
Trp Ile Asn Tyr 100 105 110 Tyr Gly Gln Gly Thr Gln Val Thr Val Ser
Ser 115 120 2123PRTLama glamaMISC_FEATURE3B4 2Gln Val Gln Leu Gln
Glu Ser Gly Gly Gly Ser Ala Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Arg Asn Phe 20 25 30 Gly
Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40
45 Ser Cys Ser Asn Val Arg Asp Gly Ser Thr Tyr Tyr Gln Ser Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Tyr Arg Asp Asn Phe Lys Asn Met
Leu Tyr 65 70 75 80 Leu Gln Met Asn Asn Leu Glu Leu Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Thr Thr Asp Cys Glu Ala Thr Ser Trp
Gly Thr Tyr Val Gly Tyr 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr
Val Ser Ser 115 120 3123PRTLama glamaMISC_FEATURE3B7 3Gln Val Gln
Leu Gln Glu Ser Gly Gly Gly Leu Val Arg Thr Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Ala Leu Ala Asn Tyr 20 25
30 Gly Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45 Ser Cys Ser Asn Val Arg Asp Gly Ser Thr Tyr Tyr Arg Asp
Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asn Asn Ile Glu
Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Ser Thr Leu Lys Pro Glu Asp
Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Thr Thr Asp Cys Glu Ala Ser
Ser Trp Gly Thr Trp Ile Asn Tyr 100 105 110 Arg Gly Gln Gly Thr Gln
Val Thr Val Ser Ser 115 120 4123PRTLama glamaMISC_FEATURE3D10 4Gln
Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Thr Leu Ser Cys Glu Ala Ser Gly Phe Arg Leu Arg Asn Phe
20 25 30 Gly Ile Gly Trp Phe Arg Gln Ala Ala Gly Lys Glu Arg Glu
Gly Val 35 40 45 Ser Cys Ser Asn Val Arg Asp Gly Thr Thr Tyr Tyr
Ala Asp Ala Val 50 55 60 Lys Gly Arg Phe Ile Ile Ser Arg Asp Asn
Thr Arg Asn Thr Leu Ser 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro
Glu Asp Thr Ala Val Tyr Ser Cys 85 90 95 Gly Thr Thr Asp Cys Glu
Ala Ser His Trp Gly Thr Tyr Val Gly Tyr 100 105 110 Phe Gly His Gly
Thr Gln Val Thr Val Ser Ser 115 120 5123PRTLama
glamaMISC_FEATURE3D2 5Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Pro Leu Val Leu Tyr 20 25 30 Gly Met Gly Trp Phe Arg Gln
Ala Pro Gly Lys Lys Arg Glu Ala Val 35 40 45 Ser Cys Ser Ser Val
Asn Asp Gly Gly Thr Tyr Tyr Ala Glu Ser Val 50 55 60 Glu Gly Arg
Phe Thr Leu Phe Arg Asp Asn Gly Ala Asn Ala Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Glu Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Thr Thr Asp Cys Glu Ala Thr Gly Trp Gly Thr Trp Thr Asn Tyr
100 105 110 Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120
6123PRTLama glamaMISC_FEATURE3D8 6Gln Val Gln Leu Gln Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Pro Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Ser Val Ala Tyr Tyr 20 25 30 Gly Met Gly Trp
Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Cys
Ile Ser Ala Leu Arg Asp Thr Thr Tyr Tyr Thr Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Leu Ser Arg Asp Asn Val Lys Asn Thr Leu Ser 65
70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Val Tyr
Tyr Cys 85 90 95 Ala Thr Thr Asp Cys Asp Ala Thr Ser Arg Met Thr
Tyr Leu Ser Tyr 100 105 110 Leu Gly Gln Gly Thr Gln Val Thr Val Ser
Ser 115 120 7123PRTLama glamaMISC_FEATURE3E6 7Gln Val Gln Leu Gln
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Thr Leu Ser
Leu Ser Cys Ala Ala Ser Gly Phe Asn Val Arg Trp Tyr 20 25 30 Gly
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40
45 Ala Cys Ile Ser Ala Leu Gln Glu Thr Thr Ala Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Leu Ser Arg Asp Asn Pro Lys Asn Thr
Leu Ser 65 70 75 80 Leu Gln Met Asn Asn Leu Gln Pro Glu Asp Thr Gly
Val Tyr Tyr Cys 85 90 95 Ala Thr Thr Asp Cys Asp Asp Ser Ser Arg
Met Thr Tyr Thr Ser Tyr 100 105 110 Leu Gly Gln Gly Thr Gln Val Thr
Val Ser Ser 115 120 8123PRTLama glamaMISC_FEATURE3F5 8Gln Val Gln
Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser
Leu Lys Val Ala Cys Ala Ala Val Gly Phe Ser Leu Arg Asn Tyr 20 25
30 Gly Ile Gly Trp Phe Arg Gln Val Pro Gly Lys Ala Arg Glu Ala Val
35 40 45 Ser Cys Thr Ser Val Asn Asp Gly Ser Thr His Tyr Gly Asp
Ser Val 50 55 60 Arg Gly Arg Phe Ser Ile Ala Arg Asp Asn Ser Lys
Asn Thr Val Phe 65 70 75 80 Leu Gln Met Asn Asp Leu Lys Pro Glu Asp
Thr Ala Val Tyr Phe Cys 85 90 95 Ala Thr Thr Asp Cys Asp Val Thr
Ser Trp Gly Thr Trp Ile Asn Tyr 100 105 110 Tyr Gly Gln Gly Thr Gln
Val Thr Val Ser Ser 115 120 9123PRTLama glamaMISC_FEATURE3F7 9Gln
Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Arg Thr Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Ala Leu Ala Asn Tyr
20 25 30 Gly Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu
Gly Val 35 40 45 Ser Cys Ser Asn Val Arg Asp Gly Ser Thr Tyr Tyr
Arg Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asn Asn
Ile Glu Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Ser Thr Leu Lys Pro
Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Thr Thr Asp Cys Glu
Ala Ser Ser Trp Gly Thr Trp Ile Asn Tyr 100 105 110 Arg Gly Gln Gly
Thr Gln Val Thr Val Ser Ser 115 120 10123PRTLama
glamaMISC_FEATURE3F9 10Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Pro Leu Lys Leu Ser Cys Ala Ala Ser
Gly Phe Ser Val Ala Tyr Tyr 20 25 30 Gly Met Gly Trp Phe Arg Gln
Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Cys Ile Ser Ala
Leu Arg Asn Thr Thr Tyr Tyr Thr Asp Ser Val 50 55 60 Gln Gly Arg
Phe Thr Leu Ser Arg Asp Asn Val Lys Asn Thr Leu Ser 65 70 75 80 Leu
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys 85 90
95 Ala Thr Thr Asp Cys Asp Thr Thr Ser Arg Met Thr Tyr Leu Ser Tyr
100 105 110 Leu Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120
11123PRTLama glamaMISC_FEATURE3G2 11Gln Val Gln Leu Gln Glu Ser Gly
Gly Gly Ser Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Thr Leu Ser Cys
Leu Ala Ser Gly Phe Ser Leu Ser Asn Tyr 20 25 30 Gly Met Gly Trp
Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ser Cys
Thr Ser Ser Pro Ser Gly His Thr Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Val Arg Asp Asn Ala Gly Asn Ser Val Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Ala Ala Val Tyr
Phe Cys 85 90 95 Ala Thr Thr Asp Cys Glu Ala Ala His Trp Gly Thr
Trp Val Asn Tyr 100 105 110 Tyr Gly Gln Gly Thr Gln Val Thr Val Ser
Ser 115 120 12123PRTLama glamaMISC_FEATURE3G4 12Gln Val Gln Leu Gln
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Thr Ser Gly Phe Pro Leu Arg Val Tyr 20 25 30 Gly
Val Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40
45 Ser Cys Ser Ser Val His Gly Ala Arg Ile His Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr
Val Phe 65 70 75 80 Leu Glu Met Asn Asp Leu Lys Pro Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Thr Thr Asp Cys Glu Ala Thr Ser Trp
Gly Thr Tyr Ile Ser Trp 100 105 110 His Gly Gln Gly Thr Gln Val Thr
Val Ser Ser 115 120 13123PRTLama glamaMISC_FEATURE3H10 13Gln Val
Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Arg Asn Tyr 20
25 30 Gly Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly
Val 35 40 45 Ser Cys Ser Asn Val Arg Asp Gly Ser Ile Tyr Tyr Ala
Asp Ser Val 50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Val Asn Val
Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Asp Leu Arg Pro Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Thr Asp Cys Glu Ala
Thr Gly Trp Gly Thr Trp Ile Gly Tyr 100 105 110 Phe Gly Gln Gly Thr
Gln Val Thr Val Ser Ser 115 120 14123PRTLama glamaMISC_FEATURE3H8
14Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Leu Val Leu
Tyr 20 25 30 Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Lys Arg
Glu Ala Val 35 40 45 Ser Cys Ser Ser Val Asn Asp Gly Gly Thr Tyr
Tyr Ala Glu Ser Val 50 55 60 Lys Gly Arg Phe Thr Leu Phe Arg Asp
Asn Gly Ala Asn Ala Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Glu
Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Thr Asp Cys
Glu Ala Thr Gly Trp Gly Thr Trp Thr Asn Tyr 100 105 110 Arg Gly Gln
Gly Thr Gln Val Thr Val Ser Ser 115 120 15123PRTLama
glamaMISC_FEATURE4A1 15Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Ser Leu Arg Tyr Phe 20 25 30 Gly Ile Gly Trp Phe Arg Gln
Ala Ala Gly Lys Glu Arg Glu Gly Val 35 40 45 Ser Cys Ser Asn Val
Arg Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Val Arg Asn Met Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Ser Cys 85 90
95 Ala Thr Thr Asp Cys Glu Ala Ala Asn Trp Gly Thr Tyr Val Ser Tyr
100 105 110 Tyr Gly Arg Gly Thr Gln Val Thr Val Ser Ser 115 120
16123PRTLama glamaMISC_FEATURE5B5 16Gln Val Gln Leu Gln Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Ser Leu Val Tyr Tyr 20 25 30 Gly Ile Gly Trp
Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ser Cys
Ser Ser Val His Asp Gly Ser Thr Tyr Tyr Ala Glu Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65
70 75 80 Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Thr Thr Asp Cys Glu Ala Thr Gly Trp Gly Thr
Trp Thr Asn Tyr 100 105 110 Arg Gly Gln Gly Thr Gln Val Thr Val Ser
Ser 115 120 17123PRTLama glamaMISC_FEATURE5B6 17Gln Val Gln Leu Gln
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Val Ser Phe 20 25 30 Gly
Ile Gly Trp Phe Arg Gln Ala Ala Gly Lys Glu Arg Glu Gly Val 35 40
45 Ser Cys Ser Asn Val Arg Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Arg Asn Gln
Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala
Val Tyr Ser Cys 85 90 95 Ala Thr Thr Asp Cys Glu Ala Thr Ser Trp
Gly Thr Tyr Arg Gly Tyr 100 105 110 Phe Gly Gln Gly Thr Gln Val Thr
Val Ser Ser 115 120 18123PRTLama glamaMISC_FEATURE5C4 18Gln Val Gln
Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Arg Tyr Phe 20 25
30 Gly Ile Gly Trp Phe Arg Gln Val Ala Gly Lys Glu Arg Glu Pro Val
35 40 45 Ser Cys Ser Asn Val Arg Asp Gly Asn Thr Tyr Tyr Ala Asp
Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Arg
Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
Thr Ala Val Tyr Ser Cys 85 90 95 Ala Thr Thr Asp Cys Glu Ala Thr
Thr Trp Gly Thr Tyr Arg Gly Tyr 100 105 110 Phe Gly Gln
Gly Thr Gln Val Thr Val Ser Ser 115 120 19123PRTLama
glamaMISC_FEATURE5C5 19Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Ser Leu Arg Asn Tyr 20 25 30 Gly Ile Gly Trp Phe Arg Gln
Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ser Cys Ser Asn Val
Arg Asp Gly Ser Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Gln Gly Arg
Leu Thr Ile Ser Arg Val Asn Val Lys Asn Thr Leu Tyr 65 70 75 80 Leu
Gln Met Asn Asp Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Thr Thr Asp Cys Glu Ala Thr Gly Trp Gly Thr Trp Ile Gly Tyr
100 105 110 Phe Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120
20123PRTLama glamaMISC_FEATURE5D4 20Gln Val Gln Leu Gln Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Asp Met Arg Arg Phe 20 25 30 Gly Ile Gly Trp
Phe Arg Gln Val Ala Gly Lys Glu Arg Glu Gly Val 35 40 45 Ser Cys
Ser Asn Val His Asp Gly Thr Thr Tyr Tyr Thr Asn Asp Val 50 55 60
Lys Gly Arg Phe Thr Ile Val Arg Asp Asn Thr Lys Asn Met Leu Tyr 65
70 75 80 Leu Gln Met Asn Lys Leu Arg Pro Glu Asp Thr Ala Val Tyr
Ser Cys 85 90 95 Ala Thr Thr Asp Cys Glu Ala Thr Ala Trp Gly Thr
Tyr Arg Gly Tyr 100 105 110 Phe Gly Gln Gly Thr Gln Val Thr Val Ser
Ser 115 120 21123PRTLama glamaMISC_FEATURE5E5 21Gln Val Gln Leu Gln
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Thr
Leu Ser Cys Thr Ala Ser Gly Phe Ala Met Arg Arg Phe 20 25 30 Gly
Ile Gly Trp Phe Arg Gln Val Val Gly Lys Glu Arg Glu Gly Val 35 40
45 Ser Cys Ser Asn Val His Asp Gly Ser Thr Tyr Tyr Ala Asn Tyr Val
50 55 60 Lys Gly Arg Phe Thr Ile Val Arg Asp Asp Thr Lys Asn Met
Leu Tyr 65 70 75 80 Leu His Met Asn Ser Leu Arg Ala Glu Asp Thr Gly
Val Tyr Ser Cys 85 90 95 Ala Thr Thr Asp Cys Glu Ala Thr Ala Trp
Gly Thr Tyr Arg Gly Tyr 100 105 110 Phe Gly Gln Gly Thr Gln Val Thr
Val Ser Ser 115 120 22123PRTLama glamaMISC_FEATURE5F5 22Gln Val Gln
Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Leu Gly Leu Tyr 20 25
30 Gly Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Ala Val
35 40 45 Ser Cys Asp Ser Val Asp Asp Gly Ser Thr Asn Tyr Leu Asp
Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Met Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Thr Asp Cys Asp Ala Lys
Ala Trp Gly Thr Trp Thr Asn Tyr 100 105 110 Arg Gly Gln Gly Thr Gln
Val Thr Val Ser Ser 115 120 23123PRTLama glamaMISC_FEATURE5G2 23Gln
Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Arg Thr Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Ala Leu Ala Asn Tyr
20 25 30 Gly Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
Gly Val 35 40 45 Ser Cys Ser Asn Val Arg Asp Gly Ser Thr Tyr Tyr
Arg Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asn Asn
Ile Glu Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Ser Thr Leu Arg Pro
Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Thr Thr Asp Cys Glu
Ala Ser Ser Trp Gly Thr Trp Ile Asn Tyr 100 105 110 Arg Gly Gln Gly
Thr Gln Val Thr Val Ser Ser 115 120 24123PRTLama
glamaMISC_FEATURE5G5 24Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Ser Leu Arg Tyr Phe 20 25 30 Gly Ile Gly Trp Phe Arg Gln
Ala Ala Gly Lys Glu Arg Glu Gly Ile 35 40 45 Ser Cys Ser Asn Val
Arg Asp Gly Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Val Arg Asn Met Leu Tyr 65 70 75 80 Leu
Gln Met Asn Asn Leu Lys Pro Asp Asp Thr Ala Val Tyr Ser Cys 85 90
95 Ala Thr Thr Asp Cys Glu Ala Thr Thr Trp Gly Thr Tyr Arg Gly Tyr
100 105 110 Phe Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120
25123PRTLama glamaMISC_FEATURE5H5 25Gln Val Gln Leu Gln Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Glu Gly Phe Ala Leu Ala Asn Tyr 20 25 30 Gly Val Gly Trp
Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Arg Ile 35 40 45 Ser Cys
Ser Ser Val Arg Asp Asn Gly Pro Tyr Tyr Ala Glu Ser Val 50 55 60
Lys Gly Arg Ser Thr Ile Ser Arg Arg Asn Ala Glu Asn Thr Leu Tyr 65
70 75 80 Leu His Met Ser Asn Leu Lys Ala Glu Asp Thr Ala Leu Tyr
Tyr Cys 85 90 95 Ala Thr Thr Asp Cys Asp Ala Thr Gly Trp Gly Thr
Trp Thr Asn Tyr 100 105 110 Arg Gly Gln Gly Thr Gln Val Thr Val Ser
Ser 115 120 26123PRTLama glamaMISC_FEATURE7A2 26Gln Val Gln Leu Gln
Glu Ser Gly Gly Gly Ser Val His Pro Gly Gly 1 5 10 15 Ser Leu Thr
Leu Ser Cys Leu Ala Ser Gly Phe Ser Leu Ser Asn Tyr 20 25 30 Gly
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Ala Val 35 40
45 Ser Cys Thr Ser Val Pro Asn Gly His Ile Tyr Tyr Ala Glu Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Val Arg Asp Asn Ala Gly Asn Ser
Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Ala Ala
Asn Tyr Phe Cys 85 90 95 Ala Thr Thr Asp Cys Glu Ala Ala His Trp
Gly Thr Trp Val Asn Tyr 100 105 110 Tyr Gly Gln Gly Thr Gln Val Thr
Val Ser Ser 115 120 27123PRTLama glamaMISC_FEATURE7C2 27Gln Val Gln
Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Phe Gly Phe Ala Leu Ala Asn Tyr 20 25
30 Gly Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Asp Arg Glu Arg Val
35 40 45 Ser Cys Asp Ser Val Asp Asp Gly Ser Thr His Tyr Ser Asn
Ser Val 50 55 60 Gln Gly Arg Phe Thr Ile Ile Arg Asp Asn Ala Lys
Asn Thr Val Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Thr Asp Cys Asp Ala Thr
Thr Trp Gly Thr Trp Ile Asn Tyr 100 105 110 Arg Gly Gln Gly Thr Gln
Val Thr Val Ser Ser 115 120 28123PRTLama glamaMISC_FEATURE7D2 28Gln
Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10
15 Ser Leu Lys Val Ala Cys Ala Ala Ala Gly Phe Ser Leu Arg Tyr Tyr
20 25 30 Gly Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
Ala Val 35 40 45 Ser Cys Thr Ser Ala Asn Asp Gly Ser Thr Tyr Tyr
Arg Asp Ser Val 50 55 60 Arg Gly Arg Phe Thr Ile Ser Arg Asp Asp
Gly Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Arg Leu Lys Pro
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Thr Asp Cys Asp
Ala Thr Ser Trp Gly Thr Trp Ile Asn Tyr 100 105 110 Tyr Gly Gln Gly
Thr Gln Val Thr Val Ser Ser 115 120 29123PRTLama
glamaMISC_FEATURE7E1_1 29Gln Val Gln Leu Gln Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Ser Leu Ser Asn Tyr 20 25 30 Gly Met Gly Trp Phe Arg
Gln Ala Pro Gly Lys Gly Arg Glu Arg Ile 35 40 45 Ser Cys Ser Ser
Val Arg Asp Asn Gly Pro Tyr Tyr Ala Glu Ser Val 50 55 60 Lys Gly
Arg Ser Thr Ile Ser Arg Arg Asn Thr Glu Asn Thr Leu Tyr 65 70 75 80
Leu His Met Ser Asn Leu Lys Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85
90 95 Ala Thr Thr Asp Cys Asp Ala Thr Gly Trp Gly Thr Trp Thr Asn
Tyr 100 105 110 Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120
30123PRTLama glamaMISC_FEATURE7F1 30Gln Val Gln Leu Gln Glu Ser Gly
Gly Gly Leu Val Gln Ala Gly Arg 1 5 10 15 Ser Leu Glu Val Ala Cys
Ala Ala His Gly Phe Ser Leu Arg Asn Tyr 20 25 30 Gly Ile Gly Trp
Phe Arg Gln Val Pro Gly Lys Ala Arg Glu Ala Val 35 40 45 Ser Cys
Thr Ser Val Asn Asp Gly Thr Thr His Tyr Gly Asp Ser Val 50 55 60
Arg Gly Arg Phe Ser Ile Ala Arg Asp Asn Ala Lys Asn Thr Val Phe 65
70 75 80 Leu Gln Met Asn Asp Leu Lys Pro Glu Asp Thr Ala Val Tyr
Phe Cys 85 90 95 Ala Thr Thr Asp Cys Asp Ala Thr Ser Trp Gly Thr
Trp Ile Asn Tyr 100 105 110 Tyr Gly Gln Gly Thr Gln Val Thr Val Ser
Ser 115 120 31123PRTLama glamaMISC_FEATURE8B10 31Gln Val Gln Leu
Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Val Ala Ser Gly Phe Pro Leu Gly Leu Tyr 20 25 30
Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Ala Val 35
40 45 Ser Cys Ser Ser Val His Asp Gly Ser Thr Tyr Tyr Ala Glu Phe
Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
Thr Met Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Ala
Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Thr Asp Cys Glu Ala Ser Ser
Trp Gly Thr Trp Ile Asn Tyr 100 105 110 Arg Gly Gln Gly Thr Gln Val
Thr Val Ser Ser 115 120 32123PRTLama glamaMISC_FEATURE8B12 32Gln
Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Val Tyr Tyr
20 25 30 Gly Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
Gly Val 35 40 45 Ala Cys Ile Ser Ala Leu Arg Asp Thr Thr Tyr Tyr
Thr Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Leu Ser Arg Asp Asn
Val Lys Asn Thr Leu Ser 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro
Glu Asp Thr Gly Val Tyr Tyr Cys 85 90 95 Ala Thr Thr Asp Cys Asp
Ala Thr Ser Arg Met Thr Tyr Leu Ser Tyr 100 105 110 Leu Gly Gln Gly
Thr Gln Val Thr Val Ser Ser 115 120 33123PRTLama
glamaMISC_FEATURE9A1 33Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Pro Leu Arg Leu Ser Cys Thr Ala Ser
Gly Phe Asn Ile Phe Tyr Tyr 20 25 30 Gly Met Gly Trp Phe Arg Gln
Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Cys Ile Ser Ala
Leu Arg Gln Ser Thr Tyr Tyr Ser Asp Ser Val 50 55 60 Glu Gly Arg
Phe Thr Leu Ser Arg Asp Asn Ala Lys Asn Thr Leu Ser 65 70 75 80 Leu
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Val Tyr Phe Cys 85 90
95 Ala Thr Thr Asp Cys Asp Ala Ala Ser Arg Met Thr Tyr Thr Ser Tyr
100 105 110 Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120
34123PRTLama glamaMISC_FEATURE9B5 34Gln Val Gln Leu Gln Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Ser Leu Arg Tyr Phe 20 25 30 Gly Ile Gly Trp
Phe Arg Gln Ala Ala Gly Lys Glu His Glu Gly Ile 35 40 45 Ser Cys
Ser Asn Val Arg Asp Gly Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Arg Asn Met Leu Tyr 65
70 75 80 Leu Gln Met Asn Asn Leu Lys Pro Asp Asp Thr Ala Val Tyr
Ser Cys 85 90 95 Ala Thr Thr Asp Cys Glu Ala Thr Thr Trp Gly Thr
Tyr Arg Gly Tyr 100 105 110 Phe Gly Gln Gly Thr Gln Val Thr Val Ser
Ser 115 120 35123PRTLama glamaMISC_FEATURE9C4 35Gln Val Gln Leu Gln
Glu Ser Gly Gly Gly Leu Val His Pro Gly Gly 1 5 10 15 Pro Leu Thr
Leu Ser Cys Ala Ala Ser Gly Phe Arg Val Glu Tyr Tyr 20 25 30 Gly
Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val 35 40
45 Ser Cys Ile Ser Ala Leu His Glu Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Asn Ala
Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly
Val Tyr Tyr Cys 85 90 95 Ala Thr Thr Asp Cys Asp Ala Thr Ser Trp
Gly Thr Trp Thr Asn Tyr 100 105 110 Arg Gly Gln Gly Thr Gln Val Thr
Val Ser Ser 115 120 36123PRTLama glamaMISC_FEATURE9D5 36Gln Val Gln
Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Thr Leu Ser Cys Val Gly His Gly Phe Gly Val Ala Asn Phe 20 25
30 Gly Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Ala Val
35 40 45 Ser Cys Asp Ser Val Asp Asp Gly Thr Ile Ala Tyr Ala Asp
Ser Val 50 55 60 Lys Gly Arg Phe Thr Leu Phe Arg Asp Asn Tyr Lys
Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Arg Leu Arg Pro Glu Asp
Thr Ala Val Tyr Phe Cys 85 90 95 Ala Thr Thr Asp Cys Asp Ala Arg
Ser Trp Gly Thr Trp Ile Asn Tyr
100 105 110 Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120
37123PRTLama glamaMISC_FEATURE9E1 37Gln Val Gln Leu Gln Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Glu Ala Ser Gly Phe Arg Leu Arg Asn Phe 20 25 30 Gly Ile Gly Trp
Phe Arg Gln Ala Ala Gly Lys Glu Arg Glu Gly Val 35 40 45 Ser Cys
Ser Asn Val Arg Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Ile Ile Ser Arg Asp Asn Thr Arg Asn Thr Leu Ser 65
70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr
Ser Cys 85 90 95 Gly Thr Thr Asp Cys Glu Ala Thr Gly Trp Gly Thr
Tyr Val Gly Tyr 100 105 110 Phe Gly Gln Gly Thr Gln Val Thr Val Ser
Ser 115 120 38123PRTLama glamaMISC_FEATURE9E4 38Gln Val Gln Leu Gln
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Val Tyr Tyr 20 25 30 Gly
Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40
45 Ser Cys Ser Ser Val His Asp Gly Ser Thr Tyr Tyr Ala Glu Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Thr Thr Asp Cys Glu Ala Thr Gly Trp
Gly Thr Trp Thr Asn Tyr 100 105 110 Arg Gly Gln Gly Thr Gln Val Thr
Val Ser Ser 115 120 39123PRTLama glamaMISC_FEATURE9F4 39Gln Val Gln
Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Leu Ser Val Tyr 20 25
30 Gly Ile Gly Trp Phe Arg Leu Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45 Ser Cys Ser Ser Val His Asp Gly Ser Thr Tyr Tyr Ala Glu
Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Asn Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Thr Asp Cys Asp Ala Ser
Ser Trp Gly Thr Trp Thr Asn Tyr 100 105 110 Arg Gly Gln Gly Thr Gln
Val Thr Val Ser Ser 115 120 40123PRTLama glamaMISC_FEATURE9H1 40Gln
Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Met Arg Arg Phe
20 25 30 Gly Ile Gly Trp Phe Arg Gln Val Ala Gly Lys Glu Arg Glu
Gly Val 35 40 45 Ser Cys Ser Asn Val His Asp Gly Thr Thr Tyr Tyr
Thr Asn Asp Val 50 55 60 Lys Gly Arg Phe Thr Ile Val Arg Asp Asn
Thr Lys Asn Met Leu Tyr 65 70 75 80 Leu Gln Met Asn Glu Leu Arg Pro
Glu Asp Thr Ala Val Tyr Ser Cys 85 90 95 Ala Thr Thr Asp Cys Glu
Ala Thr Ala Trp Gly Thr Tyr Arg Gly Tyr 100 105 110 Phe Gly Gln Gly
Thr Gln Val Thr Val Ser Ser 115 120 41123PRTLama
glamaMISC_FEATURE9H2 41Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Leu Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Ser Val Ala Tyr Tyr 20 25 30 Gly Met Gly Trp Phe Arg Gln
Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Cys Ile Ser Ala
Leu Arg Asp Thr Thr Tyr Tyr Thr Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Leu Ser Arg Asp Asn Val Lys Asn Thr Leu Ser 65 70 75 80 Leu
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys 85 90
95 Ala Thr Thr Asp Cys Asp Ala Thr Ser Arg Met Thr Tyr Leu Ser Tyr
100 105 110 Leu Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120
42124PRTLama glama 42Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu
Val Gln Ala Gly Asp 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Arg Ile Phe Ser Ala Tyr 20 25 30 Val Val Gly Trp Phe Arg Gln
Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Arg Trp
Ser Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Gln Asn Thr Val Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90
95 Ala Ala Lys Tyr Ser Gly Ser Tyr Tyr Leu Ser Ser Tyr Ala Tyr Asn
100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120
4323DNAArtificial SequencePrimer 43gtcctggctg ctcttctaca agg
234423DNAArtificial SequencePrimer 44cctggctgct cttctacaag gtg
234523DNAArtificial SequencePrimer 45ggtacgtgct gttgaactgt tcc
234629DNAArtificial SequencePrimer 46gatgtgcagc tgcaggagtc
tggrggagg 294735DNAArtificial SequencePrimer 47ggactagtgc
ggccgctgga gacggtgacc tgggt 354820DNAArtificial SequencePrimer
48ttatgcttcc ggctcgtatg 204919DNAArtificial SequencePrimer
49ccacagacag ccctcatag 19509PRTArtificial SequenceLinker 50Gly Gly
Gly Gly Ser Gly Gly Gly Ser 1 5 5111PRTArtificial SequenceTag 51Glu
Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn 1 5 10 526PRTArtificial
SequenceTag 52His His His His His His 1 5 5323PRTArtificial
SequenceSequence C-terminal of bivalent VHH construct 53Ala Ala Ala
Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Gly Ala 1 5 10 15 Ala
His His His His His His 20
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