U.S. patent application number 13/647302 was filed with the patent office on 2013-08-22 for specific delivery of agrochemicals.
This patent application is currently assigned to Erik Jongedijk. The applicant listed for this patent is Erik Jongedijk, Vib VZW, Vrije Universiteit Brussel. Invention is credited to Chris De Jonghe, Peter Verheesen.
Application Number | 20130217572 13/647302 |
Document ID | / |
Family ID | 42331002 |
Filed Date | 2013-08-22 |
United States Patent
Application |
20130217572 |
Kind Code |
A1 |
Verheesen; Peter ; et
al. |
August 22, 2013 |
SPECIFIC DELIVERY OF AGROCHEMICALS
Abstract
Described is the specific delivery of agrochemicals to plants.
More specifically, described is a composition having 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. Also described is a
binding domain that specifically binds the binding site on an
intact living plant. More specifically, described are binding
domains comprising a peptide sequence that comprises four framework
regions and three complementary-determining regions, or any
suitable fragment thereof, so that the binding domains are able to
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. Described is 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, described is a method for manufacturing a specifically
targeting agrochemical carrier.
Inventors: |
Verheesen; Peter; (Gent,
BE) ; De Jonghe; Chris; (Aartselaar, BE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Vib VZW;
Vrije Universiteit Brussel;
Jongedijk; Erik |
|
|
US
US
US |
|
|
Assignee: |
Jongedijk; Erik
Lokeren
BE
VRIJE UNIVERSITEIT BRUSSEL
Brussel
BE
VIB VZW
Gent
BE
|
Family ID: |
42331002 |
Appl. No.: |
13/647302 |
Filed: |
October 8, 2012 |
Current U.S.
Class: |
504/116.1 |
Current CPC
Class: |
A01N 25/24 20130101;
C07K 2317/92 20130101; A01N 25/28 20130101; C07K 16/16 20130101;
A61K 47/6835 20170801; A01N 25/00 20130101; A01N 3/00 20130101;
C07K 2317/569 20130101; A01N 53/00 20130101; C07K 2317/35 20130101;
A01N 25/28 20130101; A01N 25/24 20130101 |
Class at
Publication: |
504/116.1 |
International
Class: |
A01N 25/28 20060101
A01N025/28 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 6, 2010 |
EP |
10159100.6 |
Claims
1. A process for producing specifically targeting microcapsules,
the process comprising: emulsifying into a continuous aqueous phase
an organic phase in which a to be encapsulated agrochemical or
combination of agrochemicals is dissolved or dispersed to form an
emulsion of droplets of the organic phase in the continuous aqueous
phase; causing an aqueous suspension of microcapsules with polymer
walls having anchor groups at their surface to be formed; and
covalently linking at least one targeting agent to the anchor
groups at the microcapsule surface, at a ratio from about 0.01
.mu.g to about 1 .mu.g targeting agent per square centimeter
microcapsular surface.
2. The process of claim 1, further comprising: causing
polymerization of polyfunctional monomers or pre-polymers present
in the organic phase to form an aqueous suspension of microcapsules
having anchor groups at their surface.
3. The process of claim 2, further comprising: adding to the
emulsion a monomer- or pre-polymer reactant component containing
anchor groups.
4. The process of claim 1, further comprising: emulsifying into the
continuous aqueous phase an organic phase which contains a
pre-polymer or mixture of pre-polymers containing anchor groups,
causing in situ self-condensation of the pre-polymers surrounding
the droplets of organic phase to form an aqueous suspension of
microcapsules having polymer walls with anchor groups at their
surface.
5. The process of claim 1, wherein the process comprises:
emulsifying into the continuous aqueous phase an organic phase in
which a to be encapsulated agrochemical or combination of
agrochemicals is dissolved or dispersed to form an emulsion of
droplets of the organic phase in the continuous aqueous phase;
adding to the continuous aqueous phase a water-soluble pre-polymer
or mixture of pre-polymers, containing anchor groups; causing in
situ self-condensation of the pre-polymers surrounding the droplets
of organic phase to form an aqueous suspension of microcapsules
with polymer walls having anchor groups at their surface; and
covalently linking at least one targeting agent to the anchor
groups at the microcapsule surface, at a ratio from about 0.01
.mu.g to about 1 .mu.g targeting agent per square centimeter
microcapsular surface.
6. The process of claim 1, wherein the targeting agent comprises an
antigen binding protein.
7. The process of claim 6, wherein the antigen binding protein is
derived from a camelid antibody.
8. The process of claim 7, wherein the antigen binding protein is
comprised in a VHH.
9. A specifically targeting microcapsule produced of a process
comprising: emulsifying into a continuous aqueous phase an organic
phase comprising an agrochemical, together with polyfunctional
monomers or pre-polymers, to form an emulsion of droplets of the
organic phase in the continuous aqueous phase, thus forming an
aqueous suspension of microcapsules, microcapsules thereof having
polymer walls with anchor groups at each microcapsule's surface;
and covalently linking a targeting agent to the anchor groups at
the microcapsule surface, at a ratio from about 0.01 .mu.g to about
1 .mu.g targeting agent per square centimeter microcapsular
surface.
10. The specifically targeting microcapsule, of claim 9, able to
bind an agrochemical or combination of agrochemicals to a
surface.
11. The specifically targeting microcapsule of claim 9, having a
targeting agent comprising an antigen binding protein.
12. The specifically targeting microcapsule of claim 11, wherein
the antigen binding protein is derived from a camelid antibody.
13. The specifically targeting microcapsule of claim 12, wherein
the antigen binding protein is comprised in a VHH sequence.
14. An agrochemical composition comprising a suspension or
dispersion of the specifically targeting microcapsules of claim 9
in an aqueous medium.
15. A method of protecting a plant, or modulating viability, growth
or yield of a plant or plant part and/or to modulate gene
expression in a plant or plant part, the method comprising:
utilizing the agrochemical composition of claim 14 to protect a
plant and/or to modulate the viability, growth or yield of a plant
or plant part, and/or to modulate gene expression in a plant or
plant part.
16. The process of claim 1, wherein the continuous aqueous phase
comprises a surfactant.
17. A process for producing a microcapsule, the process comprising:
emulsifying into a continuous aqueous phase an organic phase
comprising an agrochemical and polyfunctional monomers or
pre-polymers, to form an emulsion of droplets of the organic phase
in the continuous aqueous phase, thus forming an aqueous suspension
of microcapsules, wherein microcapsules thereof have polymer walls
with anchor groups at the microcapsule's surface; and covalently
linking a targeting agent to the anchor groups at the microcapsule
surface, at a ratio from about 0.01 .mu.g to about 1 .mu.g
targeting agent per square centimeter microcapsular surface.
18. The process of claim 17, wherein the continuous aqueous phase
comprises a surfactant.
19. The process of claim 17, wherein the organic phase comprises a
combination of agrochemicals.
20. The process of claim 17, comprising covalently linking more
than one targeting agent to the anchor groups at the microcapsule
surface.
Description
CROSS-REFERENCE TO RELATED APPLICATION(S)
[0001] This application is a continuation-in-part of co-pending
U.S. patent application Ser. No. 13/081,435, filed Apr. 6, 2011,
which is a utility conversion of U.S. Provisional Patent
Application Ser. No. 61/341,930, filed Apr. 6, 2010, and claims
priority to European Patent Application Serial No. EP 10159100.6,
filed Apr. 6, 2010, the disclosure of each of which is hereby
incorporated herein by this reference in its entirety.
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, so that the binding domains are able to bind or retain a
carrier onto a plant. In one embodiment, the disclosure relates to
binding domains which specifically bind trichomes, stomata,
cuticle, lenticels, thorns, spines, root hairs, or wax layer. The
disclosure 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, the disclosure
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 (incorporated herein by reference)
describes composite gel microparticles that can be used to deliver
plant-protection active principles. WO 2005102045 (incorporated
herein by reference) 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. Patent
Publication 20070280981 (incorporated herein by reference)
describes carrier granules, coated with a lipophilic tackifier on
the surface, so that 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. No. 5,738,984 and U.S. Pat. No. 6,124,117 (both
incorporated herein by reference)). 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
(incorporated herein by reference) 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 (incorporated herein by reference), 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 (incorporated herein
by reference) 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:
[0008] 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.
[0009] Second, 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.
[0010] Third, 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.
[0011] 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 able 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.
[0012] 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
(incorporated herein by reference)), 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.
[0013] There is still an unmet need for 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 capable of retaining an
agrochemical or a carrier containing the agrochemical onto the
plant.
[0014] We isolated binding domains, more specifically binding
domains comprising an amino acid sequence that comprises four
framework regions (FR) and three complementary-determining regions
(CDR) (FR and CDR definitions according to Kabat), so that the
binding domains are able 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.
Preferably, 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; more preferably, 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). Preferably, 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).
DISCLOSURE
[0015] 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, so that 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.
[0016] Moreover, when comprised in a composition disclosed herein,
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.
BRIEF DESCRIPTION OF THE FIGURES
[0017] FIG. 1: Binding domains (VHH) binding to leaf surface
[0018] 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.
[0019] 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.
[0020] 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.
[0021] 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.
[0022] FIG. 1E: Without primary antibody (plain PBS) on native
potato leaf surface. Incubation with anti-histidine antibodies
directly conjugated with Alexa-488 fluorescent dye.
[0023] 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.
[0024] 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.
[0025] FIG. 2: Binding of binding domains (VHH) to intact living
plant
[0026] 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.
[0027] 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.
[0028] FIG. 3: Coupling of binding domains to microcapsules
[0029] 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.
[0030] 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.
[0031] 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.
[0032] 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.
[0033] 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.
[0034] 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
Definitions
[0035] The invention will be described with respect to particular
embodiments and with reference to certain drawings. Any reference
signs in the claims shall not be construed as limiting the scope.
The drawings described are only schematic and are non-limiting. In
the drawings, the size of some of the elements may be exaggerated
and not drawn on scale for illustrative purposes. Where the term
"comprising" is used in the present description and claims, it does
not exclude other elements or steps. Where an indefinite or
definite article is used when referring to a singular noun, e.g.,
"a" or "an," "the," this includes a plural of that noun unless
something else is specifically stated. Furthermore, the terms
first, second, third and the like in the description and in the
claims, are used for distinguishing between similar elements and
not necessarily for describing a sequential or chronological order.
It is to be understood that the terms so used are interchangeable
under appropriate circumstances and that the embodiments hereof
described herein are capable of operation in other sequences than
described or illustrated herein.
[0036] Unless otherwise defined herein, scientific and technical
terms and phrases used in connection with the disclosure shall have
the meanings that are commonly understood by those of ordinary
skill in the art. Generally, nomenclatures used in connection with,
and techniques of molecular and cellular biology, genetics and
protein and nucleic acid chemistry described herein are those
well-known and commonly used in the art. The methods and techniques
hereof are generally performed according to conventional methods
well known in the art and as described in various general and more
specific references that are cited and discussed throughout the
present specification unless otherwise indicated. See, for example,
Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed.,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1989); Ausubel et al., Current Protocols in Molecular Biology,
Greene Publishing Associates (1992, and Supplements to 2002).
[0037] As used herein, the terms "determining," "measuring,"
"assessing," "monitoring" and "assaying" are used interchangeably
and include both quantitative and qualitative determinations.
[0038] The terms "effective amount," "effective dose" and
"effective amount," as used herein, mean the amount needed to
achieve the desired result or results.
[0039] As used herein, the terms "polypeptide," "protein,"
"peptide" are used interchangeably, and refer to a polymeric form
of amino acids of any length, which can include coded and non-coded
amino acids, chemically or biochemically modified or derivatized
amino acids, and polypeptides having modified peptide
backbones.
[0040] As used herein, the terms "complementarity-determining
region" or "CDR" within the context of antibodies refer to variable
regions of either H (heavy) or L (light) chains (also abbreviated
as VH and VL, respectively) and contains the amino acid sequences
capable of specifically binding to antigenic targets. These CDR
regions account for the basic specificity of the antibody for a
particular antigenic determinant structure. Such regions are also
referred to as "hypervariable regions." The CDRs represent
non-contiguous stretches of amino acids within the variable regions
but, regardless of species, the positional locations of these
critical amino acid sequences within the variable heavy and light
chain regions have been found to have similar locations within the
amino acid sequences of the variable chains. The variable heavy and
light chains of all canonical antibodies each have three CDR
regions, each non-contiguous with the others (termed L1, L2, L3,
H1, H2, H3) for the respective light (L) and heavy (H) chains.
[0041] The term "affinity," as used herein, refers to the degree to
which a polypeptide, in particular an immunoglobulin, such as an
antibody, or an immunoglobulin fragment, such as a VHH, binds to an
antigen so as to shift the equilibrium of antigen and polypeptide
toward the presence of a complex formed by their binding. Thus, for
example, where an antigen and antibody (fragment) are combined in
relatively equal concentration, an antibody (fragment) of high
affinity will bind to the available antigen so as to shift the
equilibrium toward high concentration of the resulting complex. The
dissociation constant is commonly used to describe the affinity
between the protein-binding domain and the antigenic target.
Typically, the dissociation constant is lower than 10.sup.-5 M.
Preferably, the dissociation constant is lower than 10.sup.-6 M,
more preferably, lower than 10.sup.-7 M. Most preferably, the
dissociation constant is lower than 10.sup.-8 M.
[0042] The terms "specifically bind" and "specific binding," as
used herein, generally refers to the ability of a polypeptide, in
particular an immunoglobulin, such as an antibody, or an
immunoglobulin fragment, such as a VHH, to preferentially bind to a
particular antigen that is present in a homogeneous mixture of
different antigens. In certain embodiments, a specific binding
interaction will discriminate between desirable and undesirable
antigens in a sample, in some embodiments more than about ten- to
100-fold or more (e.g., more than about 1000- or 10,000-fold).
[0043] "Plant" as used herein, means live plants and live plant
parts, including fresh fruit, vegetables and seeds. 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, cassava,
tobacco, banana, apple, orange, lemon, olive, pineapple, avocado,
vines, lettuce, cabbage, carrot, eggplant, pepper, melon, rose,
lilies, chrysanthemum, grass-like weeds, or broadleaved weeds.
[0044] 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, etc.) 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,
it should be clear that 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.
[0045] "Crop" as used herein means a plant species or variety that
is grown to be harvested as food, livestock fodder, fuel raw
material, or for any other economic purpose. As a non-limiting
example, the crops can be maize, cereals, such as wheat, rye,
barley and oats, sorghum, rice, sugar beet and fodder beet, fruit,
such as pome fruit (e.g., apples and pears), citrus fruit (e.g.,
oranges, lemons, limes, grapefruit, or mandarins), stone fruit
(e.g., peaches, nectarines or plums), nuts (e.g., almonds or
walnuts), soft fruit (e.g., cherries, strawberries, blackberries or
raspberries), the plantain family or grapevines, leguminous crops,
such as beans, lentils, peas and soya, oil crops, such as
sunflower, safflower, rapeseed, canola, castor or olives,
cucurbits, such as cucumbers, melons or pumpkins, fiber plants,
such as cotton, flax or hemp, fuel crops, such as sugarcane,
miscanthus or switchgrass, vegetables, such as potatoes, tomatoes,
peppers, lettuce, spinach, onions, carrots, egg-plants, asparagus
or cabbage, ornamentals, such as flowers (e.g., petunias,
pelargoniums, roses, tulips, lilies, or chrysanthemums), shrubs,
broad-leaved trees (e.g., poplars or willows) and evergreens (e.g.,
conifers), grasses, such as lawn, turf or forage grass or other
useful plants, such as coffee, tea, tobacco, hops, pepper, rubber
or latex plants.
[0046] "Microbe," as used herein, means bacterium, virus, fungus,
yeast and the like and "microbial" means derived from a
microbe.
[0047] "Active substance," as used herein, means any chemical
element and its compounds, including micro-organisms, having
general or specific action against harmful organisms or on plants,
parts of plants or plant products, as they occur naturally or by
manufacture, including any impurity inevitably resulting from the
manufacturing process.
[0048] "Agrochemical," as used herein, means any active substance
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; semiochemicals,
repellants; insect baits; microbes and microbial derived products
and/or active substances 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, imazalil, fluazinam,
pyraclostrobin, epoxiconazole, chlorothalonil, copper fungicides,
trifloxystrobin, prothioconazole, difenoconazole, carbendazim,
propiconazole, thiophanate, sulphur, boscalid and other known
agrochemicals or any suitable combination(s) thereof.
[0049] The terms "agrochemical composition" and "agrochemical
formulation" are used interchangeably and refer to 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.
[0050] "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.).
[0051] "Polyfunctional monomers," as used herein, means monomeric
components with functionalities greater than two that can be
converted by chemical reaction into polymers. Examples of such
polyfunctional monomers include, but are not limited to TDI
(toluene diisocyanate) and PMPPI (Polymethylene polyphenyl
isocyanate).
[0052] "Pre-polymers," as used herein, means partially polymerized
polyfunctional monomers, containing at least one free reactive
group, which when added to a pre-polymer-reactant compound will
participate in the further polymerization reaction.
[0053] "Monomer- or pre-polymer-reactant component," as used
herein, means a component containing for example hydroxyl, amine
and/or thiol groups such that it can participate in a chemical
reaction with the polyfunctional monomers or pre-polymers.
[0054] The terms "anchor groups" and "functional groups" are used
interchangeably and refer to parts of chemical compounds, that have
such properties that (poly)peptides can be bound covalently
thereon. Examples of such anchor groups include carboxyl-,
aldehyde-, hydroxyl-, sulfhydryl-, terminal alkyne-, diene,
dienophile and azide groups.
[0055] A "targeting agent," as used herein, is a molecular
structure, preferably with a polypeptide backbone, comprising at
least one antigen-binding protein. A targeting agent in its
simplest form consists solely of one single antigen-binding
protein; however, a targeting agent can comprise more than one
antigen-binding protein and can be monovalent or multivalent and
monospecific or multispecific, as further defined. Apart from one
single or multiple antigen-binding proteins, 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 antigen-binding protein. The other
moieties include, without limitation, one or more amino acids,
including labeled amino acids (e.g., fluorescently or radioactively
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.
[0056] The terms "antigen-binding protein" and "binding domain" are
used interchangeably and refer to 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. An antigen-binding protein can
be a naturally occurring molecule, it can be derived from a
naturally occurring molecule, or it can be entirely artificially
designed. An antigen-binding protein 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
antigen-binding proteins are carbohydrate antigen-binding proteins
(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).
[0057] A "microcapsule," as used herein, is a microcarrier,
consisting of an inner liquid core, preferably containing one or
more agrochemicals, more preferably active substances, surrounded
by a solid wall or shell.
[0058] 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. Preferably, the binding
site comprises at least one antigen.
[0059] "Antigen," as used herein, means a molecule capable of
eliciting an immune response in an animal.
[0060] A "microcarrier" as used herein, means a particulate carrier
where the particles are less than 500 .mu.m in diameter, preferably
less than 250 .mu.m, even more preferable less than 100 .mu.m,
still more preferably less than 50 .mu.m, most preferably less than
20 .mu.m.
[0061] 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.
[0062] A "linking agent," as used here, may be any linking agent
known to the person skilled in the art; that allows attaching of
targeting agents, preferably by covalent binding, to the
microcapsule surface, such as, but not limited to EDC
(1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) or
the homobifunctional cross-linker ((bis[sulfosuccinimidyl]suberate)
(BS3).
[0063] "Specifically targeting microcapsule," as used herein, means
that the microcapsule can bind specifically to a binding site on a
solid surface, through the antigen-binding proteins comprised in
the targeting agents present at the microcapsule surface.
[0064] "Retain" as used herein means that the binding force
resulting from the affinity or avidity of either one single binding
protein or a combination of two or more binding proteins or
targeting agents comprising antigen-binding proteins for its or
their target molecule present at the solid surface is larger than
the combined force and torque imposed by the gravity of the
carrier, and the force and torque, if any, imposed by shear forces
caused by one or more external factors.
[0065] "VHH," as used herein, means the variable domain of heavy
chain camelid antibodies, devoid of light chains.
[0066] A first aspect of the invention is a binding domain able to
bind at least one binding site on an intact living plant.
[0067] Preferably, the binding site comprises at least one antigen.
Preferably, 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. Preferably, 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.
[0068] 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. 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.
[0069] 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.
[0070] 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.
[0071] 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.
[0072] 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.
[0073] 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.
[0074] 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.
[0075] 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 preferred
embodiment, the harsh conditions are defined by a temperature range
from 4.degree. C.-50.degree. C., more preferably a temperature
range from 0.degree. C.-55.degree. C., even more preferably a
temperature range from 0.degree. C.-60.degree. C. In another
preferred embodiment, the harsh conditions are defined by the
presence of an agrochemical formulation as defined above.
[0076] 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. 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).
[0077] 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.
[0078] 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.
[0079] 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). 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.
[0080] In one embodiment, the binding domain hereof is binding gum
arabic. In another preferred 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, the VHH comprises
two disulphide bridges.
[0081] 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, the 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.
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).
[0082] A second aspect of the invention is a targeting agent, able
to retain an agrochemical on a plant and/or a plant part.
[0083] 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.
[0084] "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.
[0085] In order to be able 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.
[0086] In one embodiment, the agrochemical is bound on or comprised
in a carrier, as defined above, so that the targeting agent is
coupled either to the carrier or to the agrochemical. The binding
domain may be coupled to the carrier. "Coupled," as used here, 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. The coupling may
be 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. As a non-limiting example, coupling by forming of a
carbodiimide bond between carboxylgroups 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.
[0087] 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. The carrier may be a microcarrier. 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.
[0088] 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 herein 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.
[0089] 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.
[0090] 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. The
particular structure may be 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 preferred 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 preferred 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.
[0091] In yet another embodiment, one or more binding domains
hereof and comprised in the targeting agent, bind to gum Arabic. In
another preferred 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, the VHH
comprises two disulphide bridges. Most preferably, the 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.
[0092] A third aspect of the invention 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.
[0093] 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.
[0094] The 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.
[0095] One particularly advantageous aspect of the disclosure 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.
[0096] 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.
[0097] 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.
[0098] 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.
[0099] 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.
[0100] A fourth aspect of the invention 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.
[0101] 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.
[0102] 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.
[0103] 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 preferred 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 preferred 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.
[0104] In yet another embodiment, the targeting agent(s) comprised
in the composition, comprise at least one binding domain that binds
to gum arabic. In another 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, the VHH comprises two disulphide
bridges. Most preferably, the 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.
[0105] 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.
[0106] 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.
[0107] 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).
[0108] In one embodiment, the composition hereof is for
agrochemical use.
[0109] 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, the invention makes it 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, in
the invention 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.
[0110] 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: [0111] 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; [0112] 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; [0113] 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; [0114] and each such composition
hereof forms a specific, but non-limiting aspect of the
invention.
[0115] In a preferred embodiment, the composition hereof is an
agrochemical composition comprising a suspension or dispersion of
specifically targeting microcapsules, as further defined, in an
aqueous medium.
[0116] It is preferred that the size distribution of the
specifically targeting microcapsules in the suspension or
dispersion falls within certain limits. Preferably, the
volume-weighted mean diameter of the specifically targeting
microcapsules of the agrochemical composition hereof is less than
about 20 microns with at least 90%, on a volume basis, of the
microcapsules having a diameter less than about 60 microns. More
preferably the volume-weighted mean diameter of the specifically
targeting microcapsules is between about 2 and about 10 microns
with at least 90%, on a volume basis, of the microcapsules having a
diameter less than about 40 microns. Even more preferably, the
volume-weighted mean diameter of the specifically targeting
microcapsules is between about 2 and about 5 microns with at least
90%, on a volume basis, of the microcapsules having a diameter less
than about 20 microns.
[0117] The aqueous medium in which the specifically targeting
microcapsules are suspended or dispersed is preferably water and
the aqueous suspension or dispersion of specifically targeting
microcapsules is preferably formulated with additional additives to
optimize its shelf life and in-use stability. Dispersants and/or
thickeners may be used to inhibit the agglomeration and settling of
microcapsules. Suitable dispersants are preferably high molecular
weight, anionic or non-ionic dispersants, such as, but not limited
to, naphthalene sulfonate sodium salt, gelatin, casein, polyvinyl
alcohol, alkylated polyvinyl pyrrolidone polymers, sodium and
calcium lignosulfonates, sulfonated naphthalene-formaldehyde
condensates, modified starches, or modified cellulosics. Thickeners
are useful in retarding the settling process by increasing the
viscosity of the aqueous phase. Preferably shear-thinning
thickeners are used, because they result in a reduction in
viscosity of the suspension or dispersion during pumping, which
facilitates the application and even coverage of the suspension or
dispersion to the field using commonly used spraying equipment.
Suitable examples of shear-thinning thickeners include, but are not
limited to, guar- or xanthan-based gums, cellulose ethers or
modified cellulosics and polymers. Anti-packing agents are useful
to redisperse or resuspend the microcapsules upon agitation when
microcapsules have settled. Suitable anti-packing agents include,
but are not limited to, microcrystalline cellulose material, clay,
silicon dioxide, or insoluble metal oxides.
[0118] A pH buffer may be used to maintain the pH of the suspension
or dispersion. Suitable buffers such as disodium phosphate may be
used to hold the pH in a range within which most of the components
are most effective. Preferably this range is between pH 4 and
9.
[0119] Other useful additives are biocides, preservatives,
anti-freeze agents and antifoam agents.
[0120] In certain embodiments, the agrochemical composition
comprising a suspension or dispersion of specifically targeting
microcapsules in an aqueous medium has a stability that allows the
composition hereof to be suitably stored and transported and (where
necessary after further dilution) applied to the intended site of
action. Preferably, the agrochemical composition hereof is stable
at least for two years at ambient temperature. Preferably, the
agrochemical composition hereof is stable at least for fourteen
days at 54.degree. C. Preferably, the agrochemical composition
hereof remains stable after at least one, preferably after more
than one, freeze-thaw cycle. "Stable," as used in this context,
means that the total content of the agrochemical active substance
present in the specifically targeting microcapsule suspension or
dispersion shall not have been decreased with more than 10%,
preferably not have been decreased with more than 5%, compared with
the initial total content of the agrochemical active substance that
was present in the specifically targeting microcapsule suspension
or dispersion before applying the storage conditions. Preferably,
in addition the free (non-encapsulated) content of the agrochemical
active substance present in the specifically targeting microcapsule
suspension or dispersion shall not have been increased with more
than 100%, more preferably not have been increased with more than
50%, most preferably not have been increased with more than 25%,
compared with the initial free content of the agrochemical active
substance that was present in the specifically targeting
microcapsule suspension or dispersion before applying the storage
conditions.
[0121] In yet another embodiment, the agrochemical or combination
of agrochemicals comprised in the specifically targeting
microcapsules comprised in the agrochemical composition hereof is
selected from the group consisting of fungicides, insecticides,
herbicides, safeners, nematicides, acaricides, bactericides,
pheromones, repellants, plant and insect growth regulators and
fertilizers.
[0122] Preferably, the characteristics of the specifically
targeting microcapsules comprised in the agrochemical composition
hereof are such that maintaining them in suspension in a tank mix
causes no difficulty and that they can withstand the pressure
applied with spraying equipment, whether this spraying is performed
with hand-applied equipment, machine-operated spraying equipment or
even aerial spraying equipment.
[0123] A fifth aspect of the invention is a composition, comprising
at least (i) one targeting agent comprising at least one binding
domain hereof and (ii) a carrier.
[0124] 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.
[0125] 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.
[0126] 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 of the invention).
[0127] 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 absorped 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).
[0128] 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).
[0129] 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.
[0130] 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.
[0131] 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, the VHH
comprises two disulphide bridges. Most preferably, the 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.
[0132] 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.
[0133] In yet another preferred 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 preferred embodiment, the composition is for
agrochemical use.
[0134] 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.
[0135] 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).
[0136] A sixth aspect of the invention 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.
[0137] "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.
[0138] 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.
[0139] In a preferred 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.
[0140] 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, so that 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 so that 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.
[0141] A seventh aspect of the invention 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.
[0142] In a preferred embodiment, the composition hereof consists
of a suspension or dispersion of specifically targeting
microcapsules containing an agrochemical or combination of
agrochemicals.
[0143] The agrochemical composition hereof may be the only material
applied to a plant, preferably a crop, or it may be blended with
other agrochemicals or additives for simultaneous application.
Examples of agrochemicals which may be blended for simultaneous
application include fertilizers, herbicide safeners, complimentary
agrochemicals and even the free form of the encapsulated active
substance. For a stand-alone application, the agrochemical
composition hereof is preferably diluted with water prior to
application to the field. Preferably, no additional additives are
required to use the agrochemical composition for application in the
field.
[0144] In a preferred 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.
Generally, application rates of agrochemicals are in the order of
grams up to kilograms of active substance per hectare. Preferably,
application rates of agrochemicals comprised in the agrochemical
composition hereof are in the range of 1 g to 1000 g of active
substance per hectare, more preferably in the range of 1 g to 500 g
of active substance per hectare, even more preferably in the range
of 1 g to 300 g of active substance per hectare, most preferably in
the range of 1 g to 200 g of active substance per hectare.
[0145] 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, so that 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 so that 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.
[0146] An eighth aspect of the invention 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.
[0147] "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.
[0148] 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.
[0149] In one embodiment, the method for manufacturing a
specifically targeting agrochemical carrier, consist of a process,
the process comprising at least the steps of: [0150] a. Emulsifying
into a continuous aqueous phase, the aqueous phase optionally
comprising a surfactant, an organic phase in which a to be
encapsulated agrochemical or combination of agrochemicals,
optionally together with polyfunctional monomers or pre-polymers,
are dissolved or dispersed to form an emulsion of droplets of the
organic phase in the continuous aqueous phase; [0151] b. Causing an
aqueous suspension of microcapsules with polymer walls having
anchor groups at their surface to be formed; and [0152] c.
Covalently linking at least one targeting agent to the anchor
groups at the microcapsule surface, at a ratio from about 0.01
.mu.g to about 1 .mu.g targeting agent per square cm microcapsule
surface.
[0153] In one embodiment, the method consists of a process
comprises the steps of: [0154] a. Emulsifying into a continuous
aqueous phase, the aqueous phase optionally comprising a
surfactant, an organic phase in which a to be encapsulated
agrochemical or combination of agrochemicals together with
polyfunctional monomers or pre-polymers are dissolved or dispersed
to form an emulsion of droplets of the organic phase in the
continuous aqueous phase; [0155] b. Optionally adding to the
emulsion a monomer- or pre-polymer-reactant component containing
anchor groups; [0156] c. Causing polymerization of the monomers or
pre-polymers to form an aqueous suspension of microcapsules with
polymer walls having anchor groups at their surface; and [0157] d.
Covalently linking at least one targeting agent to the anchor
groups at the microcapsule surface, at a ratio from about 0.01
.mu.g to about 1 .mu.g targeting agent per square cm microcapsule
surface.
[0158] The organic phase is preferably substantially
water-immiscible, meaning that the solubility of the organic phase
in the aqueous phase is less than 10% by weight, preferably less
than 5%, more preferably less than 1%, even more preferably less
than 0.5%. The substantially water-immiscible organic phase
consists preferably of a non-polar solvent that does not interfere
with the encapsulation reaction, in which the polyfunctional
monomers or pre-polymers, together with the agrochemicals to be
encapsulated can be dissolved or dispersed. Suitable solvents
include hydrocarbon solvents, such as kerosene, and alkyl benzenes,
such as toluene, xylene, benzyl benzoate, diisopropyl naphthalene,
Norpar 15, Exxsol D110 and D130, Orchex 692, Suresol 330, Aromatic
200, Citroflex A-4 and diethyl adipate.
[0159] Suitable polyfunctional monomers include dicarboxylic acid
chlorides, bis(chlorocarbonates), bis(sulfonylchlorides),
trifunctional adducts of linear aliphatic isocyanates, such as
hexamethylene 1,6-diisocyanate, 1,4-cyclohexane diisocyanate,
triethyl-hexamethylene diisocyanate, trimethylenediisocyanate,
propylene-1,2-diisocyanate, butylene-1,2-diisocyanate, isophorone
diisocyanate, Desmodur N3200, Desmodur N3300, Desmodur W, Tolonate
HDB, Tolonate HDT, or isocyanates containing at least one aromatic
moiety are used as monomers, such as
methylene-bis-diphenyldiisocyanate ("MDI"), polymeric
methylene-bis-diphenyldiisocyanate,
polymethylenepolyphenyleneisocyanate ("PMPPI") or 2,4- and
2,6-toluene diisocyanate ("TDI"), naphthalene diisocyanate,
diphenylmethane diisocyanate and triphenylmethane-p,p',p''-trityl
triisocyanate.
[0160] Pre-polymers can be prepared by polymerizing as a
non-limiting example one or more polyisocyanates with one or more
organic components having at least one isocyanate reactive hydrogen
atom, such as a polyol or a polyamine.
[0161] Preferably, the aqueous phase comprises a surfactant to
stabilize the formed emulsion. The surfactant may be ionic or
non-ionic. Examples of suitable ionic surfactants include sodium
dodecyl sulphate, sodium or potassium polyacrylate or sodium or
potassium polymethacrylate. Examples of suitable non-ionic
surfactants include polyvinlyalcohol ("PVA"), polyvinlypyrrolidone
("PVP"), poly(ethoxy)nonylphenol, polyether block copolymers, such
as Pluronic and Tetronic, polyoxyethylene adducts of fatty
alcohols, such as Brij surfactants, esters of fatty acids, such as
sorbitan monostearate, sorbitan monooleate, TWEEN.RTM.-20
(Polyoxyethylene (20) sorbitan monolaurate), TWEEN.RTM.-80
(Polyoxyethylene (80) sorbitan monooleate), sorbitan sesquioleate
or Arlacel C surfactants. The quantity of surfactant is not
critical but for convenience generally comprises from about 0.05%
to about 10% by weight of the aqueous phase.
[0162] It will be clear to the person skilled in the art how the
organic phase can be emulsified in the aqueous phase. Suitable
emulsification techniques include homogenization by any type of
agitation, but may also be performed using micro-sieving
techniques. Emulsification of the organic phase in the aqueous
phase is preferably done by high shear agitation. The agitation
rate determines the droplet size of the emulsion. Typical initial
agitation rates are from about 5000 rpm to about 20000 rpm, more
preferably from about 75000 rpm to about 15000 rpm. The agitation
is preferably slowed down prior to addition of the monomer- or
pre-polymer-reactant components to a stirring rate of about 100 rpm
to 1000 rpm, more preferably from about 200 rpm to about 500
rpm.
[0163] Preferably, as soon as possible after the emulsion has been
prepared, the monomer- or pre-polymer-reactant components are added
to the aqueous phase. In their simplest form, the monomer- or
pre-polymer-reactant components consist of water and are already
present in the aqueous phase, in which case the interfacial
polymerization reaction is initiated by hydrolysis of the
polyfunctional monomers. In a preferred embodiment, however,
monomer- or pre-polymer-reactant components comprising anchor
groups are added to the aqueous phase. In order to be reactive with
the polyfunctional monomers or pre-polymers, the reactant
components comprise preferably amine, hydroxyl and/or thiol groups.
The monomer- or pre-polymer-reactant components hereof comprise at
least one anchor group and at least one, preferably more reactive
groups which reacts during the polymerization process with one of
the polyfunctional monomers or pre-polymers. In a preferred
embodiment the anchor group does not react during the
polymerization process with one of the other reaction components.
In another preferred embodiment, the monomer- or
pre-polymer-reactant component comprises at least two reactive
groups which react during the polymerization process with the
polyfunctional monomers or pre-polymers. In this way larger amounts
of the monomer- or pre-polymer reactant component can be used since
it does not act as a chain terminator but instead as a chain
extender or cross-linker Suitable examples of such monomer- or
pre-polymer reactant components, comprise tetraethylene-pentamine
(TEPA), pentamethylene hexamine, lysine, dipeptides, including
H-Lys-Glu-OH, H-Asp-Lys-OH, H-Lys-Asp-OH, H-Glu-Lys-OH,
H-Glu-Asp-OH, propargylethanol, propargylamine,
N-propargyldiethanolamine, 2,2-di(prop-2-ynyl)propane-1,3diol
(DPPD), 1-(propargyloxy)benzene-3,5-methanol (PBM),
N-propargyldipropanol-amine, 2-propargylpropane-1,3-diol,
(2-methyl-2-propargyl)propane-diol.
[0164] One type of monomer- or pre-polymer reactant components can
be used in the process hereof or a blend of at least two,
optionally more than two, monomer- or pre-polymer reactant
components can be added. In a preferred embodiment, cross-linkers,
such as tri-, tetra- or pentamines, are added to strengthen the
microcapsule wall.
[0165] Alternative methods for presenting anchor groups at the
surface of a microcapsule are known to the person skilled in the
art, and have been disclosed, amongst others, by Mason et al., 2009
and in U.S. Pat. No. 5,011,885 and U.S. Pat. No. 6,022,501,
incorporated herein by reference.
[0166] The reaction proceeds readily at room temperature, but it
may be advantageous to operate at elevated temperatures, at about
40.degree. C. to about 70.degree. C., preferably at about
50.degree. C. to about 60.degree. C., it may as well be
advantageous to operate at slightly decreased temperatures,
preferably at about 15.degree. C.
[0167] In the finishing step of the process, at least one targeting
agent is covalently linked to the anchor groups at the microcapsule
surface, at a ratio from about 0.01 .mu.g to about 1 .mu.g
targeting agent per square cm microcapsule surface.
[0168] It will be clear to the person skilled in the art how a
targeting agent can be covalently linked to anchor groups present
at the microcapsules surface. Methods for linking proteinaceous
molecules to carboxyl or amine anchor groups have been extensively
described such as in Bioconjugate Techniques, 2nd Edition, Greg T.
Hermanson.
[0169] In one embodiment, such covalent linking is performed using
carbodiimide chemistry, by forming of a carbodiimide bond between
the anchor groups at the surface of the microcapsule and reactant
groups in the targeting agent, as a non-limiting example between
carboxylgroups on the outer surface of the microcapsule and
amine-groups of the antigen-binding domain comprised in the
targeting agent. Such covalent linking may be effectuated in a
one-step reaction, in which all reaction components are added
simultaneously, or it may be performed in a two-step reaction, in
which either the anchor group on the microcapsule surface or the
targeting agent is first activated into a highly reactive
intermediate product, after which the other reaction components are
added. Optionally, an additional stabilizing agent, such as
N-hydroxysuccinimide (NHS) or N-hydroxysulfosuccinimide
(sulfo-NHS), may be added to the reaction to stabilize the highly
reactive intermediate product and increase the reaction
efficiency.
[0170] In another preferred embodiment, the targeting agent is
covalently bound to the anchor groups on the microcapsule surface
using "click chemistry," as defined by Sharpless in Angew. Chem.
Int. Ed. 2001, 40, 2004. In this preferred embodiment, the anchor
groups are reactive unsaturated groups which do not react during
the polymerization process and are preferably selected from the
group consisting of a terminal alkyne and an azide, which are able
to participate in a Huisgen 1,3-dipolar cycloaddition reaction, or
from the group consisting of a diene and a dienophile, which are
able to participate in a Diels-Alder cycloaddition reaction.
[0171] Targeting agents or the antigen-binding proteins comprised
therein can be coupled with or without linking agents to the
microcapsules. A "linking agent," as used here, may be any linking
agent known to the person skilled in the art; that allows covalent
linking of targeting agents or the antigen-binding domains
comprised in the targeting agent to the anchor groups at the
microcapsule surface, such as, but not limited to EDC
(1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) or
the homobifunctional cross-linker ((bis[sulfosuccinimidyl]suberate)
(BS3). The linking agent can be such that it results in the
incorporation of a spacer between the targeting agent and the
microcapsule surface, in order to increase the flexibility of the
targeting agent bound to the microcapsule and thereby facilitating
the binding of the antigen-binding protein comprised in the
targeting agent to its target molecule on the solid surface.
Examples of such spacers can be found in WO0024884 and WO0140310.
In a preferred embodiment, the linking agent, however, results in a
direct covalent binding of the targeting agent to the microcapsule
surface, without the incorporation of a spacer.
[0172] In a preferred embodiment, the method for covalently linking
at least one targeting agent, or an antigen-binding protein
comprised in a targeting agent, using a linking agent to an anchor
group on the microcapsule surface, comprises the steps of: [0173]
reacting a linking agent with the targeting agent; and [0174]
reacting the microcapsule to the linking agent in a ratio in a
ratio from about 0.01 .mu.g to about 1 .mu.g targeting agent per
square cm microcapsule surface.
[0175] In another preferred embodiment, the method for covalently
linking at least one targeting agent, or an antigen-binding domain
comprised in a targeting agent, using a linking agent to an anchor
group on the microcapsule surface, comprises the steps of: [0176]
reacting the microcapsule with a linking agent; and [0177] reacting
targeting agents with the linking agent in a ratio from about 0.01
.mu.g to about 1 .mu.g targeting agent per square cm microcapsule
surface.
[0178] In one embodiment, at least one targeting agent is
covalently linked to the anchor groups at the microcapsule surface
at a ratio from about 0.01 .mu.g to about 1 .mu.g per square cm
microcapsule surface.
[0179] In more specific embodiments, at least one targeting agent
is covalently linked to the anchor groups at the microcapsule
surface at a ratio from 0.01 .mu.g to 0.05 .mu.g, from 0.01 .mu.g
to 0.1 .mu.g, from 0.01 .mu.g to 0.2 .mu.g, from 0.01 .mu.g to 0.3
.mu.g, from 0.01 .mu.g to 0.4 .mu.g, from 0.01 .mu.g to 0.5 .mu.g,
from 0.01 .mu.g to 0.6 .mu.g, from 0.01 .mu.g to 0.7 .mu.g, from
0.01 .mu.g to 0.8 .mu.g, from 0.01 .mu.g to 0.9 .mu.g, from 0.01
.mu.g to 1 .mu.g per square cm of microcapsule surface.
[0180] In yet another embodiment, at least one targeting agent is
covalently linked to the anchor groups at the microcapsule surface
at a ratio from 0.05 .mu.g to 0.1 .mu.g, from 0.05 .mu.g to 0.2
.mu.g, from 0.05 .mu.g to 0.3 .mu.g, from 0.05 .mu.g to 0.4 .mu.g,
from 0.05 .mu.g to 0.5 .mu.g, from 0.05 .mu.g to 0.6 .mu.g, from
0.05 .mu.g to 0.7 .mu.g, from 0.05 .mu.g to 0.8 .mu.g, from 0.05
.mu.g to 0.9 .mu.g, from 0.05 .mu.g to 1 .mu.g per square cm of
microcapsule surface.
[0181] In yet another embodiment, at least one targeting agent is
covalently linked to the anchor groups at the microcapsule surface
at a ratio from 0.1 .mu.g to 0.2 .mu.g, from 0.1 .mu.g to 0.3
.mu.g, from 0.1 .mu.g to 0.4 .mu.g, from 0.1 .mu.g to 0.5 .mu.g,
from 0.1 .mu.g to 0.6 .mu.g, from 0.1 .mu.g to 0.7 .mu.g, from 0.1
.mu.g to 0.8 .mu.g, from 0.1 .mu.g to 0.9 .mu.g, from 0.1 .mu.g to
1 .mu.g per square cm of microcapsule surface.
[0182] In yet another embodiment, at least one targeting agent is
covalently linked to the anchor groups at the microcapsule surface
at a ratio from 0.2 .mu.g to 0.3 .mu.g, from 0.2 .mu.g to 0.4
.mu.g, from 0.2 .mu.g to 0.5 .mu.g, from 0.2 .mu.g to 0.6 .mu.g,
from 0.2 .mu.g to 0.7 .mu.g, from 0.2 .mu.g to 0.8 .mu.g, from 0.2
.mu.g to 0.9 .mu.g, from 0.2 .mu.g to 1 .mu.g per square cm of
microcapsule surface.
[0183] In yet another embodiment, at least one targeting agent is
covalently linked to the anchor groups at the microcapsule surface
at a ratio from 0.3 .mu.g to 0.4 .mu.g, from 0.3 .mu.g to 0.5
.mu.g, from 0.3 .mu.g to 0.6 .mu.g, from 0.3 .mu.g to 0.7 .mu.g,
from 0.3 .mu.g to 0.8 .mu.g, from 0.3 .mu.g to 0.9 .mu.g, from 0.3
.mu.g to 1 .mu.g per square cm of microcapsule surface.
[0184] In yet another embodiment, at least one targeting agent is
covalently linked to the anchor groups at the microcapsule surface
at a ratio from 0.4 .mu.g to 0.5 .mu.g, from 0.4 .mu.g to 0.6
.mu.g, from 0.4 .mu.g to 0.7 .mu.g, from 0.4 .mu.g to 0.8 .mu.g,
from 0.4 .mu.g to 0.9 .mu.g, from 0.4 .mu.g to 1 .mu.g per square
cm of microcapsule surface.
[0185] In yet another embodiment, at least one targeting agent is
covalently linked to the anchor groups at the microcapsule surface
at a ratio from 0.5 .mu.g to 0.6 .mu.g, from 0.5 .mu.g to 0.7
.mu.g, from 0.5 .mu.g to 0.8 .mu.g, from 0.5 .mu.g to 0.9 .mu.g,
from 0.5 .mu.g to 1 .mu.g per square cm of microcapsule
surface.
[0186] In yet another embodiment, at least one targeting agent is
covalently linked to the anchor groups at the microcapsule surface
at a ratio from 0.6 .mu.g to 0.7 .mu.g, from 0.6 .mu.g to 0.8
.mu.g, from 0.6 .mu.g to 0.9 .mu.g, from 0.6 .mu.g to 1 .mu.g per
square cm of microcapsule surface.
[0187] In yet another embodiment, at least one targeting agent is
covalently linked to the anchor groups at the microcapsule surface
at a ratio from 0.7 .mu.g to 0.8 .mu.g, from 0.7 .mu.g to 0.9
.mu.g, from 0.7 .mu.g to 1 .mu.g per square cm of microcapsule
surface.
[0188] In yet another embodiment, at least one targeting agent is
covalently linked to the anchor groups at the microcapsule surface
at a ratio from 0.8 .mu.g to 0.9 .mu.g, from 0.8 .mu.g to 1 .mu.g
per square cm of microcapsule surface.
[0189] In yet another embodiment, at least one targeting agent is
covalently linked to the anchor groups at the microcapsule surface
at a ratio from 0.9 .mu.g to 1 .mu.g per square cm of microcapsule
surface.
[0190] The targeting agent covalently linked to the specifically
targeting microcapsules hereof 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 antigen-binding protein, or that
comprises two or more different antigen-binding proteins that each
are directed against the same binding site. Thus, a mono-specific
targeting agent is capable of binding to a single binding site,
either through a single antigen-binding protein or through multiple
antigen-binding proteins. By a "multi-specific" targeting agent is
meant a targeting agent that comprises two or more antigen-binding
proteins that are each directed against different binding sites.
Thus, a "bi-specific" targeting agent is capable of binding to two
different binding sites; a "tri-specific" targeting agent is
capable of binding to three different binding sites; 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
antigen-binding protein; the term "bivalent" is used to indicate
that the targeting agent comprises a total of two single
antigen-binding proteins; the term "trivalent" is used to indicate
that the targeting agent comprises a total of three single
antigen-binding proteins; and so on for "multivalent" targeting
agents.
[0191] Preferably, the antigen-binding proteins comprised in the
targeting agents hereof are monoclonal antigen-binding proteins. A
"monoclonal antigen-binding protein" as used herein means an
antigen-binding protein produced by a single clone of cells and
therefore a single pure homogeneous type of antigen-binding
protein. More preferably, the antigen-binding proteins comprised in
the targeting agents hereof consist of a single polypeptide chain.
Most preferably, the antigen-binding proteins comprised in the
targeting agents hereof comprise an amino acid sequence that
comprises four framework regions and three
complementary-determining regions, or any suitable fragment
thereof, and confer their binding specificity by the amino acid
sequence of three complementary-determining regions or CDRs, each
non-contiguous with the others (termed CDR1, CDR2, CDR3), which are
interspersed amongst four framework regions or FRs, each
non-contiguous with the others (termed FR1, FR2, FR3, FR4),
preferably in a sequence FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4). The
delineation of the FR and CDR sequences is based on the unique
numbering system according to Kabat. The antigen-binding proteins
comprising an amino acid sequence that comprises four framework
regions and three complementary-determining regions, are known to
the person skilled in the art and have been described, as a
non-limiting example in Wesolowski et al. (2009). The length of the
CDR3 loop is strongly variable and can vary from 0, preferably from
1, to more than 20 amino acid residues, preferably up to 25 amino
acid residues. Preferably, the antigen-binding proteins 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). Those antibodies are easy to
produce, and are far more stable than classical antibodies, which
provides a clear advantage for stable binding to naturally
occurring surfaces under conditions that deviate substantially from
physiological conditions, such as changes in temperature,
availability of water or moisture, presence of detergents, extreme
pH or salt concentration. For each of these variables VHH are
stable and often can exert binding in conditions that are
considered extreme.
[0192] In a preferred embodiment, the targeting agent consists of a
VHH, which is either C-terminally or N-terminally or even
internally fused with one or more amino acids, such as lysines, in
order to increase functionality of the targeting agent when
covalently linked to the anchor groups on the surface of the
microcapsule.
[0193] In another preferred embodiment, the method consists of a
process comprises the steps of: [0194] a. Emulsifying into a
continuous aqueous phase, the aqueous phase optionally comprising a
surfactant, an organic phase in which a to be encapsulated
agrochemical or combination of agrochemicals, together with a
pre-polymer or mixture of pre-polymers containing anchor groups, is
dissolved or dispersed to form an emulsion of droplets of the
organic phase in the continuous aqueous phase; [0195] b. Causing in
situ self-condensation of the pre-polymers surrounding the droplets
of organic phase to form an aqueous suspension of microcapsules
having polymer walls with anchor groups at their surface; and
[0196] c. Covalently linking at least one targeting agent to the
anchor groups at the microcapsule surface, at a ratio from about
0.01 .mu.g to about 1 .mu.g targeting agent per square cm
microcapsule surface.
[0197] Amino resin pre-polymers of the urea-formaldehyde,
melamine-formaldehyde, benzoguanamine-formaldehyde or
glycoluril-formaldehyde type, with a high solubility in the organic
phase and a low solubility in the aqueous phase are suitable in the
process. To impart solubility in the organic phase, the amino resin
pre-polymers are partially etherified, meaning that they have the
hydroxyl hydrogen atoms replaced by alkyl groups. Partially
etherified amino resin pre-polymers are obtained by condensation of
the pre-polymer with an alcohol. The amino resin pre-polymers can
be prepared by techniques well known to the person skilled in the
art, such as by the reaction between the amine, preferably urea or
melamine, formaldehyde and alcohol. The organic phase may further
contain solvents and polymerization catalysts, such as sulphonic
acid surfactant catalysts.
[0198] The amount of the pre-polymer in the organic phase is not
critical and can vary over a wide range depending on the desired
capsule wall strength and the desired quantity of core material in
the finished microcapsule. In a preferred embodiment, the organic
phase comprises a pre-polymer concentration from about 1% to about
70% on a weight basis, more preferably from about 5% to about
50%.
[0199] Once the organic phase has been formed, an emulsion is then
prepared by emulsifying the organic phase in an aqueous phase,
optionally containing a surfactant. The emulsion is preferably
prepared employing any suitable high shear stirring device. The
stirring rate determines the size of the emulsion droplet size. The
relative quantities of organic and aqueous phases are not critical
to the practice of this invention, and can vary over a wide range,
determined most by convenience and ease of handling. In practical
usage, the organic phase will comprise a maximum of about 55% of
the total emulsion and will consist of discrete droplets of organic
phase dispersed in the aqueous phase. Once the desired droplet size
is obtained, mild agitation is sufficient to maintain a stable
emulsion and to proceed to the curing of the microcapsules: hereto,
the emulsion is acidified to a pH between about 1 to about 4,
preferably between about 1 to about 3. This causes the pre-polymers
to polymerize by in-situ self condensation and form a polymer wall
completely enclosing each droplet. Acidification can be
accomplished by any suitable means including any water-soluble acid
such as formic, citric, hydrochloric, sulfuric, or phosphoric acid
and the like. The rate of the in situ self-condensation increases
with both acidity and temperature. The reaction can therefore be
conducted from about 20.degree. C. to about 100.degree. C.,
preferably from about 40.degree. C. to about 70.degree. C., most
preferably from about 40.degree. C. to about 60.degree. C.
[0200] In the finishing step of the process, at least one targeting
agent is covalently linked to the anchor groups at the microcapsule
surface, at a ratio from about 0.01 .mu.g to about 1 .mu.g
targeting agent per square cm microcapsule surface, as described
above.
[0201] In yet another preferred embodiment, the method consists of
a process comprises the steps of: [0202] a. Emulsifying into a
continuous aqueous phase, the aqueous phase optionally comprising a
surfactant, an organic phase in which a to be encapsulated
agrochemical or combination of agrochemicals is dissolved or
dispersed to form an emulsion of droplets of the organic phase in
the continuous aqueous phase; [0203] b. Adding to the continuous
aqueous phase a water-soluble pre-polymer or mixture of
pre-polymers, containing anchor groups; [0204] c. Causing in situ
self-condensation of the pre-polymers surrounding the droplets of
organic phase to form an aqueous suspension of microcapsules having
polymer walls with anchor groups at their surface; and [0205] d.
Covalently linking at least one targeting agent to the anchor
groups at the microcapsule surface, at a ratio from about 0.01
.mu.g to about 1 .mu.g targeting agent per square cm microcapsule
surface.
[0206] The organic phase, in which the to be encapsulated
agrochemicals or combination of agrochemicals are dissolved or
dispersed, is substantially water-immiscible, as described above.
Once the organic phase has been formed, an emulsion is then
prepared by emulsifying the organic phase in an aqueous phase,
optionally containing a surfactant. The emulsion is preferably
prepared employing any suitable high shear stirring device. The
stirring rate determines the size of the emulsion droplet size. The
relative quantities of organic and aqueous phases are not critical
to the practice of this invention, and can vary over a wide range,
determined most by convenience and ease of handling. In practical
usage, the organic phase will comprise a maximum of about 55% of
the total emulsion and will consist of discrete droplets of organic
phase dispersed in the aqueous phase. Once the desired droplet size
is obtained, mild agitation is sufficient to maintain a stable
emulsion.
[0207] In a next step of the process, a water-soluble pre-polymer
or a mixture of water-soluble pre-polymers, containing anchor
groups are added to the aqueous phase. Amino resin pre-polymers of
the urea-formaldehyde, melamine-formaldehyde,
benzoguanamine-formaldehyde or glycoluril-formaldehyde type, with a
high solubility in the aqueous phase and a low solubility in the
organic phase are suitable in the process. Such amino resin
pre-polymers can be prepared by techniques well known to the person
skilled in the art, such as by the reaction between the amine,
preferably urea or melamine, and formaldehyde. Preferably the
anchor groups are free amine, hydroxyl or aldehyde-groups. The
aqueous phase may further contain polymerization catalysts.
[0208] The amount of the pre-polymer in the aqueous phase is not
critical and can vary over a wide range depending on the desired
capsule wall strength and the desired quantity of core material in
the finished microcapsule. In a preferred embodiment, the organic
phase comprises a pre-polymer concentration from about 1% to about
70% on a weight basis, more preferably from about 5% to about
50%.
[0209] To proceed to the curing of the microcapsules, the emulsion
is acidified to a pH between about 1 to about 4, preferably between
about 1 to about 3. This causes the pre-polymers to polymerize by
in situ self-condensation and form a polymer wall containing anchor
groups completely enclosing each droplet. Acidification can be
accomplished by any suitable means including any water-soluble acid
such as formic, citric, hydrochloric, sulfuric, or phosphoric acid
and the like. The rate of the in situ self-condensation increases
with both acidity and temperature. The reaction can therefore be
conducted from about 20.degree. C. to about 100.degree. C.,
preferably from about 40.degree. C. to about 70.degree. C., most
preferably from about 40.degree. C. to about 60.degree. C.
[0210] In the finishing step of the process, at least one targeting
agent is covalently linked to the anchor groups at the microcapsule
surface, at a ratio from about 0.01 .mu.g to about 1 .mu.g
targeting agent per square cm microcapsule surface, as described
above.
[0211] Preferred agrochemicals to be encapsulated into specifically
targeting microcapsules utilizing the process hereof include
fungicides, insecticides, herbicides, nematicides, acaricides,
bactericides, pheromones, repellants, plant and insect growth
regulators and fertilizers. Optionally included with the
agrochemical or combination of agrochemicals may be additives
typically used in conjunction with agrochemicals such as
synergists, safeners, photodegradation inhibitors, adjuvants and
the like.
[0212] The concentration of the agrochemical or combination of
agrochemicals in the resultant microcapsule suspension is dependent
on the physical properties of the agrochemical(s). When the
agrochemical(s) can be dissolved in the organic phase, the
concentration of agrochemical(s) in the microcapsule suspension may
range from about 2.5% to about 70% on a weight basis, more
preferably from about 20% to about 70%, most preferably from about
40% to about 70% on a weight basis. In the event the
agrochemical(s) need to be dispersed in the organic phase, the
concentration of agrochemical(s) in the microcapsule suspension may
range from about 2.5% to about 50% on a weight basis, more
preferably from about 5% to about 30%, most preferably from about
10% to about 20% on a weight basis.
[0213] The process so described, with its preferred embodiments,
may be performed as a continuous process or it may be performed as
a batch-type of manufacturing process.
[0214] The resulting specifically targeting microcapsules have a
specific gravity of less than 1 and remain suspended or dispersed
in the aqueous phase. The suspension of specifically targeting
microcapsules thus produced may be utilized as such, and may be
packaged as capsule suspension to be used by transferring the
capsules suspension into a spray tank, in which it is mixed with
water to form a sprayable suspension. Alternatively, the suspension
of specifically targeting microcapsules may be converted into a dry
microcapsule product by spray drying or other techniques well-known
to the person skilled in the art and the resulting material may be
packaged in dry form.
[0215] A ninth aspect of the invention 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.
[0216] A tenth aspect of the invention 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.
[0217] In a preferred embodiment, the specifically targeting
agrochemical carrier is a specifically targeting microcapsule,
manufactured by the process hereof as above described.
[0218] A "specifically targeting microcapsule," as used herein,
means that the microcapsule can bind specifically to a binding site
on a solid surface, preferably a naturally occurring surface,
through the antigen-binding proteins comprised in the targeting
agents present at the microcapsule surface. Specific binding means
that the antigen-binding protein preferentially binds to its target
molecule that is present in a homogeneous or heterogeneous mixture
of different other molecules. Specificity of binding of an
antigen-binding protein can be analyzed by methods such as ELISA,
as described in examples 7-10, in which the binding of the
specifically targeting microcapsule to a surface displaying its
target molecule is compared with the binding of the specifically
targeting microcapsule to a surface displaying an unrelated
molecule and with aspecific sticking of the specifically targeting
microcapsule to the reaction vessel. In certain embodiments, a
specific binding interaction will discriminate between desirable
and undesirable target molecules on a surface, in preferred
embodiments binding to the desirable target molecule is more than
one order of magnitude stronger than to undesirable target
molecules, in even more preferred embodiments binding to the
desirable target molecule is more than two orders of magnitude
stronger than to undesirable target molecules.
[0219] Release of the agrochemical from the specifically targeting
microcapsule can be achieved in several ways: [0220] By collapse of
rupture of the microcapsule wall after dry-down of the spray
deposit; [0221] By mechanical rupture, e.g., by crawling or feeding
of an insect; [0222] By degradation of the microcapsule wall under
influence of, e.g., light, heat or pH; [0223] By diffusion of the
agrochemical through the microcapsule wall.
[0224] The release rate by a diffusional mechanism is shown in the
equation below, as defined by Scher et al., 1998:
Release rate = ( 4 .pi. r o r i ) P ( C i - C o ) r o - r i with P
= K . D ##EQU00001##
[0225] so that r=radius; r.sub.o=outer radius; r.sub.i=inner radius
of the microcapsule [0226] P=Permeability [0227] K=Solubility
coefficient [0228] D=Diffusion coefficient [0229] C=concentration
of agrochemical; C.sub.o=concentration outside microcapsule; [0230]
C.sub.i=concentration inside microcapsule
[0231] It will be clear to the person skilled in the art that since
the release rate is directly proportional to the surface area,
permeability and concentration gradient across the microcapsule
wall and inversely proportional to microcapsule wall thickness, the
release rate can be modified by varying microcapsule size (and
hence surface area), microcapsule wall thickness and the
permeability of the microcapsule wall, which is defined as the
product of the diffusion coefficient and the solubility
coefficient. The size of the microcapsules is determined by the
droplet size of the emulsion of the organic phase in the aqueous
phase and can be determined by varying the rate of the high shear
agitation when preparing the emulsion, so that the higher the
agitation rate, the smaller is the size of the resulting
microcapsules. The ratio of the weight of the shell materials
versus the weight of the core material, will, in combination with
the size of the resultant microcapsules, determine the shell
thickness. For a certain agrochemical, the diffusion coefficient
can be varied by varying the cross-linking density of the
microcapsule wall and the solubility coefficient can be varied by
varying the chemical composition of the microcapsule wall.
[0232] Preferably, the specifically targeting microcapsules 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 microcapsules may
be made of polymer materials 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 content from the microcapsule.
[0233] Preferably, the weight ratio of shell materials versus the
weight of the core material is about 3% to 30%, more preferably the
weight ratio of shell materials versus the weight of the core
material is about 5% to 20%, still more preferably, the weight
ratio of shell materials versus the weight of the core material is
about 5% to 15%.
[0234] In one embodiment, the microcapsule wall is composed of
polyurea, polyurethane, urea/formaldehyde or melamine/formaldehyde,
containing anchor groups, most preferably the microcapsule wall is
composed of polyurea containing anchor groups.
[0235] The size distribution of the specifically targeting
microcapsules can be measured with a laser light scattering
particle size analyzer, so that the diameter data is preferably
reported as a volume distribution (D[4,3]). Thus the reported mean
for a population of microcapsules will be volume-weighted, with
about one-half of the microcapsules, on a volume basis, having
diameters less than the mean diameter for the population.
Preferably, the volume-weighted mean diameter of the specifically
targeting microcapsules manufactured according to the process
hereof is less than about 20 microns with at least 90%, on a volume
basis, of the microcapsules having a diameter less than about 60
microns. More preferably the volume-weighted mean diameter of the
specifically targeting microcapsules is between about 2 and about
10 microns with at least 90%, on a volume basis, of the
microcapsules having a diameter less than about 40 microns. Even
more preferably, the volume-weighted mean diameter of the
specifically targeting microcapsules is between about 2 and about 5
microns with at least 90%, on a volume basis, of the microcapsules
having a diameter less than about 20 microns.
[0236] The specifically targeting microcapsules have a spherical
shape, their outer surface may vary from a completely smooth to a
slightly rough appearance as observable under scanning electron
microscopy (SEM).
[0237] The zeta-potential of the specifically targeting
microcapsules may differ from the zeta-potential of comparable
microcapsules, prepared without anchor groups at their surface
and/or without targeting agents covalently linked thereto (Ni et
al., 1995). In a preferred embodiment, the zeta-potential of the
specifically targeting microcapsules is higher than the
zeta-potential of comparable microcapsules, prepared without anchor
groups at their surface and/or without targeting agents covalently
linked thereto.
[0238] In one embodiment, the specifically targeting microcapsules
are able to bind an agrochemical or combination of agrochemicals to
a surface. The surface may be any surface, known to the person
skilled in the art. Preferably, the surface is a naturally
occurring surface. As a non-limiting example, the surface may be a
plant surface such as the surface of leaves, stem, roots, fruits,
seeds, cones, flowers, bulbs or tubers, or it may be an insect
surface, preferably as a part of the insect body that is accessible
from the outside, such as, but not limited to the exoskeleton of an
insect.
[0239] Preferably, the specifically targeting microcapsules are
binding so strongly that they are retained to the solid surface.
"Retain" as used herein means that the binding force resulting from
the affinity or avidity of either one single binding protein or a
combination of two or more binding proteins or targeting agents
comprising antigen-binding proteins for its or their target
molecule present at the solid surface is larger than the combined
force and torque imposed by the gravity of the carrier, and the
force and torque, if any, imposed by shear forces caused by one or
more external factors.
[0240] Another aspect is a specifically targeting microcapsule,
containing an agrochemical and comprising from about 0.01 .mu.g to
about 1 .mu.g targeting agent per square cm microcapsule surface.
Preferably, the specifically targeting microcapsule is produced
according to the process hereof. Preferably the targeting agent
comprises an antigen-binding protein. Even more preferably, the
antigen-binding protein is derived from a camelid antibody. Most
preferably, the antigen-binding domain is comprised in a VHH
sequence.
[0241] A last aspect 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.
EXAMPLES
Example 1
Generation and Selection of VHH
[0242] Immunization of Llamas with Gum Arabic, Potato Leaf
Homogenate, or Wheat Leaf Homogenate
[0243] 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. 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.
[0244] 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.
[0245] Library Construction
[0246] 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.
[0247] Phage Selections Against Gum Arabic, Plant Epidermal
Extracts, or Whole Leaves.
[0248] 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. 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. 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 pH8.3. 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.
[0249] 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.
[0250] 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 here "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.
[0251] Picking Single Colonies from Selection Outputs--
[0252] 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.l 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
[0253] Single-Point Binding ELISA--
[0254] 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.
[0255] 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 pH8.3.
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. Ten .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.
[0256] 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.
[0257] 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.
[0258] Single Colony PCR and Sequencing--
[0259] 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. Five
.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).
[0260] Antibody Production and Purification--
[0261] 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 pH7; 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.
[0262] VHH Binding to Gum Arabic in ELISA--
[0263] 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 pH9.6. 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] (.mu.g/ml) 3 3 0.75 0.1875 0.04688
0.0117188 [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
[0264] VHH Binding to Potato Lectin in ELISA
[0265] 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 VHH VHH VHH 3E6 5D4 5C4 5G5 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
[0266] VHH Binding to Leaf Discs--
[0267] 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-1C). 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 of this invention (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.
[0268] VHH Binding to Intact Living Plants--
[0269] 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 of this invention
are capable of specifically binding to intact living plants.
[0270] VHH Binding in Water--
[0271] 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 of this invention
are capable of binding leaf structures and leaf surface in
water.
[0272] VHH Binding Kinetics--
[0273] 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. o 8 mm potato leaf discs (variety
Desiree) were cut using a puncher tool and washed three times in
PBS. Five .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
of this invention show detectable binding to leaf structures, such
as trichomes and stomata and leaf surface within 10 seconds after
application.
[0274] VHH Binding at Different pH--
[0275] 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. o 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 of this invention (e.g., VHH 3E6) showed detectable
binding to leaf discs over the whole range tested from pH 2 to pH
11.
[0276] VHH Binding at Different Temperatures--
[0277] 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 of this
invention (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
[0278] Construction, Production and Purification of Bivalent
VHH--
[0279] 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 nine 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.
[0280] Coupling of VHH to Microparticles--
[0281] 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.
[0282] Different types of microparticles were prepared. Purified
VHH antibody fragments were (i) coupled to O 2.8 .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 O 2 .mu.m FluoSpheres
fluorescent microspheres (Molecular Probes, Invitrogen), both
according to the manufacturers' instructions.
[0283] Briefly, for coupling to Dynabeads M-270 carboxylic acid:
VHH were dialyzed to 50 mM MES buffer pH5.0 using Vivaspin 5 kDa
spin filter devices (Sartorius Stedim). Beads were prepared by 2
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. Sixty .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 pH7.4 and washed
three times with PBS/0.1%-TWEEN.RTM.-20 and stored at 4.degree.
C.
[0284] Briefly, for coupling to FluoSpheres fluorescent
microspheres: VHH were dialyzed to 50 mM MES buffer pH6.0 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.
[0285] Coupling of Targeting Agents to Microcapsules Containing
Fluorescent Tracer or Active Ingredient--
[0286] 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 o 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
[0287] Binding Assays with VHH-Coupled Beads or Microcapsules--
[0288] Functionality of VHH-coupled microparticles was investigated
in ELISA plates that were coated with 100 .mu.g/ml gum arabic in 50
mM carbonate pH9.6 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 Number of Fluospheres Fluospheres coupled
Fluospheres coupled Coating added with VHH 3E6 with 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
[0289] 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 unrelated coverage
microcapsules control VHH 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 100% n.d. n.d. 861 84.508 94.153 antigen unrelated
20% n.d. n.d. 601 47.906 39.218 antigen unrelated 4% n.d. n.d. 386
23.525 18.517 antigen
[0290] 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.
[0291] 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 of this
invention (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 the example with
microcapsules containing the insecticide lambda cyhalothrin.
[0292] 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.
[0293] 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 Blank Surface
Monovalent Bivalent unrelated micro- coverage VHH 3E6 VHH 3E6 VHH
capsules 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 100% 69.503
45.677 78.205 50.965 antigen unrelated 20% 27.742 22.114 30.459
17.831 antigen unrelated 4% 5.011 15.038 19.755 6.279 antigen
[0294] A leaf disc binding assay was used to evaluate the
interaction of VHH-coupled microcapsules with potato, grass and
azalea leaves. o 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.
[0295] 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 of this
invention 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.
[0296] 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) of this invention proved capable of
binding and retaining microcapsules specifically to leaf
surfaces.
[0297] 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.
[0298] 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 according to this invention,
to a microcarrier containing the agrochemical.
Example 6
Deposition and Retention of Targeting Agent-Coupled Microcapsules
on Intact Living plant surface
[0299] 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.
[0300] 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.
[0301] A rain simulation with fine droplets (SSCOTFVS2 nozzle type)
of 1 L/m.sup.2 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.
[0302] 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. 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
from improved deposition and improved retention targeting agents of
this invention 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.
Example 7
Manufacturing of Microcapsules with Carboxyl Anchor Groups Using
Lysine as the Amine Source by Interfacial Polymerization
[0303] Uvitex OB was dissolved to 1.7% (w/w) in Benzyl Benzoate.
Polymethylene polyphenyl isocyanate (PMPPI) and 2,4 Toluene
diisocyanate (TDI) (1:1) were added to 13% (w/w) and mixed. The
organic phase was emulsified in a solution of 0.5% (w/w) SDS in
water, using homogenization with an Ultra-Turrax disperser. A
solution of 17% (w/w) lysine in water was added under mixing with a
marine impeller and polymerization performed at 40.degree. C. for
30 minutes. For the production of slow release microcapsules, a
solution of 25% (w/w) DETA in water was added after the
polymerization reaction with lysine and polymerization continued at
40.degree. C. for 30 minutes. Microcapsules were washed with water
and collected. The mean volume-weighted diameter of the
microcapsules was 6.1 .mu.m.
[0304] Covalent Linking of VHH to Microcapsules--
[0305] Microcapsules were washed to appropriate amine-free buffers
using vacuum filtration and concentrated. VHH were dialyzed to the
same buffer and concentrated by spin filtration. VHH were added and
mixed with the microcapsules. A premix of EDC and Sulfo-NHS was
made immediately before use and added. Final concentration of EDC
in the reaction was 2 mM, final concentration of Sulfo-NHS in the
reaction was 5 mM. Final concentration of VHH in the coupling
reaction was 1 mg/ml or 0.5 mg/ml. The calculated maximum density
of VHH added to the coupling reactions was 1 .mu.g/cm.sup.2
(4.3E+05 VHH molecules/.mu.m.sup.2 microcapsule surface), 0.5
.mu.g/cm.sup.2 (2.1E+05 VHH molecules/.mu.m.sup.2 microcapsule
surface), or 0.25 .mu.g/cm.sup.2 (1.1E+05 VHH molecules/.mu.m.sup.2
microcapsule surface). Covalent linking reactions were performed at
room temperature for 2 hours or overnight with slow tilt agitation
or head-over-head rotation. Reactions were quenched by the addition
of amine-containing Tris or glycine solution. Reaction mixtures
were transferred to a filtration setup and non-linked VHH were
collected by vacuum filtration for analysis. VHH-coupled
microcapsules were washed twice with appropriate buffer in a
filtration setup and collected in the same buffer.
[0306] Functionality of VHH-Linked Microcapsules--
[0307] High-binding microtiter plates were coated with antigens
corresponding to the specificity of the coupled VHH. Wells coated
with unrelated antigens were used as controls. Plates were washed
and blocked with skimmed milk. A calculation was made for how many
microcapsules were to be added to a well for full coverage of the
bottom of the well. Microcapsules were added to full coverage of
the wells, or serial dilutions were made and added to the wells.
Microcapsules with antigen-specific VHH and control microcapsules
were diluted to appropriate densities in skimmed milk, added to the
wells, and allowed to bind. Non-bound microcapsules were removed by
consecutive washes. Wells were filled with wash buffer, shaken on
an ELISA shaking platform .gtoreq.900 rpm, and microcapsules in
suspension removed together with the wash buffer. Bound
microcapsules were visualized using a macrozoom microscope system
(Nikon) and counted using Volocity image analysis software
(PerkinElmer); the number of bound microcapsules per microtiter
plate well is shown in Table 7. Microcapsules coupled with
antigen-specific VHH at 1, 0.5, or 0.25 .mu.g VHH per cm.sup.2
microcapsule surface are specifically binding to antigen-containing
surfaces with the application rates tested from 0.2% to 25%
coverage. Moreover, it can be anticipated that application rates
beyond these values will also result in specific binding of
microcapsules with antigen-specific VHH.
TABLE-US-00007 TABLE 7 Carboxyl microcapsules produced with lysine
as the amine source and EDC/Sulfo-NHS mediated coupling of VHH
Antigen- Antigen- Antigen- Antigen- binding binding binding binding
Blank VHH VHH VHH VHH microcapsules VHH concentration in coupling 1
1 0.5 0.5 reaction (mg/ml) Calculated maximum density (.mu.g 1 0.5
0.5 0.25 VHH/cm.sup.2 microcapsule surface) Potato lectin coat/25%
coverage 11287 9611 8898 6978 2501 (# microcapsules) Potato lectin
coat/5% coverage 4936 3445 3605 2723 633 (# microcapsules) Potato
lectin coat/1% coverage 1109 1006 1257 833 184 (# microcapsules)
Potato lectin coat/0.2% coverage 237 181 195 160 52 (#
microcapsules) No coat/25% coverage 1758 1559 1952 1718 2641 (#
microcapsules)
[0308] In another experiment the final concentration of VHH in the
covalent linking reaction was 1 mg/ml, 0.3 mg/ml, 0.1 mg/ml, or
0.04 mg/ml. The calculated maximum density of VHH on the
microcapsule surface that was added to the reaction mixtures was 1
.mu.g/cm.sup.2 (4.3E+05 VHH molecules/.mu.m.sup.2 microcapsule
surface), 0.3 .mu.g/cm.sup.2 (1.4E+05 VHH molecules/.mu.m.sup.2
microcapsule surface), 0.1 .mu.g/cm.sup.2 (4.7E+04 VHH
molecules/.mu.m.sup.2 microcapsule surface), or 0.04 .mu.g/cm.sup.2
(1.6E+04 VHH molecules/.mu.m.sup.2 microcapsule surface).
Functionality of the microcapsules was analyzed for microcapsules
coupled with antigen-specific VHH and compared to microcapsules
coupled with a control VHH, tables 8 & 9. Microcapsules coupled
with antigen-specific VHH at 1, 0.3, 0.1, or 0.04 .mu.g VHH per
cm.sup.2 microcapsule surface are specifically binding to
antigen-containing surfaces with the application rates tested from
4% to 100% coverage. Moreover, it can be anticipated that
application rates beyond these values will also result in specific
binding of microcapsules with antigen-specific VHH.
TABLE-US-00008 TABLE 8 Carboxyl microcapsules produced with lysine
as the amine source and EDC/Sulfo-NHS mediated coupling of VHH
Antigen- Antigen- binding Control Fold binding Control Fold VHH VHH
difference VHH VHH difference VHH concentration in 1 1 0.3 0.3
coupling reaction (mg/ml) Calculated maximum 1 1 0.3 0.3 density
(.mu.g VHH/cm.sup.2 microcapsule surface) Potato lectin coat/100%
33914 1571 22 8779 1443 6.1 coverage (# microcapsules) Potato
lectin coat/20% 8992 436 21 4111 396 10 coverage (# microcapsules)
Potato lectin coat/4% 3082 94 33 1564 92 17 coverage (#
microcapsules) No coat/100% coverage 562 1104 0.5 492 971 0.5 (#
microcapsules)
TABLE-US-00009 TABLE 9 Carboxyl microcapsules produced with lysine
as the amine source and EDC/Sulfo-NHS mediated coupling of VHH
Antigen- Antigen- binding Control Fold binding Control Fold VHH VHH
difference VHH VHH difference VHH concentration in 0.1 0.1 0.04
0.04 coupling reaction (mg/ml) Calculated maximum 0.1 0.1 0.04 0.04
density (.mu.g VHH/cm.sup.2 microcapsule surface) Potato lectin
coat/100% 2079 719 2.9 565 657 0.9 coverage (# microcapsules)
Potato lectin coat/20% 2044 80 26 146 114 1.3 coverage (#
microcapsules) Potato lectin coat/4% 477 10 48 32 13 2.5 coverage
(# microcapsules) No coat/100% coverage 392 488 0.8 367 455 0.8 (#
microcapsules)
Example 8
Manufacturing of Microcapsules with Carboxyl Groups Using the
Dipeptide H-Lys-Glu-OH as the Amine Source by Interfacial
Polymerization
[0309] Uvitex OB was dissolved to 1.6% (w/w) in Benzyl Benzoate.
Polymethylene polyphenyl isocyanate (PMPPI) and 2,4 Toluene
diisocyanate (TDI) (1:1) were added to 13% (w/w) and mixed. The
organic phase was emulsified in a solution of 0.5% (w/w) SDS in
water, using homogenization with an Ultra-Turrax disperser. A
solution of 12.5% (w/w) H-Lys-Glu-OH in water was added under
mixing with a marine impeller and interfacial polymerization
performed at 40.degree. C. Microcapsules were washed with water and
collected. The mean volume-weighted diameter of the microcapsules
was 6.1 .mu.m.
[0310] Covalent Linking of VHH to Microcapsules--
[0311] Microcapsules were washed to appropriate amine-free buffers
using vacuum filtration and concentrated. VHH were dialyzed to the
same buffer and concentrated by spin filtration. VHH were added and
mixed with the microcapsules. A premix of EDC and Sulfo-NHS was
made immediately before use and added. Final concentration of EDC
in the reaction was 2 mM, final concentration of Sulfo-NHS in the
reaction was 5 mM. Final concentration of VHH in the covalent
linking reaction was 1 mg/ml. The calculated maximum density of VHH
added to the coupling reactions was 1 .mu.g/cm.sup.2 (4.3E+05 VHH
molecules/.mu.m.sup.2 microcapsule surface). Covalent linking
reactions were performed at room temperature for 2 hours with slow
tilt agitation or head-over-head rotation. Reactions were quenched
by the addition of amine-containing glycine solution. Reaction
mixtures were transferred to a filtration setup and non-linked VHH
were collected by vacuum filtration for analysis. VHH-linked
microcapsules were washed twice with appropriate buffer in a
filtration setup and collected in the same buffer. Functionality of
the microcapsules was analyzed for microcapsules coupled with
antigen-specific VHH and compared to microcapsules covalently
linked with a control VHH, Table 10. Microcapsules with
antigen-specific VHH are specifically binding to antigen-containing
surfaces over surfaces not containing the antigen. Microcapsules
with antigen-specific VHH are binding to antigen-containing
surfaces over surfaces not containing the antigen in both
application rates tested of 5% and 25% coverage. Moreover, it can
be anticipated that application rates beyond these values will also
result in specific binding of microcapsules with antigen-specific
VHH.
TABLE-US-00010 TABLE 10 Carboxyl microcapsules produced with
dipeptide H-Lys-Glu-OH as the amine source and EDC/Sulfo-NHS
mediated coupling of VHH Antigen- Control Fold binding VHH VHH
difference VHH concentration in coupling 1 1 reaction (mg/ml)
Calculated maximum density (.mu.g 1 1 VHH/cm.sup.2 microcapsule
surface) Potato lectin coat/25% coverage (# 9995 749 13
microcapsules) Potato lectin coat/5% coverage (# 3121 79 40
microcapsules) No coat/25% coverage (# 969 838 1.2 microcapsules)
No coat/5% coverage (# 144 73 2.0 microcapsules)
Example 9
Manufacturing of Microcapsules with Amine Functional Groups and VHH
Coupling Through Amine-Reactive Homobifunctional Cross-Linkers
[0312] Uvitex OB was dissolved in 1.7% (w/w) in Benzyl Benzoate.
Polymethylene polyphenyl isocyanate (PMPPI) and 2,4 Toluene
diisocyanate (TDI) (1:1) were added to 6% (w/w) and mixed. The
organic phase was emulsified in a solution of 0.5% (w/w) SDS using
homogenization with an Ultra-Turrax disperser. Alternatively
TWEEN.RTM.-80 was used as surfactant at 0.5% (w/w) concentration
and stirring performed with an overhead stirrer. A solution of 5%
(w/w) TEPA in water was added under mixing with a marine impeller
and interfacial polymerization performed at 40.degree. C. for 30
minutes. Alternatively an overhead stirrer was used, the pH
adjusted to pH 12, and interfacial polymerization performed at room
temperature overnight. Microcapsules were washed with water and
collected. The mean volume-weighted diameter of the microcapsules
obtained was .+-.10 .mu.m.
[0313] Covalent Linking of VHH to Microcapsules Using
EDC/Sulfo-NHS--
[0314] Microcapsules were washed to appropriate amine-free buffers
using vacuum filtration and concentrated. VHH were dialyzed to the
same buffer and concentrated by spin filtration. VHH were added and
mixed with the microcapsules. A premix of EDC and Sulfo-NHS was
made immediately before use and added. Final concentration of EDC
in the reaction was 2 mM, final concentration of Sulfo-NHS in the
reaction was 5 mM. Final concentration of VHH in the reaction
mixture was 1 mg/ml or 0.1 mg/ml. The calculated maximum density of
VHH added to the reaction mixtures was 1 .mu.g/cm.sup.2 (4.3E+05
VHH molecules/.mu.m.sup.2 microcapsule surface), or 0.1
.mu.g/cm.sup.2 (4.3E+04 VHH molecules/.mu.m.sup.2 microcapsule
surface). Covalent linking reactions were performed at room
temperature overnight with slow tilt agitation or head-over-head
rotation. Reactions were quenched by the addition of
amine-containing glycine solution. Coupling reactions were
transferred to a filtration setup and non-coupled VHH were
collected by vacuum filtration for analysis. VHH-coupled
microcapsules were washed twice with appropriate buffer in a
filtration setup and collected in the same buffer.
[0315] Coupling of VHH to Microcapsules Using BS3 Cross-Linker in a
One-Step Procedure--
[0316] Microcapsules were washed to appropriate amine-free buffer
using vacuum filtration and concentrated. VHH were dialyzed to the
same buffer and concentrated by spin filtration. VHH were added and
mixed with the microcapsules. BS3 ((bis[sulfosuccinimidyl]suberate)
cross-linker was dissolved immediately before use and added to the
reaction mix in ten-fold molar excess over the VHH concentration.
Final concentration of VHH in the reaction mix was 1 mg/ml or 0.1
mg/ml. The calculated maximum density of VHH added to the reaction
mixtures was 1 .mu.g/cm.sup.2 (4.3E+05 VHH molecules/.mu.m.sup.2
microcapsule surface), or 0.1 .mu.g/cm.sup.2 (4.3E+04 VHH
molecules/.mu.m.sup.2 microcapsule surface). Covalent linking
reactions were performed at room temperature overnight with slow
tilt agitation or head-over-head rotation. Reactions were quenched
by the addition of amine-containing glycine solution. Reaction
mixtures were transferred to a filtration setup and non-linked VHH
were collected by vacuum filtration for analysis. VHH-linked
microcapsules were washed twice with appropriate buffer in a
filtration setup and collected in the same buffer.
[0317] Coupling of VHH to Microcapsules Using BS3 Cross-Linker in a
Two-Step Procedure--
[0318] Microcapsules were washed to appropriate amine-free buffer
using vacuum filtration and concentrated. VHH were dialyzed to the
same buffer and concentrated by spin filtration. BS3
((bis[sulfosuccinimidyl]suberate) cross-linker was dissolved
immediately before use and added to the microcapsules in 2.5 mM
concentration and allowed to react for 30 minutes at room
temperature with slow tilt agitation or head-over-head rotation.
After incubation activated microcapsules were transferred to a
filtration setup and washed twice with appropriate buffer.
Microcapsules with activated groups were collected in the same
buffer. VHH were added immediately and mixed with the
microcapsules. Final concentration of VHH in the reaction mix was 1
mg/ml or 0.1 mg/ml. The calculated maximum density of VHH added to
the reaction mixtures was 1 .mu.g/cm.sup.2 (4.3E+05 VHH
molecules/.mu.m.sup.2 microcapsule surface), or 0.1 .mu.g/cm.sup.2
(4.3E+04 VHH molecules/.mu.m.sup.2 microcapsule surface). Covalent
linking reactions were performed at room temperature overnight with
slow tilt agitation or head-over-head rotation. Reactions were
quenched by the addition of amine-containing glycine solution.
Covalent linking reactions were transferred to a filtration setup
and non-linked VHH were collected by vacuum filtration for
analysis. VHH-linked microcapsules were washed twice with
appropriate buffer in a filtration setup and collected in the same
buffer.
[0319] Functionality of the microcapsules was analyzed for
microcapsules covalently linked with antigen-specific VHH and
compared to microcapsules covalently linked with a control VHH,
tables 11-13. Microcapsules with antigen-specific VHH covalently
linked to amine groups of the microcapsule by means of
EDC/Sulfo-NHS are specifically binding to antigen-containing
surfaces. Microcapsules covalently linked with antigen-specific VHH
at 1 or 0.1 .mu.g VHH per cm.sup.2 microcapsule surface are
specifically binding to antigen-containing surfaces with the
application rates tested from 4% to 100% coverage. Moreover, it can
be anticipated that application rates beyond these values will also
result in specific binding of microcapsules with antigen-specific
VHH.
[0320] Microcapsules with antigen-specific VHH covalently linked to
amine groups of the microcapsule by means of a BS3 homobifunctional
cross-linker in a one-step protocol are specifically binding to
antigen-containing surfaces. Microcapsules covalently linked with
antigen-specific VHH at 1 or 0.1 .mu.g VHH per cm.sup.2
microcapsule surface are specifically binding to antigen-containing
surfaces with the application rates tested from 4% to 100%
coverage. Moreover, it can be anticipated that application rates
beyond these values will also result in specific binding of
microcapsules with antigen-specific VHH.
[0321] Microcapsules with antigen-specific VHH covalently linked to
amine groups of the microcapsule by means of a BS3 homobifunctional
cross-linker in a two-step protocol are specifically binding to
antigen-containing surfaces. Microcapsules covalently linked with
antigen-specific VHH at 1 or 0.1 .mu.g VHH per cm.sup.2
microcapsule surface are specifically binding to antigen-containing
surfaces with the application rates tested from 4% to 100%
coverage. Moreover, it can be anticipated that application rates
beyond these values will also result in specific binding of
microcapsules with antigen-specific VHH. The best ratios of
specific microcapsule binding to antigen-containing surfaces are
obtained with specific VHH covalently linked to amine groups of the
microcapsule by means of a BS3 homobifunctional cross-linker in a
one-step coupling procedure.
TABLE-US-00011 TABLE 11 Amine microcapsules EDC/Sulfo-NHS coupling
Antigen- Antigen- binding Control Fold binding Control Fold
Microcapsule counts VHH VHH difference VHH VHH difference VHH
concentration in 1 1 0.1 0.1 coupling reaction (mg/ml) Calculated
maximum 1 1 0.1 0.1 density (.mu.g VHH/cm.sup.2 microcapsule
surface) Potato lectin coat/100% 2190 312 7.0 868 333 2.6 coverage
(# microcapsules) Potato lectin coat/20% 1821 64 28 610 106 5.8
coverage (# microcapsules) Potato lectin coat/4% 686 15 46 314 16
20 coverage (# microcapsules) No coat/100% coverage (# 269 315 0.9
333 258 1.3 microcapsules)
TABLE-US-00012 TABLE 12 Amine microcapsules one-step coupling BS3
Antigen- Antigen- binding Control Fold binding Control Fold VHH VHH
difference VHH VHH difference VHH concentration in 1 1 0.1 0.1
coupling reaction (mg/ml) Calculated maximum 1 1 0.1 0.1 density
(.mu.g VHH/cm.sup.2 microcapsule surface) Potato lectin coat/100%
35051 85 412 1536 627 2.4 coverage (# microcapsules) Potato lectin
coat/20% 9794 16 612 1149 212 5.4 coverage (# microcapsules) Potato
lectin coat/4% 1942 3 647 474 76 6.2 coverage (# microcapsules) No
coat/100% coverage 95 91 1.0 673 442 1.5 (# microcapsules)
TABLE-US-00013 TABLE 13 Amine microcapsules two-step coupling BS3
Antigen- Antigen- binding Control Fold binding Control Fold VHH VHH
difference VHH VHH difference VHH concentration in 1 1 0.1 0.1
coupling reaction (mg/ml) Calculated maximum 1 1 0.1 0.1 density
(.mu.g VHH/cm.sup.2 microcapsule surface) Potato lectin coat/100%
2681 380 7 1418 839 1.7 coverage (# microcapsules) Potato lectin
coat/20% 1829 163 11 851 351 2.4 coverage (# microcapsules) Potato
lectin coat/4% 790 50 16 361 119 3.0 coverage (# microcapsules) No
coat/100% coverage (# 747 379 2.0 817 1024 0.8 microcapsules)
[0322] In another experiment it was investigated how differently
functionalized microcapsules are binding to surfaces with different
antigen densities. Functionality of the microcapsules was analyzed
for microcapsules covalently linked with antigen-specific VHH and
compared to microcapsules covalently linked with a control VHH,
tables 14-17. Microcapsules with antigen-specific VHH covalently
linked to carboxyl or amine anchor groups of microcapsules by means
of different covalent linking procedures are specifically binding
to antigen-containing surfaces. Microcapsules covalently linked
with antigen-specific VHH at 1 or 0.1 .mu.g VHH per cm.sup.2
microcapsule surface are specifically binding to antigen-containing
surfaces with the application rates tested 10% or 100% coverage.
Microcapsules with antigen-specific VHH are specifically binding to
surfaces with different antigen densities. Moreover, it can be
anticipated that application rates beyond these values will also
result in specific binding of microcapsules with antigen-specific
VHH. The best ratios of specific microcapsule binding to surfaces
with different antigen densities and different application rates
are obtained with specific VHH coupled to amine groups of the
microcapsule by means of a BS3 homobifunctional cross-linker in a
one-step covalent linking procedure.
TABLE-US-00014 TABLE 14 Sample ID and coupling conditions VHH
Calculated concentra- maximum tion density in (.mu.g VHH/ coupling
cm.sup.2 Microcapsule functional reaction microcapsule Sample
groups VHH (mg/ml) surface) A Carboxyl (EDC/Sulfo-NHS Antigen 1 1
coupling) binding B Carboxyl (EDC/Sulfo-NHS Antigen 0.1 0.1
coupling) binding C Carboxyl (EDC/Sulfo-NHS Control 1 1 coupling) D
Carboxyl (EDC/Sulfo-NHS Control 0.1 0.1 coupling) E Amine (BS-3
cross-linker Antigen 1 1 one-step coupling) binding F Amine (BS-3
cross-linker Antigen 0.1 0.1 one-step coupling) binding G Amine
(BS-3 cross-linker Control 1 1 one-step coupling) H Amine (BS-3
cross-linker Control 0.1 0.1 one-step coupling) I Amine (BS-3
cross-linker Antigen 1 1 one-step coupling) binding J Amine (BS-3
cross-linker Antigen 0.1 0.1 two-step coupling) binding K Amine
(BS-3 cross-linker Control 1 1 two-step coupling) L Amine (BS-3
cross-linker Control 0.1 0.1 two-step coupling)
TABLE-US-00015 TABLE 15 Microcapsule counts A C B D A C B D Potato
lectin 100% 100% 100% 100% 10% 10% 10% 10% coat (.mu.g/ml) coverage
coverage coverage coverage coverage coverage coverage coverage 100
23696 297 4195 515 5154 55 2229 125 10 2755 265 2035 475 2752 50
1621 118 1 363 193 530 227 435 49 233 64 0 542 266 481 589 77 69
223 113 E G F H E G F H Potato lectin 100% 100% 100% 100% 10% 10%
10% 10% coat (.mu.g/ml) coverage coverage coverage coverage
coverage coverage coverage coverage 100 43052 150 2842 622 8959 36
699 225 10 35580 104 1693 780 6330 13 720 215 1 2001 46 1062 173
1572 7 284 36 0 202 190 975 973 119 67 142 196 I K J L I K J L
Potato lectin 100% 100% 100% 100% 10% 10% 10% 10% coat (.mu.g/ml)
coverage coverage coverage coverage coverage coverage coverage
coverage 100 3573 866 3244 1111 1409 285 667 248 10 2166 903 2406
787 904 197 484 186 1 1022 617 1235 860 385 215 290 116 0 1233 1163
1798 1368 319 366 227 273
TABLE-US-00016 TABLE 16 Fold difference between microcapsule
samples A over C A over C B over D B over D Potato lectin coat 100%
10% 100% 10% (.mu.g/ml) coverage coverage coverage coverage 100 80
94 8 18 10 10 55 4 14 1 2 9 2 4 0 2 1 1 2 E over G E over G F over
H F over H Potato lectin coat 100% 10% 100% 10% (.mu.g/ml) coverage
coverage coverage coverage 100 287 249 5 3 10 342 487 2 3 1 44 225
6 8 0 1 2 1 1 I over K I over K J over L J over L Potato lectin
coat 100% 10% 100% 10% (.mu.g/ml) coverage coverage coverage
coverage 100 4 5 3 3 10 2 5 3 3 1 2 2 1 3 0 1 1 1 1
Example 10
Functionality of Microcapsules with Antigen-Specific VHH for
Binding to Plant Leaves
[0323] Microcapsules with antigen-specific VHH or control VHH were
topically applied at 100%, 10%, 1%, or 0.1% coverage to leaf discs
prepared from outside-grown plants. Non-bound microcapsules were
removed by placing the leaf discs floating upside down on wells
filled with buffer and shaking on an ELISA shaking platform
.gtoreq.900 rpm for 45 minutes. Washed leaf discs were analyzed for
bound microcapsules using a macrozoom microscope system (Nikon) and
microcapsules counted using Volocity image analysis software
(PerkinElmer); the average number of microcapsules for each
condition is shown in tables 17 and 18. Microcapsules with
antigen-specific VHH covalently linked to carboxyl or amine anchor
groups of microcapsules by means of different linking methods are
specifically binding to leaves. Microcapsules covalently linked
with antigen-specific VHH are specifically binding to leaves with
the application rates tested 0.1%, 1%, 10% or 100% coverage for the
delivery of active substances (AS). This can be calculated to be
suitable for delivery of agrochemicals on greenhouse or field crops
in the range of 24 g AS/ha to 8.5 kg AS/ha (Table 19).
TABLE-US-00017 TABLE 17 Microcapsules with carboxyl anchor groups,
covalently linked in a one-step protocol with antigen-specific VHH
bound and retained on potato leaf discs Antigen-binding Control VHH
VHH Fold Average Stdev Average Stdev difference 100% coverage 25901
7307 3843 467 6.7 10% coverage 8278 3226 682 47 12 1% coverage 1680
393 161 49 10 0.1% coverage 320 44 34 6 9.3
TABLE-US-00018 TABLE 18 Microcapsules with amine anchor groups,
covalently linked in a one-step protocol using BS3 cross-linker
with antigen-specific VHH, bound and retained on potato leaf discs
Antigen-binding Control VHH VHH Fold Average Stdev Average Stdev
difference 100% coverage 25621 3285 1335 77 19 10% coverage 4270
375 588 168 7.3 1% coverage 902 216 170 68 5.3 0.1% coverage 125 46
39 24 3.2
TABLE-US-00019 TABLE 19 Calculated delivery of active substances
with microcapsules with antigen-specific VHH Microcapsules
Microcapsule Microcapsules Microcapsule counted on 0.5 cm.sup.2
amount on 0.5 cm.sup.2 counted on 0.5 cm.sup.2 amount on 0.5
cm.sup.2 leaf disc leaf disc (mg) leaf disc leaf disc (mg)
Microcapsule 100% coverage 100% coverage 0.1% coverage 0.1%
coverage diameter (.mu.m) 6.1 (carboxyl 25901 2.46E-02 320 3.05E-04
microcapsule) 10 (amine 25621 1.07E-01 125 5.22E-04 microcapsules)
Microcapsule Microcapsule Assuming active Assuming active amount
calculated amount calculated substance 40% substance 40% per
hectare (g) per hectare (g) load (g/ha) load (g/ha) Microcapsule
100% coverage 0.1% coverage 100% coverage 0.1% coverage diameter
(.mu.m) 6.1 (carboxyl 4.90E+03 6.06E+01 2.0E+03 2.4E+01
microcapsule) 10 (amine 2.14E+04 1.04E+02 8.5E+03 4.2E+01
microcapsules)
REFERENCES
[0324] Altschul S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z.
Zhang, W. Miller, and D. J. Lipman (1997). Gapped BLAST and
PSI-BLAST: a new generation of protein database search programs.
Nucleic Acids Res. 25:3389-3402. [0325] Blake A. W., L. McCartney,
J. Flint, D. N. Bolam, A. B. Boraston, H. J. Gilbert, and J. P.
Knox (2006). Understanding the biological rationale for the
diversity of cellulose-directed carbohydrate-binding molecules in
prokaryotic enzymes. J. Biol. Chem. 281; 29321-29329. [0326] Cabo
L., I. Garcia, C. Gotor, and L. C. Romero (2006). Leaf hairs
influence phytopathogenic fungus infection and confer an increased
resistance when expressing a Trichoderma .alpha.-1,3-glucanase. J.
Exp. Botany 57:3911-3920. [0327] Cozens-Roberts C., J. A. Quinn,
and D. A. Lauffenburger (1990). Receptor-mediated cell attachment
and detachment kinetics. Biophys. J. 58:857-872. [0328] Dimitrov D.
S. (2009). Engineered CH2 domains (nanoantibodies). mAbs 1:26-28.
[0329] Gage D. J. (2004). Infection and invasion of roots by
symbiotic, nitrogen fixing Rhizobia during nodulation of temperate
legumes. Microbiol. Mol. Biol. Rev. 68:280-300. [0330] Jones L., G.
B. Seymour, and J. P. Knox (1997). Localization of pectic galactan
in tomato cell walls using a monoclonal antibody specific to
(1-4)-.beta.-D-galactan. Plant Physiol. 113:1405-1412. [0331]
Kolmar H. (2008). Alternative binding proteins: biological activity
and therapeutic potential of cysteine-knot miniproteins. FEBS J.
275:2684-2690. [0332] Kuo S. C. and D. A. Lauffenburger (1993).
Relationship between receptor/ligand binding affinity and adhesion
strength. Biophys. J. 65:2191-2200. [0333] Lai A., V. Cianciolo, S.
Chiavarini and A. Sonnino (2000). Effect of glandular trichomes on
the development of Phytophtora infestans infection in potato (S.
tuberosum). Euphytica 114:165-174. [0334] Laus M. C., A. A. N. van
Brussel, and J. W. Kijne (2005). Role of cellulose fibrils and
exopolysaccharides of Rhizobium leguminosarum in attachment and
infection of vicia sativa root hairs. Mol. Plant-Microbe
Interactions 18:533-538. [0335] Mason B. P., S. M. Hira, G. F.
Strouse, and D. T. McQuade (2009). Microcapsules with three
orthogonal reactive sites. Org. Lett. 11:1479-1482. [0336] Melotto
M., W. Underwood, J. Koczan, K. Nomura, and S. Y. He (2006). Plant
stomata function in innate immunity against bacterial invasion.
Cell 126:969-980. [0337] Moller I., I. Sorensen, A. J. Bernal, C.
Blaukopf, K. Lee, J. Obro, F. Pettolino, A. Roberts, J. D.
Mikkelsen, J. P. Knox, A. Bacic, and W. G. Willats (2007). High
throughput mapping of cell-wall polymers within and between plants
using novel microarrays. Plant J. 50:1118-1128. [0338] Muyldermans
S., T. N. Baral, V. C. Retamozzo, P. De Baetselier, E. De Genst, J.
Kinne, H. Leonhardt, S. Magez, V. K. Nguyen, H. Revets, U.
Rothbauer, B. Stijlemans, S. Tillib, U. Wernery, L. Wyns, G.
Hassanzadeh-Ghassabeh, D. Saerens (2009). Camelid immunoglobulins
and nanobody technology. Vet. Immunol. Immunopathol. 128:178-83.
[0339] Ni P., M. Zhang, and N. Yan (1995). Effect of operating
varibales and monomers on the formation of polyurea microcapsules.
Journal of Membrane Science 103:51-55. [0340] Nygren P-A. (2008).
Alternative binding proteins: affibody binding proteins developed
from a small three-helix bundle scaffold. FEBS J. 275:2668-2676.
[0341] Pennell R. I., J. P. Knox, G. N. Scofield, R. R. Selvendran,
and K. Roberts (1989). A family of abundant plasma membrane
associated glycoproteins related to the arabinogalactan proteins is
unique to flowering plants. J. Cell. Biol. 108:1967-1977. [0342]
Scher H. B., M. Rodson, and K.-S. Lee (1998). Microencapsulation of
pesticides by interfacial polymerization utilizing isocyanate or
aminoplast chemistry. Pestic. Sci. 54:394-400. [0343] Schreiber L.
(2005). Polar paths of diffusion across plant cuticles: new
evidence for an old hypothesis. Ann. Bot. 95:1069-1073. [0344]
Skerra A. (2008). Alternative binding proteins:
anticalins--harnessing the structural plasticity of the lipocalin
ligand pocket to engineer novel binding activities. FEBS J.
275:2677-2683. [0345] Stump M. T., H. K. Binz, P. Amstutz (2008).
DARPins: a new generation of protein therapeutics. Drug Discov.
Today 13:695-701. [0346] Tramontano A., E. Bianchi, S. Venturini,
F. Martin, A. Pessi, and M. Sollazzo (1994). The making of the
minibody: an engineered beta-protein for the display of
confromationally constrained peptides. J. Mol. Recognition. 7:9-24.
[0347] Underwood W., M. Melotto, and S. Y. He (2007). Role of plant
stomata in bacterial invasion. Cellular Microbiol. 9:1621-1629.
[0348] Wesolowski J., V. Alzogaray, J. Reyelt, M. Unger, K. Juarez,
M. Urrutia, A. Cauerhiff, W. Danquah, B. Rissiek, F. Scheuplin, N.
Schwarz, S. Adriouch, O. Boyer, M. Seman, A. Licea, D. V. Serreze,
F. A. Goldbaum, F. Haag, and F. Koch-Nolte (2009). Single domain
antibodies: promising experimental and therapeutic tools in
infection and immunity. Med. Microbiol. Immunol. 198:157-174.
[0349] Willats W. G. and J. P. Knox (1999). Immunoprofiling of
pectic polysaccharides. Anal. Biochem. 268:143-146. [0350] Willats
W. G., S. E. Marcus, and J. P. Knox (1998). Generation of
monoclonal antibody specific to (A-5)-alpha-L-arabinan. Carbohydr.
Res. 308:149-152. [0351] Willats W. G., C. Orfila, G. Limberg, H.
C. Buchholt, G-J. W. M. van Alebeek, A. G. J. Voragen, S. E.
Marcus, T. M. I. E. Christensen, J. D. Mikkelsen, B. S. Murray, and
J. P. Knox (2001). Modulation of the degree and pattern of
methyl-esterification of pectic homogalacruronan in plant cell
walls. J. Biol. Chem. 276:19404-19413.
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
* * * * *