U.S. patent application number 14/787454 was filed with the patent office on 2016-05-26 for agrochemical compositions comprising antibodies binding to sphingolipids.
This patent application is currently assigned to AGROSAVFE N.V.. The applicant listed for this patent is AGROSAVFE N.V.. Invention is credited to Bruno Cammue, Miguel Francesco Coleta De Bolle, Chris De Jonghe, Karin Thevissen, Inge Elodie Van Daele, Joao Filipe Veloso Vieira, Peter Verheesen.
Application Number | 20160145325 14/787454 |
Document ID | / |
Family ID | 50736046 |
Filed Date | 2016-05-26 |
United States Patent
Application |
20160145325 |
Kind Code |
A1 |
Verheesen; Peter ; et
al. |
May 26, 2016 |
AGROCHEMICAL COMPOSITIONS COMPRISING ANTIBODIES BINDING TO
SPHINGOLIPIDS
Abstract
The present invention relates to agrochemical and biological
control compositions for combating pests, more specifically plant
pests, comprising at least one heavy chain variable domain of an
antibody, which specifically binds to a sphingolipid of a plant
pathogen. The invention further provides methods for protecting or
treating a plant or a part of a plant from an infection or other
biological interaction with a plant pathogen, at least comprising
the step of applying directly or indirectly to a plant or to a part
of a plant, an agrochemical composition, under conditions effective
to protect or treat a plant or a part of a plant against a
infection or biological interaction with a plant pathogen. Further
provided are methods for producing such agrochemical compositions
and formulations, to heavy chain variable domains with a specific
pesticidal activity comprised within an agrochemical formulation,
to nucleic acids encoding such heavy chain variable domains and to
plants comprising chimeric genes comprising such nucleic acids.
Inventors: |
Verheesen; Peter;
(Mariakerke, BE) ; De Jonghe; Chris; (Mortsel,
BE) ; Van Daele; Inge Elodie; (Melle, BE) ; De
Bolle; Miguel Francesco Coleta; (Baarle-Nassau, NL) ;
Veloso Vieira; Joao Filipe; (Didcot, GB) ; Thevissen;
Karin; (Bierbeek, BE) ; Cammue; Bruno;
(Alsemberg, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AGROSAVFE N.V. |
Gent |
|
BE |
|
|
Assignee: |
AGROSAVFE N.V.
Gent,
BE
|
Family ID: |
50736046 |
Appl. No.: |
14/787454 |
Filed: |
April 29, 2014 |
PCT Filed: |
April 29, 2014 |
PCT NO: |
PCT/EP2014/058771 |
371 Date: |
October 27, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61817170 |
Apr 29, 2013 |
|
|
|
Current U.S.
Class: |
424/172.1 ;
530/389.8 |
Current CPC
Class: |
A01N 37/46 20130101;
C07K 2317/569 20130101; C07K 2317/76 20130101; A01N 63/10 20200101;
C07K 16/14 20130101; C07K 2317/56 20130101; C07K 2317/22 20130101;
C07K 2317/565 20130101; A61P 31/10 20180101; C12N 15/8279
20130101 |
International
Class: |
C07K 16/14 20060101
C07K016/14; A01N 63/02 20060101 A01N063/02; A01N 37/46 20060101
A01N037/46 |
Claims
1. An agrochemical composition comprising at least one V.sub.HH,
which specifically binds to a glucosylceramide of a plant
pathogenic fungus.
2.-4. (canceled)
5. The agrochemical composition according to claim 1, wherein the
genus of said plant pathogenic fungus is selected from the group
consisting of Alternaria, Ascochyta, Botrytis, Cercospora,
Colletotrichum, Diplodia, Erysiphe, Fusarium, Leptosphaeria,
Gaeumanomyces, Helminthosporium, Macrophomina, Nectria,
Penicillium, Peronospora, Phoma, Phymatotrichum, Phytophthora,
Plasmopara, Podosphaera, Puccinia, Pyrenophora, Pyricularia,
Pythium, Rhizoctonia, Scerotium, Sclerotinia, Septoria,
Thielaviopsis, Uncinula, Venturia, Verticillium, Magnaporthe,
Blumeria, Mycosphaerella, Ustilago, Melampsora, Phakospora,
Monilinia, Mucor, Rhizopus, and Aspergillus.
6. The agrochemical composition of claim 5, wherein the at least
one V.sub.HH is present in the composition in an amount effective
to protect or treat a plant or a part of said plant from an
infection or other biological interaction with said plant
pathogenic fungus.
7. The agrochemical composition of claim 2, wherein the
concentration of the at least one V.sub.HH in the agrochemical
composition ranges from 0.0001% to 50% by weight.
8. The agrochemical composition of claim 6, which further comprises
an agrochemically suitable carrier and/or one or more suitable
adjuvants.
9. The agrochemical composition of claim 1, wherein said at least
one V.sub.HH comprises a CDR1 region having SEQ ID NO: 86, a CDR2
region having SEQ ID NO: 170, and a CDR3 region having SEQ ID NO:
254, and/or a CDR1 region having SEQ ID NO: 146, a CDR2 region
having SEQ ID NO: 230, and a CDR3 region having SEQ ID NO: 313,
and/or a CDR1 region having SEQ ID NO: 85, a CDR2 region having SEQ
ID NO: 169, and a CDR3 region having SEQ ID NO: 253, and/or a CDR1
region having SEQ ID NO: 87, a CDR2 region having SEQ ID NO: 171,
and a CDR3 region having SEQ ID NO: 255, and/or a CDR1 region
having SEQ ID NO: 88, a CDR2 region having SEQ ID NO: 172, and a
CDR3 region having SEQ ID NO: 256, and/or a CDR1 region having SEQ
ID NO: 89, a CDR2 region having SEQ ID NO: 173, and a CDR3 region
having SEQ ID NO: 257, and/or a CDR1 region having SEQ ID NO: 90, a
CDR2 region having SEQ ID NO: 174, and a CDR3 region having SEQ ID
NO: 258, and/or a CDR1 region having SEQ ID NO: 91, a CDR2 region
having SEQ ID NO: 175, and a CDR3 region having SEQ ID NO: 259,
and/or a CDR1 region having SEQ ID NO: 92, a CDR2 region having SEQ
ID NO: 176, and a CDR3 region having SEQ ID NO: 260, and/or a CDR1
region having SEQ ID NO: 93, a CDR2 region having SEQ ID NO: 177,
and a CDR3 region having SEQ ID NO: 261, and/or a CDR1 region
having SEQ ID NO: 94, a CDR2 region having SEQ ID NO: 178, and a
CDR3 region having SEQ ID NO: 262, and/or a CDR1 region having SEQ
ID NO: 95, a CDR2 region having SEQ ID NO: 179, and a CDR3 region
having SEQ ID NO: 263, and/or a CDR1 region having SEQ ID NO: 96, a
CDR2 region having SEQ ID NO: 180, and a CDR3 region having SEQ ID
NO: 264, and/or a CDR1 region having SEQ ID NO: 97, a CDR2 region
having SEQ ID NO: 181, and a CDR3 region having SEQ ID NO: 265,
and/or a CDR1 region having SEQ ID NO: 98, a CDR2 region having SEQ
ID NO: 182, and a CDR3 region having SEQ ID NO: 266, and/or a CDR1
region having SEQ ID NO: 99, a CDR2 region having SEQ ID NO: 183,
and a CDR3 region having SEQ ID NO: 267, and/or a CDR1 region
having SEQ ID NO: 100, a CDR2 region having SEQ ID NO: 184, and a
CDR3 region having SEQ ID NO: 268, and/or a CDR1 region having SEQ
ID NO: 101, a CDR2 region having SEQ ID NO: 185, and a CDR3 region
having SEQ ID NO: 269, and/or a CDR1 region having SEQ ID NO: 102,
a CDR2 region having SEQ ID NO: 186, and a CDR3 region having SEQ
ID NO: 270, and/or a CDR1 region having SEQ ID NO: 103, a CDR2
region having SEQ ID NO: 187, and a CDR3 region having SEQ ID NO:
271, and/or a CDR1 region having SEQ ID NO: 104, a CDR2 region
having SEQ ID NO: 188, and a CDR3 region having SEQ ID NO: 272,
and/or a CDR1 region having SEQ ID NO: 105, a CDR2 region having
SEQ ID NO: 189, and a CDR3 region having SEQ ID NO: 273, and/or a
CDR1 region having SEQ ID NO: 106, a CDR2 region having SEQ ID NO:
190, and a CDR3 region having SEQ ID NO: 274, and/or a CDR1 region
having SEQ ID NO: 107, a CDR2 region having SEQ ID NO: 191, and a
CDR3 region having SEQ ID NO: 275, and/or a CDR1 region having SEQ
ID NO: 108, a CDR2 region having SEQ ID NO: 192, and a CDR3 region
having SEQ ID NO: 276, and/or a CDR1 region having SEQ ID NO: 109,
a CDR2 region having SEQ ID NO: 193, and a CDR3 region having SEQ
ID NO: 277, and/or a CDR1 region having SEQ ID NO: 110, a CDR2
region having SEQ ID NO: 194, and a CDR3 region having SEQ ID NO:
278, and/or a CDR1 region having SEQ ID NO: 111, a CDR2 region
having SEQ ID NO: 195, and a CDR3 region having SEQ ID NO: 279,
and/or a CDR1 region having SEQ ID NO: 112, a CDR2 region having
SEQ ID NO: 196, and a CDR3 region having SEQ ID NO: 280, and/or a
CDR1 region having SEQ ID NO: 113, a CDR2 region having SEQ ID NO:
197, and a CDR3 region having SEQ ID NO: 281, and/or a CDR1 region
having SEQ ID NO: 114, a CDR2 region having SEQ ID NO: 198, and a
CDR3 region having SEQ ID NO: 282, and/or a CDR1 region having SEQ
ID NO: 115, a CDR2 region having SEQ ID NO: 199, and a CDR3 region
having SEQ ID NO: 283, and/or a CDR1 region having SEQ ID NO: 116,
a CDR2 region having SEQ ID NO: 200, and a CDR3 region having SEQ
ID NO: 284, and/or a CDR1 region having SEQ ID NO: 117, a CDR2
region having SEQ ID NO: 201, and a CDR3 region having SEQ ID NO:
285, and/or a CDR1 region having SEQ ID NO: 118, a CDR2 region
having SEQ ID NO: 202, and a CDR3 region having SEQ ID NO: 286,
and/or a CDR1 region having SEQ ID NO: 119, a CDR2 region having
SEQ ID NO: 203, and a CDR3 region having SEQ ID NO: 287, and/or a
CDR1 region having SEQ ID NO: 120, a CDR2 region having SEQ ID NO:
204, and a CDR3 region having SEQ ID NO: 288, and/or a CDR1 region
having SEQ ID NO: 121, a CDR2 region having SEQ ID NO: 205, and a
CDR3 region having SEQ ID NO: 289, and/or a CDR1 region having SEQ
ID NO: 122, a CDR2 region having SEQ ID NO: 206, and a CDR3 region
having SEQ ID NO: 290, and/or a CDR1 region having SEQ ID NO: 123,
a CDR2 region having SEQ ID NO: 207, and a CDR3 region having SEQ
ID NO: 291, and/or a CDR1 region having SEQ ID NO: 124, a CDR2
region having SEQ ID NO: 208, and a CDR3 region having SEQ ID NO:
292, and/or a CDR1 region having SEQ ID NO: 125, a CDR2 region
having SEQ ID NO: 209, and a CDR3 region having SEQ ID NO: 293,
and/or a CDR1 region having SEQ ID NO: 126, a CDR2 region having
SEQ ID NO: 210, and a CDR3 region having SEQ ID NO: 294, and/or a
CDR1 region having SEQ ID NO: 127, a CDR2 region having SEQ ID NO:
211, and a CDR3 region having SEQ ID NO: 295, and/or a CDR1 region
having SEQ ID NO: 128, a CDR2 region having SEQ ID NO: 212, and a
CDR3 region having SEQ ID NO: 296, and/or a CDR1 region having SEQ
ID NO: 129, a CDR2 region having SEQ ID NO: 213, and a CDR3 region
having SEQ ID NO: 297, and/or a CDR1 region having SEQ ID NO: 130,
a CDR2 region having SEQ ID NO: 214, and a CDR3 region having SEQ
ID NO: 298, and/or a CDR1 region having SEQ ID NO: 131, a CDR2
region having SEQ ID NO: 215, and a CDR3 region having SEQ ID NO:
299, and/or a CDR1 region having SEQ ID NO: 132, a CDR2 region
having SEQ ID NO: 216, and a CDR3 region having SEQ ID NO: 300,
and/or a CDR1 region having SEQ ID NO: 133, a CDR2 region having
SEQ ID NO: 217, and a CDR3 region having SEQ ID NO: 301, and/or a
CDR1 region having SEQ ID NO: 134, a CDR2 region having SEQ ID NO:
218, and a CDR3 region having SEQ ID NO: 302, and/or a CDR1 region
having SEQ ID NO: 135, a CDR2 region having SEQ ID NO: 219, and a
CDR3 region having SEQ ID NO: 303, and/or a CDR1 region having SEQ
ID NO: 136, a CDR2 region having SEQ ID NO: 220, and a CDR3 region
having SEQ ID NO: 304, and/or a CDR1 region having SEQ ID NO: 137,
a CDR2 region having SEQ ID NO: 221, and a CDR3 region having SEQ
ID NO: 305, and/or a CDR1 region having SEQ ID NO: 138, a CDR2
region having SEQ ID NO: 222, and a CDR3 region having the amino
acid sequence NRY, and/or a CDR1 region having SEQ ID NO: 139, a
CDR2 region having SEQ ID NO: 223, and a CDR3 region having SEQ ID
NO: 306, and/or a CDR1 region having SEQ ID NO: 140, a CDR2 region
having SEQ ID NO: 224, and a CDR3 region having SEQ ID NO: 307,
and/or a CDR1 region having SEQ ID NO: 141, a CDR2 region having
SEQ ID NO: 225, and a CDR3 region having SEQ ID NO: 308, and/or a
CDR1 region having SEQ ID NO: 142, a CDR2 region having SEQ ID NO:
226, and a CDR3 region having SEQ ID NO: 309, and/or a CDR1 region
having SEQ ID NO: 143, a CDR2 region having SEQ ID NO: 227, and a
CDR3 region having SEQ ID NO: 310, and/or a CDR1 region having SEQ
ID NO: 144, a CDR2 region having SEQ ID NO: 228, and a CDR3 region
having SEQ ID NO: 311, and/or a CDR1 region having SEQ ID NO: 145,
a CDR2 region having SEQ ID NO: 229, and a CDR3 region having SEQ
ID NO: 312, and/or a CDR1 region having SEQ ID NO: 147, a CDR2
region having SEQ ID NO: 231, and a CDR3 region having SEQ ID NO:
314, and/or a CDR1 region having SEQ ID NO: 148, a CDR2 region
having SEQ ID NO: 232, and a CDR3 region having SEQ ID NO: 315,
and/or a CDR1 region having SEQ ID NO: 149, a CDR2 region having
SEQ ID NO: 233, and a CDR3 region having SEQ ID NO: 316, and/or a
CDR1 region having SEQ ID NO: 150, a CDR2 region having SEQ ID NO:
234, and a CDR3 region having SEQ ID NO: 317, and/or a CDR1 region
having SEQ ID NO: 151, a CDR2 region having SEQ ID NO: 235, and a
CDR3 region having SEQ ID NO: 318, and/or a CDR1 region having SEQ
ID NO: 152, a CDR2 region having SEQ ID NO: 236, and a CDR3 region
having SEQ ID NO: 319, and/or a CDR1 region having SEQ ID NO: 153,
a CDR2 region having SEQ ID NO: 237, and a CDR3 region having SEQ
ID NO: 320, and/or a CDR1 region having SEQ ID NO: 154, a CDR2
region having SEQ ID NO: 238, and a CDR3 region having SEQ ID NO:
321, and/or a CDR1 region having SEQ ID NO: 155, a CDR2 region
having SEQ ID NO: 239, and a CDR3 region having SEQ ID NO: 322,
and/or a CDR1 region having SEQ ID NO: 156, a CDR2 region having
SEQ ID NO: 240, and a CDR3 region having SEQ ID NO: 323, and/or a
CDR1 region having SEQ ID NO: 157, a CDR2 region having SEQ ID NO:
241, and a CDR3 region having SEQ ID NO: 324, and/or a CDR1 region
having SEQ ID NO: 158, a CDR2 region having SEQ ID NO: 242, and a
CDR3 region having SEQ ID NO: 325, and/or a CDR1 region having SEQ
ID NO: 159, a CDR2 region having SEQ ID NO: 243, and a CDR3 region
having SEQ ID NO: 326, and/or a CDR1 region having SEQ ID NO: 160,
a CDR2 region having SEQ ID NO: 244, and a CDR3 region having SEQ
ID NO: 327, and/or a CDR1 region having SEQ ID NO: 161, a CDR2
region having SEQ ID NO: 245, and a CDR3 region having SEQ ID NO:
328, and/or a CDR1 region having SEQ ID NO: 162, a CDR2 region
having SEQ ID NO: 246, and a CDR3 region having SEQ ID NO: 329,
and/or a CDR1 region having SEQ ID NO: 163, a CDR2 region having
SEQ ID NO: 247, and a CDR3 region having SEQ ID NO: 330, and/or a
CDR1 region having SEQ ID NO: 164, a CDR2 region having SEQ ID NO:
248, and a CDR3 region having SEQ ID NO: 331, and/or a CDR1 region
having SEQ ID NO: 165, a CDR2 region having SEQ ID NO: 249, and a
CDR3 region having SEQ ID NO: 332, and/or a CDR1 region having SEQ
ID NO: 166, a CDR2 region having SEQ ID NO: 250, and a CDR3 region
having SEQ ID NO: 333, and/or a CDR1 region having SEQ ID NO: 167,
a CDR2 region having SEQ ID NO: 251, and a CDR3 region having SEQ
ID NO: 334, and/or a CDR1 region having SEQ ID NO: 168, a CDR2
region having SEQ ID NO: 252, and a CDR3 region having SEQ ID NO:
335.
10. The agrochemical composition of claim 1, wherein said at least
one V.sub.HH comprises a peptide selected from the group consisting
of SEQ ID NOs: 1 to 84.
11. A method for protecting or treating a plant or a part of said
plant from an infection or other biological interaction with a
plant pathogenic fungus, the method comprising: at least applying
to said plant or to a part of said plant, the agrochemical
composition of claim 1, under conditions effective to protect or
treat said plant or a part of said plant against said infection or
biological interaction with said plant pathogenic fungus.
12. The method according to claim 11, wherein said agrochemical
composition is applied to said plant or to a part of said plant by
spraying, atomizing, foaming, fogging, culturing in hydroculture,
culturing in hydroponics, coating, submerging, and/or
encrusting.
13. A post-harvest treatment method for protecting or treating a
harvested plant or a harvested part of said plant from an infection
or other biological interaction with a plant pathogenic fungus, the
method comprising: at least applying to said harvested plant or to
a harvested part of said plant, the agrochemical composition of
claim 1, under conditions effective to protect or treat said
harvested plant or a harvested part of said plant against said
infection or biological interaction with said plant pathogenic
fungus.
14. A method of protecting or treating a plant or a part of the
plant from an infection or other biological interaction with a
plant pathogenic fungus, the method comprising: utilizing the
agrochemical composition of claim 1 as an anti-pest agent.
15. A method of inhibiting the growth or killing a plant pathogenic
fungus, the method comprising: at least applying to a plant or to a
part of said plant, the agrochemical composition of claim 1.
16. The agrochemical composition of claim 1, wherein the
concentration of the at least one V.sub.HH in the agrochemical
composition is between 0.1% to 10% by weight of the agrochemical
composition.
17. The agrochemical composition of claim 5, wherein the
concentration of the at least one V.sub.HH in the agrochemical
composition is between 0.1% to 10% by weight of the agrochemical
composition.
18. The agrochemical composition of claim 6, wherein the
concentration of the at least one V.sub.HH in the agrochemical
composition is between 0.1% to 10% by weight of the agrochemical
composition.
19. A composition comprising: V.sub.HH, present in the composition
in an amount effective to protect or treat a plant or a part of the
plant from an infection or other biological interaction with a
plant pathogenic fungus, which V.sub.HH specifically binds to a
glucosylceramide of a plant pathogenic fungus selected from the
group consisting of Alternaria, Ascochyta, Botrytis, Cercospora,
Colletotrichum, Diplodia, Erysiphe, Fusarium, Leptosphaeria,
Gaeumanomyces, Helminthosporium, Macrophomina, Nectria,
Penicillium, Peronospora, Phoma, Phymatotrichum, Phytophthora,
Plasmopara, Podosphaera, Puccinia, Pyrenophora, Pyricularia,
Pythium, Rhizoctonia, Scerotium, Sclerotinia, Septoria,
Thielaviopsis, Uncinula, Venturia, Verticillium, Magnaporthe,
Blumeria, Mycosphaerella, Ustilago, Melampsora, Phakospora,
Monilinia, Mucor, Rhizopus, and Aspergillus, and an agrochemically
suitable carrier and/or one or more suitable adjuvants.
20. The composition of claim 19, wherein the amount of the V.sub.HH
in the composition is between 0.1% to 10% by weight of the
composition.
21. The composition of claim 19, wherein the V.sub.HH comprises: a
CDR1 region comprising SEQ ID NO: 86, a CDR2 region comprising SEQ
ID NO: 170, and a CDR3 region comprising SEQ ID NO: 254, and/or a
CDR1 region comprising SEQ ID NO: 146, a CDR2 region comprising SEQ
ID NO: 230, and a CDR3 region comprising SEQ ID NO: 313.
22. The composition of claim 19, wherein the V.sub.HH comprises a
peptide selected from the group consisting of SEQ ID NOs: 1 to
84.
23. A method of killing or inhibiting the growth of a plant
pathogenic fungus on a plant or plant part, the method comprising:
applying to the plant or plant part the composition of claim 19 in
an amount effective to inhibit growth of the plant pathogenic
fungus on the plant or plant part.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to effecting control of plant
pests. More specifically the invention provides agrochemical
compositions comprising polypeptide compositions of a specific
length and concentration which are useful to combat crop pests such
as insects, fungi, nematodes, bacteria and the like.
BACKGROUND
[0002] Crop protection, required for effective agriculture, relies
heavily on the use of pesticides, which are applied to the crops by
spraying them onto the crop, applying during watering of the crops
or incorporating them into the soil. Pesticides are often organic
chemical molecules and their repeated application to crops poses
toxicity threats to both agricultural workers during handling and
to the environment, due to spray drift, persistence in the soil or
washing off into surface or ground water. It would be advantageous
to be able to use alternative compounds that are less toxic to
humans and the environment, but that at the same time provide
effective control of plant pests. Proteinaceous pesticides with
specificity against a certain plant pest target may be very
advantageous in this respect, as they are expected to be
short-lived in the environment and to have less toxic off-target
effects. However, there are only a few proteinaceous or peptidergic
pesticides known. Some examples are Bt toxins, lectins, defensins,
fabatins, tachyplesin, magainin, harpin (see WO2010019442), pea
albumin 1-subunit b (PA1b). However, these proteinaceous pesticides
are either small peptides with compact structures, stabilized by
several disulphide bridges, or are larger proteins (>300 amino
acids) which occur in crystalline form (cry toxins). It is indeed
known in the field of agriculture that biologicals, and in
particular proteins, are challenging structures for developing
pesticides, as they generally have far too little stability to
maintain their pesticidal function in an agrochemical formulation,
in particular for applications in the field.
SUMMARY OF THE INVENTION
[0003] The present inventors have successfully developed
polypeptides with surprisingly high specificity, affinity and
potency against targets of plant or crop pests, in particular plant
pathogenic pests, such as but not limited to plant pathogenic
fungi. Moreover, it is shown that these polypeptides retain their
integrity, stability and activity in an agrochemical composition
(as further defined herein) and that efficacious pest or pathogenic
control can surprisingly be achieved by applying agrochemical
compositions, comprising the polypeptides as disclosed in the
present application, to crops.
[0004] The efficacy and potency of the polypeptides as disclosed
herein suggests a potential for either a lower treatment dosage
and/or a more effective treatment at the same dose. This can imply
a reduction of unwanted side-effects and reduced toxicity.
Moreover, this allows the application of lower amounts of the
polypeptides or agrochemical compositions disclosed herein per
hectare.
[0005] More particularly, the present inventors have found that
targeting a molecular structure of a plant pathogen with the
polypeptides envisaged herein allows for efficient control of that
pathogen when applied directly or indirectly on a plant or on one
or more parts of a plant.
[0006] In particular, the present inventors have developed
polypeptides or amino acid sequences that are capable of
preventing, protecting, treating or curing a plant from
(developing) an infection by a plant pathogen or from any other
biological interaction with a plant pathogen. Therefore, the
present invention demonstrates for the first time that biological
molecules, such as polypeptides or amino acid sequences, can be
used to effectively protect or treat a plant, from being damaged in
any way by or suffering from a biological interaction between the
plant and a plant pathogen, such as for instance through a plant
pathogen infection.
[0007] In a first aspect, the present invention provides
agrochemical compositions comprising at least one heavy chain
variable domain of an antibody (a V.sub.HH or a V.sub.H) or a
functional fragment thereof, which specifically binds to a
sphingolipid of a plant pathogen.
[0008] In particular embodiments, the agrochemical compositions as
disclosed herein, comprise at least one a heavy chain variable
domain of a heavy chain antibody (V.sub.HH), which is naturally
devoid of light chains or a functional fragment thereof, such as
but not limited to a heavy chain variable domain of a camelid heavy
chain antibody (camelid V.sub.HH) or a functional fragment
thereof.
[0009] In particular embodiments, the agrochemical compositions as
disclosed herein, comprise at least one camelized heavy chain
variable domain of a conventional four-chain antibody (camelized
V.sub.H), or a functional fragment thereof.
[0010] In certain particular embodiments, the agrochemical
compositions as disclosed herein, comprise at least one heavy chain
variable domain of an antibody or a functional fragment thereof,
which do not have an amino acid sequence that is exactly the same
as (i.e. as in a degree of sequence identity of 100% with) the
amino acid sequence of a naturally occurring V.sub.H domain, such
as the amino acid sequence of a naturally occurring V.sub.H domain
from a mammal, and in particular from a human being.
[0011] In further particular embodiments, the agrochemical
compositions as disclosed herein at least comprise a heavy chain
variable domain of an antibody or a functional fragment thereof,
which specifically binds to a sphingolipid of a plant pathogen,
such as for instance but not limited to glucosylceramide.
[0012] In certain particular embodiments, the agrochemical
compositions as disclosed herein at least comprise a heavy chain
variable domain of an antibody or a functional fragment thereof,
which specifically binds to a plant pathogenic fungus, such as but
not limited to a plant pathogenic fungus of a genus chosen from the
group comprising Alternaria, Ascochyta, Botrytis, Cercospora,
Colletotrichum, Diplodia, Erysiphe, Fusarium, Leptosphaeria,
Gaeumanomyces, Helminthosporium, Macrophomina, Nectria,
Penicillium, Peronospora, Phoma, Phymatotrichum, Phytophthora,
Plasmopara, Podosphaera, Puccinia, Pyrenophora, Pyricularia,
Pythium, Rhizoctonia, Scerotium, Sclerotinia, Septoria,
Thielaviopsis, Uncinula, Venturia, Verticillium, Magnaporthe,
Blumeria, Mycosphaerella, Ustilago, Melampsora, Phakospora,
Monilinia, Mucor, Rhizopus, and Aspergillus.
[0013] In particular embodiments, the agrochemical compositions as
disclosed herein at least comprise a heavy chain variable domain of
an antibody or a functional fragment thereof, which specifically
binds to a plant pathogen, which is a plant pathogen for a plant
chosen from the group comprising cereals, sorghum, rice, sugar
beet, fodder beet, fruit, nuts, the plantain family or grapevines,
leguminous crops, oil crops, cucurbits, fibre plants, fuel crops,
vegetables, ornamentals, shrubs, broad-leaved trees, evergreens,
grasses, coffee, tea, tobacco, hops, pepper, rubber and latex
plants.
[0014] In certain specific embodiments, the at least one heavy
chain variable domain of an antibody or functional fragment thereof
in the agrochemical compositions disclosed herein is present in an
amount effective to protect or treat a plant or a part of the plant
from an infection or other biological interaction with the plant
pathogen, such as for example but not limited to the concentration
of the at least one heavy chain variable domain in the agrochemical
composition ranging from 0.0001% to 50% by weight.
[0015] In further particular embodiments, the at least one heavy
chain variable domain of an antibody or functional fragment thereof
in the agrochemical compositions disclosed herein is formulated in
an aqueous solution, optionally but without limitation together
with an agrochemically suitable carrier and/or one or more suitable
adjuvants.
[0016] In still further particular embodiments, the at least one
heavy chain variable domain of an antibody or functional fragment
thereof in the agrochemical compositions disclosed herein, at least
comprises:
a CDR1 region having SEQ ID NO: 85, a CDR2 region having has SEQ ID
NO: 169, and a CDR3 region having SEQ ID NO: 253, or a CDR1 region
having SEQ ID NO: 86, a CDR2 region having has SEQ ID NO: 170, and
a CDR3 region having SEQ ID NO: 254, or a CDR1 region having SEQ ID
NO: 87, a CDR2 region having has SEQ ID NO: 171, and a CDR3 region
having SEQ ID NO: 255, or a CDR1 region having SEQ ID NO: 88, a
CDR2 region having has SEQ ID NO: 172, and a CDR3 region having SEQ
ID NO: 256, or a CDR1 region having SEQ ID NO: 89, a CDR2 region
having has SEQ ID NO: 173, and a CDR3 region having SEQ ID NO: 257,
or a CDR1 region having SEQ ID NO: 90, a CDR2 region having has SEQ
ID NO: 174, and a CDR3 region having SEQ ID NO: 258, or a CDR1
region having SEQ ID NO: 91, a CDR2 region having has SEQ ID NO:
175, and a CDR3 region having SEQ ID NO: 259, or a CDR1 region
having SEQ ID NO: 92, a CDR2 region having has SEQ ID NO: 176, and
a CDR3 region having SEQ ID NO: 260, or a CDR1 region having SEQ ID
NO: 93, a CDR2 region having has SEQ ID NO: 177, and a CDR3 region
having SEQ ID NO: 261, or a CDR1 region having SEQ ID NO: 94, a
CDR2 region having has SEQ ID NO: 178, and a CDR3 region having SEQ
ID NO: 262, or a CDR1 region having SEQ ID NO: 95, a CDR2 region
having has SEQ ID NO: 179, and a CDR3 region having SEQ ID NO: 263,
or a CDR1 region having SEQ ID NO: 96, a CDR2 region having has SEQ
ID NO: 180, and a CDR3 region having SEQ ID NO: 264, or a CDR1
region having SEQ ID NO: 97, a CDR2 region having has SEQ ID NO:
181, and a CDR3 region having SEQ ID NO: 265, or a CDR1 region
having SEQ ID NO: 98, a CDR2 region having has SEQ ID NO: 182, and
a CDR3 region having SEQ ID NO: 266, or a CDR1 region having SEQ ID
NO: 99, a CDR2 region having has SEQ ID NO: 183, and a CDR3 region
having SEQ ID NO: 267, or a CDR1 region having SEQ ID NO: 100, a
CDR2 region having has SEQ ID NO: 184, and a CDR3 region having SEQ
ID NO: 268, or a CDR1 region having SEQ ID NO: 101, a CDR2 region
having has SEQ ID NO: 185, and a CDR3 region having SEQ ID NO: 269,
or a CDR1 region having SEQ ID NO: 102, a CDR2 region having has
SEQ ID NO: 186, and a CDR3 region having SEQ ID NO: 270, or a CDR1
region having SEQ ID NO: 103, a CDR2 region having has SEQ ID NO:
187, and a CDR3 region having SEQ ID NO: 271, or a CDR1 region
having SEQ ID NO: 104, a CDR2 region having has SEQ ID NO: 188, and
a CDR3 region having SEQ ID NO: 272, or a CDR1 region having SEQ ID
NO: 105, a CDR2 region having has SEQ ID NO: 189, and a CDR3 region
having SEQ ID NO: 273, or a CDR1 region having SEQ ID NO: 106, a
CDR2 region having has SEQ ID NO: 190, and a CDR3 region having SEQ
ID NO: 274, or a CDR1 region having SEQ ID NO: 107, a CDR2 region
having has SEQ ID NO: 191, and a CDR3 region having SEQ ID NO: 275,
or a CDR1 region having SEQ ID NO: 108, a CDR2 region having has
SEQ ID NO: 192, and a CDR3 region having SEQ ID NO: 276, or a CDR1
region having SEQ ID NO: 109, a CDR2 region having has SEQ ID NO:
193, and a CDR3 region having SEQ ID NO: 277, or a CDR1 region
having SEQ ID NO: 110, a CDR2 region having has SEQ ID NO: 194, and
a CDR3 region having SEQ ID NO: 278, or a CDR1 region having SEQ ID
NO: 111, a CDR2 region having has SEQ ID NO: 195, and a CDR3 region
having SEQ ID NO: 279, or a CDR1 region having SEQ ID NO: 112, a
CDR2 region having has SEQ ID NO: 196, and a CDR3 region having SEQ
ID NO: 280, or a CDR1 region having SEQ ID NO: 113, a CDR2 region
having has SEQ ID NO: 197, and a CDR3 region having SEQ ID NO: 281,
or a CDR1 region having SEQ ID NO: 114, a CDR2 region having has
SEQ ID NO: 198, and a CDR3 region having SEQ ID NO: 282, or a CDR1
region having SEQ ID NO: 115, a CDR2 region having has SEQ ID NO:
199, and a CDR3 region having SEQ ID NO: 283, or a CDR1 region
having SEQ ID NO: 116, a CDR2 region having has SEQ ID NO: 200, and
a CDR3 region having SEQ ID NO: 284, or a CDR1 region having SEQ ID
NO: 117, a CDR2 region having has SEQ ID NO: 201, and a CDR3 region
having SEQ ID NO: 285, or a CDR1 region having SEQ ID NO: 118, a
CDR2 region having has SEQ ID NO: 202, and a CDR3 region having SEQ
ID NO: 286, or a CDR1 region having SEQ ID NO: 119, a CDR2 region
having has SEQ ID NO: 203, and a CDR3 region having SEQ ID NO: 287,
or a CDR1 region having SEQ ID NO: 120, a CDR2 region having has
SEQ ID NO: 204, and a CDR3 region having SEQ ID NO: 288, or a CDR1
region having SEQ ID NO: 121, a CDR2 region having has SEQ ID NO:
205, and a CDR3 region having SEQ ID NO: 289, or a CDR1 region
having SEQ ID NO: 122, a CDR2 region having has SEQ ID NO: 206, and
a CDR3 region having SEQ ID NO: 290, or a CDR1 region having SEQ ID
NO: 123, a CDR2 region having has SEQ ID NO: 207, and a CDR3 region
having SEQ ID NO: 291, or a CDR1 region having SEQ ID NO: 124, a
CDR2 region having has SEQ ID NO: 208, and a CDR3 region having SEQ
ID NO: 292, or a CDR1 region having SEQ ID NO: 125, a CDR2 region
having has SEQ ID NO: 209, and a CDR3 region having SEQ ID NO: 293,
or a CDR1 region having SEQ ID NO: 126, a CDR2 region having has
SEQ ID NO: 210, and a CDR3 region having SEQ ID NO: 294, or a CDR1
region having SEQ ID NO: 127, a CDR2 region having has SEQ ID NO:
211, and a CDR3 region having SEQ ID NO: 295, or a CDR1 region
having SEQ ID NO: 128, a CDR2 region having has SEQ ID NO: 212, and
a CDR3 region having SEQ ID NO: 296, or a CDR1 region having SEQ ID
NO: 129, a CDR2 region having has SEQ ID NO: 213, and a CDR3 region
having SEQ ID NO: 297, or a CDR1 region having SEQ ID NO: 130, a
CDR2 region having has SEQ ID NO: 214, and a CDR3 region having SEQ
ID NO: 298, or a CDR1 region having SEQ ID NO: 131, a CDR2 region
having has SEQ ID NO: 215, and a CDR3 region having SEQ ID NO: 299,
or a CDR1 region having SEQ ID NO: 132, a CDR2 region having has
SEQ ID NO: 216, and a CDR3 region having SEQ ID NO: 300, or a CDR1
region having SEQ ID NO: 133, a CDR2 region having has SEQ ID NO:
217, and a CDR3 region having SEQ ID NO: 301, or a CDR1 region
having SEQ ID NO: 134, a CDR2 region having has SEQ ID NO: 218, and
a CDR3 region having SEQ ID NO: 302, or a CDR1 region having SEQ ID
NO: 135, a CDR2 region having has SEQ ID NO: 219, and a CDR3 region
having SEQ ID NO: 303, or a CDR1 region having SEQ ID NO: 136, a
CDR2 region having has SEQ ID NO: 220, and a CDR3 region having SEQ
ID NO: 304, or a CDR1 region having SEQ ID NO: 137, a CDR2 region
having has SEQ ID NO: 221, and a CDR3 region having SEQ ID NO: 305,
or a CDR1 region having SEQ ID NO: 138, a CDR2 region having has
SEQ ID NO: 222, and a CDR3 region having the amino acid sequence
NRY, or a CDR1 region having SEQ ID NO: 139, a CDR2 region having
has SEQ ID NO: 223, and a CDR3 region having SEQ ID NO: 306, or a
CDR1 region having SEQ ID NO: 140, a CDR2 region having has SEQ ID
NO: 224, and a CDR3 region having SEQ ID NO: 307, or a CDR1 region
having SEQ ID NO: 141, a CDR2 region having has SEQ ID NO: 225, and
a CDR3 region having SEQ ID NO: 308, or a CDR1 region having SEQ ID
NO: 142, a CDR2 region having has SEQ ID NO: 226, and a CDR3 region
having SEQ ID NO: 309, or a CDR1 region having SEQ ID NO: 143, a
CDR2 region having has SEQ ID NO: 227, and a CDR3 region having SEQ
ID NO: 310, or a CDR1 region having SEQ ID NO: 144, a CDR2 region
having has SEQ ID NO: 228, and a CDR3 region having SEQ ID NO: 311,
or a CDR1 region having SEQ ID NO: 145, a CDR2 region having has
SEQ ID NO: 229, and a CDR3 region having SEQ ID NO: 312, or a CDR1
region having SEQ ID NO: 146, a CDR2 region having has SEQ ID NO:
230, and a CDR3 region having SEQ ID NO: 313, or a CDR1 region
having SEQ ID NO: 147, a CDR2 region having has SEQ ID NO: 231, and
a CDR3 region having SEQ ID NO: 314, or a CDR1 region having SEQ ID
NO: 148, a CDR2 region having has SEQ ID NO: 232, and a CDR3 region
having SEQ ID NO: 315, or a CDR1 region having SEQ ID NO: 149, a
CDR2 region having has SEQ ID NO: 233, and a CDR3 region having SEQ
ID NO: 316, or a CDR1 region having SEQ ID NO: 150, a CDR2 region
having has SEQ ID NO: 234, and a CDR3 region having SEQ ID NO: 317,
or a CDR1 region having SEQ ID NO: 151, a CDR2 region having has
SEQ ID NO: 235, and a CDR3 region having SEQ ID NO: 318, or a CDR1
region having SEQ ID NO: 152, a CDR2 region having has SEQ ID NO:
236, and a CDR3 region having SEQ ID NO: 319, or a CDR1 region
having SEQ ID NO: 153, a CDR2 region having has SEQ ID NO: 237, and
a CDR3 region having SEQ ID NO: 320, or a CDR1 region having SEQ ID
NO: 154, a CDR2 region having has SEQ ID NO: 238, and a CDR3 region
having SEQ ID NO: 321, or a CDR1 region having SEQ ID NO: 155, a
CDR2 region having has SEQ ID NO: 239, and a CDR3 region having SEQ
ID NO: 322, or a CDR1 region having SEQ ID NO: 156, a CDR2 region
having has SEQ ID NO: 240, and a CDR3 region having SEQ ID NO: 323,
or a CDR1 region having SEQ ID NO: 157, a CDR2 region having has
SEQ ID NO: 241, and a CDR3 region having SEQ ID NO: 324, or a CDR1
region having SEQ ID NO: 158, a CDR2 region having has SEQ ID NO:
242, and a CDR3 region having SEQ ID NO: 325, or a CDR1 region
having SEQ ID NO: 159, a CDR2 region having has SEQ ID NO: 243, and
a CDR3 region having SEQ ID NO: 326, or a CDR1 region having SEQ ID
NO: 160, a CDR2 region having has SEQ ID NO: 244, and a CDR3 region
having SEQ ID NO: 327, or a CDR1 region having SEQ ID NO: 161, a
CDR2 region having has SEQ ID NO: 245, and a CDR3 region having SEQ
ID NO: 328, or a CDR1 region having SEQ ID NO: 162, a CDR2 region
having has SEQ ID NO: 246, and a CDR3 region having SEQ ID NO: 329,
or a CDR1 region having SEQ ID NO: 163, a CDR2 region having has
SEQ ID NO: 247, and a CDR3 region having SEQ ID NO: 330, or a CDR1
region having SEQ ID NO: 164, a CDR2 region having has SEQ ID NO:
248, and a CDR3 region having SEQ ID NO: 331, or a CDR1 region
having SEQ ID NO: 165, a CDR2 region having has SEQ ID NO: 249, and
a CDR3 region having SEQ ID NO: 332, or a CDR1 region having SEQ ID
NO: 166, a CDR2 region having has SEQ ID NO: 250, and a CDR3 region
having SEQ ID NO: 333, or a CDR1 region having SEQ ID NO: 167, a
CDR2 region having has SEQ ID NO: 251, and a CDR3 region having SEQ
ID NO: 334, or a CDR1 region having SEQ ID NO: 168, a CDR2 region
having has SEQ ID NO: 252, and a CDR3 region having SEQ ID NO:
335.
[0017] In further embodiments, the at least one heavy chain
variable domain of an antibody or functional fragment thereof in
the agrochemical compositions disclosed herein, at least comprises
an amino acid sequence having a sequence chosen from any one of SEQ
ID NO's: 1 to 84.
[0018] In a further aspect, the present invention provides methods
for protecting or treating a plant or a part of a plant from an
infection or other biological interaction with a plant pathogen,
wherein the methods at least comprise the step of applying directly
or indirectly to the plant or to a part of the plant, an
agrochemical composition as disclosed herein, under conditions
effective to protect or treat the plant or a part of the plant
against infection or biological interaction with the plant
pathogen.
[0019] In particular embodiments, these methods comprise applying
directly or indirectly to the plant or to a part of the plant an
agrochemical composition as disclosed herein at an application rate
higher than 50 g of the agrochemical composition per hectare, such
as but not limited to an application rate higher than 75 g of the
agrochemical composition per hectare, such as an application rate
higher than 100 g of the agrochemical composition per hectare, or
in particular an application rate higher than 200 g of the
agrochemical composition per hectare.
[0020] In particular embodiments, these methods comprise applying
directly or indirectly to the plant or to a part of the plant an
agrochemical composition as disclosed herein at an application rate
between 50 g and 100 g of the agrochemical composition per hectare,
such as but not limited to an application rate of between 50 g and
200 g of the agrochemical composition per hectare, in particular an
application rate of between 75 g and 175 g of the agrochemical
composition per hectare, such as between 75 g and 150 g of the
agrochemical composition per hectare or between 75 g and 125 g per
hectare.
[0021] In particular embodiments, the agrochemical compositions as
disclosed herein are directly or indirectly applied to the plant or
to a part of the plant by spraying, atomizing, foaming, fogging,
culturing in hydroculture, culturing in hydroponics, coating,
submerging, and/or encrusting, optionally post-harvest.
[0022] In still a further aspect, the present invention provides
post-harvest treatment methods for protecting or treating a
harvested plant or a harvested part of the plant from an infection
or other biological interaction with a plant pathogen, at least
comprising the step of applying directly or indirectly to the
harvested plant or to a harvested part of the plant, an
agrochemical composition as disclosed herein, under conditions
effective to protect or treat the harvested plant or a harvested
part of the plant against infection or biological interaction with
the plant pathogen.
[0023] In yet a further aspect, the present invention provides the
use of an agrochemical composition as disclosed herein as an
anti-pest agent. In particular embodiments, the anti-pest agent is
a biostatic agent, a fungistatic agent, a pesticidal agent and/or a
fungicidal agent.
[0024] In yet a further aspect, the present invention provides
methods of inhibiting the growth of a plant pathogen or methods of
killing a plant pathogen, the methods comprising at least the step
of applying directly or indirectly to a plant or to a part of the
plant, an agrochemical composition as disclosed herein.
[0025] In particular embodiments of these methods, the agrochemical
compositions as disclosed herein are directly or indirectly applied
to the plant or to a part of the plant by spraying, atomizing,
foaming, fogging, culturing in hydroculture, culturing in
hydroponics, coating, submerging, and/or encrusting, optionally
post-harvest.
[0026] In yet another aspect, the present invention provides
methods for producing an agrochemical composition as disclosed
herein, the methods at least comprising the steps of: [0027]
obtaining at least one heavy chain variable domain of an antibody
(V.sub.HH or V.sub.H) or a functional fragment thereof, which
specifically binds to a sphingolipid of a plant pathogen, and
[0028] formulating the heavy chain variable domain or functional
fragment thereof in an agrochemical composition.
[0029] In particular embodiments of these methods, the step of
obtaining at least one heavy chain variable domain of an antibody
(V.sub.HH or V.sub.H) or functional fragment thereof, which
specifically binds to a sphingolipid of a plant pathogen
comprises:
(a) expressing a nucleotide sequence encoding a heavy chain
variable domain of an antibody or functional fragment thereof,
which specifically binds to a sphingolipid of a plant pathogen, and
optionally (b) isolating and/or purifying the variable domain or
functional fragment thereof.
[0030] In particular embodiments of these methods, the step of
obtaining at least one heavy chain variable domain of an antibody
or functional fragment thereof (V.sub.HH or V.sub.H), which
specifically binds to a sphingolipid of a plant pathogen comprises:
[0031] a) providing a set, collection or library of V.sub.HH
sequences or V.sub.H sequences or sequences of functional fragments
thereof; [0032] b) screening the set, collection or library of
V.sub.HH sequences or V.sub.H sequences or sequences of functional
fragments thereof for sequences that specifically bind to and/or
have affinity for a sphingolipid of a plant pathogen, and
optionally [0033] c) isolating the V.sub.HH sequences or V.sub.H
sequences or sequences of functional fragments thereof that
specifically bind to and/or have affinity for a sphingolipid of a
plant pathogen.
DETAILED DESCRIPTION OF THE INVENTION
[0034] The present invention will be described with respect to
particular embodiments but the invention is not limited
thereto.
[0035] Statements (features) and embodiments of the polypeptides,
compositions and methods as disclosed herein are set herebelow.
Each of the statements and embodiments as disclosed by the
invention so defined may be combined with any other statement
and/or embodiment unless clearly indicated to the contrary. In
particular, any feature indicated as being preferred or
advantageous may be combined with any other feature or features
indicated as being preferred or advantageous.
[0036] Numbered statements as disclosed in the present application
are:
1. An agrochemical composition for combating plant pests, which
composition comprises at least one polypeptide of between 80 and
200 amino acids as the active substance. 2. An agrochemical
composition for combating plant pests, which composition comprises
at least one polypeptide of between 80 and 200 amino acids as the
active substance, wherein the polypeptide is present in a
concentration of 0.01 to 50% (w/w) of the total weight of the
agrochemical composition. 3. The agrochemical composition according
to statements 1 or 2, wherein the polypeptide is obtained by
affinity selection to a specific plant pest target. 4. The
agrochemical composition according to statement 3, wherein the
polypeptide has an affinity for the target with a dissociation
constant below 10.sup.-6M. 5. The agrochemical composition
according to any of the statements 1 to 4, wherein the polypeptide
comprises 3 CDRs and 4 FRs. 6. The agrochemical composition
according to any of the statements 1 to 5, wherein the polypeptide
is derived from a camelid antibody. 7. The agrochemical composition
according to any of the statements 1 to 6, wherein the polypeptide
is a VHH. 8. The agrochemical composition according to any one of
the statements 1 to 7 wherein the plant pest is a fungal pathogen.
9. A method for combating plant pests, which method comprises
applying the composition according to any of the statements 1 to 8
to a crop at an application rate higher than 50 g per hectare of
the polypeptide comprised in the agrochemical composition. 10. The
method for producing an agrochemical composition according to any
of the statements 1 to 8, comprising formulating a polypeptide of
between 80 and 200 amino acids with pesticidal activity together
with at least one customary agrochemical auxiliary agent. 11. A
polypeptide of between 80 and 200 amino acids, obtained by affinity
selection to a specific plant pest target, which is able to inhibit
the growth and/or the activity of a crop pest at a minimum
inhibitory concentration of about 0.00001 to 1 .mu.M. 12. A nucleic
acid sequence encoding a polypeptide according to statement 11. 13.
A chimeric gene comprising a plant expressible promoter, a nucleic
acid sequence according to statement 12 and a terminator sequence.
14. A recombinant vector comprising a chimeric gene of statement
13. 15. A plant comprising a chimeric gene as defined in statement
14. 16. An agrochemical composition comprising at least one heavy
chain variable domain of an antibody (V.sub.HH or V.sub.H) or a
functional fragment thereof, which specifically binds to a
sphingolipid of a plant pathogen. 17. The agrochemical composition
according to any of the statements 1 to 8, which comprises at least
one heavy chain variable domain of an antibody (V.sub.HH or
V.sub.H) or a functional fragment thereof, which specifically binds
to a sphingolipid of a plant pathogen. 18. The agrochemical
composition according to any of the statements 1 to 8 and 17, which
comprises at least one heavy chain variable domain of a heavy chain
antibody (V.sub.HH) or a functional fragment thereof, which
specifically binds to a sphingolipid of a plant pathogen. 19. The
agrochemical composition according to any of the statements 1 to 8,
17 and 18, which comprises at least one camelid heavy chain
variable domain of a heavy chain antibody (camelid V.sub.HH) or a
functional fragment thereof, which specifically binds to a
sphingolipid of a plant pathogen. 20. The agrochemical composition
according to any of the statements 1 to 8 and 17, which comprises
at least one camelized heavy chain variable domain of a
conventional four-chain antibody (camelized V.sub.H) or a
functional fragment thereof, which specifically binds to a
sphingolipid of a plant pathogen. 21. The agrochemical composition
according to any of the statements 1 to 8 and 17 to 20, wherein the
sphingolipid is a ceramide. 22. The agrochemical composition
according to any of the statements 1 to 8 and 17 to 21, wherein the
sphingolipid is glucosylceramide. 23. The agrochemical composition
according to any of the statements 1 to 8 and 17 to 22, wherein the
plant pathogen is a plant pathogenic fungus. 24. The agrochemical
composition according to any of the statements 1 to 8 and 17 to 23,
wherein the genus of the plant pathogenic fungus is chosen from the
group comprising Alternaria, Ascochyta, Botrytis, Cercospora,
Colletotrichum, Diplodia, Erysiphe, Fusarium, Leptosphaeria,
Gaeumanomyces, Helminthosporium, Macrophomina, Nectria,
Penicillium, Peronospora, Phoma, Phymatotrichum, Phytophthora,
Plasmopara, Podosphaera, Puccinia, Pyrenophora, Pyricularia,
Pythium, Rhizoctonia, Scerotium, Sclerotinia, Septoria,
Thielaviopsis, Uncinula, Venturia, Verticillium, Magnaporthe,
Blumeria, Mycosphaerella, Ustilago, Melampsora, Phakospora,
Monilinia, Mucor, Rhizopus, and Aspergillus. 25. The agrochemical
composition according to any of the statements 1 to 8 and 17 to 24,
wherein the plant pathogen is a plant pathogen for a plant chosen
from the group comprising cereals, sorghum, rice, sugar beet,
fodder beet, fruit, nuts, the plantain family or grapevines,
leguminous crops, oil crops, cucurbits, fibre plants, fuel crops,
vegetables, ornamentals, shrubs, broad-leaved trees, evergreens,
grasses, coffee, tea, tobacco, hops, pepper, rubber and latex
plants. 26. The agrochemical composition according to any of the
statements 1 to 8 and 17 to 25, wherein the at least one heavy
chain variable domain is present in an amount effective to protect
or treat a plant or a part of the plant from an infection or other
biological interaction with the plant pathogen. 27. The
agrochemical composition according to any of the statements 1 to 8
and 17 to 26, wherein the concentration of the at least one heavy
chain variable domain in the agrochemical composition ranges from
0.0001% to 50% by weight. 28. The agrochemical composition
according to any of the statements 1 to 8 and 17 to 27, wherein the
at least one heavy chain variable domain is formulated in an
aqueous solution. 29. The agrochemical composition according to any
of the statements 1 to 8 and 17 to 28, which further comprises an
agrochemically suitable carrier and/or one or more suitable
adjuvants. 30. The agrochemical composition according to any of the
statements 1 to 8 and 17 to 29, wherein the at least one heavy
chain variable domain of an antibody at least comprises a CDR1
region having SEQ ID NO: 85, a CDR2 region having has SEQ ID NO:
169, and a CDR3 region having SEQ ID NO: 253, and/or a CDR1 region
having SEQ ID NO: 86, a CDR2 region having has SEQ ID NO: 170, and
a CDR3 region having SEQ ID NO: 254, and/or a CDR1 region having
SEQ ID NO: 87, a CDR2 region having has SEQ ID NO: 171, and a CDR3
region having SEQ ID NO: 255, and/or a CDR1 region having SEQ ID
NO: 88, a CDR2 region having has SEQ ID NO: 172, and a CDR3 region
having SEQ ID NO: 256, and/or a CDR1 region having SEQ ID NO: 89, a
CDR2 region having has SEQ ID NO: 173, and a CDR3 region having SEQ
ID NO: 257, and/or a CDR1 region having SEQ ID NO: 90, a CDR2
region having has SEQ ID NO: 174, and a CDR3 region having SEQ ID
NO: 258, and/or a CDR1 region having SEQ ID NO: 91, a CDR2 region
having has SEQ ID NO: 175, and a CDR3 region having SEQ ID NO: 259,
and/or a CDR1 region having SEQ ID NO: 92, a CDR2 region having has
SEQ ID NO: 176, and a CDR3 region having SEQ ID NO: 260, and/or a
CDR1 region having SEQ ID NO: 93, a CDR2 region having has SEQ ID
NO: 177, and a CDR3 region having SEQ ID NO: 261, and/or a CDR1
region having SEQ ID NO: 94, a CDR2 region having has SEQ ID NO:
178, and a CDR3 region having SEQ ID NO: 262, and/or a CDR1 region
having SEQ ID NO: 95, a CDR2 region having has SEQ ID NO: 179, and
a CDR3 region having SEQ ID NO: 263, and/or a CDR1 region having
SEQ ID NO: 96, a CDR2 region having has SEQ ID NO: 180, and a CDR3
region having SEQ ID NO: 264, and/or a CDR1 region having SEQ ID
NO: 97, a CDR2 region having has SEQ ID NO: 181, and a CDR3 region
having SEQ ID NO: 265, and/or a CDR1 region having SEQ ID NO: 98, a
CDR2 region having has SEQ ID NO: 182, and a CDR3 region having SEQ
ID NO: 266, and/or a CDR1 region having SEQ ID NO: 99, a CDR2
region having has SEQ ID NO: 183, and a CDR3 region having SEQ ID
NO: 267, and/or a CDR1 region having SEQ ID NO: 100, a CDR2 region
having has SEQ ID NO: 184, and a CDR3 region having SEQ ID NO: 268,
and/or a CDR1 region having SEQ ID NO: 101, a CDR2 region having
has SEQ ID NO: 185, and a CDR3 region having SEQ ID NO: 269, and/or
a CDR1 region having SEQ ID NO: 102, a CDR2 region having has SEQ
ID NO: 186, and a CDR3 region having SEQ ID NO: 270, and/or a CDR1
region having SEQ ID NO: 103, a CDR2 region having has SEQ ID NO:
187, and a CDR3 region having SEQ ID NO: 271, and/or a CDR1 region
having SEQ ID NO: 104, a CDR2 region having has SEQ ID NO: 188, and
a CDR3 region having SEQ ID NO: 272, and/or a CDR1 region having
SEQ ID NO: 105, a CDR2 region having has SEQ ID NO: 189, and a CDR3
region having SEQ ID NO: 273, and/or a CDR1 region having SEQ ID
NO: 106, a CDR2 region having has SEQ ID NO: 190, and a CDR3 region
having SEQ ID NO: 274, and/or a CDR1 region having SEQ ID NO: 107,
a CDR2 region having has SEQ ID NO: 191, and a CDR3 region having
SEQ ID NO: 275, and/or a CDR1 region having SEQ ID NO: 108, a CDR2
region having has SEQ ID NO: 192, and a CDR3 region having SEQ ID
NO: 276, and/or a CDR1 region having SEQ ID NO: 109, a CDR2 region
having has SEQ ID NO: 193, and a CDR3 region having SEQ ID NO: 277,
and/or a CDR1 region having SEQ ID NO: 110, a CDR2 region having
has SEQ ID NO: 194, and a CDR3 region having SEQ ID NO: 278, and/or
a CDR1 region having SEQ ID NO: 111, a CDR2 region having has SEQ
ID NO: 195, and a CDR3 region having SEQ ID NO: 279, and/or a CDR1
region having SEQ ID NO: 112, a CDR2 region having has SEQ ID NO:
196, and a CDR3 region having SEQ ID NO: 280, and/or a CDR1 region
having SEQ ID NO: 113, a CDR2 region having has SEQ ID NO: 197, and
a CDR3 region having SEQ ID NO: 281, and/or a CDR1 region having
SEQ ID NO: 114, a CDR2 region having has SEQ ID NO: 198, and a CDR3
region having SEQ ID NO: 282, and/or a CDR1 region having SEQ ID
NO: 115, a CDR2 region having has SEQ ID NO: 199, and a CDR3 region
having SEQ ID NO: 283, and/or a CDR1 region having SEQ ID NO: 116,
a CDR2 region having has SEQ ID NO: 200, and a CDR3 region having
SEQ ID NO: 284, and/or a CDR1 region having SEQ ID NO: 117, a CDR2
region having has SEQ ID NO: 201, and a CDR3 region having SEQ ID
NO: 285, and/or a CDR1 region having SEQ ID NO: 118, a CDR2 region
having has SEQ ID NO: 202, and a CDR3 region having SEQ ID NO: 286,
and/or a CDR1 region having SEQ ID NO: 119, a CDR2 region having
has SEQ ID NO: 203, and a CDR3 region having SEQ ID NO: 287, and/or
a CDR1 region having SEQ ID NO: 120, a CDR2 region having has SEQ
ID NO: 204, and a CDR3 region having SEQ ID NO: 288, and/or a CDR1
region having SEQ ID NO: 121, a CDR2 region having has SEQ ID NO:
205, and a CDR3 region having SEQ ID NO: 289, and/or a CDR1 region
having SEQ ID NO: 122, a CDR2 region having has SEQ ID NO: 206, and
a CDR3 region having SEQ ID NO: 290, and/or a CDR1 region having
SEQ ID NO: 123, a CDR2 region having has SEQ ID NO: 207, and a CDR3
region having SEQ ID NO: 291, and/or a CDR1 region having SEQ ID
NO: 124, a CDR2 region having has SEQ ID NO: 208, and a CDR3 region
having SEQ ID NO: 292, and/or a CDR1 region having SEQ ID NO: 125,
a CDR2 region having has SEQ ID NO: 209, and a CDR3 region having
SEQ ID NO: 293, and/or a CDR1 region having SEQ ID NO: 126, a CDR2
region having has SEQ ID NO: 210, and a CDR3 region having SEQ ID
NO: 294, and/or a CDR1 region having SEQ ID NO: 127, a CDR2 region
having has SEQ ID NO: 211, and a CDR3 region having SEQ ID NO: 295,
and/or a CDR1 region having SEQ ID NO: 128, a CDR2 region having
has SEQ ID NO: 212, and a CDR3 region having SEQ ID NO: 296, and/or
a CDR1 region having SEQ ID NO: 129, a CDR2 region having has SEQ
ID NO: 213, and a CDR3 region having SEQ ID NO: 297, and/or a CDR1
region having SEQ ID NO: 130, a CDR2 region having has SEQ ID NO:
214, and a CDR3 region having SEQ ID NO: 298, and/or a CDR1 region
having SEQ ID NO: 131, a CDR2 region having has SEQ ID NO: 215, and
a CDR3 region having SEQ ID NO: 299, and/or a CDR1 region having
SEQ ID NO: 132, a CDR2 region having has SEQ ID NO: 216, and a CDR3
region having SEQ ID NO: 300, and/or a CDR1 region having SEQ ID
NO: 133, a CDR2 region having has SEQ ID NO: 217, and a CDR3 region
having SEQ ID NO: 301, and/or a CDR1 region having SEQ ID NO: 134,
a CDR2 region having has SEQ ID NO: 218, and a CDR3 region having
SEQ ID NO: 302, and/or a CDR1 region having SEQ ID NO: 135, a CDR2
region having has SEQ ID NO: 219, and a CDR3 region having SEQ ID
NO: 303, and/or a CDR1 region having SEQ ID NO: 136, a CDR2 region
having has SEQ ID NO: 220, and a CDR3 region having SEQ ID NO: 304,
and/or a CDR1 region having SEQ ID NO: 137, a CDR2 region having
has SEQ ID NO: 221, and a CDR3 region having SEQ ID NO: 305, and/or
a CDR1 region having SEQ ID NO: 138, a CDR2 region having has SEQ
ID NO: 222, and a CDR3 region having the amino acid sequence NRY,
and/or a CDR1 region having SEQ ID NO: 139, a CDR2 region having
has SEQ ID NO: 223, and a CDR3 region having SEQ ID NO: 306, and/or
a CDR1 region having SEQ ID NO: 140, a CDR2 region having has SEQ
ID NO: 224, and a CDR3 region having SEQ ID NO: 307, and/or a CDR1
region having SEQ ID NO: 141, a CDR2 region having has SEQ ID NO:
225, and a CDR3 region having SEQ ID NO: 308, and/or a CDR1 region
having SEQ ID NO: 142, a CDR2 region having has SEQ ID NO: 226, and
a CDR3 region having SEQ ID NO: 309, and/or a CDR1 region having
SEQ ID NO: 143, a CDR2 region having has SEQ ID NO: 227, and a CDR3
region having SEQ ID NO: 310, and/or a CDR1 region having SEQ ID
NO: 144, a CDR2 region having has SEQ ID NO: 228, and a CDR3 region
having SEQ ID NO: 311, and/or a CDR1 region having SEQ ID NO: 145,
a CDR2 region having has SEQ ID NO: 229, and a CDR3 region having
SEQ ID NO: 312, and/or a CDR1 region having SEQ ID NO: 146, a CDR2
region having has SEQ ID NO: 230, and a CDR3 region having SEQ ID
NO: 313, and/or a CDR1 region having SEQ ID NO: 147, a CDR2 region
having has SEQ ID NO: 231, and a CDR3 region having SEQ ID NO: 314,
and/or a CDR1 region having SEQ ID NO: 148, a CDR2 region having
has SEQ ID NO: 232, and a CDR3 region having SEQ ID NO: 315, and/or
a CDR1 region having SEQ ID NO: 149, a CDR2 region having has SEQ
ID NO: 233, and a CDR3 region having SEQ ID NO: 316, and/or a CDR1
region having SEQ ID NO: 150, a CDR2 region having has SEQ ID NO:
234, and a CDR3 region having SEQ ID NO: 317, and/or a CDR1 region
having SEQ ID NO: 151, a CDR2 region having has SEQ ID NO: 235, and
a CDR3 region having SEQ ID NO: 318, and/or a CDR1 region having
SEQ ID NO: 152, a CDR2 region having has SEQ ID NO: 236, and a CDR3
region having SEQ ID NO: 319, and/or a CDR1 region having SEQ ID
NO: 153, a CDR2 region having has SEQ ID NO: 237, and a CDR3 region
having SEQ ID NO: 320, and/or
a CDR1 region having SEQ ID NO: 154, a CDR2 region having has SEQ
ID NO: 238, and a CDR3 region having SEQ ID NO: 321, and/or a CDR1
region having SEQ ID NO: 155, a CDR2 region having has SEQ ID NO:
239, and a CDR3 region having SEQ ID NO: 322, and/or a CDR1 region
having SEQ ID NO: 156, a CDR2 region having has SEQ ID NO: 240, and
a CDR3 region having SEQ ID NO: 323, and/or a CDR1 region having
SEQ ID NO: 157, a CDR2 region having has SEQ ID NO: 241, and a CDR3
region having SEQ ID NO: 324, and/or a CDR1 region having SEQ ID
NO: 158, a CDR2 region having has SEQ ID NO: 242, and a CDR3 region
having SEQ ID NO: 325, and/or a CDR1 region having SEQ ID NO: 159,
a CDR2 region having has SEQ ID NO: 243, and a CDR3 region having
SEQ ID NO: 326, and/or a CDR1 region having SEQ ID NO: 160, a CDR2
region having has SEQ ID NO: 244, and a CDR3 region having SEQ ID
NO: 327, and/or a CDR1 region having SEQ ID NO: 161, a CDR2 region
having has SEQ ID NO: 245, and a CDR3 region having SEQ ID NO: 328,
and/or a CDR1 region having SEQ ID NO: 162, a CDR2 region having
has SEQ ID NO: 246, and a CDR3 region having SEQ ID NO: 329, and/or
a CDR1 region having SEQ ID NO: 163, a CDR2 region having has SEQ
ID NO: 247, and a CDR3 region having SEQ ID NO: 330, and/or a CDR1
region having SEQ ID NO: 164, a CDR2 region having has SEQ ID NO:
248, and a CDR3 region having SEQ ID NO: 331, and/or a CDR1 region
having SEQ ID NO: 165, a CDR2 region having has SEQ ID NO: 249, and
a CDR3 region having SEQ ID NO: 332, and/or a CDR1 region having
SEQ ID NO: 166, a CDR2 region having has SEQ ID NO: 250, and a CDR3
region having SEQ ID NO: 333, and/or a CDR1 region having SEQ ID
NO: 167, a CDR2 region having has SEQ ID NO: 251, and a CDR3 region
having SEQ ID NO: 334, and/or a CDR1 region having SEQ ID NO: 168,
a CDR2 region having has SEQ ID NO: 252, and a CDR3 region having
SEQ ID NO: 335. 31. The agrochemical composition according to any
of the statements 1 to 8 and 17 to 30, wherein the at least one
heavy chain variable domain comprises at least one amino acid
sequence chosen from the group comprising SEQ ID NO's: 1 to 84. 32.
A method for protecting or treating a plant or a part of the plant
from an infection or other biological interaction with a plant
pathogen, at least comprising the step of applying directly or
indirectly to the plant or to a part of the plant, an agrochemical
composition according to any of the statements 1 to 8 and 17 to 31,
under conditions effective to protect or treat the plant or a part
of the plant against the infection or biological interaction with
the plant pathogen. 33. A method according to statement 9 for
protecting or treating a plant or a part of the plant from an
infection or other biological interaction with a plant pathogen, at
least comprising the step of applying directly or indirectly to the
plant or to a part of the plant, an agrochemical composition
according to any of the statements 1 to 8 and 17 to 31, under
conditions effective to protect or treat the plant or a part of the
plant against the infection or biological interaction with the
plant pathogen. 34. The method according to any of the statements
9, 32 or 33, comprising applying directly or indirectly to the
plant or to a part of the plant an agrochemical composition
according to any one of statements 1 to 8 and 17 to 31 at an
application rate higher than 50 g of the agrochemical composition
per hectare. 35. The method according to any of the statements 9 or
32 to 34, wherein the agrochemical composition is directly or
indirectly applied to the plant or to a part of the plant by
spraying, atomizing, foaming, fogging, culturing in hydroculture,
culturing in hydroponics, coating, submerging, and/or encrusting.
36. The method according to any of the statements 9 or 32 to 35,
wherein the agrochemical composition is directly or indirectly
applied to the plant or to a part of the plant, optionally
post-harvest. 37. A post-harvest treatment method for protecting or
treating a harvested plant or a harvested part of the plant from an
infection or other biological interaction with a plant pathogen, at
least comprising the step of applying directly or indirectly to the
harvested plant or to a harvested part of the plant, an
agrochemical composition according to any one of statements 1 to 8
and 17 to 31, under conditions effective to protect or treat the
harvested plant or a harvested part of the plant against the
infection or biological interaction with the plant pathogen. 38.
Use of an agrochemical composition according to any one of
statements 1 to 8 and 17 to 31 as an anti-pest agent. 39. The use
according to statement 38, wherein the anti-pest agent is a
biostatic agent. 40. The use according to statements 38 or 39,
wherein the anti-pest agent is a fungistatic agent. 41. The use
according to statement 38, wherein the anti-pest agent is a
pesticidal agent. 42. The use according to statements 38 or 41,
wherein the anti-pest agent is a fungicidal agent. 43. A method of
inhibiting the growth of a plant pathogen, comprising at least the
step of applying directly or indirectly to a plant or to a part of
the plant, an agrochemical composition according to any one of
statements 1 to 8 and 17 to 31. 44. A method of killing a plant
pathogen, comprising at least the step of applying directly or
indirectly to a plant or to a part of the plant, an agrochemical
composition according to any one of statements 1 to 8 and 17 to 31.
45. The method according to statements 43 or 44, wherein the
agrochemical composition is directly or indirectly applied to the
plant or to a part of the plant by spraying, atomizing, foaming,
fogging, culturing in hydroculture, culturing in hydroponics,
coating, submerging, and/or encrusting. 46. The method according to
any one of statements 43 to 45, wherein the agrochemical
composition is directly or indirectly applied to the plant or to a
part of the plant, optionally post-harvest. 47. A method for
producing an agrochemical composition according to any one of
statements 1 to 8 and 17 to 31, at least comprising the steps of:
[0037] obtaining at least one heavy chain variable domain of an
antibody (V.sub.HH or V.sub.H) or a functional fragment thereof,
which specifically binds to a sphingolipid of a plant pathogen, and
[0038] formulating the variable domain or functional fragment
thereof in an agrochemical composition according to any one of
statements 1 to 8 and 17 to 31. 48. A method according to statement
10 for producing an agrochemical composition according to any one
of statements 1 to 8 and 17 to 31, at least comprising the steps
of: [0039] obtaining at least one heavy chain variable domain of an
antibody (V.sub.HH or V.sub.H) or a functional fragment thereof,
which specifically binds to a sphingolipid of a plant pathogen, and
[0040] formulating the variable domain or functional fragment
thereof in an agrochemical composition according to any one of
statements 1 to 8 and 17 to 31. 49. The method according to
statements 10, 47 or 48, wherein the step of obtaining at least one
heavy chain variable domain of an antibody or functional fragment
thereof, which specifically binds to a sphingolipid of a plant
pathogen comprises: (a) expressing a nucleotide sequence encoding a
heavy chain variable domain of an antibody (V.sub.HH or V.sub.H) or
functional fragment thereof, which specifically binds to a
sphingolipid of a plant pathogen, and optionally (b) isolating
and/or purifying the variable domain or functional fragment
thereof. 50. The method according to statements 10, 47 or 48,
wherein the step of obtaining at least one heavy chain variable
domain of an antibody or functional fragment thereof, which
specifically binds to a sphingolipid of a plant pathogen comprises:
a) providing a set, collection or library of heavy chain variable
domain sequences or sequences of functional fragments thereof; b)
screening the set, collection or library of heavy chain variable
domain sequences or sequences of functional fragments thereof for
sequences that specifically bind to and/or have affinity for a
sphingolipid of a plant pathogen, and optionally c) isolating the
variable domain sequences or sequences of functional fragments
thereof that specifically bind to and/or have affinity for a
sphingolipid of a plant pathogen.
DEFINITIONS
[0041] The present invention will be described with respect to
particular embodiments but the invention is not limited thereto but
only by the claims. Any reference signs in the claims shall not be
construed as limiting the scope.
[0042] Where the term "comprising" is used in the present
description and claims, it does not exclude other elements or
steps.
[0043] 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.
[0044] The term "about" as used herein when referring to a
measurable value such as a parameter, an amount, a temporal
duration, and the like, is meant to encompass variations of +/-10%
or less, preferably +/-5% or less, more preferably +/-1% or less,
and still more preferably +/-0.1% or less of and from the specified
value, insofar such variations are appropriate to perform in the
disclosed invention. It is to be understood that the value to which
the modifier `about` refers is itself also specifically, and
preferably, disclosed.
[0045] The following terms or definitions are provided solely to
aid in the understanding of the invention. Unless specifically
defined herein, all terms used herein have the same meaning as they
would to one skilled in the art of the present invention.
Practitioners are particularly directed to Sambrook et al.,
Molecular Cloning: A Laboratory Manual, 2.sup.nd ed., Cold Spring
Harbor Press, Plainsview, N.Y. (1989); and Ausubel et al., Current
Protocols in Molecular Biology (Supplement 47), John Wiley &
Sons, New York (1999), for definitions and terms of the art. The
definitions provided herein should not be construed to have a scope
less than understood by a person of ordinary skill in the art.
[0046] Unless indicated otherwise, all methods, steps, techniques
and manipulations that are not specifically described in detail can
be performed and have been performed in a manner known per se, as
will be clear to the skilled person. Reference is for example again
made to the standard handbooks, to the general background art
referred to above and to the further references cited therein.
[0047] As used herein, the terms "polypeptide", "protein",
"peptide", and "amino acid sequence" 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.
[0048] As used herein, amino acid residues will be indicated either
by their full name or according to the standard three-letter or
one-letter amino acid code.
[0049] As used herein, the terms "nucleic acid molecule",
"polynucleotide", "polynucleic acid", "nucleic acid" are used
interchangeably and refer to a polymeric form of nucleotides of any
length, either deoxyribonucleotides or ribonucleotides, or analogs
thereof. Polynucleotides may have any three-dimensional structure,
and may perform any function, known or unknown. Non-limiting
examples of polynucleotides include a gene, a gene fragment, exons,
introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA,
ribozymes, cDNA, recombinant polynucleotides, branched
polynucleotides, plasmids, vectors, isolated DNA of any sequence,
control regions, isolated RNA of any sequence, nucleic acid probes,
and primers. The nucleic acid molecule may be linear or
circular.
[0050] As used herein, the term "homology" denotes at least
secondary structural similarity between two macromolecules,
particularly between two polypeptides or polynucleotides, from same
or different taxons, wherein said similarity is due to shared
ancestry. Hence, the term "homologues" denotes so-related
macromolecules having said secondary and optionally tertiary
structural similarity. For comparing two or more nucleotide
sequences, the `(percentage of) sequence identity` between a first
nucleotide sequence and a second nucleotide sequence may be
calculated using methods known by the person skilled in the art,
e.g. by dividing the number of nucleotides in the first nucleotide
sequence that are identical to the nucleotides at the corresponding
positions in the second nucleotide sequence by the total number of
nucleotides in the first nucleotide sequence and multiplying by
100% or by using a known computer algorithm for sequence alignment
such as NCBI Blast. In determining the degree of sequence identity
between two amino acid sequences, the skilled person may take into
account so-called `conservative` amino acid substitutions, which
can generally be described as amino acid substitutions in which an
amino acid residue is replaced with another amino acid residue of
similar chemical structure and which has little or essentially no
influence on the function, activity or other biological properties
of the polypeptide. Possible conservative amino acid substitutions
will be clear to the person skilled in the art. Amino acid
sequences and nucleic acid sequences are said to be "exactly the
same" if they have 100% sequence identity over their entire
length.
[0051] As used herein, the terms "complementarity determining
region" or "CDR" within the context of antibodies refer to variable
regions of either the H (heavy) or the L (light) chains (also
abbreviated as VH and VL, respectively) and contain 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 3 CDR regions,
each non-contiguous with the others (termed L1, L2, L3, H1, H2, H3)
for the respective light (L) and heavy (H) chains.
[0052] 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.-6 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.
[0053] 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 10 to
100-fold or more (e.g., more than about 1000- or 10,000-fold).
[0054] Accordingly, an amino acid sequence as disclosed herein is
said to "specifically bind to" a particular target when that amino
acid sequence has affinity for, specificity for and/or is
specifically directed against that target (or for at least one part
or fragment thereof).
[0055] The "specificity" of an amino acid sequence as disclosed
herein can be determined based on affinity and/or avidity.
[0056] An amino acid sequence as disclosed herein is said to be
"specific for a first target antigen of interest as opposed to a
second target antigen of interest" when it binds to the first
target antigen of interest with an affinity that is at least 5
times, such as at least 10 times, such as at least 100 times, and
preferably at least 1000 times higher than the affinity with which
that amino acid sequence as disclosed herein binds to the second
target antigen of interest. Accordingly, in certain embodiments,
when an amino acid sequence as disclosed herein is said to be
"specific for" a first target antigen of interest as opposed to a
second target antigen of interest, it may specifically bind to (as
defined herein) the first target antigen of interest, but not to
the second target antigen of interest.
[0057] As used herein, the terms "inhibiting", "reducing" and/or
"preventing" may refer to (the use of) an amino acid sequence as
disclosed herein that specifically binds to a target antigen of
interest and inhibits, reduces and/or prevents the interaction
between that target antigen of interest, and its natural binding
partner. The terms "inhibiting", "reducing" and/or "preventing" may
also refer to (the use of) an amino acid sequence as disclosed
herein that specifically binds to a target antigen of interest and
inhibits, reduces and/or prevents a biological activity of that
target antigen of interest, as measured using a suitable in vitro,
cellular or in vivo assay. Accordingly, "inhibiting", "reducing"
and/or "preventing" may also refer to (the use of) an amino acid
sequence as disclosed herein that specifically binds to a target
antigen of interest and inhibits, reduces and/or prevents one or
more biological or physiological mechanisms, effects, responses,
functions pathways or activities in which the target antigen of
interest is involved. Such an action of the amino acid sequence as
disclosed herein as an antagonist may be determined in any suitable
manner and/or using any suitable (in vitro and usually cellular or
in vivo) assay known in the art, depending on the target antigen of
interest.
[0058] Thus, more particularly, "inhibiting", "reducing" and/or
"preventing" using amino acid sequence as disclosed herein may mean
either inhibiting, reducing and/or preventing the interaction
between a target antigen of interest and its natural binding
partner, or, inhibiting, reducing and/or preventing the activity of
a target antigen of interest, or, inhibiting, reducing and/or
preventing one or more biological or physiological mechanisms,
effects, responses, functions pathways or activities in which the
target antigen of interest is involved, such as by at least 10%,
but preferably at least 20%, for example by at least 50%, at least
60%, at least 70%, at least 80%, at least 90%, at least 95% or
more, as measured using a suitable in vitro, cellular or in vivo
assay, compared to the activity of the target antigen of interest
in the same assay under the same conditions but without using the
amino acid sequence as disclosed herein. In addition, "inhibiting",
"reducing" and/or "preventing" may also mean inducing a decrease in
affinity, avidity, specificity and/or selectivity of a target
antigen of interest for one or more of its natural binding partners
and/or inducing a decrease in the sensitivity of the target antigen
of interest for one or more conditions in the medium or
surroundings in which the target antigen of interest is present
(such as pH, ion strength, the presence of co-factors, etc.),
compared to the same conditions but without the presence of the
amino acid sequence as disclosed herein. In the context of the
present invention, "inhibiting", "reducing" and/or "preventing" may
also involve allosteric inhibition, reduction and/or prevention of
the activity of a target antigen of interest.
[0059] The inhibiting or antagonizing activity or the enhancing or
agonizing activity of an amino acid sequence as disclosed herein
may be reversible or irreversible, but for agrochemical
applications will typically occur reversibly.
[0060] An amino acid sequence as disclosed herein is considered to
be "(in) essentially isolated (form)" as used herein, when it has
been extracted or purified from the host cell and/or medium in
which it is produced.
[0061] In respect of the amino acid sequences as disclosed herein,
the terms "binding region", "binding site" or "interaction site"
present on the amino acid sequences as disclosed herein shall
herein have the meaning of a particular site, region, locus, part,
or domain present on the target molecule, which particular site,
region, locus, part, or domain is responsible for binding to that
target molecule. Such binding region thus essentially consists of
that particular site, region, locus, part, or domain of the target
molecule, which is in contact with the amino acid sequence when
bound to that target molecule.
[0062] "Plant" as used herein, means live plants and live plant
parts, including fresh fruit, vegetables and seeds. Also, the term
"plant" as used herein encompasses whole plants, ancestors and
progeny of the plants and plant parts, including seeds, shoots,
stems, leaves, roots (including tubers), flowers, and tissues and
organs, wherein each of the aforementioned comprise the
gene/nucleic acid of interest. The term "plant" also encompasses
plant cells, suspension cultures, callus tissue, embryos,
meristematic regions, gametophytes, sporophytes, pollen and
microspores, again wherein each of the aforementioned comprises the
gene/nucleic acid of interest.
[0063] The choice of suitable control plants is a routine part of
an experimental setup and may include corresponding wild type
plants or corresponding plants without the gene of interest. The
control plant is typically of the same plant species or even of the
same variety as the plant to be assessed. The control plant may
also be a nullizygote of the plant to be assessed. Nullizygotes are
individuals missing the transgene by segregation. A "control plant"
as used herein refers not only to whole plants, but also to plant
parts, including seeds and seed parts.
[0064] "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, said 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, fibre 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.
[0065] A "pest", as used here, is an organism that is harmful to
plants, animals, humans or human concerns, and includes, but is not
limited to crop pests (as later defined), household pests, such as
cockroaches, ants, etc., and disease vectors, such as malaria
mosquitoes.
[0066] A "plant pest", "plant pathogen" or "crop pest", as used in
the application interchangeably, refers to organisms that
specifically cause damage to plants, plant parts or plant products,
particularly plants, plant parts or plant products, used in
agriculture. Note that the term "plant pest" or "crop pest" is used
in the meaning that the pest targets and harms plants. Pests
particularly belong to invertebrate animals (e.g. insects
(including agricultural pest insects, insect pests of ornamental
plants, insect pests of forests). Relevant crop pest examples
include, but are not limited to, aphids, caterpillars, flies,
wasps, and the like, nematodes (living freely in soil or
particularly species that parasitize plant roots, such as root-knot
nematode and cyst nematodes such as soybean cyst nematode and
potato cyst nematode), mites (such as spider mites, thread-footed
mites and gall mites) and gastropods (including slugs such as
Deroceras spp., Milax spp., Tandonia sp., Limax spp., Arion spp.
and Veronicella spp. and snails such as Helix spp., Cernuella spp.,
Theba spp., Cochlicella spp., Achatina spp., Succinea spp.,
Ovachlamys spp., Amphibulima spp., Zachrysia spp., Bradybaena spp.,
and Pomacea spp.), pathogenic fungi (including Ascomycetes (such as
Fusarium spp., Thielaviopsis spp., Verticillium spp., Magnaporthe
spp.), Basidiomycetes (such as Rhizoctonia spp., Phakospora spp.,
Puccinia spp.), and fungal-like Oomycetes (such as Pythium spp. and
Phytophthora spp.), bacteria (such as Burkholderia spp. and
Proteobacteria such as Xanthomonas spp. and Pseudomonas spp.),
Phytoplasma, Spiroplasma, viruses (such as tobacco mosaic virus and
cauliflower mosaic virus), and protozoa.
[0067] "Microbe", as used herein, means bacterium, virus, fungus,
yeast and the like and "microbial" means derived from a
microbe.
[0068] "Fungus", as used herein, means a eukaryotic organism,
belonging to the group of Eumycota. The term fungus in the present
invention also includes fungal-like organisms such as the Oomycota.
Oomycota (or oomycetes) form a distinct phylogenetic lineage of
fungus-like eukaryotic microorganisms. This group was originally
classified among the fungi but modern insights support a relatively
close relationship with the photosynthetic organisms such as brown
algae and diatoms, within the group of heterokonts.
[0069] "Pest infection" or "pest disease" as used herein refers to
any inflammatory condition, disease or disorder in a living
organism, such as a plant, animal or human, which is caused by a
pest.
[0070] "Fungal infection" or "fungal disease" as used herein refers
to any inflammatory condition, disease or disorder in a living
organism, such as a plant, animal or human, which is caused by a
fungus.
[0071] "Active substance", "active ingredient" or "active
principle", as used interchangeably herein, means any biological,
biochemical or chemical element and its derivatives, fragments or
compounds based thereon, including micro-organisms, having general
or specific action against harmful organisms on a subject, and in
particular on plants, parts of plants or on plant products, as they
occur naturally or by manufacture, including any impurity
inevitably resulting from the manufacturing process.
[0072] "Agrochemical", as used herein, means suitable for use in
the agrochemical industry (including agriculture, horticulture,
floriculture and home and garden uses, but also products intended
for non-crop related uses such as public health/pest control
operator uses to control undesirable insects and rodents, household
uses, such as household fungicides and insecticides and agents, for
protecting plants or parts of plants, crops, bulbs, tubers, fruits
(e.g. from harmful organisms, diseases or pests); for controlling,
preferably promoting or increasing, the growth of plants; and/or
for promoting the yield of plants, crops or the parts of plants
that are harvested (e.g. its fruits, flowers, seeds etc.). Examples
of such substances will be clear to the skilled person and may for
example include compounds that are active as insecticides (e.g.
contact insecticides or systemic insecticides, including
insecticides for household use), herbicides (e.g. contact
herbicides or systemic herbicides, including herbicides for
household use), fungicides (e.g. contact fungicides or systemic
fungicides, including fungicides for household use), nematicides
(e.g. contact nematicides or systemic nematicides, including
nematicides for household use) and other pesticides or biocides
(for example agents for killing insects or snails); as well as
fertilizers; growth regulators such as plant hormones;
micro-nutrients, safeners, pheromones; repellants; insect baits;
and/or active principles that are used to modulate (i.e. increase,
decrease, inhibit, enhance and/or trigger) gene expression (and/or
other biological or biochemical processes) in or by the targeted
plant (e.g. the plant to be protected or the plant to be
controlled), such as nucleic acids (e.g., single stranded or double
stranded RNA, as for example used in the context of RNAi
technology) and other factors, proteins, chemicals, etc. known per
se for this purpose, etc. Examples of such agrochemicals will be
clear to the skilled person; and for example include, without
limitation: glyphosate, paraquat, metolachlor, acetochlor,
mesotrione, 2,4-D,atrazine, glufosinate, sulfosate, fenoxaprop,
pendimethalin, picloram, trifluralin, bromoxynil, clodinafop,
fluroxypyr, nicosulfuron, bensulfuron, imazetapyr, dicamba,
imidacloprid, thiamethoxam, fipronil, chlorpyrifos, deltamethrin,
lambda-cyhalotrin, endosulfan, methamidophos, carbofuran,
clothianidin, cypermethrin, abamectin, diflufenican, spinosad,
indoxacarb, bifenthrin, tefluthrin, azoxystrobin, thiamethoxam,
tebuconazole, mancozeb, cyazofamid, fluazinam, pyraclostrobin,
epoxiconazole, chlorothalonil, copper fungicides, trifloxystrobin,
prothioconazole, difenoconazole, carbendazim, propiconazole,
thiophanate, sulphur, boscalid and other known agrochemicals or any
suitable combination(s) thereof.
[0073] An "agrochemical composition" as used herein means a
composition for agrochemical use, as further defined, comprising at
least one active substance, optionally with one or more additives
favoring optimal dispersion, atomization, deposition, leaf wetting,
distribution, retention and/or uptake of agrochemicals. It will
become clear from the further description herein that an
agrochemical composition as used herein includes biological control
agents or biological pesticides (including but not limited to
biological biocidal, biostatic, fungistatic and fungicidal agents)
and these terms will be interchangeably used in the present
application. Accordingly, an agrochemical composition as used
herein includes compositions comprising at least one biological
molecule as an active ingredient, substance or principle for
controlling pests in plants or in other agro-related settings (such
for example in soil). Non-limiting examples of biological molecules
being used as active principles in the agrochemical compositions
disclosed herein are proteins (including antibodies and fragments
thereof, such as but not limited to heavy chain variable domain
fragments of antibodies, including VHH's), nucleic acid sequences,
(poly-)saccharides, lipids, vitamins, hormones glycolipids,
sterols, and glycerolipids.
[0074] As a non-limiting example, the additives in the agrochemical
compositions disclosed herein may include but are 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.
[0075] A "biostatic composition" or a "biostatic agent" as used
herein means any active ingredient, substance or principle or a
composition comprising any active ingredient, substance or
principle for biostatic use (as further defined herein) comprising
at least one active biostatic substance or ingredient, optionally
combined with one or more additives favoring optimal dispersion,
atomization, deposition, leaf wetting, distribution, retention
and/or uptake of the active substance or ingredient. As a
non-limiting examples such additives are diluents, solvents,
adjuvants, (ionic) surfactants, wetting agents, spreading agents,
oils, stickers, thickeners, penetrants, buffering agents,
acidifiers, anti-settling agents, anti-freeze agents,
photo-protectors, defoaming agents, biocides, protease inhibitors
and/or drift control agents.
[0076] A "biocidal composition" or a "biocidal agent" as used
herein means any active ingredient, substance or principle or a
composition comprising any active ingredient, substance or
principle for biocidal use (as further defined herein) comprising
at least one active biocidal substance or ingredient, optionally
combined with one or more additives favoring optimal dispersion,
atomization, deposition, leaf wetting, distribution, retention
and/or uptake of the active substance or ingredient. As a
non-limiting examples such additives are diluents, solvents,
adjuvants, (ionic) surfactants, wetting agents, spreading agents,
oils, stickers, thickeners, penetrants, buffering agents,
acidifiers, anti-settling agents, anti-freeze agents,
photo-protectors, defoaming agents, biocides, protease inhibitors
and/or drift control agents.
[0077] A "fungistatic composition" or a "fungistatic agent" as used
herein means any active ingredient, substance or principle or a
composition comprising any active ingredient, substance or
principle for fungistatic use (as further defined herein)
comprising at least one active fungistatic substance or ingredient,
optionally combined with one or more additives favoring optimal
dispersion, atomization, deposition, leaf wetting, distribution,
retention and/or uptake of the active substance or ingredient. As a
non-limiting examples such additives are diluents, solvents,
adjuvants, (ionic) surfactants, wetting agents, spreading agents,
oils, stickers, thickeners, penetrants, buffering agents,
acidifiers, anti-settling agents, anti-freeze agents,
photo-protectors, defoaming agents, biocides, protease inhibitors
and/or drift control agents.
[0078] A "fungicidal composition" or a "fungicidal agent" as used
herein means any active ingredient, substance or principle or a
composition comprising any active ingredient, substance or
principle for fungicidal use (as further defined herein) comprising
at least one active fungicidal substance or ingredient, optionally
combined with one or more additives favoring optimal dispersion,
atomization, deposition, leaf wetting, distribution, retention
and/or uptake of the active substance or ingredient. As a
non-limiting examples such additives are diluents, solvents,
adjuvants, (ionic) surfactants, wetting agents, spreading agents,
oils, stickers, thickeners, penetrants, buffering agents,
acidifiers, anti-settling agents, anti-freeze agents,
photo-protectors, defoaming agents, biocides, protease inhibitors
and/or drift control agents.
[0079] "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.).
[0080] "Biostatic (effect)" or "biostatic use", as used herein,
includes any effect or use of an active substance (optionally
comprised in a biostatic, biocidal, fungicidal or fungistatic
composition as defined herein) for controlling, modulating or
interfering with the harmful activity of a pest, such as a plant
pest or a plant pathogen, including but not limited to inhibiting
the growth or activity of the pest, altering the behavior of the
pest, and repelling or attracting the pest in plants, plant parts
or in other agro-related settings, such as for example for
household uses or in soil.
[0081] "Biocidal (effect)" or "biocidal use", as used herein,
includes any effect or use of an active substance (optionally
comprised in a biocidal or fungicidal composition as defined
herein) for controlling, modulating or interfering with the harmful
activity of a pest, such as a plant pest or a plant pathogen,
including but not limited to killing the pest, inhibiting the
growth or activity of the pest, altering the behavior of the pest,
and repelling or attracting the pest in plants, plant parts or in
other agro-related settings, such as for example for household uses
or in soil.
[0082] "Fungistatic (effect)" or "Fungistatic use", as used herein,
includes any effect or use of an active substance (optionally
comprised in a fungicidal or fungistatic composition as defined
herein) for controlling, modulating or interfering with the harmful
activity of a fungus, including but not limited to inhibiting the
growth or activity of the fungus, altering the behavior of the
fungus, and repelling or attracting the fungus in plants, plant
parts or in other agro-related settings, such as for example for
household uses or in soil.
[0083] "Fungicidal (effect)" or "Fungicidal use", as used herein,
includes any effect or use of an active substance (optionally
comprised in a fungicidal composition as defined herein) for
controlling, modulating or interfering with the harmful activity of
a fungus, including but not limited to killing the fungus,
inhibiting the growth or activity of the fungus, altering the
behavior of the fungus, and repelling or attracting the fungus in
plants, plant parts or in other agro-related settings, such as for
example for household uses or in soil.
[0084] "Pesticidal activity" or "biocidal activity", as used
interchangeably herein, means to interfere with the harmful
activity of a pest, including but not limited to killing the pest,
inhibiting the growth or activity of the pest, altering the
behavior of the pest, repelling or attracting the pest.
[0085] "Biostatic activity", as used herein, means to interfere
with the harmful activity of a pest, including but not limited to
inhibiting the growth or activity of the pest, altering the
behavior of the pest, repelling or attracting the pest.
[0086] Pesticidal, biocidal, or biostatic activity of an active
ingredient, substance or principle or a composition or agent
comprising a pesticidal, biocidal, or biostatic active ingredient,
substance or principle, can be expressed as the minimium inhibitory
activity (MIC) of an agent (expressed in units of concentration
such as e.g. mg/mL), without however being restricted thereto.
[0087] "Fungicidal activity", as used herein, means to interfere
with the harmful activity of a fungus, including but not limited to
killing the fungus, inhibiting the growth or activity of the
fungus, altering the behavior of the fungus, and repelling or
attracting the fungus.
[0088] "Fungistatic activity", as used herein, means to interfere
with the harmful activity of a fungus, including but not limited to
inhibiting the growth or activity of the fungus, altering the
behavior of the fungus, and repelling or attracting the fungus.
[0089] Fungicidal or fungistatic activity of an active ingredient,
substance or principle or a composition or agent comprising a
pesticidal, biocidal, or biostatic active ingredient, substance or
principle, can be expressed as the minimium inhibitory activity
(MIC) of an agent (expressed in units of concentration such as e.g.
mg/mL), without however being restricted thereto.
[0090] 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.
[0091] As used herein, the term "antibody" refers to polyclonal
antibodies, monoclonal antibodies, humanized antibodies,
single-chain antibodies, and fragments thereof such as Fab F(ab)2,
Fv, and other fragments that retain the antigen binding function of
the parent antibody. As such, an antibody may refer to an
immunoglobulin or glycoprotein, or fragment or portion thereof, or
to a construct comprising an antigen-binding portion comprised
within a modified immunoglobulin-like framework, or to an
antigen-binding portion comprised within a construct comprising a
non-immunoglobulin-like framework or scaffold.
[0092] As used herein, the term "monoclonal antibody" refers to an
antibody composition having a homogeneous antibody population. The
term is not limited regarding the species or source of the
antibody, nor is it intended to be limited by the manner in which
it is made. The term encompasses whole immunoglobulins as well as
fragments such as Fab, Fab)2, Fv, and others that retain the
antigen binding function of the antibody. Monoclonal antibodies of
any mammalian species can be used in this invention. In practice,
however, the antibodies will typically be of rat or murine origin
because of the availability of rat or murine cell lines for use in
making the required hybrid cell lines or hybridomas to produce
monoclonal antibodies.
[0093] As used herein, the term "polyclonal antibody" refers to an
antibody composition having a heterogeneous antibody population.
Polyclonal antibodies are often derived from the pooled serum from
immunized animals or from selected humans.
[0094] "Heavy chain variable domain of an antibody or a functional
fragment thereof", as used herein, means (i) the variable domain of
the heavy chain of a heavy chain antibody, which is naturally
devoid of light chains (also indicated hereafter as V.sub.HH),
including but not limited to the variable domain of the heavy chain
of heavy chain antibodies of camelids or sharks or (ii) the
variable domain of the heavy chain of a conventional four-chain
antibody (also indicated hereafter as V.sub.H), including but not
limited to a camelized (as further defined herein) variable domain
of the heavy chain of a conventional four-chain antibody (also
indicated hereafter as camelized V.sub.H).
[0095] As further described hereinbelow, the amino acid sequence
and structure of a heavy chain variable domain of an antibody can
be considered, without however being limited thereto, to be
comprised of four framework regions or "FR's", which are referred
to in the art and hereinbelow as "framework region 1" or "FR1"; as
"framework region 2" or "FR2"; as "framework region 3" or "FR3";
and as "framework region 4" or "FR4", respectively, which framework
regions are interrupted by three complementary determining regions
or "CDR's", which are referred to in the art as "complementarity
determining region 1" or "CDR1"; as "complementarity determining
region 2" or "CDR2"; and as "complementarity determining region 3"
or "CDR3", respectively.
[0096] As also further described hereinbelow, the total number of
amino acid residues in a heavy chain variable domain of an antibody
(including a V.sub.HH or a V.sub.H) can be in the region of
110-130, is preferably 112-115, and is most preferably 113. It
should however be noted that parts, fragments or analogs of a heavy
chain variable domain of an antibody are not particularly limited
as to their length and/or size, as long as such parts, fragments or
analogs retain (at least part of) the functional activity, such as
the pesticidal, biocidal, biostatic activity, fungicidal or
fungistatic activity (as defined herein) and/or retain (at least
part of) the binding specificity of the original a heavy chain
variable domain of an antibody from which these parts, fragments or
analogs are derived from. Parts, fragments or analogs retaining (at
least part of) the functional activity, such as the pesticidal,
biocidal, biostatic activity, fungicidal or fungistatic activity
(as defined herein) and/or retaining (at least part of) the binding
specificity of the original heavy chain variable domain of an
antibody from which these parts, fragments or analogs are derived
from are also further referred to herein as "functional fragments"
of a heavy chain variable domain.
[0097] The amino acid residues of a variable domain of a heavy
chain variable domain of an antibody (including a V.sub.HH or a
V.sub.H) are numbered according to the general numbering for heavy
chain variable domains given by Kabat et al. ("Sequence of proteins
of immunological interest", US Public Health Services, NIH
Bethesda, Md., Publication No. 91), as applied to V.sub.HH domains
from Camelids in the article of Riechmann and Muyldermans, referred
to above (see for example FIG. 2 of said reference). According to
this numbering, FR1 of a heavy chain variable domain comprises the
amino acid residues at positions 1-30, CDR1 of a heavy chain
variable domain comprises the amino acid residues at positions
31-36, FR2 of a heavy chain variable domain comprises the amino
acids at positions 36-49, CDR2 of a heavy chain variable domain
comprises the amino acid residues at positions 50-65, FR3 of a
heavy chain variable domain comprises the amino acid residues at
positions 66-94, CDR3 of a heavy chain variable domain comprises
the amino acid residues at positions 95-102, and FR4 of a heavy
chain variable domain comprises the amino acid residues at
positions 103-113. [In this respect, it should be noted that--as is
well known in the art for V.sub.HH domains--the total number of
amino acid residues in each of the CDR's may vary and may not
correspond to the total number of amino acid residues indicated by
the Kabat numbering (that is, one or more positions according to
the Kabat numbering may not be occupied in the actual sequence, or
the actual sequence may contain more amino acid residues than the
number allowed for by the Kabat numbering). This means that,
generally, the numbering according to Kabat may or may not
correspond to the actual numbering of the amino acid residues in
the actual sequence. Generally, however, it can be said that,
according to the numbering of Kabat and irrespective of the number
of amino acid residues in the CDR's, position 1 according to the
Kabat numbering corresponds to the start of FR1 and visa versa,
position 36 according to the Kabat numbering corresponds to the
start of FR2 and visa versa, position 66 according to the Kabat
numbering corresponds to the start of FR3 and visa versa, and
position 103 according to the Kabat numbering corresponds to the
start of FR4 and visa versa.].
[0098] Alternative methods for numbering the amino acid residues of
heavy chain variable domains are the method described by Chothia et
al. (Nature 342, 877-883 (1989)), the so-called "AbM definition"
and the so-called "contact definition". However, in the present
description, claims and figures, the numbering according to Kabat
as applied to V.sub.HH domains by Riechmann and Muyldermans will be
followed, unless indicated otherwise.
[0099] For a general description of heavy chain antibodies and the
variable domains thereof, reference is inter alia made to the
following references, which are mentioned as general background
art: WO 94/04678, WO 95/04079 and WO 96/34103 of the Vrije
Universiteit Brussel; WO 94/25591, WO 99/37681, WO 00/40968, WO
00/43507, WO 00/65057, WO 01/40310, WO 01/44301, EP 1134231 and WO
02/48193 of Unilever; WO 97/49805, WO 01/21817, WO 03/035694, WO
03/054016 and WO 03/055527 of the Vlaams Instituut voor
Biotechnologie (VIB); WO 03/050531 of Algonomics N.V. and Ablynx
NV; WO 01/90190 by the National Research Council of Canada; WO
03/025020 (=EP 1 433 793) by the Institute of Antibodies; as well
as WO 04/041867, WO 04/041862, WO 04/041865, WO 04/041863, WO
04/062551 by Ablynx NV and the further published patent
applications by Ablynx NV; Hamers-Casterman et al., Nature 1993
Jun. 3; 363 (6428): 446-8; Davies and Riechmann, FEBS Lett. 1994
Feb. 21; 339(3): 285-90; Muyldermans et al., Protein Eng. 1994
September; 7(9): 1129-3; Davies and Riechmann, Biotechnology (NY)
1995 May; 13(5): 475-9; Gharoudi et al., 9th Forum of Applied
Biotechnology, Med. Fac. Landbouw Univ. Gent. 1995; 60/4a part I:
2097-2100; Davies and Riechmann, Protein Eng. 1996 June; 9(6):
531-7; Desmyter et al., Nat Struct Biol. 1996 September; 3(9):
803-11; Sheriff et al., Nat Struct Biol. 1996 September; 3(9):
733-6; Spinelli et al., Nat Struct Biol. 1996 September; 3(9):
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521-6; Vu et al., Mol. Immunol. 1997 November-December; 34(16-17):
1121-31; Atarhouch et al., Journal of Carnel Practice and Research
1997; 4: 177-182; Nguyen et al., J. Mol. Biol. 1998 Jan. 23;
275(3): 413-8; Lauwereys et al., EMBO J. 1998 Jul. 1; 17(13):
3512-20; Frenken et al., Res Immunol. 1998 July-August;
149(6):589-99; Transue et al., Proteins 1998 Sep. 1; 32(4): 515-22;
Muyldermans and Lauwereys, J. Mol. Recognit. 1999 March-April; 12
(2): 131-40; van der Linden et al., Biochim. Biophys. Acta 1999
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[0100] Generally, it should be noted that the term "heavy chain
variable domain" as used herein in its broadest sense is not
limited to a specific biological source or to a specific method of
preparation. For example, as will be discussed in more detail
below, the heavy chain variable domains of the invention can be
obtained (1) by isolating the V.sub.HH domain of a naturally
occurring heavy chain antibody; (2) by isolating the V.sub.H domain
of a naturally occurring four-chain antibody (3) by expression of a
nucleotide sequence encoding a naturally occurring V.sub.HH domain;
(4) by expression of a nucleotide sequence encoding a naturally
occurring V.sub.H domain (5) by "camelization" (as described below)
of a naturally occurring V.sub.H domain from any animal species, in
particular a species of mammal, such as from a human being, or by
expression of a nucleic acid encoding such a camelized V.sub.H
domain; (6) by "camelisation" of a "domain antibody" or "Dab" as
described by Ward et al (supra), or by expression of a nucleic acid
encoding such a camelized V.sub.H domain (7) using synthetic or
semi-synthetic techniques for preparing proteins, polypeptides or
other amino acid sequences; (8) by preparing a nucleic acid
encoding a V.sub.HH or a V.sub.H using techniques for nucleic acid
synthesis, followed by expression of the nucleic acid thus
obtained; and/or (9) by any combination of the foregoing. Suitable
methods and techniques for performing the foregoing will be clear
to the skilled person based on the disclosure herein and for
example include the methods and techniques described in more detail
hereinbelow.
[0101] However, according to a specific embodiment, the heavy chain
variable domains as disclosed herein do not have an amino acid
sequence that is exactly the same as (i.e. as a degree of sequence
identity of 100% with) the amino acid sequence of a naturally
occurring V.sub.H domain, such as the amino acid sequence of a
naturally occurring V.sub.H domain from a mammal, and in particular
from a human being.
[0102] The terms "effective amount" and "effective dose", as used
herein, mean the amount needed to achieve the desired result or
results.
[0103] As used herein, the terms "determining", "measuring",
"assessing", "monitoring" and "assaying" are used interchangeably
and include both quantitative and qualitative determinations.
[0104] All documents cited in the present specification are hereby
incorporated by reference in their entirety. Unless otherwise
defined, all terms used in disclosing the invention, including
technical and scientific terms, have the meaning as commonly
understood by one of ordinary skill in the art to which this
invention belongs. By means of further guidance, term definitions
are included to better appreciate the teaching of the present
invention.
[Compositions Comprising at Least One Heavy Chain Variable Domain
of an Antibody]
[0105] In one aspect, the present inventors have identified
agrochemical compositions comprising at least one variable domain
of an antibody, which can specifically bind to a sphingolipid of a
plant pest. Importantly, through this interaction with a specific
molecular structure of the pest, the compositions disclosed herein
are capable of controlling, modulating, inhibiting, preventing or
reducing one or more biological activities of the plant pathogen,
such that the growth of the plant pathogen is controlled,
modulated, inhibited, prevented or reduced. In certain embodiments,
the agrochemical compositions as disclosed herein are capable of
killing a plant pest through the specific interaction of at least
one variable domain of an antibody, which can specifically bind to
a sphingolipid of a plant pest and which is comprised in the
compositions.
[0106] Accordingly, the agrochemical compositions as disclosed
herein can be used to modulate, such as to change, decrease or
inhibit, the biological function of a plant pest by binding to a
binding site present on a sphingolipid target of that plant pest
thereby affecting the natural biological activities (such as, but
not limited to, growth) of the pest and/or one or more biological
pathways in which the structural target of that pest is
involved.
[0107] Furthermore, the compositions comprising at least one heavy
chain variable domain as disclosed herein have several additional
advantages over the traditional immunoglobulin and
non-immunoglobulin binding agents known in the art. Indeed, in
certain embodiments, the amino acid sequences as disclosed herein
are isolated heavy chain immunoglobulin variable domains, which are
more potent and more stable than conventional four-chain
antibodies, leading to (1) lower dosage forms, less frequent dosage
and thus less side effects; and (2) improved stability resulting in
a broader choice of administration routes. Because of their small
size, heavy chain immunoglobulin variable domains have the ability
to cross membranes and penetrate into physiological compartments,
tissues and organs not accessible to other, larger polypeptides and
proteins.
[0108] In one specific, but non-limiting embodiment, the
compositions comprising at least one heavy chain variable domain as
disclosed herein are capable of specific binding (as defined
herein) to a plant pest target or a plant pest antigen; and more
preferably capable of binding to a pest or plant pathogen target or
a plant pest antigen or plant pathogen antigen with an affinity
(suitably measured and/or expressed as a K.sub.D-value (actual or
apparent), a K.sub.A-value (actual or apparent), a k.sub.on-rate
and/or a k.sub.off-rate, or alternatively as an IC.sub.50 value, as
further described herein) that is as defined herein.
[0109] In particular embodiments, the invention provides an
agrochemical composition or a biological pesticide composition for
combating plant pests, more particularly a plant fungus, which
composition comprises at least one polypeptide or amino acid
sequence of between 80 and 200 amino acids as the active
substance.
[0110] In certain further embodiment, the invention provides an
agrochemical composition for combating plant pests, which
composition comprises at least two polypeptides or at least two
amino acid sequences of between 80 and 200 amino acids as the
active substance.
[0111] In still further embodiments, the invention provides an
agrochemical composition for combating plant pests, which
composition comprises at least three polypeptides or at least three
amino acid sequences of between 80 and 200 amino acids as the
active substance.
[0112] The agrochemical composition according to the invention is
an agrochemical composition, as defined herein, for combating plant
pests, as defined before, meaning that the agrochemical
composition, more in particular the active substance, as defined
before, comprised in the agrochemical composition, is able to
interfere with, preferably to reduce or to arrest, the harmful
effects of one or more plantpests on one or more plants, preferably
crops.
[0113] Thus, in one embodiment, the agrochemical composition
comprises a polypeptide of between 80 and 200 amino acids as the
active substance.
[0114] In more specific embodiments the agrochemical composition
comprises a polypeptide of between 80-100 amino acids, 800-120
amino acids, 80-140 amino acids, 80-160 amino acids or 80-180 amino
acids.
[0115] In yet another embodiment the agrochemical composition
comprises a polypeptide of between 100-200 amino acids, 100-180
amino acids, 100-160 amino acids, 100-150 amino acids, 100-140
amino acids or 100-120 amino acids.
[0116] In yet another embodiment the agrochemical composition
comprises a polypeptide of between 110-200 amino acids, 110-180
amino acids, 110-160 amino acids, 110-140 amino acids or 110-130
amino acids.
[0117] In yet another embodiment, the agrochemical composition
comprises a polypeptide of between 120-200 amino acids, 120-180
amino acids, 120-160 amino acids, or 120-140 amino acids.
[0118] In yet another embodiment, the agrochemical composition
comprises a polypeptide of between 140-200 amino acids, 140-180
amino acids, or 140-160 amino acids.
[0119] In yet another embodiment, the agrochemical composition
comprises a polypeptide of between 160-200 amino acids or 160-180
amino acids.
[0120] The at least one heavy chain variable domain of an antibody
comprised in the compositions disclosed herein can be derived from
a naturally occurring polypeptide, or alternatively they can be
entirely artificially designed. Non-limiting examples of such
naturally occurring polypeptides include heavy chain antibodies
(hcAb).
[0121] In particular, at least one heavy chain variable domain of
an antibody comprised in the compositions disclosed herein consists
of a single polypeptide chain and is not post-translationally
modified. More particularly, the at least one heavy chain variable
domain of an antibody comprised in the compositions disclosed
herein is derived from an innate or adaptive immune system,
preferably from a protein of an innate or adaptive immune system.
Still more particularly, the at least one heavy chain variable
domain of an antibody comprised in the compositions disclosed
herein as disclosed herein is derived from an immunoglobulin. Most
particularly, the at least one heavy chain variable domain of an
antibody comprised in the compositions disclosed herein comprises 4
framework regions and 3 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). In particular, the at least one
heavy chain variable domain of an antibody comprised in the
compositions disclosed herein is easy to produce at high yield,
preferably in a microbial recombinant expression system, and
convenient to isolate and/or purify subsequently.
[0122] According to particular embodiments, the invention provides
a number of stretches of amino acid residues (i.e. small peptides)
that are particularly suited for binding to a sphingolipid antigen
or a sphingolipid target, such as but not limited to a fungal
sphingolipid antigen or a fungal sphingolipid target.
[0123] These stretches of amino acid residues may be present in,
and/or may be incorporated into, the heavy chain variable domains
as disclosed herein, in particular in such a way that they form
(part of) the antigen binding site of that heavy chain variable
domain. As these stretches of amino acid residues were first
generated as CDR sequences of antibodies, such as heavy chain
antibodies, or of V.sub.H or V.sub.HH sequences that were raised
against a sphingolipid target (or may be based on and/or derived
from such CDR sequences, as further described herein), they will
also generally be referred to herein as "CDR sequences" (i.e. as
CDR1 sequences, CDR2 sequences and CDR3 sequences, respectively).
It should however be noted that the invention in its broadest sense
is not limited to a specific structural role or function that these
stretches of amino acid residues may have in the heavy chain
variable domains as disclosed herein, as long as these stretches of
amino acid residues allow the variable domains as disclosed herein
to specifically bind to a sphingolipid target. Thus, generally, the
invention in its broadest sense relates to agrochemical
compositions comprising a heavy chain variable domain of an
antibody that is capable of binding to a sphingolipid target and
that comprises a combination of CDR sequences as described
herein.
[0124] Thus, in particular, but non-limiting embodiments, the heavy
chain variable domain sequences as disclosed herein may be heavy
chain variable domains that comprise at least one amino acid
sequence that is chosen from the group consisting of the CDR1
sequences, CDR2 sequences and CDR3 sequences that are described
herein. In particular, a heavy chain variable domain as disclosed
herein may comprise at least one antigen binding site, wherein said
antigen binding site comprises at least one combination of a CDR1
sequence, a CDR2 sequence and a CDR3 sequence that are described
herein.
[0125] Any heavy chain variable domain comprised in the
agrochemical compositions as disclosed herein and having one these
CDR sequence combinations is preferably such that it can
specifically bind (as defined herein) to a sphingolipid target or a
sphingolipid antigen, and more in particular such that it
specifically binds to a sphingolipid of a plant pathogen, in
particular with dissociation constant (Kd) of 10.sup.-8 moles/liter
or less of said variable domain in solution. Specific binding of a
heavy chain variable domain to a sphingolipid target can be
determined in any suitable manner known per se, including, for
example biopanning, Scatchard analysis and/or competitive binding
assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA)
and sandwich competition assays, and the different variants thereof
known in the art.
[0126] In a preferred embodiment, the polypeptide of between 80 and
200 amino acids, is obtained by affinity selection against a
particular pest target molecule and said polypeptide has a high
affinity for said pest target molecule: typically, the dissociation
constant of the binding between the polypeptide and its pest target
molecule is lower than 10.sup.-6 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.
[0127] In particular embodiments, the at least one heavy chain
variable domain of an antibody comprised in the compositions
disclosed herein has a minimum inhibitory concentration (MIC) value
for said plant pathogenic fungus of 1.0 .mu.g/mL or less of said
variable domain in solution. Also disclosed herein are polypeptides
of between 80 and 200 amino acids or a sub-range as disclosed
herein before, obtained by affinity selection to a specific plant
pest target, which is able to inhibit the growth and/or the
activity of a crop pest at a minimum inhibitory concentration of
about 0.00001 to 1 .mu.M. In specific embodiments the minimum
inhibitory concentrations are between 0.0001 to 1 .mu.M, are
between 0.001 to 1 .mu.M, between 0.01 to 1 .mu.M, between 0.1 to 1
.mu.M, between 0.0001 to 0.1 .mu.M, between 0.001 to 0.1 .mu.M,
between 0.01 to 0.1 .mu.M, between 0.00001 to 0.01 .mu.M, between
0.0001 to 0.01 .mu.M, between 0.001 to 0.01 .mu.M.
[0128] The Minimal Inhibitory Concentration or the MIC value is the
lowest concentration of an agent such as a polypeptide that
inhibits the visible growth of the crop or plant pest after
incubation. For example the minimum fungicidal concentration (MFC)
is considered as the lowest concentration of polypeptide which
prevents growth and reduces the fungal inoculum by a 99.90% within
24 h. MFCs (Minimal Fungal Concentrations) can be determined on
agar plates but can also be conveniently determined in fluids (e.g.
in microwell plates) depending on the type of the fungus and the
assay conditions.
[0129] In further particular embodiments, the agrochemical
compositions as disclosed herein at least comprise a heavy chain
variable domain comprising at least one combination of CDR
sequences chosen from the group comprising:
a CDR1 region having SEQ ID NO: 85, a CDR2 region having has SEQ ID
NO: 169, and a CDR3 region having SEQ ID NO: 253, and/or a CDR1
region having SEQ ID NO: 86, a CDR2 region having has SEQ ID NO:
170, and a CDR3 region having SEQ ID NO: 254, and/or a CDR1 region
having SEQ ID NO: 87, a CDR2 region having has SEQ ID NO: 171, and
a CDR3 region having SEQ ID NO: 255, and/or a CDR1 region having
SEQ ID NO: 88, a CDR2 region having has SEQ ID NO: 172, and a CDR3
region having SEQ ID NO: 256, and/or a CDR1 region having SEQ ID
NO: 89, a CDR2 region having has SEQ ID NO: 173, and a CDR3 region
having SEQ ID NO: 257, and/or a CDR1 region having SEQ ID NO: 90, a
CDR2 region having has SEQ ID NO: 174, and a CDR3 region having SEQ
ID NO: 258, and/or a CDR1 region having SEQ ID NO: 91, a CDR2
region having has SEQ ID NO: 175, and a CDR3 region having SEQ ID
NO: 259, and/or a CDR1 region having SEQ ID NO: 92, a CDR2 region
having has SEQ ID NO: 176, and a CDR3 region having SEQ ID NO: 260,
and/or a CDR1 region having SEQ ID NO: 93, a CDR2 region having has
SEQ ID NO: 177, and a CDR3 region having SEQ ID NO: 261, and/or a
CDR1 region having SEQ ID NO: 94, a CDR2 region having has SEQ ID
NO: 178, and a CDR3 region having SEQ ID NO: 262, and/or a CDR1
region having SEQ ID NO: 95, a CDR2 region having has SEQ ID NO:
179, and a CDR3 region having SEQ ID NO: 263, and/or a CDR1 region
having SEQ ID NO: 96, a CDR2 region having has SEQ ID NO: 180, and
a CDR3 region having SEQ ID NO: 264, and/or a CDR1 region having
SEQ ID NO: 97, a CDR2 region having has SEQ ID NO: 181, and a CDR3
region having SEQ ID NO: 265, and/or a CDR1 region having SEQ ID
NO: 98, a CDR2 region having has SEQ ID NO: 182, and a CDR3 region
having SEQ ID NO: 266, and/or a CDR1 region having SEQ ID NO: 99, a
CDR2 region having has SEQ ID NO: 183, and a CDR3 region having SEQ
ID NO: 267, and/or a CDR1 region having SEQ ID NO: 100, a CDR2
region having has SEQ ID NO: 184, and a CDR3 region having SEQ ID
NO: 268, and/or a CDR1 region having SEQ ID NO: 101, a CDR2 region
having has SEQ ID NO: 185, and a CDR3 region having SEQ ID NO: 269,
and/or a CDR1 region having SEQ ID NO: 102, a CDR2 region having
has SEQ ID NO: 186, and a CDR3 region having SEQ ID NO: 270, and/or
a CDR1 region having SEQ ID NO: 103, a CDR2 region having has SEQ
ID NO: 187, and a CDR3 region having SEQ ID NO: 271, and/or a CDR1
region having SEQ ID NO: 104, a CDR2 region having has SEQ ID NO:
188, and a CDR3 region having SEQ ID NO: 272, and/or a CDR1 region
having SEQ ID NO: 105, a CDR2 region having has SEQ ID NO: 189, and
a CDR3 region having SEQ ID NO: 273, and/or a CDR1 region having
SEQ ID NO: 106, a CDR2 region having has SEQ ID NO: 190, and a CDR3
region having SEQ ID NO: 274, and/or a CDR1 region having SEQ ID
NO: 107, a CDR2 region having has SEQ ID NO: 191, and a CDR3 region
having SEQ ID NO: 275, and/or a CDR1 region having SEQ ID NO: 108,
a CDR2 region having has SEQ ID NO: 192, and a CDR3 region having
SEQ ID NO: 276, and/or a CDR1 region having SEQ ID NO: 109, a CDR2
region having has SEQ ID NO: 193, and a CDR3 region having SEQ ID
NO: 277, and/or a CDR1 region having SEQ ID NO: 110, a CDR2 region
having has SEQ ID NO: 194, and a CDR3 region having SEQ ID NO: 278,
and/or a CDR1 region having SEQ ID NO: 111, a CDR2 region having
has SEQ ID NO: 195, and a CDR3 region having SEQ ID NO: 279, and/or
a CDR1 region having SEQ ID NO: 112, a CDR2 region having has SEQ
ID NO: 196, and a CDR3 region having SEQ ID NO: 280, and/or a CDR1
region having SEQ ID NO: 113, a CDR2 region having has SEQ ID NO:
197, and a CDR3 region having SEQ ID NO: 281, and/or a CDR1 region
having SEQ ID NO: 114, a CDR2 region having has SEQ ID NO: 198, and
a CDR3 region having SEQ ID NO: 282, and/or a CDR1 region having
SEQ ID NO: 115, a CDR2 region having has SEQ ID NO: 199, and a CDR3
region having SEQ ID NO: 283, and/or a CDR1 region having SEQ ID
NO: 116, a CDR2 region having has SEQ ID NO: 200, and a CDR3 region
having SEQ ID NO: 284, and/or a CDR1 region having SEQ ID NO: 117,
a CDR2 region having has SEQ ID NO: 201, and a CDR3 region having
SEQ ID NO: 285, and/or a CDR1 region having SEQ ID NO: 118, a CDR2
region having has SEQ ID NO: 202, and a CDR3 region having SEQ ID
NO: 286, and/or a CDR1 region having SEQ ID NO: 119, a CDR2 region
having has SEQ ID NO: 203, and a CDR3 region having SEQ ID NO: 287,
and/or a CDR1 region having SEQ ID NO: 120, a CDR2 region having
has SEQ ID NO: 204, and a CDR3 region having SEQ ID NO: 288, and/or
a CDR1 region having SEQ ID NO: 121, a CDR2 region having has SEQ
ID NO: 205, and a CDR3 region having SEQ ID NO: 289, and/or a CDR1
region having SEQ ID NO: 122, a CDR2 region having has SEQ ID NO:
206, and a CDR3 region having SEQ ID NO: 290, and/or a CDR1 region
having SEQ ID NO: 123, a CDR2 region having has SEQ ID NO: 207, and
a CDR3 region having SEQ ID NO: 291, and/or a CDR1 region having
SEQ ID NO: 124, a CDR2 region having has SEQ ID NO: 208, and a CDR3
region having SEQ ID NO: 292, and/or a CDR1 region having SEQ ID
NO: 125, a CDR2 region having has SEQ ID NO: 209, and a CDR3 region
having SEQ ID NO: 293, and/or a CDR1 region having SEQ ID NO: 126,
a CDR2 region having has SEQ ID NO: 210, and a CDR3 region having
SEQ ID NO: 294, and/or a CDR1 region having SEQ ID NO: 127, a CDR2
region having has SEQ ID NO: 211, and a CDR3 region having SEQ ID
NO: 295, and/or a CDR1 region having SEQ ID NO: 128, a CDR2 region
having has SEQ ID NO: 212, and a CDR3 region having SEQ ID NO: 296,
and/or a CDR1 region having SEQ ID NO: 129, a CDR2 region having
has SEQ ID NO: 213, and a CDR3 region having SEQ ID NO: 297, and/or
a CDR1 region having SEQ ID NO: 130, a CDR2 region having has SEQ
ID NO: 214, and a CDR3 region having SEQ ID NO: 298, and/or a CDR1
region having SEQ ID NO: 131, a CDR2 region having has SEQ ID NO:
215, and a CDR3 region having SEQ ID NO: 299, and/or a CDR1 region
having SEQ ID NO: 132, a CDR2 region having has SEQ ID NO: 216, and
a CDR3 region having SEQ ID NO: 300, and/or a CDR1 region having
SEQ ID NO: 133, a CDR2 region having has SEQ ID NO: 217, and a CDR3
region having SEQ ID NO: 301, and/or a CDR1 region having SEQ ID
NO: 134, a CDR2 region having has SEQ ID NO: 218, and a CDR3 region
having SEQ ID NO: 302, and/or a CDR1 region having SEQ ID NO: 135,
a CDR2 region having has SEQ ID NO: 219, and a CDR3 region having
SEQ ID NO: 303, and/or a CDR1 region having SEQ ID NO: 136, a CDR2
region having has SEQ ID NO: 220, and a CDR3 region having SEQ ID
NO: 304, and/or a CDR1 region having SEQ ID NO: 137, a CDR2 region
having has SEQ ID NO: 221, and a CDR3 region having SEQ ID NO: 305,
and/or a CDR1 region having SEQ ID NO: 138, a CDR2 region having
has SEQ ID NO: 222, and a CDR3 region having the amino acid
sequence NRY, and/or a CDR1 region having SEQ ID NO: 139, a CDR2
region having has SEQ ID NO: 223, and a CDR3 region having SEQ ID
NO: 306, and/or a CDR1 region having SEQ ID NO: 140, a CDR2 region
having has SEQ ID NO: 224, and a CDR3 region having SEQ ID NO: 307,
and/or a CDR1 region having SEQ ID NO: 141, a CDR2 region having
has SEQ ID NO: 225, and a CDR3 region having SEQ ID NO: 308, and/or
a CDR1 region having SEQ ID NO: 142, a CDR2 region having has SEQ
ID NO: 226, and a CDR3 region having SEQ ID NO: 309, and/or a CDR1
region having SEQ ID NO: 143, a CDR2 region having has SEQ ID NO:
227, and a CDR3 region having SEQ ID NO: 310, and/or a CDR1 region
having SEQ ID NO: 144, a CDR2 region having has SEQ ID NO: 228, and
a CDR3 region having SEQ ID NO: 311, and/or a CDR1 region having
SEQ ID NO: 145, a CDR2 region having has SEQ ID NO: 229, and a CDR3
region having SEQ ID NO: 312, and/or a CDR1 region having SEQ ID
NO: 146, a CDR2 region having has SEQ ID NO: 230, and a CDR3 region
having SEQ ID NO: 313, and/or a CDR1 region having SEQ ID NO: 147,
a CDR2 region having has SEQ ID NO: 231, and a CDR3 region having
SEQ ID NO: 314, and/or a CDR1 region having SEQ ID NO: 148, a CDR2
region having has SEQ ID NO: 232, and a CDR3 region having SEQ ID
NO: 315, and/or a CDR1 region having SEQ ID NO: 149, a CDR2 region
having has SEQ ID NO: 233, and a CDR3 region having SEQ ID NO: 316,
and/or a CDR1 region having SEQ ID NO: 150, a CDR2 region having
has SEQ ID NO: 234, and a CDR3 region having SEQ ID NO: 317, and/or
a CDR1 region having SEQ ID NO: 151, a CDR2 region having has SEQ
ID NO: 235, and a CDR3 region having SEQ ID NO: 318, and/or a CDR1
region having SEQ ID NO: 152, a CDR2 region having has SEQ ID NO:
236, and a CDR3 region having SEQ ID NO: 319, and/or a CDR1 region
having SEQ ID NO: 153, a CDR2 region having has SEQ ID NO: 237, and
a CDR3 region having SEQ ID NO: 320, and/or a CDR1 region having
SEQ ID NO: 154, a CDR2 region having has SEQ ID NO: 238, and a CDR3
region having SEQ ID NO: 321, and/or a CDR1 region having SEQ ID
NO: 155, a CDR2 region having has SEQ ID NO: 239, and a CDR3 region
having SEQ ID NO: 322, and/or a CDR1 region having SEQ ID NO: 156,
a CDR2 region having has SEQ ID NO: 240, and a CDR3 region having
SEQ ID NO: 323, and/or a CDR1 region having SEQ ID NO: 157, a CDR2
region having has SEQ ID NO: 241, and a CDR3 region having SEQ ID
NO: 324, and/or a CDR1 region having SEQ ID NO: 158, a CDR2 region
having has SEQ ID NO: 242, and a CDR3 region having SEQ ID NO: 325,
and/or a CDR1 region having SEQ ID NO: 159, a CDR2 region having
has SEQ ID NO: 243, and a CDR3 region having SEQ ID NO: 326, and/or
a CDR1 region having SEQ ID NO: 160, a CDR2 region having has SEQ
ID NO: 244, and a CDR3 region having SEQ ID NO: 327, and/or a CDR1
region having SEQ ID NO: 161, a CDR2 region having has SEQ ID NO:
245, and a CDR3 region having SEQ ID NO: 328, and/or a CDR1 region
having SEQ ID NO: 162, a CDR2 region having has SEQ ID NO: 246, and
a CDR3 region having SEQ ID NO: 329, and/or a CDR1 region having
SEQ ID NO: 163, a CDR2 region having has SEQ ID NO: 247, and a CDR3
region having SEQ ID NO: 330, and/or a CDR1 region having SEQ ID
NO: 164, a CDR2 region having has SEQ ID NO: 248, and a CDR3 region
having SEQ ID NO: 331, and/or a CDR1 region having SEQ ID NO: 165,
a CDR2 region having has SEQ ID NO: 249, and a CDR3 region having
SEQ ID NO: 332, and/or a CDR1 region having SEQ ID NO: 166, a CDR2
region having has SEQ ID NO: 250, and a CDR3 region having SEQ ID
NO: 333, and/or a CDR1 region having SEQ ID NO: 167, a CDR2 region
having has SEQ ID NO: 251, and a CDR3 region having SEQ ID NO: 334,
and/or a CDR1 region having SEQ ID NO: 168, a CDR2 region having
has SEQ ID NO: 252, and a CDR3 region having SEQ ID NO: 335.
[0130] In particular embodiments, the heavy chain variable domains
in the compositions as disclosed herein are heavy chain variable
domains that essentially consist of four framework regions (FR1 to
FR4 respectively) and three complementarity determining regions
(CDR1 to CDR3 respectively); or any suitable fragment of such an
heavy chain variable domain (which will then usually contain at
least some of the amino acid residues that form at least one of the
CDR's, as further described herein).
[0131] The heavy chain variable domains as disclosed herein may in
particular be a heavy chain variable domain sequence that is
derived from a conventional four-chain antibody (such as, without
limitation, a V.sub.H sequence that is derived from a human
antibody) or be a so-called V.sub.HH-sequence (as defined herein)
that is derived from a so-called "heavy chain antibody" (as defined
herein).
[0132] In particular embodiments, the compositions as disclosed
herein, at least comprise an heavy chain variable domain sequence
derived from an antibody or a functional fragment thereof, such as
but not limited to a camelid heavy chain antibody or a functional
fragment thereof, which variable domain sequence thus may be for
instance a heavy chain variable domain of a camelid heavy chain
antibody (V.sub.HH).
[0133] However, it should be noted that the invention is not
limited as to the origin of the heavy chain variable domain
sequence comprised in the compositions disclosed herein (or of the
nucleotide sequence of the invention used to express it), nor as to
the way that the heavy chain variable domain or nucleotide sequence
thereof is (or has been) generated or obtained. Thus, the heavy
chain variable domains din the compositions disclosed herein may be
naturally occurring heavy chain variable domains (from any suitable
species) or synthetic or semi-synthetic heavy chain variable
domains. In a specific but non-limiting embodiment of the
invention, the heavy chain variable domain is a naturally occurring
immunoglobulin sequence (from any suitable species) or a synthetic
or semi-synthetic immunoglobulin sequence, including but not
limited to "camelized" immunoglobulin sequences, as well as
immunoglobulin sequences that have been obtained by techniques such
as affinity maturation (for example, starting from synthetic,
random or naturally occurring immunoglobulin sequences), CDR
grafting, veneering, combining fragments derived from different
immunoglobulin sequences, PCR assembly using overlapping primers,
and similar techniques for engineering immunoglobulin sequences
well known to the skilled person; or any suitable combination of
any of the foregoing.
[0134] The heavy chain variable domain sequences of the
compositions disclosed herein may in particular be a domain
antibody (or an heavy chain variable domain that is suitable for
use as a domain antibody), a single domain antibody (or an heavy
chain variable domain that is suitable for use as a single domain
antibody), or a "dAb" (or an heavy chain variable domain that is
suitable for use as a dAb); other single variable domains, or any
suitable fragment of any one thereof. For a general description of
(single) domain antibodies, reference is also made to the prior art
cited above, as well as to EP 0 368 684. For the term "dAb's",
reference is for example made to Ward et al. (Nature 1989 Oct. 12;
341 (6242): 544-6), to Holt et al., Trends Biotechnol., 2003,
21(11):484-490; as well as to for example WO 06/030220, WO
06/003388 and other published patent applications of Domantis
Ltd.
[0135] Thus, in particular embodiments, the present invention
provides heavy chain variable domains with the (general) structure
[0136] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in which FR1 to FR4 refer to
framework regions 1 to 4, respectively, and in which CDR1 to CDR3
refer to the complementarity determining regions 1 to 3,
respectively, and are as further defined herein.
[0137] SEQ ID NO's: 1 to 84 (see Table 1) give the amino acid
sequences of a number of heavy chain variable domains that have
been raised against a sphingolipid target, in particular against
glucosylceramide.
TABLE-US-00001 TABLE 1 VHH sequences Name SEQ ID VHH Amino acid
sequence 40F07 1
QVQLQESGGGLVQAGGSLRLSCVASGTTFSSYTMGWYRQAPGKQRELLASIEGGGNTDY
ADSVKGRFTISRDNARNTVYLQMNSLKTEDTAVYYCNAARTWSIFRNYWGQGTQVTVSS 41D01 2
QVQLQESGGGLVQAGGSLRLSCAASGRTFSRYGMGWFRQLPGKQRELVTSITRGGTTTY
ADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNARSIWRDYWGQGTQVTVSS 41D06 3
QVQLQESGGGLVQAGGSLRLSCAASGGIFGINAMRWYRQAPGKQRELVASISSGGNTNY
SESVKGRFTISRDDANYTVYLQMNSLKPEDTAVYYCNFVRLWFPDYWGQGTQVTVSS 41G10 4
QVQLQESGGGLVQPGGSLTLSCAATKTGFSINAMGWYRQAPGKQREMVATITSGGTTNY
ADSVKGRFAISRDNAKNTVSLQMNTLKPEDTALYYCNTEARRYFTRASQVYWGQGTQVT VSS
41H05 5 QVQLQESGGGLVQPGGSLRLSCAASGGIFSINAMGWYRQDPGKQREMVATITSGANTNY
TDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNAVGRRWYGGYVELWGQGTQVTV SS
42C11 6 QVQLQESGGGLVQPGGSLRLSCAASGSIFSTYVMGWYRQAIGKQRELVATITSSGKTNY
AASVKGRFTVSRDITKNTMYLQMNSLKPEDTAVYYCGADRWVLTRWSNYWGQGTQVTVS S 42C12
7 QVQLQESGGGLVQPGGSLRLSCAASGSISSLGWYRQAPGKQREFVASATSGGDTTYADS
VKGRFTISRDNSKNTVYLQMNSLKPEDTAVYYCKGQRGVAWTRKEYWGQGTQVTVSS 50D03 8
QVQLQESGGGLVQPGGSLRLSCAASGSIFSTYAMGWYRQAIGKQRELVATITSSGKTNY
AASVKGRFTISRDITKNTMYLQMNSLKPEDTAVYYCGADRWVLTRWSNYWGQGTQVTVS S 50D07
9 QVQLQESGGGLVQPGGSLRLSCTASGNIVNIRDMGWYRQVPGKQRELVATITSDQSTNY
ADSVKGRFTTTRDNAKKTVYLQMDSLKPEDTAGYYCNARVRTVLRGWRDYWGQGTQVTV SS
50E02 10
QVQLQESGGGLVQPGGSLRLSCAASGSIFSINAMGWYRQAPGKQRELVAAITSDGSTNY
ADSVKGRFTISRDNAKNTAYLQMNSLKPEDTAVYYCNLRRRTFLKSSDYWGQGTQVTVS S 51B08
11 QVQLQESGGGLVQAGDSLRLSCAASGRRFGSYAMGWFRQVPGKERELVAGISSGGSTKY
ADSVRGRFTISRDNAKNTVSLQMKSLKPEDTAVYYCNAKYGRWTYTGRPEYDSWGQGTQ VTVSS
51C06 12
QVQLQESGGGLVQPGGSLRLSCAASGSIFSSDTMGWYRRAPGKQRELVAAITTGGNTNY
ADSVKGRFTISRDNAKNTVYLQMNSLQPEDTAVYYCNCRRRWSRDFWGQGTQVTVSS 51C08 13
QVQLQESGGGLVQPGGSLRLSCAASGTIFSIKTMGWYRQAPGKQRELVATISNGGSTNY
ADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNARQQFIGAPYEYWGQGTQVTVS S 52A01
14 QVQLQESGGGLVQAGGSLRLSCTASGAITFSLGTMGWYRQAPGKQRELVASISTGSTNY
ADSVKGRFTISRDIIKNILYLQMNSLKPEDTAVYSCNARLLWSNYWGQGTQVTVSS 52B01 15
QVQLQESGGGLVQAGESLRLSCAASGSTFSINVMGWYRQAPGEQRELVATISRGGSTNY
ADSVKGRFTISRDNAKVTVYLQMDSLKPEDTAVYYCNAAGWVGVTNYWGQGTQVTVSS 52G05 16
QVQLQESGGGLVQAGGSLRLSCAASGSTGSISAMGWYRQAPGKQRELVASITRRGSTNY
ADSVKDRFTISRDNAWNTVYLQMNSLKPEDTAVYYCNARRYYTRNDYWGQGTQVTVSS 53A01 17
QVQLQESGGGLGQAGGSLRLSCEVSGTTFSINTMGWHRQAPGKQRELVASISSGGWTNY
ADSVKGRFTISRDNAKKTVYLQMNNLKPEDTAVYYCRWGAIGNWYGQGTQVTVSS 53F05 18
QVQLQESGGGLVQPGGSLRLSCAASVRIFGLNAMGWYRQGPGKQRELVASITTGGSTNY
AEPVKGRFTISRDNANNTVYLQMNNLKPEDTAVYYCNAERRWGLPNYWGQGTQVTVSS 54A02 19
QVQLQESGGGLVEAGGSLRLSCAASGRTFSRYGMGWFRQAPGKEREFVAANRWSGGSTY
YADSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAYAHITAWGMRNDYEYDYWGQ
GTQVTVSS 54B01 20
QVQLQESGGGLVQAGGSLRLSCAATGRTFSRYTMGWFRQAPGKERDFVAGITWTGGSTD
YADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAGNLLRLAGQLRRGYDSWGQG TQVTVSS
54C01 21
QVQLQESGGGLVQAGGSLRLSCAASGRTGSRYAMGWFRQAPGKEREFVAAISWSGGSTY
YADSVKDRFTISRDNAKNTVYLQMHSLKPEDTAVYYCATRNRAGPHYSRGYTAGQEYDY
WGQGTQVTVSS 54C04 22
QVQLQESGGGLVQPGGSLRLSCAASGRIFSINAMGWYRQGPGKERELVVDMTSGGSINY
ADSVSGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCHANLRTAFWRNGNDYWGQGTQVT VSS
54C08 23
QVQLQESGGGLVQPGGSLRLSCAASGSISSINAMGWYRQAPGKQRELVASITSGGSTNY
ADSVKGRFTISRDNAKNTVNLQMNSLKPEDTAVYYCSAGPWYRRSWGRGTQVTVSS 54C10 24
QVQLQESGGGLVQPGESLRLSCAASASIFWVNDMGWYRQAPGKQRELVAQITRRGSTNY
ADSVKGRFTISRDNAKDEVYLQMNSLKPEDTAVYYCNADLAVRGRYWGQGTQVTVSS 54C11 25
QVQLQESGGGLVQPGGSLRLSCAASGSFFPVNDMAWYRQALGNERELVANITRGGSTNY
ADSVKGRFTISRDNAKNTVYLQMNTLKPEDTAVYYCNVRIGFGWTAKAYWGQGTQVTVS S 54D03
26 QVQLQESGGGLVQPGGSLRLSCAASGGIFGINAMRWYRQAPGKQRELVASISSGGNTNY
SESVKGRFTISRDDANYTVYLQMNSLKPEDTAVYYCNFVRLWFPDYWGQGTQVTVSS 54D06 27
QVQLQESGGGLVQPGGSLRLSCAASGSTIRINAMGWYRQAPGKQRELVATITRGGITNY
ADSVKGRFTISRDNAKFTVYLQMNSLKPEDTAVYYCNARSWVGPEYWGQGTQVTVSS 54D10 28
QVQLQESGGGLVQPGGSLRLSCAASGMTYSIHAMGWYRQAPGKERELVAITSTSGTTDY
TDSVKGRFTISRDGANNTVYLQMNSLKSEDTAVYYCHVKTRTWYNGKYDYWGQGTQVTV SS
54E01 29
QVQLQESGGGLVQPGGSLRLSCTASGSIFSINPMGWYRQAPGKQRELVAAITSGGSTNY
ADYVKGRFTISRDNAKNVVYLQMNSLKPEDTAVYYCNGRSTLWRRDYWGQGTQVTVSS 54E05 30
QVQLQESGGGLVQPGGSLRLSCAASGSIFSINTMGWYRQAPGKQRELVAAITNRGSTNY
ADFVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCNAHRSWPRYDSWGQGTQVTVSS 54E10 31
QVQLQESGGGLVQPGGSLRLSCAASGSIFSFNAMGWYRQAPGKQRELVAAITRGGSTNY
ADSVKGRFTISRDNANNTVYLQMNSLKPEDTAVYYCNAESRIFRRYDYWGPGTQVTVSS 54F01
32 QVQLQESGGGLVQPGGSLRLSCVTSGSIFGLNLMGWYRQAPGKQRELVATITRGGSTNY
ADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCNVDRGWSSYWGQGTQVTVSS 54F02 33
QVQLQESGGGLVQPGGSLRLSCVTSGSIRSINTMGWYRQAPGNERELVATITSGGTTNY
ADSVKNRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNLHQRAWARSYVYWGQGTQVTVS S 54G01
34 QVQLQESGGGSVQPGGSLRLSCAASGSIFAVNAMGWYRQAPGHQRELVAIISSNSTSNY
ADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYFCYAKRSWFSQEYWGQGTQVTVSS 54G08 35
QVQLQESGGGLVQPGGSLRLSCAASGSIFSFNLMGWYRQAPGKQRELVAAITSSSNTNY
ADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNAQYTITPWGIKKDYWGQGTQVT VSS
54G09 36
QVQLQESGGGLMQPGGSLRLSCTASGNIVNIRDMGWYRQVPGKQRELVATITSDQSTNY
ADSVKGRFTTTRDNAKKTVYLQMDSLKPEDTAGYYCNARVRTVLRGWRDYWGQGTQVTV SS
55B02 37
QVQLQESGGGLVQPGESLRLSCVGSGSIFNINSMNWYRQASGKQRELVADMRSDGSTNY
ADSVKGRFTISRDNARKTVYLQMNSLKPEDTAVYYCHANSIFRSRDYWGQGTQVTVSS 55B05 38
QVQLQESGGGVVQAGDSLRLSCAASGRTFGGYTVAWFRQAPGKEREFVARISWSGIMAY
YAESVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCASRSQIRSPWSSLDDYDRWGQG TQVTVSS
55C05 39
QVQLQESGGGLVQPGGSLRLSCVVSGSISSMKAMGWHRQAPGKERELVAQITRGDSTNY
ADSVKGRFTISRDNAKNTVYLQMNSLKPDDTGVYYCNADRFFGRDYWGKGTQVTVSS 55D08 40
QVQLQESGGGLVQPGGSLRLSCAASRSILSISAMGWYRQGPGKQREPVATITSAGSSNY
SDSVKGRFTISRDNAKNTAYLQMNSLKPEDTAVYYCKTVYSRPLLGPLEVWGQGTQVTV SS
55E02 41
QVQLQESGGGLVQTGGSLRLSCVASGSMFSSNAMAWYRQAPGKQRELVARILSGGSTNY
ADSVKGRFTISRGNAKNTVYLQMNSLKPEDTAVYYCNAVRYLVNYWGQGTQVTVSS 55E07 42
QVQLQESGGGSVQVGDSLTLSCVASGRSLDIYGMGWFRQAPGKEREFVARITSGGSTYY
ADSVKGRFTLSRDNAKNTVYLQMNSLKPEDTAVYYCAAGVVVATSPKFYAYWGQGTQVT VSS
55E09 43
QVQLQESGGGLVQAGGSLRLSCAASKRIFSTYTMGWFRQAPGKEREFVAAIIWSGGRTR
YADSVKGRFTISRDNARNTVHLQMNSLEPEDTAVYYCYTRRLGTGYWGQGTQVTVSS 55E10 44
QVQLQESGGGLVQAGGSLRLSCAASGSTFSIQTIGWYRQAPGKQRDRVATISSGGSTNY
ADSVKGRFTISRDNAKKTVYLQMNNLKPEDTAVYYCNLRYWFRDYWGQGTQVTVSS 55F04 45
QVQLQESGGGLVQPGGSLRLSCAASGSTFSINVRGWYRQAPGKQRELVATITSDGSTNY
ADSVKGRFTISRDNAKNTAYLQMNSLKPEDTAVYYCNAVRLFRQYWGQGTQVTVSS 55F09 46
QVQLQESGGGLVQPGGSLRLSCAASGSIFRLNAMGWYRQAPGKQRELVAAITPGGGNTT
YADSVKGRFTISRDNALNTIYLQMNSLKPEDTAVYYCNAGGSSRWYSSRYYPGGYWGQG TQVTVSS
55F10 47
QVQLQESGGGLVQAGGSLRLSCATSGGTFSRYAMGWFRQAPGKERELVATIRRSGSSTY
YLDSTKGRFTISRDNAKNTVYLQMNSLKLEDTAVYYCAADSSARALVGGPGNRWDYWGQ
GTQVTVSS 55G02 48
QVQLQESGGGLVQPGGSLRLSCAASGSIGSINVMGWYRQYPGKQRELVAFITSGGITNY
TDSVKGRFAISRDNAQNTVYLQMNSLTPEDTAVYYCHLKNAKNVRPGYWGQGTQVTVSS 55G08
49 QVQLQESGGGLVQPGGSLRLSCRASGGIFGINAMRWYRQAPGKQRELVASISSGGTTDY
VESVKGRFTISRDNATNTVDLQMSALKPEDTAVYYCNFVRFWFPDYWGQGTQVTVSS 56A05 50
QVQLQESGGGLVQAGGSLRLSCAASGITFMSNTMGWYRQAPGKQRELVASISSGGSTNY
ADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCNARRNVFISSWGQGTQVTVSS 56A06 51
QVQLQESGGGLVQPGGSLRLSCVASGSISVYGMGWYRQAPGKQRELVARITNIGTTNYA
DSVKGRFTISRDNAKNTVYLQMNSLQPEDTAVYYCNLRRLGRDYWGQGTQVTVSS 56A09 52
QVQLQESGGGLVQPGGSLRLSCAASRTALRLNSMGWYRQAPGSQRELVATITRGGTTNY
ADSVKGRFTISREIGNNTVYLQMNSLEPEDTAVYYCNANFGILVGREYWGKGTQVTVSS 56C09
53 QVQLQESGGGLVQAGGSLRLSCAVSGSIFSILSMAWYRQTPGKQRELVANITSVGSTNY
ADSVKGRFTISRDIAKKTLYLQMNNLKPEDTAIYYCNTRMPFLGDSWGQGTQVTVSS 56C12 54
QVQLQESGGGLVQAGGSLRLSCAVSAFSFSNRAVSWYRQAPGKSREWVASISGIRITTY
TNSVKGRFIISRDNAKKTVYLQMNDLRPEDTGVYRCYMNRYSGQGTQVTVSS 56D06 55
QVQLQESGGGSVQPGGSLRLSCAASGTVFFSISAMGWYRQAPGKQRELVAGISRGGSTK
YGDFVKGRFTISRDNGKKTIWLQMNNLQPEDTAIYYCRLTSITGTYLWGQGTQVTVSS 56D07 56
QVQLQESGGGLVQPGGSLRLSCAASGSIFSMKVMGWYRQGPGKLRELVAVITSGGRTNY
AESVKGRFTISRDNAKNTVSLQMNSLQPEDTAVYYCYYKTIRPYWGQGTQVTVSS 56D10 57
QVQLQESGGGLVQAGGSLRLSCAASGITFRITTMGWYRQAPGKQRELVASSSSGGTTNY
ASSVKGRFTSRDNAKNTVYLQMNSLRPEDTAVYYCNARKFITTPWSTDYWGQGTQVTV SS 56E04
58 QVQLQESGGGLVQPGDSLRLSCTPSGSIFNHKATGWYRQAPGSQRELVAKITTGGTTNY
ADSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCNAERYFATTLWGQGTQVTVSS 56E05 59
QVQLQESGGGLVQAGGSLRLSCAASGITFSNNAGGWYRQAPGQQRELVARISSGGNTNY
TDSVKGRFTISRDITKNTLSLQMNNLKPEDSAVYYCNAQRRVILGPRNYWGQGTQVTVS S 56E08
60 QVQLQESGGGLVQAGGSLRLSCAASGNIFRINDMGWYRQAPGNQRELVATITSANITNY
ADSVKGRFTISRDNAKNTVYLQMNSLNPEDTAVYYCTAQAKKWRIGPWSDYWGQGTQVT VSS
56F07 61
QVQLQESGGGLVQPGGSLRLSCAASGRIFSINDMAWYRQAPGKQRELVAIITNDDSTTY
ADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNADINTAIWRRKYWGQGTQVTVS S 56F11
62 QVQLQESGGGLVQSGGSLRLSCVHSKTTFTRNAMGWYRQALGKERELVATITSGGTTNY
ADSVKGRFTISMDSAKNTVYLQMNSLKPEDTAVYYCNVNTRRIFGGTVREYWGQGTQVT VSS
56G07 63
QVQLQESGGGLVQPGGSLRLSCAVSGSRIFIHDMGWHRQAPGEPRELVATITPFGRRNY
SEYVKGRFTVSRDIARNTMSLQMSNLKAEDTGMYYCNVRVNGVDYWGQGTQVTVSS 56G08 64
QVQLQESGGGLVQAGGSLRLSCAISGITFRRPFGISRMGWYRQAPGKERELVATLSRAG
TSRYVDSVKGRFTISRDDAKNTLYLQMVSLNPEDTAVYYCYIAQLGTDYWGQGTQVTVS S 56G10
65 QVQLQESGGGLVQAGGSLRLSCVASGITLRMYQVGWYRQAPGKQRELVAEISSRGTTMY
ADSVKGRFTISRDGAKNIVYLQMNSLEPEDTAVYYCNARAFAFGRNSWGQGTQVTVSS 56H04 66
QVQLQESGGGSVQAGGSLRLSCAVSGGTFSNKAMGWYRQSSGKQRALVARISTVGTAHY
ADSVKGRFTVSKDNAGNTLYLQMNSLKPEDTAVYYCNAQAGRLYLRNYWGQGTQVTVSS 56H05
67 QVQLQESGGGLVQPGESLRLSCVAAASTSITTFNTMAWYRQAPGKQRELVAQINNRDNT
EYADSVKGRFIISRGNAKNTSNLQMNDLKSEDTGIYYCNAKRWSWSTGFWGQGTQVTVS S 56H07
68 QVQLQESGGGLVQAGGSLRLSCTASGLTFALGTMGWYRQAPGKQRELVASISTGSTNYA
DSVKGRFTISRDIIKNILYLQMNSLKPEDTAVYSCNARLWWSNYWGQGTQVTVSS 56H08 69
QVQLQESGGGLVQAGGSLRLSCTASGRTSSVNPMGWYRQAPGKQRELVAVISSDGSTNY
ADSVKGRFTVSRDNAKNTLYLQMNSLKPEDTAVYYCNANRRWSWGSEYWGQGTQVTVSS 57A06
70 QVQLQESGGGLVQAGGSLRLSCAASGITFTNNAGGWYRQAPGQQRELVARISSGGNTNY
TDSVKGRFTISRDITKNTLSLQMNNLKPEDSAVYYCNAQRRVILGPRNYWGQGTQVTVS S 57B01
71 QVQLQESGGGLVQAGGSLRLSCEAPVSTFNINAMAWYRQAPGKSRELVARISSGGSTNY
ADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYICYVNRHWGWDYWGQGTQVTVSS 57B07 72
QVQLQESGGGLVQPGGTLRLSCVASGSFRSINAMGWYRQAPGKQRELVATVDSGGYTNY
ADSVKGRFTISRDNAKNTVYLQMSSLTPEDTAVYYCYAGIYKWPWSVDARDYWGQGTQV
TVSS 57B11 73
QVQLQESGGGLVQAGGSLRLSCAASGSSISMNSMGWYRQAPGKERERVALIRSSGGTYY
ADSVKGRFTISRDNAKNTVYLQMNNLKPEDTAVYYCQARRTWLSSESWGQGTQVTVSS 57C07 74
QVQLQESGGGLVQAGGSLRLSCAVSGSTFGINTMGWYRQAPEKQRELVASISRGGMTNY
ADSVKGRFIISRDNAKNTVYLQMNSLKPEDTAVYVCNAGIRSRWYGGPITTYWGQGTQV TVSS
57C09 75
QVQLQESGGGLVQAGGSLRLSCAASGSTGSINAMGWYRQGPGKQRDLVASISSGGATNY
ADSVKGRFTISRDNSKNTVYLQMSSLKPEDTAVYYCNAKKSRWSWSIVHDYWGQGTQVT VSS
57D02 76
QVQLQESGGGSVQTGGSLTLSCTTSGSIFGRSDMGWYRQAPGKQRELVATITRRSRTNY
AEFVKGRFTISRDSAKNLVTLQMNSLKPEDTNVYYCNARWGAGGIFSTWGQGTQVTVSS 57D09
77 QVQLQESGGGLVQPGESLRLSCAASGSMSIDAMGWYRQAPGDQRELVASITTGGSTNYA
DSVKGRFTISRDNAKNTVWLQMNSLKPEDTAVYYCNAKVRLRWFRPPSDYWGQGTQVTV SS
57D10 78
QVQLQESGGGLVQPGGSLRLSCAASGRLLSISTMGWYRRTPEDQREMVASITKDGTTNY
ADSVKGRLTISRDNAKNTVYLQMNSLKPDDTAVYVCNARATTWVPYRRDAEFWGQGTQV TVSS
57E07 79
QVQLQESGGGLVQAGGSLRLSCAASGSIFGINDMGWYRQAPGKQRDLVADITRSGSTHY
VDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNADSGSHWWNRRDYWGQGTQVTV SS
57E11 80
QVQLQESGGGLVQPGGSLKLSCAASGFTFSINTMGWYRQAPGKQRELVARISRLRVTNY
ADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNAANWGLAGNEYWGQGTQVTVSS 57G01
81 QVQLQESGGGLVQAGGSLRPSCTASGSTLLINSMGWYRQAPGKQRELVATISNSGTTNY
VDAVKGRFAISRDNANHTVYLQMNSLEPEDTAVYYCNAQTFWRRNYWGQGTQVTVSS 57G07 82
QVQLQESGGGLVQAGGSLRLSCAVSGSTSRINAMGWYRQAPGKKRESVATIRRGGNTKY
ADSVKGRFTISRDNANNTVYLQLNSLKPEDTAVYYCNAHSWLDYDYWGRGTQVTVSS 57G08 83
QVQLQESGGGLVQAGGSLRLSCASRRRINGITMGWYRQAPGKQRELVATIDIHNSTKYA
DSVKGRFIISRDNGKSMLYLQMNSLKPEDTAVYYCNRIPTFGRYWGQGTQVTVSS 57H08 84
QVQLQESGGGLVQAGGSLRLSCVASGSTFYTFSTKNVGWYRQAPGKQRELVAQQRYDGS
TNYADSLQGRFTSRDNAKRTVYLQMNSLKPEDTAVYICNVNRGFISYWGQGTQVTVSS
[0138] In particular, the invention in some specific embodiments
provides agrochemical compositions comprising at least one heavy
chain variable domain that is directed against a sphingolipid
target and that has at least 80%, preferably at least 85%, such as
90% or 95% or more sequence identity with at least one of the heavy
chain variable domains of SEQ ID NO's: 1 to 84 (see Table 1), and
nucleic acid sequences that encode such heavy chain variable
domains.
[0139] Some particularly preferred heavy chain variable domain
sequences as disclosed herein are those which can bind to and/or
are directed against a sphingolipid of a plant pathogen and which
have at least 90% amino acid identity with at least one of the
heavy chain variable domains of SEQ ID NO's: 1 to 84 (see Table 1),
in which for the purposes of determining the degree of amino acid
identity, the amino acid residues that form the CDR sequences are
disregarded. [0140] In these heavy chain variable domains, the CDR
sequences (see Table 2) are generally as further defined
herein.
TABLE-US-00002 [0140] TABLE 2 CDR sequences CDR1 SEQ CDR2 SEQ CDR3
SEQ Name sequence ID sequence ID sequence ID 40F07 SYTMG 85
SIEGGGNTDYADSVKG 169 ARTWSIFRNY 253 41D01 RYGMG 86 SITRGGTTTYADSVKG
170 RSIWRDY 254 41D06 INAMR 87 SISSGGNTNYSESVKG 171 VRLWFPDY 255
41G10 INAMG 88 TITSGGTTNYADSVKG 172 EARRYFTRASQVY 256 41H05 INAMG
89 TITSGANTNYTDSVKG 173 VGRRWYGGYVEL 257 42C11 TYVMG 90
TITSSGKTNYAASVKG 174 DRWVLTRWSNY 258 42C12 ISSLG 91
SATSGGDTTYADSVKG 175 QRGVAWTRKEY 259 50D03 TYAMG 92
TITSSGKTNYAASVKG 176 DRWVLTRWSNY 260 50D07 IRDMG 93
TITSDQSTNYADSVKG 177 RVRTVLRGWRDY 261 50E02 INAMG 94
AITSDGSTNYADSVKG 178 RRRTFLKSSDY 262 51B08 SYAMG 95
GISSGGSTKYADSVRG 179 KYGRWTYTGRPEYDS 263 51C06 SDTMG 96
AITTGGNTNYADSVKG 180 RRRWSRDF 264 51C08 IKTMG 97 TISNGGSTNYADSVKG
181 RQQFIGAPYEY 265 52A01 LGTMG 98 SISTGSTNYADSVKG 182 RLLWSNY 266
52B01 INVMG 99 TISRGGSTNYADSVKG 183 AGWVGVTNY 267 52G05 ISAMG 100
SITRRGSTNYADSVKD 184 RRYYTRNDY 268 53A01 INTMG 101 SISSGGWTNYADSVKG
185 GAIGNW 269 53F05 LNAMG 102 SITTGGSTNYAEPVKG 186 ERRWGLPNY 270
54A02 RYGMG 103 ANRWSGGSTYYADSVRG 187 YAHITAWGMRNDYEYEDY 271 54B01
RYTMG 104 GITWTGGSTDYADSVKG 188 GNLLRLAGQLRRGYDS 272 54C01 RYAMG
105 AISWSGGSTYYADSVKD 189 RNRAGPHYSRGYTAGQEYDY 273 54C04 INAMG 106
DMTSGGSINYADSVSG 190 NLRTAFWRNGNDY 274 54C08 INAMG 107
SITSGGSTNYADSVKG 191 GPWYRRS 275 54C10 VNDMG 108 QITRRGSTNYADSVKG
192 DLAVRGRY 276 54C11 VNDMA 109 NITRGGSTNYADSVKG 193 RIGFGWTAKAY
277 54D03 INAMR 110 SISSGGNTNYSESVKG 194 VRLWFPDY 278 54D06 INAMG
111 TITRGGITNYADSVKG 195 RSWVGPEY 279 54D10 IHAMG 112
ITSTSGTTDYTDSVKG 196 KTRTWYNGKYDY 280 54E01 INPMG 113
AITSGGSTNYADYVKG 197 RSTLWRRDY 281 54E05 INTMG 114 AITNRGSTNYADFVKG
198 HRSWPRYDS 282 54E10 FNAMG 115 AITRGGSTNYADSVKG 199 ESRIFRRYDY
283 54F01 LNLMG 116 TITRGGSTNYADSVKG 200 DRGWSSY 284 54F02 INTMG
117 TITSGGTTNYADSVKN 201 HQRAWARSYVY 285 54G01 VNAMG 118
IISSNSTSNYADSVKG 202 KRSWFSQEY 286 54G08 FNLMG 119 AITSSSNTNYADSVKG
203 QYTITPWGIKKDY 287 54G09 IRDMG 120 TITSDQSTNYADSVKG 204
RVRTVLRGWRDY 288 55B02 INSMN 121 DMRSDGSTNYADSVKG 205 NSIFRSRDY 289
55B05 GYTVA 122 RISWSGIMAYYAESVKG 206 RSQIRSPWSSLDDYDR 290 55C05
MKAMG 123 QITRGDSTNYADSVKG 207 DRFFGRDY 291 55D08 ISAMG 124
TITSAGSSNYSDSVKG 208 VYSRPLLGPLEV 292 55E07 IYGMG 126
RITSGGSTYYADSVKG 210 GVVVATSPKFYAY 294 55E09 TYTMG 127
AIIWSGGRTRYADSVKG 211 RRLGTGY 295 55E10 IQTIG 128 TISSGGSTNYADSVKG
212 RYWFRDY 296 55F04 INVRG 129 TITSDGSTNYADSVKG 213 VRLFRQY 297
55F09 LNAMG 130 AITPGGGNTTYADSVKG 214 GGSSRWYSSRYYPGGY 298 55F10
RYAMG 131 TIRRSGSSTYYLDSTKG 215 DSSARALVGGPGNRWDY 299 55G02 INVMG
132 FITSGGITNYTDSVKG 216 KNAKNVRPGY 300 55G08 INAMR 133
SISSGGTTDYVESVKG 217 VRFWFPDY 301 56A05 SNTMG 134 SISSGGSTNYADSVKG
218 RRNVFISS 302 56A06 VYGMG 135 RITNIGTTNYADSVKG 219 RRLGRDY 303
56A09 LNSMG 136 TITRGGTTNYADSVKG 220 NFGILVGREY 304 56C09 ILSMA 137
NITSVGSTNYADSVKG 221 RMPFLGDS 305 56C12 NRAVS 138 SISGIRITTYTNSVKG
221 NRY 56D06 ISAMG 139 GISRGGSTKYGDFVKG 223 TSITGTYL 306 56D07
MKVMG 140 VITSGGRTNYAESVKG 224 KTIRPY 307 56D10 ITTMG 141
SSSSGGTTNYASSVKG 225 RKFITTPWSTDY 308 56E04 HKATG 142
KITTGGTTNYADSVKG 226 ERYFATTL 309 56E05 NNAGG 143 RISSGGNTNYTDSVKG
227 QRRVILGPRNY 310 56E08 INDMG 144 TITSANITNYADSVKG 228
QAKKWRIGPWSDY 311 56F07 INDMA 145 IITNDDSTTYADSVKG 229 DINTAIWRRKY
312 56F11 RNAMG 146 TITSGGTTNYADSVKG 230 NTRRIFGGTVREY 313 56G07
IHDMG 147 TITPFGRRNYSEYVKG 231 RVNGVDY 314 56G08 ISRMG 148
TLSRAGTSRYVDSVKG 232 AQLGTDY 315 56G10 MYQVG 149 EISSRGTTMYADSVKG
233 RAFAFGRNS 316 56H04 NKAMG 150 RISTVGTAHYADSVKG 234 QAGRLYLRNY
317 56H05 FNTMA 151 QINNRDNTEYADSVKG 235 KRWSWSTGF 318 56H07 LGTMG
152 SISTGSTNYADSVKG 236 RLWWSNY 319 56H08 VNPMG 153
VISSDGSTNYADSVKG 237 NRRWSWGSEY 320 57A06 NNAGG 154
RISSGGNTNYTDSVKG 238 QRRVILGPRNY 321 57B01 INAMA 155
RISSGGSTNYADSVKG 239 NRHWGWDY 322 57B07 INAMG 156 TVDSGGYTNYADSVKG
240 GIYKWPWSVDARDY 323 57B11 MNSMG 157 LIRSSGGTYYADSVKG 241
RRTWLSSES 324 57C07 INTMG 158 SISRGGMTNYADSVKG 242 GIRSRWYGGPITTY
325 57C09 INAMG 159 SISSGGATNYADSVKG 243 KKSRWSWSIVHDY 326 57D02
RSDMG 160 TITRRSRTNYAEFVKG 244 RWGAGGIFST 327 57D09 IDAMG 161
SITTGGSTNYADSVKG 245 KVRLRWFRPPSDY 328 57D10 ISTMG 162
SITKDGTTNYADSVKG 246 RATTWVPYRRDAEF 329 57E07 INDMG 163
DITRSGSTHYVDSVKG 247 DSGSHWWNRRDY 330 57E11 INTMG 164
RISRLRVTNYADSVKG 248 ANWGLAGNEY 331 57G01 INSMG 165
TISNSGTTNYVDAVKG 249 QTFWRRNY 332 57G07 INAMG 166 TIRRGGNTKYADSVKG
250 HSWLDYDY 333 57G08 GITMG 167 TIDIHNSTKYADSVKG 251 IPTFGRY 334
57H08 TKNVG 168 QQRYDGSTNYADSLQG 252 NRGFISY 335
[0141] Again, such heavy chain variable domains may be derived in
any suitable manner and from any suitable source, and may for
example be naturally occurring V.sub.HH sequences (i.e. from a
suitable species of Camelid) or synthetic or semi-synthetic heavy
chain variable domains, including but not limited to "camelized"
immunoglobulin sequences (and in particular camelized heavy chain
variable domain sequences), as well as those that have been
obtained by techniques such as affinity maturation (for example,
starting from synthetic, random or naturally occurring
immunoglobulin sequences), CDR grafting, veneering, combining
fragments derived from different immunoglobulin sequences, PCR
assembly using overlapping primers, and similar techniques for
engineering immunoglobulin sequences well known to the skilled
person; or any suitable combination of any of the foregoing as
further described herein.
[0142] It is understood that the agrochemical compositions or the
biological control compositions as disclosed herein are stable,
both during storage and during utilization, meaning that the
integrity of the agrochemical composition is maintained under
storage and/or utilization conditions of the agrochemical
composition, 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
polypeptide of between 80 and 200 amino acids, and the various
sub-ranges described herein, remain stable in the agrochemical
composition, meaning that the integrity and the pesticidal activity
of the polypeptide is maintained under storage and/or utilization
conditions of the agrochemical composition, which may include
elevated temperatures, freeze-thaw cycles, changes in pH or in
ionic strength, UV-irradiation, presence of harmful chemicals and
the like. Most preferably, said polypeptide of between 80 and 200
amino acids, and the various sub-ranges described herein, remain
stable in the agrochemical composition when the agrochemical
composition is stored at ambient temperature for a period of two
years or when the agrochemical composition is stored at 54.degree.
C. for a period of two weeks. Preferably, the agrochemical
composition of the present invention retains at least about 70%
activity, more preferably at least about 70% to 80% activity, most
preferably about 80% to 90% activity or more. Optionally, the
polypeptide may be comprised in a carrier, as defined, to protect
the polypeptide from harmful effects caused by other components in
the agrochemical composition or from harmful effects during storage
or during application. Examples of suitable carriers include, but
are not limited to alginates, gums, starch, .beta.-cyclodextrins,
celluloses, polyurea, polyurethane, polyester, microbial cells or
clay.
[0143] The agrochemical composition may occur in any type of
formulation, preferred formulations are powders, wettable powders,
wettable granules, water dispersible granules, emulsions,
emulsifiable concentrates, dusts, suspensions, suspension
concentrates, suspoemulsions (mixtures of suspensions and
emulsions), capsule suspensions, aqueous dispersions, oil
dispersions, aerosols, pastes, foams, slurries or flowable
concentrates.
[0144] The polypeptide of between 80 and 200 amino acids, and the
various sub-ranges described herein before, may be the only active
substance in the agrochemical or biological control composition
according to the invention; however, it is also possible that the
agrochemical composition comprises one or more additional
agrochemicals, as defined, in addition to the polypeptide or amino
acid sequence (or the at least one, at least two or at least three
polypeptides or amino acid sequences as disclosed herein). Such
additional agrochemicals or biological control compositions may
have a different effect on plant pests as the polypeptide or amino
acid sequence, they may have a synergistic effect with the
polypeptide or amino acid sequence, or they may even modify the
activity of the polypeptide or amino acid sequence on certain
plants. Suitable additional agrochemicals can be herbicides,
insecticides, fungicides, nematicides, acaricides, bactericides,
viricides, plant growth regulators, safeners and the like and
include, but are not limited to glyphosate, paraquat, metolachlor,
acetochlor, mesotrione, 2,4-D,atrazine, glufosinate, sulfosate,
fenoxaprop, pendimethalin, picloram, trifluralin, bromoxynil,
clodinafop, fluroxypyr, nicosulfuron, bensulfuron, imazetapyr,
dicamba, imidacloprid, thiamethoxam, fipronil, chlorpyrifos,
deltamethrin, lambda-cyhalotrin, endosulfan, methamidophos,
carbofuran, clothianidin, cypermethrin, abamectin, diflufenican,
spinosad, indoxacarb, bifenthrin, tefluthrin, azoxystrobin,
thiamethoxam, tebuconazole, mancozeb, cyazofamid, fluazinam,
pyraclostrobin, epoxiconazole, chlorothalonil, copper fungicides,
trifloxystrobin, prothioconazole, difenoconazole, carbendazim,
propiconazole, thiophanate, sulphur, boscalid and other known
agrochemicals or any suitable combination(s) thereof.
[Compositions Comprising Variants of Heavy Chain Variable Domain
Sequences]
[0145] In a certain aspects, the heavy chain variable domains
comprised in the agrochemical compositions as disclosed herein may
be optionally linked to one or more further groups, moieties, or
residues via one or more linkers. These one or more further groups,
moieties or residues can serve for binding to other targets of
interest. It should be clear that such further groups, residues,
moieties and/or binding sites may or may not provide further
functionality to the heavy chain variable domains as disclosed
herein (and/or to the composition in which it is present) and may
or may not modify the properties of the heavy chain variable domain
as disclosed herein. Such groups, residues, moieties or binding
units may also for example be chemical groups which can be
biologically active.
[0146] These groups, moieties or residues are, in particular
embodiments, linked N- or C-terminally to the heavy chain variable
domain in the compositions as disclosed herein.
[0147] In particular embodiments, the heavy chain variable domains
in the agrochemical compositions as disclosed herein may also have
been chemically modified. For example, such a modification may
involve the introduction or linkage of one or more functional
groups, residues or moieties into or onto the heavy chain variable
domain. These groups, residues or moieties may confer one or more
desired properties or functionalities to the heavy chain variable
domain. Examples of such functional groups will be clear to the
skilled person.
[0148] For example, the introduction or linkage of such functional
groups to a heavy chain variable domain can result in an increase
in the solubility and/or the stability of the heavy chain variable
domain, in a reduction of the toxicity of the heavy chain variable
domain, or in the elimination or attenuation of any undesirable
side effects of the heavy chain variable domain, and/or in other
advantageous properties.
[0149] In particular embodiments, the one or more groups, residues,
moieties are linked to the heavy chain variable domain via one or
more suitable linkers or spacers.
[0150] In further particular embodiments, two or more
target-specific heavy chain variable domains in the agrochemical
compositions disclosed herein may be linked to each other or may be
interconnected. In particular embodiments, the two or more heavy
chain variable domains are linked to each other via one or more
suitable linkers or spacers. Suitable spacers or linkers for use in
the coupling of different heavy chain variable domains as disclosed
herein will be clear to the skilled person and may generally be any
linker or spacer used in the art to link peptides and/or
proteins.
[0151] Some particularly suitable linkers or spacers include for
example, but are not limited to, polypeptide linkers such as
glycine linkers, serine linkers, mixed glycine/serine linkers,
glycine- and serine-rich linkers or linkers composed of largely
polar polypeptide fragments, or homo- or heterobifunctional
chemical crosslinking compounds such as glutaraldehyde or,
optionally PEG-spaced, maleimides or NHS esters.
[0152] For example, a polypeptide linker or spacer may be a
suitable amino acid sequence having a length between 1 and 50 amino
acids, such as between 1 and 30, and in particular between 1 and 10
amino acid residues. It should be clear that the length, the degree
of flexibility and/or other properties of the linker(s) may have
some influence on the properties of the heavy chain variable
domains, including but not limited to the affinity, specificity or
avidity for the fungal target. It should be clear that when two or
more linkers are used, these linkers may be the same or different.
In the context and disclosure of the present invention, the person
skilled in the art will be able to determine the optimal linkers
for the purpose of coupling heavy chain variable domains as
disclosed herein without any undue experimental burden.
[[Compositions Comprising Fragments of Heavy Chain Variable
Domains]
[0153] The present invention also encompasses parts, fragments,
analogs, mutants, variants, and/or derivatives of the heavy chain
variable domains comprised in the compositions as disclosed herein
and/or polypeptides comprising or essentially consisting of one or
more of such parts, fragments, analogs, mutants, variants, and/or
derivatives, as long as these parts, fragments, analogs, mutants,
variants, and/or derivatives are suitable for the purposes
envisaged herein. Such parts, fragments, analogs, mutants,
variants, and/or derivatives according to the invention are still
capable of specifically binding to the sphingolipid target.
[Targets]
[0154] In particular embodiments, the heavy chain variable domains
comprised in the compositions disclosed herein are obtained by
affinity selection against a particular pest target. Obtaining
suitable polypeptides by affinity selection against a particular
pest target may for example be performed by screening a set,
collection or library of cells that express polypeptides on their
surface (e.g. bacteriophages) for binding against a pest target
molecule, which molecule is known in the art to be a target for a
pesticide; all of which may be performed in a manner known per se,
essentially comprising the following non-limiting steps: a)
obtaining an isolated solution or suspension of a pest target
molecule, which molecule is known to be a target for a pesticide;
b) bio-panning phages or other cells from a polypeptide library
against said target molecule; c) isolating the phages or other
cells binding to the target molecule; d) determining the nucleotide
sequence encoding the polypeptide insert from individual binding
phages or other cells; e) producing an amount of polypeptide
according to this sequence using recombinant protein expression and
f) determining the affinity of said polypeptide for said pest
target and optionally g) testing the pesticidal activity of said
polypeptide in a bio-assay for said pest. Various methods may be
used to determine the affinity between the polypeptide and the pest
target molecule, including for example, enzyme linked immunosorbent
assays (ELISA) or Surface Plasmon Resonance (SPR) assays, which are
common practice in the art, for example, as described in Sambrook
et al. (2001), Molecular Cloning, A Laboratory Manual. Third
Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y. The dissociation constant is commonly used to describe the
affinity between a polypeptide and its pest target molecule.
Typically, the dissociation constant of the binding between the
polypeptide and its pest target molecule is lower than 10.sup.-6 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.
[0155] Pest target molecules as disclosed herein are molecules
occurring in or on pest organisms and which, when bound and/or
inhibited, kill or arrest, inhibit or reduce the growth or
pesticidal activity of said pest organism. Such suitable target
molecules are readily available from existing literature or patent
databases for the skilled person and include, without limitation
secreted parasitism proteins such as 16D10 as suitable pest target
molecules for root knot nematodes (Huang et al (2006) PNAS 103:
14302-14306), the V-ATPase proton pump as suitable pest target
molecule for coleopteran, hemipteran, dipteran insect species and
nematodes (Knight A J and Behm C A (2011) Ex. Parasitol. September
19), the tetraspanin PLS1 as suitable fungal pest target molecule
for B. cinerea and M. grisea (Gourgues et al (2002) Biochem.
Biophys. Res. Commun. 297: 1197) or the proton-pumping-ATPase as
antifungal target (Manavathu E K et al (1999) Antimicrob Agents and
Chemotherapy, December p. 2950). It is understood that preferred
pest target molecules are accessible in the extra-cellular space
(as opposed to intracellular pest targets).
[0156] More particularly, the sphingolipid targets to which the at
least on heavy chain variable domain of the agrochemical
compositions as disclosed herein bind, constitute a distinctive
group of membrane lipids characterized by a long-chain
(monounsaturated), di-hydroxy amine structure (sphingosine).
Sphingolipids are essential components of the plasma membrane of
cells where they are typically found in the outer leaflet. They are
membrane constituents of some bacterial groups, particularly
anaerobes. These groups include Bacteroides, Prevotella,
Porphyromonas, Fusobacterium, Sphingomonas, Sphingobacterium,
Bdellovibrio, Cystobacter, Mycoplasma, Flectobacillus, and possibly
Acetobacter. Fungi in which sphingolipids have been found comprise
Saccharomyces, Candida, Histoplasma, Phytophthora, Cryptococcus,
Aspergillus, Neurospora, Schizosaccharomyces, Fusicoccum,
Shizophyllum, Amanita, Hansenula, Lactarius, Lentinus, Penicillium,
Clitocybe, Paracoccidioides, Agaricus, Sporothrix, and oomycete
plant pathogens.
[0157] The basic building block of fungal sphingolipids is
sphinganine, which can be converted either to ceramide and finally
to ceramide monohexosides (CMH; cerebrosides), or to phytoceramide
and finally to ceramide dihexosides (CDH) or to glycoinositol
phosphorylceramides (GIPCs). Non-limiting examples of
sphinglolipids against which the at least one heavy chain variable
antibody domains of the compositions as disclosed herein are
directed include for instance 9-methyl 4,8-sphingadienine,
glycosylceramides, glucosylceramide, monoglucosylceramides,
oligoglucosylceramides, gangliosides, sulfatides, ceramides,
sphingosine-1-phosphate, ceramide-1-phosphate, galactosylceramide,
inositol-phosphorylceramide (IPC),
mannosyl-inositol-phosphorylceramide (MIPC),
galactosyl-inositol-phosphorylceramide,
mannosyl-(inositol-phosphoryl).sub.2-ceramide (M(IP).sub.2C),
dimannosyl-inositol-phosphorylceramide (M2IPC),
galactosyl-dimannosyl-inositol-phosphorylceramide (GaIM2IPC),
mannosyl-di-inositol-diphosphorylceramide,
di-inositol-diphosphorylceramide,
trigalactosyl-glycosylceramide.
[0158] Non-limiting examples of sphingolipids against which the at
least one heavy chain variable antibody domains of the compositions
as disclosed herein are directed include for instance
glycosylceramides, glucosylceramide, sphingomyelin,
monoglycosylceramides, oligoglycosylceramides, gangliosides,
sulfatides, ceramides, sphingosine-1-phosphate and
ceramide-1-phosphate.
[0159] In certain specific embodiments, the target to which the
variable domains in the agrochemical compositions of the present
invention bind is not chitin.
[0160] In a preferred embodiment, the plant pest(s) that is/are
combated by the agrochemical composition or biological control
composition as disclosed herein is a fungus, such as a plant
pathogenic fungus, as defined before. Fungi can be highly
detrimental for plants and can cause substantial harvest losses in
crops. Plant pathogenic fungi include necrotrophic fungi and
biotrophic fungi, and include ascomycetes, basidiomycetes and
oomycetes.
[0161] Examples of plant pathogenic fungi are known in the art and
include, but are not limited to, those selected from the group
consisting of the Genera: Alternaria; Ascochyta; Botrytis;
Cercospora; Colletotrichum; Diplodia; Erysiphe; Fusarium;
Leptosphaeria; Gaeumanomyces; Helminthosporium; Macrophomina;
Nectria; Peronospora; Phoma; Phymatotrichum; Phytophthora;
Plasmopara; Podosphaera; Puccinia; Puthium; Pyrenophora;
Pyricularia; Pythium; Rhizoctonia; Scerotium; Sclerotinia;
Septoria; Thielaviopsis; Uncinula; Venturia; and Verticillium.
Specific examples of plant fungi infections which may be combated
with the agrochemical compositions of the invention include,
Erysiphe graminis in cereals, Erysiphe cichoracearum and
Sphaerotheca fuliginea in cucurbits, Podosphaera leucotricha in
apples, Uncinula necator in vines, Puccinia sp. in cereals,
Rhizoctonia sp. in cotton, potatoes, rice and lawns, Ustilago sp.
in cereals and sugarcane, Venturia inaequalis (scab) in apples,
Helminthosporium sp. in cereals, Septoria nodorum in wheat,
Septoria tritici in wheat, Rhynchosporium secalis on barley,
Botrytis cinerea (gray mold) in strawberries, tomatoes and grapes,
Cercospora arachidicola in groundnuts, Peronospora tabacina in
tobacco, or other Peronospora in various crops, Pseudocercosporella
herpotrichoides in wheat and barley, Pyrenophera teres in barley,
Pyricularia oryzae in rice, Phytophthora infestans in potatoes and
tomatoes, Fusarium sp. (such as Fusarium oxysporum) and
Verticillium sp. in various plants, Plasmopara viticola in grapes,
Alternaria sp. in fruit and vegetables, Pseudoperonospora cubensis
in cucumbers, Mycosphaerella fijiensis in banana, Ascochyta sp. in
chickpeas, Leptosphaeria sp. on canola, and Colleotrichum sp. in
various crops. The compositions according to the invention are
active against normally sensitive and resistant species and against
all or some stages in the life cycle of the plant pathogenic
fungus.
[0162] In particular embodiments, the agrochemical compositions as
disclosed herein are directed against a plant pathogenic fungus
from the genus chosen from the group comprising Alternaria,
Ascochyta, Botrytis, Cercospora, Colletotrichum, Diplodia,
Erysiphe, Fusarium, Leptosphaeria, Gaeumanomyces, Helminthosporium,
Macrophomina, Nectria, Penicillium, Peronospora, Phoma,
Phymatotrichum, Phytophthora, Plasmopara, Podosphaera, Puccinia,
Pyrenophora, Pyricularia, Pythium, Rhizoctonia, Scerotium,
Sclerotinia, Septoria, Thielaviopsis, Uncinula, Venturia,
Verticillium, Magnaporthe, Blumeria, Mycosphaerella, Ustilago,
Melampsora, Phakospora, Monilinia, Mucor, Rhizopus, and
Aspergillus.
[0163] In certain particular embodiments, the compositions as
disclosed herein at least comprise a heavy chain variable domain,
which specifically binds to a sphingolipid of a fungus from the
fungal species Botrytis, Fusarium or Penicillium. In further
particular embodiments, the fungal sphingolipid is a ceramide, such
as in particular glucosylceramide.
[0164] Indeed, in particular embodiments, the present invention
provides agrochemical compositions comprising heavy chain variable
domains that are specifically directed against a structural
molecular component of the fungus, i.e. a fungal sphingolipid. The
inventors have surprisingly succeeded in identifying such heavy
chain variable domains while it is generally described in the art
that it is (technically) difficult to generate proteins or amino
acid sequences having a unique and specific interaction with
non-protein molecular structures.
[0165] Based on the present teaching, further non-limiting examples
of suitable fungal pest target molecules can be envisaged by the
person skilled in the art and comprise for example chitin synthase,
.beta.-1,3-glucan synthase, succinate dehydrogenase, fungal
glycosylceramides, or the tetraspanin PLS1.
[0166] Also disclosed herein are plant pathogenic bacteria
including, but not limited to, Acidovorax avenae subsp. avenae
(causing bacterial brown stripe of rice), Acidovorax avenae subsp.
cattleyae (causing bacterial brown spot of cattleya), Acidovorax
konjaci Konnyaku (causing bacterial leaf blight), Agrobacterium
rhizogenes (causing hairy root of melon), Agrobacterium tumefaciens
(causing crown gall), Burkholderia andropogonis (causing bacterial
spot of carnation), Burkholderia caryophylli (causing bacterial
wilt of carnation), Burkholderia cepacia (causing bacterial brown
spot of cymbidium), Burkholderia gladioli pv. gladioli (causing
neck rot of gladiolus), Burkholderia glumae (causing bacterial
grain rot of rice), Burkholderia plantarii (causing bacterial
seedling blight of rice), Clavibacter michiganensis subsp.
michiganensis (causing bacterial canker of tomato), Clavibacter
michiganensis subsp. sepedonicus (causing ring rot of potato),
Clostridium spp. (causing slimy rot of potato), Curtobacterium
flaccumfaciens (causing bacterial canker of onion), Erwinia
amylovora (causing fire blight of pear), Erwinia ananas (causing
bacterial palea browning of rice), Erwinia carotovora subsp.
atroseptica (causing black leg of potato), Erwinia carotovora
subsp. carotovora (causing bacterial soft rot of vegetables),
Erwinia chrysanthemi (causing bacterial seedling blight of taro),
Erwinia chrysanthemi pv. zeae (causing bacterial foot rot of rice),
Erwinia herbicola pv. millettiae (causing bacterial gall of
wisteria), Pseudomonas cichorii (causing bacterial spot of
chrysanthemum), Pseudomonas corrugate Pith (causing necrosis of
tomato), Pseudomonas fuscovaginae (causing sheath brown rot of
rice), Pseudomonas marginalis pv. marginalis (causing soft rot of
cabbage) Pseudomonas rubrisubalbicans (causing mottled stripe of
sugar cane), Pseudomonas syringae pv. aptata (causing bacterial
blight of sugar beet), Pseudomonas syringae pv. atropurpurea
(causing halo blight of ryegrass), Pseudomonas syringae pv.
castaneae (causing bacterial canker of chestnut), Pseudomonas
syringae pv. glycinea (causing bacterial blight of soybean),
Pseudomonas syringae pv. lachrymans (causing bacterial spot of
cucumber), Pseudomonas syringae pv. maculicola (causing bacterial
black spot of cabbage), Pseudomonas syringae pv. mori (causing
bacterial blight of mulberry), Pseudomonas syringae pv.
morsprunorum (causing bacterial canker of plums), Pseudomonas
syringae pv. oryzae (causing halo blight of rice), Pseudomonas
syringae pv. phaseolicola (causing halo blight of kidney bean),
Pseudomonas syringae pv. pisi (causing bacterial blight of garden
pea), Pseudomonas syringae pv. sesame (causing bacterial spot of
sesame), Pseudomonas syringae pv. striafaciens (causing bacterial
stripe blight of oats), Pseudomonas syringae pv. syringae (causing
bacterial brown spot of small red bead), Pseudomonas syringae pv.
tabaci (causing wild fire of tobacco), Pseudomonas syringae pv.
theae (causing bacterial shoot blight of tea), Pseudomonas syringae
pv. tomato (causing bacterial leaf spot of tomato), Pseudomonas
viridiflava (causing bacterial brown spot of kidney bean),
Ralstonia solanacearum (causing bacterial wilt), Rathayibacter
rathayi (causing bacterial head blight of orchardgrass),
Streptomyces scabies (causing common scab of potato), Streptomyces
ipomoea (causing soil rot of sweet potato), Xanthomonas albilineans
(causing white streak of sugar cane), Xanthomonas campestris pv.
cerealis (causing bacterial streak of rye), Xanthomonas campestris
pv. campestris (causing black rot), Xanthomonas campestris pv.
citri (causing canker of citrus), Xanthomonas campestris pv.
cucurbitae (causing bacterial brown spot of cucumber), Xanthomonas
campestris pv. glycines (causing bacterial pastule of soybean),
Xanthomonas campestris pv. incanae (causing black rot of stock),
Xanthomonas campestris pv. (causing angular leaf spot of cotton
malvacearum), Xanthomonas campestris pv. (causing bacterial canker
of mango), Mangiferaeindicae Xanthomonas campestris pv. mellea
(causing wisconsin bacterial leaf spot of tobacco), Xanthomonas
campestris pv. (causing bacterial spot of great nigromaculans
burdock), Xanthomonas campestris pv. phaseoli (causing bacterial
pastule of kidney bean), Xanthomonas campestris pv. pisi (causing
bacterial stem-rot of kidney bean), Xanthomonas campestris pv.
pruni (causing bacterial shot hole of peach), Xanthomonas
campestris pv. raphani (causing bacterial spot of Japanese radish),
Xanthomonas campestris pv. ricini (causing bacterial spot of
castor-oil plant), Xanthomonas campestris pv. theicola (causing
canker of tea), Xanthomonas campestris pv. translucens (causing
bacterial blight of orchardgrass), Xanthomonas campestris pv.
vesicatoria (causing bacterial spot of tomato), Xanthomonas oryzae
pv. oryzae (causing bacterial leaf blight of rice).
[0167] Also disclosed herein are plant pests such as insects,
arachnids, helminths, viruses, nematodes and molluscs encountered
in agriculture, in horticulture, in forests, in gardens and in
leisure facilities. The compositions according to the invention are
active against normally sensitive and resistant species and against
all or some stages of development. These plant pests include: pests
from the phylum: Arthropoda, in particular from the class of the
arachnids, for example Acarus spp., Aceria sheldoni, Aculops spp.,
Aculus spp., Amblyomma spp., Amphitetranychus viennensis, Argas
spp., Boophilus spp., Brevipalpus spp., Bryobia praetiosa,
Centruroides spp., Chorioptes spp., Dermanyssus gallinae,
Dermatophagoides pteronyssius, Dermatophagoides farinae,
Dermacentor spp., Eotetranychus spp., Epitrimerus pyri,
Eutetranychus spp., Eriophyes spp., Halotydeus destructor,
Hemitarsonemus spp., Hyalomma spp., Ixodes spp., Latrodectus spp.,
Loxosceles spp., Metatetranychus spp., Nuphersa spp., Oligonychus
spp., Ornithodorus spp., Ornithonyssus spp., Panonychus spp.,
Phyllocoptruta oleivora, Polyphagotarsonemus latus, Psoroptes spp.,
Rhipicephalus spp., Rhizoglyphus spp., Sarcoptes spp., Scorpio
maurus, Stenotarsonemus spp., Tarsonemus spp., Tetranychus spp.,
Vaejovis spp., Vasates lycopersici. Still other examples are from
the order of the Anoplura (Phthiraptera), for example, Damalinia
spp., Haematopinus spp., Linognathus spp., Pediculus spp., Ptirus
pubis, Trichodectes spp.
[0168] Still other examples are from the order of the Chilopoda,
for example, Geophilus spp., Scutigera spp.
[0169] Still other examples are from the order of the Coleoptera,
for example, Acalymma vittatum, Acanthoscelides obtectus, Adoretus
spp., Agelastica alni, Agriotes spp., Alphitobius diaperinus,
Amphimallon solstitialis, Anobium punctatum, Anoplophora spp.,
Anthonomus spp., Anthrenus spp., Apion spp., Apogonia spp.,
Atomaria spp., Attagenus spp., Bruchidius obtectus, Bruchus spp.,
Cassida spp., Cerotoma trifurcata, Ceutorrhynchus spp., Chaetocnema
spp., Cleonus mendicus, Conoderus spp., Cosmopolites spp.,
Costelytra zealandica, Ctenicera spp., Curculio spp.,
Cryptorhynchus lapathi, Cylindrocopturus spp., Dermestes spp.,
Diabrotica spp., Dichocrocis spp., Diloboderus spp., Epilachna
spp., Epitrix spp., Faustinus spp., Gibbium psylloides, Hellula
undalis, Heteronychus arator, Heteronyx spp., Hylamorpha elegans,
Hylotrupes bajulus, Hypera postica, Hypothenemus spp., Lachnosterna
consanguinea, Lema spp., Leptinotarsa decemlineata, Leucoptera
spp., Lissorhoptrus oryzophilus, Lixus spp., Luperodes spp., Lyctus
spp., Megascelis spp., Melanotus spp., Meligethes aeneus,
Melolontha spp., Migdolus spp., Monochamus spp., Naupactus
xanthographus, Niptus hololeucus, Oryctes rhinoceros, Oryzaephilus
surinamensis, Oryzaphagus oryzae, Otiorrhynchus spp., Oxycetonia
jucunda, Phaedon cochleariae, Phyllophaga spp., Phyllotreta spp.,
Popillia japonica, Premnotrypes spp., Prostephanus truncatus,
Psylliodes spp., Ptinus spp., Rhizobius ventralis, Rhizopertha
dominica, Sitophilus spp., Sphenophorus spp., Stegobium paniceum,
Sternechus spp., Symphyletes spp., Tanymecus spp., Tenebrio
molitor, Tribolium spp., Trogoderma spp., Tychius spp., Xylotrechus
spp., Zabrus spp.
[0170] Still other examples are from the order of the Collembola,
for example, Onychiurus armatus.
[0171] Still other examples are from the order of the Diplopoda,
for example, Blaniulus guttulatus.
[0172] Still other examples are from the order of the Diptera, for
example, Aedes spp., Agromyza spp., Anastrepha spp., Anopheles
spp., Asphondylia spp., Bactrocera spp., Bibio hortulanus,
Calliphora erythrocephala, Ceratitis capitata, Chironomus spp.,
Chrysomyia spp., Chrysops spp., Cochliomyia spp., Contarinia spp.,
Cordylobia anthropophaga, Culex spp., Culicoides spp., Culiseta
spp., Cuterebra spp., Dacus oleae, Dasyneura spp., Delia spp.,
Dermatobia hominis, Drosophila spp., Echinocnemus spp., Fannia
spp., Gasterophilus spp., Glossina spp., Haematopota spp.,
Hydrellia spp., Hylemyia spp., Hyppobosca spp., Hypoderma spp.,
Liriomyza spp., Lucilia spp., Lutzomia spp., Mansonia spp., Musca
spp., Nezara spp., Oestrus spp., Oscinella frit, Pegomyia spp.,
Phlebotomus spp., Phorbia spp., Phormia spp., Prodiplosis spp.,
Psila rosae, Rhagoletis spp., Sarcophaga spp., Simulium spp.,
Stomoxys spp., Tabanus spp., Tannia spp., Tetanops spp., Tipula
spp.
[0173] Still other examples are from the order of the Heteroptera,
for example, Anasa tristis, Antestiopsis spp., Boisea spp., Blissus
spp., Calocoris spp., Campylomma livida, Cavelerius spp., Cimex
spp., Collaria spp., Creontiades dilutus, Dasynus piperis,
Dichelops furcatus, Diconocoris hewetti, Dysdercus spp., Euschistus
spp., Eurygaster spp., Heliopeltis spp., Horcias nobilellus,
Leptocorisa spp., Leptoglossus phyllopus, Lygus spp., Macropes
excavatus, Miridae, Monalonion atratum, Nezara spp., Oebalus spp.,
Pentomidae, Piesma quadrata, Piezodorus spp., Psallus spp.,
Pseudacysta persea, Rhodnius spp., Sahlbergella singularis,
Scaptocoris castanea, Scotinophora spp., Stephanitis nashi, Tibraca
spp., Triatoma spp.
[0174] Still other examples are from the order of the Homoptera,
for example, Acyrthosipon spp., Acrogonia spp., Aeneolamia spp.,
Agonoscena spp., Aleurodes spp., Aleurolobus barodensis,
Aleurothrixus spp., Amrasca spp., Anuraphis cardui, Aonidiella
spp., Aphanostigma pin, Aphis spp., Arboridia apicalis, Aspidiella
spp., Aspidiotus spp., Atanus spp., Aulacorthum solani, Bemisia
spp., Brachycaudus helichrysii, Brachycolus spp., Brevicoryne
brassicae, Calligypona marginata, Carneocephala fulgida,
Ceratovacuna lanigera, Cercopidae, Ceroplastes spp., Chaetosiphon
fragaefolii, Chionaspis tegalensis, Chlorita onukii, Chromaphis
juglandicola, Chrysomphalus ficus, Cicadulina mbila, Coccomytilus
halli, Coccus spp., Cryptomyzus ribis, Dalbulus spp., Dialeurodes
spp., Diaphorina spp., Diaspis spp., Drosicha spp., Dysaphis spp.,
Dysmicoccus spp., Empoasca spp., Eriosoma spp., Erythroneura spp.,
Euscelis bilobatus, Ferrisia spp., Geococcus coffeae, Hieroglyphus
spp., Homalodisca coagulata, Hyalopterus arundinis, lcerya spp.,
Idiocerus spp., Idioscopus spp., Laodelphax striatellus, Lecanium
spp., Lepidosaphes spp., Lipaphis erysimi, Macrosiphum spp.,
Mahanarva spp., Melanaphis sacchari, Metcalfiella spp.,
Metopolophium dirhodum, Monellia costalis, Monelliopsis pecanis,
Myzus spp., Nasonovia ribisnigri, Nephotettix spp., Nilaparvata
lugens, Oncometopia spp., Orthezia praelonga, Parabemisia myricae,
Paratrioza spp., Parlatoria spp., Pemphigus spp., Peregrinus
maidis, Phenacoccus spp., Phloeomyzus passerinii, Phorodon humuli,
Phylloxera spp., Pinnaspis aspidistrae, Planococcus spp.,
Protopulvinaria pyriformis, Pseudaulacaspis pentagona, Pseudococcus
spp., Psylla spp., Pteromalus spp., Pyrilla spp., Quadraspidiotus
spp., Quesada gigas, Rastrococcus spp., Rhopalosiphum spp.,
Saissetia spp., Scaphoides titanus, Schizaphis graminum,
Selenaspidus articulatus, Sogata spp., Sogatella furcifera,
Sogatodes spp., Stictocephala festina, Tenalaphara malayensis,
Tinocallis caryaefoliae, Tomaspis spp., Toxoptera spp.,
Trialeurodes spp., Trioza spp., Typhlocyba spp., Unaspis spp.,
Viteus vitifolii, Zygina spp.
[0175] Still other examples are from the order of the Hymenoptera,
for example, Acromyrmex spp., Athalia spp., Atta spp., Diprion
spp., Hoplocampa spp., Lasius spp., Monomorium pharaonis,
Solenopsis invicta, Tapinoma spp., Vespa spp.
[0176] Still other examples are from the order of the Isopoda, for
example, Armadillidium vulgare, Oniscus asellus, Porcellio
scaber.
[0177] Still other examples are from the order of the Isoptera, for
example, Coptotermes spp., Cornitermes cumulans, Cryptotermes spp.,
Incisitermes spp., Microtermes obesi, Odontotermes spp.,
Reticulitermes spp.
[0178] Still other examples are from the order of the Lepidoptera,
for example, Acronicta major, Adoxophyes spp., Aedia leucomelas,
Agrotis spp., Alabama spp., Amyelois transitella, Anarsia spp.,
Anticarsia spp., Argyroploce spp., Barathra brassicae, Borbo
cinnara, Bucculatrix thurberiella, Bupalus piniarius, Busseola
spp., Cacoecia spp., Caloptilia theivora, Capua reticulana,
Carpocapsa pomonella, Carposina niponensis, Chematobia brumata,
Chilo spp., Choristoneura spp., Clysia ambiguella, Cnaphalocerus
spp., Cnephasia spp., Conopomorpha spp., Conotrachelus spp.,
Copitarsia spp., Cydia spp., Dalaca noctuides, Diaphania spp.,
Diatraea saccharalis, Earias spp., Ecdytolopha aurantium,
Elasmopalpus lignosellus, Eldana saccharina, Ephestia spp.,
Epinotia spp., Epiphyas postvittana, Etiella spp., Eulia spp.,
Eupoecilia ambiguella, Euproctis spp., Euxoa spp., Feltia spp.,
Galleria mellonella, Gracillaria spp., Grapholitha spp., Hedylepta
spp., Helicoverpa spp., Heliothis spp., Hofmannophila
pseudospretella, Homoeosoma spp., Homona spp., Hyponomeuta padella,
Kakivoria flavofasciata, Laphygma spp., Laspeyresia molesta,
Leucinodes orbonalis, Leucoptera spp., Lithocolletis spp.,
Lithophane antennata, Lobesia spp., Loxagrotis albicosta, Lymantria
spp., Lyonetia spp., Malacosoma neustria, Maruca testulalis,
Mamestra brassicae, Mocis spp., Mythimna separata, Nymphula spp.,
Oiketicus spp., Oria spp., Orthaga spp., Ostrinia spp., Oulema
oryzae, Panolis flammea, Parnara spp., Pectinophora spp.,
Perileucoptera spp., Phthorimaea spp., Phyllocnistis citrella,
Phyllonorycter spp., Pieris spp., Platynota stultana, Plodia
interpunctella, Plusia spp., Plutella xylostella, Prays spp.,
Prodenia spp., Protoparce spp., Pseudaletia spp., Pseudoplusia
includens, Pyrausta nubilalis, Rachiplusia nu, Schoenobius spp.,
Scirpophaga spp., Scotia segetum, Sesamia spp., Sparganothis spp.,
Spodoptera spp., Stathmopoda spp., Stomopteryx subsecivella,
Synanthedon spp., Tecia solanivora, Thermesia gemmatalis, Tinea
pellionella, Tineola bisselliella, Tortrix spp., Trichophaga
tapetzella, Trichoplusia spp., Tuta absoluta, Virachola spp.
[0179] Still other examples are from the order of the Orthoptera,
for example, Acheta domesticus, Blatta orientalis, Blattella
germanica, Dichroplus spp., Gryllotalpa spp., Leucophaea maderae,
Locusta spp., Melanoplus spp., Periplaneta spp., Pulex irritans,
Schistocerca gregaria, Supella longipalpa.
[0180] Still other examples are from the order of the Siphonaptera,
for example, Ceratophyllus spp., Ctenocephalides spp., Tunga
penetrans, Xenopsylla cheopis.
[0181] Still other examples are from the order of the Symphyla, for
example, Scutigerella spp.
[0182] Still other examples are from the order of the Thysanoptera,
for example, Anaphothrips obscurus, Baliothrips biformis,
Drepanothris reuteri, Enneothrips flavens, Frankliniella spp.,
Heliothrips spp., Hercinothrips femoralis, Rhipiphorothrips
cruentatus, Scirtothrips spp., Taeniothrips cardamoni, Thrips
spp.
[0183] Still other examples are from the order of the Zygentoma
(=Thysanura), for example, Lepisma saccharina, Thermobia domestica.
for example Lepisma saccharina, Thermobia domestica.
[0184] In another embodiment pests of the phylum Mollusca, in
particular from the class of the Bivalvia, for example Dreissena
spp. are also important plant pests.
[0185] In another embodiment pests of the class of the Gastropoda
are important plant pests, for example, Anion spp., Biomphalaria
spp., Bulinus spp., Deroceras spp., Galba spp., Lymnaea spp.,
Oncomelania spp., Pomacea spp., Succinea spp.
[0186] In yet another embodiment plant pests are from the phylum
Nematoda are important plant pests, i.e. phytoparasitic nematodes,
thus meaning plant parasitic nematodes that cause damage to plants.
Plant nematodes encompass plant parasitic nematodes and nematodes
living in the soil. Plant parasitic nematodes include, but are not
limited to, ectoparasites such as Xiphinema spp., Longidorus spp.,
and Trichodorus spp.; semiparasites such as Tylenchulus spp.;
migratory endoparasites such as Pratylenchus spp., Radopholus spp.,
and Scutellonerna. spp.; sedentary parasites such as Heterodera
spp., Globodera spp., and Meloidogyne spp., and stem and leaf
endoparasites such as Ditylenchus spp., Aphelenchoides spp., and
Hirshmaniella spp. In addition, harmful root parasitic soil
nematodes are cyst-forming nematodes of the genera Heterodera or
Globodera, and/or root knot nematodes of the genus Meloidogyne.
Harmful species of these genera are for example Meloidogyne
incognata, Heterodera glycines (soybean cyst nematode), Globodera
pallida and Globodera rostochiensis (potato cyst nematode). Still
other important genera of importance as plant pests comprise
Rotylenchulus spp., Paratriclodorus spp., Pratylenchus penetrans,
Radolophus simuli, Ditylenchus dispaci, Tylenchulus semipenetrans,
Xiphinema spp., Bursaphelenchus spp., and the like. in particular
Aphelenchoides spp., Bursaphelenchus spp., Ditylenchus spp.,
Globodera spp., Heterodera spp., Longidorus spp., Meloidogyne spp.,
Pratylenchus spp., Radopholus similis, Trichodorus spp.,
Tylenchulus semipenetrans, Xiphinema spp.
[0187] Also disclosed herein as being plant pests are plant viruses
selected from an alfamovirus, an allexivirus, an alphacryptovirus,
an anulavirus, an apscaviroid, an aureusvirus, an avenavirus, an
aysunviroid, a badnavirus, a begomovirus, a benyvirus, a
betacryptovirus, a betaflexiviridae, a bromovirus, a bymovirus, a
capillovirus, a carlavirus, a carmovirus, a caulimovirus, a
cavemovirus, a cheravirus, a closterovirus, a cocadviroid, a
coleviroid, a comovirus, a crinivirus, a cucumovirus, a curtovirus,
a cytorhabdovirus, a dianthovirus, an enamovirus, an umbravirus
& B-type satellite virus, a fabavirus, a fijivirus, a
furovirus, a hordeivirus, a hostuviroid, an idaeovirus, an
ilarvirus, an ipomovirus, a luteovirus, a machlomovirus, a
macluravirus, a marafivirus, a mastrevirus, a nanovirus, a
necrovirus, a nepovirus, a nucleorhabdovirus, an oleavirus, an
ophiovirus, an oryzavirus, a panicovirus, a pecluvirus, a
petuvirus, a phytoreovirus, a polerovirus, a pomovirus, a
pospiviroid, a potexvirus, a potyvirus, a reovirus, a rhabdovirus,
a rymovirus, a sadwavirus, a SbCMV-like virus, a sequivirus, a
sobemovirus, a tenuivirus, a TNsatV-like satellite virus, a
tobamovirus, a topocuvirus, a tospovirus, a trichovirus, a
tritimovirus, a tungrovirus, a tymovirus, an umbravirus, a
varicosavirus, a vitivirus, or a waikavirus.
[Forms of Target Antigen]
[0188] It will be appreciated based on the disclosure herein that
for agrochemical and biological control applications, the heavy
chain variable domains of the compositions as disclosed herein will
in principle be directed against or specifically bind to several
different forms of the sphingolipid target. It is also expected
that the heavy chain variable domains of the compositions as
disclosed herein will bind to a number of naturally occurring or
synthetic analogs, variants, mutants, alleles, parts and fragments
of their sphingolipid target. More particularly, it is expected
that the heavy chain variable domains of the compositions as
disclosed herein will bind to at least to those analogs, variants,
mutants, alleles, parts and fragments of the sphingolipid target
that (still) contain the binding site, part or domain of the
natural sphingolipid target to which those heavy chain variable
domains bind.
[Formulations]
[0189] It is envisaged that the polypeptide content contained in
the agrochemical or biological control composition as disclosed
herein may vary within a wide range and it is generally up to the
manufacturer to modify the concentration range of a particular
polypeptide according to specific crop pest which is to be
attenuated.
[0190] In particular embodiments, the present invention provides
agrochemical compositions comprising at least one heavy chain
variable domain, wherein said heavy chain variable domain is
present in an amount effective to protect or treat a plant or a
part of said plant from an infection or other biological
interaction with said plant pathogen.
[0191] In a specific embodiment the concentration of the heavy
chain variable domain or polypeptide contained in the agrochemical
composition may be from 0.0001% to 50% by weight.
[0192] In particular embodiments, the present invention provides
agrochemical compositions comprising at least one heavy chain
variable domain, wherein the concentration of the at least one
variable domain in the agrochemical composition ranges from 0.001%
to 50% by weight.
[0193] In yet another specific embodiment the concentration of the
heavy chain variable domain or polypeptide contained in the
agrochemical composition may be from 0.001% to 50% by weight.
[0194] In yet another specific embodiment the concentration of the
heavy chain variable domain or polypeptide contained in the
agrochemical composition may be from 0.01% to 50% by weight.
[0195] In yet another specific embodiment the concentration of the
heavy chain variable domain or polypeptide contained in the
agrochemical composition may be from 0.1% to 50% by weight.
[0196] In yet another specific embodiment the concentration of the
heavy chain variable domain or polypeptide contained in the
agrochemical composition may be from 1% to 50% by weight. In yet
another specific embodiment the concentration of the heavy chain
variable domain or polypeptide contained in the agrochemical
composition may be from 10% to 50% by weight. In yet another
specific embodiment the concentration of the heavy chain variable
domain or polypeptide contained in the agrochemical composition may
be from 0.0001% to 40% by weight. In yet another specific
embodiment the concentration of the heavy chain variable domain or
polypeptide contained in the agrochemical composition may be from
0.001% to 40% by weight. In yet another specific embodiment the
concentration of the heavy chain variable domain or polypeptide
contained in the agrochemical composition may be from 0.01% to 40%
by weight. In yet another specific embodiment the concentration of
the heavy chain variable domain or polypeptide contained in the
agrochemical composition may be from 0.1% to 40% by weight. In yet
another specific embodiment the concentration of the heavy chain
variable domain or polypeptide contained in the agrochemical
composition may be from 1% to 40% by weight. In yet another
specific embodiment the concentration of the heavy chain variable
domain or polypeptide contained in the agrochemical composition may
be from 0.0001% to 30% by weight. In yet another specific
embodiment the concentration of the heavy chain variable domain or
polypeptide contained in the agrochemical composition may be from
0.001% to 30% by weight. In yet another specific embodiment the
concentration of the heavy chain variable domain or polypeptide
contained in the agrochemical composition may be from 0.01% to 30%
by weight. In yet another specific embodiment the concentration of
the heavy chain variable domain or polypeptide contained in the
agrochemical composition may be from 0.1% to 30% by weight. In yet
another specific embodiment the concentration of the heavy chain
variable domain or polypeptide contained in the agrochemical
composition may be from 1% to 30% by weight. In yet another
specific embodiment the concentration of the heavy chain variable
domain or polypeptide contained in the agrochemical composition may
be from 0.0001% to 10% by weight. In yet another specific
embodiment the concentration of the heavy chain variable domain or
polypeptide contained in the agrochemical composition may be from
0.001% to 10% by weight. In yet another specific embodiment the
concentration of the heavy chain variable domain or polypeptide
contained in the agrochemical composition may be from 0.01% to 10%
by weight. In yet another specific embodiment the concentration of
the heavy chain variable domain or polypeptide contained in the
agrochemical composition may be from 0.1% to 10% by weight. In yet
another specific embodiment the concentration of the heavy chain
variable domain or polypeptide contained in the agrochemical
composition may be from 1% to 10% by weight. In yet another
specific embodiment the concentration of the heavy chain variable
domain or polypeptide contained in the agrochemical composition may
be from 0.0001% to 1% by weight. In yet another specific embodiment
the concentration of the heavy chain variable domain or polypeptide
contained in the agrochemical composition may be from 0.001% to 1%
by weight. In yet another specific embodiment the concentration of
the heavy chain variable domain or polypeptide contained in the
agrochemical composition may be from 0.01% to 1% by weight. In yet
another specific embodiment the concentration of the heavy chain
variable domain or polypeptide contained in the agrochemical
composition may be from 0.1% to 1% by weight.
[0197] In particular embodiments, the agrochemical compositions
disclosed herein comprise at least one heavy chain variable domain,
which is formulated in an aqueous solution.
[0198] In further particular embodiments, the agrochemical
compositions disclosed herein comprise at least one heavy chain
variable domain and further comprise an agrochemically suitable
carrier and/or one or more suitable adjuvants.
[0199] The compositions according to the invention may comprise, in
addition to the anti-pest variable domains described above, solid
or liquid carriers which are acceptable in the pest treatment of
plants and/or parts of plants and/or surfactants which are also
acceptable in the pest treatment of plants and/or parts of plants.
In particular, there may be used inert and customary carriers and
customary surfactants. These compositions cover not only
compositions ready to be applied to the plants and/or parts of
plants to be treated by immersion or using a suitable device, but
also the commercial concentrated compositions which have to be
diluted before application to the plants and/or parts of
plants.
[0200] These agrochemical compositions according to the invention
may also contain any sort of other ingredients such as, for
example, protective colloids, adhesives, thickeners, thixotropic
agents, penetrating agents, stabilizers, sequestrants, texturing
agents, flavouring agents, taste enhancers, sugars, sweeteners,
colorants and the like. More generally, the active substances, i.e.
the at least one heavy chain variable domain, may be combined with
any solid or liquid additives corresponding to the usual
formulation techniques.
[0201] The term "carrier", in the present disclosure, denotes a
natural or synthetic organic or inorganic substance with which the
anti-pest active substance is combined to facilitate its
application to plants and/or one or more plant parts. This carrier
is therefore generally inert and should be acceptable in the
agri-sector. The carrier may be solid (clays, natural or synthetic
silicates, silica, resins, waxes, solid fertilizers, and the like)
or liquid (water, alcohols, in particular butanol, and the
like).
[0202] The surfactant may be an emulsifying agent, a dispersing
agent or a wetting agent of the ionic or nonionic type or a mixture
of such surfactants. There may be mentioned, for example, salts of
polyacrylic acids, salts of lignosulphonic acids, salts of
phenolsulphonic or naphthalenesulphonic acids, polycondensates of
ethylene oxide with fatty alcohols or with fatty acids or with
fatty amines, substituted phenols (in particular alkylphenols or
arylphenols), salts of esters of sulphosuccinic acids, derivatives
of taurine (in particular alkyl taurates), phosphoric esters of
polyoxyethylated phenols or alcohols, esters of fatty acids and
polyols, sulphate, sulphonate and phosphate functional
group-containing derivatives of the above compounds. The presence
of at least one surfactant is generally essential when the inert
carrier is not soluble in water and when the vector agent for
application is water.
[0203] The agrochemical compositions as disclosed herein are
themselves in fairly diverse, solid or liquid, forms.
[0204] As solid composition forms, there may be mentioned dustable
powders (content of active substance which may be up to 100%) and
granules, in particular those obtained by extrusion, by compacting,
by impregnation of a granulated carrier, by granulation using a
powder as starting material (the content of active substance in
these granules being between 0.5 and 80% for these latter cases).
Such solid compositions may be optionally used in the form of a
liquid which is viscous to a greater or lesser degree, depending on
the type of application desired, for example by diluting in
water.
[0205] As liquid composition forms or forms intended to constitute
liquid compositions during application, there may be mentioned
solutions, in particular water-soluble concentrates, emulsions,
suspension concentrates, wettable powders (or spraying powder),
oils and waxes.
[0206] The suspension concentrates, which can be applied by
spraying, are prepared so as to obtain a stable fluid product which
does not form a deposit and they usually contain from 10 to 75% of
active substance, from 0.5 to 15% of surfactants, from 0.1 to 10%
of thixotropic agents, from 0 to 10% of appropriate additives, such
as antifoams, corrosion inhibitors, stabilizers, penetrating agents
and adhesives and, as carrier, water or an organic liquid in which
the active substance is not or not very soluble: some organic
solids or inorganic salts may be dissolved in the carrier to help
prevent sedimentation or as antigels for water.
[0207] The agrochemical compositions as disclosed herein can be
used as such, in form of their formulations or as the use forms
prepared therefrom, such as aerosol dispenser, capsule suspension,
cold fogging concentrate, hot fogging concentrate, encapsulated
granule, fine granule, flowable concentrate for seed treatment,
ready-to-use solutions, dustable powder, emulsifiable concentrate,
emulsion oil in water, emulsion water in oil, macrogranule,
macrogranule, oil dispersible powder, oil miscible flowable
concentrate, oil miscible liquid, froths, paste, seed coated with a
pesticide, suspension concentrate (flowable concentrate),
suspensions-emulsions-concentrates, soluble concentrate,
suspensions, soluble powder, granule, water soluble granules or
tablets, water soluble powder for seed treatment, wettable powder,
natural and synthetic materials impregnated with active compound,
micro-encapsulation in polymeric materials and in jackets for seed,
as well as ULV-cold and hot fogging formulations, gas (under
pressure), gas generating product, plant rodlet, powder for dry
seed treatment, solution for seed treatment, ultra low volume (ULV)
liquid, ultra low volume (ULV) suspension, water dispersible
granules or tablets, water dispersible powder for slurry
treatment.
[0208] These formulations are prepared in a known manner by mixing
the active compounds or active compound combinations with customary
additives, such as, for example, customary extenders and also
solvents or diluents, emulsifiers, dispersants, and/or bonding or
fixing agent, wetting agents, water repellents, if appropriate
siccatives and UV stabilisers, colorants, pigments, defoamers,
preservatives, secondary thickeners, adhesives, gibberellins and
water as well further processing auxiliaries.
[0209] These compositions include not only compositions which are
ready to be applied to the plant or seed to be treated by means of
a suitable device, such as a spraying or dusting device, but also
concentrated commercial compositions which must be diluted before
application to the crop.
[Methods of Plant Protection or Treatment]
[0210] In certain aspects, the present invention provides methods
for protecting or treating a plant or a part of a plant from an
infection or other biological interaction with a plant pathogen, at
least comprising the step of applying directly or indirectly to the
plant or to a part of the plant, an agrochemical composition as
disclosed herein, under conditions effective to protect or treat
the plant or a part of the plant against that infection or
biological interaction with the plant pathogen.
[0211] In particular embodiments, these methods comprise applying
directly or indirectly to the plant or to a part of the plant an
agrochemical composition as disclosed herein at an application rate
higher than 50 g of the agrochemical composition per hectare, such
as but not limited to an application rate higher than 75 g of the
agrochemical composition per hectare, such as an application rate
higher than 100 g of the agrochemical composition per hectare, or
in particular an application rate higher than 200 g of the
agrochemical composition per hectare.
[0212] In particular embodiments, these methods comprise applying
directly or indirectly to the plant or to a part of the plant an
agrochemical composition as disclosed herein at an application rate
between 50 g and 100 g of the agrochemical composition per hectare,
such as but not limited to an application rate of between 50 g and
200 g of the agrochemical composition per hectare, in particular an
application rate of between 75 g and 175 g of the agrochemical
composition per hectare, such as between 75 g and 150 g of the
agrochemical composition per hectare or between 75 g and 125 g per
hectare.
[0213] In yet another embodiment, the invention provides methods
for combating plant pests, which methods comprise applying an
agrochemical or biological control composition according to the
invention to a plant, such as a crop, or a part of a plant or a
crop, at an application rate below 50 g of said polypeptide per
hectare. In specific embodiments the application rate is below 45
g/ha, below 40 g/ha, below 35 g/ha, below 30 g/ha, below 25 g/ha,
below 20 g/ha, below 15 g/ha, below 10 g/ha, below 5 g/ha, below 1
g/ha or even lower amounts of polypeptide/ha.
[0214] It is understood depending on the crop and the environmental
pressure of the plant pests that the farmer can vary the
application rate. These application rates variances are specified
in the technical sheet delivered with the specific agrochemical
composition.
[0215] In yet another embodiment, the invention provides the use of
the agrochemical or biological control compositions of the
invention for combating plant pests.
[0216] Applying an agrochemical or biological control composition
according to the invention to a crop may be done using any suitable
method for applying an agrochemical or biological control
composition to a crop, including, but not limited to spraying
(including high volume (HV), low volume (LV) and ultra low volume
(ULV) spraying), brushing, dressing, dripping, coating, dipping,
immersing, spreading, fogging, applying as small droplets, a mist
or an aerosol.
[0217] Thus, in particular embodiments, the methods for protecting
or treating a plant or a part of a plant from an infection or other
biological interaction with a plant pathogen as disclosed herein,
comprise applying the agrochemical composition directly or
indirectly to the plant or to a part of the plant by spraying,
atomizing, foaming, fogging, culturing in hydroculture, culturing
in hydroponics, coating, submerging, and/or encrusting.
[0218] In certain particular embodiments, the present invention
provides methods of inhibiting, preventing, reducing or controlling
the growth of a plant pathogen, comprising at least the step of
applying directly or indirectly to a plant or to a part of said
plant, an agrochemical composition as disclosed herein.
[0219] In certain other embodiments, the present invention provides
methods for of killing a plant pathogen, comprising at least the
step of applying directly or indirectly to a plant or to a part of
said plant, an agrochemical composition as disclosed herein.
[0220] The application rate of the agrochemical composition
according to the invention, meaning the amount of the agrochemical
composition that is applied to the crop, is such that less than 50
g, 45 g, 40 g, 35 g, 30 g, 25 g, 20 g, 20 g, 15 g, 10 g, 5 g, 1 g
or even lower than 1 g of the polypeptide, comprised in the
agrochemical or biological control composition according to the
invention, is applied to the crop per hectare.
[0221] According to the methods as disclosed herein, the
agrochemical or biological control composition can be applied once
to a crop, or it can be applied two or more times after each other
with an interval between every two applications. According to the
method of the present invention, the agrochemical or biological
control composition according to the invention can be applied alone
or in mixture with other materials, preferably other agrochemical
or biological control compositions, to the crop; alternatively, the
agrochemical or biological control composition according to the
invention can be applied separately to the crop with other
materials, preferably other agrochemical or biological control
compositions, applied at different times to the same crop.
According to the method of the present invention, the agrochemical
or biological control composition according to the invention may be
applied to the crop prophylactically, or alternatively, may be
applied once target pests have been identified on the particular
crop to be treated.
[0222] The agrochemical compositions as disclosed herein can be
applied directly to a plant, a crop or to one or more parts of the
plant by the above mentioned methods, such as directly to the
entire plant or directly to one or more parts of the plant, either
in a pre-harvest or in a post-harvest stage. In certain further
embodiments, the agrochemical compositions as disclosed herein can
be applied directly to one or more parts of the plant by the above
mentioned methods, such as directly to the stalks, leafs, tubers,
stems, shoots, the seeds, the fruits, the roots, the flowers,
grains, the buds etc.
[0223] The method of treatment as disclosed herein can also be used
in the field of protecting storage goods against attack of plant
pathogens. According to the present invention, the term "storage
goods" is understood to denote natural substances of vegetable or
animal origin and their processed forms, which have been taken from
the natural life cycle and for which long-term protection is
desired. Storage goods of vegetable origin, such as plants or parts
thereof, for example stalks, leafs, tubers, seeds, fruits or
grains, can be protected in the freshly harvested state or in
processed form, such as pre-dried, moistened, comminuted, ground,
pressed or roasted. Also falling under the definition of storage
goods is timber, whether in the form of crude timber, such as
construction timber, electricity pylons and barriers, or in the
form of finished articles, such as furniture or objects made from
wood. Storage goods of animal origin are hides, leather, furs,
hairs and the like. The combinations according the present
invention can prevent disadvantageous effects such as decay,
discoloration or mold. Preferably "storage goods" is understood to
denote natural substances of vegetable origin and their processed
forms, more preferably fruits and their processed forms, such as
pomes, stone fruits, soft fruits and citrus fruits and their
processed forms.
[0224] The agrochemical compositions as disclosed herein can also
be applied indirectly to a plant, a crop or to one or more parts of
the plant by the above mentioned methods, such as indirectly to the
entire plant or indirectly to one or more parts of the plant,
either in a pre-harvest or in a post-harvest stage. Thus, in
certain embodiments, the agrochemical compositions as disclosed
herein can be applied indirectly to a plant, a crop or to one or
more parts of the plant by the above mentioned methods, such as by
applying the agrochemical composition to the surroundings or to the
medium in which the plant or the one or more parts of the plant are
growing or are stored, such as for instance but not limited to the
air, the soil, the hydroponic culture, the hydroculture, or the
liquid medium, such as for instance the aqueous liquid medium or
water, in which the plant or the one or more parts of the plant are
growing or are stored.
[0225] It thus should be generally understood in the context of
this application that the treatment of plants and plant parts with
the agrochemical compositions as disclosed herein is carried out
directly or by action on their environment, habitat or storage area
by means of the normal treatment methods, for example by watering
(drenching), drip irrigation, spraying, vaporizing, atomizing,
broadcasting, dusting, foaming, spreading-on, and as a powder. It
is furthermore possible to apply the compositions by the ultra-low
volume method, or to inject the active compound preparation or the
active compound itself into the soil.
[0226] In particular embodiments, the methods for protecting or
treating a plant or a part of a plant from an infection or other
biological interaction with a plant pathogen as disclosed herein,
comprise applying the agrochemical composition directly or
indirectly to the plant or to a part of the plant either in a
pre-harvest or in a post-harvest stage.
[0227] According to specific embodiments, the harvested produce is
a fruit, flower, nut or vegetable, a fruit or vegetable with
inedible peel, preferably selected from avocados, bananas,
plantains, lemons, grapefruits, melons, oranges, pineapples, kiwi
fruits, guavas, mandarins, mangoes and pumpkin, is preferred, more
preferably bananas, oranges, lemons and peaches, in particular
bananas. According to further specific embodiments, the harvested
produce is a cut flower from ornamental plants, preferably selected
from Alstroemeria, Carnation, Chrysanthemum, Freesia, Gerbera,
Gladiolus, baby's breath (Gypsophila spec), Helianthus, Hydrangea,
Lilium, Lisianthus, roses and summer flowers.
[0228] The plant species to which the agrochemical compositions as
disclosed herein can be applied can for example be but are not
limited to maize, soya bean, alfalfa, cotton, sunflower, Brassica
oil seeds such as Brassica napus (e.g. canola, rape-seed), Brassica
rapa, B. juncea (e.g. (field) mustard) and Brassica carinata,
Arecaceae sp. (e.g. oilpalm, coconut), rice, wheat, sugar beet,
sugar cane, oats, rye, barley, millet and sorghum, triticale, flax,
nuts, grapes and vine and various fruit and vegetables from various
botanic taxa, e.g. Rosaceae sp. (e.g. pome fruits such as apples
and pears, but also stone fruits such as apricots, cherries,
almonds, plums and peaches, and berry fruits such as strawberries,
raspberries, red and black currant and gooseberry), Ribesioidae
sp., Juglandaceae sp., Betulaceae sp., Anacardiaceae sp., Fagaceae
sp., Moraceae sp., Oleaceae sp. (e.g. olive tree), Actinidaceae
sp., Lauraceae sp. (e.g. avocado, cinnamon, camphor), Musaceae sp.
(e.g. banana trees and plantations), Rubiaceae sp. (e.g. coffee),
Theaceae sp. (e.g. tea), Sterculiceae sp., Rutaceae sp. (e.g.
lemons, oranges, mandarins and grapefruit); Solanaceae sp. (e.g.
tomatoes, potatoes, peppers, capsicum, aubergines, tobacco),
Liliaceae sp., Compositae sp. (e.g. lettuce, artichokes and
chicory--including root chicory, endive or common chicory),
Umbelliferae sp. (e.g. carrots, parsley, celery and celeriac),
Cu-curbitaceae sp. (e.g. cucumbers--including gherkins, pumpkins,
watermelons, calabashes and melons), Alliaceae sp. (e.g. leeks and
onions), Cruciferae sp. (e.g. white cabbage, red cabbage, broccoli,
cauliflow-er, Brussels sprouts, pak choi, kohlrabi, radishes,
horseradish, cress and Chinese cabbage), Leguminosae sp. (e.g.
peanuts, peas, lentils and beans--e.g. common beans and broad
beans), Chenopodiaceae sp. (e.g. Swiss chard, fodder beet, spinach,
beetroot), Linaceae sp. (e.g. hemp), Cannabeacea sp. (e.g.
cannabis), Malvaceae sp. (e.g. okra, cocoa), Papaveraceae (e.g.
poppy), Asparagaceae (e.g. asparagus); useful plants and ornamental
plants in the garden and woods including turf, lawn, grass and
Stevia rebaudiana; and in each case genetically modified types of
these plants.
[0229] In a preferred embodiment of the treatment methods disclosed
herein, the crop is selected from the group consisting of field
crops, grasses, fruits and vegetables, lawns, trees and ornamental
plants.
[0230] In certain aspects, the present invention thus also provides
post-harvest treatment methods for protecting or treating a
harvested plant or a harvested part of the plant from an infection
or other biological interaction with a plant pathogen, at least
comprising the step of applying directly or indirectly to the
harvested plant or to a harvested part of the plant, an
agrochemical composition as disclosed herein, under conditions
effective to protect or treat the harvested plant or a harvested
part of the plant against the infection or biological interaction
with the plant pathogen. According to specific embodiments, the
harvested produce is a fruit, flower, nut or vegetable, a fruit or
vegetable with inedible peel, preferably selected from avocados,
bananas, plantains, lemons, grapefruits, melons, oranges,
pineapples, kiwi fruits, guavas, mandarins, mangoes and pumpkin, is
preferred, more preferably bananas, oranges, lemons and peaches, in
particular bananas. According to further specific embodiments, the
harvested produce is a cut flower from ornamental plants,
preferably selected from Alstroemeria, Carnation, Chrysanthemum,
Freesia, Gerbera, Gladiolus, baby's breath (Gypsophila spec),
Helianthus, Hydrangea, Lilium, Lisianthus, roses and summer
flowers. According to further specific embodiments, the harvested
produce is cut grass or wood.
[0231] Post-harvest disorders are e.g. lenticel spots, scorch,
senescent breakdown, bitter pit, scald, water core, browning,
vascular breakdown, C0.sub.2 injury, C0.sub.2 or 0.sub.2
deficiency, and softening. Fungal diseases may be caused for
example by the following fungi: Mycosphaerella spp., Mycosphaerella
musae, Mycosphaerella frag a ae, Mycosphaerella citri; Mucor spp.,
e.g. Mucor piriformis; Monilinia spp., e.g. Monilinia fructigena,
Monilinia laxa; Phomopsis spp., Phomopsis natalensis;
Colletotrichum spp., e.g. Colletotrichum musae, Colletotrichum
gloeosporioides, Colletotrichum coccodes; Verticillium spp., e.g.
Verticillium theobromae; Nigrospora spp.; Botrytis spp., e.g.
Botrytis cinerea; Diplodia spp., e.g. Diplodia citri; Pezicula
spp.; Alternaria spp., e.g. Alternaria citri, Alternaria alternata;
Septoria spp., e.g. Septoria depressa; Venturia spp., e.g. Venturia
inaequalis, Venturia pyrina; Rhizopus spp., e.g. Rhizopus
stolonifer, Rhizopus oryzae; Glomerella spp., e.g. Glomerella
cingulata; Sclerotinia spp., e.g. Sclerotinia fruiticola;
Ceratocystis spp., e.g. Ceratocystis paradoxa; Fusarium spp., e.g.
Fusarium semitectum, Fusarium moniliforme, Fusarium solani,
Fusarium oxysporum; Cladosporium spp., e.g. Cladosporium fulvum,
Cladosporium cladosporioides, Cladosporium cucumerinum,
Cladosporium musae; Penicillium spp., e.g. Penicillium funiculosum,
Penicillium expansum, Penicillium digitatum, Penicillium italicum;
Phytophthora spp., e.g. Phytophthora citrophthora, Phytophthora
fragariae, Phytophthora cactorum, Phytophthora parasitica;
Phacydiopycnis spp., e.g. Phacydiopycnis malirum; Gloeosporium
spp., e.g. Gloeosporium album, Gloeosporium perennans, Gloeosporium
fructigenum, Gloeosporium singulata; Geotrichum spp., e.g.
Geotrichum candidum; Phlyctaena spp., e.g. Phlyctaena vagabunda;
Cylindrocarpon spp., e.g. Cylindrocarpon mail; Stemphyllium spp.,
e.g. Stemphyllium vesica um; Thielaviopsis spp., e.g. Thielaviopsis
paradoxy; Aspergillus spp., e.g. Aspergillus niger, Aspergillus
carbonari us; Nectria spp., e.g. Nectria galligena; Cercospora
spp., e.g. Cercospora angreci, Cercospora apii, Cercospora
atrofiliformis, Cercospora musae, Cercospora zeae-maydis.
[0232] In further aspects, the present invention provides uses of
the agrochemical compositions as disclosed herein as an anti-pest
agent, such as for instance a biostatic agent or a pesticidal
agent, including but not limited to a fungistatic or a fungicidal
agent.
[0233] In a particular embodiment, the plant pests combated by the
method according to the present invention are plant pathogenic
fungi, as defined before. Lesion number, lesion size, and extent of
sporulation of fungal pathogens may all be decreased as a result of
the application of the method according to the present
invention.
[Methods of Production and Manufacturing of Heavy Chain Variable
Domain Sequences]
[0234] The invention further provides methods for preparing or
generating the heavy chain variable domain sequences, as well as
methods for producing nucleic acids encoding these and host cells,
products and compositions comprising these heavy chain variable
domain sequences. Some preferred but non-limiting examples of such
methods will become clear from the further description herein.
[0235] As will be clear to the skilled person, one particularly
useful method for preparing heavy chain variable domain sequences
as disclosed herein generally comprises the steps of: [0236] (a)
expressing a nucleotide sequence encoding a heavy chain variable
domain sequence as disclosed herein or a vector or genetic
construct a nucleotide sequence encoding that heavy chain variable
domain sequence and [0237] (b) optionally isolating and/or
purifying the heavy chain variable domain sequence.
[0238] In particular embodiments envisaged herein, the
pest-specific a heavy chain variable domain sequences can be
obtained by methods which involve generating a random library of
amino acid sequences and screening this library for an amino acid
sequence capable of specifically binding to a sphingolipid target.
[0239] Accordingly, in particular embodiments, methods for
preparing a heavy chain variable domain sequence as disclosed
herein comprise the steps of [0240] a) providing a set, collection
or library of amino acid sequences of a heavy chain variable domain
sequences; and [0241] b) screening said set, collection or library
of amino acid sequences for amino acid sequences that can bind to
and/or have affinity for the sphingolipid target. and [0242] c)
isolating the amino acid sequence(s) that can bind to and/or have
affinity for the sphingolipid target.
[0243] In such a method, the set, collection or library of amino
acid sequences may be any suitable set, collection or library of
amino acid sequences. For example, the set, collection or library
of amino acid sequences may be a set, collection or library of
immunoglobulin fragment sequences (as described herein), such as a
naive set, collection or library of immunoglobulin fragment
sequences; a synthetic or semi-synthetic set, collection or library
of immunoglobulin fragment sequences; and/or a set, collection or
library of immunoglobulin fragment sequences that have been
subjected to affinity maturation.
[0244] In particular embodiments of this method, the set,
collection or library of amino acid sequences may be an immune set,
collection or library of immunoglobulin fragment sequences, for
example derived from a mammal that has been suitably immunized with
a sphingolipid target or with a suitable antigenic determinant
based thereon or derived therefrom, such as an antigenic part,
fragment, region, domain, loop or other epitope thereof. In one
particular aspect, said antigenic determinant may be an
extracellular part, region, domain, loop or other extracellular
epitope(s).
[0245] In the above methods, the set, collection or library of
amino acid sequences may be displayed on a phage, phagemid,
ribosome or suitable micro-organism (such as yeast), such as to
facilitate screening. Suitable methods, techniques and host
organisms for displaying and screening (a set, collection or
library of) amino acid sequences will be clear to the person
skilled in the art, for example on the basis of the further
disclosure herein. Reference is also made to the review by
Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).
[0246] In other embodiments, the methods for generating the heavy
chain variable domain sequences as disclosed herein comprises at
least the steps of: [0247] a) providing a collection or sample of
cells expressing heavy chain variable domain amino acid sequences;
[0248] b) screening said collection or sample of cells for cells
that express an amino acid sequence that can bind to and/or have
affinity for a sphingolipid target; and [0249] c) either (i)
isolating said amino acid sequence; or (ii) isolating from said
cell a nucleic acid sequence that encodes said amino acid sequence,
followed by expressing said amino acid sequence.
[0250] The collection or sample of cells may for example be a
collection or sample of B-cells. Also, in this method, the sample
of cells may be derived from a mammal that has been suitably
immunized with a fungal target or with a suitable antigenic
determinant based thereon or derived therefrom, such as an
antigenic part, fragment, region, domain, loop or other epitope
thereof. In one particular embodiment, the antigenic determinant
may be an extracellular part, region, domain, loop or other
extracellular epitope(s).
[0251] In other embodiments, the method for generating a heavy
chain variable domain sequence directed against a sphingolipid
target may comprise at least the steps of: [0252] a) providing a
set, collection or library of nucleic acid sequences encoding a
heavy chain variable domain amino acid sequence; [0253] b)
screening said set, collection or library of nucleic acid sequences
for nucleic acid sequences that encode an amino acid sequence that
can bind to and/or has affinity for the sphingolipid target; and
[0254] c) isolating said nucleic acid sequence, followed by
expressing said amino acid sequence.
[0255] In the above methods, the set, collection or library of
nucleic acid sequences encoding amino acid sequences may for
example be a set, collection or library of nucleic acid sequences
encoding a naive set, collection or library of immunoglobulin
fragment sequences; a set, collection or library of nucleic acid
sequences encoding a synthetic or semi-synthetic set, collection or
library of immunoglobulin fragment sequences; and/or a set,
collection or library of nucleic acid sequences encoding a set,
collection or library of immunoglobulin fragment sequences that
have been subjected to affinity maturation.
[0256] In particular, in such a method, the set, collection or
library of nucleic acid sequences encodes a set, collection or
library of heavy chain variable domains (such as V.sub.H domains or
V.sub.HH domains). For example, the set, collection or library of
nucleic acid sequences may encode a set, collection or library of
domain antibodies or single domain antibodies, or a set, collection
or library of amino acid sequences that are capable of functioning
as a domain antibody or single domain antibody. In specific
embodiments, the set, collection or library of nucleotide sequences
encodes a set, collection or library of V.sub.HH sequences.
[0257] In the above methods, the set, collection or library of
nucleotide sequences may be displayed on a phage, phagemid,
ribosome or suitable micro-organism (such as yeast), such as to
facilitate screening. Suitable methods, techniques and host
organisms for displaying and screening (a set, collection or
library of) nucleotide sequences encoding amino acid sequences will
be clear to the person skilled in the art, for example on the basis
of the further disclosure herein. Reference is also made to the
review by Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116
(2005).
[0258] The invention also relates to amino acid sequences that are
obtainable or obtained by the above methods, or alternatively by a
method that comprises one of the above methods and in addition at
least the steps of determining the nucleotide sequence or amino
acid sequence of said immunoglobulin sequence; and of expressing or
synthesizing said amino acid sequence in a manner known per se,
such as by expression in a suitable host cell or host organism or
by chemical synthesis.
[Isolation of Heavy Chain Variable Domains]
[0259] In some cases, the methods for producing the amino acid
sequences binding specifically to a fungal target as envisaged
herein may further comprise the step of isolating from the amino
acid sequence library at least one heavy chain variable domain
having detectable binding affinity for, or detectable in vitro
effect on a sphingolipid target.
[0260] These methods may further comprise the step of amplifying a
sequence encoding at least one heavy chain variable domain having
detectable binding affinity for, or detectable in vitro effect on
the activity of a sphingolipid target. For example, a phage clone
displaying a particular amino acid sequence, obtained from a
selection step of a method described herein, may be amplified by
reinfection of a host bacteria and incubation in a growth
medium.
[0261] In particular embodiments, these methods may encompass
determining the sequence of the one or more amino acid sequences
capable of binding to a sphingolipid target.
[0262] Where a heavy chain variable domain sequence, comprised in a
set, collection or library of amino acid sequences, is displayed on
a suitable cell or phage or particle, it is possible to isolate
from said cell or phage or particle, the nucleotide sequence that
encodes that amino acid sequence. In this way, the nucleotide
sequence of the selected amino acid sequence library member(s) can
be determined by a routine sequencing method.
[0263] In further particular embodiments, the methods for producing
a heavy chain variable domain as envisaged herein comprise the step
of expressing said nucleotide sequence(s) in a host organism under
suitable conditions, so as to obtain the actual desired amino acid
sequence. This step can be performed by methods known to the person
skilled in the art.
[0264] In addition, the obtained heavy chain variable domain
sequences having detectable binding affinity for, or detectable in
vitro effect on the activity of a sphingolipid target, may be
synthesized as soluble protein construct, optionally after their
sequence has been identified.
[0265] For instance, the heavy chain variable domain sequences
obtained, obtainable or selected by the above methods can be
synthesized using recombinant or chemical synthesis methods known
in the art. Also, the amino acid sequences obtained, obtainable or
selected by the above methods can be produced by genetic
engineering techniques. Thus, methods for synthesizing the heavy
chain variable domain sequences obtained, obtainable or selected by
the above methods may comprise transforming or infecting a host
cell with a nucleic acid or a vector encoding an amino acid
sequence having detectable binding affinity for, or detectable in
vitro effect on the activity of a sphingolipid target. Accordingly,
the amino acid sequences having detectable binding affinity for, or
detectable in vitro effect on the activity of a sphingolipid target
can be made by recombinant DNA methods. DNA encoding the amino acid
sequences can be readily synthesized using conventional procedures.
Once prepared, the DNA can be introduced into expression vectors,
which can then be transformed or transfected into host cells such
as E. coli or any suitable expression system, in order to obtain
the expression of amino acid sequences in the recombinant host
cells and/or in the medium in which these recombinant host cells
reside.
[0266] It should be understood, as known by someone skilled in the
art of protein expression and purification, that the heavy chain
variable domain produced from an expression vector using a suitable
expression system may be tagged (typically at the N-terminal or
C-terminal end of the amino acid sequence) with e.g. a His-tag or
other sequence tag for easy purification.
[0267] Transformation or transfection of nucleic acids or vectors
into host cells may be accomplished by a variety of means known to
the person skilled in the art including calcium phosphate-DNA
co-precipitation, DEAE-dextran-mediated transfection,
polybrene-mediated transfection, electroporation, microinjection,
liposome fusion, lipofection, protoplast fusion, retroviral
infection, and biolistics.
[0268] Suitable host cells for the expression of the desired heavy
chain variable domain sequences may be any eukaryotic or
prokaryotic cell (e.g., bacterial cells such as E. coli, yeast
cells, mammalian cells, avian cells, amphibian cells, plant cells,
fish cells, and insect cells), whether located in vitro or in vivo.
For example, host cells may be located in a transgenic plant.
[0269] Thus, the application also provides methods for the
production of heavy chain variable domain sequences having
detectable binding affinity for, or detectable in vitro effect on
the activity of a sphingolipid target comprising transforming,
transfecting or infecting a host cell with nucleic acid sequences
or vectors encoding such amino acid sequences and expressing the
amino acid sequences under suitable conditions.
[0270] In yet another embodiment, the invention further provides
methods for the manufacture Or the production of which is
equivalent wording) an agrochemical or biological control
composition as disclosed herein.
[0271] In particular embodiments, the invention provides methods
for producing an agrochemical composition as disclosed herein, at
least comprising the steps of: [0272] obtaining at least one heavy
chain variable domain of an antibody (V.sub.HH or V.sub.H) or a
functional fragment thereof, which specifically binds to a
sphingolipid of a plant pathogen, and [0273] formulating said heavy
chain variable domain or functional fragment thereof in an
agrochemical composition.
[0274] In particular embodiments of these methods, the step of
obtaining at least one heavy chain variable domain or functional
fragment thereof, which specifically binds to a sphingolipid of a
plant pathogen comprises:
(a) expressing a nucleotide sequence encoding a heavy chain
variable domain or functional fragment thereof, which specifically
binds to a sphingolipid of a plant pathogen, and optionally (b)
isolating and/or purifying the heavy chain variable domain or
functional fragment thereof.
[0275] In other particular embodiments of these methods, the step
of obtaining at least one heavy chain variable domain or functional
fragment thereof, which specifically binds to a sphingolipid of a
plant pathogen comprises: [0276] a) providing a set, collection or
library of heavy chain variable domain sequences or functional
fragments of heavy chain variable domain sequences; [0277] b)
screening said set, collection or library of heavy chain variable
domain sequences or sequences of functional fragments thereof for
sequences that specifically bind to and/or have affinity for a
sphingolipid of a plant pathogen, and optionally [0278] c)
isolating the heavy chain variable domain sequences or sequences of
functional fragments thereof that specifically bind to and/or have
affinity for a sphingolipid of a plant pathogen.
[0279] The present application further discloses methods for the
manufacture Or the production of which is equivalent wording) an
agrochemical or biological control composition as disclosed herein,
comprising formulating an amino acid sequence or polypeptide of
between 80 and 200 amino acids, or other suitable sub-ranges as
defined herein before, with pesticidal activity together with at
least one customary agrochemical auxiliary agent.
[0280] Suitable manufacturing methods are known in the art and
include, but are not limited to, high or low shear mixing, wet or
dry milling, drip-casting, encapsulating, emulsifying, coating,
encrusting, pilling, 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, fluid bed coating, pan coating,
melting, passive or active absorption or adsorption.
[0281] Specifically, the amino acid sequences or polypeptides of
between 80 and 200 amino acids as disclosed herein, or other
suitable sub-ranges as defined herein before, may be prepared by
chemical synthesis.
[0282] It is further disclosed that the amino acid sequences or
polypeptides of between 80 and 200 amino acids, or other suitable
sub-ranges as defined herein before, may be prepared by recombinant
microbial expression systems in vitro and isolated for further use.
Such amino acid sequences or polypeptides may be either in crude
cell lysates, suspensions, colloids, etc., or alternatively may be
purified, refined, buffered and/or further processed before
formulating together with customary agrochemical auxiliary
agents.
[0283] Specifically recombinant methodologies generally involve
inserting a DNA molecule expressing an amino acid sequence, protein
or polypeptide of interest into an expression system to which the
DNA molecule is heterologous (i.e. not normally present in the
host). The heterologous DNA molecule is inserted into the
expression system or vector in proper sense orientation and correct
reading frame. The vector contains the necessary elements for the
transcription and translation of the inserted protein-coding
sequences. Transcription of DNA is dependent upon the presence of a
promoter. Similarly, translation of mRNA in prokaryotes depends
upon the presence of the proper prokaryotic signals which differ
from those of eukaryotes. For a review on maximizing gene
expression, see Roberts and Lauer, Methods in Enzymology 68:473
(1979. Regardless of the specific regulatory sequences employed,
the DNA molecule is cloned into the vector using standard cloning
procedures in the art, as described by Sambrook et al, Molecular
Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs
Harbor, N.Y. (1989). Once the isolated DNA molecule encoding the
protein has been cloned into an expression system, it is ready to
be incorporated into a host cell. Such incorporation can be carried
out by the various forms of transformation, depending upon the
vector/host cell system. Suitable host cells include, but are not
limited to, bacteria, virus, yeast, mammalian cells, insect, plant,
and the like. Optionally, the recombinant host cells can be host
cells that express a native or recombinant, functional type III
secretion system. This is described in detail in U.S. Pat. No.
6,596,509. As a consequence of expressing the functional type III
secretion system, the cells will express the polypeptide and then
secrete the protein into the culture medium. This can simplify
isolation and purification of the polypeptide. The recombinant host
cells can be grown in appropriate fermentation chambers, preferably
under temperature and nutrient conditions that optimize growth of
the host cells and the expression of the polypeptide. Persons of
skill in the art are able to identify optimal conditions for
particular host cells. After fermentation, for example the
bacterial suspension may be diluted in, e.g. about 2 to 5 fold
volume of a buffer to adjust the pH between about 5.5 to 10, more
preferably to a pH of between about 7 to 9, and even more
preferably to a pH of about 8.0. Suitable buffers are well-known in
the art and may include, for example, potassium phosphate buffer or
a Tris-EDTA buffer. The concentration of the buffer can be from
about 0.001 mM to about 0.5 M. Following the pH adjustment, the
(bacterial) suspension solution is heat treated to a temperature of
about 60-130.degree. C., preferably to a temperature of about
95-125.degree. C. Heat treatment may be carried out for any
suitable period of time. In one embodiment, heat treatment is
carried out for a period of about five minutes up to about 30
minutes. The heated suspension solution is then cooled. A suitable
cool down temperature is, without limitation, about 35-55.degree.
C., preferably about 45.degree. C. Following cooling, bacterial
cells in the bacterial suspension are lysed, if required, to
liberate the polypeptide. Cell lysis may be carried out, e.g. by
contacting the bacterial suspension with a lysozyme. The
concentration of lysozyme may be anywhere from about 2 ppm to 100
ppm. Alternatively, cell lysis may involve non-chemical methods,
such as high pressure or sonication, both of which are well known
by persons of ordinary skill in the art. It may be desirable, after
cell lysis, to incubate the bacterial suspension. Suitable
incubation times may vary. For example, it may be desirable to
incubate the bacterial suspension for a period of about 30-45
minutes at a temperature of about 40-42.degree. C. After lysing,
the desired polypeptide can be further extracted by removing the
cell debris and the denatured proteins resulting from the previous
heat treatment step. In one embodiment, the extract is centrifuged
for about 10-20 minutes to remove some of the cell debris. Suitable
centrifuge speeds may be from about 4,000 to 20,000 rpm and the
spinning down time can be from about 10 minutes to 20 minutes.
Further cell debris may then be removed by heat treating and
centrifuging the supernatant to obtain a liquid extract that is
substantially free of cellular debris by removing more than about
60%, 70%, 80%, 90%, or 95% of total solids. This subsequent heat
treatment may be carried out at a temperature of about 60.degree.
C. for up to about two hours, at about 100.degree. C. for about 10
minutes, or at about 121.degree. C. with 15 psi of pressure for
about 5 minutes. These temperatures and times may vary depending on
other conditions. The method of making a stable liquid composition
containing an amino acid sequence or polypeptide as disclosed
herein further involves introducing into the liquid extract a
biocidal agent and, optionally, one or both of a protease inhibitor
and a non-ionic surfactant, thereby obtaining a liquid composition
comprising the polypeptide. In one embodiment, a protease inhibitor
is introduced into the liquid extract without a non-ionic
surfactant. In another embodiment, a non-ionic surfactant is
introduced into the liquid extract without a protease inhibitor. In
a further embodiment, both a protease inhibitor and a non-ionic
surfactant are introduced into the liquid extract. In yet another
embodiment, neither a protease inhibitor nor a non-ionic surfactant
are introduced into the liquid extract. Alternatively, the
stability of the liquid composition as disclosed herein can be
assessed using, e.g., HPLC analysis or other suitable procedures
that can identify quantity of a specific protein or polypeptide.
The stability of the amino acid sequences or polypeptides in a
composition as disclosed herein can be determined by comparing the
quantity of the protein in the aged liquid composition to that of a
recently prepared liquid composition or to a prior quantitation
performed on the same composition. The measurement of protein
stability strongly correlates with a retention of its activity.
[0284] Customary agrochemical auxiliary agents are well-known in
the art and include, but are not limited to aqueous or organic
solvents, buffering agents, acidifiers, surfactants, wetting
agents, spreading agents, tackifiers, stickers, carriers, fillers,
thickeners, emulsifiers, dispersants, sequestering agents,
anti-settling agents, coalescing agents, rheology modifiers,
defoaming agents, photo-protectors, anti-freeze agents, biocides,
penetrants, mineral or vegetable oils, pigments and drift control
agents or any suitable combination thereof.
[0285] In yet another embodiment, the invention provides a
polypeptide of between 80 and 200 amino acids or the sub-ranges
disclosed herein before, obtained by affinity selection to a
certain plant pest target, which is able to inhibit the growth
and/or the activity of a plant pest at a minimum inhibitory
concentration of about 0.00001 to 1 .mu.M.
[0286] In particular embodiments of the methods as disclosed herein
for protecting, preventing, curing or treating a plant from an
infection by a fungus or from another biological interaction with a
fungus, the heavy chain variable domain sequences sequences,
polypeptides or compositions as disclosed herein are directly or
indirectly applied to the plant by spraying, atomizing, foaming,
fogging, in hydroculture/hydroponics, coating, submerging, and/or
encrusting.
[Nucleic Acid Sequences]
[0287] In a further aspect, the present invention provides nucleic
acid sequences encoding the heavy chain variable domain amino acid
sequences in the compositions as disclosed herein (or suitable
fragments thereof). These nucleic acid sequences can also be in the
form of a vector or a genetic construct or polynucleotide. The
nucleic acid sequences as disclosed herein may be synthetic or
semi-synthetic sequences, nucleotide sequences that have been
isolated from a library (and in particular, an expression library),
nucleotide sequences that have been prepared by PCR using
overlapping primers, or nucleotide sequences that have been
prepared using techniques for DNA synthesis known per se.
[Constructs, Vectors, Host Cells]
[0288] The genetic constructs as disclosed herein may be DNA or
RNA, and are preferably double-stranded DNA. The genetic constructs
of the invention may also be in a form suitable for transformation
of the intended host cell or host organism in a form suitable for
integration into the genomic DNA of the intended host cell or in a
form suitable for independent replication, maintenance and/or
inheritance in the intended host organism. For instance, the
genetic constructs of the invention may be in the form of a vector,
such as for example a plasmid, cosmid, YAC, a viral vector or
transposon. In particular, the vector may be an expression vector,
i.e., a vector that can provide for expression in vitro and/or in
vivo (e.g. in a suitable host cell, host organism and/or expression
system).
[0289] Accordingly, in another further aspect, the present
invention also provides vectors comprising one or more nucleic acid
sequences of the invention.
[0290] In still a further aspect, the present invention provides
hosts or host cells that express or are capable of expressing one
or more amino acid sequences as disclosed herein. Suitable examples
of hosts or host cells for expression of the amino acid sequences,
polypeptides of the invention will be clear to the skilled
person.
[0291] The application also discloses, polypeptides of between 80
and 200 amino acids or the sub-ranges discussed herein before,
remain stable in an agrochemical or biological control composition,
as defined, meaning that the integrity and the pesticidal activity,
as defined, of the polypeptide is maintained under storage and/or
utilization conditions of the agrochemical composition, which may
include elevated temperatures, freeze-thaw cycles, changes in pH or
in ionic strength, UV-irradiation, presence of harmful chemicals
and the like. Most preferably, these polypeptides of between 80 and
200 amino acids remains stable in the agrochemical composition when
the agrochemical composition is stored at ambient temperature for a
period of two years or when the agrochemical composition is stored
at 54.degree. C. for a period of two weeks. Particularly, the
polypeptides of between 80 and 200 amino acids comprised in an
agrochemical composition retains at least about 70% activity, more
particularly at least about 70% to 80% activity, most particularly
about 80% to 90% activity, after having been stored in the
agrochemical composition at ambient temperature for a period of two
years or when the agrochemical composition containing the
polypeptide is stored at 54.degree. C. for a period of two
weeks.
[0292] In yet another embodiment, for use in the methods disclosed
herein, the application discloses nucleic acid sequences encoding a
polypeptides of between 80 and 200 amino acids, wherein
polypeptides are obtained by affinity selection to a specific plant
pathogenic target, which polypeptide is able to inhibit the growth
and/or the activity of a crop pest at a minimum inhibitory
concentration of about 0.00001 to 1 .mu.M.
[0293] Also disclosed are chimeric genes comprising the following
operably linked DNA elements: a) a plant expressible promoter, b) a
DNA region which when transcribed yields a mRNA molecule capable of
being translated into a polypeptide and c) a 3' end region
comprising transcription termination and polyadenylation signals
functioning in cells of said plant.
[0294] A "chimeric gene" or "chimeric construct" is a recombinant
nucleic acid sequence in which a promoter (e.g. a plant expressible
promoter) or regulatory nucleic acid sequence is operatively linked
to, or associated with, a nucleic acid sequence that codes for an
mRNA, such that the regulatory nucleic acid sequence is able to
regulate transcription or expression of the associated nucleic acid
coding sequence when introduced into a cell such as a plant cell.
The regulatory nucleic acid sequence of the chimeric gene is not
normally operatively linked to the associated nucleic acid sequence
as found in nature.
[0295] In the present invention, a "plant promoter" comprises
regulatory elements, which mediate the expression of a coding
sequence segment in plant cells. For expression in plants, the
nucleic acid molecule must be linked operably to or comprise a
suitable promoter which expresses the gene at the right point in
time and with the required spatial expression pattern.
[0296] The term "operably linked" as used herein refers to a
functional linkage between the promoter sequence and the gene of
interest, such that the promoter sequence is able to initiate
transcription of the gene of interest.
[0297] Plant expressible promoters comprise nucleic acid sequences
which are able to direct the expression of a transgene in a plant.
Examples of plant expressible promoters are constitutive promoters
which are transcriptionally active during most, but not necessarily
all, phases of growth and development and under most environmental
conditions, in at least one cell, tissue or organ, other promoters
are inducible promoters, other examples are tissue specific
promoters, still other examples are abiotic stress inducible
promoters.
[0298] The chimeric gene (or the expression cassette) when
transformed in a plant expresses a nucleic acid which results in
expression of a protein.
[0299] Also disclosed is a recombinant vector which comprises an
expression cassette (or a chimeric gene) as herein described
before.
[0300] The term "terminator" encompasses a control sequence which
is a DNA sequence at the end of a transcriptional unit which
signals 3' processing and polyadenylation of a primary transcript
and termination of transcription. The terminator can be derived
from the natural gene, from a variety of other plant genes, or from
T-DNA. The terminator to be added may be derived from, for example,
the nopaline synthase or octopine synthase genes, or alternatively
from another plant gene, or less preferably from any other
eukaryotic gene.
[0301] "Selectable marker", "selectable marker gene" or "reporter
gene" includes any gene that confers a phenotype on a cell in which
it is expressed to facilitate the identification and/or selection
of cells that are transfected or transformed with a nucleic acid
construct of the invention. These marker genes enable the
identification of a successful transfer of the nucleic acid
molecules via a series of different principles. Suitable markers
may be selected from markers that confer antibiotic or herbicide
resistance, that introduce a new metabolic trait or that allow
visual selection. Examples of selectable marker genes include genes
conferring resistance to antibiotics (such as nptII that
phosphorylates neomycin and kanamycin, or hpt, phosphorylating
hygromycin, or genes conferring resistance to, for example,
bleomycin, streptomycin, tetracyclin, chloramphenicol, ampicillin,
gentamycin, geneticin (G418), spectinomycin or blasticidin), to
herbicides (for example bar which provides resistance to
Basta.RTM.; aroA or gox providing resistance against glyphosate, or
the genes conferring resistance to, for example, imidazolinone,
phosphinothricin or sulfonylurea), or genes that provide a
metabolic trait (such as manA that allows plants to use mannose as
sole carbon source or xylose isomerase for the utilisation of
xylose, or antinutritive markers such as the resistance to
2-deoxyglucose). Expression of visual marker genes results in the
formation of colour (for example .beta.-glucuronidase, GUS or
.beta.-galactosidase with its coloured substrates, for example
X-Gal), luminescence (such as the luciferin/luceferase system) or
fluorescence (Green Fluorescent Protein, GFP, and derivatives
thereof). This list represents only a small number of possible
markers. The skilled worker is familiar with such markers.
Different markers are preferred, depending on the organism and the
selection method.
[0302] It is known that upon stable or transient integration of
nucleic acids into plant cells, only a minority of the cells takes
up the foreign DNA and, if desired, integrates it into its genome,
depending on the expression vector used and the transfection
technique used. To identify and select these integrants, a gene
coding for a selectable marker (such as the ones described above)
is usually introduced into the host cells together with the gene of
interest. These markers can for example be used in mutants in which
these genes are not functional by, for example, deletion by
conventional methods. Furthermore, nucleic acid molecules encoding
a selectable marker can be introduced into a host cell on the same
vector that comprises the sequence encoding the polypeptides of the
invention or used in the methods of the invention, or else in a
separate vector. Cells which have been stably transfected with the
introduced nucleic acid can be identified for example by selection
(for example, cells which have integrated the selectable marker
survive whereas the other cells die).
[0303] Since the marker genes, particularly genes for resistance to
antibiotics and herbicides, are no longer required or are undesired
in the transgenic host cell once the nucleic acids have been
introduced successfully, the process according to the invention for
introducing the nucleic acids advantageously employs techniques
which enable the removal or excision of these marker genes. One
such a method is what is known as co-transformation. The
co-transformation method employs two vectors simultaneously for the
transformation, one vector bearing the nucleic acid according to
the invention and a second bearing the marker gene(s). A large
proportion of transformants receives or, in the case of plants,
comprises (up to 40% or more of the transformants), both vectors.
In case of transformation with Agrobacteria, the transformants
usually receive only a part of the vector, i.e. the sequence
flanked by the T-DNA, which usually represents the expression
cassette. The marker genes can subsequently be removed from the
transformed plant by performing crosses. In another method, marker
genes integrated into a transposon are used for the transformation
together with desired nucleic acid (known as the Ac/Ds technology).
The transformants can be crossed with a transposase source or the
transformants are transformed with a nucleic acid construct
conferring expression of a transposase, transiently or stable. In
some cases (approx. 10%), the transposon jumps out of the genome of
the host cell once transformation has taken place successfully and
is lost. In a further number of cases, the transposon jumps to a
different location. In these cases the marker gene must be
eliminated by performing crosses. In microbiology, techniques were
developed which make possible, or facilitate, the detection of such
events. A further advantageous method relies on what is known as
recombination systems; whose advantage is that elimination by
crossing can be dispensed with. The best-known system of this type
is what is known as the Cre/lox system. Cre1 is a recombinase that
removes the sequences located between the loxP sequences. If the
marker gene is integrated between the loxP sequences, it is removed
once transformation has taken place successfully, by expression of
the recombinase. Further recombination systems are the HIN/HIX,
FLP/FRT and REP/STB system (Tribble et al., J. Biol. Chem., 275,
2000: 22255-22267; Velmurugan et al., J. Cell Biol., 149, 2000:
553-566). A site-specific integration into the plant genome of the
nucleic acid sequences according to the invention is possible.
[0304] For the purposes of the invention, "transgenic", "transgene"
or "recombinant" means with regard to, for example, a nucleic acid
sequence, an expression cassette, gene construct or a vector
comprising the nucleic acid sequence or an organism transformed
with the nucleic acid sequences, expression cassettes or vectors
according to the invention.
[0305] A transgenic plant for the purposes of the invention is thus
understood as meaning, as above, that the nucleic acids used in the
method of the invention are not present in, or originating from,
the genome of said plant, or are present in the genome of said
plant but not at their natural locus in the genome of said plant,
it being possible for the nucleic acids to be expressed
homologously or heterologously. However, as mentioned, transgenic
also means that, while the nucleic acids according to the invention
or used in the inventive method are at their natural position in
the genome of a plant, the sequence has been modified with regard
to the natural sequence, and/or that the regulatory sequences of
the natural sequences have been modified. Transgenic is preferably
understood as meaning the expression of the nucleic acids according
to the invention at an unnatural locus in the genome, i.e.
homologous or, heterologous expression of the nucleic acids takes
place. Preferred transgenic plants are mentioned herein.
[0306] The term "expression" or "gene expression" means the
transcription of a specific gene or specific genes or specific
genetic construct. The term "expression" or "gene expression" in
particular means the transcription of a gene or genes or genetic
construct into structural RNA (rRNA, tRNA) or mRNA with or without
subsequent translation of the latter into a protein. The process
includes transcription of DNA and processing of the resulting mRNA
product.
[0307] The term "increased expression" or "overexpression" as used
herein means any form of expression that is additional to the
original wild-type expression level. For the purposes of this
invention, the original wild-type expression level might also be
zero, i.e. absence of expression or immeasurable expression.
[0308] Methods for increasing expression of genes or gene products
are well documented in the art and include, for example,
overexpression driven by appropriate promoters (as described herein
before), the use of transcription enhancers or translation
enhancers. Isolated nucleic acids which serve as promoter or
enhancer elements may be introduced in an appropriate position
(typically upstream) of a non-heterologous form of a polynucleotide
so as to upregulate expression of a nucleic acid encoding the
polypeptide of interest. If polypeptide expression is desired, it
is generally desirable to include a polyadenylation region at the
3'-end of a polynucleotide coding region. The polyadenylation
region can be derived from the natural gene, from a variety of
other plant genes, or from T-DNA. The 3' end sequence to be added
may be derived from, for example, the nopaline synthase or octopine
synthase genes, or alternatively from another plant gene, or less
preferably from any other eukaryotic gene.
[0309] An intron sequence may also be added to the 5' untranslated
region (UTR) or the coding sequence of the partial coding sequence
to increase the amount of the mature message that accumulates in
the cytosol. Inclusion of a spliceable intron in the transcription
unit in both plant and animal expression constructs has been shown
to increase gene expression at both the mRNA and protein levels up
to 1000-fold (Buchman and Berg (1988) Mol. Cell biol. 8: 4395-4405;
Callis et al. (1987) Genes Dev 1:1 183-1200). Such intron
enhancement of gene expression is typically greatest when placed
near the 5' end of the transcription unit. Use of the maize introns
Adh1-S intron 1, 2, and 6, the Bronze-1 intron are known in the
art. For general information see: The Maize Handbook, Chapter 1 16,
Freeling and Walbot, Eds., Springer, N.Y. (1994).
[0310] The term "introduction" or "transformation" as referred to
herein encompass the transfer of an exogenous polynucleotide or
chimeric gene (or expression cassette) into a host cell,
irrespective of the method used for transfer. Plant tissue capable
of subsequent clonal propagation, whether by organogenesis or
embryogenesis, may be transformed with a genetic construct of the
present invention and a whole plant regenerated there from. The
particular tissue chosen will vary depending on the clonal
propagation systems available for, and best suited to, the
particular species being transformed. Exemplary tissue targets
include leaf disks, pollen, embryos, cotyledons, hypocotyls,
megagametophytes, callus tissue, existing meristematic tissue
(e.g., apical meristem, axillary buds, and root meristems), and
induced meristem tissue (e.g., cotyledon meristem and hypocotyl
meristem). The polynucleotide may be transiently or stably
introduced into a host cell and may be maintained non-integrated,
for example, as a plasmid. Alternatively, it may be integrated into
the host genome. The resulting transformed plant cell may then be
used to regenerate a transformed plant in a manner known to persons
skilled in the art.
[0311] The transfer of foreign genes into the genome of a plant is
called transformation. Transformation of plant species is now a
fairly routine technique. Advantageously, any of several
transformation methods may be used to introduce the gene of
interest into a suitable ancestor cell. The methods described for
the transformation and regeneration of plants from plant tissues or
plant cells may be utilized for transient or for stable
transformation. Transformation methods include the use of
liposomes, electroporation, chemicals that increase free DNA
uptake, injection of the DNA directly into the plant, particle gun
bombardment, transformation using viruses or pollen and
microprojection. Methods may be selected from the
calcium/polyethylene glycol method for protoplasts (Krens, F. A. et
al., (1982) Nature 296, 72-74; Negrutiu I et al. (1987) Plant Mol
Biol 8: 363-373); electroporation of protoplasts (Shillito R. D. et
al. (1985) Bio/Technol 3, 1099-1 102); microinjection into plant
material (Crossway A et al., (1986) Mol. Gen Genet 202: 179-185);
DNA or RNA-coated particle bombardment (Klein T M et al., (1987)
Nature 327: 70) infection with (non-integrative) viruses and the
like. Transgenic plants, including transgenic crop plants, are
preferably produced via Agrobacterium-mediated transformation. An
advantageous transformation method is the transformation in planta.
To this end, it is possible, for example, to allow the agrobacteria
to act on plant seeds or to inoculate the plant meristem with
agrobacteria. It has proved particularly expedient in accordance
with the invention to allow a suspension of transformed
agrobacteria to act on the intact plant or at least on the flower
primordia. The plant is subsequently grown on until the seeds of
the treated plant are obtained (Clough and Bent, Plant J. (1998)
16, 735-743). Methods for Agrobacterium-mediated transformation of
rice include well known methods for rice transformation, such as
those described in any of the following: European patent
application EP1198985, Aldemita and Hodges (Planta 199: 612-617,
1996); Chan et al. (Plant Mol Biol 22 (3): 491-506, 1993), Hiei et
al. (Plant J 6 (2): 271-282, 1994), which disclosures are
incorporated by reference herein as if fully set forth. In the case
of corn transformation, the preferred method is as described in
either Ishida et al. (Nat. Biotechnol 14(6): 745-50, 1996) or Frame
et al. (Plant Physiol 129(1): 13-22, 2002), which disclosures are
incorporated by reference herein as if fully set forth. Said
methods are further described by way of example in B. Jenes et al.,
Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1,
Engineering and Utilization, eds. S. D. Kung and R. Wu, Academic
Press (1993) 128-143 and in Potrykus Annu. Rev. Plant Physiol.
Plant Molec. Biol. 42 (1991) 205-225). The nucleic acids or the
construct to be expressed is preferably cloned into a vector, which
is suitable for transforming Agrobacterium tumefaciens, for example
pBin19 (Bevan et al (1984) Nucl. Acids Res. 12-8711). Agrobacteria
transformed by such a vector can then be used in known manner for
the transformation of plants, such as plants used as a model, like
Arabidopsis (Arabidopsis thaliana is within the scope of the
present invention not considered as a crop plant), or crop plants
such as, by way of example, tobacco plants, for example by
immersing bruised leaves or chopped leaves in an agrobacterial
solution and then culturing them in suitable media. The
transformation of plants by means of Agrobacterium tumefaciens is
described, for example, by Hofgen and Willmitzer in Nucl. Acid Res.
(1988) 16, 9877 or is known inter alia from F. F. White, Vectors
for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1,
Engineering and Utilization, eds. S. D. Kung and R. Wu, Academic
Press, 1993, pp. 15-38.
[0312] In addition to the transformation of somatic cells, which
then have to be regenerated into intact plants, it is also possible
to transform the cells of plant meristems and in particular those
cells which develop into gametes. In this case, the transformed
gametes follow the natural plant development, giving rise to
transgenic plants. Thus, for example, seeds of Arabidopsis are
treated with agrobacteria and seeds are obtained from the
developing plants of which a certain proportion is transformed and
thus transgenic [Feldman, K A and Marks M D (1987). Mol Gen Genet
208:1-9; Feldmann K (1992). In: C Koncz, N-H Chua and J Shell, eds,
Methods in Arabidopsis Research. Word Scientific, Singapore, pp.
274-289]. Alternative methods are based on the repeated removal of
the inflorescences and incubation of the excision site in the
center of the rosette with transformed agrobacteria, whereby
transformed seeds can likewise be obtained at a later point in time
(Chang (1994). Plant J. 5: 551-558; Katavic (1994). Mol Gen Genet,
245: 363-370). However, an especially effective method is the
vacuum infiltration method with its modifications such as the
"floral dip" method. In the case of vacuum infiltration of
Arabidopsis, intact plants under reduced pressure are treated with
an agrobacterial suspension [Bechthold, N (1993). CR Acad Sci Paris
Life Sci, 316: 1 194-1 199], while in the case of the "floral dip"
method the developing floral tissue is incubated briefly with a
surfactant-treated agrobacterial suspension [Clough, S J and Bent A
F (1998) The Plant J. 16, 735-743]. A certain proportion of
transgenic seeds are harvested in both cases, and these seeds can
be distinguished from non-transgenic seeds by growing under the
above-described selective conditions. In addition the stable
transformation of plastids is of advantages because plastids are
inherited maternally is most crops reducing or eliminating the risk
of transgene flow through pollen. The transformation of the
chloroplast genome is generally achieved by a process which has
been schematically displayed in Klaus et al., 2004 [Nature
Biotechnology 22 (2), 225-229]. Briefly the sequences to be
transformed are cloned together with a selectable marker gene
between flanking sequences homologous to the chloroplast genome.
These homologous flanking sequences direct site specific
integration into the plastome. Plastidal transformation has been
described for many different plant species and an overview is given
in Bock (2001) Transgenic plastids in basic research and plant
biotechnology. J Mol Biol. 2001 Sep. 21; 312 (3):425-38 or Maliga,
P (2003) Progress towards commercialization of plastid
transformation technology. Trends Biotechnol. 21, 20-28. Further
biotechnological progress has recently been reported in form of
marker free plastid transformants, which can be produced by a
transient co-integrated maker gene (Klaus et al., 2004, Nature
Biotechnology 22(2), 225-229).
[0313] The genetically modified plant cells can be regenerated via
all methods with which the skilled worker is familiar. Suitable
methods can be found in the abovementioned publications by S. D.
Kung and R. Wu, Potrykus or Hofgen and Willmitzer.
[0314] Generally after transformation, plant cells or cell
groupings are selected for the presence of one or more markers
which are encoded by plant-expressible genes co-transferred with
the gene of interest, following which the transformed material is
regenerated into a whole plant. To select transformed plants, the
plant material obtained in the transformation is, as a rule,
subjected to selective conditions so that transformed plants can be
distinguished from untransformed plants. For example, the seeds
obtained in the above-described manner can be planted and, after an
initial growing period, subjected to a suitable selection by
spraying. A further possibility consists in growing the seeds, if
appropriate after sterilization, on agar plates using a suitable
selection agent so that only the transformed seeds can grow into
plants. Alternatively, the transformed plants are screened for the
presence of a selectable marker such as the ones described above.
Following DNA transfer and regeneration, putatively transformed
plants may also be evaluated, for instance using Southern analysis,
for the presence of the gene of interest, copy number and/or
genomic organisation. Alternatively or additionally, expression
levels of the newly introduced DNA may be monitored using Northern
and/or Western analysis, both techniques being well known to
persons having ordinary skill in the art.
[0315] The generated transformed plants may be propagated by a
variety of means, such as by clonal propagation or classical
breeding techniques. For example, a first generation (or T1)
transformed plant may be selfed and homozygous second-generation
(or T2) transformants selected, and the T2 plants may then further
be propagated through classical breeding techniques. The generated
transformed organisms may take a variety of forms. For example,
they may be chimeras of transformed cells and non-transformed
cells; clonal transformants (e.g., all cells transformed to contain
the expression cassette); grafts of transformed and untransformed
tissues (e.g., in plants, a transformed rootstock grafted to an
untransformed scion).
[0316] The following non-limiting Examples describe methods and
means according to the invention. Unless stated otherwise in the
Examples, all techniques are carried out according to protocols
standard in the art. The following examples are included to
illustrate embodiments of the invention. Those of skill in the art
should, in light of the present disclosure, appreciate that many
changes can be made in the specific embodiments which are disclosed
and still obtain a like or similar result without departing from
the concept, spirit and scope of the invention. More specifically,
it will be apparent that certain agents which are both chemically
and physiologically related may be substituted for the agents
described herein while the same or similar results would be
achieved. All such similar substitutes and modifications apparent
to those skilled in the art are deemed to be within the spirit,
scope and concept of the invention as defined by the appended
claims.
[0317] Thus, the Figures, Sequence Listing and the Experimental
Part/Examples are only given to further illustrate the invention
and should not be interpreted or construed as limiting the scope of
the invention and/or of the appended claims in any way, unless
explicitly indicated otherwise herein.
[0318] The above disclosure will now be further described by means
of the following non-limiting Examples and Figures, in which the
figures show:
[0319] FIG. 1: Binding of VHH as crude VHH-containing periplasmic
extracts to coated fungal GlcCer from Pleurotus citrinopileatus.
Anti-GlcCer VHH bind to fungal GlcCer, no binding is observed for
unrelated VHH.
[0320] FIG. 2: Binding specificity of VHH 41D01. Binding of
purified VHH 41D01 at 0.1 .mu.g/ml to coated fungal GlcCer from
Fusarium oxysporum or Pleurotus citrinopileatus, and non-fungal
GlcCer from plant (soy), or mammal (pork). Bars represent average
OD 405 nm values, error bars represent standard errors of the mean
of n=6. Anti-GlcCer VHH 41 D01 specifically binds fungal GlcCer and
not plant or mammalian GlcCer.
[0321] FIG. 3A: Binding specificity of VHH. Binding of purified VHH
at 1 .mu.g/ml to coated fungal GlcCer from Fusarium oxysporum or
Pleurotus citrinopileatus. Different anti-GlcCer VHH specifically
bind to different fungal GlcCer.
[0322] FIG. 3B: Binding specificity of VHH. Binding of purified VHH
at 1 .mu.g/ml to coated non-fungal GlcCer from plant (soy).
Different anti-GlcCer VHH do not bind plant GlcCer.
[0323] FIG. 3C: Binding specificity of VHH. Binding of purified VHH
at 1 .mu.g/ml to coated non-fungal mammalian GlcCer (pork).
Different anti-GlcCer VHH do not bind mammalian GlcCer.
[0324] FIG. 4: Real-time measurement of the antibody-antigen
interaction between VHH 41 D01 and fungal GlcCer. VHH 41 D01 binds
fungal GlcCer. A slow dissociation of GlcCer from VHH 41 D01 is
observed. Unrelated VHH_A does not bind fungal GlcCer.
[0325] FIG. 5: Cross-reactivity and specificity of VHH 41 D01 and
VHH 56F11. Binding of purified VHH 41 D01 at 0.1 .mu.g/ml and VHH
56F11 at 1 .mu.g/ml to coated fungal lipid extracts, GlcCer from
Pleurotus citrinopileatus, and unrelated compounds: apple pectin,
citrus pectin, or potato lectin. Bars represent average OD 405 nm
values, error bars represent standard errors of the mean of n=2.
Anti-GlcCer VHH 41 D01 and VHH 56F11 specifically bind each of the
fungal lipid extracts tested. Anti-GlcCer VHH 41D01 and VHH 56F11
do not show binding to unrelated coated compounds or non-coated
wells.
[0326] FIG. 6: Binding of VHH 41D01 in different compositions to
fungal GlcCer from Fusarium oxysporum. Aqueous compositions
containing anti-GlcCer VHH 41D01 at 0.1 .mu.g/ml and protease
inhibitors and/or non-ionic surfactant and/or preservative were
tested for binding to fungal GlcCer. GlcCer-specific VHH 41D01
binds to fungal GlcCer in all compositions tested without adverse
effects of any of the additives.
[0327] FIG. 7A: Visual scoring of fungal growth. Serial dilution of
VHH (anti-GlcCer VHH's 41D01, 56E05, 56F11, and 57A06 as well as
unrelated VHH_A or unrelated VHH_B) were inoculated with Botrytis
cinerea spores (1E+05/ml) and incubated at room temperature. Effect
on fungal growth of anti-GlcCer VHH's 41D01, 56E05, 56F11, and
57A06, unrelated VHH_A or unrelated VHH_B was quantified based on a
set of photographic standards. Bars represent average % of growth,
error bars represent standard errors of the mean of at least 3
replicas.
[0328] FIG. 7B: Visual scoring of fungal growth. Serial dilution of
VHH (anti-GlcCer VHH's 56C09, 56H07, 57C09, 57E07, 57E11 as well as
unrelated VHH_A or unrelated VHH_B) were inoculated with Botrytis
cinerea spores (1E+05/ml) and incubated at room temperature. Effect
on fungal growth of anti-GlcCer VHH's 56C09, 56H07, 57C09, 57E07,
57E11, unrelated VHH_A or unrelated VHH_B was quantified based on a
set of photographic standards. Bars represent average % of growth,
error bars represent standard errors of the mean of at least 3
replicas.
[0329] FIG. 7C: Visual scoring of fungal growth. Serial dilution of
VHH (anti-GlcCer VHH's 54C08, 54C11, 56A05, 56A09 as well as
unrelated VHH_A or unrelated VHH_B) were inoculated with Botrytis
cinerea spores (1E+05/ml) and incubated at room temperature. Effect
on fungal growth of anti-GlcCer VHH's 54C08, 54C11, 56A05, 56A09,
unrelated VHH_A or unrelated VHH_B was quantified based on a set of
photographic standards. Bars represent average % of growth, error
bars represent standard errors of the mean of at least 3
replicas.
[0330] FIG. 8A: Visual scoring of fungal growth of different fungal
species. Two-fold serial dilutions of VHH (anti-GlcCer VHH or
unrelated VHH) are incubated with spores (1E+05/ml) of Alternaria
brassicicola at room temperature. Effect on fungal growth of VHH
and control compounds was based on a set of photographic standards.
Bars represent average % growth, error bars represent standard
errors of the mean of n=2.
[0331] FIG. 8B: Visual scoring of fungal growth of different fungal
species. Two-fold serial dilutions of VHH (anti-GlcCer VHH or
unrelated VHH) are incubated with spores (1E+05/ml) of Cercospora
beticola at room temperature. Effect on fungal growth of VHH and
control compounds was based on a set of photographic standards.
Bars represent average % growth, error bars represent standard
errors of the mean of n=2.
[0332] FIG. 8C: Visual scoring of fungal growth of different fungal
species. Two-fold serial dilutions of VHH (anti-GlcCer VHH or
unrelated VHH) are incubated with spores (1E+05/ml) of Fusarium
culmorum at room temperature. Effect on fungal growth of VHH and
control compounds was based on a set of photographic standards.
Bars represent average % growth, error bars represent standard
errors of the mean of n=2.
[0333] FIG. 8D: Visual scoring of fungal growth of different fungal
species. Two-fold serial dilutions of VHH (anti-GlcCer VHH or
unrelated VHH) are incubated with spores (1E+05/ml) of Verticillium
dahliae at room temperature. Effect on fungal growth of VHH and
control compounds was based on a set of photographic standards.
Bars represent average % growth, error bars represent standard
errors of the mean of n=2.
[0334] FIG. 9: In-vitro antifungal assay using Penicillium
expansum. Two-fold serial dilutions of VHH were inoculated with P.
expansum spores (1E+03/ml) at room temperature. Anti-GlcCer VHH
41D01, unrelated VHH_A, BSA, unrelated hlgG, anti-GlcCer mouse
monoclonal antibody and water were tested. Luminescence (RLU) was
measured after 24 h incubation. % RLU of treated spores are
expressed versus untreated spores. Values represent average % RLU,
error bars represent standard errors of the mean of n=4.
[0335] FIG. 10: Disease severity was measured on tomato leaves
preventively treated with anti-GclCer VHH 41D01, unrelated VHH_A,
or water, and inoculated with Botrytis cinerea spores (6E+06
spores/ml). Bars represent average lesion diameter (mm) scored at 6
days post infection, error bars represent standard errors of the
mean of n=5.
[0336] FIG. 11: Disease severity was measured on tomato leaves
curatively treated with anti-GclCer VHH 41D01, unrelated VHH_A, or
BSA, and inoculated with Botrytis cinerea spores (6E+06 spores/ml).
Bars represent average lesion diameter (mm) scored at 5 days post
infection, error bars represent standard errors of the mean of
n=5.
[0337] FIG. 12: Disease severity was measured on pears preventively
treated with anti-GclCer VHH 41D01, unrelated VHH_A, or water, and
inoculated with Botrytis cinerea spores (1E+04 spores/ml). Bars
represent average lesion diameter (mm) scored at 4 days post
infection, error bars represent standard errors of the mean of
n=5
EXAMPLES AND MATERIALS AND METHODS
Example 1
Isolation of Nucleic Acid Sequences Encoding Peptides with Affinity
for Fungal Glucosylceramide
Animal Immunizations:
[0338] VHH's were generated from llamas immunized with fungal
glucosylceramide (GlcCer). Llamas were immunized according to
standard protocols with 6 boosts of thin Layer Chromatography
(TLC)-purified (99%) glucosylceramide (GlcCer) from Pleurotus
citrinopileatus (Nacalai Tesque). Purified GlcCer was dissolved in
a water:methanol:chloroform mixture and spotted on a TLC silica
glass plate. Silica with adsorbed GlcCer was scraped from the plate
and suspended in phosphate buffer. The suspension was sonicated,
mixed with Freund incomplete adjuvant, and used for subcutaneous
injections. VHH were also generated from llamas immunized with
native germinated fungal or oomycete spores. Llamas were immunized
according to standard protocols with 6 boosts of native germinated
spores of Botrytis cinerea or Phytophthora infestans by
subcutaneous injections. All llamas remained healthy throughout the
immunization process and blood samples were taken before and after
immunizations.
Library Construction:
[0339] A phage library of antibodies is a phage population in which
each individual phage exposes a unique antigen-binding antibody
domain on its surface as a part of a chimeric pill protein.
Peripheral blood mononuclear cells were prepared from blood samples
of the immunized llamas using Ficoll-Hypaque according to the
manufacturer's instructions. Total RNA was extracted from these
cells and used as starting material for RT-PCR to amplify VHH
encoding gene fragments. These fragments were cloned into phagemid
vector pASF20. pASF20 is an expression vector that is derived from
pUC119 which contains the lacZ promotor, a synthetic leader
sequence, a multiple cloning site, a coliphage pill protein coding
sequence, a resistance gene for ampicillin, and an M13 phage origin
for single strand production. In frame with the VHH conding
sequence, the vector codes for a C-terminal (His)6 peptide tag and
c-myc peptide tag. Phage were prepared according to standard
methods (Phage Display of Peptides and Proteins: A Laboratory
Manual; Brian K. Kay, Jill Winter, Dr. John McCafferty). 4
libraries each with a clonal diversity equal to or greater than
1E+08 were obtained and phage were produced ensuring presentation
of the antibody diversity.
VHH Selections by Phage Display:
[0340] Phage expressing antigen-binding antibody domains specific
for a particular antigen were isolated by selecting the phage in
the library for binding to the antigen. Fungal GlcCer were
immobilized on polystyrene Maxisorp multiwell plates by dissolving
fungal GlcCer in a water:methanol:chloroform mixture or methanol at
different concentrations, adding dissolved fungal GlcCer to wells
of the multiwell plate, and allowing to dry overnight at room
temperature. Wells with coated fungal GlcCer were washed and
blocked with 1% fish gelatin in preparation of VHH selections by
phage display. VHH library phage were allowed to bind for two hours
at room temperature to wells of 96-well plate coated with fungal
GlcCer. To specifically select for phage binding to fungal GlcCer
phage were pre-incubated with 1% fish gelatin and/or BSA and/or
skimmed milk and/or plant GlcCer and/or mammalian GlcCer. Non-bound
phage were removed by extensive washing and bound phage were eluted
by competitive elution with RsAFP2 (Osborn et al., 1995) or with
trypsin. One to three consecutive rounds of selection were
performed, and the titers of phage from fungal GlcCer-coated wells
were compared to titers of phage from blank wells and non-target
pathogen sphingolipids for enrichment and specificity,
respectively. Enrichments were observed in first and subsequent
rounds of selection, and phage populations after one or more
selection rounds already showed specificity for fungal GlcCer in
ELISA (not shown). Individual clones were picked from first, second
and/or third round selections for further characterization by
sequence analysis and primary binding assays.
VHH Characterization by Sequencing and Binding Assays:
[0341] The diversity of the obtained antibody or antibody domain
population can be rapidly determined using high-throughput DNA
sequencing and allows precise quantification of clonal diversity.
Antibody or antibody domain binding and specificity of binding to
an antigen can be analyzed in assays for binding to that antigen
and compared to related and unrelated controls. Each antibody or
antibody domain can bind to a specific antigen and possibly to
antigenic variants of that antigen. Specificity is the degree to
which the binding of an antibody or antibody domain discriminates
between antigenic variants. From individual VHH clones that were
picked from first, second or third round phage display selections
the DNA was amplified in a colony PCR and PCR products were
sequenced by Sanger-sequencing. After sequence analysis and based
on sequence diversity, VHH were selected for further
characterization. To check for species specificity, fungal and
non-fungal GlcCer from target and non-target species were used in
binding assays. Primary binding assays to identify which clones
were functionally selected from the libraries were performed with
TLC-purified (99%) GlcCer or GlcCer-enriched Glycosphingolipids
(GSL) fractions from A. brassicicola, B. cinerea, C. beticola, F.
culmorum, F. graminearum, F. oxysporum, P. citrinopileatus P.
digitatum, P. expansum, or V. dahlia (prepared as described in
Ternes et al., 2011 JBC 286:11401-14). GlcCer from soybean and
porcine GlcCer were purchased from Avanti Polar Lipids. VHH were
produced in 96-well deep-well plates and the binding profile of
diluted crude VHH-containing periplasmic extracts was assessed in
ELISA format. In the same way, binding assays were performed with
purified VHH.
[0342] From the primary binding assays 130 VHH-containing
periplasmic extracts showed to bind fungal GlcCer with higher OD
405 nm values than the unrelated VHH_A, unrelated VHH_B and blank.
OD 405 nm values demonstrating the specific binding of several of
these fungal GlcCer binding VHH's are shown in FIG. 1. Sequence
analysis revealed 84 unique sequences from the identified set of
anti-GlcCer VHH.
Further Characterization by Differential Binding Screens:
[0343] For further characterization, VHH belonging to the
abovementioned lead panel were produced in E. coli in culture
flasks according to standard procedures. Hexahistidine-tagged VHH
were purified from the periplasmic extract with TALON metal
affinity resin (Clontech), according to the manufacturer's
instructions. Purified VHH were concentrated and dialyzed to PBS.
VHH were also purified using automated purification systems using a
combination of immobilized Nickel IMAC and desalting columns. VHH
of the lead panel that scored positively in primary binding assays,
were subsequently tested for their specificity towards GlcCer or
cell wall fractions from different fungal phytopathogens.
[0344] As demonstrated in FIGS. 2, 3A, 3B and 3C, GlcCer-specific
VHH showed specific binding to fungal GlcCer (Pleurotus
citrinopileatus, Fusarium oxysporum) and not to other non-fungal
GlcCer or blank non-coated well.
Surface Plasmon Resonance:
[0345] Binding of VHH to fungal GlcCer was characterised by surface
plasmon resonance in a Biacore 3000 instrument. Anti-GlcCer VHH 41
D01 or unrelated VHH_A were covalently bound to CM5 sensor chips
surface via amine coupling until an increase of 1000 response units
was reached. Remaining reactive groups were inactivated. A range of
concentrations of in solution Fusarium oxysporum GlcCer prepared
according to Salio et al., 2013 PNAS 110, E4753-E4761 was injected
for 2 minutes at a flow rate of 30 .mu.l/min to allow for binding
to chip-bound VHH. Running buffer without GlcCer was injected over
the chip at the same flow rate to allow spontaneous dissociation of
bound fungal GlcCer for 10 minutes. A Koff-value was calculated
from the sensorgrams obtained for the different fungal GlcCer
concentrations with 1:1 Langmuir dissociation global fitting
model.
[0346] For anti-GlcCer VHH a slow off-rate of 4.86*1E-4/s was
calculated. As shown in FIG. 4, an unrelated VHH did not bind
fungal GlcCer.
[0347] Plant (soy), mammalian (pork) and fungal (Fusarim oxysporum)
GlcCer in solution were sequentially injected for 2 minutes at a
flow rate of 30 .mu.l/min to allow for binding to chip-bound VHH
(anti-GlcCer VHH 41D01 or unrelated VHH_A). Running buffer without
GlcCer was injected over the chip between each injection at the
same flow rate to allow spontaneous dissociation of bound
GlcCer.
[0348] No plant or mammalian GlcCer binding to anti-GlcCer VHH
41D01 or unrelated VHH_A was observed. Specific binding of fungal
GlcCer was observed for anti-GlcCer VHH 41 D01 and not for
unrelated VHH_A.
Differential Binding to Different Fungal Lipid Extracts:
[0349] The binding of anti-GlcCer VHH compositions to different
fungal lipid extracts compared to unrelated compounds.
[0350] Fungal extracts were prepared according to Rodrigues et al.
2000 Infection and Immunity 68 (12): 7049-60. Briefly, mycelium
from Botrytis cinerea B05-10, Botrytis cinerea MUCL401, Botrytis
cinerea R16, Botrytis cinerea (own pear isolate), Fusarium culmorum
MUCL555, Fusarium graminearum MUCL53451, Penicillium digitatum
MUCL43-410, Penicillium digitatum (own lemon isolate) or
Penicillium expansum CBS 146.45 were harvested from fungi grown in
agar plates and lipids were extracted with chloroform/methanol 2:1
(vol/vol) and 1:2 (vol/vol); crude lipid extract was partitioned
according to Folch et al. 1957. Journal of Biological Chemistry 226
(1): 497-509. Fungal lipid extracts were recovered from Folch's
lower phase. Binding of anti-GlcCer VHH 41D01 (0.1 .mu.g/ml) and
anti-GlcCer VHH 56F11 (1 .mu.g/ml) was evaluated to wells coated
with the extracted fungal lipids (each in 1/20 dilution), purified
Fusarium oxysporum GlcCer, purified Pleurotus citrinopileatus
GlcCer and unrelated compounds: apple pectin (Apple pectin high
esterified 70-75%, Sigma, cat#: 76282), citrus pectin (Citrus
pectin low esterified 20-34%, Sigma, cat# P9311) or potato lectin
(Solanum Tuberosum Lectin, Vector labs, cat#: L-1160) or a blank
non-coated well. Binding was measured after consecutive incubation
with enzyme-conjugated detection antibodies adding substrate and
measuring absorbance at 405 nm. Bars represent average OD 405 nm
values, error bars represent standard errors of the mean of
n=2.
[0351] As shown in FIG. 5, anti-GlcCer VHH 41 D01 and 56F11
specifically recognized all the fungi lipid extracts tested.
Anti-GlcCer VHH 41D01 and 56F11 did not show binding to unrelated
coated compounds or non-coated wells. The binding of the
anti-GlcCer VHH compositions to a wide array of fungal lipids
extracts potentiates a variety of applications for the anti-GlcCer
VHH compositions as disclosed herein against different fungi.
Binding of Anti-GlcCer VHH to Fungal GlcCer in Different Aqueous
Compositions:
[0352] Aqueous compositions containing anti-GlcCer VHH 41 D01
and/or protease inhibitors and/or non-ionic surfactants and/or
preservatives were prepared. Composition A1 (protease inhibitors:
0.06 .mu.g/ml aprotinin (Roche, cat#: 10236624001), 0.5 .mu.g/ml
leupeptin (Roche, cat#: 11017101001), 24 .mu.g/ml 4-benzenesulfonyl
fluoride hydrochloride (Sigma, A8456), 1 mM EDTA (Carl-Roth,
cat#8040.1) and non-ionic surfactant: 0.00001% Polysorbate 20
(Tween.sup.20, Sigma, cat# P2287); Composition A2 (protease
inhibitors: 1 .mu.g/ml aprotinin, 2.5 .mu.g/ml leupeptin, 100
.mu.g/ml 4-benzenesulfonyl fluoride hydrochloride, 1 mM EDTA and
non-ionic surfactant: 0.05% Polysorbate 20); Composition A3
(protease inhibitors: 2 .mu.g/ml aprotinin, 5 .mu.g/ml leupeptin,
240 .mu.g/ml 4-benzenesulfonyl fluoride hydrochloride, 1 mM EDTA
and non-ionic surfactant: 5% Polysorbate 20), Composition B1
(non-ionic surfactant: 0.0001%% Polysorbate 20), Composition B2
(non-ionic surfactant: 0.05% Polysorbate 20), Composition B3
(non-ionic surfactant: 5% Polysorbate 20) and Composition C1
(preservative: 0.05% sodium benzoate (Sigma, cat# B3420)). Binding
of anti-GlcCer VHH (at 0.1 .mu.g/ml) to fungal GlcCer in different
aqueous compositions was tested in ELISA with coated GlcCer from F.
oxysporum and compared to blank non-coated wells. Binding was
measured after consecutive incubation with enzyme-conjugated
detection antibodies, adding substrate and measuring absorbance at
405 nm.
[0353] In FIG. 6, values of GlcCer-specific VHH 41 D01 in the
different compositions were compared with 41 D01 in solution
without other additives. It is shown in FIG. 6 that GlcCer-specific
VHH 41 D01 was capable of specifically binding to fungal GlcCer in
all tested compositions.
Example 2
In Vitro Evaluation of the Antifungal Activity of Anti-GlcCer VHH
Compositions
In Vitro Evaluation of the Antifungal Activity of VHH:
[0354] The antifungal activity of the anti-GlcCer-VHH was tested
using antifungal assays in liquid media and on agar plates as
described in Thevissen et al., 2011, Bioorg. Med. Chem. Lett.
21(12): 3686-92; Francois et al., 2009, J. Biol. Chem. 284(47):
32680-5; Aerts et al., 2009, FEBS Lett. 583(15): 25143-6. The
minimal inhibitory concentration (MIC) was determined for the VHH
on in vitro growth of Botrytis cinerea and Phytophthora
infestans.
[0355] An in vitro assay to test fungal growth in liquid media in
96-well plate format can also be used to directly screen different
VHH that are generated against integral fungal material and
selected against molecular antigens, different from GlcCer, for
antifungal activity. This screening is performed on crude
VHH-containing periplasmic extracts of E. coli cells in which the
VHH are produced, or with purified VHH.
In Vitro Evaluation of the Antifungal Activity of Anti-GlcCer VHH
Compositions Against Different Plant Pathogenic Fungi:
[0356] The antifungal activity of anti-GlcCer VHH compositions was
assessed in vitro against a number of plant pathogenic fungi and
compared with the antifungal activity of unrelated VHH.
[0357] Two-fold dilutions of the aqueous VHH compositions in water
(starting at 1.5 mg VHH/ml) were prepared in 96-well microtiter
plates. To 20 .mu.l of these dilutions and to 20 .mu.l of water as
a control, 80 .mu.l of fungal spores suspension (1 E+05 spores/ml
in half strength potato dextrose broth (PDB)) were added. The
fungal test strains were Alternaria brassicicola MUCL20297,
Botrytis cinerea R16, Cercospora beticola (own sugar beet isolate),
Fusarium culmorum MUCL555 and Verticillium dahliae MUCL6963. The
test plates were incubated for 72 h at room temperature in the dark
and the antifungal activity of the test compounds was scored
microscopically and quantified based on photographic standards,
whereby a score of 0 or 100 referred to no or maximal fungal
growth, respectively. All tests were performed in at least 2
replicas.
[0358] The results of the antifungal activity assays, shown in
FIGS. 7A, 7B, 7C, 8A, 8B, 8C and 8D indicated a clear difference
between the growth inhibition pattern, expressed as the % fungal
growth in function of VHH concentration (.mu.g/ml), of the
anti-GlcCer VHH (including 41D01, 56F11, 56E05 or 57A06) and the
unrelated VHH (VHH_A and VHH_B). This difference was clear
irrespective of the species of the test fungus. Generally, at a
test concentration of 100 .mu.g/ml, all the anti-GlcCer VHH didn't
allow more than 20% fungal growth, whereas at 100 .mu.g/ml the
unrelated VHH showed very weak or no antifungal activity (80% or
more fungal growth). From all the different tested anti-GlcCer VHH,
41D01 showed the most prominent antifungal activity, for several
test strains, even at test concentrations lower than 50 .mu.g/ml
fungal growth was less than 20%.
[0359] The results show the antifungal potency of anti-GlcCer VHH
compared to unrelated VHH. Moreover, the results reveal a
broad-spectrum of antifungal activity of anti-GlcCer VHH
compositions towards at least 5 different fungal plant pathogens
and indicate that the spectrum of antifungal activity of the
selected anti-GlcCer VHH can be broadened to other plant pathogenic
fungi.
In Vitro Evaluation of the Antifungal Activity of Anti-GlcCer VHH
Compositions Against Penicillium expansum Using Luminescence:
[0360] The in vitro antifungal activity of anti-GlcCer VHH 41D01
composition was assessed against the plant pathogen fungus
Penicillium expansum CBS 146.45 and compared with the antifungal
activity of unrelated VHH_A, a mouse monoclonal anti-GlcCer
antibody (mouse MAb anti-GlcCer), human immunoglobulin G (hlgG) or
bovine serum albumin (BSA) as controls using luminescence as
read-out.
[0361] Two-fold serial dilutions of all the test compositions in
water (starting at 1.5 mg/ml) were prepared in 96-well microtiter
plates. To 20 .mu.l of these dilutions and to 20 .mu.l of water as
a control, 80 .mu.l of fungal spores suspension (1 E+03 spores/ml
in 4-fold PDB) were added. The test plates were incubated for 24 h
at room temperature in the dark and the spore viability was
determined at 24 post inoculation (hpi) using luminescence
according to the supplier's instructions (BacTiter Glo; Promega).
The relative light units (RLU) were determined (Tecan luminometer)
and the RLU measured for anti-GlcCer VHH 41D01, unrelated VHH_A,
hlgG, mouse MAb anti-GlcCer or BSA treated fungal spores were
expressed versus the RLU determined for the untreated fungal spores
as % RLU. Four replicas were included in the test (n=4).
[0362] As shown in FIG. 9, the % RLU determined upon anti-GlcCer
VHH 41D01 composition treatment differed clearly from the % RLU
recorded upon unrelated VHH_A, mouse MAb anti-GlcCer, hlgG or BSA
treatments. Particularly, the effect of 41 D01 treatment on fungal
spores, expressed as % RLU versus non-treated control was less than
25% at 300 .mu.g/ml or 150 .mu.g/ml of 41 D01, and less than 50% at
75 .mu.g/ml, 37.5 .mu.g/ml and 19 .mu.g/ml. In contrast, the effect
of all the other test compositions, expressed as % RLU versus
non-treated control was generally 100% for all the tested
concentrations.
[0363] These results show that the specific anti-GlcCer VHH 41 D01
composition had a clear antifungal effect on the plant pathogenic
fungus Penicillium expansum down to 19 .mu.g/ml and is
outperforming non-related VHH_A, mouse MAb anti-GlcCer, hlgG, or
BSA. As such, anti-GlcCer VHH compositions can be used to protect
plants against plant pathogenic fungi.
Example 3
Formulation of VHH into Agricultural Formulations
[0364] Anti-GlcCer VHH were produced as recombinant proteins in a
suitable E. coli production strain. Anti-GlcCer VHH were purified
from the media and/or the periplasm and/or the E. coli cells were
killed and lysed at the end of the fermentation process.
Anti-GlcCer VHH can also be produced as recombinant proteins in
Pichia pastoris, or Saccharomyces cerevisiae and secreted into the
fermentation media. Anti-GlcCer VHH are then purified from media
components and cell constituents by diafiltration.
[0365] The resulting protein solution is diluted in a suitable
buffer, such as phosphate buffered saline, to adjust the pH to
about 7. Optionally a biocidal agent, such as sodium azide in a
concentration of about 0.0001% to 0.1% and a non-ionic detergent,
such as Tween20 in a concentration of about 0.0001% to 5%, is added
to the buffered protein solution.
[0366] Alternatively, the resulting protein solution is admixed
with a suitable wetting and dispersing agent in the presence of a
customary filler material before being spray dried into wettable
granules.
Example 4
Evaluation of Antifungal Activity of VHH on Crops
[0367] The efficacy of the VHH with potent in vitro antifungal
activity against B. cinerea and P. infestans is further evaluated
in planta via disease bio-assays on (i) detached leaves from tomato
and potato plants and (ii) on greenhouse-grown tomato and potato
plants.
[0368] Detached leaf disease assays are performed by using the
model pathosystems tomato-B. cinerea and potato-P. infestans.
Greenhouse-grown tomato and potato plants are sprayed in a spraying
cabinet with an aqueous VHH solution in a volume equivalent to 300
liter per ha and with an application rate below 50 g VHH per
hectare. After spraying, the spray deposit is allowed to dry on the
plants and composite leaves are subsequently detached from the
plants and placed on water agar-plates. The leaves on the
water-agar-plates are drop-inoculated at different time points with
a spore suspension of B. cinerea or P. infestans (5.times.10.sup.5
spores/ml). Disease development is monitored visually and/or
digitally via measuring lesion diameter and image analysis
software, respectively (Assess, Lamari 2002, St. Paul, Minn., USA:
APS Press).
Example 5
In Planta Evaluation of the Antifungal Activity of Anti-GlcCer VHH
Composition to Protect Crops Against Fungal Infection
[0369] Efficacy of Anti-GlcCer VHH Compositions on Tomato Leaves
Inoculated with Botrytis cinerea: Preventive Treatment:
[0370] The effect of a preventive treatment with anti-GlcCer VHH
compositions on the disease severity of Botrytis. cinerea B05-10
inoculated tomato leaves was evaluated and compared with the
effects of unrelated VHH, water or a formulated commercial chemical
fungicide.
[0371] Detached leaves from greenhouse grown tomato plants were
treated with 10 .mu.l of an aqueous VHH composition (anti-GlcCer or
an unrelated VHH at 5 mg/ml), and, water and Scala (1 mg
pyrimethanil/ml, as recommended by the manufacturer) as controls.
Upon drying of the applied compositions, 10 .mu.l drops of a
Botrytis cinerea spores suspension (6 E+06 spores/ml in 4-fold
diluted PDB) were applied on the treated surfaces. Treated and
inoculated leaves were incubated at high relative humidity and at
room temperature in small plant propagators. Disease severity was
scored measuring the bidirectional diameter at 6 days post
inoculation (dpi).
[0372] As shown in FIG. 10, preventive treatment with the
anti-GlcCer VHH composition resulted in an average lesion diameter
of 6 mm (+/-1.4 mm), whereas treatment with an unrelated VHH or
water showed an average lesion diameter of 13.4 mm (+/-4 mm) or 15
mm (+/-4 mm), respectively. In the control treatment with a
formulated commercial chemical fungicide, tomato leaves were
effectively protected against Botrytis cinerea infection (without a
visible lesion).
[0373] As also shown in FIG. 10, preventive treatment of tomato
leaves with the application of the anti-GlcCer VHH composition
clearly resulted in a 2-fold reduction of disease severity compared
with the treatment with an unrelated VHH or water. Therefore, the
specific anti-GlcCer VHH, yet applied as an unformulated aqueous
composition at 5 mg/ml, showed the potency of specific anti-GlcCer
VHH to be used as antifungal compounds to protect crops against
fungal pathogens in agricultural applications.
Efficacy of Anti-GlcCer VHH Compositions on Tomato Leaves
Inoculated with Botrytis cinerea: Curative Treatment:
[0374] The effect of a curative treatment with anti-GlcCer VHH
compositions on the disease severity of Botrytis cinerea B05-10
inoculated tomato leaves was evaluated and compared with the effect
of unrelated VHH, bovine serum albumin (BSA) or a formulated
commercial chemical fungicide.
[0375] Detached leaves from greenhouse-grown tomato plants were
inoculated with 10 .mu.l drops of a Botrytis cinerea spores
suspension ((6 E+06 spores/ml) in 4-fold diluted potato dextrose
broth). One hour after inoculation, the inoculated spots on the
leaves were treated with 10 .mu.l of an aqueous VHH composition
(anti-GlcCer and unrelated VHH at 1.6 mg/ml), and, BSA at 1.6 mg/ml
and Scala (1 mg pyrimethanil/ml, as recommended by the
manufacturer) as controls. Inoculated and treated leaves were
incubated at high relative humidity and at room temperature in
small plant propagators. Disease severity was scored measuring the
bidirectional diameter at 5 dpi.
[0376] As shown in FIG. 11, curative treatment with the anti-GlcCer
VHH composition resulted in an average lesion diameter of 3 mm
(+/-0.8 mm), whereas treatment with an unrelated VHH or BSA showed
an average lesion diameter of 15 mm (+/-3.5 mm) or 13 mm (+/-3.5
mm), respectively. In the control treatment with a formulated
commercial chemical fungicide, tomato leaves were effectively
protected against Botrytis cinerea infection (without a visible
lesion).
[0377] As also shown in FIG. 11, curative treatment of tomato
leaves with the application of the anti-GlcCer VHH composition
clearly resulted in a 4-fold reduction of disease severity compared
with the treatment of unrelated VHH or BSA. Therefore, the specific
anti-GlcCer VHH, yet applied as an unformulated aqueous composition
at 1.6 mg/ml, showed the potency of specific anti-GlcCer VHH to be
used as antifungal compounds to protect crops against fungal
pathogens in agricultural applications.
Efficacy of Anti-GlcCer VHH Compositions on Pears Inoculated with
Botrytis cinerea: Preventive Treatment:
[0378] The effect of a preventive treatment with anti-GlcCer VHH
compositions on the disease severity of Botrytis cinerea (own
isolate from pears) inoculated pears was evaluated and compared
with the effect of unrelated VHH, water, or a formulated commercial
chemical fungicide.
[0379] Pears (variety Williams) from biological agriculture,
previously confirmed as untreated, were treated with 10 .mu.l of
aqueous VHH compositions (containing anti-GlcCer VHH or an
unrelated VHH at 5 mg/ml), and, water and Scala (1 mg
pyrimethanil/ml, as recommended by the manufacturer) as controls.
Upon drying of the applied solutions, 10 .mu.l drops of a Botrytis
cinerea spores suspension (1 E+04 spores/ml in water) were applied
on the treated surfaces. Treated and inoculated pears were
incubated at high relative humidity and at room temperature in
small containers. Disease severity was scored measuring the
bidirectional diameter at 4 dpi.
[0380] As shown in FIG. 12, preventive treatment with the
anti-GlcCer VHH composition resulted in an average lesion diameter
of 3 mm (+/-2 mm), whereas treatment with an unrelated VHH or water
showed an average lesion diameter of 9.6 mm (+/-0.8 mm) or 6.6 mm
(+/-1.6 mm), respectively. In the control preventive treatment with
a formulated commercial chemical fungicide pears were effectively
protected against Botrytis cinerea infection (without a visible
lesion).
[0381] As also shown in FIG. 12, preventive treatment of pears with
the application of the anti-GlcCer VHH composition clearly resulted
in an at least 2-fold reduction of disease severity compared with
the treatment of an unrelated VHH or water. Therefore, the specific
anti-GlcCer VHH, yet applied as an unformulated aqueous solution at
5 mg/ml, showed the potency of specific anti-GlcCer VHH to be used
as an antifungal compounds to protect crops against fungal
pathogens in agricultural applications.
Anti-GlcCer VHH Composition to Protect Plant Seeds Against Fungal
Infection:
[0382] The effect of an anti-GlcCer VHH composition on the
protection of plant seeds against pathogenic fungi can be evaluated
as follows. Surface-sterile plant seeds, treated with an
anti-GlcCer VHH, an unrelated VHH, water or a formulated commercial
chemical fungicide are put on top of a potato dextrose agar plate
containing 1 E+03 spores/ml of the test fungus Fusarium
graminearum. Test plates are incubated at room temperature and the
fungal growth inhibition zones (mm) surrounding the seeds can be
measured allowing comparing the effect of the different
treatments.
Anti-GlcCer VHH Composition to Protect Plant Roots Against Fungal
Infection in Hydroponics:
[0383] The effect of an anti-GlcCer VHH composition on the
protection of plant roots against pathogenic fungi and on plant
health in general can be evaluated as follows. Tomato plants are
grown with their roots in a mineral nutrient solution or on inert
media such as perlite supplemented or drenched, respectively with
an anti-GlcCer VHH composition, an unrelated VHH, water or a
formulated commercial chemical fungicide. Verticillium dahliae (1
E+03 spores/ml) can be used to inoculate the plant roots and the
effect of the different treatments is scored at harvest measuring
disease severity on the plants based on an arbitrary scale of
diseases classes: 0=no symptoms, 1=slight yellowing of leaf,
stunting, or wilting, 2=moderate yellowing of leaf, stunting, or
wilting, 3=severe yellowing of leaf, stunting, or wilting, and
4=leaf death (as described by Fakhro et al., 2010).
Anti-GlcCer VHH Composition to Protect Plant Flowers Against Fungal
Infection:
[0384] The effect of an anti-GlcCer VHH composition on the
protection of plant flowers against pathogenic fungi can be
evaluated using cereals or Arabidopsis thaliana and Fusarium
culmorum or Fusarium graminearum as test fungi (as described by
Urban et al., 2002). In short, flowering plants are
spray-inoculated with 1 E+05 spores/ml) of Fusarium culmorum or
Fusarium graminearum followed by a treatment with an anti-GlcCer
VHH composition, an unrelated VHH, water or a formulated commercial
chemical fungicide (curative treatment) or vice versa (preventive
treatment). Plants are incubated and the disease scoring is
performed as described by Urban et al. (2002) and allows
quantifying the effect of the different treatments.
Sequence CWU 1
1
3351118PRTArtificial SequenceVHH sequence 40F07 1Gln Val Gln Leu
Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Val Ala Ser Gly Thr Thr Phe Ser Ser Tyr 20 25 30
Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Leu 35
40 45 Ala Ser Ile Glu Gly Gly Gly Asn Thr Asp Tyr Ala Asp Ser Val
Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Thr
Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala
Val Tyr Tyr Cys Asn 85 90 95 Ala Ala Arg Thr Trp Ser Ile Phe Arg
Asn Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115
2115PRTArtificial SequenceVHH sequence 41D01 2Gln Val Gln Leu Gln
Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Arg Tyr 20 25 30 Gly
Met Gly Trp Phe Arg Gln Leu Pro Gly Lys Gln Arg Glu Leu Val 35 40
45 Thr Ser Ile Thr Arg Gly Gly Thr Thr Thr Tyr Ala Asp Ser Val Lys
50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val
Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val
Tyr Tyr Cys Asn 85 90 95 Ala Arg Ser Ile Trp Arg Asp Tyr Trp Gly
Gln Gly Thr Gln Val Thr 100 105 110 Val Ser Ser 115
3116PRTArtificial SequenceVHH sequence 41D06 3Gln Val Gln Leu Gln
Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Gly Ile Phe Gly Ile Asn 20 25 30 Ala
Met Arg Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40
45 Ala Ser Ile Ser Ser Gly Gly Asn Thr Asn Tyr Ser Glu Ser Val Lys
50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Asn Tyr Thr Val
Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val
Tyr Tyr Cys Asn 85 90 95 Phe Val Arg Leu Trp Phe Pro Asp Tyr Trp
Gly Gln Gly Thr Gln Val 100 105 110 Thr Val Ser Ser 115
4121PRTArtificial SequenceVHH sequence 41G10 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 Ala Ala Thr Lys Thr Gly Phe Ser Ile Asn 20 25 30 Ala
Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Met Val 35 40
45 Ala Thr Ile Thr Ser Gly Gly Thr Thr Asn Tyr Ala Asp Ser Val Lys
50 55 60 Gly Arg Phe Ala Ile Ser Arg Asp Asn Ala Lys Asn Thr Val
Ser Leu 65 70 75 80 Gln Met Asn Thr Leu Lys Pro Glu Asp Thr Ala Leu
Tyr Tyr Cys Asn 85 90 95 Thr Glu Ala Arg Arg Tyr Phe Thr Arg Ala
Ser Gln Val Tyr Trp Gly 100 105 110 Gln Gly Thr Gln Val Thr Val Ser
Ser 115 120 5120PRTArtificial SequenceVHH sequence 41H05 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 Gly Ile Phe Ser Ile Asn 20
25 30 Ala Met Gly Trp Tyr Arg Gln Asp Pro Gly Lys Gln Arg Glu Met
Val 35 40 45 Ala Thr Ile Thr Ser Gly Ala Asn Thr Asn Tyr Thr Asp
Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp
Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Ala Val Gly Arg Arg Trp Tyr
Gly Gly Tyr Val Glu Leu Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr
Val Ser Ser 115 120 6119PRTArtificial SequenceVHH sequence 42C11
6Gln 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 Ser Ile Phe Ser Thr
Tyr 20 25 30 Val Met Gly Trp Tyr Arg Gln Ala Ile Gly Lys Gln Arg
Glu Leu Val 35 40 45 Ala Thr Ile Thr Ser Ser Gly Lys Thr Asn Tyr
Ala Ala Ser Val Lys 50 55 60 Gly Arg Phe Thr Val Ser Arg Asp Ile
Thr Lys Asn Thr Met Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro
Glu Asp Thr Ala Val Tyr Tyr Cys Gly 85 90 95 Ala Asp Arg Trp Val
Leu Thr Arg Trp Ser Asn Tyr Trp Gly Gln Gly 100 105 110 Thr Gln Val
Thr Val Ser Ser 115 7116PRTArtificial SequenceVHH sequence 42C12
7Gln 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 Ser Ile Ser Ser Leu
Gly 20 25 30 Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Phe Val
Ala Ser Ala 35 40 45 Thr Ser Gly Gly Asp Thr Thr Tyr Ala Asp Ser
Val Lys Gly Arg Phe 50 55 60 Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Val Tyr Leu Gln Met Asn 65 70 75 80 Ser Leu Lys Pro Glu Asp Thr
Ala Val Tyr Tyr Cys Lys Gly Gln Arg 85 90 95 Gly Val Ala Trp Thr
Arg Lys Glu Tyr Trp Gly Gln Gly Thr Gln Val 100 105 110 Thr Val Ser
Ser 115 8119PRTArtificial SequenceVHH sequence 50D03 8Gln 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 Ser Ile Phe Ser Thr Tyr 20 25
30 Ala Met Gly Trp Tyr Arg Gln Ala Ile Gly Lys Gln Arg Glu Leu Val
35 40 45 Ala Thr Ile Thr Ser Ser Gly Lys Thr Asn Tyr Ala Ala Ser
Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Ile Thr Lys Asn
Thr Met Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr
Ala Val Tyr Tyr Cys Gly 85 90 95 Ala Asp Arg Trp Val Leu Thr Arg
Trp Ser Asn Tyr Trp Gly Gln Gly 100 105 110 Thr Gln Val Thr Val Ser
Ser 115 9120PRTArtificial SequenceVHH sequence 50D07 9Gln 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 Thr Ala Ser Gly Asn Ile Val Asn Ile Arg 20 25
30 Asp Met Gly Trp Tyr Arg Gln Val Pro Gly Lys Gln Arg Glu Leu Val
35 40 45 Ala Thr Ile Thr Ser Asp Gln Ser Thr Asn Tyr Ala Asp Ser
Val Lys 50 55 60 Gly Arg Phe Thr Thr Thr Arg Asp Asn Ala Lys Lys
Thr Val Tyr Leu 65 70 75 80 Gln Met Asp Ser Leu Lys Pro Glu Asp Thr
Ala Gly Tyr Tyr Cys Asn 85 90 95 Ala Arg Val Arg Thr Val Leu Arg
Gly Trp Arg Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val
Ser Ser 115 120 10119PRTArtificial SequenceVHH sequence 50E02 10Gln
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 Ser Ile Phe Ser Ile Asn
20 25 30 Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu
Leu Val 35 40 45 Ala Ala Ile Thr Ser Asp Gly Ser Thr Asn Tyr Ala
Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
Lys Asn Thr Ala Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu
Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Leu Arg Arg Arg Thr Phe
Leu Lys Ser Ser Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Gln Val Thr
Val Ser Ser 115 11123PRTArtificial SequenceVHH sequence 51B08 11Gln
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 Arg Phe Gly Ser Tyr
20 25 30 Ala Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu
Leu Val 35 40 45 Ala Gly Ile Ser Ser Gly Gly Ser Thr Lys Tyr Ala
Asp Ser Val Arg 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
Lys Asn Thr Val Ser Leu 65 70 75 80 Gln Met Lys Ser Leu Lys Pro Glu
Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Ala Lys Tyr Gly Arg Trp
Thr Tyr Thr Gly Arg Pro Glu Tyr Asp Ser 100 105 110 Trp Gly Gln Gly
Thr Gln Val Thr Val Ser Ser 115 120 12116PRTArtificial SequenceVHH
sequence 51C06 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 Ala Ser Gly
Ser Ile Phe Ser Ser Asp 20 25 30 Thr Met Gly Trp Tyr Arg Arg Ala
Pro Gly Lys Gln Arg Glu Leu Val 35 40 45 Ala Ala Ile Thr Thr Gly
Gly Asn Thr Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met
Asn Ser Leu Gln Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95
Cys Arg Arg Arg Trp Ser Arg Asp Phe Trp Gly Gln Gly Thr Gln Val 100
105 110 Thr Val Ser Ser 115 13119PRTArtificial SequenceVHH sequence
51C08 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 Thr Ile Phe
Ser Ile Lys 20 25 30 Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys
Gln Arg Glu Leu Val 35 40 45 Ala Thr Ile Ser Asn Gly Gly Ser Thr
Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu
Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Ala Arg Gln
Gln Phe Ile Gly Ala Pro Tyr Glu Tyr Trp Gly Gln Gly 100 105 110 Thr
Gln Val Thr Val Ser Ser 115 14115PRTArtificial SequenceVHH sequence
52A01 14Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Ala Ile Thr
Phe Ser Leu 20 25 30 Gly Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly
Lys Gln Arg Glu Leu 35 40 45 Val Ala Ser Ile Ser Thr Gly Ser Thr
Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg
Asp Ile Ile Lys Asn Ile Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu
Lys Pro Glu Asp Thr Ala Val Tyr Ser Cys Asn 85 90 95 Ala Arg Leu
Leu Trp Ser Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr 100 105 110 Val
Ser Ser 115 15117PRTArtificial SequenceVHH sequence 52B01 15Gln Val
Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Glu 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Ser Ile Asn 20
25 30 Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Glu Gln Arg Glu Leu
Val 35 40 45 Ala Thr Ile Ser Arg Gly Gly Ser Thr Asn Tyr Ala Asp
Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Val Thr Val Tyr Leu 65 70 75 80 Gln Met Asp Ser Leu Lys Pro Glu Asp
Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Ala Ala Gly Trp Val Gly Val
Thr Asn Tyr Trp Gly Gln Gly Thr Gln 100 105 110 Val Thr Val Ser Ser
115 16117PRTArtificial SequenceVHH sequence 52G05 16Gln Val Gln Leu
Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Gly Ser Ile Ser 20 25 30
Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35
40 45 Ala Ser Ile Thr Arg Arg Gly Ser Thr Asn Tyr Ala Asp Ser Val
Lys 50 55 60 Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Trp Asn Thr
Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala
Val Tyr Tyr Cys Asn 85 90 95 Ala Arg Arg Tyr Tyr Thr Arg Asn Asp
Tyr Trp Gly Gln Gly Thr Gln 100 105 110 Val Thr Val Ser Ser 115
17114PRTArtificial SequenceVHH sequence 53A01 17Gln Val Gln Leu Gln
Glu Ser Gly Gly Gly Leu Gly Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Glu Val Ser Gly Thr Thr Phe Ser Ile Asn 20 25 30 Thr
Met Gly Trp His Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40
45 Ala Ser Ile Ser Ser Gly Gly Trp Thr Asn Tyr Ala Asp Ser Val Lys
50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Thr Val
Tyr Leu 65 70 75 80 Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val
Tyr Tyr Cys Arg 85 90 95 Trp Gly Ala Ile Gly Asn Trp Tyr Gly Gln
Gly Thr Gln Val Thr Val 100 105 110 Ser Ser 18117PRTArtificial
SequenceVHH sequence 53F05 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 Val Arg Ile Phe Gly Leu Asn 20 25 30 Ala Met Gly Trp Tyr
Arg Gln Gly Pro Gly Lys Gln Arg Glu Leu Val 35 40 45 Ala Ser Ile
Thr Thr Gly Gly Ser Thr Asn Tyr Ala Glu Pro Val Lys 50 55 60 Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Asn Asn Thr Val Tyr Leu 65 70
75 80 Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
Asn 85 90 95 Ala Glu Arg Arg Trp Gly Leu Pro Asn Tyr Trp Gly Gln
Gly Thr Gln 100 105 110 Val Thr Val Ser Ser 115 19126PRTArtificial
SequenceVHH sequence 54A02 19Gln Val Gln Leu Gln Glu Ser Gly Gly
Gly Leu Val Glu Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Arg Thr Phe Ser Arg Tyr 20 25 30 Gly Met Gly Trp Phe
Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Asn
Arg Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr 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 Ala Tyr Ala His Ile Thr Ala Trp Gly Met Arg
Asn Asp Tyr Glu 100 105 110 Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val
Thr Val Ser Ser 115 120 125 20125PRTArtificial SequenceVHH sequence
54B01 20Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Thr Gly Arg Thr Phe
Ser Arg Tyr 20 25 30 Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys
Glu Arg Asp Phe Val 35 40 45 Ala Gly Ile Thr Trp Thr Gly Gly Ser
Thr Asp Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Thr 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 Ala Gly
Asn Leu Leu Arg Leu Ala Gly Gln Leu Arg Arg Gly Tyr 100 105 110 Asp
Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125
21129PRTArtificial SequenceVHH sequence 54C01 21Gln Val Gln Leu Gln
Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Arg Thr Gly Ser Arg Tyr 20 25 30 Ala
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40
45 Ala Ala Ile Ser Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60 Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr
Val Tyr 65 70 75 80 Leu Gln Met His Ser Leu Lys Pro Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Thr Arg Asn Arg Ala Gly Pro His Tyr
Ser Arg Gly Tyr Thr Ala 100 105 110 Gly Gln Glu Tyr Asp Tyr Trp Gly
Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser 22121PRTArtificial
SequenceVHH sequence 54C04 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 Arg Ile Phe Ser Ile Asn 20 25 30 Ala Met Gly Trp Tyr
Arg Gln Gly Pro Gly Lys Glu Arg Glu Leu Val 35 40 45 Val Asp Met
Thr Ser Gly Gly Ser Ile Asn Tyr Ala Asp Ser Val Ser 50 55 60 Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70
75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
His 85 90 95 Ala Asn Leu Arg Thr Ala Phe Trp Arg Asn Gly Asn Asp
Tyr Trp Gly 100 105 110 Gln Gly Thr Gln Val Thr Val Ser Ser 115 120
23115PRTArtificial SequenceVHH sequence 54C08 23Gln 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 Ser Ile Ser Ser Ile Asn 20 25 30 Ala
Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40
45 Ala Ser Ile Thr Ser Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys
50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val
Asn Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val
Tyr Tyr Cys Ser 85 90 95 Ala Gly Pro Trp Tyr Arg Arg Ser Trp Gly
Arg Gly Thr Gln Val Thr 100 105 110 Val Ser Ser 115
24116PRTArtificial SequenceVHH sequence 54C10 24Gln Val Gln Leu Gln
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Glu 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Ala Ser Ile Phe Trp Val Asn 20 25 30 Asp
Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40
45 Ala Gln Ile Thr Arg Arg Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys
50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asp Glu Val
Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val
Tyr Tyr Cys Asn 85 90 95 Ala Asp Leu Ala Val Arg Gly Arg Tyr Trp
Gly Gln Gly Thr Gln Val 100 105 110 Thr Val Ser Ser 115
25119PRTArtificial SequenceVHH sequence 54C11 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 Ser Gly Ser Phe Phe Pro Val Asn 20 25 30 Asp
Met Ala Trp Tyr Arg Gln Ala Leu Gly Asn Glu Arg Glu Leu Val 35 40
45 Ala Asn Ile Thr Arg Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys
50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val
Tyr Leu 65 70 75 80 Gln Met Asn Thr Leu Lys Pro Glu Asp Thr Ala Val
Tyr Tyr Cys Asn 85 90 95 Val Arg Ile Gly Phe Gly Trp Thr Ala Lys
Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Gln Val Thr Val Ser Ser 115
26116PRTArtificial SequenceVHH sequence 54D03 26Gln 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 Gly Ile Phe Gly Ile Asn 20 25 30 Ala
Met Arg Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40
45 Ala Ser Ile Ser Ser Gly Gly Asn Thr Asn Tyr Ser Glu Ser Val Lys
50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Asn Tyr Thr Val
Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val
Tyr Tyr Cys Asn 85 90 95 Phe Val Arg Leu Trp Phe Pro Asp Tyr Trp
Gly Gln Gly Thr Gln Val 100 105 110 Thr Val Ser Ser 115
27116PRTArtificial SequenceVHH sequence 54D06 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 Ser Gly Ser Thr Ile Arg Ile Asn 20 25 30 Ala
Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40
45 Ala Thr Ile Thr Arg Gly Gly Ile Thr Asn Tyr Ala Asp Ser Val Lys
50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Phe Thr Val
Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val
Tyr Tyr Cys Asn 85 90 95 Ala Arg Ser Trp Val Gly Pro Glu Tyr Trp
Gly Gln Gly Thr Gln Val 100 105 110 Thr Val Ser Ser 115
28120PRTArtificial SequenceVHH sequence 54D10 28Gln 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 Met Thr Tyr Ser Ile His 20 25 30 Ala
Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40
45 Ala Ile Thr Ser Thr Ser Gly Thr Thr Asp Tyr Thr Asp Ser Val Lys
50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Gly Ala Asn Asn Thr Val
Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val
Tyr Tyr Cys His 85 90 95 Val Lys Thr Arg Thr Trp Tyr Asn Gly Lys
Tyr Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser
115 120 29117PRTArtificial SequenceVHH sequence 54E01 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 Thr Ala Ser Gly Ser Ile Phe Ser Ile Asn 20 25
30 Pro Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val
35 40 45 Ala Ala Ile Thr Ser Gly Gly Ser Thr Asn Tyr Ala Asp Tyr
Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
Val Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr
Ala Val Tyr Tyr Cys Asn 85 90 95 Gly Arg Ser Thr Leu Trp Arg Arg
Asp Tyr Trp Gly Gln Gly Thr Gln 100 105 110 Val Thr Val Ser Ser 115
30117PRTArtificial SequenceVHH sequence 54E05 30Gln 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 Ser Ile Phe Ser Ile Asn 20 25 30 Thr
Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40
45 Ala Ala Ile Thr Asn Arg Gly Ser Thr Asn Tyr Ala Asp Phe Val Lys
50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val
Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Asp Asp Thr Ala Val
Tyr Tyr Cys Asn 85 90 95 Ala His Arg Ser Trp Pro Arg Tyr Asp Ser
Trp Gly Gln Gly Thr Gln 100 105 110 Val Thr Val Ser Ser 115
31118PRTArtificial SequenceVHH sequence 54E10 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 Ala Ala Ser Gly Ser Ile Phe Ser Phe Asn 20 25 30 Ala
Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40
45 Ala Ala Ile Thr Arg Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys
50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Asn Asn Thr Val
Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val
Tyr Tyr Cys Asn 85 90 95 Ala Glu Ser Arg Ile Phe Arg Arg Tyr Asp
Tyr Trp Gly Pro Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115
32115PRTArtificial SequenceVHH sequence 54F01 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 Val Thr Ser Gly Ser Ile Phe Gly Leu Asn 20 25 30 Leu
Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40
45 Ala Thr Ile Thr Arg Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys
50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Thr Val
Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val
Tyr Tyr Cys Asn 85 90 95 Val Asp Arg Gly Trp Ser Ser Tyr Trp Gly
Gln Gly Thr Gln Val Thr 100 105 110 Val Ser Ser 115
33119PRTArtificial SequenceVHH sequence 54F02 33Gln 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 Thr Ser Gly Ser Ile Arg Ser Ile Asn 20 25 30 Thr
Met Gly Trp Tyr Arg Gln Ala Pro Gly Asn Glu Arg Glu Leu Val 35 40
45 Ala Thr Ile Thr Ser Gly Gly Thr Thr Asn Tyr Ala Asp Ser Val Lys
50 55 60 Asn Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val
Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val
Tyr Tyr Cys Asn 85 90 95 Leu His Gln Arg Ala Trp Ala Arg Ser Tyr
Val Tyr Trp Gly Gln Gly 100 105 110 Thr Gln Val Thr Val Ser Ser 115
34117PRTArtificial SequenceVHH sequence 54G01 34Gln Val Gln Leu Gln
Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ala Val Asn 20 25 30 Ala
Met Gly Trp Tyr Arg Gln Ala Pro Gly His Gln Arg Glu Leu Val 35 40
45 Ala Ile Ile Ser Ser Asn Ser Thr Ser Asn Tyr Ala Asp Ser Val Lys
50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val
Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val
Tyr Phe Cys Tyr 85 90 95 Ala Lys Arg Ser Trp Phe Ser Gln Glu Tyr
Trp Gly Gln Gly Thr Gln 100 105 110 Val Thr Val Ser Ser 115
35121PRTArtificial SequenceVHH sequence 54G08 35Gln 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 Ser Ile Phe Ser Phe Asn 20 25 30 Leu
Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40
45 Ala Ala Ile Thr Ser Ser Ser Asn Thr Asn Tyr Ala Asp Ser Val Lys
50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val
Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val
Tyr Tyr Cys Asn 85 90 95 Ala Gln Tyr Thr Ile Thr Pro Trp Gly Ile
Lys Lys Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Gln Val Thr Val Ser
Ser 115 120 36120PRTArtificial SequenceVHH sequence 54G09 36Gln Val
Gln Leu Gln Glu Ser Gly Gly Gly Leu Met Gln Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Asn Ile Val Asn Ile Arg 20
25 30 Asp Met Gly Trp Tyr Arg Gln Val Pro Gly Lys Gln Arg Glu Leu
Val 35 40 45 Ala Thr Ile Thr Ser Asp Gln Ser Thr Asn Tyr Ala Asp
Ser Val Lys 50 55 60 Gly Arg Phe Thr Thr Thr Arg Asp Asn Ala Lys
Lys Thr Val Tyr Leu 65 70 75 80 Gln Met Asp Ser Leu Lys Pro Glu Asp
Thr Ala Gly Tyr Tyr Cys Asn 85 90 95 Ala Arg Val Arg Thr Val Leu
Arg Gly Trp Arg Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr
Val Ser Ser 115 120 37117PRTArtificial SequenceVHH sequence 55B02
37Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Glu 1
5 10 15 Ser Leu Arg Leu Ser Cys Val Gly Ser Gly Ser Ile Phe Asn Ile
Asn 20 25 30 Ser Met Asn Trp Tyr Arg Gln Ala Ser Gly Lys Gln Arg
Glu Leu Val 35 40 45 Ala Asp Met Arg Ser Asp Gly Ser Thr Asn Tyr
Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ala Arg Lys Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro
Glu Asp Thr Ala Val Tyr Tyr Cys His 85 90
95 Ala Asn Ser Ile Phe Arg Ser Arg Asp Tyr Trp Gly Gln Gly Thr Gln
100 105 110 Val Thr Val Ser Ser 115 38125PRTArtificial SequenceVHH
sequence 55B05 38Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val
Gln Ala Gly Asp 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Arg Thr Phe Gly Gly Tyr 20 25 30 Thr Val Ala Trp Phe Arg Gln Ala
Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Arg Ile Ser Trp Ser
Gly Ile Met Ala Tyr Tyr Ala Glu Ser Val 50 55 60 Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr 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 Ser Arg Ser Gln Ile Arg Ser Pro Trp Ser Ser Leu Asp Asp Tyr 100
105 110 Asp Arg Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120
125 39116PRTArtificial SequenceVHH sequence 55C05 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 Val Val Ser Gly Ser Ile Ser Ser Met Lys 20 25 30
Ala Met Gly Trp His Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val 35
40 45 Ala Gln Ile Thr Arg Gly Asp Ser Thr Asn Tyr Ala Asp Ser Val
Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr
Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Asp Asp Thr Gly
Val Tyr Tyr Cys Asn 85 90 95 Ala Asp Arg Phe Phe Gly Arg Asp Tyr
Trp Gly Lys Gly Thr Gln Val 100 105 110 Thr Val Ser Ser 115
40120PRTArtificial SequenceVHH sequence 55D08 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 Arg Ser Ile Leu Ser Ile Ser 20 25 30 Ala
Met Gly Trp Tyr Arg Gln Gly Pro Gly Lys Gln Arg Glu Pro Val 35 40
45 Ala Thr Ile Thr Ser Ala Gly Ser Ser Asn Tyr Ser Asp Ser Val Lys
50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Ala
Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val
Tyr Tyr Cys Lys 85 90 95 Thr Val Tyr Ser Arg Pro Leu Leu Gly Pro
Leu Glu Val Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser
115 120 41115PRTArtificial SequenceVHH sequence 55E02 41Gln Val Gln
Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Val Ala Ser Gly Ser Met Phe Ser Ser Asn 20 25
30 Ala Met Ala Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val
35 40 45 Ala Arg Ile Leu Ser Gly Gly Ser Thr Asn Tyr Ala Asp Ser
Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Gly Asn Ala Lys Asn
Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr
Ala Val Tyr Tyr Cys Asn 85 90 95 Ala Val Arg Tyr Leu Val Asn Tyr
Trp Gly Gln Gly Thr Gln Val Thr 100 105 110 Val Ser Ser 115
42121PRTArtificial SequenceVHH sequence 55E07 42Gln Val Gln Leu Gln
Glu Ser Gly Gly Gly Ser Val Gln Val Gly Asp 1 5 10 15 Ser Leu Thr
Leu Ser Cys Val Ala Ser Gly Arg Ser Leu Asp Ile Tyr 20 25 30 Gly
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40
45 Ala Arg Ile Thr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60 Gly Arg Phe Thr Leu Ser Arg Asp Asn Ala Lys Asn Thr Val
Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val
Tyr Tyr Cys Ala 85 90 95 Ala Gly Val Val Val Ala Thr Ser Pro Lys
Phe Tyr Ala Tyr Trp Gly 100 105 110 Gln Gly Thr Gln Val Thr Val Ser
Ser 115 120 43116PRTArtificial SequenceVHH sequence 55E09 43Gln Val
Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Lys Arg Ile Phe Ser Thr Tyr 20
25 30 Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe
Val 35 40 45 Ala Ala Ile Ile Trp Ser Gly Gly Arg Thr Arg Tyr Ala
Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
Arg Asn Thr Val His 65 70 75 80 Leu Gln Met Asn Ser Leu Glu Pro Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Tyr Thr Arg Arg Leu Gly Thr
Gly Tyr Trp Gly Gln Gly Thr Gln Val 100 105 110 Thr Val Ser Ser 115
44115PRTArtificial SequenceVHH sequence 55E10 44Gln Val Gln Leu Gln
Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Ser Ile Gln 20 25 30 Thr
Ile Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Asp Arg Val 35 40
45 Ala Thr Ile Ser Ser Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys
50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Thr Val
Tyr Leu 65 70 75 80 Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val
Tyr Tyr Cys Asn 85 90 95 Leu Arg Tyr Trp Phe Arg Asp Tyr Trp Gly
Gln Gly Thr Gln Val Thr 100 105 110 Val Ser Ser 115
45115PRTArtificial SequenceVHH sequence 55F04 45Gln 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 Ser Thr Phe Ser Ile Asn 20 25 30 Val
Arg Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40
45 Ala Thr Ile Thr Ser Asp Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys
50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Ala
Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val
Tyr Tyr Cys Asn 85 90 95 Ala Val Arg Leu Phe Arg Gln Tyr Trp Gly
Gln Gly Thr Gln Val Thr 100 105 110 Val Ser Ser 115
46125PRTArtificial SequenceVHH sequence 55F09 46Gln 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 Ser Ile Phe Arg Leu Asn 20 25 30 Ala
Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40
45 Ala Ala Ile Thr Pro Gly Gly Gly Asn Thr Thr Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Leu Asn Thr
Ile Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Asn Ala Gly Gly Ser Ser Arg Trp Tyr Ser
Ser Arg Tyr Tyr Pro Gly 100 105 110 Gly Tyr Trp Gly Gln Gly Thr Gln
Val Thr Val Ser Ser 115 120 125 47126PRTArtificial SequenceVHH
sequence 55F10 47Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val
Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly
Gly Thr Phe Ser Arg Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala
Pro Gly Lys Glu Arg Glu Leu Val 35 40 45 Ala Thr Ile Arg Arg Ser
Gly Ser Ser Thr Tyr Tyr Leu Asp Ser Thr 50 55 60 Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln
Met Asn Ser Leu Lys Leu Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Ala Asp Ser Ser Ala Arg Ala Leu Val Gly Gly Pro Gly Asn Arg 100
105 110 Trp Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115
120 125 48118PRTArtificial SequenceVHH sequence 55G02 48Gln 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 Ser Ile Gly Ser Ile Asn 20 25
30 Val Met Gly Trp Tyr Arg Gln Tyr Pro Gly Lys Gln Arg Glu Leu Val
35 40 45 Ala Phe Ile Thr Ser Gly Gly Ile Thr Asn Tyr Thr Asp Ser
Val Lys 50 55 60 Gly Arg Phe Ala Ile Ser Arg Asp Asn Ala Gln Asn
Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Thr Pro Glu Asp Thr
Ala Val Tyr Tyr Cys His 85 90 95 Leu Lys Asn Ala Lys Asn Val Arg
Pro Gly Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser
115 49116PRTArtificial SequenceVHH sequence 55G08 49Gln 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 Arg Ala Ser Gly Gly Ile Phe Gly Ile Asn 20 25 30
Ala Met Arg Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35
40 45 Ala Ser Ile Ser Ser Gly Gly Thr Thr Asp Tyr Val Glu Ser Val
Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Thr Asn Thr
Val Asp Leu 65 70 75 80 Gln Met Ser Ala Leu Lys Pro Glu Asp Thr Ala
Val Tyr Tyr Cys Asn 85 90 95 Phe Val Arg Phe Trp Phe Pro Asp Tyr
Trp Gly Gln Gly Thr Gln Val 100 105 110 Thr Val Ser Ser 115
50116PRTArtificial SequenceVHH sequence 56A05 50Gln Val Gln Leu Gln
Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Met Ser Asn 20 25 30 Thr
Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40
45 Ala Ser Ile Ser Ser Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys
50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Thr Val
Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val
Tyr Tyr Cys Asn 85 90 95 Ala Arg Arg Asn Val Phe Ile Ser Ser Trp
Gly Gln Gly Thr Gln Val 100 105 110 Thr Val Ser Ser 115
51114PRTArtificial SequenceVHH sequence 56A06 51Gln 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 Ser Ile Ser Val Tyr Gly 20 25 30 Met
Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Ala 35 40
45 Arg Ile Thr Asn Ile Gly Thr Thr Asn Tyr Ala Asp Ser Val Lys Gly
50 55 60 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
Leu Gln 65 70 75 80 Met Asn Ser Leu Gln Pro Glu Asp Thr Ala Val Tyr
Tyr Cys Asn Leu 85 90 95 Arg Arg Leu Gly Arg Asp Tyr Trp Gly Gln
Gly Thr Gln Val Thr Val 100 105 110 Ser Ser 52118PRTArtificial
SequenceVHH sequence 56A09 52Gln 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 Arg Thr Ala Leu Arg Leu Asn 20 25 30 Ser Met Gly Trp Tyr
Arg Gln Ala Pro Gly Ser Gln Arg Glu Leu Val 35 40 45 Ala Thr Ile
Thr Arg Gly Gly Thr Thr Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly
Arg Phe Thr Ile Ser Arg Glu Ile Gly Asn Asn Thr Val Tyr Leu 65 70
75 80 Gln Met Asn Ser Leu Glu Pro Glu Asp Thr Ala Val Tyr Tyr Cys
Asn 85 90 95 Ala Asn Phe Gly Ile Leu Val Gly Arg Glu Tyr Trp Gly
Lys Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115
53116PRTArtificial SequenceVHH sequence 56C09 53Gln Val Gln Leu Gln
Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Val Ser Gly Ser Ile Phe Ser Ile Leu 20 25 30 Ser
Met Ala Trp Tyr Arg Gln Thr Pro Gly Lys Gln Arg Glu Leu Val 35 40
45 Ala Asn Ile Thr Ser Val Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys
50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Ile Ala Lys Lys Thr Leu
Tyr Leu 65 70 75 80 Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Ile
Tyr Tyr Cys Asn 85 90 95 Thr Arg Met Pro Phe Leu Gly Asp Ser Trp
Gly Gln Gly Thr Gln Val 100 105 110 Thr Val Ser Ser 115
54111PRTArtificial SequenceVHH sequence 56C12 54Gln Val Gln Leu Gln
Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Val Ser Ala Phe Ser Phe Ser Asn Arg 20 25 30 Ala
Val Ser Trp Tyr Arg Gln Ala Pro Gly Lys Ser Arg Glu Trp Val 35 40
45 Ala Ser Ile Ser Gly Ile Arg Ile Thr Thr Tyr Thr Asn Ser Val Lys
50 55 60 Gly Arg Phe Ile Ile Ser Arg Asp Asn Ala Lys Lys Thr Val
Tyr Leu 65 70 75 80 Gln Met Asn Asp Leu Arg Pro Glu Asp Thr Gly Val
Tyr Arg Cys Tyr 85 90 95 Met Asn Arg Tyr Ser Gly Gln Gly Thr Gln
Val Thr Val Ser Ser 100 105 110 55117PRTArtificial SequenceVHH
sequence 56D06 55Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val
Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Thr Val Phe Phe Ser Ile 20 25 30 Ser Ala Met Gly Trp Tyr Arg Gln
Ala Pro Gly Lys Gln Arg Glu Leu 35 40 45 Val Ala Gly Ile Ser Arg
Gly Gly Ser Thr Lys Tyr Gly Asp Phe Val 50 55 60 Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Gly Lys Lys Thr Ile Trp 65 70 75 80 Leu Gln
Met Asn Asn Leu Gln Pro Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95
Arg Leu Thr Ser Ile Thr Gly Thr Tyr Leu Trp Gly Gln Gly Thr Gln 100
105 110 Val Thr Val Ser Ser 115 56114PRTArtificial SequenceVHH
sequence 56D07 56Gln 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
Ser Ile Phe Ser Met Lys 20 25 30 Val Met Gly Trp Tyr Arg Gln
Gly
Pro Gly Lys Leu Arg Glu Leu Val 35 40 45 Ala Val Ile Thr Ser Gly
Gly Arg Thr Asn Tyr Ala Glu Ser Val Lys 50 55 60 Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Ser Leu 65 70 75 80 Gln Met
Asn Ser Leu Gln Pro Glu Asp Thr Ala Val Tyr Tyr Cys Tyr 85 90 95
Tyr Lys Thr Ile Arg Pro Tyr Trp Gly Gln Gly Thr Gln Val Thr Val 100
105 110 Ser Ser 57120PRTArtificial SequenceVHH sequence 56D10 57Gln
Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Arg Ile Thr
20 25 30 Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu
Leu Val 35 40 45 Ala Ser Ser Ser Ser Gly Gly Thr Thr Asn Tyr Ala
Ser Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Pro Glu
Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Ala Arg Lys Phe Ile Thr
Thr Pro Trp Ser Thr Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Gln Val
Thr Val Ser Ser 115 120 58116PRTArtificial SequenceVHH sequence
56E04 58Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
Asp 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Pro Ser Gly Ser Ile Phe
Asn His Lys 20 25 30 Ala Thr Gly Trp Tyr Arg Gln Ala Pro Gly Ser
Gln Arg Glu Leu Val 35 40 45 Ala Lys Ile Thr Thr Gly Gly Thr Thr
Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Ser Ser Leu
Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Ala Glu Arg
Tyr Phe Ala Thr Thr Leu Trp Gly Gln Gly Thr Gln Val 100 105 110 Thr
Val Ser Ser 115 59119PRTArtificial SequenceVHH sequence 56E05 59Gln
Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Asn Asn
20 25 30 Ala Gly Gly Trp Tyr Arg Gln Ala Pro Gly Gln Gln Arg Glu
Leu Val 35 40 45 Ala Arg Ile Ser Ser Gly Gly Asn Thr Asn Tyr Thr
Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Ile Thr
Lys Asn Thr Leu Ser Leu 65 70 75 80 Gln Met Asn Asn Leu Lys Pro Glu
Asp Ser Ala Val Tyr Tyr Cys Asn 85 90 95 Ala Gln Arg Arg Val Ile
Leu Gly Pro Arg Asn Tyr Trp Gly Gln Gly 100 105 110 Thr Gln Val Thr
Val Ser Ser 115 60121PRTArtificial SequenceVHH sequence 56E08 60Gln
Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asn Ile Phe Arg Ile Asn
20 25 30 Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Asn Gln Arg Glu
Leu Val 35 40 45 Ala Thr Ile Thr Ser Ala Asn Ile Thr Asn Tyr Ala
Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Asn Pro Glu
Asp Thr Ala Val Tyr Tyr Cys Thr 85 90 95 Ala Gln Ala Lys Lys Trp
Arg Ile Gly Pro Trp Ser Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Gln
Val Thr Val Ser Ser 115 120 61119PRTArtificial SequenceVHH sequence
56F07 61Gln 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 Arg Ile Phe
Ser Ile Asn 20 25 30 Asp Met Ala Trp Tyr Arg Gln Ala Pro Gly Lys
Gln Arg Glu Leu Val 35 40 45 Ala Ile Ile Thr Asn Asp Asp Ser Thr
Thr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu
Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Ala Asp Ile
Asn Thr Ala Ile Trp Arg Arg Lys Tyr Trp Gly Gln Gly 100 105 110 Thr
Gln Val Thr Val Ser Ser 115 62121PRTArtificial SequenceVHH sequence
56F11 62Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ser Gly
Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val His Ser Lys Thr Thr Phe
Thr Arg Asn 20 25 30 Ala Met Gly Trp Tyr Arg Gln Ala Leu Gly Lys
Glu Arg Glu Leu Val 35 40 45 Ala Thr Ile Thr Ser Gly Gly Thr Thr
Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Met
Asp Ser Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu
Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Val Asn Thr
Arg Arg Ile Phe Gly Gly Thr Val Arg Glu Tyr Trp Gly 100 105 110 Gln
Gly Thr Gln Val Thr Val Ser Ser 115 120 63115PRTArtificial
SequenceVHH sequence 56G07 63Gln 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
Val Ser Gly Ser Arg Ile Phe Ile His 20 25 30 Asp Met Gly Trp His
Arg Gln Ala Pro Gly Glu Pro Arg Glu Leu Val 35 40 45 Ala Thr Ile
Thr Pro Phe Gly Arg Arg Asn Tyr Ser Glu Tyr Val Lys 50 55 60 Gly
Arg Phe Thr Val Ser Arg Asp Ile Ala Arg Asn Thr Met Ser Leu 65 70
75 80 Gln Met Ser Asn Leu Lys Ala Glu Asp Thr Gly Met Tyr Tyr Cys
Asn 85 90 95 Val Arg Val Asn Gly Val Asp Tyr Trp Gly Gln Gly Thr
Gln Val Thr 100 105 110 Val Ser Ser 115 64119PRTArtificial
SequenceVHH sequence 56G08 64Gln Val Gln Leu Gln Glu Ser Gly Gly
Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala
Ile Ser Gly Ile Thr Phe Arg Arg Pro 20 25 30 Phe Gly Ile Ser Arg
Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu 35 40 45 Arg Glu Leu
Val Ala Thr Leu Ser Arg Ala Gly Thr Ser Arg Tyr Val 50 55 60 Asp
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Asn 65 70
75 80 Thr Leu Tyr Leu Gln Met Val Ser Leu Asn Pro Glu Asp Thr Ala
Val 85 90 95 Tyr Tyr Cys Tyr Ile Ala Gln Leu Gly Thr Asp Tyr Trp
Gly Gln Gly 100 105 110 Thr Gln Val Thr Val Ser Ser 115
65117PRTArtificial SequenceVHH sequence 56G10 65Gln Val Gln Leu Gln
Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Val Ala Ser Gly Ile Thr Leu Arg Met Tyr 20 25 30 Gln
Val Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40
45 Ala Glu Ile Ser Ser Arg Gly Thr Thr Met Tyr Ala Asp Ser Val Lys
50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Gly Ala Lys Asn Ile Val
Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Glu Pro Glu Asp Thr Ala Val
Tyr Tyr Cys Asn 85 90 95 Ala Arg Ala Phe Ala Phe Gly Arg Asn Ser
Trp Gly Gln Gly Thr Gln 100 105 110 Val Thr Val Ser Ser 115
66118PRTArtificial SequenceVHH sequence 56H04 66Gln Val Gln Leu Gln
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Val Ser Gly Gly Thr Phe Ser Asn Lys 20 25 30 Ala
Met Gly Trp Tyr Arg Gln Ser Ser Gly Lys Gln Arg Ala Leu Val 35 40
45 Ala Arg Ile Ser Thr Val Gly Thr Ala His Tyr Ala Asp Ser Val Lys
50 55 60 Gly Arg Phe Thr Val Ser Lys Asp Asn Ala Gly Asn Thr Leu
Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val
Tyr Tyr Cys Asn 85 90 95 Ala Gln Ala Gly Arg Leu Tyr Leu Arg Asn
Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115
67119PRTArtificial SequenceVHH sequence 56H05 67Gln Val Gln Leu Gln
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Glu 1 5 10 15 Ser Leu Arg
Leu Ser Cys Val Ala Ala Ala Ser Thr Ser Ile Thr Thr 20 25 30 Phe
Asn Thr Met Ala Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu 35 40
45 Leu Val Ala Gln Ile Asn Asn Arg Asp Asn Thr Glu Tyr Ala Asp Ser
50 55 60 Val Lys Gly Arg Phe Ile Ile Ser Arg Gly Asn Ala Lys Asn
Thr Ser 65 70 75 80 Asn Leu Gln Met Asn Asp Leu Lys Ser Glu Asp Thr
Gly Ile Tyr Tyr 85 90 95 Cys Asn Ala Lys Arg Trp Ser Trp Ser Thr
Gly Phe Trp Gly Gln Gly 100 105 110 Thr Gln Val Thr Val Ser Ser 115
68114PRTArtificial SequenceVHH sequence 56H07 68Gln Val Gln Leu Gln
Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Thr Ala Ser Gly Leu Thr Phe Ala Leu Gly 20 25 30 Thr
Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40
45 Ala Ser Ile Ser Thr Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly
50 55 60 Arg Phe Thr Ile Ser Arg Asp Ile Ile Lys Asn Ile Leu Tyr
Leu Gln 65 70 75 80 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr
Ser Cys Asn Ala 85 90 95 Arg Leu Trp Trp Ser Asn Tyr Trp Gly Gln
Gly Thr Gln Val Thr Val 100 105 110 Ser Ser 69118PRTArtificial
SequenceVHH sequence 56H08 69Gln Val Gln Leu Gln Glu Ser Gly Gly
Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr
Ala Ser Gly Arg Thr Ser Ser Val Asn 20 25 30 Pro Met Gly Trp Tyr
Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45 Ala Val Ile
Ser Ser Asp Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly
Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70
75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
Asn 85 90 95 Ala Asn Arg Arg Trp Ser Trp Gly Ser Glu Tyr Trp Gly
Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115
70119PRTArtificial SequenceVHH sequence 57A06 70Gln Val Gln Leu Gln
Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Thr Asn Asn 20 25 30 Ala
Gly Gly Trp Tyr Arg Gln Ala Pro Gly Gln Gln Arg Glu Leu Val 35 40
45 Ala Arg Ile Ser Ser Gly Gly Asn Thr Asn Tyr Thr Asp Ser Val Lys
50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Ile Thr Lys Asn Thr Leu
Ser Leu 65 70 75 80 Gln Met Asn Asn Leu Lys Pro Glu Asp Ser Ala Val
Tyr Tyr Cys Asn 85 90 95 Ala Gln Arg Arg Val Ile Leu Gly Pro Arg
Asn Tyr Trp Gly Gln Gly 100 105 110 Thr Gln Val Thr Val Ser Ser 115
71116PRTArtificial SequenceVHH sequence 57B01 71Gln Val Gln Leu Gln
Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Glu Ala Pro Val Ser Thr Phe Asn Ile Asn 20 25 30 Ala
Met Ala Trp Tyr Arg Gln Ala Pro Gly Lys Ser Arg Glu Leu Val 35 40
45 Ala Arg Ile Ser Ser Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys
50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val
Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val
Tyr Ile Cys Tyr 85 90 95 Val Asn Arg His Trp Gly Trp Asp Tyr Trp
Gly Gln Gly Thr Gln Val 100 105 110 Thr Val Ser Ser 115
72122PRTArtificial SequenceVHH sequence 57B07 72Gln Val Gln Leu Gln
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Thr Leu Arg
Leu Ser Cys Val Ala Ser Gly Ser Phe Arg Ser Ile Asn 20 25 30 Ala
Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40
45 Ala Thr Val Asp Ser Gly Gly Tyr Thr Asn Tyr Ala Asp Ser Val Lys
50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val
Tyr Leu 65 70 75 80 Gln Met Ser Ser Leu Thr Pro Glu Asp Thr Ala Val
Tyr Tyr Cys Tyr 85 90 95 Ala Gly Ile Tyr Lys Trp Pro Trp Ser Val
Asp Ala Arg Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val
Ser Ser 115 120 73117PRTArtificial SequenceVHH sequence 57B11 73Gln
Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ser Ile Ser Met Asn
20 25 30 Ser Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu
Arg Val 35 40 45 Ala Leu Ile Arg Ser Ser Gly Gly Thr Tyr Tyr Ala
Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Asn Leu Lys Pro Glu
Asp Thr Ala Val Tyr Tyr Cys Gln 85 90 95 Ala Arg Arg Thr Trp Leu
Ser Ser Glu Ser Trp Gly Gln Gly Thr Gln 100 105 110 Val Thr Val Ser
Ser 115 74122PRTArtificial SequenceVHH sequence 57C07 74Gln Val Gln
Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Val Ser Gly Ser Thr Phe Gly Ile Asn 20 25
30 Thr Met Gly Trp Tyr Arg Gln Ala Pro Glu Lys Gln Arg Glu Leu Val
35 40 45 Ala Ser Ile Ser Arg Gly Gly Met Thr Asn Tyr Ala Asp Ser
Val Lys 50 55 60 Gly Arg Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn
Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr
Ala Val Tyr Val Cys Asn 85 90 95 Ala Gly Ile Arg Ser Arg Trp Tyr
Gly
Gly Pro Ile Thr Thr Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr
Val Ser Ser 115 120 75121PRTArtificial SequenceVHH sequence 57C09
75Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Gly Ser Ile
Asn 20 25 30 Ala Met Gly Trp Tyr Arg Gln Gly Pro Gly Lys Gln Arg
Asp Leu Val 35 40 45 Ala Ser Ile Ser Ser Gly Gly Ala Thr Asn Tyr
Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Ser Ser Leu Lys Pro
Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Ala Lys Lys Ser Arg
Trp Ser Trp Ser Ile Val His Asp Tyr Trp Gly 100 105 110 Gln Gly Thr
Gln Val Thr Val Ser Ser 115 120 76118PRTArtificial SequenceVHH
sequence 57D02 76Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val
Gln Thr Gly Gly 1 5 10 15 Ser Leu Thr Leu Ser Cys Thr Thr Ser Gly
Ser Ile Phe Gly Arg Ser 20 25 30 Asp Met Gly Trp Tyr Arg Gln Ala
Pro Gly Lys Gln Arg Glu Leu Val 35 40 45 Ala Thr Ile Thr Arg Arg
Ser Arg Thr Asn Tyr Ala Glu Phe Val Lys 50 55 60 Gly Arg Phe Thr
Ile Ser Arg Asp Ser Ala Lys Asn Leu Val Thr Leu 65 70 75 80 Gln Met
Asn Ser Leu Lys Pro Glu Asp Thr Asn Val Tyr Tyr Cys Asn 85 90 95
Ala Arg Trp Gly Ala Gly Gly Ile Phe Ser Thr Trp Gly Gln Gly Thr 100
105 110 Gln Val Thr Val Ser Ser 115 77120PRTArtificial SequenceVHH
sequence 57D09 77Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Glu 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Ser Met Ser Ile Asp Ala 20 25 30 Met Gly Trp Tyr Arg Gln Ala Pro
Gly Asp Gln Arg Glu Leu Val Ala 35 40 45 Ser Ile Thr Thr Gly Gly
Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly 50 55 60 Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Thr Val Trp Leu Gln 65 70 75 80 Met Asn
Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala 85 90 95
Lys Val Arg Leu Arg Trp Phe Arg Pro Pro Ser Asp Tyr Trp Gly Gln 100
105 110 Gly Thr Gln Val Thr Val Ser Ser 115 120 78122PRTArtificial
SequenceVHH sequence 57D10 78Gln 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 Arg Leu Leu Ser Ile Ser 20 25 30 Thr Met Gly Trp Tyr
Arg Arg Thr Pro Glu Asp Gln Arg Glu Met Val 35 40 45 Ala Ser Ile
Thr Lys Asp Gly Thr Thr Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly
Arg Leu Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70
75 80 Gln Met Asn Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Val Cys
Asn 85 90 95 Ala Arg Ala Thr Thr Trp Val Pro Tyr Arg Arg Asp Ala
Glu Phe Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115
120 79120PRTArtificial SequenceVHH sequence 57E07 79Gln Val Gln Leu
Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Gly Ile Asn 20 25 30
Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Asp Leu Val 35
40 45 Ala Asp Ile Thr Arg Ser Gly Ser Thr His Tyr Val Asp Ser Val
Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr
Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala
Val Tyr Tyr Cys Asn 85 90 95 Ala Asp Ser Gly Ser His Trp Trp Asn
Arg Arg Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Ser
Ser 115 120 80118PRTArtificial SequenceVHH sequence 57E11 80Gln Val
Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Asn 20
25 30 Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu
Val 35 40 45 Ala Arg Ile Ser Arg Leu Arg Val Thr Asn Tyr Ala Asp
Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp
Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Ala Ala Asn Trp Gly Leu Ala
Gly Asn Glu Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser
Ser 115 81116PRTArtificial SequenceVHH sequence 57G01 81Gln Val Gln
Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser
Leu Arg Pro Ser Cys Thr Ala Ser Gly Ser Thr Leu Leu Ile Asn 20 25
30 Ser Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val
35 40 45 Ala Thr Ile Ser Asn Ser Gly Thr Thr Asn Tyr Val Asp Ala
Val Lys 50 55 60 Gly Arg Phe Ala Ile Ser Arg Asp Asn Ala Asn His
Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Glu Pro Glu Asp Thr
Ala Val Tyr Tyr Cys Asn 85 90 95 Ala Gln Thr Phe Trp Arg Arg Asn
Tyr Trp Gly Gln Gly Thr Gln Val 100 105 110 Thr Val Ser Ser 115
82116PRTArtificial SequenceVHH sequence 57G07 82Gln Val Gln Leu Gln
Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Val Ser Gly Ser Thr Ser Arg Ile Asn 20 25 30 Ala
Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Lys Arg Glu Ser Val 35 40
45 Ala Thr Ile Arg Arg Gly Gly Asn Thr Lys Tyr Ala Asp Ser Val Lys
50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Asn Asn Thr Val
Tyr Leu 65 70 75 80 Gln Leu Asn Ser Leu Lys Pro Glu Asp Thr Ala Val
Tyr Tyr Cys Asn 85 90 95 Ala His Ser Trp Leu Asp Tyr Asp Tyr Trp
Gly Arg Gly Thr Gln Val 100 105 110 Thr Val Ser Ser 115
83114PRTArtificial SequenceVHH sequence 57G08 83Gln Val Gln Leu Gln
Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ser Arg Arg Arg Ile Asn Gly Ile Thr 20 25 30 Met
Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Ala 35 40
45 Thr Ile Asp Ile His Asn Ser Thr Lys Tyr Ala Asp Ser Val Lys Gly
50 55 60 Arg Phe Ile Ile Ser Arg Asp Asn Gly Lys Ser Met Leu Tyr
Leu Gln 65 70 75 80 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr
Tyr Cys Asn Arg 85 90 95 Ile Pro Thr Phe Gly Arg Tyr Trp Gly Gln
Gly Thr Gln Val Thr Val 100 105 110 Ser Ser 84118PRTArtificial
SequenceVHH sequence 57H08 84Gln Val Gln Leu Gln Glu Ser Gly Gly
Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val
Ala Ser Gly Ser Thr Phe Tyr Thr Phe 20 25 30 Ser Thr Lys Asn Val
Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg 35 40 45 Glu Leu Val
Ala Gln Gln Arg Tyr Asp Gly Ser Thr Asn Tyr Ala Asp 50 55 60 Ser
Leu Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg Thr 65 70
75 80 Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val
Tyr 85 90 95 Ile Cys Asn Val Asn Arg Gly Phe Ile Ser Tyr Trp Gly
Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115
855PRTArtificial SequenceCDR1 of VHH sequence 40F07 85Ser Tyr Thr
Met Gly 1 5 865PRTArtificial SequenceCDR1 of VHH sequence 41D01
86Arg Tyr Gly Met Gly 1 5 875PRTArtificial SequenceCDR1 of VHH
sequence 41D06 87Ile Asn Ala Met Arg 1 5 885PRTArtificial
SequenceCDR1 of VHH sequence 41G10 88Ile Asn Ala Met Gly 1 5
895PRTArtificial SequenceCDR1 of VHH sequence 41H05 89Ile Asn Ala
Met Gly 1 5 905PRTArtificial SequenceCDR1 of VHH sequence 42C11
90Thr Tyr Val Met Gly 1 5 915PRTArtificial SequenceCDR1 of VHH
sequence 42C12 91Ile Ser Ser Leu Gly 1 5 925PRTArtificial
SequenceCDR1 of VHH sequence 50D03 92Thr Tyr Ala Met Gly 1 5
935PRTArtificial SequenceCDR1 of VHH sequence 50D07 93Ile Arg Asp
Met Gly 1 5 945PRTArtificial SequenceCDR1 of VHH sequence 50E02
94Ile Asn Ala Met Gly 1 5 955PRTArtificial SequenceCDR1 of VHH
sequence 51B08 95Ser Tyr Ala Met Gly 1 5 965PRTArtificial
SequenceCDR1 of VHH sequence 51C06 96Ser Asp Thr Met Gly 1 5
975PRTArtificial SequenceCDR1 of VHH sequence 51C08 97Ile Lys Thr
Met Gly 1 5 985PRTArtificial SequenceCDR1 of VHH sequence 52A01
98Leu Gly Thr Met Gly 1 5 995PRTArtificial SequenceCDR1 of VHH
sequence 52B01 99Ile Asn Val Met Gly 1 5 1005PRTArtificial
SequenceCDR1 of VHH sequence 52G05 100Ile Ser Ala Met Gly 1 5
1015PRTArtificial SequenceCDR1 of VHH sequence 53A01 101Ile Asn Thr
Met Gly 1 5 1025PRTArtificial SequenceCDR1 of VHH sequence 53F05
102Leu Asn Ala Met Gly 1 5 1035PRTArtificial SequenceCDR1 of VHH
sequence 54A02 103Arg Tyr Gly Met Gly 1 5 1045PRTArtificial
SequenceCDR1 of VHH sequence 54B01 104Arg Tyr Thr Met Gly 1 5
1055PRTArtificial SequenceCDR1 of VHH sequence 54C01 105Arg Tyr Ala
Met Gly 1 5 1065PRTArtificial SequenceCDR1 of VHH sequence 54C04
106Ile Asn Ala Met Gly 1 5 1075PRTArtificial SequenceCDR1 of VHH
sequence 54C08 107Ile Asn Ala Met Gly 1 5 1085PRTArtificial
SequenceCDR1 of VHH sequence 54C10 108Val Asn Asp Met Gly 1 5
1095PRTArtificial SequenceCDR1 of VHH sequence 54C11 109Val Asn Asp
Met Ala 1 5 1105PRTArtificial SequenceCDR1 of VHH sequence 54D03
110Ile Asn Ala Met Arg 1 5 1115PRTArtificial SequenceCDR1 of VHH
sequence 54D06 111Ile Asn Ala Met Gly 1 5 1125PRTArtificial
SequenceCDR1 of VHH sequence 54D10 112Ile His Ala Met Gly 1 5
1135PRTArtificial SequenceCDR1 of VHH sequence 54E01 113Ile Asn Pro
Met Gly 1 5 1145PRTArtificial SequenceCDR1 of VHH sequence 54E05
114Ile Asn Thr Met Gly 1 5 1155PRTArtificial SequenceCDR1 of VHH
sequence 54E10 115Phe Asn Ala Met Gly 1 5 1165PRTArtificial
SequenceCDR1 of VHH sequence 54F01 116Leu Asn Leu Met Gly 1 5
1175PRTArtificial SequenceCDR1 of VHH sequence 54F02 117Ile Asn Thr
Met Gly 1 5 1185PRTArtificial SequenceCDR1 of VHH sequence 54G01
118Val Asn Ala Met Gly 1 5 1195PRTArtificial SequenceCDR1 of VHH
sequence 54G08 119Phe Asn Leu Met Gly 1 5 1205PRTArtificial
SequenceCDR1 of VHH sequence 54G09 120Ile Arg Asp Met Gly 1 5
1215PRTArtificial SequenceCDR1 of VHH sequence 55B02 121Ile Asn Ser
Met Asn 1 5 1225PRTArtificial SequenceCDR1 of VHH sequence 55B05
122Gly Tyr Thr Val Ala 1 5 1235PRTArtificial SequenceCDR1 of VHH
sequence 55C05 123Met Lys Ala Met Gly 1 5 1245PRTArtificial
SequenceCDR1 of VHH sequence 55D08 124Ile Ser Ala Met Gly 1 5
1255PRTArtificial SequenceCDR1 of VHH sequence 55E02 125Ser Asn Ala
Met Ala 1 5 1265PRTArtificial SequenceCDR1 of VHH sequence 55E07
126Ile Tyr Gly Met Gly 1 5 1275PRTArtificial SequenceCDR1 of VHH
sequence 55E09 127Thr Tyr Thr Met Gly 1 5 1285PRTArtificial
SequenceCDR1 of VHH sequence 55E10 128Ile Gln Thr Ile Gly 1 5
1295PRTArtificial SequenceCDR1 of VHH sequence 55F04 129Ile Asn Val
Arg Gly 1 5 1305PRTArtificial SequenceCDR1 of VHH sequence 55F09
130Leu Asn Ala Met Gly 1 5 1315PRTArtificial SequenceCDR1 of VHH
sequence 55F10 131Arg Tyr Ala Met Gly 1 5 1325PRTArtificial
SequenceCDR1 of VHH sequence 55G02 132Ile Asn Val Met Gly 1 5
1335PRTArtificial SequenceCDR1 of VHH sequence 55G08 133Ile Asn Ala
Met Arg 1 5 1345PRTArtificial SequenceCDR1 of VHH sequence 56A05
134Ser Asn Thr Met Gly 1 5 1355PRTArtificial SequenceCDR1 of VHH
sequence 56A06 135Val Tyr Gly Met Gly 1 5 1365PRTArtificial
SequenceCDR1 of VHH sequence 56A09 136Leu Asn Ser Met Gly 1 5
1375PRTArtificial SequenceCDR1 of VHH sequence 56C09 137Ile Leu Ser
Met Ala 1 5 1385PRTArtificial SequenceCDR1 of VHH sequence 56C12
138Asn Arg Ala Val Ser 1 5 1395PRTArtificial SequenceCDR1 of VHH
sequence 56D06 139Ile Ser Ala Met Gly 1 5 1405PRTArtificial
SequenceCDR1 of VHH sequence 56D07 140Met Lys Val Met Gly 1 5
1415PRTArtificial SequenceCDR1 of VHH sequence 56D10 141Ile Thr Thr
Met Gly 1 5 1425PRTArtificial SequenceCDR1 of VHH sequence 56E04
142His Lys Ala Thr Gly 1 5 1435PRTArtificial SequenceCDR1 of VHH
sequence 56E05 143Asn Asn Ala Gly Gly 1 5 1445PRTArtificial
SequenceCDR1 of VHH sequence 56E08 144Ile Asn Asp Met Gly 1 5
1455PRTArtificial SequenceCDR1 of VHH sequence 56F07 145Ile Asn Asp
Met Ala 1 5 1465PRTArtificial SequenceCDR1 of VHH sequence 56F11
146Arg Asn Ala Met Gly 1 5 1475PRTArtificial SequenceCDR1 of VHH
sequence 56G07 147Ile His Asp Met Gly 1 5 1485PRTArtificial
SequenceCDR1 of VHH sequence 56G08 148Ile Ser Arg Met Gly 1 5
1495PRTArtificial SequenceCDR1 of VHH sequence 56G10 149Met Tyr Gln
Val Gly 1 5 1505PRTArtificial SequenceCDR1 of VHH sequence 56H04
150Asn Lys Ala Met Gly 1 5 1515PRTArtificial SequenceCDR1 of VHH
sequence 56H05 151Phe Asn Thr Met Ala 1 5 1525PRTArtificial
SequenceCDR1 of VHH sequence 56H07 152Leu Gly Thr Met Gly 1 5
1535PRTArtificial SequenceCDR1 of VHH sequence 56H08 153Val Asn Pro
Met Gly 1 5 1545PRTArtificial SequenceCDR1 of VHH sequence 57A06
154Asn Asn Ala Gly Gly 1 5 1555PRTArtificial SequenceCDR1 of VHH
sequence 57B01 155Ile Asn Ala Met Ala 1 5 1565PRTArtificial
SequenceCDR1 of VHH sequence 57B07 156Ile Asn Ala Met Gly 1 5
1575PRTArtificial SequenceCDR1 of VHH sequence 57B11 157Met Asn Ser
Met Gly 1 5 1585PRTArtificial SequenceCDR1 of VHH sequence 57C07
158Ile Asn Thr Met Gly 1 5 1595PRTArtificial SequenceCDR1 of VHH
sequence 57C09 159Ile Asn Ala Met Gly 1 5 1605PRTArtificial
SequenceCDR1 of VHH sequence 57D02 160Arg Ser Asp Met Gly 1 5
1615PRTArtificial SequenceCDR1 of VHH sequence 57D09 161Ile Asp Ala
Met Gly 1 5 1625PRTArtificial SequenceCDR1 of VHH sequence 57D10
162Ile Ser Thr Met Gly 1 5 1635PRTArtificial SequenceCDR1 of VHH
sequence 57E07 163Ile Asn Asp Met Gly 1 5 1645PRTArtificial
SequenceCDR1 of VHH sequence 57E11 164Ile Asn Thr Met Gly 1 5
1655PRTArtificial SequenceCDR1 of VHH sequence 57G01 165Ile Asn Ser
Met Gly 1 5 1665PRTArtificial SequenceCDR1 of VHH sequence
57G07 166Ile Asn Ala Met Gly 1 5 1675PRTArtificial SequenceCDR1 of
VHH sequence 57G08 167Gly Ile Thr Met Gly 1 5 1685PRTArtificial
SequenceCDR1 of VHH sequence 57H08 168Thr Lys Asn Val Gly 1 5
16916PRTArtificial SequenceCDR2 of VHH sequence 40F07 169Ser Ile
Glu Gly Gly Gly Asn Thr Asp Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
17016PRTArtificial SequenceCDR2 of VHH sequence 41D01 170Ser Ile
Thr Arg Gly Gly Thr Thr Thr Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
17116PRTArtificial SequenceCDR2 of VHH sequence 41D06 171Ser Ile
Ser Ser Gly Gly Asn Thr Asn Tyr Ser Glu Ser Val Lys Gly 1 5 10 15
17216PRTArtificial SequenceCDR2 of VHH sequence 41G10 172Thr Ile
Thr Ser Gly Gly Thr Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
17316PRTArtificial SequenceCDR2 of VHH sequence 41H05 173Thr Ile
Thr Ser Gly Ala Asn Thr Asn Tyr Thr Asp Ser Val Lys Gly 1 5 10 15
17416PRTArtificial SequenceCDR2 of VHH sequence 42C11 174Thr Ile
Thr Ser Ser Gly Lys Thr Asn Tyr Ala Ala Ser Val Lys Gly 1 5 10 15
17516PRTArtificial SequenceCDR2 of VHH sequence 42C12 175Ser Ala
Thr Ser Gly Gly Asp Thr Thr Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
17616PRTArtificial SequenceCDR2 of VHH sequence 50D03 176Thr Ile
Thr Ser Ser Gly Lys Thr Asn Tyr Ala Ala Ser Val Lys Gly 1 5 10 15
17716PRTArtificial SequenceCDR2 of VHH sequence 50D07 177Thr Ile
Thr Ser Asp Gln Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
17816PRTArtificial SequenceCDR2 of VHH sequence 50E02 178Ala Ile
Thr Ser Asp Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
17916PRTArtificial SequenceCDR2 of VHH sequence 51B08 179Gly Ile
Ser Ser Gly Gly Ser Thr Lys Tyr Ala Asp Ser Val Arg Gly 1 5 10 15
18016PRTArtificial SequenceCDR2 of VHH sequence 51C06 180Ala Ile
Thr Thr Gly Gly Asn Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
18116PRTArtificial SequenceCDR2 of VHH sequence 51C08 181Thr Ile
Ser Asn Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
18215PRTArtificial SequenceCDR2 of VHH sequence 52A01 182Ser Ile
Ser Thr Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
18316PRTArtificial SequenceCDR2 of VHH sequence 52B01 183Thr Ile
Ser Arg Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
18416PRTArtificial SequenceCDR2 of VHH sequence 52G05 184Ser Ile
Thr Arg Arg Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Asp 1 5 10 15
18516PRTArtificial SequenceCDR2 of VHH sequence 53A01 185Ser Ile
Ser Ser Gly Gly Trp Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
18616PRTArtificial SequenceCDR2 of VHH sequence 53F05 186Ser Ile
Thr Thr Gly Gly Ser Thr Asn Tyr Ala Glu Pro Val Lys Gly 1 5 10 15
18717PRTArtificial SequenceCDR2 of VHH sequence 54A02 187Ala Asn
Arg Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Arg 1 5 10 15
Gly 18817PRTArtificial SequenceCDR2 of VHH sequence 54B01 188Gly
Ile Thr Trp Thr Gly Gly Ser Thr Asp Tyr Ala Asp Ser Val Lys 1 5 10
15 Gly 18917PRTArtificial SequenceCDR2 of VHH sequence 54C01 189Ala
Ile Ser Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10
15 Asp 19016PRTArtificial SequenceCDR2 of VHH sequence 54C04 190Asp
Met Thr Ser Gly Gly Ser Ile Asn Tyr Ala Asp Ser Val Ser Gly 1 5 10
15 19116PRTArtificial SequenceCDR2 of VHH sequence 54C08 191Ser Ile
Thr Ser Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
19216PRTArtificial SequenceCDR2 of VHH sequence 54C10 192Gln Ile
Thr Arg Arg Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
19316PRTArtificial SequenceCDR2 of VHH sequence 54C11 193Asn Ile
Thr Arg Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
19416PRTArtificial SequenceCDR2 of VHH sequence 54D03 194Ser Ile
Ser Ser Gly Gly Asn Thr Asn Tyr Ser Glu Ser Val Lys Gly 1 5 10 15
19516PRTArtificial SequenceCDR2 of VHH sequence 54D06 195Thr Ile
Thr Arg Gly Gly Ile Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
19616PRTArtificial SequenceCDR2 of VHH sequence 54D10 196Ile Thr
Ser Thr Ser Gly Thr Thr Asp Tyr Thr Asp Ser Val Lys Gly 1 5 10 15
19716PRTArtificial SequenceCDR2 of VHH sequence 54E01 197Ala Ile
Thr Ser Gly Gly Ser Thr Asn Tyr Ala Asp Tyr Val Lys Gly 1 5 10 15
19816PRTArtificial SequenceCDR2 of VHH sequence 54E05 198Ala Ile
Thr Asn Arg Gly Ser Thr Asn Tyr Ala Asp Phe Val Lys Gly 1 5 10 15
19916PRTArtificial SequenceCDR2 of VHH sequence 54E10 199Ala Ile
Thr Arg Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
20016PRTArtificial SequenceCDR2 of VHH sequence 54F01 200Thr Ile
Thr Arg Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
20116PRTArtificial SequenceCDR2 of VHH sequence 54F02 201Thr Ile
Thr Ser Gly Gly Thr Thr Asn Tyr Ala Asp Ser Val Lys Asn 1 5 10 15
20216PRTArtificial SequenceCDR2 of VHH sequence 54G01 202Ile Ile
Ser Ser Asn Ser Thr Ser Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
20316PRTArtificial SequenceCDR2 of VHH sequence 54G08 203Ala Ile
Thr Ser Ser Ser Asn Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
20416PRTArtificial SequenceCDR2 of VHH sequence 54G09 204Thr Ile
Thr Ser Asp Gln Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
20516PRTArtificial SequenceCDR2 of VHH sequence 55B02 205Asp Met
Arg Ser Asp Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
20617PRTArtificial SequenceCDR2 of VHH sequence 55B05 206Arg Ile
Ser Trp Ser Gly Ile Met Ala Tyr Tyr Ala Glu Ser Val Lys 1 5 10 15
Gly 20716PRTArtificial SequenceCDR2 of VHH sequence 55C05 207Gln
Ile Thr Arg Gly Asp Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10
15 20816PRTArtificial SequenceCDR2 of VHH sequence 55D08 208Thr Ile
Thr Ser Ala Gly Ser Ser Asn Tyr Ser Asp Ser Val Lys Gly 1 5 10 15
20916PRTArtificial SequenceCDR2 of VHH sequence 55E02 209Arg Ile
Leu Ser Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
21016PRTArtificial SequenceCDR2 of VHH sequence 55E07 210Arg Ile
Thr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
21117PRTArtificial SequenceCDR2 of VHH sequence 55E09 211Ala Ile
Ile Trp Ser Gly Gly Arg Thr Arg Tyr Ala Asp Ser Val Lys 1 5 10 15
Gly 21216PRTArtificial SequenceCDR2 of VHH sequence 55E10 212Thr
Ile Ser Ser Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10
15 21316PRTArtificial SequenceCDR2 of VHH sequence 55F04 213Thr Ile
Thr Ser Asp Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
21417PRTArtificial SequenceCDR2 of VHH sequence 55F09 214Ala Ile
Thr Pro Gly Gly Gly Asn Thr Thr Tyr Ala Asp Ser Val Lys 1 5 10 15
Gly 21517PRTArtificial SequenceCDR2 of VHH sequence 55F10 215Thr
Ile Arg Arg Ser Gly Ser Ser Thr Tyr Tyr Leu Asp Ser Thr Lys 1 5 10
15 Gly 21616PRTArtificial SequenceCDR2 of VHH sequence 55G02 216Phe
Ile Thr Ser Gly Gly Ile Thr Asn Tyr Thr Asp Ser Val Lys Gly 1 5 10
15 21716PRTArtificial SequenceCDR2 of VHH sequence 55G08 217Ser Ile
Ser Ser Gly Gly Thr Thr Asp Tyr Val Glu Ser Val Lys Gly 1 5 10 15
21816PRTArtificial SequenceCDR2 of VHH sequence 56A05 218Ser Ile
Ser Ser Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
21916PRTArtificial SequenceCDR2 of VHH sequence 56A06 219Arg Ile
Thr Asn Ile Gly Thr Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
22016PRTArtificial SequenceCDR2 of VHH sequence 56A09 220Thr Ile
Thr Arg Gly Gly Thr Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
22116PRTArtificial SequenceCDR2 of VHH sequence 56C09 221Asn Ile
Thr Ser Val Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
22216PRTArtificial SequenceCDR2 of VHH sequence 56C12 222Ser Ile
Ser Gly Ile Arg Ile Thr Thr Tyr Thr Asn Ser Val Lys Gly 1 5 10 15
22316PRTArtificial SequenceCDR2 of VHH sequence 56D07 223Val Ile
Thr Ser Gly Gly Arg Thr Asn Tyr Ala Glu Ser Val Lys Gly 1 5 10 15
22416PRTArtificial SequenceCDR2 of VHH sequence 56D07 224Val Ile
Thr Ser Gly Gly Arg Thr Asn Tyr Ala Glu Ser Val Lys Gly 1 5 10 15
22516PRTArtificial SequenceCDR2 of VHH sequence 56D10 225Ser Ser
Ser Ser Gly Gly Thr Thr Asn Tyr Ala Ser Ser Val Lys Gly 1 5 10 15
22616PRTArtificial SequenceCDR2 of VHH sequence 56E04 226Lys Ile
Thr Thr Gly Gly Thr Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
22716PRTArtificial SequenceCDR2 of VHH sequence 56E05 227Arg Ile
Ser Ser Gly Gly Asn Thr Asn Tyr Thr Asp Ser Val Lys Gly 1 5 10 15
22816PRTArtificial SequenceCDR2 of VHH sequence 56E08 228Thr Ile
Thr Ser Ala Asn Ile Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
22916PRTArtificial SequenceCDR2 of VHH sequence 56F07 229Ile Ile
Thr Asn Asp Asp Ser Thr Thr Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
23016PRTArtificial SequenceCDR2 of VHH sequence 56F11 230Thr Ile
Thr Ser Gly Gly Thr Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
23116PRTArtificial SequenceCDR2 of VHH sequence 56G07 231Thr Ile
Thr Pro Phe Gly Arg Arg Asn Tyr Ser Glu Tyr Val Lys Gly 1 5 10 15
23216PRTArtificial SequenceCDR2 of VHH sequence 56G08 232Thr Leu
Ser Arg Ala Gly Thr Ser Arg Tyr Val Asp Ser Val Lys Gly 1 5 10 15
23316PRTArtificial SequenceCDR2 of VHH sequence 56G10 233Glu Ile
Ser Ser Arg Gly Thr Thr Met Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
23416PRTArtificial SequenceCDR2 of VHH sequence 56H04 234Arg Ile
Ser Thr Val Gly Thr Ala His Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
23516PRTArtificial SequenceCDR2 of VHH sequence 56H05 235Gln Ile
Asn Asn Arg Asp Asn Thr Glu Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
23615PRTArtificial SequenceCDR2 of VHH sequence 56H07 236Ser Ile
Ser Thr Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
23716PRTArtificial SequenceCDR2 of VHH sequence 56H08 237Val Ile
Ser Ser Asp Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
23816PRTArtificial SequenceCDR2 of VHH sequence 57A06 238Arg Ile
Ser Ser Gly Gly Asn Thr Asn Tyr Thr Asp Ser Val Lys Gly 1 5 10 15
23916PRTArtificial SequenceCDR2 of VHH sequence 57B01 239Arg Ile
Ser Ser Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
24016PRTArtificial SequenceCDR2 of VHH sequence 57B07 240Thr Val
Asp Ser Gly Gly Tyr Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
24116PRTArtificial SequenceCDR2 of VHH sequence 57B11 241Leu Ile
Arg Ser Ser Gly Gly Thr Tyr Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
24216PRTArtificial SequenceCDR2 of VHH sequence 57C07 242Ser Ile
Ser Arg Gly Gly Met Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
24316PRTArtificial SequenceCDR2 of VHH sequence 57C09 243Ser Ile
Ser Ser Gly Gly Ala Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
24416PRTArtificial SequenceCDR2 of VHH sequence 57D02 244Thr Ile
Thr Arg Arg Ser Arg Thr Asn Tyr Ala Glu Phe Val Lys Gly 1 5 10 15
24516PRTArtificial SequenceCDR2 of VHH sequence 57D09 245Ser Ile
Thr Thr Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
24616PRTArtificial SequenceCDR2 of VHH sequence 57D10 246Ser Ile
Thr Lys Asp Gly Thr Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
24716PRTArtificial SequenceCDR2 of VHH sequence 57E07 247Asp Ile
Thr Arg Ser Gly Ser Thr His Tyr Val Asp Ser Val Lys Gly 1 5 10 15
24816PRTArtificial SequenceCDR2 of VHH sequence 57E11 248Arg Ile
Ser Arg Leu Arg Val Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
24916PRTArtificial SequenceCDR2 of VHH sequence 57G01 249Thr Ile
Ser Asn Ser Gly Thr Thr Asn Tyr Val Asp Ala Val Lys Gly 1 5 10 15
25016PRTArtificial SequenceCDR2 of VHH sequence 57G07 250Thr Ile
Arg Arg Gly Gly Asn Thr Lys Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
25116PRTArtificial SequenceCDR2 of VHH sequence 57G08 251Thr Ile
Asp Ile His Asn Ser Thr Lys Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
25216PRTArtificial SequenceCDR2 of VHH sequence 57H08 252Gln Gln
Arg Tyr Asp Gly Ser Thr Asn Tyr Ala Asp Ser Leu Gln Gly 1 5 10 15
25310PRTArtificial SequenceCDR3 of VHH sequence 40F07 253Ala Arg
Thr Trp Ser Ile Phe Arg Asn Tyr 1 5 10 2547PRTArtificial
SequenceCDR3 of VHH sequence 41D01 254Arg Ser Ile Trp Arg Asp Tyr 1
5 2558PRTArtificial SequenceCDR3 of VHH sequence 41D06 255Val Arg
Leu Trp Phe Pro Asp Tyr 1 5 25613PRTArtificial SequenceCDR3 of VHH
sequence 41G10 256Glu Ala Arg Arg Tyr Phe Thr Arg Ala Ser Gln Val
Tyr 1 5 10 25712PRTArtificial SequenceCDR3 of VHH sequence 41H05
257Val Gly Arg Arg Trp Tyr Gly Gly Tyr Val Glu Leu 1 5 10
25811PRTArtificial SequenceCDR3 of VHH sequence 42C11 258Asp Arg
Trp Val Leu Thr Arg Trp Ser Asn Tyr 1 5 10 25911PRTArtificial
SequenceCDR3 of VHH sequence 42C12 259Gln Arg Gly Val Ala Trp Thr
Arg Lys Glu Tyr 1 5 10 26011PRTArtificial SequenceCDR3 of VHH
sequence 50D03 260Asp Arg Trp Val Leu Thr Arg Trp Ser Asn Tyr 1 5
10 26112PRTArtificial SequenceCDR3 of VHH sequence 50D07 261Arg Val
Arg Thr Val Leu Arg Gly Trp Arg Asp Tyr 1 5 10 26211PRTArtificial
SequenceCDR3 of VHH sequence 50E02 262Arg Arg Arg Thr Phe Leu Lys
Ser Ser Asp Tyr 1 5 10 26315PRTArtificial SequenceCDR3 of VHH
sequence 51B08 263Lys Tyr Gly Arg Trp Thr Tyr Thr Gly Arg Pro Glu
Tyr Asp Ser 1 5 10 15 2648PRTArtificial SequenceCDR3 of VHH
sequence 51C06 264Arg Arg Arg Trp Ser Arg Asp Phe 1 5
26511PRTArtificial SequenceCDR3 of VHH sequence 51C08 265Arg Gln
Gln Phe Ile Gly Ala Pro Tyr Glu Tyr 1 5 10 2667PRTArtificial
SequenceCDR3 of VHH sequence 52A01 266Arg Leu Leu Trp Ser Asn Tyr 1
5 2679PRTArtificial SequenceCDR3 of VHH sequence 52B01 267Ala Gly
Trp Val Gly Val Thr Asn Tyr 1 5 2689PRTArtificial SequenceCDR3 of
VHH sequence 52G05 268Arg Arg Tyr Tyr Thr Arg Asn Asp Tyr 1 5
2696PRTArtificial SequenceCDR3 of VHH
sequence 53A01 269Gly Ala Ile Gly Asn Trp 1 5 2709PRTArtificial
SequenceCDR3 of VHH sequence 53F05 270Glu Arg Arg Trp Gly Leu Pro
Asn Tyr 1 5 27117PRTArtificial SequenceCDR3 of VHH sequence 54A02
271Tyr Ala His Ile Thr Ala Trp Gly Met Arg Asn Asp Tyr Glu Tyr Asp
1 5 10 15 Tyr 27216PRTArtificial SequenceCDR3 of VHH sequence 54B01
272Gly Asn Leu Leu Arg Leu Ala Gly Gln Leu Arg Arg Gly Tyr Asp Ser
1 5 10 15 27320PRTArtificial SequenceCDR3 of VHH sequence 54C01
273Arg Asn Arg Ala Gly Pro His Tyr Ser Arg Gly Tyr Thr Ala Gly Gln
1 5 10 15 Glu Tyr Asp Tyr 20 27413PRTArtificial SequenceCDR3 of VHH
sequence 54C04 274Asn Leu Arg Thr Ala Phe Trp Arg Asn Gly Asn Asp
Tyr 1 5 10 2757PRTArtificial SequenceCDR3 of VHH sequence 54C08
275Gly Pro Trp Tyr Arg Arg Ser 1 5 2768PRTArtificial SequenceCDR3
of VHH sequence 54C10 276Asp Leu Ala Val Arg Gly Arg Tyr 1 5
27711PRTArtificial SequenceCDR3 of VHH sequence 54C11 277Arg Ile
Gly Phe Gly Trp Thr Ala Lys Ala Tyr 1 5 10 2788PRTArtificial
SequenceCDR3 of VHH sequence 54D03 278Val Arg Leu Trp Phe Pro Asp
Tyr 1 5 2798PRTArtificial SequenceCDR3 of VHH sequence 54D06 279Arg
Ser Trp Val Gly Pro Glu Tyr 1 5 28012PRTArtificial SequenceCDR3 of
VHH sequence 54D10 280Lys Thr Arg Thr Trp Tyr Asn Gly Lys Tyr Asp
Tyr 1 5 10 2819PRTArtificial SequenceCDR3 of VHH sequence 54E01
281Arg Ser Thr Leu Trp Arg Arg Asp Tyr 1 5 2829PRTArtificial
SequenceCDR3 of VHH sequence 54E05 282His Arg Ser Trp Pro Arg Tyr
Asp Ser 1 5 28310PRTArtificial SequenceCDR3 of VHH sequence 54E10
283Glu Ser Arg Ile Phe Arg Arg Tyr Asp Tyr 1 5 10 2847PRTArtificial
SequenceCDR3 of VHH sequence 54F01 284Asp Arg Gly Trp Ser Ser Tyr 1
5 28511PRTArtificial SequenceCDR3 of VHH sequence 54F02 285His Gln
Arg Ala Trp Ala Arg Ser Tyr Val Tyr 1 5 10 2869PRTArtificial
SequenceCDR3 of VHH sequence 54G01 286Lys Arg Ser Trp Phe Ser Gln
Glu Tyr 1 5 28713PRTArtificial SequenceCDR3 of VHH sequence 54G08
287Gln Tyr Thr Ile Thr Pro Trp Gly Ile Lys Lys Asp Tyr 1 5 10
28812PRTArtificial SequenceCDR3 of VHH sequence 54G09 288Arg Val
Arg Thr Val Leu Arg Gly Trp Arg Asp Tyr 1 5 10 2899PRTArtificial
SequenceCDR3 of VHH sequence 55B02 289Asn Ser Ile Phe Arg Ser Arg
Asp Tyr 1 5 29016PRTArtificial SequenceCDR3 of VHH sequence 55B05
290Arg Ser Gln Ile Arg Ser Pro Trp Ser Ser Leu Asp Asp Tyr Asp Arg
1 5 10 15 2918PRTArtificial SequenceCDR3 of VHH sequence 55C05
291Asp Arg Phe Phe Gly Arg Asp Tyr 1 5 29212PRTArtificial
SequenceCDR3 of VHH sequence 55D08 292Val Tyr Ser Arg Pro Leu Leu
Gly Pro Leu Glu Val 1 5 10 2937PRTArtificial SequenceCDR3 of VHH
sequence 55E02 293Val Arg Tyr Leu Val Asn Tyr 1 5
29413PRTArtificial SequenceCDR3 of VHH sequence 55E07 294Gly Val
Val Val Ala Thr Ser Pro Lys Phe Tyr Ala Tyr 1 5 10
2957PRTArtificial SequenceCDR3 of VHH sequence 55E09 295Arg Arg Leu
Gly Thr Gly Tyr 1 5 2967PRTArtificial SequenceCDR3 of VHH sequence
55E10 296Arg Tyr Trp Phe Arg Asp Tyr 1 5 2977PRTArtificial
SequenceCDR3 of VHH sequence 55F04 297Val Arg Leu Phe Arg Gln Tyr 1
5 29816PRTArtificial SequenceCDR3 of VHH sequence 55F09 298Gly Gly
Ser Ser Arg Trp Tyr Ser Ser Arg Tyr Tyr Pro Gly Gly Tyr 1 5 10 15
29917PRTArtificial SequenceCDR3 of VHH sequence 55F10 299Asp Ser
Ser Ala Arg Ala Leu Val Gly Gly Pro Gly Asn Arg Trp Asp 1 5 10 15
Tyr 30010PRTArtificial SequenceCDR3 of VHH sequence 55G02 300Lys
Asn Ala Lys Asn Val Arg Pro Gly Tyr 1 5 10 3018PRTArtificial
SequenceCDR3 of VHH sequence 55G08 301Val Arg Phe Trp Phe Pro Asp
Tyr 1 5 3028PRTArtificial SequenceCDR3 of VHH sequence 56A05 302Arg
Arg Asn Val Phe Ile Ser Ser 1 5 3037PRTArtificial SequenceCDR3 of
VHH sequence 56A06 303Arg Arg Leu Gly Arg Asp Tyr 1 5
30410PRTArtificial SequenceCDR3 of VHH sequence 56A09 304Asn Phe
Gly Ile Leu Val Gly Arg Glu Tyr 1 5 10 3058PRTArtificial
SequenceCDR3 of VHH sequence 56C09 305Arg Met Pro Phe Leu Gly Asp
Ser 1 5 3068PRTArtificial SequenceCDR3 of VHH sequence 56D06 306Thr
Ser Ile Thr Gly Thr Tyr Leu 1 5 3076PRTArtificial SequenceCDR3 of
VHH sequence 56D07 307Lys Thr Ile Arg Pro Tyr 1 5
30812PRTArtificial SequenceCDR3 of VHH sequence 56D10 308Arg Lys
Phe Ile Thr Thr Pro Trp Ser Thr Asp Tyr 1 5 10 3098PRTArtificial
SequenceCDR3 of VHH sequence 56E04 309Glu Arg Tyr Phe Ala Thr Thr
Leu 1 5 31011PRTArtificial SequenceCDR3 of VHH sequence 56E05
310Gln Arg Arg Val Ile Leu Gly Pro Arg Asn Tyr 1 5 10
31113PRTArtificial SequenceCDR3 of VHH sequence 56E08 311Gln Ala
Lys Lys Trp Arg Ile Gly Pro Trp Ser Asp Tyr 1 5 10
31211PRTArtificial SequenceCDR3 of VHH sequence 56F07 312Asp Ile
Asn Thr Ala Ile Trp Arg Arg Lys Tyr 1 5 10 31313PRTArtificial
SequenceCDR3 of VHH sequence 56F11 313Asn Thr Arg Arg Ile Phe Gly
Gly Thr Val Arg Glu Tyr 1 5 10 3147PRTArtificial SequenceCDR3 of
VHH sequence 56G07 314Arg Val Asn Gly Val Asp Tyr 1 5
3157PRTArtificial SequenceCDR3 of VHH sequence 56G08 315Ala Gln Leu
Gly Thr Asp Tyr 1 5 3169PRTArtificial SequenceCDR3 of VHH sequence
56G10 316Arg Ala Phe Ala Phe Gly Arg Asn Ser 1 5 31710PRTArtificial
SequenceCDR3 of VHH sequence 56H04 317Gln Ala Gly Arg Leu Tyr Leu
Arg Asn Tyr 1 5 10 3189PRTArtificial SequenceCDR3 of VHH sequence
56H05 318Lys Arg Trp Ser Trp Ser Thr Gly Phe 1 5 3197PRTArtificial
SequenceCDR3 of VHH sequence 56H07 319Arg Leu Trp Trp Ser Asn Tyr 1
5 32010PRTArtificial SequenceCDR3 of VHH sequence 56H08 320Asn Arg
Arg Trp Ser Trp Gly Ser Glu Tyr 1 5 10 32111PRTArtificial
SequenceCDR3 of VHH sequence 57A06 321Gln Arg Arg Val Ile Leu Gly
Pro Arg Asn Tyr 1 5 10 3228PRTArtificial SequenceCDR3 of VHH
sequence 57B01 322Asn Arg His Trp Gly Trp Asp Tyr 1 5
32314PRTArtificial SequenceCDR3 of VHH sequence 57B07 323Gly Ile
Tyr Lys Trp Pro Trp Ser Val Asp Ala Arg Asp Tyr 1 5 10
3249PRTArtificial SequenceCDR3 of VHH sequence 57B11 324Arg Arg Thr
Trp Leu Ser Ser Glu Ser 1 5 32514PRTArtificial SequenceCDR3 of VHH
sequence 57C07 325Gly Ile Arg Ser Arg Trp Tyr Gly Gly Pro Ile Thr
Thr Tyr 1 5 10 32613PRTArtificial SequenceCDR3 of VHH sequence
57C09 326Lys Lys Ser Arg Trp Ser Trp Ser Ile Val His Asp Tyr 1 5 10
32710PRTArtificial SequenceCDR3 of VHH sequence 57D02 327Arg Trp
Gly Ala Gly Gly Ile Phe Ser Thr 1 5 10 32813PRTArtificial
SequenceCDR3 of VHH sequence 57D09 328Lys Val Arg Leu Arg Trp Phe
Arg Pro Pro Ser Asp Tyr 1 5 10 32914PRTArtificial SequenceCDR3 of
VHH sequence 57D10 329Arg Ala Thr Thr Trp Val Pro Tyr Arg Arg Asp
Ala Glu Phe 1 5 10 33012PRTArtificial SequenceCDR3 of VHH sequence
57E07 330Asp Ser Gly Ser His Trp Trp Asn Arg Arg Asp Tyr 1 5 10
33110PRTArtificial SequenceCDR3 of VHH sequence 57E11 331Ala Asn
Trp Gly Leu Ala Gly Asn Glu Tyr 1 5 10 3328PRTArtificial
SequenceCDR3 of VHH sequence 57G01 332Gln Thr Phe Trp Arg Arg Asn
Tyr 1 5 3338PRTArtificial SequenceCDR3 of VHH sequence 57G07 333His
Ser Trp Leu Asp Tyr Asp Tyr 1 5 3347PRTArtificial SequenceCDR3 of
VHH sequence 57G08 334Ile Pro Thr Phe Gly Arg Tyr 1 5
3357PRTArtificial SequenceCDR3 of VHH sequence 57H08 335Asn Arg Gly
Phe Ile Ser Tyr 1 5
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