Anti-insect cda nanobodies and uses thereof

Abstract

Anti-insect CDA nanobodies are provided. Thus, a nanobody is provided that specifically binds to a non-linear epitope of insect CDA, said non-linear epitope being in the catalytic and / or chitin-binding domain of insect CDA, wherein binding of said nanobody to said insect CDA confers insect control activity to said nanobody. Anti-insect CDA nanobodies defined by specific CDR sequences are also provided. Polynucleotides encoding the nanobodies, host cells expressing the nanobodies, and methods of using the same are also provided.

Description

[0001] Related applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 623,274, filed January 21, 2024, the entire contents of which are incorporated herein by reference.

[0002] Sequence list declaration An XML file titled 101891.xml, created on January 14, 2025 and filed concurrently with this application (comprising 431,963 bytes), is incorporated herein by reference. Technical Field

[0003] In some embodiments of the present invention, therein relates to anti-insect CDA nanobodies and their uses. Background Technology

[0004] For the past 70 years, insect pest management has primarily relied on the application of synthetic pesticides. Since Paul Müller discovered the insecticidal properties of DDT in 1939, hundreds of insecticidal compounds have been developed, and pesticide use has steadily increased. Most chemical pesticides currently in use have a high potential to disrupt ecosystems; they are toxic to humans directly or through the food chain, and their use is hampered by the development of genetic resistance in insects. The most prominent biological solutions currently in use include beneficial organisms or natural enemies that reduce insect damage, pheromones that act as bait or disrupt reproduction, the release of sterile males, the spraying of Bacillus thuringiensis toxins, or the use of genetically modified crops containing genes for Bacillus thuringiensis toxins that are lethal to insects but harmless to humans. However, these biological solutions also have drawbacks, including low efficiency, the risk of disrupting the natural ecological balance, and the development of genetic insect resistance (e.g., in the case of using Bacillus toxins).

[0005] Nanobodies, also known as VHH antibodies, are single-domain antibodies that actually contain the heavy chain (HCAb) of the antibody and have no light chain at all. They were discovered by Raymond Hammers in camel blood, a discovery he made in 1989 at the Free University of Brussels. Nanobodies are the smallest available intact antigen-binding fragments (Cortez–Retamozo et al., 2004; Revets et al., 2005), with a size of approximately 15 kDa. Nanobodies offer significant advantages, including high yields across a wide range of expression systems, their minimal size allowing for high accessibility to their epitopes, high physicochemical stability, reversible refolding and high solubility in aqueous solutions, high homogeneity, no signs of spontaneous dimerization, and the ability to specifically recognize unique epitopes with sub-nanomolar affinity. The use of nanobodies as insecticides has been previously suggested (see, for example, EP patent application publications: EP3415010 and EP2609116; US patent publication: US9516879; US patent application publication: US9803003B2; and international patent application publication WO2014191146).

[0006] Chitin deacetylases (CDA) are enzymes that convert chitin into chitosan, thereby affecting the mechanical and permeability of structures such as the cuticle and peritrophic membrane matrix. Most CDA-like proteins have a putative signal peptide, consistent with their role in modifying extracellular chitin in the cuticle and peritrophic membrane during morphogenesis and molting [Dixit R. et al. (2008) Insect Biochem Mol Biol. 38(4):440-51]. Defects in CDA can disrupt feeding and digestion in insects, inhibit their growth, impair their development, and even lead to death [Yinchen et al. (2021) J Pestic Sci. 46(1):43–52]. In fact, inhibition of insect CDA by small molecules or RNA interference has been shown to induce developmental defects, abnormal growth and molting, and increase mortality [Yingchen et al. (2021) J Pestic Sci. 46(1):43–52; Jian-Jian Wu et al. (2018) Insect Molecular Biology 28(1):52-64; Chikate et al. (2016) Pestic Biochem Physiol. 133:44-51; Zhao et al. (2021) Pesticide Biochemistry and Physiology 173:104770]. Anti-insect CDA nanobodies and their use as insecticides have also been disclosed (International Patent Application Publication No. WO2021 / 095031). Summary of the Invention

[0007] According to one aspect of some embodiments of the present invention, a nanobody is provided that specifically binds to a nonlinear epitope of insect CDA, the nonlinear epitope being located in a catalytic and / or chitin-binding domain of insect CDA, wherein the binding of the nanobody to insect CDA endows the nanobody with insect control activity.

[0008] According to one aspect of some embodiments of the present invention, a nanobody is provided that specifically binds to a nonlinear epitope of insect CDA, the nonlinear epitope comprising amino acid residues selected from the group consisting of D170, D175, E360, D361, D365, D384, Y387, N388, N391, H392, D395, Y398, E399, E419, E422, E430, S433 and S434 of SEQ ID NO:1, or corresponding amino acid residues in orthologous polypeptides, wherein the binding of the nanobody to insect CDA confers insect control activity on the nanobody.

[0009] According to some embodiments of the present invention, the nonlinear epitopes of insect CDA include all amino acid residues.

[0010] According to some embodiments of the present invention, the amino acid residues are selected from the group consisting of E360, D361, D365, D384, Y387, N388, N391, H392, D395, Y398, E399, E419, E422, E430, S433 and S434 of SEQ ID NO:1, or the corresponding amino acid residues in orthologous polypeptides.

[0011] According to some embodiments of the present invention, the nonlinear epitopes of insect CDA include amino acid residues E360, D361, D365, D384, Y387, N388, N391, H392, D395, Y398, E399, E419, E422, E430, S433 and S434 of SEQ ID NO:1, or corresponding amino acid residues in orthologous polypeptides.

[0012] According to some embodiments of the present invention, the nonlinear epitopes of insect CDA include amino acid residues E360, D361, D365, D384, H392, E419 and E422 of SEQ ID NO:1, or corresponding amino acid residues in orthologous polypeptides.

[0013] According to one aspect of some embodiments of the present invention, a nanobody is provided that specifically binds to a nonlinear epitope of insect CDA, the nonlinear epitope comprising selected from V22, E23, L24, C25, D46, I48, I68, E99, P100, L101, C102, R286, A287, G293, G294, N295, N296, Q297, M301, E302, and E30. 3. Q304, L307, Y308, D309, S310, T311, I312, P319, P320, P323, Y324, T325, Y327, F328, H332 , R333, C334, H335, G336, N337, L338, Q339, S340, C341, P342, T343, R344, W349, E350, M351, V352, N354, E355, D357, R358, E360, P362, N364, E366, M373, V374, D375, N391, H392, D395, Y 398, E399, A403, P404, L405, G406, R415, S434, M443, T444, Q445, V446, I447, Q448, P475, C4 The amino acid residues of the group consisting of 77, W478, V479, P480, S482, C483, K484, L485, T499, C500, V501, R502, P508, L510, N511, D512, D516, G517, H518 and Y519, or the corresponding amino acid residues in orthologous polypeptides, wherein the binding of nanobodies to insect CDA endows the nanobodies with insect control activity.

[0014] According to some embodiments of the present invention, the nonlinear epitopes of insect CDA include all amino acid residues.

[0015] According to some embodiments of the present invention, the amino acid residues are selected from the group consisting of W349, E350, M351, V352, N354, E355, D357, R358, E360, P362, N364, E366, M373, V374, D375, N391, H392, D395, Y398, E399, A403, P404, L405, G406, R415, S434, M443, T444, Q445, V446, I447 and Q448 of SEQ ID NO:1, or the corresponding amino acid residues in orthologous polypeptides.

[0016] According to some embodiments of the present invention, the nonlinear epitopes of insect CDA include amino acid residues W349, E350, M351, V352, N354, E355, D357, R358, E360, P362, N364, E366, M373, V374, D375, N391, H392, D395, Y398, E399, A403, P404, L405, G406, R415, S434, M443, T444, Q445, V446, I447, and Q448 of SEQ ID NO:1, or corresponding amino acid residues in orthologous polypeptides.

[0017] According to some embodiments of the present invention, the amino acid residues are selected from the group consisting of C25, D46, I48, and I68 of SEQ ID NO:1 or the corresponding amino acid residues in orthologous polypeptides.

[0018] According to some embodiments of the present invention, the nonlinear epitopes of insect CDA include the corresponding amino acid residues of amino acid residues C25, D46, I48, I68 of SEQ ID NO:1 or orthologous polypeptides.

[0019] According to some embodiments of the present invention, the nonlinear epitopes of insect CDA include amino acid residues E350, M351 and V352 of SEQ ID NO:1 or corresponding amino acid residues in orthologous polypeptides.

[0020] According to one aspect of some embodiments of the present invention, a nanobody that specifically binds to insect CDA is provided, the nanobody comprising SEQ ID NO: 3-5, 7-9, 11-13, 15-17, 19-21, 23-25, 27-29, 31-33, 35-37, 39-41, 43-45, 47-49, 51-53, 55-57, 59-61, 63-65, 67-69, 71-73, 75-77, 79-81, 83-85, 87-89, 91-93, 95-97, 99-101, 103-105, 107-109, 111 The nanobody contains three complementarity-determining regions (CDRs) listed in -113, 115-117, 119-121, 123-125, 127-129, 131-133, 135-137, 139-141, 143-145, 147-149, 151-153, 155-157, or 159-161. These three CDRs are arranged on the nanobody in order from N to C. The binding of the nanobody to the insect CDA confers insect control activity on the nanobody.

[0021] According to some embodiments of the present invention, the nanobody includes three CDRs listed in SEQ ID NO:15-17, 83-85 or 95-97, which are arranged on the nanobody in order from N to C.

[0022] According to some embodiments of the present invention, the nanobody includes three CDRs listed in SEQ ID NO:15-17, which are arranged on the nanobody in order from N to C.

[0023] According to some embodiments of the present invention, the nanobody includes three CDRs listed in SEQ ID NO:83-85, which are arranged on the nanobody in order from N to C.

[0024] According to some embodiments of the present invention, nanobodies further include a heterologous toxin portion having insect control activity.

[0025] According to some embodiments of the present invention, nanobodies downregulate the activity of insect CDA.

[0026] According to one aspect of some embodiments of the present invention, a polynucleotide encoding a nanobody is provided.

[0027] According to one aspect of some embodiments of the present invention, a nucleic acid construct is provided, comprising a polynucleotide and a cis-regulatory element for guiding the expression of the polynucleotide.

[0028] According to one aspect of some embodiments of the present invention, a host cell is provided comprising a nanobody or a polynucleotide or nucleic acid construct encoding a nanobody.

[0029] According to one aspect of some embodiments of the present invention, a method for producing insect control nanobodies is provided, the method comprising culturing cells.

[0030] According to some embodiments of the present invention, the method includes isolating nanobodies.

[0031] According to one aspect of some embodiments of the present invention, an insect control method is provided, the method comprising contacting an insect with a nanobody, a polynucleotide or nucleic acid construct encoding a nanobody, or a host cell expressing a nanobody.

[0032] According to some embodiments of the present invention, contact includes applying nanobodies directly to insects.

[0033] According to some embodiments of the invention, contact includes applying nanobodies to an organism or surface that may come into contact with insects.

[0034] According to some embodiments of the present invention, nanobodies are formulated for delivery by spraying, irrigation, and / or fumigation.

[0035] According to some embodiments of the present invention, nanobodies are formulated as liquid preparations.

[0036] According to some embodiments of the present invention, nanobodies are formulated as dry preparations.

[0037] According to one aspect of some embodiments of the present invention, a plant is provided comprising nanobodies or polynucleotide or nucleic acid constructs encoding nanobodies.

[0038] According to some embodiments of the present invention, the plant is a genetically modified plant.

[0039] According to one aspect of some embodiments of the present invention, a commercial product comprising nanobodies is provided.

[0040] According to some embodiments of the present invention, the commercial product is produced from the plant described in any one of claims 31-32.

[0041] According to some embodiments of the present invention, the insects are selected from the group consisting of moths, whiteflies, aphids, stink bugs, leafhoppers, beetles, and bees.

[0042] According to some embodiments of the present invention, the insect is a moth.

[0043] According to some embodiments of the present invention, the moth is the fall armyworm (Spodoptera frugiperda).

[0044] According to some embodiments of the present invention, the insect is a whitefly.

[0045] According to some embodiments of the present invention, the whitefly is the tobacco whitefly.

[0046] According to some embodiments of the present invention, the insect is an aphid.

[0047] According to some embodiments of the present invention, the aphid is the peach aphid.

[0048] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While similar or equivalent methods and materials may be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification (including definitions) shall prevail. Furthermore, materials, methods, and examples are illustrative only and are not necessarily limiting. Attached Figure Description

[0049] This document describes some embodiments of the invention by way of example only, with reference to the accompanying drawings. Referring now to the drawings in detail, it should be emphasized that the details shown are merely examples and are used to illustrate the embodiments of the invention. In this regard, the description taken in conjunction with the accompanying drawings will make it clear to those skilled in the art how to practice the embodiments of the invention.

[0050] In the attached diagram: Figure 1 This figure illustrates the effects of anti-CDA nanobodies CDA-SF-1D (SEQ ID NO:14), CDA-SF-2C (SEQ ID NO:82), and CDA-SF-3B (SEQ ID NO:94) at concentrations of 2–4 mg / ml (0.1 ml) on the mortality rate of fall armyworm in a cotton leaf disc bioassay, compared to the PBS control. In three independent studies, one 1–2 stage larva was added to each well, with 12 replicates per treatment; mortality was assessed 4 days after treatment. Data are expressed as mean ± standard error (SE).

[0051] Figure 2 This is a dose-response graph showing the effect of anti-CDA nanobody CDA-SF-1D (SEQ ID NO:14) on the mortality rate of peach aphids in a liquid bioassay after feeding with nanobody at concentrations between 0.01 and 2 mg / ml. Each sample included 10 wingless nymphal aphids, with 3 replicates per treatment; and mortality was assessed after 48 hours of incubation.

[0052] Figure 3 This is a dose-response graph showing the effect of anti-CDA nanobody CDA-SF-1D (SEQ ID NO:14) on whitefly mortality in a liquid bioassay after feeding with nanobody at concentrations between 0.05 and 2.5 mg / ml. Each sample included 50 adult aphids, with three replicates per treatment; and mortality was assessed after 48 hours of incubation.

[0053] Figure 4 This is a dose-response graph showing the effect of anti-CDA nanobody CDA-SF-2C (SEQ ID NO:82) on the mortality rate of peach aphids in a liquid bioassay after feeding with nanobody at concentrations between 0.05 and 2.5 mg / ml. Each sample included 10 wingless nymphal aphids, with 3 replicates per treatment; and mortality was assessed after 48 hours of incubation.

[0054] Figure 5This figure illustrates the effect of anti-CDA nanobody CDA-SF-1D (SEQ ID NO:14) at a concentration of 0.6 mg / ml on the mortality rate of peach aphids in a pepper plant bioassay (where the nanobody was applied before insect introduction), compared to the PBS control. Twenty N2-N3 stage aphids were inoculated per plant, with four replicates per treatment; mortality was assessed four days after treatment. Data are expressed as mean ± standard error.

[0055] Figure 6 This figure illustrates the effect of anti-CDA nanobody CDA-SF-2C (SEQ ID NO:82) at a concentration of 2 mg / ml (1.4 ml) on the mortality rate of peach aphids in a pepper plant bioassay (where the nanobody was applied before insect introduction), compared to a PBS control. Twenty N2-N3 stage aphids were inoculated per plant, with four replicates per treatment; mortality was assessed four days after nanobody treatment. Data are expressed as mean ± standard error.

[0056] Figure 7 This figure illustrates the effect of anti-CDA nanobody CDA-SF-2C (SEQ ID NO:82) on the mortality rate of peach aphids in a greenhouse plant bioassay for chili peppers (where nanobodies were applied after insect introduction), using 0.8 ml of nanobody at concentrations of 1 mg / ml or 3 mg / ml. In three independent studies, each plant was inoculated with 20 nymphal aphids before nanobody treatment, with 3–10 independent replicates per treatment; mortality was assessed on day 1 and day 5 after nanobody treatment. Data are expressed as mean ± standard error, ***-p≤0.001.

[0057] Figure 8 This study demonstrates the effect of the anti-CDA nanobody CDA-SF-2C (SEQ ID NO:82) on whitefly mortality in a greenhouse plant bioassay for chili peppers (where nanobodies were applied after insect introduction). The nanobody was sprayed twice, on days 0 and 6, using 0.8 ml of nanobody at final concentrations of 1 mg / ml or 3 mg / ml. In two independent studies, 100 adult aphids were inoculated per plant prior to the first nanobody treatment, with 4–6 independent replicates per treatment; mortality was assessed on days 1, 5, 7, and 12 following the first nanobody treatment. Data are expressed as mean ± standard error.

[0058] Figure 9This figure illustrates the effect of the anti-CDA nanobody CDA-SF-2C (SEQ ID NO:82) on the mortality rate of fall armyworm in a cotton greenhouse plant bioassay (where nanobodies were applied after insect introduction), using 1.5 ml of nanobody at a final concentration of 1 mg / ml or 3 mg / ml. In four independent studies, each plant was inoculated with 16 L2-stage larvae prior to nanobody treatment, with 3–10 independent replicates per treatment; mortality was assessed on days 1 and 5 after nanobody treatment. Data are expressed as mean ± standard error, *-p≤0.05), **-p≤0.01 and ****-p≤0.001. Detailed Implementation

[0059] In some embodiments of the present invention, therein relates to anti-insect CDA nanobodies and their uses.

[0060] Before explaining at least one embodiment of the present invention in detail, it should be understood that the application of the present invention is not necessarily limited to the details set forth in the following description or illustrated by examples. The present invention can have other embodiments, or can be practiced or implemented in various ways.

[0061] For the past 70 years, pest management has primarily relied on the application of synthetic pesticides, most of which have a high potential to damage ecosystems. These pesticides are toxic to humans directly or through the food chain, and their use is hampered by the development of genetic resistance in insects. Currently used biological solutions also have many drawbacks, including low efficiency, the risk of disrupting natural ecological balance, and the development of genetic resistance in insects.

[0062] The invention also discloses anti-insect CDA nanobodies and their use as insecticides (International Patent Application Publication No. WO2021 / 095031).

[0063] While practicing specific embodiments of the present invention, the inventors have now developed nanobodies with insect control activity targeting insect CDA (Examples 1 and 4 in the following Examples section and Tables 1-2 below). The killing effect is shown to be dose-dependent. Therefore, specific embodiments of this teaching suggest its use as an insecticide.

[0064] It should be understood that the nanobodies disclosed herein are not intended to include any anti-CDA nanobodies disclosed in International Patent Application Publication No. WO2021 / 095031.

[0065] According to specific embodiments, the nanobodies disclosed herein have superior performance compared to other insecticidal anti-CDA targeting agents described in the prior art (e.g., nanobodies described in International Patent Application Publication No. WO2021 / 095031), such as, but not limited to, increased stability, insecticidal efficacy, and / or affinity for the target.

[0066] According to specific embodiments, the nanobody is not SEQ ID NO: 166, 170, 174, 178, 182, 186, 190, 194, 198, 202, 206, 210, 214, 218, 222, 226, 230, 234, 238, 242, 246, 250, 254, 258, 262, 266, 270, 274, 278, 282, 286, 290, 294, 298, 302, 306 Any nanobody listed in 310, 314, 318, 322, 326, 330, 331, 335, 336, 340, 344, 345, 346, 350, 354, 358, 359, 363, 367, 371, 372, 376, 380, 384, 388, 392, 396, 400, 404, 408, 412, 416, 420, or 424.

[0067] According to specific embodiments, the amino acid sequences of the nanobody are identical to those in SEQ ID NO: 166, 170, 174, 178, 182, 186, 190, 194, 198, 202, 206, 210, 214, 218, 222, 226, 230, 234, 238, 242, 246, 250, 254, 258, 262, 266, 270, 274, 278, 282, 286, 290, 294, 298, 302, 306, 310, 3 Any nanobody listed in 14, 318, 322, 326, 330, 331, 335, 336, 340, 344, 345, 346, 350, 354, 358, 359, 363, 367, 371, 372, 376, 380, 384, 388, 392, 396, 400, 404, 408, 412, 416, 420, or 424 has less than 70% identity.

[0068] Sequence identity or homology can be determined using any protein or nucleic acid sequence alignment algorithm, such as Blast, ClustalW, and MUSCLE.

[0069] According to specific embodiments, the nanobody does not include SEQ ID NO: 167-169, 171-173, 175-177, 179-181, 183-185, 187-189, 191-193, 195-197, 199-201, 203-205, 207-209, 211-213, 215-217, 219-221, 223-225, 227-229, 2 arranged in order from N to C on the nanobody. 31-233, 235-237, 239-241, 243-245, 247-249, 251-253, 255-257, 259-261, 263-265, 267-269, 271-273, 275-277, 279-281, 283-285, 287-289, 291-293, 295-597 299-301, 303-305, 307-309, 311-313, 315-317, 319-321, 323-325, 327-329, 332-334, 337-339, 341-343, 347-349, 351-353, 355-357, 360-362, 364-366, 368-370 Any one of the three complementary determination regions (CDRs) listed in 373-375, 377-379, 381-383, 385-387, 389-391, 393-395, 397-399, 401-403, 405-407, 409-411, 413-415, 417-419, 421-423, or 425-427.

[0070] According to a specific embodiment, the three CDR amino acid sequences of the nanobody are arranged in order from N to C on the nanobody as SEQ ID NO: 167-169, 171-173, 175-177, 179-181, 183-185, 187-189, 191-193, 195-197, 199-201, 203-205, 207-209, 211-213, 215-217, 219-221, 223-225, 227-229, 23 1-233, 235-237, 239-241, 243-245, 247-249, 251-253, 255-257, 259-261, 263-265, 267-269, 271-273, 275-277, 279-281, 283-285, 287-289, 291-293, 295-597 299-301, 303-305, 307-309, 311-313, 315-317, 319-321, 323-325, 327-329, 332-334, 337-339, 341-343, 347-349, 351-353, 355-357, 360-362, 364-366, 368- The three CDRs listed in 370, 373-375, 377-379, 381-383, 385-387, 389-391, 393-395, 397-399, 401-403, 405-407, 409-411, 413-415, 417-419, 421-423, and 425-427 have less than 70% identity.

[0071] According to specific embodiments, the CDR amino acid sequence of any nanobody is related to the SEQ ID NOs arranged on the nanobody in order from N to C: 167-169, 171-173, 175-177, 179-181, 183-185, 187-189, 191-193, 195-197, 199-201, 203-205, 207-209, 211-213, 215-217, 219-221, 223-225, 227-229, 23 1-233, 235-237, 239-241, 243-245, 247-249, 251-253, 255-257, 259-261, 263-265, 267-269, 271-273, 275-277, 279-281, 283-285, 287-289, 291-293, 295-597 299-301, 303-305, 307-309, 311-313, 315-317, 319-321, 323-325, 327-329, 332-334, 337-339, 341-343, 347-349, 351-353, 355-357, 360-362, 364-366, 368- The CDRs listed in 370, 373-375, 377-379, 381-383, 385-387, 389-391, 393-395, 397-399, 401-403, 405-407, 409-411, 413-415, 417-419, 421-423, or 425-427 have less than 70% identity.

[0072] Therefore, according to one aspect of the present invention, a nanobody specifically binding to insect CDA is provided, said nanobody comprising SEQ ID NO: 3-5, 7-9, 11-13, 15-17, 19-21, 23-25, 27-29, 31-33, 35-37, 39-41, 43-45, 47-49, 51-53, 55-57, 59-61, 63-65, 67-69, 71-73, 75-77, 79-81, 83-85, 87-89, 91-93, 95-97, 99-101, 103-105, 10 The nanobody contains three complementary determinant regions (CDRs) listed in 7-109, 111-113, 115-117, 119-121, 123-125, 127-129, 131-133, 135-137, 139-141, 143-145, 147-149, 151-153, 155-157, or 159-161, wherein the binding of the nanobody to the insect CDA confers insect control activity on the nanobody.

[0073] According to another or alternative aspect of the invention, a composition is provided comprising a nanobody that specifically binds to insect CDA and a toxin portion having insect control activity, wherein the nanobody comprises SEQ ID NOs arranged in order from N to C: 3-5, 7-9, 11-13, 15-17, 19-21, 23-25, 27-29, 31-33, 35-37, 39-41, 43-45, 47-49, 51-53, 55-57, 59-61, 63-65, 67-69, 71-73, 75-77, 79-81, 83-85, 87-89, 91-93, 95-97. The three complementary determination regions (CDRs) listed in 99-101, 103-105, 107-109, 111-113, 115-117, 119-121, 123-125, 127-129, 131-133, 135-137, 139-141, 143-145, 147-149, 151-153, 155-157, or 159-161.

[0074] Epitope mapping analysis (Example 2 in the following examples section) shows that the anti-CDA nanobody CDA-SF-1D (SEQ ID NO:14) binds to the fall armyworm CDA (SEQ ID NO:14). The nonlinear epitopes of NO:1 include amino acids: ASP(D)170, ASP(D)175, GLU(E)360, ASP(D)361, ASP(D)365, ASP(D)384, TYR(Y)387, ASN(N)388, ASN(N)391, HIS)H(392, ASP(D)395, TYR(Y)398, GLU(E)399, GLU(E)419, GLU(E)422, GLU(E)430, SER(S)433 and SER(S)434; wherein the CRD3 region of the nanobody binds GLU(E)360, ASP(D)361, ASP(D)365, ASP(D)384, HIS)H(392, GLU(E)419 and GLU(E)422.

[0075] Therefore, according to one aspect of the present invention, a nanobody that specifically binds to a nonlinear epitope of insect CDA is provided, said nonlinear epitope comprising an amino acid residue or a corresponding amino acid residue of an orthologous polypeptide selected from the group consisting of D170, D175, E360, D361, D365, D384, Y387, N388, N391, H392, D395, Y398, E399, E419, E422, E430, S433 and S434 of SEQ ID NO:1, wherein the binding of the nanobody to the insect CDA confers insect control activity upon the nanobody.

[0076] According to another or alternative aspect of the invention, a composition is provided comprising a nanobody specifically binding to a nonlinear epitope of an insect CDA and a toxin moiety having insect control activity, wherein the nonlinear epitope comprises an amino acid residue selected from the group consisting of D170, D175, E360, D361, D365, D384, Y387, N388, N391, H392, D395, Y398, E399, E419, E422, E430, S433, and S434 of SEQ ID NO:1, or a corresponding amino acid residue from an orthologous polypeptide.

[0077] Epitope mapping analysis (Example 3 in the following examples section) shows that the anti-CDA nanobody CDA-SF-2C (SEQ ID NO:82) binds to the nonlinear epitope of fall armyworm CDA (SEQ ID NO:1), which includes the following amino acids: V22, E23, L24, C25, D46, I48, I68, E99, P100, L101, C102, R286, A287, G293, G294, N295, N296, Q297, M301, E302, E303, Q304, L307, Y308, D309. , S310, T311, I312, P319, P320, P323, Y324, T325, Y327, F328, H332, R333, C334, H33 5. G336, N337, L338, Q339, S340, C341, P342, T343, R344, W349, E350, M351, V352, N3 54. E355, D357, R358, E360, P362, N364, E366, M373, V374, D375, N391, H392, D395, Y398, E399, A403, P404, L405, G406, R415, S434, M443, T444, Q445, V446, I447, Q448 P475, C477, W478, V479, P480, S482, C483, K484, L485, T499, C500, V501, R502, P508, L510, N511, D512, D516, G517, H518 and Y519; wherein the CRD3 region of the nanobody binds E350, M351 and V352.

[0078] Therefore, according to one aspect of the present invention, a nanobody specifically binding to a nonlinear epitope of insect CDA is provided, said nonlinear epitope comprising amino acid residues selected from: V22, E23, L24, C25, D46, I48, I68, E99, P100, L101, C102, R286, A287, G293, G294, N295, N296, Q297, M301, E30 2. E303, Q304, L307, Y308, D309, S310, T311, I312, P319, P320, P323, Y324, T325, Y327, F328 , H332, R333, C334, H335, G336, N337, L338, Q339, S340, C341, P342, T343, R344, W349, E350, M 351, V352, N354, E355, D357, R358, E360, P362, N364, E366, M373, V374, D375, N391, H392, D3 95, Y398, E399, A403, P404, L405, G406, R415, S434, M443, T444, Q445, V446, I447, Q448, P47 5. C477, W478, V479, P480, S482, C483, K484, L485, T499, C500, V501, R502, P508, L510, N511, D512, D516, G517, H518 and Y519, or corresponding amino acid residues in orthologous polypeptides, wherein the binding of the nanobody to the insect CDA endows the nanobody with insect control activity.

[0079] According to another or alternative aspect of the invention, a composition is provided comprising a nanobody specifically binding to a nonlinear epitope of insect CDA and a toxin moiety having insecticidal activity, wherein the nonlinear epitope comprises amino acid residues selected from the group consisting of: V22, E23, L24, C25, D46, I48, I68, E99, P100, L101, C102, R286, A287, G293, G2 94, N295, N296, Q297, M301, E302, E303, Q304, L307, Y308, D309, S310, T311, I312, P319, P3 20. P323, Y324, T325, Y327, F328, H332, R333, C334, H335, G336, N337, L338, Q339, S340, C3 41. P342, T343, R344, W349, E350, M351, V352, N354, E355, D357, R358, E360, P362, N364, E3 66. M373, V374, D375, N391, H392, D395, Y398, E399, A403, P404, L405, G406, R415, S434, M4 43, T444, Q445, V446, I447, Q448, P475, C477, W478, V479, P480, S482, C483, K484, L485, T499, C500, V501, R502, P508, L510, N511, D512, D516, G517, H518 and Y519, or the corresponding amino acid residues in orthologous polypeptides.

[0080] Next, BLAST analysis of the CDA sequence of fall armyworm in NCBI showed that the nanobodies CDA-SF-1D (SEQ ID NO:14) and CDA-CF-2C (SEQ ID ID:82) bind nonlinear amino acid residues in the catalytic and / or chitin-binding domains of CDA (Examples 2-3 in the following Examples section).

[0081] Therefore, according to one aspect of the present invention, a nanobody is provided that specifically binds to a nonlinear epitope of insect CDA, the nonlinear epitope being located in a catalytic and / or chitin-binding domain of the insect CDA, wherein the binding of the nanobody to the insect CDA imparts insect control activity to the nanobody.

[0082] According to another or alternative aspect of the invention, a composition is provided comprising a nanobody that specifically binds to a nonlinear epitope of insect CDA and a toxin moiety having insect control activity, wherein the nonlinear epitope is located in a catalytic and / or chitin-binding domain of the insect CDA.

[0083] As used herein, the term "nanobody" refers to a single-domain antigen-binding fragment.

[0084] According to specific implementations, nanobodies are single variable domains derived from the natural heavy chains of antibodies. Nanobodies are typically derived from heavy-chain-only antibodies (excluding light chains) found in camelids (Hamers-Casterman et al., 1993, Nature 363:446-448; Desmyter et al., 1996, Nat. Structur. Biol. 803-811), and are therefore commonly referred to as VHH antibodies, VHH sequences, or immunoglobulin single variable domains (ISVDs). Camelidae include Old World camels (Bactrian camels...). Camelus bactrianus ) and dromedary camel ( Camelus dromedarius And New World camel family plants (e.g., alpacas) Lama paccos ), Alpaca ( Lama glama ), Log camel ( Lama guanicoe ) and llamas ( Lama vicugna Non-limiting examples of camels include dromedary camels, Bactrian camels, wild Bactrian camels, llamas, alpacas, vicuñas, and guanacos. According to a specific implementation, the camel is the llama.

[0085] NANOBODY® and NANOBODIES® are registered trademarks of Ablynx NV (Belgium).

[0086] For a further description of VHH or nanobodies, please refer to the bibliography “Single domain antibodies,” Methods in Molecular Biology, eds. Saerens and Muyldermans, 2012, Vol. 911, particularly the chapters of Vincke and Muyldermans (2012), and a non-limiting list of patent applications mentioned as general background art, including: WO94 / 04678, WO95 / 04079, WO96 / 34103 from the Free University of Brussels; WO94 / 25591, WO99 / 37681, WO00 / 40968, WO00 / 43507, WO00 / 65057, WO01 / 40310, WO01 / 44301, EP1134231, and WO02 / 48193 from Unilever; and Vlaams Institute for Biotechnology. WO97 / 49805, WO01 / 21817, WO03 / 035694, WO03 / 054016 and WO03 / 055527 of voorBiotechnologie (VIB); WO04 / 041867, WO04 / 041862, WO04 / 041865, WO04 / 041863, WO04 / 062551, WO05 / 044858, WO06 / 40153, WO06 / 079372, WO06 / 122786, WO06 / 122787 and WO06 / 122825 of Ablynx NV, and further disclosed patent applications of Ablyynx NV. As those skilled in the art will know, nanobodies are characterized by the presence of one or more Camelidae “marker residues” in one or more framework sequences (according to Kabat numbering), as described, for example, in Table A-3 on page 75 of WO08 / 020079, which is incorporated herein by reference.

[0087] According to specific implementations, nanobodies refer to complete molecules (i.e., including 4 framework regions and 3 complementarity-determining regions) or functional fragments of molecules capable of binding to antigenic epitopes bound to complete molecules.

[0088] As used herein, the term "epitaph" refers to any antigenic determinant on an antigen to which the complementary site of a nanobody binds. Epitope determinants typically consist of chemically active molecular surface groups such as amino acid or carbohydrate side chains and usually possess specific three-dimensional structural features and specific charge characteristics. Epitopes can be linear or non-linear, as further described below.

[0089] According to a specific implementation, the epitope is located within the catalytic domain of CDA.

[0090] According to a specific implementation, the epitope is located within the chitin-binding structural domain of CDA.

[0091] Depending on the specific implementation method, the nanobody is a monolithic or complete nanobody.

[0092] According to the specific implementation method, a nanobody is a nanobody fragment.

[0093] Depending on the specific implementation method, the size of the nanobody is 5-30kDa, 10-30kDa, or 10-20kDa.

[0094] According to the specific implementation method, the size of the nanobody is approximately 15 kDa.

[0095] The term "nanobody" also includes natural or synthetic analogs, homologs, mutants, and variants of nanobodies.

[0096] Typically, a complete nanobody consists of three complementarity-determining regions (CDRs) (CDR1, CDR2, and CDR3).

[0097] As used herein, the terms “complementarity-determining region” or “CDR” are used interchangeably to refer to the antigen-binding region found within the variable region of a heavy chain polypeptide. The identity of amino acid residues in a specific nanobody that constitutes the CDR can be determined using methods known in the art, including methods such as sequence variability as defined by Kabat et al. (see, e.g., Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington DC), and methods applied to V from camels, as described in the article by Riechmann and Muyldermans (1999). HHThe location of structural domains, such as the structural ring regions defined by Chothia et al. (see, e.g., Chothia et al., Nature 342:877-883, 1989), the available complex crystal structures defined by the trade-off between Kabat and Chothia using Oxford Molecular's AbM antibody modeling software (now Accelrys®, see Martin et al., 1989, Proc. Natl Acad Sci USA. 86:9268; and www(dot)bioinf-org(dot)uk / abs), by contact definition (see MacCallum et al., J.Mol.Biol. 262:732-745 1996) and “conformation definition” (e.g., see Makabe et al., Journal of Biological Chemistry, 283:1156-1166 2008).

[0098] As used herein, “CDR” may refer to a CDR defined by any method known in the art, including combinations of methods.

[0099] According to specific implementation methods, CDR, as Kabat et al. in Riechmann and Muyldermans (1999), was applied to V from camelidae animals. HH Defined by the structural domain.

[0100] Nanobodies can be monospecific (capable of recognizing one epitope or protein), bispecific (capable of binding to two epitopes or proteins), or multispecific (capable of recognizing multiple epitopes or proteins).

[0101] According to the specific implementation method, the nanobody is a single-specific nanobody.

[0102] Depending on the specific implementation method, nanobodies are multispecific, such as bispecific, trispecific, or quadrispecific.

[0103] According to the specific implementation, the nanobody is a bispecific nanobody. Methods for generating bispecific nanobodies are known in the art, for example in Deffar K, Shi H, Li L, Wang X, Zhu X (2009) Afr J Biotechnol 8 (12): 2645–2652; and Zhu, Y. et al., (2017). Scientific reports , 7 (1), disclosed in 2602; its contents are incorporated herein by reference in their entirety.

[0104] This article discloses nanobodies that specifically bind to insect CDA.

[0105] Preferably, the nanobody specifically binds to at least one epitope of insect CDA.

[0106] Assays used to test binding are well known in the art, including but not limited to ELISA, radioimmunoassay (RIA), flow cytometry, BiaCore, biolayer interference Blitz® assay, and HPLC.

[0107] According to specific implementation methods, nanobodies with Kd≤10 -6 M, ≤10 -7 M, ≤10 -8 M, ≤10 -9 M combines insect CDA, and each possibility represents a separate implementation of the invention.

[0108] According to specific implementations, nanobodies can bind insect CDA under open or controlled conditions (e.g., greenhouse).

[0109] The term "insect" as used herein has a broad and popular meaning, encompassing all species within the superphylum Panarthropoda (classification Systema Naturale, Brands, SJ (comp.) 1989–2005. Systema Naturae 2000. Amsterdam, Netherlands, [www.sn2000.taxonomy.nl / ]), including the phyla Arthropoda, Tardigrada, and Onychophora; and including all different stages of the life cycle, such as, but not limited to, egg, larva, nymph, pupa, and adult. Depending on the specific implementation, insects may belong to the phylum Arthropoda (including but not limited to Lepidoptera, Hemiptera, Archaeognatha, Thysanura, Paleoptera, and Neoptera, and also ticks, mites, and spiders), or more preferably, the class Insecta.

[0110] According to the specific implementation method, the insect belongs to the order Lepidoptera.

[0111] According to the specific implementation method, the insect belongs to the order Hemiptera.

[0112] Non-limiting examples of insects include moths, aphids, whiteflies, bedbugs, houseflies, beetles, grasshoppers, caterpillars, mosquitoes, fleas, horseflies, wasps, cockroaches, and ants, such as, but not limited to: • From the order Lepidoptera, such as: *Acleris* spp., *Adoxophyes* spp., *Agrotis* spp., *Alabama argillacea*, *Amyelois* spp., *Anticarsia gemmatalis* spp., *Archips* spp., *Argyrotaenia* spp., *Autographa* spp., *Busseolafusca*, *Cadra cautella*, *Carposina nipponensis*, *Chilosuppressalis*, *Chilo* spp., *Choristoneura conflictana*, *Choristoneura*. fumiferana, western spruce leafroller (Choristoneura occidentalis), beard leafroller (Choristoneura rosaceana), color leafroller (Choristoneura spp.), privet leafroller (Clysia ambiguella), leaf roller (Cnaphalocrocis spp.), cloud leafroller (Cnephasia spp.), striped leafroller (Cochylis spp.), sheath moth (Coleophora spp.), large cabbage moth (Crocidolomiabinotalis), apple leafroller (Cryptophlebia leucotreta), boxwood leafroller (Cydalimaperspectalis), apple fruit moth (Cydia inopinata), small leafroller (Cydia spp.), stem moth (Diatraea spp.), Sudan cotton bollworm (Diparopsis castanea), night borer (Earias) spp.), Ephestia spp., Eucosma spp., Eupoecilia ambiguella, Euproctis spp., Euxoa spp., Grapholita prunivora, Grapholita spp.The following are listed: Apple brown leafroller (Hedya nubiferanal), cotton bollworm (Helicoverpa armigera), corn armyworm (Helicoverpa zea), *Helicoverpa* spp., *Heliothis* spp., Chinese cabbage moth (Hellula undalis), American white tiger moth (Hyphantria cunea), tomato stem moth (Keiferia lycopersicella), spiral leafminer (Leucoptera scitella), *Lithocolletis* spp., grape flower leafroller (Lobesia botrana), tussock moth (Lymantria spp.), leafminer (Lyonetia spp.), tent caterpillar (Malacosoma spp.), cabbage leafminer (Mamestrabrassicae), tobacco hawk moth (Manduca sexta), pear leafminer (Numonia) *Pyrivorella*, *Operophtera* spp., *Opogona sacchari*, *Ostrinia nubilalis*, *Pammene* spp., *Pandemis* spp., *Panolis flammea*, *Paysandisia archon*, *Pectinophora gossypiella*, *Phthorimaea operculella*, *Phyllonorycter* spp., *Pieris rapae*, *Pieris* spp., *Platynota rostrana*, *Plutella xylostella*, *Prays* spp., *Scirpophaga* spp., *Sesamia* The genera *Sesia* (spp.), *Sparganothis* (spp.), and *Sesia* (spp.) are mentioned.The following are listed: * *Spodoptera dolichos*, *Spodoptera eridania*, *Spodoptera exigua*, *Spodopterafrugiperda*, *Spodoptera littoralis*, *Spodoptera litura*, *Spodoptera* spp., *Synanthedon* spp., *Teciasolanivora*, *Thaumatotibia leucotreta*, *Thaumetopoea processionea*, *Thaumetopoea* spp., *Tortrix* spp., *Trichoplusia ni*, and *Yponomeuta* spp. • From the order Coleoptera, for example, *Agrilus anxius*, *Agrilus planipennis*, *Agriotes* spp., *Anomala orientalis*, *Anoplophora chinensis*, *Anoplophora glabripennis*, *Anoplophora* spp., strawberry weevil *Anthonomus bisignifer*, pepper weevil *Anthonomuseugenii*, cotton boll weevil *Anthonomus grandis*, four-spotted flower weevil *Anthonomus quadrigibbus*, strawberry flower weevil *Anthonomus signatus*, and flower weevil *Anthonomus* spp., longhorn beetle *Aprionaspp.*, oak longhorn weevil *Arrhenodes minutus*, beet gnater *Atomaria linearis*, beet flea beetle *Chaetocnema* tibialis), Plum weevil (Conotrachelus nenuphar), Weevil (Cosmopolites spp.), Weevil (Curculio spp.), Great spruce bark beetle (Dendroctonus micans), Siberian pine caterpillar (Dendrolimus sibiricus), Dermestids (Dermestes spp.), Maize root leaf beetle (Diabrotica virgifera), Maize root leaf beetle subspecies (Diabrotica virgifera zeae), Maize root leaf beetle (Diabrotica virgifera), Striped cucumber beetle (Diabrotica balteata), Northern maize root leaf beetle (Diabrotica barberi), Eleven-spotted cucumber beetle (Diabrotica undecimpunctata howardi), Eleven-spotted cucumber beetle (Diabrotica undecimpunctata tenella), Eleven-spotted cucumber beetle (Diabrotica undecimpunctata), Eleven-spotted cucumber beetle (Diabrotica *Undecimpunctata*, *Diabroticaspp.*, *Epilachna varivestis*, and *Epilachna spp.*The following are listed: cucumber flea beetle (Epitrix cucumeris), cereal weevil (Eremnus cerealis), weevil (Eremnus spp.), eucalyptus leaf beetle (Gonipterus scutellatus), small pine bark beetle (Ips amitinus), larch bark beetle (Ips cembrae), double-toothed bark beetle (Ips duplicatus), six-toothed bark beetle (Ips sexdentatus), spruce bark beetle (Ips typographus), tooth beetle (Ips spp.), potato beetle (Leptinotarsa ​​decemlineata), false potato beetle (Leptinotarsa ​​juncta), Texas potato beetle (Leptinotarsa ​​texana), rice water weevil (Lissorhoptrus spp.), Argentine stem weevil (Listronotus bonariensis), and scarab beetle (Melolontha). spp.), Monochamus spp., Naupactus leucoloma, Oryzaephilus spp., Otiorhynchus spp., Phlyctinus spp., Pissodes nemorensis, Pissodes strobi, Pissodes terminalis, Pissodes spp., Popilia japonica, Popilia spp., Premnotrypes spp., Pseudopityophthorus minutissimus, Pseudopityophthorus pruinosus, Psylliodes spp.), *Rhizopertha* spp., *Rhynchophorus ferrugineus*, *Rhynchophorus palmarum*, Scarabaeidae family spp., Scolytidae family spp., *Sitophilus* spp., *Sitotrogas* spp., *Sternochetus mangiferae*, *Tenebrio* spp.), Tribolium castaneum, Tribolium spp., and Trogoderma spp.; • From the order Orthoptera, such as the genera *Gryllotalpa*, *Locusta*, and *Schistocerca*. • From the order Blattodea, for example, the genera *Blatta*, *Blattella*, *Periplaneta*, and *Leucophaea maderae*. • From the order Isoptera, for example, the genera *Coptotermes* and *Reticulitermes*. • From the order Psittaciformes, for example, the genus *Liposcelis*. • From the suborder Ciprophagia of the order Tetranychiformes, such as the genera *Haematopinus* spp., *Linognathus* spp., *Pediculus* spp., and *Trichodectes* spp.; • From the order Tetranychiformes, suborder Mechanomorpha, for example, the genus *Damalinia* spp.; • From the order Thysanoptera, for example, western flower thrips (Frankliniella occidentalis), Frankliniella platensis, genus Frankliniella spp., genus Herciothrips spp., genus Taeniothrips spp., thrips palmi, thrips tabaci, citrus thrips aurantii, citrus thrips citri, scirtothrips dorsalis, and genus scirtothrips spp.; • From the suborder Heteroptera of the order Hemiptera, such as the genera *Cimex*, *Distantiellatheobroma*, *Dysdercus*, *Euschistus*, *Eurygaster*, *Leptocorisa*, *Nezara*, *Piesma*, *Rhodnius*, *Sahlbergella singularis*, *Scotinophara*, *Triatoma*, and the family Miridae, such as *Lygus hesperus* and *Lygus*. The family Lygaeidae (e.g., Blissus leucopterus) and the family Pentatomidae (spp.) are also mentioned.

[0113] • From the suborder Thyrocoracida of the order Hemiptera, such as the spiny whitefly (Aleurocanthus spiniferus), the citrus black spiny whitefly (Aleurocanthus woglumi), the genus *Aleurocanthus*, the cottony whitefly (Aleurothrixus floccosus), the cabbage whitefly (Aleyrodes brassicae), the yellow round-hoofed scale (Aonidella citrina), the genus *Aonidiella*, the family Aphididae, the genus *Acyrthosiphon*, the black bean aphid (Aphis fabae), the soybean aphid (Aphis glycines), the cotton aphid (Aphis gossypii), the genus *Aphis*, the genus *Aspidiotus*, and the tobacco whitefly (Bemisia). *Ceroplastes* spp., *Chrysomphalus aonidium*, *Chrysomphalus dictyospermi*, *Coccus hesperidum*, *Daktulosphaira vitifoliae*, *Diaphorina citri*, *Eriosoma larigerum*, *Gascardia* spp., *Lacanium corni*, *Lepidosaphes* spp., *Lopholeucaspis japonica*, *Macrosiphus* spp., *Margarodes prieskaensis*, *Margarodes vitis*, *Margarodes furdendal* The genera *Pteris vredendalensis*, *Myzus persicae*, *Myzus spp.*, *Parasaissetia nigra*, *Pemphigus spp.*, *Phylloxera spp.*, *Planococcus spp.*, *Pseudaulacaspis spp.*, *Pseudococcus spp.*, and *Psylla spp.* are all related to the aphids *P. vredendalensis*, *Myzus persicae*, *Myzus spp.*, *Parasaissetia nigra*, *Pemphigus spp.*, *Phylloxera spp.*, *Planococcus spp.*, *Pseudaulacaspis spp.*, *Pseudococcus spp.*, and *Psylla spp.*.), Ethiopian cottony mealybug (Pulvinaria aethiopica), kidney shield scale (Quadraspidiotus spp.), constrictor aphid (Rhopalosiphum spp.), hibiscus root mealybug (Ripersiella hibisci), soft scale (Saissetia spp.), two-pronged aphid (Schizaphis spp.), grain web aphid (Sitobion spp.), citrus aphid (Toxopteracitricida), greenhouse whitefly (Trialeurodes vaporariorum), African citrus psyllid (Trioza erytreae), and citrus arrowhead scale (Unaspis citri). • From the order Hemiptera, suborder Cephalomyla, such as *Circulifer haematoceps*, *Circulifer tenellus*, *Draeculacephala minerva*, *Empoascaspp.*, *Erythroneura spp.*, *Graphocephala atropunctata*, *Hishimonus phycitis*, *Myndus crudus*, *Laodelphax spp.*, *Nephotettix spp.*, *Nilaparvata spp.*, *Scaphoideus luteolus*, *Scaphoideus spp.*, and *Xyphon fulgida*. • From the order Hymenoptera, for example, genera such as *Acromyrmex*, *Atta*, *Cephus*, a genus of the family Diprionidae such as *Diprions* and *Gilpinia polytoma*, *Hoplocampa*, *Lasius*, *Monomorium pharaonis*, *Neodiprion*, a genus of the family Formicidae such as *Solenopsis*, and *Vespa*. • From the order Diptera, for example: *Aedes albopictus*, *Aedes cinereus*, *Aedes polynesiensis*, *Aedes* spp., *Amauromyzamaculosa*, *Anastrepha fraterculus*, *Anastrepha ludens*, *Anastrepha obliqua*, *Anastrepha suspensa*, *Anastrepha* spp., *Anopheles gambiae*, *Anopheles* spp., *Aschistonyx eppoi*, *Atherigona soccata*, *Bactrocera* spp., *Bibio hortulanus*, *Calliphora* *Cephalcia lariciphila*, *Ceratitis rosa*, *Ceratitis* spp., *Chrysomyia* spp., *Culex* spp., *Cuterebra* spp., *Dacus* spp., *Drosophila melanogaster*, *Dryocosmus kuriphilus*, *Euphranta canadensis*, *Euphranta japonica*, *Fannia* spp., *Gastrophilus* spp., *Gilpinia hercyniae*, *Glossina* spp., *Hypoderma* spp., *Hippobosca* *Liriomyza spp.*, *Liriomyza bryoniae*, *Liriomyza huidobrensis*, *Liriomyza sativae*, *Liriomyza trifolii*, *Liriomyza spp.*, *Lucilia spp.*, *Melanagromyza spp.*, *Musca spp.*The genera *Oestrus* spp., *Orseolia* spp., *Oscinella frit*, *Pardalaspis cyanescens*, *Pardalaspis quinaria*, *Pegomyia hyoscyami*, *Phorbia* spp., *Rhagoletis pomonella*, *Rhagoletis* spp., *Sciara* spp., *Stomoxys* spp., *Tabanus* spp., and *Tipula* spp. • From the order Fleas, for example, the genera *Ceratophyllus* spp. and *Xenopsyllacheopis*; and • From the order Lepisma, subclass Thysanura, for example, silverfish (Lepisma saccharina).

[0114] In this specific implementation, insects are considered pests. As used herein, the term "pest" refers to agricultural pests, including but not limited to whiteflies, aphids, grasshoppers, caterpillars, beetles, moths, stink bugs, thrips; household pests such as cockroaches, ants, wasps, flies, house crickets, bedbugs, wood borers, mealworms, earwigs, silverfish, termites; and blood-sucking pests such as mosquitoes, fleas, and lice. In this specific implementation, insects are agricultural pests.

[0115] According to the specific implementation method, the insects selected are a group consisting of moths, whiteflies, aphids, stink bugs, leafhoppers, beetles, and bees.

[0116] Depending on the specific implementation method, the insect is a sucking pest.

[0117] Non-limiting examples of sucking pests include whiteflies, aphids, stink bugs, and leafhoppers.

[0118] According to the specific implementation method, the insect is a moth.

[0119] Non-limiting examples of moths include cotton bollworm, codling moth (Cydia pomonella), fall armyworm, armyworm (Mythimna unipuncta), tobacco budworm (Helicoverpa virescens), five-spotted hawk moth (Manducaquinquemaculata), European corn borer (Ostrinia nubilalis), and codling moth (Cydia pomonella).

[0120] According to the specific implementation method, the moth is the fall armyworm.

[0121] According to the specific implementation method, the insect is a whitefly.

[0122] Non-limiting examples of whiteflies include tobacco whitefly, greenhouse whitefly (Trialeurodes vaporariorum), spiny whitefly (Aleurocanthus spiniferus), cabbage whitefly (Alleyrodes proletella), and citrus whitefly (Dialenodes citri).

[0123] According to the specific implementation method, the whitefly is the tobacco whitefly.

[0124] According to specific implementations, whiteflies include biotypes B and / or Q (see, for example, De Barro et al. (2003) Molecular Ecology Notes, 3(1), 40-43, the contents of which are incorporated herein by reference in their entirety).

[0125] According to the specific implementation method, the insect is an aphid.

[0126] Non-limiting examples of aphids include the peach aphid, cotton aphid, broad bean aphid, orange aphid (Toxoptera citricida), wheat aphid (Schizaphis graminum), eggplant aphid (Aulacorthum solani), and rice aphid (Rhopalosiphum padi).

[0127] According to the specific implementation method, the aphid is the peach aphid.

[0128] As used herein, the term "specifically bind" refers to the ability of a nanobody to bind insect CDA with a higher affinity in a physiological environment (e.g., within an insect under physiological conditions) compared to other peptides in the environment.

[0129] According to the specific implementation method, the nanobody binds to insect CDA and does not cross-react with non-insect peptides (such as plant or human peptides).

[0130] According to the specific implementation method, the nanobody binds to insect CDA and does not cross-react with non-insect CDA.

[0131] According to the specific implementation method, the nanobody specifically binds to insect CDA and does not cross-react with other insect peptides.

[0132] According to a specific implementation, the nanobody binds to the CDA of the fall armyworm with higher affinity than the CDA sequence, wherein the CDA sequence has less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, or less than 40% homology with SEQ ID NO:1.

[0133] As used in this article, “higher affinity” means a difference of at least 1.2 times, at least 1.5 times, at least 2 times, at least 5 times, at least 10 times, at least 100 times or more using the same affinity assay.

[0134] According to the specific implementation method, the nanobody specifically binds to the CDA of the fall armyworm and does not cross-react with the CDA sequence of SEQ ID NO:1 which has less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, or less than 40% homology.

[0135] As used herein, the term “insect chitin deacetylase (CDA)” refers to the amino acid sequence of insect enzyme ECNo. 3.5.1.41 (an enzyme that catalyzes the conversion of chitin and H2O into chitosan and acetate) or an antigenic fragment capable of evoking a specific immune response against said fragment.

[0136] Non-limiting examples of insect CDAs that can be used in specific embodiments of the claimed invention include CDAs for beet armyworm, silkworm, red flour beetle, and cotton bollworm (see Tables 4-5 below).

[0137] According to specific implementation methods, the insect CDA is the fall armyworm CDA, such as those provided in GenBank accession numbers XM_035590027 (SEQ ID NO:1), XP_035444320, or XP_050561891.

[0138] According to a specific implementation, the insect CDA includes SEQ ID NO:1.

[0139] According to a specific implementation, the insect CDA consists of SEQ ID NO:1.

[0140] Typically, CDA includes a chitin-binding domain and a catalytic domain (catalyzing chemical reactions).

[0141] As used herein, the term "chitin-binding domain" refers to the amino acid domain of insect CDA (a linear polysaccharide consisting of (1->4)-β-linked D-glucosamine residues, most of which are N-acetylated) capable of binding insect chitin. According to a specific embodiment, the chitin-binding domain is the perifeeding membrane protein A domain.

[0142] As used herein, the term "enveloping membrane protein A domain," also known as "type 2 chitin-binding domain (ChtBD2)," pfam PF01607, refers to an amino acid sequence containing six cysteine ​​residues forming three disulfide bonds. According to specific embodiments, ChtBD2 has the following common sequence: CX 11-30 CX 5-6 CX 9-24 CX 12-17 CX 6-12 C (SEQ ID NO:428).

[0143] The amino acid residues constituting the chitin-binding domain of insect CDA are known to those skilled in the art. For example, the chitin-binding domain of the fall armyworm CDA is defined by amino acid residues C25 to C82 of SEQ ID NO:1. Tables 4-5 below provide other non-limiting examples of chitin-binding domains of other insect CDA.

[0144] As used herein, the term "catalytic domain" refers to the amino acid domain of insect CDA, which catalyzes the conversion of chitin and H2O into chitosan and acetate.

[0145] The amino acid residues constituting the catalytic domain of insect CDA are known to those skilled in the art. For example, the catalytic domain of the CDA of the fall armyworm is defined by amino acid residues S345 to P453 of SEQ ID NO:1. Tables 4-5 below provide other non-limiting examples of catalytic domains of CDA from other insects.

[0146] According to a specific implementation method, nanobodies bind to nonlinear epitopes of insect CDA.

[0147] As used herein, the term “nonlinear epitope” (also known as “conformational epitope”) refers to a discontinuous amino acid sequence that forms a three-dimensional structure within insect CDA and binds to nanobodies.

[0148] According to specific implementation methods, the nonlinear epitope is located in the catalytic and / or chitin-binding domain of insect CDA.

[0149] According to a specific implementation, the nonlinear epitope is at least in the catalytic domain of insect CDA.

[0150] According to specific implementation methods, nonlinear epitopes are not only found in the chitin-binding structural domain of insect CDA.

[0151] The amino acid residues constituting the catalytic and chitin-binding domains of insect CDA are known to those skilled in the art. Several non-limiting examples of the catalytic and chitin-binding domains of insect CDA are further described above and provided in Tables 4-5 below. Furthermore, methods for detecting epitopes bound to nanobodies are known to those skilled in the art, including, for example, computational modeling, peptide scanning, alanine sequencing, site-directed mutagenesis, X-ray crystallography and NMR spectroscopy, ELISA, etc.

[0152] According to specific embodiments, the nonlinear epitope includes amino acid residues or corresponding amino acid residues in orthologous polypeptides selected from the group consisting of D170, D175, E360, D361, D365, D384, Y387, N388, N391, H392, D395, Y398, E399, E419, E422, E430, S433 and S434 of SEQ ID NO:1.

[0153] According to specific embodiments, the nonlinear epitope includes the corresponding amino acid residues in the group consisting of E360, D361, D365, D384, Y387, N388, N391, H392, D395, Y398, E399, E419, E422, E430, S433 and S434 of SEQ ID NO:1, or the corresponding amino acid residues in orthologous polypeptides.

[0154] According to specific embodiments, the nonlinear epitope includes an amino acid residue selected from the group consisting of E360, D361, D365, D384, H392, E419 and E422 of SEQ ID NO:1, or a corresponding amino acid residue in a homologous polypeptide.

[0155] According to specific embodiments, the nonlinear epitopes include amino acid residues selected from the following: V22, E23, L24, C25, D46, I48, I68, E99, P100, L101, C102, R286, A287, G293, G294, N295, N296, Q297, M301, E302, E303, Q304, L307, Y308, D309, S310, T3 of SEQ ID NO:1. 11. I312, P319, P320, P323, Y324, T325, Y327, F328, H332, R333, C334, H335, G3 36. N337, L338, Q339, S340, C341, P342, T343, R344, W349, E350, M351, V352, N3 54. E355, D357, R358, E360, P362, N364, E366, M373, V374, D375, N391, H392, D3 95, Y398, E399, A403, P404, L405, G406, R415, S434, M443, T444, Q445, V446, I4 47, Q448, P475, C477, W478, V479, P480, S482, C483, K484, L485, T499, C500, V501, R502, P508, L510, N511, D512, D516, G517, H518 and Y519, or the corresponding amino acid residues in orthologous polypeptides.

[0156] According to specific embodiments, the nonlinear epitopes include amino acid residues selected from the following: W349, E350, M351, V352, N354, E355, D357, R358, E360, P362, N364, E366, M373, V374, D375, N391, H392, D395, Y398, E399, A403, P404, L405, G406, R415, S434, M443, T444, Q445, V446, I447, and Q448 of SEQ ID NO:1, or corresponding amino acid residues in orthologous polypeptides.

[0157] According to specific embodiments, nonlinear epitopes include amino acid residues selected from the group consisting of C25, D46, I48, and I68 of SEQ ID NO:1, or corresponding amino acid residues in orthologous polypeptides.

[0158] According to specific embodiments, the nonlinear epitope includes an amino acid residue selected from the group consisting of E350, M351 and V352 of SEQ ID NO:1 or a corresponding amino acid residue in a homologous polypeptide.

[0159] The phrase “corresponding amino acid residues in orthologous polypeptides” or “corresponding to SEQ ID NO:1” is intended to include the amino acid residues specifically identified in SEQ ID NO:1, as well as the corresponding amino acid residues relative to any other insect CDA amino acid sequence. Methods for determining the corresponding amino acids in such polypeptides are known to those skilled in the art, including, for example, gene annotation databases such as NCBI combined with sequence alignment algorithms such as BLAST, ClustalW, MUSCLE, etc. Tables 4-5 below provide non-limiting examples of such polypeptides and their respective amino acid residues.

[0160] According to the specific implementation method, the amino acid residues are specifically identified as shown in SEQ ID NO:1.

[0161] According to other specific embodiments, the nanobody interacts with at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least five, at least sixteen, at least twenty, at least twenty-five, at least thirty, at least thirty-two, at least thirty-five, at least forty, at least fifty, at least sixty, at least seventy, at least eighty, at least ninety, at least ninety-five, or at least one hundred amino acid residues of the CDA as described herein, each possibility representing a separate embodiment of the invention.

[0162] According to specific embodiments, the nonlinear epitopes include amino acid residues D170, D175, E360, D361, D365, D384, Y387, N388, N391, H392, D395, Y398, E399, E419, E422, E430, S433 and S434 of SEQ ID NO:1 or the corresponding amino acid residues in orthologous polypeptides.

[0163] According to specific embodiments, the nonlinear epitopes include amino acid residues E360, D361, D365, D384, Y387, N388, N391, H392, D395, Y398, E399, E419, E422, E430, S433, and S434 of SEQ ID NO:1 or corresponding amino acid residues in orthologous polypeptides.

[0164] According to specific embodiments, the nonlinear epitopes include amino acid residues E360, D361, D365, D384, H392, E419 and E422 of SEQ ID NO:1 or corresponding amino acid residues in orthologous polypeptides.

[0165] According to specific embodiments, the nonlinear epitopes include the following amino acid residues: V22, E23, L24, C25, D46, I48, I68, E99, P100, L101, C102, R286, A287, G293, G294, N295, N296, Q297, M301, E302, E303, Q304, L307, Y30 8. D309, S310, T311, I312, P319, P320, P323, Y324, T325, Y327, F328, H332, R333, C334, H335, G336, N337, L338, Q339, S340, C341, P342, T343, R344, W349, E350, M35 1. V352, N354, E355, D357, R358, E360, P362, N364, E366, M373, V374, D375, N391, H392, D395, Y398, E399, A403, P404, L405, G406, R415, S434, M443, T444, Q445, V4 46, I447, Q448, P475, C477, W478, V479, P480, S482, C483, K484, L485, T499, C500, V501, R502, P508, L510, N511, D512, D516, G517, H518 and Y519, or the corresponding amino acid residues in orthologous polypeptides.

[0166] According to specific embodiments, the nonlinear epitopes include amino acid residues W349, E350, M351, V352, N354, E355, D357, R358, E360, P362, N364, E366, M373, V374, D375, N391, H392, D395, Y398, E399, A403, P404, L405, G406, R415, S434, M443, T444, Q445, V446, I447, and Q448 of SEQ ID NO:1, or corresponding amino acid residues in orthologous polypeptides.

[0167] According to specific embodiments, the nonlinear epitopes include amino acid residues C25, D46, I48, I68 of SEQ ID NO:1 or the corresponding amino acid residues in orthologous polypeptides.

[0168] According to specific embodiments, the nonlinear epitopes include amino acid residues E350, M351, and V352 of SEQ ID NO:1 or corresponding amino acid residues in orthologous polypeptides.

[0169] Table 2 below shows non-limiting examples of nanobodies that specifically bind insect CDA and their respective CDRs and can be used in specific embodiments of the present invention.

[0170] According to a specific embodiment, the nanobody specifically binds to insect CDA and includes three CDRs arranged in order from N to C on the nanobody, the amino acid sequences of which are identical to those in SEQ ID NO: 3-5, 7-9, 11-13, 15-17, 19-21, 23-25, 27-29, 31-33, 35-37, 39-41, 43-45, 47-49, 51-53, 55-57, 59-61, 63-65, 67-69, 71-73, 75-77, 79-81, 83-85, 87-89, 91-93, 95-97, 99-101, 103-105, 107-109, 111-113, 115-117, 11 The amino acid sequences listed in 9-121, 123-125, 127-129, 131-133, 135-137, 139-141, 143-145, 147-149, 151-153, 155-157 or 159-161 have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity, each possibility representing a separate embodiment of the invention.

[0171] According to a specific embodiment, the nanobody specifically binds to insect CDA and includes three CDRs arranged in order from N to C on the nanobody, the amino acid sequence of which has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequences listed in SEQ ID NO:15-17, 83-85, or 95-97.

[0172] According to a specific embodiment, the nanobody specifically binds to insect CDA and includes three CDRs arranged in order from N to C on the nanobody, the amino acid sequence of which has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequences listed in SEQ ID NO:15-17.

[0173] According to a specific embodiment, the nanobody specifically binds to insect CDA and includes three CDRs arranged in order from N to C on the nanobody, the amino acid sequence of which has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequences listed in SEQ ID NO:83-85.

[0174] According to a specific embodiment, the nanobody specifically binds to insect CDA and includes SEQ ID NOs arranged in order from N to C on the nanobody: 3-5, 7-9, 11-13, 15-17, 19-21, 23-25, 27-29, 31-33, 35-37, 39-41, 43-45, 47-49, 51-53, 55-57, 59-61, 63-65, 67-69, 71-73, 75-77, 79-81, 83-85, 87-89, 91-93, 95-97, 99-101. The three CDRs listed in 103-105, 107-109, 111-113, 115-117, 119-121, 123-125, 127-129, 131-133, 135-137, 139-141, 143-145, 147-149, 151-153, 155-157, or 159-161 each represent a single embodiment of the invention.

[0175] According to a specific embodiment, the nanobody specifically binds to insect CDA and includes three CDRs listed in SEQ ID NO:15-17, 83-85 or 95-97, which are arranged on the nanobody in order from N to C.

[0176] According to a specific embodiment, the nanobody specifically binds to insect CDA and includes three CDRs listed in SEQ ID NO:15-17, which are arranged on the nanobody in order from N to C.

[0177] According to a specific embodiment, the nanobody specifically binds to insect CDA and includes three CDRs listed in SEQ ID NO:83-85, which are arranged on the nanobody in order from N to C.

[0178] According to a specific embodiment, the nanobody specifically binds to insect CDA and includes three CDRs listed in SEQ ID NO:95-97, which are arranged on the nanobody in order from N to C.

[0179] According to specific embodiments, the nanobody comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with an amino acid sequence selected from the group consisting of SEQ ID NO:2, 6, 10, 14, 18, 22, 26, 30, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, and 146, each possibility representing a separate embodiment of the invention.

[0180] According to a specific embodiment, the nanobody includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 14, 82, and 94.

[0181] According to a specific embodiment, the nanobody includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:14.

[0182] According to a specific embodiment, the nanobody includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:82.

[0183] According to a specific embodiment, the nanobody includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:94.

[0184] According to a specific embodiment, the nanobody comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, and 146.

[0185] According to a specific embodiment, the nanobody comprises an amino acid sequence selected from the group consisting of SEQ ID NO:14, 82 and 94.

[0186] According to a specific implementation, the nanobody includes SEQ ID NO:14.

[0187] According to a specific implementation, the nanobody includes SEQ ID NO:82.

[0188] According to a specific implementation, the nanobody includes SEQ ID NO:94.

[0189] According to a specific embodiment, the nanobody is composed of an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, and 146.

[0190] According to a specific implementation, the nanobody is composed of an amino acid sequence selected from the group consisting of SEQ ID NO:14, 82 and 94.

[0191] According to a specific implementation, the nanobody is composed of SEQ ID NO:14.

[0192] According to a specific implementation, the nanobody is composed of SEQ ID NO:82.

[0193] According to a specific implementation, the nanobody is composed of SEQ ID NO:94.

[0194] It should be noted that nanobodies of some embodiments of the present invention are not limited in their broadest sense to specific biological sources or specific preparation methods. For example, nanobodies can generally be obtained by: (1) by isolating the VHH domain of a naturally occurring heavy chain antibody; (2) by expressing a nucleotide sequence encoding a naturally occurring VHH domain; (3) by “humanizing” a naturally occurring VHH domain or by expressing a nucleic acid encoding such a humanized VHH domain; (4) by “camelizing” a natural VH domain from any animal species, and especially from mammalian species such as humans, or by expressing a nucleic acid encoding such a camelized VH domain; (5) by “camelizing” a “domain antibody” or “Dab” as described in the art, or by expressing a nucleic acid encoding such a camelized VH domain; (6) by preparing a protein, polypeptide, or other amino acid sequence known per se using synthetic or semi-synthetic techniques; (7) by preparing a nucleic acid encoding a nanobodies using nucleic acid synthesis techniques known per se, and then expressing the nucleic acid obtained therefrom; and / or (8) by any combination of one or more of the above. Further descriptions of nanobodies, including humanization and / or camelification, can be found, for example, in WO08 / 101985 and WO08 / 142164 and in this paper. McMahon, Conor, et al. Nature structural&molecular biology A recently reported fully in vitro platform for nanobody discovery based on yeast surface display is disclosed in 25.3 (2018): 289.

[0195] According to a specific implementation, the nanobody is "camelized." For example, "camelization" can be achieved by providing a nucleotide sequence encoding a naturally occurring VH domain and then altering one or more codons in the nucleotide sequence in a manner known per se, such that the new nucleotide sequences encode "camelized" nanobodies. This nucleic acid can then be expressed in a manner known per se, thereby providing the desired nanobody.

[0196] Those skilled in the art will understand that it is used from naturally occurring VH sequences or preferred V sequences. H Other suitable methods and techniques for obtaining nanobodies and / or nucleic acid sequences encoding nanobodies, starting with the H sequence, may include, for example, combining a portion or more of one or more naturally occurring VH sequences (such as one or more FR sequences and / or CDR sequences) in a suitable manner, one or more naturally occurring V... H H sequences (e.g., one or more FR sequences or CDR sequences, and / or one or more synthetic or semi-synthetic sequences) to provide nanobodies or nucleic acid sequences encoding them.

[0197] This article describes a specific method for producing nanobodies.

[0198] According to one aspect of the present invention, a method for producing nanobodies is provided, the method comprising immunizing a camelid with recombinant or purified insect CDA listed in SEQ ID NO:1, wherein the insect CDA has a purity of at least 80% in the insect polypeptide formulation.

[0199] As used herein, the phrase “purified insect CDA” refers to CDA peptides purified from insects such that their purity is at least 80% compared to other peptides present in protein formulations.

[0200] According to specific embodiments, the purity of insect CDA in the purified insect protein preparation is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, each possibility representing a separate embodiment of the invention.

[0201] According to a specific implementation, the method also includes separating nanobodies.

[0202] Methods for isolating nanobodies are well known and are disclosed, for example, in Pardon et al., Nat Protoc. 2014 Mar; 9(3): 674–693, the contents of which are incorporated herein by reference in their entirety and in the following Examples section.

[0203] Once nanobodies are obtained, their binding to target insect CDA, their effect on target insect CDA activity, and / or the bioactivity of the nanobodies (e.g., insect control activity) can be determined in vivo or in vitro. This method is known in the art and is further disclosed above and below.

[0204] Therefore, according to a specific embodiment, the method further includes selecting nanobodies that exhibit insect control activity in a bioassay. Such an assay is known in the art and is further described below.

[0205] According to a specific implementation, the method includes testing the catalytic and / or chitin-binding activity of insect CDA after contact with the nanobody.

[0206] Therefore, according to another or alternative aspect of the present invention, a method for producing nanobodies is provided, the method comprising: (i) Immunize camelids with recombinant or purified insect CDA, wherein the purity of the insect CDA in the insect polypeptide preparation is at least 80%; (ii) Isolation of nanobodies after immunization; and (ii) Test the catalytic and / or chitin-binding activity of insect CDA after contact with the nanobody.

[0207] According to a specific embodiment, the method further includes selecting nanobodies that downregulate the catalytic and / or chitin-binding activity of insect CDA. Such assays are known in the art and are further described below.

[0208] According to the specific implementation method, the nanobody is a naked nanobody.

[0209] As used herein, the term "naked nanobody" refers to a nanobody that does not include heterologous effector components (such as toxin components or detectable components).

[0210] According to specific embodiments, nanobodies include heterologous effector portions, such as toxic portions or detectable portions. The effector portions can be protein- or non-protein-based (e.g., small molecule compounds); the latter are typically generated using functional groups on nanobodies and conjugated spouses.

[0211] Therefore, for example, various types of detectable or reportable portions can be bound to the nanobodies of the present invention. These include, but are not limited to, radioisotopes (e.g.,

[125] Iodine), phosphorescent chemicals, chemiluminescent chemicals, fluorescent chemicals or peptides (e.g., phycoerythrin (PE), fluorescein isothiocyanate (FITC), cytochrome C (Cy-chrome), rhodamine, green fluorescent protein (GFP), blue fluorescent protein (BFP), Texas red, Cy5, PE-Cy5, etc.), enzymes (e.g., horseradish peroxidase (HPR), β-galactosidase and alkaline phosphatase (AP)), affinity tags [e.g., antigens that can be identified by the corresponding antibody (e.g., digitoxin (DIG) antibodies identified by anti-DIG) or molecules with high affinity for the tag (e.g., streptoacidin and biotin)], and molecules that can be detected by positron emission tomography (PET) or magnetic resonance imaging (MRI) (contrast agents).

[0212] Depending on the specific implementation, nanobodies include toxins.

[0213] As used herein, the term "toxin" or "toxin fraction" refers to a compound that has insect control activity (as defined below), including but not limited to polypeptides, polynucleotides, and small molecules.

[0214] Non-limiting examples of the toxin portion include delta-endotoxins [e.g., Cry1A, Cry1B, Cry1C, Cry1D, Cry1E, Cry1F, Cry1G, Cry1H, Cry1I, Cry1J, Cry1K, Cry2A, Cry7B, Cry8D, Cry9A, Cry9B, Cry9C, Cry9D, Cry9E, Cry15A, Cry22A, Cry32A, Cry51A, Cyt1A (Crickmore et al., 1998; van Frankenhuyzen, 2009), coliforms (e.g., coliform E1, coliform Ia, coliform toxin A, coliform protein N), and actinomycins (e.g., equinatoxin II, sticholysin II, fragaceatoxin). C) ClyA family toxins (e.g., hemolysin A, non-hemolytic triple enterotoxin, hemolysin BL), hemolysins (e.g., α-hemolysin, γ-hemolysin, leukocidin, necrotizing enterotoxin B, δ-toxin, Vibrio cholerae hemolysin and Vibrio vulnificus hemolysin), Aeromonas lysin family toxins (e.g., Aeromonas lysin, α-toxin, hydrolysin, ε-toxin, enterotoxin, hemolysin, Kysenin), cholesterol-dependent cytolysins (e.g., Clostridium perfringens hemolysin O, Streptococcus suis hemolysin, intermediate cytolysin, Listeria monocytogenes hemolysin O, lectin cytolysin, anthrax cytolysin, streptococcal hemolysin), membrane attack complex components / perforin (e.g., Plu-MACPF, Bth-MACPF), repeating sequence toxins (e.g., HlyA, bifunctional hemolysin-adenosyl cyclase toxin, MARTX) (DalPeraro and van (der Goot, 2016), spider venom, scorpion venom, potato glycoprotein (patatin), Bacillus thuringiensis insecticidal protein, pathogenic bacillus insecticidal protein, luminescent bacillus insecticidal protein, Bacillus laterosporus insecticidal protein, Bacillus bronchiseptica insecticidal protein, Bacillus spheroides insecticidal protein, and double-stranded RNA for insect control.

[0215] Effector portions, such as toxin portions or detectable portions, can be attached to or conjugated to the nanobodies of the present invention in various ways, depending on the context, application, and purpose.

[0216] The effector portion can be conjugated to the nanobody directly or indirectly (e.g., when included in a carrier). The conjugation can be covalent or non-covalent.

[0217] When the effector moiety is a polypeptide, immunoconjugates can be generated recombinantly. For example, a nucleic acid sequence encoding a toxin or fluorescent protein can be linked in a framework with a nucleic acid sequence encoding a nanobody and expressed in a host cell to produce a recombinant antibody conjugate. Alternatively, the effector moiety can be synthesized chemically, for example, by stepwise addition of one or more amino acid residues in a prescribed order, such as in solid-phase peptide synthesis.

[0218] The effector moiety can also be attached to the nanobody using standard chemical synthesis techniques widely practiced in the art [see, for example, worldwideweb(dot)chemistry(dot)org / portal / chemistry], such as using any suitable chemical linker, directly or indirectly, such as via peptide bonds (when the effector moiety is a polypeptide), or via covalent bonds to intermediate linker elements, such as linking peptides or other chemical moieties, such as organic polymers. Chimeric peptides can be linked by binding at the carboxyl (C) or amino (N) terminus of the peptide, or by binding to internal chemical groups such as straight-chain, branched, or cyclic side chains, internal carbon or nitrogen atoms, etc. A detailed description of fluorescent labeling is provided in U.S. Patent Nos. 3,940,475, 4,289,747, and 4,376,110.

[0219] Exemplary methods for conjugating peptides to nanobodies include, but are not limited to, SPDP conjugation, glutaraldehyde conjugation, and carbodiimide conjugation.

[0220] Nanobodies can also be attached to particles or carriers that include effector moieties. Methods for covalently binding nanobodies to encapsulated particles are known in the art, for example, as disclosed in U.S. Patents 5,171,578, 5,204,096, and 5,258,499.

[0221] Any peptide described herein (e.g., nanobodies and protein compositions) may be encoded by polynucleotides. These polynucleotides can be used on their own or for the recombinant production of the peptides disclosed herein.

[0222] Therefore, according to one aspect of the present invention, a polynucleotide encoding a nanobody or a composition comprising a nanobody and a toxin is provided.

[0223] As used herein, the term "polynucleotide" refers to a single-stranded or double-stranded nucleic acid sequence isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence, and / or a complex polynucleotide sequence (e.g., a combination of the above).

[0224] According to specific embodiments, any polynucleotide and nucleic acid sequences disclosed herein may include conserved nucleic acid substitutions. A conserved modified polynucleotide refers to a nucleic acid encoding the same or substantially the same amino acid sequence, or, where the nucleic acid does not encode an amino acid sequence, a substantially identical or related (e.g., naturally continuous) sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode most proteins. For example, codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Therefore, at each position of alanine specified by a codon, the codon can be changed to another corresponding codon without altering the encoded polypeptide. This nucleic acid variation is a “silent variation” and is one type of conserved modified polynucleotide. According to specific embodiments, any polynucleotide and nucleic acid sequences encoding polypeptides described herein also describe silent variations of nucleic acids. Those skilled in the art will recognize that, in some cases, each codon in a nucleic acid (except AUG and TGG, where AUG is typically the only codon for methionine and TGG is typically the only codon for tryptophan) can be modified to produce a functionally identical molecule. Therefore, silent variations of polynucleotides encoding polypeptides are implicit in the sequence of the expressed product.

[0225] According to specific implementation methods, the nucleic acid sequences disclosed herein are optimized for codons expressed, for example, in mammals or plants.

[0226] Methods for codon optimization are known in the art and are disclosed, for example, in the following literature: Codon Usage Database via DNA Library of NIAS (National Institute of Agro-biosciences), Japan (www.kazusa.org.jp / codon / ); International Patent Application No. WO 93 / 07278; and Grote et al., Nucleic Acids Res. (2005) July 1; 33 (Web Server Special Issue): W526–W531.

[0227] Therefore, some embodiments of the present invention include the nucleic acid sequences described above; fragments thereof, sequences hybridized with thereof, sequences homologous to thereof, sequences orthologous to thereof, sequences encoding similar polypeptides with different codon usages, and altered sequences characterized by mutations, such as deletions, insertions, or substitutions of one or more nucleotides, whether naturally occurring or artificially induced, whether random or targeted.

[0228] Non-limiting examples of polynucleotides encoding nanobodies of some embodiments of the present invention are provided in SEQ ID NO:429-468.

[0229] To express a foreign polypeptide in cells, it is preferable to link the polynucleotide sequence encoding the polypeptide to a nucleic acid construct suitable for intracellular expression. This nucleic acid construct includes a promoter sequence for constitutively or inducibly directing the transcription of the polynucleotide sequence in the cell.

[0230] Therefore, according to one aspect of the invention, a nucleic acid construct is provided, comprising a polynucleotide and a cis-regulatory element for guiding the expression of said polynucleotide.

[0231] According to the specific implementation method, the control element is a heterogeneous control element.

[0232] Some embodiments of the nucleic acid constructs of the present invention (also referred to herein as "expression vectors") include additional sequences that make the vector suitable for replication and integration in prokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors). Furthermore, typical cloning vectors may also contain transcription and translation initiation sequences, transcription and translation terminators, and polyadenylation signals. For example, such constructs typically include a 5' LTR, an tRNA binding site, a packaging signal, a start point for second-strand DNA synthesis, and a 3' LTR or a portion thereof.

[0233] Nucleic acid constructs in some embodiments of the present invention typically include a signal sequence for secreting the peptide from a cell where the peptide is placed. According to a specific embodiment, the signal sequence is a natural signal sequence of the peptide (e.g., a nanobody) in some embodiments of the present invention.

[0234] Eukaryotic promoters typically contain two types of recognition sequences: the TATA box and upstream promoter elements. The TATA box, located 25-30 base pairs upstream of the transcription start site, is believed to be involved in guiding RNA polymerase to initiate RNA synthesis. Other upstream promoter elements determine the rate of transcription initiation.

[0235] Preferably, the promoters used in the nucleic acid constructs of some embodiments of the present invention are active in specific cell populations transformed. Examples of cell type-specific and / or tissue-specific promoters include promoters such as liver-specific albumin [Pinkert et al. (1987) Genes Dev. 1:268-277], lymphocyte-specific promoters [Calame et al. (1988) Adv. Immunol. 43:235-275]; particularly promoters of T-cell receptors [Winoto et al. (1989) EMBOJ. 8:729-733] and immunoglobulins [Banerji et al. (1983) Cell 33729-740], pancreas-specific promoters [Edlunch et al. (1985) Science 230:912-916] or breast-specific promoters, such as whey promoters (US Patent No. 4873316 and European Application Publication No. 264166).

[0236] When using plant expression vectors, the expression of coding sequences can be driven by a number of promoters. For example, viral promoters can be used, such as the 35S RNA and 19S RNA promoters of CaMV [Brisson et al. (1984) Nature 310:511-514], or the capsid protein promoter of TMV [Takamatsu et al. (1987) EMBO J.6:307-311]. Alternatively, plant promoters such as small subunits of RUBISCO [Coruzzi et al. (1984) EMBO J.3:1671-1680 and Brogli et al. (1984) Science 224:838-843] can be used, including AlcR / AlcA (ethanol-inducible); GR fusion, GVG and pOp / LhGR (dexamethasone-inducible); XVE / OlexA (β-estradiol-inducible); or heat shock promoters such as soybean hsp17.5-e or hsp17.3-B [Gurley et al. (1986) Mol.Cell.Biol.6:559-565].

[0237] Enhancer elements can stimulate transcription up to 1,000-fold from connected homologous or heterologous promoters. They are active when the enhancer is positioned downstream or upstream of the transcription start site. Many viral-derived enhancer elements have a broad host range and are active in a variety of tissues. For example, the SV40 early gene enhancer is suitable for many cell types. Other enhancer / promoter combinations suitable for some embodiments of the invention include those derived from polyomaviruses, human or mouse cytomegaloviruses (CMV), various retroviruses such as mouse leukemia virus, mouse or Rous sarcoma virus, and long terminal repeat sequences of HIV. See Enhancers and Eukaryotic Expression, Cold Spring Harbor Press, Cold Spring Harbor, New York, 1983, which is incorporated herein by reference. Enhancers specific to different neuronal cell types are also described in this field. These enhancers can be included in AAV expression vectors to obtain specificity in the absence of Cre-driven strains, for example, as described by Hrvatin et al. (doi:www(dot)doi(dot)org / 10.1101 / 570895), which is incorporated herein by reference.

[0238] When constructing expression vectors, the promoter is preferably located at approximately the same distance from the heterologous transcription start site, just as it is in its natural environment. However, as is known in the art, some variations in this distance can be adapted without loss of promoter function.

[0239] Polyadenylation sequences can also be added to expression vectors to improve the efficiency of mRNA translation. Accurate and efficient polyadenylation requires two distinct sequence elements: a GU- or U-rich sequence downstream of the polyadenylation site, and a highly conserved 6-nucleotide sequence, AAUAAA, located 11-30 nucleotides upstream of it. Termination and polyadenylation signals applicable to some embodiments of the invention include signals derived from SV40.

[0240] In addition to the elements already described, expression vectors in some embodiments of the present invention may typically contain other specialized elements designed to enhance the expression level of clonal nucleic acids or facilitate the identification of cells carrying recombinant DNA. For example, many animal viruses contain DNA sequences that can promote extrachromosomal replication of the viral genome in permissive cell types. Plasmids carrying these viral replicons will replicate peripherally, provided that the genes carried on the plasmid or the host cell's genome provides the appropriate factors.

[0241] The vector may or may not include a eukaryotic replicon. If a eukaryotic replicon is present, the vector can be amplified in eukaryotic cells using appropriate selective markers. If the vector does not include a eukaryotic replicon, free amplification is not possible. Instead, the recombinant DNA is integrated into the genome of the engineered cell, where a promoter directs the expression of the desired nucleic acid.

[0242] The expression vectors of some embodiments of the present invention may further include additional polynucleotide sequences that allow, for example, the translation of several proteins from a single mRNA, such as internal ribosome entry sites (IRES), and sequences for promoter chimeric polypeptide genome integration.

[0243] It should be understood that the various elements included in an expression vector can be arranged in various configurations. For example, enhancer elements, promoters, and even polynucleotide sequences (one or more) encoding polypeptides can be arranged head-to-tail, existing as anticomplements, or existing as antiparallel strands in complementary configurations. While such diverse configurations are more likely to occur in the non-coding elements of the expression vector, alternative configurations of coding sequences within the expression vector can also be envisioned.

[0244] Examples of mammalian expression vectors include, but are not limited to: pcDNA3, pcDNA3.1 (+ / -), pGL3, pZeoSV2 (+ / -), pSecTag2, pDisplay, pEF / myc / cyto, pCMV / myc / cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1, pNMT41, pNMT81 (available from Invitrogen), pCI (available from Promega), pMbac, pPbac, pBK-RSV and pBK-CMV (available from Strategene), pTRES (available from Clontech) and their derivatives.

[0245] Expression vectors containing regulatory elements of eukaryotic viruses (such as retroviruses) can also be used. SV40 vectors include pSVT7 and pMT2. Vectors derived from bovine papillomavirus include pBV-1MTHA, and vectors derived from Epstein-Barr virus include pHEBO and p2O5. Other exemplary vectors include pMSG and pAV009 / A. + pMTO10 / A + pMAMneo-5, baculovirus pDSVE, and any other vector that allows protein expression under the guidance of the SV-40 early promoter, SV-40 late promoter, metallothionein promoter, mouse mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrosis protein promoter, or other promoters that are effectively expressed in eukaryotic cells.

[0246] As mentioned above, viruses are highly specialized infectious agents, and in many cases, they have evolved to evade host defense mechanisms. Typically, viruses infect and spread in specific cell types. The targeting specificity of viral vectors utilizes their natural specificity to target a predetermined cell type, thereby introducing recombinant genes into the infected cells. Therefore, the type of vector used in some embodiments of the present invention will depend on the type of cell being transformed. The ability to select a suitable vector based on the type of cell being transformed is entirely within the capabilities of those skilled in the art, and therefore, a general description of the selection considerations is not provided herein.

[0247] Cells can be stably or transiently transformed using nucleic acid constructs according to some embodiments of the present invention. In stable transformation, the nucleic acid molecule according to some embodiments of the present invention is integrated into the cell genome, and therefore represents a stable and heritable trait. In transient transformation, the nucleic acid molecule is expressed by the transformed cell, but it is not integrated into the genome, and therefore represents a transient trait.

[0248] Various methods can be used to introduce expression vectors of some embodiments of the present invention into cells. Such methods are generally described in the following literature: Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSprings Harbor Laboratory, New York (1989, 1992); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989); Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995); Vega et al., Gene Targeting, CRC Press, Ann Arbor, Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988); and Gilboa et al., [Biotechniques 4(6): 504-512, 1986], and include, for example, stable or transient transfection, liposome transfection, electroporation, and infection with recombinant viral vectors. In addition, for methods of positive and negative selection, please refer to U.S. Patent Nos. 5,464,764 and 5,487,992.

[0249] When using plant expression vectors, constructs can be introduced into plant cells using Ti plasmids, Ri plasmids, plant viral vectors, direct DNA transformation, microinjection, electroporation, and other techniques well known to those skilled in the art. See, for example, Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, New York, Section VIII, pp. 421-463.

[0250] Introducing nucleic acids via viral infection offers several advantages over other methods, such as lipid infection and electroporation, because higher transfection efficiency can be achieved due to the infectivity of the virus.

[0251] Currently preferred in vivo nucleic acid transfer techniques include transfection with viral or non-viral constructs, such as adenovirus, lentivirus, type I herpes simplex virus, or adeno-associated virus (AAV), and lipid-based systems. Useful lipids for lipid-mediated gene transfer include, for example, DOTMA, DOPE, and DC-Chol [Tonkinson et al., Cancer Investigation, 14(1): 54-65 (1996)]. The most preferred constructs for gene therapy are viruses, most preferably adenovirus, AAV, lentivirus, or retrovirus. Viral constructs, such as retroviral constructs, include at least one transcription promoter / enhancer or locus-defining element (one or more), or other elements that control gene expression by other means, such as alternating splicing, nuclear RNA output, or post-translational modifications of messengers. Such vector constructs also include packaging signals, long terminal repeats (LTRs) or portions thereof, and positive and negative strand primer binding sites suitable for the virus used, unless they are already present in the viral construct. In addition, such constructs typically include signaling sequences for secreting peptides from host cells where the peptides are placed. Preferably, the signal sequence used for this purpose is a mammalian signal sequence or a signal sequence of a peptide variant of some embodiments of the invention. Optionally, the construct may also include a signal guiding polyadenylation, as well as one or more restriction sites and translation termination sequences. For example, such a construct typically includes a 5' LTR, an tRNA binding site, a packaging signal, a start point for second-strand DNA synthesis, and a 3' LTR or a portion thereof. Other non-viral vectors, such as cationic lipids, polylysine, and dendritic polymers, may be used.

[0252] In addition to the elements required for transcription and translation containing the inserted coding sequence, expression constructs of some embodiments of the present invention may also include engineered sequences to enhance the stability, production, purification, yield, or toxicity of the expressed peptide. For example, the expression of a fusion protein or cleavable fusion protein comprising a peptide and a heterologous protein of some embodiments of the present invention can be engineered. Such fusion proteins can be engineered such that they can be readily separated by affinity chromatography, for example by immobilization on a column specific to the heterologous protein. In the case of engineered cleavage sites between the peptide and the heterologous protein, the peptide can be released from the column by treatment with an appropriate enzyme or reagent that disrupts the cleavage site [e.g., see Booth et al. (1988) Immunol. Lett. 19:65-70; and Gardella et al. (1990) J. Biol. Chem. 265:15854-15859].

[0253] The present invention also considers cells including the polypeptides, polynucleotides and nucleic acid constructs described herein.

[0254] Therefore, according to one aspect of the invention, a host cell is provided comprising a nanobody or a composition comprising a nanobody and a toxin, or a polynucleotide or nucleic acid construct encoding a nanobody.

[0255] As described above, various prokaryotic or eukaryotic cells can be used as host expression systems to express peptides of some embodiments of the present invention. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant phage DNA, plasmid DNA, or copious DNA expression vectors containing coding sequences; yeast transformed with recombinant yeast expression vectors containing coding sequences; plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus CaMV; tobacco mosaic virus TMV); or plant cells transformed with recombinant plasmid expression vectors containing coding sequences (e.g., Ti plasmid). Mammalian expression systems can also be used to express peptides of some embodiments of the present invention.

[0256] According to the specific implementation method, the cell is a mammalian cell.

[0257] According to the specific implementation method, the cells are camel cells.

[0258] Suitable mammalian cells include primary cells and immortalized cell lines.

[0259] According to other specific implementations, mammalian cells are immortalized cell lines.

[0260] Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) CCL-2), CHO cells (e.g., ATCC CRL9618, CCL61, CRL9096), HEK293 cells (e.g., ATCC CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC CCL10), PC12 cells (ATCC CRL1721), COS cells, COS-7 cells (ATCC CRL1651), RAT1 cells, mouse L cells (ATCC CCLI.3), human embryonic kidney (HEK) cells (ATCC CRL1573), HLHepG2 cells, NSO, Sp2 / O, BHK, Namalwa, etc.

[0261] According to the specific implementation, the cells are Escherichia coli, such as SHuffle T7 Express and BL21.

[0262] According to the specific implementation method, the cell is a plant cell.

[0263] According to one aspect of the present invention, a method for producing insect control nanobodies is provided, the method comprising expressing the polynucleotide or nucleic acid constructs disclosed herein in a host cell.

[0264] According to another or alternative aspect of the invention, a method for producing insect control nanobodies is provided, the method comprising introducing a polynucleotide or nucleic acid construct encoding an insect control nanobodies disclosed herein into a host cell.

[0265] According to another or alternative aspect of the invention, a method for producing insect control nanobodies is provided, the method comprising culturing cells comprising the insect control nanobodies disclosed herein or polynucleotide or nucleic acid constructs encoding them.

[0266] According to the specific implementation method, expression, introduction and / or culture are carried out under conditions that allow for the expression of insect control nanobodies.

[0267] These conditions can be, for example, appropriate temperature (e.g., 37°C), atmosphere (e.g., air with 5% CO2), pH, light, culture medium, supplements, etc.

[0268] Depending on the specific implementation method, expression and / or introduction may be performed in vitro or ex vivo.

[0269] According to a specific implementation, the method further includes separating nanobodies.

[0270] The isolation or recovery of any recombinant polypeptide (e.g., nanobodies) can be performed by any method known in the art. According to specific embodiments, the recovery or isolation of the recombinant polypeptide is performed after an appropriate period of culture. The phrase "recovery of recombinant polypeptide" or "isolation of recombinant polypeptide" refers to the collection of the entire fermentation medium containing the polypeptide and does not imply any additional isolation or purification steps. Nevertheless, polypeptides of some embodiments of the present invention can be purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reversed-phase chromatography, concanavalin A chromatography, mixed-mode chromatography, metal affinity chromatography, lectin affinity chromatography, chromatographic focusing, and differential dissolution.

[0271] According to specific embodiments, after synthesis and purification, the binding and / or insect control activity of the nanobodies can be determined in vivo or in vitro. These methods are known in the art and are further disclosed above and below.

[0272] The compositions disclosed herein (e.g., nanobodies, compositions comprising nanobodies and toxins, polynucleotide and nucleic acid constructs encoding them, and host cells expressing them) can be formulated in a suitable manner, such as lyophilization, freeze-drying, drying, or in an aqueous carrier, culture medium, or suitable diluent such as saline or another buffer. Depending on the specific embodiment, the formulated compositions may be in the form of dust or particulate material, powder, gel, cream, paste, pill, tablet, or oil (vegetable or mineral) suspension, or water or oil / water emulsion, capsule suspension, emulsion concentrate, or wettable powder, wettable granules, water-dispersible granules, aerosol, foam, slurry, or flowable concentrate.

[0273] According to specific embodiments, the composition is formulated as a liquid concentrate, dry powder, tablet, capsule suspension, slurry, or “wet cake”, which may be appropriately diluted, dispersed, suspended, emulsified, or otherwise reconstituted by the end user prior to final use.

[0274] According to a specific embodiment, the composition is formulated as a liquid preparation.

[0275] According to a specific embodiment, the composition is formulated as a dry preparation.

[0276] According to a specific embodiment, the composition is formulated for delivery by spraying, irrigation, and / or fumigation.

[0277] According to specific embodiments, the compositions disclosed herein (e.g., nanobodies, compositions comprising nanobodies and toxins, polynucleotide and nucleic acid constructs encoding them, and host cells expressing them) are stable during storage and use, meaning that the integrity of the composition is maintained under storage and / or use conditions, which may include elevated temperatures, freeze-thaw cycles, changes in pH or ionic strength, UV irradiation, the presence of hazardous chemicals, etc.

[0278] According to specific embodiments, the integrity and activity of the composition are maintained under storage and / or use conditions, which may include elevated temperature, freeze-thaw cycles, changes in pH or ionic strength, UV irradiation, presence of harmful chemicals, etc.

[0279] According to specific embodiments, the integrity and activity of the composition are maintained under open or controlled conditions (e.g., greenhouse).

[0280] It should be noted that the compositions disclosed herein can be formulated with various carriers designed to enhance, for example, delivery, stability, permeability, etc.

[0281] As used herein, “carrier” means any solid, semi-solid, or liquid carrier in which a compound (e.g., nanobodies and / or toxins, including compositions thereof) may be suitably incorporated, included, immobilized, adsorbed, absorbed, bound, encapsulated, embedded, attached, or contained therein or on. Non-limiting examples of such carriers include nanocapsules, microcapsules, nanospheres, microspheres, nanoparticles, microparticles, liposomes, vesicles, beads, gels, weakly ionized resin particles, liposomes, cochlear delivery carriers, small particles, granules, nanotubes, buckyballs, water droplets as part of a water-in-oil emulsion, oil droplets as part of an oil-in-water emulsion, organic materials (such as cork, wood, or other plant-derived materials, such as seed husks, sawdust, 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.

[0282] According to specific embodiments, the carrier is such that it has immediate, gradual, or sustained-release properties over minutes, hours, days, or weeks. Furthermore, the carrier may be made of a material (e.g., a polymer) that breaks down or slowly degrades over time (e.g., over minutes, hours, days, or weeks) (e.g., due to prolonged exposure to high or low temperatures, sunlight, high or low humidity, or other environmental factors or conditions), thereby releasing compounds (e.g., nanobodies and / or toxins, including compositions thereof) from the carrier. According to specific embodiments, the carrier is coupled, bound, linked, or otherwise attached or associated with the compound. According to specific embodiments, the carrier is covalently coupled to the compound.

[0283] The compositions disclosed herein (e.g., nanobodies, compositions including nanobodies and toxins, polynucleotide and nucleic acid constructs encoding them, and host cells expressing them) can be formulated in compositions, such as agrochemical or pesticide compositions, wherein they are mixed with suitable physiologically acceptable carriers or excipients.

[0284] In this paper, the term "active ingredient" refers to nanobodies responsible for biological effects, compositions including nanobodies and toxins, polynucleotide and nucleic acid constructs encoding them, and host cells expressing them.

[0285] As used in this article, “agricultural chemical formulation” refers to an agricultural composition comprising one or more of the aforementioned active ingredients, as well as other chemical components, such as agriculturally acceptable carriers and excipients.

[0286] In the following text, the phrase "physiologically acceptable carrier" refers to a carrier or diluent that does not cause significant irritation to the organism and does not eliminate the biological activity and properties of the given compound. These phrases include adjuvants.

[0287] According to a specific embodiment, the composition is formulated with an agriculturally acceptable carrier. Suitable agricultural carriers can be solid or liquid and are well known in the art. The term "agriculturally acceptable carrier" encompasses all adjuvants, such as inert components, dispersants, surfactants, thickeners, binders, etc., which are commonly used in insecticide formulation technology and are well known to those skilled in the art.

[0288] In this document, the term "excipient" refers to an inert substance added to a composition to further facilitate the application of the active ingredient. Examples of excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and starches, cellulose derivatives, surfactants, gelatin, vegetable oils, polyethylene glycol, wetting agents, spreading agents, buffers, acidifiers, etc.

[0289] According to specific embodiments, the compositions disclosed herein (e.g., nanobodies, compositions comprising nanobodies and toxins, polynucleotide and nucleic acid constructs encoding them, and host cells expressing them) may be the only active substance in the composition.

[0290] According to other specific embodiments, in addition to the compositions disclosed herein (e.g., nanobodies, compositions comprising nanobodies and toxins, polynucleotide and nucleic acid constructs encoding them, and host cells expressing them), the composition may also include one or more additional active agents. Non-limiting examples of such agents include herbicides, insecticides, plant growth regulators, insecticidal nanobodies, safeners, etc.

[0291] According to specific embodiments, the compositions disclosed herein and other active agents are in the same formulation.

[0292] According to specific embodiments, the compositions disclosed herein and additional active agents are in separate containers.

[0293] According to other specific embodiments, the composition may include an insect attractant. The attractant may be a pheromone, such as, for example, a male or female pheromone. For example, the pheromone mentioned in the book "Insect Pheromones and their use in Pest Management" (Howse et al., Chapman and Hall, 1998) may be used in this invention.

[0294] Attractants can be present in the formulation or applied separately to ensure that insects are attracted to the site where the formulation is applied.

[0295] Nanobodies and compositions comprising nanobodies and toxins in some embodiments of the present invention have insect control activity.

[0296] As used herein, the term “insect control” refers to preventing, inhibiting, or reducing the ability of insects to feed, grow, move, spread, develop, survive, and / or reproduce, and / or limiting insect-related damage, which may manifest itself, for example, by killing insects, reducing insect survival or lifespan, reducing insect fertility and / or reproductive capacity, reducing or stopping insect feeding, reducing or stopping insect growth, reducing or stopping insect development, reducing or stopping insect activity, and / or preventing insect infestation.

[0297] Depending on the specific implementation method, insect control activities are carried out by killing insects.

[0298] Methods for determining insect control activity are well known to those skilled in the art and are also disclosed in the following examples section, including but not limited to in vitro rearing insects in the presence of nanobodies or compositions comprising nanobodies and determining mortality, development, weight, length, activity, etc., compared to the absence of nanobodies or compositions.

[0299] According to the specific implementation method, the insect control activity is manifested by an increase in insect mortality in the presence of nanobodies compared to the absence of nanobodies.

[0300] According to the specific implementation method, the increase in mortality rate is statistically significant.

[0301] According to the specific implementation, compared with the case where there are no nanobodies, the increased mortality rate is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, at least 2 times, at least 5 times, at least 10 times, or at least 100 times.

[0302] The nanobodies disclosed in this paper can have insect control activity themselves, or they can exert their insect control activity by delivering toxins to insects.

[0303] Therefore, according to specific implementation methods, the binding of nanobodies to insect CDA endows the nanobodies with insect control activity.

[0304] According to a specific implementation method, nanobodies downregulate the activity of insect CDA.

[0305] As used herein, “downregulate activity” means that the biological function of an insect polypeptide (i.e., CDA) is reduced by at least 5% in the presence of the nanobody compared to its biological function in the absence of the nanobody, as determined by methods suitable for determining the activity of the insect polypeptide. Since CDA comprises a chitin-binding domain (e.g., amino acid residues C25 to C82 of SEQ ID NO:1) and a catalytic domain (e.g., amino acid residues S345 to P453 of SEQ ID NO:1), nanobodies of some embodiments of the present invention can bind to and downregulate the activity of the binding domain and / or the catalytic domain. Therefore, for example, the activity of CDA can be determined by, for example, ELISA, Western blot analysis, immunoprecipitation, flow cytometry, colorimetric or fluorescent ATPase assays, or chaperone activity assays.

[0306] According to other specific embodiments, the reduction is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% compared to the absence of nanobodies, which can be determined by, for example, any of the methods described above.

[0307] According to other or alternative embodiments, nanobodies are targeting agents used to specifically deliver to insects, for example, toxins with insect control activity.

[0308] The above text further describes non-limiting examples of toxins and methods for conjugating them with nanobodies.

[0309] Since nanobodies and compositions comprising nanobodies and toxins in some embodiments of the present invention have insect control activity, the present invention also includes methods for insect control.

[0310] Therefore, according to one aspect of the present invention, an insect control method is provided, the method comprising contacting an insect with a nanobody or a composition comprising a nanobody and a toxin, a polynucleotide or nucleic acid construct encoding the nanobody or a host cell expressing the nanobody or a toxin.

[0311] According to a specific embodiment, contact includes directly applying nanobodies or a composition comprising nanobodies and toxins to insects.

[0312] According to a specific embodiment, contact includes applying nanobodies or a composition including nanobodies and toxins to an organism or surface that may come into contact with the insect.

[0313] According to another aspect of the present invention, a method for preventing insect infestation of commercial products is provided, the method comprising adding nanobodies or a composition comprising nanobodies and toxins to the product.

[0314] According to another aspect of the present invention, a method for preventing insect infestation of commercial products is provided, the method comprising packaging the product in packaging material comprising nanobodies or a composition comprising nanobodies and toxins.

[0315] Contact or addition can be achieved using any suitable method known in the art, including but not limited to spraying (including high-volume (HV), low-volume (LV), and ultra-low-volume (ULV) sprays), atomizing, brushing, dressing, dripping, coating, impregnation, soaking, immersion, encrusting, spreading, foaming, atomizing, injecting, adding to cultures, irrigating, applying in the form of droplets, mist, or aerosol, or recombinantly expressing nucleic acid constructs in somatic cells of organisms (as further disclosed above).

[0316] Depending on the specific implementation method, contact or addition may be achieved through spraying, irrigation, and / or fumigation.

[0317] According to specific implementation methods, contact or addition is achieved by introducing polynucleotide or nucleic acid constructs into the cells of an organism.

[0318] According to the specific implementation method, the organism is a plant.

[0319] As used herein, the term "surface" refers to any object that may come into contact with insects. Non-limiting surfaces include nets (e.g., mosquito nets), light sources, colored objects, shapes or outlines that stand out against a contrasting background, greenhouses, outdoor camping facilities, soil, etc.

[0320] According to the specific implementation method, the product is produced from plants.

[0321] As used in this article, the term "plant" includes the whole plant, grafted plants, ancestral and descendant plants, and plant parts, including seeds, buds, stems, roots (including tubers), rootstocks, scions, fruits, vegetables, flowers, and plant cells, tissues, and organs. Plants can be in any form, including suspension cultures, embryos, meristematic zones, callus, leaves, gametophytes, sporophytes, pollen, and microspores.

[0322] According to the specific implementation, the plant is a crop. As used herein, "crop" refers to a plant species or variety cultivated for food, livestock feed, fuel, or any other economic purpose. As non-limiting examples, a crop can be corn, cereals such as wheat, rye, barley, and oats, sorghum, rice, sugar beets and forage beets, fruits such as pome fruits (e.g., apples and pears), citrus fruits (e.g., oranges, lemons, limes, grapefruits, or tangerines), stone fruits (e.g., peaches, nectarines, or plums), nuts (e.g., almonds or walnuts), soft fruits (e.g., cherries, strawberries, blackberries, or raspberries), plantain plants or grapevines, legumes such as beans, lentils, peas, and soybeans, oilseed crops such as sunflowers, safflowers, rapeseed, canola, castor beans, or olives, and cucurbitaceous plants such as cucumbers and melons. Or pumpkins, fiber plants such as cotton, flax or hemp, fuel crops such as sugarcane, miscanthus or switchgrass, vegetables such as potatoes, tomatoes, peppers, lettuce, spinach, onions, carrots, eggplants, asparagus or cabbage, ornamental plants such as flowers (e.g., petunias, geraniums, roses, tulips, lilies or chrysanthemums), herbs (e.g., basil, mint), shrubs, broad-leaved trees (e.g., poplars or willows) and evergreen trees (e.g., conifers), grasses such as lawns, turf or forage, or other useful plants such as coffee, tea, tobacco, hops, pepper, medicinal plants (e.g., hemp), rubber or latex plants.

[0323] According to specific implementation methods, plants may be selected from the group consisting of: corn, soybean, alfalfa, cotton, sunflower, Brassica oilseeds such as rapeseed (e.g., rapeseed oil), Chinese rapeseed, mustard (e.g., field mustard) and Ethiopian mustard, Arecaceae species (e.g., oil palm, coconut), rice, wheat, sugar beets, sugarcane, oats, rye, barley, millet and sorghum, triticale, flax, nuts, grapes and vines, as well as various fruits and vegetables from various plant taxonomic units, such as Rosaceae species (e.g., pomegranates). Examples include apples and pears, but there are also stone fruits such as apricots, cherries, almonds, plums, and peaches, as well as berries such as strawberries, raspberries, red currants, black currants, and currants. Species include those from the following families: *Rhizophoraceae*, *Juglans*, *Betulaceae*, *Anacardiaceae*, *Fagaceae*, *Moraceae*, *Oleaceae* (e.g., olive trees), *Actinidia*, *Lauraceae* (e.g., avocados, cinnamon, camphor), *Musaceae* (e.g., banana trees and plantations), *Rutaceae* (e.g., coffee), *Theaceae* (e.g., tea), *Sterculiaceae*, and *Rutaceae*. (e.g., lemons, oranges, tangerines, and grapefruits); Solanaceae (e.g., tomatoes, potatoes, peppers, field peppers, eggplants, tobacco), Liliaceae (e.g., lettuce, artichokes, and chicory, including root chicory, chicory, or common chicory), Apiaceae (e.g., carrots, parsley, celery, and root celery), Cucurbitaceae (e.g., cucumbers, including small cucumbers, squash, watermelons, gourds, and cantaloupes), Allium (e.g., leeks and onions), Brassicaceae (e.g., white cabbage, red cabbage, broccoli, cauliflower, Brussels sprouts, bok choy, etc.). Turnips, radishes, horseradishes, watercress, and Chinese cabbage); legumes (e.g., peanuts, peas, lentils, and beans such as common beans and broad beans); gooseberries (e.g., Swiss chard, forage beets, spinach, beetroot); linnaceae (e.g., hemp); cannabinaceae (e.g., cannabis); malvaceae (e.g., okra, cocoa); poppies (e.g., poppies); asparagus (e.g., asparagus); useful and ornamental plants in gardens and woodlands, including turf, lawns, grasses, and stevia, and genetically modified types of these plants.

[0324] According to the specific implementation, the plant can be the harvestable part of a plant, selected from the group consisting of: fruits, flowers, nuts, vegetables, fruits or vegetables with inedible peels, such as avocados, bananas, plantains, lemons, grapefruits, melons, oranges, pineapples, kiwis, guavas, citrus fruits, mangoes, and pumpkins.

[0325] According to the specific implementation, the plant is a forest plant, such as those described in www.fao.org / 3 / i0640e / i0640e13.pdf.

[0326] According to the specific implementation method, the plant is an ornamental plant.

[0327] According to the specific implementation method, the plant can be cut flowers of ornamental plants, preferably selected from the group consisting of: six-petaled flowers, carnations, chrysanthemums, freesias, gerberas, gladioli, baby's breath (a species of the genus Dianthus), sunflowers, hydrangeas, lilies, lisianthus, roses, and summer flowers.

[0328] Depending on the specific implementation method, the plant is cut grass or wood.

[0329] According to the specific implementation method, the plant is cotton.

[0330] According to the specific implementation method, the plant is a genetically modified plant.

[0331] According to a specific embodiment, the plant is a transgenic plant that recombinantly expresses nanobodies or a composition including nanobodies and toxins.

[0332] Methods for producing transgenic plants are well known in the art and have been described above.

[0333] Next, the present invention also includes products comprising nanobodies or compositions comprising nanobodies and toxins, polynucleotide or nucleic acid constructs encoding them, or host cells expressing them.

[0334] According to specific embodiments, this product is more resistant to insect infestation or damage compared to products that do not include nanobodies or include compositions containing nanobodies and toxins, polynucleotide or nucleic acid constructs encoding them, or host cells expressing them.

[0335] Therefore, according to one aspect of the invention, a plant is provided comprising nanobodies or a composition comprising nanobodies and toxins, or a polynucleotide or nucleic acid construct encoding therethe.

[0336] According to another or alternative aspect of the invention, a commercial product is provided that comprises nanobodies or a composition comprising nanobodies and toxins.

[0337] According to another or alternative aspect of the invention, a surface covered with nanobodies or a composition comprising nanobodies and toxins is provided.

[0338] According to another or alternative aspect of the invention, a packaged product is provided, comprising a commodity product contained in packaging material comprising nanobodies or a composition comprising nanobodies and toxins.

[0339] Tables 1-2 below list CDA peptides and nanobodies that can be used in specific embodiments of the present invention.

[0340] Table 1: Amino acid sequence of chitin deacetylase (CDA) of the target fall armyworm. Table 2: Amino acid sequence of anti-CDA nanobodies Table 3: Epitope mapping showing amino acid residues in the fall armyworm CDA (SEQ ID NO:1) that interact with the anti-CDA nanobody (in bold). Table 4: Amino acid sequences of CDA from different insects. Underlined segments indicate catalytic domains, and bold segments indicate amino acid residues corresponding to the amino acid residues in the CDA of the fall armyworm (SEQ ID NO:1), which interact with the anti-CDA nanobody CDA-SF-1D. Table 5: Amino acid sequences of CDA from different insects. Underlined parts indicate chitin-binding and catalytic domains. Bold parts indicate amino acid residues corresponding to the amino acid residues of CDA from the fall armyworm (SEQ ID NO:1), which interact with the anti-CDA nanobody CDA-SF-2C. As used in this article, the term “about” means ±10%.

[0341] The terms “comprise,” “comprising,” “include,” “including,” and “having” mean “including, but not limited to.”

[0342] The phrase “consisting of” means “including and limited to”.

[0343] The term "consisting essentially of" means that the composition, method, or structure may include additional components, steps, and / or portions, provided that such additional components, steps, or portions do not substantially alter the essential and novel features of the claimed composition, method, or structure.

[0344] As used herein, unless the context clearly specifies otherwise, the singular forms “a,” “an,” and “the” include plural references. For example, the terms “compound” or “at least one compound” can include a variety of compounds, including mixtures thereof.

[0345] In this application, various embodiments of the invention may be presented in a range format. It should be understood that the range format is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the invention. Therefore, the description of a range should be considered as specifically disclosing all possible subranges and the individual values ​​within that range. For example, a description of a range such as 1 to 6 should be considered as specifically disclosing subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and the individual numbers within that range, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0346] Whenever a range of numbers is indicated herein, it means to include any referenced numbers (fractions or integers) within the indicated range. The phrases “range between the first indicated number and the second indicated number” and “range from the first indicated number to the second indicated number” are used interchangeably herein and are intended to include the first indicated number and the second specified number, as well as all fractions and integers in between.

[0347] As used herein, the term "method" refers to the manner, means, techniques, and procedures used to accomplish a given task, including but not limited to those manner, means, techniques, and procedures known to or readily derived from known manner, means, techniques, and procedures by practitioners in the fields of chemistry, pharmacology, biology, biochemistry, and medicine.

[0348] When referring to a specific list of sequences, this reference should be understood to also include sequences that substantially correspond to their complementary sequences, including other minor sequence variations caused by, for example, sequencing errors, cloning errors, or changes resulting in base substitutions, deletions, or additions, provided that the frequency of such variations is less than 1 / 50 nucleotide, alternatively less than 1 / 100 nucleotide, alternatively less than 1 / 200 nucleotide, alternatively less than 1 / 500 nucleotide, alternatively less than 1 / 1000 nucleotide, alternatively less than 1 / 5,000 nucleotide, and alternatively less than 1 / 10,000 nucleotide.

[0349] It should be understood that, for clarity, certain features of the invention described in the context of a single embodiment may also be provided in combination in a single embodiment. Conversely, for brevity, various features of the invention described in the context of a single embodiment may also be provided individually, or in any suitable sub-combination, or appropriately provided in any other described embodiment of the invention. Certain features described in the context of various embodiments should not be considered as essential features of those embodiments unless the embodiment is inoperable without these elements.

[0350] The various embodiments and aspects of the invention described above and claimed in the claims section below are experimentally supported in the following examples.

[0351] Example Referring now to the following embodiments, which, together with the foregoing description, illustrate some implementations of the invention in a non-limiting manner. Generally, the nomenclature used herein and the laboratory procedures used in the invention include molecular, biochemical, microbiological, and recombinant DNA technologies.

[0352] Materials and Methods Protein target formulations for immunization- The DNA sequence encoding the target CDA of fall armyworm was optimized and synthesized (see Table 1 above). The synthesized sequence and His tag were cloned into pET-28a or pET-30a vectors and expressed in Escherichia coli strain BL21-Star(DE3). Single colonies were inoculated into LB medium containing kanamycin; the cultures were incubated at 37°C at 200 rpm and then induced with isopropyl β-D1-thiogalactopyranoside (IPTG). SDS-PAGE analysis was used to monitor protein expression. Subsequently, BL21-Star(DE3) stored in glycerol was inoculated into TB medium containing kanamycin and cultured at 37°C. When the OD600 reached 1.2, the cells were induced with IPTG at 15°C for 16 hours. Cells were collected by centrifugation and the particles were resuspended in lysis buffer (20 mM NaPi pH 7.4, 500 mM NaCl, 20 mM imidazole, a mixture of protease inhibitors - cOmplete™, and a mixture of EDTA-free protease inhibitors - Roch cat #4693132001), followed by sonication and centrifugation. The precipitate was dissolved in urea; and the denatured supernatant was reserved for future purification. Subsequently, proteins were purified from the supernatant using a Ni-NTA column (GE Healthcare, catalog number 17-5318-02): the column was washed with 10 bed volumes of wash buffer (20 mM NaPi pH 7.4, 500 mM NaCl, 50 mM imidazole), and proteins were eluted with elution buffer (20 mM NaPi pH 7.4, 500 mM NaCl, 500 mM imidazole). After elution, the fraction was placed in a 3.5 kDa MEGA tube (Tivan Biotech MEGA3-50) and dialyzed overnight in 1 L of pH 7.4 PBS at 4°C. A second round of dialysis was then performed under the same conditions. Protein purity and molecular weight were determined by standard SDS-PAGE.

[0353] immunity - Subcutaneous injections of approximately 125 or 150 µg of the target protein were administered to llamas on days 0, 7, 14, 21, 28, and 35, in combination with Gerbu adjuvant P (Gerbu Biotech, #3111-0025). On day 40, approximately 100 ml of anticoagulated blood was collected from the llamas for lymphocyte preparation.

[0354] Construction of the VHH Library- A VHH library was constructed from alpaca lymphocytes to screen for the presence of antigen-specific nanobodies. For this purpose, total RNA from peripheral blood lymphocytes was used as a template for first-strand cDNA synthesis using oligonucleotide (dT) primers. Using this cDNA, the VHH coding sequence was amplified by PCR, digested with PstI (#ER0611 from Thermofischer) and NotI (#11037668001 from Sigma), and cloned into the PstI and NotI sites of the phageparticle vector pMECS. The VHH library contained approximately 10... 8 Each of the individual transducers contains a carrier of the correct insertion size in approximately 80-92% of them.

[0355] Isolation and sequencing of specific target nanobodies - The library was panned three times on a solid phase coated with the target antigen (100 µg / ml, in 100 mM NaHCO3, pH 8.2). After each panning round, the enrichment of antigen-specific phages was assessed by comparing the number of phage particles eluted from the antigen-coated wells with the number eluted from the negative control (uncoated, closed) wells. These experiments showed that the phage population was enriched with antigen-specific phages in the third round. Uncoated, closed wells were used as negative controls, and the antigen used for panning and ELISA screening was the same as that used for immunization. The amino acid sequences of the resulting nanobodies are shown in Table 2 above.

[0356] Nanobody expression and purification- The nanobody DNA sequence was optimized and synthesized using GenScript (provided in SEQ ID NO:429-468). The synthesized sequence was cloned with a His tag into the pET-28b+ vector for protein expression in *E. coli*. Subsequently, *E. coli* SHuffle T7 expression was transformed with the recombinant plasmid. Single colonies were inoculated into TB medium containing kanamycin and incubated overnight at 30°C and 200 rpm. Subsequently, the SHuffle T7 expression initiator was inoculated into a larger volume of TB medium containing kanamycin and cultured at 30°C and 200 rpm. When the OD600 reached 0.6–0.8, the cell cultures were induced with 0.7 mM IPTG at 30°C for 20–22 hours. Cells were harvested by centrifugation, and the cell pellet was resuspended in lysis buffer (20 mM NaPi pH 7.5, 500 mM NaCl, 20 mM imidazole), followed by sonication and centrifugation. The supernatant was retained for purification. Nanobodies were purified using a Ni-Sepharose column. The column was washed with 5 bed volumes of wash buffer (20 mM NaPi pH 7.5, 500 mM NaCl, 50 mM imidazole), and the protein was eluted with elution buffer (20 mM NaPi pH 7.5, 500 mM NaCl, 500 mM imidazole). The eluted fractions were then desalted using a column containing Sepharose G-25 beads and PBS elution buffer (pH 7.4). The fractions were collected and their concentrations determined using Nanodrop. Protein purity and molecular weight were determined by standard SDS-PAGE, followed by Coomassie staining.

[0357] Insect cultureFall armyworm colonies were collected from cotton fields in Israel and reared in the laboratory under conditions of 24°C, 65% relative humidity, and 10 hours of daylight / 14 hours of nightlight. The collected larvae were fed a synthetic diet containing Ward's diet powder (Stonefly Heliothis diet, product number 38-0600, Ward's Natural Science, Rochester, New York), supplemented with a vitamin blend (niacin 40µM, calcium pantothenate 5µM, riboflavin 5µM, thiamine hydrochloride 4µM, pyridoxine hydrochloride 5µM, folic acid 2.5µM, D-biotin 4µM, and cyanocobalamin 5 nM), sorbic acid 0.2%, methylparaben 0.4%, ascorbic acid 0.25%, brewer's yeast 1%, white vinegar 4%, and linseed oil 0.5%. Whitefly and peach aphid colonies were collected outdoors and preserved separately in growth chambers under continuous light at 22±3°C. Whiteflies live on cotton plants, and peach aphids live on pepper plants. The whitefly communities used in this study were a mixture of B and Q biotypes, and the procedures described in De Barro et al. (2003) Molecular Ecology Notes, 3(1), 40-43 were followed.

[0358] Bioassay of the cotton leaf disc of the fall armyworm —Add 1 mL of sterile co-agar to each well of a 12-well plate. After the agar cools, place a 2.0 cm diameter cotton disc leaf into each well and cover it with 50 µl of the test nanobody suspension at a concentration of 2–4 mg / mL or PBS control. Add one 24–48 hour old larva to each well, cover the plate with polyolefin sealing foil (HJ-BIOANALITIC, catalog number 900371), and incubate in the dark at 24°C and 70% relative humidity in the laboratory. After 4 days of incubation, transfer the larvae to 12-well culture dishes containing a 2.0 cm diameter cotton disc leaf and 50 µl of the test nanobody. Larval mortality was measured after 4 and 7 days of incubation. Each study included 12 replicates for each treatment, and each study was performed three times.

[0359] Liquid bioassay of whiteflies and peach aphids—The test was conducted using pouches / bags containing liquid feed made from stretched paraffin film (“parafilmM”). These were handmade and manually filled using pipettes, with each pouch containing approximately 100 µl of spiked feed. The artificial liquid feed consisted of IPL41 (x1) insect culture medium (Gibco, CAS 11405-081) supplemented with diluted 30% sucrose and 0.5% arginine, diluted 1:1 with PBS stock solution containing 1 mg / ml nanobody or as a control. For dose range testing, the nanobody material was diluted to the desired level by mixing it with PBS before incorporating it into the liquid feed. The pouches were then sealed to prevent leakage of test material and placed in an acrylic tube (approximately 22 mm outer diameter × 22 mm height) along with a test population of 10 wingless nymphal aphids. The open end of the tube was sealed with paraffin film.

[0360] Bioassay of peach aphids on pepper plants (applying nanobodies before introducing aphids) Pepper plants were grown under climatic conditions of an average daily temperature of 25°C, 57% humidity, and natural light as hosts. When the plants reached six to eight weeks of age, they were sprayed with 0.6 to 1.4 ml of the test nanobody at a concentration of 1 to 4 mg / ml or PBS as a control. The sprayed plants were then transferred to plastic insect cages (BugDorm-6E610). Twenty N2-N3 stage aphid nymphs were added to each plant and then incubated in a laboratory at 24°C. Aphid mortality was measured at 24, 48, and 72 hours after incubation. Each study had four replicates for each treatment.

[0361] Bioassay of Pepper Greenhouse Plants by Peach Aphids (Applying Nanobodies After Aphid Introduction) Pepper plants were grown under climate-controlled conditions with an average daily temperature of 25°C, humidity of 57%, and natural light. When the plants reached 6-8 weeks of age, 20 N2-N3 stage aphid nymphs were introduced per plant. After a one-day acclimatization period, the plants were sprayed with 0.8 ml of the test nanobody solution or water at a final concentration of 1 mg / ml or 3 mg / ml as a negative control. The plants were then transferred to plastic insect cages (BugDorm-6E610) and incubated in a climate-controlled greenhouse. Aphid mortality was measured on days 1 and 5 after spraying. Each study was conducted three times, with 3-10 independent replicates per treatment.

[0362] Bioassay of whiteflies on pepper greenhouse plants (application of nanobodies after aphid introduction)Chili pepper plants were grown under controlled climate conditions with an average daily temperature of 25°C, humidity of 57%, and natural light. When the plants reached 6 to 8 weeks of age, they were transferred to plastic insect cages (BugDorm-6E610). Each plant was then inoculated with 100 adult aphids; and after a one-day acclimatization period, the plants were sprayed twice as negative controls on days 0 and 6 with 0.8 ml of the test nanobody solution or water at final concentrations of 1 mg / ml or 3 mg / ml. Whitefly mortality was measured on days 1, 5, 7, and 12 post-spraying. Each study was conducted twice, with four independent replicates for each treatment.

[0363] Bioassay of fall armyworm on cotton greenhouse plants (application of nanobodies after larval introduction) Cotton plants were grown under climate-controlled conditions with a daily average temperature of 25°C, humidity of 57%, and natural light. When the plants reached 6–8 weeks of age, 16 L2-stage larvae were introduced per plant (72 hours post-hatching). After a one-day acclimatization period, plants were sprayed as a negative control with 1.5 ml of the test nanobody solution or water at a final concentration of 1 mg / ml or 3 mg / ml. The plants were then transferred to plastic insect cages (BugDorm-6E610) and incubated in a climate-controlled greenhouse. Fall armyworm mortality was measured on days 1 and 5 post-spraying. Each study was conducted four times, with 3–10 independent replicates per treatment.

[0364] Epitope plotting - The interaction between the target CDA (SEQ ID NO:1) and the nanobody was analyzed using computational modeling tools to identify the specific amino acids involved in their binding. In short, the 3D structure of the nanobody and the target CDA sequence were calculated using homology modeling based on known protein structures. In the next step, the initial binding conformation of the protein-nanobody pair was predicted via the Alphafold 2 multimer model. Subsequently, the nanobody homology model was aligned with the expected conformation of AF2, and the combined structure was refined using Rosetta Snugdock and Rosetta relax. Finally, all selected best models were combined and quality-assured through MolProbity and manual checks. The results are high-resolution 3D structures of the target CDA sequence and the nanobody pair, as well as the identification of their amino acid interactions and relationships.

[0365] Example 1 Insecticidal activity of anti-CDA nanobodies Several nanobodies were generated against the fall armyworm CDA (see Tables 1-2 above). The insecticidal activity of the generated nanobodies was evaluated in cotton leaf disc bioassay, liquid bioassay, and pepper plant bioassay (where the nanobodies were applied prior to insect introduction) using three different insects: fall armyworm, whitefly, and peach aphid.

[0366] like Figure 1-6 As shown, in all assays performed, the anti-CDA nanobodies CDA-SF-1D (SEQ ID NO:14), CDA-SF-2C (SEQ ID NO:82), and CDA-SF-3B (SEQ ID NO:94) induced significant mortality. Although the observed effects were clearly dose-dependent, a mortality rate exceeding 80% was observed at a dose of 1 mg / ml.

[0367] Example 2 Epitope mapping of anti-CDA-SF-1D nanobody As detailed above, computational modeling tools were used to identify the amino acids in the fall armyworm CDA (SEQ ID NO:1) that interact with the anti-CDA nanobody CDA-SF-1D (SEQ ID NO:14). This analysis showed that the nanobody CDA-SF-1D interacts with the following amino acids: ASP(D)170, ASP(D)175, GLU(E)360, ASP(D)361, ASP(D)365, ASP(D)384, TYR(Y)387, ASN(N)388, ASN(N)391, HIS(H)392, ASP(D)395, TYR(Y)398, GLU(E)399. 9. GLU(E)419, GLU(E)422, GLU(E)430, SER(S)433 and SER(S)434, see Table 3 above. More specifically, the CRD3 region of CDA-SF-1D binds the following 7 amino acids: GLU(E)360, ASP(D)361, ASP(D)365, ASP(D)384, HIS(H)392, GLU(E)419 and GLU(E)422.

[0368] Chitin deacetylase (CDA, EC 3.5.1.41) is a secreted metalloprotein belonging to the extracellular chitin-modifying enzyme family. It catalyzes the N-deacetylation of chitin (a β-1,4-linked N-acetylglucosamine polymer) to form chitosan (a polymer of β-(1,4)-linked d-glucosamine residues). CDA has been isolated and characterized from a variety of insect, bacterial, and fungal species and belongs to the large carbohydrate esterase 4 (CE4) superfamily. Analysis of the CDA sequence in NCBI using BLAST revealed that amino acid residues SER(S)345 to PRO(P)453 contain the catalytic site of CE4_CDA_like_1, which was identified as the putative catalytic domain of a chitin deacetylase-like protein with additional chitin-binding peritrophic membrane protein-A domain (ChBD) and / or low-density lipoprotein receptor class A domain (LDLa). Since CDA-SF-1D binds to 16 amino acids in this catalytic region, it can be hypothesized that it blocks the enzyme's catalytic region.

[0369] Example 3 Epitope mapping of anti-CDA-SF-2C nanobody As described above, computational modeling tools were used to identify the amino acids in the fall armyworm CDA (SEQ ID NO:1) that interact with the anti-CDA nanobody CDA-SF-2C (SEQ ID ID:82). This analysis showed that the nanobody CDA-SF-2C interacts with the following amino acids: VAL(V)22, GLU(E)23, LEU(L)24, CYS(C)25, ASP(D)46, IIE(I)48, IIE(I)68, GLU(E)99, PRO(P)100, LEU(L)101, CYS(C)102, ARG(R)286, AAL(A)287, GLY(G)293, GLY(G)294, ASN(N)295, ASN(N)296, GLN(Q)297, MET(M)301, GLU(E)302, GLU(E)297. 303, GLN (Q) 304, LEU (L) 307, TYR (Y) 308, ASP (D) 309, SER (S) 310, THR (T) 311, IIE (I) 312, PRO (P) 319, PRO (P) 320, PRO (P) 323, TYR (Y) 324, TYR (T) 325, TYR (Y) 327, PHE (F) 328, HIS (H) 332, ARG (R) 333, CYS (C) 334, HIS (H) 335, GLY (G) 336, ASN (N) 337, LEU (L) 338, GLN (Q) 339, SER ( S) 340, CYS (C) 341, PRO (P) 342, THR (T) 343, ARG (R) 344, TRP (W) 349, GLU (E) 350, MET (M) 351, VAL (V) 352, ASN (N) 354, GLU (E) 355, ASP (D) 35 7. ARG (R) 358, GLU (E) 360, PRO (P) 362, ASN (N) 364, GLU (E) 366, MET (M) 373, VAL (V) 374, ASP (D) 375, ASN (N) 391, HIS (H) 392, ASP (D) 395, TY R(Y)398, GLU(E)399, AAL(A)403, PRO(P)404, LEU(L)405, GLY(G)406, ARG(R)415, SER(S)434, MET(M)443, TYR(T)444, GLN(Q)445, VAL(V) 446, IIE (I) 447, GLN (Q) 448, PRO (P) 475, CYS (C) 477, TRP (W) 478, VAL (V) 479, PRO (P) 480, SER (S) 482, CYS (C) 483, LYS (K) 484, LEU (L) 485,TYR(T) 499, CYS(C) 500, VAL(V) 501, ARG(R) 502, PRO(P) 508, LEU(L) 510, ASN(N) 511, ASP(D) 512, ASP(D) 516, GLY(G) 517, HIS(H) 518, and TYR(Y) 519, see Table 3 above. More specifically, the CRD3 region of CDA-SF-2C binds to the following three amino acids: GLU(E) 350, MET(M) 351, and VAL(V) 352.

[0370] BLAST analysis of the CDA sequence in NCBI revealed that amino acid residues CYS(C)25 to CYS(C)82 include the CBM_14 chitin-binding perifeeding membrane protein-A domain, referred to as the perifeeding membrane protein-A domain, found in chitin-binding proteins, particularly in insect perifeeding matrix proteins. Furthermore, amino acid residues SER(S)345 to PRO(P)453 include the catalytic site CE4_CDA_like_1, which has been identified as the putative catalytic domain of a chitin deacetylase-like protein containing an additional chitin-binding perifeeding membrane protein-A domain (ChBD) and / or a low-density lipoprotein receptor class A domain (LDLa). Since CDA-SF-2C binds to 4 amino acids in the chitin-binding site region and 32 amino acids in the catalytic region, it can be hypothesized that it blocks the enzyme's chitin-binding site and / or catalytic region.

[0371] Example 4 Insecticidal activity of anti-CDA nanobodies The insecticidal activity of the anti-CDA nanobody CDA-SF-2C (SEQ ID NO:82) was evaluated in greenhouse plant bioassays of pepper and cotton using three different insects: peach aphid, tobacco whitefly, and fall armyworm (where the nanobody was applied after insect introduction).

[0372] like Figure 7-9 As shown, the anti-CDA nanobody CDA-SF-2C (SEQ ID NO:82) induced significant mortality in all assays. Although the observed effects were clearly dose-dependent, mortality was observed at a dose of 1 mg / ml.

[0373] Although the invention has been described in conjunction with specific embodiments thereof, it will be apparent to those skilled in the art that many alternatives, modifications, and variations will be apparent. Therefore, the invention is intended to cover all such alternatives, modifications, and variations falling within the spirit and broad scope of the appended claims.

[0374] The applicant intends that all publications, patents, and patent applications mentioned in this specification be incorporated herein by reference in their entirety, just as each individual publication, patent, or patent application is specifically and separately indicated to be incorporated herein by reference. Furthermore, any reference or identification of any reference in this application should not be construed as an admission that such reference is prior art to the invention. The use of section headings should not be construed as an inherent limitation. In addition, the entire contents of any priority document of this application are incorporated herein by reference.

Claims

1. A nanobody that specifically binds to a nonlinear epitope of insect CDA, wherein the nonlinear epitope is located in a catalytic and / or chitin-binding domain of the insect CDA, and wherein the binding of the nanobody to the insect CDA endows the nanobody with insect control activity.

2. A nanobody that specifically binds to a nonlinear epitope of insect CDA, wherein the nonlinear epitope comprises amino acid residues selected from the group consisting of D170, D175, E360, D361, D365, D384, Y387, N388, N391, H392, D395, Y398, E399, E419, E422, E430, S433, and S434 of SEQ ID NO:1, or corresponding amino acid residues in orthologous polypeptides, wherein the binding of the nanobody to the insect CDA confers insect control activity upon the nanobody.

3. The nanobody according to claim 2, wherein the nonlinear epitope of the insect CDA comprises all of the amino acid residues.

4. The nanobody according to claim 2, wherein the amino acid residues are selected from the group consisting of E360, D361, D365, D384, Y387, N388, N391, H392, D395, Y398, E399, E419, E422, E430, S433 and S434 of SEQ ID NO:1, or the corresponding amino acid residues in the orthologous polypeptide.

5. The nanobody according to claim 2, wherein the nonlinear epitope of the insect CDA comprises the amino acid residues E360, D361, D365, D384, Y387, N388, N391, H392, D395, Y398, E399, E419, E422, E430, S433 and S434 of SEQ ID NO:1, or the corresponding amino acid residues in the orthologous polypeptide.

6. The nanobody according to claim 2, wherein the nonlinear epitope of the insect CDA comprises the amino acid residues E360, D361, D365, D384, H392, E419 and E422 of SEQ ID NO:1 or the corresponding amino acid residues in the orthologous polypeptide.

7. A nanobody that specifically binds to a nonlinear epitope of insect CDA, said nonlinear epitope comprising V22, E23, L24, C25, D46, I48, I68, E99, P100, L101, C102, R286, A287, G293, G294, N295, N296, Q297, M301, E302, E303, Q304, L307, Y308, D309, S310, T311, I312, and P31 selected from SEQ ID NO:

1.

9. P320, P323, Y324, T325, Y327, F328, H332, R333, C334, H335, G336, N337, L338, Q33 9. S340, C341, P342, T343, R344, W349, E350, M351, V352, N354, E355, D357, R358, E360 , P362, N364, E366, M373, V374, D375, N391, H392, D395, Y398, E399, A403, P404, L405 , G406, R415, S434, M443, T444, Q445, V446, I447, Q448, P475, C477, W478, V479, P480, The nanobody comprises the amino acid residues of the group consisting of S482, C483, K484, L485, T499, C500, V501, R502, P508, L510, N511, D512, D516, G517, H518 and Y519, or the corresponding amino acid residues in orthologous polypeptides, wherein the binding of the nanobody to the insect CDA endows the nanobody with insect control activity.

8. The nanobody of claim 7, wherein the nonlinear epitope of the insect CDA comprises all of the amino acid residues.

9. The nanobody according to claim 7, wherein the amino acid residues are selected from the group consisting of W349, E350, M351, V352, N354, E355, D357, R358, E360, P362, N364, E366, M373, V374, D375, N391, H392, D395, Y398, E399, A403, P404, L405, G406, R415, S434, M443, T444, Q445, V446, I447, and Q448 of SEQ ID NO:1, or the corresponding amino acid residues in the orthologous polypeptide.

10. The nanobody of claim 7, wherein the nonlinear epitope of the insect CDA comprises the amino acid residues W349, E350, M351, V352, N354, E355, D357, R358, E360, P362, N364, E366, M373, V374, D375, N391, H392, D395, Y398, E399, A403, P404, L405, G406, R415, S434, M443, T444, Q445, V446, I447, and Q448 of the SEQ ID NO:1, or the corresponding amino acid residues in the orthologous polypeptide.

11. The nanobody according to claim 7, wherein the amino acid residues are selected from the group consisting of C25, D46, I48, and I68 of SEQ ID NO:1 or the corresponding amino acid residues in the orthologous polypeptide.

12. The nanobody according to claim 2, wherein the nonlinear epitope of the insect CDA comprises the amino acid residues C25, D46, I48, I68 of SEQ ID NO:1 or the corresponding amino acid residues in the orthologous polypeptide.

13. The nanobody of claim 7, wherein the nonlinear epitope of the insect CDA comprises the amino acid residues E350, M351 and V352 of SEQ ID NO:1 or the corresponding amino acid residues in the orthologous polypeptide.

14. A nanobody that specifically binds to insect CDA, said nanobody comprising SEQ ID NO: 3-5, 7-9, 11-13, 15-17, 19-21, 23-25, 27-29, 31-33, 35-37, 39-41, 43-45, 47-49, 51-53, 55-57, 59-61, 63-65, 67-69, 71-73, 75-77, 79-81, 83-85, 87-89, 91-93, 95-97, 99-101, 103-105, 107-109, 111-113, The nanobody contains three complementary determinant regions (CDRs) listed in 115-117, 119-121, 123-125, 127-129, 131-133, 135-137, 139-141, 143-145, 147-149, 151-153, 155-157, or 159-161, arranged in order from N to C, wherein the binding of the nanobody to the insect CDA confers insect control activity upon the nanobody.

15. The nanobody of claim 14, wherein the nanobody comprises three CDRs listed in SEQ ID NO:15-17, 83-85 or 95-97, the three CDRs being arranged on the nanobody in order from N to C.

16. The nanobody according to any one of claims 1-6 and 14, wherein the nanobody comprises three CDRs listed in SEQ ID NO:15-17, the three CDRs being arranged on the nanobody in order from N to C.

17. The nanobody according to any one of claims 7-13 and 14, wherein the nanobody comprises three CDRs listed in SEQ ID NO:83-85, the three CDRs being arranged on the nanobody in order from N to C.

18. The nanobody according to any one of claims 1-17, further comprising a heterologous toxin portion having insect control activity.

19. The nanobody according to any one of claims 1-18, wherein the nanobody downregulates the activity of the insect CDA.

20. A polynucleotide encoding a nanobody according to any one of claims 1-19.

21. A nucleic acid construct comprising the polynucleotide of claim 20 and a cis-regulatory element for directing the expression of the polynucleotide.

22. A host cell comprising a nanobody or a polynucleotide or nucleic acid construct encoding the nanobody according to any one of claims 1-19.

23. A method for producing insect control nanobodies, the method comprising culturing cells according to claim 22.

24. The method of claim 23, further comprising isolating the nanobody.

25. An insect control method, the method comprising contacting the insect with a nanobody according to any one of claims 1-19, a polynucleotide or nucleic acid construct encoding the nanobody, or a host cell expressing the nanobody.

26. The method of claim 25, wherein the contact comprises applying the nanobody directly to the insect.

27. The method of claim 25, wherein the contact comprises applying the nanobody to an organism or surface that may come into contact with the insect.

28. The nanobody according to any one of claims 1-19 or the method according to any one of claims 25-27, wherein the nanobody is formulated for delivery by spraying, irrigation and / or fumigation.

29. The nanobody or method according to any one of claims 1-19 and 25-28, wherein the nanobody is formulated as a liquid preparation.

30. The nanobody or method according to any one of claims 1-19 and 25-28, wherein the nanobody is formulated as a dry preparation.

31. A plant comprising a nanobody or a polynucleotide or nucleic acid construct encoding the nanobody according to any one of claims 1-19.

32. The plant according to claim 31, wherein it is a transgenic plant.

33. A commercial product comprising nanobodies according to any one of claims 1-19.

34. The product according to claim 33, wherein the product is produced from the plant according to any one of claims 31-32.

35. The nanobody, polynucleotide, nucleic acid construct, host cell, plant, product or method according to any one of claims 1-34, wherein the insect is selected from the group consisting of moths, whiteflies, aphids, stink bugs, leafhoppers, beetles and bees.

36. The nanobody, polynucleotide, nucleic acid construct, host cell, plant, product or method according to any one of claims 1-34, wherein the insect is a moth.

37. The nanobody, polynucleotide, nucleic acid construct, host cell, plant, product or method according to any one of claims 35-36, wherein the moth is the fall armyworm.

38. The nanobody, polynucleotide, nucleic acid construct, host cell, plant, product or method according to any one of claims 1-34, wherein the insect is a whitefly.

39. The nanobody, polynucleotide, nucleic acid construct, host cell, plant, product or method according to any one of claims 35 and 38, wherein the whitefly is a tobacco whitefly.

40. The nanobody, polynucleotide, nucleic acid construct, host cell, plant, product or method according to any one of claims 1-34, wherein the insect is an aphid.

41. The nanobody, polynucleotide, nucleic acid construct, host cell, plant, product or method according to any one of claims 35 and 40, wherein the aphid is the peach aphid.

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