Anti-insect hsp70 nanobodies and uses thereof

Abstract

Anti-insect HSP70 nanobodies are provided. Accordingly, a nanobody is provided that specifically binds to insect HSP70, wherein binding of the nanobody to insect HSP70 confers insect control activity to the nanobody. Also provided are polynucleotides encoding the nanobodies, host cells expressing the nanobodies, and methods of using the same.

Description

[0001] Related applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 623,275, filed January 21, 2024, the entire contents of which are incorporated herein by reference.

[0002] Sequence list declaration An XML file titled 101893.xml, created on January 14, 2025 and filed concurrently with this application (comprising 212,062 bytes), is incorporated herein by reference. Technical Field

[0003] In some embodiments of the present invention, therein relates to anti-insect HSP70 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 publications WO2014191146 and WO2021 / 095031).

[0006] Heat shock protein 70 (HSP70) is a molecular chaperone expressed in a variety of organisms, from bacteria to humans, and plays a crucial role in cellular homeostasis and stress response. This chaperone protein contributes to the proper folding of other proteins, protein translocation, and multimeric complex translocation, helping to maintain cellular homeostasis under adverse conditions. Insect HSP70 is significantly induced under various stresses, including extreme temperatures, pathogen infection, and environmental toxins. It has been shown that large amounts of HSP70 are synthesized under high-temperature stress to protect insects from heat damage. Furthermore, insect HSP70 has been shown to be associated with cellular protection against pesticides and other exogenous stresses. [Bai et al. (2021) International Journal of Biological Macromolecules: Part A, 193, 933-940; Wolfe et al. (1998) Journal of Insect Physiology, 44, 597-603; Liu F et al. (2008) Pestic. Biochem. Physiol., 91: 45-52]. Furthermore, it was shown that knocking out HSP70 via RNA interference led to increased susceptibility of wingless adult peach aphids to pyrethroids [Dong et al. (2022) Pestic Biochem Physiol Feb 181]; and increased mortality of whiteflies [Vyas M et al. (2017) PLOS ONE 12(1): e0168921].

[0007] Other background technologies include: International patent application publication numbers WO2023056361 and WO2021195557; and Chinese patent application publication number CN116355090. Summary of the Invention

[0008] According to one aspect of some embodiments of the present invention, a nanobody that specifically binds to insect HSP70 is provided, wherein the binding of the nanobody to insect HSP70 endows the nanobody with insect control activity.

[0009] According to some embodiments of the present invention, nanobodies further include a heterologous toxin portion having insect control activity.

[0010] According to some embodiments of the present invention, nanobodies downregulate the activity of insect HSP70.

[0011] According to some embodiments of the present invention, the nanobody comprises 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, 111-1 The three complementarity-determining regions (CDRs) listed in 13, 115-117, 119-121, 123-125, 127-129, 131-133, 135-137, 139-141, 143-145, 147-149, 151-153, 155-157, 159-161, 163-165, 167-169, 171-173, or 175-177 are arranged on the nanobody in order from N to C.

[0012] According to some embodiments of the present invention, the nanobody includes three complementarity-determining regions (CDRs) listed in SEQ ID NO:15-17, 31-33, 55-57 or 59-61, which are arranged on the nanobody in order from N to C.

[0013] According to some embodiments of the present invention, the nanobody includes three complementarity-determining regions (CDRs) listed in SEQ ID NO:15-17, which are arranged on the nanobody in order from N to C.

[0014] According to one aspect of some embodiments of the present invention, a polynucleotide encoding a nanobody is provided.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] According to some embodiments of the present invention, the method includes isolating nanobodies.

[0019] 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.

[0020] According to some embodiments of the present invention, contact includes applying nanobodies directly to insects.

[0021] According to some embodiments of the invention, contact includes applying nanobodies to an organism or surface that may come into contact with insects.

[0022] According to some embodiments of the present invention, nanobodies are formulated for delivery by spraying, irrigation, and / or fumigation.

[0023] According to some embodiments of the present invention, nanobodies are formulated as liquid preparations.

[0024] According to some embodiments of the present invention, nanobodies are formulated as dry preparations.

[0025] 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.

[0026] According to some embodiments of the present invention, the plant is a genetically modified plant.

[0027] According to one aspect of some embodiments of the present invention, a commercial product comprising nanobodies is provided.

[0028] According to some embodiments of the present invention, the commodity product is produced from plants.

[0029] According to one aspect of some embodiments of the present invention, a method for producing nanobodies is provided, the method comprising immunizing a camelid with recombinant or purified insect HSP70, wherein the purity of the insect HSP70 in the insect polypeptide preparation is at least 80%.

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

[0031] According to some embodiments of the present invention, the camel is the alpaca.

[0032] According to some embodiments of the present invention, the insects are selected from the group consisting of whiteflies, aphids, moths, stink bugs, leafhoppers, beetles and bees.

[0033] According to some embodiments of the present invention, the insect is a whitefly.

[0034] According to some embodiments of the present invention, the whitefly is the tobacco whitefly.

[0035] According to some embodiments of the present invention, the insect is an aphid.

[0036] According to some embodiments of the present invention, the aphid is the peach aphid.

[0037] According to some embodiments of the present invention, the insect is a moth.

[0038] According to some embodiments of the present invention, the moth is the fall armyworm (Spodoptera frugiperda).

[0039] 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

[0040] 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 illustrately discuss 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 embodiments of the invention.

[0041] In the attached diagram: Figure 1This figure illustrates the effect of anti-HSP70 nanobodies HSP70-BT-1D (SEQ ID NO:14), HSP70-BT-1H (SEQ ID NO:30), HSP70-BT-2A (SEQ ID NO:54), and HSP-BT-2B (SEQ ID NO:58) on the mortality rate of peach aphids in a liquid bioassay after feeding with nanobodies at a concentration of 1 mg / ml. Each sample included 10 wingless nymphal aphids, with 3 replicates per treatment; mortality was assessed after 48 hours of incubation. Data are expressed as mean ± standard error (SE).

[0042] Figure 2 This is a dose-response graph showing the effect of anti-HSP70 nanobody HSP70-BT-1D (SEQ ID NO:14) on whitefly mortality in a liquid bioassay after feeding with nanobody at concentrations between 0.05 and 4 mg / ml. Each sample included 50 adult aphids, with 3 replicates per treatment; and mortality was assessed after 48 hours of incubation.

[0043] Figure 3 This figure shows the effect of 1 ml of 2 mg / ml anti-HSP70 nanobody HSP70-BT-1D (SEQ ID NO:14) on the mortality rate of peach aphids in a pepper leaf disc bioassay, compared with the PBS control. Fifteen N2-N3 nymphs and adults were transferred into each cup, with four replicates per treatment; mortality was assessed 4 days after treatment. Data are expressed as mean ± standard error.

[0044] Figure 4 This figure shows the effect of 0.5 ml of 3 mg / ml anti-HSP70 nanobody HSP70-BT-1D (SEQ ID NO:14) on the mortality rate of whiteflies in detached pepper leaf disc assays compared to the PBS control. Each sample included 50 whiteflies, and each treatment was performed in quadruplicate; mortality was assessed 4 days after treatment. Data are expressed as mean ± standard error.

[0045] Figure 5 This figure illustrates the effect of the anti-HSP70 nanobody HSP70-BT-1D (SEQ ID NO:14) on the mortality rate of peach aphids in a greenhouse plant bioassay for chili peppers, using nanobodies at concentrations of 1 mg / ml or 3 mg / ml (0.8 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.

[0046] Figure 6 This study demonstrates the effect of the anti-HSP70 nanobody HSP70-BT-1D (SEQ ID NO:14) on whitefly mortality in a greenhouse plant bioassay for chili peppers, using 0.8 ml of nanobody at final concentrations of 1 mg / ml or 3 mg / ml, sprayed twice on days 0 and 6. 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.

[0047] Figure 7 This figure illustrates the effect of the anti-HSP70 nanobody HSP70-BT-1D (SEQ ID NO:14) on the mortality rate of fall armyworm in a cotton greenhouse plant bioassay, 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 day 1 and day 5 after nanobody treatment. Data are expressed as mean ± standard error, ****-p≤0.001. Detailed Implementation

[0048] In some embodiments of the present invention, therein relates to anti-insect HSP70 nanobodies and their uses.

[0049] 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.

[0050] 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.

[0051] While practicing specific embodiments of the present invention, the inventors have now developed nanobodies with insect control activity targeting insect HSP70 (Examples 1-2, 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.

[0052] Therefore, according to one aspect of the present invention, a nanobody that specifically binds to insect HSP70 is provided, wherein the binding of the nanobody to the insect HSP70 endows the nanobody with insect control activity.

[0053] According to another or alternative aspect of the invention, a composition is provided comprising a nanobody that specifically binds to insect HSP70 and a toxin portion having insect control activity.

[0054] As used herein, the term "nanobody" refers to a single-domain antigen-binding fragment.

[0055] 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.

[0056] NANOBODY® and NANOBODIES® are registered trademarks of Ablynx NV (Belgium).

[0057] 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.

[0058] 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.

[0059] As used herein, the term "epitope" 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.

[0060] Epitopes can be linear or non-linear.

[0061] According to a specific implementation, the epitope is located within the catalytic and / or binding domain of HSP70.

[0062] According to a specific implementation, the epitope is located within the ATPase domain of HSP70.

[0063] According to a specific implementation, the epitope is located within the substrate binding region of the HSP70.

[0064] Depending on the specific implementation method, the nanobody is a monolithic or complete nanobody.

[0065] According to the specific implementation method, a nanobody is a nanobody fragment.

[0066] Depending on the specific implementation method, the size of the nanobody is 5-30kDa, 10-30kDa, or 10-20kDa.

[0067] According to the specific implementation method, the size of the nanobody is approximately 15 kDa.

[0068] The term "nanobody" also includes natural or synthetic analogs, homologs, mutants, and variants of nanobodies.

[0069] Typically, a complete nanobody consists of three complementarity-determining regions (CDRs) (CDR1, CDR2, and CDR3).

[0070] 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 structural ring regions as 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).

[0071] As used herein, “CDR” may refer to a CDR defined by any method known in the art, including combinations of methods.

[0072] 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.

[0073] 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).

[0074] According to the specific implementation method, the nanobody is a single-specific nanobody.

[0075] Depending on the specific implementation method, nanobodies are multispecific, such as bispecific, trispecific, or quadrispecific.

[0076] 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.

[0077] This article discloses a nanobody that specifically binds to insect HSP70.

[0078] Preferably, the nanobody specifically binds to at least one epitope of insect HSP70.

[0079] 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.

[0080] According to specific implementation methods, nanobodies with Kd≤10 -6 M, ≤10 -7 M, ≤10 -8 M, ≤10 -9 M combines with insect HSP70, each possibility representing a separate embodiment of the invention.

[0081] According to specific embodiments, nanobodies can bind to insect HSP70 under open or controlled conditions (e.g., greenhouse).

[0082] 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, Hemiptera, Archaeognatha, Thysanura, Paleoptera, and Neoptera, and also ticks, mites, and spiders), or more preferably, the class Insecta.

[0083] According to the specific implementation method, the insect belongs to the order Hemiptera.

[0084] Non-limiting examples of insects include aphids, whiteflies, bedbugs, houseflies, moths, beetles, grasshoppers, caterpillars, mosquitoes, fleas, horseflies, wasps, cockroaches, and ants, such as, but not limited to: • From 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.

[0085] • From the suborder Thyracoidea 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*, the tobacco whitefly, the genus *Ceroplastes*, and the red round scale (Chrysomphalus). *Aonidium*, *Chrysomphalus dictyospermi*, *Coccushesperidum*, *Daktulosphaira vitifoliae*, *Diaphorina citri*, *Eriosoma larigerum*, *Gascardia* spp., *Lacaniumcorni*, *Lepidosaphes* spp., *Lopholeucaspis japonica*, *Macrosiphus* spp., *Margarodes prieskaensis*, *Margarodes vitis*, *Margarodes vredendalensis*, *Myzuspersicae*, *Myzus* spp.), Parasaissetia nigra, Pemphigus spp., Phylloxera spp., Planococcus spp., Pseudaulacaspis spp., Pseudococcus spp., Psylla spp., Pulvinaria aethiopica, Quadraspidiotus spp.The genera *Rhopalosiphum* spp., *Ripersiella hibisci*, *Saissetia* spp., *Schizaphis* spp., *Sitobion* spp., *Toxopteracitricida*, *Trialeurodes vaporariorum*, *Trioza erytreae*, and *Unaspis citri* are mentioned. • 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).

[0086] 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.

[0087] According to the specific implementation method, the insects were selected from the group consisting of whiteflies, aphids, moths, stink bugs, leafhoppers, beetles, and bees.

[0088] According to the specific implementation method, the insect is a moth.

[0089] 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).

[0090] According to the specific implementation method, the moth is the fall armyworm.

[0091] Depending on the specific implementation method, the insect is a sucking pest.

[0092] Non-limiting examples of sucking pests include whiteflies, aphids, stink bugs, and leafhoppers.

[0093] According to the specific implementation method, the insect is a whitefly.

[0094] 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).

[0095] According to the specific implementation method, the whitefly is the tobacco whitefly.

[0096] 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).

[0097] According to the specific implementation method, the insect is an aphid.

[0098] Non-limiting examples of aphids include the peach aphid, cotton aphid, broad bean aphid, citrus aphid (Toxoptera citricida), wheat aphid (Schizaphis graminum), eggplant aphid (Aulacorthum solani), and rice constrictor aphid (Rhopalosiphum padi).

[0099] According to the specific implementation method, the aphid is the peach aphid.

[0100] As used herein, the term "specifically bind" refers to the ability of a nanobody to bind insect HSP70 with a higher affinity in a physiological environment (e.g., in an insect under physiological conditions) compared to other peptides in the environment.

[0101] According to the specific implementation method, the nanobody binds to insect HSP70 and does not cross-react with non-insect peptides (such as plant or human peptides).

[0102] According to the specific implementation method, the nanobody binds to insect HSP70 and does not cross-react with non-insect HSP70.

[0103] According to the specific implementation method, the nanobody specifically binds to insect HSP70 and does not cross-react with other insect peptides.

[0104] According to a specific embodiment, the nanobody binds to the HSP70 of the whitefly (Bemisia tabaci) with higher affinity than the HSP70 sequence, wherein the HSP70 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.

[0105] 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.

[0106] According to the specific implementation method, the nanobody specifically binds to the HSP70 of the whitefly and does not cross-react with the HSP70 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.

[0107] As used herein, the term "insect HSP70," also known as "heat shock 70 kDa protein," refers to the polypeptide encoded by the HSP70 gene in insects. HSP70 (pfam PF00012) consists of two regions: an N-terminal ATPase domain and a C-terminal substrate-binding domain.

[0108] According to the specific implementation method, HSP70 is insect HSP70.

[0109] According to specific embodiments, the insect HSP70 is the whitefly HSP70, such as those provided in GenBank accession numbers ADO14473, ACH85197, AAZ17399, QHB15581.

[0110] According to a specific implementation, the insect HSP70 includes SEQ ID NO:1.

[0111] According to a specific implementation, the insect HSP70 consists of SEQ ID NO:1.

[0112] Table 2 below shows non-limiting examples of nanobodies that specifically bind to insect HSP70 and their respective CDRs.

[0113] According to a specific embodiment, the nanobody specifically binds to insect HSP70 and includes three CDRs arranged in order from N to C on the nanobody, the amino acid sequence of which corresponds to 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, 119-121, 123-125, 12 The amino acid sequences listed in 7-129, 131-133, 135-137, 139-141, 143-145, 147-149, 151-153, 155-157, 159-161, 163-165, 167-169, 171-173, or 175-177 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.

[0114] According to a specific embodiment, the nanobody specifically binds to insect HSP70 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, 31-33, 55-57, or 59-61.

[0115] According to a specific embodiment, the nanobody specifically binds to insect HSP70 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.

[0116] According to a specific embodiment, the nanobody specifically binds to insect HSP70 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, 103-105, 107-109. The three CDRs listed in 111-113, 115-117, 119-121, 123-125, 127-129, 131-133, 135-137, 139-141, 143-145, 147-149, 151-153, 155-157, 159-161, 163-165, 167-169, 171-173, or 175-177 each represent a single embodiment of the invention.

[0117] According to a specific embodiment, the nanobody specifically binds to insect HSP70 and includes three CDRs listed in SEQ ID NO:15-17, 31-33, 55-57 or 59-61, which are arranged on the nanobody in order from N to C.

[0118] According to a specific embodiment, the nanobody specifically binds to insect HSP70 and includes three CDRs listed in SEQ ID NO:15-17, which are arranged on the nanobody in order from N to C.

[0119] 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, 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, 146, 150, 154, 158, 162, 166, 170, and 174, each possibility representing a separate embodiment of the invention.

[0120] According to a specific embodiment, 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: 14, 30, 54, and 58.

[0121] 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.

[0122] 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, 146, 150, 154, 158, 162, 166, 170, and 174.

[0123] According to a specific embodiment, the nanobody comprises an amino acid sequence selected from the group consisting of SEQ ID NO:14, 30, 54 and 58.

[0124] According to a specific implementation, the nanobody includes SEQ ID NO:14.

[0125] 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, 146, 150, 154, 158, 162, 166, 170, and 174.

[0126] According to a specific embodiment, the nanobody is composed of an amino acid sequence selected from the group consisting of SEQ ID NO: 14, 30, 54 and 58.

[0127] According to a specific implementation, the nanobody is composed of SEQ ID NO:14.

[0128] 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.

[0129] 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.

[0130] 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.

[0131] This article describes a specific method for producing nanobodies.

[0132] 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 HSP70, wherein the insect HSP70 has a purity of at least 80% in the insect polypeptide formulation.

[0133] As used herein, the phrase “purified insect HSP70” refers to an HSP70 polypeptide purified from insects such that its purity is at least 80% compared to other polypeptides present in protein formulations.

[0134] According to specific embodiments, the purity of insect HSP70 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.

[0135] According to a specific implementation, the method also includes separating nanobodies.

[0136] 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.

[0137] Once nanobodies are obtained, their binding and / or biological activity (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.

[0138] Therefore, according to a specific embodiment, the method further includes selecting nanobodies that exhibit insect control activity in bioassays.

[0139] Such determinations are known in the art and are described further below.

[0140] According to the specific implementation method, the nanobody is a naked nanobody.

[0141] 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).

[0142] 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.

[0143] 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).

[0144] Depending on the specific implementation, nanobodies include toxins.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] 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.

[0151] Exemplary methods for conjugating peptides to nanobodies include, but are not limited to, SPDP conjugation, glutaraldehyde conjugation, and carbodiimide conjugation.

[0152] 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.

[0153] 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.

[0154] 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.

[0155] 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).

[0156] 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.

[0157] According to specific implementation methods, the nucleic acid sequences disclosed herein are optimized for codons expressed, for example, in mammals or plants.

[0158] 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.

[0159] 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.

[0160] Non-limiting examples of polynucleotides encoding nanobodies of some embodiments of the present invention are provided in SEQ ID NO:178-221.

[0161] 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.

[0162] 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 the polynucleotide.

[0163] According to the specific implementation method, the control element is a heterogeneous control element.

[0164] 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.

[0165] 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.

[0166] 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.

[0167] 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).

[0168] 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].

[0169] 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.

[0170] 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.

[0171] 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.

[0172] 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.

[0173] 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.

[0174] 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.

[0175] 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.

[0176] 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.

[0177] 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.

[0178] 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.

[0179] 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.

[0180] 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.

[0181] 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.

[0182] 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.

[0183] 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.

[0184] 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].

[0185] The present invention also considers cells including the polypeptides, polynucleotides and nucleic acid constructs described herein.

[0186] 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.

[0187] 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.

[0188] According to the specific implementation method, the cell is a mammalian cell.

[0189] According to the specific implementation method, the cells are camel cells.

[0190] Suitable mammalian cells include primary cells and immortalized cell lines.

[0191] According to other specific embodiments, mammalian cells are immortalized cell lines.

[0192] 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.

[0193] According to the specific implementation, the cells are Escherichia coli, such as SHuffle T7 Express and BL21.

[0194] According to the specific implementation method, the cell is a plant cell.

[0195] 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.

[0196] 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.

[0197] 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.

[0198] 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.

[0199] 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.

[0200] Depending on the specific implementation method, expression and / or introduction may be performed in vitro or ex vivo.

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

[0202] 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.

[0203] 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.

[0204] 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.

[0205] 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.

[0206] According to a specific embodiment, the composition is formulated as a liquid preparation.

[0207] According to a specific embodiment, the composition is formulated as a dry preparation.

[0208] According to a specific embodiment, the composition is formulated for delivery by spraying, irrigation, and / or fumigation.

[0209] 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.

[0210] 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.

[0211] According to specific embodiments, the integrity and activity of the composition are maintained under open or controlled conditions (e.g., greenhouse).

[0212] It should be noted that the compositions disclosed herein can be formulated with various carriers designed to enhance, for example, delivery, stability, permeability, etc.

[0213] 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.

[0214] 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.

[0215] 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.

[0216] 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.

[0217] 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.

[0218] 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.

[0219] 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.

[0220] 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.

[0221] 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.

[0222] 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, toxins, safeners, insecticidal nanobodies, etc.

[0223] According to specific embodiments, the compositions disclosed herein and other active agents are in the same formulation.

[0224] According to specific embodiments, the compositions disclosed herein and additional active agents are in separate containers.

[0225] 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.

[0226] Attractants can be present in the formulation or applied separately to ensure that insects are attracted to the site where the formulation is applied.

[0227] Nanobodies and compositions comprising nanobodies and toxins in some embodiments of the present invention have insect control activity.

[0228] 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.

[0229] Depending on the specific implementation method, insect control activities are carried out by killing insects.

[0230] 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.

[0231] 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.

[0232] According to the specific implementation method, the increase in mortality rate is statistically significant.

[0233] 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.

[0234] 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.

[0235] Therefore, according to specific implementation methods, the binding of nanobodies to insect HSP70 endows the nanobodies with insect control activity.

[0236] According to a specific implementation method, nanobodies downregulate the activity of insect HSP70.

[0237] As used herein, "downregulate activity" means that the biological function of an insect polypeptide (i.e., HSP70) 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 HSP70 includes both an ATPase domain and a substrate-binding domain, nanobodies of some embodiments of the present invention can bind to and downregulate the activity of the ATPase domain and / or the binding domain. Therefore, for example, the activity of HSP70 can be determined by, for example, ELISA, Western blot analysis, immunoprecipitation, flow cytometry, colorimetric or fluorescent ATPase assays, or chaperone activity assays.

[0238] 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.

[0239] According to other or alternative embodiments, nanobodies are targeting agents used to specifically deliver to insects, for example, toxins with insect control activity.

[0240] The above text further describes non-limiting examples of toxins and methods for conjugating them with nanobodies.

[0241] 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.

[0242] 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.

[0243] According to a specific embodiment, contact includes directly applying nanobodies or a composition comprising nanobodies and toxins to insects.

[0244] 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.

[0245] 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.

[0246] 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.

[0247] 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).

[0248] Depending on the specific implementation method, contact or addition may be achieved through spraying, irrigation, and / or fumigation.

[0249] According to specific implementation methods, contact or addition is achieved by introducing polynucleotide or nucleic acid constructs into the cells of an organism.

[0250] According to the specific implementation method, the organism is a plant.

[0251] 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.

[0252] According to the specific implementation method, the product is produced from plants.

[0253] 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.

[0254] 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.

[0255] 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.

[0256] 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.

[0257] According to the specific implementation, the plant is a forest plant, such as those described in www.fao.org / 3 / i0640e / i0640e13.pdf.

[0258] According to the specific implementation method, the plant is an ornamental plant.

[0259] 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.

[0260] Depending on the specific implementation method, the plant is cut grass or wood.

[0261] According to the specific implementation method, the plant is cotton.

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

[0263] According to a specific embodiment, the plant is a transgenic plant that recombinantly expresses nanobodies or a composition including nanobodies and toxins.

[0264] Methods for producing transgenic plants are well known in the art and have been described above.

[0265] 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.

[0266] 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.

[0267] 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.

[0268] According to another or alternative aspect of the invention, a commercial product is provided that comprises nanobodies or a composition comprising nanobodies and toxins.

[0269] According to another or alternative aspect of the invention, a surface covered with nanobodies or a composition comprising nanobodies and toxins is provided.

[0270] 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.

[0271] Tables 1-2 below list the HSP70 peptides and nanobodies that can be used in specific embodiments of the present invention.

[0272] Table 1: Amino acid sequence of HSP70 of the target whitefly Table 2: Amino acid sequence of anti-HSP70 nanobody As used in this article, the term “about” means ±10%.

[0273] The terms “comprise,” “comprising,” “include,” “including,” and “having” mean “including, but not limited to.”

[0274] The phrase “consisting of” means “including and limited to”.

[0275] 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.

[0276] 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.

[0277] 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.

[0278] 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.

[0279] 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.

[0280] 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.

[0281] 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.

[0282] The various embodiments and aspects of the invention described above and claimed in the claims section below are experimentally supported in the following examples.

[0283] 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.

[0284] Materials and Methods Protein target formulations for immunization- The DNA sequence encoding the target HSP70 of the whitefly 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 *E. 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.

[0285] 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.

[0286] 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.

[0287] 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.

[0288] Nanobody expression and purification- The nanobody DNA sequence was optimized and synthesized using GenScript (provided in SEQ ID NO: 178-221). 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.

[0289] Insect culture- Colonies of whiteflies and peach aphids were collected outdoors and stored separately in growth chambers under continuous light at 22±3°C. Whiteflies grow on cotton plants, and peach aphids grow on pepper plants. The whitefly communities used in this study were a mixture of B and Q biotypes, following the procedures described by De Barro et al. [Molecular Ecology Notes (2003) 3(1), 40-43]. Fall armyworm colonies were collected from cotton fields in Israel and reared in the laboratory under 24°C, 65% relative humidity, and 10 hours day / 14 hours night light conditions. 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 flaxseed oil 0.5%.

[0290] 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 an IPL41 (x1) insect culture medium (Gibco, CAS 11405-081) supplemented with 30% sucrose and 0.5% arginine, diluted 1:1 with PBS 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 the test material and placed in an acrylic tube (approximately 22 mm outer diameter × 22 mm height) along with a test population of 10 nymphs and adult aphids. The open end of the tube was sealed with paraffin film.

[0291] Bioassay of peach aphid on pepper leaf disc—1 mL of sterile co-agar was added to a plastic cup 4 cm high and 2.0 cm in diameter. After the agar cooled, a 2.0 cm diameter cotton disc leaf was placed in each cup, and then covered with 100 µl of the test nanobody suspension at a concentration of 2 mg / mL or PBS control. Next, 15 N2-N3 nymphs were transferred into each cup, and the cup was sealed with a mesh cup with 150 holes per square inch / 89 µm (referred to as the “150-mesh cup” in this paper). The cups were incubated in the laboratory at 25 °C for 2 to 5 days; and aphid mortality was measured after 4 days of incubation. Each study was performed in quadruplicate for each treatment, with at least two replicates.

[0292] Bioassay of whiteflies on detached pepper leaf discs —Extracted pepper leaves were placed in glass test tubes containing 3 ml of water. Then, 500 µl of the tested nanobody suspension was applied to the leaves. Each test tube was then placed in a 500 ml plastic container, and 60 adult aphids were added. The container was sealed with a 150-mesh screen. The containers were incubated in the laboratory at 25°C for 2 to 5 days; aphid mortality was measured after 4 days of incubation. Each study had four replicates for each treatment, with at least two repetitions.

[0293] Biological assay of pepper plant by peach aphid Pepper plants were used as hosts under climatic conditions of an average daily temperature of 25°C, 57% humidity, and natural light. When the plants reached six to eight weeks of age, they were sprayed with either the test nanobody or PBS at a concentration of 1 to 4 mg / ml as a control, at a concentration of 0.6 to 1.4 ml. The sprayed plants were then transferred to plastic insect cages (BugDorm-6E610). Twenty N2-N3 stage aphid nymphs were added to each plant and 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.

[0294] Biological assay of pepper greenhouse plants by peach aphids 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.

[0295] Bioassay of whiteflies on pepper greenhouse plantsPepper 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 BugDorm-6E610 insect cages. 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 a final concentration 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 4–6 independent replicates per treatment.

[0296] Bioassay of fall armyworm on cotton greenhouse plants 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.

[0297] Example 1 Insecticidal effect of anti-HSP70 nanobodies Several nanobodies were generated against the whitefly HSP70 (see Tables 1-2 above). The insecticidal activity of the generated nanobodies was evaluated using two different insects, the whitefly and the peach aphid, in three different bioassays: liquid bioassay, pepper leaf disc bioassay, and pepper detached leaf disc bioassay.

[0298] like Figure 1-4 As shown, in all assays performed, the anti-HSP70 nanobodies HSP70-BT-1D (SEQ ID NO:14), HSP70-BT-1H (SEQ ID NO:30), HSP70-BT-2A (SEQ ID NO:54), and HSP-BT-2B (SEQ ID NO:58) all induced significant mortality. These findings indicate that targeting insect HSP70 effectively induces significant mortality in sucking pests, making it a viable option for protecting crops from these pests.

[0299] Example 2 Insecticidal effect of anti-HSP70 nanobodies The insecticidal activity of the anti-HSP70 nanobody HSP70-BT-1D (SEQ ID NO:14) 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).

[0300] like Figure 5-7 As shown, the anti-HSP70 nanobody HSP70-BT-1D (SEQ ID NO:14) induced significant mortality in all assays. Although the observed effects were clearly dose-dependent, mortality was observed at a dose of 1 mg / ml.

[0301] 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.

[0302] 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 insect HSP70, wherein the binding of the nanobody to the insect HSP70 endows the nanobody with insect control activity.

2. The nanobody according to claim 1, further comprising a heterologous toxin portion having insect control activity.

3. The nanobody according to claim 1, wherein the nanobody downregulates the activity of the insect HSP70.

4. The nanobody according to any one of claims 1-3, wherein the nanobody comprises 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 three complementarity-determining 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, 159-161, 163-165, 167-169, 171-173, or 175-177 are arranged on the nanobody in order from N to C.

5. The nanobody according to any one of claims 1-3, wherein the nanobody comprises three complementarity-determining regions (CDRs) listed in SEQ ID NO: 15-17, 31-33, 55-57 or 59-61, the three complementarity-determining regions being arranged on the nanobody in order from N to C.

6. The nanobody according to any one of claims 1-3, wherein the nanobody comprises three complementarity-determining regions (CDRs) listed in SEQ ID NO:15-17, the three complementarity-determining regions being arranged on the nanobody in order from N to C.

7. A polynucleotide encoding a nanobody according to any one of claims 1-6.

8. A nucleic acid construct comprising the polynucleotide of claim 7 and a cis-regulatory element for guiding the expression of the polynucleotide.

9. A host cell comprising a nanobody or a polynucleotide or nucleic acid construct encoding the nanobody according to any one of claims 1-6.

10. A method for producing insect control nanobodies, the method comprising culturing cells according to claim 9.

11. The method of claim 10, further comprising isolating the nanobody.

12. An insect control method, the method comprising contacting the insect with a nanobody according to any one of claims 1-6, a polynucleotide or nucleic acid construct encoding the nanobody, or a host cell expressing the nanobody.

13. The method of claim 12, wherein the contact comprises applying the nanobody directly to the insect.

14. The method of claim 12, wherein the contact comprises applying the nanobody to an organism or surface that may come into contact with the insect.

15. The nanobody according to any one of claims 1-6 or the method according to any one of claims 12-14, wherein the nanobody is formulated for delivery by spraying, irrigation and / or fumigation.

16. The nanobody or method according to any one of claims 1-6 and 12-15, wherein the nanobody is formulated as a liquid preparation.

17. The nanobody or method according to any one of claims 1-6 and 12-15, wherein the nanobody is formulated as a dry preparation.

18. A plant comprising a nanobody or a polynucleotide or nucleic acid construct encoding the nanobody according to any one of claims 1-6.

19. The plant according to claim 18, wherein it is a transgenic plant.

20. A commercial product comprising nanobodies according to any one of claims 1-6.

21. The product according to claim 20, wherein the product is produced from the plant of any one of claims 18-19.

22. A method for producing nanobodies, the method comprising immunizing a camelid with recombinant or purified insect HSP70, wherein the purity of the insect HSP70 in the insect polypeptide preparation is at least 80%.

23. The method of claim 22, comprising separating the nanobody after immunization.

24. The method according to any one of claims 22 to 23, wherein the camel is a llama.

25. The nanobody, polynucleotide, nucleic acid construct, host cell, plant, product or method according to any one of claims 1-24, wherein the insect is selected from the group consisting of whiteflies, aphids, moths, stink bugs, leafhoppers, beetles and bees.

26. The nanobody, polynucleotide, nucleic acid construct, host cell, plant, product or method according to any one of claims 1-24, wherein the insect is a whitefly.

27. The nanobody, polynucleotide, nucleic acid construct, host cell, plant, product or method according to any one of claims 25-26, wherein the whitefly is a tobacco whitefly.

28. The nanobody, polynucleotide, nucleic acid construct, host cell, plant, product or method according to any one of claims 1-24, wherein the insect is an aphid.

29. The nanobody, polynucleotide, nucleic acid construct, host cell, plant, product or method according to any one of claims 25 and 28, wherein the aphid is the peach aphid.

30. The nanobody, polynucleotide, nucleic acid construct, host cell, plant, product or method according to any one of claims 1-24, wherein the insect is a moth.

31. The nanobody, polynucleotide, nucleic acid construct, host cell, plant, product or method according to any one of claims 25 and 28, wherein the moth is the fall armyworm.

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