Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
  • A01N63/60—Isolated nucleic acids
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
  • A01P7/00—Arthropodicides
  • A01P7/04—Insecticides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation

Definitions

• Nanobodies also known as VHH antibodies, are single domain antibodies which practically contain the heavy chain of an antibody (HCAb) and completely lack the light chain. They were discovered in the blood of camels by Raymond Hamers who was credited with this discovery in 1989 at Vrije Universiteit Brussel. Nanobodies are the smallest available intact antigen binding fragment (Cortez-Retamozo et al., 2004; Revets et al., 2005) with a size of approximately 15 kDa.
• Heat shock protein 70 is a molecular chaperone expressed in diverse organisms, from bacteria to humans, that plays a pivotal role in cellular homeostasis and stress response. This chaperone protein aids in the proper folding of other proteins, protein translocation, and multimeric complex translocation, contributing to the maintenance of cellular homeostasis under adverse conditions. Insect HSP70 is prominently induced in response to various stressors such as temperature extremes, pathogen infections, and environmental toxins. It was shown that under high-temperature stress, a large amount of HSP70 is synthesized to protect the insect from high- temperature damage. In addition, insect HSP70 was shown to be related to cellular protection against insecticide and other xenobiotic stresses.
• the nanobody downregulates activity of the insect HSP70.
• the nanobody comprises three complementarity determining regions (CDRs) as set forth in SEQ ID NOs: 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, 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
• CDRs complementarity determining regions
• the nanobody comprises three complementarity determining regions (CDRs) as set forth in SEQ ID NOs: 15-17 arranged in a sequential order from N to C on the nanobody.
• CDRs complementarity determining regions
• a polynucleotide encoding the nanobody According to an aspect of some embodiments of the present invention there is provided a polynucleotide encoding the nanobody.
• nucleic acid construct comprising the polynucleotide and a cis-acting regulatory element for directing expression of the polynucleotide.
• a host cell comprising the nanobody or a polynucleotide or a nucleic acid construct encoding it.
• a method of producing an insect control nanobody comprising culturing the cell.
• the method comprising isolating the nanobody.
• a method of insect control comprising contacting the insect with the nanobody, a polynucleotide or a nucleic acid construct encoding same or a host cell expressing same.
• the contacting comprises applying the nanobody directly to the insect.
• the nanobody is formulated for delivery by spraying, irrigation and/or fumigation. According to some embodiments of the invention, the nanobody is formulated as a liquid formulation.
• the nanobody is formulated as a dry formulation.
• a plant comprising the nanobody or a polynucleotide or a nucleic acid construct encoding same.
• the plant being a transgenic plant.
• a commodity product comprising the nanobody.
• the commodity product is produced from the plant.
• a method of producing a nanobody comprising immunizing a camelid with a recombinant or purified insect HSP70, wherein purity of the insect HSP70 in an insect polypeptide preparation is at least 80 %.
• the method comprising isolating the nanobody following the immunizing.
• the camelid is a llama.
• the insect is a whitefly.
• the whitefly is Bemisia tabaci.
• the insect is an aphid.
• the aphid is Myzus persicae.
• the insect is a moth.
• the moth is Spodoptera friigiperda.
• FIG. 1 is a graph demonstrating the effect of 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) on Myzus persicae mortality in the liquid bioassay, following feeding with each of the nanobodies in a concentration of 1 mg / ml. Each sample included 10 non winged nymph aphids with 3 repetitions for each treatment; and mortality was evaluated following 48 hours of incubation. Data is presented as average ⁇ SE.
• FIG. 2 is a dose-respone graph demonstrating the effect of the anti-HSP70 nanobody HSP70-BT-1D (SEQ ID NO: 14) on Bemisia tabaci mortality in the liquid bioassay, following feeding with the nanobody in concentrations between 0.05 to 2.4 mg / ml. Each sample included 50 adult aphids with 3 repetitions for each treatment; and mortality was evaluated following 48 hours of incubation.
• FIG. 3 is a graph demonstrating the effect of the anti-HSP70 nanobody HSP70-BT-1D (SEQ ID NO: 14) on Myzus persicae mortality, in the pepper leaf disc bioassay, using 0.1 ml nanobody at a concentration of 2 mg / ml as compared to PBS control. Transfer 15 N2-N3 nymphs and adults were added to each cup, with 4 repetitions for each treatment; and mortality was evaluated following 4 days of treatment. Data is presented as average ⁇ SE.
• FIG. 4 is a graph demonstrating the effect of the anti-HSP70 nanobody HSP70-BT-1D (SEQ ID NO: 14) on Bemisia tabaci mortality, in the pepper detached leaf disc bioassay, using 0.5 ml nanobody at a concentration of 3 mg / ml as compared to PBS control. Each sample included 50 whiteflies, with 4 repetitions for each treatment; and mortality was evaluated following 4 days of treatment. Data is presented as average ⁇ SE.
• FIG. 5 is a graph demonstrating the effect of the anti-HSP70 nanobody HSP70-BT-1D (SEQ ID NO: 14) on Myzus persicae mortality, in the pepper green-house plant bioassay, using 0.8 ml nanobody at a concentration of 1 mg / ml or 3 mg / ml.
• Each plant was inoculated with 20 nymph stage aphids prior to nanobody treatment, with 3-10 independent repetitions for each treatmemt in 3 independent studies; and mortality was evaluated on days 1 and 5 after treatment with the nanobody. Data is presented as average ⁇ SE, *** - p ⁇ 0.001.
• FIG. 6 is a graph demonstrating the effect of the anti-HSP70 nanobody HSP70-BT-1D (SEQ ID NO: 14) on Bemisia tabaci mortality, in the pepper green-house plant bioassay, using 0.8 ml nanobody at a final concentration of 1 mg / ml or 3 mg / ml sprayed twice on days 0 and 6.
• Each plant was inoculated with 100 adult aphids prior to first nanobody treatment, with 4-6 independent repetitions for each treatmemt in 2 independent studies; and mortality was evaluated on days 1, 5, 7 and 12 after first treatment with the nanobody. Data is presented as average ⁇ SE.
• FIG. 7 is a graph demonstrating the effect of the anti-HSP70 nanobody HSP70-BT-1D (SEQ ID NO: 14) on Spodoptera frugiperda mortality, in the cotton green-house plant bioassay, using 1.5 ml nanobody at a final concentration of 1 mg / ml or 3 mg / ml.
• Each plant was inoculated with 16 L2-stage larvae prior to nanobody treatment, with 3-10 independent repetitions for each treatment in 4 independent studies; and mortality was evaluated on days 1 and 5 after treatment with the nanobody. Data is presented as average ⁇ SE, *** - p ⁇ 0.001.
• the present invention in some embodiments thereof, relates to anti-insect HSP70 nanobodies and uses thereof.
• a nanobody which specifically binds to an insect HSP70, wherein binding of said nanobody to said insect HSP70 confers an insect control activity to said nanobody.
• a composition comprising a nanobody which specifically binds to an insect HSP70, and a toxin moiety having an insect control activity.
• nanobody refers to a single-domain antigen binding fragment.
• the nanobody is a single variable domain derived from naturally occurring heavy chain of an antibody.
• Nanobodies are usually derived from heavy chain only antibodies (devoid of light chains) seen in camelids (Hamers-Casterman et al., 1993, Nature 363: 446-448; Desmyter et al., 1996, Nat. Struct. Biol. 803-811) and consequently are often referred to as VHH antibody, VHH sequence or immunoglobulin single variable domain (ISVD).
• Camelids comprise old world camelids (Camelus bactrianus and Camelus dromedarius) and new world camelids (for example, Lama paccos, Lama glama, Lama guanicoe and Lama vicugna).
• Non-limiting examples of camelids include dromedary camels, Bactrian camels, wild Bactrian camels, llamas, alpacas, vicunas, and guanacos.
• the camelid is a llama.
• NANOBODY® and NANOBODIES® are registered trademarks of Ablynx NV (Belgium).
• the nanobody refers to an intact molecule (i.e. comprising 4 frameworks regions and 3 complementarity-determining regions) or a functional fragment thereof capable of binding to an epitope of the antigen to which the intact molecule binds.
• epitope refers to any antigenic determinant on an antigen to which the paratope of a nanobody binds.
• Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or carbohydrate side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics.
• the epitope is within the catalytic and/or binding domain of HSP70.
• the nanobody is a whole or intact nanobody.
• the nanobody is a nanobody fragment.
• the size of the nanobody is about 15 kDa.
• nanobody also encompasses natural or synthetic analogs, homologous, mutants and variants of a nanobody.
• CDR complementarity-determining region
• the identity of the amino acid residues in a particular nanobody that make up a CDR can be determined using methods well known in the art and include 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 D.C.), as applied to VHH domains from Camelids in the article of Riechmann and Muyldermans (1999), location of the structural loop regions as defined by Chothia et al.
• the nanobody is a mono-specific nanobody.
• the nanobodies disclosed herein specifically bind an insect HSP70.
• the nanobody specifically binds at least one epitope of an insect HSP70.
• Assays for testing binding are well known in the art and include, but not limited to ELISA, radioimmunoassays (RIA), flow cytometry, BiaCore, bio-layer interferometry Blitz® assay, HPLC.
• the insect belongs to the phylum Arthropoda (including, but not limited to the orders Hemiptera, Archaeognatha, Thysanura, Paleoptera and Neoptera, also ticks, mites and spiders), even more preferably to the class Insecta.
• Trogoderma spp. from the order Orthoptera, for example, Gryllotalpa spp., Locusta spp., and Schistocerca spp.; from the order Blattaria, from example, Blatta spp., Blattella spp., Periplaneta spp., and Eeucophaea maderae, from the order Isoptera, for example, Coptotermes spp. and Reticulitermes spp.; from the order Psocoptera, for example, Liposcelis spp.
• Phthiraptera suborder Anoplura, for example, Haematopinus spp., Einognathus spp., and Pediculus spp., and Trichodectes spp.; from the order Phthiraptera, suborder Ischnocera, for example, Damalinia spp.; from the order Thysanoptera, for example, Frankliniella occidentalis, Frankliniella platensis, Frankliniella spp., Hercinothrips spp., Taeniothrips spp., Thrips palmi, Thrips tabaci, Scirtothrips aurantii, Scirtothrips citri, Scirtothrips dorsalis, and Scirtothrips spp.; from the order Hemiptera, suborder Heteroptera, for example, Cimex spp., Distantiella theobroma, Dysdercus spp., Euschist
• Eygus hesperus and Eygus lineoloris such as Eygus hesperus and Eygus lineoloris, Eygaeidae family spp. such as Blissus leucopterus, and Pentatomidae family spp.; from the order Hemiptera, suborder Sternorrhyncha, for example, Aleurocanthus spiniferus, Aleurocanthus woglumi, Aleurocanthus spp., Aleurothrixus floccosus, Aleyrodes brassicae, Aonidella citrina, Aonidiella spp., Aphididae family spp., Acyrthosiphon spp., Aphis fabae, Aphis glycines, Aphis gossypii, Aphis spp., Aspidiotus spp., Bemisia tabaci, Ceroplastes
• the insect is considered as a pest.
• pest refers to an agricultural pest organism, including but not limited to whiteflies, aphids, grasshoppers, caterpillars, beetles, moths, stinkbugs, thrips, household pest organisms, such as cockroaches, ants, wasps, flies, house crickets, bed bugs, wood worms, mealworm beetles, earwigs, silverfish, termites, blood-feeding pest insects such as mosquitos, fleas and lice etc.
• the insect is an agricultural pest organism.
• the insect is selected from the group consisting of whitefly, aphid, moth, stinkbug, hopper, beetle and honeybee.
• the moth is Spodoptera frugiperda.
• the insect is a sucking pest.
• the Bemisia tabaci comprises B and/or Q biotypes (see e.g. by De Barro et al. (2003) Molecular Ecology Notes, 3(1), 40-43, the contents of which are fully incorporated herein by reference).
• Non-limiting examples of aphids include Myzus persicae, Aphis gossypii, Aphis fabae, Toxoptera citricida, Schizaphis graminum, Aulacorthum solani and Rhopalosiphum padi.
• HSP70 also known as “heat shock 70 kDa protein” refers to the polypeptide encoded by the HSP70 gene in an insect.
• HSP70, pfam PF00012 is made up of two regions: the amino terminus is the ATPase domain and the carboxyl terminus is the substrate binding region.
• the insect HSP70 is the Bemisia tabaci HSP70, such as provided e.g. in GeneBank Accession No. ADO14473, ACH85197, AAZ17399, QHB15581.
• the insect HSP70 comprises SEQ ID NO: 1.
• the nanobody specifically binds insect HSP70 and comprises three CDRs as set forth in SEQ ID NOs: 15-17, 31-33, 55-57, or 59-61 arranged in a sequential order from N to C on said nanobody
• the nanobody specifically binds insect HSP70 and comprises three CDRs as set forth in SEQ ID NOs: 15-17 arranged in a sequential order from N to C on said nanobody.
• 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 to an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 30, 54 and 58.
• 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 to SEQ ID NO: 14.
• the nanobody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 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.
• the nanobody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 30, 54 and 58.
• the nanobody consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 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.
• the nanobody consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 30, 54 and 58.
• the method further comprising isolating the nanobody.
• the binding and/or the biological activity (e.g. insect control activity) of the nanobody can be assayed either in vivo or in vitro.
• the method further comprises selecting a nanobody demonstrating an insect control activity in a biological assay.
• assays are known in the art and are further described hereinbelow.
• the nanobody comprises a heterologous effector moiety e.g. toxin moiety, detectable moiety.
• the effector moiety can be proteinaceous or non-proteinaceous (e.g. small molecule chemical compounds); the latter generally being generated using functional groups on the nanobody and on the conjugate partner.
• detectable or reporter moieties may be conjugated to the nanobody of the invention.
• a radioactive isotope such as [125] iodine
• a phosphorescent chemical such as [125] iodine
• a chemiluminescent chemical such as a fluorescent chemical or polypeptide
• fluorescent chemical or polypeptide e.g. phycoerythrin (PE), fluorescein isothiocyanate (FITC), Cy-chrome, rhodamine, green fluorescent protein (GFP), blue fluorescent protein (BFP), Texas red, Cy5, PE-Cy5, and the like
• an enzyme e.g.
• toxin or “toxin moiety” refers to a compound having an insect control activity (as defined hereinbelow) including, but not limited to, a polypeptide, a polynucleotide, a small molecule, etc.
• the effector moiety may be coupled directly or indirectly (e.g. when comprised in a carrier) to the nanobody.
• the coupling can be a covalent or non-covalent binding.
• polypeptides e.g. nanobodies and proteinaceous compositions
• polypeptides described herein can be encoded from a polynucleotide.
• These polynucleotides can be used per se or in the recombinant production of the polypeptides disclosed herein.
• a polynucleotide encoding the nanobody or the composition comprising the nanobody and the toxin.
• polynucleotide refers to a single or double stranded nucleic acid sequence which is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence and/or a composite polynucleotide sequence (e.g., a combination of the above).
• any of the polynucleotides and nucleic acid sequences disclosed herein may comprise conservative nucleic acid substitutions.
• Conservatively modified polynucleotides refer to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical or associated (e.g., naturally contiguous) sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode most proteins. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
• nucleic acid sequences disclosed herein are codon optimized for e.g. mammalian or plant expression.
• some embodiments of the invention encompasse nucleic acid sequences described hereinabove; fragments thereof, sequences hybridizable therewith, sequences homologous thereto, sequences orthologous thereto, sequences encoding similar polypeptides with different codon usage, altered sequences characterized by mutations, such as deletion, insertion or substitution of one or more nucleotides, either naturally occurring or man induced, either randomly or in a targeted fashion.
• Non-limiting examples of polynucleotides encoding the nanobody of some embodiments of the invention are provided in SEQ ID NOs: 178-221.
• a polynucleotide sequence encoding the polypeptide is preferably ligated into a nucleic acid construct suitable for expression in the cell.
• a nucleic acid construct includes a promoter sequence for directing transcription of the polynucleotide sequence in the cell in a constitutive or inducible manner.
• the nucleic acid construct of some embodiments of the invention typically includes a signal sequence for secretion of the polypeptide from a cell in which it is placed.
• the signal sequence is the native signal sequence of the polypeptide (e.g. nanobody) of some embodiments of the invention.
• pancreas-specific promoters [Edlunch et al. (1985) Science 230:912-916] or mammary gland- specific promoters such as the milk whey promoter (U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166).
• the expression of the coding sequence can be driven by a number of promoters.
• viral promoters such as the 35S RNA and 19S RNA promoters of CaMV [Brisson et al. (1984) Nature 310:511-514], or the coat protein promoter to TMV [Takamatsu et al. (1987) EMBO J. 3:17-311] can be used.
• plant promoters such as the small subunit of RUBISCO [Coruzzi et al. (1984) EMBO J.
• the promoter is preferably positioned approximately the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function.
• the expression vector of some embodiments of the invention may typically contain other specialized elements intended to increase the level of expression of cloned nucleic acids or to facilitate the identification of cells that carry the recombinant DNA.
• a number of animal viruses contain DNA sequences that promote the extra chromosomal replication of the viral genome in permissive cell types. Plasmids bearing these viral replicons are replicated episomally as long as the appropriate factors are provided by genes either carried on the plasmid or with the genome of the host cell.
• the expression vector of some embodiments of the invention can further include additional polynucleotide sequences that allow, for example, the translation of several proteins from a single mRNA such as an internal ribosome entry site (IRES) and sequences for genomic integration of the promoter-chimeric polypeptide.
• IRS internal ribosome entry site
• 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, pNMTl, pNMT41, pNMT81, which are available from Invitrogen, pCI which is available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which are available from Strategene, pTRES which is available from Clontech, and their derivatives.
• Expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses can be also used.
• SV40 vectors include pSVT7 and pMT2.
• Vectors derived from bovine papilloma virus include pBV-lMTHA, and vectors derived from Epstein Bar virus include pHEBO, and p2O5.
• the cells may be transformed stably or transiently with the nucleic acid constructs of some embodiments of the invention.
• stable transformation the nucleic acid molecule of some embodiments of the invention is integrated into the cell genome and as such it represents a stable and inherited trait.
• transient transformation the nucleic acid molecule is expressed by the cell transformed but it is not integrated into the genome and as such, it represents a transient trait.
• the constructs can be introduced into plant cells using Ti plasmid, Ri plasmid, plant viral vectors, direct DNA transformation, microinjection, electroporation and other techniques well known to the skilled artisan. See, for example, Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463.
• Introduction of nucleic acids by viral infection offers several advantages over other methods such as lipofection and electroporation, since higher transfection efficiency can be obtained due to the infectious nature of viruses.
• the construct may also include a signal that directs polyadenylation, as well as one or more restriction sites and a translation termination sequence.
• a signal that directs polyadenylation will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3' LTR or a portion thereof.
• Other vectors can be used that are non-viral, such as cationic lipids, polylysine, and dendrimers.
• the expression construct of some embodiments of the invention can also include sequences engineered to enhance stability, production, purification, yield or toxicity of the expressed peptide.
• the expression of a fusion protein or a cleavable fusion protein comprising the polypeptide of some embodiments of the invention and a heterologous protein can be engineered.
• Such a fusion protein can be designed so that the fusion protein can be readily isolated by affinity chromatography; e.g., by immobilization on a column specific for the heterologous protein.
• the polypeptide can be released from the chromatographic column by treatment with an appropriate enzyme or agent 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].
• an appropriate enzyme or agent 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].
• the present invention also contemplates cells comprising the polypeptides, polynucleotides and nucleic acid constructs described herein.
• a host cell comprising the nanobody or the composition comprising the nanobody and the toxin or a polynucleotide or a nucleic acid construct encoding same.
• prokaryotic or eukaryotic cells can be used as hostexpression systems to express the polypeptides of some embodiments of the invention.
• hostexpression systems include, but are not limited to, microorganisms, such as bacteria transformed with a recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vector containing the coding sequence; yeast transformed with recombinant yeast expression vectors containing the coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors, such as Ti plasmid, containing the coding sequence.
• Mammalian expression systems can also be used to express the polypeptides of some embodiments of the invention.
• the cell is a mammalian cell.
• the cell is a camelid cell.
• Suitable mammalian cells include primary cells and immortalized cell lines.
• the mammalian cell is an immortalized cell line.
• Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), HEK293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RATI cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, NSO, Sp2/0, BHK, Namalwa, and the like.
• HeLa cells e.g., American Type Culture Collection (ATCC) No. CCL
• the cell is E. coli e.g. SHuffle T7 Express & BL21.
• the cell is a plant cell.
• an insect control nanobody comprising expressing in a host cell the polynucleotide or the nucleic acid construct disclosed herein.
• Isolation or recovery of any of the recombinant polypeptides may be effected by any method known in the art. According to specific embodiments, recovery or isolation of the recombinant polypeptide is effected following an appropriate time in culture.
• recovery or isolation of the recombinant polypeptide is effected following an appropriate time in culture.
• the phrase "recovering the recombinant polypeptide” or “isolating the recombinant polypeptide” refers to collecting the whole fermentation medium containing the polypeptide and need not imply additional steps of separation or purification.
• the formulated compositions may be in the form of a dust or granular material, powder, gel, cream, paste, pellet, tablet or a suspension in oil (vegetable or mineral), or water or oil/water emulsions, capsule suspension, emulsifiable concentrate, or as a wettable powder, wettable granules, water dispersible granules, aerosols, foam, slurries or flowable concentrates.
• the composition is formulated as a liquid concentrate, dry powder, tablet, capsule suspension, slurry or "wet cake", which can be suitably diluted, dispersed, suspended, emulsified or otherwise suitably reconstituted by the end user prior to final use.
• the composition is formulated as a dry formulation.
• the composition is formulated for delivery by spraying, irrigation and/or fumigation.
• the compositions disclosed herein are stable, both during storage and during utilization, meaning that the integrity of the composition is maintained under storage and/or utilization conditions of the composition, which may include elevated temperatures, freeze-thaw cycles, changes in pH or in ionic strength, UV-irradiation, presence of harmful chemicals and the like.
• the integrity and activity of the composition is maintained under open field or controlled conditions, e.g., greenhouse.
• compositions disclosed herein can be formulated with various carriers designed to increase e.g. delivery, stability, permeability and the like.
• a “carrier”, as used herein, means any solid, semi-solid or liquid carrier in or on(to) which a compound (e.g. nanobody and/or toxin, composition comprising same) can be suitably incorporated, included, immobilized, adsorbed, absorbed, bound, encapsulated, embedded, attached, or comprised.
• a compound e.g. nanobody and/or toxin, composition comprising same
• Non-limiting examples of such carriers include nanocapsules, microcapsules, nanospheres, microspheres, nanoparticles, microparticles, liposomes, vesicles, beads, a gel, weak ionic resin particles, liposomes, cochleate delivery vehicles, small granules, granulates, nano-tubes, bucky-balls, water droplets that are part of an water-in-oil emulsion, oil droplets that are part of an oil-in-water emulsion, organic materials such as cork, wood or other plant-derived materials (e.g.
• 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.
• the carriers are such that they have immediate or gradual or slow-release characteristics, for example over several minutes, several hours, several days or several weeks.
• the carriers may be made of materials (e.g. polymers) that rupture or slowly degrade (for example, due to prolonged exposure to high or low temperature, sunlight, high or low humidity or other environmental factors or conditions) over time (e.g. over minutes, hours, days or weeks) and so release the compound (e.g. nanobody and/or toxin, composition comprising same) from the carrier.
• the carrier is coupled, bound, linked or otherwise attached to or associated with the compound.
• the carrier is covalently coupled to the compound.
• physiologically acceptable carrier refers to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
• An adjuvant is included under these phrases.
• excipient refers to an inert substance added to a composition to further facilitate administration of an active ingredient.
• excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, surfactant, gelatin, vegetable oils, polyethylene glycols, wetting agents, spreading agents, buffering agents, acidifiers and the like.
• the composition comprises one or more additional active agents in addition to the compositions disclosed herein (e.g. the nanobodies, the composition comprising the nanobody and the toxin, polynucleotides and nucleic acid constructs encoding it and host cells expressing it).
• additional active agents include herbicides, insecticides, plant growth regulators, toxins, safeners, insecticidal nanobodies and the like.
• composition disclosed herein and the additional active agent are in a co-formulation.
• composition disclosed herein and the additional active agent are in separate containers.
• nanobody and the compositions comprising the nanobody and the toxin of some embodiments of the invention are endowed with an insect control activity.
• the insect control activity is manifested by increased mortality of the insect in the presence of the nanobody in comparison to its mortality in the absence of the nanobody.
• the nanobody disclosed herein may have an insect control activity by itself or it may exert its insect control activity by delivering a toxin to an insect.
• binding of the nanobody to the insect HSP70 confers an insect control activity to the nanobody.
• the nanobody downregulates activity of the insect HSP70.
• determining activity refers to a decrease of at least 5 % in biological function of the insect polypeptide (i.e. HSP70) in the presence of the nanobody in comparison to its biological function in the absence of the nanobody, as determined by a method suitable for determining activity of the insect polypeptide.
• HSP70 comprises both an ATPase domain and a substrate binding domain
• the nanobody of some embodiments of the invention may bind and downregulate activity of the ATPase domain and/or the binding domain.
• determining the activity of HSP70 may be effected by e.g. ELISA, Western blot analysis, immunoprecipitation, flow cytometry, a colorimetric or fluorometric ATPase assay, or a chaperone activity assay.
• the decrease is by 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 % as compared to same in the absence of the nanobody, as may be determined by e.g. any of the methods described hereinabove.
• Non-limiting examples of toxins and methods of coupling them to the nanobody are further described hereinabove.
• the present invention also encompasses methods of insect control.
• the contacting comprises applying the nanobody or the composition comprising the nanobody and the toxin to an organism or a surface, which may be in contact with said insect.
• a method of preventing insect infestation of a commodity product comprising adding to the product the nanobody or the composition comprising the nanobody and the toxin.
• a method of preventing insect infestation of a commodity product comprising packaging the product in a packaging material comprising the nanobody or the composition comprising the nanobody and the toxin.
• the contacting or the adding may be effected 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) spraying), atomizing, brushing, dressing, dripping, coating, dipping, immersing, submerging, encrusting, spreading, foaming, fogging, injecting, adding to a culture, irrigating, applying as small droplets, a mist or an aerosol, recombinantly expressing the nucleic acid construct in a cell of an organism (as further disclosed hereinabove).
• spraying including high volume (HV), low volume (LV) and ultra low volume (ULV) spraying
• atomizing brushing
• dressing dripping
• coating dipping
• immersing submerging
• encrusting spreading
• foaming fogging
• injecting adding to a culture, irrigating, applying as small droplets, a mist or an aerosol, recombinantly expressing
• contacting or adding is effected by introducing the polynucleotide or the nucleic acid construct into a cell of the organism.
• the organism is a plant.
• plant encompasses whole plants, a grafted plant, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, roots (including tubers), rootstock, scion, fruits, vegetables, flowers and plant cells, tissues and organs.
• the plant may be in any form including suspension cultures, embryos, meristematic regions, callus tissue, leaves, gametophytes, sporophytes, pollen, and microspores.
• the plant is a crop.
• “Crop” as used herein means a plant species or variety that is grown to be harvested as food, livestock fodder, fuel raw material, or for any other economic purpose.
• the crops can be maize, cereals, such as wheat, rye, barley and oats, sorghum, rice, sugar beet and fodder beet, fruit, such as pome fruit (e.g., apples and pears), citrus fruit (e.g., oranges, lemons, limes, grapefruit, or mandarins), stone fruit (e.g., peaches, nectarines or plums), nuts (e.g., almonds or walnuts), soft fruit (e.g., cherries, strawberries, blackberries or raspberries), the plantain family or grapevines, leguminous crops, such as beans, lentils, peas and soya, oil crops, such as sunflower, safflower, rapeseed, canola,
• the plant may be selected from the group consisting of maize, soybean, alfalfa, cotton, sunflower, Brassica oil seeds such as Brassica napus (e.g. canola, rape- seed), Brassica rapa, Brassica juncea (e.g. (field) mustard) and Brassica carinata, Arecaceae sp. (e.g. oilpalm, coconut), rice, wheat, sugarbeet, sugarcane, oats, rye, barley, millet and sorghum, triticale, flax, nuts, grapes and vine and various fruit and vegetables from various botanic taxa, e.g. Rosaceae sp. (e.g.
• pome fruits such as apples and pears, but also stone fruits such as apricots, cherries, almonds, plums and peaches, and berry fruits such as strawberries, raspberries, red and black currant and gooseberry), Ribesioidae sp., Juglandaceae sp., Betulaceae sp., Anacardiaceae sp., Fagaceae sp., Moraceae sp., Oleaceae sp. (e.g. olive tree), Actinidaceae sp., Lauraceae sp. (e.g. avocado, cinnamon, camphor), Musaceae sp. (e.g.
• Rubiaceae sp. e.g. coffee
• Theaceae sp. e.g. tea
• Sterculiceae sp. e.g. lemons, oranges, mandarins and grapefruit
• Solanaceae sp. e.g. tomatoes, potatoes, peppers, capsicum, aubergines, tobacco
• Liliaceae sp. Compositae sp. (e.g. lettuce, artichokes and chicory - including root chicory, endive or common chicory), Umbelliferae sp. (e.g.
• Cucurbitaceae sp. e.g. cucumbers - including gherkins, pumpkins, watermelons, calabashes and melons
• Alliaceae sp. e.g. leeks and onions
• Cruciferae sp. e.g. white cabbage, red cabbage, broccoli, cauliflower, Brussels sprouts, pak choi, kohlrabi, radishes, horseradish, cress and Chinese cabbage
• Leguminosae sp. e.g. peanuts, peas, lentils and beans - e.g. common beans and broad beans
• Chenopodiaceae sp. e.g.
• the plant is an ornamental plant.
• the plant may be a cut flower of ornamental plants, preferably selected from Alstroemeria, carnation, Chrysanthemum, Freesia, Gerbera, Gladiolus, baby's breath (Gypsophila spp.), Helianthus, Hydrangea, Lilium, Lisianthus, roses and summer flowers.
• the plant is a cut grass or wood.
• the plant is cotton.
• the plant is a transgenic plant.
• the plant is a transgenic plant recombinantly expressing the nanobody or the composition comprising the nanobody and the toxin.
• the present invention also encompasses products comprising the nanobody or the composition comprising the nanobody and the toxin, polynucleotides or nucleic acid constructs encoding same or host cells expressing same.
• such products are more resistant to insect infestation or damage as compared to products not comprising the nanobody or the composition comprising the nanobody and the toxin, polynucleotides or nucleic acid constructs encoding same or host cells expressing same.
• a plant comprising the nanobody or the composition comprising the nanobody and the toxin, or a polynucleotide or a nucleic acid construct encoding same.
• a surface covered with the nanobody or the composition comprising the nanobody and the toxin.
• compositions, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
• range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
• a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range.
• the phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first, indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.
• the term "method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
• BL21 Star stored in glycerol was inoculated into TB medium containing kanamycin and cultured at 37 °C.
• OD600 reached 1.2
• cell culture was induced with IPTG at 15 °C for 16 hours.
• Cells were harvested by centrifugation and pellets were resuspended with lysis buffer (20mM NaPi pH7.4, 500mM NaCl, 20mM Imidazole, Protease Inhibitor cocktail - cOmpleteTM, EDTA-free Protease Inhibitor Cocktail - Roche cat#4693132001) followed by sonication and centrifugation.
• the precipitate was dissolved using urea; and the denatured supernatant was kept for future purification.
• the protein was purified from the supernatant using a Ni-NTA column (GE-Healthcare, Cat No. 17-5318-02): The column was washed with 10 bed volumes of wash buffer (20 mM NaPi pH7.4, 500 mM NaCl, 50 mM Imidazole) and the protein was eluted with elution buffer (20 mM NaPi pH7.4, 500 mM NaCl, 500 mM Imidazole).
• the fractions were placed in MEGA tubes with membrane cutoff of 3.5 kDa (Tivan Biotech MEGA3-50) and dialyzed in IE PBS pH7.4 at 4 °C overnight, followed by a second round of dialysis under the same conditions.
• the protein purity and molecular weight were determined by a standard SDS-PAGE.
• a llama was subcutaneously injected on days 0, 7, 14, 21, 28 and 35, each time with about 125 or 150 pg target protein in combination with a Gerbu adjuvant P (Gerbu Biotech, #3111-0025). On day 40, about 100 ml anti-coagulated blood was collected from the llama for lymphocyte preparation.
• VHH library A VHH library was constructed from the llama lymphocytes to screen for the presence of antigen- specific nanobodies. To this end, total RNA from peripheral blood lymphocytes was used as a template for first strand cDNA synthesis with an oligo(dT) primer. Using this cDNA, the VHH encoding sequences were amplified by PCR, digested with PstI (#ER0611 from Thermofischer) and Notl (#11037668001 from Sigma), and cloned into the PstI & Notl sites of the phagemid vector pMECS. The VHH library comprised about 10 8 independent transformants, with about 80-92 % of transformants harboring the vector with the right insert size.
• the cell culture was induced with 0.7mM IPTG at 30 °C for 20-22 hours.
• Cells were harvested by centrifugation and cell pellets were resuspended with lysis buffer (20 mM NaPi pH7.5, 500 mM NaCl, 20 mM imidazole) followed by sonication and centrifugation. The supernatant was kept for purification.
• the nanobodies were purified with a Ni-Sepharose column.
• Insect culture - Bemisia tabaci and Myzus persicae colonies were collected outdoors and maintained separately in growth chambers at 22 ⁇ 3°C under continuous lighting.
• the Bemisia tabaci were grown on cotton plants and Myzus persicae on pepper plants.
• the whitefly colony of Bemisia tabaci that was used in this study was a mix of B and Q biotypes using the procedure described by De Barro et ai. [Molecular Ecology Notes (2003) 3(1), 40-43].
• Spodoptera frugiperda colonies were collected from cotton fields in Israel and reared in the laboratory at 24 °C with 65 % relative humidity on a 10 hours day / 14 hours night photoperiod.
• the collected larvae were kept on an artificial diet with Ward’s diet powder Stonefly Heliothis Diet (Product No. 38-0600, Ward’s Natural Science, Rochester, NY) supplemented with a vitamin mixture (Nicotinic acid 40 pM, Calcium pantothenate 5 pM, Riboflavin 5 pM, Thiamine hydrochloride 4 pM, Pyridoxine hydrochloride 5 pM, Folic acid 2.5 pM, D-biotin 4 pM and Cyanocobalamin 5 nM), Sorbic acid 0.2 %, Methylparaben 0.4 %, Ascorbic acid 0.25 %, Brewer Yeast 1 %, white vinegar 4 % and Linseed oil 0.5 %.
• a vitamin mixture Naturalicotinic acid 40 pM, Calcium pantothenate 5 pM, Riboflavin 5 pM, Thiamine hydrochloride 4 pM, Pyridoxine hydrochloride 5 p
• the sachet is then sealed to prevent leakage of the test material and is placed in a perspex tube (approx. 22 mm outside diameter x 22 mm height) together with a test population of 10 nymph and/or adult aphids.
• a perspex tube approximately 22 mm outside diameter x 22 mm height
• the open ends of the tubes are closed with a parafilm seal.
• Pepper detached leaf disc bioassay for Bemisia tabaci - Detached pepper leaves were placed in glass test tubes with 3 ml of water. Following, the leaf was overlaid with a 500 pl suspension of the tested nanobody. Each tube was then placed in a 500 ml plastic container and 60 adult aphids were added and the container was closed with a 150 mesh cup. The container was incubated in the laboratory at 25 °C for 2 to 5 days; and aphid mortality was measured following 4 days of incubation. Each study was done with 4 replicates of each treatment and repeated at least twice.
• Pepper green-house plant bioassay for Myzus persicae - Pepper plants were cultivated under climate-controlled conditions at a mean daily temperature of 25 °C and 57 % humidity, with natural light exposure. When plants reached an age of six to eight weeks, 20 aphids nymph stages N2-N3 were introduced to each plant. Following a one-day acclimation period, plants were sprayed with 0.8 ml of the tested nanobody solution at a final concentration of 1 mg / ml or 3 mg / ml, or with water as a negative control. The plants were then transferred into plastic Insect Cages (BugDorm-6E610) and incubated in a climate-controlled greenhouse.
• plastic Insect Cages BugDorm-6E610
• Cotton green-house plant bioassay for Spodoptera frugiperda - Cotton plants were cultivated under climate-controlled conditions at a mean daily temperature of 25 °C and 57 % humidity, with natural light exposure. When plants reached an age of six to eight weeks, 16 Larvae L2 stage (72 hours after hatching), were introduced to each plant. Following a one-day acclimation period, plants were sprayed with 1.5 ml of the tested nanobody solution at a final concentration of 1 mg / ml or 3 mg / ml, or with water as a negative control. The plants were then transferred into plastic Insect Cages (BugDorm-6E610) and incubated in the climate-controlled greenhouse. Spodoptera frugiperda mortality was measured on days 1 and 5 post-spraying. Each study was conducted 4 times, with 3-10 independent replicates of each treatment.
• nanobodies were generated against the Bemisia tabaci HSP70 (see Tables 1-2 hereinabove).
• the insecticidal activity of the generated nanobodies was evaluated in three different bioassays, namely a liquid bioassay, a pepper leaf disc bioassay and a pepper detached leaf disc bioassay, using two different insects, Bemisia tabaci and Myzus persicae.
• HSP70-BT-1D SEQ ID NO: 14
• HSP70-BT-1H SEQ ID NO: 30
• HSP70-BT-2A SEQ ID NO: 54
• HSP-BT-2B SEQ ID NO: 58
• the insecticidal activity of the anti-HSP70 nanobody HSP70-BT-1D was evaluated in a pepper and cotton green-house plant bioassays (in which the nanobodies were applied post insect introduction), using three different insects, Myzus persicae, Bemisia tabaci and Spodoptera frugiperda.
• the anti-HSP70 nanobody HSP70-BT-1D (SEQ ID NO: 14) induced significant mortality in all assays performed. While the observed effect was clearly dose- dependent, mortality appeared already at a dose of 1 mg / ml.

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